**Meet the editors**

António Manuel Jordão is currently an assistant professor of Oenology at Polytechnic Institute of Viseu, Agrarian Higher School and member of the Chemistry Research Centre, Vila Real. He is a graduate of Agro-Industrial Engineering and has a Master's degree in Food Science and Technology from Technical University of Lisbon. In 2006, he obtained a PhD degree in Agro-In-

dustrial Engineering at Agronomy Higher Institute of Technical University of Lisbon. His research interests are phenolic compounds from grapes and wines, wine technology, and, in particular, the topic of wine aging process. He is a scientific editor of two books of enology. He is the author/coauthor of 40 articles in peer-reviewed scientific journals, 10 book chapters, and 65 communications in scientific congress. He is also a member of the editorial board of scientific journals, a supervisor of several Master's theses in enology and has developed consultancy services for wine companies and evaluation of research projects.

Maria Fernanda Gil Cosme Martins is a graduate of Agro-Industrial Engineering from Technical University of Lisbon and a PhD degree holder in Food Science at University of Trás-os-Montes and Alto Douro (UTAD). Currently, she is an assistant professor of Food Science/ Oenology at UTAD and a member of the Chemistry Research Centre, Vila Real. Her main research lines

include grape composition, wine technology, and wine stabilization and aging, namely fining, phenolic compounds, polysaccharides, and proteins. She is a scientific editor of 1 book of Oenology and author/coauthors of 22 articles in ISI journals, 4 articles in open access peer-reviewed international scientific journals, 10 book chapters, and 79 papers in conference proceedings. She has participated in 10 funded research projects and supervised PhD and MSc student thesis in the fields of Food Science and Oenology.

Contents

**Preface IX**

**Section 1 Viticulture and Environmental Conditions 1**

Carles Jiménez and Julio García

**Wine Vineyards in Chile 43**

Ximena Besoain

Manuel Oliveira

Leivas

Valdés

Chapter 1 **Recovering Ancient Grapevine Varieties: From Genetic**

**Variability to In Vitro Conservation, A Case Study 3** Carmina Gisbert, Rosa Peiró, Tania San Pedro, Antonio Olmos,

Chapter 2 **Fingerprints of Anthocyanins and Flavonols in Wild Grapes (Vitis vinifera L. ssp. sylvestris (Gmelin) Hegi) 23** Eugenio Revilla, Rosa Arroyo-Garcia, Alberto Bellido, David

Chapter 3 **Grapevine Trunk Diseases (GTDs): Impact on Table Grapes and**

Chapter 4 **Viticulture in Warmer Climates: Mitigating Environmental**

Antônio Heriberto de Castro Teixeira, Jorge Tonietto and Janice F.

Ester Gamero, Francisco Espinosa, Daniel Moreno, David Uriarte, María Henar Prieto, Inmaculada Garrido and María Esperanza

**Stress in Douro Region, Portugal 59**

Chapter 5 **Water Balance Indices for Tropical Wine Grapes 77**

Chapter 6 **Convenience of Applying of Viticulture Technique as a**

**Function of the Water Status of the Vine-Stock 91**

Carrasco, Anna Puig and Leonor Ruiz-Garcia

## Contents

#### **Preface XIII**


**Function of the Water Status of the Vine-Stock 91** Ester Gamero, Francisco Espinosa, Daniel Moreno, David Uriarte, María Henar Prieto, Inmaculada Garrido and María Esperanza Valdés

#### **X** Contents

#### **Section 2 Wine Production and Characterization 117**

Chapter 7 **The Evolution of Polyphenols from Grapes to Wines 119** Violeta-Carolina Niculescu, Nadia Paun and Roxana-Elena Ionete Chapter 16 **Revalorization of Grape Seed Oil for Innovative Non-Food**

Juan Carlos de Haro, Juan Francisco Rodríguez, Manuel Carmona

Contents **VII**

**Applications 337**

and Ángel Pérez


#### Chapter 16 **Revalorization of Grape Seed Oil for Innovative Non-Food Applications 337**

**Section 2 Wine Production and Characterization 117**

**Oenology 143** Anca Şipoş

**VI** Contents

Guadalupe

Domingo Rios-Mesa

Alexandra Marta-Costa

Hidalgo and Imma Andorrà

Chapter 7 **The Evolution of Polyphenols from Grapes to Wines 119**

Chapter 8 **Current State and Perspective in the Models Applicable to**

Chapter 9 **Microbiological, Physical, and Chemical Procedures to Elaborate High-Quality SO2-Free Wines 171**

Chapter 10 **Influence of Wine Chemical Compounds on the Foaming**

Chapter 11 **Occurrence and Analysis of Sulfur Compounds in Wine 225**

Chapter 13 **Novel Memory-Based Sensory Approach to Assess Large-Scale**

**Section 3 Economic Analysis and Valorization of Wine Products 305**

Chapter 14 **Wine Firm's Size and Economic Performance: Evidence from Traditional Portuguese Wine Regions 307**

Chapter 15 **Potential for Use of the Residues of the Wine Industry in Human Nutrition and as Agricultural Input 325** Renato Vasconcelos Botelho, Gabriela Datsch Bennemann, Yohandra Reyes Torres and Alessandro Jefferson Sato

Leticia Martínez-Lapuente, Belén Ayestarán and Zenaida

**Properties of Sparkling Wines 195**

Daniela Fracassetti and Ileana Vigentini

Chapter 12 **Characterization of Red Wines from Macaronesia 253**

Violeta-Carolina Niculescu, Nadia Paun and Roxana-Elena Ionete

Raúl Ferrer-Gallego, Miquel Puxeu, Laura Martín, Enric Nart, Claudio

Jesús Heras-Roger, Carlos Díaz-Romero, Jacinto Darias-Martín and

**Typicality: The Case of Mainland Portugal Red Wines 275** Anibal Jose-Coutinho, Patricia Avila and Jorge M. Ricardo-da-Silva

João Rebelo, Sofia Gouveia, Lina Lourenço-Gomes and Ana

Juan Carlos de Haro, Juan Francisco Rodríguez, Manuel Carmona and Ángel Pérez

Preface

quality of the different wine products.

the economic analysis of the wine sector.

and valorization of grapes and wines.

persistent advancements in all parts of the production chain.

Grape and wine sector has probably inspired more research and publications than any other agricultural and food area. In fact, through their passion for vine, wine, and derived prod‐ ucts, a great number of scientists not only have contributed to the development of viticultur‐ al practices but also have made advances in the winemaking technological process, in the wine aging conditions, in the valorization of wine products and their by-products, in the grape and wine characterization, and finally in the economic characterization of the wine sector. In addition, in the last years, the impact of climatic changes on viticulture practices has also been an important research line in order to analyze how the vine adapts and resists to new environmental conditions that affect several wine regions. Each applied develop‐ ment in grapes and wines has led to a better control of the physicochemical and sensory

The main goal of vine and wine production is to ensure a high production level combined with a high quality standard of grapes and wine products. During the whole life of the vine cycle, as well as, during all stages of grape development, different factors affect the grape‐ vine and the development and composition of the grapes, namely, climate, soil, wine-grow‐ ing practices, and the genetic potential that each grape variety presents. In addition, wine production and preservation also involve numerous factors of microbiological, chemical, and technological innovation, which will contribute decisively to the wine quality and de‐ rived products. In addition, the grape and wine production is a very important economic activity and, as such, economic and social dimension is still a central theme for the sustaina‐ bility of the sector. Thus, the determined evolution of the grape and wine industry demands

The goal of this book is to summarize in a concise manner the accumulated information about the most recent developments in grape and wine production, since the different ori‐ gins and location of grape production, environmental factors that determine their produc‐ tion, the vine sanitation problems and the diverse cultural practices, the production, characterization and conservation of wines, the valorization of wine by-products, and finally

This book is composed of three different general sections: (1) Viticulture and Environmental Conditions, (2) Wine Production and Characterization, and (3) Economic Analysis and Val‐ orization of Wine Products. Inside these 3 general sections, 16 different chapters provide current research on different topics of recent advances on production, processing, analysis,

Thus, inside the first section (Viticulture and Environmental Conditions), Chapter 1 focuses on an example of the recovery of two ancient Spanish grapevine varieties taking into consid‐

## Preface

Grape and wine sector has probably inspired more research and publications than any other agricultural and food area. In fact, through their passion for vine, wine, and derived prod‐ ucts, a great number of scientists not only have contributed to the development of viticultur‐ al practices but also have made advances in the winemaking technological process, in the wine aging conditions, in the valorization of wine products and their by-products, in the grape and wine characterization, and finally in the economic characterization of the wine sector. In addition, in the last years, the impact of climatic changes on viticulture practices has also been an important research line in order to analyze how the vine adapts and resists to new environmental conditions that affect several wine regions. Each applied develop‐ ment in grapes and wines has led to a better control of the physicochemical and sensory quality of the different wine products.

The main goal of vine and wine production is to ensure a high production level combined with a high quality standard of grapes and wine products. During the whole life of the vine cycle, as well as, during all stages of grape development, different factors affect the grape‐ vine and the development and composition of the grapes, namely, climate, soil, wine-grow‐ ing practices, and the genetic potential that each grape variety presents. In addition, wine production and preservation also involve numerous factors of microbiological, chemical, and technological innovation, which will contribute decisively to the wine quality and de‐ rived products. In addition, the grape and wine production is a very important economic activity and, as such, economic and social dimension is still a central theme for the sustaina‐ bility of the sector. Thus, the determined evolution of the grape and wine industry demands persistent advancements in all parts of the production chain.

The goal of this book is to summarize in a concise manner the accumulated information about the most recent developments in grape and wine production, since the different ori‐ gins and location of grape production, environmental factors that determine their produc‐ tion, the vine sanitation problems and the diverse cultural practices, the production, characterization and conservation of wines, the valorization of wine by-products, and finally the economic analysis of the wine sector.

This book is composed of three different general sections: (1) Viticulture and Environmental Conditions, (2) Wine Production and Characterization, and (3) Economic Analysis and Val‐ orization of Wine Products. Inside these 3 general sections, 16 different chapters provide current research on different topics of recent advances on production, processing, analysis, and valorization of grapes and wines.

Thus, inside the first section (Viticulture and Environmental Conditions), Chapter 1 focuses on an example of the recovery of two ancient Spanish grapevine varieties taking into consid‐ eration several topics from genetic variability to *In vitro* conservation. Chapter 2 evaluates the anthocyanin and flavonol composition of wild grapes and determines whether some wild genotypes present some genetic characters of interest related to anthocyanin and flavo‐ nol accumulation during grape maturation. Chapter 3 discusses the impact of the main grapevine trunk diseases that are affecting *Vitis vinifera* in Chile, while Chapter 4 discusses the viticulture in warmer climates and the use of several mitigation techniques (irrigation, canopy shading, water nebulization, and kaolin coating) to counteract the worse effects of adverse weather conditions associated to the climate changes in a specific Portuguese winedemarcated region. Other researchers discuss in Chapter 5 the elaboration and application of water balance indices and improving them by using a geographic information system in the wine grape–growing regions located in semiarid regions of Brazil. Finally, Chapter 6, analyzes the effects of the combination of water deficit and thinning on the constitution and composition of the *Tempranillo* grape variety, namely on the lipid peroxidation, oxidant and antioxidant enzyme activities and their interactions on phenolic profile, and chromatic char‐ acteristic of the wines.

of grape and wine research and discovering the most recent tendencies of production, proc‐

Finally, it was with great pleasure that we accepted the opportunity offered by InTechOpen Editors to assemble and edit this book. We are greatly indebted to the authors that gener‐ ously shared their scientific knowledge and experience with others through their contribu‐

**Prof. António Manuel Jordão (M.Sc., PhD)**

Department of Food Industries, Portugal

Assistant Professor of Food Science and Oenology Chemical Research Centre of Vila Real (CQ-VR) University of Trás-os-Montes and Alto Douro School of Life Science and Environment, Portugal

Assistant Professor of Oenology Polytechnic Institute of Viseu Agrarian Higher School

Preface XI

**Prof. Fernanda Cosme (PhD)**

essing, analysis, and valorization of grape and wine products.

tion to this book.

In the second section (Wine Production and Characterization), several perspectives of wine production and characterization are exposed. Thus, Chapter 7 focuses on the chemical struc‐ tures of the main polyphenols in grape and wines, followed by their identification and quantification, while in Chapter 8, the thematic of the application of mathematical models applicable to enology, in particular, the nonlinear models for white wine alcoholic fermenta‐ tion process, is approached. Other researchers discuss in Chapter 9 an important subject in modern oenology, which is the elaboration of SO2-free wines and the microbiological, physi‐ cal, and chemical factors that influence it. In Chapter 10, particular attention is given to spar‐ kling wines. The authors of this chapter analyze the influence of wine chemical compounds on the foaming properties of sparkling wines. Chapter 11 discusses the aromatic profile of wines, in particular the occurrence and analysis of sulfur compounds in wines, while in Chapter 12 the thematic of the chemical characterization of red wines produced in eight At‐ lantic islands from Canary and Cape Verde archipelago and also a comparative analysis with the red wines produced in other Atlantic islands (Azores and Madeira) are discussed. Finally, in Chapter 13, the authors approach the thematic of wine sensory analyses by the use of a new novel memory-based sensory approach to asses a large-scale typicality of main‐ land Portuguese red wines.

In the last section (Economic Analysis and Valorization of Wine Products), several stand‐ points of wine economy and the potential valorization of wine products are exposed. Thus, in Chapter 14, the authors analyze the economic performance of wineries (from two Portu‐ guese wine regions), using indicators widely employed in economic and business literature, and assess the potential influence of size on a firm´s performance. The authors in Chapter 15 focus on the potential use of winery residues as nutrition for humans and as agricultural input, and finally other researchers discuss in Chapter 16 the potential utilization of grape seed oil for producing bio-based materials through environmentally friendly processes that could substitute petroleum-derived products.

All of these chapters are written by a group of international researchers, in order to provide up-to-date reviews, overviews, and summaries of current research on the different dimen‐ sions of grape and wine production. This book is not only for technicians actively engaged in the field but also for students attending technical schools and/or universities and other professionals that might be interested in reading and learning about some fascinating areas of grape and wine research and discovering the most recent tendencies of production, proc‐ essing, analysis, and valorization of grape and wine products.

eration several topics from genetic variability to *In vitro* conservation. Chapter 2 evaluates the anthocyanin and flavonol composition of wild grapes and determines whether some wild genotypes present some genetic characters of interest related to anthocyanin and flavo‐ nol accumulation during grape maturation. Chapter 3 discusses the impact of the main grapevine trunk diseases that are affecting *Vitis vinifera* in Chile, while Chapter 4 discusses the viticulture in warmer climates and the use of several mitigation techniques (irrigation, canopy shading, water nebulization, and kaolin coating) to counteract the worse effects of adverse weather conditions associated to the climate changes in a specific Portuguese winedemarcated region. Other researchers discuss in Chapter 5 the elaboration and application of water balance indices and improving them by using a geographic information system in the wine grape–growing regions located in semiarid regions of Brazil. Finally, Chapter 6, analyzes the effects of the combination of water deficit and thinning on the constitution and composition of the *Tempranillo* grape variety, namely on the lipid peroxidation, oxidant and antioxidant enzyme activities and their interactions on phenolic profile, and chromatic char‐

In the second section (Wine Production and Characterization), several perspectives of wine production and characterization are exposed. Thus, Chapter 7 focuses on the chemical struc‐ tures of the main polyphenols in grape and wines, followed by their identification and quantification, while in Chapter 8, the thematic of the application of mathematical models applicable to enology, in particular, the nonlinear models for white wine alcoholic fermenta‐ tion process, is approached. Other researchers discuss in Chapter 9 an important subject in modern oenology, which is the elaboration of SO2-free wines and the microbiological, physi‐ cal, and chemical factors that influence it. In Chapter 10, particular attention is given to spar‐ kling wines. The authors of this chapter analyze the influence of wine chemical compounds on the foaming properties of sparkling wines. Chapter 11 discusses the aromatic profile of wines, in particular the occurrence and analysis of sulfur compounds in wines, while in Chapter 12 the thematic of the chemical characterization of red wines produced in eight At‐ lantic islands from Canary and Cape Verde archipelago and also a comparative analysis with the red wines produced in other Atlantic islands (Azores and Madeira) are discussed. Finally, in Chapter 13, the authors approach the thematic of wine sensory analyses by the use of a new novel memory-based sensory approach to asses a large-scale typicality of main‐

In the last section (Economic Analysis and Valorization of Wine Products), several stand‐ points of wine economy and the potential valorization of wine products are exposed. Thus, in Chapter 14, the authors analyze the economic performance of wineries (from two Portu‐ guese wine regions), using indicators widely employed in economic and business literature, and assess the potential influence of size on a firm´s performance. The authors in Chapter 15 focus on the potential use of winery residues as nutrition for humans and as agricultural input, and finally other researchers discuss in Chapter 16 the potential utilization of grape seed oil for producing bio-based materials through environmentally friendly processes that

All of these chapters are written by a group of international researchers, in order to provide up-to-date reviews, overviews, and summaries of current research on the different dimen‐ sions of grape and wine production. This book is not only for technicians actively engaged in the field but also for students attending technical schools and/or universities and other professionals that might be interested in reading and learning about some fascinating areas

acteristic of the wines.

X Preface

land Portuguese red wines.

could substitute petroleum-derived products.

Finally, it was with great pleasure that we accepted the opportunity offered by InTechOpen Editors to assemble and edit this book. We are greatly indebted to the authors that gener‐ ously shared their scientific knowledge and experience with others through their contribu‐ tion to this book.

#### **Prof. António Manuel Jordão (M.Sc., PhD)**

Assistant Professor of Oenology Polytechnic Institute of Viseu Agrarian Higher School Department of Food Industries, Portugal

#### **Prof. Fernanda Cosme (PhD)**

Assistant Professor of Food Science and Oenology Chemical Research Centre of Vila Real (CQ-VR) University of Trás-os-Montes and Alto Douro School of Life Science and Environment, Portugal

**Section 1**

**Viticulture and Environmental Conditions**

**Viticulture and Environmental Conditions**

**Chapter 1**

Provisional chapter

**Recovering Ancient Grapevine Varieties: From Genetic**

DOI: 10.5772/intechopen.71133

Recovering Ancient Grapevine Varieties: From Genetic

A great number of varieties have been described in grapevine; however, few of them are currently in use. The increasing concern on varietal diversity loss has encouraged actions for recovering and preserving grapevine germplasm, which represents valuable resources for breeding as well as for diversification in grapevine-derived products. On the other hand, it is expected that this important crop, which is distributed in warm areas worldwide, will suffer the climate changes. Therefore, it is also convenient the identification of intravarietal variability and the recovery of accessions well adapted to particular environments. In this chapter, we will contribute to highlight the importance of recovering ancient materials, the usefulness of SSR markers to determine their molecular profile, the importance to analyze their virus status, and the possibilities that offer biotechnological tools for virus sanitation and in vitro storage as a complement of field preservation. In this context, we have evaluated different grapevine accessions and developed in vitro culture protocols for micropropagation, sanitation, and storage grapevine cultivars. In this work, we report the results obtained for the historic variety "Valencí Blanc" (or "Beba") and the historic and endangered variety "Esclafagerres"

Keywords: Valencí Blanc, Beba, Esclafagerres, Esclafacherres, virus, sanitation,

Grapevine (Vitis vinifera L.) is a crop of major economic importance distributed in warm areas worldwide [1] with a wine production of 2910 million hectoliters in 2014 and 75,866 square kilometers dedicated to grapevine culture [2]. The majority of the world's wine-producing

> © The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and eproduction in any medium, provided the original work is properly cited.

© 2018 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

**Variability to In Vitro Conservation, A Case Study**

Variability to In Vitro Conservation, A Case Study

Carmina Gisbert, Rosa Peiró, Tania San Pedro,

Carmina Gisbert, Rosa Peiró, Tania San Pedro,

Additional information is available at the end of the chapter

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/intechopen.71133

("Esclafacherres" or "Esclafacherris").

varietal identification, in vitro culture

Julio García

Abstract

1. Introduction

Antonio Olmos, Carles Jiménez and

Antonio Olmos, Carles Jiménez and Julio García

#### **Recovering Ancient Grapevine Varieties: From Genetic Variability to In Vitro Conservation, A Case Study** Recovering Ancient Grapevine Varieties: From Genetic Variability to In Vitro Conservation, A Case Study

DOI: 10.5772/intechopen.71133

Carmina Gisbert, Rosa Peiró, Tania San Pedro, Antonio Olmos, Carles Jiménez and Julio García Carmina Gisbert, Rosa Peiró, Tania San Pedro, Antonio Olmos, Carles Jiménez and

Additional information is available at the end of the chapter Julio García Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/intechopen.71133

#### Abstract

A great number of varieties have been described in grapevine; however, few of them are currently in use. The increasing concern on varietal diversity loss has encouraged actions for recovering and preserving grapevine germplasm, which represents valuable resources for breeding as well as for diversification in grapevine-derived products. On the other hand, it is expected that this important crop, which is distributed in warm areas worldwide, will suffer the climate changes. Therefore, it is also convenient the identification of intravarietal variability and the recovery of accessions well adapted to particular environments. In this chapter, we will contribute to highlight the importance of recovering ancient materials, the usefulness of SSR markers to determine their molecular profile, the importance to analyze their virus status, and the possibilities that offer biotechnological tools for virus sanitation and in vitro storage as a complement of field preservation. In this context, we have evaluated different grapevine accessions and developed in vitro culture protocols for micropropagation, sanitation, and storage grapevine cultivars. In this work, we report the results obtained for the historic variety "Valencí Blanc" (or "Beba") and the historic and endangered variety "Esclafagerres" ("Esclafacherres" or "Esclafacherris").

Keywords: Valencí Blanc, Beba, Esclafagerres, Esclafacherres, virus, sanitation, varietal identification, in vitro culture

## 1. Introduction

Grapevine (Vitis vinifera L.) is a crop of major economic importance distributed in warm areas worldwide [1] with a wine production of 2910 million hectoliters in 2014 and 75,866 square kilometers dedicated to grapevine culture [2]. The majority of the world's wine-producing

© 2018 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

© The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and eproduction in any medium, provided the original work is properly cited.

regions are found between the temperate latitudes of 30 and 50 in both hemispheres [3]. Grapes are mainly used for making wine but also can be eaten fresh as table grapes or used for making jam, juice, jelly, grape seed extract, raisins, vinegar, and grape seed oil. Approximately 71% of world grape production is used for wine, 27% as fresh fruit, and 2% as dried fruit. In the Vitis International Variety Catalog (VIVC; http://www.vivc.de/), supported by Biodiversity and the International Organization of Vine and Wine (OIV), there are around 24,500 accessions which include cultivars, breeding lines, and different Vitis species. Around 50% (12,679) of the varieties correspond to Vitis vinifera Linné Subsp. vinifera (or sativa), and 30% (7714) correspond to Vitis interspecific crossing. Around 25% of the cultivars were registered in France (5602), followed by the United States (2401) and Italy (2348) with approximately 10% each one. Spain has registered a total of 734 varieties, being most of them (631) V. vinifera. According to Lacombe [4], a total of 1902 grape varieties (both scions and rootstocks) are officially authorized for cultivation in at least one country of the European Union. Around 65% of these grape varieties are registered only in one country, meaning the responsibility to preserve these varieties is too focused. On the other hand, four varieties ("Cabernet Sauvignon," "Merlot," "Chardonnay Blanc," and "Sauvignon Blanc") were maintained in at least 60 different institutions. Nowadays, not only these cultivars but also "Syrah" (or "Shiraz") dominates vineyards worldwide [5]. Considering that most major wine-producing regions could become by 2050 unsuitable for currently grown cultivars [6, 7], the preservation of genetic variability and the selection and/or development of cultivars well adapted to upcoming climate changes are important. The long juvenile period of grapevine makes breeding a slow process; therefore, the knowledge of the raw material and their availability is very important to speed up breeding programs.

that were also affected by the phylloxera aphid (Daktulosphaira vitifoliae Fitch) introduced from North America during the second part of the nineteenth century [26]. The phylloxera pest devastated the vineyards in all Europe. Since that time, grapevines need to be grafted onto phylloxera-resistant rootstocks. This fact reduced the number of grapevine cultivars used as scions which provoke genetic erosion [12]. On the other hand, the creation of denominations of origin (DO), each one including a reduced number of authorized varieties, has also contributed to reduce the varieties cultured in a specific area. Therefore, the preservation of grapevine minor cultivar and that on risk of disappearance together with Vitis vinifera subsp. sylvestris is a major

Recovering Ancient Grapevine Varieties: From Genetic Variability to In Vitro Conservation, A Case Study

http://dx.doi.org/10.5772/intechopen.71133

5

The importance of germplasm preservation focused on their putative use, in the present or in the future. It is the source of genes to face new pathogens or climate constraints. Genetic diversity of grapevine is maintained normally as living plants in the field [27, 28]. Several important ex situ grapevine collections exist like "The Domaine de Vassal" in Montpellier (France), the "Julius Kühn Institute" in Siebeldingen (Germany), and "La colección de vides de El Encín" in the IMIDRA (Instituto Madrileño de Investigación y Desarrollo Rural, Agrario y Alimentario) center of Alcalá de Henares (Spain). The French collection houses 7800 accessions ca 50 countries, representing 2300 different grape varieties, including wild species, rootstocks, hybrids, and mutants. Its transfer to the INRA Pech Rouge Experimental Unit (Gruissan, Aude) is under progress (https://www6.montpellier.inra.fr/vassal\_eng/). The German collection of the Institute for Grapevine Breeding Geilweilerhof holds more than 3000 accessions of cultivars and wild species as well as important breeding lines. Beyond many others old and neglected cultivars from Germany, Switzerland, and Austria and rare germplasm from Eastern Europe can be found (https://www.julius-kuehn.de/en/grapevine-breeding/fieldsof-activity/genetic-resources-and-information-centre-vine-and-wine/). The Spanish collection consists of 3532 accessions that are grouped into 852 rootstocks; 69 interspecific hybrids; 111 Vitis spp.; 1852 V. vinifera varieties, of which 1178 are for wine use and 674 for table use; and 648 V. vinifera sylvestris (http://www.madrid.org/coleccionvidencin/index.php?option=com\_content&view=article&id=9&Itemid=2). In order to avoid the loss of the stored materials which are exposed to environmental disasters and pest attacks, the duplication of accessions for storage in

different collections is a common strategy, although limited by budget constraints.

Another complementary strategy very useful in vegetative propagated plants is the in vitro preservation that offers the possibility to maintain plants under controlled and slow growing conditions and their micropropagation and transference to the field when need it. Although the first attempts to store grapevine under in vitro culture conditions were reported in the 1980s [29–31], this strategy is not usual in grapevine, although it is commonly used in other vegetative propagated crops like banana (preserved both through standard in vitro conditions and cryopreservation in the International Network for the Improvement of Banana and Plantain germplasm bank, in Leuven, Belgium). Cryopreservation is the storage of viable tissues, generally meristems or embryos, at ultralow temperature [32]. The success of in vitro conservation is tightly related to the choice of

stake in grapevine preservation.

1.2. Grapevine preservation

#### 1.1. Grapevine: gain and loss of diversity

Vitis vinifera subsp. vinifera was domesticated in the Neolithic period (ca 8500–4000 BC) [8] from wild grapevines (Vitis vinifera L. subsp. sylvestris (Gmelin) Hegi) [9–12]. Grapevine domestication appears to have occurred between the seventh and the fourth millennia BC, in a geographical area between the Black Sea and Iran [8, 13–16], and the earliest evidence for large-scale winemaking was found in the North of Zagros Mountains and in the Caucasian region around 6000–5000 BC [17]. Cuttings of cultivated grapevines would have been spread by humans in the Near East, Middle East, and Central Europe. As a result, these areas may have constituted secondary domestication centers [18, 19] where spontaneous hybridizations among cultivars or local wild plants generated the pattern of admixture that is observed in current cultivars [19–23]. In consequence, genetic variability of grapevine has increased due to the contribution of different genetic pools in the process of grapevine spreading. The appearance of spontaneous mutations [24] and the selective pressure by humans which depended on the different uses of grapevine (fresh consumption, raisin, or wine production) [25] were also contributed to increase the genetic variability of this crop.

Along the years, genetic erosion has occurred in both cultivated and wild grapevines. Anthropogenic pressure on the wild natural habitats greatly decreased the wild grapevine populations that were also affected by the phylloxera aphid (Daktulosphaira vitifoliae Fitch) introduced from North America during the second part of the nineteenth century [26]. The phylloxera pest devastated the vineyards in all Europe. Since that time, grapevines need to be grafted onto phylloxera-resistant rootstocks. This fact reduced the number of grapevine cultivars used as scions which provoke genetic erosion [12]. On the other hand, the creation of denominations of origin (DO), each one including a reduced number of authorized varieties, has also contributed to reduce the varieties cultured in a specific area. Therefore, the preservation of grapevine minor cultivar and that on risk of disappearance together with Vitis vinifera subsp. sylvestris is a major stake in grapevine preservation.

#### 1.2. Grapevine preservation

regions are found between the temperate latitudes of 30 and 50 in both hemispheres [3]. Grapes are mainly used for making wine but also can be eaten fresh as table grapes or used for making jam, juice, jelly, grape seed extract, raisins, vinegar, and grape seed oil. Approximately 71% of world grape production is used for wine, 27% as fresh fruit, and 2% as dried fruit. In the Vitis International Variety Catalog (VIVC; http://www.vivc.de/), supported by Biodiversity and the International Organization of Vine and Wine (OIV), there are around 24,500 accessions which include cultivars, breeding lines, and different Vitis species. Around 50% (12,679) of the varieties correspond to Vitis vinifera Linné Subsp. vinifera (or sativa), and 30% (7714) correspond to Vitis interspecific crossing. Around 25% of the cultivars were registered in France (5602), followed by the United States (2401) and Italy (2348) with approximately 10% each one. Spain has registered a total of 734 varieties, being most of them (631) V. vinifera. According to Lacombe [4], a total of 1902 grape varieties (both scions and rootstocks) are officially authorized for cultivation in at least one country of the European Union. Around 65% of these grape varieties are registered only in one country, meaning the responsibility to preserve these varieties is too focused. On the other hand, four varieties ("Cabernet Sauvignon," "Merlot," "Chardonnay Blanc," and "Sauvignon Blanc") were maintained in at least 60 different institutions. Nowadays, not only these cultivars but also "Syrah" (or "Shiraz") dominates vineyards worldwide [5]. Considering that most major wine-producing regions could become by 2050 unsuitable for currently grown cultivars [6, 7], the preservation of genetic variability and the selection and/or development of cultivars well adapted to upcoming climate changes are important. The long juvenile period of grapevine makes breeding a slow process; therefore, the knowledge of the raw material and their availability is very important to speed up

4 Grapes and Wines - Advances in Production, Processing, Analysis and Valorization

Vitis vinifera subsp. vinifera was domesticated in the Neolithic period (ca 8500–4000 BC) [8] from wild grapevines (Vitis vinifera L. subsp. sylvestris (Gmelin) Hegi) [9–12]. Grapevine domestication appears to have occurred between the seventh and the fourth millennia BC, in a geographical area between the Black Sea and Iran [8, 13–16], and the earliest evidence for large-scale winemaking was found in the North of Zagros Mountains and in the Caucasian region around 6000–5000 BC [17]. Cuttings of cultivated grapevines would have been spread by humans in the Near East, Middle East, and Central Europe. As a result, these areas may have constituted secondary domestication centers [18, 19] where spontaneous hybridizations among cultivars or local wild plants generated the pattern of admixture that is observed in current cultivars [19–23]. In consequence, genetic variability of grapevine has increased due to the contribution of different genetic pools in the process of grapevine spreading. The appearance of spontaneous mutations [24] and the selective pressure by humans which depended on the different uses of grapevine (fresh consumption, raisin, or wine production) [25] were also contributed to increase the genetic vari-

Along the years, genetic erosion has occurred in both cultivated and wild grapevines. Anthropogenic pressure on the wild natural habitats greatly decreased the wild grapevine populations

breeding programs.

ability of this crop.

1.1. Grapevine: gain and loss of diversity

The importance of germplasm preservation focused on their putative use, in the present or in the future. It is the source of genes to face new pathogens or climate constraints. Genetic diversity of grapevine is maintained normally as living plants in the field [27, 28]. Several important ex situ grapevine collections exist like "The Domaine de Vassal" in Montpellier (France), the "Julius Kühn Institute" in Siebeldingen (Germany), and "La colección de vides de El Encín" in the IMIDRA (Instituto Madrileño de Investigación y Desarrollo Rural, Agrario y Alimentario) center of Alcalá de Henares (Spain). The French collection houses 7800 accessions ca 50 countries, representing 2300 different grape varieties, including wild species, rootstocks, hybrids, and mutants. Its transfer to the INRA Pech Rouge Experimental Unit (Gruissan, Aude) is under progress (https://www6.montpellier.inra.fr/vassal\_eng/). The German collection of the Institute for Grapevine Breeding Geilweilerhof holds more than 3000 accessions of cultivars and wild species as well as important breeding lines. Beyond many others old and neglected cultivars from Germany, Switzerland, and Austria and rare germplasm from Eastern Europe can be found (https://www.julius-kuehn.de/en/grapevine-breeding/fieldsof-activity/genetic-resources-and-information-centre-vine-and-wine/). The Spanish collection consists of 3532 accessions that are grouped into 852 rootstocks; 69 interspecific hybrids; 111 Vitis spp.; 1852 V. vinifera varieties, of which 1178 are for wine use and 674 for table use; and 648 V. vinifera sylvestris (http://www.madrid.org/coleccionvidencin/index.php?option=com\_content&view=article&id=9&Itemid=2). In order to avoid the loss of the stored materials which are exposed to environmental disasters and pest attacks, the duplication of accessions for storage in different collections is a common strategy, although limited by budget constraints.

Another complementary strategy very useful in vegetative propagated plants is the in vitro preservation that offers the possibility to maintain plants under controlled and slow growing conditions and their micropropagation and transference to the field when need it. Although the first attempts to store grapevine under in vitro culture conditions were reported in the 1980s [29–31], this strategy is not usual in grapevine, although it is commonly used in other vegetative propagated crops like banana (preserved both through standard in vitro conditions and cryopreservation in the International Network for the Improvement of Banana and Plantain germplasm bank, in Leuven, Belgium). Cryopreservation is the storage of viable tissues, generally meristems or embryos, at ultralow temperature [32]. The success of in vitro conservation is tightly related to the choice of

an adequate conservation method with the development of the corresponding methodology [33].

In grapevine, it is convenient to check by molecular markers that the variety to be stored really corresponds to it because homonymies (similar name for different cultivars) and/or synonymies (different names for a same cultivar) are commonly found. The identification of homonymies is important to avoid the loss of variability (loss of genotypes). On the contrary, the detection of synonymies avoids the maintenance of duplicated materials that do not contribute to increase variability but increase the cost. It is also very important to check the sanitary status of the plants, sanitize them if necessary, and provide suboptimal culture conditions that limit and slow down plant development, without causing physiological damage to the plant material. Grapevine can be infected by numerous viruses [34], and a high incidence of virus infection is commonly found in autochthonous cultivars [35, 36]. The EU Directive 2002/11/EC rules require that the initial plant material for vegetative propagation is free of Grapevine fanleaf virus (GFLV), Arabis mosaic virus (ArMV), Grapevine fleck virus (GFkV), Grapevine leafroll–associated virus-1 (GLRaV-1), and Grapevine leafroll–associated virus-3 (GLRaV-3). When samples to be storage are virus infected, different approaches to regenerated virus-free plants can be used. Since the middle late of the twentieth century, meristem culture and thermotherapy were applied with this aim for grapevine sanitation [37, 38]. Other techniques such as chemotherapy, electrotherapy, cryotherapy, and somatic embryogenesis were also reported [39–41].

For in vitro preservation under standard or limiting conditions, the development of protocols adjusted to the variety to be preserved is necessary. For this kind of storage, it is important to choose the adequate culture medium and environmental conditions in order to reduce the number of subcultures and hence minimize the cost and the putative errors that could arise in each subculture. Protocols for storage grapevine under in vitro culture have been reported by several authors [42, 43]. Recently, we reported the effectiveness of the MW medium to store a broad spectrum of grapevine cultivars, including endangered varieties, as well as the modifications of this medium (reduction of sucrose or elimination of indole-3-butyric acid (IBA) in the medium) to reduce growth in the faster growing varieties [44]. Cryopreservation protocols have also been developed for grapevine [45– 47]. When developing cryopreservation protocols, the evaluation of the cryopreservation solution toxicity in the varieties to be preserved is required. Pre-culture in culture media that facilitate the dehydration of tissues (i.e., in medium with high content of sugars) is also a common step.

2. Recovering ancient germplasm at the Comunitat Valenciana: the

The Comunitat Valenciana which includes the provinces of Alicante, Valencia, and Catellón is located in the Mediterranean coast of Spain and has been an important viticulture area since historic times to nowadays. For instance, in the archeological place of "L'Alt de Benimaquia" (Denia, Alicante), dated back to the end of the seventh century BC, significant quantities of vinification residues (tartaric acid and seeds) were found [49]. Important vineyards must be also located in the Requena-Utiel plateau (Valencia) as evidenced by the big presses dated in the fifth century BC found at Las Pilillas site [50]. Nowadays there exist three DO for wine production (DO Alicante (http://www.vinosalicantedop.org/), DO Valencia (http://www.dovalencia.info/), and DO Utiel-Requena (http://utielrequena.org/)), one DO for table grape (DO Uva de mesa del Vinalopó (http://uva-vinalopo.org/wp/)) and one protected geographical indication in Castellón

Recovering Ancient Grapevine Varieties: From Genetic Variability to In Vitro Conservation, A Case Study

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7

historic varieties "Valencí Blanc" and "Esclafagerres"

Figure 1. Scheme including the main steps required for germplasm storage.

2.1. Richness of grapevine germplasm in the Comunitat Valenciana

(http://www.igpcastello.com/).

Finally, studies of genetic variability among varieties under conservation are important because they allow the detection of mutations and the study of relationships among them. The determination of molecular profiles is also of great importance in the development of core collections which represent the higher variability present in a whole collection in a reduced number of accessions. The core collections are very useful for breeders because studies on the core collection provide an overall view of the properties to be found in the whole collection [48]. In the following schema, the different steps to be carried out for germplasm storage are shown (Figure 1).

Recovering Ancient Grapevine Varieties: From Genetic Variability to In Vitro Conservation, A Case Study http://dx.doi.org/10.5772/intechopen.71133 7

Figure 1. Scheme including the main steps required for germplasm storage.

an adequate conservation method with the development of the corresponding metho-

6 Grapes and Wines - Advances in Production, Processing, Analysis and Valorization

In grapevine, it is convenient to check by molecular markers that the variety to be stored really corresponds to it because homonymies (similar name for different cultivars) and/or synonymies (different names for a same cultivar) are commonly found. The identification of homonymies is important to avoid the loss of variability (loss of genotypes). On the contrary, the detection of synonymies avoids the maintenance of duplicated materials that do not contribute to increase variability but increase the cost. It is also very important to check the sanitary status of the plants, sanitize them if necessary, and provide suboptimal culture conditions that limit and slow down plant development, without causing physiological damage to the plant material. Grapevine can be infected by numerous viruses [34], and a high incidence of virus infection is commonly found in autochthonous cultivars [35, 36]. The EU Directive 2002/11/EC rules require that the initial plant material for vegetative propagation is free of Grapevine fanleaf virus (GFLV), Arabis mosaic virus (ArMV), Grapevine fleck virus (GFkV), Grapevine leafroll–associated virus-1 (GLRaV-1), and Grapevine leafroll–associated virus-3 (GLRaV-3). When samples to be storage are virus infected, different approaches to regenerated virus-free plants can be used. Since the middle late of the twentieth century, meristem culture and thermotherapy were applied with this aim for grapevine sanitation [37, 38]. Other techniques such as chemotherapy, electrotherapy, cryotherapy, and somatic

For in vitro preservation under standard or limiting conditions, the development of protocols adjusted to the variety to be preserved is necessary. For this kind of storage, it is important to choose the adequate culture medium and environmental conditions in order to reduce the number of subcultures and hence minimize the cost and the putative errors that could arise in each subculture. Protocols for storage grapevine under in vitro culture have been reported by several authors [42, 43]. Recently, we reported the effectiveness of the MW medium to store a broad spectrum of grapevine cultivars, including endangered varieties, as well as the modifications of this medium (reduction of sucrose or elimination of indole-3-butyric acid (IBA) in the medium) to reduce growth in the faster growing varieties [44]. Cryopreservation protocols have also been developed for grapevine [45– 47]. When developing cryopreservation protocols, the evaluation of the cryopreservation solution toxicity in the varieties to be preserved is required. Pre-culture in culture media that facilitate the dehydration of tissues (i.e., in medium with high content of sugars) is

Finally, studies of genetic variability among varieties under conservation are important because they allow the detection of mutations and the study of relationships among them. The determination of molecular profiles is also of great importance in the development of core collections which represent the higher variability present in a whole collection in a reduced number of accessions. The core collections are very useful for breeders because studies on the core collection provide an overall view of the properties to be found in the whole collection [48]. In the following schema, the different steps to be carried out for

dology [33].

embryogenesis were also reported [39–41].

germplasm storage are shown (Figure 1).

also a common step.

## 2. Recovering ancient germplasm at the Comunitat Valenciana: the historic varieties "Valencí Blanc" and "Esclafagerres"

#### 2.1. Richness of grapevine germplasm in the Comunitat Valenciana

The Comunitat Valenciana which includes the provinces of Alicante, Valencia, and Catellón is located in the Mediterranean coast of Spain and has been an important viticulture area since historic times to nowadays. For instance, in the archeological place of "L'Alt de Benimaquia" (Denia, Alicante), dated back to the end of the seventh century BC, significant quantities of vinification residues (tartaric acid and seeds) were found [49]. Important vineyards must be also located in the Requena-Utiel plateau (Valencia) as evidenced by the big presses dated in the fifth century BC found at Las Pilillas site [50]. Nowadays there exist three DO for wine production (DO Alicante (http://www.vinosalicantedop.org/), DO Valencia (http://www.dovalencia.info/), and DO Utiel-Requena (http://utielrequena.org/)), one DO for table grape (DO Uva de mesa del Vinalopó (http://uva-vinalopo.org/wp/)) and one protected geographical indication in Castellón (http://www.igpcastello.com/).

The richness in grapevine cultivars before the arrival to the phylloxera pest in the provinces of Alicante and mainly, in that of Valencia, is well documented [51, 52]. In a report about grapevine varieties cultured in Spain in 1889, it is pointed that more than 150 varieties were cultured in different locations of the Valencia province. These varieties included varieties with berries of black, white, and red color. In comparison with other provinces that also appeared in this report, Valencia was one of the richest [52]. The phylloxera aphids that devastated European vineyards invaded Spain in 1878 from three areas (Girona, Málaga, and Portuguese border). Its arrival to the Comunitat Valenciana, with the consequent loss of grapevine variability, occurred in 1912 when the aphids spread to Sagunto, Líria, and Requena [53].

In the context of the research project CGL2015–70843-R, we initiated different approaches in order to contribute in the recovering of ancient varieties in the risk of disappearance from Alicante and Valencia provinces. The objectives of this project include the analysis of grapevine germplasm diversity and the development of protocols for virus sanitation and in vitro conservation. Different prospections have been performed in order to rescue ancient varieties. The determination of SSR profiles is being useful to confirm the identity and to detect synonymies and homonymies that are very common in grapevine. The analysis of the genetic variability will identify accessions which may carry useful mutations for adaptation to specific environments. As occurred in other areas, grapevine cultivars were found commonly infected by the viruses GFLV, GLRaV, and GFkV. Sanitation of cultivars to be preserved in vitro is being carried out through meristem culture, although other alternative sanitation procedures are being developed [42, 54]. In this work, we report the SSR profiles of different accessions of the historic variety "Valencí Blanc" or "Beba" and the historic and endangered variety "Esclafagerres" (or "Esclafacherres/is"). The methodologies used for their sanitation and in vitro conservation are also summarized.

easy to found this homonym. In the VIVC database, 71 synonymies for "Beba" appeared. However, some of these ("Chelva," "Hebén," "Mantúo," "Teta de Vaca," and "Uva de Planta") were rejected as they did not share the same SSR profile [64]. Probably other of those proposed are also false synonymies. On the other hand, in the report about grapevine varieties cultured in Spain in 1889 [52], the variety "Valencí" was included in the groups of cultivars with black and white berries. The comparison among the SSR profiles of some accessions of "Valencí" with white grapes ("Valencí Blanc") and with black grapes ("Valencí Tinto" or "Valencí Negre" in the Comunitat Valenciana) indicates that they are not mutant for berry color but resulting from different crosses. Comparing the SSR profile of the variety "Heben" (or "Gibi"), proposed as parent of "Valencí Blanc," with the SSR profile of the accession of "Valencí Tinto" hold in the VIVC database, no relationship between them was observed. However, one or two alleles were shared between "Valencí Blanc" and "Valencí Tinto." Therefore, the unknown parent from "Valencí Blanc" could be the parent of "Valencí Tinto." The name of "Valensi Chaselas" also appeared in the report of grapevines cultured in 1889, concretely as cultured in the Valencian

Recovering Ancient Grapevine Varieties: From Genetic Variability to In Vitro Conservation, A Case Study

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Figure 2. Grapes of "Valencí Blanc" (A) and "Esclafagerres" (B) varieties.

"Esclafagerres" variety (Figure 2B), which name means that which bursts the jars, is also an ancient variety with white berries commonly grown on the Alicante and Valencia provinces. Some old references that mentioned the culture of this variety in the Alicante province are reported by several authors [65–67]. In DGAIC [52], the culture of "Esclafagerres" appeared in the Alicante and in the Valencian locations of Albaida, Onteniente, and Sagunto, where it was included among the varieties with white and also with black berries. This variety was usually mixed with other grapevine varieties like "Merseguera" for wine production. The "Esclafagerres" variety gives high yields (probably the meaning of the name is related to this) and has grapes with

In this work, we report the assays performed with both varieties in order to determine their genetic profiles and resume the strategies performed for virus sanitation and in vitro conservation.

A total of 14 samples of "Valencí Blanc" from different locations (Table 1) and two samples of "Esclafagerres" from La Mata and Monforte del Cid were used for DNA extraction and SSR

low sugar content despite it was commonly cultured under dry land.

location of Gandía.

2.3. Determining the SSR profiles

#### 2.2. The historic varieties "Valencí Blanc" and "Esclafagerres"

The "Valencí Blanc" variety also known as "Beba" is a minor cultivar usually used as white table grape (Figure 2A). Despite in the past it was used for wine and raisin production [52, 55], today it is cultured as table grape for minor consume, and it is authorized for wine production in DO Ribera del Guadiana (Spain), where it is also named as "Eva" (http://riberadelguadiana.eu/esp/). The origin of this variety is unknown although it is proposed an oriental or North African origin [56]. The name "Valencí" ("Valensi" and "Balansí" in older reports) remembers to the name of Valencia City [57]. Oliver-Fuster (1980) cited by [58] proposed that this variety maybe was introduced by Balearic people who emigrate to Argelia. The most antique synonymy assigned for this variety is "Calop" [59] although other synonymies like "Ain el Kelp," "Tebourbi," "Panse the Provence" and "Grumer" are reported [56, 58–60]. In a report about grapevines cultivated in 1889 [52], the culture of "Grumer" is noted, among other 18 grapevine varieties in the Alicante province and in Albaida and Onteniente (locations nearest to the Alicante province). In the same report, the culture of "Valensí" in Alberique and Enguera (nearest to Valencia City) appeared. Recently, with the name of "Grumer," we have identified some accessions from the Alicante province that do not correspond to "Valencí" but grouped with "Muscat Istambul" [61]. Lacombe et al. [62] proposed as the origin of "Muscat Istambul" the cross of "Muscat of Alexandria" "Valencí Blanc" which was confirmed by Mena et al. [63]. Therefore, it could be

Recovering Ancient Grapevine Varieties: From Genetic Variability to In Vitro Conservation, A Case Study http://dx.doi.org/10.5772/intechopen.71133 9

Figure 2. Grapes of "Valencí Blanc" (A) and "Esclafagerres" (B) varieties.

The richness in grapevine cultivars before the arrival to the phylloxera pest in the provinces of Alicante and mainly, in that of Valencia, is well documented [51, 52]. In a report about grapevine varieties cultured in Spain in 1889, it is pointed that more than 150 varieties were cultured in different locations of the Valencia province. These varieties included varieties with berries of black, white, and red color. In comparison with other provinces that also appeared in this report, Valencia was one of the richest [52]. The phylloxera aphids that devastated European vineyards invaded Spain in 1878 from three areas (Girona, Málaga, and Portuguese border). Its arrival to the Comunitat Valenciana, with the consequent loss of grapevine vari-

8 Grapes and Wines - Advances in Production, Processing, Analysis and Valorization

ability, occurred in 1912 when the aphids spread to Sagunto, Líria, and Requena [53].

2.2. The historic varieties "Valencí Blanc" and "Esclafagerres"

also summarized.

In the context of the research project CGL2015–70843-R, we initiated different approaches in order to contribute in the recovering of ancient varieties in the risk of disappearance from Alicante and Valencia provinces. The objectives of this project include the analysis of grapevine germplasm diversity and the development of protocols for virus sanitation and in vitro conservation. Different prospections have been performed in order to rescue ancient varieties. The determination of SSR profiles is being useful to confirm the identity and to detect synonymies and homonymies that are very common in grapevine. The analysis of the genetic variability will identify accessions which may carry useful mutations for adaptation to specific environments. As occurred in other areas, grapevine cultivars were found commonly infected by the viruses GFLV, GLRaV, and GFkV. Sanitation of cultivars to be preserved in vitro is being carried out through meristem culture, although other alternative sanitation procedures are being developed [42, 54]. In this work, we report the SSR profiles of different accessions of the historic variety "Valencí Blanc" or "Beba" and the historic and endangered variety "Esclafagerres" (or "Esclafacherres/is"). The methodologies used for their sanitation and in vitro conservation are

The "Valencí Blanc" variety also known as "Beba" is a minor cultivar usually used as white table grape (Figure 2A). Despite in the past it was used for wine and raisin production [52, 55], today it is cultured as table grape for minor consume, and it is authorized for wine production in DO Ribera del Guadiana (Spain), where it is also named as "Eva" (http://riberadelguadiana.eu/esp/). The origin of this variety is unknown although it is proposed an oriental or North African origin [56]. The name "Valencí" ("Valensi" and "Balansí" in older reports) remembers to the name of Valencia City [57]. Oliver-Fuster (1980) cited by [58] proposed that this variety maybe was introduced by Balearic people who emigrate to Argelia. The most antique synonymy assigned for this variety is "Calop" [59] although other synonymies like "Ain el Kelp," "Tebourbi," "Panse the Provence" and "Grumer" are reported [56, 58–60]. In a report about grapevines cultivated in 1889 [52], the culture of "Grumer" is noted, among other 18 grapevine varieties in the Alicante province and in Albaida and Onteniente (locations nearest to the Alicante province). In the same report, the culture of "Valensí" in Alberique and Enguera (nearest to Valencia City) appeared. Recently, with the name of "Grumer," we have identified some accessions from the Alicante province that do not correspond to "Valencí" but grouped with "Muscat Istambul" [61]. Lacombe et al. [62] proposed as the origin of "Muscat Istambul" the cross of "Muscat of Alexandria" "Valencí Blanc" which was confirmed by Mena et al. [63]. Therefore, it could be easy to found this homonym. In the VIVC database, 71 synonymies for "Beba" appeared. However, some of these ("Chelva," "Hebén," "Mantúo," "Teta de Vaca," and "Uva de Planta") were rejected as they did not share the same SSR profile [64]. Probably other of those proposed are also false synonymies. On the other hand, in the report about grapevine varieties cultured in Spain in 1889 [52], the variety "Valencí" was included in the groups of cultivars with black and white berries. The comparison among the SSR profiles of some accessions of "Valencí" with white grapes ("Valencí Blanc") and with black grapes ("Valencí Tinto" or "Valencí Negre" in the Comunitat Valenciana) indicates that they are not mutant for berry color but resulting from different crosses. Comparing the SSR profile of the variety "Heben" (or "Gibi"), proposed as parent of "Valencí Blanc," with the SSR profile of the accession of "Valencí Tinto" hold in the VIVC database, no relationship between them was observed. However, one or two alleles were shared between "Valencí Blanc" and "Valencí Tinto." Therefore, the unknown parent from "Valencí Blanc" could be the parent of "Valencí Tinto." The name of "Valensi Chaselas" also appeared in the report of grapevines cultured in 1889, concretely as cultured in the Valencian location of Gandía.

"Esclafagerres" variety (Figure 2B), which name means that which bursts the jars, is also an ancient variety with white berries commonly grown on the Alicante and Valencia provinces. Some old references that mentioned the culture of this variety in the Alicante province are reported by several authors [65–67]. In DGAIC [52], the culture of "Esclafagerres" appeared in the Alicante and in the Valencian locations of Albaida, Onteniente, and Sagunto, where it was included among the varieties with white and also with black berries. This variety was usually mixed with other grapevine varieties like "Merseguera" for wine production. The "Esclafagerres" variety gives high yields (probably the meaning of the name is related to this) and has grapes with low sugar content despite it was commonly cultured under dry land.

In this work, we report the assays performed with both varieties in order to determine their genetic profiles and resume the strategies performed for virus sanitation and in vitro conservation.

#### 2.3. Determining the SSR profiles

A total of 14 samples of "Valencí Blanc" from different locations (Table 1) and two samples of "Esclafagerres" from La Mata and Monforte del Cid were used for DNA extraction and SSR


Table 1. SSR profiles for 14 accessions of "Valencí Blanc" (Vb-Pe0, Vb-Pe1, Vb-Pe2, Vb-Pe3, Vb-Pe4, Vb-Pe5, Vb-Pe10, Vb-Be1, Vb-Al1, Vb-FF1, Vb-FA1,Vb-Cu1) and two of "Esclafagerres" (Es-Ma1, Es-Mo1) varieties.

analysis. DNA was extracted from fully expanded leaves using the commercial DNeasy Plant Mini Kit (Qiagen) according to the manufacturers' instructions. DNA quality and quantity were assessed using gel electrophoresis and spectrophotometry. Fifteen SSR markers (VVS2, VVMD5, VVMD6, VVMD7, VVMD24, VVMD25, VVMD27, VVMD28, VVMD32, VrZAG62, VRzAG64, VrZAG79, VrZAG83, and VMC1b11) were analyzed using two sets of multiplex PCR reactions. Each multiplex was carefully assembled according to the compatibility of the SSRs during PCR and the molecular size of their amplicons. The forward primer of the SSR markers was labeled with one of the four fluorescent dyes: carboxyfluorescein (FAM), carboxytetramethylrhodamine (TAMRA), hexachloro-6 carboxyfluorescein (HEX), or 6-carboxytetramethyl rhodamine (ROX) [61]. Multiplex PCR was carried out in a total volume of 11.00 μL, using 1.25 μL of commercial Master Mix PCR Multiplex (Takara Multiplex Hot Short PCR, Takara), 20–40 ng of genomic DNA, 0.1 μL of Takara Taq Hot Start, and labeled multiplexed SSR primers (from 5.5 to 35.0 μmol). The amplification was performed in an ABI 9700 thermocycler, and the amplification conditions were 95C for 14 min followed by 30 cycles of 95C for 30 s, 55C for 90 s, and 72C for 60 s and a final extension of 72C for 30 min. Previous to PCR fragment size determination, the multiplex PCR product was previsualized using gel electrophoresis. The electrophoresis was carried out on an ABI 3100 platform (Applied Biosystems, Foster City, CA, USA). For PCR fragment size determinations, 0.13 μL of an internal size standard (GeneScan™ 500 LIZ, Applied Biosystems) was mixed with 1.00 μL of PCR product and 10.87 μL of formamide. The mixture was heated at 94C for 3 min and then cooled in icy water. The size of the SSR fragments was determined with the software package GeneScan

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11

The SSR profiles of the analyzed accessions are shown in Table 1. Whereas similar SSR profiles were found for both accessions of "Esclafacherre," some variability is found among "Valencí Blanc" accessions. Among the 15 SSRs analyzed, differences in the SSR VVMD32 were found: the accessions collected in the province of Alicante have two alleles in this loci VVMD32 (254; 270), whereas the rest of accessions from the provinces Badajoz, Cuenca, and Valencia have the allele 254 in homozygosity. We can consider that among the analyzed germplasm, there are two variants of "Valencí Blanc" as in other cases in which two plants showed identical SSR profiles for all the SSR markers studied except for one or two alleles. This could be attributable to slight clonal polymorphism [68]. They may have originated in a similar place and then spread to different areas. The accession of "Valencí Blanc" in the VIVC database (accession number 22710) and that reported by Lacombe et al. [62] had the same profile for the comparable SSRs (including the VVMD32) to the accessions from Alicante. Similarly, accessions from Alicante showed also identical SSR profile to two accessions of "Beba" from El Encín grape-

The comparison of the SSR profile of the "Esclafagerres" accessions to SSR profiles in the VIVC database (including 3265 accessions), those in the International Vitis database (including 3430 accessions), as well as those reported in several publications [62, 63, 69] did not match with any of the included varieties. No matches were found when the SSR profile of "Esclafagerres" was blasted to the Italian Vitis database. Therefore, this profile should correspond with that of the

3.7 (Applied Biosystems).

vine collection analyzed by Mena [64].

"Esclafagerres" variety which has not been reported before.

analysis. DNA was extracted from fully expanded leaves using the commercial DNeasy Plant Mini Kit (Qiagen) according to the manufacturers' instructions. DNA quality and quantity were assessed using gel electrophoresis and spectrophotometry. Fifteen SSR markers (VVS2, VVMD5, VVMD6, VVMD7, VVMD24, VVMD25, VVMD27, VVMD28, VVMD32, VrZAG62, VRzAG64, VrZAG79, VrZAG83, and VMC1b11) were analyzed using two sets of multiplex PCR reactions. Each multiplex was carefully assembled according to the compatibility of the SSRs during PCR and the molecular size of their amplicons. The forward primer of the SSR markers was labeled with one of the four fluorescent dyes: carboxyfluorescein (FAM), carboxytetramethylrhodamine (TAMRA), hexachloro-6 carboxyfluorescein (HEX), or 6-carboxytetramethyl rhodamine (ROX) [61]. Multiplex PCR was carried out in a total volume of 11.00 μL, using 1.25 μL of commercial Master Mix PCR Multiplex (Takara Multiplex Hot Short PCR, Takara), 20–40 ng of genomic DNA, 0.1 μL of Takara Taq Hot Start, and labeled multiplexed SSR primers (from 5.5 to 35.0 μmol). The amplification was performed in an ABI 9700 thermocycler, and the amplification conditions were 95C for 14 min followed by 30 cycles of 95C for 30 s, 55C for 90 s, and 72C for 60 s and a final extension of 72C for 30 min. Previous to PCR fragment size determination, the multiplex PCR product was previsualized using gel electrophoresis. The electrophoresis was carried out on an ABI 3100 platform (Applied Biosystems, Foster City, CA, USA). For PCR fragment size determinations, 0.13 μL of an internal size standard (GeneScan™ 500 LIZ, Applied Biosystems) was mixed with 1.00 μL of PCR product and 10.87 μL of formamide. The mixture was heated at 94C for 3 min and then cooled in icy water. The size of the SSR fragments was determined with the software package GeneScan 3.7 (Applied Biosystems).

The SSR profiles of the analyzed accessions are shown in Table 1. Whereas similar SSR profiles were found for both accessions of "Esclafacherre," some variability is found among "Valencí Blanc" accessions. Among the 15 SSRs analyzed, differences in the SSR VVMD32 were found: the accessions collected in the province of Alicante have two alleles in this loci VVMD32 (254; 270), whereas the rest of accessions from the provinces Badajoz, Cuenca, and Valencia have the allele 254 in homozygosity. We can consider that among the analyzed germplasm, there are two variants of "Valencí Blanc" as in other cases in which two plants showed identical SSR profiles for all the SSR markers studied except for one or two alleles. This could be attributable to slight clonal polymorphism [68]. They may have originated in a similar place and then spread to different areas. The accession of "Valencí Blanc" in the VIVC database (accession number 22710) and that reported by Lacombe et al. [62] had the same profile for the comparable SSRs (including the VVMD32) to the accessions from Alicante. Similarly, accessions from Alicante showed also identical SSR profile to two accessions of "Beba" from El Encín grapevine collection analyzed by Mena [64].

The comparison of the SSR profile of the "Esclafagerres" accessions to SSR profiles in the VIVC database (including 3265 accessions), those in the International Vitis database (including 3430 accessions), as well as those reported in several publications [62, 63, 69] did not match with any of the included varieties. No matches were found when the SSR profile of "Esclafagerres" was blasted to the Italian Vitis database. Therefore, this profile should correspond with that of the "Esclafagerres" variety which has not been reported before.

Code

 Origin

VVMD27 VVMD5 VVS2 A1 A2 A1 A2 A1 A2 A1 A2 A1 A2 A1 A2 A1 A2 A1 A2 A1 A2 A1 A2 A1 A2 A1 A2 A1 A2 A1 A2 A1 A2

> Vb-Pe0

Vb-Pe1 Vb-Pe2 Vb-Pe3 Vb-Pe4 Vb-Pe5 Vb-Pe10

Vb-Be1 Vb-Al1 Vb-FF1 La Font de la Figuera

(Valencia)

Vb-FA1

Fontanars dels

> Alforins (Valencia)

> > Vb-On1

Vb-Ba1

Fuente del Maestre

(Badajoz)

Vb-Cu1

Campillo de

> Altobuey (Cuenca)

Es-Ma1 La Mata (Alicante)

Es-Mo1

Monforte del Cid

(Alicante)

Allele sizes are expressed as base pairs. Table 1. SSR profiles for 14 accessions of "Valencí Blanc"

Vb-Cu1) and two of

"Esclafagerres"

(Es-Ma1, Es-Mo1) varieties.

(Vb-Pe0, Vb-Pe1, Vb-Pe2, Vb-Pe3, Vb-Pe4, Vb-Pe5, Vb-Pe10, Vb-Be1, Vb-Al1, Vb-FF1, Vb-FA1, Vb-On1, Vb-Ba1,

Ontinyent (Valencia)

Alicante

Benirarres (Alicante)

Penàguila (Alicante)

10 Grapes and Wines - Advances in Production, Processing, Analysis and Valorization

Penàguila (Alicante)

Penàguila (Alicante)

Penàguila (Alicante)

Penàguila (Alicante)

Penàguila (Alicante)

Penàguila (Alicante)

 VrZAG83 VrZAG79 VrZAG62 VrZAG64 VVMD7 VVMD24 VVMD32 VVMD25 VMC1b11

 VVMD28 VVMD6 VVMD21

#### 2.4. Virus analysis and sanitation

To analyze the putative virus infection in the original samples, the methodology described by López-Fabuel et al. [70] was used. Briefly, extracts were prepared from leaves 1/20 in phosphate buffered saline (PBS) buffer, pH 7.2, supplemented with 0.2% diethyldithiocarbamic acid (DIECA) and 2% polyvinylpyrrolidone-10 (PVP-10) in individual plastic bags with a heavy net (Plant Print Diagnostics). Total RNA was extracted from 200 μL of crude extract using an Ultraclean Plant RNA isolation kit (Mobio) following the manufacturer's instructions. The real-time multiplex reverse transcription polymerase chain reaction (RT-PCR) was performed for the simultaneous detection of ArMV, GFLV, GFkV, GLRaV-1, and GLRaV-3 using a StepOnePlus thermocycler (Applied Biosystems) and a reaction mixture containing 1 AgPath-ID One-step RT-PCR buffer (Ambion) and 1.5 AgPath-ID One-step RT-PCR enzyme mix (Ambion); 5 μL of sample; 400 nM of GFLV, ArMV, GFkV, and GLRaV-1 primers; 800 nM of GLRaV-3 primers; and 200 nM of each probe. The amplification protocol consisted of an RT step at 45C for 10 min and a denaturation step at 95C for 10 min, followed by 45 cycles of amplification (95C, 15 s; 50C, 15 s; and 60C, 60 s). As positive controls viral isolates maintained at the Instituto Valenciano de Investigaciones Agrarias (IVIA) were included. When amplification was observed for a specific virus, it was confirmed by real-time uniplex RT-PCR using the corresponding primers.

Meristem culture was used in a previous project (RTA2011–00067-C04) to obtain virus-free plants of "Esclafagerres." In the context of the project CGL2015–70843-R, "Valencí Blanc" (sample Vb-Pe0), which resulted in infection with GFkV and GLRaV-3, was sanitized through both meristem culture and somatic embryogenesis. Meristems (n = 35) from plants of "Valencí Blanc" were extracted using a binocular lent and cultured in vitro on plates (90 15 mm) containing the medium MW, selected for "Monastrell" micropropagation [71] supplemented with 6-benzylaminopurine (BAP) at 1.8 μM. Low light conditions were used for the two first weeks of culture. Only 54.3% of meristems grew after 20 days of culture (Figure 3A), and two develop into plants after transferring to tubes with MW, 70 days after (Figure 3B). Damage of meristem during extraction and/or the composition of the culture medium that could need to be enriched with other nutrients is putatively the cause of the low and slow regeneration of meristems. The analysis for virus presence of these two plants was carried out as described before, and one of them (50%) resulted free for both viruses. Therefore, from this plant, clones were obtained for in vitro conservation. Despite the fact that meristem culture is an efficient technology for virus sanitation, it is needed to obtain an adequate size of the meristem in order to avoid virus transmission allowing meristem development.

Ten developed plants were analyzed for evaluating the success of virus cleaning. All plants regenerated from somatic embryos resulted virus-free for both GFkV and GLRaV-3 viruses. Therefore, 100% of sanitation was obtained. The result obtained for GFkV was expected because this virus is not seed transmitted [75]. With respect to GLRaV-3, it is not clear if it is present in seeds [34, 75], but we have found this virus in some regenerated plants resulting from somatic embryos induced from seeds of other infected grapevines, which would indicate its presence in the seeds [54]. Induction of somatic embryos from stamens or pistils was also reported in grapevine to cure plants of GLRaV [76, 77]. We also analyzed the SSR profile of regenerated virus-free plants in order to select those regenerated from mother tissues of the seeds, which will show the mother genotype. The 15 SSRs used for determining the SSR profile of "Valencí Blanc" accessions were used. One of ten analyzed plants showed the same SSR profile as the mother plant, that is, a 10% of regenerated plants were obtained from mother

Figure 3. Strategies for virus sanitation of "Valencí Blanc" (accession Vb-Pe0). (A) Meristems cultured on MW supplemented with 6-benzylaminopurine (BAP) at 1.8 μM and without indole butyric acid (IBA) after 20 days of culture. (B) Plants from meristems cultured in MW medium after 90 days of culture. (C) Cut seeds cultured in EIM2 medium at day 0. (D) Seeds with

Recovering Ancient Grapevine Varieties: From Genetic Variability to In Vitro Conservation, A Case Study

http://dx.doi.org/10.5772/intechopen.71133

13

some somatic embryos after 45 days of culture. (E) Plants regenerated from somatic embryos.

Sanitized plants of "Valencí Blanc" and "Esclafagerres" are maintained in tubes with MW medium in an in vitro culture growth chamber under standard conditions (25 2C; 16 h light). The MW medium is adequate to storage a broad spectrum of grapevine cultivars including "Valencí Blanc" and "Esclafagerres" [44]. Both cultivars grew less than 4 cm after 40 days of culture in this medium. We consider that this speed of growth is acceptable to maintain these cultivars under standard conditions with small number of subcultures. A reduction of sugar or the elimination of IBA in the culture medium is used for maintaining cultivars that grew faster [44]. The higher the number of subcultures, the higher the cost and

tissue and not from the embryo.

the higher the possibility to make nomenclature errors [78].

2.5. In vitro storage

The other methodology used for virus sanitation was the induction of somatic embryos as reported in Peiró et al. [41]. Briefly, seeds of "Valencí Blanc" were extracted from grapes and disinfected and cut previously to be cultured on the embryogenesis induction medium (EIM2) which contained TDZ (thidiazuron) at 0.9 μM. Thirteen percent of explants responded after 60 days of culture on this medium (Figure 3C–D). Despite in grapevine a high percentage of somatic embryos are not able to develop into normal plants [72–74], in our work, germination of somatic embryos occurred directly in the induction medium and grew correctly (Figure 3E).

Recovering Ancient Grapevine Varieties: From Genetic Variability to In Vitro Conservation, A Case Study http://dx.doi.org/10.5772/intechopen.71133 13

Figure 3. Strategies for virus sanitation of "Valencí Blanc" (accession Vb-Pe0). (A) Meristems cultured on MW supplemented with 6-benzylaminopurine (BAP) at 1.8 μM and without indole butyric acid (IBA) after 20 days of culture. (B) Plants from meristems cultured in MW medium after 90 days of culture. (C) Cut seeds cultured in EIM2 medium at day 0. (D) Seeds with some somatic embryos after 45 days of culture. (E) Plants regenerated from somatic embryos.

Ten developed plants were analyzed for evaluating the success of virus cleaning. All plants regenerated from somatic embryos resulted virus-free for both GFkV and GLRaV-3 viruses. Therefore, 100% of sanitation was obtained. The result obtained for GFkV was expected because this virus is not seed transmitted [75]. With respect to GLRaV-3, it is not clear if it is present in seeds [34, 75], but we have found this virus in some regenerated plants resulting from somatic embryos induced from seeds of other infected grapevines, which would indicate its presence in the seeds [54]. Induction of somatic embryos from stamens or pistils was also reported in grapevine to cure plants of GLRaV [76, 77]. We also analyzed the SSR profile of regenerated virus-free plants in order to select those regenerated from mother tissues of the seeds, which will show the mother genotype. The 15 SSRs used for determining the SSR profile of "Valencí Blanc" accessions were used. One of ten analyzed plants showed the same SSR profile as the mother plant, that is, a 10% of regenerated plants were obtained from mother tissue and not from the embryo.

#### 2.5. In vitro storage

2.4. Virus analysis and sanitation

12 Grapes and Wines - Advances in Production, Processing, Analysis and Valorization

RT-PCR using the corresponding primers.

development.

To analyze the putative virus infection in the original samples, the methodology described by López-Fabuel et al. [70] was used. Briefly, extracts were prepared from leaves 1/20 in phosphate buffered saline (PBS) buffer, pH 7.2, supplemented with 0.2% diethyldithiocarbamic acid (DIECA) and 2% polyvinylpyrrolidone-10 (PVP-10) in individual plastic bags with a heavy net (Plant Print Diagnostics). Total RNA was extracted from 200 μL of crude extract using an Ultraclean Plant RNA isolation kit (Mobio) following the manufacturer's instructions. The real-time multiplex reverse transcription polymerase chain reaction (RT-PCR) was performed for the simultaneous detection of ArMV, GFLV, GFkV, GLRaV-1, and GLRaV-3 using a StepOnePlus thermocycler (Applied Biosystems) and a reaction mixture containing 1 AgPath-ID One-step RT-PCR buffer (Ambion) and 1.5 AgPath-ID One-step RT-PCR enzyme mix (Ambion); 5 μL of sample; 400 nM of GFLV, ArMV, GFkV, and GLRaV-1 primers; 800 nM of GLRaV-3 primers; and 200 nM of each probe. The amplification protocol consisted of an RT step at 45C for 10 min and a denaturation step at 95C for 10 min, followed by 45 cycles of amplification (95C, 15 s; 50C, 15 s; and 60C, 60 s). As positive controls viral isolates maintained at the Instituto Valenciano de Investigaciones Agrarias (IVIA) were included. When amplification was observed for a specific virus, it was confirmed by real-time uniplex

Meristem culture was used in a previous project (RTA2011–00067-C04) to obtain virus-free plants of "Esclafagerres." In the context of the project CGL2015–70843-R, "Valencí Blanc" (sample Vb-Pe0), which resulted in infection with GFkV and GLRaV-3, was sanitized through both meristem culture and somatic embryogenesis. Meristems (n = 35) from plants of "Valencí Blanc" were extracted using a binocular lent and cultured in vitro on plates (90 15 mm) containing the medium MW, selected for "Monastrell" micropropagation [71] supplemented with 6-benzylaminopurine (BAP) at 1.8 μM. Low light conditions were used for the two first weeks of culture. Only 54.3% of meristems grew after 20 days of culture (Figure 3A), and two develop into plants after transferring to tubes with MW, 70 days after (Figure 3B). Damage of meristem during extraction and/or the composition of the culture medium that could need to be enriched with other nutrients is putatively the cause of the low and slow regeneration of meristems. The analysis for virus presence of these two plants was carried out as described before, and one of them (50%) resulted free for both viruses. Therefore, from this plant, clones were obtained for in vitro conservation. Despite the fact that meristem culture is an efficient technology for virus sanitation, it is needed to obtain an adequate size of the meristem in order to avoid virus transmission allowing meristem

The other methodology used for virus sanitation was the induction of somatic embryos as reported in Peiró et al. [41]. Briefly, seeds of "Valencí Blanc" were extracted from grapes and disinfected and cut previously to be cultured on the embryogenesis induction medium (EIM2) which contained TDZ (thidiazuron) at 0.9 μM. Thirteen percent of explants responded after 60 days of culture on this medium (Figure 3C–D). Despite in grapevine a high percentage of somatic embryos are not able to develop into normal plants [72–74], in our work, germination of somatic embryos occurred directly in the induction medium and grew correctly (Figure 3E). Sanitized plants of "Valencí Blanc" and "Esclafagerres" are maintained in tubes with MW medium in an in vitro culture growth chamber under standard conditions (25 2C; 16 h light). The MW medium is adequate to storage a broad spectrum of grapevine cultivars including "Valencí Blanc" and "Esclafagerres" [44]. Both cultivars grew less than 4 cm after 40 days of culture in this medium. We consider that this speed of growth is acceptable to maintain these cultivars under standard conditions with small number of subcultures. A reduction of sugar or the elimination of IBA in the culture medium is used for maintaining cultivars that grew faster [44]. The higher the number of subcultures, the higher the cost and the higher the possibility to make nomenclature errors [78].

Another strategy to germplasm ex situ storage using in vitro culture is cryopreservation. With this methodology, the metabolism is greatly reduced, and few requirements are needed for the maintenance of tissue samples. Meristems of "Esclafagerres" from micropropagated virus-free plants maintained in vitro were used to initiate the cryopreservation assays using the methodology described in Gisbert et al. [79]. The first results indicated that 50 min of incubation in the plant vitrification solution 2 (PVS2) is adequate for recuperating cryopreserved meristems of "Esclafagerres" (Figure 2). Recently, Pathirana et al. [47] have reported a positive effect on grapevine regeneration when a pretreatment with salicylic acid was performed prior to cryopreservation. In both works [47, 79], the droplet vitrification protocol was used.

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#### 3. Conclusion

As a result of different actions performed in the context of the projects CGL2015-70843-R and RTA2011-00067-C04, a broad spectrum of grapevine varieties are being evaluated in order to determine the varietal identification and their variability and also their capacity for in vitro culture, plant regeneration, and germplasm storage. Different strategies for virus cleaning have been developed and applied to rescue virus-free plants. Among the analyzed materials, the historic varieties "Valencí Blanc" and "Esclafagerres" were sanitized and currently are maintained under in vitro culture conditions. Differences for the microsatellite VVMD32 were found among "Valencí Blanc" accessions, clustering the accessions from Alicante and that of other origins. The SSR profile for the variety "Esclafagerres" was firstly reported in the present work.

#### Acknowledgements

The authors thank the support of the projects CGL2015-70843-R and RTA2011-0020067-C04 both co-funded by FEDER Funds.

#### Author details

Carmina Gisbert<sup>1</sup> \*, Rosa Peiró<sup>1</sup> , Tania San Pedro<sup>2</sup> , Antonio Olmos<sup>2</sup> , Carles Jiménez<sup>3</sup> and Julio García<sup>4</sup>

\*Address all correspondence to: cgisbert@btc.upv.es

1 Polytechnic University of Valencia (Universitat Politècnica de València), Valencia, Spain


4 Conselleria of Agriculture, Environment, Climate Change and Rural Development, Alicante, Spain

## References

Another strategy to germplasm ex situ storage using in vitro culture is cryopreservation. With this methodology, the metabolism is greatly reduced, and few requirements are needed for the maintenance of tissue samples. Meristems of "Esclafagerres" from micropropagated virus-free plants maintained in vitro were used to initiate the cryopreservation assays using the methodology described in Gisbert et al. [79]. The first results indicated that 50 min of incubation in the plant vitrification solution 2 (PVS2) is adequate for recuperating cryopreserved meristems of "Esclafagerres" (Figure 2). Recently, Pathirana et al. [47] have reported a positive effect on grapevine regeneration when a pretreatment with salicylic acid was performed prior to cryo-

As a result of different actions performed in the context of the projects CGL2015-70843-R and RTA2011-00067-C04, a broad spectrum of grapevine varieties are being evaluated in order to determine the varietal identification and their variability and also their capacity for in vitro culture, plant regeneration, and germplasm storage. Different strategies for virus cleaning have been developed and applied to rescue virus-free plants. Among the analyzed materials, the historic varieties "Valencí Blanc" and "Esclafagerres" were sanitized and currently are maintained under in vitro culture conditions. Differences for the microsatellite VVMD32 were found among "Valencí Blanc" accessions, clustering the accessions from Alicante and that of other origins. The

The authors thank the support of the projects CGL2015-70843-R and RTA2011-0020067-C04

, Antonio Olmos<sup>2</sup>

, Carles Jiménez<sup>3</sup> and

preservation. In both works [47, 79], the droplet vitrification protocol was used.

14 Grapes and Wines - Advances in Production, Processing, Analysis and Valorization

SSR profile for the variety "Esclafagerres" was firstly reported in the present work.

, Tania San Pedro<sup>2</sup>

2 Valencian Institute of Agrarian Investigations (IVIA), Valencia, Spain

3 Plant Health Service of the Generalitat Valenciana, Valencia, Spain

1 Polytechnic University of Valencia (Universitat Politècnica de València), Valencia, Spain

4 Conselleria of Agriculture, Environment, Climate Change and Rural Development, Alicante,

3. Conclusion

Acknowledgements

Author details

Carmina Gisbert<sup>1</sup>

Julio García<sup>4</sup>

Spain

both co-funded by FEDER Funds.

\*, Rosa Peiró<sup>1</sup>

\*Address all correspondence to: cgisbert@btc.upv.es


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**Chapter 2**

Provisional chapter

(Gmelin) Hegi)

**Fingerprints of Anthocyanins and Flavonols in Wild**

DOI: 10.5772/intechopen.70861

Fingerprints of Anthocyanins and Flavonols in Wild

L. ssp.

**Grapes (***Vitis vinifera* **L. ssp.** *sylvestris* **(Gmelin) Hegi)**

Phenolic compounds are a group of natural products that play an important role in the quality of wines. Most phenolic compounds present in wine are derived from those contained in grapes and extracted from skins, seeds, and pulp during the initial steps of winemaking. Among them, anthocyanins and flavonols are involved in the color of red wines as pigments or copigments and also as precursors of polymeric pigments after reaction with other phenols. Biosynthesis of those phenolics in grapes is regulated by different genes; thus, each grape genotype presents a characteristic phenolic fingerprint, which is modulated by different environmental conditions. In this chapter, the anthocyanins and flavonols composition of different genotypes of wild grapes preserved at El Encin Germplasm Bank has been examined in detail. Wild grapevines are a remarkable genetic resource that may be used in breeding programs to improve the phenolic com-

Anthocyanins and flavonols are two families of phenolic compounds that play important roles in Enology. Free anthocyanins are the pigments responsible for the coloration of young red wines and take part in the reactions leading to the formation of stable polymeric pigments responsible for the coloration of aged red wines [1]. On the other hand, flavonols are involved in copigmentation of the flavylium form of anthocyanins in young red wines [2]. Moreover, flavonols present antioxidant properties that pose positive effects on human health [3]. The pathways involved in the biosynthesis of these molecules are well-known, and the core structural genes of those pathways, leading to the formation of primitive anthocyanins (delphinidin-3-O-glucoside

> © The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and eproduction in any medium, provided the original work is properly cited.

© 2018 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

sylvestris

Eugenio Revilla, Rosa Arroyo-Garcia, Alberto Bellido, David Carrasco, Anna Puig and Leonor Ruiz-Garcia

position of cultivated grapes and, hence, the quality of red wines.

Keywords: wild grapes, anthocyanins, flavonols, fingerprint, HPLC

Additional information is available at the end of the chapter

Additional information is available at the end of the chapter

Alberto Bellido, David Carrasco, Anna Puig

vinifera

http://dx.doi.org/10.5772/intechopen.70861

Eugenio Revilla, Rosa Arroyo-Garcia,

Vitis

and Leonor Ruiz-Garcia

Abstract

1. Introduction

Grapes (

#### **Fingerprints of Anthocyanins and Flavonols in Wild Grapes (***Vitis vinifera* **L. ssp.** *sylvestris* **(Gmelin) Hegi)** Fingerprints of Anthocyanins and Flavonols in Wild Grapes (Vitis vinifera L. ssp. sylvestris(Gmelin) Hegi)

DOI: 10.5772/intechopen.70861

Eugenio Revilla, Rosa Arroyo-Garcia, Alberto Bellido, David Carrasco, Anna Puig and Leonor Ruiz-Garcia Eugenio Revilla, Rosa Arroyo-Garcia, Alberto Bellido, David Carrasco, Anna Puig

Additional information is available at the end of the chapter Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/intechopen.70861

#### Abstract

and Leonor Ruiz-Garcia

Phenolic compounds are a group of natural products that play an important role in the quality of wines. Most phenolic compounds present in wine are derived from those contained in grapes and extracted from skins, seeds, and pulp during the initial steps of winemaking. Among them, anthocyanins and flavonols are involved in the color of red wines as pigments or copigments and also as precursors of polymeric pigments after reaction with other phenols. Biosynthesis of those phenolics in grapes is regulated by different genes; thus, each grape genotype presents a characteristic phenolic fingerprint, which is modulated by different environmental conditions. In this chapter, the anthocyanins and flavonols composition of different genotypes of wild grapes preserved at El Encin Germplasm Bank has been examined in detail. Wild grapevines are a remarkable genetic resource that may be used in breeding programs to improve the phenolic composition of cultivated grapes and, hence, the quality of red wines.

Keywords: wild grapes, anthocyanins, flavonols, fingerprint, HPLC

## 1. Introduction

Anthocyanins and flavonols are two families of phenolic compounds that play important roles in Enology. Free anthocyanins are the pigments responsible for the coloration of young red wines and take part in the reactions leading to the formation of stable polymeric pigments responsible for the coloration of aged red wines [1]. On the other hand, flavonols are involved in copigmentation of the flavylium form of anthocyanins in young red wines [2]. Moreover, flavonols present antioxidant properties that pose positive effects on human health [3]. The pathways involved in the biosynthesis of these molecules are well-known, and the core structural genes of those pathways, leading to the formation of primitive anthocyanins (delphinidin-3-O-glucoside

© The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, © 2018 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

distribution, and eproduction in any medium, provided the original work is properly cited.

and cyanidin-3-O-glucoside) and flavonol aglycones, like myricetin and quercetin, have been cloned and characterized [4, 5]. Moreover, several O-methyltransferases involved in the methylation of anthocyanins and flavonol glycosides have been identified [6, 7]; it has been demonstrated that the color exhibited by different grape cultivars may be associated with the VvmybA1 and VvmybA2 regulatory genes [8–10] that activate the expression of structural genes involved in the late steps of the anthocyanins biosynthetic pathway.

Anthocyanins are red pigments accumulated in skins during grape maturation (and also in pulp in teinturier cultivars), and their content has been related to several agroecological factors [11, 12], especially light and temperature, light being indispensable for anthocyanin biosynthesis and accumulation in the skins of berries and for phenylalanine ammonia lyase activity. Thus, their concentration is quite variable, even if the same cultivar or the same clone grown in a given location has been examined in several consecutive years [13, 14]. Nevertheless, the proportion of different anthocyanins, or anthocyanin fingerprint, is quite similar in the late stages of grape maturation of a cultivar grown in a given location from year to year [14]. On the other hand, the accumulation of flavonols (that are yellow pigments predominantly synthesized in grape skins [15]), is affected by shading treatments. The studies carried out in Shiraz grapes suggest that the branch of flavonoid biosynthetic pathway leading to flavonol biosynthesis is light-dependent, in contrast to anthocyanin and flavanol biosynthesis, which are little affected by shading treatments [16].

Cultivated grapevines are thought to be domesticated from genotypes of Vitis vinifera L. ssp. sylvestris (Gmelin) Hegi, which are present in small, isolated wild populations, located in riverbanks from the Western Himalayas to Western Europe [17, 18]. The sanitary status of those populations and their morphological and genetic characteristics have been recently studied [19–22]. Mature fruits of wild grapevines usually show high acidity, low pH, and a high intensity of color if compared with cultivated grapes [23, 24]; these features might be used to adapt Viticulture to the new climatic conditions, mitigating the potential effects of global warming on grape production.

on 110R and trained to cordon Royat, were collected in October 2008 at optimum stage of maturation (between 200 and 240 g of glucose + fructose by kg of must). Each sample consisted of four clusters, as only two plants of each genotype were available. Those genotypes, grown in El Encin, were originally collected from natural populations located in different Spanish regions (see Figure 1); 10 of them came from Northern Spain (Asturias, Cantabria, Castilla-León, Basque Country, and Navarra), the other 15 from Southern Spain (Andalousie, Castilla-La Mancha, and Extremadura). Every natural population was identified by two letters and by one or two numbers, and each genotype was identified with the population code and an additional number, as well as the suffix bis in some cases. Once in the laboratory, samples

Fingerprints of Anthocyanins and Flavonols in Wild Grapes (*Vitis vinifera* L. ssp. *sylvestris* (Gmelin) Hegi)

http://dx.doi.org/10.5772/intechopen.70861

25

Figure 1. Location of natural populations of wild grapes where genotypes under study were originally collected.

Fifty berries were randomly selected and weighed once berries were separated from clusters, and grape skins were removed from pulps and seeds and stored at 20C in methanol. Afterward, grape skins were grinded in a Kinematica PCU-2 blender for 1 minute. Then, they were sequentially extracted, using 25 mL of solvent for each extraction step: methanol for 16 hours at 25C, 80% methanol for 4 hours at room temperature, 50% methanol for 4 hours at room temperature, deionized water for 16 hours at 25C, and 75% acetone for 1 hour at

were stored at 20C until sample preparation.

3. Sample preparation

The qualitative and quantitative anthocyanin composition of wild grape accessions preserved at El Encin Germplasm Bank has been examined by our research group after 2006 [25, 26], as well as their flavonol fingerprint [27–29]. The main objective of this study is to evaluate whether the anthocyanin and flavonol composition of wild grapes differs from that presented by cultivated grapes, and to determine whether some wild genotypes present some genetic characters of interest related to anthocyanins and flavonols accumulation during grape maturation. For this purpose, different female genotypes preserved at El Encin Germplasm Bank, that were collected in various natural populations located in different Spanish regions, were sampled in 2008 and have been fully examined for anthocyanins and flavonols content by HPLC.

## 2. Sampling of grapes

Samples of 25 genotypes of wild grapevines from different Spanish natural populations preserved at El Encin Grapevine Germplasm Bank (IMIDRA, Alcalá de Henares, Spain), grafted Fingerprints of Anthocyanins and Flavonols in Wild Grapes (*Vitis vinifera* L. ssp. *sylvestris* (Gmelin) Hegi) http://dx.doi.org/10.5772/intechopen.70861 25

Figure 1. Location of natural populations of wild grapes where genotypes under study were originally collected.

on 110R and trained to cordon Royat, were collected in October 2008 at optimum stage of maturation (between 200 and 240 g of glucose + fructose by kg of must). Each sample consisted of four clusters, as only two plants of each genotype were available. Those genotypes, grown in El Encin, were originally collected from natural populations located in different Spanish regions (see Figure 1); 10 of them came from Northern Spain (Asturias, Cantabria, Castilla-León, Basque Country, and Navarra), the other 15 from Southern Spain (Andalousie, Castilla-La Mancha, and Extremadura). Every natural population was identified by two letters and by one or two numbers, and each genotype was identified with the population code and an additional number, as well as the suffix bis in some cases. Once in the laboratory, samples were stored at 20C until sample preparation.

#### 3. Sample preparation

and cyanidin-3-O-glucoside) and flavonol aglycones, like myricetin and quercetin, have been cloned and characterized [4, 5]. Moreover, several O-methyltransferases involved in the methylation of anthocyanins and flavonol glycosides have been identified [6, 7]; it has been demonstrated that the color exhibited by different grape cultivars may be associated with the VvmybA1 and VvmybA2 regulatory genes [8–10] that activate the expression of structural genes involved in

Anthocyanins are red pigments accumulated in skins during grape maturation (and also in pulp in teinturier cultivars), and their content has been related to several agroecological factors [11, 12], especially light and temperature, light being indispensable for anthocyanin biosynthesis and accumulation in the skins of berries and for phenylalanine ammonia lyase activity. Thus, their concentration is quite variable, even if the same cultivar or the same clone grown in a given location has been examined in several consecutive years [13, 14]. Nevertheless, the proportion of different anthocyanins, or anthocyanin fingerprint, is quite similar in the late stages of grape maturation of a cultivar grown in a given location from year to year [14]. On the other hand, the accumulation of flavonols (that are yellow pigments predominantly synthesized in grape skins [15]), is affected by shading treatments. The studies carried out in Shiraz grapes suggest that the branch of flavonoid biosynthetic pathway leading to flavonol biosynthesis is light-dependent, in contrast to anthocyanin and flavanol biosynthesis, which

Cultivated grapevines are thought to be domesticated from genotypes of Vitis vinifera L. ssp. sylvestris (Gmelin) Hegi, which are present in small, isolated wild populations, located in riverbanks from the Western Himalayas to Western Europe [17, 18]. The sanitary status of those populations and their morphological and genetic characteristics have been recently studied [19–22]. Mature fruits of wild grapevines usually show high acidity, low pH, and a high intensity of color if compared with cultivated grapes [23, 24]; these features might be used to adapt Viticulture to the new climatic conditions, mitigating the potential effects of global

The qualitative and quantitative anthocyanin composition of wild grape accessions preserved at El Encin Germplasm Bank has been examined by our research group after 2006 [25, 26], as well as their flavonol fingerprint [27–29]. The main objective of this study is to evaluate whether the anthocyanin and flavonol composition of wild grapes differs from that presented by cultivated grapes, and to determine whether some wild genotypes present some genetic characters of interest related to anthocyanins and flavonols accumulation during grape maturation. For this purpose, different female genotypes preserved at El Encin Germplasm Bank, that were collected in various natural populations located in different Spanish regions, were sampled in

2008 and have been fully examined for anthocyanins and flavonols content by HPLC.

Samples of 25 genotypes of wild grapevines from different Spanish natural populations preserved at El Encin Grapevine Germplasm Bank (IMIDRA, Alcalá de Henares, Spain), grafted

the late steps of the anthocyanins biosynthetic pathway.

24 Grapes and Wines - Advances in Production, Processing, Analysis and Valorization

are little affected by shading treatments [16].

warming on grape production.

2. Sampling of grapes

Fifty berries were randomly selected and weighed once berries were separated from clusters, and grape skins were removed from pulps and seeds and stored at 20C in methanol. Afterward, grape skins were grinded in a Kinematica PCU-2 blender for 1 minute. Then, they were sequentially extracted, using 25 mL of solvent for each extraction step: methanol for 16 hours at 25C, 80% methanol for 4 hours at room temperature, 50% methanol for 4 hours at room temperature, deionized water for 16 hours at 25C, and 75% acetone for 1 hour at room temperature [30]. At the end of each extraction step, the liquid was centrifugated at 3500 rpm for 20 minutes in a Rotofix 32A centrifuge, and the residue was submitted to extraction again. The volume of the combined liquid extracts was raised between 125 and 200 mL with methanol. Then, the extracts were stored at 20C prior to analysis.

Total anthocyanins were determined in grape skins extracts, using the procedure described by Niketic-Aleksic and Hrzadina [32] using a BOECO S-22 UV–Vis spectrophotometer. Quantitative analysis of flavonols was carried out by HPLC, considering the surface of the different peaks, using standard solutions of quercetin-3-O-glucoside in the range of 20–100 mg/L.

Fingerprints of Anthocyanins and Flavonols in Wild Grapes (*Vitis vinifera* L. ssp. *sylvestris* (Gmelin) Hegi)

The HPLC analysis of anthocyanins extracted from wild grape skins permits the separation of 15 different anthocyanins. Table 1 shows name, abbreviation, and number of peaks for each compound considered. The anthocyanin fingerprint of wild grapes revealed the presence of three groups of wild grapes genotypes, as it has been previously reported [25, 26]. Figure 2

The three groups of genotypes differ in different aspects linked to the pathways involved in anthocyanin biosynthesis [33] that are shown in Figures 3 and 4. First, the presence or absence of acylated anthocyanins, which implies important differences in the expression of genes involved in acyltransferase activity. Second, the prevalence of anthocyanins derived from delphinidin (Dp) or from cyanidin (Cy), which implies the differential expression of genes that

of methylation of Dp-3-gl and Cy-3-gl, due to the differential expression of genes controlling

Anthocyanin Abbreviation Number of peaks

Delphinidin-3-O-glucoside Dp-3-gl 1 Cyanidin-3-O-glucoside Cy-3-gl 2 Petunidin-3-O-glucoside Pt-3-gl 3 Peonidin-3-glucoside Pn3-gl 4 Malvidin-3-glucoside Mv-3-gl 5 Delphinidin-3-acetyl-glucoside Dp-3-acgl 6 Cyanidin-3-acetylglucoside Cy-3-acgl 7 Petunidin-3-acetylglucoside Pt-3-acgl 8 Delphinidin-3-p-coumarylglucoside Mv-3-cmgl 9 Peonidin-3-acetylglucoside Pn-3-acgl 10 Malvidin-3-acetylglucoside Mv-3-acgl 11 Petunidin-3-p-coumarylglucoside Pt-3-cmgl 12 Malvidin-3-caffeoylglucoside Mv-3-cfgl 13 Peonidin-3-p-coumarylglucoside Pn-3-cmgl 14 Malvidin-3-p-coumarylglucoside Mv-3-cmgl 15

Table 1. Name, abbreviation, and number of peaks for the different anthocyanins analyzed by HPLC.

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27

5. Anthocyanin fingerprint of wild grapes

control flavonoid-3<sup>0</sup>

O-methyltransferase activity.

displays three typical chromatograms of those groups of genotypes.


Flavonols were isolated prior to HPLC analysis to avoid interferences caused by anthocyanins, using solid-phase extraction on Oasis MCX cartridges (6 mL capacity) filled with 500 mg of an adsorbent containing a mixture of reverse-phase and cationic-exchanger materials (Waters Corp., Milford, MA), following a procedure described previously [31]. For this purpose, 3 mL grape skins extract was dried in a rotary evaporator (40C) and resolved in 0.1 M hydrochloric acid (3 mL). Then, it passed through the MCX cartridges, previously conditioned with methanol (5 mL) and water (5 mL). After washing with 0.1 M hydrochloric acid (5 mL) and water (5 mL), the flavonol fraction was eluted with methanol (3 5 mL). This fraction also contained other neutral or acidic polyphenols. Fixed anthocyanins were removed using 2% ammonia in 80% methanol (3 5 mL). Finally, the cationic-exchanger material was regenerated with 0.52 M hydrochloric acid in 80% methanol (3 5 mL). Subsequent conditioning of the cartridge with methanol and water allows its reuse at least four or five more times. The eluate containing flavonols was dried in a rotary evaporator (30C) and resolved in 1 mL of methanol.

#### 4. Analytical procedures

The anthocyanin and flavonol fingerprints of skin extracts were obtained with HPLC-DAD [27], using a Waters Corp. liquid chromatograph consisting of a 600 quaternary pump, a 717 automatic injector, a TC2 controller for a column oven, a 996 photodiode array detector, and a Millennium 32 workstation. The separations were performed using a Waters Nova-Pak C18 steel cartridge (3.9 250 mm), filled with 5-μm particles, and furnished with a Waters Sentry Nova-Pack C18 guard cartridge (20 3.9 mm), both thermostated at 55C. Water/acetonitrile (95:5) adjusted to pH 1.3 with trifluoroacetic acid (solvent A), and water/acetonitrile (50:50) adjusted to pH 1.3 with trifluoroacetic acid (solvent B) were used as mobile phases. Elution was performed at a 0.8 mL/min flow rate. For anthocyanins, a linear gradient from 15% B to 35% B in 20 min, from 35% B to 50% B in 10 min, 50% B for 6 min, from 50% B to 100% B in 5 min, 100% B for 5 min, 100% B to 15% B in 1 min was used. A linear gradient from 10% B to 35% B in 30 min, from 35% B to 50% B in 6 min, from 50% B to 100% B in 8 min, 100% B for 3 min, and from 100% B to 10% B in 1 min was used for flavonols. Samples (20 μL) were injected in triplicate. Spectra were recorded every second between 250 and 600 nm, with a bandwidth of 1.2 nm. Samples, standard solutions, and mobile phases were filtered before analysis through a 0.45-μm pore size membrane. The identity of the different anthocyanins and flavonols was elucidated by HPLC-MS, using an 1100 HPLC system (Agilent Technologies, Santa Clara, CA) with a PDA UV–Vis detector coupled to a QTOF mass spectrometer (AB SCiex, Framingham, MA). Chromatographic conditions were those used for the HPLC-DAD analysis. The MS analysis was carried out in the ESI+ mode, scanning from m/z 50 to 2000, with the following conditions: spray voltage, 5500 V; gas pressure, 80 psi; declustering potential, 50 V; focus potential, 210 V; CAD, 3 psi.

Total anthocyanins were determined in grape skins extracts, using the procedure described by Niketic-Aleksic and Hrzadina [32] using a BOECO S-22 UV–Vis spectrophotometer. Quantitative analysis of flavonols was carried out by HPLC, considering the surface of the different peaks, using standard solutions of quercetin-3-O-glucoside in the range of 20–100 mg/L.

## 5. Anthocyanin fingerprint of wild grapes

room temperature [30]. At the end of each extraction step, the liquid was centrifugated at 3500 rpm for 20 minutes in a Rotofix 32A centrifuge, and the residue was submitted to extraction again. The volume of the combined liquid extracts was raised between 125 and

Flavonols were isolated prior to HPLC analysis to avoid interferences caused by anthocyanins, using solid-phase extraction on Oasis MCX cartridges (6 mL capacity) filled with 500 mg of an adsorbent containing a mixture of reverse-phase and cationic-exchanger materials (Waters Corp., Milford, MA), following a procedure described previously [31]. For this purpose, 3 mL grape skins extract was dried in a rotary evaporator (40C) and resolved in 0.1 M hydrochloric acid (3 mL). Then, it passed through the MCX cartridges, previously conditioned with methanol (5 mL) and water (5 mL). After washing with 0.1 M hydrochloric acid (5 mL) and water (5 mL), the flavonol fraction was eluted with methanol (3 5 mL). This fraction also contained other neutral or acidic polyphenols. Fixed anthocyanins were removed using 2% ammonia in 80% methanol (3 5 mL). Finally, the cationic-exchanger material was regenerated with 0.52 M hydrochloric acid in 80% methanol (3 5 mL). Subsequent conditioning of the cartridge with methanol and water allows its reuse at least four or five more times. The eluate containing flavonols was dried in a rotary evaporator (30C) and resolved in 1 mL of methanol.

The anthocyanin and flavonol fingerprints of skin extracts were obtained with HPLC-DAD [27], using a Waters Corp. liquid chromatograph consisting of a 600 quaternary pump, a 717 automatic injector, a TC2 controller for a column oven, a 996 photodiode array detector, and a Millennium 32 workstation. The separations were performed using a Waters Nova-Pak C18 steel cartridge (3.9 250 mm), filled with 5-μm particles, and furnished with a Waters Sentry Nova-Pack C18 guard cartridge (20 3.9 mm), both thermostated at 55C. Water/acetonitrile (95:5) adjusted to pH 1.3 with trifluoroacetic acid (solvent A), and water/acetonitrile (50:50) adjusted to pH 1.3 with trifluoroacetic acid (solvent B) were used as mobile phases. Elution was performed at a 0.8 mL/min flow rate. For anthocyanins, a linear gradient from 15% B to 35% B in 20 min, from 35% B to 50% B in 10 min, 50% B for 6 min, from 50% B to 100% B in 5 min, 100% B for 5 min, 100% B to 15% B in 1 min was used. A linear gradient from 10% B to 35% B in 30 min, from 35% B to 50% B in 6 min, from 50% B to 100% B in 8 min, 100% B for 3 min, and from 100% B to 10% B in 1 min was used for flavonols. Samples (20 μL) were injected in triplicate. Spectra were recorded every second between 250 and 600 nm, with a bandwidth of 1.2 nm. Samples, standard solutions, and mobile phases were filtered before analysis through a 0.45-μm pore size membrane. The identity of the different anthocyanins and flavonols was elucidated by HPLC-MS, using an 1100 HPLC system (Agilent Technologies, Santa Clara, CA) with a PDA UV–Vis detector coupled to a QTOF mass spectrometer (AB SCiex, Framingham, MA). Chromatographic conditions were those used for the HPLC-DAD analysis. The MS analysis was carried out in the ESI+ mode, scanning from m/z 50 to 2000, with the following conditions: spray voltage, 5500 V; gas pressure, 80 psi; declustering poten-

200 mL with methanol. Then, the extracts were stored at 20C prior to analysis.

26 Grapes and Wines - Advances in Production, Processing, Analysis and Valorization

4. Analytical procedures

tial, 50 V; focus potential, 210 V; CAD, 3 psi.

The HPLC analysis of anthocyanins extracted from wild grape skins permits the separation of 15 different anthocyanins. Table 1 shows name, abbreviation, and number of peaks for each compound considered. The anthocyanin fingerprint of wild grapes revealed the presence of three groups of wild grapes genotypes, as it has been previously reported [25, 26]. Figure 2 displays three typical chromatograms of those groups of genotypes.

The three groups of genotypes differ in different aspects linked to the pathways involved in anthocyanin biosynthesis [33] that are shown in Figures 3 and 4. First, the presence or absence of acylated anthocyanins, which implies important differences in the expression of genes involved in acyltransferase activity. Second, the prevalence of anthocyanins derived from delphinidin (Dp) or from cyanidin (Cy), which implies the differential expression of genes that control flavonoid-3<sup>0</sup> -hydroxylase and flavonoid-3<sup>0</sup> ,50 -hydroxylase activities. Finally, the extent of methylation of Dp-3-gl and Cy-3-gl, due to the differential expression of genes controlling O-methyltransferase activity.


Table 1. Name, abbreviation, and number of peaks for the different anthocyanins analyzed by HPLC.

Figure 2. Chromatograms registered at 520 nm for three grape skins extracts representative of phenotypic groups A, B, and C. For key to peaks, see Table 1.

Figure 3. Biosynthesis of Dp-3-gl and Cy-3-gl. CH3, chalcone synthase; CHI, chalcone isomerase; F3H, flavanone-3-

H, flavonoid-3<sup>0</sup>

ANS, anthocyanidin synthase; UFGT, UDP-Glc-flavonoid 3-O-glucosyltransferase. See full compound names in Table 1.

Figure 4. Biosynthesis of anthocyanins derived from Dp-3-gl and Cy-3-gl. MT, methyltransferase; AT, acyltransferase. See

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Fingerprints of Anthocyanins and Flavonols in Wild Grapes (*Vitis vinifera* L. ssp. *sylvestris* (Gmelin) Hegi)


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29

50

hydroxylase; F3<sup>0</sup>

full compound names in Table 1.

H, flavonoid-3<sup>0</sup>


Genotypes of group A (tree samples) did not contain acylated anthocyanins (Figure 2A). This character is unusual in cultivated grapevines, occurring primarily in cv. Pinot Noir and its colored mutants [34, 35]. In these genotypes, genes encoding or regulating acyltransferase activity is neither presented nor expressed. To our knowledge, this type of anthocyanin fingerprint has not been described in grape cultivars usually considered of Spanish origin [35–37]. Table 2 displays the percentages of several groups of anthocyanins presented in these genotypes. As can be observed, one genotype (BI-1.3bis) contained a remarkable amount of Cyderived anthocyanins, over 50%, and the extent of methylation was very high (over 60%) in two genotypes (BI-1.3bis and LE-1.2). Similar trends were observed in several wild grapevine accessions that do not contain acylated anthocyanins in a previous report [26].

Fingerprints of Anthocyanins and Flavonols in Wild Grapes (*Vitis vinifera* L. ssp. *sylvestris* (Gmelin) Hegi) http://dx.doi.org/10.5772/intechopen.70861 29

Figure 3. Biosynthesis of Dp-3-gl and Cy-3-gl. CH3, chalcone synthase; CHI, chalcone isomerase; F3H, flavanone-3 hydroxylase; F3<sup>0</sup> H, flavonoid-3<sup>0</sup> -hydroxylase; F3<sup>0</sup> 50 H, flavonoid-3<sup>0</sup> ,50 -hydroxylase; DFR, dihydroflavonol-4-reductase; ANS, anthocyanidin synthase; UFGT, UDP-Glc-flavonoid 3-O-glucosyltransferase. See full compound names in Table 1.

Genotypes of group A (tree samples) did not contain acylated anthocyanins (Figure 2A). This character is unusual in cultivated grapevines, occurring primarily in cv. Pinot Noir and its colored mutants [34, 35]. In these genotypes, genes encoding or regulating acyltransferase activity is neither presented nor expressed. To our knowledge, this type of anthocyanin fingerprint has not been described in grape cultivars usually considered of Spanish origin [35–37]. Table 2 displays the percentages of several groups of anthocyanins presented in these genotypes. As can be observed, one genotype (BI-1.3bis) contained a remarkable amount of Cyderived anthocyanins, over 50%, and the extent of methylation was very high (over 60%) in two genotypes (BI-1.3bis and LE-1.2). Similar trends were observed in several wild grapevine

Figure 2. Chromatograms registered at 520 nm for three grape skins extracts representative of phenotypic groups A, B,

accessions that do not contain acylated anthocyanins in a previous report [26].

28 Grapes and Wines - Advances in Production, Processing, Analysis and Valorization

and C. For key to peaks, see Table 1.

Figure 4. Biosynthesis of anthocyanins derived from Dp-3-gl and Cy-3-gl. MT, methyltransferase; AT, acyltransferase. See full compound names in Table 1.


Table 2. Percentages of Dp-derived, Cy-derived, and methylated anthocyanins in genotypes of group A.

Genotypes of group B (six samples) contained acylated anthocyanins and a high proportion of Cy-derived anthocyanins (Figure 2B). This character is rare in cultivated grapevines, was observed only in 12 cultivars among 64 studied by Mattivi et al. [34], and it has been reported only in a cultivar considered of Spanish origin, cv. Brancellao [38]. Most cultivars of this type are gray or rosé cultivars, or even mutants of white cultivars. Table 3 displays the percentages of several groups of anthocyanins presented in these genotypes. The percentage of Cy-derived anthocyanins ranged between 37 and 68%, and usually the percentage of methylated anthocyanins was up to 50%. Thus, Pn-3-gl usually was the major anthocyanin; the most remarkable exception was genotype SS-6.5bis. In this genotype, Cy-3-gl was the major anthocyanin (40.82%), as methylation was not very intense. Acylation was quite variable; it was too low in genotype CO-5.1, but considerably high in genotypes CA-13.3 and H-6.1. Moreover, most acylated anthocyanins were p-coumarylated derivatives; this character is quite common in red cultivars usually considered as Spanish, like Garnacha and Tempranillo [35].

Genotypes of group C (16 samples) contained acylated anthocyanins and a high proportion of delphinidin-derived anthocyanins (Figure 2C), as do most grapevine cultivars [33–37]; this implies that the expression of genes controlling flavonoid-3<sup>0</sup> ,50 -hydroxylase is too high if compared with that of genes controlling flavonoid-3<sup>0</sup> -hydroxylase. The percentages of several groups of anthocyanins presented in these genotypes are displayed in Table 4. As can be noted, these genotypes also presented a high extent of methylation; the percentage of methylated anthocyanins was higher than 60%, except in four genotypes (BA-1.1, NA-1.4bis, SE-3.4,

and SS-3.5). In these late genotypes, Dp-3-gl was the major anthocyanin, but in the other 11 genotypes, the major genotype was Mv-3-gl. Sometimes, its content was higher than 90%.

Table 4. Percentages of Dp-derived, Cy-derived, methylated, acylated, acetylated, and p-coumarylated anthocyanins in

Genotype Dp-derived Cy-derived Methylated Acylated Acetylated p-Coumarylated

Fingerprints of Anthocyanins and Flavonols in Wild Grapes (*Vitis vinifera* L. ssp. *sylvestris* (Gmelin) Hegi)

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BA-1.1 85.75 14.25 59.97 3.56 1.85 1.61 CA-4.1 82.69 17.31 71.97 14.70 4.76 9.01 CA-9.7 87.90 12.10 93.26 6.33 2.62 1.90 CA-11.3 83.92 16.08 65.99 34.26 27.74 6.06 CO-2.2 87.13 12.87 68.84 15.40 7.60 6.55 CO-3.7 81.95 18.05 73.27 16.32 8.00 7.08 CR-1.6 91.87 8.13 84.54 10.73 5.12 4.16 H-1.1 79.67 20.33 72.15 3.65 1.97 1.21 J-2.4 88.23 11.77 90.76 9.01 4.08 3.90 NA-1.4bis 73.82 26.18 43.96 2.82 1.26 1.56 O-1.5bis 80.48 19.52 62.43 6.64 3.26 3.15 S-1.3bis 88.95 11.05 66.03 16.43 7.42 8.10 S-1.9 90.97 9.03 65.99 8.84 4.37 4.11 SE-1.5 92.97 7.03 86.35 25.39 11.12 12.96 SE-3.4 84.65 15.35 58.70 16.42 6.52 9.59 SS-3.5 78.68 21.32 54.09 21.27 17.41 3.58

The extent of acylation among genotypes included in group C was quite variable and not related to the extent of methylation. The percentage of acylated anthocyanins ranged from less than 3% (NA-1.4bis, 2.82%) to nearly 35% (CA-11.3, 34.26%). In two genotypes with a high extent of acylation (CA-11.3 and SS-3.5), acetylated anthocyanins were much more abundant than p-coumarylated anthocyanins. This character is well-documented in several French cultivars (e.g., Cabernet Sauvignon and Merlot), but is rare in Spanish cultivars. Most genotypes present less than 15% acylated anthocyanins, and percentages of acetylated and p-coumarylated anthocyanins were quite similar, as it has been observed in many grape cultivars

Data reported in Table 5 point out that the total content of anthocyanins was quite variable, ranging from 273 to 3534 mg/kg, but, there is a remarkable difference among genotypes collected in populations located in Northern Spain and those from populations located in Southern Spain. As can be noted, genotypes from Northern Spain contained a higher amount of anthocyanins than those originated in Southern Spain (p < 0.05). As it is well-known, the accumulation of anthocyanins in grapes, that take place after veraison, is affected, at a great extent, by day-night thermal contrast [12], which can be considered neutral in our study, as all

considered of Spanish origin [35].

See full compound names in Table 1.

genotypes of group C.


See full compound names in Table 1.

Table 3. Percentages of Dp-derived, Cy-derived, methylated, acylated, acetylated, and p-coumarylated anthocyanins in genotypes of group B.

Fingerprints of Anthocyanins and Flavonols in Wild Grapes (*Vitis vinifera* L. ssp. *sylvestris* (Gmelin) Hegi) http://dx.doi.org/10.5772/intechopen.70861 31


See full compound names in Table 1.

Genotypes of group B (six samples) contained acylated anthocyanins and a high proportion of Cy-derived anthocyanins (Figure 2B). This character is rare in cultivated grapevines, was observed only in 12 cultivars among 64 studied by Mattivi et al. [34], and it has been reported only in a cultivar considered of Spanish origin, cv. Brancellao [38]. Most cultivars of this type are gray or rosé cultivars, or even mutants of white cultivars. Table 3 displays the percentages of several groups of anthocyanins presented in these genotypes. The percentage of Cy-derived anthocyanins ranged between 37 and 68%, and usually the percentage of methylated anthocyanins was up to 50%. Thus, Pn-3-gl usually was the major anthocyanin; the most remarkable exception was genotype SS-6.5bis. In this genotype, Cy-3-gl was the major anthocyanin (40.82%), as methylation was not very intense. Acylation was quite variable; it was too low in genotype CO-5.1, but considerably high in genotypes CA-13.3 and H-6.1. Moreover, most acylated anthocyanins were p-coumarylated derivatives; this character is quite common in red cultivars usually considered as Spanish, like Garnacha and

Genotype Dp-derived Cy-derived Methylated

BI-1.3bis 48.14 51.86 62.25 LE-1.2 80.61 19.39 82.14 SS-3.5bis 70.10 29.90 36.75

30 Grapes and Wines - Advances in Production, Processing, Analysis and Valorization

Table 2. Percentages of Dp-derived, Cy-derived, and methylated anthocyanins in genotypes of group A.

Genotypes of group C (16 samples) contained acylated anthocyanins and a high proportion of delphinidin-derived anthocyanins (Figure 2C), as do most grapevine cultivars [33–37]; this

groups of anthocyanins presented in these genotypes are displayed in Table 4. As can be noted, these genotypes also presented a high extent of methylation; the percentage of methylated anthocyanins was higher than 60%, except in four genotypes (BA-1.1, NA-1.4bis, SE-3.4,

Genotype Dp-derived Cy-derived Methylated Acylated Acetylated p-Coumarylated

Table 3. Percentages of Dp-derived, Cy-derived, methylated, acylated, acetylated, and p-coumarylated anthocyanins in

CA-6.1 32.98 67.02 91.26 4.84 1.58 2.83 CA-13.3 54.35 45.65 80.84 14.77 4.42 9.97 CO-5.1 48.04 51.96 55.07 1.78 0.63 1.06 H-6.1 62.83 37.17 54.62 17.41 9.05 8.29 SS-6.5bis 42.70 57.30 31.17 4.46 2.09 2.38 VI-2.1bis 58.34 41.66 52.55 6.63 2.96 3.58

,50



implies that the expression of genes controlling flavonoid-3<sup>0</sup>

compared with that of genes controlling flavonoid-3<sup>0</sup>

Tempranillo [35].

See full compound names in Table 1.

genotypes of group B.

See full compound names in Table 1.

Table 4. Percentages of Dp-derived, Cy-derived, methylated, acylated, acetylated, and p-coumarylated anthocyanins in genotypes of group C.

and SS-3.5). In these late genotypes, Dp-3-gl was the major anthocyanin, but in the other 11 genotypes, the major genotype was Mv-3-gl. Sometimes, its content was higher than 90%.

The extent of acylation among genotypes included in group C was quite variable and not related to the extent of methylation. The percentage of acylated anthocyanins ranged from less than 3% (NA-1.4bis, 2.82%) to nearly 35% (CA-11.3, 34.26%). In two genotypes with a high extent of acylation (CA-11.3 and SS-3.5), acetylated anthocyanins were much more abundant than p-coumarylated anthocyanins. This character is well-documented in several French cultivars (e.g., Cabernet Sauvignon and Merlot), but is rare in Spanish cultivars. Most genotypes present less than 15% acylated anthocyanins, and percentages of acetylated and p-coumarylated anthocyanins were quite similar, as it has been observed in many grape cultivars considered of Spanish origin [35].

Data reported in Table 5 point out that the total content of anthocyanins was quite variable, ranging from 273 to 3534 mg/kg, but, there is a remarkable difference among genotypes collected in populations located in Northern Spain and those from populations located in Southern Spain. As can be noted, genotypes from Northern Spain contained a higher amount of anthocyanins than those originated in Southern Spain (p < 0.05). As it is well-known, the accumulation of anthocyanins in grapes, that take place after veraison, is affected, at a great extent, by day-night thermal contrast [12], which can be considered neutral in our study, as all


Table 5. Range, mean value, and standard deviation for the content of total anthocyanins (mg/kg of grapes) in genotypes originated in northern Spain and southern Spain.

genotypes grew in the same environment. Thus, differences observed in anthocyanin content can be considered of genetic nature. The most probable explanation is that genotypes from Northern Spain have evolved in oceanic climate environments, where veraison takes place at the end of summer, and day-night thermal contrast is smaller than that in the Mediterranean climate environments in which evolved those genotypes collected from Southern Spain. Thus, it is probable that wild grapes in Northern Spain have evolved to accumulate enough anthocyanins capable of attracting birds and other animals to facilitate the dispersion of seeds, despite the limiting weather conditions for anthocyanin accumulation. Thus, when genotypes from Northern Spain grow in a warmer environment, like that of El Encin Germplasm Bank, the accumulation of anthocyanins may be very high.

#### 6. Flavonol fingerprint of wild grapes

Six different flavonols were fully identified by HPLC-MS: a myricetin derivative (3-O-glucoside, My-3-gl), three quercetin derivatives (3-O-glucoside, Qu-3-gl; 3-O-glucuronide, Qu-3-gr; 3-O-rhamnoside, Qu-3-rh), a laricitrin derivative (3-O-glucoside, La-3-gl), and a syringetin derivative (3-O-glucoside, Sy-3-gl). All these flavonols have been identified in berries of several red grapevine cultivars [31, 38], and their presence in wild grapes should be expected. The flavonols tentatively identified were 3-O-galactosides of myricetin (My-3-gal) and quercetin (Qu-3-gal), which have been previously detected in red grape skins [38].

Among those flavonols, the most abundant were My-3-gl (trihydroxysusbtituted in B-ring, analogous to Dp-3-gl) and two quercetin derivatives (Qu-3-gl and Qu-3-gr, analogous to Cy-3 gl because they are dihydroxysubstituted in B-ring). Other myricetin derivatives, like laricitrin and syringetin derivatives (La-3-gl and Sy-3-gl), were minor components, and some of them were absent in several samples. Figure 5 displays the chromatogram registered at 350 nm for an extract of genotype H-6.1, with three major peaks, corresponding to My-3-gl, Qu-3-gr, and Qu-3-gl.

the exceptions being genotypes CA-11.3 and SE-3.4. In these two genotypes, methylation of My-3-gl by action of a O-methyltransferase is blocked. Finally, group 3 includes eight genotypes presenting My-3-gl as a major flavonol (Table 8). In most cases, these genotypes contained several minor flavonoids, including La-3-gl and Sy-3-gl, the exceptions being genotypes O-1.5bis and SS-3.5. Like in genotypes CA-11.3 and SE-3.4, methylation of My-3-gl by

Table 6. Content of flavonols (mg/kg of grapes) in genotypes that did not contain My-3-gl.

Genotype My-3-gl Qu-3-gr Qu-3-gl Other flavonols Total CA-6.1 nd 27.0 60.8 14.0 101.9 CA-13.3 nd 11.5 47.7 9.0 68.2 H-6.1 nd 54.2 73.6 17.3 145.1 NA-1.4bis nd 18.0 10.7 0.0 28.7 S-1.3bis nd 10.0 6.8 12.5 29.4 SS-3.5bis nd 21.1 26.5 nd 47.6 SS-6.5bis nd 26.1 61.5 nd 87.5 VI-2.1bis nd 22.3 44.9 nd 67.2

Figure 5. Chromatogram registered at 350 nm for a grape skin extract. My-3-gl, myricetin-3-O-glucoside; Qu-3-gr,

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Data reported in Tables 6–8 point out that the total content of flavonols was quite variable, ranging from 29 to 324 mg/kg. Nevertheless, there is a remarkable difference among genotypes from populations located in Northern Spain and those from populations located in Southern Spain (Table 9). As can be noted, genotypes from Northern Spain contained a lower amount of flavonols than those originated in Southern Spain. The accumulation of flavonols in grapes

action of a methyltransferase is blocked.

nd: not detected. See full compound names in Figure 5.

quercetin-3-O-glucuronide; Qu-3-gl, quercetin-3-O-glucoside.

Three phenotypic groups of wild grapes have been considered, taking into account the amounts of My-3-gl, Qu-3-gl, and Qu-3-gr. Group 1 includes eight genotypes, which did not contain My-3-gl (Table 6). This fact implies that, in these genotypes, dihydroxylation of dihydrokaempferol by flavonoid-3<sup>0</sup> ,50 -dihydroxylase is blocked (Figure 6). In these genotypes, the major flavonol was Qu-3-gl or Qu-3-gr, and in some cases, contained very small amounts of other flavonol; anyway, La-3-gl and Sy-3-gl were absent (Table 6). Group 2 is formed by nine genotypes, which contain My-3-gl, but major flavonol was Qu-3-gl or Qu-3-gr (Table 7). These genotypes usually contained several minor flavonoids, including La-3-gl and Sy-3-gl,

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genotypes grew in the same environment. Thus, differences observed in anthocyanin content can be considered of genetic nature. The most probable explanation is that genotypes from Northern Spain have evolved in oceanic climate environments, where veraison takes place at the end of summer, and day-night thermal contrast is smaller than that in the Mediterranean climate environments in which evolved those genotypes collected from Southern Spain. Thus, it is probable that wild grapes in Northern Spain have evolved to accumulate enough anthocyanins capable of attracting birds and other animals to facilitate the dispersion of seeds, despite the limiting weather conditions for anthocyanin accumulation. Thus, when genotypes from Northern Spain grow in a warmer environment, like that of El Encin Germplasm Bank,

Table 5. Range, mean value, and standard deviation for the content of total anthocyanins (mg/kg of grapes) in genotypes

Northern Spain 956–3078 2088 693 Southern Spain 273–3534 1697 797

32 Grapes and Wines - Advances in Production, Processing, Analysis and Valorization

Range Mean value Standard deviation

Six different flavonols were fully identified by HPLC-MS: a myricetin derivative (3-O-glucoside, My-3-gl), three quercetin derivatives (3-O-glucoside, Qu-3-gl; 3-O-glucuronide, Qu-3-gr; 3-O-rhamnoside, Qu-3-rh), a laricitrin derivative (3-O-glucoside, La-3-gl), and a syringetin derivative (3-O-glucoside, Sy-3-gl). All these flavonols have been identified in berries of several red grapevine cultivars [31, 38], and their presence in wild grapes should be expected. The flavonols tentatively identified were 3-O-galactosides of myricetin (My-3-gal) and quercetin

Among those flavonols, the most abundant were My-3-gl (trihydroxysusbtituted in B-ring, analogous to Dp-3-gl) and two quercetin derivatives (Qu-3-gl and Qu-3-gr, analogous to Cy-3 gl because they are dihydroxysubstituted in B-ring). Other myricetin derivatives, like laricitrin and syringetin derivatives (La-3-gl and Sy-3-gl), were minor components, and some of them were absent in several samples. Figure 5 displays the chromatogram registered at 350 nm for an extract of genotype H-6.1, with three major peaks, corresponding to My-3-gl, Qu-3-gr, and

Three phenotypic groups of wild grapes have been considered, taking into account the amounts of My-3-gl, Qu-3-gl, and Qu-3-gr. Group 1 includes eight genotypes, which did not contain My-3-gl (Table 6). This fact implies that, in these genotypes, dihydroxylation of

the major flavonol was Qu-3-gl or Qu-3-gr, and in some cases, contained very small amounts of other flavonol; anyway, La-3-gl and Sy-3-gl were absent (Table 6). Group 2 is formed by nine genotypes, which contain My-3-gl, but major flavonol was Qu-3-gl or Qu-3-gr (Table 7). These genotypes usually contained several minor flavonoids, including La-3-gl and Sy-3-gl,


(Qu-3-gal), which have been previously detected in red grape skins [38].

,50

the accumulation of anthocyanins may be very high.

6. Flavonol fingerprint of wild grapes

originated in northern Spain and southern Spain.

Qu-3-gl.

dihydrokaempferol by flavonoid-3<sup>0</sup>

Figure 5. Chromatogram registered at 350 nm for a grape skin extract. My-3-gl, myricetin-3-O-glucoside; Qu-3-gr, quercetin-3-O-glucuronide; Qu-3-gl, quercetin-3-O-glucoside.


Table 6. Content of flavonols (mg/kg of grapes) in genotypes that did not contain My-3-gl.

the exceptions being genotypes CA-11.3 and SE-3.4. In these two genotypes, methylation of My-3-gl by action of a O-methyltransferase is blocked. Finally, group 3 includes eight genotypes presenting My-3-gl as a major flavonol (Table 8). In most cases, these genotypes contained several minor flavonoids, including La-3-gl and Sy-3-gl, the exceptions being genotypes O-1.5bis and SS-3.5. Like in genotypes CA-11.3 and SE-3.4, methylation of My-3-gl by action of a methyltransferase is blocked.

Data reported in Tables 6–8 point out that the total content of flavonols was quite variable, ranging from 29 to 324 mg/kg. Nevertheless, there is a remarkable difference among genotypes from populations located in Northern Spain and those from populations located in Southern Spain (Table 9). As can be noted, genotypes from Northern Spain contained a lower amount of flavonols than those originated in Southern Spain. The accumulation of flavonols in grapes

Figure 6. Biosynthesis of flavonol aglicones. CH3, chalcone synthase; CHI, chalcone isomerase; F3H, flavanone-3β-hydroxylase; FLS, flavonol synthase; F3<sup>0</sup> H, flavonoid-3<sup>0</sup> -hydroxylase; F3<sup>0</sup> 50 H, flavonoid-3<sup>0</sup> ,50 -hydroxylase; MT, methyltransferase.

oceanic climate environments, where sunlight exposure is lower than in the Mediterranean

Table 9. Range, mean value, and standard deviation for the content of total flavonols (mg/kg of grapes) in genotypes

Range Mean value Standard deviation

Genotype My-3-gl Qu-3-gr Qu-3-gl Other flavonols Total BA-1.1 84.8 47.0 42.0 9.5 183.3 CA-4.1 22.7 15.2 15.2 1.3 54.4 CO-2.2 48.7 14.6 22.8 14.0 100.1 CO-3.7 26.8 24.9 9.8 23.5 84.9 H-1.1 40.4 11.1 18.0 25.2 94.7 O-1.5bis 77.7 35.1 59.2 nd 172.1 S-1.9 58.0 30.1 28.3 12.2 128.6 SS-3.5 22.9 11.2 14.0 nd 48.1

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7. Comparison between anthocyanin fingerprint and flavonol fingerprint

The anthocyanin and flavonol fingerprints of wild grape genotypes are quite different, taking into account the pathways involved in their biosynthesis. Thus, in most genotypes B-ring trisubstituted anthocyanins (Dp-derived) predominate, but B-ring disubstituted flavonols (Qu derivatives) are more abundant than My derivatives (B-ring trisubstituted). Moreover, some genotypes do not present B-ring trisubstituted flavonols (Table 7), but they always present Dpderived anthocyanins, sometimes in a high proportion (e.g., genotype S-1.3bis, see Table 4). On the other hand, some genotypes presenting a very low amount of Cy-derived anthocyanins (e.g., CR-1.6 and SE-1.5, see Table 4) contain a remarkable amount of Qu derivatives (Table 7). These data suggest that flavonol synthase activities linked to the formation of Qu and My are regulated in a different way than enzymatic activities linked to the formation of Dp-3-gl and Cy-3-gl from the corresponding dihydroflavonols. Other relevant biosynthetic difference among flavonols and anthocyanins is B-ring O-methylation. This reaction seems to be more intense for anthocyanins than for flavonols; thus, in most genotypes, methylated anthocyanins predominate. This fact suggests that primitive anthocyanins (Cy-3-gl and Dp-3-gl) are better substrates for O-methyltransferases (OMT) than quercetin and myricietin, as pointed out

climate environments in which evolved genotypes from Southern Spain.

Table 8. Content of flavonols (mg/kg of grapes) in genotypes in which My-3-gl was the major flavonol.

Northern Spain 28.7–172.1 69.7 47.3 Southern Spain 39.8–324.1 121.0 87.3

nd: not detected. See full compound names in Figure 5.

originated in northern Spain and southern Spain.

previously [34].


Table 7. Content of flavonols (mg/kg of grapes) in genotypes that contained My-3-gl, being Qu-3-gl or Qu-3-gr the major flavonol.

increases with sun exposure, as demonstrated by other authors [16]. This factor can be considered as neutral in our study because all genotypes grown in the same environment; thus, differences observed in flavonols content can be considered of genetic nature. The most probable explanation is that genotypes originally collected in Northern Spain have evolved in

Fingerprints of Anthocyanins and Flavonols in Wild Grapes (*Vitis vinifera* L. ssp. *sylvestris* (Gmelin) Hegi) http://dx.doi.org/10.5772/intechopen.70861 35


nd: not detected. See full compound names in Figure 5.

increases with sun exposure, as demonstrated by other authors [16]. This factor can be considered as neutral in our study because all genotypes grown in the same environment; thus, differences observed in flavonols content can be considered of genetic nature. The most probable explanation is that genotypes originally collected in Northern Spain have evolved in

Table 7. Content of flavonols (mg/kg of grapes) in genotypes that contained My-3-gl, being Qu-3-gl or Qu-3-gr the major

Figure 6. Biosynthesis of flavonol aglicones. CH3, chalcone synthase; CHI, chalcone isomerase; F3H, flavanone-3β-hydrox-

Genotype My-3-gl Qu-3-gr Qu-3-gl Other flavonols Total BI-1.3bis 1.4 14.0 14.3 3.1 32.8 CA-9.7 2.9 13.8 9.4 13.7 39.8 CA-11.3 10.3 25.8 27.4 nd 63.4 CO-5.1 4.8 37.8 74.1 6.7 123.4 CR-1.6 6.3 31.4 20.3 25.2 83.2 J-2.4 8.1 14.4 7.8 14.2 44.5 LE-1.2 3.2 16.2 11.5 24.0 54.8 SE-1.5 80.8 106.1 71.4 46.2 304.4 SE-3.4 98.3 113.4 112.5 nd 324.1

50

H, flavonoid-3<sup>0</sup>

,50



H, flavonoid-3<sup>0</sup>

34 Grapes and Wines - Advances in Production, Processing, Analysis and Valorization

ylase; FLS, flavonol synthase; F3<sup>0</sup>

flavonol.

nd: not detected. See full compound names in Figure 5.

Table 8. Content of flavonols (mg/kg of grapes) in genotypes in which My-3-gl was the major flavonol.


Table 9. Range, mean value, and standard deviation for the content of total flavonols (mg/kg of grapes) in genotypes originated in northern Spain and southern Spain.

oceanic climate environments, where sunlight exposure is lower than in the Mediterranean climate environments in which evolved genotypes from Southern Spain.

#### 7. Comparison between anthocyanin fingerprint and flavonol fingerprint

The anthocyanin and flavonol fingerprints of wild grape genotypes are quite different, taking into account the pathways involved in their biosynthesis. Thus, in most genotypes B-ring trisubstituted anthocyanins (Dp-derived) predominate, but B-ring disubstituted flavonols (Qu derivatives) are more abundant than My derivatives (B-ring trisubstituted). Moreover, some genotypes do not present B-ring trisubstituted flavonols (Table 7), but they always present Dpderived anthocyanins, sometimes in a high proportion (e.g., genotype S-1.3bis, see Table 4). On the other hand, some genotypes presenting a very low amount of Cy-derived anthocyanins (e.g., CR-1.6 and SE-1.5, see Table 4) contain a remarkable amount of Qu derivatives (Table 7). These data suggest that flavonol synthase activities linked to the formation of Qu and My are regulated in a different way than enzymatic activities linked to the formation of Dp-3-gl and Cy-3-gl from the corresponding dihydroflavonols. Other relevant biosynthetic difference among flavonols and anthocyanins is B-ring O-methylation. This reaction seems to be more intense for anthocyanins than for flavonols; thus, in most genotypes, methylated anthocyanins predominate. This fact suggests that primitive anthocyanins (Cy-3-gl and Dp-3-gl) are better substrates for O-methyltransferases (OMT) than quercetin and myricietin, as pointed out previously [34].

In general, phenylpropanoid biosynthesis and subsequent flavonoid production are tightly linked to primary metabolism through phenylalanine as a precursor of flavonoids. Catalyzing the first committed step into the flavonoid biosynthetic pathway, chalcone synthase (CHS) plays a pivotal role to provide a common chalcone precursor for the production of all intermediates and final products of the flavonoid biosynthetic pathway which are therefore biogenetically and structurally related (Figures 3 and 6). In most plants, including grapevine, flavanones are preferentially used as substrates for flavanone-3β-hydroxylase (F3H), which produces dihydroflavonols as an important branch point flavonoid and an essential substrate for all classes of downstream compounds (Figures 3 and 6). The biosynthesis of flavonol aglycones through flavonol synthase 1, FLS4 [39, 40]; as well as the biosynthesis of proanthocyanidins and anthocyanin precursors through dihydroflavonol 4-reductase (DFR) employs dihydroflavonols as substrates thereby directly competing for the same substrate (Figures 3 and 6). DFR reshuffles substrates away from flavonol biosynthesis and converts dihydroflavonols to leucoanthocyanidins, which are precursors for proanthocyanidin and anthocyanin biosynthesis [41]. While DFR is specific for the anthocyanin/proanthocyanidin pathway, flavonoid-3<sup>0</sup> -hydroxylase (F3<sup>0</sup> H) and flavonoid-3<sup>0</sup> ,50 -hydroxylase (F3<sup>0</sup> 50 H) gene products are necessary for the production of all subclasses, namely flavonols, anthocyanins, and proanthocyanidins. In general, hydroxylation of the B-ring of dihydroflavonols, flavanones, and flavones changes the color of the resulting anthocyanin-derived pigment and increases dramatically the chemodiversity of flavonols, proanthocyanidins, and anthocyanins [42].

acyltransferase, anthocyanin-3-O-glucoside-600-O-acyltransferase (3), which is capable of pro-

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The anthocyanin fingerprint of wild grapes skins, considering the relative amount of 15 anthocyanins, showed a considerable variability, being possible to distinguish three phenotypic groups. Differences into those groups are related with the predominance of delphinidinor cyanidin-derived anthocyanins and the expression of genes involved in acyltransferase activities. Moreover, it has been possible to separate 12 flavonol glycosides, eight of them were successfully identified. Major flavonols were Qu-3-gl, Qu-3-gr, and My-3-gl. The diversity and number of flavonols differed for each genotype. In most genotypes, Qu-3-gl or Qu-3-gr was the major flavonol, and My-3-gl was absent in some genotypes. Quantitative analysis of anthocyanins and flavonols revealed that genotypes collected in wild grapevine populations located in Northern Spain were richer in anthocyanins and poorer in flavonols than those collected in populations located in Southern Spain. This difference may be explained by the different expression level of the structural genes and transcriptional factors in the biosynthesis pathway in relation with the impact of climatic conditions on the evolution of wild grapes in different

The authors acknowledge the financial support of Spain's Ministry of Economy and Competitiveness (Project RTA2014-0016-C003-01, MINECO-INIA-CCAA). AB holds an INIA fellow-

, Alberto Bellido<sup>2</sup>

1 Departament of Agricultural and Food Chemistry, Universidad Autonoma de Madrid,

2 Center for Plant Biotechnology and Genomics, CBGP-INIA, Pozuelo de Alarcón, Spain

4 Murcia Institute for Agricultural and Food Research and Development, IMIDA, Murcia,

3 Catalonian Grape and Wine Institute, INCAVI-IRTA, Vilafranca del Penedès, Spain

, David Carrasco<sup>2</sup>

, Anna Puig<sup>3</sup> and

ducing the common acylated anthocyanins found in grape berries [46].

8. Conclusions

environments.

Acknowledgements

ship for this project.

Author details

Eugenio Revilla<sup>1</sup>

Madrid, Spain

Spain

Leonor Ruiz-Garcia<sup>4</sup>

\*, Rosa Arroyo-Garcia<sup>2</sup>

\*Address all correspondence to: eugenio.revilla@uam.es

The known biosynthetic pathway of flavonoids shares common enzymatic steps, whereas the activities of enzymes specific for anthocyanins or flavonols lead exclusively to the biosynthesis of the respective flavonoid by competing for common substrates (Figures 3 and 6). The accumulation of flavonol compounds in the berry is mediated by an increase of transcripts encoding FLS (VvFLS4 or VvFLS5) under the regulation of the transcriptional factor VvMYF [40]. Later during veraison, the anthocyanins are synthesized through the flavonoid pathway in grapevine cultivars that harbor the wild-type VvmybA1 transcription factor for the expression of UFGT [43]. The encoded enzyme UFGT catalyzes the glycosylation of unstable anthocyanidin aglycones into pigmented anthocyanins (Figure 3). Two primitive anthocyanins (Cy-3-gl and Dp-3-gl) are synthesized in the cytosol of berry epidermal cells. The B-ring of Cy-3-gl is dihydroxylated at the 3<sup>0</sup> and 4<sup>0</sup> positions, whereas Dp-3-gl has a tri-hydroxylated B-ring because of an additional hydroxyl group at the 5<sup>0</sup> position. The 3<sup>0</sup> position of Cy-3-gl and Dp-3-gl and sequentially the 5<sup>0</sup> position of Dp-3-gl can be methoxylated by Omethyltransferase (VvOMT), generating Pn-3-gl, Pt-3-gl, and Mv-3-gl, respectively [44]. Anthocyanins can be further modified by acyltransferases, which produce 3-O-acetyl-, 3-Ocoumaroyl-, and 3-O-caffeoyl-monoglucosides by attaching acyl groups to the C6<sup>00</sup> position of the glucose moiety [45].

Taking into consideration the regulations and biosynthesis pathway, we can suggest that the differences in the anthocyanin and flavonol fingerprints of wild grapes are putative due to the different expression level of the structural genes F3'H and F3'5'H, which are essential in the breach point for the final anthocyanin and flavonol compounds and the transcriptional factor involved in the pathway. Finally, the different level of methylation of Cy-3-gl and Dp-3-gl could be due to different expression of OMT genes and the gene encoding an anthocyanin acyltransferase, anthocyanin-3-O-glucoside-600-O-acyltransferase (3), which is capable of producing the common acylated anthocyanins found in grape berries [46].

## 8. Conclusions

In general, phenylpropanoid biosynthesis and subsequent flavonoid production are tightly linked to primary metabolism through phenylalanine as a precursor of flavonoids. Catalyzing the first committed step into the flavonoid biosynthetic pathway, chalcone synthase (CHS) plays a pivotal role to provide a common chalcone precursor for the production of all intermediates and final products of the flavonoid biosynthetic pathway which are therefore biogenetically and structurally related (Figures 3 and 6). In most plants, including grapevine, flavanones are preferentially used as substrates for flavanone-3β-hydroxylase (F3H), which produces dihydroflavonols as an important branch point flavonoid and an essential substrate for all classes of downstream compounds (Figures 3 and 6). The biosynthesis of flavonol aglycones through flavonol synthase 1, FLS4 [39, 40]; as well as the biosynthesis of proanthocyanidins and anthocyanin precursors through dihydroflavonol 4-reductase (DFR) employs dihydroflavonols as substrates thereby directly competing for the same substrate (Figures 3 and 6). DFR reshuffles substrates away from flavonol biosynthesis and converts dihydroflavonols to leucoanthocyanidins, which are precursors for proanthocyanidin and anthocyanin biosynthesis [41]. While

DFR is specific for the anthocyanin/proanthocyanidin pathway, flavonoid-3<sup>0</sup>

50

of all subclasses, namely flavonols, anthocyanins, and proanthocyanidins. In general, hydroxylation of the B-ring of dihydroflavonols, flavanones, and flavones changes the color of the resulting anthocyanin-derived pigment and increases dramatically the chemodiversity of flavo-

The known biosynthetic pathway of flavonoids shares common enzymatic steps, whereas the activities of enzymes specific for anthocyanins or flavonols lead exclusively to the biosynthesis of the respective flavonoid by competing for common substrates (Figures 3 and 6). The accumulation of flavonol compounds in the berry is mediated by an increase of transcripts encoding FLS (VvFLS4 or VvFLS5) under the regulation of the transcriptional factor VvMYF [40]. Later during veraison, the anthocyanins are synthesized through the flavonoid pathway in grapevine cultivars that harbor the wild-type VvmybA1 transcription factor for the expression of UFGT [43]. The encoded enzyme UFGT catalyzes the glycosylation of unstable anthocyanidin aglycones into pigmented anthocyanins (Figure 3). Two primitive anthocyanins (Cy-3-gl and Dp-3-gl) are synthesized in the cytosol of berry epidermal cells. The B-ring of Cy-3-gl is dihydroxylated at the 3<sup>0</sup> and 4<sup>0</sup> positions, whereas Dp-3-gl has a tri-hydroxylated B-ring because of an additional hydroxyl group at the 5<sup>0</sup> position. The 3<sup>0</sup> position of Cy-3-gl and Dp-3-gl and sequentially the 5<sup>0</sup> position of Dp-3-gl can be methoxylated by Omethyltransferase (VvOMT), generating Pn-3-gl, Pt-3-gl, and Mv-3-gl, respectively [44]. Anthocyanins can be further modified by acyltransferases, which produce 3-O-acetyl-, 3-Ocoumaroyl-, and 3-O-caffeoyl-monoglucosides by attaching acyl groups to the C6<sup>00</sup> position of

Taking into consideration the regulations and biosynthesis pathway, we can suggest that the differences in the anthocyanin and flavonol fingerprints of wild grapes are putative due to the different expression level of the structural genes F3'H and F3'5'H, which are essential in the breach point for the final anthocyanin and flavonol compounds and the transcriptional factor involved in the pathway. Finally, the different level of methylation of Cy-3-gl and Dp-3-gl could be due to different expression of OMT genes and the gene encoding an anthocyanin


36 Grapes and Wines - Advances in Production, Processing, Analysis and Valorization

(F3<sup>0</sup>

H) and flavonoid-3<sup>0</sup>

the glucose moiety [45].

,50

nols, proanthocyanidins, and anthocyanins [42].


H) gene products are necessary for the production

The anthocyanin fingerprint of wild grapes skins, considering the relative amount of 15 anthocyanins, showed a considerable variability, being possible to distinguish three phenotypic groups. Differences into those groups are related with the predominance of delphinidinor cyanidin-derived anthocyanins and the expression of genes involved in acyltransferase activities. Moreover, it has been possible to separate 12 flavonol glycosides, eight of them were successfully identified. Major flavonols were Qu-3-gl, Qu-3-gr, and My-3-gl. The diversity and number of flavonols differed for each genotype. In most genotypes, Qu-3-gl or Qu-3-gr was the major flavonol, and My-3-gl was absent in some genotypes. Quantitative analysis of anthocyanins and flavonols revealed that genotypes collected in wild grapevine populations located in Northern Spain were richer in anthocyanins and poorer in flavonols than those collected in populations located in Southern Spain. This difference may be explained by the different expression level of the structural genes and transcriptional factors in the biosynthesis pathway in relation with the impact of climatic conditions on the evolution of wild grapes in different environments.

## Acknowledgements

The authors acknowledge the financial support of Spain's Ministry of Economy and Competitiveness (Project RTA2014-0016-C003-01, MINECO-INIA-CCAA). AB holds an INIA fellowship for this project.

## Author details

Eugenio Revilla<sup>1</sup> \*, Rosa Arroyo-Garcia<sup>2</sup> , Alberto Bellido<sup>2</sup> , David Carrasco<sup>2</sup> , Anna Puig<sup>3</sup> and Leonor Ruiz-Garcia<sup>4</sup>

\*Address all correspondence to: eugenio.revilla@uam.es

1 Departament of Agricultural and Food Chemistry, Universidad Autonoma de Madrid, Madrid, Spain

2 Center for Plant Biotechnology and Genomics, CBGP-INIA, Pozuelo de Alarcón, Spain

3 Catalonian Grape and Wine Institute, INCAVI-IRTA, Vilafranca del Penedès, Spain

4 Murcia Institute for Agricultural and Food Research and Development, IMIDA, Murcia, Spain

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UNSP 01018. DOI: 10.1051/bioconf/20160701018

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[14] Revilla E, García-Beneytez E, Cabello F. Anthocyanin composition of several clones of Tempranillo grapes and wines made with them. Australian Journal of Grape and Wine Research. 2009;15:70-78. DOI: 10.1111/j.1755-0238.2008.00037.x

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**Chapter 3**

Provisional chapter

**Grapevine Trunk Diseases (GTDs): Impact on Table**

DOI: 10.5772/intechopen.72607

This chapter involves the description and information about the main grapevine trunk diseases (GTDs) affecting Vitis vinifera in Chile. There is a complete description of Escatype disease, bot canker disease affecting more than 7-year-old plants, and Petri disease affecting young plants. Symptom descriptions of GTD diseases are done, and also the principal species as causal agents and the importance of them are analyzed. The dispersal of Botryosphaeriaceae spores is considered, and finally, the principal management measures can be taken in order to avoid or control these important diseases. The information is contrasted with different researches done in the most important countries where these

Keywords: bot canker, Esca-like disease, Diplodia seriata, D. mutila, Botryosphaeriaceae,

In Chile, the first notice of a disease caused by Botryosphaeriaceae occurred in 1986 [1], where Botryosphaeria dothidea was reported causing damage to a grapevine grown in a Spanish trellis for table grape production. Since the 1960s, worldwide studies in grapevine trunk diseases (GTDs) have expanded incrementally; four important types of diseases have been recognized: Esca disease, bot canker disease, Eutypa dieback, and Phomopsis dieback [2], and actually, the reported studies had acquired an exponential curve, but up to date, there is not a satisfactory field control of these important diseases. In the study done by Fontaine et al. [2], there is an important contribution to understanding these important diseases in many countries, but the

> © 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and eproduction in any medium, provided the original work is properly cited.

> © 2018 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Grapevine Trunk Diseases (GTDs): Impact on

Table Grapes and Wine Vineyards in Chile

**Grapes and Wine Vineyards in Chile**

Additional information is available at the end of the chapter

Phaeomoniella chlamydospora, chlorotic leaf roll

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/intechopen.72607

Ximena Besoain

Abstract

diseases are relevant.

1. Introduction

Ximena Besoain

#### **Grapevine Trunk Diseases (GTDs): Impact on Table Grapes and Wine Vineyards in Chile** Grapevine Trunk Diseases (GTDs): Impact on Table Grapes and Wine Vineyards in Chile

DOI: 10.5772/intechopen.72607

Ximena Besoain Ximena Besoain

Additional information is available at the end of the chapter Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/intechopen.72607

#### Abstract

This chapter involves the description and information about the main grapevine trunk diseases (GTDs) affecting Vitis vinifera in Chile. There is a complete description of Escatype disease, bot canker disease affecting more than 7-year-old plants, and Petri disease affecting young plants. Symptom descriptions of GTD diseases are done, and also the principal species as causal agents and the importance of them are analyzed. The dispersal of Botryosphaeriaceae spores is considered, and finally, the principal management measures can be taken in order to avoid or control these important diseases. The information is contrasted with different researches done in the most important countries where these diseases are relevant.

Keywords: bot canker, Esca-like disease, Diplodia seriata, D. mutila, Botryosphaeriaceae, Phaeomoniella chlamydospora, chlorotic leaf roll

## 1. Introduction

In Chile, the first notice of a disease caused by Botryosphaeriaceae occurred in 1986 [1], where Botryosphaeria dothidea was reported causing damage to a grapevine grown in a Spanish trellis for table grape production. Since the 1960s, worldwide studies in grapevine trunk diseases (GTDs) have expanded incrementally; four important types of diseases have been recognized: Esca disease, bot canker disease, Eutypa dieback, and Phomopsis dieback [2], and actually, the reported studies had acquired an exponential curve, but up to date, there is not a satisfactory field control of these important diseases. In the study done by Fontaine et al. [2], there is an important contribution to understanding these important diseases in many countries, but the

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and eproduction in any medium, provided the original work is properly cited. © 2018 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Chilean situation was not included; therefore, this chapter contributes to understand the actual status of GTDs in Chile.

Chile is the principal fruit exporter of fresh table grapes in the world. In addition, it is important as wine producer. An analysis of the main table grapes and wine producer regions is done, with special association to GTD predisposition. The economic importance of GTDs and potential damage that it causes is described. The principal species associated to Botryosphaeriaceae canker and a disease called Esca-like, caused mainly by Phaeomoniella chlamydospora in Chile, are descripted in detail, analyzing previous published information [3]. The symptomatology associated to bot canker and Esca disease causal agents is detailed, and a comparison of the situation for the main wine- and table grape-producing countries is done. Furthermore, the importance of these causal agents affecting another high-value species or plants associated to natural habitats, commonly interacting with vineyard's and table grape's orchards, is considered. The factors associated to GTDs will be described, including the analysis associated to climatic conditions. A spatial analysis will be done including different stressaffecting plants and the importance of the Botryosphaeriaceae family as endophytic pathogens. The chapter includes information about the tissues commonly colonized by Botryosphaeriaceae and by Phaeomoniella, considering analysis of their preference and age of the colonized tissue. Finally, the chapter includes a description of a complete integrated management and future trends for control of GTDs, with special emphasis in bot canker and Esca diseases. Up to date there is no official report of Eutypa disease affecting Vitis vinifera in Chile [3, 4], and Phomopsis dieback was reported, but the prevalence of its causal agent, Phomopsis viticola, is very low [3]. So, this chapter includes an analysis of bot canker and Escatype disease.

The ability of GTD-associated species to be endophytes is specially a threat for the quarantine programs worldwide, which can be overlooked considering its latent phase. This is particularly important because most of the causal agents have the ability to rapidly express pathogenicity when their hosts are under stress [5]. This is particularly important considering that the Servicio Agrícola y Ganadero (SAG) of Chile requires a 2-year period of quarantine when propagation material is introduced from foreign countries, and not always these types of diseases will express in this period. In addition, it is also important to consider that under the climate change, there is an increase in drought periods in many places, with an increase in stress for many plant communities and an increase of GTD-type diseases.

Region, there are about 8515.9 ha, mainly grown for pisco (distilled liqueur) production. In relation to the wine industry, Chile is the sixth most important wine producer and the fourth most important wine exporter. Chile exported in 2016 about 9,100,000 hectoliters, corresponding to 8.74% of the total volume exported in the world [6]. The main wineproducing regions in Chile considering the cultivated area are Maule (37.94%), O'Higgins (32.81%), Bío Bío (10.65%), Metropolitana (9.2%), and Valparaíso (7.1%) (Figure 1) with a total of 141,918 ha in 2015 [7]. So, this delineates the significance of GTDs affecting vineyards and

Grapevine Trunk Diseases (GTDs): Impact on Table Grapes and Wine Vineyards in Chile

http://dx.doi.org/10.5772/intechopen.72607

45

In Chile Petri disease [8] has been detected affecting young Vitis plants produced for the wine industry, mainly associated to Phaeomoniella and Phaeoacremonium genera. In vineyards more than 7-year-old, the main diseases affecting plants are Botryosphaeria dieback (or bot canker)

the importance of prevention, detection, diagnosis, and opportune control.

Figure 1. Map of Chile; in detail the five main producing regions with vineyards for wine.

2.2. Main diseases and economic importance of GTDs in Chile

## 2. Grapevine trunk diseases (GTDs) in Chile

#### 2.1. Chilean table grape and wine industry

Considering world table grape production, Chile is the seventh main producer worldwide, but considering the export to other countries, Chile is the first exporter, doubling the exports of the second most important country, the United States. Actually, there are 52,234 ha for table grape fresh fruit production in Chile, with vineyards developed in seven different regions, from the North in the Atacama Region and to the South up to the Bío Bío Region. In the Atacama Grapevine Trunk Diseases (GTDs): Impact on Table Grapes and Wine Vineyards in Chile http://dx.doi.org/10.5772/intechopen.72607 45

Chilean situation was not included; therefore, this chapter contributes to understand the actual

44 Grapes and Wines - Advances in Production, Processing, Analysis and Valorization

Chile is the principal fruit exporter of fresh table grapes in the world. In addition, it is important as wine producer. An analysis of the main table grapes and wine producer regions is done, with special association to GTD predisposition. The economic importance of GTDs and potential damage that it causes is described. The principal species associated to Botryosphaeriaceae canker and a disease called Esca-like, caused mainly by Phaeomoniella chlamydospora in Chile, are descripted in detail, analyzing previous published information [3]. The symptomatology associated to bot canker and Esca disease causal agents is detailed, and a comparison of the situation for the main wine- and table grape-producing countries is done. Furthermore, the importance of these causal agents affecting another high-value species or plants associated to natural habitats, commonly interacting with vineyard's and table grape's orchards, is considered. The factors associated to GTDs will be described, including the analysis associated to climatic conditions. A spatial analysis will be done including different stressaffecting plants and the importance of the Botryosphaeriaceae family as endophytic pathogens. The chapter includes information about the tissues commonly colonized by Botryosphaeriaceae and by Phaeomoniella, considering analysis of their preference and age of the colonized tissue. Finally, the chapter includes a description of a complete integrated management and future trends for control of GTDs, with special emphasis in bot canker and Esca diseases. Up to date there is no official report of Eutypa disease affecting Vitis vinifera in Chile [3, 4], and Phomopsis dieback was reported, but the prevalence of its causal agent, Phomopsis viticola, is very low [3]. So, this chapter includes an analysis of bot canker and Esca-

The ability of GTD-associated species to be endophytes is specially a threat for the quarantine programs worldwide, which can be overlooked considering its latent phase. This is particularly important because most of the causal agents have the ability to rapidly express pathogenicity when their hosts are under stress [5]. This is particularly important considering that the Servicio Agrícola y Ganadero (SAG) of Chile requires a 2-year period of quarantine when propagation material is introduced from foreign countries, and not always these types of diseases will express in this period. In addition, it is also important to consider that under the climate change, there is an increase in drought periods in many places, with an increase in

Considering world table grape production, Chile is the seventh main producer worldwide, but considering the export to other countries, Chile is the first exporter, doubling the exports of the second most important country, the United States. Actually, there are 52,234 ha for table grape fresh fruit production in Chile, with vineyards developed in seven different regions, from the North in the Atacama Region and to the South up to the Bío Bío Region. In the Atacama

stress for many plant communities and an increase of GTD-type diseases.

2. Grapevine trunk diseases (GTDs) in Chile

2.1. Chilean table grape and wine industry

status of GTDs in Chile.

type disease.

Region, there are about 8515.9 ha, mainly grown for pisco (distilled liqueur) production. In relation to the wine industry, Chile is the sixth most important wine producer and the fourth most important wine exporter. Chile exported in 2016 about 9,100,000 hectoliters, corresponding to 8.74% of the total volume exported in the world [6]. The main wineproducing regions in Chile considering the cultivated area are Maule (37.94%), O'Higgins (32.81%), Bío Bío (10.65%), Metropolitana (9.2%), and Valparaíso (7.1%) (Figure 1) with a total of 141,918 ha in 2015 [7]. So, this delineates the significance of GTDs affecting vineyards and the importance of prevention, detection, diagnosis, and opportune control.

#### 2.2. Main diseases and economic importance of GTDs in Chile

In Chile Petri disease [8] has been detected affecting young Vitis plants produced for the wine industry, mainly associated to Phaeomoniella and Phaeoacremonium genera. In vineyards more than 7-year-old, the main diseases affecting plants are Botryosphaeria dieback (or bot canker) and a disease similar to Esca found associated to Diatrypaceae fungi and with Basidiomycete fungi associated to wood spongy tissues [1, 3, 4, 8].

Black dead arm (BDA) disease was first reported by Lehoczky [15] associated to B. stevensii (formerly named as D. mutila). This pathogen did not infect the green parts of the vines or the chesters. He reported that the phloem and xylem tissues were black discolored; therefore, he named the disease as "black dead arm disease" instead of the "dead arm disease" caused by Phomopsis viticola, previously described by Pine [16]. Actually, there are four major diseases in vineyards. And, their names are Esca, Eutypa dieback, Botryosphaeria canker, and Phomopsis

Grapevine Trunk Diseases (GTDs): Impact on Table Grapes and Wine Vineyards in Chile

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47

According to the 10th International Workshop on Grapevine Trunk Diseases (2017) taken place in Reims, France, in the introduction done by Dr. José Ramón Úrbez-Torres "Main achievements and future prospects in GTDs Pathogen Characterization and Identification," he analyzed that 133 species from 34 genera are associated to GTDs worldwide. Considering these information, the analysis of this type of diseases affecting grapevines is much complex to organize. Fortunately, in Chile the situation is more easy considering that there are only five species causing bot canker [3, 4, 8, 10, 17], two main species associated to Petri and Esca-like diseases [3, 9, 18], plus three species of Basidiomycetes [3]. Phomopsis dieback is very infrequently seen, and black foot also seems to be associated with problems at the plantation site of

In table grape and wine vineyard research done in Chile, only five Botryosphaeriaceae species have been reported to affect these crops: Diplodia seriata De Not. ( Botryosphaeria obtusa (Schwein.) Shoemaker); D. mutila (Fr.) Mont. (= Botryosphaeria stevensii Shoemaker); Neofusicoccum parvum (Pennycook & Samuels) Crous, Slippers & A.J.L. Phillips (= Botryosphaeria parva) Pennycook & Samuels; N. australe (Slippers, Crous & M.J. Wingf.) Crous, Slippers & A.J.L. Phillips (= Botryosphaeria australis Slippers, Crous & M.J. Wingf.); and Spencermartinsia viticola (A.J.L. Phillips & J. Luque) A.J.L. Phillips, A. Alves & Crous (= Botryosphaeria viticola A.J.L. Phillips & J. Luque) [1, 3, 4, 8, 11, 17]. The names in parentheses are in accordance with the name

In accordance with Díaz et al. [3] from a total of 694 analyzed samples obtained from different varieties grown for table grapes or wine industry, 56.3% of them were Botryosphaeriaceae species, and from 85% of these samples, the principal recovered species was Phaeomoniella chlamydospora (Pa. chlamydospora) (W. Gams, Crous, M.J. Wingf. & L. Mugnai) Crous & W. Gams, obtained from vascular streaks. But considering cankers with V-shaped form, 91% of the recovered species were Botryosphaeriaceae, and from these 68% of them was Diplodia seriata. From a soft-spongy canker (n = 314), the main species isolated in 96% of the samples was Inocutis sp. In pathogenicity tests carried out by Díaz et al. [3], Inocutis was able to cause damage but significantly less than that

Considering Úrbez-Torres' [20] review of species affecting Vitis vinifera, he could recognize 21 different Botryosphaeriaceae species affecting this crop in California. In France the disease known as Esca complex is caused by Pa. chlamydospora and by Phaeoacremonium spp. plus the

dieback [2].

2.3. GTD causal agents

grafted material and with very low prevalence [3].

and authors described by Phillips et al. [19].

caused by Botryosphaeriaceae or Phaeomoniella species.

basidiomycete Fomitiporia mediterranea [2].

Pathogenicity of bot canker has been consistently proven in Chile [4] as well as necrotic strikes associated to Phaeomoniella chlamydospora [3, 9]. But there is an important disease called chlorotic leaf roll (enrollamiento clorótico) of the vines that is frequently associated to V-shaped necrosis and necrotic streaks in the xylem and also with spongy tissues mainly associated to Basidiomycetes colonization, which Koch's postulates have not been done. So, this type of disease and symptoms needed to be solved by the Chilean plant pathologists.

The economic importance of diseases affecting grapevines has been partially studied in Chile. Morales et al. [4] found that bot canker disease affects table grape vineyards and that the disease increases with the age of the plants. With an increasing incidence of the disease of 22% in an 11-year-old vineyard to 70% in the 22-year-old vineyard, and with maximum severity up to 22% of damage index in a 16-year-old table grape vineyard. In these table grape vineyards, the main species and detected prevalence (%) were Diplodia seriata (83.3%), D. mutila (8.3%), and Spencermartinsia viticola (8.3%). In a national survey done by Díaz et al. [3], with about 694 wood samples being analyzed obtained from 67 Chilean vineyards, from Copiapó (27 18'S) to Los Angeles (3742'S), a total of 1363 fungal isolates were obtained from diseased plants with different symptoms, and the most prevalent identified species were Phaeomoniella chlamydospora (in 85% of the samples); Botryosphaeriaceae (in 56% of the samples) with the main detected species Diplodia seriata, D. mutila, Neofusicoccum parvum, and Spencermartinsia viticola; and Diatrypaceae (in 4.8% of the samples, but Eutypa lata was not found). Phomopsis viticola and Cylindrocarpon sp. were only found in 0.4% of the analyzed samples. Finally, Valencia et al. [10] analyzing conidial dissemination in four vineyards from the Valparaíso Region found in the spore traps conidia from D. seriata, S. viticola, and Neofusicoccum australe, but the greater peaks found corresponded to D. seriata.

On the other hand, Torres et al. [11] reported the prevalence and damage observed in 10 vineyards cv. Cabernet Sauvignon during the 2010–2011 growing season. In the research done in 14 blocks of the O'Higgins (n = 8) and Maule (n = 6) regions, the average damages associated with bot canker were 36 and 48% for each region, respectively, with an average of production loss estimated at 5800 kg ha<sup>1</sup> . The most prevalent species detected were Diplodia seriata (69.2%), followed by D. mutila (12.8%), and Neofusicoccum parvum (10.2%).

GTDs are the main biotic factors affecting grapes wherever are grown and affecting vineyard productivity and longevity [12]. In France, there is a national crisis with 12% of the vineyards not economically viable, causing annual losses for about 1 billion euros [13]. In China, where Botryosphaeria dieback is a serious disease, Yan et al. [14] made a survey in 72 vineyards of 20 grape-growing regions; they found that dieback occurs in 18/20 provinces, and they confirmed the presence of B. dothidea, D. seriata, L. theobromae, and N. parvum. These species were spatially distributed: L. theobromae and N. parvum only occurring in subtropical monsoon climate regions, Diplodia seriata only occurring in temperate climatic regions, and B. dothidea occurring in both types of climates. Of the 25 cultivars most grown in China, none was resistant to the four (taxa) species.

Black dead arm (BDA) disease was first reported by Lehoczky [15] associated to B. stevensii (formerly named as D. mutila). This pathogen did not infect the green parts of the vines or the chesters. He reported that the phloem and xylem tissues were black discolored; therefore, he named the disease as "black dead arm disease" instead of the "dead arm disease" caused by Phomopsis viticola, previously described by Pine [16]. Actually, there are four major diseases in vineyards. And, their names are Esca, Eutypa dieback, Botryosphaeria canker, and Phomopsis dieback [2].

#### 2.3. GTD causal agents

and a disease similar to Esca found associated to Diatrypaceae fungi and with Basidiomycete

Pathogenicity of bot canker has been consistently proven in Chile [4] as well as necrotic strikes associated to Phaeomoniella chlamydospora [3, 9]. But there is an important disease called chlorotic leaf roll (enrollamiento clorótico) of the vines that is frequently associated to V-shaped necrosis and necrotic streaks in the xylem and also with spongy tissues mainly associated to Basidiomycetes colonization, which Koch's postulates have not been done. So, this type of disease and symptoms needed to be solved by the Chilean plant pathologists.

The economic importance of diseases affecting grapevines has been partially studied in Chile. Morales et al. [4] found that bot canker disease affects table grape vineyards and that the disease increases with the age of the plants. With an increasing incidence of the disease of 22% in an 11-year-old vineyard to 70% in the 22-year-old vineyard, and with maximum severity up to 22% of damage index in a 16-year-old table grape vineyard. In these table grape vineyards, the main species and detected prevalence (%) were Diplodia seriata (83.3%), D. mutila (8.3%), and Spencermartinsia viticola (8.3%). In a national survey done by Díaz et al. [3], with about 694 wood samples being analyzed obtained from 67 Chilean vineyards, from Copiapó (27 18'S) to Los Angeles (3742'S), a total of 1363 fungal isolates were obtained from diseased plants with different symptoms, and the most prevalent identified species were Phaeomoniella chlamydospora (in 85% of the samples); Botryosphaeriaceae (in 56% of the samples) with the main detected species Diplodia seriata, D. mutila, Neofusicoccum parvum, and Spencermartinsia viticola; and Diatrypaceae (in 4.8% of the samples, but Eutypa lata was not found). Phomopsis viticola and Cylindrocarpon sp. were only found in 0.4% of the analyzed samples. Finally, Valencia et al. [10] analyzing conidial dissemination in four vineyards from the Valparaíso Region found in the spore traps conidia from D. seriata, S. viticola, and Neofusicoccum australe, but the greater peaks

On the other hand, Torres et al. [11] reported the prevalence and damage observed in 10 vineyards cv. Cabernet Sauvignon during the 2010–2011 growing season. In the research done in 14 blocks of the O'Higgins (n = 8) and Maule (n = 6) regions, the average damages associated with bot canker were 36 and 48% for each region, respectively, with an average of production

GTDs are the main biotic factors affecting grapes wherever are grown and affecting vineyard productivity and longevity [12]. In France, there is a national crisis with 12% of the vineyards not economically viable, causing annual losses for about 1 billion euros [13]. In China, where Botryosphaeria dieback is a serious disease, Yan et al. [14] made a survey in 72 vineyards of 20 grape-growing regions; they found that dieback occurs in 18/20 provinces, and they confirmed the presence of B. dothidea, D. seriata, L. theobromae, and N. parvum. These species were spatially distributed: L. theobromae and N. parvum only occurring in subtropical monsoon climate regions, Diplodia seriata only occurring in temperate climatic regions, and B. dothidea occurring in both types of climates. Of the 25 cultivars most grown in China, none was

(69.2%), followed by D. mutila (12.8%), and Neofusicoccum parvum (10.2%).

. The most prevalent species detected were Diplodia seriata

fungi associated to wood spongy tissues [1, 3, 4, 8].

46 Grapes and Wines - Advances in Production, Processing, Analysis and Valorization

found corresponded to D. seriata.

loss estimated at 5800 kg ha<sup>1</sup>

resistant to the four (taxa) species.

According to the 10th International Workshop on Grapevine Trunk Diseases (2017) taken place in Reims, France, in the introduction done by Dr. José Ramón Úrbez-Torres "Main achievements and future prospects in GTDs Pathogen Characterization and Identification," he analyzed that 133 species from 34 genera are associated to GTDs worldwide. Considering these information, the analysis of this type of diseases affecting grapevines is much complex to organize. Fortunately, in Chile the situation is more easy considering that there are only five species causing bot canker [3, 4, 8, 10, 17], two main species associated to Petri and Esca-like diseases [3, 9, 18], plus three species of Basidiomycetes [3]. Phomopsis dieback is very infrequently seen, and black foot also seems to be associated with problems at the plantation site of grafted material and with very low prevalence [3].

In table grape and wine vineyard research done in Chile, only five Botryosphaeriaceae species have been reported to affect these crops: Diplodia seriata De Not. ( Botryosphaeria obtusa (Schwein.) Shoemaker); D. mutila (Fr.) Mont. (= Botryosphaeria stevensii Shoemaker); Neofusicoccum parvum (Pennycook & Samuels) Crous, Slippers & A.J.L. Phillips (= Botryosphaeria parva) Pennycook & Samuels; N. australe (Slippers, Crous & M.J. Wingf.) Crous, Slippers & A.J.L. Phillips (= Botryosphaeria australis Slippers, Crous & M.J. Wingf.); and Spencermartinsia viticola (A.J.L. Phillips & J. Luque) A.J.L. Phillips, A. Alves & Crous (= Botryosphaeria viticola A.J.L. Phillips & J. Luque) [1, 3, 4, 8, 11, 17]. The names in parentheses are in accordance with the name and authors described by Phillips et al. [19].

In accordance with Díaz et al. [3] from a total of 694 analyzed samples obtained from different varieties grown for table grapes or wine industry, 56.3% of them were Botryosphaeriaceae species, and from 85% of these samples, the principal recovered species was Phaeomoniella chlamydospora (Pa. chlamydospora) (W. Gams, Crous, M.J. Wingf. & L. Mugnai) Crous & W. Gams, obtained from vascular streaks. But considering cankers with V-shaped form, 91% of the recovered species were Botryosphaeriaceae, and from these 68% of them was Diplodia seriata. From a soft-spongy canker (n = 314), the main species isolated in 96% of the samples was Inocutis sp. In pathogenicity tests carried out by Díaz et al. [3], Inocutis was able to cause damage but significantly less than that caused by Botryosphaeriaceae or Phaeomoniella species.

Considering Úrbez-Torres' [20] review of species affecting Vitis vinifera, he could recognize 21 different Botryosphaeriaceae species affecting this crop in California. In France the disease known as Esca complex is caused by Pa. chlamydospora and by Phaeoacremonium spp. plus the basidiomycete Fomitiporia mediterranea [2].

#### 2.3.1. Reproductive structures

Taking into account Botryosphaeria dieback disease, all of the pathogens involved produce their asexual spores or conidia in pycnidia, and depending on the species, it is the shape and color of the conidia (Figure 2A–C). They are formed in conidiogenous cells, but some species have conidiophores [19]. In their sexual stage (if present), the ascospores are produced in a bitunicate asci, and this is inside an ascomata, most of them gregarious. In Chile, there is no report of sexual stage of Botryosphaeriaceae occurring in Vitis.

2.4. Principal symptoms associated to GTDs

reproducing the symptoms in mature plants.

2.5. Cultivar susceptibility

According to the research carried out by Díaz et al. [3, 4], Esca-like symptoms and their causal agents are associated to brown streaking in the xylem of the trunk and cordon of young grapevines (≤ 10 years old) (Figure 2A–C). Cordons and trunk from young grapevines showed this brown strike with the appearance of necrotic mottling in cross sections or dark brown necrotic streaking in longitudinal sections. They reproduce part of the symptoms associated to

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On the other hand, brown V-shaped cankers were consistently associated in table grape vineyards to four Botryosphaeriaceae species by Díaz et al. [3] and Morales et al. [4]. In a vast survey done along the main viticulture regions, Díaz et al. [3] found a total of 12 genera including Diplodia, Dothiorella, Neofusicoccum, Spencermartinsia, Cryptovalsa, Eutypella, Cylindrocarpon, Phomopsis, Inocutis, Phaeoacremonium, Phaeomoniella, and Seimatosporium. When the fungal isolates obtained from these genera were correlated to the principal symptoms observed in the field, these authors associated dark brown streaking symptoms to Pa. chlamydospora (85.2% of the samples), D. mutila (3.8% of samples), and Inocutis sp. (3.7% of samples). Symptoms of brown hard V-shaped canker (Figure 3A) were associated to Diplodia seriata (68.1% of samples), Neofusicoccum parvum (8.3% of samples), Spencermartinsia viticola (8.1% of samples), and Pa. chlamydospora (5.3%), and yellowish soft-spongy canker (Figure 3B) was associated to Inocutis sp. (96.2% of samples), Diplodia seriata (2.5% of samples), and Pa. chlamydospora (1.3% of samples). In vineyards,

the presence of dead spurs that show a grayish color is frequently observed (Figure 3C).

The causal agent of leaf chlorotic roll symptom associated to decay plants of table grape and vineyard cultivars (Figure 2D) has not solved up to date. This symptom is frequently associated to cankers caused by Botryosphaeriaceae species, but up to date, no one has reproduced these symptoms in Chile. It consists in a leaf malformation and chlorosis of the leaf lamina, short internodes of the shoots or canes, death of spurs, and dieback commonly associated to cordon and/or trunk dieback [3, 4]. In Argentina, a similar situation was studied by Gatica et al. [21] with the disease called "hoja de malvón," where they found similar symptoms in association to mature vines with Phellinus sp. that prevailed over Phaeoacremonium aleophilum, Pm. parasiticum, and Phaeomoniella chlamydospora. In a previous study done by Gatica et al. [22], they made an in vitro pathogenicity test with Phellinus sp., Botryodiplodia sp., Phaeoacremonium aleophilum, and Phaeomoniella chlamydospora, where all these were pathogenic and cause decline and death of young plants, but the chlorotic leaf symptoms with reddish edges were produced by Phellinus sp. and by P. chlamydospora. Nevertheless, this situation needed to be studied

The first report of a Botryosphaeria species affecting the Vitis genus in Chile was done by Latorre et al. [1], where these authors report a severe attack of a canker disease affecting 2-year-old plants of cv. Flame Seedless grown in a Spanish trellis for fresh table grape production. Extensive cankers were found in the trunk, causing mild to moderate chlorosis and leaf necrosis and a poor seasonal growth. All that time, the authors classified the causal agent as

Esca in Europe [2] but not the characteristics foliar symptoms of this disease.

The Phaeomoniella genus produces yeast-like colonies that were olivaceous with sparse aerial mycelium and erect non-branched, smooth, and septate conidiophores [18]. The conidia are cellular, slightly pigmented, smooth, oblong to ellipsoid, and with small dimensions (3.0–4.2 μm large). Black globose pycnidia were produced on pine needles. White dense cirrus was produced on pine needles.

Figure 2. Grapevine trunk symptoms associated to bot canker and Esca-like disease symptoms in Chile. (A) V-shaped canker on the main branch of Vitis. (B) Main brunch associated with canker necrosis, yellow soft-spongy canker, and mottling in the trunk. (C) Dead spur attached to a diseased brunch with a typical grayish color. (D) Table grape vineyard with symptoms of distorted leaf and necrosis in the margin associated to chlorotic leaf roll.

#### 2.4. Principal symptoms associated to GTDs

2.3.1. Reproductive structures

on pine needles.

Taking into account Botryosphaeria dieback disease, all of the pathogens involved produce their asexual spores or conidia in pycnidia, and depending on the species, it is the shape and color of the conidia (Figure 2A–C). They are formed in conidiogenous cells, but some species have conidiophores [19]. In their sexual stage (if present), the ascospores are produced in a bitunicate asci, and this is inside an ascomata, most of them gregarious. In Chile, there is no

The Phaeomoniella genus produces yeast-like colonies that were olivaceous with sparse aerial mycelium and erect non-branched, smooth, and septate conidiophores [18]. The conidia are cellular, slightly pigmented, smooth, oblong to ellipsoid, and with small dimensions (3.0–4.2 μm large). Black globose pycnidia were produced on pine needles. White dense cirrus was produced

Figure 2. Grapevine trunk symptoms associated to bot canker and Esca-like disease symptoms in Chile. (A) V-shaped canker on the main branch of Vitis. (B) Main brunch associated with canker necrosis, yellow soft-spongy canker, and mottling in the trunk. (C) Dead spur attached to a diseased brunch with a typical grayish color. (D) Table grape vineyard

with symptoms of distorted leaf and necrosis in the margin associated to chlorotic leaf roll.

report of sexual stage of Botryosphaeriaceae occurring in Vitis.

48 Grapes and Wines - Advances in Production, Processing, Analysis and Valorization

According to the research carried out by Díaz et al. [3, 4], Esca-like symptoms and their causal agents are associated to brown streaking in the xylem of the trunk and cordon of young grapevines (≤ 10 years old) (Figure 2A–C). Cordons and trunk from young grapevines showed this brown strike with the appearance of necrotic mottling in cross sections or dark brown necrotic streaking in longitudinal sections. They reproduce part of the symptoms associated to Esca in Europe [2] but not the characteristics foliar symptoms of this disease.

On the other hand, brown V-shaped cankers were consistently associated in table grape vineyards to four Botryosphaeriaceae species by Díaz et al. [3] and Morales et al. [4]. In a vast survey done along the main viticulture regions, Díaz et al. [3] found a total of 12 genera including Diplodia, Dothiorella, Neofusicoccum, Spencermartinsia, Cryptovalsa, Eutypella, Cylindrocarpon, Phomopsis, Inocutis, Phaeoacremonium, Phaeomoniella, and Seimatosporium. When the fungal isolates obtained from these genera were correlated to the principal symptoms observed in the field, these authors associated dark brown streaking symptoms to Pa. chlamydospora (85.2% of the samples), D. mutila (3.8% of samples), and Inocutis sp. (3.7% of samples). Symptoms of brown hard V-shaped canker (Figure 3A) were associated to Diplodia seriata (68.1% of samples), Neofusicoccum parvum (8.3% of samples), Spencermartinsia viticola (8.1% of samples), and Pa. chlamydospora (5.3%), and yellowish soft-spongy canker (Figure 3B) was associated to Inocutis sp. (96.2% of samples), Diplodia seriata (2.5% of samples), and Pa. chlamydospora (1.3% of samples). In vineyards, the presence of dead spurs that show a grayish color is frequently observed (Figure 3C).

The causal agent of leaf chlorotic roll symptom associated to decay plants of table grape and vineyard cultivars (Figure 2D) has not solved up to date. This symptom is frequently associated to cankers caused by Botryosphaeriaceae species, but up to date, no one has reproduced these symptoms in Chile. It consists in a leaf malformation and chlorosis of the leaf lamina, short internodes of the shoots or canes, death of spurs, and dieback commonly associated to cordon and/or trunk dieback [3, 4]. In Argentina, a similar situation was studied by Gatica et al. [21] with the disease called "hoja de malvón," where they found similar symptoms in association to mature vines with Phellinus sp. that prevailed over Phaeoacremonium aleophilum, Pm. parasiticum, and Phaeomoniella chlamydospora. In a previous study done by Gatica et al. [22], they made an in vitro pathogenicity test with Phellinus sp., Botryodiplodia sp., Phaeoacremonium aleophilum, and Phaeomoniella chlamydospora, where all these were pathogenic and cause decline and death of young plants, but the chlorotic leaf symptoms with reddish edges were produced by Phellinus sp. and by P. chlamydospora. Nevertheless, this situation needed to be studied reproducing the symptoms in mature plants.

#### 2.5. Cultivar susceptibility

The first report of a Botryosphaeria species affecting the Vitis genus in Chile was done by Latorre et al. [1], where these authors report a severe attack of a canker disease affecting 2-year-old plants of cv. Flame Seedless grown in a Spanish trellis for fresh table grape production. Extensive cankers were found in the trunk, causing mild to moderate chlorosis and leaf necrosis and a poor seasonal growth. All that time, the authors classified the causal agent as

5-year-old shoots, the most aggressive species of the Botryosphaeriaceae tested was also Diplodia mutila (Figure 3B), and Diplodia seriata (Figure 3C) was less aggressive in all of the tested tissues considering the necrotic lesion. However, considering vascular streak length, Diplodia seriata was more aggressive than D. mutila. This is not in accordance with another pathogenicity tests reported by Úrbez-Torres and Gubler [23], where the most virulent species was Lasiodiplodia theobromae, followed by Neofusicoccum luteum, N. parvum, and N. australe, and they considered Diplodia mutila and D. seriata as weakly virulent. Maybe, the climatic conditions make the difference in the pathogenicity of the isolates or the age of the tissue done in the pathogenicity test. On the other hand, in pathogenicity field trials done in two regions, Sonoma and Colusa, employing adult plants of Chardonnay and Zinfandel, respectively, infecting nine different GTD causal agents, the most infectious were the four Botryosphaeriaceae species tested, while Pa. chlamydospora was in an intermediate position, and the less infective were Togninia

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In young tissue the most aggressive species are Neofusicoccum and Lasiodiplodia. Also, it is important to note that in Chile only five species of Botryosphaeriaceae have been detected affecting Vitis vinifera [1, 4, 8, 17]. Nevertheless, these species are capable to cause almost 50% of less yield in some regions [11]. Up to date, Lasiodiplodia theobromae, an important causal agent described in different countries, has not been detected or described affecting table grape

In order to understand how Botryosphaeriaceae species can arrive to Vitis plants, one way could be in the association of different species crops in a same orchard or in surrounding orchards. If we analyze the statement done by De Wet et al. [25], this authors classify different species of Botryosphaeriaceae associated to gymnosperms or angiosperms: Diplodia species occurred mainly in gymnosperms; Dothiorella species restricted to angiosperms; Lasiodiplodia occurred equally on gymnosperms or angiosperms, and Fusicoccum anamorphs occurred mostly on angiosperms with rare reports in gymnosperms. Nevertheless, in the case of Vitis vinifera, this species has been affected by all the different genera of the Botryosphaeriaceae family. In Chile, other important hosts for Botryosphaeriaceae are almond trees [26], loquat

In the trunk a depletion of starch reserves in woody tissues is not only associated with fungal colonization but also affects leaves (where no colonization is achieved) affecting photosynthetic rate. This can cause a decrease of plant development and vigor during the subsequent year. Also, lipid and amino acid are downregulated [2]. There is a response of the affected plants resulting in an accumulation of tyloses and gummosis, not only causing the blockage of the limiting fungal invasion but also causing disease. But, their progression in the wood is also inhibited by the formation of polyphenol-rich reaction zones and by the accumulation of pathogenesis-related (PR) proteins and the production of oxidative damage and reactive oxy-

[27], blueberry [28], avocado trees [29], and araucaria trees [30], among others.

minima, Phaeoacremonium parasiticum, P. richardsiae, and Eutypa lata [24].

vines or wine cultivars in Chile [3, 4, 10].

2.6. Botryosphaeriaceae main hosts

2.7. Plant physiology damage

gen species (ROS) production.

Figure 3. Pathogenicity tests done in shoots of 1-, 2-, and 5-year-old Vitis plants and the spores of different Botryosphaeriaceae. (A) Lessons and spores of Diplodia seriata. (B) Lessons and spores of D. mutila. (C) Lessons and spores of Spencermartinsia (= Dothiorella) viticola.

being B. dothidea, but with the increasing knowledge in the genera, the detected species correspond to D. mutila. Considering the most important table grape cultivars grown in Chile, in trials done by Morales et al. [4] with excised shoots of cvs. Thompson Seedless, Red Globe, and Flame Seedless, the most aggressive species was D. mutila. In a field trial done with 25-year-old plants of the cv. Flame Seedless, inoculating three different tissues, 1-, 2-, and 5-year-old shoots, the most aggressive species of the Botryosphaeriaceae tested was also Diplodia mutila (Figure 3B), and Diplodia seriata (Figure 3C) was less aggressive in all of the tested tissues considering the necrotic lesion. However, considering vascular streak length, Diplodia seriata was more aggressive than D. mutila. This is not in accordance with another pathogenicity tests reported by Úrbez-Torres and Gubler [23], where the most virulent species was Lasiodiplodia theobromae, followed by Neofusicoccum luteum, N. parvum, and N. australe, and they considered Diplodia mutila and D. seriata as weakly virulent. Maybe, the climatic conditions make the difference in the pathogenicity of the isolates or the age of the tissue done in the pathogenicity test. On the other hand, in pathogenicity field trials done in two regions, Sonoma and Colusa, employing adult plants of Chardonnay and Zinfandel, respectively, infecting nine different GTD causal agents, the most infectious were the four Botryosphaeriaceae species tested, while Pa. chlamydospora was in an intermediate position, and the less infective were Togninia minima, Phaeoacremonium parasiticum, P. richardsiae, and Eutypa lata [24].

In young tissue the most aggressive species are Neofusicoccum and Lasiodiplodia. Also, it is important to note that in Chile only five species of Botryosphaeriaceae have been detected affecting Vitis vinifera [1, 4, 8, 17]. Nevertheless, these species are capable to cause almost 50% of less yield in some regions [11]. Up to date, Lasiodiplodia theobromae, an important causal agent described in different countries, has not been detected or described affecting table grape vines or wine cultivars in Chile [3, 4, 10].

#### 2.6. Botryosphaeriaceae main hosts

In order to understand how Botryosphaeriaceae species can arrive to Vitis plants, one way could be in the association of different species crops in a same orchard or in surrounding orchards. If we analyze the statement done by De Wet et al. [25], this authors classify different species of Botryosphaeriaceae associated to gymnosperms or angiosperms: Diplodia species occurred mainly in gymnosperms; Dothiorella species restricted to angiosperms; Lasiodiplodia occurred equally on gymnosperms or angiosperms, and Fusicoccum anamorphs occurred mostly on angiosperms with rare reports in gymnosperms. Nevertheless, in the case of Vitis vinifera, this species has been affected by all the different genera of the Botryosphaeriaceae family. In Chile, other important hosts for Botryosphaeriaceae are almond trees [26], loquat [27], blueberry [28], avocado trees [29], and araucaria trees [30], among others.

#### 2.7. Plant physiology damage

being B. dothidea, but with the increasing knowledge in the genera, the detected species correspond to D. mutila. Considering the most important table grape cultivars grown in Chile, in trials done by Morales et al. [4] with excised shoots of cvs. Thompson Seedless, Red Globe, and Flame Seedless, the most aggressive species was D. mutila. In a field trial done with 25-year-old plants of the cv. Flame Seedless, inoculating three different tissues, 1-, 2-, and

Figure 3. Pathogenicity tests done in shoots of 1-, 2-, and 5-year-old Vitis plants and the spores of different Botryosphaeriaceae. (A) Lessons and spores of Diplodia seriata. (B) Lessons and spores of D. mutila. (C) Lessons and spores

of Spencermartinsia (= Dothiorella) viticola.

50 Grapes and Wines - Advances in Production, Processing, Analysis and Valorization

In the trunk a depletion of starch reserves in woody tissues is not only associated with fungal colonization but also affects leaves (where no colonization is achieved) affecting photosynthetic rate. This can cause a decrease of plant development and vigor during the subsequent year. Also, lipid and amino acid are downregulated [2]. There is a response of the affected plants resulting in an accumulation of tyloses and gummosis, not only causing the blockage of the limiting fungal invasion but also causing disease. But, their progression in the wood is also inhibited by the formation of polyphenol-rich reaction zones and by the accumulation of pathogenesis-related (PR) proteins and the production of oxidative damage and reactive oxygen species (ROS) production.

It has been demonstrated that fresh pruning wounds are the main infection route for grape trunk disease pathogens, bot canker disease [3, 4], and Esca-like disease [3].

Taking into account Díaz and Latorre's [18] work, they reported that they could not find Pa. chlamydospora in pruning debris samples (n = 168), sap samples (n = 82), soil samples (n = 100), or weed samples (n = 293) that were collected during 2010–2012. Maybe, infection of

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Studies carried out by Úrbez-Torres et al. [36], evaluating the conidial germination of different Botryosphaeriaceae at different temperatures found that conidia did not germinate at 5C with the exception of Botryosphaeria dothidea and Neofusicoccum parvum. On the other hand, only B. dothidea, Diplodia seriata, and Lasiodiplodia theobromae germinate at 40C. Optimum germination at 25C was for B. dothidea and Dothiorella iberica; 25–30C for Spencermartinsia viticola; 30C for D. corticola, D. mutila, D. seriata, and Neofusicoccum parvum; and 40C for pigmented L. theobromae. This is not strange considering the observations done by Yan et al. [14] that L.

Considering the recent statement done by Gramaje et al. [38] about the establishment, progress, severity, and spread of GTDs have been associated with (i) drought, (ii) limited availability of effective fungicides, (iii) pressure to increase yields, (iv) lack of pruning wound protection, and (iv) poor low-quality propagation material. All the efforts should first be done

In order to avoid early infection of grapevines by Phaeomoniella or Phaeoacremonium species, the main treatments considered are during the production of plants in the nursery. The program should start with an adequate clean mother plants and use of fungicides in the hydration process when the new plants are produced. Details of this process can be followed in Auger

The disease's integrated management of this disease should start with an efficient cleaning process of all the diseased or dead shoots, arms, and trunks present in a vineyard. This material should be immediately taken out of the vineyard and efficiently destroyed in order to avoid the formation of reproductive structures such as pycnidia or ascomata of Botryosphaeriaceae and also to avoid the colonization of diseased tissues associated to Basid-

A cultural management interesting to note is that the actual pruning cordon system produces many little injuries that are difficult to protect, more over if a mechanical pruning is used. Something that should be evaluated, especially in regions where GTDs are highly prevalent,

Botryosphaeriaceae can cause cankers and dieback in grapevines, but different species affect grapevines in different grape-growing regions and countries. There are regions or countries

theobromae was found in Vitis under subtropical monsoon climatic conditions.

this species is mainly associated to young plants.

2.10. Disease management

2.10.1. Petri disease

2.10.2. Bot canker control

iomycetes like Inocutis spp.

in order to avoid these types of situation.

et al. [39]. This is complemented with the heat treatment [40].

the use of double Guyot pruning system should be considered.

#### 2.8. Endophytic species

Many authors highlight the fact that these fungi are common endophytes mainly in woody plants and that these fungi have the ability to cause serious diseases, particularly when plants are predisposing due to environmental stress [5, 31]. In general, these species have been recognized as natural opening colonizers. The first report of a forest pathogen isolated from stem and xylem healthy tissues was done by Petrini and Fisher [32]. Later, Johnson et al. [33] could recover Fusicoccum, Neofusicoccum, Pseudofusicoccum, and Lasiodiplodia from healthy mango plant parts.

Muruamendiaraz and Legorburu [34] could recover D. seriata from asymptomatic tissues of Vitis plants. This finding could agree with the endophytic character of this species. In the same work, E. lata was rarely found in asymptomatic vines. On the other hand, Phaeomoniella chlamydospora and Phaeoacremonium spp. were present in healthy rootstock propagation material as endophytes [35].

#### 2.9. GTD spore's dispersion and optimal temperature

In Chile according to Valencia et al.'s [10] work, the dispersal of Botryosphaeriaceae conidia was studied in two vineyards in the Valparaíso Region of Chile, where semiarid Mediterranean conditions prevail. Due to the climate situation in this region characterized by the habitual rainy period mainly occurring in winter months, peaks of inoculum also occurred in the season associated to precipitation events equal or greater than 2 mm. The main species associated to the spore traps were D. seriata, Spencermartinsia (= Dothiorella) viticola, Neofusicoccum, and probably N. australe. In this work no association was observed between the volume of water used in the application of agrichemical products (≤1000 L ha<sup>1</sup> ) and peaks of Botryosphaeriaceae.

Considering the dispersion of Botryosphaeriaceae species, there are mainly two works done in other countries: Úrbez-Torres et al.'s [36] work done in California, United States, and Amponsah et al.'s [37] work done in Canterbury, New Zealand. In California, Úrbez-Torres et al. [36] found a strong regression between the spore release and precipitation and that more spores were trapped during the winter period. Amponsah et al. [37] work on the dispersal of spores of Botryosphaeriaceae that are responsible for dieback in grapevines. They work in one vineyard, with the Vaseline-coated slide trap. During all the trapping period, 59.8% of the total species trapped were Neofusicoccum spp., and 40.2% of the rest were Diplodia spp. The spores were detected during the entire period but most abundant during December, January, and February associated to high temperatures and rainfall, where routine trimming of mature canes provides wounds for infection. Under these conditions, the main collected species were Neofusicoccum luteum, N. australe, N. parvum, and Diplodia mutila. These species infect mainly trunks, canes, green shoots, and plant debris. In this region, rainfall occurs not only in the winter period, mainly June, July, and August but also in the rest of the year.

Taking into account Díaz and Latorre's [18] work, they reported that they could not find Pa. chlamydospora in pruning debris samples (n = 168), sap samples (n = 82), soil samples (n = 100), or weed samples (n = 293) that were collected during 2010–2012. Maybe, infection of this species is mainly associated to young plants.

Studies carried out by Úrbez-Torres et al. [36], evaluating the conidial germination of different Botryosphaeriaceae at different temperatures found that conidia did not germinate at 5C with the exception of Botryosphaeria dothidea and Neofusicoccum parvum. On the other hand, only B. dothidea, Diplodia seriata, and Lasiodiplodia theobromae germinate at 40C. Optimum germination at 25C was for B. dothidea and Dothiorella iberica; 25–30C for Spencermartinsia viticola; 30C for D. corticola, D. mutila, D. seriata, and Neofusicoccum parvum; and 40C for pigmented L. theobromae. This is not strange considering the observations done by Yan et al. [14] that L. theobromae was found in Vitis under subtropical monsoon climatic conditions.

#### 2.10. Disease management

It has been demonstrated that fresh pruning wounds are the main infection route for grape

Many authors highlight the fact that these fungi are common endophytes mainly in woody plants and that these fungi have the ability to cause serious diseases, particularly when plants are predisposing due to environmental stress [5, 31]. In general, these species have been recognized as natural opening colonizers. The first report of a forest pathogen isolated from stem and xylem healthy tissues was done by Petrini and Fisher [32]. Later, Johnson et al. [33] could recover Fusicoccum, Neofusicoccum, Pseudofusicoccum, and Lasiodiplodia from healthy

Muruamendiaraz and Legorburu [34] could recover D. seriata from asymptomatic tissues of Vitis plants. This finding could agree with the endophytic character of this species. In the same work, E. lata was rarely found in asymptomatic vines. On the other hand, Phaeomoniella chlamydospora and Phaeoacremonium spp. were present in healthy rootstock propagation material

In Chile according to Valencia et al.'s [10] work, the dispersal of Botryosphaeriaceae conidia was studied in two vineyards in the Valparaíso Region of Chile, where semiarid Mediterranean conditions prevail. Due to the climate situation in this region characterized by the habitual rainy period mainly occurring in winter months, peaks of inoculum also occurred in the season associated to precipitation events equal or greater than 2 mm. The main species associated to the spore traps were D. seriata, Spencermartinsia (= Dothiorella) viticola, Neofusicoccum, and probably N. australe. In this work no association was observed between

Considering the dispersion of Botryosphaeriaceae species, there are mainly two works done in other countries: Úrbez-Torres et al.'s [36] work done in California, United States, and Amponsah et al.'s [37] work done in Canterbury, New Zealand. In California, Úrbez-Torres et al. [36] found a strong regression between the spore release and precipitation and that more spores were trapped during the winter period. Amponsah et al. [37] work on the dispersal of spores of Botryosphaeriaceae that are responsible for dieback in grapevines. They work in one vineyard, with the Vaseline-coated slide trap. During all the trapping period, 59.8% of the total species trapped were Neofusicoccum spp., and 40.2% of the rest were Diplodia spp. The spores were detected during the entire period but most abundant during December, January, and February associated to high temperatures and rainfall, where routine trimming of mature canes provides wounds for infection. Under these conditions, the main collected species were Neofusicoccum luteum, N. australe, N. parvum, and Diplodia mutila. These species infect mainly trunks, canes, green shoots, and plant debris. In this region, rainfall occurs not only in the

) and peaks

the volume of water used in the application of agrichemical products (≤1000 L ha<sup>1</sup>

winter period, mainly June, July, and August but also in the rest of the year.

trunk disease pathogens, bot canker disease [3, 4], and Esca-like disease [3].

52 Grapes and Wines - Advances in Production, Processing, Analysis and Valorization

2.8. Endophytic species

mango plant parts.

as endophytes [35].

of Botryosphaeriaceae.

2.9. GTD spore's dispersion and optimal temperature

Considering the recent statement done by Gramaje et al. [38] about the establishment, progress, severity, and spread of GTDs have been associated with (i) drought, (ii) limited availability of effective fungicides, (iii) pressure to increase yields, (iv) lack of pruning wound protection, and (iv) poor low-quality propagation material. All the efforts should first be done in order to avoid these types of situation.

#### 2.10.1. Petri disease

In order to avoid early infection of grapevines by Phaeomoniella or Phaeoacremonium species, the main treatments considered are during the production of plants in the nursery. The program should start with an adequate clean mother plants and use of fungicides in the hydration process when the new plants are produced. Details of this process can be followed in Auger et al. [39]. This is complemented with the heat treatment [40].

#### 2.10.2. Bot canker control

The disease's integrated management of this disease should start with an efficient cleaning process of all the diseased or dead shoots, arms, and trunks present in a vineyard. This material should be immediately taken out of the vineyard and efficiently destroyed in order to avoid the formation of reproductive structures such as pycnidia or ascomata of Botryosphaeriaceae and also to avoid the colonization of diseased tissues associated to Basidiomycetes like Inocutis spp.

A cultural management interesting to note is that the actual pruning cordon system produces many little injuries that are difficult to protect, more over if a mechanical pruning is used. Something that should be evaluated, especially in regions where GTDs are highly prevalent, the use of double Guyot pruning system should be considered.

Botryosphaeriaceae can cause cankers and dieback in grapevines, but different species affect grapevines in different grape-growing regions and countries. There are regions or countries where the rainfall period is concentrated mainly in winter time, while there are some regions or countries where rainfall conditions occur around the whole year. According to the climatic specific conditions of each area, the management will be oriented to prevent the disease mainly in the winter period or during the whole year. In Chile, according to the work published by Díaz et al. [3] and Valencia et al. [10], the main risk period for bot canker is in winter, where pruning management takes place, so the main effort should be oriented to protect pruning wounds. Nevertheless, the wine-producing area has expanded to the southern regions of Chile, where it usually rains during the summer months, so at these locations the problem probably expands to another species and attack green shoots that currently occurs in New Zealand [37] or in subtropical regions of China [14].

(cyproconazole and iodocarb). All of these fungicides were effective in reducing the lessons caused by this GTD species, but the most effective according to the authors was Topsin M.

Grapevine Trunk Diseases (GTDs): Impact on Table Grapes and Wine Vineyards in Chile

http://dx.doi.org/10.5772/intechopen.72607

55

In Portugal, Rego et al. [43] in a 3-year field trial conducted in 15-year-old vineyard cv. Aragonez evaluate the efficacy of six different treatments against Botryosphaeria canker and Phomopsis dieback, applying three sprays each year, and the most efficient treatment was the

In Chile the most important diseases affecting vineyards for table grapes and wine production are bot canker and Esca-like disease. In addition, for young plants the main problem is Petri disease. There are several studies done in determining causal agents, epidemiology, and control strategies, but there still remaining work to be done, for example, the fulfillment of Koch's postulates in order to determine the etiology of chlorotic leaf roll disease. Also, the convenience of the use of a double Guyot pruning system instead of a cordon pruning system, in order to avoid so many exposed fresh cuts during the pruning period, should be analyzed. Something that is important to note is that some management change occurred worldwide that makes a point of inflection in the increase of GTDs. In Europe some change was that sodium arsenite was excluded in 2001 for Esca control, but in Chile sodium arsenite was not used. In the management of vineyards, a change in the gray mold control was done, mainly associated with the discontinuous use of benzimidazoles because of the increase of resistant Botrytis cinerea isolates. It is important to note that in many trials done in different countries, benzimid-

These considerations are something that should be approached in an interdisciplinary work

School of Agronomy, Pontifical Catholic University of Valparaíso (Escuela de Agronomía,

[1] Latorre BA, Besoain X, Flores V. Botryosphaeria canker of table grapes. Phytopathology.

application of Bion plus Score (difenoconazole).

azoles were highly efficient in controlling most of the GTD agents.

done with different researchers from different countries.

Address all correspondence to: ximena.besoain@pucv.cl

Pontificia Universidad Católica de Valparaíso), Quillota, Chile

Author details

Ximena Besoain

References

1986;76(10):1112. (Abstract)

3. Conclusion and future trends

One of the first works done in evaluating different fungicides under in vitro conditions was the work done by Amponsah et al. [41]. They evaluate mycelial and conidial inhibition and also if they were able to protect pruning wounds against infection. In this work the tested species differed in their response to the different fungicides, and considering all the evaluated species, N. luteum was the least sensitive. The most effective fungicides were flusilazole, thiophanatemethyl, and mancozeb. According to the in vitro mycelial inhibition, flusilazole was the most effective (0.002 ppm) but was less effective considering conidial germination inhibition. For the in vivo trial, the best treatments were carbendazim, mancozeb, iprodione, and flusilazole. Under field conditions the best treatment was based on flusilazole active ingredient.

In the work done in Chile by Torres et al. [42], they evaluate demethylation-inhibiting (DMI) fungicides against D. mutila, D. seriata, N. australe, and N. parvum. The results demonstrate that the fungicides tebuconazole, myclobutanil, prochloraz, and prochloraz plus epoxiconazole were effective under in vitro conditions in inhibit mycelial and conidial germination for all the evaluated species.

Díaz and Latorre [18] evaluate the efficacy of benomyl, pyraclostrobin, tebuconazole, and thiophanate-methyl, applied as paste or liquid, in pre- or postinoculation. The paste application provides a better control than the sprayed form. Table 1 shows the actual registered fungicides to be used for GTD control in Chile.

In California, USA, Rolshausen et al. [24] probed four fungicides (previously registered for use in vineyards) and their effect in the control of nine different GTD causal agents. The fungicides were Topsin M (thiophanate-methyl), Biopaste (boron), Cabrio EG (pyraclostrobin), and Garrison


Table 1. Commercial products actually commercialized in Chile for GTD control in vineyards.

(cyproconazole and iodocarb). All of these fungicides were effective in reducing the lessons caused by this GTD species, but the most effective according to the authors was Topsin M.

In Portugal, Rego et al. [43] in a 3-year field trial conducted in 15-year-old vineyard cv. Aragonez evaluate the efficacy of six different treatments against Botryosphaeria canker and Phomopsis dieback, applying three sprays each year, and the most efficient treatment was the application of Bion plus Score (difenoconazole).

## 3. Conclusion and future trends

where the rainfall period is concentrated mainly in winter time, while there are some regions or countries where rainfall conditions occur around the whole year. According to the climatic specific conditions of each area, the management will be oriented to prevent the disease mainly in the winter period or during the whole year. In Chile, according to the work published by Díaz et al. [3] and Valencia et al. [10], the main risk period for bot canker is in winter, where pruning management takes place, so the main effort should be oriented to protect pruning wounds. Nevertheless, the wine-producing area has expanded to the southern regions of Chile, where it usually rains during the summer months, so at these locations the problem probably expands to another species and attack green shoots that currently occurs in New

One of the first works done in evaluating different fungicides under in vitro conditions was the work done by Amponsah et al. [41]. They evaluate mycelial and conidial inhibition and also if they were able to protect pruning wounds against infection. In this work the tested species differed in their response to the different fungicides, and considering all the evaluated species, N. luteum was the least sensitive. The most effective fungicides were flusilazole, thiophanatemethyl, and mancozeb. According to the in vitro mycelial inhibition, flusilazole was the most effective (0.002 ppm) but was less effective considering conidial germination inhibition. For the in vivo trial, the best treatments were carbendazim, mancozeb, iprodione, and flusilazole.

In the work done in Chile by Torres et al. [42], they evaluate demethylation-inhibiting (DMI) fungicides against D. mutila, D. seriata, N. australe, and N. parvum. The results demonstrate that the fungicides tebuconazole, myclobutanil, prochloraz, and prochloraz plus epoxiconazole were effective under in vitro conditions in inhibit mycelial and conidial germination for all

Díaz and Latorre [18] evaluate the efficacy of benomyl, pyraclostrobin, tebuconazole, and thiophanate-methyl, applied as paste or liquid, in pre- or postinoculation. The paste application provides a better control than the sprayed form. Table 1 shows the actual registered

In California, USA, Rolshausen et al. [24] probed four fungicides (previously registered for use in vineyards) and their effect in the control of nine different GTD causal agents. The fungicides were Topsin M (thiophanate-methyl), Biopaste (boron), Cabrio EG (pyraclostrobin), and Garrison

Podexal Pyraclostrobin BASF Plateado, Phaeomoniella, and Botryosphaeria obtusa Podastik Max Tebuconazole Arysta LifeScience Silver leaf, Nectria, Neofusicoccum spp., and black

Pasta Poda TPN-50 Chlorothalonil Anasac Plateado, Phomopsis, and European canker

dead arm of Vitis

Anasac Neofusicoccum parvum and Phaeomoniella spp.

Commercial product Active ingredient Manufacturer Pathogens controlled

Table 1. Commercial products actually commercialized in Chile for GTD control in vineyards.

Under field conditions the best treatment was based on flusilazole active ingredient.

Zealand [37] or in subtropical regions of China [14].

54 Grapes and Wines - Advances in Production, Processing, Analysis and Valorization

fungicides to be used for GTD control in Chile.

Kresoxim-methyl

Pasta Poda Full Tebuconazole +

the evaluated species.

In Chile the most important diseases affecting vineyards for table grapes and wine production are bot canker and Esca-like disease. In addition, for young plants the main problem is Petri disease. There are several studies done in determining causal agents, epidemiology, and control strategies, but there still remaining work to be done, for example, the fulfillment of Koch's postulates in order to determine the etiology of chlorotic leaf roll disease. Also, the convenience of the use of a double Guyot pruning system instead of a cordon pruning system, in order to avoid so many exposed fresh cuts during the pruning period, should be analyzed.

Something that is important to note is that some management change occurred worldwide that makes a point of inflection in the increase of GTDs. In Europe some change was that sodium arsenite was excluded in 2001 for Esca control, but in Chile sodium arsenite was not used. In the management of vineyards, a change in the gray mold control was done, mainly associated with the discontinuous use of benzimidazoles because of the increase of resistant Botrytis cinerea isolates. It is important to note that in many trials done in different countries, benzimidazoles were highly efficient in controlling most of the GTD agents.

These considerations are something that should be approached in an interdisciplinary work done with different researchers from different countries.

## Author details

Ximena Besoain

Address all correspondence to: ximena.besoain@pucv.cl

School of Agronomy, Pontifical Catholic University of Valparaíso (Escuela de Agronomía, Pontificia Universidad Católica de Valparaíso), Quillota, Chile

## References

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[15] Lehoczky J. Black dead arm disease caused by Botryosphaeria stevensii infection. Acta

Grapevine Trunk Diseases (GTDs): Impact on Table Grapes and Wine Vineyards in Chile

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57

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[11] Torres C, Camps R, Latorre BA, Besoain X. Prevalence, damage and potential yield loss of vineyards cv. Cabernet Sauvignon in two regions of Chile. In: 10th International Workshop on Grapevine Trunk Diseases. Book of Abstracts. Reims, July 4–7, 2017. p. 73 (Abstract) [12] Bertsch C, Ramírez-Suero M, Magnin-Robert M, Larignon P, Chong J, Abou-Mansour E, Spagnolo A, Clément C, Fontaine F. Grapevine trunk diseases: Complex and still poorly understood. Plant Pathology. 2013;62:243-265. DOI: 10.1111/j.1365-3059.2012.02674.x [13] Lorsch W. Fatal wood diseases affect 12 percent of French vineyards. 2014. Available from: http://www.wine-searcher.com/m/2014/10/fatal-wooddiseases-affect-12-percent-offrench-

[14] Yan JY, Xie Y, Zhang W, Wang Y, Liu JK, Hyde KD, Seem RC, Zhang GZ, Wang ZY, Yao SW, Bai XJ, Dissanayake AJ, Peng YL, Li XH. Species of Botryosphaeriaceae involved in grapevine dieback in China. Fungal Diversity. 2013;61:221-236. DOI: 10.1007/s13225-013-

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0251-8

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com/top-ten-wine-exporting-countries/ [Accessed: June 15, 2017]

superficie-y-produccion-3/. [Accessed: June 30, 2017]


[30] Besoain X, Camps R, Guajardo J. First report of Diplodia mutila causing gummy canker in Araucaria araucana in Chile. Plant Disease. 2017;101:1328. DOI: 10.1094/PDIS-12-16-1730-PDN **Chapter 4**

**Provisional chapter**

**Viticulture in Warmer Climates: Mitigating**

**Viticulture in Warmer Climates: Mitigating** 

Additional information is available at the end of the chapter

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/intechopen.71155

cooling techniques, climate change

Manuel Oliveira

**Abstract**

**1. Introduction**

Manuel Oliveira

**Environmental Stress in Douro Region, Portugal**

**Environmental Stress in Douro Region, Portugal**

DOI: 10.5772/intechopen.71155

There is evidence that temperatures all over the planet are increasing slowly but steadily. The projections for the Mediterranean basin indicate a strong warming and decreased precipitation, especially in summer, and they are not favorable for agriculture in general and, in particular, for viticulture, which is a very climate-sensitive system. The vineyards of Douro Demarcated Region, located in the northeastern Portugal, are grown in marginal conditions, and the expected daily higher temperatures and decreased rainfall are bound to affect the yield and wine quality. Touriga Nacional and Tinta Roriz are two of the most valuable varieties for high-end wines of Douro, and they were subjected to a number of experiments on mitigation techniques to counteract the worse effects of adverse weather conditions. Irrigation, canopy shading, water nebulization, and kaolin coating can mitigate the losses caused by increasing stressful conditions, but they have detrimental effects on some wine characteristics. They even might not be sufficient to

protect the already producing vineyards from aggravated weather conditions.

**Keywords:** viticulture, temperature stress, water stress, irrigation, canopy management,

Today there is a large consensus that the earth's average temperature has been increasing slowly but steadily. The planet's average surface temperature has risen about 1.1°C since the late nineteenth century, and most of the warming occurred in the past 35 years; 2016 was the warmest year on record and every month from January through September, except for June, which was the warmest on record for those respective months [1]. Both projections from general circulation and regional climate models show that Southern Europe and the Mediterranean basin will experience strong warming and decreased precipitation,

> © 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution,

© 2018 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

and reproduction in any medium, provided the original work is properly cited.


**Provisional chapter**

## **Viticulture in Warmer Climates: Mitigating Environmental Stress in Douro Region, Portugal Environmental Stress in Douro Region, Portugal**

**Viticulture in Warmer Climates: Mitigating** 

DOI: 10.5772/intechopen.71155

Manuel Oliveira Manuel Oliveira Additional information is available at the end of the chapter

[30] Besoain X, Camps R, Guajardo J. First report of Diplodia mutila causing gummy canker in Araucaria araucana in Chile. Plant Disease. 2017;101:1328. DOI: 10.1094/PDIS-12-16-1730-PDN

[31] Crous PW, Slippers B, Groenwald JZ, Wingfield MJ. Botryosphaeriaceae: Systematics, pathology, and genetics. Fungal Biology. 2017;121:305-306. DOI: 10.1016/j.funbio.2017.01.003

[32] Petrini O, Fisher PJ. A comparative study of fungal endophytes in xylem and whole stem of Pinus sylvestris and Fagus sylvatica. Transactions of the British Mycological Society.

[33] Johnson GI, Mead AJ, Cooke AW, Dean JR. Mango stem end rot pathogens – Fruit infection by endophytic colonization of the inflorescence and pedicel. Annals of Applied

[34] Muruamendiaraz A, Legorburu J. Suitable of an increment borer as a sampling device for grapevine trunk disease. Phytopathologia Mediterranea. 2009;48:145-149. DOI: 10.14601/

[35] Haleen F, Crous PW, Petrini O. Fungi associated with healthy grapevine cuttings in nurseries, with special reference to pathogens involved in the decline of young vines.

[36] Úrbez-Torres JR, Bruez E, Hurtado J, Gubler WD. Effect of temperature on conidial germination of Botryosphaeriaceae species infecting grapevines. Plant Disease. 2010;94:

[37] Amponsah NT, Jones EE, Ridgway HJ, Jasper MV. Rainwater dispersal of Botryosphaeria conidia from infected grapevines. New Zealand Plant Protection. 2009;62:228-233

[38] Gramaje D, Baumgastnerk K, Halleen F, Mostert L, Sosnowski MR, Úrbez-Torres JR, Armengol J. Fungal trunk diseases: A problem beyond grapevines? Plant Pathology. 2016;

[39] Auger J, Pérez I, Esterio M. Enfermedades causadas por hongos de la madera de la vid. Red agrícola. 2017. Available from: www.redagricola.com/manejo-control-enfermedades-

[40] Elena G, Di Bella V, Armengol J, Luque J. Viability of Botryosphaeriaceae species pathogenic to grapevine after hot water treatment. Phytopathologia Mediterranea 2015;54:325-

[41] Amponsah N, Jones E, Ridway HJ, Jaspers MV. Evaluation of fungicides for the management of Botryosphaeria dieback diseases of grapevines. Pest Management Science. 2012;68:

[42] Torres C, Latorre B, Undurraga P, Besoain X. Evaluation of DMI fungicides against species of Diplodia and Neofusicoccum associated with Botryosphaeria canker of grapevine. Ciencia e Investigación Agraria. 2013;40(1):131-138. DOI: 10.7764/rcia.v40i1.457 [43] Rego C, Reis P, Dias A, Correia L. Field evaluation of fungicides against Botryosphaeria canker and Phomopsis cane and leaf spot. Phytopathologia Mediterranea. 2014;53:581-

1988;91:233-238. DOI: 10.1016/S0007-1536(88)80210-9

58 Grapes and Wines - Advances in Production, Processing, Analysis and Valorization

Phytopathol\_Mediterr-2884

1476-1484. DOI: 10.1094/ PDIS-06-10-0423

65:355-356. DOI: 10.1111/ppa.12486

causadas-hongos-la-madera-la vid/

676-683. DOI: 10.1002/ps.2309

334. DOI: 10.14601/Phytopathol\_Mediterr-15526

582. DOI: 10.14601/Phytopathol\_Mediterr-15167

Biology. 1992;120:225-234. DOI: 10.1111/j.1744-7348.1992.tb03420.x

Australasian Plant Pathology. 2003;32(1):47-52. DOI: 10.1071/AP02062

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/intechopen.71155

#### **Abstract**

There is evidence that temperatures all over the planet are increasing slowly but steadily. The projections for the Mediterranean basin indicate a strong warming and decreased precipitation, especially in summer, and they are not favorable for agriculture in general and, in particular, for viticulture, which is a very climate-sensitive system. The vineyards of Douro Demarcated Region, located in the northeastern Portugal, are grown in marginal conditions, and the expected daily higher temperatures and decreased rainfall are bound to affect the yield and wine quality. Touriga Nacional and Tinta Roriz are two of the most valuable varieties for high-end wines of Douro, and they were subjected to a number of experiments on mitigation techniques to counteract the worse effects of adverse weather conditions. Irrigation, canopy shading, water nebulization, and kaolin coating can mitigate the losses caused by increasing stressful conditions, but they have detrimental effects on some wine characteristics. They even might not be sufficient to protect the already producing vineyards from aggravated weather conditions.

**Keywords:** viticulture, temperature stress, water stress, irrigation, canopy management, cooling techniques, climate change

## **1. Introduction**

Today there is a large consensus that the earth's average temperature has been increasing slowly but steadily. The planet's average surface temperature has risen about 1.1°C since the late nineteenth century, and most of the warming occurred in the past 35 years; 2016 was the warmest year on record and every month from January through September, except for June, which was the warmest on record for those respective months [1]. Both projections from general circulation and regional climate models show that Southern Europe and the Mediterranean basin will experience strong warming and decreased precipitation,

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2018 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

especially in summer, and more frequent and longer extreme climate events such as heat waves and prolonged drought [2, 3].

1.0 to 2.2°C, while precipitation during the bud break to veraison period declined significantly,

Viticulture in Warmer Climates: Mitigating Environmental Stress in Douro Region, Portugal

http://dx.doi.org/10.5772/intechopen.71155

61

Facing the most likely scenarios of climate change, wine farmers must adapt strategies to continue producing quality wines and to preserve their typicity from each growing location [18]. As a perennial crop, grapevines require a few years to reach maturity and can remain economically productive for several decades; thus, adaptation strategies must account for both short- and long-term impacts of a shifting environment. Perennial practices, such as choice of the planting site, choice of grapevine and rootstock variety, and vineyard layout, must be decided prior to planting. For those vineyards already under production, winegrowers will have to adapt using suitable short-term mitigation strategies [19], such as irrigation, canopy

Alto Douro has a total area of 120,000 ha with an average elevation of 420 m, but the best prized vintages are produced at lower elevations where records for the period 1950–2000 show an annual precipitation ranging from 600 to 800 mm, but only 200 to 300 mm occur during the growing season from April to October, while annual temperatures ranged between 14 and 16°C and growing season oscillated from 19 to over 21°C [20]. Located in Alto Douro, close to Douro river (41° 08′ North, 7° 08′ West), some mitigation techniques have been experimented to protect *Vitis vinifera* cv Touriga Nacional and cv Tinta Roriz (Syn. Aragonêz, Tempranillo) from intense abiotic stresses that might prove useful under more extreme conditions eventu-

Touriga Nacional is one of the most valuable premium varieties of DDR. It is considered one of the best red varieties to produce quality wines with a unique aroma profile that can fetch high market prices [21]. It has good adaptation to environmental stresses, withstanding high light intensities that allow for better adjustment to warm conditions, provided an adequate water supply [22]. Tinta Roriz can be cultivated in a variety of soils and climatic conditions, because it resists well to moderate drought. It has a thick-skinned berry with a high anthocyanin concentration that makes for deep-colored wines with moderate tannins and moderate acidity. It provides some of the best red wines of Portugal and Spain [23, 24]. Despite Touriga Nacional and Tinta Roriz good adaptability, they can suffer serious yield losses under unfavorable weather conditions. An on-site automatic weather station provided records for the period 2004 to 2016 (**Table 1**). Average annual temperature was 16.1 and 21°C for the growing season, whereas total precipitation was 594 mm annually with 284 mm for the growing season.

ring during the growing season (1631.3 mm) creating an imbalance between ET0

limit that is considered adequate for quality wine grape production [26].

of 1349.3 and 1321.4 mm annually and for the growing season, respectively. Temperature was higher and rainfall was lower than the average values recorded for Alto Douro during the period 1950–2000. The average temperature for the growing season is already at the upper

) [25] averaged 1946.8 mm a year, mostly occur-

and rainfall

indicating more intense soil moisture stress during this critical growth stage [10].

management, and cooling techniques.

ally brought about by climate change.

Calculated reference evapotranspiration (ET0

**2. Weather conditions in Alto Douro**

Such scenarios are not favorable for agriculture for an economic activity dependent upon and deeply connected to climate and weather. The cultivation of grapevines (*Vitis vinifera*), the fruit in Europe that is primarily used for winemaking and is a climate-sensitive agricultural system that has been used as an indicator of both historic and contemporary climate change [4]. Despite adaptation of *Vitis vinifera* to a wide range of climatic conditions, individual varieties, in particular premium wine grapes, have narrow climate ranges making the system very susceptible to minor changes in climate [5, 6]. Climate, especially air temperature, together with soil properties are considered the most important factors in defining the suitability of an area for wine production, affecting physiological behavior of the grapevine, the phenological phases of vine, the formation and maturity of the berry and its chemical changes, and finally, the characteristics of wines produced in a particular area [7, 8].

Portugal is expected to face aggravated weather conditions in coming years because the territory has suffered a pronounced decrease in precipitation in the spring, in particular in March, since the early 1950s [9] while a warming trend has been observed revealing a significant increase in extreme heat events for both spring and summer seasons, and a decrease in extreme cold events in winter [10]. All Portuguese wine regions are expected to experience an upper shift in temperature and lower precipitation. The Douro Demarcated Region (DDR), located in the northeastern Portugal, is classified as a Denomination of Controlled Origin (DOC), the highest Portuguese wine classification, given its distinctive climatic, topographic, and soil characteristics. It is of particular concern given its economic preeminence in Portuguese wine industry. Its strong Mediterranean climate influence shows the typical variability in precipitation along with high evapotranspiration during summer, the factors that limit grapevine development, yield, and quality components [11]. In these regions, meteorological averages from the period 1950–2000 show that during active vine development from April to September, rainfall varies from 190 to 326 mm but as low as 50 to 85 mm during the ripening stage (from July to September) coupled with high temperatures and intense solar radiation that give rise to very stressful conditions, particularly in the eastern end of the region (Alto Douro) [12] where grapevines can be subjected to a difference between evapotranspiration and precipitation as high as 730–750 mm from bud break to harvest [13]. Under these conditions, vineyards of DDR are, in general, grown in marginal conditions for agriculture production [14] that contribute to large fluctuations in interannual production and low yields averaging 30 hl ha−1 of wine well below the legal limit of 55 hl ha−1 permitted for DOC wine [12]. The expected higher daily temperatures and decreased rainfall [15] are bound to affect yield and wine quality.

Similar trends were observed in other wine regions worldwide where average temperatures have increased by 1.7°C during the growing seasons since 1950 and are expected to rise by an average of 2°C by 2050 leaving some regions at or near their optimum growing-season temperatures [16]. There are forecasts of further warming in Greek wine regions, pushing some of them past suitability for viticulture [17]. Three wine regions of the Northeastern Spain (Alt Penedès, Priorat, and Segrià) have experienced, from 1952 to 2006, overall growing-season warming of 1.0 to 2.2°C, while precipitation during the bud break to veraison period declined significantly, indicating more intense soil moisture stress during this critical growth stage [10].

Facing the most likely scenarios of climate change, wine farmers must adapt strategies to continue producing quality wines and to preserve their typicity from each growing location [18]. As a perennial crop, grapevines require a few years to reach maturity and can remain economically productive for several decades; thus, adaptation strategies must account for both short- and long-term impacts of a shifting environment. Perennial practices, such as choice of the planting site, choice of grapevine and rootstock variety, and vineyard layout, must be decided prior to planting. For those vineyards already under production, winegrowers will have to adapt using suitable short-term mitigation strategies [19], such as irrigation, canopy management, and cooling techniques.

## **2. Weather conditions in Alto Douro**

especially in summer, and more frequent and longer extreme climate events such as heat

Such scenarios are not favorable for agriculture for an economic activity dependent upon and deeply connected to climate and weather. The cultivation of grapevines (*Vitis vinifera*), the fruit in Europe that is primarily used for winemaking and is a climate-sensitive agricultural system that has been used as an indicator of both historic and contemporary climate change [4]. Despite adaptation of *Vitis vinifera* to a wide range of climatic conditions, individual varieties, in particular premium wine grapes, have narrow climate ranges making the system very susceptible to minor changes in climate [5, 6]. Climate, especially air temperature, together with soil properties are considered the most important factors in defining the suitability of an area for wine production, affecting physiological behavior of the grapevine, the phenological phases of vine, the formation and maturity of the berry and its chemical changes, and finally,

Portugal is expected to face aggravated weather conditions in coming years because the territory has suffered a pronounced decrease in precipitation in the spring, in particular in March, since the early 1950s [9] while a warming trend has been observed revealing a significant increase in extreme heat events for both spring and summer seasons, and a decrease in extreme cold events in winter [10]. All Portuguese wine regions are expected to experience an upper shift in temperature and lower precipitation. The Douro Demarcated Region (DDR), located in the northeastern Portugal, is classified as a Denomination of Controlled Origin (DOC), the highest Portuguese wine classification, given its distinctive climatic, topographic, and soil characteristics. It is of particular concern given its economic preeminence in Portuguese wine industry. Its strong Mediterranean climate influence shows the typical variability in precipitation along with high evapotranspiration during summer, the factors that limit grapevine development, yield, and quality components [11]. In these regions, meteorological averages from the period 1950–2000 show that during active vine development from April to September, rainfall varies from 190 to 326 mm but as low as 50 to 85 mm during the ripening stage (from July to September) coupled with high temperatures and intense solar radiation that give rise to very stressful conditions, particularly in the eastern end of the region (Alto Douro) [12] where grapevines can be subjected to a difference between evapotranspiration and precipitation as high as 730–750 mm from bud break to harvest [13]. Under these conditions, vineyards of DDR are, in general, grown in marginal conditions for agriculture production [14] that contribute to large fluctuations in interannual production and low yields averaging 30 hl ha−1 of wine well below the legal limit of 55 hl ha−1 permitted for DOC wine [12]. The expected higher daily temperatures and decreased rainfall [15] are bound to

Similar trends were observed in other wine regions worldwide where average temperatures have increased by 1.7°C during the growing seasons since 1950 and are expected to rise by an average of 2°C by 2050 leaving some regions at or near their optimum growing-season temperatures [16]. There are forecasts of further warming in Greek wine regions, pushing some of them past suitability for viticulture [17]. Three wine regions of the Northeastern Spain (Alt Penedès, Priorat, and Segrià) have experienced, from 1952 to 2006, overall growing-season warming of

waves and prolonged drought [2, 3].

affect yield and wine quality.

the characteristics of wines produced in a particular area [7, 8].

60 Grapes and Wines - Advances in Production, Processing, Analysis and Valorization

Alto Douro has a total area of 120,000 ha with an average elevation of 420 m, but the best prized vintages are produced at lower elevations where records for the period 1950–2000 show an annual precipitation ranging from 600 to 800 mm, but only 200 to 300 mm occur during the growing season from April to October, while annual temperatures ranged between 14 and 16°C and growing season oscillated from 19 to over 21°C [20]. Located in Alto Douro, close to Douro river (41° 08′ North, 7° 08′ West), some mitigation techniques have been experimented to protect *Vitis vinifera* cv Touriga Nacional and cv Tinta Roriz (Syn. Aragonêz, Tempranillo) from intense abiotic stresses that might prove useful under more extreme conditions eventually brought about by climate change.

Touriga Nacional is one of the most valuable premium varieties of DDR. It is considered one of the best red varieties to produce quality wines with a unique aroma profile that can fetch high market prices [21]. It has good adaptation to environmental stresses, withstanding high light intensities that allow for better adjustment to warm conditions, provided an adequate water supply [22]. Tinta Roriz can be cultivated in a variety of soils and climatic conditions, because it resists well to moderate drought. It has a thick-skinned berry with a high anthocyanin concentration that makes for deep-colored wines with moderate tannins and moderate acidity. It provides some of the best red wines of Portugal and Spain [23, 24]. Despite Touriga Nacional and Tinta Roriz good adaptability, they can suffer serious yield losses under unfavorable weather conditions. An on-site automatic weather station provided records for the period 2004 to 2016 (**Table 1**). Average annual temperature was 16.1 and 21°C for the growing season, whereas total precipitation was 594 mm annually with 284 mm for the growing season. Calculated reference evapotranspiration (ET0 ) [25] averaged 1946.8 mm a year, mostly occurring during the growing season (1631.3 mm) creating an imbalance between ET0 and rainfall of 1349.3 and 1321.4 mm annually and for the growing season, respectively. Temperature was higher and rainfall was lower than the average values recorded for Alto Douro during the period 1950–2000. The average temperature for the growing season is already at the upper limit that is considered adequate for quality wine grape production [26].


The time frequency generated data in **Figure 1** shows that the minimum SW content is lower than the permanent wilting point (0.19), bringing the SW content of the important period from flowering to veraison to a critical level when irrigation will be most useful. As climate becomes warmer, grapevines in Douro show a clear tendency toward earlier phenological events [18, 29] a phenomenon also observed in Touriga Nacional and Tinta Roriz for the period 2004–2016, and in 2017, flowering occurred about 15–20 days earlier than usual across Alto Douro according to empirical observations. Earlier events could overlap with periods of higher SW content, but ripening might also occur in time of higher temperatures that are detrimental to quality [30, 31]. As rainfall is expected to diminish, SW will become scarcer and a short growing season might not bring any benefit. Under present conditions, rainfed vineyards in Alto Douro produce as little as 0.7 kg per plant and even less in drier years [32], not enough to cover production costs. Irrigation is then essential to ensure a stable production and minimize the risk of drought damage [33]. Experimental fields were set up in DDR to study the effects of irrigation in grapevines [32–35]. The main experimental field was a 2.5-hectare commercial vineyard (*Vitis vinifera* cv. Tinta Roriz) where six treatments comprising three adjacent grapevine rows each, 120 plants per treatment, laid out in a random design pattern and trained to a vertical shoot position (VSP). Each grapevine row had its own drip irrigation line and they were nonirrigated, irrigated from flowering to veraison at rates of 4 (total of 160 mm) and 8 mm a day (total of 320 mm), irrigated from veraison to commercial maturation at rates of 4 (total of 225 mm) and 8 mm a day (total of 450 mm), and irrigated from flowering to commercial maturation at a rate of 8 mm a day (total of 720 mm). Water use efficiency (WUE) was calculated as the ratio between a given parameter (*X*) and crop evapo-

Viticulture in Warmer Climates: Mitigating Environmental Stress in Douro Region, Portugal

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63

*WUE* = *X*/*Ec* (1)

*Ec* was determined by soil water balance [37, 38] using SW data measured with TDR probes.

(*hi* − *hi*−1) = (*P* + *I*) − (*Ec* + *R* + *D*) (2)

where *h* is the soil water storage at measurement time *i* and at preceding measurement time *i-*1, *P* is precipitation, *I* is irrigation, *Ec* is the water used by plant transpiration plus evaporation from soil, *R* is the surface run-off, and *D* is the soil drainage below the root zone (all units in mm). *R* and *D* can be safely ignored because low SW content and sparse precipitation dur-

At commercial harvest, three samples were collected from every treatment for laboratory analysis; each sample was a composite of berries collected from 10 randomly chosen grape-

Irrigation caused a significant increase in yield (**Table 2**) due to larger berries, rather than to a greater number of clusters or berries per cluster, and simultaneously, the compounds related to must quality (color, aroma, and soluble solids) were either diluted or their amount significantly reduced compared with musts from rainfed plants. On the other hand, there were no

ing growing season, conditions similar to those found by other authors [39, 40].

significant differences in pH, free anthocyanins, and total flavonols.

transpiration (*Ec*) [36].

vines (18 analyses repeated over 3 years).

**Table 1.** Total solar radiation (rad), air temperature (T), precipitation (R), and reference evapotranspiration (ET0 ) averages for the period 2004–2016 at location in Alto Douro 41° 08′ North, 7° 08′ West (on-site meteorological station).

#### **2.1. Mitigation techniques in Alto Douro**

#### *2.1.1. Irrigation*

Historically, vineyards of DDR in general and Alto Douro in particular are water stressed during the growing season, given the high atmospheric demand and the scarcity of rainfall that limits the soil water (SW) availability. SW has a complex relationship with air temperature that, when it rises, increases vapor pressure deficit, and more water is evapotranspirated from the soil; eventually, water availability becomes critical. In [27], SW will be the limiting resource in vineyards of Douro and it will become relatively more important than temperature for maintaining grapevine production. A model of SW annual distribution in Douro valley shows a sinusoidal pattern (**Figure 1**) that is consistent with temperature and rainfall distributions [28].

**Figure 1.** Time frequency (Fourier series) generated data of soil water content (m<sup>3</sup> m−3) based on 364 historical observations from 1991 to 1997 and annual occurrence of main phenological stages of the grapevines: bud breaking, flowering, veraison, and harvest. (Adapted from Oliveira [28]).

The time frequency generated data in **Figure 1** shows that the minimum SW content is lower than the permanent wilting point (0.19), bringing the SW content of the important period from flowering to veraison to a critical level when irrigation will be most useful. As climate becomes warmer, grapevines in Douro show a clear tendency toward earlier phenological events [18, 29] a phenomenon also observed in Touriga Nacional and Tinta Roriz for the period 2004–2016, and in 2017, flowering occurred about 15–20 days earlier than usual across Alto Douro according to empirical observations. Earlier events could overlap with periods of higher SW content, but ripening might also occur in time of higher temperatures that are detrimental to quality [30, 31]. As rainfall is expected to diminish, SW will become scarcer and a short growing season might not bring any benefit. Under present conditions, rainfed vineyards in Alto Douro produce as little as 0.7 kg per plant and even less in drier years [32], not enough to cover production costs. Irrigation is then essential to ensure a stable production and minimize the risk of drought damage [33]. Experimental fields were set up in DDR to study the effects of irrigation in grapevines [32–35]. The main experimental field was a 2.5-hectare commercial vineyard (*Vitis vinifera* cv. Tinta Roriz) where six treatments comprising three adjacent grapevine rows each, 120 plants per treatment, laid out in a random design pattern and trained to a vertical shoot position (VSP). Each grapevine row had its own drip irrigation line and they were nonirrigated, irrigated from flowering to veraison at rates of 4 (total of 160 mm) and 8 mm a day (total of 320 mm), irrigated from veraison to commercial maturation at rates of 4 (total of 225 mm) and 8 mm a day (total of 450 mm), and irrigated from flowering to commercial maturation at a rate of 8 mm a day (total of 720 mm). Water use efficiency (WUE) was calculated as the ratio between a given parameter (*X*) and crop evapotranspiration (*Ec*) [36].

**2.1. Mitigation techniques in Alto Douro**

Budbreaking

flowering, veraison, and harvest. (Adapted from Oliveira [28]).

Historically, vineyards of DDR in general and Alto Douro in particular are water stressed during the growing season, given the high atmospheric demand and the scarcity of rainfall that limits the soil water (SW) availability. SW has a complex relationship with air temperature that, when it rises, increases vapor pressure deficit, and more water is evapotranspirated from the soil; eventually, water availability becomes critical. In [27], SW will be the limiting resource in vineyards of Douro and it will become relatively more important than temperature for maintaining grapevine production. A model of SW annual distribution in Douro valley shows a sinusoidal pattern (**Figure 1**) that is consistent with temperature and rainfall

**Rad (Kw m−2) T (°C) R (mm) ET0**

Annual 1757.8 16.1 594.0 1946.2 Nov–Mar 346.8 9.2 309.9 315.6 Apr–Oct 1411.0 21.0 284.2 1631.3

62 Grapes and Wines - Advances in Production, Processing, Analysis and Valorization

**Table 1.** Total solar radiation (rad), air temperature (T), precipitation (R), and reference evapotranspiration (ET0

for the period 2004–2016 at location in Alto Douro 41° 08′ North, 7° 08′ West (on-site meteorological station).

 **(mm)**

) averages

13579 11 13 15 17 19 21 23 25 27 29 31 33 35 37 39 41 43 45 47 49 51 53 **Weeks** 

**Figure 1.** Time frequency (Fourier series) generated data of soil water content (m<sup>3</sup> m−3) based on 364 historical observations from 1991 to 1997 and annual occurrence of main phenological stages of the grapevines: bud breaking,

Veraison Harvest

Flowering

*2.1.1. Irrigation*

distributions [28].

0.10

0.15

0.20

0.25

**Soil water content (m3m-3)**

0.30

0.35

$$\text{WUE} = X/\text{Ec} \tag{1}$$

*Ec* was determined by soil water balance [37, 38] using SW data measured with TDR probes.

$$\left(h\_{l} - h\_{l+1}\right) = \left(P + D\right) - \left(Ec + R + D\right) \tag{2}$$

where *h* is the soil water storage at measurement time *i* and at preceding measurement time *i-*1, *P* is precipitation, *I* is irrigation, *Ec* is the water used by plant transpiration plus evaporation from soil, *R* is the surface run-off, and *D* is the soil drainage below the root zone (all units in mm). *R* and *D* can be safely ignored because low SW content and sparse precipitation during growing season, conditions similar to those found by other authors [39, 40].

At commercial harvest, three samples were collected from every treatment for laboratory analysis; each sample was a composite of berries collected from 10 randomly chosen grapevines (18 analyses repeated over 3 years).

Irrigation caused a significant increase in yield (**Table 2**) due to larger berries, rather than to a greater number of clusters or berries per cluster, and simultaneously, the compounds related to must quality (color, aroma, and soluble solids) were either diluted or their amount significantly reduced compared with musts from rainfed plants. On the other hand, there were no significant differences in pH, free anthocyanins, and total flavonols.


polyphenolic content of must and wine, and it relates to color stability of red wines as it ages; irrigated plants produced musts with lower phenolic potential that contribute for producing red wines with lower aging potential irrespective of the irrigation schedule. Accumulation of anthocyanins (glucosides and aglycones) in grape skin is responsible for the dark color of red grapes, and their concentration is related to the berry size that increases with irrigation, resulting in musts with lower concentration of anthocyanins as the amount of available water is higher, especially if irrigation occurs close to harvest date. CI is directly related to TAn, and

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65

It is then clear that irrigation rate and its schedule had a significant influence on yield and berry quality. Irrigated grapevines had better yields but, in general, the must characteristics revealed lower quality. The must quality of grapevines receiving water before veraison was better than from those irrigated after veraison. Excessive water stress before veraison causes poor fruit setting and reduced metabolic functions; berry growth is stunted and essential metabolites, such as organic acids, do not accumulate. Irrigation starting after veraison cannot make up for the losses already sustained. If water stress before veraison is moderate, berries still can attain size and composition adequate for winemaking but increasing the SW content after veraison might dilute soluble solids, acids, anthocyanins, and phenols, turning the must

For grape growers, profits are made up of both yield and quality and very low yield, no matter how the quality is, which is not profitable. The best balance between quality and yield and still attaining acceptable water use efficiency was reached with irrigation from flowering to veraison amounting to about 50% of the reference evapotranspiration of that period. When irrigation is halted at veraison, SW content declines slowly until harvest and probably there is enough water to sustain the vine metabolic functions and yield at higher levels than rainfed grapevines [32, 35]. In addition, it also permits a larger foliar area providing additional shade

Tinta Roriz had lower water use efficiency as more water is added (**Tables 2** and **3**), an important consideration in scenarios of scarcer water resources and competing uses. In dry weather conditions and sparse soil cover as vineyards, direct evaporation from soil becomes an increasingly larger proportion of evapotranspiration, and when more water is available, it results in higher crop water use that is not directly proportional to plant yield and to its metabolic functions; thus, WUE lowers in irrigated plants. Again, a compromise between yield, quality, and WUE can be achieved with deficit irrigation before veraison. Deficit irrigation at critical moments contribute to moderate water stress during the entire growing season to

High solar radiation intensity and elevated temperatures damage the grape berries. The fruit shrivels and can desiccate completely (raisining), making it inappropriate for winemaking. This is a phenomenon very common in Douro, also in other wine regions [42], given the actual climate conditions (**Table 1**), and it is bound to increase in the future. A north-south row orientation allows the fruits, on both sides of the canopy, reach a balance in photosynthetic

produce quality musts, acceptable yields, and still keep a judicious use of water.

its values in must of irrigated plants follow the pattern of TAn.

inadequate to high-quality wine.

to partially offset rising temperatures.

*2.1.2. Canopy management*

Different superscript letters on same column indicate a significant difference at α ≤ 0.05 (Tukey's HSD test). *Vitis vinifera* cv Tinta Roriz, Alto Douro (adapted from Refs. [32, 35]).

\* WUE × 10.

\*\*WUE × 100.

**Table 2.** Effect of irrigation on crop evapotranspiration (Ec), yield (Y), total soluble solids (TSS), titrable acidity (TA), tartaric acid (TAc), malic acid (Mac), and respective water use efficiency of each parameter (in parenthesis).

Titrable acidity increased with irrigation, but it is still too low for high-end wines with a preferred acidity of 6–7 mg L−1 that is equivalent of tartaric acid [41], which is a manifestation of the influence of high temperatures on degrading the main organic acids of the must, namely, malic and tartaric acids (**Table 2**).

Total soluble solids (TSS), polyphenol index (PI), total anthocyanins (TAn), and color index (CI) significantly decreased with irrigation (**Table 3**). TSS is very important for grape growers and winemakers because it determines how much sugar is available for conversion into alcohol. The relationship between irrigation and TSS is complex; more available water induces higher photosynthetic rate and accumulation of soluble solids but also augments yields and the berry size that can dilute the soluble solids, and in the end, their lower concentration produces a total volume of wine poorer in alcohol. PI is a phenolic parameter that measures the


Different superscript letters on same column indicate a significant difference at α ≤ 0.05 (Tukey's HSD test). *Vitis vinifera* cv Tinta Roriz, Alto Douro (adapted from Oliveira et al. [35]).

\* WUE × 10.

\*\*WUE × 100.

**Table 3.** Effect of irrigation treatments on total soluble solids (TSS), polyphenol index (PI), total anthocyanins (TAn), color intensity (CI), and respective water use efficiency of each parameter (in parenthesis).

polyphenolic content of must and wine, and it relates to color stability of red wines as it ages; irrigated plants produced musts with lower phenolic potential that contribute for producing red wines with lower aging potential irrespective of the irrigation schedule. Accumulation of anthocyanins (glucosides and aglycones) in grape skin is responsible for the dark color of red grapes, and their concentration is related to the berry size that increases with irrigation, resulting in musts with lower concentration of anthocyanins as the amount of available water is higher, especially if irrigation occurs close to harvest date. CI is directly related to TAn, and its values in must of irrigated plants follow the pattern of TAn.

It is then clear that irrigation rate and its schedule had a significant influence on yield and berry quality. Irrigated grapevines had better yields but, in general, the must characteristics revealed lower quality. The must quality of grapevines receiving water before veraison was better than from those irrigated after veraison. Excessive water stress before veraison causes poor fruit setting and reduced metabolic functions; berry growth is stunted and essential metabolites, such as organic acids, do not accumulate. Irrigation starting after veraison cannot make up for the losses already sustained. If water stress before veraison is moderate, berries still can attain size and composition adequate for winemaking but increasing the SW content after veraison might dilute soluble solids, acids, anthocyanins, and phenols, turning the must inadequate to high-quality wine.

For grape growers, profits are made up of both yield and quality and very low yield, no matter how the quality is, which is not profitable. The best balance between quality and yield and still attaining acceptable water use efficiency was reached with irrigation from flowering to veraison amounting to about 50% of the reference evapotranspiration of that period. When irrigation is halted at veraison, SW content declines slowly until harvest and probably there is enough water to sustain the vine metabolic functions and yield at higher levels than rainfed grapevines [32, 35]. In addition, it also permits a larger foliar area providing additional shade to partially offset rising temperatures.

Tinta Roriz had lower water use efficiency as more water is added (**Tables 2** and **3**), an important consideration in scenarios of scarcer water resources and competing uses. In dry weather conditions and sparse soil cover as vineyards, direct evaporation from soil becomes an increasingly larger proportion of evapotranspiration, and when more water is available, it results in higher crop water use that is not directly proportional to plant yield and to its metabolic functions; thus, WUE lowers in irrigated plants. Again, a compromise between yield, quality, and WUE can be achieved with deficit irrigation before veraison. Deficit irrigation at critical moments contribute to moderate water stress during the entire growing season to produce quality musts, acceptable yields, and still keep a judicious use of water.

#### *2.1.2. Canopy management*

Titrable acidity increased with irrigation, but it is still too low for high-end wines with a preferred acidity of 6–7 mg L−1 that is equivalent of tartaric acid [41], which is a manifestation of the influence of high temperatures on degrading the main organic acids of the must, namely,

**Table 2.** Effect of irrigation on crop evapotranspiration (Ec), yield (Y), total soluble solids (TSS), titrable acidity (TA),

Different superscript letters on same column indicate a significant difference at α ≤ 0.05 (Tukey's HSD test). *Vitis vinifera*

**Irrigation (mm) Ec (mm) Y (g plant−1) TSS (°Brix) TA (g L−1) TAc (g L−1) MAc (g L−1)**

)\* 2.3a

)\* 2.4b

\* 2.7c

)\* 2.4b

)\* 3.0d(0.4d)

(1.2a

(0.8b

(0.5c

(0.8b )

(0.5c

)\*\* 6.6a

)\*\* 6.5a

)\*\* 6.5a

\*\* 6.9b

)\*\* 7.0b

\*\* 6.8b

(2.3a

(2.1b

(1.2c )

(2.2a

(1.3 c

)\*\* 1.5a

)\*\* 2.2c

\*\* 2.7e

)\*\* 2.2c

(0.9 d)\*\* 2.1b

)\*\* 2.3d(0.7b

(0.8a )\*\*

(0.7b )\*\*

(0.5c ) \*\*

(0.4c )\*\*

(0.3d)\*\*

)\*\*

(1.5a

(0.9b

 (0.5c )

(0.9b

) 25d (0.4d)\* 2.6c

(0.3e

tartaric acid (TAc), malic acid (Mac), and respective water use efficiency of each parameter (in parenthesis).

Total soluble solids (TSS), polyphenol index (PI), total anthocyanins (TAn), and color index (CI) significantly decreased with irrigation (**Table 3**). TSS is very important for grape growers and winemakers because it determines how much sugar is available for conversion into alcohol. The relationship between irrigation and TSS is complex; more available water induces higher photosynthetic rate and accumulation of soluble solids but also augments yields and the berry size that can dilute the soluble solids, and in the end, their lower concentration produces a total volume of wine poorer in alcohol. PI is a phenolic parameter that measures the

)\* 13.8a

)\* 11.9b

\* 12.3b

)\* 10.6c

Different superscript letters on same column indicate a significant difference at α ≤ 0.05 (Tukey's HSD test). *Vitis vinifera*

**Table 3.** Effect of irrigation treatments on total soluble solids (TSS), polyphenol index (PI), total anthocyanins (TAn),

)\* 9.1d (1.2d)

(7.9a

(4.1b

 (2.4c )

(3.8b

(3.2d)\* 9.2d (1.7d)\*\* 0.15d (2.8d)\*\*

)\*\* 0.25a

)\*\* 0.21b

\*\* 0.18c

\*\* 0.13e

)\*\* 0.16d (5.3c

 (12.0a ) \*\*

 (6.9b ) \*\*

(3.5d)\*\*

 (1.5e ) \*\*

)\*\*

**Irrigation TSS (°Brix) PI TAn (mg berry−1) CI**

(13.4a

(7.2b

(3.9d)

(6.1c

)\* 233a

\* 203b

\* 196b

)\* 176c

)\* 174c

(0.3d)\* 138d (1.8e

color intensity (CI), and respective water use efficiency of each parameter (in parenthesis).

malic and tartaric acids (**Table 2**).

0 193 721a

160 302 1002b

320 522 1003b

225 308 865c

450 548 889c

\* WUE × 10. \*\*WUE × 100.

720 744 1224d (1.6e

cv Tinta Roriz, Alto Douro (adapted from Refs. [32, 35]).

(3.7a

(3.3b

64 Grapes and Wines - Advances in Production, Processing, Analysis and Valorization

(1.9c

(1.6e

) 30a

) 28b

) 26c

) 24e

(2.8d) 27c

(1.5a

 (0.9b )

 (0.5c )

(0.9b

cv Tinta Roriz, Alto Douro (adapted from Oliveira et al. [35]).

0 30a

160 28b

320 26c

225 27c

720 24e

\* WUE × 10. \*\*WUE × 100.

450 25d (0.4c

High solar radiation intensity and elevated temperatures damage the grape berries. The fruit shrivels and can desiccate completely (raisining), making it inappropriate for winemaking. This is a phenomenon very common in Douro, also in other wine regions [42], given the actual climate conditions (**Table 1**), and it is bound to increase in the future. A north-south row orientation allows the fruits, on both sides of the canopy, reach a balance in photosynthetic efficiency, and their exposure to radiation [43] and shade propitiated by a denser canopy can minimize the losses. However, slope might not permit a north-south orientation and additional shade provided by a denser canopy actually is not enough to prevent significant yield reductions. Shading the plants can reduce their exposure to radiation and heat but creates an imbalance in carbon budget, reducing vine biomass, and affects negatively the canopy photosynthesis [44]. To avoid these negative effects and still protect a rainfed commercial vineyard of *Vitis vinifera* cv Touriga Nacional in Alto Douro, planted in east-west oriented rows and VSP trained, had the lower third of their south-facing side (from ground to 20 cm above the insertion point of grape clusters) partially covered with a vertical double layer of white plastic netting attached to iron spikes driven into the ground at 3-m intervals [45]. At about 30 randomly laid out rows with 70 vines, each was divided into three groups with equal number of rows. One group was shaded after fruit setting, another after veraison, and the third was nonshaded (control). The net was removed just prior to harvest. The net reduced the total solar radiation by 23% and photosynthetic active radiation by 27%.

the must. Must pH was higher (3.9) in nonshaded vines against shaded (3.8). These results suggest that photosynthetic carbon acquisition was not significantly different for all groups of plants, and they are consistent with values reported for the same region and they are typical for hot climate vineyards [47, 48]. The shaded berries were approximately 1°C cooler than nonshaded ones, a difference probably due to less radiation reaching the clusters [49]. There is evidence that berry temperature affects its composition, but only differences in 3°C or above are reported as causing changes [50, 51]. On the other hand, the biosynthesis of anthocyanins is sensitive to light environment and it decreases with lower light intensity [52, 53], and in fact, shaded berries from Touriga Nacional had lower concentration of total anthocyanins and

Viticulture in Warmer Climates: Mitigating Environmental Stress in Douro Region, Portugal

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67

Lower concentrations of anthocyanins, responsible for the red color intensity of wines, can be detrimental to the must quality [54, 55] with loss in market value but that might be offset by

According to the net maker, the net can last to 10–12 years. The grower did support the cost of the net, 3000 euros per hectare, plus an expense outlay of 150 euros per hectare for handling of the net. These added expenses look reasonable for vineyards already planted in an inconvenient layout that can sustain heavy losses under spells of extreme weather conditions.

The temperature of plant leaves are closely related to air temperature, and the rising temperatures reduce stomatal conductance [55], decrease photosynthesis rate significantly due to the increase in respiration [56], restrict carbon assimilation per unit leaf area, affecting negatively the vegetative growth and yield [57], and impair several physiological processes, notably sugar accumulation [58]. Berries after veraison are very sensitive to a combination of intense solar radiation and high temperature, reducing in size and in soluble solid concentration [59]. High temperatures and light intensity cause berries to ripe more slowly and contribute to berry sunburn and shriveling as cells of the mesocarp die [60]. The negative effects of high-temperature events on vine physiology are more severe in vines experiencing water

Two mitigation techniques are actually being experimented in Alto Douro region with rainfed Touriga Nacional to reduce the effects of adverse temperature and radiation. Nontreated

Different superscript letters on same column indicate a significant difference at α ≤ 0.05 (Tukey's HSD test). *Vitis vinifera*

**Table 5.** Effect of shading on total anthocyanins (TAn) and extractable anthocyanins (EAn) for each shading treatment.

**Shading TAn (mg L−1) EAn (mg L−1)**

Nonshaded 1534a 825a After fruit setting 990b 679b After veraison 1240b 726b

cv Touriga Nacional, Alto Douro (adapted from Oliveira et al. [45]).

extractable anthocyanins than nonshaded berries (**Table 5**).

*2.1.3. Cooling techniques*

stress [61].

an additional yield of 2300 kg ha−1 in favor of shaded grapevines.

From the end of flowering to commercial harvest, every week on clear sky days at noon, two well lit, adult leaves of each five randomly chosen plants on each group were used to measure stomatal conductance (porometry) and leaf water potential (pressure chamber) (300 readings repeated over 3 years). On the same days and time but only after veraison, the temperature (infrared thermometry) of one cluster on 10 randomly chosen plants per treatment was measured (180 readings repeated over 3 years). At harvest, the same procedure described for irrigation section was used to sample berries for laboratory analysis (nine analyses repeated over 3 years).

Shading did not influence significatively either water status (leaf water potential) of the plants or their stomatal conductance; thus, it is reasonable to assume that they did not affect the net photosynthesis per unit of leaf area. Shaded vines consistently had significant higher yields (2100 g plant−1) than nonshaded vines (1500 g plant−1) because the percentage of shriveled berries per cluster was about 6.5% against 14%, respectively (**Table 4**). The number of clusters, weight, and size of the berries showed no significant differences. Shading does not totally prevent berry shriveling because the phenomenon is also caused by other factors like water stress [46].

Shading did not alter significatively the concentrations of total soluble solids (24.3 °Brix), tartaric acid (4.3 g L−1), malic acid (1.7 g L−1), glucose (118.2 g L−1), and fructose (110 g L−1) in


Different superscript letters on same column indicate a significant difference at α ≤ 0.05 (Tukey's HSD test). *Vitis vinifera* cv Touriga Nacional, Alto Douro (adapted from Oliveira et al. [45]).

\* In parenthesis calculated yield kg ha−1.

**Table 4.** Effect of shading on yield, percentage of shriveled berries per cluster, weight, and volume of 200 berries for each shading treatment.

the must. Must pH was higher (3.9) in nonshaded vines against shaded (3.8). These results suggest that photosynthetic carbon acquisition was not significantly different for all groups of plants, and they are consistent with values reported for the same region and they are typical for hot climate vineyards [47, 48]. The shaded berries were approximately 1°C cooler than nonshaded ones, a difference probably due to less radiation reaching the clusters [49]. There is evidence that berry temperature affects its composition, but only differences in 3°C or above are reported as causing changes [50, 51]. On the other hand, the biosynthesis of anthocyanins is sensitive to light environment and it decreases with lower light intensity [52, 53], and in fact, shaded berries from Touriga Nacional had lower concentration of total anthocyanins and extractable anthocyanins than nonshaded berries (**Table 5**).

Lower concentrations of anthocyanins, responsible for the red color intensity of wines, can be detrimental to the must quality [54, 55] with loss in market value but that might be offset by an additional yield of 2300 kg ha−1 in favor of shaded grapevines.

According to the net maker, the net can last to 10–12 years. The grower did support the cost of the net, 3000 euros per hectare, plus an expense outlay of 150 euros per hectare for handling of the net. These added expenses look reasonable for vineyards already planted in an inconvenient layout that can sustain heavy losses under spells of extreme weather conditions.

#### *2.1.3. Cooling techniques*

efficiency, and their exposure to radiation [43] and shade propitiated by a denser canopy can minimize the losses. However, slope might not permit a north-south orientation and additional shade provided by a denser canopy actually is not enough to prevent significant yield reductions. Shading the plants can reduce their exposure to radiation and heat but creates an imbalance in carbon budget, reducing vine biomass, and affects negatively the canopy photosynthesis [44]. To avoid these negative effects and still protect a rainfed commercial vineyard of *Vitis vinifera* cv Touriga Nacional in Alto Douro, planted in east-west oriented rows and VSP trained, had the lower third of their south-facing side (from ground to 20 cm above the insertion point of grape clusters) partially covered with a vertical double layer of white plastic netting attached to iron spikes driven into the ground at 3-m intervals [45]. At about 30 randomly laid out rows with 70 vines, each was divided into three groups with equal number of rows. One group was shaded after fruit setting, another after veraison, and the third was nonshaded (control). The net was removed just prior to harvest. The net reduced the total

From the end of flowering to commercial harvest, every week on clear sky days at noon, two well lit, adult leaves of each five randomly chosen plants on each group were used to measure stomatal conductance (porometry) and leaf water potential (pressure chamber) (300 readings repeated over 3 years). On the same days and time but only after veraison, the temperature (infrared thermometry) of one cluster on 10 randomly chosen plants per treatment was measured (180 readings repeated over 3 years). At harvest, the same procedure described for irrigation section

Shading did not influence significatively either water status (leaf water potential) of the plants or their stomatal conductance; thus, it is reasonable to assume that they did not affect the net photosynthesis per unit of leaf area. Shaded vines consistently had significant higher yields (2100 g plant−1) than nonshaded vines (1500 g plant−1) because the percentage of shriveled berries per cluster was about 6.5% against 14%, respectively (**Table 4**). The number of clusters, weight, and size of the berries showed no significant differences. Shading does not totally prevent berry

Shading did not alter significatively the concentrations of total soluble solids (24.3 °Brix), tartaric acid (4.3 g L−1), malic acid (1.7 g L−1), glucose (118.2 g L−1), and fructose (110 g L−1) in

(6000)\* 14a 270a 136a

(8690)\* 6b 257a 131a

(8300)\* 7b 256a 131a

Different superscript letters on same column indicate a significant difference at α ≤ 0.05 (Tukey's HSD test). *Vitis vinifera*

**Table 4.** Effect of shading on yield, percentage of shriveled berries per cluster, weight, and volume of 200 berries for

**Weight 200 berries (g) Volume 200 berries (cm3 )**

was used to sample berries for laboratory analysis (nine analyses repeated over 3 years).

shriveling because the phenomenon is also caused by other factors like water stress [46].

**per cluster**

solar radiation by 23% and photosynthetic active radiation by 27%.

66 Grapes and Wines - Advances in Production, Processing, Analysis and Valorization

**Shading Yield (g plant−1) % Shriveled berries** 

cv Touriga Nacional, Alto Douro (adapted from Oliveira et al. [45]).

Nonshaded 1504a

After fruit setting 2172b

After veraison 2076b

each shading treatment.

In parenthesis calculated yield kg ha−1.

\*

The temperature of plant leaves are closely related to air temperature, and the rising temperatures reduce stomatal conductance [55], decrease photosynthesis rate significantly due to the increase in respiration [56], restrict carbon assimilation per unit leaf area, affecting negatively the vegetative growth and yield [57], and impair several physiological processes, notably sugar accumulation [58]. Berries after veraison are very sensitive to a combination of intense solar radiation and high temperature, reducing in size and in soluble solid concentration [59].

High temperatures and light intensity cause berries to ripe more slowly and contribute to berry sunburn and shriveling as cells of the mesocarp die [60]. The negative effects of high-temperature events on vine physiology are more severe in vines experiencing water stress [61].

Two mitigation techniques are actually being experimented in Alto Douro region with rainfed Touriga Nacional to reduce the effects of adverse temperature and radiation. Nontreated


Different superscript letters on same column indicate a significant difference at α ≤ 0.05 (Tukey's HSD test). *Vitis vinifera* cv Touriga Nacional, Alto Douro (adapted from Oliveira et al. [45]).

**Table 5.** Effect of shading on total anthocyanins (TAn) and extractable anthocyanins (EAn) for each shading treatment.

grapevines (control) are compared with plants treated from the end of flowering for 2 weeks before the forecast harvest date: (1): coated with a suspension of kaolin [Al<sup>4</sup> Si4 O10(OH)8 ] at a rate of 12.5 kg ha−1 for every 20–25 days and (2): nebulized with water from misters located above the canopy for 30 seconds every 30 minutes as long as air temperature is higher than 32°C (The aperture of electro valves is automatically triggered by temperature records of the weather station.)

The sampling procedures described on above sections (irrigation, canopy management) were followed from the end of flowering to commercial harvest, to measure canopy temperature (average temperature of both sides of the canopy), cluster temperature, stomatal conductance, and berry sampling for laboratory analysis.

Infrared thermometry shows that nebulization keeps the canopies cooler at least 1°C than coated and control canopies (**Table 6**). Despite the reflective characteristics of kaolin, temperature of coated canopy is not significantly different from canopy temperature of control plants. Reduced stomatal opening, conducive to lower stomatal conductance, higher resistance of leaf boundary layer, and low photosynthetic photon flux density during part of the day are pointed out as explanations for these results [62, 63]. Stomatal conductance (gs , cm s−1) of nebulized plants (3.11) is significantly higher than of nontreated plants (2.14) (**Table 6**). Stomatal conductance is negatively correlated with leaf temperature [55], and as temperature raises, the plant reduces the opening of its stomata to conserve water and this mechanism reduces transpiration, a mechanism that rises the leaf temperature.

temperatures and, consequently, higher stomatal conductance (**Table 6**) that might permit an increased photosynthetic rate as observed by other authors [55, 56]. In turn, increased photosynthetic activity contributes to larger berry sugar content, evaluated as total soluble solids,

Different superscript letters on same column indicate a significant difference at α ≤ 0.05 (Tukey's HSD test). *Vitis vinifera*

**Table 7.** Effect of nebulization and kaolin coating on yield, percentage of sunburnt berries, and total soluble solids (TSS).

Control 868a 3470 12a 24.7a Nebulization 983b 3930 7b 26.1b Kaolin coating 1012b 4050 5b 24.6a

**Yield Sunburnt berries TSS g plant−1 Kg ha−1 % °Brix**

Viticulture in Warmer Climates: Mitigating Environmental Stress in Douro Region, Portugal

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69

However, other characteristics of the must (pH, titrable acidity, tartaric acid, malic acid, total tannins, total polyphenols, and total anthocianins) do not show significant differences among

The cost of installation (2500 euros ha−1) and running the water nebulization system (100 euros ha−1) might be impeditive of its adoption. Infection by fungus and insect repellency are factors associated to water spray and kaolin coating, respectively, that must be considered

Meteorological records and grapevine phenological occurrences in DDR are in agreement with forecasts of higher temperatures, lower precipitations, and advances of phenological stages. The environment of Douro is expected to become more stressful in coming years, and adaptations are imperative to save the viticulture as an economic activity. For the vineyards already in commercial production, efficient irrigation associated with shading and cooling techniques can reduce significatively water and temperature stress on grapevines and they can maintain acceptable yields with marketable quality wine. However, these techniques have some drawbacks in must quality and might not suffice to a large shift in temperature and precipitation given the actual conditions already at the limit of tolerance of the best grape

Financial support was provided by Quinta do Vale Meão (Olazabal & Sons, LDA), COTESI: Companhia de Texteis, S.A., and UTAD. My special thanks go to Francisco Olazabal, Pedro

as seen in **Table 7** and corroborated in other works [58, 59].

but they have not been assessed at the time of this report.

treated and control plants (data not shown).

**3. Conclusions**

cv Touriga Nacional, Alto Douro.

varieties.

**Acknowledgements**

Barbosa, André Guedes, and Frederico Lobão.

Evaporative cooling and kaolin coating had the same effect on cluster temperature, measured after veraison (infrared thermometry). Clusters of treated plants show a reduced temperature in relation to control plants, which are at least 1.3°C warmer (**Table 6**). Here, the scattering of radiation caused by kaolin is not counter acted by reduced transpiration and cluster temperature was maintained lower than air temperature.

Excessive temperature and radiation that cause fruit sunburn, a major factor in loss of production in grapevines of Alto Douro region, were effectively avoided by nebulization and kaolin coating (**Table 7**). Treated plants have 5–7% of shriveled berries per cluster compared with 12–13% in control vines. Reduced berry loss contributed to higher yield per plant.

Total soluble solids are 26.1°Brix for nebulized vines, and it is significantly higher than kaolin coated and control, 24.6 and 24.7°Brix, respectively (**Table 7**). Nebulized canopies show lower


Different superscript letters on same column indicate a significant difference at α ≤ 0.05 (Tukey's HSD test). *Vitis vinifera* cv Touriga Nacional, Alto Douro.

**Table 6.** Effect of nebulization and kaolin coating on average canopy temperature and stomatal conductance (g<sup>s</sup> ) from flowering to harvest and for cluster temperature from veraison to harvest.


Different superscript letters on same column indicate a significant difference at α ≤ 0.05 (Tukey's HSD test). *Vitis vinifera* cv Touriga Nacional, Alto Douro.

**Table 7.** Effect of nebulization and kaolin coating on yield, percentage of sunburnt berries, and total soluble solids (TSS).

temperatures and, consequently, higher stomatal conductance (**Table 6**) that might permit an increased photosynthetic rate as observed by other authors [55, 56]. In turn, increased photosynthetic activity contributes to larger berry sugar content, evaluated as total soluble solids, as seen in **Table 7** and corroborated in other works [58, 59].

However, other characteristics of the must (pH, titrable acidity, tartaric acid, malic acid, total tannins, total polyphenols, and total anthocianins) do not show significant differences among treated and control plants (data not shown).

The cost of installation (2500 euros ha−1) and running the water nebulization system (100 euros ha−1) might be impeditive of its adoption. Infection by fungus and insect repellency are factors associated to water spray and kaolin coating, respectively, that must be considered but they have not been assessed at the time of this report.

## **3. Conclusions**

grapevines (control) are compared with plants treated from the end of flowering for 2 weeks

of 12.5 kg ha−1 for every 20–25 days and (2): nebulized with water from misters located above the canopy for 30 seconds every 30 minutes as long as air temperature is higher than 32°C (The aperture of electro valves is automatically triggered by temperature records of the weather station.) The sampling procedures described on above sections (irrigation, canopy management) were followed from the end of flowering to commercial harvest, to measure canopy temperature (average temperature of both sides of the canopy), cluster temperature, stomatal conductance,

Infrared thermometry shows that nebulization keeps the canopies cooler at least 1°C than coated and control canopies (**Table 6**). Despite the reflective characteristics of kaolin, temperature of coated canopy is not significantly different from canopy temperature of control plants. Reduced stomatal opening, conducive to lower stomatal conductance, higher resistance of leaf boundary layer, and low photosynthetic photon flux density during part of the day are

ulized plants (3.11) is significantly higher than of nontreated plants (2.14) (**Table 6**). Stomatal conductance is negatively correlated with leaf temperature [55], and as temperature raises, the plant reduces the opening of its stomata to conserve water and this mechanism reduces

Evaporative cooling and kaolin coating had the same effect on cluster temperature, measured after veraison (infrared thermometry). Clusters of treated plants show a reduced temperature in relation to control plants, which are at least 1.3°C warmer (**Table 6**). Here, the scattering of radiation caused by kaolin is not counter acted by reduced transpiration and cluster tempera-

Excessive temperature and radiation that cause fruit sunburn, a major factor in loss of production in grapevines of Alto Douro region, were effectively avoided by nebulization and kaolin coating (**Table 7**). Treated plants have 5–7% of shriveled berries per cluster compared with

Total soluble solids are 26.1°Brix for nebulized vines, and it is significantly higher than kaolin coated and control, 24.6 and 24.7°Brix, respectively (**Table 7**). Nebulized canopies show lower

**Canopy Cluster**

Different superscript letters on same column indicate a significant difference at α ≤ 0.05 (Tukey's HSD test). *Vitis vinifera*

**Table 6.** Effect of nebulization and kaolin coating on average canopy temperature and stomatal conductance (g<sup>s</sup>

Control 30.3a 32.1a 2.14a Nebulization 28.4b 31.1b 3.11b Kaolin coating 30.1a 31.5b 2.09a

flowering to harvest and for cluster temperature from veraison to harvest.

**Temperature (°C) gs**

12–13% in control vines. Reduced berry loss contributed to higher yield per plant.

pointed out as explanations for these results [62, 63]. Stomatal conductance (gs

transpiration, a mechanism that rises the leaf temperature.

ture was maintained lower than air temperature.

cv Touriga Nacional, Alto Douro.

Si4

O10(OH)8

] at a rate

, cm s−1) of neb-

 **(cm s−1)**

) from

before the forecast harvest date: (1): coated with a suspension of kaolin [Al<sup>4</sup>

68 Grapes and Wines - Advances in Production, Processing, Analysis and Valorization

and berry sampling for laboratory analysis.

Meteorological records and grapevine phenological occurrences in DDR are in agreement with forecasts of higher temperatures, lower precipitations, and advances of phenological stages. The environment of Douro is expected to become more stressful in coming years, and adaptations are imperative to save the viticulture as an economic activity. For the vineyards already in commercial production, efficient irrigation associated with shading and cooling techniques can reduce significatively water and temperature stress on grapevines and they can maintain acceptable yields with marketable quality wine. However, these techniques have some drawbacks in must quality and might not suffice to a large shift in temperature and precipitation given the actual conditions already at the limit of tolerance of the best grape varieties.

## **Acknowledgements**

Financial support was provided by Quinta do Vale Meão (Olazabal & Sons, LDA), COTESI: Companhia de Texteis, S.A., and UTAD. My special thanks go to Francisco Olazabal, Pedro Barbosa, André Guedes, and Frederico Lobão.

## **Author details**

Manuel Oliveira

\*Address all correspondence to: mto@utad.pt

CITAB – UTAD Department of Agronomy, Vila Real, Portugal

## **References**

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Manuel Oliveira

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**Chapter 5**

**Provisional chapter**

**Water Balance Indices for Tropical Wine Grapes**

**Water Balance Indices for Tropical Wine Grapes**

DOI: 10.5772/intechopen.72751

Over the last few decades, the Brazilian semiarid region has appeared as one of the main tropical wine production areas in the country. The aim of this research was the elaboration and application of water balance indices to upscale them in the wine grape growing regions of the Petrolina and Juazeiro counties in the states of Pernambuco (PE) and Bahia (BA), respectively, simulating different pruning dates along the year. Previous energy balance measurements were used for relating the crop coefficient (Kc) with the accumulated degree-days (DDac). The model was applied to upscale the water balance indices during the growing seasons (GS). It was concluded that if irrigation water is available, the best pruning periods are for GS from May to July because of better natural thermal and moisture conditions. Much care should be taken for pruning done in other periods of the year, with regard to the effect of increasing thermal conditions on wine quality. The classifications and delimitations done, joined with other environmental characteristics, are important for a rational planning of the commercial tropical wine production expansion, mainly in the actual situations of climate and land use changes together with rising water

**Keywords:** evapotranspiration, crop coefficient, vineyard adaptation, water resources,

The influence of climatic variables during the vineyard growing seasons on wine quality is well known because they influence the grapevine growth and then the berry composition. For wine grape crops, plant phenology, wine quality, and yield are very dependent on climate at regional, local and microclimatic scales [1–4]. For any of these spatial scales, considerations of grape site selection, cultural practices, and water management are important, which are very

> © 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution,

© 2018 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

and reproduction in any medium, provided the original work is properly cited.

Antônio Heriberto de Castro Teixeira, Jorge Tonietto and Janice F. Leivas

and Janice F. Leivas

**Abstract**

*Vitis vinifera*

**1. Introduction**

http://dx.doi.org/10.5772/intechopen.72751

Additional information is available at the end of the chapter

Additional information is available at the end of the chapter

Antônio Heriberto de Castro Teixeira, Jorge Tonietto

competition along the years in the Brazilian semiarid region.

**Provisional chapter**

## **Water Balance Indices for Tropical Wine Grapes**

**Water Balance Indices for Tropical Wine Grapes**

DOI: 10.5772/intechopen.72751

Antônio Heriberto de Castro Teixeira, Jorge Tonietto and Janice F. Leivas and Janice F. Leivas Additional information is available at the end of the chapter

Additional information is available at the end of the chapter

Antônio Heriberto de Castro Teixeira, Jorge Tonietto

http://dx.doi.org/10.5772/intechopen.72751

#### **Abstract**

Over the last few decades, the Brazilian semiarid region has appeared as one of the main tropical wine production areas in the country. The aim of this research was the elaboration and application of water balance indices to upscale them in the wine grape growing regions of the Petrolina and Juazeiro counties in the states of Pernambuco (PE) and Bahia (BA), respectively, simulating different pruning dates along the year. Previous energy balance measurements were used for relating the crop coefficient (Kc) with the accumulated degree-days (DDac). The model was applied to upscale the water balance indices during the growing seasons (GS). It was concluded that if irrigation water is available, the best pruning periods are for GS from May to July because of better natural thermal and moisture conditions. Much care should be taken for pruning done in other periods of the year, with regard to the effect of increasing thermal conditions on wine quality. The classifications and delimitations done, joined with other environmental characteristics, are important for a rational planning of the commercial tropical wine production expansion, mainly in the actual situations of climate and land use changes together with rising water competition along the years in the Brazilian semiarid region.

**Keywords:** evapotranspiration, crop coefficient, vineyard adaptation, water resources, *Vitis vinifera*

## **1. Introduction**

The influence of climatic variables during the vineyard growing seasons on wine quality is well known because they influence the grapevine growth and then the berry composition. For wine grape crops, plant phenology, wine quality, and yield are very dependent on climate at regional, local and microclimatic scales [1–4]. For any of these spatial scales, considerations of grape site selection, cultural practices, and water management are important, which are very

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2018 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

important issues for potential adaptations to different climate scenarios; however, large-scale climate has been the focus for assessing the climate change impacts [5–8].

used. For the vineyard climatic suitability determination, one can apply water balance indices by using GIS and long-term weather data [4, 18, 21, 28]. For the vineyard water balance indi-

which may be considered as a hypothetical grass surface with specific characteristics, while ET is the real water flux occurring from the surfaces in a specific situation involving all envi-

This chapter aims to elaborate on and apply water balance indices to be scaled up by using a GIS in the wine grape growing regions of Petrolina and Juazeiro counties, located respectively in the semiarid regions of Pernambuco (PE) and Bahia (BA) states, simulating different pruning dates along the year. These indices were delimitated and analyzed, generating criteria for a rational expansion of irrigated and rain-fed vineyards with higher probability of success for tropical wine elaboration. The results generate criteria for a rational expansion of irrigated and rain-fed vineyards with higher probability of success for the Brazilian tropical wine elaboration, under the actual scenario of rising water competition by irrigated agriculture and

A net of agrometeorological stations was used throughout interpolation processes in a GIS environment. The stations are spread in the Petrolina (PE) and Juazeiro (BA) counties, with seven of them inside irrigated farms and the other seven in the natural vegetation, called "Caatinga." The gridded weather data well characterize the horizontal thermohydrological

According to Teixeira [19], in the Brazilian Northeast semiarid region, disturbed currents from the South, North, East, and West influence the climatology. Excluding the places of high

of 33°C and the average minimum of 19°C. The warmest months are October and November when the Sun is close to the zenith position with low cloud cover, and the coldest ones are June and July at winter solstice in the Southern hemisphere. The thermal homogeneity strongly contrasts with the spatial and temporal heterogeneity of the rainfall regime, with the rainy period from November to April (90% of the annual total), the period January to April representing 68% of the annual rainfall. The weather variables in the current study involved a 10-year period (2003–2012). Monthly data were used to calculate the reference evapotranspi-

Previous Bowen ratio energy balance data from Teixeira et al. [26] for the cv. *Syrah* in the Brazilian semiarid region were used. **Figure 2** shows the details of the field experiment car-

higher than 24°C, with the average maximum

is the water flux from a reference surface, not a shortage of water,

) and actual (ET)

79

Water Balance Indices for Tropical Wine Grapes http://dx.doi.org/10.5772/intechopen.72751

ces used in this chapter, distinctions are important between reference (ET0

evapotranspiration. ET0

ronmental conditions [29].

nonagricultural sectors.

**2. Materials and methods**

**2.1. Study region and data set**

contrast between these mixed agroecosystems (**Figure 1**).

altitude, all areas present long-term annual Ta

ration (ET0) by the Penman-Monteith method [29].

**2.2. Modeling vineyard water balance indices**

ried out in Petrolina (PE), Northeast of Brazil.

The optimal vineyard response to air temperature (Ta ) ranges between 20 and 35°C [9]. According to Fraga et al. [3], a 10°C basal temperature (Tb ) is needed for the growing season onset. However, high thermal conditions contribute to increase sugar content in grapes, resulting in large alcohol concentration and low acidity in wines, rising pH [2, 10–12]. There are also important secondary thermal effects, such as increase of pest and disease risks, like downy and powdery mildew, especially under rainy conditions [4, 13, 14].

Rising thermal conditions (Ta > 30°C) should increase suspended solid concentrations, but high Brix levels may be attributed to large evapotranspiration (ET) rates [15]. According to Webb et al. [16], high Ta during the harvest period may reduce berry quality due to increasing ET. These issues make the rational water management an important issue for controlling water deficiencies and excesses, and the need of vineyard water requirements (WR) quantification [12, 17, 18].

Taking into account the water balance, on the one hand, warming conditions can directly affect the vineyard WR, which, together with low precipitation (P) amounts, promote high levels of aridity and water demand. On the other hand, high soil moisture throughout the growing season may cause excessive vigor, increased risks of pest and diseases, and other problems related to wine quality and to the balance of its chemical components [4, 9, 19–21]. All these thermohydrological effects during the vineyard growing seasons on wine quality and production show the importance of water accounting to delimitate areas and seasons with suitable climatic aptitude for winemaking processes.

A large number of climate models have been used worldwide to classify winemaking regions by using different methodologies. For aptitude delimitation, aiming at grape and wine production, one can apply bioclimatic indices based on the thermohydrological requirements. The multicriteria climatic classification (MCC) system proposed by Tonietto and Carbonneau [22] has been used under temperate climate conditions in Europe [23] and in South America [24, 25]. However, the method has worked well considering a single, 6-month growing season per year under temperate climate conditions.

Over the last years, the Brazilian semiarid region has appeared among the main tropical wineproducing areas in the country, typically growing under irrigation conditions and trained mainly in vertical shoot-positioning systems. With proper irrigation and cultural management practices, the farmers can produce grapes and carry out winemaking at any time of the year, allowing a potential average of between two and three vineyard-growing cycles per year, in accordance with and depending on each variety. The Ta rising with a consequent increase in ET rates and aridity in this region will affect both the wine grape quality and vineyard water requirements.

The coupled effect of increasing water consumption and decreasing precipitation, together with land use change, makes it important to elaborate and upscale indices for subsidizing winemaking adaptations and water productivity improvements. Vineyard water variables have been quantified in this region by point measurements [26, 27] but to upscale these punctual results, tools such as remote sensing and geographic information system (GIS) can be used. For the vineyard climatic suitability determination, one can apply water balance indices by using GIS and long-term weather data [4, 18, 21, 28]. For the vineyard water balance indices used in this chapter, distinctions are important between reference (ET0 ) and actual (ET) evapotranspiration. ET0 is the water flux from a reference surface, not a shortage of water, which may be considered as a hypothetical grass surface with specific characteristics, while ET is the real water flux occurring from the surfaces in a specific situation involving all environmental conditions [29].

This chapter aims to elaborate on and apply water balance indices to be scaled up by using a GIS in the wine grape growing regions of Petrolina and Juazeiro counties, located respectively in the semiarid regions of Pernambuco (PE) and Bahia (BA) states, simulating different pruning dates along the year. These indices were delimitated and analyzed, generating criteria for a rational expansion of irrigated and rain-fed vineyards with higher probability of success for tropical wine elaboration. The results generate criteria for a rational expansion of irrigated and rain-fed vineyards with higher probability of success for the Brazilian tropical wine elaboration, under the actual scenario of rising water competition by irrigated agriculture and nonagricultural sectors.

## **2. Materials and methods**

important issues for potential adaptations to different climate scenarios; however, large-scale

son onset. However, high thermal conditions contribute to increase sugar content in grapes, resulting in large alcohol concentration and low acidity in wines, rising pH [2, 10–12]. There are also important secondary thermal effects, such as increase of pest and disease risks, like

Rising thermal conditions (Ta > 30°C) should increase suspended solid concentrations, but high Brix levels may be attributed to large evapotranspiration (ET) rates [15]. According to

ing ET. These issues make the rational water management an important issue for controlling water deficiencies and excesses, and the need of vineyard water requirements (WR) quantifi-

Taking into account the water balance, on the one hand, warming conditions can directly affect the vineyard WR, which, together with low precipitation (P) amounts, promote high levels of aridity and water demand. On the other hand, high soil moisture throughout the growing season may cause excessive vigor, increased risks of pest and diseases, and other problems related to wine quality and to the balance of its chemical components [4, 9, 19–21]. All these thermohydrological effects during the vineyard growing seasons on wine quality and production show the importance of water accounting to delimitate areas and seasons

A large number of climate models have been used worldwide to classify winemaking regions by using different methodologies. For aptitude delimitation, aiming at grape and wine production, one can apply bioclimatic indices based on the thermohydrological requirements. The multicriteria climatic classification (MCC) system proposed by Tonietto and Carbonneau [22] has been used under temperate climate conditions in Europe [23] and in South America [24, 25]. However, the method has worked well considering a single, 6-month growing season

Over the last years, the Brazilian semiarid region has appeared among the main tropical wineproducing areas in the country, typically growing under irrigation conditions and trained mainly in vertical shoot-positioning systems. With proper irrigation and cultural management practices, the farmers can produce grapes and carry out winemaking at any time of the year, allowing a potential average of between two and three vineyard-growing cycles

increase in ET rates and aridity in this region will affect both the wine grape quality and vine-

The coupled effect of increasing water consumption and decreasing precipitation, together with land use change, makes it important to elaborate and upscale indices for subsidizing winemaking adaptations and water productivity improvements. Vineyard water variables have been quantified in this region by point measurements [26, 27] but to upscale these punctual results, tools such as remote sensing and geographic information system (GIS) can be

during the harvest period may reduce berry quality due to increas-

) ranges between 20 and 35°C [9].

) is needed for the growing sea-

rising with a consequent

climate has been the focus for assessing the climate change impacts [5–8].

downy and powdery mildew, especially under rainy conditions [4, 13, 14].

The optimal vineyard response to air temperature (Ta

Webb et al. [16], high Ta

cation [12, 17, 18].

According to Fraga et al. [3], a 10°C basal temperature (Tb

78 Grapes and Wines - Advances in Production, Processing, Analysis and Valorization

with suitable climatic aptitude for winemaking processes.

per year, in accordance with and depending on each variety. The Ta

per year under temperate climate conditions.

yard water requirements.

#### **2.1. Study region and data set**

A net of agrometeorological stations was used throughout interpolation processes in a GIS environment. The stations are spread in the Petrolina (PE) and Juazeiro (BA) counties, with seven of them inside irrigated farms and the other seven in the natural vegetation, called "Caatinga." The gridded weather data well characterize the horizontal thermohydrological contrast between these mixed agroecosystems (**Figure 1**).

According to Teixeira [19], in the Brazilian Northeast semiarid region, disturbed currents from the South, North, East, and West influence the climatology. Excluding the places of high altitude, all areas present long-term annual Ta higher than 24°C, with the average maximum of 33°C and the average minimum of 19°C. The warmest months are October and November when the Sun is close to the zenith position with low cloud cover, and the coldest ones are June and July at winter solstice in the Southern hemisphere. The thermal homogeneity strongly contrasts with the spatial and temporal heterogeneity of the rainfall regime, with the rainy period from November to April (90% of the annual total), the period January to April representing 68% of the annual rainfall. The weather variables in the current study involved a 10-year period (2003–2012). Monthly data were used to calculate the reference evapotranspiration (ET0) by the Penman-Monteith method [29].

#### **2.2. Modeling vineyard water balance indices**

Previous Bowen ratio energy balance data from Teixeira et al. [26] for the cv. *Syrah* in the Brazilian semiarid region were used. **Figure 2** shows the details of the field experiment carried out in Petrolina (PE), Northeast of Brazil.

**Figure 1.** Petrolina and Juazeiro counties, respectively in Pernambuco (PE) and Bahia (BA) states, in the Brazilian Northeast, together with the agrometeorological stations used for the interpolation processes.

The energy balance equation for the wine grape can be expressed by means of bulk energy and heat fluxes:

$$\mathbf{R}\_n - \lambda \mathbf{E} - \mathbf{H} - \mathbf{G} = \mathbf{0} \tag{1}$$

Actual evapotranspiration (ET) was derived from the latent heat of vaporization (λ), density of water, and λE. The field experiment was close (3 km) to Bebedouro station (**Figure 1**), which ET0 data allowed the acquirement of the crop coefficient (Kc) along the crop stages [29]:

**Figure 2.** Measurements of gradients of air temperature, vapor pressure and wind speed; incident and reflected

shortwave; net radiation; acquisition data system; and soil moisture in Bowen ratio system of wine grape.

where a = −2 × 10−7, b = 4 × 10−4 and c = 0.54 are the regression coefficients (R<sup>2</sup> > 0.70).

Further, Kc was used to obtain the ET under potential conditions, which in turn considered the vineyard water requirements (WR), using the cv. *Syrah* as a reference wine grape in the study region. WR for a growing season (GS) was acquired by simulating different pruning date and considering a 4-month mean GS duration under the Brazilian semiarid conditions:

WRGS = Kc GSE T 0GS (6)

Five Kc values were taken into account, being DDac zero at the start of a GS, while the other

five-Kc averaged values and the total ET0 for a GS (ET0GS) were considered for acquiring WRGS. Another indicator, the water balance difference (WBd), was applied to quantify the magnitude of excess or deficiencies of water in the vineyards on large scales for a GS, where PGS is

WBdGS = PGS − WRGS (7)

ET0 (4)

Water Balance Indices for Tropical Wine Grapes http://dx.doi.org/10.5772/intechopen.72751 81

<sup>2</sup> + bDDac + c (5)

for the subsequent months. The

) of 10°C, Kc was related with the accumulated degree

Kc = \_\_\_\_ ET

Kc = aDDac

DDac values were calculated, along the GS, with the average Ta

Considering a base temperature (Tb

the total growing season precipitation:

days DDac [30]:

where Rn is the net radiation, λE is the latent heat flux, H is the sensible heat flux, and G is the soil heat flux.

The vineyard λE was obtained by a partitioning parameter:

$$
\lambda \mathbf{E} = \frac{\mathbf{R}\_n - \mathbf{G}}{1 + \beta} \tag{2}
$$

where β is the Bowen ratio:

$$
\beta = \gamma \left(\frac{\Delta T}{\Delta \mathbf{e}}\right) \tag{3}
$$

and γ (kPa °C−1) is the psychrometric constant, ΔT(°C) the temperature gradient measured by dry thermocouples, and ∆e (kPa) is the water vapor pressure gradient measured by the difference between dry and wet thermocouples over the height interval above the vineyard canopy surface.

**Figure 2.** Measurements of gradients of air temperature, vapor pressure and wind speed; incident and reflected shortwave; net radiation; acquisition data system; and soil moisture in Bowen ratio system of wine grape.

Actual evapotranspiration (ET) was derived from the latent heat of vaporization (λ), density of water, and λE. The field experiment was close (3 km) to Bebedouro station (**Figure 1**), which ET0 data allowed the acquirement of the crop coefficient (Kc) along the crop stages [29]:

$$\mathbf{Kc = \frac{ET}{ET0}}\tag{4}$$

Considering a base temperature (Tb ) of 10°C, Kc was related with the accumulated degree days DDac [30]:

$$\text{Kc = } \text{aD} \\ \text{D}\_{\text{uc}} \text{ }^2 \text{+bD} \\ \text{D}\_{\text{uc}} \text{ +c} \tag{5}$$

where a = −2 × 10−7, b = 4 × 10−4 and c = 0.54 are the regression coefficients (R<sup>2</sup> > 0.70).

The energy balance equation for the wine grape can be expressed by means of bulk energy

**Figure 1.** Petrolina and Juazeiro counties, respectively in Pernambuco (PE) and Bahia (BA) states, in the Brazilian

Northeast, together with the agrometeorological stations used for the interpolation processes.

Rn − λE − H − G = 0 (1)

where Rn is the net radiation, λE is the latent heat flux, H is the sensible heat flux, and G is the

\_\_\_ ΔT

and γ (kPa °C−1) is the psychrometric constant, ΔT(°C) the temperature gradient measured by dry thermocouples, and ∆e (kPa) is the water vapor pressure gradient measured by the difference between dry and wet thermocouples over the height interval above the vineyard

<sup>1</sup> <sup>+</sup> <sup>β</sup> (2)

Δe) (3)

The vineyard λE was obtained by a partitioning parameter:

80 Grapes and Wines - Advances in Production, Processing, Analysis and Valorization

λE <sup>=</sup> Rn <sup>−</sup> <sup>G</sup> \_\_\_\_\_

β = γ(

and heat fluxes:

soil heat flux.

canopy surface.

where β is the Bowen ratio:

Further, Kc was used to obtain the ET under potential conditions, which in turn considered the vineyard water requirements (WR), using the cv. *Syrah* as a reference wine grape in the study region. WR for a growing season (GS) was acquired by simulating different pruning date and considering a 4-month mean GS duration under the Brazilian semiarid conditions:

$$\text{WR}\_{\text{GS}} = \text{Kc}\_{\text{cs}} \to \text{T} \, \text{0}\_{\text{cs}} \tag{6}$$

Five Kc values were taken into account, being DDac zero at the start of a GS, while the other DDac values were calculated, along the GS, with the average Ta for the subsequent months. The five-Kc averaged values and the total ET0 for a GS (ET0GS) were considered for acquiring WRGS.

Another indicator, the water balance difference (WBd), was applied to quantify the magnitude of excess or deficiencies of water in the vineyards on large scales for a GS, where PGS is the total growing season precipitation:

$$\text{WBad}\_{\text{GS}} = \text{P}\_{\text{GS}} - \text{WR}\_{\text{GS}} \tag{7}$$

Neglecting the water storage in the root zones in the vineyard water balance, difficult to consider in large-scale analyses, positive WBd values are a quantification of vineyard water excess, while the negative ones are related to the vineyard water deficiencies.

The WRGS together with PGS values allowed the development and application of the water balance ratio (WBr) indicator:

$$\mathbf{W} \mathbf{B} \mathbf{r}\_{\rm CS} = \frac{\mathbf{P}\_{\rm CS}}{\mathbf{W} \mathbf{R}\_{\rm cs}} \tag{8}$$

solstice time in the Southern hemisphere, while the highest ones are when the Sun is around the zenith position over the Brazilian tropical wine grape growing region. For pruning done during the coldest periods, several pixels present TGS values lower than 24°C, while one can

Water Balance Indices for Tropical Wine Grapes http://dx.doi.org/10.5772/intechopen.72751 83

There are no thermal limitations for wine grape crop in the Brazilian semiarid region, with pruning dates in the middle of the year. On the one hand, for all pruning periods, TGS pixels are below 30°C, which conditions around or above this value should increase suspended solid concentrations [15]. On the other hand, TGS values are not below the threshold of 10°C, which could introduce a dormancy stage in temperate climates [3]. The TGS values in the study area are between 23°C and 28°C, inside the optimum thermal range pointed by Gouveia et al. [9]. However, when the pruning is done from September to December, many areas with TGS above 27°C often occur and could affect negatively the wine quality. These latter conditions will contribute to high sugar content in grapes but wines with increasing levels of alcohol, low acidity, and large pH values. These effects together will promote a wine unbalance with instability for

**Figure 4** shows the PGS maps, for different wine grape pruning dates along the year, consider-

The pruning dates with the highest PGS are those from December to February, with several pixel values larger than 300 mm GS−1 in the Petrolina County. During this period, the largest moisture spatial variation is also verified according to the SD values. The lowest PGS are for pruning between May and July. High pixel values occur in the northwestern side of Petrolina

**Figure 4.** Spatial averages of total precipitation for a 4-month wine grape-growing season (PGS), and a 10-year period (2003–2012), simulating different pruning dates, in the Petrolina (PE) and Juazeiro (BA) counties, Northeast Brazil. The

see all the areas with TGS higher than 26°C, for pruning in the hottest months.

the phenolic and aromatic composition [2, 3, 10–12].

mean pixel values and standard deviations are also indicated.

ing a mean 4-month GS, and the 10-year period from 2003 to 2012.

(PE), while the lowest ones occur in the southwestern area of Juazeiro (BA).

WBr takes into account the thermohydrological conditions, and it is a measure of the water availability in the vineyard root zone. When it is around 1.00, imply the feasibility for rain-fed wine grape, while those much higher should indicate moisture excess problems, independently of the absence or not of irrigation. Low WBr values mean possibility of natural water deficiencies and the degree of irrigation needs according to the pruning dates.

#### **3. Results and discussion**

**Figure 3** shows the TGS maps, for different wine grape pruning dates along the year, considering a mean 4-month GS, and the 10-year period from 2003 to 2012.

The coldest and the hottest growing seasons (GS) are those for pruning dates between April and July and from September to December, respectively. Considering the standard deviation (SD) values, there are low thermal spatial variations, due to the proximity of the counties to the equator. The lowest air temperatures for a growing season (TGS) occur at the winter

**Figure 3.** Spatial averages of mean air temperature values for a 4-month wine grape-growing season (TGS), and a 10-year period (2003–2012), simulating different pruning dates, in the Petrolina (PE) and Juazeiro (BA) counties, Northeast Brazil. The mean pixel values and standard deviations are also indicated.

solstice time in the Southern hemisphere, while the highest ones are when the Sun is around the zenith position over the Brazilian tropical wine grape growing region. For pruning done during the coldest periods, several pixels present TGS values lower than 24°C, while one can see all the areas with TGS higher than 26°C, for pruning in the hottest months.

Neglecting the water storage in the root zones in the vineyard water balance, difficult to consider in large-scale analyses, positive WBd values are a quantification of vineyard water

The WRGS together with PGS values allowed the development and application of the water

WBr takes into account the thermohydrological conditions, and it is a measure of the water availability in the vineyard root zone. When it is around 1.00, imply the feasibility for rain-fed wine grape, while those much higher should indicate moisture excess problems, independently of the absence or not of irrigation. Low WBr values mean possibility of natural water

**Figure 3** shows the TGS maps, for different wine grape pruning dates along the year, consider-

The coldest and the hottest growing seasons (GS) are those for pruning dates between April and July and from September to December, respectively. Considering the standard deviation (SD) values, there are low thermal spatial variations, due to the proximity of the counties to the equator. The lowest air temperatures for a growing season (TGS) occur at the winter

**Figure 3.** Spatial averages of mean air temperature values for a 4-month wine grape-growing season (TGS), and a 10-year period (2003–2012), simulating different pruning dates, in the Petrolina (PE) and Juazeiro (BA) counties, Northeast

Brazil. The mean pixel values and standard deviations are also indicated.

WRGS

(8)

excess, while the negative ones are related to the vineyard water deficiencies.

deficiencies and the degree of irrigation needs according to the pruning dates.

ing a mean 4-month GS, and the 10-year period from 2003 to 2012.

balance ratio (WBr) indicator:

**3. Results and discussion**

WBrGS <sup>=</sup> P\_\_\_\_\_ GS

82 Grapes and Wines - Advances in Production, Processing, Analysis and Valorization

There are no thermal limitations for wine grape crop in the Brazilian semiarid region, with pruning dates in the middle of the year. On the one hand, for all pruning periods, TGS pixels are below 30°C, which conditions around or above this value should increase suspended solid concentrations [15]. On the other hand, TGS values are not below the threshold of 10°C, which could introduce a dormancy stage in temperate climates [3]. The TGS values in the study area are between 23°C and 28°C, inside the optimum thermal range pointed by Gouveia et al. [9].

However, when the pruning is done from September to December, many areas with TGS above 27°C often occur and could affect negatively the wine quality. These latter conditions will contribute to high sugar content in grapes but wines with increasing levels of alcohol, low acidity, and large pH values. These effects together will promote a wine unbalance with instability for the phenolic and aromatic composition [2, 3, 10–12].

**Figure 4** shows the PGS maps, for different wine grape pruning dates along the year, considering a mean 4-month GS, and the 10-year period from 2003 to 2012.

The pruning dates with the highest PGS are those from December to February, with several pixel values larger than 300 mm GS−1 in the Petrolina County. During this period, the largest moisture spatial variation is also verified according to the SD values. The lowest PGS are for pruning between May and July. High pixel values occur in the northwestern side of Petrolina (PE), while the lowest ones occur in the southwestern area of Juazeiro (BA).

**Figure 4.** Spatial averages of total precipitation for a 4-month wine grape-growing season (PGS), and a 10-year period (2003–2012), simulating different pruning dates, in the Petrolina (PE) and Juazeiro (BA) counties, Northeast Brazil. The mean pixel values and standard deviations are also indicated.

Taking into account all pruning dates, the rainfall amounts in Petrolina (PE) are 61% larger than in Juazeiro (BA). Thus, in the first county, there are more possibilities of matching the vineyard water requirements with rainfall together with supplementary irrigations, whenever irrigation water is available. However, as a first guess, risks of pest and diseases and other problems related to wine quality and to the balance between its chemical components are higher for pruning done from December to February [4, 9, 19–21].

found throughout field experiments in different wine grapes growing regions of the world [27, 30, 31], bringing confidence to the upscaling techniques applied in the current case study. **Table 1** resumes the averages and SD values of the vineyard water balance indices for each 4-month pruning date per county, for the wine grape, cv. *Syrah* considering the period of weather data from 2003 to 2012 in the growing region of Petrolina – Pet (PE) e Juazeiro – Jua

Water Balance Indices for Tropical Wine Grapes http://dx.doi.org/10.5772/intechopen.72751 85

No significant differences arise among the TGS mean values from Petrolina (PE) and those from Juazeiro (BA) with average for all pruning periods of 26°C GS−1 for both counties; however, the second one presents larger spatial thermal variation, according to the SD values.

In case of PGS, the values for Juazeiro (BA) are lower those for Petrolina (PE), indicating higher possibility of rainfall water use by the vineyards in the second county. As the vineyard thermal conditions between them did not differ so much, the WRGS values for Juazeiro (BA) were, in average, 90% of those for Petrolina (PE). These differences could be attributed to the effect of relative humidity (RH) in the ET0 calculations, as lower PGS reduce RH in Juazeiro (BA)

Keeping in mind that the wine quality depends on both thermal and water conditions, these conditions were analyzed throughout the mean pixel values and standard deviations (SD)

**Pet Jua Pet Jua Pet Jua**

**Table 1.** Mean values and standard deviations (SD) of the vineyard water balance indices for the wine grape, cv. *Syrah*, considering a 4-month average growing season (GS) and a 10-year period (2003–2012), in the Petrolina—Pet (PE) and

**Pruning date TGS (°C) PGS (mm GS−1) WRGS (mm GS−1)**

Air temperature (TGS); Precipitation (PGS); and Water requirements (WRGS).

Juazeiro—Jua (BA) counties, Northeast Brazil.

January 26.7 ± 0.1 26.8 ± 0.3 396 ± 65 253 ± 52 398 ± 23 362 ± 20 February 26.2 ± 0.1 26.3 ± 0.3 329 ± 57 209 ± 47 372 ± 22 337 ± 18 March 25.6 ± 0.1 25.6 ± 0.3 224 ± 47 139 ± 35 342 ± 19 313 ± 18 April 25.0 ± 0.1 24.9 ± 0.3 130 ± 28 78 ± 22 340 ± 19 310 ± 19 May 24.6 ± 0.2 24.4 ± 0.3 58 ± 14 32 ± 11 361 ± 22 327 ± 20 June 24.8 ± 0.2 24.5 ± 0.3 40 ± 10 17 ± 7 399 ± 25 360 ± 22 July 25.5 ± 0.2 25.3 ± 0.3 44 ± 8 18 ± 7 439 ± 28 396 ± 24 August 26.5 ± 0.2 26.3 ± 0.3 65 ± 11 37 ± 7 461 ± 29 416 ± 24 September 27.3 ± 0.2 27.2 ± 0.3 105 ± 17 62 ± 12 461 ± 29 417 ± 23 October 27.7 ± 0.1 27.7 ± 0.3 191 ± 28 121 ± 21 448 ± 27 404 ± 22 November 27.6 ± 0.1 27.6 ± 0.3 292 ± 40 189 ± 33 441 ± 26 396 ± 22 December 27.2 ± 0.1 27.3 ± 0.3 365 ± 57 233 ± 45 420 ± 24 368 ± 21 Mean 26.1 ± 0.1 26.2 ± 0.3 187 ± 32 116 ± 25 407 ± 24 367 ± 21

(BA), in the semiarid region of Northeast Brazil.

when comparing with Petrolina (PE).

Considering the cv. *Syrah* as reference for wine grapes in the growing regions of Petrolina (PE) and Juazeiro (BA), and the long-term weather conditions (2003–2012), the WRGS spatial values for a 4-month mean GS are presented in **Figure 5**.

The pruning dates with the highest WRGS are from August to October, with average pixel values larger than 420 mm GS−1, when, according to the SD values, there are also the highest spatial variations. Pruning done from March to May will promote the lowest water consumptions, with mean WRGS below 350 mm GS−1, and the smallest SD values, below 25 mm in March and April. Large WRGS occur in the northwestern side of Petrolina (PE), what might correspond to good grape yield and wine quality if water is available together with techniques to avoid natural water excesses in the root zones [4, 12]. However, special attention should be given under water scarcity conditions, when there is ample room for water productivity improvements in situations of lower atmospheric demands [21].

Taking into account all pruning dates along a year, the water demands in the Petrolina (PE) county are 10% larger, when comparing with those for Juazeiro (BA) one. Daily average WR values in the study region were between 2.7 and 3.6 mm day−1, being similar to the ET rates

**Figure 5.** Spatial averages of the water requirements for a 4-month wine grape-growing season (WRGS), and 10-year period (2003–2012), simulating different pruning dates, in the Petrolina (PE) and Juazeiro (BA) counties, Northeast Brazil. The mean pixel values and standard deviations are also indicated.

found throughout field experiments in different wine grapes growing regions of the world [27, 30, 31], bringing confidence to the upscaling techniques applied in the current case study.

Taking into account all pruning dates, the rainfall amounts in Petrolina (PE) are 61% larger than in Juazeiro (BA). Thus, in the first county, there are more possibilities of matching the vineyard water requirements with rainfall together with supplementary irrigations, whenever irrigation water is available. However, as a first guess, risks of pest and diseases and other problems related to wine quality and to the balance between its chemical components

Considering the cv. *Syrah* as reference for wine grapes in the growing regions of Petrolina (PE) and Juazeiro (BA), and the long-term weather conditions (2003–2012), the WRGS spatial values

The pruning dates with the highest WRGS are from August to October, with average pixel values larger than 420 mm GS−1, when, according to the SD values, there are also the highest spatial variations. Pruning done from March to May will promote the lowest water consumptions, with mean WRGS below 350 mm GS−1, and the smallest SD values, below 25 mm in March and April. Large WRGS occur in the northwestern side of Petrolina (PE), what might correspond to good grape yield and wine quality if water is available together with techniques to avoid natural water excesses in the root zones [4, 12]. However, special attention should be given under water scarcity conditions, when there is ample room for water produc-

Taking into account all pruning dates along a year, the water demands in the Petrolina (PE) county are 10% larger, when comparing with those for Juazeiro (BA) one. Daily average WR values in the study region were between 2.7 and 3.6 mm day−1, being similar to the ET rates

**Figure 5.** Spatial averages of the water requirements for a 4-month wine grape-growing season (WRGS), and 10-year period (2003–2012), simulating different pruning dates, in the Petrolina (PE) and Juazeiro (BA) counties, Northeast

Brazil. The mean pixel values and standard deviations are also indicated.

are higher for pruning done from December to February [4, 9, 19–21].

84 Grapes and Wines - Advances in Production, Processing, Analysis and Valorization

tivity improvements in situations of lower atmospheric demands [21].

for a 4-month mean GS are presented in **Figure 5**.

**Table 1** resumes the averages and SD values of the vineyard water balance indices for each 4-month pruning date per county, for the wine grape, cv. *Syrah* considering the period of weather data from 2003 to 2012 in the growing region of Petrolina – Pet (PE) e Juazeiro – Jua (BA), in the semiarid region of Northeast Brazil.

No significant differences arise among the TGS mean values from Petrolina (PE) and those from Juazeiro (BA) with average for all pruning periods of 26°C GS−1 for both counties; however, the second one presents larger spatial thermal variation, according to the SD values.

In case of PGS, the values for Juazeiro (BA) are lower those for Petrolina (PE), indicating higher possibility of rainfall water use by the vineyards in the second county. As the vineyard thermal conditions between them did not differ so much, the WRGS values for Juazeiro (BA) were, in average, 90% of those for Petrolina (PE). These differences could be attributed to the effect of relative humidity (RH) in the ET0 calculations, as lower PGS reduce RH in Juazeiro (BA) when comparing with Petrolina (PE).

Keeping in mind that the wine quality depends on both thermal and water conditions, these conditions were analyzed throughout the mean pixel values and standard deviations (SD)


**Table 1.** Mean values and standard deviations (SD) of the vineyard water balance indices for the wine grape, cv. *Syrah*, considering a 4-month average growing season (GS) and a 10-year period (2003–2012), in the Petrolina—Pet (PE) and Juazeiro—Jua (BA) counties, Northeast Brazil.

pixel values lower than −340 mm GS−1 and also the lowest SD, around 23 mm GS−1. The less negative WBd values obtained for pruning dates are from December to February, when the average was above −150 mm GS−1. These last thermohydrological conditions indicated the feasibility of rain-fed wine grape with supplementary irrigation. Natural water deficiency in Petrolina is 87% of that for Juazeiro, with better chances of success for rain-fed wine grapes,

As, in average, PGS in Petrolina is 61% higher than that for Juazeiro, but WRGS is only 11% larger (**Table 1**), the differences regarding rainfall amounts will affect more the water balance than the different evapotranspiration rates between the counties. Similarly to the WBd index, pruning

1.00, meaning that rainfall amounts met from 60 to 100% of the vineyard water demands during these pruning periods; however, SD values for both of them are around 0.10 (**Figure 6b**). Although rainy conditions having the beneficial aspect of natural water availability, increasing soil moisture may reduce the ripening capacity of grapes, and the difficulty of water stress management is unfavorable for the organoleptic wine quality. In this sense, care should be also taken for improving drainage for both irrigated and rain-fed vineyards during the peri-

The natural climate dryness conditions occur when the pruning is done from May to August. Under these circumstances, the WBr values are around 0.10 with almost no spatial variation, favoring more the irrigated vineyards. These conditions avoid plant diseases, root respiration problems, and direct damage to the berries promoted by excess of precipitation, favoring the

Water balance indices are successfully developed and applied, allowing the large-scale analyses of the thermohydrological conditions for wine grape production under the semiarid con-

On the one hand, under irrigation conditions, the best wine grape pruning dates in the Brazilian Northeast are from May to August, with the thermohydrological conditions favoring a better tropical wine quality. On the other hand, the most problematic pruning periods for irrigated crops are from December to February because the joint effects of higher air temperatures and precipitations. Considering the possibility of rainfed crops, this last period should be considered in situations with the possibility of supplementary irrigation applying

The spatial delimitations carried out in the current research, joined with other environmental characteristics, are important for the success of the commercial tropical wine production expansion, considering also the sustainability of the activity in the Brazilian semiarid region, where the land use and climate changes are happening together with water competition dur-

ditions of the Brazilian Northeast, considering different pruning dates along the year.

with averages ranging from 0.60 to

Water Balance Indices for Tropical Wine Grapes http://dx.doi.org/10.5772/intechopen.72751 87

once rainfall-water storage techniques are applied.

quality of must and wine [3, 4, 9, 12, 20, 21].

rainfall water storage techniques.

ing the last decades.

ods of high WBr.

**4. Conclusions**

dates from December to February present the highest WBr,

**Figure 6.** Mean pixel values and standard deviations (SD) for the water balance indices, considering a 10-year period (2003–2012) and an average 4-month wine grape-growing season, cv. *Syrah* according to the simulated pruning dates, in the Petrolina—Pet (PE) and Juazeiro—Jua (BA) counties, Northeast Brazil: (a) water balance difference (WBd) and (b) water balance ratio (WBr).

of WBd and WBr for a 4-month average growing season and according to the pruning dates considering the 10-year period of weather data (2003–2012) (**Figure 6**).

According to **Figure 6a**, there are no positive WBd mean values, meaning that considering the whole area and the average conditions; in general, there is absence of vineyard water excesses for any pruning dates. Disregarding the water storage in the root zones in the vineyard water balance, the pruning periods with the highest water deficiencies (the most negative WBd) are from June to September, when both counties presented average WBd pixel values lower than −340 mm GS−1 and also the lowest SD, around 23 mm GS−1. The less negative WBd values obtained for pruning dates are from December to February, when the average was above −150 mm GS−1. These last thermohydrological conditions indicated the feasibility of rain-fed wine grape with supplementary irrigation. Natural water deficiency in Petrolina is 87% of that for Juazeiro, with better chances of success for rain-fed wine grapes, once rainfall-water storage techniques are applied.

As, in average, PGS in Petrolina is 61% higher than that for Juazeiro, but WRGS is only 11% larger (**Table 1**), the differences regarding rainfall amounts will affect more the water balance than the different evapotranspiration rates between the counties. Similarly to the WBd index, pruning dates from December to February present the highest WBr, with averages ranging from 0.60 to 1.00, meaning that rainfall amounts met from 60 to 100% of the vineyard water demands during these pruning periods; however, SD values for both of them are around 0.10 (**Figure 6b**).

Although rainy conditions having the beneficial aspect of natural water availability, increasing soil moisture may reduce the ripening capacity of grapes, and the difficulty of water stress management is unfavorable for the organoleptic wine quality. In this sense, care should be also taken for improving drainage for both irrigated and rain-fed vineyards during the periods of high WBr.

The natural climate dryness conditions occur when the pruning is done from May to August. Under these circumstances, the WBr values are around 0.10 with almost no spatial variation, favoring more the irrigated vineyards. These conditions avoid plant diseases, root respiration problems, and direct damage to the berries promoted by excess of precipitation, favoring the quality of must and wine [3, 4, 9, 12, 20, 21].

## **4. Conclusions**

of WBd and WBr for a 4-month average growing season and according to the pruning dates

**Figure 6.** Mean pixel values and standard deviations (SD) for the water balance indices, considering a 10-year period (2003–2012) and an average 4-month wine grape-growing season, cv. *Syrah* according to the simulated pruning dates, in the Petrolina—Pet (PE) and Juazeiro—Jua (BA) counties, Northeast Brazil: (a) water balance difference (WBd) and (b) water

According to **Figure 6a**, there are no positive WBd mean values, meaning that considering the whole area and the average conditions; in general, there is absence of vineyard water excesses for any pruning dates. Disregarding the water storage in the root zones in the vineyard water balance, the pruning periods with the highest water deficiencies (the most negative WBd) are from June to September, when both counties presented average WBd

considering the 10-year period of weather data (2003–2012) (**Figure 6**).

86 Grapes and Wines - Advances in Production, Processing, Analysis and Valorization

balance ratio (WBr).

Water balance indices are successfully developed and applied, allowing the large-scale analyses of the thermohydrological conditions for wine grape production under the semiarid conditions of the Brazilian Northeast, considering different pruning dates along the year.

On the one hand, under irrigation conditions, the best wine grape pruning dates in the Brazilian Northeast are from May to August, with the thermohydrological conditions favoring a better tropical wine quality. On the other hand, the most problematic pruning periods for irrigated crops are from December to February because the joint effects of higher air temperatures and precipitations. Considering the possibility of rainfed crops, this last period should be considered in situations with the possibility of supplementary irrigation applying rainfall water storage techniques.

The spatial delimitations carried out in the current research, joined with other environmental characteristics, are important for the success of the commercial tropical wine production expansion, considering also the sustainability of the activity in the Brazilian semiarid region, where the land use and climate changes are happening together with water competition during the last decades.

## **Acknowledgements**

The authors acknowledge the National Council for Scientific and Technological Development – CNPq (Processe 404229/2013-1), for the financial support to a project. The research deals with agricultural water productivity in hydrological basins with land use changes in Brazil.

[8] Tóth JP, Végvári Z. Future of winegrape growing regions in Europe. Australian Journal of Grape and Wine Research. 2016;**22**:64-72. DOI: 10.1111/ajgw.12303, 10.1111/ajgw.12138

Water Balance Indices for Tropical Wine Grapes http://dx.doi.org/10.5772/intechopen.72751 89

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[11] Orduña RM. Climate change associated effects on grape and wine quality and production. Food Research International. 2010;**43**:1844-1845. DOI: 10.1016/j.foodres.2010.05.001

[12] Maxwell JT, Ficklin DL, Harley GL, Jones GV. Projecting future winegrape yields using a combination of *Vitis vinifera* L. growth rings and soil moisture simulations, northern California, UAS. Australian Journal of Grape and Wine Research. 2016;**22**:73-80. DOI:

[13] Centeno A, Baeza P, Lissarrague JR. Relationship between soil and plant water status in wine grapes under various water deficits regimes. HortTechnology. 2010;**20**:585-593.

[14] Caffarra A, Rinaldi MF, Eccel E, Rossi V, Pertot I. Modelling the impact of climate change on the interaction between grapevine and its pests and pathogens: European grapevine moth and powdery mildew. Agriculture Ecosystems and Environments. 2012;**148**:89-

[15] Keller M. Managing grapevines to optimize fruit development in a challenging environment: A climate change primer for viticulturists. Australian Journal of Grape and Wine

[16] Webb LB, Whetton PH, Bhend J, Darbyshire R, Briggs PR, Barlow EWR. Earlier winegrape ripening driven by climatic warming and drying and management practices.

[17] Anderson K, Findlay C, Fuentes S, Tyerman S. Garmaute Climate Change Review: Viticulture, Wine and Climate Change. Adelaide: University of Adelaide; 2008

[18] Teixeira AH de C, Hernandez FBT, Lopes HL. Up scaling table grape water requirements in the low-middle São Francisco River basin. Acta Horticulturae. 2014;**1038**:655-

[19] Teixeira AH de C.Water Productivity Assessments from Field to Large Scale: A Case Study in the Brazilian Semi-Arid Region. Saarbrücken: LAP Lambert Academic Publishing;

[20] Santos JA, Grätsch SD, Karremann MK, Jones GV, Pinto JG. Ensemble projections for wine production in the Douro Valley of Portugal. Climate Change. 2013;**117**:211-225.

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662. DOI: 10.17660/ActaHortic.2014.1038.83

2009. 226 p. ISBN: 978-3838327785

DOI: 10.1007/s10584-012-0538-x

## **Author details**

Antônio Heriberto de Castro Teixeira1 \*, Jorge Tonietto<sup>2</sup> and Janice F. Leivas<sup>3</sup>

\*Address all correspondence to: heriberto.teixeira@embrapa.br

1 Embrapa Coastal Tablelands, Aracaju-SE, Brazil

2 Embrapa Grape and Wine, Bento Gonçalves, Rio Grande do Sul, Brazil

3 Embrapa Satellite Monitoring, Campinas, São Paulo, Brazil

## **References**


[8] Tóth JP, Végvári Z. Future of winegrape growing regions in Europe. Australian Journal of Grape and Wine Research. 2016;**22**:64-72. DOI: 10.1111/ajgw.12303, 10.1111/ajgw.12138

**Acknowledgements**

**Author details**

**References**

fes3.14

s10113-015-0883-1

Antônio Heriberto de Castro Teixeira1

\*Address all correspondence to: heriberto.teixeira@embrapa.br

88 Grapes and Wines - Advances in Production, Processing, Analysis and Valorization

3 Embrapa Satellite Monitoring, Campinas, São Paulo, Brazil

2 Embrapa Grape and Wine, Bento Gonçalves, Rio Grande do Sul, Brazil

1 Embrapa Coastal Tablelands, Aracaju-SE, Brazil

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The authors acknowledge the National Council for Scientific and Technological Development – CNPq (Processe 404229/2013-1), for the financial support to a project. The research deals with agricultural water productivity in hydrological basins with land use changes in Brazil.

\*, Jorge Tonietto<sup>2</sup>

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**Chapter 6**

**Provisional chapter**

**Convenience of Applying of Viticulture Technique as a**

This study determined the effect that the irrigation dosage and cluster thinning showed over the antioxidant activities and total phenols in grapes, and phenolic profile and chromatic characteristics of cv. Tempranillo wines. The experiment was carried out over two consecutive seasons at an experimental vineyard in Extremadura (Spain). The plants were subjected to two post-*véraison* irrigation treatments, one at 100% (R100) and the other at 25% (R25) of crop evapotranspiration (ETc). Two crop loads (C and T) were additionally established for each irrigation treatment by cluster thinning at *véraison*. The winemaking process involved separate use of the grapes from each of the four resulting treatment groups (R100C, R100T, R25C and R25T) and followed a common protocol. In grapes, the effect of thinning enhanced when combined with water deficit, resulting in increases phenylpropanoids and flavonoids at the harvest, while leaving the polyphenol oxidase´s activity unaffected. In wines, the higher post-*véraison* water stress of the R25 treatments resulted in higher values of dimeric flavonol and flavanol concentrations. The wine hue was affected by water status. The cluster thinning caused additional increases in the concentrations of monomeric flava-

nols and anthocyanins, as well as resulting in stronger wine colour intensity.

**Keywords:** irrigation, crop load level, antioxidant activity, phenolic composition,

**Convenience of Applying of Viticulture Technique as a** 

DOI: 10.5772/intechopen.72799

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution,

© 2018 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

and reproduction in any medium, provided the original work is properly cited.

Because of the high dependence of fruit quality on various environmental and endogenous factors [1, 2], several agronomic techniques have been used to improve the quality of the berries. Appropriate viticultural practices that enhance ripening, by creating favourable mesoclimates,

**Function of the Water Status of the Vine-Stock**

**Function of the Water Status of the Vine-Stock**

Ester Gamero, Francisco Espinosa, Daniel Moreno,

Ester Gamero, Francisco Espinosa, Daniel Moreno, David Uriarte, María Henar Prieto, Inmaculada Garrido

Inmaculada Garrido and María Esperanza Valdés

Additional information is available at the end of the chapter

Additional information is available at the end of the chapter

David Uriarte, María Henar Prieto,

and María Esperanza Valdés

http://dx.doi.org/10.5772/intechopen.72799

**Abstract**

wine, grapes

**1. Introduction**


**Provisional chapter**

## **Convenience of Applying of Viticulture Technique as a Function of the Water Status of the Vine-Stock Function of the Water Status of the Vine-Stock**

**Convenience of Applying of Viticulture Technique as a** 

DOI: 10.5772/intechopen.72799

Ester Gamero, Francisco Espinosa, Daniel Moreno, David Uriarte, María Henar Prieto, Inmaculada Garrido and María Esperanza Valdés David Uriarte, María Henar Prieto, Inmaculada Garrido and María Esperanza Valdés Additional information is available at the end of the chapter

Ester Gamero, Francisco Espinosa, Daniel Moreno,

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/intechopen.72799

#### **Abstract**

[21] Teixeira AH de C, Hernandez FBT, Scherer-Warren M, Andtrade RG, Victoria D de C, Bolfe EL, Thenkabail PS, Franco RAM. Water productivity studies from earth observation data: Characterization, modeling, and mapping water use and water productivity. In: Prasad ST, editor. Remote Sensing of Water Resources, Disasters, and Urban Studies. Vol. III. 1st ed. Boca Raton: Taylor and Francis; 2015. pp. 101-126. ISBN: 9781482217919

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[24] Ferrer M, Pedocchi R, Michelazzo M, Gonzalez-Neves G, Carbonneau A. Delimitación y descripción de regiones vitícolas del Uruguay en base al método de clasificación climática multicriterio utilizando índices bioclimáticos adaptados a las condiciones del

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[27] Azevedo PV, Soares JM, Silva V de P, Silva BB, Nascimento T. Evapotranspiration of "superior" grapevines under intermitente irrigation. Agricultural Water Management.

[28] Teixeira AH de C, Scherer-Warren M, Hernandez FBT, Andrade RG, Leivas JF. Largescale water productivity assessments with MODIS images in a changing semi-arid environment: A Brazilian case study. Remote Sensing. 2013;**5**:5783-5804. DOI: 10.3390/

[29] Allen RG, Pereira LS, Raes D, Smith M. Crop Evapotranspiration: Guidelines for Computing Crop Water Requirements. Rome: Food and Agriculture Organization of the United

[30] Williams LE, Ayars JE. Grapevine water use and the crop coefficient are linear functions of the shaded area measured beneath the canopy. Agricultural and Forest Meteorology.

[31] Ortega-Farias S, Carrasco M, Olioso A, Acevedo C, Poblete C. Latent heat flux over Cabernet Sauvignon vineyard using the Shuttleworth and Wallace model. Irrigation

cultivo. Agrociencia. 2007;**11**:47-56. ISSN: 1405-3195

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2008;**95**:301-308. DOI: 10.1016/j.agwat.2007.10.011

2005;**132**:201-211. DOI: 10.1016/j.agrformet.2005.07.010

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agrformet.2003.06.001

10.1111/j.1755-0238.2011.00165.x

rs5115783

Nations; 1998. 300 p

This study determined the effect that the irrigation dosage and cluster thinning showed over the antioxidant activities and total phenols in grapes, and phenolic profile and chromatic characteristics of cv. Tempranillo wines. The experiment was carried out over two consecutive seasons at an experimental vineyard in Extremadura (Spain). The plants were subjected to two post-*véraison* irrigation treatments, one at 100% (R100) and the other at 25% (R25) of crop evapotranspiration (ETc). Two crop loads (C and T) were additionally established for each irrigation treatment by cluster thinning at *véraison*. The winemaking process involved separate use of the grapes from each of the four resulting treatment groups (R100C, R100T, R25C and R25T) and followed a common protocol. In grapes, the effect of thinning enhanced when combined with water deficit, resulting in increases phenylpropanoids and flavonoids at the harvest, while leaving the polyphenol oxidase´s activity unaffected. In wines, the higher post-*véraison* water stress of the R25 treatments resulted in higher values of dimeric flavonol and flavanol concentrations. The wine hue was affected by water status. The cluster thinning caused additional increases in the concentrations of monomeric flavanols and anthocyanins, as well as resulting in stronger wine colour intensity.

**Keywords:** irrigation, crop load level, antioxidant activity, phenolic composition, wine, grapes

## **1. Introduction**

Because of the high dependence of fruit quality on various environmental and endogenous factors [1, 2], several agronomic techniques have been used to improve the quality of the berries. Appropriate viticultural practices that enhance ripening, by creating favourable mesoclimates,

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2018 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

or by achieving adequate but not excessive vine vigour, will improve wine quality [3]. Soil water availability is a critical factor for vine performance and wine composition. Reports show that severe water stress can influence various physiological and metabolic plant processes, including growth, photosynthesis, and respiration and might be detrimental to fruit quality because of a poor canopy development and reduced leaf assimilation rate thus leading to an inadequate vine capacity to ripen the crop [4–6], particularly under high yield levels [7]. These, in turn, can affect the production, composition and characteristics of the must [8] and, in consequence, the quality of the resulting wine [9]. However, controversy remains as to whether there is any direct effect on berry metabolism other than inhibition of growth [10]. In semi-arid regions, irrigation is often applied as a technique to increase yield and in occasions to achieve a proper supply-demand vine balance. Regulated deficit irrigation (RDI) consists of applying short episodes of water restriction, typically starting after bloom (anthesis), whereby irrigation water is supplied at amounts below those lost to vineyard or crop evapotranspiration (ETc) [11]. Several studies have reported that water stress during the growing season has some beneficial effects (increasing anthocyanin and polyphenol concentrations and soluble solids content) on grape berry quality [12–15]. Water stress applied during the period from fruit set to *véraison*, heavily reduces fruit size [16] and in the late season water restriction may reduce fruit cell enlargement and water accumulation [17]. The timing and intensity of water deficits influence the extent of alterations occurring in berry metabolism and therefore in wine colour and flavour [18–20]. Irrigation is now a commonly used technique in vine-growing in Spain, where cv. Tempranillo is the most widespread cultivar for red wine production. Previous studies have been conducted to analyse the effect of irrigation in phenolic profile of grapes [9, 21–23]. In another hand, it is widely accepted that very high crop yields delay ripening and reduce fruit and wine quality [24]. This leads to increases in the required substrate levels for the synthesis and accumulation in thinned vine berries of, amongst others, phenolic substances [25–29]. Water restriction in Tempranillo grapes led to alterations in the levels of peroxidation and peroxidase activity [30].

as some compounds, such as the organic acids [39]. In addition, the result of cluster thinning can depend to a large extent on the climatological conditions during the season, with contrasting and even opposite results being obtained depending on the varieties and yields [36, 37]. The overall effect of irrigation might change according to other cultural practices particularly those affecting the crop level [24, 40]. Vines with higher crop level seem to benefit more of a higher amount of irrigation both in terms of yield [41] and of fruit composition [42]. Numerous studies exist in the effects of water deficit and control of yield by eliminating fruits (thinning) on berry composition, but less research has been developed to the combination of both techniques in its composition of wines. Determination of such relationships between composition and chromatic characteristics allow greater understanding of how interactions between cultural practices and chemical composition are reflected in the perception of colour. This type of study is particularly important in vineyards with resource limitations, as would be the case in deficit irrigated grapevines with high crop loads. Such studies are also relevant in relation to red grapevine cultivars, since the repercussions of these practices on the phenolic and chromatic characteristics of the wines obtained need to be analysed. Up to new, few studies have analysed how the combined practice of these techniques affects wine phenolic composition, and the results are not conclusive as to how the water status of

Convenience of Applying of Viticulture Technique as a Function of the Water Status of the…

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93

the vine impacts on the effect of cluster thinning or vice-versa [18, 19, 39, 43, 44].

concentrations of both stable pigments and substances that can act as cofactors.

In plants, increased synthesis of phenols is a common response to stress [52] and the same environmental conditions that cause oxidative stress are associated with the induction of phenylpropanoid metabolism. Phenylpropanoids and flavonoids are involved in the

The phenolic compounds from the grape are responsible for some of the main organoleptic characteristics of the resulting wine, including its colour, astringency and aroma. In the red wine making process, the phenolic compounds of the skin and seeds are transferred to the must during the fermentation and maceration process [45]. Phenolic compounds can be divided into flavonoid (anthocyanins, flavan-3-ols and flavonols) and non-flavonoid (stilbenes and *hydroxybenzoic and hydroxycinnamic* acids). Given the importance of colour in the quality of red wines, phenolic compound concentration can be used as a parameter for the evaluation of grape, must and wine quality [46]. Besides the variety, other factors such as location, climate, soil type, berry maturity and the vine-growing practices affect the phenolic composition of the grape berries [47]. Improved vine management techniques can therefore be used to modify phenolic content and enhance wine quality [48]. The question of grape berry size and its effect on wine composition has produced inconclusive results, and the relationship between grape berry size and the phenolic composition of wine remains unclear [49]. Wine colour is linked to the accumulation of anthocyanins in the grape berries and particularly in the skin. However, it is not only the anthocyanin concentration and profile that is responsible for wine colour: copigmentation can account for 30–50% of colour in young wines [50]. Copigmentation in wine results from molecular interactions between anthocyanic pigments and other organic molecules, called cofactors, which form molecular associations or complexes. For copigmentation in young wines to increase, there must be sufficient quantities of substances that can act as copigments. The most common cofactors are compounds such as phenolic acids, flavonoids, and in particular, flavonol and flavone derivatives [51]. Good quality grapes should have high

In another hand, it is widely accepted that very high crop yields delay ripening and reduce fruit and wine quality [24]. The crop level is a determining factor in berry quality and one of the goals of modern viticulture is to establish field practices which are able to limit vineyard yield and improve grapevine composition [31, 32]. The cluster thinning is a common practice designed to control yield and ripening under adverse conditions of climate. This practice influences the sink-source ratio, restricting part of the yield without lowering leaf surface area. Thinning allows the plant to concentrate its activity on the remaining clusters, making it an effective method of regulating production and modifying the composition of the berries [31, 33, 34]. Several authors have found that cluster thinning enhances berry ripening, affecting the contents of sugars, acids, polyphenols and aromas of the harvested grape and therefore positively affecting the quality of the wine [34, 35]. Cluster thinning is in cases applied to regulate the yield levels and to help ripening the crop under poor climatic conditions or excessive crop demand. However, results presented in literature have reported contrasting results, with cluster thinning leading to better fruit quality in some cases [36, 37], but with no clear effect in some others [33, 38]. Studies on cluster thinning have also shown the importance of the time when it is performed. When thinning is performed at *véraison*, the effect on berry weight and cluster size is lower than when performed immediately after fruit set, since it is at *véraison* that potential berry size and the number of berries per cluster are determined, as well as some compounds, such as the organic acids [39]. In addition, the result of cluster thinning can depend to a large extent on the climatological conditions during the season, with contrasting and even opposite results being obtained depending on the varieties and yields [36, 37].

or by achieving adequate but not excessive vine vigour, will improve wine quality [3]. Soil water availability is a critical factor for vine performance and wine composition. Reports show that severe water stress can influence various physiological and metabolic plant processes, including growth, photosynthesis, and respiration and might be detrimental to fruit quality because of a poor canopy development and reduced leaf assimilation rate thus leading to an inadequate vine capacity to ripen the crop [4–6], particularly under high yield levels [7]. These, in turn, can affect the production, composition and characteristics of the must [8] and, in consequence, the quality of the resulting wine [9]. However, controversy remains as to whether there is any direct effect on berry metabolism other than inhibition of growth [10]. In semi-arid regions, irrigation is often applied as a technique to increase yield and in occasions to achieve a proper supply-demand vine balance. Regulated deficit irrigation (RDI) consists of applying short episodes of water restriction, typically starting after bloom (anthesis), whereby irrigation water is supplied at amounts below those lost to vineyard or crop evapotranspiration (ETc) [11]. Several studies have reported that water stress during the growing season has some beneficial effects (increasing anthocyanin and polyphenol concentrations and soluble solids content) on grape berry quality [12–15]. Water stress applied during the period from fruit set to *véraison*, heavily reduces fruit size [16] and in the late season water restriction may reduce fruit cell enlargement and water accumulation [17]. The timing and intensity of water deficits influence the extent of alterations occurring in berry metabolism and therefore in wine colour and flavour [18–20]. Irrigation is now a commonly used technique in vine-growing in Spain, where cv. Tempranillo is the most widespread cultivar for red wine production. Previous studies have been conducted to analyse the effect of irrigation in phenolic profile of grapes [9, 21–23]. In another hand, it is widely accepted that very high crop yields delay ripening and reduce fruit and wine quality [24]. This leads to increases in the required substrate levels for the synthesis and accumulation in thinned vine berries of, amongst others, phenolic substances [25–29]. Water restriction in Tempranillo grapes led to alterations in the levels of peroxidation and peroxidase activity [30]. In another hand, it is widely accepted that very high crop yields delay ripening and reduce fruit and wine quality [24]. The crop level is a determining factor in berry quality and one of the goals of modern viticulture is to establish field practices which are able to limit vineyard yield and improve grapevine composition [31, 32]. The cluster thinning is a common practice designed to control yield and ripening under adverse conditions of climate. This practice influences the sink-source ratio, restricting part of the yield without lowering leaf surface area. Thinning allows the plant to concentrate its activity on the remaining clusters, making it an effective method of regulating production and modifying the composition of the berries [31, 33, 34]. Several authors have found that cluster thinning enhances berry ripening, affecting the contents of sugars, acids, polyphenols and aromas of the harvested grape and therefore positively affecting the quality of the wine [34, 35]. Cluster thinning is in cases applied to regulate the yield levels and to help ripening the crop under poor climatic conditions or excessive crop demand. However, results presented in literature have reported contrasting results, with cluster thinning leading to better fruit quality in some cases [36, 37], but with no clear effect in some others [33, 38]. Studies on cluster thinning have also shown the importance of the time when it is performed. When thinning is performed at *véraison*, the effect on berry weight and cluster size is lower than when performed immediately after fruit set, since it is at *véraison* that potential berry size and the number of berries per cluster are determined, as well

92 Grapes and Wines - Advances in Production, Processing, Analysis and Valorization

The overall effect of irrigation might change according to other cultural practices particularly those affecting the crop level [24, 40]. Vines with higher crop level seem to benefit more of a higher amount of irrigation both in terms of yield [41] and of fruit composition [42]. Numerous studies exist in the effects of water deficit and control of yield by eliminating fruits (thinning) on berry composition, but less research has been developed to the combination of both techniques in its composition of wines. Determination of such relationships between composition and chromatic characteristics allow greater understanding of how interactions between cultural practices and chemical composition are reflected in the perception of colour. This type of study is particularly important in vineyards with resource limitations, as would be the case in deficit irrigated grapevines with high crop loads. Such studies are also relevant in relation to red grapevine cultivars, since the repercussions of these practices on the phenolic and chromatic characteristics of the wines obtained need to be analysed. Up to new, few studies have analysed how the combined practice of these techniques affects wine phenolic composition, and the results are not conclusive as to how the water status of the vine impacts on the effect of cluster thinning or vice-versa [18, 19, 39, 43, 44].

The phenolic compounds from the grape are responsible for some of the main organoleptic characteristics of the resulting wine, including its colour, astringency and aroma. In the red wine making process, the phenolic compounds of the skin and seeds are transferred to the must during the fermentation and maceration process [45]. Phenolic compounds can be divided into flavonoid (anthocyanins, flavan-3-ols and flavonols) and non-flavonoid (stilbenes and *hydroxybenzoic and hydroxycinnamic* acids). Given the importance of colour in the quality of red wines, phenolic compound concentration can be used as a parameter for the evaluation of grape, must and wine quality [46]. Besides the variety, other factors such as location, climate, soil type, berry maturity and the vine-growing practices affect the phenolic composition of the grape berries [47]. Improved vine management techniques can therefore be used to modify phenolic content and enhance wine quality [48]. The question of grape berry size and its effect on wine composition has produced inconclusive results, and the relationship between grape berry size and the phenolic composition of wine remains unclear [49]. Wine colour is linked to the accumulation of anthocyanins in the grape berries and particularly in the skin. However, it is not only the anthocyanin concentration and profile that is responsible for wine colour: copigmentation can account for 30–50% of colour in young wines [50]. Copigmentation in wine results from molecular interactions between anthocyanic pigments and other organic molecules, called cofactors, which form molecular associations or complexes. For copigmentation in young wines to increase, there must be sufficient quantities of substances that can act as copigments. The most common cofactors are compounds such as phenolic acids, flavonoids, and in particular, flavonol and flavone derivatives [51]. Good quality grapes should have high concentrations of both stable pigments and substances that can act as cofactors.

In plants, increased synthesis of phenols is a common response to stress [52] and the same environmental conditions that cause oxidative stress are associated with the induction of phenylpropanoid metabolism. Phenylpropanoids and flavonoids are involved in the protection against oxidative stress [53]. The level of lipid peroxidation and how the total antioxidant capacity and the phenolic compound content evolve can both be used as indicators of stress and the response to it. One response to stressors during ripening is an increase in the amount of phenolic compounds. These include flavonoids and phenylpropanoid glycosides, compounds that are part of the cellular antioxidant system, and which are involved in the elimination of ROS [52–55].

48 vines across 6 rows. The experiment comprised 768 vines in total. The irrigation regimes,

Convenience of Applying of Viticulture Technique as a Function of the Water Status of the…

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95

ETc was calculated using the equation ETc = ETo × Kc, where ETc was estimated as the product of reference evapotranspiration (ETo), measured by a weather station at the site, and crop coefficient (Kc), following the methodology of Allen et al. [62]. Irrigation was initiated when stem water potential (Ψs) reached −0.5 MPa and stopped after harvest, in mid-September. Irrigation was applied with pressure-compensated emitters supplying 4 L h−1 and spaced

• Cluster-thinning treatments (T), in which the load was adjusted to 4–5 clusters m−2 of plant-

**Table 1** represents the ETo, ETc and Kc, as well as the irrigation amount applied in the R100 and R25 treatments for each of the study years. Also shown is the water status of the plant during the growing period in the two campaigns (stem water potential during the flowering-

**Table 2** represents the physico-chemical characteristics of the grapes at harvest and the chromatic characteristics of the wines. The winemaking protocol and analytic methodology can be

Phenols, flavonoids and phenylpropanoid glycosides were assayed colourimetrically. First, grapes were homogenised with methanol, chloroform and 1% NaCl (1:1:0.5). The homogenate was filtered and centrifuged at 3200 × g for 10 min. Total phenols (expressed as μg caffeic acid g−1 FW) were determined at 765 nm with Folin:Ciocalteu reagent according to the method of singleton et al. [63]. Total flavonoids (expressed as μg rutin g−1 FW) were determined according to the method of Kim et al. [64], calculating the content on the basis of the rutin standard curve. Phenylpropanoids (expressed as μg verbascoside g−1 FW) were determined at 525 nm based on estimating an *O*-dihydroxycinnamic derivative using the Arnow reagent as described in Gálvez et al. [65], calculating the content on the basis of the 3,4-dihydroxyphe-

Lipid peroxidation was determined by measuring malondialdehyde (MDA) formation using the thiobarbituric acid (TBA) method as described by Madhava Rao and Sresty [66]. The MDA

• Full irrigation (R100), corresponding to 100% of crop evapotranspiration (ETc).

120 cm apart. Two cluster load levels were established for each irrigation regime:

• Control treatment (C) without cluster thinning (7–9 clusters m−2 of planting area)

**2.2. Physico-chemical composition of grapes and wine chromatic characteristics**

**2.3. Biochemical assay and enzymatic activities of the grapes**

applied during *véraison* and maturation, were as follows:

• Deficit irrigation (R25) corresponding to 25% of ETc

ing area by removing clusters at *véraison*.

*véraison* and *véraison*-harvest periods) [18].

consulted in Gamero et al. [19].

nylalanine standard curve.

ROS have been implicated in plant growth and stress responses. The production and detoxification of ROS are both highly regulated processes, and ROS levels are kept under tight control. The antioxidant system plays an important part in ROS homeostasis [56, 57]. It includes such enzymes as peroxidase (POX), catalase (CAT) and superoxide dismutase (SOD). Stresses and physiological processes in plants both induce the development of a complex network of oxidant/antioxidant reactions that modulate the resulting oxidative response [58, 59]. Thus, a key role in the response to stresses is played by the activities involved in ROS production and elimination, and they are also involved in such physiological processes as ripening [60]. In this regard, while the participation of peroxidases, catalases and superoxide dismutase is crucial for the control of the production or elimination of ROS, their activities are affected by water stress and defoliation [30].

Tempranillo is the most common variety of red grape in Spain comprising a total of 205,975 ha (20% of Spain's total vineyard area), though most of these are cultivated under rainfed conditions with typically low yields (≤ 6500 kg ha−1). This cultivar is originally from northern, cool regions of Spain and today is the most widely cultivated for production of red wines all over Spain. It is reputedly to be sensitive to water stress and prone to early leaf senescence [61].

This chapter analyses and studies the effects of the combination of water deficit and thinning on the constitution and composition of the Tempranillo grape, namely on the lipid peroxidation, oxidant and antioxidant enzymes activities and their interactions on phenolic profile and chromatic characteristic from the wines. Also, relations between wine chromatic parameters and phenolic substances have been investigated.

## **2. Materials and methods**

#### **2.1. Vineyard site and experimental design**

The experiment was conducted in a *Vitis vinifera* L. cv. Tempranillo vineyard in Finca La Orden (owned by the Regional Government of Extremadura) in Extremadura, Spain, over the 2007 and 2008 vintages. The vineyard was planted in 2001 on Richter 110 rootstock at a spacing of 2.5 by 1.2 m (3333 vines ha−1). Row orientation was north-south and vines were trained to a bilateral cordon system and vertical trellis.

The experimental design was a complete randomised block with 16 experimental plots, 4 replicates by 4 treatments (irrigation and cluster thinning). Experimental plots consisted of 48 vines across 6 rows. The experiment comprised 768 vines in total. The irrigation regimes, applied during *véraison* and maturation, were as follows:


protection against oxidative stress [53]. The level of lipid peroxidation and how the total antioxidant capacity and the phenolic compound content evolve can both be used as indicators of stress and the response to it. One response to stressors during ripening is an increase in the amount of phenolic compounds. These include flavonoids and phenylpropanoid glycosides, compounds that are part of the cellular antioxidant system, and which are involved

ROS have been implicated in plant growth and stress responses. The production and detoxification of ROS are both highly regulated processes, and ROS levels are kept under tight control. The antioxidant system plays an important part in ROS homeostasis [56, 57]. It includes such enzymes as peroxidase (POX), catalase (CAT) and superoxide dismutase (SOD). Stresses and physiological processes in plants both induce the development of a complex network of oxidant/antioxidant reactions that modulate the resulting oxidative response [58, 59]. Thus, a key role in the response to stresses is played by the activities involved in ROS production and elimination, and they are also involved in such physiological processes as ripening [60]. In this regard, while the participation of peroxidases, catalases and superoxide dismutase is crucial for the control of the production or elimination of ROS, their activities are affected by water stress

Tempranillo is the most common variety of red grape in Spain comprising a total of 205,975 ha (20% of Spain's total vineyard area), though most of these are cultivated under rainfed conditions with typically low yields (≤ 6500 kg ha−1). This cultivar is originally from northern, cool regions of Spain and today is the most widely cultivated for production of red wines all over Spain. It is reputedly to be sensitive to water stress and prone to early leaf

This chapter analyses and studies the effects of the combination of water deficit and thinning on the constitution and composition of the Tempranillo grape, namely on the lipid peroxidation, oxidant and antioxidant enzymes activities and their interactions on phenolic profile and chromatic characteristic from the wines. Also, relations between wine chromatic parameters

The experiment was conducted in a *Vitis vinifera* L. cv. Tempranillo vineyard in Finca La Orden (owned by the Regional Government of Extremadura) in Extremadura, Spain, over the 2007 and 2008 vintages. The vineyard was planted in 2001 on Richter 110 rootstock at a spacing of 2.5 by 1.2 m (3333 vines ha−1). Row orientation was north-south and vines were trained to a

The experimental design was a complete randomised block with 16 experimental plots, 4 replicates by 4 treatments (irrigation and cluster thinning). Experimental plots consisted of

in the elimination of ROS [52–55].

94 Grapes and Wines - Advances in Production, Processing, Analysis and Valorization

and defoliation [30].

senescence [61].

and phenolic substances have been investigated.

**2.1. Vineyard site and experimental design**

bilateral cordon system and vertical trellis.

**2. Materials and methods**

ETc was calculated using the equation ETc = ETo × Kc, where ETc was estimated as the product of reference evapotranspiration (ETo), measured by a weather station at the site, and crop coefficient (Kc), following the methodology of Allen et al. [62]. Irrigation was initiated when stem water potential (Ψs) reached −0.5 MPa and stopped after harvest, in mid-September. Irrigation was applied with pressure-compensated emitters supplying 4 L h−1 and spaced 120 cm apart. Two cluster load levels were established for each irrigation regime:


**Table 1** represents the ETo, ETc and Kc, as well as the irrigation amount applied in the R100 and R25 treatments for each of the study years. Also shown is the water status of the plant during the growing period in the two campaigns (stem water potential during the flowering*véraison* and *véraison*-harvest periods) [18].

#### **2.2. Physico-chemical composition of grapes and wine chromatic characteristics**

**Table 2** represents the physico-chemical characteristics of the grapes at harvest and the chromatic characteristics of the wines. The winemaking protocol and analytic methodology can be consulted in Gamero et al. [19].

#### **2.3. Biochemical assay and enzymatic activities of the grapes**

Phenols, flavonoids and phenylpropanoid glycosides were assayed colourimetrically. First, grapes were homogenised with methanol, chloroform and 1% NaCl (1:1:0.5). The homogenate was filtered and centrifuged at 3200 × g for 10 min. Total phenols (expressed as μg caffeic acid g−1 FW) were determined at 765 nm with Folin:Ciocalteu reagent according to the method of singleton et al. [63]. Total flavonoids (expressed as μg rutin g−1 FW) were determined according to the method of Kim et al. [64], calculating the content on the basis of the rutin standard curve. Phenylpropanoids (expressed as μg verbascoside g−1 FW) were determined at 525 nm based on estimating an *O*-dihydroxycinnamic derivative using the Arnow reagent as described in Gálvez et al. [65], calculating the content on the basis of the 3,4-dihydroxyphenylalanine standard curve.

Lipid peroxidation was determined by measuring malondialdehyde (MDA) formation using the thiobarbituric acid (TBA) method as described by Madhava Rao and Sresty [66]. The MDA


Effective seasonal rain, reference evapotranspiration (ETo), crop evapotranspiration (ETc), crop coefficient (Kc) from April to September and mean values in each season of stem water potential (Ψs). Irrigation: Volumes of water applied in the 2007 and 2008 treatments. In 2007 and 2008, the ETc values were obtained by means of a lysimeter installed in the vineyard.1 R100 = Full irrigation (100% ETc); R25 = Water deficit (25% ETc)

**Table 1.** Water balance variables in Tempranillo vineyard, Extremadura (Spain).

concentration (expressed as nmol MDA g−1 FW) was calculated using an extinction coefficient of ε = 155 mM−1 cm−1.

Enzymatic activities were determined on a crude extract of the grapes. The grapes (2 g mL−1) were homogenised at 4°C in 50 mM phosphate buffer, pH 6.0. The homogenate was filtered and centrifuged at 39,000 × g for 30 min at 4°C. The pellet was discarded, and the supernatant filtered and collected for the enzyme assays. The protein content was determined by the method of Bradford [67].

Peroxidase (EC 1.11.1.7) activity, POX, was measured at 590 nm (ε = 47.6 mM−1 cm−1) [68], and the coniferyl alcohol (CA) peroxidase activity, CA-POX, was determined by measuring the decrease in absorbance at 265 nm of a reaction medium consisting of the enzyme extract and 0.1 mM CA in 25 mM acetate buffer pH 5.0 (ε = 7.5 mM−1 cm−1). A unit of CA-POX is defined as the amount of enzyme required to cause the oxidation of 1 nmol CA per minute at 25°C, pH 5.0. The superoxide dismutase (SOD) (EC 1.15.1.1) activity was determined from the absorbance at 560 nm according to [69]. A unit of SOD is defined as the amount of enzyme required to cause 50% inhibition of NBT reduction. Polyphenol oxidase (PPO) (EC 1.14.18.1) activity was determined from the absorbance at 390 nm [70]. A unit of PPO is defined as the amount of enzyme required to cause a decrease in absorption of 0.001 units min−1.

#### **2.4. Individual phenolic composition of the grapes analysed by HPLC-DAD-FLD**

Low molecular weight phenols were analysed by HPLC-DAD, following the method described in Gómez-Alonso [71], though with some slight modifications to enhance the resolution. After filtering (0.25 m diameter Chromafil filters, Düren, Germany), 10 μl of wine were directly injected into a LC-Agilent 1200 (Agilent Technologies, Palo Alto, CA, USA) chromatographic system. Separation was performed in an Ace® 5 C18 250 × 4.6 mm (Advanced Chromatography Technologies, Aberdeen, Scotland) column used as stationary phase.

The various phenolic compounds were identified by order of elution and the retention times of their respective standards. A total of 42 phenolic compounds were identified, distributed between simple and conjugated anthocyanins and monomeric and dimeric flavonols and flavanols. The anthocyanins were identified in the monoglucoside, acetyl-glucoside and *p*-coumaroyl-glucoside forms of delphinidin, cyanidin, petunidin, peonidin and malvidin. The quantified flavanols were (+)-catechin, (−)-epicatechin and the procyanidins (B1, B2 and B3). The analysed flavonols were myricetin, quercetin, kaempherol and isorhamnetin in free form

**Table 2.** Mean values of 2007 and 2008 seasons combined for yield, berry composition and wine chromatic characteristics of cv. Tempranillo wines, subjected to different irrigation dosage levels with cluster-thinning treatment (T) and without

**Parameters Treatment Irrigation dosage1,3 Significance level<sup>2</sup>**

Yield (kg ha−1) C 21403.75a 18749.58a **ns \*\* ns**

Berry weight (g) C 2.17a 2.00<sup>b</sup> **ns \*\* ns**

Total soluble solids (°Brix) C 22.75ab 21.93<sup>b</sup> **ns \*\*\* \***

Titratable acidity (g tartaric acid L−1) C 6.43a 5.51ab **\*\*\* \*\*\* ns**

Maturity index<sup>4</sup> C 3.60<sup>b</sup> 4.08<sup>b</sup> **\* \*\* \***

Colour intensity (AU) C 6.69<sup>b</sup> 6.71<sup>b</sup> **ns \*\*\* \*\*\***

Colour hue C 0.65a 0.62<sup>b</sup> **\* ns ns**

% red C 52.81<sup>b</sup> 54.00a **\*\*\* ns ns**

% blue C 13.00a 12.31<sup>b</sup> **\* ns ns**

% yellow C 34.18a 33.67<sup>b</sup> **\* ns ns**

deficit without cluster thinning and R25A = Water deficit with cluster thinning

Significance level: ns = not significant; \* = *p* ≤ 0.05; \*\* = *p* ≤ 0.01; \*\*\* = *p* ≤ 0.001

Maturity index = Total soluble solids/titratable acidity

cluster-thinning treatment (C) in Extremadura (Spain).

Values followed by different letters indicate significant differences between them (*p* ≤ 0.5)

1

2

3

4

T 14987.03<sup>b</sup> 14704.16<sup>b</sup>

Convenience of Applying of Viticulture Technique as a Function of the Water Status of the…

T 2.03<sup>b</sup> 1.90<sup>b</sup>

T 23.87ab 24.37a

T 5.12ab 4.32<sup>b</sup>

T 4.66ab 5.81a

T 9.51a 9.70a

T 0.67a 0.60<sup>b</sup>

T 52.06<sup>b</sup> 54.88a

T 13.20a 12.08<sup>b</sup>

T 34.73a 33.02<sup>b</sup>

R100C = Full irrigation (R100) without cluster thinning (C); R100A = Full irrigation with cluster thinning; R25C = Water

**R100 R25 R T R × T**

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1 R100C = Full irrigation (R100) without cluster thinning (C); R100A = Full irrigation with cluster thinning; R25C = Water deficit without cluster thinning and R25A = Water deficit with cluster thinning 2 Significance level: ns = not significant; \* = *p* ≤ 0.05; \*\* = *p* ≤ 0.01; \*\*\* = *p* ≤ 0.001 3 Values followed by different letters indicate significant differences between them (*p* ≤ 0.5)

4 Maturity index = Total soluble solids/titratable acidity

concentration (expressed as nmol MDA g−1 FW) was calculated using an extinction coefficient

**(mm)**

2008 129 1026 755 0.74 611 153 −0.52 −0.76 −0.49 −0.89

Effective seasonal rain, reference evapotranspiration (ETo), crop evapotranspiration (ETc), crop coefficient (Kc) from April to September and mean values in each season of stem water potential (Ψs). Irrigation: Volumes of water applied in the 2007 and 2008 treatments. In 2007 and 2008, the ETc values were obtained by means of a lysimeter installed in the

**Ψs flowering-***véraison*

**Kc R1001 R25 R100 R25 R100 R25**

**Ψs** *véraison***-harvest** 

**(MPa)**

−0.58 −0.56 −0.77

**(MPa)**

−0.81–0.51

Enzymatic activities were determined on a crude extract of the grapes. The grapes (2 g mL−1) were homogenised at 4°C in 50 mM phosphate buffer, pH 6.0. The homogenate was filtered and centrifuged at 39,000 × g for 30 min at 4°C. The pellet was discarded, and the supernatant filtered and collected for the enzyme assays. The protein content was determined by the

Peroxidase (EC 1.11.1.7) activity, POX, was measured at 590 nm (ε = 47.6 mM−1 cm−1) [68], and the coniferyl alcohol (CA) peroxidase activity, CA-POX, was determined by measuring the decrease in absorbance at 265 nm of a reaction medium consisting of the enzyme extract and 0.1 mM CA in 25 mM acetate buffer pH 5.0 (ε = 7.5 mM−1 cm−1). A unit of CA-POX is defined as the amount of enzyme required to cause the oxidation of 1 nmol CA per minute at 25°C, pH 5.0. The superoxide dismutase (SOD) (EC 1.15.1.1) activity was determined from the absorbance at 560 nm according to [69]. A unit of SOD is defined as the amount of enzyme required to cause 50% inhibition of NBT reduction. Polyphenol oxidase (PPO) (EC 1.14.18.1) activity was determined from the absorbance at 390 nm [70]. A unit of PPO is defined as the amount of enzyme required to cause a decrease in absorp-

**2.4. Individual phenolic composition of the grapes analysed by HPLC-DAD-FLD**

Low molecular weight phenols were analysed by HPLC-DAD, following the method described in Gómez-Alonso [71], though with some slight modifications to enhance the resolution. After filtering (0.25 m diameter Chromafil filters, Düren, Germany), 10 μl of wine were directly injected into a LC-Agilent 1200 (Agilent Technologies, Palo Alto, CA, USA) chromatographic system. Separation was performed in an Ace® 5 C18 250 × 4.6 mm (Advanced Chromatography Technologies, Aberdeen, Scotland) column used as station-

of ε = 155 mM−1 cm−1.

vineyard.1

**Rainfall (mm)**

**Year Seasonal measurements Irrigation** 

**ETc (mm)**

2007 182 975 508 0.52 172 62 −0.54

96 Grapes and Wines - Advances in Production, Processing, Analysis and Valorization

R100 = Full irrigation (100% ETc); R25 = Water deficit (25% ETc)

**Table 1.** Water balance variables in Tempranillo vineyard, Extremadura (Spain).

**ETo (mm)**

method of Bradford [67].

tion of 0.001 units min−1.

ary phase.

**Table 2.** Mean values of 2007 and 2008 seasons combined for yield, berry composition and wine chromatic characteristics of cv. Tempranillo wines, subjected to different irrigation dosage levels with cluster-thinning treatment (T) and without cluster-thinning treatment (C) in Extremadura (Spain).

The various phenolic compounds were identified by order of elution and the retention times of their respective standards. A total of 42 phenolic compounds were identified, distributed between simple and conjugated anthocyanins and monomeric and dimeric flavonols and flavanols. The anthocyanins were identified in the monoglucoside, acetyl-glucoside and *p*-coumaroyl-glucoside forms of delphinidin, cyanidin, petunidin, peonidin and malvidin. The quantified flavanols were (+)-catechin, (−)-epicatechin and the procyanidins (B1, B2 and B3). The analysed flavonols were myricetin, quercetin, kaempherol and isorhamnetin in free form and their respective 3-glucosides. Also quantified were the quercetin and kaempherol rutinosides, quercetin-3-glucuronide and quercetin-3-galactoside.

The concentration was calculated in mg L−1 of malvidin-3-glucoside, myricetin-3-glucoside and catechin for anthocyanins, flavonols and flavanols, respectively.

#### **2.5. Wine colour composition**

Wine colour was evaluated using the indices proposed by Glories [72]: colour intensity (CI) was calculated by adding the absorbance readings at 420, 520 and 620 nm, while hue (CT) was determined as the ratio of absorbance readings at 420 and 520 nm, and percentages of red, blue and yellow (red %, blue % and yellow %). The wines were centrifuged for 3 min at 1100 × g before spectrophotometric analysis. Measurements were taken using a Shimadzu UV-1700 spectrophotometer (Shimadzu, Japan).

#### **2.6. Statistical analysis**

Data were analysed using a general linear model, with water status (two levels), cluster thinning (two levels) and their interactions as factors. Differences between treatments were obtained using Tukey's test for significant differences (*p* ≤ 0.05).

A principal component analysis (PCA) was carried out to observe the distribution of the wines of the different treatments as a function of their phenolic composition. A correlation analysis was performed to determine the relationships between phenolic compounds and agronomic factors and Tukey's test was used to evaluate significant correlations (*p* < 0.05).

The partial least squares (PLS) approach was used to analyse the relationships between wine colour and HPLC-quantified phenolic compounds. Collinearity between X variables was discarded as significant correlations were not found between them. The statistical analyses were performed with the XLSTAT-Pro (Addinsoft 2009, París, France) statistical software package.

## **3. Results**

#### **3.1. Effect of treatments on the phenolic content and oxidant/antioxidant activities in grapes**

**Table 3** shows the different values obtained for oxidant and antioxidant activities, and phenolic content in grapes at harvest. With thinning, all these phenolic components were found in higher or equal concentrations, but not lower. The greater value of lipid peroxidation corresponds to CT (rainfed and thinned). The SOD activity was always lower than in the unthinned treatments. It stands out that the lowest values of SOD activity corresponded to CT. Regarding the activity of PPO, an enzyme which catalyses the formation of *o*-quinones, at harvest, the PPO activity levels were similar in all treatments, although showing a minimum value in CT. The lowest POX values correspond to the thinning treatments (CT and R100T). The activity levels of CA-POX, an enzyme which is involved in processes of lignification, were slightly higher with thinning than without thinning.

**Treatment\* Lipid**  **peroxidation**

**Superoxide** 

**Peroxidase** 

**Conyferilalcohol** 

**Polyphenoloxidase** 

**Total Phenols**

**Total** 

**Total flavonoids**

**μg rutín g−1 FW**

**Phenylpropanoid** 

**glycosides**

**μg verbascoside g−1**

**FW**

**μg caffeic acid** 

**g−1 FW**

**(PPO)**

**peroxidase**

**dismutase** 

**(POX)**

**U mg−1 prot**

**(CA-POX)**

**U mg−1 prot**

**U mg−1 prot**

**(SOD)**

**nmol MDA** 

**g−1 FW**

C CT R100C R100T

91.4 ± 24.3b

Data were the means

 ± column indicate significant difference, *p*

Full irrigation with cluster thinning

R100T =

**Table 3.** Tempranillo at harvest.

 ≤ 0.05)1

C =

Rainfed without cluster thinning; R100C

 = Average values (2007 and 2008 vintage) of lipid peroxidation, antioxidant activities, total phenols, phenylpropanoid glycosides and flavonoids on grapes cv.

Full irrigation without cluster thinning; CT

 =

Rainfed with cluster thinning;

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81.5 ± 20.1b

2.6 ± 1.0a

79.0 ± 22.1a

1389.0 ± 340.0b SD of at least 10 replicates obtained from 5 different experiments. The values were subjected to the Tukey test (different letters within each

966.0 ± 144.5a

1924.4 ± 171.3b

88.3 ± 18.4b

158.7 ± 22.8a

5.2 ± 1.2b

78.2 ± 20.8a

1510–3 ± 312.5b

1045.6 ± 100.1a

1865.0 ± 366.0ab

130.1 ± 23.0a

71.6 ± 18.0b

4.2 ± 1.5b

66.2 ± 19.3ab

900.3 ± 189.0a

1108.9 ± 158.0a

2013.5 ± 188.3b

94.6 ± 20.5b

89.3 ± 21.7b

4.9 ± 1.2b

53.6 ± 16.5b

1415.5 ± 615.4b

988.6 ± 142.2a

1689.9 ± 290.5b

1122.3 ± 211.0b 1589.7 ± 132.4a

1325.6 ± 126.1ab

1159.0 ± 206.6b

**U mg−1 prot**


and their respective 3-glucosides. Also quantified were the quercetin and kaempherol rutino-

The concentration was calculated in mg L−1 of malvidin-3-glucoside, myricetin-3-glucoside

Wine colour was evaluated using the indices proposed by Glories [72]: colour intensity (CI) was calculated by adding the absorbance readings at 420, 520 and 620 nm, while hue (CT) was determined as the ratio of absorbance readings at 420 and 520 nm, and percentages of red, blue and yellow (red %, blue % and yellow %). The wines were centrifuged for 3 min at 1100 × g before spectrophotometric analysis. Measurements were taken using a Shimadzu

Data were analysed using a general linear model, with water status (two levels), cluster thinning (two levels) and their interactions as factors. Differences between treatments were

A principal component analysis (PCA) was carried out to observe the distribution of the wines of the different treatments as a function of their phenolic composition. A correlation analysis was performed to determine the relationships between phenolic compounds and agronomic

The partial least squares (PLS) approach was used to analyse the relationships between wine colour and HPLC-quantified phenolic compounds. Collinearity between X variables was discarded as significant correlations were not found between them. The statistical analyses were performed with the XLSTAT-Pro (Addinsoft 2009, París, France) statistical software package.

**3.1. Effect of treatments on the phenolic content and oxidant/antioxidant activities in grapes**

**Table 3** shows the different values obtained for oxidant and antioxidant activities, and phenolic content in grapes at harvest. With thinning, all these phenolic components were found in higher or equal concentrations, but not lower. The greater value of lipid peroxidation corresponds to CT (rainfed and thinned). The SOD activity was always lower than in the unthinned treatments. It stands out that the lowest values of SOD activity corresponded to CT. Regarding the activity of PPO, an enzyme which catalyses the formation of *o*-quinones, at harvest, the PPO activity levels were similar in all treatments, although showing a minimum value in CT. The lowest POX values correspond to the thinning treatments (CT and R100T). The activity levels of CA-POX, an enzyme which is involved in processes of lignification, were

factors and Tukey's test was used to evaluate significant correlations (*p* < 0.05).

sides, quercetin-3-glucuronide and quercetin-3-galactoside.

98 Grapes and Wines - Advances in Production, Processing, Analysis and Valorization

UV-1700 spectrophotometer (Shimadzu, Japan).

obtained using Tukey's test for significant differences (*p* ≤ 0.05).

slightly higher with thinning than without thinning.

**2.5. Wine colour composition**

**2.6. Statistical analysis**

**3. Results**

and catechin for anthocyanins, flavonols and flavanols, respectively.

**Table 3.** Average values (2007 and 2008 vintage) of lipid peroxidation, antioxidant activities, total phenols, phenylpropanoid glycosides and flavonoids on grapes cv. Tempranillo at harvest.

Full irrigation with cluster thinning

R100T =

#### **3.2. Effect of treatments on the phenolic profile of wines**

**Table 4A** and **B** shows the mean concentration values over the 2 year study period of anthocyanin and non-anthocyanin flavonoid substances (flavanols and flavonols) of the R100C, R100T, R25C and R25T wines, as well as the results of the statistical analysis.

**Table 4A** shows the amount of monoglucosides (G), acetates (A) and coumaroyl derivatives (C) and the amounts of delphinidin (Dp), cyanidin (Cy), petunidin (Pt), peonidin (Pn) and malvidin (Mv) compounds.

In the case of the flavanols (**Table 4B**), as well as the mean concentration values of (+)-catechin (Cat) and (−)-epicatechin (Ep), also shown are the global mean values of these monomers (Mon) and, as well as the individual mean of (−)-epicatechin-(4ß-8)-(+)-catechin (PB1), (−)-epicatechin- (4ß-8)-(−)-epicatechin (PB2) and (+)-catechin-(4ß-8)-(+)-catechin (PB3) values, the combined mean values of these dimers (Dim). The flavonols are grouped together by the flavonol molecule present, such that myricetin (My), quercetin (Qc), kaempherol (Kp) and isorhamnetin (Ir) glucosides represent the total values, respectively.

Results of the general linear model analysis only found statistical significance of the irrigation and thinning interaction in Cy, PB1, PB3, Dim and total flavonoid substances. A statistical analysis was then performed of the overall effect of irrigation dosage and its individual effect on each crop load, as well as of the overall effect of cluster thinning and its effect on each water status.

#### *3.2.1. Effect of irrigation*

As can be seen in **Table 4A**, the different irrigation dosages had almost no effect on wine anthocyanin concentration values. Slightly lower concentrations of these substances were found in the wine from R25 treatment compared to the wine from R100 treatment, but the differences were non-significant (*p* > 0.05). This trend was also observed when studying separately the effect of irrigation dosage on the different crop loads (R100T vs. R25T and R100C vs. R25C).

However, the opposite trend was observed for the rest of the flavonoid families (flavanols and flavonols). The R25 wines contained significantly higher concentrations of Cat, the flavonol compounds My, Qc, Kp and Ir, Mon and total flavanols (**Table 4B**). An analysis of the effect of irrigation dosage on the different crop loads revealed significant differences only in the wines from the cluster-thinned treatments (R25T vs. R100T).

the concentration of anthocyanin substances present in the wines. In addition, when compared to the C-treatment wines, the T-treatment wines had lower concentrations (*p* < 0.001) of

**Table 4A** and **B** also shows the results of the combined statistical analysis of the four treatments. In concordance with the results described above, the highest and lowest values for anthocyanin substances were found in the 100T and 25C wines, respectively. Of these substances, Cy (the sum total of cyanidin compounds) is a special case as both the lower irrigation

Cat, and higher concentrations of My and Qc (**Table 4B**).

**Compound Load Water status2 Significance level<sup>1</sup>**

**T** 111.08a 100.19a

**T** 11.28a 11.2a

**T** 14.45a 14.32ab

**T** 100.35a 92.06a

T 18.73a 17.58ab

T 11.80a 11.06ab

T 3.93a 3.49a

T 2.01<sup>b</sup> 1.53<sup>b</sup>

T 136.82a 125.67a

**Table 4A.** Average values (2007 and 2008 vintage) of anthocyanins (mg L−1

significant; \* = *p* ≤ 0.05; \*\* = *p* ≤ 0.01; \*\*\* = *p* ≤ 0.001

R T Irrigation Thinning R and T

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) in Tempranillo wines from vines subjected to

**R100 R25 in C in T in R25 in R100**

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**∑ Glucosides C** 90.21ab 83.35<sup>b</sup> **ns \* ns ns ns \*\* \***

**∑ Acetates C** 11.96a 10.26a **ns ns ns ns ns ns ns**

**∑ Cumarates C** 10.18<sup>b</sup> 11.65<sup>b</sup> **ns \*\* ns ns ns ns \***

**∑ Malvidins C** 82.88a 79.59a **ns \*\* ns ns ns \*\* ns**

**∑ Petunidins C** 15.04ab 13.23<sup>b</sup> **ns \*\*\* ns ns ns \*\* \***

**∑ Delphinids** C 7.97<sup>b</sup> 8.11ab **ns** \*\*\* **ns ns ns** \*\*\* **\*\***

**∑ Peonidins** C 3.32a 2.62a **ns** \* **ns ns ns** ns **ns**

**∑ Cyanidins** C 3.15a 1.70<sup>b</sup> **Significant interaction** \*

**Total Anthocyanins** C 112.36a 105.25a **ns** \*\* **ns ns ns** \* **ns**

ANOVA test significance level. R: irrigation; A: cluster thinning; R and A: combination of both treatments. ns = not

For the same compound, values followed by different letters indicate significant differences between treatments (*p* ≤ 0.5)

different irrigation dosages and with different crop level loads, cluster thinned (T) and non-thinned (C), in Extremadura

*3.2.3. Effect of treatments*

1

2

(Spain).

#### *3.2.2. Effect of cluster thinning*

In terms of wine anthocyanin composition, the effect of thinning was greater than of irrigation, with the A-treatment wines having significantly higher values of all the substances of this family, except for the acetyl-acetate derivatives. The biggest differences were found in the G (18%), Mv (15%) and Dp (29%). **Table 4A** also shows the separate analysis made of the effect of thinning on the R25 and R100 wines, with a greater and more significant response in R100. These results confirm the importance of vine water status in the effect of cluster thinning on


1 ANOVA test significance level. R: irrigation; A: cluster thinning; R and A: combination of both treatments. ns = not significant; \* = *p* ≤ 0.05; \*\* = *p* ≤ 0.01; \*\*\* = *p* ≤ 0.001

2 For the same compound, values followed by different letters indicate significant differences between treatments (*p* ≤ 0.5)

**Table 4A.** Average values (2007 and 2008 vintage) of anthocyanins (mg L−1 ) in Tempranillo wines from vines subjected to different irrigation dosages and with different crop level loads, cluster thinned (T) and non-thinned (C), in Extremadura (Spain).

the concentration of anthocyanin substances present in the wines. In addition, when compared to the C-treatment wines, the T-treatment wines had lower concentrations (*p* < 0.001) of Cat, and higher concentrations of My and Qc (**Table 4B**).

#### *3.2.3. Effect of treatments*

**3.2. Effect of treatments on the phenolic profile of wines**

100 Grapes and Wines - Advances in Production, Processing, Analysis and Valorization

(Ir) glucosides represent the total values, respectively.

from the cluster-thinned treatments (R25T vs. R100T).

malvidin (Mv) compounds.

*3.2.1. Effect of irrigation*

R100C vs. R25C).

*3.2.2. Effect of cluster thinning*

**Table 4A** and **B** shows the mean concentration values over the 2 year study period of anthocyanin and non-anthocyanin flavonoid substances (flavanols and flavonols) of the R100C,

**Table 4A** shows the amount of monoglucosides (G), acetates (A) and coumaroyl derivatives (C) and the amounts of delphinidin (Dp), cyanidin (Cy), petunidin (Pt), peonidin (Pn) and

In the case of the flavanols (**Table 4B**), as well as the mean concentration values of (+)-catechin (Cat) and (−)-epicatechin (Ep), also shown are the global mean values of these monomers (Mon) and, as well as the individual mean of (−)-epicatechin-(4ß-8)-(+)-catechin (PB1), (−)-epicatechin- (4ß-8)-(−)-epicatechin (PB2) and (+)-catechin-(4ß-8)-(+)-catechin (PB3) values, the combined mean values of these dimers (Dim). The flavonols are grouped together by the flavonol molecule present, such that myricetin (My), quercetin (Qc), kaempherol (Kp) and isorhamnetin

Results of the general linear model analysis only found statistical significance of the irrigation and thinning interaction in Cy, PB1, PB3, Dim and total flavonoid substances. A statistical analysis was then performed of the overall effect of irrigation dosage and its individual effect on each crop load, as well as of the overall effect of cluster thinning and its effect on each water status.

As can be seen in **Table 4A**, the different irrigation dosages had almost no effect on wine anthocyanin concentration values. Slightly lower concentrations of these substances were found in the wine from R25 treatment compared to the wine from R100 treatment, but the differences were non-significant (*p* > 0.05). This trend was also observed when studying separately the effect of irrigation dosage on the different crop loads (R100T vs. R25T and

However, the opposite trend was observed for the rest of the flavonoid families (flavanols and flavonols). The R25 wines contained significantly higher concentrations of Cat, the flavonol compounds My, Qc, Kp and Ir, Mon and total flavanols (**Table 4B**). An analysis of the effect of irrigation dosage on the different crop loads revealed significant differences only in the wines

In terms of wine anthocyanin composition, the effect of thinning was greater than of irrigation, with the A-treatment wines having significantly higher values of all the substances of this family, except for the acetyl-acetate derivatives. The biggest differences were found in the G (18%), Mv (15%) and Dp (29%). **Table 4A** also shows the separate analysis made of the effect of thinning on the R25 and R100 wines, with a greater and more significant response in R100. These results confirm the importance of vine water status in the effect of cluster thinning on

R100T, R25C and R25T wines, as well as the results of the statistical analysis.

**Table 4A** and **B** also shows the results of the combined statistical analysis of the four treatments. In concordance with the results described above, the highest and lowest values for anthocyanin substances were found in the 100T and 25C wines, respectively. Of these substances, Cy (the sum total of cyanidin compounds) is a special case as both the lower irrigation


dosage as well as cluster thinning resulted in lower concentration values, with the consequent concentration value for the R100C wines being significantly higher than for the other wines.

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Values of the PB1 and PB3 dimers increased as irrigation dosage fell, with the 25T and 100C wines having the minimum and maximum values, respectively. Finally, and given the sensitivity of flavonols to both irrigation dosage and crop load, the R25T wines had significantly

To determine the degree of correlation between the agronomic parameters (*véraison*-harvest Ψs and yield values of the different treatments), berry weight and berry ripeness indicators (TSS—total soluble solids and MI—maturity index) and the values of the different phenolic families of the vines, the mean values for the 2 years of the study were subjected to a Pearson's

No significant correlation was found between *véraison*-harvest Ψs and wine phenolic values. However, significant negative correlations were found between yield and concentration

**Figure 1.** Principal component analysis (PCA) between the different parameters studied (stem water potential Ψs, yield, ° brix and maturity index (MI) and wine phenolic compounds. G: ∑ Glycosides, Cm: ∑ Coumarates, Pt: ∑ Petunidins, Dp: ∑ Delphinids, Cy: ∑ Cyanidins, My: ∑ Myricetins, Qc: ∑ Quercetins; Kp: ∑ Kaempherols; Ir: ∑ Isorhamnetin; Mon:∑

*3.2.4. Correlation between agronomic parameters and grape berry ripeness indicators and* 

higher values than the R100C.

correlation and a PCA.

*concentration of wine phenolic substances*

Monomers; Dim: ∑ Dimers, TA: total acidity.

1 ANOVA test significance level. R: irrigation; A: cluster thinning; R and A: combination of both treatments. ns = not significant; \* = *p* ≤ 0.05; \*\* = *p* ≤ 0.01; \*\*\* = *p* ≤ 0.001

2 For the same compound, values followed by different letters indicate significant differences between treatments (*p* ≤ 0.5)

**Table 4B.** Average values (2007 and 2008 vintage) of flavanols, flavonols, (mg L−1 ) in cv. Tempranillo wines from vines subjected to different irrigation dosages and with different crop load levels, cluster thinned (T) and non-thinned (C) in Extremadura (Spain).

dosage as well as cluster thinning resulted in lower concentration values, with the consequent concentration value for the R100C wines being significantly higher than for the other wines.

Values of the PB1 and PB3 dimers increased as irrigation dosage fell, with the 25T and 100C wines having the minimum and maximum values, respectively. Finally, and given the sensitivity of flavonols to both irrigation dosage and crop load, the R25T wines had significantly higher values than the R100C.

#### *3.2.4. Correlation between agronomic parameters and grape berry ripeness indicators and concentration of wine phenolic substances*

**Flavanols**

1

2

Extremadura (Spain).

Compound Load Water status2 Significance level1

102 Grapes and Wines - Advances in Production, Processing, Analysis and Valorization

T 16.35c 23.08<sup>b</sup>

T 5.42a 4.31a

T 40.95ab 71.18a

T 6.00<sup>b</sup> 8.24a

T 21.96a 18.27<sup>b</sup>

T 21.77c 27.38<sup>b</sup>

T 71.04<sup>b</sup> 97.69a

T 92.81c 125.07a

A 11.46<sup>b</sup> 14.06a

A 7.84bc 10.5a

A 1.55bc 2.17a

A 1.24<sup>b</sup> 1.62a

A 22.09<sup>b</sup> 28.37a

**Table 4B.** Average values (2007 and 2008 vintage) of flavanols, flavonols, (mg L−1

significant; \* = *p* ≤ 0.05; \*\* = *p* ≤ 0.01; \*\*\* = *p* ≤ 0.001

R T Irrigation Thinning R and T

R100 R25 in C in T in R25 in R100

**(+)-catechin** C 25.67<sup>b</sup> 31.89a **\*\* \*\*\* ns \*\*\* \* \*\*\* \*\*\***

**(−)-epicatechin** C 4.35a 4.01a **ns ns ns \* ns ns ns**

**Procyanidin 1 (PB1)** C 34.54c 49.44<sup>b</sup> **Significant interaction \*\*\***

**Procyanidin 2 (PB2)** C 6.64<sup>b</sup> 6.00<sup>b</sup> **ns \*\*\* ns ns \*\* \*\*\* \*\***

**Procyanidin 3 (PB3)** C 17.08bc 16.14c **Significant interaction \*\*\***

**Monomers** C 30.03<sup>b</sup> 35.91a **\*\*\* \*\*\* ns \*\* \* \*\* \*\*\***

**Dimers** C 58.26c 71.59<sup>b</sup> **Significant interaction \*\*\***

**Total Flavanols** C 88.29c 107.49<sup>b</sup> **\*\*\* \*\* \*\*\* \*\*\* \*\* ns \*\*\***

**∑ Myricetins** C 8.06c 9.64<sup>b</sup> **\*\*\* \*\*\* ns \* \*\*\* \*\*\* \*\*\***

**∑ Quercetins** C 6.52c 8.20<sup>b</sup> **\*\*\* \*\*\* ns \*\* \*\* \*\* \*\*\***

**∑ Kaempherols** C 1.40c 1.74<sup>b</sup> **\* ns \*\*\* ns \*\*\* \*\*\* \*\*\***

**∑ Isorhamnetins** C 0.96c 1.28<sup>b</sup> **\*\*\* \*\*\* \* \* \*\* \*\* \*\*\***

**Total Flavonols** C 16.94c 20.85<sup>b</sup> **\*\*\* \*\*\* ns \*\* \*\*\* \*\*\* \*\*\***

ANOVA test significance level. R: irrigation; A: cluster thinning; R and A: combination of both treatments. ns = not

For the same compound, values followed by different letters indicate significant differences between treatments (*p* ≤ 0.5)

subjected to different irrigation dosages and with different crop load levels, cluster thinned (T) and non-thinned (C) in

) in cv. Tempranillo wines from vines

To determine the degree of correlation between the agronomic parameters (*véraison*-harvest Ψs and yield values of the different treatments), berry weight and berry ripeness indicators (TSS—total soluble solids and MI—maturity index) and the values of the different phenolic families of the vines, the mean values for the 2 years of the study were subjected to a Pearson's correlation and a PCA.

No significant correlation was found between *véraison*-harvest Ψs and wine phenolic values. However, significant negative correlations were found between yield and concentration

**Figure 1.** Principal component analysis (PCA) between the different parameters studied (stem water potential Ψs, yield, ° brix and maturity index (MI) and wine phenolic compounds. G: ∑ Glycosides, Cm: ∑ Coumarates, Pt: ∑ Petunidins, Dp: ∑ Delphinids, Cy: ∑ Cyanidins, My: ∑ Myricetins, Qc: ∑ Quercetins; Kp: ∑ Kaempherols; Ir: ∑ Isorhamnetin; Mon:∑ Monomers; Dim: ∑ Dimers, TA: total acidity.

of C (anthocyanin coumarates, r = −0.997) and between Qc and Ih (r = −0.961 and −0.984, respectively) and berry weight. This latter parameter was also positively correlated with Cy (r = 0.955). Conversely, berry acidity was negatively correlated with dimer values (r = −0.956) and all the flavonols except Kp. Finally, berry ripeness as reflected by the MI was positively correlated with dimers and My (r = 0.955 and 0.997 respectively).

yellow and dA%, and the phenolic compounds with significant inter-treatment differences. The chromatic data were taken as Y variables and phenolic substance concentrations as X variables. The data were also standardised to approximately the same scale to better reveal the correlations between the colour and chemical variables. Two new variables (axes) were

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**Figure 2** shows how CI was high and positively correlated with the presence of G, C, Dp, Pt and My, with this correlation being higher and more intense in the R100T and R25A wines. Conversely, %dA and %red were positively correlated with Kp (sum total of kaempherol glucosides) and the overall amount of flavonol, with this correlation being higher in the R25T wines. These results allow to make wine colour predictions based on wine phenolic composition. Accordingly, colour intensity can be predicted by the amount of certain phenolic compounds in the wines: G and C, Dp and Pt, and My. In the same way, the %dA and %red of a

The results obtained for phenols, flavonoids and PPGs in grapes at harvest are consistent with those obtained by Tardáguila et al. [73] who describe increased overall amounts of phenols and anthocyanins in grapes from thinned cv. Tempranillo vines. Likewise, Diago et al. [27] also describe an increase in these compounds caused by thinning in this variety, although the treatment was much less effective in the Garnacha variety. Esteban et al. [74] and Ojeda et al. [75] describe similar results in the content of total phenols, flavonoids, anthocyanins and flavonols as a result of water stress. Again, the combination of water deficit and thinning is the treatment with the greatest incidence on the evolution and content of these phenolic compounds. The SOD activity was always lower than in the unthinned treatments, an aspect which could have been the cause of the observed greater level of lipid peroxidation in the water deficit and thinned treatment. The nonspecific peroxidase activity was very low, especially in the thinned treatments. On the contrary, in the harvest the CA-POX activity's levels were slightly higher with thinning than without thinning. This enzyme might be involved in

[30], it does not appear to be affected by either the stage of ripening or the different treatments

Most studies on the effect of irrigation on grape berry and wine anthocyanin composition have found a decrease in the concentration of different anthocyanin derivatives and in their overall concentration as irrigation dosage is increased [77, 78]. Until a few years ago, these results were explained as being secondary effects of irrigation, including modification of berry size and consequently of skin/pulp ratio, which were responsible for the decrease in the concentration of these substances in the berry [75]. In this respect, sufficiently large increases in R100 berry size were not obtained in our study (**Table 2**) to generate, by dilution effect, a decrease in the concentration of these substances in the wines. However, recent studies have found that these substances are synthesised in the biosynthetic phenylpropanoid pathway, and it has been shown that it is the pre-*véraison* deficit which regulates the expression of genes involved in this biosynthesis [79]. Accordingly, the lack of response to irrigation in the G, and

during the formation of lignins [76]. According to our previous results

obtained through this technique which explains together 97% of total variance.

wine can be predicted by the concentration of Kp and flavonols.

**4. Discussion**

the production of H2

(thinning and water deficit).

O2

**Figure 1** represents the projection of the variables and samples in the plane defined by the first two principal components. The two principal components explain 96.65% of total variance. Principal component 1, which explains 71.87% of the variance, allows to distinguish the T wines from the C wines. This component is characterised by titratable acidity (TA), yield and berry weight on the positive side of the axis and by practically all the phenolic substances except G and Pt on the negative side, such that as we shift from left to right the wines are poorer in phenolic substances. The second principal component explains 24.78% of the variance and allows to distinguish the R100 from the R25 wines and is characterised by high monomer concentrations on the positive side of the axis and by *véraison*-harvest Ψs and G values on the negative side.

#### *3.2.5. Correlation between wine phenolic composition and chromatic parameters*

A PLS regression was performed to determine correlations between the chromatic parameters of the different wines, Colour intensity and Colour hue (CI, CT), percentage of red, blue and

**Figure 2.** Partial least squares (PSA) regression between the colorimetric parameters and wine phenolic compounds. G: ∑ Glycosides, Cm: ∑ Coumarates, Pt: ∑ Petunidins, Dp: ∑ Delphinids, Cy: ∑ Cyanidins, My: ∑ Myricetins, Qc: ∑ Quercetins; Kp: ∑ Kaempherols; Ir: ∑ Isorhamnetin; Mon:∑ Monomers; Dim: ∑ Dimers.

yellow and dA%, and the phenolic compounds with significant inter-treatment differences. The chromatic data were taken as Y variables and phenolic substance concentrations as X variables. The data were also standardised to approximately the same scale to better reveal the correlations between the colour and chemical variables. Two new variables (axes) were obtained through this technique which explains together 97% of total variance.

**Figure 2** shows how CI was high and positively correlated with the presence of G, C, Dp, Pt and My, with this correlation being higher and more intense in the R100T and R25A wines. Conversely, %dA and %red were positively correlated with Kp (sum total of kaempherol glucosides) and the overall amount of flavonol, with this correlation being higher in the R25T wines. These results allow to make wine colour predictions based on wine phenolic composition. Accordingly, colour intensity can be predicted by the amount of certain phenolic compounds in the wines: G and C, Dp and Pt, and My. In the same way, the %dA and %red of a wine can be predicted by the concentration of Kp and flavonols.

## **4. Discussion**

**Figure 2.** Partial least squares (PSA) regression between the colorimetric parameters and wine phenolic compounds. G: ∑ Glycosides, Cm: ∑ Coumarates, Pt: ∑ Petunidins, Dp: ∑ Delphinids, Cy: ∑ Cyanidins, My: ∑ Myricetins, Qc: ∑

of C (anthocyanin coumarates, r = −0.997) and between Qc and Ih (r = −0.961 and −0.984, respectively) and berry weight. This latter parameter was also positively correlated with Cy (r = 0.955). Conversely, berry acidity was negatively correlated with dimer values (r = −0.956) and all the flavonols except Kp. Finally, berry ripeness as reflected by the MI was positively

**Figure 1** represents the projection of the variables and samples in the plane defined by the first two principal components. The two principal components explain 96.65% of total variance. Principal component 1, which explains 71.87% of the variance, allows to distinguish the T wines from the C wines. This component is characterised by titratable acidity (TA), yield and berry weight on the positive side of the axis and by practically all the phenolic substances except G and Pt on the negative side, such that as we shift from left to right the wines are poorer in phenolic substances. The second principal component explains 24.78% of the variance and allows to distinguish the R100 from the R25 wines and is characterised by high monomer concentrations on the positive side of the axis and by *véraison*-harvest Ψs and

A PLS regression was performed to determine correlations between the chromatic parameters of the different wines, Colour intensity and Colour hue (CI, CT), percentage of red, blue and

correlated with dimers and My (r = 0.955 and 0.997 respectively).

104 Grapes and Wines - Advances in Production, Processing, Analysis and Valorization

*3.2.5. Correlation between wine phenolic composition and chromatic parameters*

G values on the negative side.

Quercetins; Kp: ∑ Kaempherols; Ir: ∑ Isorhamnetin; Mon:∑ Monomers; Dim: ∑ Dimers.

The results obtained for phenols, flavonoids and PPGs in grapes at harvest are consistent with those obtained by Tardáguila et al. [73] who describe increased overall amounts of phenols and anthocyanins in grapes from thinned cv. Tempranillo vines. Likewise, Diago et al. [27] also describe an increase in these compounds caused by thinning in this variety, although the treatment was much less effective in the Garnacha variety. Esteban et al. [74] and Ojeda et al. [75] describe similar results in the content of total phenols, flavonoids, anthocyanins and flavonols as a result of water stress. Again, the combination of water deficit and thinning is the treatment with the greatest incidence on the evolution and content of these phenolic compounds. The SOD activity was always lower than in the unthinned treatments, an aspect which could have been the cause of the observed greater level of lipid peroxidation in the water deficit and thinned treatment. The nonspecific peroxidase activity was very low, especially in the thinned treatments. On the contrary, in the harvest the CA-POX activity's levels were slightly higher with thinning than without thinning. This enzyme might be involved in the production of H2 O2 during the formation of lignins [76]. According to our previous results [30], it does not appear to be affected by either the stage of ripening or the different treatments (thinning and water deficit).

Most studies on the effect of irrigation on grape berry and wine anthocyanin composition have found a decrease in the concentration of different anthocyanin derivatives and in their overall concentration as irrigation dosage is increased [77, 78]. Until a few years ago, these results were explained as being secondary effects of irrigation, including modification of berry size and consequently of skin/pulp ratio, which were responsible for the decrease in the concentration of these substances in the berry [75]. In this respect, sufficiently large increases in R100 berry size were not obtained in our study (**Table 2**) to generate, by dilution effect, a decrease in the concentration of these substances in the wines. However, recent studies have found that these substances are synthesised in the biosynthetic phenylpropanoid pathway, and it has been shown that it is the pre-*véraison* deficit which regulates the expression of genes involved in this biosynthesis [79]. Accordingly, the lack of response to irrigation in the G, and T and C forms, as well as in the Mv, Pt, Df and Pn derived compounds can be explained based on the different irrigation dosages applied in the post-*véraison* period.

The increased concentrations of catechin and procyanidin B1 are also important given their relation to colour and body stability through these copigmentation and polymerisation reac-

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Similar to the results from other studies [19, 26, 39], wines from the cluster-thinned treatments had higher CI values. Colour hue variation between wines as a function of vine water status has also been described in other studies, though with contradictory results [21]. In our study, the greater presence of cyanidin due to the later ripening of the R100C grapes may have been

Many studies have used statistical techniques to find correlations between phenolic compounds and colour parameters during the maturation and ageing processes of red wine [90]. In one interesting work, Monagas et al. [91] showed that chromatic attributes of red wines could be predicted by their phenolic profile using polynomial regression techniques. The substances which provided the best fitting model in that study were the anthocyanin compounds, and in the specific case of cv. Tempranillo wines the pyruvic adducts. According to the PLS regression which we preformed, the CI of Tempranillo wines can also be predicted as a function of anthocyanin compounds, specifically C, G, and Df and Pt compounds. Along the same line, our work also confirms the findings of Escudero-Gilete et al. [92]. They studied the correlations between colour and anthocyanin composition of wines made from a blend of cv. Tempranillo and cv. Graciano grapes and found that the petunidin compounds (monoglucoside, acetyl-acetate and acetyl-coumarate) were the ones most closely related to the chromaticity of these wines. Similarly, the negative correlation between the percentages of yellow and blue and the flavanols which were also found by the PLS regression concur with the findings of the previously cited work of Monagas et al. [91]. From all of the above, it can be concluded that the thinning treatment generated, most notable, an increase in wine colour intensity and that vine water stress modified, most notably, wine hue as a result of differences in flavonol concentrations.

This work contributes to our understanding of how vine-growing practices can affect the compounds responsible for the sensory attributes of wines. It is shown how modification of water status and crop load through deficit irrigation and cluster-thinning techniques can induce variations in the phenolic composition and consequently the chromatic characteristics of a wine. It can be concluded from the results that post-*véraison* water stress increased the concentration values of non-anthocyanin flavonoids, though the extent of the increase varied depending on the compound under consideration and, in the case of flavanols, on the crop load. Consequently, special care should be taken to adapt and control irrigation dosages in regions of very dry and hot summers where post-*véraison* irrigation is an absolute necessity. The results of our study also show an increase in the phenolic composition of wines as a result of the cluster thinning of irrigated vines and, above all, show how the extent and significance of this technique depends in the case of anthocyanins and flavanols on vine water status. Furthermore, our work demonstrates how these practices can affect wine colour. With the increase in anthocyanin content, thinning augmented wine colour intensity, while the different levels of vine

responsible for the higher percentage of blue hues (*p* < 0.05) of these wines.

tions with other flavonols.

**5. Conclusion**

In a similar study to ours conducted in Requena (Spain) with Tempranillo vines, Intrigliolo et al. [9] found increased anthocyanin concentrations in wines from the most irrigated vines. They explained their results as the consequence of the greater water requirements and the inability of cv. Tempranillo, typically grown in colder regions, to adapt to edapho-climatic conditions. This could also be the case in our study, as the highest values were observed in the R100 wines. This result might also suggest a greater synchrony between sugar and anthocyanin accumulation in the berry of the higher irrigation treatments [18].

The significant differences that were found in the flavanol- and flavonol-type substances are not a decisive factor for these substances. Studies have shown that while water deficit has a moderate effect on flavonol synthesis, the effect of the irrigation application period is almost negligible [23, 80]. This would explain the significant increase in My, Qc, Kp and Ir in the R25 wines compared to the R100 wines. The synthesis of these substances is light dependent, so the lower leaf area observed in the R25 treatment may also have contributed to this increase. In this respect, Matthews et al. [81] reported that the decrease in berry weight was higher, and consequently the increase in flavonoid concentration was higher, when water stress was imposed to pre-*véraison*.

Given the higher content of anthocyanin substance in the A-treatment musts [18], the formation of copigments must have taken place in them in a greater proportion during fermentation, which would in turn have resulted in greater flavonol extraction. This may be one of the reasons why, when the effect of irrigation was analysed separately on the two load levels, significant differences were only found in My, Qc and Ir values in the R100T versus R25T comparison. This result is very important, as copigments stabilise the colour of young red wines [50].

The results shown in **Table 4A** and **B** concurs with those from previous studies which show that cluster thinning is a useful tool to increase the concentration of different flavonoid compounds in grapes and wines [29, 37, 82]. Oenologically speaking, and in full concurrence with the results observed by Peña-Neira et al. (29), the most important and notable effects of cluster thinning are those related to the increase in phenolic compound values via the phenylpropanoid pathway.

Among the anthocyanin substances, the increase in the coumarate forms is particularly important, as these are more stable than the monoglucosides [83, 84] and offer greater stability to wine colour. Also notable is the low significance of cluster thinning on the R25 wines. In a similar study performed with the Syrah grape [82], cluster thinning led to significant increases in the coumarate forms of malvidin and peonidin. In our study, the maximum anthocyanin content in the R25 berries may have been generated at lower TSS values than those at which the berries were harvested (24.37°Brix), so that anthocyanin material may have been lost in these berries.

The increase in flavonols is especially interesting, since these contribute to astringency and also play an important role in anthocyanin copigmentation and in the formation of more complex pigments during the ageing process [85, 86]. In the copigmentation phenomenon which contributes to the stabilisation of the colour of red wines [50, 87], Baranac et al. [88] reported that flavonol substances are amongst the best copigments, especially quercetin [89].

The increased concentrations of catechin and procyanidin B1 are also important given their relation to colour and body stability through these copigmentation and polymerisation reactions with other flavonols.

Similar to the results from other studies [19, 26, 39], wines from the cluster-thinned treatments had higher CI values. Colour hue variation between wines as a function of vine water status has also been described in other studies, though with contradictory results [21]. In our study, the greater presence of cyanidin due to the later ripening of the R100C grapes may have been responsible for the higher percentage of blue hues (*p* < 0.05) of these wines.

Many studies have used statistical techniques to find correlations between phenolic compounds and colour parameters during the maturation and ageing processes of red wine [90]. In one interesting work, Monagas et al. [91] showed that chromatic attributes of red wines could be predicted by their phenolic profile using polynomial regression techniques. The substances which provided the best fitting model in that study were the anthocyanin compounds, and in the specific case of cv. Tempranillo wines the pyruvic adducts. According to the PLS regression which we preformed, the CI of Tempranillo wines can also be predicted as a function of anthocyanin compounds, specifically C, G, and Df and Pt compounds. Along the same line, our work also confirms the findings of Escudero-Gilete et al. [92]. They studied the correlations between colour and anthocyanin composition of wines made from a blend of cv. Tempranillo and cv. Graciano grapes and found that the petunidin compounds (monoglucoside, acetyl-acetate and acetyl-coumarate) were the ones most closely related to the chromaticity of these wines. Similarly, the negative correlation between the percentages of yellow and blue and the flavanols which were also found by the PLS regression concur with the findings of the previously cited work of Monagas et al. [91]. From all of the above, it can be concluded that the thinning treatment generated, most notable, an increase in wine colour intensity and that vine water stress modified, most notably, wine hue as a result of differences in flavonol concentrations.

## **5. Conclusion**

T and C forms, as well as in the Mv, Pt, Df and Pn derived compounds can be explained based

In a similar study to ours conducted in Requena (Spain) with Tempranillo vines, Intrigliolo et al. [9] found increased anthocyanin concentrations in wines from the most irrigated vines. They explained their results as the consequence of the greater water requirements and the inability of cv. Tempranillo, typically grown in colder regions, to adapt to edapho-climatic conditions. This could also be the case in our study, as the highest values were observed in the R100 wines. This result might also suggest a greater synchrony between sugar and antho-

The significant differences that were found in the flavanol- and flavonol-type substances are not a decisive factor for these substances. Studies have shown that while water deficit has a moderate effect on flavonol synthesis, the effect of the irrigation application period is almost negligible [23, 80]. This would explain the significant increase in My, Qc, Kp and Ir in the R25 wines compared to the R100 wines. The synthesis of these substances is light dependent, so the lower leaf area observed in the R25 treatment may also have contributed to this increase. In this respect, Matthews et al. [81] reported that the decrease in berry weight was higher, and consequently the increase in flavonoid concentration was higher, when water stress was

Given the higher content of anthocyanin substance in the A-treatment musts [18], the formation of copigments must have taken place in them in a greater proportion during fermentation, which would in turn have resulted in greater flavonol extraction. This may be one of the reasons why, when the effect of irrigation was analysed separately on the two load levels, significant differences were only found in My, Qc and Ir values in the R100T versus R25T comparison. This result is very important, as copigments stabilise the colour of young red wines [50]. The results shown in **Table 4A** and **B** concurs with those from previous studies which show that cluster thinning is a useful tool to increase the concentration of different flavonoid compounds in grapes and wines [29, 37, 82]. Oenologically speaking, and in full concurrence with the results observed by Peña-Neira et al. (29), the most important and notable effects of cluster thinning are those related to the increase in phenolic compound values via the phenylpropanoid pathway. Among the anthocyanin substances, the increase in the coumarate forms is particularly important, as these are more stable than the monoglucosides [83, 84] and offer greater stability to wine colour. Also notable is the low significance of cluster thinning on the R25 wines. In a similar study performed with the Syrah grape [82], cluster thinning led to significant increases in the coumarate forms of malvidin and peonidin. In our study, the maximum anthocyanin content in the R25 berries may have been generated at lower TSS values than those at which the berries were harvested (24.37°Brix), so that anthocyanin material may have been lost in these berries. The increase in flavonols is especially interesting, since these contribute to astringency and also play an important role in anthocyanin copigmentation and in the formation of more complex pigments during the ageing process [85, 86]. In the copigmentation phenomenon which contributes to the stabilisation of the colour of red wines [50, 87], Baranac et al. [88] reported that flavonol substances are amongst the best copigments, especially quercetin [89].

on the different irrigation dosages applied in the post-*véraison* period.

106 Grapes and Wines - Advances in Production, Processing, Analysis and Valorization

cyanin accumulation in the berry of the higher irrigation treatments [18].

imposed to pre-*véraison*.

This work contributes to our understanding of how vine-growing practices can affect the compounds responsible for the sensory attributes of wines. It is shown how modification of water status and crop load through deficit irrigation and cluster-thinning techniques can induce variations in the phenolic composition and consequently the chromatic characteristics of a wine. It can be concluded from the results that post-*véraison* water stress increased the concentration values of non-anthocyanin flavonoids, though the extent of the increase varied depending on the compound under consideration and, in the case of flavanols, on the crop load. Consequently, special care should be taken to adapt and control irrigation dosages in regions of very dry and hot summers where post-*véraison* irrigation is an absolute necessity. The results of our study also show an increase in the phenolic composition of wines as a result of the cluster thinning of irrigated vines and, above all, show how the extent and significance of this technique depends in the case of anthocyanins and flavanols on vine water status. Furthermore, our work demonstrates how these practices can affect wine colour. With the increase in anthocyanin content, thinning augmented wine colour intensity, while the different levels of vine water status affected colour hue by modifying the concentration of flavonols which participate in the reactions that help to stabilise wine colour.

[6] Procházková D, Jan S, Abd-Allah EF, Ahmad P. Water stress in grapevine (*Vitis vinifera* L.). In: Ahmad P, editor. Water Stress and Crop Plants: A Sustainable Approach.

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## **Acknowledgements**

This research was supported by funds from INIA, FEDER and Junta de Extremadura, Projects RTA2005-00038-C06 and RTA2008-00037-C04. The authors gratefully acknowledge the technical assistance of Dra. Mar Vilanova (Misión Biológica de Galicia, CSIC) in statistical analysis.

## **Author details**

Ester Gamero1 , Francisco Espinosa2 \*, Daniel Moreno1 , David Uriarte<sup>3</sup> , María Henar Prieto<sup>3</sup> , Inmaculada Garrido2 and María Esperanza Valdés1

\*Address all correspondence to: espinosa@unex.es

1 Technological Institute of Food and Agriculture, INTAEX Junta of Extremadura, Badajoz, Spain

2 U. Plant Physiology, Facultad de Ciencias, University of Extremadura, Badajoz, Spain

3 Institute of Agricultural Research Finca La Orden-Valdesequera, CICYTEX Junta of Extremadura, Badajoz, Spain

## **References**


[6] Procházková D, Jan S, Abd-Allah EF, Ahmad P. Water stress in grapevine (*Vitis vinifera* L.). In: Ahmad P, editor. Water Stress and Crop Plants: A Sustainable Approach. Chichester: Wiley; 2016

water status affected colour hue by modifying the concentration of flavonols which participate

This research was supported by funds from INIA, FEDER and Junta de Extremadura, Projects RTA2005-00038-C06 and RTA2008-00037-C04. The authors gratefully acknowledge the technical assistance of Dra. Mar Vilanova (Misión Biológica de Galicia, CSIC) in statistical analysis.

\*, Daniel Moreno1

1 Technological Institute of Food and Agriculture, INTAEX Junta of Extremadura, Badajoz,

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**Section 2**

**Wine Production and Characterization**

**Wine Production and Characterization**

**Chapter 7**

**Provisional chapter**

**The Evolution of Polyphenols from Grapes to Wines**

Polyphenols play an important role in the quality of wines, due to their contribution to the wine sensory properties: color, astringency and bitterness. They act as antioxidants, having positive role in human health. They can be divided into non-flavonoid (hydroxybenzoic and hydroxycinnamic acids and stilbenes) and flavonoid compounds (anthocyanins, flavan-3-ols and flavonols). Anthocyanins are responsible for the color of red grapes and wines, hydroxycinnamic and hydroxybenzoic acids act as copigments, stilbenes as antioxidants and the flavan-3-ols are mainly responsible for the astringency, bitterness and structure of wines, being involved also in the color stabilization during aging. This chapter will focus on the chemical structures of the main polyphenols, their identification and quantification in grapes and wines by advanced analytical techniques, highlighting also the maceration and aging impact on the polyphenols evolution. The factors influencing the phenolic accumulation in grapes are also reviewed, emphasizing as well the relationship between phenolic content in grapes versus wine. Polyphenolic changes during the wine making process are highlighted along with the main polyphenol extraction methods and analysis techniques. This research will contribute to the improvement in the knowledge of polyphenols: their presence in grapes, the relationship with wine quality

**The Evolution of Polyphenols from Grapes to Wines**

DOI: 10.5772/intechopen.72800

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution,

© 2018 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

and reproduction in any medium, provided the original work is properly cited.

First, a simple question can be addressed: what are phenolic compounds? For a simple question, a simple answer is that they are compounds that have one or more hydroxyl groups attached directly to an aromatic ring. Phenol (**Figure 1**) is the basic structure, the aromatic ring being

Violeta-Carolina Niculescu, Nadia Paun and

Violeta-Carolina Niculescu, Nadia Paun and

Additional information is available at the end of the chapter

and the influence of the external factors on their evolution.

**Keywords:** grapes, liquid chromatography, polyphenols, chemistry, wines

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/intechopen.72800

Roxana-Elena Ionete

**Abstract**

**1. Introduction**

benzene.

Roxana-Elena Ionete

### **The Evolution of Polyphenols from Grapes to Wines The Evolution of Polyphenols from Grapes to Wines**

DOI: 10.5772/intechopen.72800

Violeta-Carolina Niculescu, Nadia Paun and Violeta-Carolina Niculescu, Nadia Paun and Roxana-Elena Ionete

Roxana-Elena Ionete

Additional information is available at the end of the chapter Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/intechopen.72800

#### **Abstract**

Polyphenols play an important role in the quality of wines, due to their contribution to the wine sensory properties: color, astringency and bitterness. They act as antioxidants, having positive role in human health. They can be divided into non-flavonoid (hydroxybenzoic and hydroxycinnamic acids and stilbenes) and flavonoid compounds (anthocyanins, flavan-3-ols and flavonols). Anthocyanins are responsible for the color of red grapes and wines, hydroxycinnamic and hydroxybenzoic acids act as copigments, stilbenes as antioxidants and the flavan-3-ols are mainly responsible for the astringency, bitterness and structure of wines, being involved also in the color stabilization during aging. This chapter will focus on the chemical structures of the main polyphenols, their identification and quantification in grapes and wines by advanced analytical techniques, highlighting also the maceration and aging impact on the polyphenols evolution. The factors influencing the phenolic accumulation in grapes are also reviewed, emphasizing as well the relationship between phenolic content in grapes versus wine. Polyphenolic changes during the wine making process are highlighted along with the main polyphenol extraction methods and analysis techniques. This research will contribute to the improvement in the knowledge of polyphenols: their presence in grapes, the relationship with wine quality and the influence of the external factors on their evolution.

**Keywords:** grapes, liquid chromatography, polyphenols, chemistry, wines

## **1. Introduction**

First, a simple question can be addressed: what are phenolic compounds? For a simple question, a simple answer is that they are compounds that have one or more hydroxyl groups attached directly to an aromatic ring. Phenol (**Figure 1**) is the basic structure, the aromatic ring being benzene.

and reproduction in any medium, provided the original work is properly cited.

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, © 2018 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

**Figure 1.** Phenol chemical structure.

Due to the aromatic ring, the hydrogen of the hydroxyl group is labile, which makes phenols weak acids. Polyphenols are compounds that have more than one phenolic hydroxyl group attached to one or more aromatic rings. The term is somewhat misleading since it tends to make people think of polymers of individual phenol molecules.

The general term 'phenolic' covers a very large and diverse group of chemical compounds, and they can be classified in many ways. Phenolic compounds are well connected with several functions in plants like protection against invading pathogens or from UV radiation, pigmentation, or attraction of pollinators and seed dispersers. These compounds were classified into groups based on the number of carbons in the molecule (**Table 1**) [1].

Phenolic compounds play an important role on the sensory characteristics of both grapes and wine because they are responsible for some sensory properties: aroma, color, flavor, bitterness and astringency [4]. The chemical structure enables them to act as antioxidants, scavenging and neutralizing free radicals, and wine polyphenols have been extensively studied in rela-

The Evolution of Polyphenols from Grapes to Wines http://dx.doi.org/10.5772/intechopen.72800 121

During the grape ripening phase, the physiological and biochemical changes determine the quality of the fruit, the relative proportions of the grape components and wines, being influenced considerably by site (altitude, geological features, soil type, sunlight exposure), climate (cool climate or warm climate), time of harvesting (fungi and bacteria present in grapes), and viticultural practice. The studies about the formation and development of grapes showed that environmental stress factors like heat, drought and light intensity drive to changes in the development of grapes, their chemical composition and phenolic metabolism. The first period of grapes' growth consists mostly of cell division and expansion, followed by a rapid growth phase during which the berry is formed and the seed embryos are produced. In this period, several compounds accumulate in the berry, especially the tartaric and malic acids, conferring the acidity of the future wine. During the first growth period, other compounds accumulate: hydroxycinnamic acids––in the flesh and skin of the grapes; tannins and catechins––in the skin and seed tissues of the grapes; minerals, amino acids and amino acids. The most important changes in grapes' composition occur during the second growth phase (the ripening phase). Grapes switch from small, hard and acidic berries, to larger, softer, sweeter, less acidic, flavored and colored one. The majority of the solutes accumulated during the first growing period remain at harvest. During the second period, the malic acid is metabolized and used as an energy source, its proportion decreasing toward the tartaric acid concentration, which remains almost unchanged. Tannins also

tion to their positive role in human health [5].

**Structure Class**

C6, C10, C14 Quinines C18 Betacyanins

Lignins Polymers

Phlobaphenes Polymers

**Table 1.** Classification of phenolic compounds.

Lignans, neolignans Dimmers or oligomers

Tannins Oligomers or polymers

C6-C1-C6, C6-C2-C6 Benzophenones, xanthones, stilbenes

decrease considerably after *véraison*.

**2. Grape and wine polyphenols: a chemical perspective**

An alternative classification has been used by Swain and Bate-Smith [2]. They grouped the phenols in 'common' and 'less common' categories. Also, phenols were grouped into three families [3]:




**Table 1.** Classification of phenolic compounds.

Due to the aromatic ring, the hydrogen of the hydroxyl group is labile, which makes phenols weak acids. Polyphenols are compounds that have more than one phenolic hydroxyl group attached to one or more aromatic rings. The term is somewhat misleading since it tends to

The general term 'phenolic' covers a very large and diverse group of chemical compounds, and they can be classified in many ways. Phenolic compounds are well connected with several functions in plants like protection against invading pathogens or from UV radiation, pigmentation, or attraction of pollinators and seed dispersers. These compounds were classified

An alternative classification has been used by Swain and Bate-Smith [2]. They grouped the phenols in 'common' and 'less common' categories. Also, phenols were grouped into three families [3]:

**1.** Widely distributed phenols––ubiquitous to all plants, or of importance in a specific plant

**2.** Phenols that are less widely distributed––limited number of compounds known

make people think of polymers of individual phenol molecules.

120 Grapes and Wines - Advances in Production, Processing, Analysis and Valorization

**3.** Phenolic constituents present as polymers

C6-C1 Phenolics acids and related compounds C6-C2 Acetophenones and phenylacetic acids

C6-C3 Coumarins, isocoumarins, chromones C15 Chalcones, aurones, dihydrochalcones

C6-C3 Cinnamic acids, cinnamyl aldehydes, cinnamyl alcohols

C6 Simple phenolics

**Structure Class**

C15 Flavans C15 Flavones C15 Flavanones C15 Flavanonols C15 Anthocyanidins C15 Anthocyanins C30 Biflavonyls

**Figure 1.** Phenol chemical structure.

into groups based on the number of carbons in the molecule (**Table 1**) [1].

Phenolic compounds play an important role on the sensory characteristics of both grapes and wine because they are responsible for some sensory properties: aroma, color, flavor, bitterness and astringency [4]. The chemical structure enables them to act as antioxidants, scavenging and neutralizing free radicals, and wine polyphenols have been extensively studied in relation to their positive role in human health [5].

## **2. Grape and wine polyphenols: a chemical perspective**

During the grape ripening phase, the physiological and biochemical changes determine the quality of the fruit, the relative proportions of the grape components and wines, being influenced considerably by site (altitude, geological features, soil type, sunlight exposure), climate (cool climate or warm climate), time of harvesting (fungi and bacteria present in grapes), and viticultural practice. The studies about the formation and development of grapes showed that environmental stress factors like heat, drought and light intensity drive to changes in the development of grapes, their chemical composition and phenolic metabolism. The first period of grapes' growth consists mostly of cell division and expansion, followed by a rapid growth phase during which the berry is formed and the seed embryos are produced. In this period, several compounds accumulate in the berry, especially the tartaric and malic acids, conferring the acidity of the future wine. During the first growth period, other compounds accumulate: hydroxycinnamic acids––in the flesh and skin of the grapes; tannins and catechins––in the skin and seed tissues of the grapes; minerals, amino acids and amino acids. The most important changes in grapes' composition occur during the second growth phase (the ripening phase). Grapes switch from small, hard and acidic berries, to larger, softer, sweeter, less acidic, flavored and colored one. The majority of the solutes accumulated during the first growing period remain at harvest. During the second period, the malic acid is metabolized and used as an energy source, its proportion decreasing toward the tartaric acid concentration, which remains almost unchanged. Tannins also decrease considerably after *véraison*.

Winemaking techniques involve the extraction of juice from ripe grapes and fermentation with yeast, changes in polyphenolic composition occurring due to the participation of these compounds in various reactions such as copigmentation, cycloaddition, polymerization and oxidation. The reactions begin after grapes crushing, followed by fermentation and aging, contributing to the sensory properties of wines, mainly color and astringency.

The understanding of the relationship between the quality of a particular wine and its phenolic composition is, at the moment, one of the major challenges in oenology research. For example, the anthocyanin fingerprints of varietal wines are proposed as an analytical tool for authenticity certification [6].

Polyphenolics must be also considered from the taxonomical point of view, knowing that the patterns of some classes of flavonoids are under strict genetic control and that their distribution varies considerably among different grape cultivars [7]. There are several factors with impact on the nonvolatile wine phenolic compounds, including the '*terroir,*' the grape variety and its degree of maturation before harvesting, or the winemaking process with its specific conditions of fermentation or aging [8]. Certain technological procedures such as addition of sulfur dioxide (SO<sup>2</sup> ) and/or ascorbic acid (C<sup>6</sup> H8 O6 ) prior to crushing the grapes, maceration, yeast strain utilization and alcoholic fermentation, oxidation or adsorption, can also influence the levels of phenolic during the winemaking process [9].

The investigation of phenolic composition in grapes and wine may provide some specific biomarkers that allow us to better assess the chemical evolution of grapes during growth and maturation periods but also to improve the knowledge on wine authentication by developing and implementing new/improved control methods. Phenolic compound identification and quantification fit authentication purpose, protecting consumers against fraud [10].

> tannins. Hydroxycinnamic acids (HCA) are found in both grapes and wine, the most referenced compounds among them being *p*-coumaric, caffeic, ferulic and sinapic acids (**Figure 2**),

The Evolution of Polyphenols from Grapes to Wines http://dx.doi.org/10.5772/intechopen.72800 123

**Figure 2.** Chemical structures of hydroxybenzoic, hydroxycinnamic and hydroxycinnamoyltartaric acids.

Cinnamic acids exist as *cis* or *trans* isomeric forms, which are convertible either enzymatically or through the action of light. Other hydroxycinnamic acids detected in grapes and wines are *p*-coutaric acid, fertaric acid and caftaric acid (**Figure 2**). Caftaric and fertaric acids exist in their *trans* form, mainly localized in the grape pulp and, during the grape pressing, being quickly released into the juice, while a negligible fraction of the *cis* isomer has been found for *p*-coutaric acid. Contrary to this, the *trans* and *cis* isomers of *p*-coutaric acid are less extractable since they are mostly localized in the grape skin, being partially responsible for the astringent properties of both grapes and wines [14]. Although the white wines have a lower concentration of phenolic compounds compared to the red ones, they contain, in turn, a high quantity of caftaric acid. Hydroxycinnamic acids and their tartaric esters constitute the main class of nonflavonoid phenolics in red wines and the main class of phenolic compounds in white wines [14]. During the fermentation process, these esters are partially hydrolyzed, resulting free hydroxycinnamic acids, transformed then into ethyl coumarate

which are associated with the browning of wine [13].

and ethyl caffeate [15].

There is a great chemical diversity in the nonvolatile phenolic composition of grapes and wines, due not only to the varieties of grapes, but also to the fact that they exist in both the free and conjugated forms, as they may be bound to one or more sugar molecules (glucose, galactose, sucrose and mannose) [11]. From the flavonoid compounds, the main phenolics involved in the red color of grapes and wines are the anthocyanins, while the flavan-3-ols are most linked with the astringency, bitterness and structure of wines, but also with the stabilization of color during aging. The nonflavonoid compounds act as copigments (hydroxycinnamic and hydroxybenzoic acids) and antioxidants (stilbenes).

#### **2.1. Phenolic acids**

They include two main groups, the benzoic acids, containing seven carbon atoms (C6-C1), and the cinnamic acids, with nine carbon atoms (C6-C3), and they exist mainly as hydroxybenzoic and hydroxycinnamic acids, in either the free or the conjugated form. Various types of hydroxybenzoic acids (HBA) have been identified in both grapes and wines, among them parahydroxybenzoic, protocatechuic, vanillic, gallic and syringic acids (**Figure 2**) [12]. Gallic acid is considered the most important phenolic acid, being the precursor of all hydrolyzable

Winemaking techniques involve the extraction of juice from ripe grapes and fermentation with yeast, changes in polyphenolic composition occurring due to the participation of these compounds in various reactions such as copigmentation, cycloaddition, polymerization and oxidation. The reactions begin after grapes crushing, followed by fermentation and aging, contributing to the sensory properties of wines, mainly color

The understanding of the relationship between the quality of a particular wine and its phenolic composition is, at the moment, one of the major challenges in oenology research. For example, the anthocyanin fingerprints of varietal wines are proposed as an analytical tool for

Polyphenolics must be also considered from the taxonomical point of view, knowing that the patterns of some classes of flavonoids are under strict genetic control and that their distribution varies considerably among different grape cultivars [7]. There are several factors with impact on the nonvolatile wine phenolic compounds, including the '*terroir,*' the grape variety and its degree of maturation before harvesting, or the winemaking process with its specific conditions of fermentation or aging [8]. Certain technological procedures such as addition of

> H8 O6

yeast strain utilization and alcoholic fermentation, oxidation or adsorption, can also influence

The investigation of phenolic composition in grapes and wine may provide some specific biomarkers that allow us to better assess the chemical evolution of grapes during growth and maturation periods but also to improve the knowledge on wine authentication by developing and implementing new/improved control methods. Phenolic compound identification and quantification fit authentication purpose, protecting consumers against

There is a great chemical diversity in the nonvolatile phenolic composition of grapes and wines, due not only to the varieties of grapes, but also to the fact that they exist in both the free and conjugated forms, as they may be bound to one or more sugar molecules (glucose, galactose, sucrose and mannose) [11]. From the flavonoid compounds, the main phenolics involved in the red color of grapes and wines are the anthocyanins, while the flavan-3-ols are most linked with the astringency, bitterness and structure of wines, but also with the stabilization of color during aging. The nonflavonoid compounds act as copigments (hydroxycin-

They include two main groups, the benzoic acids, containing seven carbon atoms (C6-C1), and the cinnamic acids, with nine carbon atoms (C6-C3), and they exist mainly as hydroxybenzoic and hydroxycinnamic acids, in either the free or the conjugated form. Various types of hydroxybenzoic acids (HBA) have been identified in both grapes and wines, among them parahydroxybenzoic, protocatechuic, vanillic, gallic and syringic acids (**Figure 2**) [12]. Gallic acid is considered the most important phenolic acid, being the precursor of all hydrolyzable

) prior to crushing the grapes, maceration,

) and/or ascorbic acid (C<sup>6</sup>

namic and hydroxybenzoic acids) and antioxidants (stilbenes).

the levels of phenolic during the winemaking process [9].

122 Grapes and Wines - Advances in Production, Processing, Analysis and Valorization

and astringency.

sulfur dioxide (SO<sup>2</sup>

fraud [10].

**2.1. Phenolic acids**

authenticity certification [6].

**Figure 2.** Chemical structures of hydroxybenzoic, hydroxycinnamic and hydroxycinnamoyltartaric acids.

tannins. Hydroxycinnamic acids (HCA) are found in both grapes and wine, the most referenced compounds among them being *p*-coumaric, caffeic, ferulic and sinapic acids (**Figure 2**), which are associated with the browning of wine [13].

Cinnamic acids exist as *cis* or *trans* isomeric forms, which are convertible either enzymatically or through the action of light. Other hydroxycinnamic acids detected in grapes and wines are *p*-coutaric acid, fertaric acid and caftaric acid (**Figure 2**). Caftaric and fertaric acids exist in their *trans* form, mainly localized in the grape pulp and, during the grape pressing, being quickly released into the juice, while a negligible fraction of the *cis* isomer has been found for *p*-coutaric acid. Contrary to this, the *trans* and *cis* isomers of *p*-coutaric acid are less extractable since they are mostly localized in the grape skin, being partially responsible for the astringent properties of both grapes and wines [14]. Although the white wines have a lower concentration of phenolic compounds compared to the red ones, they contain, in turn, a high quantity of caftaric acid. Hydroxycinnamic acids and their tartaric esters constitute the main class of nonflavonoid phenolics in red wines and the main class of phenolic compounds in white wines [14]. During the fermentation process, these esters are partially hydrolyzed, resulting free hydroxycinnamic acids, transformed then into ethyl coumarate and ethyl caffeate [15].

#### **2.2. Flavonoids**

They are a chemical class with a basic structure of 15 carbon atoms, including two aromatic rings bound through a three-carbon chain (C6-C3-C6), which is responsible for the chemical. Flavonoids are grouped into several classes, differing from the oxidation degree of the central pyran ring, except of chalcones, including flavanols, flavonols, flavononols, flavones, flavanones, flavanes, anthocyanidins and anthocyanins, chalcones and dihydrochalcones (**Figure 3I**) [12].

In grapes, the highest flavonol concentrations were found at flowering, decreasing as the grapes maturate. Some of the flavonoids present in both grapes and wine are represented in **Figure 3II**. In the flavonoid class, the chemical diversity *versus* complexity is related to the high variety of aglycones and glycosides, as well as to the occurrence of condensation reactions, three distinct categories being usually accepted: glycosylated flavonoids, flavonoid aglycones and anthocyanidin glycosides (anthocyanins) [16].

Flavanols (**Figure 3I**) are benzopyrans with a saturated carbon chain between C2 and C3, a hydroxyl function in C3, and no carbonyl group in C4. The most abundant flavan-3-ols in nature are catechin and its enantiomer epicatechin (**Figure 3II**), and they can be found in the grape skin, seeds and stems, as well as in wine. Some catechin derivatives, such as gallocatechin, epigallocatechin, epicatechin gallate and epigallocatechin gallate, were identified in grapes and wine [17].

Flavonols (**Figure 3I**) have a double bond between C2 and C3 atoms and a hydroxyl group in C3, being often named 3-hydroxyflavones. Approximately 90% of the flavonols are hydroxylated in C3, C5 and C7, being designated as 3,5,7-trihydroxylated derivatives [18]. In grapes, there were identified: quercetin (3′,4′-diOH), kaempferol (4′-OH), myricetin (3′,4′,5′-triOH), isorhamnetin (3′-MeO analog of quercetin), laricitrin (3′-MeO analog of myricetin) and syringetin (3′,5′-MeO analog of myricetin) (**Figure 3II**). In *Vitis vinifera* grapes, there was detected the simultaneous presence of these aglycones [19]. The most abundant condensed flavonoids are the *O*-glucosides, the *O*-sulfates and the derivatives containing acylated sugars and aliphatic or aromatic acid groups in their structure [20]. White wine contains only quercetin, kaempferol and isorhamnetin [20]. Depending on the nature of sugar moiety bound at position C-3, there are three different complete series for flavonol 3-*O*-glycosides in red grapes. The 3-*O*-glucosides are the main derivative of the flavonol aglycones, namely kaempferol, quercetin, isorhamnetin, myricetin, laricitrin and syringetin, while the minor ones are the 3-*O*-galactoside derivatives.

Flavononols (**Figure 3I**) are characterized by the presence of a hydroxyl group in the C3 position and the absence of a double bond in the heterocyclic ring, being also named 3-hydroxyflavonones or dihydroflavonols (e.g., taxifolin, astilbin and dihydromyricetin 3-*O*-rhamnoside) [16].

Flavones (**Figure 3I**) have a double bond between carbons C2 and C3, and the hydroxyl group is absent in the C3 position. Although they are widely encountered in plants, as aglycones or glycosides, they are not present in grapes in significant amounts, except for luteolin (**Figure 3II**) [8].

Flavanones (**Figure 3I**) have a saturated carbon chain between atoms C2 and C3, being often named dihydroflavones (e.g., eriodictyol, a flavanone that has been extracted from grapes) [21].

**Figure 3.** General chemical structure of flavonoid subgroups.

The Evolution of Polyphenols from Grapes to Wines http://dx.doi.org/10.5772/intechopen.72800 125

**Figure 3.** General chemical structure of flavonoid subgroups.

**2.2. Flavonoids**

grapes and wine [17].

3-*O*-galactoside derivatives.

3-*O*-rhamnoside) [16].

They are a chemical class with a basic structure of 15 carbon atoms, including two aromatic rings bound through a three-carbon chain (C6-C3-C6), which is responsible for the chemical. Flavonoids are grouped into several classes, differing from the oxidation degree of the central pyran ring, except of chalcones, including flavanols, flavonols, flavononols, flavones, flavanones, flavanes,

In grapes, the highest flavonol concentrations were found at flowering, decreasing as the grapes maturate. Some of the flavonoids present in both grapes and wine are represented in **Figure 3II**. In the flavonoid class, the chemical diversity *versus* complexity is related to the high variety of aglycones and glycosides, as well as to the occurrence of condensation reactions, three distinct categories being usually accepted: glycosylated flavonoids, flavonoid

Flavanols (**Figure 3I**) are benzopyrans with a saturated carbon chain between C2 and C3, a hydroxyl function in C3, and no carbonyl group in C4. The most abundant flavan-3-ols in nature are catechin and its enantiomer epicatechin (**Figure 3II**), and they can be found in the grape skin, seeds and stems, as well as in wine. Some catechin derivatives, such as gallocatechin, epigallocatechin, epicatechin gallate and epigallocatechin gallate, were identified in

Flavonols (**Figure 3I**) have a double bond between C2 and C3 atoms and a hydroxyl group in C3, being often named 3-hydroxyflavones. Approximately 90% of the flavonols are hydroxylated in C3, C5 and C7, being designated as 3,5,7-trihydroxylated derivatives [18]. In grapes, there were identified: quercetin (3′,4′-diOH), kaempferol (4′-OH), myricetin (3′,4′,5′-triOH), isorhamnetin (3′-MeO analog of quercetin), laricitrin (3′-MeO analog of myricetin) and syringetin (3′,5′-MeO analog of myricetin) (**Figure 3II**). In *Vitis vinifera* grapes, there was detected the simultaneous presence of these aglycones [19]. The most abundant condensed flavonoids are the *O*-glucosides, the *O*-sulfates and the derivatives containing acylated sugars and aliphatic or aromatic acid groups in their structure [20]. White wine contains only quercetin, kaempferol and isorhamnetin [20]. Depending on the nature of sugar moiety bound at position C-3, there are three different complete series for flavonol 3-*O*-glycosides in red grapes. The 3-*O*-glucosides are the main derivative of the flavonol aglycones, namely kaempferol, quercetin, isorhamnetin, myricetin, laricitrin and syringetin, while the minor ones are the

Flavononols (**Figure 3I**) are characterized by the presence of a hydroxyl group in the C3 position and the absence of a double bond in the heterocyclic ring, being also named 3-hydroxyflavonones or dihydroflavonols (e.g., taxifolin, astilbin and dihydromyricetin

Flavones (**Figure 3I**) have a double bond between carbons C2 and C3, and the hydroxyl group is absent in the C3 position. Although they are widely encountered in plants, as aglycones or glycosides, they are not present in grapes in significant amounts, except for luteolin (**Figure 3II**) [8]. Flavanones (**Figure 3I**) have a saturated carbon chain between atoms C2 and C3, being often named dihydroflavones (e.g., eriodictyol, a flavanone that has been extracted from grapes) [21].

anthocyanidins and anthocyanins, chalcones and dihydrochalcones (**Figure 3I**) [12].

aglycones and anthocyanidin glycosides (anthocyanins) [16].

124 Grapes and Wines - Advances in Production, Processing, Analysis and Valorization

Flavanes (**Figure 3I**) contain a saturated carbon chain between C2 and C3, and no carbonyl group in C4 [22].

Chalcones have two aromatic rings linked by a carbonylic α,β-insaturated system (**Figure 3I**). Dihydrochalcones are obtained from chalcones through a reduction process [23].

Different families of anthocyanin derivatives have been reported in grapes, wine and winelike solutions. Anthocyanidins and anthocyanins (**Figure 3I**) have as nucleus the 2-phenylbenzopyrylium (the flavylium) cation. Anthocyanins are anthocyanidin glycosides. They are responsible for the color of grapes and wines. In red grapes and wines, six anthocyanidins have been detected: cyanidin (orange red), peonidin (red), delphinidin (bluish red), pelargonidin (orange), petunidin and malvidin (bluish red) [24], the last one being the most representative compound in *V. vinifera* [25, 26]. Also, the 3-*O*-monoglucosides of the mentioned six anthocyanidins have already been detected in grapes [24, 25, 27]. The anthocyanins are mainly found in the skin of grapes, while the other flavonoids occur in both skin and seeds. The amount of anthocyanins and other flavonoids extracted during vinification depends on the duration of the process, the temperature or the extent of disruption of the grapes [16]. In the grape vines, anthocyanins accumulate in the leaves during senescence and they determine the coloration of the grape skin in red and rose cultivars. The anthocyanidin composition of grapes is affected by factors such as the grape-growing origin (*terroir*) and varieties, the degree of maturity and the weather conditions. The profiles of anthocyanins for each grape variety are relatively stable, while absolute concentrations can vary widely between different vintages due to environmental and agronomical factors [11].

(proanthocyanidins) are polymeric compounds that are transformed into anthocyanidins, and they can be found in residual amounts of the solid grape components (e.g., seeds, skin) or in the pulp [27]. They are transferred into the must during winemaking operations such as crushing, maceration and fermentation [27]. Proanthocyanidins generally occur as oligomers and polymers of flavan-3-ols. The proportion of the polymer units and the average number of proanthocyanidins have been highlighted by LC–MS/MS and thiolytic degradation [31]. *V. vinifera* grapes contain mainly proanthocyanidins as oligomers and polymers of (+)-catechin and (−)-epicatechin linked through C4/C8 bonds [32]. The procyanidins and prodelphinidins, leading to cyanidin and delphinidin, are the most abundant condensed tannins in grapes and wine [32]. The relationship between monomeric, oligomeric and flavan-3-ol composition has been intensively studied as a prerequisite for improving wine quality, being showed that flavan-3-ols and their monomers ((+)-catechin and (−)-epicatechin) as predominant phenolic compounds extracted from grape seeds have a highly positive correlation with antioxidant activity. In *V. vinifera*, oligomers with a maximum degree of polymerization of 16 have been identified. The identification and quantification of proanthocyanidins is an important subject, since these compounds are mainly responsible for the sensory characteristics of wine and they play an important role in the wine aging process.

The Evolution of Polyphenols from Grapes to Wines http://dx.doi.org/10.5772/intechopen.72800 127

**Figure 4.** Chemical structures of resveratrol and resveratrol glycoside.

**3. Polyphenolic changes in the winemaking and polyphenol's** 

The total extractable phenolic content in grapes is encountered in the pulp (about 10% or less), in the seeds (60–70%) and in the skin (28–35%). In the seeds, the phenolic content may range

**extraction techniques**

Stilbenes have two aromatic rings linked by an ethene bridge, resveratrol (3,5,4′-trihydroxystilbene, **Figure 4**) being the most important in grapes and wine. Also, it was identified in vine leaf and in the grapes skin, and its concentration is known to decrease significantly upon grape maturation [28]. Due to the presence of resveratrol in the grapes skin, the wineprocessing methods determine its concentration in the final product. Wines from grapes with longer maturation periods have an increased content of resveratrol, its concentration being higher in red wines compared to white wines [28]. The stilbenes in wines can contribute to the correlation of their profiles with winemaking procedures and with the grape wine varieties.

Tannins are polyphenols with astringent properties, able to cause protein precipitation. They are usually divided in two classes: the hydrolyzable and the nonhydrolyzable or condensed tannins [22]. Most of the natural tannins present in grapes and wine are the condensed type. In young wines, tannins occur mainly in the form of dimers or trimers. However, their concentration decreases with aging due to oxidation and precipitation processes [29]. Hydrolyzable tannins can be degraded through pH changes as well as by enzymatic or nonenzymatic hydrolysis into smaller fragments, mainly sugars and phenolic acids. Their basic unit is gallic acid (**Figure 2**) and its derivatives (e.g., ellagic acid). These acids are usually esterified with D-glucose, resulting 500–2800 molecular weight species. Aging in oak barrels promotes the extraction of low molecular weight phenolic compounds, mainly from ellagitannins, into wine. For example, ellagic acid and myricetin are probably the major phenolic compounds present in muscadine grapes (*Vitis rotundifolia)* [30]. Condensed tannins

**Figure 4.** Chemical structures of resveratrol and resveratrol glycoside.

Flavanes (**Figure 3I**) contain a saturated carbon chain between C2 and C3, and no carbonyl

Chalcones have two aromatic rings linked by a carbonylic α,β-insaturated system (**Figure 3I**).

Different families of anthocyanin derivatives have been reported in grapes, wine and winelike solutions. Anthocyanidins and anthocyanins (**Figure 3I**) have as nucleus the 2-phenylbenzopyrylium (the flavylium) cation. Anthocyanins are anthocyanidin glycosides. They are responsible for the color of grapes and wines. In red grapes and wines, six anthocyanidins have been detected: cyanidin (orange red), peonidin (red), delphinidin (bluish red), pelargonidin (orange), petunidin and malvidin (bluish red) [24], the last one being the most representative compound in *V. vinifera* [25, 26]. Also, the 3-*O*-monoglucosides of the mentioned six anthocyanidins have already been detected in grapes [24, 25, 27]. The anthocyanins are mainly found in the skin of grapes, while the other flavonoids occur in both skin and seeds. The amount of anthocyanins and other flavonoids extracted during vinification depends on the duration of the process, the temperature or the extent of disruption of the grapes [16]. In the grape vines, anthocyanins accumulate in the leaves during senescence and they determine the coloration of the grape skin in red and rose cultivars. The anthocyanidin composition of grapes is affected by factors such as the grape-growing origin (*terroir*) and varieties, the degree of maturity and the weather conditions. The profiles of anthocyanins for each grape variety are relatively stable, while absolute concentrations can vary widely between different vintages due to environmental and agronomical factors [11].

Stilbenes have two aromatic rings linked by an ethene bridge, resveratrol (3,5,4′-trihydroxystilbene, **Figure 4**) being the most important in grapes and wine. Also, it was identified in vine leaf and in the grapes skin, and its concentration is known to decrease significantly upon grape maturation [28]. Due to the presence of resveratrol in the grapes skin, the wineprocessing methods determine its concentration in the final product. Wines from grapes with longer maturation periods have an increased content of resveratrol, its concentration being higher in red wines compared to white wines [28]. The stilbenes in wines can contribute to the correlation of their profiles with winemaking procedures and with the grape wine

Tannins are polyphenols with astringent properties, able to cause protein precipitation. They are usually divided in two classes: the hydrolyzable and the nonhydrolyzable or condensed tannins [22]. Most of the natural tannins present in grapes and wine are the condensed type. In young wines, tannins occur mainly in the form of dimers or trimers. However, their concentration decreases with aging due to oxidation and precipitation processes [29]. Hydrolyzable tannins can be degraded through pH changes as well as by enzymatic or nonenzymatic hydrolysis into smaller fragments, mainly sugars and phenolic acids. Their basic unit is gallic acid (**Figure 2**) and its derivatives (e.g., ellagic acid). These acids are usually esterified with D-glucose, resulting 500–2800 molecular weight species. Aging in oak barrels promotes the extraction of low molecular weight phenolic compounds, mainly from ellagitannins, into wine. For example, ellagic acid and myricetin are probably the major phenolic compounds present in muscadine grapes (*Vitis rotundifolia)* [30]. Condensed tannins

Dihydrochalcones are obtained from chalcones through a reduction process [23].

126 Grapes and Wines - Advances in Production, Processing, Analysis and Valorization

group in C4 [22].

varieties.

(proanthocyanidins) are polymeric compounds that are transformed into anthocyanidins, and they can be found in residual amounts of the solid grape components (e.g., seeds, skin) or in the pulp [27]. They are transferred into the must during winemaking operations such as crushing, maceration and fermentation [27]. Proanthocyanidins generally occur as oligomers and polymers of flavan-3-ols. The proportion of the polymer units and the average number of proanthocyanidins have been highlighted by LC–MS/MS and thiolytic degradation [31]. *V. vinifera* grapes contain mainly proanthocyanidins as oligomers and polymers of (+)-catechin and (−)-epicatechin linked through C4/C8 bonds [32]. The procyanidins and prodelphinidins, leading to cyanidin and delphinidin, are the most abundant condensed tannins in grapes and wine [32]. The relationship between monomeric, oligomeric and flavan-3-ol composition has been intensively studied as a prerequisite for improving wine quality, being showed that flavan-3-ols and their monomers ((+)-catechin and (−)-epicatechin) as predominant phenolic compounds extracted from grape seeds have a highly positive correlation with antioxidant activity. In *V. vinifera*, oligomers with a maximum degree of polymerization of 16 have been identified. The identification and quantification of proanthocyanidins is an important subject, since these compounds are mainly responsible for the sensory characteristics of wine and they play an important role in the wine aging process.

## **3. Polyphenolic changes in the winemaking and polyphenol's extraction techniques**

The total extractable phenolic content in grapes is encountered in the pulp (about 10% or less), in the seeds (60–70%) and in the skin (28–35%). In the seeds, the phenolic content may range between 5% and 8%, by weight [33]. There are a great number of methods for polyphenol extraction from grapes. **Table 2** summarizes the most applied extraction techniques.

ultrapure water, stirring continuously, then adding 125 mL ethanol and adjusting the final volume to 1 L with ultrapure water. Often, extracts have to be concentrated by solid phase extraction (SPE) before performing analysis by high-performance liquid chromatography (HPLC) or spectrophotometry. As soon as the grapes were harvested, the seeds and skins of 50 berries must be separated from the others parts of the berry, immersed in two solutions of 50 mL tartrate buf-

heated at room temperature and kept for 4 h; the extracts must be then homogenized and centrifuged; the supernatant transferred in a 100-mL volumetric flask, pellets being resuspended twice in 15–20 mL tartrate buffer; after centrifugation, the liquid phases can be collected in the same volumetric flask; the final volume must be adjusted to 100 mL with tartrate buffer [33].

When using acetone/water solutions for the extraction, skins from 300 berries are rinsed with ultrapure water, after separation from the flashes, immersed in 100 mL of aqueous solution con-

be laid into a 500-mL Erlenmeyer flask, over which 200 mL of acetone is added. Next, the flask is covered and placed for 24 h on a shaker, at 20°C and 100 rpm. After this procedure, the acetone is removed at 40°C, under vacuum; the solution is centrifuged, and the supernatant is recovered and diluted with ultrapure water, at 100 mL, and finally stored at −20°C until analysis [35].

Another method for extracting polyphenols from seeds and skins is by immersion of the fine powder (previously obtained by grinding the grape skins and seeds in liquid nitrogen) in an acetone/water 7:3 (v/v) solution containing 0.1% ascorbic acid added to prevent oxidation

Other methods use methanol for extraction: 250 mg skins must be stored at −80°C, then grounded with mortar and pestle in liquid nitrogen and soaked in 30 mL methanol at 4°C; the

Ethanol/water/chloroform solutions can be also used for phenolic compound extraction; the skins and seeds are freeze-dried until obtaining 1 g (dry weight) and 0.5 g (dry weight), respectively, and subjected to an extraction procedure using 20 mL ethanol/water/chloroform 1:1:2 (v/v/v) for 2 min. The 50% upper layer of ethanol containing the phenolic compounds is separated by the chloroform lower layer which contains undesirable compounds like chlorophylls and lipids; the ethanol is removed under vacuum; the resulted aqueous solution is then filtered through a glass membrane, diluted with water up to 100 mL and stored at −20°C

Another method proposes that 1 g of freeze-dried grape seeds and skins is to be extracted, using a blender, with 5 × 20 mL with ethanol/water 1:1 (v/v). Subsequently, the obtained extracts are adjusted to a final volume of 100 mL and pH 4.0 and then purified and fractionated on a 250 × 5 mm column, type Toyopearl Gel HW-40(s), using as eluent the methanol [39].

Monomer phenols in grapes and wines are usually analyzed by high-performance liquid chromatography (HPLC) using a reverse phase C18 column (usually 250 × 4 mm, 5 μm) oper-

**4. Advanced techniques for polyphenol analysis**

ating close to or at room temperature.

extract is then concentrated to almost dryness and dissolved in 1 mL methanol [37].

and immediately frozen. Before analysis, the samples must be

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and stored at 4°C. In order to perform the extraction, the sample must

fer solutions containing 1 g/L SO<sup>2</sup>

taining 100 mg/L of SO<sup>2</sup>

[36].

until analysis [38].

The main phenolic compounds in musts from white grapes are hydroxycinnamic tartaric acid esters (HCTA). Catechins and proanthocyanidins are found mainly in the skins of white grapes. They can sustain oxidation; as a consequence, the grapes must often be protected by limiting the contact with oxygen and inhibiting polyphenoloxidase (PPO) by SO<sup>2</sup> addition. Due to the oxidation, the phenolic profile of the musts is characterized from lower levels of HCTA and flavanols, and high 2-S-glutationyl caffeyl tartaric acid (GRP). Red wines are produced by the must fermentation in the presence of the grape skins and seeds. During the process, phenolic compounds such as anthocyanins are subjected to various reactions, such as enzymatic oxidation, electrophilic substitution, degradation of anthocyanins, cyclo-accession of the carbonyl compounds to anthocyanins and formation of vitisins (A, B and C), hydrolysis of proanthocyanidins and formation of carbocation in C4 position which attacks the positions C6 and C8 of the proanthocyanidins and anthocyanins, attack of C4 carbocation of anthocyanin to C6 or C8 of proanthocyanidins, attack of acetaldehyde to C6 and C8 of proanthocyanidins and formation of structures with flavanol linked to anthocyanin by ethyl bridge [33].

Polyphenol extraction from grapes can be achieved by using alcoholic or hydroalcoholic solutions either containing, or not containing, mineral acids, or using acetone/water solutions. The use of a mineral acid (usually methanol containing HCl 1% by volume) allows extraction of all phenolic compounds. The use of nonalcoholic acid containing solutions does not guarantee satisfying extraction of HCTA. Grapes' freezing after the harvest, followed by heating at room temperature before performing extraction, allows PPO to be in touch with and to oxidize the substrates [34].

A satisfying extraction method for all polyphenols is the use of a model wine solution containing: tartrate buffer pH 3.2 (with 12% ethanol, v/v) and sodium metabisulfite 2 g/L. This can be prepared by adding 5 g tartaric acid, 22.2 mL 1 M NaOH solution and 2 g of Na<sup>2</sup> S2 O5 to 500 mL


**Table 2.** Polyphenol extraction methods.

ultrapure water, stirring continuously, then adding 125 mL ethanol and adjusting the final volume to 1 L with ultrapure water. Often, extracts have to be concentrated by solid phase extraction (SPE) before performing analysis by high-performance liquid chromatography (HPLC) or spectrophotometry. As soon as the grapes were harvested, the seeds and skins of 50 berries must be separated from the others parts of the berry, immersed in two solutions of 50 mL tartrate buffer solutions containing 1 g/L SO<sup>2</sup> and immediately frozen. Before analysis, the samples must be heated at room temperature and kept for 4 h; the extracts must be then homogenized and centrifuged; the supernatant transferred in a 100-mL volumetric flask, pellets being resuspended twice in 15–20 mL tartrate buffer; after centrifugation, the liquid phases can be collected in the same volumetric flask; the final volume must be adjusted to 100 mL with tartrate buffer [33].

between 5% and 8%, by weight [33]. There are a great number of methods for polyphenol

The main phenolic compounds in musts from white grapes are hydroxycinnamic tartaric acid esters (HCTA). Catechins and proanthocyanidins are found mainly in the skins of white grapes. They can sustain oxidation; as a consequence, the grapes must often be protected by

Due to the oxidation, the phenolic profile of the musts is characterized from lower levels of HCTA and flavanols, and high 2-S-glutationyl caffeyl tartaric acid (GRP). Red wines are produced by the must fermentation in the presence of the grape skins and seeds. During the process, phenolic compounds such as anthocyanins are subjected to various reactions, such as enzymatic oxidation, electrophilic substitution, degradation of anthocyanins, cyclo-accession of the carbonyl compounds to anthocyanins and formation of vitisins (A, B and C), hydrolysis of proanthocyanidins and formation of carbocation in C4 position which attacks the positions C6 and C8 of the proanthocyanidins and anthocyanins, attack of C4 carbocation of anthocyanin to C6 or C8 of proanthocyanidins, attack of acetaldehyde to C6 and C8 of proanthocyanidins and formation of structures with flavanol linked to anthocyanin by ethyl bridge [33]. Polyphenol extraction from grapes can be achieved by using alcoholic or hydroalcoholic solutions either containing, or not containing, mineral acids, or using acetone/water solutions. The use of a mineral acid (usually methanol containing HCl 1% by volume) allows extraction of all phenolic compounds. The use of nonalcoholic acid containing solutions does not guarantee satisfying extraction of HCTA. Grapes' freezing after the harvest, followed by heating at room temperature before performing extraction, allows PPO to be in touch with and to oxidize the

A satisfying extraction method for all polyphenols is the use of a model wine solution containing: tartrate buffer pH 3.2 (with 12% ethanol, v/v) and sodium metabisulfite 2 g/L. This can be

SPE

MSPD

**Extraction Clean-up isolation Instrumental analysis**

based fragmentation:

Column chromatography/acidity-

Thin-layer chromatography (TLC),

High-speed countercurrent chromatography (HSCCC)

prepared by adding 5 g tartaric acid, 22.2 mL 1 M NaOH solution and 2 g of Na<sup>2</sup>

Supercritical fluid extraction (SFE)/solid phase extraction (SPE)/solid phase microextraction (SPME)

Accelerated solvent extraction/ pressurized liquid extraction

Matrix solid phase dispersion

Soxhlet Solid-liquid Liquid-liquid

(ASE/PLE) Microwave

(MSPD) Sonication

**Table 2.** Polyphenol extraction methods.

addition.

S2 O5

CE

Liquid Chromatography (LC): UV, FLUD, ECD, NMR, MS, MS<sup>n</sup>

Gas Chromatography (GC): FID, ECD, MS, MS<sup>n</sup>

to 500 mL

extraction from grapes. **Table 2** summarizes the most applied extraction techniques.

128 Grapes and Wines - Advances in Production, Processing, Analysis and Valorization

limiting the contact with oxygen and inhibiting polyphenoloxidase (PPO) by SO<sup>2</sup>

substrates [34].

**Sample preparation**

Freeze-drying Air drying Centrifugation Filtration

When using acetone/water solutions for the extraction, skins from 300 berries are rinsed with ultrapure water, after separation from the flashes, immersed in 100 mL of aqueous solution containing 100 mg/L of SO<sup>2</sup> and stored at 4°C. In order to perform the extraction, the sample must be laid into a 500-mL Erlenmeyer flask, over which 200 mL of acetone is added. Next, the flask is covered and placed for 24 h on a shaker, at 20°C and 100 rpm. After this procedure, the acetone is removed at 40°C, under vacuum; the solution is centrifuged, and the supernatant is recovered and diluted with ultrapure water, at 100 mL, and finally stored at −20°C until analysis [35].

Another method for extracting polyphenols from seeds and skins is by immersion of the fine powder (previously obtained by grinding the grape skins and seeds in liquid nitrogen) in an acetone/water 7:3 (v/v) solution containing 0.1% ascorbic acid added to prevent oxidation [36].

Other methods use methanol for extraction: 250 mg skins must be stored at −80°C, then grounded with mortar and pestle in liquid nitrogen and soaked in 30 mL methanol at 4°C; the extract is then concentrated to almost dryness and dissolved in 1 mL methanol [37].

Ethanol/water/chloroform solutions can be also used for phenolic compound extraction; the skins and seeds are freeze-dried until obtaining 1 g (dry weight) and 0.5 g (dry weight), respectively, and subjected to an extraction procedure using 20 mL ethanol/water/chloroform 1:1:2 (v/v/v) for 2 min. The 50% upper layer of ethanol containing the phenolic compounds is separated by the chloroform lower layer which contains undesirable compounds like chlorophylls and lipids; the ethanol is removed under vacuum; the resulted aqueous solution is then filtered through a glass membrane, diluted with water up to 100 mL and stored at −20°C until analysis [38].

Another method proposes that 1 g of freeze-dried grape seeds and skins is to be extracted, using a blender, with 5 × 20 mL with ethanol/water 1:1 (v/v). Subsequently, the obtained extracts are adjusted to a final volume of 100 mL and pH 4.0 and then purified and fractionated on a 250 × 5 mm column, type Toyopearl Gel HW-40(s), using as eluent the methanol [39].

## **4. Advanced techniques for polyphenol analysis**

Monomer phenols in grapes and wines are usually analyzed by high-performance liquid chromatography (HPLC) using a reverse phase C18 column (usually 250 × 4 mm, 5 μm) operating close to or at room temperature.

For example, in order to obtain the anthocyanin monomer profile of grapes, a Cabernet Sauvignon grape skin extract was used, the chromatogram being displayed in **Figure 5**. A column type C18 (250 × 4 mm, 5 μm), at 45°C, using a binary solvent mixture of formic acid/ water 10:90 (v/v) and formic acid/methanol/water 10:50:40 (v/v/v) and detection at 520 nm wavelength were used for the analysis. For compound elution from the column, a gradient program was applied. The sample volume injected is 20 μL [33].

Peaks of some typical anthocyanins for *V. vinifera* varieties, namely 3-glucosides, 3-glucoside acetates, 3-glucoside para-coumarates and malvidin 3-glucoside caffeate, are highlighted in the **Figure 5**. Even if different separation column is used, the sequence for compound elution from a reverse phase column is due to their polarity and is always the same. The values of single anthocyanin concentration can be expressed as area percentage on the total area amount of the peaks of all identified anthocyanins or in mg/kg grapes of malvidin-3-glucoside (compound used as external standard).

HPLC with UV-Vis detection makes possible the analysis of some anthocyanin derivatives in wines, such as vitisins, several pyranoanthocyanins and flavanol-ethyl-anthocyanin derivatives. Due to the lack of commercially available standards, identification and quantification of these compounds is difficult. Usually, the column is a reverse-phase polystyrene divinylbenzene (250 × 4.6 mm, 5 μm) operating at 30°C, elution being performed by a binary mobile phase mixture composed of aqueous H3 PO4 1.5% (w/w) and aqueous H3 PO4 /acetonitrile 20:80 (v/v) with a gradient program, at 520 nm, the compounds being quantified as malvidin-3-glucoside. Analysis can be performed by direct injection of the sample or, to increase the sensibility, after concentration on a C18 cartridge [40].

Due to the lack of commercially available standards for HCTA, chlorogenic acid or free hydroxycinnamic acids are used as external standards for quantitative analysis. Quercetin and myricetin glucoside and other flavonols glycoside (e.g., rutin) are standards of flavonols

For the HCTA and flavonols in wines, analysis can be performed on the acidified sample

The typical HPLC chromatograms of grape skins and red wines for the identification of *trans*-resveratrol in grape skins and red wines are presented in **Figures 7** and **8**, respectively. *Trans*-resveratrol was identified by the comparison with a commercial standard of *trans*resveratrol. An Aquasil C18 (250 × 4.6 mm, 5 μm) analytical column was used for separation. Chromatographic conditions included a mixed mobile phase of water:acetonitrile:acetic acid

to 4.5 mL of wine and filtration on 0.45 μm

The Evolution of Polyphenols from Grapes to Wines http://dx.doi.org/10.5772/intechopen.72800 131

PO4

membrane. The HPLC conditions used are the same reported for skin extract.

**Figure 7.** HPLC chromatogram of grape skin (*V. vinifera*, Pinot Noir) extract and standard [28].

**Figure 6.** HPLC profile of flavonols of a Cabernet Sauvignon grape skin extract recorded at 360 nm [33].

commercially available.

prepared by the addition of 0.5 mL 1 M H3

HCTA and flavonols can be simultaneously detected in a single run using a C18 (250 × 4 mm, 5 μm) column operating at 40°C, performing elution with a binary solvent mixture composed of H<sup>3</sup> PO4 10−3 M and methanol, and an elution gradient program. **Figure 6** shows a typical chromatogram for flavonols, respectively, recorded in the analysis of a Cabernet Sauvignon grape skin extract. Detection wavelength was 360 nm for flavonols (sample volume injected 25 μL). Identification of compounds is performed on the elution order from the column and by recording UV-Vis spectra on the basis of maximum wavelengths [33].

**Figure 5.** HPLC anthocyanin profile of Cabernet Sauvignon grape skin extract.

**Figure 6.** HPLC profile of flavonols of a Cabernet Sauvignon grape skin extract recorded at 360 nm [33].

For example, in order to obtain the anthocyanin monomer profile of grapes, a Cabernet Sauvignon grape skin extract was used, the chromatogram being displayed in **Figure 5**. A column type C18 (250 × 4 mm, 5 μm), at 45°C, using a binary solvent mixture of formic acid/ water 10:90 (v/v) and formic acid/methanol/water 10:50:40 (v/v/v) and detection at 520 nm wavelength were used for the analysis. For compound elution from the column, a gradient

Peaks of some typical anthocyanins for *V. vinifera* varieties, namely 3-glucosides, 3-glucoside acetates, 3-glucoside para-coumarates and malvidin 3-glucoside caffeate, are highlighted in the **Figure 5**. Even if different separation column is used, the sequence for compound elution from a reverse phase column is due to their polarity and is always the same. The values of single anthocyanin concentration can be expressed as area percentage on the total area amount of the peaks of all identified anthocyanins or in mg/kg grapes of malvidin-3-glucoside

HPLC with UV-Vis detection makes possible the analysis of some anthocyanin derivatives in wines, such as vitisins, several pyranoanthocyanins and flavanol-ethyl-anthocyanin derivatives. Due to the lack of commercially available standards, identification and quantification of these compounds is difficult. Usually, the column is a reverse-phase polystyrene divinylbenzene (250 × 4.6 mm, 5 μm) operating at 30°C, elution being performed by a binary mobile phase

with a gradient program, at 520 nm, the compounds being quantified as malvidin-3-glucoside. Analysis can be performed by direct injection of the sample or, to increase the sensibility,

HCTA and flavonols can be simultaneously detected in a single run using a C18 (250 × 4 mm, 5 μm) column operating at 40°C, performing elution with a binary solvent mixture composed

chromatogram for flavonols, respectively, recorded in the analysis of a Cabernet Sauvignon grape skin extract. Detection wavelength was 360 nm for flavonols (sample volume injected 25 μL). Identification of compounds is performed on the elution order from the column and

1.5% (w/w) and aqueous H3

10−3 M and methanol, and an elution gradient program. **Figure 6** shows a typical

PO4

/acetonitrile 20:80 (v/v)

program was applied. The sample volume injected is 20 μL [33].

130 Grapes and Wines - Advances in Production, Processing, Analysis and Valorization

PO4

by recording UV-Vis spectra on the basis of maximum wavelengths [33].

**Figure 5.** HPLC anthocyanin profile of Cabernet Sauvignon grape skin extract.

(compound used as external standard).

mixture composed of aqueous H3

of H<sup>3</sup> PO4

after concentration on a C18 cartridge [40].

Due to the lack of commercially available standards for HCTA, chlorogenic acid or free hydroxycinnamic acids are used as external standards for quantitative analysis. Quercetin and myricetin glucoside and other flavonols glycoside (e.g., rutin) are standards of flavonols commercially available.

For the HCTA and flavonols in wines, analysis can be performed on the acidified sample prepared by the addition of 0.5 mL 1 M H3 PO4 to 4.5 mL of wine and filtration on 0.45 μm membrane. The HPLC conditions used are the same reported for skin extract.

The typical HPLC chromatograms of grape skins and red wines for the identification of *trans*-resveratrol in grape skins and red wines are presented in **Figures 7** and **8**, respectively. *Trans*-resveratrol was identified by the comparison with a commercial standard of *trans*resveratrol. An Aquasil C18 (250 × 4.6 mm, 5 μm) analytical column was used for separation. Chromatographic conditions included a mixed mobile phase of water:acetonitrile:acetic acid

**Figure 7.** HPLC chromatogram of grape skin (*V. vinifera*, Pinot Noir) extract and standard [28].

**Figure 8.** HPLC chromatogram of red wine (*V. vinifera*, Pinot Noir) and standard [28].

of 70:29.9:0.1, flowing through the system at the rate of 1 mL/min; the injection volume was 20 μL; the detection was set at 310 nm [28].

The phenolic profile in grape skins of red grape varieties grown in Southeastern Romania in 2013 season, under the same agronomic conditions and microclimate, showed significant differences. Each variety had a different ripening trend. The ripening influenced the phenolic composition of red grape skins, higher amounts of phenolic compounds being found on the last sampling week for Cabernet Sauvignon, Feteasca Neagra and Merlot grape cultivars, while phenolic maturity was reached for Mamaia and Pinot Noir. This observation must be taken into consideration in winemaking process, in order to obtain wines with high phenolic content [41]. Low molecular weight and volatile phenols are usually identified and quantified by Gas Chromatography coupled with Mass Spectrometry (GC-MS). For analysis and structural characterization of more polar compounds such as polyphenols, Liquid Chromatography Mass Spectrometry (LC-MS) and Multiple Mass Spectrometry (MS/MS and MS<sup>n</sup>) techniques are used [42]. These methods are the most effective techniques to characterize individual polyphenols in grape extracts and wine, due to the soft ionization conditions and minor sample purification required. Opposed to the LC methods coupled with spectrophotometric detection which require hydrolysis or thiolysis for compounds identification, the LC-MS is a more reliable and advanced technique to be applied in the study of compound structure linked with the color-changing of red wines during aging formed by reactions of anthocyanins with other compounds. This technique also allows the characterization of proanthocyanidins (procyanidins, prodelphinidins) also called condensed tannins. Moreover, for the particular case of studying compounds, the MS/MS approach is a very powerful tool that allows character-

**Syringic acid Catechin Epicatechin Rutin Trans-resveratrol**

The Evolution of Polyphenols from Grapes to Wines http://dx.doi.org/10.5772/intechopen.72800 133

**Grape cultivar Phenolic compound in grape skins**

Cabernet Sauvignon Average 6.1 44.2 20.3 30.0 70.6 Merlot Average 11.5 52.6 41.5 63.5 151.6 Feteasca Neagra Average 1.1 24.4 18.3 15.2 112.7 Mamaia Average 3.4 14.4 13.6 18.3 18.1 Pinot Noir Average Not detected 28.1 38.1 88.2 203.6

In order to analyze the grape flavonols, the extraction of berries with acidified methanol (0.01% 12 N HCl) must be performed. The extract is then filtered, the solvent removed under vacuum and the residue dissolved in 0.1 M citric acid buffer at pH 3.5. Polyphenolics are fractionated on the basis of their affinity to a C18 cartridge and then on a Sephadex LH-20 cartridge, the fractions being then eluted by ethyl acetate and methanol. To perform analysis of the isolate, ethyl acetate is evaporated and the residue is redissolved in the pH 3.5 buffer. The isolates can be analyzed by Liquid-Chromatography-Electrospray-Ionization-Mass Spectrometry. By connecting the LC/ESI-MS system to the probe of the mass spectrometer via the UV cell outlet, LC-UV chromatograms and spectra can also be recorded. Compounds such as vanillin and phenolic acid present in wine can be detected

ization of aglycone and sugar moiety.

**Table 3.** Phenolic compound in grape skins.

by LC/ESI-MS analysis [43].

In order to evaluate the difference in phenolic composition of grape skins, 35 samples of five red grape varieties (*V. vinifera L.*), namely Pinot Noir, Merlot, Cabernet Sauvignon, Feteasca Neagra and Mamaia cultivated in Southeastern Romania, were investigated by reversed-phase high-performance liquid chromatography (RP-HPLC) using a system with: a diode array detector set at 280 nm; the mobile phase––water with 0.1% formic acid and acetonitrile with 0.1% formic acid; and an Accuacore PFP (100 × 2.1 mm, 2.6 μm) column, operated at 30°C. The concentrations of phenolic compounds in the extracts were calculated as mg/L using external calibration curves generated from standards, which were obtained for each phenolic compound (**Figure 9** and **Table 3**) [41].

**Figure 9.** HPLC chromatogram of Cabernet Sauvignon grape skin extract [41].


**Table 3.** Phenolic compound in grape skins.

of 70:29.9:0.1, flowing through the system at the rate of 1 mL/min; the injection volume was

In order to evaluate the difference in phenolic composition of grape skins, 35 samples of five red grape varieties (*V. vinifera L.*), namely Pinot Noir, Merlot, Cabernet Sauvignon, Feteasca Neagra and Mamaia cultivated in Southeastern Romania, were investigated by reversed-phase high-performance liquid chromatography (RP-HPLC) using a system with: a diode array detector set at 280 nm; the mobile phase––water with 0.1% formic acid and acetonitrile with 0.1% formic acid; and an Accuacore PFP (100 × 2.1 mm, 2.6 μm) column, operated at 30°C. The concentrations of phenolic compounds in the extracts were calculated as mg/L using external calibration curves generated from standards, which were obtained for each phenolic compound (**Figure 9** and **Table 3**) [41].

20 μL; the detection was set at 310 nm [28].

**Figure 8.** HPLC chromatogram of red wine (*V. vinifera*, Pinot Noir) and standard [28].

132 Grapes and Wines - Advances in Production, Processing, Analysis and Valorization

**Figure 9.** HPLC chromatogram of Cabernet Sauvignon grape skin extract [41].

The phenolic profile in grape skins of red grape varieties grown in Southeastern Romania in 2013 season, under the same agronomic conditions and microclimate, showed significant differences. Each variety had a different ripening trend. The ripening influenced the phenolic composition of red grape skins, higher amounts of phenolic compounds being found on the last sampling week for Cabernet Sauvignon, Feteasca Neagra and Merlot grape cultivars, while phenolic maturity was reached for Mamaia and Pinot Noir. This observation must be taken into consideration in winemaking process, in order to obtain wines with high phenolic content [41].

Low molecular weight and volatile phenols are usually identified and quantified by Gas Chromatography coupled with Mass Spectrometry (GC-MS). For analysis and structural characterization of more polar compounds such as polyphenols, Liquid Chromatography Mass Spectrometry (LC-MS) and Multiple Mass Spectrometry (MS/MS and MS<sup>n</sup>) techniques are used [42]. These methods are the most effective techniques to characterize individual polyphenols in grape extracts and wine, due to the soft ionization conditions and minor sample purification required. Opposed to the LC methods coupled with spectrophotometric detection which require hydrolysis or thiolysis for compounds identification, the LC-MS is a more reliable and advanced technique to be applied in the study of compound structure linked with the color-changing of red wines during aging formed by reactions of anthocyanins with other compounds. This technique also allows the characterization of proanthocyanidins (procyanidins, prodelphinidins) also called condensed tannins. Moreover, for the particular case of studying compounds, the MS/MS approach is a very powerful tool that allows characterization of aglycone and sugar moiety.

In order to analyze the grape flavonols, the extraction of berries with acidified methanol (0.01% 12 N HCl) must be performed. The extract is then filtered, the solvent removed under vacuum and the residue dissolved in 0.1 M citric acid buffer at pH 3.5. Polyphenolics are fractionated on the basis of their affinity to a C18 cartridge and then on a Sephadex LH-20 cartridge, the fractions being then eluted by ethyl acetate and methanol. To perform analysis of the isolate, ethyl acetate is evaporated and the residue is redissolved in the pH 3.5 buffer. The isolates can be analyzed by Liquid-Chromatography-Electrospray-Ionization-Mass Spectrometry. By connecting the LC/ESI-MS system to the probe of the mass spectrometer via the UV cell outlet, LC-UV chromatograms and spectra can also be recorded. Compounds such as vanillin and phenolic acid present in wine can be detected by LC/ESI-MS analysis [43].

To achieve structural and semiquantitative information on the anthocyanins from grapes, ESI-MS/MS direct-injection analysis of extract can be performed. An example of an ESI-MS spectrum is reported in **Figure 10**, the peaks being identified and quantified in **Table 4**.

**Peak number RT (min) m/z Mass assignment (M+**

495

617

617

8 23.2 779/287

10 25.1 763

11 25.8 807

and electrospray ionization-MS.

 13.9 633 Malvidin-Delphinidin (oligomeric form) 1.87 15.1 617 Malvidin-Cyanidin (oligomeric form) 1.87 16.2 469/303 Delphinidin-3-*O*-glucoside 40.64 17.6 453/287 Cyanidin-3-*O*-glucoside 17.82 18.2 453/287 Cyanidin-3-*O*-galactoside 5.75 19.7 467/303 Delphinidin-3-*O*-glucoside 5.44 21.5 511/303 Delphinidin-3-(6-*O*-acetylglucoside) 4.25

9 24.7 763 Cyanidin-3-glucoside-ethyl-catechin 1.16

12 26.8 601 Malvidin-3-(6-*O*-acetylglucoside) pyruvate 6.01 13 28.1 645 Malvidin-Petunidin (oligomeric form) 0.16

**Figure 11.** LC-ESI-MS of flavan-3-ols from a wine sample analysis performed in negative ionization mode.

 Protocatechuic acid 153 (109) 109 (153) Protocatechuic aldehyde 137 137 (108) *p*-Hydroxibenzoic acid 137 (93) 93 (137) *p*-Hydroxibenzoic aldehyde 121 121 (92)

168 Vanillic acid 167 108 (167, 123, 91) 152 Vanillin 151 (136) 136 (151)

**MW Compound Main ions observed (m/z)**

**Table 4.** Peak assignments, retention time, mass special data, and percentage of grape skin anthocyanins detected by LC

 **sau [M + H]+**

Malvidin-Cyanidin + glucoside Malvidin-3-*O*-glucoside

Cyanidin-3-glucoside-ethyl-catechin Malvidin-Cyanidin (oligomeric form)

Malvidin-3-glucoside-8-vinyl(epi)catechin Malvidin-Cyanidin (oligomeric form)

**Fragm. 60 V Fragm. 120 V**

**) Anthocyanins content (%)**

135

The Evolution of Polyphenols from Grapes to Wines http://dx.doi.org/10.5772/intechopen.72800

4.75

0.53

9.71

Quantification by ESI/MS of anthocyanins present in grape extract can be done by constructing a calibration curve using two commercially available standard compounds, the Mv-3- *O*-glucoside (M+ species at m/z 493) for monoglucosides and the Mv-3,5-*O*-diglucoside (M+ species at m/z 655) for diglucosides [44].

Operating in the positive ionization mode, the ESI is also effective for analysis of flavan-3-ols. But since most phenolic acids in wine are not detectable in this mode, the negative ionization mode is preferable to work with (see an example in **Figure 11**) [43].

By operating with a cone voltage of 60 V, these compounds show high formation of the [M–H]− ion. The sample under investigation can be prepared by the liquid-liquid extraction of 50 mL wine, using diethyl ether (3 × 5 mL) and ethyl acetate (3 × 15 mL), after previous concentrating to 15 mL, at 30°C, under vacuum, to eliminate the ethanol. Next, the organic phases are combined and the resulting solution dried over Na<sup>2</sup> SO<sup>4</sup> . The solvent is removed under vacuum, then the residue is dissolved in 2 mL of methanol/water (1:1) and the solution is filtrated prior to analysis [45]. The compounds identified by LC-ESI-MS are reported in **Table 5** [43].

**Figure 10.** LC-ESI-MS anthocyanin profile of Othello grape skin extract.


**Table 4.** Peak assignments, retention time, mass special data, and percentage of grape skin anthocyanins detected by LC and electrospray ionization-MS.

To achieve structural and semiquantitative information on the anthocyanins from grapes, ESI-MS/MS direct-injection analysis of extract can be performed. An example of an ESI-MS spectrum is reported in **Figure 10**, the peaks being identified and quantified in **Table 4**.

Quantification by ESI/MS of anthocyanins present in grape extract can be done by constructing a calibration curve using two commercially available standard compounds, the Mv-3- *O*-glucoside (M+ species at m/z 493) for monoglucosides and the Mv-3,5-*O*-diglucoside (M+

Operating in the positive ionization mode, the ESI is also effective for analysis of flavan-3-ols. But since most phenolic acids in wine are not detectable in this mode, the negative ionization

By operating with a cone voltage of 60 V, these compounds show high formation of the [M–H]− ion. The sample under investigation can be prepared by the liquid-liquid extraction of 50 mL wine, using diethyl ether (3 × 5 mL) and ethyl acetate (3 × 15 mL), after previous concentrating to 15 mL, at 30°C, under vacuum, to eliminate the ethanol. Next, the organic phases are com-

then the residue is dissolved in 2 mL of methanol/water (1:1) and the solution is filtrated prior

to analysis [45]. The compounds identified by LC-ESI-MS are reported in **Table 5** [43].

SO<sup>4</sup>

. The solvent is removed under vacuum,

mode is preferable to work with (see an example in **Figure 11**) [43].

134 Grapes and Wines - Advances in Production, Processing, Analysis and Valorization

species at m/z 655) for diglucosides [44].

bined and the resulting solution dried over Na<sup>2</sup>

**Figure 10.** LC-ESI-MS anthocyanin profile of Othello grape skin extract.

**Figure 11.** LC-ESI-MS of flavan-3-ols from a wine sample analysis performed in negative ionization mode.



and the skins are kept cool enough, and maceration will occur in an aqueous medium. A key decision is when to separate wine and skins after fermentation has finished. After the wine has been separated from the skins, a pulp containing a mix of juice, skins and pips remains. This mixture is introduced in a press to squeeze out the remaining juice. The applied force and the type of the press determine the quality of the wine. This juice may be finished off separately from the rest of the wine or blended back into it. By pressing too hard, high concentrations of bitter compounds from the skins and seeds can be extracted,

The Evolution of Polyphenols from Grapes to Wines http://dx.doi.org/10.5772/intechopen.72800 137

The phenolic composition of the wine also changes along the wine aging process, reflecting in the color and astringency degree of the final product. The relative anthocyanin content decreases upon aging although this chemical modification is associated with a very clear change in color, this characteristic being often used as a quality standard for aged wines. One

The stability of the phenolics present in the wine is different from the one in situ (nonharvested grapes), several chemical changes, beginning in the grapes, and reaching completion only after the processing period. In general, the chemical composition of the final product is much more complex than the one of the raw material, due to the formation of a variety of new

Although procyanidins are related to grape composition, in wine, they were found to evolve during wine aging. In fact, they show a remarkable stability and resistance to several winemaking processes, such as sulfite bleaching. Oxygenated wines display characteristic color changes, along with a significant increase in the concentration of pyranoanthocyanins and related pigments [29]. In summary, one can say that a diversity of products can be obtained

A main conclusion can be displayed: the resulted wine is a very complex medium influenced by winemaking techniques applied to different batches of grapes. The anthocyanin content of grapes at harvest is of vital importance in achieving quality wines. Tannins are the largest contributor, their content and composition in grapes probably determining the final wine quality. Presently, tannins are not routinely measured, the total phenolic content being determined and linked to the tannin content of grapes and wine, although it is not a specific assay. The wine phenolics can be used as a fingerprint for their differentiation, according to the geographical origin, vine variety and vintage. Phenolics compounds can be used as markers to discriminate the origin of wine (e.g., geographical, vine variety and vintage). For selection of winemaking practices aimed to improve the wine quality, the knowledge of intrinsic physicochemical characteristics of the grape cultivar employed in the vinification is required, but also the influence of different technological procedure to the final product, the wine. Since there is an increasing demand for producing and protecting high-quality wines, extensive studies on the factors

influencing the chemical composition and biological effects of wine are still a necessity.

of the main factors responsible for anthocyanin loss is the storage temperature [24].

from condensation reactions between anthocyanins and tannins.

affecting the wine quality.

compounds.

**6. Conclusions**

**Table 5.** Some wine polyphenols determined by negative ionization mode.

#### **5. Factors influencing phenolic content and composition of wine**

Wine phenolic profile depends on the grapes and on the vinification technique, although other variables such as cultivar, viticulture practices can also affect it. The variety, growing season, environmental and climatic conditions, soil type and even maturity influence the concentration of phenolic compounds within grapes. The qualitative changes in the phenolic composition of the final wine, compared to the composition of the corresponding grapes, are mainly due to the production of new derivatives (such as flavenes, tyrosol and free phenolic acids––gallic, paracoumaric, caffeic, and so on). The environment in which both the must and the wine are produced can contribute to the generation of new phenolics [21]. Since wine composition is in constant evolution, the aging in bottles also seems to contribute to changes in the flavonol content, through the interaction of flavonols with other constituents [3]. It was found that the effect of vintage was significantly more pronounced on the anthocyanins than on the flavonols [46].

The total phenolic content in grapes is clearly affected by several factors: the cultivar, the year of production, the geographic origin of grapes, soil chemistry and the degree of maturation, many studies focusing on defining the effects of growing conditions on grape and wine phenolic composition and of the impact of light and temperature conditions on berry flavonoid composition [35]. The results revealed a combined effect of solar radiation and temperature on the compositional profile of flavonoids. It was observed that moderate exposure and temperature were favorable for the accumulation of anthocyanins, while an enhancement of skin tannins was noted in the grape berries from bunches exposed to sunlight versus those from shaded bunches or dense canopies. In fact, maturation is associated with polymerization of phenols, which leads to a marked decrease of astringency [16].

The phenolic composition, the relative proportions of anthocyanins and flavonols, changes mainly in the first steps of vinification and continues during storage, the applied techniques (maceration, fermentation, clarification, aging, etc.) influencing significantly both the concentration and the composition of phenolics and, therefore, also the color intensity and hue of red wines. Extraction of the beneficial components from the grape skins can occur before fermentation, during it, or after it. Fermentation can be delayed, if the must and the skins are kept cool enough, and maceration will occur in an aqueous medium. A key decision is when to separate wine and skins after fermentation has finished. After the wine has been separated from the skins, a pulp containing a mix of juice, skins and pips remains. This mixture is introduced in a press to squeeze out the remaining juice. The applied force and the type of the press determine the quality of the wine. This juice may be finished off separately from the rest of the wine or blended back into it. By pressing too hard, high concentrations of bitter compounds from the skins and seeds can be extracted, affecting the wine quality.

The phenolic composition of the wine also changes along the wine aging process, reflecting in the color and astringency degree of the final product. The relative anthocyanin content decreases upon aging although this chemical modification is associated with a very clear change in color, this characteristic being often used as a quality standard for aged wines. One of the main factors responsible for anthocyanin loss is the storage temperature [24].

The stability of the phenolics present in the wine is different from the one in situ (nonharvested grapes), several chemical changes, beginning in the grapes, and reaching completion only after the processing period. In general, the chemical composition of the final product is much more complex than the one of the raw material, due to the formation of a variety of new compounds.

Although procyanidins are related to grape composition, in wine, they were found to evolve during wine aging. In fact, they show a remarkable stability and resistance to several winemaking processes, such as sulfite bleaching. Oxygenated wines display characteristic color changes, along with a significant increase in the concentration of pyranoanthocyanins and related pigments [29]. In summary, one can say that a diversity of products can be obtained from condensation reactions between anthocyanins and tannins.

## **6. Conclusions**

**5. Factors influencing phenolic content and composition of wine**

 Ferulic acid 193 (134, 149) 134 (193) Syringic aldehyde 181 (166) 181 (166, 151) *p*-Coumaric acid 147 (103) 147 (103) Caffeic acid 179 (135) 135 (179) Esculetin 177 177 (133)

**MW Compound Main ions observed (m/z)**

136 Grapes and Wines - Advances in Production, Processing, Analysis and Valorization

**Table 5.** Some wine polyphenols determined by negative ionization mode.

on the flavonols [46].

Wine phenolic profile depends on the grapes and on the vinification technique, although other variables such as cultivar, viticulture practices can also affect it. The variety, growing season, environmental and climatic conditions, soil type and even maturity influence the concentration of phenolic compounds within grapes. The qualitative changes in the phenolic composition of the final wine, compared to the composition of the corresponding grapes, are mainly due to the production of new derivatives (such as flavenes, tyrosol and free phenolic acids––gallic, paracoumaric, caffeic, and so on). The environment in which both the must and the wine are produced can contribute to the generation of new phenolics [21]. Since wine composition is in constant evolution, the aging in bottles also seems to contribute to changes in the flavonol content, through the interaction of flavonols with other constituents [3]. It was found that the effect of vintage was significantly more pronounced on the anthocyanins than

**Fragm. 60 V Fragm. 120 V**

The total phenolic content in grapes is clearly affected by several factors: the cultivar, the year of production, the geographic origin of grapes, soil chemistry and the degree of maturation, many studies focusing on defining the effects of growing conditions on grape and wine phenolic composition and of the impact of light and temperature conditions on berry flavonoid composition [35]. The results revealed a combined effect of solar radiation and temperature on the compositional profile of flavonoids. It was observed that moderate exposure and temperature were favorable for the accumulation of anthocyanins, while an enhancement of skin tannins was noted in the grape berries from bunches exposed to sunlight versus those from shaded bunches or dense canopies. In fact, maturation is associated with polymerization of

The phenolic composition, the relative proportions of anthocyanins and flavonols, changes mainly in the first steps of vinification and continues during storage, the applied techniques (maceration, fermentation, clarification, aging, etc.) influencing significantly both the concentration and the composition of phenolics and, therefore, also the color intensity and hue of red wines. Extraction of the beneficial components from the grape skins can occur before fermentation, during it, or after it. Fermentation can be delayed, if the must

phenols, which leads to a marked decrease of astringency [16].

A main conclusion can be displayed: the resulted wine is a very complex medium influenced by winemaking techniques applied to different batches of grapes. The anthocyanin content of grapes at harvest is of vital importance in achieving quality wines. Tannins are the largest contributor, their content and composition in grapes probably determining the final wine quality. Presently, tannins are not routinely measured, the total phenolic content being determined and linked to the tannin content of grapes and wine, although it is not a specific assay. The wine phenolics can be used as a fingerprint for their differentiation, according to the geographical origin, vine variety and vintage. Phenolics compounds can be used as markers to discriminate the origin of wine (e.g., geographical, vine variety and vintage). For selection of winemaking practices aimed to improve the wine quality, the knowledge of intrinsic physicochemical characteristics of the grape cultivar employed in the vinification is required, but also the influence of different technological procedure to the final product, the wine. Since there is an increasing demand for producing and protecting high-quality wines, extensive studies on the factors influencing the chemical composition and biological effects of wine are still a necessity.

The use of advanced analytical techniques leads to the identification of some structures derived from tannins and anthocyanins in wine and determined how they are formed, the diversity of methods and experimental procedures reflecting the complexity of phenolics in grapes and wine. Improvement is still necessary, since each species is present in very small amounts and too many unidentified compounds still remain, especially with the polymeric fraction. Also, various extraction and analysis techniques have been developed as an alternative to conventional procedures, offering advantages with respect to analysis time, solvent consumption, extraction yields and reproducibility, each method providing specific advantages. Currently, the best approach to properly characterize the phenolic composition of the grapes and wines is using some assay combinations.

[8] Fang F, Li JM, Zhang P, Tang K, Wang W, Pan Q-H, Huang W-D. Effects of grape variety, harvest date, fermentation vessel and wine ageing on flavonoid concentration in red wines. Food Research International. 2008;**41**(1):53-60. DOI: 10.1016/j.foodres.2007.09.004

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[9] Saucier C. How do wine polyphenols evolve during wine ageing? Cerevisia. 2010;**35**(1):

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[11] Cheynier V, Schneider R, Salmon J, Fulcrand H. Chemistry of wine. In: Mander L, Liu HW, editors. Comprehensive Natural Products II. Oxford: Elsevier; 2010. pp. 1119-1172

[12] Baderschneider B, Winterhalter P. Isolation and characterization of novel benzoates, cinnamates, flavonoids, and lignans from Riesling wine and screening for antioxidant activity. Journal of Agricultural and Food Chemistry. 2001;**49**(6):2788-2798. DOI: 10.1021/jf010396d

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[15] Pozo-Bayon MA, Hernandez MT, Martin-Alvarez PJ, Polo MC. Study of low molecular weight phenolic compounds during the ageing of sparkling wines manufactured with red and white grape varieties. Journal of Agricultural and Food Chemistry. 2003;

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## **Author details**

Violeta-Carolina Niculescu\*, Nadia Paun and Roxana-Elena Ionete

\*Address all correspondence to: violeta.niculescu@icsi.ro

National Research and Development Institute for Cryogenics and Isotopic Technologies—ICSI Ramnicu Valcea, Ramnicu Valcea, Romania

## **References**


[8] Fang F, Li JM, Zhang P, Tang K, Wang W, Pan Q-H, Huang W-D. Effects of grape variety, harvest date, fermentation vessel and wine ageing on flavonoid concentration in red wines. Food Research International. 2008;**41**(1):53-60. DOI: 10.1016/j.foodres.2007.09.004

The use of advanced analytical techniques leads to the identification of some structures derived from tannins and anthocyanins in wine and determined how they are formed, the diversity of methods and experimental procedures reflecting the complexity of phenolics in grapes and wine. Improvement is still necessary, since each species is present in very small amounts and too many unidentified compounds still remain, especially with the polymeric fraction. Also, various extraction and analysis techniques have been developed as an alternative to conventional procedures, offering advantages with respect to analysis time, solvent consumption, extraction yields and reproducibility, each method providing specific advantages. Currently, the best approach to properly characterize the phenolic composition of the

grapes and wines is using some assay combinations.

138 Grapes and Wines - Advances in Production, Processing, Analysis and Valorization

Violeta-Carolina Niculescu\*, Nadia Paun and Roxana-Elena Ionete

Technologies—ICSI Ramnicu Valcea, Ramnicu Valcea, Romania

National Research and Development Institute for Cryogenics and Isotopic

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**Author details**

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**Chapter 8**

Provisional chapter

**Current State and Perspective in the Models Applicable**

DOI: 10.5772/intechopen.71711

Current State and Perspective in the Models Applicable

Modeling, simulation, and control of the alcoholic fermentation of grape juice into wine are still not a totally resolved problem. A model that makes it possible to predict alcoholic fermentation development would be a valuable instrument to its technical and economical implications. Considering the field of bioprocess used in food production at the industrial scale, the chapter will be centered on models applicable to oenology. On the first part, the chapter proposes the different approaches that have been taken: "knowledge-based" models, non-physiological mathematical descriptions, behavior prediction models, and empirical models. The second part will deal with a nonlinear model for white wine alcoholic fermentation process which, besides the detailed kinetic model, involves equations corresponding to the physiological phases of yeast cells, the inhibitory effect of ethanol, heat transfer equations, and the dependence

Keywords: modeling, simulation, advanced control of technological process

conducted in order to find ways of improving the food safety quality.

If the twentieth century belonged to electronics and computer science, the twenty-first century would belong to biology and biotechnologies [1]. Bioprocesses are the foundation of life and especially of human health. Therefore, on an international scale, there are studies being

A new science was born at the turn of two centuries of technical and scientific revolution: bioinformatics. The methods and the concepts of computer science began to hold the interest of many biologists, especially to the molecular biology specialists [2]. These methods are essential for various problems such as the analysis of evolutionary processes, the complex molecular structures shaping, and the simulating of some biological aspects. Researchers are currently

> © The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and eproduction in any medium, provided the original work is properly cited.

© 2018 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

**to Oenology**

to Oenology

Abstract

1. Introduction

Additional information is available at the end of the chapter

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/intechopen.71711

of kinetic parameters on temperature.

Anca Şipoş

Anca Şipoş

## **Current State and Perspective in the Models Applicable to Oenology** Current State and Perspective in the Models Applicable to Oenology

DOI: 10.5772/intechopen.71711

## Anca Şipoş Anca Şipoş

Additional information is available at the end of the chapter Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/intechopen.71711

#### Abstract

Modeling, simulation, and control of the alcoholic fermentation of grape juice into wine are still not a totally resolved problem. A model that makes it possible to predict alcoholic fermentation development would be a valuable instrument to its technical and economical implications. Considering the field of bioprocess used in food production at the industrial scale, the chapter will be centered on models applicable to oenology. On the first part, the chapter proposes the different approaches that have been taken: "knowledge-based" models, non-physiological mathematical descriptions, behavior prediction models, and empirical models. The second part will deal with a nonlinear model for white wine alcoholic fermentation process which, besides the detailed kinetic model, involves equations corresponding to the physiological phases of yeast cells, the inhibitory effect of ethanol, heat transfer equations, and the dependence of kinetic parameters on temperature.

Keywords: modeling, simulation, advanced control of technological process

#### 1. Introduction

If the twentieth century belonged to electronics and computer science, the twenty-first century would belong to biology and biotechnologies [1]. Bioprocesses are the foundation of life and especially of human health. Therefore, on an international scale, there are studies being conducted in order to find ways of improving the food safety quality.

A new science was born at the turn of two centuries of technical and scientific revolution: bioinformatics. The methods and the concepts of computer science began to hold the interest of many biologists, especially to the molecular biology specialists [2]. These methods are essential for various problems such as the analysis of evolutionary processes, the complex molecular structures shaping, and the simulating of some biological aspects. Researchers are currently

© The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and eproduction in any medium, provided the original work is properly cited.

overwhelmed by the enormous data quantity that comes from the multiple projects belonging to genomics, transcriptomics, proteomics, and metabolomics. The necessity of using computerized methods in manipulating and assembling these extremely complex high-level data is obvious. But the great challenge lies not only in the genetic sequencing and cartography but especially in understanding what do transcriptomics, proteomics, and metabolomics mean, when associated with certain life conditions and with a certain hereditary program at a given time. The identification of the structure and function of the proteins that were biosynthesized by the organisms, the complex mechanisms that allow the development of life, is therefore required. In order to achieve this, theoretical informatics—through some of its domains, which have reached a certain level of maturity, formal languages, data structures, and artificial intelligence—offer viable theoretical models.

others. Second, there is the mass transfer, caused by the liquid or gas interchanges between the reactor and the environment or with other reactors. In this context, the mathematical model of a bioprocess is a mathematical description of two types of phenomena, in which chemical and biochemical reactions generate the kinetics of the bioprocess, while the mass transfer phenom-

Current State and Perspective in the Models Applicable to Oenology

http://dx.doi.org/10.5772/intechopen.71711

145

Another important aspect in connection to the control of the bioprocesses is the lack of transducers and on-line biosensors, specific to biochemical and micro-biochemical sizes, that characterize these kinds of processes [13, 14]. Attempts of obtaining these biosensors are made [11,

At the same time, the general model of a bioprocessor can provide a series of structural properties which might be useful for the resolution of some identification, state estimation (state observers development), or bioprocesses leading problems. The need for the state observers is imposed by the absence of reliable and cheap biosensors, capable of making online measurements of the biochemical and biological variables, used in implementing some convenient methods of monitoring and leading biotechnological processes [1]. The acquisition of biomass, sublayer, and the metabolism products are made through lab analysis; this method makes the leading of the bioreactors difficult (as for the direct adjustment of these sizes). The lab analysis requires taking a sample from the contents of the bioreactor, and this represents a higher risk of contaminating the culture. Also, lab models for determining the number of microorganisms, the concentration of the sublayer, as well as the concentrations of metabolism products are quite imprecise, thus generating uncertainties in appreciating the evolution of the abovementioned sizes. These problems are strongly amplified in the industrial environment, especially because of the lack of adequate equipment and sufficient staff in order to realize quality measurements in the lab. Normally, under these conditions, there are maximum three-

The estimation of sizes of the biotechnological process is considered to be a way of avoiding the various drawbacks connected to the acquisition of data from the bioreactors. The state estimator (also known as software sensor or observer in specialized works) is an algorithm used for determining some measurements of the process, which are not measurable in real time, based

An essential problem, specific to industrial scale food industry bioprocesses, is the fact that the technological background results are very variable [1]. In many cases, the variability of raw materials used in the industrial processes leads to a non-reproducibility of the charges. Taking into consideration these aspects, the use of the technical operator's experience for leading the process is recommended, and it is possible by systems based on system expert type of knowledge. Also, heat transfer aspects led to the development of models and automatic control techniques

All these research and achievements are proper to the examined bioprocesses and usually have a low degree of reproducibility. A generalization has not been achieved at the moment; it is

four reliable analyses during the unfolding of a biotechnological process.

regarding the optimization of the thermal regime of the bioprocessors [17].

very difficult to achieve and this can only be made to a certain extent.

on other accessible measurements as related to their acquisition.

ena define the transport dynamics.

15, 16], but the research is far from being finished.

At the same time, the beginning of our century is righteously marked by biology, determined and largely conditioned by the revolution within the biological sciences [1]. Therefore, modern biology begins to determine the main directions of some interdisciplinary developments, constantly calling on the discoveries of chemistry, physics, mathematics, and engineering. A new, promising branch of science was born at a specific meeting point: bioengineering. Bioengineering especially has developed in connection with biotransformation processes (biosynthesisbiodegradation), in order to obtain antibiotics, enzymes, vitamins, amino acids, organic acids, bicarbonates, biopolymers, as a result of the cooperation among microbiologist, biochemist, chemical, food industry and mechanical engineer, operator and computer scientist, in a domain which is called Microbial Engineering and Biochemical Engineering [3]. Food safety and its quality is a primary field in European and global policy and legislation of the twenty-first century because it concerns the required conditions for a healthy population. The key issues for improving the biochemical and micro-biochemical safety and the quality of food reside in knowing, understanding, and leading the bioprocess used in food production as well as possible [4]. The volume of bioprocesses in the technical development and implementation of the processes of obtaining new food products and a significant number of non-food items based on microbiological processes and enzyme technology have grown significantly in the last years.

It is very difficult to monitor, design, and control a bioprocess [5]. For example, in the last years, an impressive number of kinetic models were developed only for fermentation processes and the various phenomena that influence fermentation kinetics were taken into account. That is why these models needed the estimation of a large number of sizes, which are often very hard to identify [6–8]. A series of artificial intelligence-based control and optimization models and techniques were also developed (fuzzy and neuro-fuzzy techniques) [9–12]. These models allowed a more realistic definition of some model sizes, which carry a high degree of uncertainty.

The mathematical model of a biotechnological process is generally represented using differential equation systems, which are obtained by writing, for each component, the mass and energy balance equations. In these equations, the components or the concentrations of the components involved in the reactions that take place in the bioreactor appear. The dynamics of the temporal variation of each component is expressed by two types of phenomena. First, there are chemical and biochemical reactions that transform some components into some others. Second, there is the mass transfer, caused by the liquid or gas interchanges between the reactor and the environment or with other reactors. In this context, the mathematical model of a bioprocess is a mathematical description of two types of phenomena, in which chemical and biochemical reactions generate the kinetics of the bioprocess, while the mass transfer phenomena define the transport dynamics.

overwhelmed by the enormous data quantity that comes from the multiple projects belonging to genomics, transcriptomics, proteomics, and metabolomics. The necessity of using computerized methods in manipulating and assembling these extremely complex high-level data is obvious. But the great challenge lies not only in the genetic sequencing and cartography but especially in understanding what do transcriptomics, proteomics, and metabolomics mean, when associated with certain life conditions and with a certain hereditary program at a given time. The identification of the structure and function of the proteins that were biosynthesized by the organisms, the complex mechanisms that allow the development of life, is therefore required. In order to achieve this, theoretical informatics—through some of its domains, which have reached a certain level of maturity, formal languages, data structures, and artificial

At the same time, the beginning of our century is righteously marked by biology, determined and largely conditioned by the revolution within the biological sciences [1]. Therefore, modern biology begins to determine the main directions of some interdisciplinary developments, constantly calling on the discoveries of chemistry, physics, mathematics, and engineering. A new, promising branch of science was born at a specific meeting point: bioengineering. Bioengineering especially has developed in connection with biotransformation processes (biosynthesisbiodegradation), in order to obtain antibiotics, enzymes, vitamins, amino acids, organic acids, bicarbonates, biopolymers, as a result of the cooperation among microbiologist, biochemist, chemical, food industry and mechanical engineer, operator and computer scientist, in a domain which is called Microbial Engineering and Biochemical Engineering [3]. Food safety and its quality is a primary field in European and global policy and legislation of the twenty-first century because it concerns the required conditions for a healthy population. The key issues for improving the biochemical and micro-biochemical safety and the quality of food reside in knowing, understanding, and leading the bioprocess used in food production as well as possible [4]. The volume of bioprocesses in the technical development and implementation of the processes of obtaining new food products and a significant number of non-food items based on microbiological pro-

It is very difficult to monitor, design, and control a bioprocess [5]. For example, in the last years, an impressive number of kinetic models were developed only for fermentation processes and the various phenomena that influence fermentation kinetics were taken into account. That is why these models needed the estimation of a large number of sizes, which are often very hard to identify [6–8]. A series of artificial intelligence-based control and optimization models and techniques were also developed (fuzzy and neuro-fuzzy techniques) [9–12]. These models allowed a more realistic definition of some model sizes, which carry a high degree of uncertainty.

The mathematical model of a biotechnological process is generally represented using differential equation systems, which are obtained by writing, for each component, the mass and energy balance equations. In these equations, the components or the concentrations of the components involved in the reactions that take place in the bioreactor appear. The dynamics of the temporal variation of each component is expressed by two types of phenomena. First, there are chemical and biochemical reactions that transform some components into some

cesses and enzyme technology have grown significantly in the last years.

intelligence—offer viable theoretical models.

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Another important aspect in connection to the control of the bioprocesses is the lack of transducers and on-line biosensors, specific to biochemical and micro-biochemical sizes, that characterize these kinds of processes [13, 14]. Attempts of obtaining these biosensors are made [11, 15, 16], but the research is far from being finished.

At the same time, the general model of a bioprocessor can provide a series of structural properties which might be useful for the resolution of some identification, state estimation (state observers development), or bioprocesses leading problems. The need for the state observers is imposed by the absence of reliable and cheap biosensors, capable of making online measurements of the biochemical and biological variables, used in implementing some convenient methods of monitoring and leading biotechnological processes [1]. The acquisition of biomass, sublayer, and the metabolism products are made through lab analysis; this method makes the leading of the bioreactors difficult (as for the direct adjustment of these sizes). The lab analysis requires taking a sample from the contents of the bioreactor, and this represents a higher risk of contaminating the culture. Also, lab models for determining the number of microorganisms, the concentration of the sublayer, as well as the concentrations of metabolism products are quite imprecise, thus generating uncertainties in appreciating the evolution of the abovementioned sizes. These problems are strongly amplified in the industrial environment, especially because of the lack of adequate equipment and sufficient staff in order to realize quality measurements in the lab. Normally, under these conditions, there are maximum threefour reliable analyses during the unfolding of a biotechnological process.

The estimation of sizes of the biotechnological process is considered to be a way of avoiding the various drawbacks connected to the acquisition of data from the bioreactors. The state estimator (also known as software sensor or observer in specialized works) is an algorithm used for determining some measurements of the process, which are not measurable in real time, based on other accessible measurements as related to their acquisition.

An essential problem, specific to industrial scale food industry bioprocesses, is the fact that the technological background results are very variable [1]. In many cases, the variability of raw materials used in the industrial processes leads to a non-reproducibility of the charges. Taking into consideration these aspects, the use of the technical operator's experience for leading the process is recommended, and it is possible by systems based on system expert type of knowledge.

Also, heat transfer aspects led to the development of models and automatic control techniques regarding the optimization of the thermal regime of the bioprocessors [17].

All these research and achievements are proper to the examined bioprocesses and usually have a low degree of reproducibility. A generalization has not been achieved at the moment; it is very difficult to achieve and this can only be made to a certain extent.

Another aspect is the improvement of the technological performances concerning the energy consumption. As a result, optimizing the energy consumption through the introduction of advanced leading systems leads to a conservation of fossil fuels. It can be affirmed that, indirectly, a bio-economy is being advocated for. In Bio-economy versus biodiversity, Hall [18] says "The bio-economy agenda is especially attractive to fossil fuel companies who want to be seen pursuing an exit- from-oil strategy; and to biotechnology companies desperately in need of a Trojan horse to provide safe passage for risky and unpopular new technologies."

biomass, is introduced in the bioreactor at the beginning of the fermentation. During the fermentation, the bioreactor will not be fed with any substrate and the process will be completed after the substrate has been sufficiently consumed. The result is that the entire quantity of biomass is collected, from which then the desire product will be extracted. The possibilities of process control in this case are very limited and could be resumed with some physical variables: temperature, pH, energetically consumption, length of fermenta-

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• Fed-batch mode—The fed-batch bioreactors type or with semi-continuous operating is a reactor with cyclical working, with a continuous or intermittent feeding and without an exit. In the vessel, both a small quantity of substrate and a biomass are initially introduced. Then, during the fermentation process, and in the function of the microorganisms' necessities, the reactor will be fed with a controlled flow of substrate. Therefore, the possibilities of process control of this kind of bioreactors are more diverse than that of the batch type. Aside from the physical variables, the biological variables: substrate

• Continuous mode—This mode is more efficient from the economical point of view and the bioreactors are stirred, with continuous operation, eventually with separation and recycling, as well as the gas-liquid bioreactors, used especially in the industry for obtaining a great volume of biomass (i.e., unicellular proteins) or for the biological treatment of residual water. In such a reactor, the biomass is evacuated with a flow equal with that of the substrate

The mathematical model of a biotechnological process is formed by an equation system in which the components or the concentrations of components implied in reactions that take place in bioreactor appear, equations which are obtained by writing, for each component, the mass and energetic balances. The dynamic or variation in time of the quantity of each component is expressed by two phenomena. Primarily, there are chemical, biochemical, and biological reactions which transform certain components in others. Secondarily, mass transfer due to the liquid and gas interchanges between the reactor and the environment or with other bioreactors exists. Within this context, the mathematical model of a bioprocess represents a mathematical description of two types of phenomena, where the chemical, biochemical, and biological reactions cause the bioprocess kinetics while the mass transfer phenomena define the

If the process is passed off in a reactor with perfect stirring, which means a reactor in which the culture medium composition is supposed to be homogeneous, the dynamics of the two types of phenomena can be represented by a unitary description, by differential equations system which involve the reaction components concentrations, as well the pH, temperature, and so on, variables which can be organized like a state vector. There are also bioreactors without perfect stirred and in which a set of concentration gradients, temperature, pressure, and so on, appear. Such types of bioreactors are tubular, bioreactors with fix layers, and so on. These reactors are described by mathematical models which contain partial derivate equations, where, in the

excepted the temporal variables, at least a spatial variable will appear.

concentration, biomass concentration, and so on, can also be controlled.

tion, and so on.

feeding.

dynamics of transport.

After a brief introduction regarding the modeling of bioprocesses, on the first part, the chapter proposes the different approaches that have been taken: "knowledge-based" models, nonphysiological mathematical descriptions, behavior prediction models, and empirical models. The second part will deal with a nonlinear model for white wine alcoholic fermentation process which, besides the detailed kinetic model, involves equations corresponding to the physiological phases of yeast cells, the inhibitory effect of ethanol, heat transfer equations, and the dependence of kinetic parameters on temperature.

## 2. Current stage in fermentation processes modeling

#### 2.1. The biotechnological processes modeling

A biotechnological process implies a development (growth) of a microorganism population (culture medium), biomass, in a bioreactor (vessel) by the consumption of some nutrients (carbon, nitrogen, oxygen, vitamins, etc.) that represent the substrate, if the physical and chemical conditions (temperature, pH, aeration, etc.) are favorable. Customarily, the microorganisms' growth takes place in a liquid medium (aqua). It is obvious that in a bioreactor many biochemical and biological reactions take place simultaneously. Each elementary reaction is, usually, catalyzed by a protein (enzyme) and can form a specific product or a metabolite. The aim of such a culture can be



Because the microbial mechanism of growth that involves alive organisms is very complex, a detailed modeling is not possible or is very complicated. Usually, the bioreactor assumes a perfect stirred and is described by a number of macro-scope variables, such as biomass, substrate, product, oxygen concentrations, pH, temperature, etc. Function of the fermentation type could also be defined by another concentration.

The mathematical model of a bioreactor depends on its operating mode. In this way, in practice, for these types of bioreactors three operating modes could be defined:

• Batch mode—A batch bioreactor is a reactor with a cyclical operating, without feed, and exit flows. The entire quantity of substrate and nutrients, with a small quantity of biomass, is introduced in the bioreactor at the beginning of the fermentation. During the fermentation, the bioreactor will not be fed with any substrate and the process will be completed after the substrate has been sufficiently consumed. The result is that the entire quantity of biomass is collected, from which then the desire product will be extracted. The possibilities of process control in this case are very limited and could be resumed with some physical variables: temperature, pH, energetically consumption, length of fermentation, and so on.

Another aspect is the improvement of the technological performances concerning the energy consumption. As a result, optimizing the energy consumption through the introduction of advanced leading systems leads to a conservation of fossil fuels. It can be affirmed that, indirectly, a bio-economy is being advocated for. In Bio-economy versus biodiversity, Hall [18] says "The bio-economy agenda is especially attractive to fossil fuel companies who want to be seen pursuing an exit- from-oil strategy; and to biotechnology companies desperately in need

After a brief introduction regarding the modeling of bioprocesses, on the first part, the chapter proposes the different approaches that have been taken: "knowledge-based" models, nonphysiological mathematical descriptions, behavior prediction models, and empirical models. The second part will deal with a nonlinear model for white wine alcoholic fermentation process which, besides the detailed kinetic model, involves equations corresponding to the physiological phases of yeast cells, the inhibitory effect of ethanol, heat transfer equations, and

A biotechnological process implies a development (growth) of a microorganism population (culture medium), biomass, in a bioreactor (vessel) by the consumption of some nutrients (carbon, nitrogen, oxygen, vitamins, etc.) that represent the substrate, if the physical and chemical conditions (temperature, pH, aeration, etc.) are favorable. Customarily, the microorganisms' growth takes place in a liquid medium (aqua). It is obvious that in a bioreactor many biochemical and biological reactions take place simultaneously. Each elementary reaction is, usually, catalyzed by a protein (enzyme) and can form a specific product or a metabolite. The aim of such a culture


The mathematical model of a bioreactor depends on its operating mode. In this way, in

• Batch mode—A batch bioreactor is a reactor with a cyclical operating, without feed, and exit flows. The entire quantity of substrate and nutrients, with a small quantity of

of a Trojan horse to provide safe passage for risky and unpopular new technologies."

the dependence of kinetic parameters on temperature.

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2.1. The biotechnological processes modeling


type could also be defined by another concentration.

can be

2. Current stage in fermentation processes modeling


practice, for these types of bioreactors three operating modes could be defined:


The mathematical model of a biotechnological process is formed by an equation system in which the components or the concentrations of components implied in reactions that take place in bioreactor appear, equations which are obtained by writing, for each component, the mass and energetic balances. The dynamic or variation in time of the quantity of each component is expressed by two phenomena. Primarily, there are chemical, biochemical, and biological reactions which transform certain components in others. Secondarily, mass transfer due to the liquid and gas interchanges between the reactor and the environment or with other bioreactors exists. Within this context, the mathematical model of a bioprocess represents a mathematical description of two types of phenomena, where the chemical, biochemical, and biological reactions cause the bioprocess kinetics while the mass transfer phenomena define the dynamics of transport.

If the process is passed off in a reactor with perfect stirring, which means a reactor in which the culture medium composition is supposed to be homogeneous, the dynamics of the two types of phenomena can be represented by a unitary description, by differential equations system which involve the reaction components concentrations, as well the pH, temperature, and so on, variables which can be organized like a state vector. There are also bioreactors without perfect stirred and in which a set of concentration gradients, temperature, pressure, and so on, appear. Such types of bioreactors are tubular, bioreactors with fix layers, and so on. These reactors are described by mathematical models which contain partial derivate equations, where, in the excepted the temporal variables, at least a spatial variable will appear.

The modeling of a biotechnological system can be done progressive. It begins with the information that could be easily obtained, namely the reaction of the components involved in the bioprocess, the reactions which take place in the bioreactor and the liquid or/and gas interconnections of the system, elements which define the reaction schema and the system architecture. With these information, the general structure of the model is settled out. The general model of the bioprocess can wise up a set of structural properties useful for solving some issues concerning the state estimation or the control of the bioprocesses.

#### 2.2. The reaction kinetics modeling

#### 2.2.1. The kinetic of microorganisms growing

The kinetic of microorganisms' growth (Figure 1) is necessary from the point of view of knowing the process phases (Table 1), the duration of these phases, the factors which influence them in order to choose then the type of vessel, and the process control mode for the bioreactor.

#### 2.2.2. The types of kinetic models

The phenomena that take place in a bioreactor are complex and coupled. The complexity is given by the heterogenic medium three phases (solid, liquid, and gas), in which the biosynthesis process takes place; the three phases medium is in a dynamic evolution and has a nonlinear character. The processes are coupled among them, an operational variable (the feed flow with substrate, the pressure, the temperature, the stirred, etc.) or the way in which the oxygen reaches at the microorganisms, are important both individually and for the whole microorganisms, for the biomass process control [19].

> In Figure 2, the main phenomena are presented, the interactions and variables which influence the kinetic behavior of the population cells. The two systems, the biological system (represented by the microbial population) and the physical and chemical system (external medium), are in close correlation; the cells consume nutrients and transform the substrate in reaction products. The cells generate heat which warms the medium, and the medium temper-

Figure 2. The main phenomena, interactions, and variables which influence the kinetic of the microbial populations.

The specific growth rate μ comes down. It appears when the feed with an essential nutrient

Generally, this phase has a reduced practical importance. For this reason, in order to eliminate this phase, more generation of cells are cultivated on the respective medium aiming to accustom the cells with it.

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This phase presents a practical importance when we have the obtaining of biomass in view. The metabolical activity from the exponential phase is in fact the primary

becomes insufficient or an element necessary for growth has been run out or intermediary substances have been

The secondary metabolism is typical for the stationary

At one point, it is possible that an easy growth of biomass takes place, due to the alive cells' consumption of the nutrients. The nutrients have been delivered by the lysated cells. For a valorous culture, the decline

metabolite; it corresponds with trot phase.

accumulated in the medium.

phase must be eliminated.

phase; it corresponds to idiophase.

Phase Characterization Observations

The regeneration processes of the hyphes or the germination of the spores, the function of the

The specific growth rate μ is constant and the

concentration, the proportion between alive and death cells number remains constant. The quantity of limiting nutrient influences this quantity of biomass, named total production.

Table 1. The microbial population growing phases (adapted from [19]).

inoculums type take place.

The volume of the cells grows fast.

biomass growth is exponential.

V. Stationary The microbial cells reach a maximum

VI. Decay The cells die, the autolysis takes place, and the quantity of biomass comes down.

I. Latent (adaptation)

II. Beginning of growth

III. Exponential growth

IV. Deceleration growing

ature influences the cell behavior.

Figure 1. The microorganisms number evolution versus time.


Table 1. The microbial population growing phases (adapted from [19]).

The modeling of a biotechnological system can be done progressive. It begins with the information that could be easily obtained, namely the reaction of the components involved in the bioprocess, the reactions which take place in the bioreactor and the liquid or/and gas interconnections of the system, elements which define the reaction schema and the system architecture. With these information, the general structure of the model is settled out. The general model of the bioprocess can wise up a set of structural properties useful for solving some issues concerning

The kinetic of microorganisms' growth (Figure 1) is necessary from the point of view of knowing the process phases (Table 1), the duration of these phases, the factors which influence them in order to choose then the type of vessel, and the process control mode for the bioreactor.

The phenomena that take place in a bioreactor are complex and coupled. The complexity is given by the heterogenic medium three phases (solid, liquid, and gas), in which the biosynthesis process takes place; the three phases medium is in a dynamic evolution and has a nonlinear character. The processes are coupled among them, an operational variable (the feed flow with substrate, the pressure, the temperature, the stirred, etc.) or the way in which the oxygen reaches at the microorganisms, are important both individually and for the whole microorgan-

I II III IV V VI VII

0 2 4 6 8 24 26 28

Time, [*h*]

the state estimation or the control of the bioprocesses.

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2.2. The reaction kinetics modeling

2.2.2. The types of kinetic models

2.2.1. The kinetic of microorganisms growing

isms, for the biomass process control [19].

0.4

Figure 1. The microorganisms number evolution versus time.

0.6

0.8

1.0

Optical density, [*AU*] Specific rate of growing, [*h-1*]

0.0

0.4

In Figure 2, the main phenomena are presented, the interactions and variables which influence the kinetic behavior of the population cells. The two systems, the biological system (represented by the microbial population) and the physical and chemical system (external medium), are in close correlation; the cells consume nutrients and transform the substrate in reaction products. The cells generate heat which warms the medium, and the medium temperature influences the cell behavior.

Figure 2. The main phenomena, interactions, and variables which influence the kinetic of the microbial populations.

The mechanical interactions are due to the hydrostatic pressure and to the flow effects of the medium on the cells and also to the medium viscosity changes, caused by the cells accumulation and/or by the cellular metabolites.

place simultaneously in every cell, which involves an internal complex control; this control allows the cell to adapt its activity and even to the biochemical type reaction in function of the

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The dissimilarity among the cells is given by the variation in time and space of the cells' age (some cells are just born while others are dying or dividing); the cells with different ages are

In the cultivation for longer period, many spontaneous mutations of some individuals of cells

Analyzing all these aspects, it becomes obvious that, in practice, a kinetic model that may include all the phenomena and interactions of the system cannot be formulated, a first simplification concerning the medium being necessary. It is considered that even a single component of medium, which is in quite great quantities, can influence the microbial growth of the medium. Sometimes, it could be necessary to include other components of medium such as the products with inhibitory role, which are accumulated in medium, in order to obtain a

From the kinetics point of view, in order to construct a model it is necessary to study the rates and mechanisms of the physical, biochemical, and microbiological processes, in which microorganisms are involved (growing, cellular cycle, the components produced by reaction, the

The specific growth rate, μ, represents the variation in time of the microbial cells concentration in

X ¼ X<sup>0</sup> � e

The specific growth rate depends, among other elements, on the limiting substrate concentra-

<sup>μ</sup> <sup>¼</sup> <sup>μ</sup>max � <sup>S</sup>

];

dt <sup>¼</sup> <sup>μ</sup> (1)

<sup>μ</sup>�<sup>t</sup> (2)

];

KS <sup>þ</sup> <sup>S</sup> (3)

];

1 X � dX

where X represents the microbial cells concentration in biosynthesis medium [mol/m3

tion (i.e., the glucose concentration), S, taking place after the following equation:

X0 is the steady-state operation point of microbial concentration [mol/m3

μ is the specific growth rate in exponential phase, [s�<sup>1</sup>

often characterized by various types of activities and metabolical functions.

suitable description of the kinetics of the cells population.

2.2.3. The unstructured kinetics models 2.2.3.1. Models based on Monod equation

medium effects, and the biological interactions).

external medium.

population may appear.

synthesis medium:

or in integrating form:

t is the development time [s];

The medium is a multi-composition system that must contain the necessary nutrients for the growth of the microorganisms (carbon, nitrogen sources, mineral salts, vitamins, growing factors, oxygen, etc.) and in which different products of cellular metabolism (primary and secondary metabolites) are accumulated while the cells grow on.

In solution reactions that could modify the structure of the final products (i.e., the penicillin hydrolyses) could take place.

Often, the cells consume or produce components that could influence the medium acidity and the interrelation between cell needful and acid-base equilibrium determinates the medium pH, which, in turn, influences the cells' activity and the transport processes.

During the cellular reactions, the medium temperature, pH, ionic strength, and rheological properties can change in time.

A complex model of a bioreactor is multi-phases; it consists of a medium with solid particles among which liquid and gas particles (at the surface being the microbial culture) are dispersed or from a liquid medium in which gas bulls are dispersed. An example of three phases model, with two physical phases, gassy and liquid phases and a biotical one—the microorganisms culture, is presented in Figure 3, model which could be considered a model of a fermentation process.

Because of the great volume of bioreactors, of the higher viscosity and of the non-Newtonian nature of medium, in most cases, the technological conditions from vessel can vary from point to point.

Every individual of microbial population is a complex component of the system, frequently nonhomogeneous, even at the level of a single cell. Many independent biochemical reactions take

Figure 3. A bioreactor model with three-structured phases.

place simultaneously in every cell, which involves an internal complex control; this control allows the cell to adapt its activity and even to the biochemical type reaction in function of the external medium.

The dissimilarity among the cells is given by the variation in time and space of the cells' age (some cells are just born while others are dying or dividing); the cells with different ages are often characterized by various types of activities and metabolical functions.

In the cultivation for longer period, many spontaneous mutations of some individuals of cells population may appear.

Analyzing all these aspects, it becomes obvious that, in practice, a kinetic model that may include all the phenomena and interactions of the system cannot be formulated, a first simplification concerning the medium being necessary. It is considered that even a single component of medium, which is in quite great quantities, can influence the microbial growth of the medium. Sometimes, it could be necessary to include other components of medium such as the products with inhibitory role, which are accumulated in medium, in order to obtain a suitable description of the kinetics of the cells population.

#### 2.2.3. The unstructured kinetics models

The mechanical interactions are due to the hydrostatic pressure and to the flow effects of the medium on the cells and also to the medium viscosity changes, caused by the cells accumula-

The medium is a multi-composition system that must contain the necessary nutrients for the growth of the microorganisms (carbon, nitrogen sources, mineral salts, vitamins, growing factors, oxygen, etc.) and in which different products of cellular metabolism (primary and

In solution reactions that could modify the structure of the final products (i.e., the penicillin

Often, the cells consume or produce components that could influence the medium acidity and the interrelation between cell needful and acid-base equilibrium determinates the medium pH,

During the cellular reactions, the medium temperature, pH, ionic strength, and rheological

A complex model of a bioreactor is multi-phases; it consists of a medium with solid particles among which liquid and gas particles (at the surface being the microbial culture) are dispersed or from a liquid medium in which gas bulls are dispersed. An example of three phases model, with two physical phases, gassy and liquid phases and a biotical one—the microorganisms culture, is presented in Figure 3, model which could be considered a model of a fermentation process.

Because of the great volume of bioreactors, of the higher viscosity and of the non-Newtonian nature of medium, in most cases, the technological conditions from vessel can vary from point

Every individual of microbial population is a complex component of the system, frequently nonhomogeneous, even at the level of a single cell. Many independent biochemical reactions take

**Gassy phase Liquid phase Biomass**

**Biological phase** 

**Continues biological phase** 

**Gassy phase Liquid phase Biomass** 

**Exterior**

**The system temperature** 

**Wall** 

tion and/or by the cellular metabolites.

hydrolyses) could take place.

properties can change in time.

to point.

**The system temperature** 

secondary metabolites) are accumulated while the cells grow on.

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which, in turn, influences the cells' activity and the transport processes.

**Continues gassy phase** 

**Dispersed gassy phase** 

Figure 3. A bioreactor model with three-structured phases.

#### 2.2.3.1. Models based on Monod equation

From the kinetics point of view, in order to construct a model it is necessary to study the rates and mechanisms of the physical, biochemical, and microbiological processes, in which microorganisms are involved (growing, cellular cycle, the components produced by reaction, the medium effects, and the biological interactions).

The specific growth rate, μ, represents the variation in time of the microbial cells concentration in synthesis medium:

$$\frac{1}{X} \cdot \frac{dX}{dt} = \mu \tag{1}$$

or in integrating form:

$$X = X\_0 \cdot e^{\mu \cdot t} \tag{2}$$

where X represents the microbial cells concentration in biosynthesis medium [mol/m3 ];

X0 is the steady-state operation point of microbial concentration [mol/m3 ];

t is the development time [s];

μ is the specific growth rate in exponential phase, [s�<sup>1</sup> ];

The specific growth rate depends, among other elements, on the limiting substrate concentration (i.e., the glucose concentration), S, taking place after the following equation:

$$
\mu = \frac{\mu\_{\text{max}} \cdot \mathcal{S}}{K\_{\mathcal{S}} + \mathcal{S}} \tag{3}
$$

with the name Monod equation where S is the limiting substrate concentration (glucose concentration); μmax is the maximum specific growth rate, achieving when S>>Ks and the concentration of all the nutrients is unchanged; Ks is a constant which has a concentration dimension and represents the value of limited nutrient concentration at which <sup>μ</sup> <sup>¼</sup> <sup>1</sup> <sup>2</sup> � μmax.

The values of the two variables depend on the microorganism, on the work conditions (pH and temperature), and on the substrate complexity. The typical curves of biomass evolution and substrate consumption in time are presented in Figure 4, for a batch fermentation system. The value of substrate concentration <sup>S</sup> was pointed out for which <sup>μ</sup> <sup>¼</sup> <sup>1</sup> <sup>2</sup> � μmax.

Ks can be considered a measure of the microorganisms affinity against substrate:



The specific forming rate of the metabolism product:

$$
\eta\_p = \frac{r\_p}{X} \tag{4}
$$

Introducing Eq. (5) in Eq. (4), the specific rate of metabolism product can be determined:

The forming rate of the product doesn't depend on the biomass X but on the growing rate of the cellular mass rx at the obtained primary metabolites (ethanol, acetic acid, gluconic acid,

If the development of microorganisms is limited by the concentration of a biosynthesis medium component, the microorganisms quantity that is formed is proportionally with the used quantity

dS <sup>¼</sup> mols of biomass produced

dS <sup>¼</sup> mols of metabolism produced

where ms is a forming coefficient ½ � mol substrate=ð Þ mol biomass � s , specific for microorganism. With the growth in the anaerobic conditions, the energy necessary to cell is obtained by the substrate transformation in reaction products; therefore, the metabolism products forming is a

> �rs <sup>¼</sup> rx Y0 xs

rp <sup>¼</sup> <sup>Y</sup><sup>0</sup> ps Y0 xs

where mp is a coefficient that describes the product forming during the growth ½ � mol substrate=ð Þ mol biomass � s . This coefficient can be determined from ms on the basis that the

For S>0, the forming rate of metabolites in anaerobe conditions is

In aerobic conditions, at the biomass forming, the rate consume of substrate is

�rs <sup>¼</sup> rx Y0 xs þ rp Y0 ps

butyric alcohol, acetone, etc.). Therefore, k1= 0 and Eq. (5) become

In case of obtaining secondary metabolites, rp depends only by biomass X:

of substrate. Therefore, the efficiency coefficient for biomass producing can be defined:

where μ ¼ rx=X.

Y0 xs <sup>¼</sup> dX

Y0 ps <sup>¼</sup> dP

consequence of biomass growing:

and the efficiency coefficient for metabolite producing:

qp ¼ k<sup>1</sup> þ k<sup>2</sup> � μ (6)

Current State and Perspective in the Models Applicable to Oenology

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153

rp ¼ k<sup>2</sup> � rx (7)

rp ¼ k<sup>1</sup> � X (8)

þ ms � X (11)

þ ms � X (12)

� rs þ mp � X (13)

mols of substrate consumed for biomass producing (9)

mols of substrate consumed for metabolism producing (10)

where

$$r\_p = k\_1 \cdot X + k\_2 \cdot r\_x \tag{5}$$

is the forming rate of metabolism product mol<sup>=</sup> <sup>m</sup><sup>3</sup> � <sup>s</sup> ; <sup>X</sup> is the biomass concentration; rx is the growing rate of cellular mass mol<sup>=</sup> <sup>m</sup><sup>3</sup> � <sup>s</sup> ; <sup>k</sup><sup>1</sup> is the proportionality factor between the forming rate of product and biomass concentration ½ � mol product=ð Þ mol biomass � s ; k2 is the proportionality factor between the forming rate of the product and the growing rate of the cellular mass ½ � mol product=mol biomass .

Figure 4. The evolution of biomass and substrate consume versus time, for a fermentation in batch system (a); for the same batch system-specific growth rate μ as a function of limiting substrate concentration, S (b).

Introducing Eq. (5) in Eq. (4), the specific rate of metabolism product can be determined:

$$q\_p = k\_1 + k\_2 \cdot \mu \tag{6}$$

where μ ¼ rx=X.

with the name Monod equation where S is the limiting substrate concentration (glucose concentration); μmax is the maximum specific growth rate, achieving when S>>Ks and the concentration of all the nutrients is unchanged; Ks is a constant which has a concentration

The values of the two variables depend on the microorganism, on the work conditions (pH and temperature), and on the substrate complexity. The typical curves of biomass evolution and substrate consumption in time are presented in Figure 4, for a batch fermentation system. The



qp <sup>¼</sup> rp

is the forming rate of metabolism product mol<sup>=</sup> <sup>m</sup><sup>3</sup> � <sup>s</sup> ; <sup>X</sup> is the biomass concentration; rx is the growing rate of cellular mass mol<sup>=</sup> <sup>m</sup><sup>3</sup> � <sup>s</sup> ; <sup>k</sup><sup>1</sup> is the proportionality factor between the forming rate of product and biomass concentration ½ � mol product=ð Þ mol biomass � s ; k2 is the proportionality factor between the forming rate of the product and the growing rate of the

10

Time, [*h*]

same batch system-specific growth rate μ as a function of limiting substrate concentration, S (b).

*S=Ks*

7.5

5

Substrate concentration (*S*)

1

0.8

0

0.2

*S=Ks*

0.4

0.6 Specific growth rate, [*h-1*]

2.5

0

Figure 4. The evolution of biomass and substrate consume versus time, for a fermentation in batch system (a); for the

<sup>2</sup> � μmax.

<sup>2</sup> � μmax.

<sup>X</sup> (4)

**b.**

0 2 4 6 8 10

maximum of specific growth rate

Substrate concentration (*S*)

rp ¼ k<sup>1</sup> � X þ k<sup>2</sup> � rx (5)

dimension and represents the value of limited nutrient concentration at which <sup>μ</sup> <sup>¼</sup> <sup>1</sup>

Ks can be considered a measure of the microorganisms affinity against substrate:

very small substrate concentrations (small dilution at continuous processes);

value of substrate concentration <sup>S</sup> was pointed out for which <sup>μ</sup> <sup>¼</sup> <sup>1</sup>

152 Grapes and Wines - Advances in Production, Processing, Analysis and Valorization

slowly, even if the substrate concentrations are great. The specific forming rate of the metabolism product:

cellular mass ½ � mol product=mol biomass .

10 10

*S*

**a.** 

0 2 4 6 8

*X* 

where

0.01

0.1

Biomass concentration (*X*)

1

The forming rate of the product doesn't depend on the biomass X but on the growing rate of the cellular mass rx at the obtained primary metabolites (ethanol, acetic acid, gluconic acid, butyric alcohol, acetone, etc.). Therefore, k1= 0 and Eq. (5) become

$$r\_p = k\_2 \cdot r\_x \tag{7}$$

In case of obtaining secondary metabolites, rp depends only by biomass X:

$$r\_p = k\_1 \cdot X \tag{8}$$

If the development of microorganisms is limited by the concentration of a biosynthesis medium component, the microorganisms quantity that is formed is proportionally with the used quantity of substrate. Therefore, the efficiency coefficient for biomass producing can be defined:

$$Y\_{xs}^{\prime} = \frac{dX}{dS} = \frac{\text{mols of biomass produced}}{\text{mols of substrate consumed for biomass producing}} \tag{9}$$

and the efficiency coefficient for metabolite producing:

$$Y\_{ps}^{\prime} = \frac{dP}{d\mathbb{S}} = \frac{\text{mols of metalolism produced}}{\text{mols of substrate consumed for metalolism producing}} \tag{10}$$

In aerobic conditions, at the biomass forming, the rate consume of substrate is

$$-r\_s = \frac{r\_x}{Y\_{xs}'} + \frac{r\_p}{Y\_{ps}'} + m\_s \cdot X \tag{11}$$

where ms is a forming coefficient ½ � mol substrate=ð Þ mol biomass � s , specific for microorganism.

With the growth in the anaerobic conditions, the energy necessary to cell is obtained by the substrate transformation in reaction products; therefore, the metabolism products forming is a consequence of biomass growing:

$$-r\_s = \frac{r\_\pi}{Y\_{\infty}'} + m\_s \cdot X \tag{12}$$

For S>0, the forming rate of metabolites in anaerobe conditions is

$$r\_p = \frac{Y\_{ps}'}{Y\_{xs}'} \cdot r\_s + m\_p \cdot X \tag{13}$$

where mp is a coefficient that describes the product forming during the growth ½ � mol substrate=ð Þ mol biomass � s . This coefficient can be determined from ms on the basis that the reaction of the stoichiometric coefficients substrate consumed formed product. From Eqs. (5) and (3), the k<sup>1</sup> and k<sup>2</sup> coefficients could be obtained in anaerobic conditions.

The total efficiencies of biomass and metabolic products are used in practice.

During the aerobic processes, considering Eqs. (5) and (11), the result is

$$Y\_{xs} = \frac{r\_x}{-r\_s} = \frac{r\_x}{\frac{r\_x}{Y'\_{xs}} + \frac{r\_p}{Y'\_{ps}} + m\_s \cdot X} = \frac{\mu \cdot X}{\frac{\mu \cdot X}{Y'\_{xs}} + m\_s \cdot X} = \frac{\mu}{\frac{\mu}{Y'\_{xs}} + \frac{k\_1 + k\_2 \cdot \mu}{Y'\_{ps}} + m\_s} \tag{14}$$

<sup>μ</sup> <sup>¼</sup> <sup>μ</sup>max � <sup>S</sup> Ks <sup>þ</sup> <sup>S</sup> <sup>þ</sup> <sup>S</sup><sup>2</sup> Ki

Contois model, which takes into consideration the inhibitory effect of the biomass on the

Because of the greater concentration of biomass, the biotical phase can be a substantial part from the total volume of the bioreactor and thus it is difficult to assimilate the substances by the biomass. Anyway, it is heavy to imagine in which mode the cells' concentration can inhibit its own growth and, probably, the ability of Contois kinetics to be in concordance with the

Another model that takes into consideration the inhibitory effect of the biomass on the specific

Ks <sup>þ</sup> <sup>S</sup> � <sup>1</sup> � <sup>S</sup>

where Ki is the inhibitory constant and Smax is the substrate concentration at which the

A similar variant of the specific growth rate of Monod biomass in which the substrate S

<sup>μ</sup> <sup>¼</sup> <sup>μ</sup>max � Sn

On the case of bioreactors with two substrates (S1 and S2), the models with Eqs. (3) and (19)–

K<sup>1</sup> þ S<sup>1</sup>

K<sup>1</sup> þ S<sup>1</sup>

When a metabolic product inhibits the growth, the specific growth rate has the following

� <sup>S</sup><sup>2</sup> K<sup>2</sup> þ S<sup>2</sup>

� <sup>S</sup><sup>2</sup> <sup>K</sup><sup>2</sup> <sup>þ</sup> <sup>S</sup><sup>2</sup> <sup>þ</sup> <sup>S</sup><sup>2</sup>

2 Ki

<sup>μ</sup> <sup>¼</sup> <sup>μ</sup>max � <sup>S</sup><sup>1</sup>

<sup>μ</sup> <sup>¼</sup> <sup>μ</sup>max � <sup>S</sup><sup>1</sup>

S

μ ¼ μmax

experimental data is explained by the toxic effect of some metabolic products.

<sup>μ</sup> <sup>¼</sup> <sup>μ</sup>max � <sup>S</sup>

ffiffiffiffi KS Ki � � q

.

<sup>S</sup> <sup>þ</sup> KSX (21)

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<sup>S</sup>max � � (22)

KS <sup>þ</sup> <sup>S</sup><sup>n</sup> (23)

where Ki is the inhibitory factor and μ<sup>0</sup> ¼ μmax 1 þ

microorganisms are in a stationary growth phase.

appears at power n (empirical) is Moser model:

(23) are combined. For example:

Monod-Monod model

Monod-Andrews model

expression:

specific growth rate, is often used [1].

growth rate is Luong model:

Contois model

(20)

155

(24)

(25)

At the specific growth very high rates, Yxs=Y'xs

$$Y\_{ps} = \frac{r\_p}{-r\_s} = \frac{r\_p}{\frac{r\_s}{Y\_{ss}'} + \frac{r\_p}{Y\_{ps}'} + m\_s \cdot X} = \frac{k\_1 \cdot X + k\_2 \cdot r\_x}{\frac{\mu \cdot X}{Y\_{ss}'} + m\_s \cdot X} = \frac{k\_1 + k\_2 \cdot \mu}{\frac{\mu}{Y\_{ss}'} + \frac{k\_1 + k\_2 \cdot \mu}{Y\_{ps}'} + m\_s} \tag{15}$$

For the anaerobic processes:

$$Y\_{xs} = \frac{r\_x}{-r\_s} = \frac{r\_x}{\frac{r\_r}{Y'\_{xs}} + m\_s \cdot X} = \frac{Y'\_{xs}}{1 + \frac{m\_s}{\mu} \cdot Y'\_{xs}} \tag{16}$$

$$Y\_{ps} = \frac{r\_p}{-r\_s} = \frac{\frac{Y\_{ps}'}{Y\_{rs}'} \cdot r\_x + m\_p \cdot X}{\frac{r\_s}{Y\_{rs}'} + m\_s \cdot X} = \frac{Y\_{ps}' + \frac{m\_p}{\mu} \cdot Y\_{xs}'}{1 + \frac{m\_s}{\mu} \cdot Y\_{xs}'} \tag{17}$$

There is a simplified modality to express the efficiencies with the following equations:

$$Y\_{xs} = \frac{X - X\_0}{S\_0 - S} \quad \text{and} \quad Y\_{ps} = \frac{P - P\_0}{S\_0 - S} \tag{18}$$

#### 2.2.4. Alternative variants of models are based on Monod equation.

It is expected that the specific growth rate of biomass, expressed by Monod equation, does not match for all the fermentation processes. Many authors have tried to improve the Monod model and some examples will be presented in the subsequent part.

#### 2.2.4.1. Teissier model

$$
\mu = \mu\_{\text{max}} \cdot \left(1 - e^{-\frac{S}{K\_S}}\right) \tag{19}
$$

A disadvantage of the Monod and Teissier models is that these models do not take into consideration the inhibitory factor of the substrate when it is in excess. Andrews model abolishes this disadvantage by adding S<sup>2</sup> =Ki at the denominator of biomass specific growth rate expression.

#### 2.2.4.2. Andrews model

Therefore, a great quantity of substrate inhibits the cells' growth (i.e., glucose in quantities of >800 mol/m<sup>3</sup> ) and the following equation can be used:

Current State and Perspective in the Models Applicable to Oenology http://dx.doi.org/10.5772/intechopen.71711 155

$$\mu = \frac{\mu\_{\text{max}} \cdot \mathcal{S}}{\mathcal{K}\_s + \mathcal{S} + \frac{\mathcal{S}^2}{\mathcal{K}\_i}} \tag{20}$$

where Ki is the inhibitory factor and μ<sup>0</sup> ¼ μmax 1 þ ffiffiffiffi KS Ki � � q .

Contois model, which takes into consideration the inhibitory effect of the biomass on the specific growth rate, is often used [1].

Contois model

reaction of the stoichiometric coefficients substrate consumed formed product. From Eqs. (5)

<sup>þ</sup> ms � <sup>X</sup> <sup>¼</sup> <sup>μ</sup> � <sup>X</sup> μ�X Y0 xs

<sup>þ</sup> ms � <sup>X</sup> <sup>¼</sup> <sup>k</sup><sup>1</sup> � <sup>X</sup> <sup>þ</sup> <sup>k</sup><sup>2</sup> � rx μ�X Y0 xs

<sup>þ</sup> ms � <sup>X</sup> <sup>¼</sup> <sup>Y</sup><sup>0</sup>

<sup>¼</sup> rx rx Y0 xs

> rx Y0 xs

Yxs <sup>¼</sup> <sup>X</sup> � <sup>X</sup><sup>0</sup>

There is a simplified modality to express the efficiencies with the following equations:

� rx þ mp � X

<sup>þ</sup> ms � <sup>X</sup> <sup>¼</sup> <sup>Y</sup><sup>0</sup>

<sup>S</sup><sup>0</sup> � <sup>S</sup> and Yps <sup>¼</sup> <sup>P</sup> � <sup>P</sup><sup>0</sup>

It is expected that the specific growth rate of biomass, expressed by Monod equation, does not match for all the fermentation processes. Many authors have tried to improve the Monod

μ ¼ μmax � 1 � e

A disadvantage of the Monod and Teissier models is that these models do not take into consideration the inhibitory factor of the substrate when it is in excess. Andrews model abolishes this

Therefore, a great quantity of substrate inhibits the cells' growth (i.e., glucose in quantities of

� S KS 

=Ki at the denominator of biomass specific growth rate expression.

<sup>þ</sup> ms � <sup>X</sup> <sup>¼</sup> <sup>μ</sup> μ Y0 xs

<sup>þ</sup> ms � <sup>X</sup> <sup>¼</sup> <sup>k</sup><sup>1</sup> <sup>þ</sup> <sup>k</sup><sup>2</sup> � <sup>μ</sup> μ Y0 xs

> xs <sup>1</sup> <sup>þ</sup> ms <sup>μ</sup> � Y<sup>0</sup> xs

> > ps <sup>þ</sup> mp <sup>μ</sup> � Y<sup>0</sup> xs

<sup>1</sup> <sup>þ</sup> ms <sup>μ</sup> � Y<sup>0</sup> xs

<sup>þ</sup> <sup>k</sup>1þk2�<sup>μ</sup> Y0 ps

> <sup>þ</sup> <sup>k</sup>1þk2�<sup>μ</sup> Y0 ps

þ ms

þ ms

<sup>S</sup><sup>0</sup> � <sup>S</sup> (18)

(14)

(15)

(16)

(17)

(19)

and (3), the k<sup>1</sup> and k<sup>2</sup> coefficients could be obtained in anaerobic conditions. The total efficiencies of biomass and metabolic products are used in practice.

During the aerobic processes, considering Eqs. (5) and (11), the result is

<sup>¼</sup> rx rx Y0 xs <sup>þ</sup> rp Y0 ps

154 Grapes and Wines - Advances in Production, Processing, Analysis and Valorization

<sup>¼</sup> rp rx Y0 xs <sup>þ</sup> rp Y0 ps

> Yxs <sup>¼</sup> rx �rs

Yps <sup>¼</sup> rp �rs ¼ Y0 ps Y0 xs

2.2.4. Alternative variants of models are based on Monod equation.

model and some examples will be presented in the subsequent part.

) and the following equation can be used:

Yxs <sup>¼</sup> rx �rs

Yps <sup>¼</sup> rp �rs

For the anaerobic processes:

2.2.4.1. Teissier model

disadvantage by adding S<sup>2</sup>

2.2.4.2. Andrews model

>800 mol/m<sup>3</sup>

At the specific growth very high rates, Yxs=Y'xs

$$
\mu = \mu\_{\text{max}} \frac{\mathcal{S}}{\mathcal{S} + K\_{\mathcal{S}} X} \tag{21}
$$

Because of the greater concentration of biomass, the biotical phase can be a substantial part from the total volume of the bioreactor and thus it is difficult to assimilate the substances by the biomass. Anyway, it is heavy to imagine in which mode the cells' concentration can inhibit its own growth and, probably, the ability of Contois kinetics to be in concordance with the experimental data is explained by the toxic effect of some metabolic products.

Another model that takes into consideration the inhibitory effect of the biomass on the specific growth rate is Luong model:

$$
\mu = \frac{\mu\_{\text{max}} \cdot S}{K\_s + S} \cdot \left(1 - \frac{S}{S\_{\text{max}}}\right) \tag{22}
$$

where Ki is the inhibitory constant and Smax is the substrate concentration at which the microorganisms are in a stationary growth phase.

A similar variant of the specific growth rate of Monod biomass in which the substrate S appears at power n (empirical) is Moser model:

$$
\mu = \frac{\mu\_{\text{max}} \cdot \mathcal{S}^{\text{v}}}{K\_{\text{S}} + \mathcal{S}^{\text{v}}} \tag{23}
$$

On the case of bioreactors with two substrates (S1 and S2), the models with Eqs. (3) and (19)– (23) are combined. For example:

Monod-Monod model

$$
\mu = \mu\_{\text{max}} \cdot \frac{\text{S}\_1}{\text{K}\_1 + \text{S}\_1} \cdot \frac{\text{S}\_2}{\text{K}\_2 + \text{S}\_2} \tag{24}
$$

Monod-Andrews model

$$
\mu = \mu\_{\text{max}} \cdot \frac{\text{S}\_1}{\text{K}\_1 + \text{S}\_1} \cdot \frac{\text{S}\_2}{\text{K}\_2 + \text{S}\_2 + \frac{\text{S}\_2^2}{\text{K}\_i}} \tag{25}
$$

When a metabolic product inhibits the growth, the specific growth rate has the following expression:

$$
\mu = \mu\_{\text{max}} \cdot \frac{\text{S}}{\text{K}\_{\text{s}} + \text{S}} \cdot \frac{\text{K}\_{\text{p}}}{\text{K}\_{\text{p}} + \text{P}} \tag{26}
$$

$$
\mu = \mu\_{\text{max}} \cdot \frac{S}{K\_s + S} \cdot \left(1 - \frac{P}{P\_{\text{max}}}\right) \tag{27}
$$

$$
\mu\_{\text{max}} = A \cdot e^{-\frac{\nu\_a}{k \cdot \overline{r}^0}} \tag{28}
$$

$$\mu\_{\text{max}} = \frac{A \cdot e^{-\frac{E}{R \cdot T^0}}}{1 + B \cdot e^{-\frac{M\_d}{R \cdot T^0}}} \tag{29}$$

$$\mu\_{\text{max}} = \begin{cases} a\_1 \cdot e^{-\frac{E\_1}{RT^0}} - a\_2 \cdot e^{-\frac{E\_2}{RT^0}} - b, & \text{if } T\_1^0 \le T^0 \le T\_2^0 \\ 0 & \text{, if } T^0 < T\_1^0 \text{ or } T^0 > T\_2^0 \end{cases} \tag{30}$$

$$
\mu\_{\text{max}} = a \cdot pH^2 + b \cdot pH + c \tag{31}
$$

$$\mu = \mu\_{\text{max}} \cdot f\_1(pH) \cdot f\_2(T) \cdot f\_3(X) \cdot \prod\_{j=1}^{ms} \varphi\_j(\mathbb{S}\_j) \cdot \prod\_{j=1}^{mp} \psi\_j(P\_j) \tag{32}$$

$$\begin{array}{cccccc}\hline\\\hline\\\hline\\\hline\end{array}\begin{array}{cccccc}\hline\\\hline\\\hline\end{array}\begin{array}{cccccc}\hline\\\hline\end{array}\begin{array}{cccccc}\hline\\\hline\end{array}\begin{array}{cccccc}\hline\\\hline\end{array}\begin{array}{cccccc}\hline\\\hline\end{array}\begin{array}{cccccc}\hline\\\hline\end{array}\begin{array}{cccccc}\hline\\\hline\end{array}\begin{array}{cccccc}\hline\\\hline\end{array}\begin{array}{cccccc}\hline\\\hline\end{array}\begin{array}{cccccc}\hline\\\hline\end{array}\begin{array}{cccccc}\hline\\\hline\end{array}\begin{array}{cccccc}\hline\\\hline\end{array}\begin{array}{cccccc}\hline\\\hline\end{array}\begin{array}{cccccc}\hline\\\hline\end{array}\begin{array}{cccccc}\hline\\\hline\end{array}\begin{array}{cccccc}\hline\\\hline\end{array}\begin{array}{cccccc}\hline\end{array}\begin{array}{cccccc}\begin{array}{cccccc}\hline\\\hline\end{$$

#### 2.2.5. A batch bioreactor modeling

A batch bioreactor is a closed system that is fed at the beginning of the process with sterile medium (with S0 initial concentration) and pure culture of the microorganisms with X0 initial concentration. The microorganisms will grow up by multiplication, and eventually they will produce metabolisms products by substrate consummation. Inside the bioreactor, the conditions for multiplication will be established: pH, temperature, oxygen feeding (at aerobic processes), and stirring (at processes with liquid substrate).

Suppose that the growth rate of the biomass depends only by the cells' mass, the mass balance equations could be written for

$$\text{biomass:} \frac{dX}{dt} = r\_x;$$

substrate: dS dt ¼ rs;

metabolism product: dP dt ¼ rp.

One of the simplest models that respect the previous equation is those of Malthus model:

$$r\_{\mathbf{x}} = \mu \cdot \mathbf{X} \tag{33}$$

If the substrate is in excess (in exponential growing phase), this means S>>Ks and the process issues with maximum growth rate of biomass, the model is more simple. The total efficiency equation of the biomass, at maximum growth rates and constant forming efficiency of the

> dt ¼ � <sup>μ</sup> � <sup>X</sup> Y0 xs

<sup>S</sup> <sup>¼</sup> <sup>S</sup><sup>0</sup> � <sup>X</sup> � <sup>X</sup><sup>0</sup> Y0 xs

<sup>μ</sup>max � dt <sup>¼</sup> KS <sup>þ</sup> <sup>S</sup><sup>0</sup> � <sup>X</sup>�X<sup>0</sup>

<sup>S</sup><sup>0</sup> � <sup>X</sup>�X<sup>0</sup> Y0 xs � � � <sup>X</sup>

> � ln <sup>X</sup> X0 � �

and by integrating it between t=0, X=X0 and t and X the named Monod integrated equation

In order to model this type of the processes, Eqs. (35)–(37) will be used. If μ will be considered constant and if the total efficiencies of the biomass and product will be introduced, the variation of the substrate quantity will be given by Eq. (36) in which the Y'xs efficiency has

> dt ¼ � <sup>μ</sup> � <sup>X</sup> Y0 xs

and the equation that expresses the reaction product quantity variation will be obtained from

The equations that characterize the cultivation model in the aerobiosis conditions of the microorganisms, where the products of the reaction (usually primary metabolites) are formed

Y0 xs

� ln

" # !

<sup>S</sup><sup>0</sup> � <sup>X</sup>�X<sup>0</sup> Y0 xs S0

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� μ � X (44)

(39)

159

(40)

(42)

(43)

� dX (41)

dS

By combining the above equation with Eq. (35), the following could be obtained:

will be obtained, by which the biomass variation against time can be determined:

Ks <sup>S</sup><sup>0</sup> <sup>þ</sup> <sup>X</sup><sup>0</sup> Y0 xs

dS

dP dt <sup>¼</sup> <sup>Y</sup><sup>0</sup> ps Y0 xs

<sup>μ</sup>max � <sup>t</sup> <sup>¼</sup> ln <sup>X</sup>

2.2.5.1. The metabolism products forming

been introduced, which means Eq. (39):

2.2.5.1.1. Aerobic processes

2.2.5.1.2. Anaerobic processes

X0 � �

the total efficiency of the product equation, Y'ps, and Eq. (43):

always, are the mass balance equations, as follows:

þ

biomass, becomes

From Eq. (18) results:

where μ is constant; the equation corresponds to exponential growing phase.

The specific growth rate μ can be described by Monod model:

$$
\mu = \frac{\mu\_{\text{max}} \cdot \text{S}}{K\_s + \text{S}} \tag{34}
$$

with the application of correction factors at the inhibitory process produce by the substrate or metabolism product.

With the forming rate of biomass rx (33), the consuming rate of the substrate rs and the forming rate of the metabolite rp, which have been presented at the kinetics model, could be written by the following equations:

$$\text{biomass}: \frac{dX}{dt} = \mu \cdot X \tag{35}$$

$$\text{Substrate}: \frac{dS}{dt} = -\frac{\mu \cdot X}{Y\_{\text{xs}}'} - \frac{k\_1 \cdot X + k\_2 \cdot \mu \cdot X}{Y\_{\text{ps}}'} - m\_s \cdot X \tag{36}$$

$$\text{Ametabolis product}: \frac{dP}{dt} = k\_1 \cdot X + k\_2 \cdot \mu \cdot X \tag{37}$$

The biomass forming

It takes place in the aerobic conditions and in Eq. (36) the factor of metabolism products formed will not appear:

$$\frac{d\mathbf{S}}{dt} = -\frac{\mu \cdot \mathbf{X}}{Y\_{\mathbf{x}s}'} - m\_{\mathbf{s}} \cdot \mathbf{X} \tag{38}$$

If the substrate is in excess (in exponential growing phase), this means S>>Ks and the process issues with maximum growth rate of biomass, the model is more simple. The total efficiency equation of the biomass, at maximum growth rates and constant forming efficiency of the biomass, becomes

$$\frac{d\mathcal{S}}{dt} = -\frac{\mu \cdot X}{Y\_{\infty}'}\tag{39}$$

From Eq. (18) results:

2.2.5. A batch bioreactor modeling

equations could be written for

dt ¼ rx;

dt ¼ rs;

metabolism product: dP

metabolism product.

the following equations:

The biomass forming

will not appear:

biomass: dX

substrate: dS

cesses), and stirring (at processes with liquid substrate).

158 Grapes and Wines - Advances in Production, Processing, Analysis and Valorization

dt ¼ rp.

A batch bioreactor is a closed system that is fed at the beginning of the process with sterile medium (with S0 initial concentration) and pure culture of the microorganisms with X0 initial concentration. The microorganisms will grow up by multiplication, and eventually they will produce metabolisms products by substrate consummation. Inside the bioreactor, the conditions for multiplication will be established: pH, temperature, oxygen feeding (at aerobic pro-

Suppose that the growth rate of the biomass depends only by the cells' mass, the mass balance

One of the simplest models that respect the previous equation is those of Malthus model:

<sup>μ</sup> <sup>¼</sup> <sup>μ</sup>max � <sup>S</sup>

with the application of correction factors at the inhibitory process produce by the substrate or

With the forming rate of biomass rx (33), the consuming rate of the substrate rs and the forming rate of the metabolite rp, which have been presented at the kinetics model, could be written by

dX

dP

It takes place in the aerobic conditions and in Eq. (36) the factor of metabolism products formed

� <sup>k</sup><sup>1</sup> � <sup>X</sup> <sup>þ</sup> <sup>k</sup><sup>2</sup> � <sup>μ</sup> � <sup>X</sup> Y0 ps

biomass :

dt ¼ � <sup>μ</sup> � <sup>X</sup> Y0 xs

where μ is constant; the equation corresponds to exponential growing phase.

The specific growth rate μ can be described by Monod model:

substrate :

dS

metabolism product :

dS

dt ¼ � <sup>μ</sup> � <sup>X</sup> Y0 xs

rx ¼ μ � X (33)

Ks <sup>þ</sup> <sup>S</sup> (34)

dt <sup>¼</sup> <sup>μ</sup> � <sup>X</sup> (35)

dt <sup>¼</sup> <sup>k</sup><sup>1</sup> � <sup>X</sup> <sup>þ</sup> <sup>k</sup><sup>2</sup> � <sup>μ</sup> � <sup>X</sup> (37)

� ms � X (38)

� ms � X (36)

$$S = S\_0 - \frac{X - X\_0}{Y\_{\infty}'} \tag{40}$$

By combining the above equation with Eq. (35), the following could be obtained:

$$
\mu\_{\text{max}} \cdot dt = \frac{K\_S + S\_0 - \frac{X - X\_0}{Y'\_{\text{ns}}}}{\left(S\_0 - \frac{X - X\_0}{Y'\_{\text{ns}}}\right) \cdot X} \cdot dX \tag{41}
$$

and by integrating it between t=0, X=X0 and t and X the named Monod integrated equation will be obtained, by which the biomass variation against time can be determined:

$$\mu\_{\text{max}} \cdot t = \ln\left(\frac{X}{X\_0}\right) + \frac{K\_s}{S\_0 + \frac{X\_0}{Y'\_{xs}}} \cdot \left[ \ln\left(\frac{X}{X\_0}\right) - \ln\left(\frac{S\_0 - \frac{X - X\_0}{Y'\_{xs}}}{S\_0}\right) \right] \tag{42}$$

#### 2.2.5.1. The metabolism products forming

#### 2.2.5.1.1. Aerobic processes

In order to model this type of the processes, Eqs. (35)–(37) will be used. If μ will be considered constant and if the total efficiencies of the biomass and product will be introduced, the variation of the substrate quantity will be given by Eq. (36) in which the Y'xs efficiency has been introduced, which means Eq. (39):

$$\frac{d\mathcal{S}}{dt} = -\frac{\mu \cdot X}{Y\_{\infty}'}\tag{43}$$

and the equation that expresses the reaction product quantity variation will be obtained from the total efficiency of the product equation, Y'ps, and Eq. (43):

$$\frac{dP}{dt} = \frac{Y\_{ps}'}{Y\_{xs}'} \cdot \mu \cdot X \tag{44}$$

#### 2.2.5.1.2. Anaerobic processes

The equations that characterize the cultivation model in the aerobiosis conditions of the microorganisms, where the products of the reaction (usually primary metabolites) are formed always, are the mass balance equations, as follows:

$$\text{biomass}: \frac{dX}{dt} = \mu \cdot X \tag{45}$$

3. Study case—modeling of a white wine alcoholic fermentation process

Wine making is a complex ecological and biochemical process involving many interactions such as grape variety, microbiota, and several technological operations. The process' variables are often controlled empirically and traditionally. There are some factors that strongly affect the alcoholic fermentation. The most important ones are fermentation temperature, grape juice composition, anaerobic conditionsdue to CO2 production, low media pH, sulfur dioxide concentration level, selected yeasts inoculation, and interaction with other microorganisms [21]. The models developed for these cases consequently have various domains of applications but none of them include the whole oenological aspects of the process. The majority of the models are of "knowledge-based" models type and they take into consideration a great number of phenomena that have an important effect on the kinetics of the process fermentation [22].

This part of the chapter proposes a complex nonlinear wine fermentation model based on

To evaluate the total fermentation yield losses under different operating conditions, four experiments were carried out and based on the data obtained within these experiments, a mathematical model was proposed. The strain and the culture medium, the equipment and the experimental conditions together with the measurements of the fermentation parameters were presented by Sipos and coworkers [23–25]. For the experiments, the Saccharomyces cerevisiae YEPD wine yeast was used, being seeded on a culture medium with the following composition:

.

Mauzac must (sterilized through flash pasteurization). The sugar content of the grape must was supplemented with sucrose up to 180 g/L and 40 mL/h tiaminol was added. The SO2 content reached 50 mg/L and the pH was adjusted at 3.8 mg/L H3PO4. Both the fermentation medium and the bioreactor were autoclaved for 20 min at 393 K. A New-Brunswick continuously stirred

The following operating conditions were as follows: working volume, 8 L; temperature, 291 and 301 K; stirring speed, 150 rpm; pH, 3.8; influent glucose concentrations, 180 and 210 g/L;

The mathematical model of the alcoholic fermentation process was determined on the basis of the approach of the zone modeling principle, taking into consideration the evolution of the viable biomass (Xv(t)). Based on the analysis of the phenomenological aspects of the alcoholic fermentation process, the evolution of Xv(t) was divided into three parts (Figure 5) as follows:

7H2O, 1 g/L yeast extract, 50 g/L glucose, and

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161

previous researches by the author [23–25].

5 g/L KH2PO4, 2 g/L (NH4)2SO4, 0.4 g/L (MgSO4)

bioreactor equipped with pH and temperature sensors was used.

without aeration, the necessary oxygen was dissolved in must.

3.1. Experimental conditions

3.2. The mathematical model

• latent phase (1);

• decay phase (3).

• growing phase (2);

$$\text{Substrate}: \frac{dS}{dt} = -\frac{\mu \cdot X}{Y\_{\text{xs}}'} - m\_s \cdot X \tag{46}$$

$$\text{Ametaloolism product}: \frac{dP}{dt} = \frac{Y\_{ps}'}{Y\_{xs}'} \cdot \mu \cdot X + m\_p \cdot X \tag{47}$$

The biomass and metabolism production will be completed when the substrate will be wasted.

In case the specific growth rate remains constant, simplifications can be made. Keeping tabs of total biomass and metabolism products efficiencies, Eqs. (46) and (47) become

$$\frac{d\mathcal{S}}{dt} = -\frac{\mu \cdot X}{Y\_{\infty}}\tag{48}$$

$$\frac{dP}{dt} = \frac{Y\_{\rm ps}}{Y\_{\infty}} \cdot \mu \cdot X \tag{49}$$

which are identical with the equations that describe the aerobic fermentation. Unfortunately, during the anaerobic fermentation the biomass growth rate is inhibited by the metabolism products and the model becomes more complex. An example is the inhibition of the biomass growth in an alcoholic fermentation by the concentration of formed ethanol:

$$
\mu = \frac{\mu\_{\text{max}} \cdot \text{S}}{\text{K}\_s + \text{S}} \cdot \left(1 - \frac{P}{P\_{\text{max}}}\right) \tag{50}
$$

where Pmax represents the maximum concentration of ethylic alcohol at which the biomass production is stopped.

#### 2.2.5.1.3. Energetically model for the batch bioreactor

The balance energy for reaction medium and bioreactor's jacket can been written as

$$\frac{\Delta Hr \cdot \frac{d\mathbf{S}}{dt}}{\rho \cdot c\_p} - \frac{\mathbf{K}\_T \cdot A\_T}{V \cdot \rho \cdot c\_p} \left( T^0 - T\_{\rm ag}^0 \right) = \frac{dT^0}{dt} \tag{51}$$

$$\frac{F\_{\rm ag}}{V\_{\rm ag}} \left( T\_{\rm agi}^{0} - T\_{\rm ag}^{0} \right) + \frac{K\_{T} A\_{T}}{V\_{\rm ag} \cdot \rho\_{\rm ag} \cdot c\_{\rm pgg}} \left( T^{0} - T\_{\rm ag}^{0} \right) = \frac{d T\_{\rm ag}^{0}}{dt} \tag{52}$$

where ΔHr is the reaction heat of the fermentation [J/mol of the produced alcohol]; KT is the heat transfer coefficient [W/m2 .K] ; AT is the heat transfer area [m2 ]; r, rag is the density of the mass of the reaction, respectively, of the cooling agent [kg/m3 ]; cp, cpag is the heat capacity of the mass of the reaction, respectively, of the cooling agent [J/kg.K]; V, Vagis the fermentation medium volume, volume of the jacket [m3 ]; Fag is the flow of the cooling agent [m3 /h]; T0 and T0 agare the temperature of the fermentation medium and temperature of the cooling agent in the jacket [K].

## 3. Study case—modeling of a white wine alcoholic fermentation process

Wine making is a complex ecological and biochemical process involving many interactions such as grape variety, microbiota, and several technological operations. The process' variables are often controlled empirically and traditionally. There are some factors that strongly affect the alcoholic fermentation. The most important ones are fermentation temperature, grape juice composition, anaerobic conditionsdue to CO2 production, low media pH, sulfur dioxide concentration level, selected yeasts inoculation, and interaction with other microorganisms [21]. The models developed for these cases consequently have various domains of applications but none of them include the whole oenological aspects of the process. The majority of the models are of "knowledge-based" models type and they take into consideration a great number of phenomena that have an important effect on the kinetics of the process fermentation [22].

This part of the chapter proposes a complex nonlinear wine fermentation model based on previous researches by the author [23–25].

#### 3.1. Experimental conditions

biomass :

dS

substrate :

160 Grapes and Wines - Advances in Production, Processing, Analysis and Valorization

metabolism product :

total biomass and metabolism products efficiencies, Eqs. (46) and (47) become

dS

dP dt <sup>¼</sup> Yps Yxs

growth in an alcoholic fermentation by the concentration of formed ethanol:

production is stopped.

transfer coefficient [W/m2

volume of the jacket [m3

2.2.5.1.3. Energetically model for the batch bioreactor

Fag Vag

T0 agi � <sup>T</sup><sup>0</sup> ag 

the reaction, respectively, of the cooling agent [kg/m3

<sup>μ</sup> <sup>¼</sup> <sup>μ</sup>max � <sup>S</sup>

The balance energy for reaction medium and bioreactor's jacket can been written as

� KT � AT V � r � cp

.K] ; AT is the heat transfer area [m2

þ

<sup>Δ</sup>Hr � dS dt r � cp

dX

dt ¼ � <sup>μ</sup> � <sup>X</sup> Y0 xs

> dP dt <sup>¼</sup> <sup>Y</sup><sup>0</sup> ps Y0 xs

The biomass and metabolism production will be completed when the substrate will be wasted. In case the specific growth rate remains constant, simplifications can be made. Keeping tabs of

> dt ¼ � <sup>μ</sup> � <sup>X</sup> Yxs

which are identical with the equations that describe the aerobic fermentation. Unfortunately, during the anaerobic fermentation the biomass growth rate is inhibited by the metabolism products and the model becomes more complex. An example is the inhibition of the biomass

Ks <sup>þ</sup> <sup>S</sup> � <sup>1</sup> � <sup>P</sup>

where Pmax represents the maximum concentration of ethylic alcohol at which the biomass

KTAT Vag � rag � cpag

where ΔHr is the reaction heat of the fermentation [J/mol of the produced alcohol]; KT is the heat

the reaction, respectively, of the cooling agent [J/kg.K]; V, Vagis the fermentation medium volume,

]; Fag is the flow of the cooling agent [m3

ature of the fermentation medium and temperature of the cooling agent in the jacket [K].

Pmax 

<sup>T</sup><sup>0</sup> � <sup>T</sup><sup>0</sup> ag  <sup>¼</sup> dT<sup>0</sup>

<sup>¼</sup> dT<sup>0</sup> ag

<sup>T</sup><sup>0</sup> � <sup>T</sup><sup>0</sup> ag 

dt (51)

]; r, rag is the density of the mass of

]; cp, cpag is the heat capacity of the mass of

/h]; T0 and T0

dt (52)

agare the temper-

dt <sup>¼</sup> <sup>μ</sup> � <sup>X</sup> (45)

� ms � X (46)

� μ � X þ mp � X (47)

� μ � X (49)

(48)

(50)

To evaluate the total fermentation yield losses under different operating conditions, four experiments were carried out and based on the data obtained within these experiments, a mathematical model was proposed. The strain and the culture medium, the equipment and the experimental conditions together with the measurements of the fermentation parameters were presented by Sipos and coworkers [23–25]. For the experiments, the Saccharomyces cerevisiae YEPD wine yeast was used, being seeded on a culture medium with the following composition: 5 g/L KH2PO4, 2 g/L (NH4)2SO4, 0.4 g/L (MgSO4) . 7H2O, 1 g/L yeast extract, 50 g/L glucose, and Mauzac must (sterilized through flash pasteurization). The sugar content of the grape must was supplemented with sucrose up to 180 g/L and 40 mL/h tiaminol was added. The SO2 content reached 50 mg/L and the pH was adjusted at 3.8 mg/L H3PO4. Both the fermentation medium and the bioreactor were autoclaved for 20 min at 393 K. A New-Brunswick continuously stirred bioreactor equipped with pH and temperature sensors was used.

The following operating conditions were as follows: working volume, 8 L; temperature, 291 and 301 K; stirring speed, 150 rpm; pH, 3.8; influent glucose concentrations, 180 and 210 g/L; without aeration, the necessary oxygen was dissolved in must.

#### 3.2. The mathematical model

The mathematical model of the alcoholic fermentation process was determined on the basis of the approach of the zone modeling principle, taking into consideration the evolution of the viable biomass (Xv(t)). Based on the analysis of the phenomenological aspects of the alcoholic fermentation process, the evolution of Xv(t) was divided into three parts (Figure 5) as follows:


X Biomass concentration g/L S Substrate concentration g/L P Alcohol concentration g/L k Kinetic constant 1/h

data)

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data)

data)

1/h

163

J/mol

K

<sup>g</sup>cells. h

0.435<sup>c</sup> g/g

A Pre-exponential factor in Arrhenius' equation 148 (calculated using experimental

Ea Activation energy 21,424 (calculated using experimental

A1 Pre-exponential factor in Arrhenius' equation 9.5 <sup>10</sup>8 a 1/h Ea1 Activation energy 55,000a J/mol A2 Pre-exponential factor in Arrhenius' equation 2.551033 1/h Ea2 Activation energy 220,000<sup>a</sup> J/mol R Universal gas constant 8.31 J/mol.

T0 Temperature in bioreactor 291 and 301 K Ks Substrate limitation constant 0.2<sup>a</sup> g/L

η Efficiency in alcohol of fermentation reaction 48b % S0 Steady-state operation point of substrate 180 g/L P0 Steady-state operation point of alcohol 0 g/L t Time h μmax Maximum specific growth rate 1/h KP Alcohol limitation constant 0.14c g/L qpmax Maximum specific alcohol production rate 1.02c g/

KSP Constant in the substrate term for ethanol production 1.68c g/L KPP Constant of fermentation inhibition by ethanol 0.07d g/L YXS Ratio of cell produced per glucose consumed for growth 0.607<sup>d</sup> g/g

d Pseudo-constant of the biomass 1.67 (calculated using experimental

f Pseudo-constant of the biomass 0.34 α Pseudo-order of the substrate 0.69b β Pseudo-order of the alcohol 0.32b

YPS Ratio of ethanol produced per glucose consumed for

Table 4. Variables and parameters of the kinetic model.

fermentation

a [6, 27, 28] b [26] c [29] d [16]

Figure 5. Evolution of the viable biomass concentration Xv(t).

Table 3 presents the equations of the model. The parameters are adjusted through the nonlinear programming method, which compares the model predictions with experimental data and minimizes the errors.

Tables 4 and 5 present the list of the variables and parameters of the mathematical model.


Table 3. The model of the alcoholic fermentation process.


[29] d [16]

Table 3 presents the equations of the model. The parameters are adjusted through the nonlinear programming method, which compares the model predictions with experimental

**2 3**

2 8 4 6 10 12 14

Time, [*days*]

*Xv(t)* 

16

Tables 4 and 5 present the list of the variables and parameters of the mathematical model.

<sup>T</sup><sup>0</sup> þ b




dCCO<sup>2</sup>

ΔHr� dS dt <sup>r</sup>�cp � KT �AT

Fag Vag <sup>T</sup><sup>0</sup> agi � <sup>T</sup><sup>0</sup> ag 

dt <sup>¼</sup> <sup>μ</sup>max � <sup>S</sup>

dt <sup>¼</sup> qpmax � <sup>S</sup> KSPþ<sup>S</sup>

dt ¼ f � X � k; k ¼ A � e

dt ¼ �<sup>k</sup> � <sup>S</sup><sup>α</sup> � <sup>P</sup><sup>β</sup>

dt <sup>¼</sup> <sup>g</sup> � CCO<sup>2</sup> � <sup>k</sup> � <sup>S</sup> KSPþ<sup>S</sup> � ln <sup>k</sup> � <sup>S</sup> KSPþ<sup>S</sup> � <sup>t</sup>

ag 

<sup>þ</sup> KT AT

<sup>¼</sup> dT<sup>0</sup> dt

Vag �rag �cpag <sup>T</sup><sup>0</sup> � <sup>T</sup><sup>0</sup>

dt ¼ � <sup>1</sup> YXS � dX dt 


<sup>V</sup>�r�cp <sup>T</sup><sup>0</sup> � <sup>T</sup><sup>0</sup>


KSþS 

� <sup>e</sup>�Kp �<sup>P</sup> � <sup>X</sup>; <sup>μ</sup>max <sup>¼</sup> <sup>A</sup><sup>1</sup> � <sup>e</sup>

� <sup>e</sup>�Kpp �<sup>P</sup> � <sup>X</sup>

� Ea <sup>R</sup>�T<sup>0</sup>

ag  <sup>¼</sup> dT<sup>0</sup> ag dt

� <sup>1</sup> YPS � dP dt  � Ea<sup>1</sup> <sup>R</sup>�T<sup>0</sup> � A<sup>2</sup> � e

� Ea<sup>2</sup> <sup>R</sup>�T<sup>0</sup>

data and minimizes the errors.

Kinetic model

Energetic model

25

**1**

Figure 5. Evolution of the viable biomass concentration Xv(t).

Current phase Model equations

Latent phase [23, 24] tlat <sup>¼</sup> <sup>a</sup>

Exponential growing phase [1] - biomass: dX

Decay phase [23–26] - biomass: dX

All phases [23, 26] - for the bioreactor:

Table 3. The model of the alcoholic fermentation process.

All phases - carbon dioxide concentration:

100

125

162 Grapes and Wines - Advances in Production, Processing, Analysis and Valorization

75

50

Yeast population, [*millions/mL*]

Table 4. Variables and parameters of the kinetic model.


corresponds to the real one. The non-physiological model proposed by Bovée and Strehaiano [26] was chosen because it accurately describes the substrate consumption and the evolution of the alcohol concentration in the growing and decay phases. This model proposes the use of a semi-empirical model in which the velocity of sugar consumption is described by a chemical law that depends on substrate and product contents. The parameters of the model are adjusted by means of nonlinear programming methods, which compare model predictions with experimental data and minimize errors [23–25]. The Bovée and Strehaiano model is capable of predicting the fermented sugar (and thus thermal planning) within an error of 3.3% [25]. Thus, the model offers a good qualitative and quantitative description of the behavior of the alco-

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165

Figures 6–8 show the simulation results of the model presented in Table 3 considering the following initial values: the initial substrate concentration was 210 g/L and the fermentation

The equation of the latent phase is valid for a time interval [0, 100 h] and the model has been tested for a grape juice variety with an initial concentration of the substrate varying between 180 and 210 g/L, a fermentation temperature between 299 and 303 K and without

Figure 6. Evolution of glucose and alcohol concentrations; a comparison between experimental values (o—glucose and +

holic fermentation process.

temperature was 301 K.

—alcohol) and simulation results (continuous lines).

aeration.

c [30]

Table 5. Parameters of the kinetic model.

#### 3.3. Result and discussion regarding the mathematical model simulation

The nonlinear mathematical model of the batch fermentation process (Table 3) used in this work contains the following equations:


The model proposed by Aiba [1] includes the inhibitory effects of the fermentation product (alcohol). In the growing phase, the value of the maximum specific growth rate of the biomass corresponds to the real one. The non-physiological model proposed by Bovée and Strehaiano [26] was chosen because it accurately describes the substrate consumption and the evolution of the alcohol concentration in the growing and decay phases. This model proposes the use of a semi-empirical model in which the velocity of sugar consumption is described by a chemical law that depends on substrate and product contents. The parameters of the model are adjusted by means of nonlinear programming methods, which compare model predictions with experimental data and minimize errors [23–25]. The Bovée and Strehaiano model is capable of predicting the fermented sugar (and thus thermal planning) within an error of 3.3% [25]. Thus, the model offers a good qualitative and quantitative description of the behavior of the alcoholic fermentation process.

Figures 6–8 show the simulation results of the model presented in Table 3 considering the following initial values: the initial substrate concentration was 210 g/L and the fermentation temperature was 301 K.

The equation of the latent phase is valid for a time interval [0, 100 h] and the model has been tested for a grape juice variety with an initial concentration of the substrate varying between 180 and 210 g/L, a fermentation temperature between 299 and 303 K and without aeration.

3.3. Result and discussion regarding the mathematical model simulation

biomass, alcohol production, and substrate consumption;

work contains the following equations:

Table 5. Parameters of the kinetic model.

posed by Sipos in [23–25]);

(dT<sup>0</sup>

T0

T0

a [17] b

c [30]

experimental data

jacket.

(the model proposed by Sipos);

phase time of the process temperature;

The nonlinear mathematical model of the batch fermentation process (Table 3) used in this

KT Heat transfer coefficient 3.6105 a J/ m2.K.

AT Heat transfer area 0.8<sup>b</sup> m<sup>2</sup> Fag Flow of cooling agent 0.01b m<sup>3</sup>

164 Grapes and Wines - Advances in Production, Processing, Analysis and Valorization

Vag Volume of the jacket 0.2<sup>b</sup> m<sup>3</sup> V Volume of the mass of reaction 1b m<sup>3</sup>

agi Temperature of cooling agent entering to the jacket 278b K <sup>Δ</sup>Hr Reaction heat of fermentation <sup>98465</sup><sup>c</sup> J/mol r Density of the mass of reaction 1100b kg/m<sup>3</sup> rag Density of cooling agent 999.8<sup>a</sup> kg/m<sup>3</sup> cp Heat capacity of mass of reaction 3391b J/kg.

cpag Heat capacity of cooling agent 4217a J/kg.

ag Temperature of cooling agent in the jacket K

h

/h

K

K

• an equation for the latent phase of fermentation that describes the dependence of the

• the model proposed by Aiba [1] for the growing phase with the three equations of

• the model presented by Bovée-Strehaiano [26] for the decay phase with two equations:

• an equation that describes the biomass behavior along the phase no. 3 (the model pro-

• an equation that describes the carbon dioxide concentration behavior along all the phases

• an energy balance model in which the rate of change of the medium's temperature

The model proposed by Aiba [1] includes the inhibitory effects of the fermentation product (alcohol). In the growing phase, the value of the maximum specific growth rate of the biomass

/dt) is a result of the balance between the rate of the heat generation due to fermentation and the rate of the heat transfer to the cooling medium inside the bioreactor

one for the substrate consumption and the other for alcohol formation;

Figure 6. Evolution of glucose and alcohol concentrations; a comparison between experimental values (o—glucose and + —alcohol) and simulation results (continuous lines).

Author details

Address all correspondence to: anca.sipos@ulbsibiu.ro

[1] Caraman S, Ceangă E, Frangu L, Mencinicopschi G. Modelarea şi conducerea proceselor biotehnologice (The modelling and control of biotechnological processes). Bucharest:

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167

[2] Mitrana V. Bioinformatica – unde se întâlnesc: biologia, informatica, matematica? (Bioinformatics - where is met: the biology, informatics and mathematic?). Bucharest: Editura L

[3] Vogel HC, Tadaro CL. Fermentation and biochemical engineering handbook – Principles. 2nd ed. Process Design and Equipment. Noyes: William Andrew Publishing; 1996. Chap.1 [4] Xiong Z, Zhang J. Mint. Modelling and optimal control of fed-batch processes using a novel control affine feedforward neural network. Neurocomputing. 2004;61:317-337. DOI: 10.1016/

[5] Tijskens LMM, Hertog MLATM, Nicolaï BM. Food process modeling. Abigton Hall, Cambridge: Woodhead Publishing Series in Food Science, Technology and Nutrition; 2001 [6] Valentinotti S, Srinivasan B, Holmberg U, Bonvin D, Cannizzaro C, Rhiel M, von Stockar U. Optimal operation of fed-batch fermentation via adaptive control of overflow metabolite. Control Engineering Practice. 2003;11:665-674. DOI: 10.1016/S0967-0661(02)00172-7

[7] Szederkenyi G, Kristensen NR, Hangos KM, Bay Jorgensen S. Nonlinear analysis and control of a continuous fermentation process. Computers and Chemical Engineering.

[8] Zhang Y, Henson MA, Kevrekidis YG. Nonlinear model reduction for dynamic analysis of cell population models. Chemical Engineering Science. 2003;58:429-445. DOI: 10.1016/

[9] Vlassides S, Ferrier JG, Block DE. Using historical data for bioprocess optimization: Modeling wine characteristics using artificial neural networks and archived process information. Biotechnology and Bioengineering. 2001;73:55-68. DOI: 10.1002/1097-0290

[10] Banga JR, Balsa-Canto E, Moles CG, Alonso AA. Improving food processing using modern optimization methods; Trends in Food Science and Technology. 2003;14:131-144. DOI:

2002;26:659-670. DOI: 10.1016/S0098-1354(01)00793-1

(20010405)73:1%3C55::AID-BIT1036%3E3.0.CO;2-5/full

"Lucian Blaga" University of Sibiu, Romania

Editura Didactică şi Pedagogică; 2002

and S INFOMAT; 1998

j.neucom.2003.11.006

S0009-2509(02)00439-6

10.1016/S0924-2244(03)00048-7

Anca Şipoş

References

Figure 7. Comparison between the biomass simulation results (continuous line) and experimental data (o).

Figure 8. Comparison between the carbon dioxide concentration simulation results (continuous line) and experimental data (o).

## Author details

Anca Şipoş

Address all correspondence to: anca.sipos@ulbsibiu.ro

"Lucian Blaga" University of Sibiu, Romania

## References

Figure 8. Comparison between the carbon dioxide concentration simulation results (continuous line) and experimental

Figure 7. Comparison between the biomass simulation results (continuous line) and experimental data (o).

166 Grapes and Wines - Advances in Production, Processing, Analysis and Valorization

data (o).


[11] Harms P, Kostov Y, Rao G. Bioprocess monitoring. Current Opinion in Biotechnology. 2002;13:124-127. DOI: 10.1016/S0958-1669(02)00295-1

[25] Șipoș A, Agachi ŞP. Direct sensitivity analysis of a white wine alcoholic fermentation

Current State and Perspective in the Models Applicable to Oenology

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[26] Bovée JP, Strehaiano P, Goma G, Sevely Y. Alcoholic fermentation: modelling based on sole substrate and product measurement. Biotechnology and Bioengineering. 1984;26:328-334.

[27] Krothapally M, Palanki S. A neural network strategy for end-point optimization of batch processes. ISA Transactions. 1999;38:383. DOI: 10.1016/S0019-0578(99)00031-2

[28] Lei F, Rotboll M, Jorgensen SB. A biochemically structured model for Saccharomyces cerevisiae. Journal of Biotechnology. 2001;88:205-221. DOI: 10.1016/S0168-1656(01)00269-3

[29] Dengfeng L, Ling X, Weili X, Hong-Tao Z, Chi-Chung L, Lihua J, Baoguo X. Hindawi fermentation process modeling with Levenberg-Marquardt algorithm and Runge-Kutta method on ethanol production by Saccharomyces cerevisiae. Mathematical Problems in

[30] Costa AC, Atala DIP, Maugeri F, Maciel R. Factorial design and simulation for the optimization and determination of control structures for an extractive alcoholic fermentation. Process Biochemistry. 2001;37:125-137. DOI: 10.1016/S0032-9592(01)00188-1

process. Studia Universitatis Babeș-Bolyai Chemia. 2015;60:125-141

Engineering. 2014;2014:1-10. DOI: 10.1155/2014/289492

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[25] Șipoș A, Agachi ŞP. Direct sensitivity analysis of a white wine alcoholic fermentation process. Studia Universitatis Babeș-Bolyai Chemia. 2015;60:125-141

[11] Harms P, Kostov Y, Rao G. Bioprocess monitoring. Current Opinion in Biotechnology.

[12] Karakuzu C, Turker M, Ozturk S: Modelling, on-line state estimation and fuzzy control of production scale fed-batch baker's yeast fermentation. Control Engineering Practice.

[13] Kress-Rogers E, Brimelow CJB. Instrumentation and sensors for the food industry. 2nd ed. Abigton Hall, Cambridge: Woodhead Publishing Series in Food Science, Technology

[14] Ferreira LS, De Souza MB Jr, Trierweiler JO, Broxtermann O, Folly ROM, Hitzmann B. Aspects concerning the use of biosensors for process control: experimental and simulation investigations; Computers and Chemical Engineering. 2003;27:1165-1173. DOI:

[15] Ferreira LS, De Souza MB Jr, Folly ROM. Development of an alcohol fermentation control system based on biosensor measurements interpreted by neural networks; Sensors and

[16] Torija Ma J, Rozes N, Poblet M, Guillamon JM, Mas A. Effects of fermentation temperature on the strain population of Saccharomyces cerevisiae. International Journal of Food

[17] El-Mansi EMT, Bryce CFA. Fermentation Microbiology and Biotechnology. London: CRC

[18] Hall R, Zacune J. Bio-economies: the EU's Real 'Green Economy' Agenda? World Devel-

[19] Bastin G, Dochain D. On-line estimation and adaptive control of bioreactors. In: Process Measurement and Control. Amsterdam: Elsevier Science Publishers B.V.; 1990. Chap. 5

[20] Roels JA. Energetics and Kinetics in Biotechnology. Amsterdam: Elsevier Biomedical

[21] Ribereau-Gayon P, Dubourdieu D, Doneche B, Lonvaud A. Handbook of Enology. The Microbiology of Wine and Vinifications; vol. I; West Sussex, England: Wiley; 2000 [22] Svendsen C, Skov T, van den Berg FWJ. Monitoring fermentation processes using inprocess measurements of different orders. Journal of Chemical Technology and Biotech-

[23] Sipos A, Meyer XM, Strehaiano P. Development of a non-linear, dynamic mathematical model for the alcoholic fermentation. Acta Alimentaria. 2007;34:429-438. DOI: 10.1556/

[24] Șipoș A, Imre-Lucaci A. Statistical processing and dynamic modeling of an alcoholic fermentation process. Studia Universitatis Babeș-Bolyai Chemia. 2014;3:17-28

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AAlim.2007.0014


**Chapter 9**

**Provisional chapter**

content in wines is

) has become indispensable in the food

has been displayed by consum-

prevents undesir-

implicates a wide

in wines is a decisive

**Microbiological, Physical, and Chemical Procedures to**

**-Free Wines**

) is the most preservative used in the wine industry and has been widely


applied, as antioxidant and antibacterial agent. However, the use of sulfur dioxide implicates

one of the most important challenges for scientist and winemakers. This book chapter gives an overview regarding different microbiological, physical, and chemical alternatives to elab-

review articles and results obtained by the research group of the Wine Technology Center (VITEC) are shown. This study provides useful information related to this novel and healthy type of wines, highlighting the development of winemaking strategies and procedures.

industry. This substance is widely applied as antioxidant and antibacterial in many processed

able sensory properties and the spoilage of wines produced by chemical or microbiological

range of adverse health consequences, such as allergic reactions and cumulative harmful effects [1]. Therefore, negative perceptions toward sulfites have been induced, and a signifi-

a range of adverse clinical effects. Therefore, the replacement of the SO<sup>2</sup>

**Keywords:** food safety, grape juice, sensory analysis, sulfur dioxide, wine

foods, being the most preservative used in the wine industry. In wines, SO2

agents. However, in recent times, it has been shown that the intake of SO2

ers in the last years [2]. For this reason, reducing the amount of SO2

**Microbiological, Physical, and Chemical Procedures to** 

DOI: 10.5772/intechopen.71627

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution,

© 2018 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

and reproduction in any medium, provided the original work is properly cited.

strategy for the wine industry and one of the current topics on the oenological science.

**Elaborate High-Quality SO2-Free Wines**

Raúl Ferrer-Gallego, Miquel Puxeu, Laura Martín, Enric Nart, Claudio Hidalgo and Imma Andorrà

Raúl Ferrer-Gallego, Miquel Puxeu, Laura Martín, Enric Nart, Claudio Hidalgo and Imma Andorrà

Additional information is available at the end of the chapter

In the last decades, the use of the sulfur dioxide (SO2

cant increase on the demand of wines with low content of SO2

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/intechopen.71627

**Abstract**

**1. Introduction**

Sulfur dioxide (SO2

orate high-quality SO2

**Elaborate High-Quality SO<sup>2</sup>**

**Provisional chapter**

#### **Microbiological, Physical, and Chemical Procedures to Elaborate High-Quality SO2-Free Wines Elaborate High-Quality SO<sup>2</sup> -Free Wines**

**Microbiological, Physical, and Chemical Procedures to** 

DOI: 10.5772/intechopen.71627

Raúl Ferrer-Gallego, Miquel Puxeu, Laura Martín, Enric Nart, Claudio Hidalgo and Imma Andorrà Enric Nart, Claudio Hidalgo and Imma Andorrà Additional information is available at the end of the chapter

Raúl Ferrer-Gallego, Miquel Puxeu, Laura Martín,

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/intechopen.71627

#### **Abstract**

Sulfur dioxide (SO2 ) is the most preservative used in the wine industry and has been widely applied, as antioxidant and antibacterial agent. However, the use of sulfur dioxide implicates a range of adverse clinical effects. Therefore, the replacement of the SO<sup>2</sup> content in wines is one of the most important challenges for scientist and winemakers. This book chapter gives an overview regarding different microbiological, physical, and chemical alternatives to elaborate high-quality SO2 -free wines. In the present chapter, original research articles as well as review articles and results obtained by the research group of the Wine Technology Center (VITEC) are shown. This study provides useful information related to this novel and healthy type of wines, highlighting the development of winemaking strategies and procedures.

**Keywords:** food safety, grape juice, sensory analysis, sulfur dioxide, wine

## **1. Introduction**

In the last decades, the use of the sulfur dioxide (SO2 ) has become indispensable in the food industry. This substance is widely applied as antioxidant and antibacterial in many processed foods, being the most preservative used in the wine industry. In wines, SO2 prevents undesirable sensory properties and the spoilage of wines produced by chemical or microbiological agents. However, in recent times, it has been shown that the intake of SO2 implicates a wide range of adverse health consequences, such as allergic reactions and cumulative harmful effects [1]. Therefore, negative perceptions toward sulfites have been induced, and a significant increase on the demand of wines with low content of SO2 has been displayed by consumers in the last years [2]. For this reason, reducing the amount of SO2 in wines is a decisive strategy for the wine industry and one of the current topics on the oenological science.

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2018 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

In wines, SO2 is composed by total SO2 , bound SO2 , free SO2 , and molecular SO2 . Proper adjustment of the SO2 dosage is difficult because it depends on the equilibrium between its free and bound forms. The active form is molecular SO2, which depends on the concentration of free SO2 and the pH [3]. This active form has the antimicrobial and antioxidant properties. In terms of antimicrobial, an insufficient addition of SO<sup>2</sup> will not ensure the wine protection, increasing the risk of yeast and bacteria proliferation. In terms of antioxidant, an inadequate dosage will allow an excessive oxidation of aromas and flavors, compromising the quality of wines [4]. Contrary, excessive dosages in wines may cause organoleptic alterations and also health reactions in consumers. Taking this into account, the International Organization of Vine and Wine (OIV) has progressively reduced the maximum limits of the total SO2 in wines, which is nowadays 150 mg/L for red wines and 200 mg/L for white wines, with some exceptions depending on the sugar content (Regulation (EC) No 607/2009).

sulfur (naturally available as sulfate in grape juice) is used by yeasts in the synthesis of amino acids. In particular, *Saccharomyces cerevisiae* produces sulfite as an intermediate product during the assimilatory reduction of sulfate to sulfide, via adenosine-5′-phosphosulfate [6, 8]. The available sulfide (S2−) can be used in the synthesis of amino acids, as well as being

from 0 to 115 mg/L [10–14]. Most strains of *S. cerevisiae* produce between 10 and 30 mg/L of

called "low sulfite-forming strains" [6]. On the opposite side, "high sulfite-forming strains" are able to produce more than 100 mg/L. These classifications according to their ability to

and indigenous yeast strains. In 1985, Suzzi et al*.* [13] investigated the biological sulfite role in the stabilization of white wines by comparing 1700 strains of *Saccharomyces* isolated from spontaneous fermentations. The majority of them produced less than 10 mg/L of total SO<sup>2</sup>

around 350 produced between 10 and 20 mg/L, 52 strains produced between 20 and 40 mg/L, and just two strains produced more than 40 mg/L. More recently, an experiment carried out at industrial scale by Werner et al*.* [14] showed two distinguishable groups of yeasts, among

produced between 10 and 20 mg/L. Significant differences among yeasts strains in produc-

Querétaro (Mexico), and the obtained results ranged from 37 to 115 mg/L. More recently,

cial and indigenous). Fermentations were conducted using Muscat grape juice at 18 and

results in agreement with other works which also showed diverse yeast strains are able

considerations should be taking into account, such as the organoleptic properties and micro-

the alcoholic fermentations due to its unpleasant smell and taste. It should be noted that the

S and SO2

In the last years, the use of yeast strains with a low capacity to produce SO2

22 commercial strains. The first one produced under 10 mg/L of total SO<sup>2</sup>

. However, some of them may produce less than 10 mg/L, which were commonly

during the alcoholic fermentation have been reported by several authors over the

produced during alcoholic fermentation by different commercial

) were also described by Wells and Osborne [7]. In this case,

S). Eventually, the sulfur amino acid biosynthesis (SAAB)

Microbiological, Physical, and Chemical Procedures to Elaborate High-Quality SO2-Free Wines

and in the resistance of yeasts against this additive [9].

, estimating a total average content ranged

in wines [15]. Several studies have com-

were observed. In 2015, Miranda-Castilleja

of 21 selected yeast strains (commer-


of 52 indigenous species of *Saccharomyces* from

production lesser than 10 mg/L in all cases. The

(<1.4 mg/L) [16, 17]. Thus, several commercial and

S is one of the most undesirable metabolites derived from

are linked [18, 19]. As occurs in the case of

by yeasts is influenced by a complex interaction of genetic, physio-

S varies widely depend on the yeast strains [20, 21]. The release of H<sup>2</sup>

2−) into the cell, as well as in

http://dx.doi.org/10.5772/intechopen.71627

has been one of

, and the second one

,

173

S

excreted as hydrogen sulfide (H<sup>2</sup>

total SO2

form SO2

tion of SO2

time [6, 7, 12, 14].

pared the amount of SO2

the reduction and production of SO2

Yeast strains differ in their capacity to form SO<sup>2</sup>

the most used strategies to reduce the amount of SO2

(free and bound-SO2

et al*.* [11] studied the production of total SO2

25°C. These results showed a total SO<sup>2</sup>

to produce small amounts of total SO2

bial stability of this type of wines.

chemical, and metabolic factors. H2

biosynthesis and the production of H2

The formation of SO<sup>2</sup>

SO2, the formation of H2

values ranged from 25 to 60 mg/L of bound-SO<sup>2</sup>

VITEC has investigated the natural production of SO<sup>2</sup>

indigenous yeast strains have proved to be able to produce SO2

pathway plays a crucial role in the active transport of sulfate (SO4

Today, there is not a commercial product or recipe able to replace the widespread SO<sup>2</sup> actions. Consequently, diverse technological strategies should be considered by winemakers in each stage of the winemaking process, according to the type of wine to be produced and the winery capabilities. From our point of view, these strategies should be addressed from three joint perspectives; microbiological strategies, physical technologies, and chemical treatments. In this sense, the Wine Technology Centre (VITEC) has been working in this research field since 2012. Our studies have been focused in red and white wines, especially regarding Tempranillo and Albariño grape varieties.

#### **2. Microbiological strategies to elaborate SO2 -free wines**

From a microbiological point of view, many factors should be taken into account to reduce the quantity of SO2 in wines. First, it should be considered that an endogenous content of SO2 is naturally produced by yeasts during alcoholic fermentation. Second, grape juice composition, yeast nutrition, and fermentation management may strongly influence the ability of yeasts to produce sulfites. Finally, microbiological stability of the SO<sup>2</sup> -free wines remains uncertain yet.

As mentioned above, the European Union regulates the levels of total sulfites in wines following the Regulation (EC) 607/2009. Therefore, wines must be labeled with the indication "contains sulfites," when the total content of SO<sup>2</sup> is over 10 mg/L, either exogenous or endogenous. Most organisms produce sulfites as a normal intermediate during digestion or synthesis of the sulfur-containing amino acids, such as methionine and cysteine [5]. Sulfites are minor by-products of yeast fermentation, and therefore, they are natural wine constituents. The ability of yeasts to form SO<sup>2</sup> has been reported in different types of wines and geographical areas, and it was known long time ago and investigated intensively over the years [6, 7].

One of the most important factors to elaborate SO2 -free wines is the choice of the suitable yeast strains used for the development of the alcoholic fermentation. During winemaking process, sulfur (naturally available as sulfate in grape juice) is used by yeasts in the synthesis of amino acids. In particular, *Saccharomyces cerevisiae* produces sulfite as an intermediate product during the assimilatory reduction of sulfate to sulfide, via adenosine-5′-phosphosulfate [6, 8]. The available sulfide (S2−) can be used in the synthesis of amino acids, as well as being excreted as hydrogen sulfide (H<sup>2</sup> S). Eventually, the sulfur amino acid biosynthesis (SAAB) pathway plays a crucial role in the active transport of sulfate (SO4 2−) into the cell, as well as in the reduction and production of SO2 and in the resistance of yeasts against this additive [9]. Yeast strains differ in their capacity to form SO<sup>2</sup> , estimating a total average content ranged from 0 to 115 mg/L [10–14]. Most strains of *S. cerevisiae* produce between 10 and 30 mg/L of total SO2 . However, some of them may produce less than 10 mg/L, which were commonly called "low sulfite-forming strains" [6]. On the opposite side, "high sulfite-forming strains" are able to produce more than 100 mg/L. These classifications according to their ability to form SO2 during the alcoholic fermentation have been reported by several authors over the time [6, 7, 12, 14].

In wines, SO2

adjustment of the SO2

maximum limits of the total SO2

(Regulation (EC) No 607/2009).

Albariño grape varieties.

the quantity of SO2

uncertain yet.

SO2

tration of free SO2

is composed by total SO2

172 Grapes and Wines - Advances in Production, Processing, Analysis and Valorization

**2. Microbiological strategies to elaborate SO2**

"contains sulfites," when the total content of SO<sup>2</sup>

One of the most important factors to elaborate SO2

The ability of yeasts to form SO<sup>2</sup>

of yeasts to produce sulfites. Finally, microbiological stability of the SO<sup>2</sup>

properties. In terms of antimicrobial, an insufficient addition of SO<sup>2</sup>

, bound SO2

its free and bound forms. The active form is molecular SO2, which depends on the concen-

wine protection, increasing the risk of yeast and bacteria proliferation. In terms of antioxidant, an inadequate dosage will allow an excessive oxidation of aromas and flavors, compromising the quality of wines [4]. Contrary, excessive dosages in wines may cause organoleptic alterations and also health reactions in consumers. Taking this into account, the International Organization of Vine and Wine (OIV) has progressively reduced the

and 200 mg/L for white wines, with some exceptions depending on the sugar content

Consequently, diverse technological strategies should be considered by winemakers in each stage of the winemaking process, according to the type of wine to be produced and the winery capabilities. From our point of view, these strategies should be addressed from three joint perspectives; microbiological strategies, physical technologies, and chemical treatments. In this sense, the Wine Technology Centre (VITEC) has been working in this research field since 2012. Our studies have been focused in red and white wines, especially regarding Tempranillo and

From a microbiological point of view, many factors should be taken into account to reduce

As mentioned above, the European Union regulates the levels of total sulfites in wines following the Regulation (EC) 607/2009. Therefore, wines must be labeled with the indication

enous. Most organisms produce sulfites as a normal intermediate during digestion or synthesis of the sulfur-containing amino acids, such as methionine and cysteine [5]. Sulfites are minor by-products of yeast fermentation, and therefore, they are natural wine constituents.

cal areas, and it was known long time ago and investigated intensively over the years [6, 7].

strains used for the development of the alcoholic fermentation. During winemaking process,

 is naturally produced by yeasts during alcoholic fermentation. Second, grape juice composition, yeast nutrition, and fermentation management may strongly influence the ability

Today, there is not a commercial product or recipe able to replace the widespread SO<sup>2</sup>

, free SO2

dosage is difficult because it depends on the equilibrium between

in wines, which is nowadays 150 mg/L for red wines

**-free wines**

has been reported in different types of wines and geographi-

is over 10 mg/L, either exogenous or endog-


in wines. First, it should be considered that an endogenous content of

and the pH [3]. This active form has the antimicrobial and antioxidant

, and molecular SO2

. Proper

actions.


will not ensure the

In the last years, the use of yeast strains with a low capacity to produce SO2 has been one of the most used strategies to reduce the amount of SO2 in wines [15]. Several studies have compared the amount of SO2 produced during alcoholic fermentation by different commercial and indigenous yeast strains. In 1985, Suzzi et al*.* [13] investigated the biological sulfite role in the stabilization of white wines by comparing 1700 strains of *Saccharomyces* isolated from spontaneous fermentations. The majority of them produced less than 10 mg/L of total SO<sup>2</sup> , around 350 produced between 10 and 20 mg/L, 52 strains produced between 20 and 40 mg/L, and just two strains produced more than 40 mg/L. More recently, an experiment carried out at industrial scale by Werner et al*.* [14] showed two distinguishable groups of yeasts, among 22 commercial strains. The first one produced under 10 mg/L of total SO<sup>2</sup> , and the second one produced between 10 and 20 mg/L. Significant differences among yeasts strains in production of SO2 (free and bound-SO2 ) were also described by Wells and Osborne [7]. In this case, values ranged from 25 to 60 mg/L of bound-SO<sup>2</sup> were observed. In 2015, Miranda-Castilleja et al*.* [11] studied the production of total SO2 of 52 indigenous species of *Saccharomyces* from Querétaro (Mexico), and the obtained results ranged from 37 to 115 mg/L. More recently, VITEC has investigated the natural production of SO<sup>2</sup> of 21 selected yeast strains (commercial and indigenous). Fermentations were conducted using Muscat grape juice at 18 and 25°C. These results showed a total SO<sup>2</sup> production lesser than 10 mg/L in all cases. The results in agreement with other works which also showed diverse yeast strains are able to produce small amounts of total SO2 (<1.4 mg/L) [16, 17]. Thus, several commercial and indigenous yeast strains have proved to be able to produce SO2 -free wines. However, other considerations should be taking into account, such as the organoleptic properties and microbial stability of this type of wines.

The formation of SO<sup>2</sup> by yeasts is influenced by a complex interaction of genetic, physiochemical, and metabolic factors. H2 S is one of the most undesirable metabolites derived from the alcoholic fermentations due to its unpleasant smell and taste. It should be noted that the biosynthesis and the production of H2 S and SO2 are linked [18, 19]. As occurs in the case of SO2, the formation of H2 S varies widely depend on the yeast strains [20, 21]. The release of H<sup>2</sup> S by yeast during the fermentation is a long-standing problem that has been extensively studied in comparison to the SO2 production. There has been an ever-growing interest in wine yeasts with low production in H2 S. The selection of suitable strains has so far been the principal way of limiting excessive H2 S formation. Other engineering strategies have been used for limiting its production, which generally consisted of overexpression or inactivation of some genes involved in the sulfate reduction pathway [22–24].

Both sulfites and hydrogen sulfides are produced during the biosynthesis of the sulfur containing amino acids, methionine, and cysteine, starting from sulfate assimilation. Given the metabolic link between H2 S and SO2 , such kind of biotechnological and engineering strategies firstly applied to reduce H<sup>2</sup> S production could also be applied to decrease SO2 formation by yeasts. Nonetheless, few works have been aimed to obtain both low SO<sup>2</sup> and low H2 S production. Three strains with low SO<sup>2</sup> production (SO2 < 10 mg/L) and with reduced H2 S production were selected by De Vero et al [25]. These authors proposed a strategy that combines sexual recombination and specific selective pressure to generate nongenetically-modified *S. cerevisiae* with desired oenological characteristics. More recently, new insight into the regulation of sulfur metabolism in wine yeasts by the identification of variants of MET2 and SKP2 genes within SAAB has been reported to modulate the production of sulfites and sulfides [26]. These results provide novel targets for the improvement of wine yeast strains orientated to produce SO2 -free wines. This knowledge on the sulfate pathway provides a chance to successfully apply engineering strategies to select "low sulfite-forming" yeast strains. However, as we previously highlighted, the production of sulfites by yeast during fermentation not only depend on metabolic factors but also on the environment, including nutrients and fermentation management, among others. Hence, grape juices composition is an imperative factor that should be considered in order to elaborate this type of wines. The insoluble solids contained in the grape juice also appeared to have an effect on the SO<sup>2</sup> content, and wines with the higher insoluble solids obtained lower values of SO2 [27]. In contrast, results obtained in our experimental cellar showed that grapes with higher content of soluble solids produced higher content of total SO2 (**Figure 1**). The biplot of the principal component analysis (PCA) shows that the amount of SO2 produced during the alcoholic fermentation is mainly favored by a high amount of sugars and a low quantity of nitrogen. Furthermore, musts fermented at low temperatures (18°C), and a low titratable acidity may contribute on the production of SO<sup>2</sup> .

of SO2

SO2

facilitate SO2

 under higher yeast assimilable nitrogen (YAN) quantities [7, 29]. The supplementation on nitrogen using ammonium salts (sulfate or phosphate) allows higher growth rates and biomass yielding and also the stimulation of the fermentative activity [30, 31]. The addition

Microbiological, Physical, and Chemical Procedures to Elaborate High-Quality SO2-Free Wines

http://dx.doi.org/10.5772/intechopen.71627

175

kinetics of fermentation and aroma profile of wine [32]. In the last 5 years, VITEC has been

by yeast along of the alcoholic fermentation. Results obtained showed that the addition of

sulfates and DAP using low sulfite-forming strains to ferment musts showed no significant differences. In the case of musts fermented by "high sulfite-forming" strains, the addition of

holic fermentation. In this sense, it has been stated that temperature has several effects on biochemical and physiological properties in yeast cells. Some changes in the sulfur assimilation pathway by *S. cerevisiae* depending on temperature may occur [34]. Our results are in agreement with other authors, who reported that at low temperature, the

[33].

uptake) and concentration of some minerals (copper and zinc) and vitamins,

studying the effect of ammonium sulfate and DAP addition on the amount of SO<sup>2</sup>

and H2

S production and improves the

in wines. The addition of ammonium


S production is also affected by pH (acidic pH

produced

of diammonium phosphate (DAP) significantly decreases H<sup>2</sup>

**Figure 1.** Biplot performed by 74 wines produced from Tempranillo and Albariño musts.

the N-sources slightly increases the total content of SO<sup>2</sup>

DAP significantly increased the total content of SO<sup>2</sup>

Other important consideration to elaborate SO2

production increases [26]. SO2

In addition, the supplementation of musts with amino acids can significantly affect SO<sup>2</sup> and H2 S production depending on the amount added, the time of addition, and the nitrogen concentration [26, 28]. Individual amino acids such as methionine, cysteine, asparagine, and arginine have been shown to influence sulfite formation [18, 28]. Higher the concentration of methionine and cysteine in the grape must, lower the formation of SO2 [6]. Under ammonia limitations, the addition of nonsulfur amino acids tended to increase the formation of SO2 (but inhibits the formation of H2 S). The addition of cysteine seems to increase the H<sup>2</sup> S content but inhibits the sulfite formation, and the addition of methionine inhibits both SO<sup>2</sup> and H2 S formation [28]. More recently, it was stated that methionine repressed the cysteine-induced increase in the H2 S production but had no effect on the formation of SO<sup>2</sup> . Both compounds were produced in greater quantities by yeast when grown in the presence of increasing concentrations of cysteine [18]. It has been reported that yeasts produce higher concentrations

by yeast during the fermentation is a long-standing problem that has been extensively studied

its production, which generally consisted of overexpression or inactivation of some genes

Both sulfites and hydrogen sulfides are produced during the biosynthesis of the sulfur containing amino acids, methionine, and cysteine, starting from sulfate assimilation. Given the

tion were selected by De Vero et al [25]. These authors proposed a strategy that combines sexual recombination and specific selective pressure to generate nongenetically-modified *S. cerevisiae* with desired oenological characteristics. More recently, new insight into the regulation of sulfur metabolism in wine yeasts by the identification of variants of MET2 and SKP2 genes within SAAB has been reported to modulate the production of sulfites and sulfides [26]. These results provide novel targets for the improvement of wine yeast strains orientated to

fully apply engineering strategies to select "low sulfite-forming" yeast strains. However, as we previously highlighted, the production of sulfites by yeast during fermentation not only depend on metabolic factors but also on the environment, including nutrients and fermentation management, among others. Hence, grape juices composition is an imperative factor that should be considered in order to elaborate this type of wines. The insoluble solids contained

experimental cellar showed that grapes with higher content of soluble solids produced higher

high amount of sugars and a low quantity of nitrogen. Furthermore, musts fermented at low temperatures (18°C), and a low titratable acidity may contribute on the production of SO<sup>2</sup>

S production depending on the amount added, the time of addition, and the nitrogen concentration [26, 28]. Individual amino acids such as methionine, cysteine, asparagine, and arginine have been shown to influence sulfite formation [18, 28]. Higher the concentration of

limitations, the addition of nonsulfur amino acids tended to increase the formation of SO2

formation [28]. More recently, it was stated that methionine repressed the cysteine-induced

S production but had no effect on the formation of SO<sup>2</sup>

were produced in greater quantities by yeast when grown in the presence of increasing concentrations of cysteine [18]. It has been reported that yeasts produce higher concentrations

but inhibits the sulfite formation, and the addition of methionine inhibits both SO<sup>2</sup>

In addition, the supplementation of musts with amino acids can significantly affect SO<sup>2</sup>

methionine and cysteine in the grape must, lower the formation of SO2

S production could also be applied to decrease SO2


(**Figure 1**). The biplot of the principal component analysis (PCA) shows

produced during the alcoholic fermentation is mainly favored by a

S). The addition of cysteine seems to increase the H<sup>2</sup>

production (SO2 < 10 mg/L) and with reduced H2

production. There has been an ever-growing interest in wine yeasts

S formation. Other engineering strategies have been used for limiting

S. The selection of suitable strains has so far been the principal way

, such kind of biotechnological and engineering strategies

formation by

S produc-

S produc-

.

S content

S

and H2

[6]. Under ammonia

. Both compounds

and

and low H2

content, and wines with the

[27]. In contrast, results obtained in our

in comparison to the SO2

of limiting excessive H2

with low production in H2

metabolic link between H2

produce SO2

content of total SO2

H2

that the amount of SO2

(but inhibits the formation of H2

increase in the H2

firstly applied to reduce H<sup>2</sup>

tion. Three strains with low SO<sup>2</sup>

involved in the sulfate reduction pathway [22–24].

S and SO2

174 Grapes and Wines - Advances in Production, Processing, Analysis and Valorization

in the grape juice also appeared to have an effect on the SO<sup>2</sup>

higher insoluble solids obtained lower values of SO2

yeasts. Nonetheless, few works have been aimed to obtain both low SO<sup>2</sup>

**Figure 1.** Biplot performed by 74 wines produced from Tempranillo and Albariño musts.

of SO2 under higher yeast assimilable nitrogen (YAN) quantities [7, 29]. The supplementation on nitrogen using ammonium salts (sulfate or phosphate) allows higher growth rates and biomass yielding and also the stimulation of the fermentative activity [30, 31]. The addition of diammonium phosphate (DAP) significantly decreases H<sup>2</sup> S production and improves the kinetics of fermentation and aroma profile of wine [32]. In the last 5 years, VITEC has been studying the effect of ammonium sulfate and DAP addition on the amount of SO<sup>2</sup> produced by yeast along of the alcoholic fermentation. Results obtained showed that the addition of the N-sources slightly increases the total content of SO<sup>2</sup> in wines. The addition of ammonium sulfates and DAP using low sulfite-forming strains to ferment musts showed no significant differences. In the case of musts fermented by "high sulfite-forming" strains, the addition of DAP significantly increased the total content of SO<sup>2</sup> [33].

Other important consideration to elaborate SO2 -free wines is the management of the alcoholic fermentation. In this sense, it has been stated that temperature has several effects on biochemical and physiological properties in yeast cells. Some changes in the sulfur assimilation pathway by *S. cerevisiae* depending on temperature may occur [34]. Our results are in agreement with other authors, who reported that at low temperature, the SO2 production increases [26]. SO2 and H2 S production is also affected by pH (acidic pH facilitate SO2 uptake) and concentration of some minerals (copper and zinc) and vitamins, such as pantothenate or thiamine [9, 26, 35]. Thiamine is a vitamin used as a co-enzyme in the alcoholic fermentation pathway. It stimulates yeast growth, speeds up fermentation, and reduces production of SO2 binding compounds. Thiamine supplementation allows the transformation of pyruvic acid to acetaldehyde and limits the accumulation of ketonic compounds on wine being considered a factor to reduce the SO2 amount on wines [36]. A deficiency in thiamine may reduce yeast growth, slow fermentation, and promote the accumulation of pyruvic acid and acetaldehyde, the components responsible of wine oxidation. The effect of major SO<sup>2</sup> binding compounds (acetaldehyde, pyruvic, and α-ketoglutarate) on the production of SO<sup>2</sup> by different yeasts strains is still poorly understood, and more studies should be performed to better understand their role on the SO2 production [7]. In this way, the results obtained in VITEC are in agreement with the results obtained by Comuzzo and Zironi [33, 36], who showed that the addition of DAP + thiamine reduced the production of α-ketoglutarate.

#### **3. Physical technologies to replace the use of SO2 in the wine industry**

From a physical point of view, different technologies have been used to ensure the wine microbiological stability and to prevent oxidations [37]. The main advantage of using physical methods is the nonaddition of chemical substances that may affect human health. By these technologies, the preservation of the organoleptic properties of wines and the antimicrobial effect should be produced at the same time. Pulsed electric fields (PEF), ultraviolet radiation (UV), high hydrostatic pressure (HHP), and flash-pasteurization lead an antimicrobial result, while the use of ultrasounds (US) or inert gases does not share this property [38–41]. The PEF consists in the application of short electric pulses of high intensity between two electrodes, producing electroporation of the cell membranes increasing their permeability. It has been shown that this technique is effective to inactivate both bacteria and yeasts [42]. Thus, PEF may be applied to eliminate undesirable microorganisms at different winemaking stages, for example, before bottling. It has been stated that the treatments with PEF also reduces the activity of enzymes, such as polyphenol oxidases and peroxidases, increases the extraction of phenolic compounds and affects the aromas of white wines [42, 43]. VITEC has evaluated the antimicrobial effect of PEF, HHP, US, and EMR (electromagnetic radiation). **Figure 2** shows the obtained results after the quantification of viable yeasts and acetic acid bacteria (AAB) in Petri dishes culture. The PEF conditions were electric field 35 kV/cm, voltage 23 kV, pulse rate 0.65 kHz, pulse duration 2.5 μS, initial conductivity 5.04 mS/cm, flow 25 l/h, and initial temperature 20.8°C. The PEF 1 and PEF 2 differed on the final temperature of the treatment which was 23 and 31°C, respectively. Worthy results of PEF as antimicrobial technique were obtained, although high colony-forming units of yeast were observed in the case of PEF 1.

applied but also on the variety and the type of microorganisms (**Figure 2**). Tempranillo and Albariño yeast growth were inhibited by all pressures and times applied. However, in the case of acetic acid bacteria, the HHP treatment was very efficient for Tempranillo but not for Albariño wines. Even so, low levels of viable AAB (10<sup>2</sup> cfu/100 mL) were found. According to Bartowsky et al*.* [44], AAB populations from either spoiled or unspoiled wines ranged

**Figure 2.** Evaluation of different physical treatments in Tempranillo and Albariño wines (at the end of the alcoholic fermentation) by the quantification of viable yeasts and acetic acid bacteria in Petri dishes culture (cfu, colony forming

the polyphenol oxidase, although lower values of pressure are enough to inactivate yeasts and bacteria [45]. In our experiments, HHP results as a very effective technique against yeast and lactic acid bacteria and a lesser extent against AAB. At the studied conditions, HPP and PEF showed a noteworthy preservation of the organoleptic properties of wines (data not shown),

Other techniques, such as ultrasounds (US) and EMR, were also evaluated. The EMR is one of the most recent physical technologies evaluated in wines, which has shown a good potential in food processing, such as fruits, vegetables, and juices. This technique allows increasing the wine temperature for a short time period without any external heating source. EMR allows achieving the reduction of microorganisms with low effect on the organoleptic properties of wines, when compared with other heating techniques, such as flash pasteurization. However, recently studies have shown that the application of lower power microwave exposures may increase the growth of *Bretttanomyces* cells [48]. In agreement, **Figure 2** shows an increase on AAB after the treatment with EMR in both cases. The application of US at different conditions considering time of application (from 1 to 3 min)

and 103 cfu/mL. According to the literature, pressures above 700 MPa may inhibit

Microbiological, Physical, and Chemical Procedures to Elaborate High-Quality SO2-Free Wines

http://dx.doi.org/10.5772/intechopen.71627

177

between 102

units).

according to other authors [45–47].

The use of high hydrostatic pressures (HHP) was evaluated in our studies at different pressures (from 400 to 600 MPa) and times (1, 3, 5, and 10 min). HHP results showed that the inhibition of microorganism by this methodology depends not only on the time and pressure Microbiological, Physical, and Chemical Procedures to Elaborate High-Quality SO2-Free Wines http://dx.doi.org/10.5772/intechopen.71627 177

such as pantothenate or thiamine [9, 26, 35]. Thiamine is a vitamin used as a co-enzyme in the alcoholic fermentation pathway. It stimulates yeast growth, speeds up fermenta-

allows the transformation of pyruvic acid to acetaldehyde and limits the accumulation

wines [36]. A deficiency in thiamine may reduce yeast growth, slow fermentation, and promote the accumulation of pyruvic acid and acetaldehyde, the components responsible

understood, and more studies should be performed to better understand their role on the

From a physical point of view, different technologies have been used to ensure the wine microbiological stability and to prevent oxidations [37]. The main advantage of using physical methods is the nonaddition of chemical substances that may affect human health. By these technologies, the preservation of the organoleptic properties of wines and the antimicrobial effect should be produced at the same time. Pulsed electric fields (PEF), ultraviolet radiation (UV), high hydrostatic pressure (HHP), and flash-pasteurization lead an antimicrobial result, while the use of ultrasounds (US) or inert gases does not share this property [38–41]. The PEF consists in the application of short electric pulses of high intensity between two electrodes, producing electroporation of the cell membranes increasing their permeability. It has been shown that this technique is effective to inactivate both bacteria and yeasts [42]. Thus, PEF may be applied to eliminate undesirable microorganisms at different winemaking stages, for example, before bottling. It has been stated that the treatments with PEF also reduces the activity of enzymes, such as polyphenol oxidases and peroxidases, increases the extraction of phenolic compounds and affects the aromas of white wines [42, 43]. VITEC has evaluated the antimicrobial effect of PEF, HHP, US, and EMR (electromagnetic radiation). **Figure 2** shows the obtained results after the quantification of viable yeasts and acetic acid bacteria (AAB) in Petri dishes culture. The PEF conditions were electric field 35 kV/cm, voltage 23 kV, pulse rate 0.65 kHz, pulse duration 2.5 μS, initial conductivity 5.04 mS/cm, flow 25 l/h, and initial temperature 20.8°C. The PEF 1 and PEF 2 differed on the final temperature of the treatment which was 23 and 31°C, respectively. Worthy results of PEF as antimicrobial technique were obtained, although high colony-forming units of yeast were observed in the case of PEF 1.

The use of high hydrostatic pressures (HHP) was evaluated in our studies at different pressures (from 400 to 600 MPa) and times (1, 3, 5, and 10 min). HHP results showed that the inhibition of microorganism by this methodology depends not only on the time and pressure

 production [7]. In this way, the results obtained in VITEC are in agreement with the results obtained by Comuzzo and Zironi [33, 36], who showed that the addition of DAP +

of ketonic compounds on wine being considered a factor to reduce the SO2

binding compounds. Thiamine supplementation

binding compounds (acetaldehyde, pyruvic,

 **in the wine** 

by different yeasts strains is still poorly

amount on

tion, and reduces production of SO2

of wine oxidation. The effect of major SO<sup>2</sup>

SO2

**industry**

and α-ketoglutarate) on the production of SO<sup>2</sup>

thiamine reduced the production of α-ketoglutarate.

176 Grapes and Wines - Advances in Production, Processing, Analysis and Valorization

**3. Physical technologies to replace the use of SO2**

**Figure 2.** Evaluation of different physical treatments in Tempranillo and Albariño wines (at the end of the alcoholic fermentation) by the quantification of viable yeasts and acetic acid bacteria in Petri dishes culture (cfu, colony forming units).

applied but also on the variety and the type of microorganisms (**Figure 2**). Tempranillo and Albariño yeast growth were inhibited by all pressures and times applied. However, in the case of acetic acid bacteria, the HHP treatment was very efficient for Tempranillo but not for Albariño wines. Even so, low levels of viable AAB (10<sup>2</sup> cfu/100 mL) were found. According to Bartowsky et al*.* [44], AAB populations from either spoiled or unspoiled wines ranged between 102 and 103 cfu/mL. According to the literature, pressures above 700 MPa may inhibit the polyphenol oxidase, although lower values of pressure are enough to inactivate yeasts and bacteria [45]. In our experiments, HHP results as a very effective technique against yeast and lactic acid bacteria and a lesser extent against AAB. At the studied conditions, HPP and PEF showed a noteworthy preservation of the organoleptic properties of wines (data not shown), according to other authors [45–47].

Other techniques, such as ultrasounds (US) and EMR, were also evaluated. The EMR is one of the most recent physical technologies evaluated in wines, which has shown a good potential in food processing, such as fruits, vegetables, and juices. This technique allows increasing the wine temperature for a short time period without any external heating source. EMR allows achieving the reduction of microorganisms with low effect on the organoleptic properties of wines, when compared with other heating techniques, such as flash pasteurization. However, recently studies have shown that the application of lower power microwave exposures may increase the growth of *Bretttanomyces* cells [48]. In agreement, **Figure 2** shows an increase on AAB after the treatment with EMR in both cases. The application of US at different conditions considering time of application (from 1 to 3 min) and wavelengths (12, 43 and 75 μm) inhibited the yeasts growth but not the bacteria population (**Figure 2**). The effectiveness of US resulted lower than HHP, at least at the experimental conditions studied. As occurred with EMR treatment, an increase on the colony-forming units was observed after the treatment with US. Ultraviolet radiation reduces the population of wine microorganisms, but different resistances to the radiation have been stated depending on species. It appears to be an effective method against *Brettanomyces*, *Saccharomyces*, *Acetobacter*, *Lactobacillus*, and *Pediococcus* [46]. Furthermore, it has been described that phenolic compounds can absorb UV radiation and is therefore less effective in red wines. This technique seems to be more effective in white wines at the end of fermentation, when wines present low turbidity. In order to increase the total polyphenol, it could be also applied at maceration stage [38, 49].

In general, all the physical treatments assessed clearly affect the viability of lactic acid bacteria in Tempranillo and Albariño varieties. In both cases, only viable lactic acid bacteria were detected in the control (data not shown). The employed treatments reduced the viability of yeasts and lactic and acetic acid bacteria. However, in this study, both US and EMR were not effective enough to reduce the population of viable acetic acid bacteria. According to the results, AAB were more resistant to the treatments than lactic acid bacteria (LAB). Regarding techniques, a higher antimicrobial effect of HHP and EMR was observed in comparison to the other methodologies employed. Besides, some wines produced by US and EMR showed oxidation characteristics. As occurred in the antimicrobial assays, the optimization of methods and experimental conditions is an imperative action to avoid adverse effects on the sensory quality of wines. It should be noted that some of these physical techniques are commonly used in food industry, but their implementation on the wine sector is so far to be available for a daily work routine, mainly due to economic and technique questions.

because it is a very oxidative process. To ensure a correct conservation of the SO<sup>2</sup>

). \* Significant differences by HSD Tukey test (*p* < 0.05).

experience, between 0.5 and 1.5 mg/L of dissolved O2

and sulfur dioxide (SO2

wines, the amount of oxygen incorporated into wine should be controlled, especially at bottling, where concentrations from 0.2 to 4 mg/L may be incorporated, depending on conditions [51]. The amount of oxygen incorporated at bottling is the sum of the dissolved oxygen and the headspace oxygen, which is called TPO (total packaged oxygen). By our

**Figure 3.** Comparison of the sensory evaluation of Tempranillo wines elaborated using argon (Ar), carbon dioxide (CO<sup>2</sup>

Microbiological, Physical, and Chemical Procedures to Elaborate High-Quality SO2-Free Wines

cess. Moreover, the oxygen in the headspace changes depending on the type of closure. In submerged caps, the headspace height is commonly 1–2 cm, and the normal values of dissolved oxygen ranged from 0.5 mg/L (with the use of inert gases) to 2 mg/L (without inertization). In the case of screw caps, the headspace height is higher, about 4 to 6 cm, and the oxygen values ranged from 2 to 6 mg/L. In summary, in submerged caps, values of TPO around 1 or 2 mg/L could be optimum, but values over 3 mg/L are not suitable. In screw caps, TPO values around 2.5 mg/L are optimum, but values over 7 mg/L are not suitable. The type of caps employed not only changes the amount of oxygen incorporated at bottling but also is the ultimate barrier physic to protect wines during the storage period. Thus, a correct cap should be selected depending on the type of wine, and also its permeability to oxygen should be measured to estimate the optimum storage period. The measure of the oxygen transmission rate (OTR) helps to carried out these purposes. **Figure 4** shows "high" and "low" oxygen permeability of different types of caps measured in VITEC by the MOCON® equipment. The OTR measurement corresponds to two natural corks stoppers. As can be seen in the figure, the cork stopper represented in green reached the stability of the oxygen permeability at 24 h, while the stopper represented in red did not reach this stability until the third day. Moreover, once reached the stability, the values of OTR


)

179

is usually incorporated at this pro-

http://dx.doi.org/10.5772/intechopen.71627

The oxidation is one of the main processes that affect SO<sup>2</sup> -free wines. Apart from the mentioned technologies and despite of its antimicrobial effect is limited, the use of inert gases is more and more applied throughout the winemaking process. The oxygen control by the management of the inert gases during the winemaking process must be considered because they have an important impact on the organoleptic properties. Caps are the ultimate physical barrier to preserve wines during storage, and so their oxygen permeability should be considered. The long-term protection is one of the most concerns for wineries in bottled wines with reduced SO2 content [50]. The assays carried out in VITEC using argon and carbon dioxide showed valuable sensory results (**Figure 3**). The SO<sup>2</sup> -free red wines produced by the use of Ar and CO<sup>2</sup> showed higher significant color intensity, tannic intensity, and dryness. Greater aroma intensity and mouthfeel were also found, although values did not show significant differences. In general, Tempranillo-bottled SO<sup>2</sup> -free wines obtained higher global punctuations than wines with SO2 addition.

The oxygen control during all the production process of this type of wines is an imperative engagement. It is important to take into account that wines without sulfite addition are exposed to physicochemical and microbiological alterations. Considering the techniques available in any winery, to avoid microbiological alterations, sterilizing filtration may be an alternative. However, this technique could reduce the sensorial quality of the wine Microbiological, Physical, and Chemical Procedures to Elaborate High-Quality SO2-Free Wines http://dx.doi.org/10.5772/intechopen.71627 179

and wavelengths (12, 43 and 75 μm) inhibited the yeasts growth but not the bacteria population (**Figure 2**). The effectiveness of US resulted lower than HHP, at least at the experimental conditions studied. As occurred with EMR treatment, an increase on the colony-forming units was observed after the treatment with US. Ultraviolet radiation reduces the population of wine microorganisms, but different resistances to the radiation have been stated depending on species. It appears to be an effective method against *Brettanomyces*, *Saccharomyces*, *Acetobacter*, *Lactobacillus*, and *Pediococcus* [46]. Furthermore, it has been described that phenolic compounds can absorb UV radiation and is therefore less effective in red wines. This technique seems to be more effective in white wines at the end of fermentation, when wines present low turbidity. In order to increase the total polyphenol, it could be also applied at

In general, all the physical treatments assessed clearly affect the viability of lactic acid bacteria in Tempranillo and Albariño varieties. In both cases, only viable lactic acid bacteria were detected in the control (data not shown). The employed treatments reduced the viability of yeasts and lactic and acetic acid bacteria. However, in this study, both US and EMR were not effective enough to reduce the population of viable acetic acid bacteria. According to the results, AAB were more resistant to the treatments than lactic acid bacteria (LAB). Regarding techniques, a higher antimicrobial effect of HHP and EMR was observed in comparison to the other methodologies employed. Besides, some wines produced by US and EMR showed oxidation characteristics. As occurred in the antimicrobial assays, the optimization of methods and experimental conditions is an imperative action to avoid adverse effects on the sensory quality of wines. It should be noted that some of these physical techniques are commonly used in food industry, but their implementation on the wine sector is so far to be available for a daily work routine, mainly due to economic and

tioned technologies and despite of its antimicrobial effect is limited, the use of inert gases is more and more applied throughout the winemaking process. The oxygen control by the management of the inert gases during the winemaking process must be considered because they have an important impact on the organoleptic properties. Caps are the ultimate physical barrier to preserve wines during storage, and so their oxygen permeability should be considered. The long-term protection is one of the most concerns for wineries in bottled wines with

aroma intensity and mouthfeel were also found, although values did not show significant dif-

The oxygen control during all the production process of this type of wines is an imperative engagement. It is important to take into account that wines without sulfite addition are exposed to physicochemical and microbiological alterations. Considering the techniques available in any winery, to avoid microbiological alterations, sterilizing filtration may be an alternative. However, this technique could reduce the sensorial quality of the wine

content [50]. The assays carried out in VITEC using argon and carbon dioxide

showed higher significant color intensity, tannic intensity, and dryness. Greater




maceration stage [38, 49].

technique questions.

reduced SO2

Ar and CO<sup>2</sup>

than wines with SO2

The oxidation is one of the main processes that affect SO<sup>2</sup>

178 Grapes and Wines - Advances in Production, Processing, Analysis and Valorization

showed valuable sensory results (**Figure 3**). The SO<sup>2</sup>

ferences. In general, Tempranillo-bottled SO<sup>2</sup>

addition.

**Figure 3.** Comparison of the sensory evaluation of Tempranillo wines elaborated using argon (Ar), carbon dioxide (CO<sup>2</sup> ) and sulfur dioxide (SO2 ). \* Significant differences by HSD Tukey test (*p* < 0.05).

because it is a very oxidative process. To ensure a correct conservation of the SO<sup>2</sup> -free wines, the amount of oxygen incorporated into wine should be controlled, especially at bottling, where concentrations from 0.2 to 4 mg/L may be incorporated, depending on conditions [51]. The amount of oxygen incorporated at bottling is the sum of the dissolved oxygen and the headspace oxygen, which is called TPO (total packaged oxygen). By our experience, between 0.5 and 1.5 mg/L of dissolved O2 is usually incorporated at this process. Moreover, the oxygen in the headspace changes depending on the type of closure. In submerged caps, the headspace height is commonly 1–2 cm, and the normal values of dissolved oxygen ranged from 0.5 mg/L (with the use of inert gases) to 2 mg/L (without inertization). In the case of screw caps, the headspace height is higher, about 4 to 6 cm, and the oxygen values ranged from 2 to 6 mg/L. In summary, in submerged caps, values of TPO around 1 or 2 mg/L could be optimum, but values over 3 mg/L are not suitable. In screw caps, TPO values around 2.5 mg/L are optimum, but values over 7 mg/L are not suitable. The type of caps employed not only changes the amount of oxygen incorporated at bottling but also is the ultimate barrier physic to protect wines during the storage period. Thus, a correct cap should be selected depending on the type of wine, and also its permeability to oxygen should be measured to estimate the optimum storage period. The measure of the oxygen transmission rate (OTR) helps to carried out these purposes. **Figure 4** shows "high" and "low" oxygen permeability of different types of caps measured in VITEC by the MOCON® equipment. The OTR measurement corresponds to two natural corks stoppers. As can be seen in the figure, the cork stopper represented in green reached the stability of the oxygen permeability at 24 h, while the stopper represented in red did not reach this stability until the third day. Moreover, once reached the stability, the values of OTR

Chitosan is a natural polymer formed by deacetylation of chitin, which has a wide range of applications in different field research, such as agriculture, food, and pharmaceutical industry, among others [59]. The use of this polysaccharide in oenology was approved in 2009 by the OIV to fining musts (OIV-OENO 336A-2009). Moreover, it also used as antimicrobial and antioxidant. Chitosan allows the growth of *Saccharomyces* strains but is an antimicrobial against *Brettanomyces*, acetic, and lactic acid bacteria [60–63]. Commonly, it is used to preserve wine from oxidation and also as fining agent for white wine protein stabilization [64, 65]. **Figure 5** shows the potential of chitosan as antimicrobial. In this case, a significant decrease on yeasts, LAB, and AAB after the addition of 10 g/hL of chitosan to Tempranillo wines (after alcoholic fermentation) was observed. This effectiveness was

Microbiological, Physical, and Chemical Procedures to Elaborate High-Quality SO2-Free Wines

http://dx.doi.org/10.5772/intechopen.71627

181

cfu/100 mL.

Dimethyl dicarbonate (DMDC) was also accepted by European Union to be used in wine with a maximum limit amount of 200 mg/L (Regulation (EC) No 643/2006). DMDC is an organic chemical compound, which acts inhibiting the growth of microorganisms [9, 66]. When it is added to wines, it is quickly transformed to methanol and produces certain content on methyl and alkyl carbonates as products reaction by polyphenols or organic acids. These products are usually found at a low concentration, and so the quality of wine, flavors and aromas, should not be affected [67]. DMDC seems to be more effective against yeasts than against

**Figure 5.** Viable yeasts, lactic acid bacteria (LAB), and acetic acid bacteria (AAB) quantified in Petri dishes culture (cfu; colony-forming units) from Tempranillo wines before and after a treatment with chitosan (10 g/hL). \*Significant

greater for yeasts, decreasing up to 1 × 104

differences by HSD Tukey test (*p* < 0.05).

**Figure 4.** Representative oxygen transmission rate (OTR) of caps with different oxygen permeability.

were 4 times higher for "red" stopper than for "green". It can be also observed a great decrease in the case of the "red" stopper, likely due to higher content of oxygen inside of the cork and therefore higher porosity.

#### **4. Chemical treatments to elaborate SO2 -free wines**

The addition of chemical substances to wines is the most used alternative to reduce the SO<sup>2</sup> addition in wines. Over the years, the addition of several chemical substances has been allowed by the OIV with different purposes. Accordingly, new antioxidant and antimicrobial additives have been evaluated as possible alternatives to the use of the SO2 [37, 52]. Particularly, the addition of dry yeasts enriched in glutathione, chitosan, and dimethyl dicarbonate, and different hydrolyzed and condensed tannins were evaluated by our research group. The most relevant results and some considerations related to these practices are summarized below.

In the last years, the potential application of glutathione (GSH) has increased the attention of many winemakers and researchers. The addition of reduced glutathione to grape juices or wines is allowed by OIV up to 20 mg/L (OIV OENO 445/2015). The use of GSH in the wine production was reviewed in 2013 by several authors [36, 53]. Following studies also demonstrated that the combination of SO2 and GSH involves a notable protective effect in wines [54]. Recent studies have shown that the addition of glutathione-rich dry inactivated yeast to grape juices modifies the white wine aroma influencing the concentrations of some volatile compounds and precursors with some benefits on its preservation [55–57]. The GSH amount of wine changes depending on the winemaking period. Hence, this compound decreases after wine aging and storage; at pressing could increase its content up to 20 times [58].

Chitosan is a natural polymer formed by deacetylation of chitin, which has a wide range of applications in different field research, such as agriculture, food, and pharmaceutical industry, among others [59]. The use of this polysaccharide in oenology was approved in 2009 by the OIV to fining musts (OIV-OENO 336A-2009). Moreover, it also used as antimicrobial and antioxidant. Chitosan allows the growth of *Saccharomyces* strains but is an antimicrobial against *Brettanomyces*, acetic, and lactic acid bacteria [60–63]. Commonly, it is used to preserve wine from oxidation and also as fining agent for white wine protein stabilization [64, 65]. **Figure 5** shows the potential of chitosan as antimicrobial. In this case, a significant decrease on yeasts, LAB, and AAB after the addition of 10 g/hL of chitosan to Tempranillo wines (after alcoholic fermentation) was observed. This effectiveness was greater for yeasts, decreasing up to 1 × 104 cfu/100 mL.

Dimethyl dicarbonate (DMDC) was also accepted by European Union to be used in wine with a maximum limit amount of 200 mg/L (Regulation (EC) No 643/2006). DMDC is an organic chemical compound, which acts inhibiting the growth of microorganisms [9, 66]. When it is added to wines, it is quickly transformed to methanol and produces certain content on methyl and alkyl carbonates as products reaction by polyphenols or organic acids. These products are usually found at a low concentration, and so the quality of wine, flavors and aromas, should not be affected [67]. DMDC seems to be more effective against yeasts than against

were 4 times higher for "red" stopper than for "green". It can be also observed a great decrease in the case of the "red" stopper, likely due to higher content of oxygen inside of

**Figure 4.** Representative oxygen transmission rate (OTR) of caps with different oxygen permeability.

The addition of chemical substances to wines is the most used alternative to reduce the SO<sup>2</sup> addition in wines. Over the years, the addition of several chemical substances has been allowed by the OIV with different purposes. Accordingly, new antioxidant and antimicrobial addi-

the addition of dry yeasts enriched in glutathione, chitosan, and dimethyl dicarbonate, and different hydrolyzed and condensed tannins were evaluated by our research group. The most relevant results and some considerations related to these practices are summarized below.

In the last years, the potential application of glutathione (GSH) has increased the attention of many winemakers and researchers. The addition of reduced glutathione to grape juices or wines is allowed by OIV up to 20 mg/L (OIV OENO 445/2015). The use of GSH in the wine production was reviewed in 2013 by several authors [36, 53]. Following studies also demon-

[54]. Recent studies have shown that the addition of glutathione-rich dry inactivated yeast to grape juices modifies the white wine aroma influencing the concentrations of some volatile compounds and precursors with some benefits on its preservation [55–57]. The GSH amount of wine changes depending on the winemaking period. Hence, this compound decreases after

wine aging and storage; at pressing could increase its content up to 20 times [58].

tives have been evaluated as possible alternatives to the use of the SO2

**-free wines**

and GSH involves a notable protective effect in wines

[37, 52]. Particularly,

the cork and therefore higher porosity.

strated that the combination of SO2

**4. Chemical treatments to elaborate SO2**

180 Grapes and Wines - Advances in Production, Processing, Analysis and Valorization

**Figure 5.** Viable yeasts, lactic acid bacteria (LAB), and acetic acid bacteria (AAB) quantified in Petri dishes culture (cfu; colony-forming units) from Tempranillo wines before and after a treatment with chitosan (10 g/hL). \*Significant differences by HSD Tukey test (*p* < 0.05).

**Figure 6.** Viable yeasts, lactic acid bacteria (LAB), and acetic acid bacteria (AAB) quantified in Petri dishes culture (cfu, colony-forming units) from Albariño musts treated with dimethyl dicarbonate (DMDC = 20 g/hL). \*Significant differences by HSD Tukey test (*p* < 0.05).

studies carried out in VITEC have recently shown that the addition of tannins mixed with

**Figure 7.** Sensory profile of Tempranillo wines elaborated by different enological tannin additions to grape juices. SO<sup>2</sup>

Wine control. ST: Grape seed tannins (40 g/hL), SKT: Grape skin tannins (30 g/hL), GAL: Tara tannin (20 g/hL), OAK: oak

Microbiological, Physical, and Chemical Procedures to Elaborate High-Quality SO2-Free Wines

analysis of Tempranillo wines with addition of grape seed tannins (ST), grape skin tannins (SKT), oak tannins (OAK), and tara tannins (GAL). In general, the sensory profiles of wines produced with the addition of different tannins were similar (and even better) than wines elaborated by addition of SO2. Significant higher color intensity was observed between control and treated wines. Treated wines also obtained significant dryness and tannic intensity. Astringency and mouthfeel reached higher values but not significant. Lower persistence and higher aroma intensity can also be observed. Low differences between treatments were found, which may be due not only to the different quantity of tannins added but also to their qualitative profile. Recent studies performed by other authors have confirmed the importance of the anthocyanin/tannin ratio on the wine oxidation process and especially on the acetaldehyde formation. Wines with higher tannin addition showed lower production of

Other chemical substances, such as ascorbic acid and lysozyme, may also be able alterna-

of phenolic compounds occurs. It is a highly efficient antioxidant in combination with sulfur

acid is low [76]. The reaction between ascorbic acid and oxygen results in dehydroascorbic acid and hydrogen peroxide, which would be removed by sulfites. Under certain condi-

ratio [4]. In wines, it is generally employed in winemaking stages with high oxygen dissolution, such as grape crushing, after racking or just before bottling. The addition of ascorbic

dioxide; nonetheless, a pro-oxidation effect may occur when the content of SO<sup>2</sup>

tions, ascorbic acid both accelerates oxygen removal and reduces the O2

. Ascorbic acid has the ability to scavenge molecular oxygen before the oxidation

[74]. **Figure 7** shows the sensory

http://dx.doi.org/10.5772/intechopen.71627

:

183

and ascorbic

molar reaction

:SO2

glutathione may be an effective alternative to the use of SO<sup>2</sup>

tannins (30 g/hL). \*Significant differences by HSD Tukey test (*p* < 0.05).

acetaldehyde [75].

tives to SO2

bacteria, although its activity depends on several factors, such as the pH [66–68]. In this sense, **Figure 6** shows the results obtained by the addition of DMDC to Albariño musts. The abovementioned antimicrobial effect can be observed in yeast, LAB, and AAB. However and as occurred with chitosan, DMDC treatment was clearly more effective in yeasts than in bacteria.

The addition of oenological tannins to wine is an accepted practice by the OIV (OENO 12/2002 and revisions OENO 5/2008, OENO 6/2008, OENO 352/2009, and OENO 554/2015), which mainly aims the color stabilization and the improvement of the wine mouthfeel and flavor. Quite a few studies have evaluated the influence of the tannin addition on the chemical and sensory properties of wines. However, the results obtained are not as promising as expected. In 2005, Bautista-Ortiz et al. [69] did not observe any improvement on the chromatic and sensory properties of wines treated with different oenological tannins. Harbertson and co-workers [70] observed that some additions may be unjustified and have limited or negative impacts on the wine quality. A wide range of commercial tannins exists on the market; nonetheless, a lack of information about the composition and origin of the product is a common pattern. This fact could lead to technological problems according to the expected final wine [71]. The antioxidant properties of tannins, with related health beneficial effects, and their benefits when added to wines are also well known [72]. Both characteristics make tannins a very attractive alternative to the use of SO<sup>2</sup> in wine. Some studies showed hopeful results when mixed with antimicrobials, such as lysozyme [17, 73]. The Microbiological, Physical, and Chemical Procedures to Elaborate High-Quality SO2-Free Wines http://dx.doi.org/10.5772/intechopen.71627 183

**Figure 7.** Sensory profile of Tempranillo wines elaborated by different enological tannin additions to grape juices. SO<sup>2</sup> : Wine control. ST: Grape seed tannins (40 g/hL), SKT: Grape skin tannins (30 g/hL), GAL: Tara tannin (20 g/hL), OAK: oak tannins (30 g/hL). \*Significant differences by HSD Tukey test (*p* < 0.05).

studies carried out in VITEC have recently shown that the addition of tannins mixed with glutathione may be an effective alternative to the use of SO<sup>2</sup> [74]. **Figure 7** shows the sensory analysis of Tempranillo wines with addition of grape seed tannins (ST), grape skin tannins (SKT), oak tannins (OAK), and tara tannins (GAL). In general, the sensory profiles of wines produced with the addition of different tannins were similar (and even better) than wines elaborated by addition of SO2. Significant higher color intensity was observed between control and treated wines. Treated wines also obtained significant dryness and tannic intensity. Astringency and mouthfeel reached higher values but not significant. Lower persistence and higher aroma intensity can also be observed. Low differences between treatments were found, which may be due not only to the different quantity of tannins added but also to their qualitative profile. Recent studies performed by other authors have confirmed the importance of the anthocyanin/tannin ratio on the wine oxidation process and especially on the acetaldehyde formation. Wines with higher tannin addition showed lower production of acetaldehyde [75].

bacteria, although its activity depends on several factors, such as the pH [66–68]. In this sense, **Figure 6** shows the results obtained by the addition of DMDC to Albariño musts. The abovementioned antimicrobial effect can be observed in yeast, LAB, and AAB. However and as occurred with chitosan, DMDC treatment was clearly more effective in yeasts than in bacteria. The addition of oenological tannins to wine is an accepted practice by the OIV (OENO 12/2002 and revisions OENO 5/2008, OENO 6/2008, OENO 352/2009, and OENO 554/2015), which mainly aims the color stabilization and the improvement of the wine mouthfeel and flavor. Quite a few studies have evaluated the influence of the tannin addition on the chemical and sensory properties of wines. However, the results obtained are not as promising as expected. In 2005, Bautista-Ortiz et al. [69] did not observe any improvement on the chromatic and sensory properties of wines treated with different oenological tannins. Harbertson and co-workers [70] observed that some additions may be unjustified and have limited or negative impacts on the wine quality. A wide range of commercial tannins exists on the market; nonetheless, a lack of information about the composition and origin of the product is a common pattern. This fact could lead to technological problems according to the expected final wine [71]. The antioxidant properties of tannins, with related health beneficial effects, and their benefits when added to wines are also well known [72]. Both char-

**Figure 6.** Viable yeasts, lactic acid bacteria (LAB), and acetic acid bacteria (AAB) quantified in Petri dishes culture (cfu, colony-forming units) from Albariño musts treated with dimethyl dicarbonate (DMDC = 20 g/hL). \*Significant

showed hopeful results when mixed with antimicrobials, such as lysozyme [17, 73]. The

in wine. Some studies

acteristics make tannins a very attractive alternative to the use of SO<sup>2</sup>

differences by HSD Tukey test (*p* < 0.05).

182 Grapes and Wines - Advances in Production, Processing, Analysis and Valorization

Other chemical substances, such as ascorbic acid and lysozyme, may also be able alternatives to SO2 . Ascorbic acid has the ability to scavenge molecular oxygen before the oxidation of phenolic compounds occurs. It is a highly efficient antioxidant in combination with sulfur dioxide; nonetheless, a pro-oxidation effect may occur when the content of SO<sup>2</sup> and ascorbic acid is low [76]. The reaction between ascorbic acid and oxygen results in dehydroascorbic acid and hydrogen peroxide, which would be removed by sulfites. Under certain conditions, ascorbic acid both accelerates oxygen removal and reduces the O2 :SO2 molar reaction ratio [4]. In wines, it is generally employed in winemaking stages with high oxygen dissolution, such as grape crushing, after racking or just before bottling. The addition of ascorbic acid in white wines improves color and flavor retention during bottling aging [77]. Certain carbonyl compounds, such as furfural, acetaldehyde, glyoxal, and diacetyl, formed from the oxidation of ascorbic acid may involve the formation of brown pigments by reacting with phenolic compounds. Higher browning was observed in catechin model solutions containing ascorbic acid than in model solutions containing sulfite [78]. These oxidation products of ascorbic acid bind to SO2 reducing in some extent the ratio between free and total SO2 content [76]. The mixture of ascorbic acid together with SO<sup>2</sup> seems to be a better antioxidant combination than the use of SO2 alone, avoiding the oxidation of wine and preserving the aroma profile. In white wines, ascorbic acid provides considerable protection against oxidation under conditions of low oxygen [79]. However, it should be highlighted that the impact of the addition of ascorbic acid to wine composition and sensory characters is far to be clarified [36, 77].

indigenous yeasts strains can be used with this purpose. However, factors as grape juice composition, the management of the fermentation, and musts supplementation will be decisive. Different physical technologies and methodologies can be used to elaborate this type of wines. The replacement of the antioxidant and antimicrobial action of the SO<sup>2</sup>

a complex mission. However, the combination of different physical techniques together with a good management of inert gases to control oxygen appears to be a suitable practice to achieve this purpose. In addition, some chemical treatments will help to complete the effects caused by these practices. In general, chemical treatments should be combined at different wine production stages to complete their respective actions. The combination of

should be noted that still today, there is a lack on the knowledge of the microbiological

new additives, especially regarding the wine stability after storage and the effects on the

In summary, multidisciplinary approaches should be considered to elaborate high-quality

eral physical and chemical treatments have shown their antioxidant and antimicrobial effect.

research should be done to adapt winemaking procedures according to the particular work-

Thanks are due to the Spanish MICINN for their financial support of VINNO\_SO<sup>2</sup>

Raúl Ferrer-Gallego\*, Miquel Puxeu, Laura Martín, Enric Nart, Claudio Hidalgo and

(Ref. IPT-2012-0967-060000). The authors also thank AGROVIN S.A. for supplying the yeast strains, Bodegas RODA S.A. (Haro, La Rioja, Spain) and Adegas Valmiñor S.L. (O Rosal, Pontevedra) for supplying the grape samples. We also thank Programa de Desenvolupament



Microbiological, Physical, and Chemical Procedures to Elaborate High-Quality SO2-Free Wines

may help to reduce its use during the winemaking. It

during alcoholic fermentations (<10 mg/L), and sev-

amount in wine production may be achieved. Nonetheless, more

in wines as well as the use of

http://dx.doi.org/10.5772/intechopen.71627

chemical additions even with SO2

are able to generate low quantities of SO2

ing conditions and the desired product of each winery.

Rural de Catalunya 2014–2020 (N° expdte. 56 30032 2017 2A).

\*Address all correspondence to: raul.ferrer@vitec.cat VITEC, Wine Technology Center, Tarragona, Spain

Therefore, reducing the SO<sup>2</sup>

**Acknowledgements**

**Author details**

Imma Andorrà

better understand the effect of the low concentration of SO<sup>2</sup>

stability of SO2

human health.

SO2

is

185

Project

Lysozyme belongs to glycoside hydrolases, which is a type of enzyme that catalyzes the hydrolysis of bonds between N-acetyl muramic acid and N-acetyl-D-glucosamine residues in peptidoglycans, and it is found in the cell walls of bacteria, especially in Gram-positive bacteria. These enzymes are therefore destructive to many bacteria like lactic acid bacteria (LAB). In winemaking, indigenous LAB, such as *Lactobacillus brevis*, *Oenococcus oeni, Lactobacillus kunkeei*, *Pediococcus parvulus* and *Pediococcus damnosus,* can be completely inhibited by lysozyme, being this efficacy strongly affected by winemaking and dosage [80, 81]. The addition of lysozyme did not have any negative effect on yeast growth and sugar reduction and may prevent the increase of volatile acidity during the stuck/sluggish of the alcoholic fermentation [17, 81]. This substance had little or no effect on the content of alcohol, titratable acidity, and pH value and did not cause important changes on the sensory characteristics of wines. Nonetheless, it may produce esters in certain wines, contributing to their complexity [73, 82]. Lysozyme may involve changes on yeast nitrogen consumption and the amino nitrogen metabolism, although it does not appear to have an effect on the formation of biogenic amines [16]. The addition of lysozyme may produce a color loss associate with the formation of precipitates in red wines and may induce protein haze in white wines [82]. Lysozyme does not possess an antioxidant activity and therefore does not prevent the wine oxidation. Hence, it becomes necessary the addition of antioxidants, such as proanthocyanidins, in combination with lysozyme to replace the SO2 actions [16, 73]. A critical point of lysozyme is the safety of wines treated with this additive, since it is an egg allergen (allergen Gal d 4 according to the International Allergen Code) that remains in bottled wine. The OIV issued limitation of 500 mg/L [83], and this quantity is removed by an efficient fining treatment using, for example, bentonite or metatartaric acid [84].

#### **5. Conclusions**

The use of yeast strains with a low capacity to produce SO<sup>2</sup> , during the alcoholic fermentation is essential to reduce the final amount of SO<sup>2</sup> in wines. Both commercial and indigenous yeasts strains can be used with this purpose. However, factors as grape juice composition, the management of the fermentation, and musts supplementation will be decisive. Different physical technologies and methodologies can be used to elaborate this type of wines. The replacement of the antioxidant and antimicrobial action of the SO<sup>2</sup> is a complex mission. However, the combination of different physical techniques together with a good management of inert gases to control oxygen appears to be a suitable practice to achieve this purpose. In addition, some chemical treatments will help to complete the effects caused by these practices. In general, chemical treatments should be combined at different wine production stages to complete their respective actions. The combination of chemical additions even with SO2 may help to reduce its use during the winemaking. It should be noted that still today, there is a lack on the knowledge of the microbiological stability of SO2 -free wines during the aging period. Therefore, more research is needed to better understand the effect of the low concentration of SO<sup>2</sup> in wines as well as the use of new additives, especially regarding the wine stability after storage and the effects on the human health.

In summary, multidisciplinary approaches should be considered to elaborate high-quality SO2 -free wines. The combination of microbiological strategies, physical methods, and chemical treatments becomes indispensable to achieve this ambitious purpose. Several yeast strains are able to generate low quantities of SO2 during alcoholic fermentations (<10 mg/L), and several physical and chemical treatments have shown their antioxidant and antimicrobial effect. Therefore, reducing the SO<sup>2</sup> amount in wine production may be achieved. Nonetheless, more research should be done to adapt winemaking procedures according to the particular working conditions and the desired product of each winery.

## **Acknowledgements**

acid in white wines improves color and flavor retention during bottling aging [77]. Certain carbonyl compounds, such as furfural, acetaldehyde, glyoxal, and diacetyl, formed from the oxidation of ascorbic acid may involve the formation of brown pigments by reacting with phenolic compounds. Higher browning was observed in catechin model solutions containing ascorbic acid than in model solutions containing sulfite [78]. These oxidation products

the aroma profile. In white wines, ascorbic acid provides considerable protection against oxidation under conditions of low oxygen [79]. However, it should be highlighted that the impact of the addition of ascorbic acid to wine composition and sensory characters is far to

Lysozyme belongs to glycoside hydrolases, which is a type of enzyme that catalyzes the hydrolysis of bonds between N-acetyl muramic acid and N-acetyl-D-glucosamine residues in peptidoglycans, and it is found in the cell walls of bacteria, especially in Gram-positive bacteria. These enzymes are therefore destructive to many bacteria like lactic acid bacteria (LAB). In winemaking, indigenous LAB, such as *Lactobacillus brevis*, *Oenococcus oeni, Lactobacillus kunkeei*, *Pediococcus parvulus* and *Pediococcus damnosus,* can be completely inhibited by lysozyme, being this efficacy strongly affected by winemaking and dosage [80, 81]. The addition of lysozyme did not have any negative effect on yeast growth and sugar reduction and may prevent the increase of volatile acidity during the stuck/sluggish of the alcoholic fermentation [17, 81]. This substance had little or no effect on the content of alcohol, titratable acidity, and pH value and did not cause important changes on the sensory characteristics of wines. Nonetheless, it may produce esters in certain wines, contributing to their complexity [73, 82]. Lysozyme may involve changes on yeast nitrogen consumption and the amino nitrogen metabolism, although it does not appear to have an effect on the formation of biogenic amines [16]. The addition of lysozyme may produce a color loss associate with the formation of precipitates in red wines and may induce protein haze in white wines [82]. Lysozyme does not possess an antioxidant activity and therefore does not prevent the wine oxidation. Hence, it becomes necessary the addition of antioxidants, such as proanthocyanidins, in combination with

wines treated with this additive, since it is an egg allergen (allergen Gal d 4 according to the International Allergen Code) that remains in bottled wine. The OIV issued limitation of 500 mg/L [83], and this quantity is removed by an efficient fining treatment using, for

reducing in some extent the ratio between free and total SO2

actions [16, 73]. A critical point of lysozyme is the safety of

alone, avoiding the oxidation of wine and preserving

seems to be a better antioxi-

, during the alcoholic fer-

in wines. Both commercial and

of ascorbic acid bind to SO2

lysozyme to replace the SO2

**5. Conclusions**

example, bentonite or metatartaric acid [84].

The use of yeast strains with a low capacity to produce SO<sup>2</sup>

mentation is essential to reduce the final amount of SO<sup>2</sup>

be clarified [36, 77].

dant combination than the use of SO2

content [76]. The mixture of ascorbic acid together with SO<sup>2</sup>

184 Grapes and Wines - Advances in Production, Processing, Analysis and Valorization

Thanks are due to the Spanish MICINN for their financial support of VINNO\_SO<sup>2</sup> Project (Ref. IPT-2012-0967-060000). The authors also thank AGROVIN S.A. for supplying the yeast strains, Bodegas RODA S.A. (Haro, La Rioja, Spain) and Adegas Valmiñor S.L. (O Rosal, Pontevedra) for supplying the grape samples. We also thank Programa de Desenvolupament Rural de Catalunya 2014–2020 (N° expdte. 56 30032 2017 2A).

## **Author details**

Raúl Ferrer-Gallego\*, Miquel Puxeu, Laura Martín, Enric Nart, Claudio Hidalgo and Imma Andorrà

\*Address all correspondence to: raul.ferrer@vitec.cat

VITEC, Wine Technology Center, Tarragona, Spain

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**Chapter 10**

Provisional chapter

**Influence of Wine Chemical Compounds on the**

Influence of Wine Chemical Compounds on the Foaming

DOI: 10.5772/intechopen.70859

The foam of a sparkling wine is a key parameter of its quality, and the main characteristic differentiating sparkling wines from the so-called still wines. Both foam formation and duration are directly related to the chemical composition of sparkling wines. This chapter reviews the most recent studies made to determine the influence of chemical compounds on the foamability and foam stability of sparkling wines. Foam properties of sparkling wines are ruled by a large number of molecules, but some compounds seem to be more relevant than others to explain their behavior. The content of total amino acids, polysaccharides, anthocyanins, coumaric acid, and isorhamnetin showed high correlation values with foam quality parameters. The alcohol content and the concentration of acid polysaccharides, proanthocyanidins and free SO2 are the factors which most negatively affect foam quality. A recent study, by means of prediction models, has concluded that the different forms of malvidin show the highest influence on the foamability parameters in rosé sparkling wines, followed by amino acid compounds, while foam stability model was only predicted by polysaccharides rich in arabinose and galactose. These research findings provide industry with a better understanding of the composi-

**Foaming Properties of Sparkling Wines**

tional factors influencing the foam quality of sparkling wines.

Keywords: sparkling wine, foaming properties, quality, chemical composition,

Nowadays the economic impact of sparkling wine shows a fast growth in the world wine trade because of its high added value. According to the report published in the year 2014 by the

> © The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and eproduction in any medium, provided the original work is properly cited.

© 2018 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Leticia Martínez-Lapuente, Belén Ayestarán and

Additional information is available at the end of the chapter

Additional information is available at the end of the chapter

Leticia Martínez-Lapuente, Belén Ayestarán

Properties of Sparkling Wines

http://dx.doi.org/10.5772/intechopen.70859

Zenaida Guadalupe

Abstract

predictive models

1. Introduction

and Zenaida Guadalupe

## **Influence of Wine Chemical Compounds on the Foaming Properties of Sparkling Wines** Provisional chapter Influence of Wine Chemical Compounds on the Foaming

DOI: 10.5772/intechopen.70859

Leticia Martínez-Lapuente, Belén Ayestarán and

Properties of Sparkling Wines

#### Zenaida Guadalupe Leticia Martínez-Lapuente, Belén Ayestarán

Additional information is available at the end of the chapter and Zenaida Guadalupe

http://dx.doi.org/10.5772/intechopen.70859 Additional information is available at the end of the chapter

#### Abstract

The foam of a sparkling wine is a key parameter of its quality, and the main characteristic differentiating sparkling wines from the so-called still wines. Both foam formation and duration are directly related to the chemical composition of sparkling wines. This chapter reviews the most recent studies made to determine the influence of chemical compounds on the foamability and foam stability of sparkling wines. Foam properties of sparkling wines are ruled by a large number of molecules, but some compounds seem to be more relevant than others to explain their behavior. The content of total amino acids, polysaccharides, anthocyanins, coumaric acid, and isorhamnetin showed high correlation values with foam quality parameters. The alcohol content and the concentration of acid polysaccharides, proanthocyanidins and free SO2 are the factors which most negatively affect foam quality. A recent study, by means of prediction models, has concluded that the different forms of malvidin show the highest influence on the foamability parameters in rosé sparkling wines, followed by amino acid compounds, while foam stability model was only predicted by polysaccharides rich in arabinose and galactose. These research findings provide industry with a better understanding of the compositional factors influencing the foam quality of sparkling wines.

Keywords: sparkling wine, foaming properties, quality, chemical composition, predictive models

## 1. Introduction

Nowadays the economic impact of sparkling wine shows a fast growth in the world wine trade because of its high added value. According to the report published in the year 2014 by the

© 2018 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

© The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and eproduction in any medium, provided the original work is properly cited.

International Organization of Vine and Wine [1], sparkling wine production increased by 40% in the last decade and by 7% compared to global wine production.

Sparkling wines are obtained after a second fermentation of a base wine that can be carried out in closed bottles or in hermetically sealed tanks. High quality sparkling wines, such as Champagne wines in France, Cava wines in Spain or Talento in Italy, are fermented in closed bottles following the traditional method, and they remain in contact with the yeast lees in a bottle. The first fermentation transforms grape must into base wine. The essence of the traditional method is the second fermentation, which takes place in the bottle and increases the alcohol content and internal bottle pressure (up to 5–7 atmospheres). After this second alcoholic fermentation, the wine is aged on yeast lees for at least 9 months (EC Regulation N 606/2009) [2]. Autolysis of the yeast occurs during this prolonged contact and involves hydrolytic enzymes that act to release cytoplasmic (peptides, fatty acids, nucleotides, amino acids) and cell wall (mannoproteins) compounds into the wine. This aging on yeast lees leads to significant changes in wine composition and the organoleptic and foam properties of the wine are modified [3].

In sparkling wines the level of dissolved carbon dioxide (CO2) found in the liquid phase is indeed a parameter of paramount importance. CO2 is responsible for the visually appealing, and very much sought-after repetitive bubbling process (the so-called effervescence). In fact, foam is the characteristic that differentiates sparkling wines from still wines, being the first sensory attribute that tasters and consumers perceive and that determines the final quality of sparkling wines [4]. Moreover, dissolved CO2 is also responsible for the very characteristic tingling sensation in aroma and mouthfeel sensations [5]. During carbonated beverage tasting, dissolved CO2 acts on both trigeminal receptors [6], and gustatory receptors, via the conversion of dissolved CO2 to carbonic acid [7], in addition to the tactile stimulation of mechanoreceptors in the oral cavity (through bursting bubbles). Moreover, a link has been evidenced between carbonation and the release of some aroma compounds in carbonated waters [8].

composition of the film that supports it [11]. In sparkling wines, bubbles consist of gas surrounded by a film of wine constituents. These tensioactive components and other substances afford viscosity to the film, giving texture to the bubble (Figure 1) [4]. In fact, it was established that foaming properties depend on compounds that decrease surface tension and increase the viscosity of the film between the bubbles. This factor contributes to foam stabili-

Influence of Wine Chemical Compounds on the Foaming Properties of Sparkling Wines

http://dx.doi.org/10.5772/intechopen.70859

197

In brief, foam formation and persistence is directly dependent upon its chemical composition, and the synergistic interaction among numerous foam active compounds which, due to aggregation or complex formation, may modify their surface-active properties. For this reason, and in order to ascertain which compounds affect foam quality, it is necessary to evaluate as many compounds as possible. In this sense, several scientific studies have been carried out in an attempt to determine the wine compounds that could play a role in the foam properties of sparkling wines. Many of these studies carried out in model solutions and in base and sparkling wines, are summarized in the reviews made by several authors [11, 14]. The present chapter increases the knowledge on this topic and reviews the latest studies made to determine the influence of proteins, peptides, amino acids, polysaccharides, phenolic compounds, lipids,

organic acids and others on the foamability and foam stability of sparkling wines.

Most of the studies published in the literature on sparkling wine foam quality are aimed at establishing the effect of the chemical composition of grape juices, base wines and sparkling

2. Measuring of foaming properties of sparkling wines

zation and renders the bubbles more resistant to coalescence [12, 13].

Figure 1. Carbon dioxide/liquid interphase of the bubble's film. Adapted from Ref. [11].

The formation of foam, its stability and the size of the bubbles in sparkling wines are directly related to the surface tension. This can be defined as the force per unit area that maintains the bond between the molecules at the surface of the liquid. The presence of surfactants reduces the surface tension of the liquid and allows to the formation and persistence of bubbles. Secondary fermentation in sparkling wines leads to the formation of carbon dioxide, which increases the pressure inside the bottle and causes the gas to dissolve in the liquid. When the bottle is opened, the difference between the pressure in the bottle and that of the atmosphere causes the dissolved gas to spontaneously leave the liquid. Once the pressure on the surface of the liquid has been equalized with the atmospheric pressure, bubbles continue to form inside the liquid [9].

Currently, the quality of sparkling wine is linked to the formation and persistence of foam. Quality foam can be defined as one that causes a slow release of CO2, in ring shapes from the depths of the liquid, with small bubbles that contribute to the formation of a crown over the surface of the wine, covering it completely, and with bubbles two or three rows deep [10]. Foam duration is directly related to bubble stability, and stability is itself dependent on the

Figure 1. Carbon dioxide/liquid interphase of the bubble's film. Adapted from Ref. [11].

International Organization of Vine and Wine [1], sparkling wine production increased by 40%

Sparkling wines are obtained after a second fermentation of a base wine that can be carried out in closed bottles or in hermetically sealed tanks. High quality sparkling wines, such as Champagne wines in France, Cava wines in Spain or Talento in Italy, are fermented in closed bottles following the traditional method, and they remain in contact with the yeast lees in a bottle. The first fermentation transforms grape must into base wine. The essence of the traditional method is the second fermentation, which takes place in the bottle and increases the alcohol content and internal bottle pressure (up to 5–7 atmospheres). After this second alcoholic fermentation, the wine is aged on yeast lees for at least 9 months (EC Regulation N 606/2009) [2]. Autolysis of the yeast occurs during this prolonged contact and involves hydrolytic enzymes that act to release cytoplasmic (peptides, fatty acids, nucleotides, amino acids) and cell wall (mannoproteins) compounds into the wine. This aging on yeast lees leads to significant changes in wine composition and the organoleptic and foam properties of the wine are

In sparkling wines the level of dissolved carbon dioxide (CO2) found in the liquid phase is indeed a parameter of paramount importance. CO2 is responsible for the visually appealing, and very much sought-after repetitive bubbling process (the so-called effervescence). In fact, foam is the characteristic that differentiates sparkling wines from still wines, being the first sensory attribute that tasters and consumers perceive and that determines the final quality of sparkling wines [4]. Moreover, dissolved CO2 is also responsible for the very characteristic tingling sensation in aroma and mouthfeel sensations [5]. During carbonated beverage tasting, dissolved CO2 acts on both trigeminal receptors [6], and gustatory receptors, via the conversion of dissolved CO2 to carbonic acid [7], in addition to the tactile stimulation of mechanoreceptors in the oral cavity (through bursting bubbles). Moreover, a link has been evidenced between carbonation and the release of some aroma compounds in carbonated waters [8].

The formation of foam, its stability and the size of the bubbles in sparkling wines are directly related to the surface tension. This can be defined as the force per unit area that maintains the bond between the molecules at the surface of the liquid. The presence of surfactants reduces the surface tension of the liquid and allows to the formation and persistence of bubbles. Secondary fermentation in sparkling wines leads to the formation of carbon dioxide, which increases the pressure inside the bottle and causes the gas to dissolve in the liquid. When the bottle is opened, the difference between the pressure in the bottle and that of the atmosphere causes the dissolved gas to spontaneously leave the liquid. Once the pressure on the surface of the liquid has been

equalized with the atmospheric pressure, bubbles continue to form inside the liquid [9].

Currently, the quality of sparkling wine is linked to the formation and persistence of foam. Quality foam can be defined as one that causes a slow release of CO2, in ring shapes from the depths of the liquid, with small bubbles that contribute to the formation of a crown over the surface of the wine, covering it completely, and with bubbles two or three rows deep [10]. Foam duration is directly related to bubble stability, and stability is itself dependent on the

in the last decade and by 7% compared to global wine production.

196 Grapes and Wines - Advances in Production, Processing, Analysis and Valorization

modified [3].

composition of the film that supports it [11]. In sparkling wines, bubbles consist of gas surrounded by a film of wine constituents. These tensioactive components and other substances afford viscosity to the film, giving texture to the bubble (Figure 1) [4]. In fact, it was established that foaming properties depend on compounds that decrease surface tension and increase the viscosity of the film between the bubbles. This factor contributes to foam stabilization and renders the bubbles more resistant to coalescence [12, 13].

In brief, foam formation and persistence is directly dependent upon its chemical composition, and the synergistic interaction among numerous foam active compounds which, due to aggregation or complex formation, may modify their surface-active properties. For this reason, and in order to ascertain which compounds affect foam quality, it is necessary to evaluate as many compounds as possible. In this sense, several scientific studies have been carried out in an attempt to determine the wine compounds that could play a role in the foam properties of sparkling wines. Many of these studies carried out in model solutions and in base and sparkling wines, are summarized in the reviews made by several authors [11, 14]. The present chapter increases the knowledge on this topic and reviews the latest studies made to determine the influence of proteins, peptides, amino acids, polysaccharides, phenolic compounds, lipids, organic acids and others on the foamability and foam stability of sparkling wines.

## 2. Measuring of foaming properties of sparkling wines

Most of the studies published in the literature on sparkling wine foam quality are aimed at establishing the effect of the chemical composition of grape juices, base wines and sparkling wines on their foaming properties. In order to correlate the foaming properties with the physical and chemical characteristics of sparkling wines, much effort has been made to find instrumental techniques that can be used to obtain a quantifiable value for foam quality, and consequently to be able to compare sparkling wines. Among them, methods based on measuring the kinetics of CO2 discharging, gas sparging methods, and image analysis methods are some of the most often employed [15].

An automated equipment, called "Mosalux" was designed to measure the foaming properties of wines [16]. This apparatus was adapted from that described by Rudin [17] and allows measurement of the increase with time of the height of a wine foam column submitted to a definite effervescence [16]. In fact, this is an objective and normalized method based on the interruption of a beam of ultra red light by the foam produced after the injection of CO2 into the wine. Three parameters can be measured.


Figure 2 shows the description of the "Mosalux" equipment and an example of the plot generated during a foam measurement.

The "Mosalux" equipment has been the most widely used since 1990 and in addition to research laboratories. It is probably the most used instrumental system in sparkling wine cellars for foam characterization. Moreover, a good relationship has been established between the foaming properties obtained by using "Mosalux" and foam sensory analysis [18]. The "Mosalux" apparatus has also been used to determine other parameters such as the expansion of foam E, the Bickerman coefficient Σ [19] (lifetime of a bubble in dynamic conditions, when formation and destruction of bubbles are balanced), and the lifetime of foam LF [20]. When comparing the different foam parameters (HM, HS, TS, E, LF, and ∑) obtained by the gas sparging method, it was concluded that the best parameters to characterize the foam capacities of wines were HM, ∑, and TS [21]. Other variation of this system uses an ultrasound emitterdetector and a waveguide to detect foam fluctuations [22, 23] to obtain Hpeak (maximum height reached by the foam after air injection through a glass frit). Hpeak has been related to the wine's ability for foaming and Hplato (foam height stability during air injection) has been related to the average bubble lifetime. Correlation between the results obtained with this technique and sensory analysis has also been established [24].

3. Impact of wine macromolecules on the sparkling wine foam quality

(6) infrared receiver, (7) personal computer, (8) printer; (b) example of a foam profile of a sparkling wine.

wine, grape juice, base wine, sparkling wine or isolated foam.

of grape juices, base wines and sparkling wines.

Table 1 includes a summary of the compounds that have been related to foam properties in the different scientific studies published, making reference to the type of sample used: model

Figure 2. (a) Diagram of the "Mosalux" equipment. (1) Flowmeter, (2) test tube, (3) wine, (4) foam, (5) infrared emitter,

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Table 2 shows the correlations (r) at significance level (p < 0.05) between parameters that determine foam properties (HM, HS, TS, Peak H and Plateau H) and the chemical composition Influence of Wine Chemical Compounds on the Foaming Properties of Sparkling Wines http://dx.doi.org/10.5772/intechopen.70859 199

wines on their foaming properties. In order to correlate the foaming properties with the physical and chemical characteristics of sparkling wines, much effort has been made to find instrumental techniques that can be used to obtain a quantifiable value for foam quality, and consequently to be able to compare sparkling wines. Among them, methods based on measuring the kinetics of CO2 discharging, gas sparging methods, and image analysis methods are

An automated equipment, called "Mosalux" was designed to measure the foaming properties of wines [16]. This apparatus was adapted from that described by Rudin [17] and allows measurement of the increase with time of the height of a wine foam column submitted to a definite effervescence [16]. In fact, this is an objective and normalized method based on the interruption of a beam of ultra red light by the foam produced after the injection of CO2 into

• HM: maximum height reached by the foam after carbon dioxide injection through the glass frit, expressed in mm; this could represent the foam-ability, the wine's ability to

• HS: foam stability height during carbon dioxide injection, expressed in mm; this could represent the foam stability, the wine's ability to produce stable foam or persistence of

• TS: foam stability time, until all bubbles collapse, when CO2 injection is interrupted, expressed in s; this could represent the foam stability time, once effervescence has

Figure 2 shows the description of the "Mosalux" equipment and an example of the plot

The "Mosalux" equipment has been the most widely used since 1990 and in addition to research laboratories. It is probably the most used instrumental system in sparkling wine cellars for foam characterization. Moreover, a good relationship has been established between the foaming properties obtained by using "Mosalux" and foam sensory analysis [18]. The "Mosalux" apparatus has also been used to determine other parameters such as the expansion of foam E, the Bickerman coefficient Σ [19] (lifetime of a bubble in dynamic conditions, when formation and destruction of bubbles are balanced), and the lifetime of foam LF [20]. When comparing the different foam parameters (HM, HS, TS, E, LF, and ∑) obtained by the gas sparging method, it was concluded that the best parameters to characterize the foam capacities of wines were HM, ∑, and TS [21]. Other variation of this system uses an ultrasound emitterdetector and a waveguide to detect foam fluctuations [22, 23] to obtain Hpeak (maximum height reached by the foam after air injection through a glass frit). Hpeak has been related to the wine's ability for foaming and Hplato (foam height stability during air injection) has been related to the average bubble lifetime. Correlation between the results obtained with this

some of the most often employed [15].

198 Grapes and Wines - Advances in Production, Processing, Analysis and Valorization

the wine. Three parameters can be measured.

generated during a foam measurement.

technique and sensory analysis has also been established [24].

foam.

foam collar.

decreased.

Figure 2. (a) Diagram of the "Mosalux" equipment. (1) Flowmeter, (2) test tube, (3) wine, (4) foam, (5) infrared emitter, (6) infrared receiver, (7) personal computer, (8) printer; (b) example of a foam profile of a sparkling wine.

#### 3. Impact of wine macromolecules on the sparkling wine foam quality

Table 1 includes a summary of the compounds that have been related to foam properties in the different scientific studies published, making reference to the type of sample used: model wine, grape juice, base wine, sparkling wine or isolated foam.

Table 2 shows the correlations (r) at significance level (p < 0.05) between parameters that determine foam properties (HM, HS, TS, Peak H and Plateau H) and the chemical composition of grape juices, base wines and sparkling wines.


Compounds Type of sample Results References

BWa Total polysaccharides increase foam height and reduce foam

stability time

reduce foam stability

and effervescence

stability time

Grape juice Total polyphenol increase foam height

Separated foam Increase foam [34] BWa and SWa Xylose in polysaccharides increase foam stability [29] BWa Increase foam height [25] SWa Increase foam height and stability time [40]

Acid and neutral polysaccharides increase foam height

2000) increase foam height, and the Mr. 3000–2000 polysaccharide

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Grape juice and BWa Total and neutral polysaccharides increase foam height [42] BWa and SWa Increase foam height and foam height stability [22] BWa and SWa Polysaccharides (Mr of 62,000–48,000, 13,000–11,000, and 3000 to

SWa Total and acid polysaccharides decrease foam stability time [66] BWa Reduce foam height [45]

Model wine and SWa Increase foam height and foam height stability [30] Model wine Increase foam stability [56]

stability. Arabinogalactans and hydrophobic low molecular

(HMW) increase foam height, foam stability height and foam

SWa Mannoproteins increase maximum height, foam height stability

Model wine Mannoproteins with low content of protein (5%) increase foam

SWa Mannoproteins, arabinogalactans and pectic polysaccharides

Polyphenols Model wine (+)-catechin increase foamability and foam stability [61]

reduce foam stability time

weight fraction (<1 kDa) increase foamability.

Model wine Mannoproteins increase foamability [33] SWa Mannoproteins and PRAG increase foam stability time [28] BWa and SWa High molecular weight polysaccharides decrease foam height [49] BWa Increase foam stability time [50]

SWa Increase foam height and reduce foam stability [40]

BWa Non flavonoids phenols decrease foam stability time [26] Grape juice and BWa Total polyphenols, ortodiphenols, flavonoids and nonflavonoids

BWa Reduce foam height [45]

BWa Increase foam stability time [50]

Proanthocyanidins decrease foam height and foam stability height

SWa Anthocyanins increase foam height and foam stability height

Nonflavonoid phenol increase foam height Flavonoid phenol increase foam height

[26]

201

[43]

[23]

[32]

[31]

[12]

[42]

[28]

Polysaccharides Model wine Increase foam stability [38]


Compounds Type of sample Results References

Model wine Increase foam height [38] BWa Increase foam height [16] Separated foam Increase foam [34, 35] Model wine and BWa Increase foam stability [20] BWa Increase foam height and foam stability [39] BWa and SWa Reduce foam height and increase foam stability [29] BWa Increase foam height and reduce foam stability [25] SWa Increase foam height and foam stability [40] Grape juice Increase foam height [12] BWa Increase foam height and reduce foam stability [26]

Proteins Model wine Increase foam [67]

200 Grapes and Wines - Advances in Production, Processing, Analysis and Valorization

SWa Increase foam height, foam stability height and decrease

Grape juice and BWa Increase foam height [42] BWa and SWa Increase foam height and foam height stability [22] BWa and SWa Increase foam height [43] SWa Increase foam height [44] BWa Increase foam height [45] SWa Increase foam height stability [24] BWa Increase foam height and foam stability [46] SWa Increase maximum height, foam height stability and effervescence [23] BWa Increase foam stability [70] BWa and SWa Increase foam height and foam stability height [47] SWa Increase foam height and foam stability height [48] BWa and SWa Increase foam height and foam stability height [27]

Model wine Cooperative effects between mannoproteins and the proteins of grape origin to improve foamability

Peptides BWa and SWa No influence on foam height and foam height stability [22]

Amino acids BWa Decrease foam stability time [25]

Decrease foam stability time

height

SWa Proline and glutamine increase foam height and foam stability

BWa and SWa Increase foam height [49] BWa Increase foam height [50]

SWa Improve foam height stability [24]

BWa and SWa Increase foam height and foam height stability [22] SWa Increase maximum height, foam height stability and effervescence [23] SWa Increase foam height and foam stability height [28]

[41]

[33]

[41]

foam stability time


In the majority of the works shown in Tables 1 and 2, the chemical compounds have been related to the foam physical parameters obtained by the "Mosalux" device [12, 16, 25–28] or other variations of this method [20, 22–24, 29–33]. All studies have shown that the foam properties of sparkling wines mainly depend on the qualitative composition and quantitative content of surface active substances. The relation found between the foaming properties and

Compounds Type of sample Results References

SWa Residual sugars and ethanolamine increase foam height and foam

Ethyl acetate decrease foam stability time

SWa Ethanol, volatile acidity and total SO2 reduce foam height

Model wine Glycerol and glycerol plus ethyloctanoate increase foam height

and foam stability time

SWa Botrytis cinerea infection decrease foamability [44] BWa Alcohol concentration and total SO2 reduce foam height [45]

BWa Lysozyme have a protective effect on foaming properties [71] Model wine Botrytis cinerea protease activity decrease wine foaming properties [69] BWa Botrytis cinerea infection decrease foamability and foam stability [70]

BWa and SWa Ethanol content reduce foam height [49]

Volatile acidity and total SO2 reduce foam stability time

time

stability height

Free sulfur dioxide increase foam height and reduce foam stability

Influence of Wine Chemical Compounds on the Foaming Properties of Sparkling Wines

Conductivity increase foam height and reduce foam stability time

[41]

203

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[66]

[32]

Despite of the low concentration of proteins in sparkling wines (ranging from 4 to 16 mg/L) [14], previous works have shown that these compounds are largely involved in the foaming properties of sparkling wines due to their surfactant properties. Surfactant agents are inferred to stabilize foams by settling at the bubble's edge, with the hydrophobic side interacting with the gas phase and the hydrophilic side interacting with the aqueous liquid phase [34]. The behavior of proteins in the foam depends on their hydrophobicity, solubility (dependent on the isoelectric point and the pH of the wine), and molecular weight [35, 36]. The net charge of macromolecules depends on the pH [37]. The isoelectric point of the wine proteins is between 3.5 and 4.5 [35] and between 4.6 and 5.0 [29]. At the wine pH, 2.9, its proteins would be positively charged and could migrate to the wine/air interphase and to stabilize foam [20]. However, characterization of foaming proteins have showed that foam formation is dependent

on protein hydrophobicity but not on their molecular weight or isoelectric point [34].

the different wine macromolecules is detailed below.

Table 1. Compounds related to foam properties in sparkling wines.

3.1. Proteins

a

BW: base wines; SW: sparkling wines.


Table 1. Compounds related to foam properties in sparkling wines.

In the majority of the works shown in Tables 1 and 2, the chemical compounds have been related to the foam physical parameters obtained by the "Mosalux" device [12, 16, 25–28] or other variations of this method [20, 22–24, 29–33]. All studies have shown that the foam properties of sparkling wines mainly depend on the qualitative composition and quantitative content of surface active substances. The relation found between the foaming properties and the different wine macromolecules is detailed below.

#### 3.1. Proteins

Compounds Type of sample Results References Lipids BWa C8 and C10 increase collar height and reduce stability foam [16] Model wine and BWa Lipids are only foam active compounds at low alcohol

Palmitic acid increase foam height

promotion and stabilization of foam

Titratable acidity increase foam height Lactic acid decrease foam height

SWa Monoacylglycerols of palmitic and stearic acids and

Organic acids Model wine and BWa Tartaric acid increase foam [20]

pH reduce foam stability time

SWa Malic acid increase foam height and reduce stability foam

Total acidity decrease foam height

BWa Titratable acidity, malic acid increase foam height and reduce

Lactic acid decrease foam stability time

foam stability time

pH decrease foam height

Others Separated foam Iron increase foam [34]

SWa Tartaric acid increase foam height

BWa and SWa Glucose increase foam height

height

BWa Alcohol content increase foam height

C8, C10, and C12 reduce foam height. Ethyl esters of hexanoic,

octanoic, and decanoic acids increase foam height.

BWa and SWa Tartaric acid increase foam height [29]

SWa Galacturonic acid decrease foam stability time [12]

BWa Malic acid increase foam height [26]

BWa and SWa Gluconic acid reduce foam height [49]

Model wine and BWa Glycerol increase foam [20]

Total content of SO2 reduce foam stability ɤ-butyrolactone increase foam stability

Free sulfur dioxide decrease foam height

BWa Acetaldehyde, ethyl acetate, diacetaldehyde and isoamylic alcohols reduce foam stability time

Grape juice Fructose, glucose and methanol increase foam height

Lactic acid reduce foam height and increase foam stability time Citric acid and galacturonic acid reduce foam stability time

Alcohol content increase foam height and foam stability height Glucose increase foam height and fructose reduce foam height

Soluble solid concentration and maturity index increase foam

Turbidity increase foam height and reduce foam stability time

Citric and galacturonic acid reduce foam stability time

glycerylethylene glycol fatty acid derivatives increase the

[64]

[29]

[65]

[31]

[25]

[40]

[41]

[45]

[29]

[25]

[12]

[26]

concentration

202 Grapes and Wines - Advances in Production, Processing, Analysis and Valorization

BWa and SWa Linoleic acid increase foam stability

BWa Malic acid increase foam height

Grape juice pH increase foam height

BWa and separated

foam

Despite of the low concentration of proteins in sparkling wines (ranging from 4 to 16 mg/L) [14], previous works have shown that these compounds are largely involved in the foaming properties of sparkling wines due to their surfactant properties. Surfactant agents are inferred to stabilize foams by settling at the bubble's edge, with the hydrophobic side interacting with the gas phase and the hydrophilic side interacting with the aqueous liquid phase [34]. The behavior of proteins in the foam depends on their hydrophobicity, solubility (dependent on the isoelectric point and the pH of the wine), and molecular weight [35, 36]. The net charge of macromolecules depends on the pH [37]. The isoelectric point of the wine proteins is between 3.5 and 4.5 [35] and between 4.6 and 5.0 [29]. At the wine pH, 2.9, its proteins would be positively charged and could migrate to the wine/air interphase and to stabilize foam [20]. However, characterization of foaming proteins have showed that foam formation is dependent on protein hydrophobicity but not on their molecular weight or isoelectric point [34].


Compounds Type of sample HM HS TS Peak H Plateau H References

Histamine SWa 0.39 0.42 0.43 [28] GABA BWa 0.38 [25]

Arginine BWa 0.36 [25]

α alanine SWa 0.53 0.63 [22]

Β alanine SWa 0.92 0.55 [28] Tyrosine BWa 0.53 [25]

Valine BWa 0.50 [25]

Methionine BWa 0.34 [25]

Cysteine SWa 0.79 0.49 [28] Isoleucine BWa [25]

Leucine BWa 0.34 [25]

Phenylalanine BWa 0.29 [25]

Ornithine BWa 0.31 [25]

Tryptophan BWa 0.37 [25]

Lysine BWa 0.36 [25]

SWa 0.82 0.60 0.34 [28]

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SWa 0.52 0.60 [22] SWa 0.77 0.52 [28]

SWa 0.50 0.62 [22] SWa 0.83 0.65 [28]

BWa 0.37 [25] SWa 0.83 0.65 0.39 [28]

SWa 0.49 0.63 [22] SWa 0.81 0.60 [28]

SWa 0.52 0.67 [22]

SWa 0.51 0.63 [22] SWa 0.89 0.58 [28]

SWa 0.67 0.64 0.47 [28]

SWa 0.51 0.64 [22] SWa 0.42 0.55 0.55 [28]

SWa 0.42 0.62 [22] SWa 0.84 0.62 0.36 [28]

SWa 0.79 0.64 [28]

SWa 0.85 0.59 [28]

SWa 0.52 [22] SWa 0.66 0.61 [28]


Compounds Type of sample HM HS TS Peak H Plateau H References

Grape juice 0.91 [12] SWa 0.62 0.49 [22] BWa 0.31 [45] BWa and SWa 0.58 [49] BWa and SWa 0.44 [43] Grape juice 0.75 [42]

SWa 0.86 0.63 [28]

SWa 0.46 [28]

SWa 0.66 0.71 [22] SWa 0.77 0.54 0.46 [28]

SWa 0.56 0.68 [22] SWa 0.62 0.59 0.58 [28]

SWa 0.41 0.57 [22] SWa 0.79 0.68 0.45 [28]

SWa 0.41 0.57 [22] SWa 0.88 0.66 0.35 [28]

SWa 0.53 [22] SWa 0.42 [28]

SWa [22]

Proteins BWa 0.32 0.51 [25]

204 Grapes and Wines - Advances in Production, Processing, Analysis and Valorization

Total amino acids SWa 0.85 0.63 [28] Acid amino acids SWa 0.82 0.58 [28] Neutral amino acids SWa 0.85 0.68 [28] Basic amino acids SWa 0.75 0.62 [28] Total biogenic amines SWa 0.66 0.64 0.48 [28] Aspartic acid SWa 0.52 0.67 [22]

Hydroxyproline BWa 0.39 [25]

Glutamic acid BWa 0.50 [25]

Serine BWa 0.425 [25]

Asparagine BWa 0.38 [25]

Glycine BWa 0.39 [25]

Glutamine BWa 0.37 0.36 [25]

Histidine SWa 0.50 0.48 [22] Threonine SWa 0.56 0.42 [28] Proline BWa 0.34 0.58 0.69 [25]

Amino acids


Compounds Type of sample HM HS TS Peak H Plateau H References

BWa 0.33 [42]

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BWa 0.64 [42]

Nonflavonoid phenol Grape juice 0.59 [12]

Total flavan-3-ols SWa 0.50 0.42 [28] Flavonoid phenol Grape juice 0.52 [12]

Ortodiphenols BWa 0.49 [42] Total monomeric anthocyanins SWa 0.96 0.80 [28] Non-acylated anthocyanins SWa 0.97 0.81 [28] Acetyl-glucoside anthocyanins SWa 0.94 0.75 [28] Coumaryl-glucoside anthocyanins SWa 0.88 0.67 [28] delphinidin-3-glucoside SWa 0.94 0.71 [28] cyanidin-3-glucoside SWa 0.84 0.60 [28] petunidin-3-glucoside SWa 0.95 0.73 [28] peonidin-3-glucoside SWa 0.87 0.65 [28] malvidin-3-glucoside SWa 0.98 0.85 [28] delphinidin-3-(6-acetyl)-glucoside SWa 0.91 0.67 [28] cyanidin-3-(6-acetyl)-glucoside SWa 0.89 0.62 [28] petunidin-3-(6-acetyl)-glucoside SWa 0.92 0.69 [28] peonidin-3-(6-acetyl)-glucoside SWa 0.89 0.65 [28] malvidin-3-(6-acetyl)-glucoside SWa 0.89 0.92 [28] delphinidin-3-(6-p-coumaryl)-glucoside SWa 0.76 0.52 [28] cyanidin-3-(6-p-coumaryl)-glucoside SWa 0.92 0.68 [28] petunidin-3-(6-p-coumaryl)-glucoside SWa 0.78 0.55 [28] peonidin-3-(6-p-coumaryl)-glucoside SWa 0.91 0.67 [28] malvidin-3-(6-p-coumaryl)-glucoside SWa 0.94 0.76 [28] cis-caftaric SWa 0.65 [28] trans-fertaric SWa 0.35 [28] coumaric acid SWa 0.77 0.37 [28] ferulic acid SWa 0.39 0.41 [28] gallic acid SWa 0.62 [28] (+)-catechin SWa 0.50 0.42 [28] quercetin-3-rutinoside SWa 0.43 [28] myricetin SWa 0.36 [28] quercetin SWa 0.58 [28] kaempferol SWa 0.53 [28] isorhamnetin SWa 0.84 [28]


Compounds Type of sample HM HS TS Peak H Plateau H References Spermidine SWa 0.72 0.41 [28] Tyramine SWa 0.35 [28] Putrescine SWa 0.51 0.59 0.43 [28] Cadaverine SWa 0.35 [28] Phenylethylamine SWa 0.60 [28] Isoamylamine SWa 0.55 [28]

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Total polysaccharides Grape juice 0.55 [42]

Polysaccharides from yeasts SWa 0.53 [28] Polysaccharides from grapes SWa 0.68 [28] Neutral polysaccharides Grape juice 0.65 [42]

Acid polysaccharides BWa 0.76 [45] High molecular weight polysaccharides BWa and SWa 0.65 [49]

Mannoproteins SWa 0.47 [28]

Homogalacturonans SWa 0.58 [28] Glucans SWa 0.40 [28]

Absorbance 520 (nm) BWa 0.35 [25] Absorbance 280 (nm) Grape juice 0.92 [12]

Total polyphenol Grape juice 0.76 [12]

Total proanthocyanidins SWa 0.73 [28]

BWa 0.40 [42] SWa 0.80 0.68 [22] SWa 0.64 [28]

BWa 0.46 [42] SWa 0.82 0.71 [22]

BWa and SWa 0.51 [43]

BWa and SWa 0.46 [43]

BWa and SWa 0.32 [43]

SWa 0.72 [28]

BWa 0.63 [42]

BWa 0.60 [42] BWa 0.45 [45]

Polysaccharides

Polysaccharides Molecular Mass

Polysaccharides Molecular Mass

Polysaccharides Molecular Mass

Polysaccharides rich in arabinose

62,000–48,000

13,000–11,000

and galactose

Polyphenols

3000–2000


Proteins have been the most studied compounds in relation to wine foamability. Most studies indicate a positive influence of protein content on foam height in grape juices, base wines and sparkling wines [16, 20, 22–27, 38–50] (Tables 1 and 2). Some of this studies showed positive correlations between proteins and parameter HM [12, 25, 42, 43, 45, 49], Peak H, and Plateau H [22]. The highest correlations between proteins and foamability parameters were observed in juices of white grapes (r > 0.75) [12, 42]. Correlation between proteins and foamability parameters was lower in base wines and sparkling wines [22, 25, 43, 45, 49] (Table 2). The work conducted in Spanish sparkling wines was an exception to this because authors observed a

Table 2. Correlation coefficients (r) at significance levels (p < 0.05) between parameters that determine foam properties (HM, HS, TS, Peak H and Plateau H) and the chemical composition of grape juices, base wines and sparkling wines.

Compounds Type of sample HM HS TS Peak H Plateau H References

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Maturity index Grape juice 0.78 [12] Soluble solid concentration Grape juice 0.75 [12] Methanol Grape juice 0.80 [12] Free sulfur dioxide Grape juice 0.65 [12] Total sulfur dioxide BWa 0.68 [45]

The correlations between proteins and foam stability have shown contradictory results. Therefore, some proteins have been described as good foam formers but poor stabilizers, while others are poor foam formers but good stabilizers [13, 20, 25, 26, 29, 39, 41]. Inverse relationship between HM and TS [16, 25, 26] could justify that proteins may be active agents on

The influence of specific proteins on foam quality has also been studied in several research papers. Grape invertase is one of the most abundant protein present in wine (from 9 to 14% of the total protein content of a Chardonnay wine) [51]. This grape protein possesses a pI close to the pH of wine and a high hydrophobicity, potentially conferring good surface properties on this protein [51]. Significant decreases in the invertase content in base wines have been shown to correlate with decreases in foam height and foam stability [46]. Other grape proteins, such as thaumatin-like proteins and chinases, did not contribute alone to the formation and stabilization of foam; however, when synergistically acting with mannoproteins, foam height was found to be maximized [33]. On the other hand, the release of proteins from the yeast cells prior to autolysis has also been shown to contribute to foam height and foam stability height in sparkling wines [23, 24, 47].

The hydrophobicity of peptides may be related to the quality of sparkling wine foam [52, 53]. Proteins and peptides with molecular weight within 24–60 kDa, even in low concentrations,

negative relation between proteins and foam height [29].

foamability but may not sustain a foam collar for a long time.

3.2. Peptides

a

BW: base wines; SW: sparkling wines.


Table 2. Correlation coefficients (r) at significance levels (p < 0.05) between parameters that determine foam properties (HM, HS, TS, Peak H and Plateau H) and the chemical composition of grape juices, base wines and sparkling wines.

Proteins have been the most studied compounds in relation to wine foamability. Most studies indicate a positive influence of protein content on foam height in grape juices, base wines and sparkling wines [16, 20, 22–27, 38–50] (Tables 1 and 2). Some of this studies showed positive correlations between proteins and parameter HM [12, 25, 42, 43, 45, 49], Peak H, and Plateau H [22]. The highest correlations between proteins and foamability parameters were observed in juices of white grapes (r > 0.75) [12, 42]. Correlation between proteins and foamability parameters was lower in base wines and sparkling wines [22, 25, 43, 45, 49] (Table 2). The work conducted in Spanish sparkling wines was an exception to this because authors observed a negative relation between proteins and foam height [29].

The correlations between proteins and foam stability have shown contradictory results. Therefore, some proteins have been described as good foam formers but poor stabilizers, while others are poor foam formers but good stabilizers [13, 20, 25, 26, 29, 39, 41]. Inverse relationship between HM and TS [16, 25, 26] could justify that proteins may be active agents on foamability but may not sustain a foam collar for a long time.

The influence of specific proteins on foam quality has also been studied in several research papers. Grape invertase is one of the most abundant protein present in wine (from 9 to 14% of the total protein content of a Chardonnay wine) [51]. This grape protein possesses a pI close to the pH of wine and a high hydrophobicity, potentially conferring good surface properties on this protein [51]. Significant decreases in the invertase content in base wines have been shown to correlate with decreases in foam height and foam stability [46]. Other grape proteins, such as thaumatin-like proteins and chinases, did not contribute alone to the formation and stabilization of foam; however, when synergistically acting with mannoproteins, foam height was found to be maximized [33]. On the other hand, the release of proteins from the yeast cells prior to autolysis has also been shown to contribute to foam height and foam stability height in sparkling wines [23, 24, 47].

#### 3.2. Peptides

Compounds Type of sample HM HS TS Peak H Plateau H References

0.43 [65]

0.66 [65]

0.57 [65]

0.65 [65]

0.86 [65]

0.90 [65]

Grape juice 0.59 [12]

Grape juice 0.71 [12]

BWa 0.40 [45]

BWa 0.47 [45] BWa and SWa 0.92 [49]

Grape juice 0.58 [12]

Grape juice 0.73 [12]

Lipids

Organic acids

Others

C8 (n = 28) BWa and separated

C10 (n = 28) BWa and separated

C12 (n = 28) BWa and separated

Ethyl hexanoate (n) 28 BWa and separated

Ethyl octanoate (n) 28 BWa and separated

Ethyl decanoate (n) 28 BWa and separated

foam

208 Grapes and Wines - Advances in Production, Processing, Analysis and Valorization

foam

foam

foam

foam

foam

Titratable acidity BWa 0.46 [25]

pH BWa 0.32 [25]

Citric acid BWa 0.38 [25] Galacturonic acid BWa 0.42 [25] Malic acid BWa 0.46 [25]

Lactic acid BWa 0.43 [25] Gluconic acid BWa and SWa 0.36 [49]

Alcohol content BWa 0.47 0.46 [25]

Glucose BWa 0.31 [25]

Fructose BWa 0.56 0.32 [25]

Ethanolamine BWa 0.31 [25] Acetaldehide BWa 0.35 [25] Ethyl acetate BWa 0.51 [25] Diacetaldehyde BWa 0.36 [25] Isoamylic alcohols BWa 0.43 [25]

The hydrophobicity of peptides may be related to the quality of sparkling wine foam [52, 53]. Proteins and peptides with molecular weight within 24–60 kDa, even in low concentrations, provide for the foam formation in base wine [13, 31, 47] since they form adsorption layers with high mechanical strength and, as a result, increase the stability of the sparkling wine foam. In fact, a positive correlation has been reported between polypeptide molecular mass, hydrophobicity and foam stabilizing activity in beer [54, 55]. Although no correlations have been found between peptides and foam properties of sparkling wines [22], bentonite added to the tirage solution produced a reduction in both protein and peptide contents and thereby negatively affected foaming properties [24] (Table 1).

higher increase in both nitrogen compounds (proteins, peptides and amino acids) and poly-

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Contradictory results have been published on the effect of polysaccharides on foam quality. Girbau Sola et al. [45] showed a negative influence of acid polysaccharides on foam height in base wines (r = 076). The same authors showed that polysaccharides were negatively correlated with foam stability but positively with the average bubble lifetime or Bikerman's coefficient [45]. Similarly, polysaccharides with a molecular weight higher than 180 kDa have also shown a negative influence on foam height (r = 0.65), although these authors associated the negative contribution with the presence of β-glucans secreted by Botrytis cinerea and stated that

other polysaccharides probably would not have a negative effect [49] (Tables 1 and 2).

were correlated with the Bikerman coefficient [43].

In contrast with the results described above, most studies point to a positive influence of total polysaccharides on both foamability [22, 25, 26, 40, 42] and foam stability [28, 29, 50, 56] (Table 1). The relation of the molecular weight of polysaccharides and the foaming properties of wines has also been studied. Polysaccharides of molecular mass of 62 to 48 kDa; 13 to 11 kDa; and 3 to 2 kDa have been demonstrated to be active agents on foamability, and polysaccharides with molecular mass of 3 to 2 kDa might be a foam stability agent, since they

Among polysaccharides, glycoproteins like mannoproteins released by yeast during fermentation and autolysis, have been described as the major compounds affecting foaming properties [13, 23, 30, 33]. The hydrophobic nature of glycoproteins explains why they are better foam stabilizers and foam producers than non-glycosylated proteins. Glycoproteins present a protein moiety with hydrophobic and hydrophilic domains and sugar moieties, which are hydrophilic and they could interact with surface-active materials and be absorbed at the gas/liquid interface. The hydrophilic glycans are located at the liquid layer, among the bubbles, corresponding to the oxidic zone of the protein. Hence, when the layer surrounding the bubbles becomes thinner, the viscosity increases and drainage of the liquid is delayed. The hydrophobic polypeptides increase the surface tension of the bubbles, resulting in more stable foam [13]. In this sense, the literature has tried to explain the influence of mannoproteins on foaming properties. Mannoproteins also influence the viscosity of the bubble wall and reduces the drainage of the liquid [34]. Foaming may be due to their interactions with proteins [36] and their surface properties and capacity to reorientation quickly at the liquid/gas interface in the bubble when the foam is formed [20]. In fact, the proteinaceous fraction of mannoproteins is able to bind to the liquid/air interface and interact with other compounds by means of electrostatic or hydrophobic forces, hydrogen bonds, or covalent linkages [13]. These interactions could lead to the formation of a strong viscoelastic film that could be highly resistant to tension and able to withstand the film's thickness [13], preventing coalescence of bubbles and leading to more stable foams. As a matter of fact the presence of both glycocompounds and protein material deriving from macromolecular fractions of different molecular weights in the adsorption layer of

saccharides [23].

3.4. Polysaccharides

#### 3.3. Amino acids

In addition to proteins and peptides, some authors agree in considering amino acids as foaming agents. At wine pH, amino acids carry a net positive charge, so they are surfactant with hydrophilic and hydrophobic groups. This property causes amino acids to be retained in the air/liquid interface, and thus reduces the wine surface tension, improving the sparkling wine ability to foam [28].

Moreno-Arribas et al. [22] showed positive correlations between free amino acids and white sparkling wine foamability (Tables 1 and 2). The authors observed that maximum height (Peak H) was significantly correlated with most of the amino-acids, although coefficients of over 0.60 were only found for glutamic acid (r = 0.66). Moreover, Plateau H (associated to bubble lifetime) was highly correlated with glutamic acid (r = 0.71), serine (r = 0.68), valine (r = 0.67) and proline (r = 0.69). Lower positive correlations were found by Andrés-Lacueva et al. [25] in white base wines between glutamine and proline and foamability parameters.

Other study conducted by our research group in white and rosé sparkling wine showed the highest positive correlations between total amino acids and foam height (r = 0.85) and total amino acids and foam stability height (r = 0.63) [28] (Table 2). Biogenic amines showed the same behavior as amino acids, although lower correlation values were observed [28] (Table 2). When comparing the different amino acids analyzed, glycine, β-alanine, and methionine had the highest correlation with foam height (r > 0.88) [28] (Table 2). In general, amino acids with nonpolar side chains showed higher values of correlation than amino acids with polar side chains. At wine pH, amino acids are protonated and they act as cationic surfactants according to the hydrophobicity of their side chains. Their amphiphilic character could cause amino acids to become concentrated at the liquid–gas interfaces, improving the sparkling wine foamability [28].

Negative correlations have been observed between amino acids and foam stability time in base wines [25] (Tables 1 and 2). However, conflicting results have been published on the influence of amino acid on foam stability of sparkling wines. It has shown that lower levels of amino acids favors a greater stability time of foam [41]; while other authors did not found any influence of these compounds on the foam stability time [28].

It was confirmed that the autolytic capacity of yeast was important for the quality of sparkling wines [23]. The use of a mutant having accelerated autolysis showed that the second fermentation of wines with this mutant improved the foaming properties versus a control strain due to higher increase in both nitrogen compounds (proteins, peptides and amino acids) and polysaccharides [23].

#### 3.4. Polysaccharides

provide for the foam formation in base wine [13, 31, 47] since they form adsorption layers with high mechanical strength and, as a result, increase the stability of the sparkling wine foam. In fact, a positive correlation has been reported between polypeptide molecular mass, hydrophobicity and foam stabilizing activity in beer [54, 55]. Although no correlations have been found between peptides and foam properties of sparkling wines [22], bentonite added to the tirage solution produced a reduction in both protein and peptide contents and thereby negatively

In addition to proteins and peptides, some authors agree in considering amino acids as foaming agents. At wine pH, amino acids carry a net positive charge, so they are surfactant with hydrophilic and hydrophobic groups. This property causes amino acids to be retained in the air/liquid interface, and thus reduces the wine surface tension, improving the sparkling

Moreno-Arribas et al. [22] showed positive correlations between free amino acids and white sparkling wine foamability (Tables 1 and 2). The authors observed that maximum height (Peak H) was significantly correlated with most of the amino-acids, although coefficients of over 0.60 were only found for glutamic acid (r = 0.66). Moreover, Plateau H (associated to bubble lifetime) was highly correlated with glutamic acid (r = 0.71), serine (r = 0.68), valine (r = 0.67) and proline (r = 0.69). Lower positive correlations were found by Andrés-Lacueva et al. [25] in

Other study conducted by our research group in white and rosé sparkling wine showed the highest positive correlations between total amino acids and foam height (r = 0.85) and total amino acids and foam stability height (r = 0.63) [28] (Table 2). Biogenic amines showed the same behavior as amino acids, although lower correlation values were observed [28] (Table 2). When comparing the different amino acids analyzed, glycine, β-alanine, and methionine had the highest correlation with foam height (r > 0.88) [28] (Table 2). In general, amino acids with nonpolar side chains showed higher values of correlation than amino acids with polar side chains. At wine pH, amino acids are protonated and they act as cationic surfactants according to the hydrophobicity of their side chains. Their amphiphilic character could cause amino acids to become concentrated at the liquid–gas interfaces, improving the sparkling wine foamability [28]. Negative correlations have been observed between amino acids and foam stability time in base wines [25] (Tables 1 and 2). However, conflicting results have been published on the influence of amino acid on foam stability of sparkling wines. It has shown that lower levels of amino acids favors a greater stability time of foam [41]; while other authors did not found any

It was confirmed that the autolytic capacity of yeast was important for the quality of sparkling wines [23]. The use of a mutant having accelerated autolysis showed that the second fermentation of wines with this mutant improved the foaming properties versus a control strain due to

white base wines between glutamine and proline and foamability parameters.

influence of these compounds on the foam stability time [28].

affected foaming properties [24] (Table 1).

210 Grapes and Wines - Advances in Production, Processing, Analysis and Valorization

3.3. Amino acids

wine ability to foam [28].

Contradictory results have been published on the effect of polysaccharides on foam quality. Girbau Sola et al. [45] showed a negative influence of acid polysaccharides on foam height in base wines (r = 076). The same authors showed that polysaccharides were negatively correlated with foam stability but positively with the average bubble lifetime or Bikerman's coefficient [45]. Similarly, polysaccharides with a molecular weight higher than 180 kDa have also shown a negative influence on foam height (r = 0.65), although these authors associated the negative contribution with the presence of β-glucans secreted by Botrytis cinerea and stated that other polysaccharides probably would not have a negative effect [49] (Tables 1 and 2).

In contrast with the results described above, most studies point to a positive influence of total polysaccharides on both foamability [22, 25, 26, 40, 42] and foam stability [28, 29, 50, 56] (Table 1). The relation of the molecular weight of polysaccharides and the foaming properties of wines has also been studied. Polysaccharides of molecular mass of 62 to 48 kDa; 13 to 11 kDa; and 3 to 2 kDa have been demonstrated to be active agents on foamability, and polysaccharides with molecular mass of 3 to 2 kDa might be a foam stability agent, since they were correlated with the Bikerman coefficient [43].

Among polysaccharides, glycoproteins like mannoproteins released by yeast during fermentation and autolysis, have been described as the major compounds affecting foaming properties [13, 23, 30, 33]. The hydrophobic nature of glycoproteins explains why they are better foam stabilizers and foam producers than non-glycosylated proteins. Glycoproteins present a protein moiety with hydrophobic and hydrophilic domains and sugar moieties, which are hydrophilic and they could interact with surface-active materials and be absorbed at the gas/liquid interface. The hydrophilic glycans are located at the liquid layer, among the bubbles, corresponding to the oxidic zone of the protein. Hence, when the layer surrounding the bubbles becomes thinner, the viscosity increases and drainage of the liquid is delayed. The hydrophobic polypeptides increase the surface tension of the bubbles, resulting in more stable foam [13]. In this sense, the literature has tried to explain the influence of mannoproteins on foaming properties. Mannoproteins also influence the viscosity of the bubble wall and reduces the drainage of the liquid [34]. Foaming may be due to their interactions with proteins [36] and their surface properties and capacity to reorientation quickly at the liquid/gas interface in the bubble when the foam is formed [20]. In fact, the proteinaceous fraction of mannoproteins is able to bind to the liquid/air interface and interact with other compounds by means of electrostatic or hydrophobic forces, hydrogen bonds, or covalent linkages [13]. These interactions could lead to the formation of a strong viscoelastic film that could be highly resistant to tension and able to withstand the film's thickness [13], preventing coalescence of bubbles and leading to more stable foams. As a matter of fact the presence of both glycocompounds and protein material deriving from macromolecular fractions of different molecular weights in the adsorption layer of the foam of sparkling wines has been reported [56], and the presence of aggregated materials involving yeast glycoproteins and other unidentified wine components has also been indicated as contributing to the foam stability of sparkling wines [57, 58]. In this sense, reconstitution experiments performed by adding in a model solution different molecular fractions isolated from wine indicated that a synergistic effect in foamability and foam stability exists between high and low molecular weight wine compounds [31]. The fraction most responsible for foam stability was mainly influenced by mannoproteins with low content of protein (5%) and the foamability by arabinogalactans and a hydrophobic low molecular weight fraction (< 1 kDa) [32].

ranging from 0.67 to 0.97, but these compounds did not show any effect on the foam stability time. Authors attributed this effect to the interaction of anthocyanins with wine proteins through hydrophobic interactions and hydrogen bonds. Attachment of a long aliphatic chain could confer interesting surfactant behavior on flavylium cations. Therefore, the product formed could be retained in the liquid/air interface, resulting in a reduction of the interfacial tension and an increase in the foamability. On the other hand, total proanthocyanidins showed high negative correlation with sparkling wine ability to foam (r = 0.73). Since proteins play an important role on the foamability of sparkling wines, the negative correlation of proanthocyanidins with foam height could be due to the precipitation of wine proteins by tannins. Cis-caftaric was the hidroxicinamic acid most negatively correlated with foam height (r = 0.65), while coumaric acid showed the most positive effect (r = 0.77) and isorhamnetin

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Some authors describe that lipids can accumulate in the foam, reducing surface tension and stabilizing it [63]. However, the researches made in wines to establish the possible relationships among lipid content, fatty acids, and foam behavior have produced contradictory findings (Table 1). The addition of octanoic and decanoic fatty acids to wines had a negative effect on the foam stability time, but it positively influenced foam collar height [16]. However, the addition of a lipid mixture to wine did not affect their foam, but when the ethanol concentration was reduced, authors observed an adverse effect on bubble lifetime [64]. They concluded that linolenic acid and palmitic acid were, respectively, the best indicators of foam stability and foam height in base wines and sparkling wines respectively, both having a positive influence [29].

Moreover, it was studied the influence of fatty acids (free and bound as ethyl esters) on wine foaming in different white wines and separated foam (Tables 1 and 2). The free fatty acids C8, C10, and C12 were negatively correlated with foam height with values ranging from 0.43 to 0.66, whereas the ethyl esters of hexanoic, octanoic, and decanoic acids were positively related with values ranging from 0.65 to 0.90. These authors found that the value of foam height was directly proportional to the ratio of esterified to non-esterified fatty acids. So, the higher the coefficient, the greater the foamability; thus, it appeared that it was the esterified forms of fatty acids that increased foam height [65]. It was also shown that monoacylglycerols of palmitic and stearic acids and glycerylethylene glycol fatty acid derivatives were surface active compounds preferentially partitioned by the sparkling wine foam rather than the liquid phase, allowing the inference of their role as key components in the promotion and stabiliza-

With regards to organic acids (Tables 1 and 2), López-Barajas et al. [12] observed low negative correlations between titratable acidity and foamability in grape juices of white varieties (r = 0.58). However, other studies showed that tartaric acid, titratable acidity and pH

was the flavonol with a major influence on foam height (r = 0.84).

3.6. Lipids

tion of sparkling wine foam [31].

3.7. Organic acids

The specific contribution of the different families of wine polysaccharides to the wine foaming properties has been recently studied by our research group [28]. Mannoproteins, glucans, polysaccharides rich in arabinose and galactose, rhamnogalacturonans type II, and homogalacturonans did not show any influence on the foamability of sparkling wines. On the contrary, positive influence was found between foam stability time and all wine polysaccharides, with the exception of rhamnogalacturonans type II. Surprisingly, polysaccharides rich in arabinose and galactose showed higher positive correlations on foam stability (r = 0.72) than mannoproteins (r = 0.47) [28] (Table 2).

#### 3.5. Polyphenols

It is widely known that polyphenols are highly reactive compounds. Some authors have tried to establish a correlation between them and the quality of foam in grape juices, base wines and sparkling wines. Polyphenols can interact with proteins and polysaccharides [36, 37], mainly the low molecular weight polyphenols [59], which participate in the hydration layer of the proteins [60]. Moreover, the formation of hydrogen bonds between the hydroxyl groups of the phenolic compounds and the polar head groups of proteins can be particularly relevant for the interaction with the air/liquid interface of the bubble film [61, 62]. These formed compounds could adsorb at the interface and form a stabilizing film around bubbles, which could promote foam formation [28].

Most of the studies carried out to correlate the influence of phenols on foam quality of sparkling wines have shown contradictory results [12, 26, 28, 40, 42, 45, 50, 61] (Tables 1 and 2). In fact, total phenolics did not shown correlation with any foam instrumental property in sparkling wines [28], but they showed a negative correlation with foam height in base wines (r = 0.45) [45], and a high positive correlation with foam height in grape juices (r = 0.76) [12]. Moreover, most of studies refer to global measurements of phenolic compounds, which could lead to inaccurate results difficult to understand. A recent study of our group has analyzed the relation of individual phenolics with foam parameters in white and rosé sparkling wines, which could be critical for their production [28].

The study concluded that each phenolic compound exhibits different behavior patterns on foam instrumental properties (Table 2). Non acylated, acetyl glucoside and coumaryl glucoside anthocyanins showed the highest positive correlations with foamability, with values ranging from 0.67 to 0.97, but these compounds did not show any effect on the foam stability time. Authors attributed this effect to the interaction of anthocyanins with wine proteins through hydrophobic interactions and hydrogen bonds. Attachment of a long aliphatic chain could confer interesting surfactant behavior on flavylium cations. Therefore, the product formed could be retained in the liquid/air interface, resulting in a reduction of the interfacial tension and an increase in the foamability. On the other hand, total proanthocyanidins showed high negative correlation with sparkling wine ability to foam (r = 0.73). Since proteins play an important role on the foamability of sparkling wines, the negative correlation of proanthocyanidins with foam height could be due to the precipitation of wine proteins by tannins. Cis-caftaric was the hidroxicinamic acid most negatively correlated with foam height (r = 0.65), while coumaric acid showed the most positive effect (r = 0.77) and isorhamnetin was the flavonol with a major influence on foam height (r = 0.84).

#### 3.6. Lipids

the foam of sparkling wines has been reported [56], and the presence of aggregated materials involving yeast glycoproteins and other unidentified wine components has also been indicated as contributing to the foam stability of sparkling wines [57, 58]. In this sense, reconstitution experiments performed by adding in a model solution different molecular fractions isolated from wine indicated that a synergistic effect in foamability and foam stability exists between high and low molecular weight wine compounds [31]. The fraction most responsible for foam stability was mainly influenced by mannoproteins with low content of protein (5%) and the foamability by arabinogalactans and a hydrophobic low molecular weight

212 Grapes and Wines - Advances in Production, Processing, Analysis and Valorization

The specific contribution of the different families of wine polysaccharides to the wine foaming properties has been recently studied by our research group [28]. Mannoproteins, glucans, polysaccharides rich in arabinose and galactose, rhamnogalacturonans type II, and homogalacturonans did not show any influence on the foamability of sparkling wines. On the contrary, positive influence was found between foam stability time and all wine polysaccharides, with the exception of rhamnogalacturonans type II. Surprisingly, polysaccharides rich in arabinose and galactose showed higher positive correlations on foam stability (r = 0.72) than manno-

It is widely known that polyphenols are highly reactive compounds. Some authors have tried to establish a correlation between them and the quality of foam in grape juices, base wines and sparkling wines. Polyphenols can interact with proteins and polysaccharides [36, 37], mainly the low molecular weight polyphenols [59], which participate in the hydration layer of the proteins [60]. Moreover, the formation of hydrogen bonds between the hydroxyl groups of the phenolic compounds and the polar head groups of proteins can be particularly relevant for the interaction with the air/liquid interface of the bubble film [61, 62]. These formed compounds could adsorb at the interface and form a stabilizing film around bubbles, which could promote

Most of the studies carried out to correlate the influence of phenols on foam quality of sparkling wines have shown contradictory results [12, 26, 28, 40, 42, 45, 50, 61] (Tables 1 and 2). In fact, total phenolics did not shown correlation with any foam instrumental property in sparkling wines [28], but they showed a negative correlation with foam height in base wines (r = 0.45) [45], and a high positive correlation with foam height in grape juices (r = 0.76) [12]. Moreover, most of studies refer to global measurements of phenolic compounds, which could lead to inaccurate results difficult to understand. A recent study of our group has analyzed the relation of individual phenolics with foam parameters in white and rosé sparkling wines,

The study concluded that each phenolic compound exhibits different behavior patterns on foam instrumental properties (Table 2). Non acylated, acetyl glucoside and coumaryl glucoside anthocyanins showed the highest positive correlations with foamability, with values

fraction (< 1 kDa) [32].

proteins (r = 0.47) [28] (Table 2).

3.5. Polyphenols

foam formation [28].

which could be critical for their production [28].

Some authors describe that lipids can accumulate in the foam, reducing surface tension and stabilizing it [63]. However, the researches made in wines to establish the possible relationships among lipid content, fatty acids, and foam behavior have produced contradictory findings (Table 1). The addition of octanoic and decanoic fatty acids to wines had a negative effect on the foam stability time, but it positively influenced foam collar height [16]. However, the addition of a lipid mixture to wine did not affect their foam, but when the ethanol concentration was reduced, authors observed an adverse effect on bubble lifetime [64]. They concluded that linolenic acid and palmitic acid were, respectively, the best indicators of foam stability and foam height in base wines and sparkling wines respectively, both having a positive influence [29].

Moreover, it was studied the influence of fatty acids (free and bound as ethyl esters) on wine foaming in different white wines and separated foam (Tables 1 and 2). The free fatty acids C8, C10, and C12 were negatively correlated with foam height with values ranging from 0.43 to 0.66, whereas the ethyl esters of hexanoic, octanoic, and decanoic acids were positively related with values ranging from 0.65 to 0.90. These authors found that the value of foam height was directly proportional to the ratio of esterified to non-esterified fatty acids. So, the higher the coefficient, the greater the foamability; thus, it appeared that it was the esterified forms of fatty acids that increased foam height [65]. It was also shown that monoacylglycerols of palmitic and stearic acids and glycerylethylene glycol fatty acid derivatives were surface active compounds preferentially partitioned by the sparkling wine foam rather than the liquid phase, allowing the inference of their role as key components in the promotion and stabilization of sparkling wine foam [31].

#### 3.7. Organic acids

With regards to organic acids (Tables 1 and 2), López-Barajas et al. [12] observed low negative correlations between titratable acidity and foamability in grape juices of white varieties (r = 0.58). However, other studies showed that tartaric acid, titratable acidity and pH increased foam height in grape juices, base and sparkling wines [12, 20, 25, 26, 29, 66]. In fact, pH and foamability in grape juices were highly correlated (r = 0.71) [12], while titratable acidity exhibited lower influence on foam height (r = 0.46) and foam stability time (r = 0.32) in white base wines [25]. In the same way, it was observed that acidity had a marked effect on foam since it modified protein solubility; if the juice acidity was low, protein hydrophobicity would be high, the surface activity could be increased, and then juice would have a higher foamability [35].

The effect of Botrytis cinerea on the foaming characteristics of sparkling wines has also been studied [44, 69, 70]. In these works, it was concluded that this infection can cause a drastic

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Diverse studies have been published about the influence of stabilization treatments, either using clarifiers or filtrations, on the foam quality of wines [20, 24, 41, 46, 47, 71–74]. In all cases, the foams were negatively affected by these treatments, and this was directly correlated with a decrease in the protein concentration. On the contrary, lysozyme additions made to the musts and wines before and after bentonite or charcoal treatments seem to have a protective effect on

Research conducted suggest that many compounds influence foam capacity of sparkling wines (Tables 1 and 2); however, the most influencing compounds on the foaming properties have proved to be total amino acids, polysaccharides, anthocyanins, coumaric acid and isorhamnetin, all of them showing correlation coefficients higher than 0.75 (Table 2). On the contrary, the alcohol content and the concentration of acid polysaccharides, proanthocyanidins and free SO2

In view of the results shown in Tables 1 and 2, it can be concluded that foamability and foam stability is a complicated issue. In fact, the foaming capacity of wines depends on a complex equilibrium among all the compounds that favor its formation and stability and those that do not. There is not one compound or group of compounds that is responsible for making and keeping good quality foam. Instead, foam quality depends on a synergistic relationship between

Foam behavior results from the synergistic interaction between the different foam active compounds which, due to aggregation or complex formation, may modify their surface-active properties. Thus, foaming properties not only are due to the presence or absence of a specific group of compounds but also are influenced by the net balance of the number and type of compounds ranging among different chemical structures. For this reason, and in order to ascertain which compounds have a major influence on the foam quality of sparkling wines, it is necessary to evaluate as many compounds together as possible, and to study the combined effect of all them. In this sense, statistical tools of multiple linear regression [12, 22, 26, 28, 75] and partial least squares regression analysis [29] has been used by several authors in an attempt to predict the foam properties of sparkling wines, and find out the chemical com-

Most of the studies include in the models all the variables that are usually analyzed in the wineries, and try to predict values for foamability, foam stability and Bickerman coefficient Σ [12, 26, 75]. Results of these researches have shown a great influence of proteins, SO2, absorbance at 280 nm, glycerol and maturation index. Moreover, stepwise analysis showed that the foam height and Bickerman coefficient of sulphited grape juices could be predicted with a probability higher than 89.97% by the following polynomial equations (Eq. (1) and (2)) [12]:

many different compounds that when acting together result in the foaming properties.

pound that provided the best predictive model of the foam properties.

reduction in foamability, since it uses up the proteins in the medium.

the wine proteins, and thereby an increase in foamability [71].

are the factors which most negatively affect foam quality (Table 2).

4. Prediction of foaming properties

Different authors agree in pointing to malic acid as an enhancer of the foam height in base wines and sparkling wines [25, 26, 40, 45], but also stated that malic acid reduces foam stability time [26, 40]. On the contrary, lactic acid exerted the opposite effect on foam height [25, 26]. Malic acid and lactic acid showed low negative correlations with foam height [25, 45], which could indicate that malolactic fermentation is not recommended as a way to maximize foamability in sparkling wines. Moreover, conflicting results have been published on the influence of lactic acid on foam stability time. Some authors have observed a positive influence of lactic acid on foam stability in base wines [26], while others showed the opposite effect in sparkling wines [66]. Other acids such as citric and galacturonic acid reduced foam stability time in base and sparkling wines [25, 26, 41]. Moreover, the presence of gluconic acid due to Botrytis cinerea was shown to negatively affect wine foamability (r = 0.36) [49].

#### 3.8. Others

Several authors agree that sulfur dioxide negatively affect the foaming qualities of wines [12, 26, 29, 45, 66] due to SO2 is a denaturing agent of proteins [16]. In fact, negative correlations have been obtained between free and total sulfur dioxide and foam height in grape juices and base wines [12, 45].

There is some controversy about the effect of ethanol content on foaming quality. Some authors consider that ethanol has beneficial effects on foam [25, 26] while others assign it negative contribution [45, 49, 66]. The negative effect of ethanol on foam seems to be dependent on its content [67]. This could be explained by the ethanol modification of the solvent properties, the interactions between the protein and the solvent, and the structure of the adsorption layer [68]. When the alcohol content is low, other surfactants can be more active and thus more easily adsorbed at the interface, stabilizing the foam formed [20, 64]. In this sense, higher alcohol content was reported to decrease foamability [16]; however, this effect could be counteracted by other compounds produced in the second fermentation. In this regard, juices with a maturation index [ratio between soluble solids (Brix) and titratable acidity (grams of tartaric acid per litter of juice) ranging from 4 to 5.5 had high foamability [12]. In fact, it was observed a high positive correlation between foam height and maturation index (r = 0.78) [12]. Subsequently, these results were confirmed, showing that maturation indexes for foamability and stability above 5.5 provided the wine with a less optimal alcoholic content for the formation of foam than wine produced from grapes with a maturation index within the stated range [45].

Glycerol is known to contribute to the viscosity of the wines. Due to its tensoactive properties, glycerol has shown a positive influence on foamability in sparkling wines [20, 32]. On the other hand, iron [34] and residual sugars [25, 29, 41] have been related with an improvement of foamability in sparkling wines.

The effect of Botrytis cinerea on the foaming characteristics of sparkling wines has also been studied [44, 69, 70]. In these works, it was concluded that this infection can cause a drastic reduction in foamability, since it uses up the proteins in the medium.

Diverse studies have been published about the influence of stabilization treatments, either using clarifiers or filtrations, on the foam quality of wines [20, 24, 41, 46, 47, 71–74]. In all cases, the foams were negatively affected by these treatments, and this was directly correlated with a decrease in the protein concentration. On the contrary, lysozyme additions made to the musts and wines before and after bentonite or charcoal treatments seem to have a protective effect on the wine proteins, and thereby an increase in foamability [71].

Research conducted suggest that many compounds influence foam capacity of sparkling wines (Tables 1 and 2); however, the most influencing compounds on the foaming properties have proved to be total amino acids, polysaccharides, anthocyanins, coumaric acid and isorhamnetin, all of them showing correlation coefficients higher than 0.75 (Table 2). On the contrary, the alcohol content and the concentration of acid polysaccharides, proanthocyanidins and free SO2 are the factors which most negatively affect foam quality (Table 2).

## 4. Prediction of foaming properties

increased foam height in grape juices, base and sparkling wines [12, 20, 25, 26, 29, 66]. In fact, pH and foamability in grape juices were highly correlated (r = 0.71) [12], while titratable acidity exhibited lower influence on foam height (r = 0.46) and foam stability time (r = 0.32) in white base wines [25]. In the same way, it was observed that acidity had a marked effect on foam since it modified protein solubility; if the juice acidity was low, protein hydrophobicity would be high, the surface activity could be increased, and then juice would have a higher foamability [35].

214 Grapes and Wines - Advances in Production, Processing, Analysis and Valorization

Different authors agree in pointing to malic acid as an enhancer of the foam height in base wines and sparkling wines [25, 26, 40, 45], but also stated that malic acid reduces foam stability time [26, 40]. On the contrary, lactic acid exerted the opposite effect on foam height [25, 26]. Malic acid and lactic acid showed low negative correlations with foam height [25, 45], which could indicate that malolactic fermentation is not recommended as a way to maximize foamability in sparkling wines. Moreover, conflicting results have been published on the influence of lactic acid on foam stability time. Some authors have observed a positive influence of lactic acid on foam stability in base wines [26], while others showed the opposite effect in sparkling wines [66]. Other acids such as citric and galacturonic acid reduced foam stability time in base and sparkling wines [25, 26, 41]. Moreover, the presence of gluconic acid due to

Several authors agree that sulfur dioxide negatively affect the foaming qualities of wines [12, 26, 29, 45, 66] due to SO2 is a denaturing agent of proteins [16]. In fact, negative correlations have been obtained between free and total sulfur dioxide and foam height in grape juices and

There is some controversy about the effect of ethanol content on foaming quality. Some authors consider that ethanol has beneficial effects on foam [25, 26] while others assign it negative contribution [45, 49, 66]. The negative effect of ethanol on foam seems to be dependent on its content [67]. This could be explained by the ethanol modification of the solvent properties, the interactions between the protein and the solvent, and the structure of the adsorption layer [68]. When the alcohol content is low, other surfactants can be more active and thus more easily adsorbed at the interface, stabilizing the foam formed [20, 64]. In this sense, higher alcohol content was reported to decrease foamability [16]; however, this effect could be counteracted by other compounds produced in the second fermentation. In this regard, juices with a maturation index [ratio between soluble solids (Brix) and titratable acidity (grams of tartaric acid per litter of juice) ranging from 4 to 5.5 had high foamability [12]. In fact, it was observed a high positive correlation between foam height and maturation index (r = 0.78) [12]. Subsequently, these results were confirmed, showing that maturation indexes for foamability and stability above 5.5 provided the wine with a less optimal alcoholic content for the formation of foam than wine produced from grapes with a maturation index within the stated range [45]. Glycerol is known to contribute to the viscosity of the wines. Due to its tensoactive properties, glycerol has shown a positive influence on foamability in sparkling wines [20, 32]. On the other hand, iron [34] and residual sugars [25, 29, 41] have been related with an improvement of

Botrytis cinerea was shown to negatively affect wine foamability (r = 0.36) [49].

3.8. Others

base wines [12, 45].

foamability in sparkling wines.

In view of the results shown in Tables 1 and 2, it can be concluded that foamability and foam stability is a complicated issue. In fact, the foaming capacity of wines depends on a complex equilibrium among all the compounds that favor its formation and stability and those that do not. There is not one compound or group of compounds that is responsible for making and keeping good quality foam. Instead, foam quality depends on a synergistic relationship between many different compounds that when acting together result in the foaming properties.

Foam behavior results from the synergistic interaction between the different foam active compounds which, due to aggregation or complex formation, may modify their surface-active properties. Thus, foaming properties not only are due to the presence or absence of a specific group of compounds but also are influenced by the net balance of the number and type of compounds ranging among different chemical structures. For this reason, and in order to ascertain which compounds have a major influence on the foam quality of sparkling wines, it is necessary to evaluate as many compounds together as possible, and to study the combined effect of all them. In this sense, statistical tools of multiple linear regression [12, 22, 26, 28, 75] and partial least squares regression analysis [29] has been used by several authors in an attempt to predict the foam properties of sparkling wines, and find out the chemical compound that provided the best predictive model of the foam properties.

Most of the studies include in the models all the variables that are usually analyzed in the wineries, and try to predict values for foamability, foam stability and Bickerman coefficient Σ [12, 26, 75]. Results of these researches have shown a great influence of proteins, SO2, absorbance at 280 nm, glycerol and maturation index. Moreover, stepwise analysis showed that the foam height and Bickerman coefficient of sulphited grape juices could be predicted with a probability higher than 89.97% by the following polynomial equations (Eq. (1) and (2)) [12]:

$$\text{HM} = -126.80 + 1.04\\\text{Combined SO}\_2 + 16.85\\\text{OD} \\ 280 + 1.07 \\\text{Proteins} - 44.40 \\\text{Glycerol} \quad \text{(1)}$$

$$
\sum = 4.76 + 1.68
\text{Maturation index} - 5.48
\text{Tartaric acid} + 0.34
\text{Glucose} \tag{2}
$$

5. Conclusions

Author details

References

Finca La Grajera, Logroño, Spain

24.7.2009. pp. 1-59

tb00051.x

6.00001-X

In conclusion, this work shows that the foam properties of sparkling wines are ruled by a large number of molecules that act in a synergistic way. Nevertheless, some compounds seem to be

Influence of Wine Chemical Compounds on the Foaming Properties of Sparkling Wines

http://dx.doi.org/10.5772/intechopen.70859

217

Although contradictory results have sometimes been obtained, a high correlation (≥ 0.75) has been found in the literature between the foam properties of sparkling wines and the content of total amino acids, polysaccharides, anthocyanins, coumaric acid and isorhamnetin. On the contrary, the alcohol content and the concentration of acid polysaccharides, proanthocyanidins

A recent study, by means of prediction models, has also concluded that the different forms of malvidin shows the highest influence on the foam height and foam stability height parameters, followed by amino acid compounds, while foam stability model was only predicted by polysaccharides from grapes, concretely by polysaccharides rich in arabinose and galactose.

These research findings provide industry with a better understanding of the compositional

Institute of Vine and Wine Sciences (University of La Rioja, Government of La Rioja and CSIC)

[1] OIV's Focus. The sparkling wine market. [Internet]. 2017. Available from: http://www. oiv.int/public/medias/3098/les-vins-effervescents-en-complet.pdf [Accessed: 2017-05-15] [2] Commission Regulation (EC) No 606/2009 of 10 July 2009 laying down certain detailed rules for implementing Council Regulation (EC) No 479/2008 as regards the categories of grapevine products, oenological practices and the applicable restrictions. OJ L 193,

[3] Alexandre H, Guilloux-Benatier M. Yeast autolysis in sparkling wine. A review. Australian Journal of Grape and Wine Research. 2006;12:119-127. DOI: 10.1111/j.1755-0238.2006.

[4] Buxaderas S, López-Tamames E. Sparkling wines: Features and trends from tradition. Advances in Food and Nutrition Research. 2012;66:1-45. DOI: 10.1016/B978-0-12-394597-

more relevant than others to explain their foam properties.

factors influencing the foam quality of sparkling wines.

and free SO2 are the factors which most negatively affect foam quality.

Leticia Martínez-Lapuente, Belén Ayestarán and Zenaida Guadalupe\*

\*Address all correspondence to: zenaida.guadalupe@unirioja.es

Other study conducted by Pueyo et al. [29] applied PLS regression to predict foam height and foam stability in base wines and sparkling wines using 73 chemical variables analyzed. Tartaric acid, glucose, total palmitoleic acid and protein content were the most influent variables in the prediction of foam height in base wines. However, total contents of oleic, palmitic, and stearic acids, and the content of 1-hexanol were the most important variables for predicting foam height in sparkling wines. With regards to foam stability, the variables with high predictive relevance in base wines were the total content of linolenic and undecanoic acids and the free content of undecanoic acid, while the total content of SO2, the isobutanol, the total acidity, and proteins were the variables with high predictive relevance.

Moreno-Arribas et al. [22] observed that neutral polysaccharides, protein nitrogen and phenylalanine displayed high positive contribution to the prediction of maximum foam height (Peak H), and height at which the foam stabilizes in sparkling wines (Plateau H). The fitted final models, which presented the following adjusted equations (Eq. (3) and (4)), explained 76% of Peak H variation and 70% of the variation of Plateau H.

$$\text{Peak H} = 194.31 + 0.37 \text{Neutral polygons} \\ \text{Arrides} + 59.68 \text{Protein nitrogen} \tag{3}$$

$$\text{Plateau H} = 180.45 + 0.17 \text{Neutral polygons} \\ \text{chaires} + 3.80 \text{Phenyalinine} \\ \tag{4}$$

A recent work carried out in our group in 2015 used multiple linear regression analysis in white and rosé sparkling wines differentiating models which anthocyanins were included. It was concluded that the different forms of malvidin had the highest influence on the foam height and foam stability height parameters, followed by amino acid compounds ((Eq. (5) to (8)), while foam stability model was only predicted by polysaccharides from grapes, concretely by polysaccharides rich in arabinose and galactose ((Eq. (9) and (10)) [28].

$$\begin{aligned} \text{HM (ros\'e sprakling wines)} &= 84.882 + 0.065 \text{Total amino acids} \\ &+ 5.242 \text{Non-acylated antibocycles} - 0.477 \text{Total pronanthacyandins} \{ \text{R}^2 = 90.2\% \} \end{aligned} \tag{5}$$

HM white sparkling wines � � <sup>¼</sup> <sup>66</sup>:<sup>997</sup> <sup>þ</sup> <sup>0</sup>:206Total amino acids R2 <sup>¼</sup> <sup>73</sup>:3% � � (6)

HS rosé sparkling wines � � <sup>¼</sup> <sup>9</sup>:<sup>730</sup> <sup>þ</sup> <sup>0</sup>:331Basic amino acids

$$+2.492\text{Acetyl} - \text{glucose}\newline\text{ando}\newline\text{a}\newline\text{b}\newline\text{g}\newline\text{c}\newline\text{d}\newline\text{c}\newline\text{g}\newline\text{Total biogenic amines}\newline\text{ }\tag{7}$$

<sup>þ</sup> <sup>0</sup>:013Neutral amino acids <sup>R</sup><sup>2</sup> <sup>¼</sup> <sup>97</sup>:4% � �

$$\text{MS (white sparkings)} = 13.258 + 2.906\\\text{Total bigger amines} \{\text{R}^2 = 19.2\%\} \tag{8}$$

TS rosé sparkling wines � � ¼ �22:<sup>277</sup> <sup>þ</sup> <sup>0</sup>:489Polysaccharides from grapes R<sup>2</sup> <sup>¼</sup> <sup>46</sup>:7% � � (9)

$$\text{TS(white spark wins)} = -7.348 + 0.359\\\text{Polysacharides from gaps} \{ \mathbf{R}^2 = 33.9\% \} \text{ (10)}$$

## 5. Conclusions

HM ¼ �126:80 þ 1:04Combined SO2 þ 16:85OD280 þ 1:07Proteins � 44:40Glycerol (1)

Other study conducted by Pueyo et al. [29] applied PLS regression to predict foam height and foam stability in base wines and sparkling wines using 73 chemical variables analyzed. Tartaric acid, glucose, total palmitoleic acid and protein content were the most influent variables in the prediction of foam height in base wines. However, total contents of oleic, palmitic, and stearic acids, and the content of 1-hexanol were the most important variables for predicting foam height in sparkling wines. With regards to foam stability, the variables with high predictive relevance in base wines were the total content of linolenic and undecanoic acids and the free content of undecanoic acid, while the total content of SO2, the isobutanol, the

Moreno-Arribas et al. [22] observed that neutral polysaccharides, protein nitrogen and phenylalanine displayed high positive contribution to the prediction of maximum foam height (Peak H), and height at which the foam stabilizes in sparkling wines (Plateau H). The fitted final models, which presented the following adjusted equations (Eq. (3) and (4)), explained

A recent work carried out in our group in 2015 used multiple linear regression analysis in white and rosé sparkling wines differentiating models which anthocyanins were included. It was concluded that the different forms of malvidin had the highest influence on the foam height and foam stability height parameters, followed by amino acid compounds ((Eq. (5) to (8)), while foam stability model was only predicted by polysaccharides from grapes, concretely

Peak H ¼ 194:31 þ 0:37Neutral polysaccharides þ 59:68Protein nitrogen (3)

Plateau H ¼ 180:45 þ 0:17Neutral polysaccharides þ 3:80Phenylanine (4)

<sup>þ</sup> <sup>5</sup>:242Non-acylated anthocyanins � <sup>0</sup>:477Total proanthocyanidins R2 <sup>¼</sup> <sup>90</sup>:2% � � (5)

HM white sparkling wines � � <sup>¼</sup> <sup>66</sup>:<sup>997</sup> <sup>þ</sup> <sup>0</sup>:206Total amino acids R2 <sup>¼</sup> <sup>73</sup>:3% � � (6)

þ 2:492Acetyl � glucoside anthocyanins þ 0:995Total biogenic amines

HS white sparkling wines � � <sup>¼</sup> <sup>13</sup>:<sup>258</sup> <sup>þ</sup> <sup>2</sup>:906Total biogenic amines R2 <sup>¼</sup> <sup>19</sup>:2% � � (8)

TS rosé sparkling wines � � ¼ �22:<sup>277</sup> <sup>þ</sup> <sup>0</sup>:489Polysaccharides from grapes R<sup>2</sup> <sup>¼</sup> <sup>46</sup>:7% � � (9)

TS white sparkling wines � � ¼ �7:<sup>348</sup> <sup>þ</sup> <sup>0</sup>:359Polysaccharides from grapes R<sup>2</sup> <sup>¼</sup> <sup>33</sup>:9% � � (10)

total acidity, and proteins were the variables with high predictive relevance.

by polysaccharides rich in arabinose and galactose ((Eq. (9) and (10)) [28].

HS rosé sparkling wines � � <sup>¼</sup> <sup>9</sup>:<sup>730</sup> <sup>þ</sup> <sup>0</sup>:331Basic amino acids

HM rosé sparkling wines � � <sup>¼</sup> <sup>84</sup>:<sup>882</sup> <sup>þ</sup> <sup>0</sup>:065Total amino acids

<sup>þ</sup> <sup>0</sup>:013Neutral amino acids <sup>R</sup><sup>2</sup> <sup>¼</sup> <sup>97</sup>:4% � �

76% of Peak H variation and 70% of the variation of Plateau H.

216 Grapes and Wines - Advances in Production, Processing, Analysis and Valorization

<sup>X</sup> <sup>¼</sup> <sup>4</sup>:<sup>76</sup> <sup>þ</sup> <sup>1</sup>:68Maturation index � <sup>5</sup>:48Tartaric acid <sup>þ</sup> <sup>0</sup>:34Glucose (2)

In conclusion, this work shows that the foam properties of sparkling wines are ruled by a large number of molecules that act in a synergistic way. Nevertheless, some compounds seem to be more relevant than others to explain their foam properties.

Although contradictory results have sometimes been obtained, a high correlation (≥ 0.75) has been found in the literature between the foam properties of sparkling wines and the content of total amino acids, polysaccharides, anthocyanins, coumaric acid and isorhamnetin. On the contrary, the alcohol content and the concentration of acid polysaccharides, proanthocyanidins and free SO2 are the factors which most negatively affect foam quality.

A recent study, by means of prediction models, has also concluded that the different forms of malvidin shows the highest influence on the foam height and foam stability height parameters, followed by amino acid compounds, while foam stability model was only predicted by polysaccharides from grapes, concretely by polysaccharides rich in arabinose and galactose.

These research findings provide industry with a better understanding of the compositional factors influencing the foam quality of sparkling wines.

## Author details

Leticia Martínez-Lapuente, Belén Ayestarán and Zenaida Guadalupe\*

\*Address all correspondence to: zenaida.guadalupe@unirioja.es

Institute of Vine and Wine Sciences (University of La Rioja, Government of La Rioja and CSIC) Finca La Grajera, Logroño, Spain

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**Chapter 11**

**Provisional chapter**

**Occurrence and Analysis of Sulfur Compounds in Wine**

Sulfur compounds play an important role in the sensory characteristics of wine. These molecules can derive from the grape, in which the non-volatile forms are usually present as glycosylated molecules, the metabolic activities of yeast and bacteria, the chemical reactions taking place during the wine aging and storage, and the environment. The sulfur compounds include molecules positively correlated to the aromatic profile of wine, namely the volatile thiols, and are responsible for certain defects, imparting notes described as cabbage, onion, rotten egg, garlic, sulfur and rubber. Due to the low concentration of these molecules in wine, their high reactivity and the matrix complexity, the analytical methods which enable their detection and quantification represent a challenge. The solid phase microextraction (SPME) technique has been developed for sulfur compounds associated with off-flavors. The analysis of volatile thiols usually requires a derivatization followed by gas chromatography (GC)-MS or UPLC-MS methods. Besides the sulfur-containing aromas, another sulfur compound that deserves mention is the reduced glutathione (GSH) which has been widely studied due to its antioxidant properties. The analysis of GSH has been proposed using a liquid chromatography technique

(HPLC or UPLC) coupled with fluorescence, MS and UV detectors.

**Keywords:** sulfur off-flavors, volatile thiols, reduced glutathione, sample preparation,

Sulfur-containing compounds strongly affect the sensory properties of wine and must. They include aromatic molecules, off-flavors and a well-known non-volatile compound with antioxidant properties such as glutathione. The sulfur-containing compounds can be derived from the grape in which the non-volatile forms are usually present, the metabolic activities of yeast and bacteria, the chemical reactions taking place during the wine aging and storage, and the environment [1]. Their presence in wine can be the result of both enzymatic and non-enzymatic

**Occurrence and Analysis of Sulfur Compounds in Wine**

DOI: 10.5772/intechopen.72823

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution,

© 2018 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

and reproduction in any medium, provided the original work is properly cited.

Daniela Fracassetti and Ileana Vigentini

Daniela Fracassetti and Ileana Vigentini

http://dx.doi.org/10.5772/intechopen.72823

**Abstract**

analysis, wine

**1. Introduction**

Additional information is available at the end of the chapter

Additional information is available at the end of the chapter

**Provisional chapter**

## **Occurrence and Analysis of Sulfur Compounds in Wine Occurrence and Analysis of Sulfur Compounds in Wine**

DOI: 10.5772/intechopen.72823

Daniela Fracassetti and Ileana Vigentini Daniela Fracassetti and Ileana Vigentini Additional information is available at the end of the chapter

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/intechopen.72823

#### **Abstract**

Sulfur compounds play an important role in the sensory characteristics of wine. These molecules can derive from the grape, in which the non-volatile forms are usually present as glycosylated molecules, the metabolic activities of yeast and bacteria, the chemical reactions taking place during the wine aging and storage, and the environment. The sulfur compounds include molecules positively correlated to the aromatic profile of wine, namely the volatile thiols, and are responsible for certain defects, imparting notes described as cabbage, onion, rotten egg, garlic, sulfur and rubber. Due to the low concentration of these molecules in wine, their high reactivity and the matrix complexity, the analytical methods which enable their detection and quantification represent a challenge. The solid phase microextraction (SPME) technique has been developed for sulfur compounds associated with off-flavors. The analysis of volatile thiols usually requires a derivatization followed by gas chromatography (GC)-MS or UPLC-MS methods. Besides the sulfur-containing aromas, another sulfur compound that deserves mention is the reduced glutathione (GSH) which has been widely studied due to its antioxidant properties. The analysis of GSH has been proposed using a liquid chromatography technique (HPLC or UPLC) coupled with fluorescence, MS and UV detectors.

**Keywords:** sulfur off-flavors, volatile thiols, reduced glutathione, sample preparation, analysis, wine

## **1. Introduction**

Sulfur-containing compounds strongly affect the sensory properties of wine and must. They include aromatic molecules, off-flavors and a well-known non-volatile compound with antioxidant properties such as glutathione. The sulfur-containing compounds can be derived from the grape in which the non-volatile forms are usually present, the metabolic activities of yeast and bacteria, the chemical reactions taking place during the wine aging and storage, and the environment [1]. Their presence in wine can be the result of both enzymatic and non-enzymatic

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2018 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

mechanisms. In the first case, the sulfur compounds represent the products of metabolic and fermentative pathways whose substrates are both amino acids and some sulfur-containing pesticides. When yeast and bacteria metabolize these thiols, the released sulfur compounds are generally considered off-flavors [2]. Non-enzymatic processes include photochemical, thermal and other chemical reactions of sulfur compounds during winemaking and storage [3]. The reactions involving the sulfur-containing amino acids, in particular, can bring about the light-struck taste in case the bottled white wine is exposed to light greatly affecting the sensory properties [4, 5]. The sulfur-containing off-flavors impart negative notes such as cabbage, onion, rotten egg, garlic, sulfur and rubber [6]. Among them, hydrogen sulfide is probably the best-known sulfur compound in wine. Hydrogen sulfide is a very reactive species and can trigger reactions generating compounds such as mercaptans, dimethyl sulfide and polysulfide, which also have a negative impact on the wine aroma. The long-chain polyfunctional sulfur compounds, also known as volatile thiols, are one of the most important groups of aroma compounds in wine, which confer pleasant aromatic notes at trace levels; at high concentrations, these compounds can be objectionable yet [3, 7–9]. Volatile thiols such as 4-mercapto-4-methylpentan-2-one (4MMP), 3-mercaptohexan-1-ol (3MH) and 3-mercaptohexyl acetate (3MHA) are particularly important, for example, for characterizing the typical aroma of Sauvignon Blanc wine [10]. Moreover, in some wines, 4-mercapto-4-methylpentan-2-ol (4MMPOH) is also detected but its concentration is lower than the thiol-related aromas mentioned above [11].

Besides the sulfur compounds contributing to the sensory characteristics of wine, the glutathione is of particular interest due to its antioxidant properties. This tripeptide can limit the browning of white must as it reduces back the *o*-quinones to the correspondent phenols and, consequently, the formation of the brown polymers are avoided [12].

> precursors could produce hydrogen sulfite, methanthiol and dimethylsulfide during fermentation and not all these compounds are released in gaseous form. The chemistry behind the reactions and regulation mechanisms involving hydrogen sulfite and other mercaptans produced by the yeast is not well understood [17]. Methionine can be metabolized by yeast to produce fusel alcohol, methionol and 3-methylthio-1-propanol imparting cabbage and cauliflower aromas. Cysteine is a precursor of S-containing heterocycle compounds; these compounds can be metabolized by *Oenococcus oeni* and aroma descriptors such as sulfury, floral, fruity, toasted and roasted can be imparted to the wine [2, 18]. The microbial degradation of these amino acids can lead to the presence of dimethyl sulfide which has shown the property of enhancing the wine aroma due to the interactions with other volatiles, such as esters and norisoprenoids [19, 20]. Nevertheless, dimethyl sulfide imparts notes such as asparagus, corn and molasses which are undesired characters in white wine to a certain extent, although the aroma complexity is increased [21]. The levels of dimethyl sulfide, methionol, diethyl sulfide and diethyl disulfide increase as the aging time and storage temperature increase and they can influence the aroma complexity of aged wine [13, 22]. Mercaptans are responsible for reduced aroma in wine. Different strategies can be used for the removal of sulfur off-flavors. The addition of diammonium phosphate or other yeast-based nutrients leads to an increase of the readily assimilable nitrogen enabling the yeast to convert sulfur-containing amino acids

**Table 1.** Common fermentative sulfur compounds and off-flavors found in wines [3, 22, 120].

**Compound Olfactory description Perception threshold** 

Hydrogen sulfide Rotten eggs, reduced taste 0.001–150 0–370 Methanethiol Cooked cabbage, reduced taste 0.3 0–16 Ethanthiol Onion, rubber, putrefaction 1.1 0–50 Dimethyl sulfide Cabbage, asparagus, corn, molasses 10–160 0–910 Carbon disulfide Cabbage, rubber >38 0–18 Dimethyl trisulfide Cabbage, onions, cooked vegetable 0.1 0–111 Diethyl sulfide Garlic 0.93–18 0–10 Dimethyl disulfide Cooked cabbage, asparagus, onions 20–45 0–160 Diethyl disulfide Garlic, onion, burnt rubber 4.3–40 0–160 2-Mercaptoethanol Barnyard-like, poultry, farmyard 130 0–400 Methylthioacetate Sulfurous, rotten vegetables 300 0–115 S-Ethylthioacetate Sulfurous 40 0–180 2-(Methylthio)-1-ethanol Cauliflower, French bean 250 0–139 2-(Methylthio)-1-propanol Cauliflower, cooked cabbage 1200 0–5655 2-(Methylthio)-1-butanol Onion, garlic, earthy 100 0–180 Benzothiazole Rubber 50–350 0–30 5-(2-Hydroxyethyl)-4-methylthiazole Green 100–1000 5–50

**(μg/L)**

Occurrence and Analysis of Sulfur Compounds in Wine http://dx.doi.org/10.5772/intechopen.72823

> **Range in wine (μg/L)**

227

## **2. Aromatic compounds containing sulfur**

#### **2.1. Sulfur off-flavors**

Sulfur-containing compounds are mainly associated with off-odors due to the presence of hydrogen sulfide and mercaptans whose concentrations are typically low as well as their perception threshold [2]. The most abundant sulfur off-flavors found in wine are represented by hydrogen sulfide, methanethiol, ethanethiol, dimethylmercaptans (dimethylsulfide, dimethyldisulfide, dimethyltrisulfide) [13] and other sulfur-containing compounds responsible for off-flavors in wine (**Table 1**). Hydrogen sulfide is the most recognized sulfur compound associated with the rotten egg aroma, it is highly volatile and has a low perception threshold (10–80 μg/L). Even if hydrogen sulfide could be easily removed by copper treatment [14], it is very reactive specie participating in the generation of other mercaptans, such as dimethylsulfide and polysulfide, and ethanthiol, the latter due to its combination with ethanol or acetaldehyde [7]. *Saccharomyces cerevisiae* can release this compound through the degradation of sulfur-containing amino acids and the reduction of elemental sulfur, sulfite or sulfate [15]. However, its synthesis is limited when nitrogen derived from either amino acids (except for cysteine) or ammonium is added [13]. Variable amounts of hydrogen sulfite can be released during the alcoholic fermentation. Smith and co-authors [16] suggest that the sulfur-containing


**Table 1.** Common fermentative sulfur compounds and off-flavors found in wines [3, 22, 120].

mechanisms. In the first case, the sulfur compounds represent the products of metabolic and fermentative pathways whose substrates are both amino acids and some sulfur-containing pesticides. When yeast and bacteria metabolize these thiols, the released sulfur compounds are generally considered off-flavors [2]. Non-enzymatic processes include photochemical, thermal and other chemical reactions of sulfur compounds during winemaking and storage [3]. The reactions involving the sulfur-containing amino acids, in particular, can bring about the light-struck taste in case the bottled white wine is exposed to light greatly affecting the sensory properties [4, 5]. The sulfur-containing off-flavors impart negative notes such as cabbage, onion, rotten egg, garlic, sulfur and rubber [6]. Among them, hydrogen sulfide is probably the best-known sulfur compound in wine. Hydrogen sulfide is a very reactive species and can trigger reactions generating compounds such as mercaptans, dimethyl sulfide and polysulfide, which also have a negative impact on the wine aroma. The long-chain polyfunctional sulfur compounds, also known as volatile thiols, are one of the most important groups of aroma compounds in wine, which confer pleasant aromatic notes at trace levels; at high concentrations, these compounds can be objectionable yet [3, 7–9]. Volatile thiols such as 4-mercapto-4-methylpentan-2-one (4MMP), 3-mercaptohexan-1-ol (3MH) and 3-mercaptohexyl acetate (3MHA) are particularly important, for example, for characterizing the typical aroma of Sauvignon Blanc wine [10]. Moreover, in some wines, 4-mercapto-4-methylpentan-2-ol (4MMPOH) is also detected but its concentration is lower than the thiol-related aromas mentioned above [11].

226 Grapes and Wines - Advances in Production, Processing, Analysis and Valorization

Besides the sulfur compounds contributing to the sensory characteristics of wine, the glutathione is of particular interest due to its antioxidant properties. This tripeptide can limit the browning of white must as it reduces back the *o*-quinones to the correspondent phenols and,

Sulfur-containing compounds are mainly associated with off-odors due to the presence of hydrogen sulfide and mercaptans whose concentrations are typically low as well as their perception threshold [2]. The most abundant sulfur off-flavors found in wine are represented by hydrogen sulfide, methanethiol, ethanethiol, dimethylmercaptans (dimethylsulfide, dimethyldisulfide, dimethyltrisulfide) [13] and other sulfur-containing compounds responsible for off-flavors in wine (**Table 1**). Hydrogen sulfide is the most recognized sulfur compound associated with the rotten egg aroma, it is highly volatile and has a low perception threshold (10–80 μg/L). Even if hydrogen sulfide could be easily removed by copper treatment [14], it is very reactive specie participating in the generation of other mercaptans, such as dimethylsulfide and polysulfide, and ethanthiol, the latter due to its combination with ethanol or acetaldehyde [7]. *Saccharomyces cerevisiae* can release this compound through the degradation of sulfur-containing amino acids and the reduction of elemental sulfur, sulfite or sulfate [15]. However, its synthesis is limited when nitrogen derived from either amino acids (except for cysteine) or ammonium is added [13]. Variable amounts of hydrogen sulfite can be released during the alcoholic fermentation. Smith and co-authors [16] suggest that the sulfur-containing

consequently, the formation of the brown polymers are avoided [12].

**2. Aromatic compounds containing sulfur**

**2.1. Sulfur off-flavors**

precursors could produce hydrogen sulfite, methanthiol and dimethylsulfide during fermentation and not all these compounds are released in gaseous form. The chemistry behind the reactions and regulation mechanisms involving hydrogen sulfite and other mercaptans produced by the yeast is not well understood [17]. Methionine can be metabolized by yeast to produce fusel alcohol, methionol and 3-methylthio-1-propanol imparting cabbage and cauliflower aromas. Cysteine is a precursor of S-containing heterocycle compounds; these compounds can be metabolized by *Oenococcus oeni* and aroma descriptors such as sulfury, floral, fruity, toasted and roasted can be imparted to the wine [2, 18]. The microbial degradation of these amino acids can lead to the presence of dimethyl sulfide which has shown the property of enhancing the wine aroma due to the interactions with other volatiles, such as esters and norisoprenoids [19, 20]. Nevertheless, dimethyl sulfide imparts notes such as asparagus, corn and molasses which are undesired characters in white wine to a certain extent, although the aroma complexity is increased [21]. The levels of dimethyl sulfide, methionol, diethyl sulfide and diethyl disulfide increase as the aging time and storage temperature increase and they can influence the aroma complexity of aged wine [13, 22]. Mercaptans are responsible for reduced aroma in wine. Different strategies can be used for the removal of sulfur off-flavors. The addition of diammonium phosphate or other yeast-based nutrients leads to an increase of the readily assimilable nitrogen enabling the yeast to convert sulfur-containing amino acids into hydrogen sulfide [23]. The aeration of wine during the alcoholic fermentation as well as wine racking in aerated conditions showed a protective effect against the formation of sulfur off-flavors. The oxygen positively contributes to the fermentative capacity of the yeast [24, 25]. Thereafter, the oxygen presence maintains lower levels of sulfur off-flavors during storage [26] probably due to either the formation molecular mass sulfur compounds or the quinone ability for trapping the sulfur compounds [16]. The aging of the lees can have a positive effect since the mercaptans can link to the cysteinyl residues of the yeast cell wall to give disulfides [27]. Wine treatment with copper or silver showed to be affective for the removal of hydrogen sulfide as copper or silver sulfide precipitates were formed [28]. However, recent studies have indicated that copper addition post-bottling increases the level of sulfur off-flavors during aging [24, 29, 30]. The effect of copper still needs to be clarified as well as its dosages.

The sulfur-containing compounds are associated with the occurrence of light-struck taste, a defect affecting white wine bottled in clear bottles and exposed to light. The photochemical oxidations may affect different wine components including phenols, acids and alcohols [31, 32]. The formation of the light-struck taste is related to the presence of riboflavin, a vitamin highly sensitive to light and methionine. The pathway proposed by Maujean and Seguin [4] involves the photoxidative degradation of methionine to give methional and reduced riboflavin. Methional is unstable when exposed to light and decomposes to acrolein and methanethiol (**Figure 1**). Two molecules of the latter eventually yield dimethyl disulfide (DMDS). The lightstruck taste is described as cooked cabbage mainly due to the formation of methanethiol and DMDS though hydrogen sulfide may have a role [33–35]. Light exposure can also lead to the formation of other undesirable compounds, such as furfural [36] which has been positively correlated to the "cooked vegetable" aroma of white wines stored under oxidative conditions [37, 38]. Not all wines are susceptible to developing this defect. The concentration of riboflavin plays the major role [5, 39] and when it is lower than 80–100 μg/L, the risk of the light-struck taste decreases. The riboflavin content in the grape hardly exceeds a few tens of micrograms per liter and the metabolic activities of *S. cerevisiae* affect its levels in wine [40, 41]. The levels of riboflavin can be reduced by means of wine treatment with bentonite or charcoal as well as by using a low producer yeast strain of this vitamin. Finally, nutrients commonly employed for favoring yeast growth could further increase the amount of riboflavin in wine [41]. Moreover, the amounts of methionine could influence the occurrence of light-struck taste as well as the presence of oxygen [42]. The light-struck taste can be limited by the addition of antioxidant compounds. The use of gallic tannins seems to be particularly promising in protecting wine possibly due to the binding of sulfur off-flavors and the quinones present in the polyphenol-based mixture [42].

molecules if their concentration is close to the perception threshold [22, 49, 50]. 3MH and 3MHA have olfactory perception thresholds of 60 and 4 ng/L, respectively and they are responsible for passion fruit-like and grapefruit-like olfactory notes [11]. These compounds were also found in red wines [51]. The 4MMP has an olfactory perception threshold of 0.8 ng/L, and its aroma is described as box tree-like, black currant-like, or even cat urine-like when occurring at high concentration [10]. The 4MMPOH has a perception threshold of 55 ng/L in aqueous alcoholic solution and it is reminiscent of citrus zest and grapefruit [44]. 3MH is the most abundant in wine, in concentrations generally higher than its perception threshold, while 4MMPOH in wine is generally lower than its perception threshold. The level of 4MMP is dependent on the grape cultivar and it changes between different samples in the same cultivar [52]. The concentrations of 3MH and 3MHA in Sauvignon Blanc wines from different countries have been reported to be in the range 688–18,681 and 10–2507 ng/L, respectively [53–55]. Besides Sauvignon Blanc wines, the volatile thiols were detected in white wine made from different *Vitis vinifera* grape varieties, such as Gewürtztraminer, Muscat, Riesling, Sylvaner, Pinot Gris, Pinot Blanc, Colombard, Petit Manseng, botrytised Semillon and Grenache [44, 56]. Moreover, these compounds were also found in certain Italian autochthonous varieties including Verdicchio Bianco (also known as

Occurrence and Analysis of Sulfur Compounds in Wine http://dx.doi.org/10.5772/intechopen.72823 229

**Figure 1.** Reaction scheme of methional formation due to light exposure [4].

Trebbiano di Lugana) [57], Arneis [58], Grillo and Catarratto Bianco Comune [59].

The content of volatile thiols decreases during wine aging, according to the oxidative conditions. Glutathione, sulfur dioxide and anthocyanin content exert a protective effect. In contrast,

#### **2.2. Varietal thiols**

The varietal thiols including 3-mercaptohexan-1-ol (3MH), 3-mercaptohexylacetate (3MHA) and 4-mercapto-4-methylpentan-2-one (4MMP) are sulfur-containing aromas associated with the typical flavor of Sauvignon Blanc wines [11, 43, 44]. Moreover, in some wine, 4-mercapto-4-methylpentan-2-ol (4MMPOH) was also detected, but its level was lower than the volatile thiols mentioned above [44] (**Table 2**). They are characterized by some fruity aromas, like cassis [45], grapefruit [46], passion fruit [47] and guava [48]. The impact of sulfur compounds on wine aroma has been updated in literature and the varietal character is affected by several of these

**Figure 1.** Reaction scheme of methional formation due to light exposure [4].

into hydrogen sulfide [23]. The aeration of wine during the alcoholic fermentation as well as wine racking in aerated conditions showed a protective effect against the formation of sulfur off-flavors. The oxygen positively contributes to the fermentative capacity of the yeast [24, 25]. Thereafter, the oxygen presence maintains lower levels of sulfur off-flavors during storage [26] probably due to either the formation molecular mass sulfur compounds or the quinone ability for trapping the sulfur compounds [16]. The aging of the lees can have a positive effect since the mercaptans can link to the cysteinyl residues of the yeast cell wall to give disulfides [27]. Wine treatment with copper or silver showed to be affective for the removal of hydrogen sulfide as copper or silver sulfide precipitates were formed [28]. However, recent studies have indicated that copper addition post-bottling increases the level of sulfur off-flavors during

228 Grapes and Wines - Advances in Production, Processing, Analysis and Valorization

aging [24, 29, 30]. The effect of copper still needs to be clarified as well as its dosages.

of sulfur off-flavors and the quinones present in the polyphenol-based mixture [42].

The varietal thiols including 3-mercaptohexan-1-ol (3MH), 3-mercaptohexylacetate (3MHA) and 4-mercapto-4-methylpentan-2-one (4MMP) are sulfur-containing aromas associated with the typical flavor of Sauvignon Blanc wines [11, 43, 44]. Moreover, in some wine, 4-mercapto-4-methylpentan-2-ol (4MMPOH) was also detected, but its level was lower than the volatile thiols mentioned above [44] (**Table 2**). They are characterized by some fruity aromas, like cassis [45], grapefruit [46], passion fruit [47] and guava [48]. The impact of sulfur compounds on wine aroma has been updated in literature and the varietal character is affected by several of these

**2.2. Varietal thiols**

The sulfur-containing compounds are associated with the occurrence of light-struck taste, a defect affecting white wine bottled in clear bottles and exposed to light. The photochemical oxidations may affect different wine components including phenols, acids and alcohols [31, 32]. The formation of the light-struck taste is related to the presence of riboflavin, a vitamin highly sensitive to light and methionine. The pathway proposed by Maujean and Seguin [4] involves the photoxidative degradation of methionine to give methional and reduced riboflavin. Methional is unstable when exposed to light and decomposes to acrolein and methanethiol (**Figure 1**). Two molecules of the latter eventually yield dimethyl disulfide (DMDS). The lightstruck taste is described as cooked cabbage mainly due to the formation of methanethiol and DMDS though hydrogen sulfide may have a role [33–35]. Light exposure can also lead to the formation of other undesirable compounds, such as furfural [36] which has been positively correlated to the "cooked vegetable" aroma of white wines stored under oxidative conditions [37, 38]. Not all wines are susceptible to developing this defect. The concentration of riboflavin plays the major role [5, 39] and when it is lower than 80–100 μg/L, the risk of the light-struck taste decreases. The riboflavin content in the grape hardly exceeds a few tens of micrograms per liter and the metabolic activities of *S. cerevisiae* affect its levels in wine [40, 41]. The levels of riboflavin can be reduced by means of wine treatment with bentonite or charcoal as well as by using a low producer yeast strain of this vitamin. Finally, nutrients commonly employed for favoring yeast growth could further increase the amount of riboflavin in wine [41]. Moreover, the amounts of methionine could influence the occurrence of light-struck taste as well as the presence of oxygen [42]. The light-struck taste can be limited by the addition of antioxidant compounds. The use of gallic tannins seems to be particularly promising in protecting wine possibly due to the binding

molecules if their concentration is close to the perception threshold [22, 49, 50]. 3MH and 3MHA have olfactory perception thresholds of 60 and 4 ng/L, respectively and they are responsible for passion fruit-like and grapefruit-like olfactory notes [11]. These compounds were also found in red wines [51]. The 4MMP has an olfactory perception threshold of 0.8 ng/L, and its aroma is described as box tree-like, black currant-like, or even cat urine-like when occurring at high concentration [10]. The 4MMPOH has a perception threshold of 55 ng/L in aqueous alcoholic solution and it is reminiscent of citrus zest and grapefruit [44]. 3MH is the most abundant in wine, in concentrations generally higher than its perception threshold, while 4MMPOH in wine is generally lower than its perception threshold. The level of 4MMP is dependent on the grape cultivar and it changes between different samples in the same cultivar [52]. The concentrations of 3MH and 3MHA in Sauvignon Blanc wines from different countries have been reported to be in the range 688–18,681 and 10–2507 ng/L, respectively [53–55]. Besides Sauvignon Blanc wines, the volatile thiols were detected in white wine made from different *Vitis vinifera* grape varieties, such as Gewürtztraminer, Muscat, Riesling, Sylvaner, Pinot Gris, Pinot Blanc, Colombard, Petit Manseng, botrytised Semillon and Grenache [44, 56]. Moreover, these compounds were also found in certain Italian autochthonous varieties including Verdicchio Bianco (also known as Trebbiano di Lugana) [57], Arneis [58], Grillo and Catarratto Bianco Comune [59].

The content of volatile thiols decreases during wine aging, according to the oxidative conditions. Glutathione, sulfur dioxide and anthocyanin content exert a protective effect. In contrast,


*2.2.1. Varietal thiol precursors*

The varietal thiols occur in grape berry as non-volatile cysteinyl- and glutathionyl-conjugated precursors [29, 68]. 3MH bound as cysteinyl-conjugate (Cys-3MH), glutathionyl-conjugate (GSH-3MH) and also cysteinylglycin-conjugate (CysGly-3MH) have been reported [70, 71]. 4MMP occurs in the grape and must as cysteine conjugate (Cys-4MMP) and glutathione conjugate (GSH-4MMP) [72, 73]. Moreover, 3MH can be derived from either (E)-2-hexen-1-ol or (E)-2-hexenal [74, 75]. Through a lipoxygenase/lyase sequence active in the presence of oxygen, (E)-2-hexenal can be obtained from linolenic acid and converted to GSH-3MH by coupling with glutathione [75–77]. (E)-2-hexenal may act as a precursor when hydrogen sulfide is released in the early part of the fermentation [73, 74, 78]. Under aerobic conditions, *S. cerevisiae* is able to oxidize (E)-2-hexen-1-ol into (E)-2-hexenal, and the reverse process can occur under anaerobic conditions [77]. Recently, S-3-(hexan-1-al)-glutathione (GSH-3MHAl) was identified in Sauvignon Blanc juice [79] and it can be considered a precursor of thiol aromas. These compounds are split by the *S. cerevisiae* through the enzymatic activity of carbon-sulfur lyase [10, 80], the volatile thiols are liberated. 3MHA is obtained by yeast activity from 3MH esterified with acetic acid as precursor [7]. This finding correlated the ester and volatile thiol metabolism in yeast for the first time [2]. The capability of *S. cerevisiae* to liberate volatile thiols from their precursors is genetically determined; the yeast selection can represent a useful tool to favor the 3MH and 4MMP release [8, 50]. The fermentation temperature affects the varietal thiol concentrations in the resulting wines: in the range of alcoholic fermentation for white wines, at higher fermentation temperature and higher concentrations of varietal thiols is released. Among the *S. cerevisiae* strains commonly used, VL3 and EG8 release more volatile thiols, in comparison to VL1 and 522 [80]. *S. bayanus* var. *uvarum* strains and hybrids of the latter yeast and *S. cerevisiae* showed a higher ability to release both 3MH and 4MMP when compared to *S. cerevisiae* strain [81]. Furthermore, non-*Saccharomyces* yeasts, like *Torulaspora delbrueckii* and *Pichia kluyveri*, have been proposed as innovative tools to improve wine quality, being able to release varietal thiols [82, 83]. The 3MHA formation from 3MH is also dependent on the strain genetic characteristics [84]. *S. cerevisiae* strains showed the enhanced ability to hydrolyze the S-cysteinyl link rather than the ester synthetic activity. The combined use of different yeast strains, one of them having hydrolyzing ability, with stronger esterification ability, can represent a useful tool to affect the volatile thiol composition [50]. However, several researches have indicated that no clear correlation between precursor concen-

Occurrence and Analysis of Sulfur Compounds in Wine http://dx.doi.org/10.5772/intechopen.72823 231

trations in must and free thiol concentration in wine exist [8, 16, 59, 73, 80, 85–89].

The harvest has a significant effect on the content of thiol precursors in juice. Capone and Jeffery [90] showed that machine harvest and transportation for 12 h of the grapes led to higher levels of thiol precursors in comparison to hand harvest grapes immediately processed. The authors suggested the minimal berry damage results in less formation of the precursors. More than half of the total cysteine conjugates is located in the grape skin; as a consequence, an increased skin contact time augments Cys-3MH in grape musts, while it has little effect on Cys-4MMP and Cys-4MMPOH concentrations [91–93]. Stronger pressing conditions allow a major extraction of thiol precursors in comparison to free run juice. Roland and coauthors [94] demonstrated an easier extraction of Cys-3MH in comparison to GSH-3MH due to their distribution in different parts of the skin. The produced wines contained 3MH at higher concentrations when the juice was collected at the end of the pressing cycle. On the

**Table 2.** Contribution of volatile thiols in *Vitis vinifera* wines.

increased contact with oxygen, particularly in the presence of catechin derivatives, promotes their degradation [52, 60, 61]. Three different mechanisms proposed lead to their decay with a different degradation kinetics. The volatile thiols can be easily oxidized when oxygen and iron are present, forming disulfides [62, 63]. Since the volatile thiols act as electrophiles, they can react with phenolic compounds [64]. Moreover, the presence of oxygen and catalyst metals, namely iron and copper, lead the oxidation of phenols into the corresponding *o*-quinones which can bind the volatile thiols [65, 66]. A rapid decline of 3MHA is observed after 3 months of bottle storage, while a much slower decline in 3MH can be noticed. After 1 year of storage, the ester has completely disappeared, while 3MH is still present but its content is halved. The chemical structure of the volatile thiols affects their degradation: 4MMP, a tertiary thiol, results less able to react with *o*-quinones and decreases slower in comparison to 3MH, a secondary thiol, maybe due to its steric hindrance [63]. The wine composition and oxygen exposure are the major reasons for loss of volatile thiols both during winemaking and post-bottling [29, 67]. The presence of antioxidants, such as sulfur dioxide, ascorbic acid and reduced glutathione, limit the polyphenol oxidation either by removing oxygen from wine or by reversing and altering the oxidation process. In particular, the addition of glutathione, up to 20 mg/L before bottling, led to higher 3MH level after 6 months of storage in bottle [29].

#### *2.2.1. Varietal thiol precursors*

increased contact with oxygen, particularly in the presence of catechin derivatives, promotes their degradation [52, 60, 61]. Three different mechanisms proposed lead to their decay with a different degradation kinetics. The volatile thiols can be easily oxidized when oxygen and iron are present, forming disulfides [62, 63]. Since the volatile thiols act as electrophiles, they can react with phenolic compounds [64]. Moreover, the presence of oxygen and catalyst metals, namely iron and copper, lead the oxidation of phenols into the corresponding *o*-quinones which can bind the volatile thiols [65, 66]. A rapid decline of 3MHA is observed after 3 months of bottle storage, while a much slower decline in 3MH can be noticed. After 1 year of storage, the ester has completely disappeared, while 3MH is still present but its content is halved. The chemical structure of the volatile thiols affects their degradation: 4MMP, a tertiary thiol, results less able to react with *o*-quinones and decreases slower in comparison to 3MH, a secondary thiol, maybe due to its steric hindrance [63]. The wine composition and oxygen exposure are the major reasons for loss of volatile thiols both during winemaking and post-bottling [29, 67]. The presence of antioxidants, such as sulfur dioxide, ascorbic acid and reduced glutathione, limit the polyphenol oxidation either by removing oxygen from wine or by reversing and altering the oxidation process. In particular, the addition of glutathione, up to 20 mg/L before bottling, led to higher 3MH level after 6 months of storage

**Compound Structure Olfactory** 

230 Grapes and Wines - Advances in Production, Processing, Analysis and Valorization

3-mercaptohexan-1-ol

3-mercaptohexylacetate

(3MH)

(3MHA)

4-mercapto-4 methylpentan-2-one

4-mercapto-4 methylpentan-2-ol (4MMPOH)

In model wine solution [8, 10, 11, 147].

Range *Vitis vinifera* wines [11, 28, 53–55, 92, 148].

**Table 2.** Contribution of volatile thiols in *Vitis vinifera* wines.

(4MMP)

a

b

c [43]. d[8, 11]. e [8, 147]. **description**

Passion fruit, grape fruit, guavac

Passion fruit, grape fruit, box tree, guavad

Box tree, black currant, passion

fruite

**Perception threshold (ng/L)a**

60 26–18,000

4 0–2500

0.8 0–40

Citrus zestf 0–90

**Range in wine (ng/L)b**

in bottle [29].

The varietal thiols occur in grape berry as non-volatile cysteinyl- and glutathionyl-conjugated precursors [29, 68]. 3MH bound as cysteinyl-conjugate (Cys-3MH), glutathionyl-conjugate (GSH-3MH) and also cysteinylglycin-conjugate (CysGly-3MH) have been reported [70, 71]. 4MMP occurs in the grape and must as cysteine conjugate (Cys-4MMP) and glutathione conjugate (GSH-4MMP) [72, 73]. Moreover, 3MH can be derived from either (E)-2-hexen-1-ol or (E)-2-hexenal [74, 75]. Through a lipoxygenase/lyase sequence active in the presence of oxygen, (E)-2-hexenal can be obtained from linolenic acid and converted to GSH-3MH by coupling with glutathione [75–77]. (E)-2-hexenal may act as a precursor when hydrogen sulfide is released in the early part of the fermentation [73, 74, 78]. Under aerobic conditions, *S. cerevisiae* is able to oxidize (E)-2-hexen-1-ol into (E)-2-hexenal, and the reverse process can occur under anaerobic conditions [77]. Recently, S-3-(hexan-1-al)-glutathione (GSH-3MHAl) was identified in Sauvignon Blanc juice [79] and it can be considered a precursor of thiol aromas. These compounds are split by the *S. cerevisiae* through the enzymatic activity of carbon-sulfur lyase [10, 80], the volatile thiols are liberated. 3MHA is obtained by yeast activity from 3MH esterified with acetic acid as precursor [7]. This finding correlated the ester and volatile thiol metabolism in yeast for the first time [2]. The capability of *S. cerevisiae* to liberate volatile thiols from their precursors is genetically determined; the yeast selection can represent a useful tool to favor the 3MH and 4MMP release [8, 50]. The fermentation temperature affects the varietal thiol concentrations in the resulting wines: in the range of alcoholic fermentation for white wines, at higher fermentation temperature and higher concentrations of varietal thiols is released. Among the *S. cerevisiae* strains commonly used, VL3 and EG8 release more volatile thiols, in comparison to VL1 and 522 [80]. *S. bayanus* var. *uvarum* strains and hybrids of the latter yeast and *S. cerevisiae* showed a higher ability to release both 3MH and 4MMP when compared to *S. cerevisiae* strain [81]. Furthermore, non-*Saccharomyces* yeasts, like *Torulaspora delbrueckii* and *Pichia kluyveri*, have been proposed as innovative tools to improve wine quality, being able to release varietal thiols [82, 83]. The 3MHA formation from 3MH is also dependent on the strain genetic characteristics [84]. *S. cerevisiae* strains showed the enhanced ability to hydrolyze the S-cysteinyl link rather than the ester synthetic activity. The combined use of different yeast strains, one of them having hydrolyzing ability, with stronger esterification ability, can represent a useful tool to affect the volatile thiol composition [50]. However, several researches have indicated that no clear correlation between precursor concentrations in must and free thiol concentration in wine exist [8, 16, 59, 73, 80, 85–89].

The harvest has a significant effect on the content of thiol precursors in juice. Capone and Jeffery [90] showed that machine harvest and transportation for 12 h of the grapes led to higher levels of thiol precursors in comparison to hand harvest grapes immediately processed. The authors suggested the minimal berry damage results in less formation of the precursors. More than half of the total cysteine conjugates is located in the grape skin; as a consequence, an increased skin contact time augments Cys-3MH in grape musts, while it has little effect on Cys-4MMP and Cys-4MMPOH concentrations [91–93]. Stronger pressing conditions allow a major extraction of thiol precursors in comparison to free run juice. Roland and coauthors [94] demonstrated an easier extraction of Cys-3MH in comparison to GSH-3MH due to their distribution in different parts of the skin. The produced wines contained 3MH at higher concentrations when the juice was collected at the end of the pressing cycle. On the contrary, Patel et al. [95] found lower amounts of 3MH and 3MHA in wine produced from juice obtained from stronger pressing conditions in which the thiol precursors were present in higher amounts. This can be due to the higher extraction of oxidizable phenols inducing the rapid decrease of glutathione and the oxidation of varietal thiols. The pressing performed on an industrial scale caused a strong decrease of thiol precursors. The factors affecting the GSH-portion degradation of the precursors are not completely clear and mechanisms, other than oxidation or proteolysis, could induce the loss of the thiol precursors [59].

during the oxidative aging. GSH reduces the formation of both these off-flavors during storage. Moreover, GSH can have a positive effect on white wine color which appears to be more stable during aging [103, 107]. The first source of GSH is the grape in which it can exceed 200 mg/L of grape juice according to grape cultivar, environmental conditions and viticultural practices [108]

Occurrence and Analysis of Sulfur Compounds in Wine http://dx.doi.org/10.5772/intechopen.72823 233

**Figure 2.** Scheme of GRP and browning compound formation [100–102].

## **3. The reduced glutathione**

The reduced glutathione (GSH) is a tripeptide constituted by l-cysteine, γ-glutamic acid and glycine exerting antioxidant and detoxifying activities in the cell [96, 97]. This compound exerts several activities in must and wine. The antioxidant property of GSH is well known: it can reduce the *o*-quinone deriving from the enzymatic oxidation carried out by the polyphenoloxidase enzymes (PPO) on the tartaric esters of hydroxycinnamic acids. This hinders the formation polymers causing the browning of must [12]. During aging, the *o*-quinones are produced as a result of the non-enzymatic oxidation (also known as chemical oxidation) of *o*-diphenols [98]. The level of *o*-diphenols in wine is correlated to the browning of white wines [28, 98, 99]. Caffeoyl-tartaric acid (caftaric acid) and coumaric-tartaric acid (coutaric acid) are some of the most abundant hydroxycinnamic acids in must representing the substrate mainly oxidized by the enzymatic action. The GSH can reduce the oxidized caftaric acid, generating 2-*S*-glutathionyl caftaric acid, also known as Grape Reaction Product (GRP) [100]. The GRP is not a substrate of the PPO and it can trap the *o*-quinone, limiting the formation of brown polymers which are responsible for color changes of white must and wine. GRP can oxide enzymatically by the *Botrytis cinerea* laccase and chemically by the caffeoyl-tartaric acid quinone. In this way, the GRP quinone is originated; it can be a substrate of condensation reaction with the phenols. This molecule is responsible for brown compound formation. The high GSH concentration allows a second nucleophile attack, in position 5 of the benzyl ring. The 2,5-diglutathionyl caftaric acid (GRP2) is formed and is a substrate of the laccase action [12]. With low GSH concentration, the GRP can be oxidized by the excess caftaric acid quinones which can cause an intense browning (**Figure 2**) [101, 102].

GSH is able to limit the loss of the flavoring volatile thiols acting as a competitor for the reduction of the quinones [103]. In fact, GSH concentration is about a thousand times higher than that of volatile thiols which are protected against oxidation. Lavigne and Dubourdieu [103] reported that when the GSH concentration ranged from 6 to 10 mg/L it slowed down the decrease of volatile thiols. Additionally, other aromatic compounds, such as isoamyl acetate (3-methyl-1-butyl acetate), ethyl hexanoate and linalool (3,7-dimethylocta-1,6-dien-3-ol), are better protected during bottle storage [104]. GSH can limit the formation of sotolon (3-hydroxy-4,5-dimethyl-2(5H)-furanone), a compound responsible for the atypical aging character of white wine [103]. It confers aroma descriptors such as dried fig and rancid and its perception threshold is 7 μg/L [105]. Besides sotolon, 2-aminoacetophenone (1-(2-aminophenyl)-ethanone) is also responsible for the atypical aging and it has a lower perception threshold than sotolon, corresponding to 1 μg/L. Both sotolon and 2-aminoacetophenone concentrations increase due to the exposition of wine to oxygen during bottling [106]. GSH can have a protective action on the wine aroma during the oxidative aging. GSH reduces the formation of both these off-flavors during storage. Moreover, GSH can have a positive effect on white wine color which appears to be more stable during aging [103, 107]. The first source of GSH is the grape in which it can exceed 200 mg/L of grape juice according to grape cultivar, environmental conditions and viticultural practices [108]

contrary, Patel et al. [95] found lower amounts of 3MH and 3MHA in wine produced from juice obtained from stronger pressing conditions in which the thiol precursors were present in higher amounts. This can be due to the higher extraction of oxidizable phenols inducing the rapid decrease of glutathione and the oxidation of varietal thiols. The pressing performed on an industrial scale caused a strong decrease of thiol precursors. The factors affecting the GSH-portion degradation of the precursors are not completely clear and mechanisms, other

The reduced glutathione (GSH) is a tripeptide constituted by l-cysteine, γ-glutamic acid and glycine exerting antioxidant and detoxifying activities in the cell [96, 97]. This compound exerts several activities in must and wine. The antioxidant property of GSH is well known: it can reduce the *o*-quinone deriving from the enzymatic oxidation carried out by the polyphenoloxidase enzymes (PPO) on the tartaric esters of hydroxycinnamic acids. This hinders the formation polymers causing the browning of must [12]. During aging, the *o*-quinones are produced as a result of the non-enzymatic oxidation (also known as chemical oxidation) of *o*-diphenols [98]. The level of *o*-diphenols in wine is correlated to the browning of white wines [28, 98, 99]. Caffeoyl-tartaric acid (caftaric acid) and coumaric-tartaric acid (coutaric acid) are some of the most abundant hydroxycinnamic acids in must representing the substrate mainly oxidized by the enzymatic action. The GSH can reduce the oxidized caftaric acid, generating 2-*S*-glutathionyl caftaric acid, also known as Grape Reaction Product (GRP) [100]. The GRP is not a substrate of the PPO and it can trap the *o*-quinone, limiting the formation of brown polymers which are responsible for color changes of white must and wine. GRP can oxide enzymatically by the *Botrytis cinerea* laccase and chemically by the caffeoyl-tartaric acid quinone. In this way, the GRP quinone is originated; it can be a substrate of condensation reaction with the phenols. This molecule is responsible for brown compound formation. The high GSH concentration allows a second nucleophile attack, in position 5 of the benzyl ring. The 2,5-diglutathionyl caftaric acid (GRP2) is formed and is a substrate of the laccase action [12]. With low GSH concentration, the GRP can be oxidized by the

excess caftaric acid quinones which can cause an intense browning (**Figure 2**) [101, 102].

GSH is able to limit the loss of the flavoring volatile thiols acting as a competitor for the reduction of the quinones [103]. In fact, GSH concentration is about a thousand times higher than that of volatile thiols which are protected against oxidation. Lavigne and Dubourdieu [103] reported that when the GSH concentration ranged from 6 to 10 mg/L it slowed down the decrease of volatile thiols. Additionally, other aromatic compounds, such as isoamyl acetate (3-methyl-1-butyl acetate), ethyl hexanoate and linalool (3,7-dimethylocta-1,6-dien-3-ol), are better protected during bottle storage [104]. GSH can limit the formation of sotolon (3-hydroxy-4,5-dimethyl-2(5H)-furanone), a compound responsible for the atypical aging character of white wine [103]. It confers aroma descriptors such as dried fig and rancid and its perception threshold is 7 μg/L [105]. Besides sotolon, 2-aminoacetophenone (1-(2-aminophenyl)-ethanone) is also responsible for the atypical aging and it has a lower perception threshold than sotolon, corresponding to 1 μg/L. Both sotolon and 2-aminoacetophenone concentrations increase due to the exposition of wine to oxygen during bottling [106]. GSH can have a protective action on the wine aroma

than oxidation or proteolysis, could induce the loss of the thiol precursors [59].

232 Grapes and Wines - Advances in Production, Processing, Analysis and Valorization

**3. The reduced glutathione**

**Figure 2.** Scheme of GRP and browning compound formation [100–102].

and the amounts of readily assimilable nitrogen in the soil [103]. Lower levels were found in must up to 100 mg/L [108] and it can be affected by exposure to oxygen, tyrosinase activity and prefermentative grape skin maceration [93, 109]. The loss of GSH in must production can negatively affect the formation of precursors of the varietal thiol compounds [73] as well as the residual content of GSH during wine aging. Glutathione was reported to be consumed by *S. cerevisiae* at the beginning of the alcoholic fermentation and then to be released by the yeast cell lysis [103, 110]. At the beginning of the alcoholic fermentation GSH almost disappears and then its concentration increases as an effect of the yeast cell synthesis and lysis. However, winemaking on an industrial scale did not preserve GSH in must and the level of this antioxidant increased during the alcoholic fermentation due to the yeast metabolism [111]. Lavigne and Dubourdieu [103] reported the GSH level becomes stable 1 month after the alcoholic fermentation is started. On the contrary, in recent researches, the highest levels of GSH were observed after the racking [111, 112]. The concentration of GSH in wine is lower than in the juice and grapes and it ranges from 3 to 35.5 mg/L [109, 111–113] and can be increased through the choice of an adequate yeast strain [15]. No GSH is released from yeast under nitrogen starvation during the alcoholic fermentation [103, 111]. The GSH concentration decreased after the racking as well as during wine aging on lees [57, 95, 111] maybe due to the adsorption of GSH on the yeast lees as occurs for other low-molecular weight thiols during wine aging [114].

## **4. Analytical methods**

The analytical methods developed for the analysis of sulfur compounds in wine need to overcome the low concentrations and the high reactivity of these molecules as well as the complexity of the matrix. Sulfur compounds in wine are frequently divided into "light" (boiling point < 90°C) and "heavy" (boiling point > 90°C) compounds [3] indicating difficulty in using a relevant common sampling/enrichment technique.

many sources of error in Purge and Trap instruments have been reviewed [116]. As for the dynamic headspace, the choices of the proper fiber as well as sample temperature and the presence of salt increasing the ionic strength can improve extraction yield in static headspace. In particular, the use of carboxen-polydimethylsiloxane fiber (CAR-PDMS) [117, 118] or carboxen-polydimethylsiloxane-divinylbenzene (CAR-PDMS-DVB) [22, 119] has been proposed for the analysis of fermentative sulfur compounds. The use of the latter fiber has been shown to produce good results in terms of repeatability and reproducibility [22]. Moreover, the best modified ionic strength has been obtained by using magnesium sulfate. Due to the different boiling temperature of the fermentative sulfur compounds, a good compromise needs to be set to allow for the adsorption of these compounds through the SPME technique. As reported by Nguyen and co-authors [120], the incubation of the sample was carried out at 45°C for 5 min and the extraction at 45°C for 30 min under agitation. The GC equipment was coupled with MS detector and the compounds of interest were detected in single ion monitoring (SIM) mode and quantified by means of different internal standards properly chosen. The analytical methods described above are suitable for the analysis of the volatiles characterized by low

applied

**Analytical technique Advantages Disadvantages**

Increased sampling phase; good sensitivity

Cheaper and simpler instrumentation

Suggested for sulfur compounds with low boiling point

Best extraction yield for the analytical conditions Many sources of errors

Occurrence and Analysis of Sulfur Compounds in Wine http://dx.doi.org/10.5772/intechopen.72823

needs to be properly set

fiber

time)

Compromise between sensitivity and extraction/temperature conditions

235

Less versatile than CAR-PDMS-DVB

Suitable analytical method setting for increasing the extraction yield (sample temperature, ionic strength, extraction

Purge and trap—gas chromatography

(SPME)—gas chromatography

Carboxen-polydimethylsiloxane

Carboxen-polydimethylsiloxane-

**Table 3.** Analytical methods for the determination of sulfur off-flavors.

Static headspace Solid phase micro extraction

(CAR-PDMS)<sup>a</sup>

divinylbenzene (CAR-PDMS-DVB)<sup>b</sup>

boiling point (lower than 90°C) that is not the case for 3MH, 3MHA and 4MMP.

Among headspace sampling techniques, only purge and trap has been used to analyze 3MH and 3MHA in wines, reaching detection limits closer to their perception threshold [121]. Despite this, the other heavy volatile sulfur aromas were not identified at perception threshold, thus leaving derivatization procedures as the most promising technique for the extraction and analysis of volatile thiols. Due to the low concentration of these compounds, in the order of magnitude of ng/L, the sample preparation has provided a liquid/liquid extraction followed by the evaporation of the organic solvent. As the varietal thiols are present in wine in low concentration as well

**4.2. Varietal thiols**

Dynamic headspace

SPME fiber

a [117, 118]. b

[22, 119, 120].

#### **4.1. Sulfur off-flavors**

The most common technique employed for the analysis of sulfur compounds associated with off-flavors in wine is static headspace analysis by means of solid phase micro extraction (SPME) combined with gas chromatography (GC) coupled with different detectors (**Table 3**). Methods in dynamic headspace by using Purge and Trap equipment was also described as an alternative to the static headspace. In static headspace technique, the analytes reach the equilibrium state between the liquid and gas phases and then are adsorbed in a fiber. In dynamic space, the analytes in the gas phase into the headspace and the atmosphere around the sample is constantly swept away by a flow of carrier gas, taking volatile analytes with it. Through this technique, the equilibrium state is not reached and, thus, more of the volatile dispersed in the matrix will pass into the headspace whose size results in increased sampling phase. As a consequence, the trapping stage of the analysis offers good sensitivity [43, 115]. The proper combination of sorbent and temperature may permit collection and concentration of specific analytes while venting others. Despite this, the instrumentation requires more complexity and it is more expensive than other sampling techniques, such as the static headspace. Moreover,


**Table 3.** Analytical methods for the determination of sulfur off-flavors.

many sources of error in Purge and Trap instruments have been reviewed [116]. As for the dynamic headspace, the choices of the proper fiber as well as sample temperature and the presence of salt increasing the ionic strength can improve extraction yield in static headspace. In particular, the use of carboxen-polydimethylsiloxane fiber (CAR-PDMS) [117, 118] or carboxen-polydimethylsiloxane-divinylbenzene (CAR-PDMS-DVB) [22, 119] has been proposed for the analysis of fermentative sulfur compounds. The use of the latter fiber has been shown to produce good results in terms of repeatability and reproducibility [22]. Moreover, the best modified ionic strength has been obtained by using magnesium sulfate. Due to the different boiling temperature of the fermentative sulfur compounds, a good compromise needs to be set to allow for the adsorption of these compounds through the SPME technique. As reported by Nguyen and co-authors [120], the incubation of the sample was carried out at 45°C for 5 min and the extraction at 45°C for 30 min under agitation. The GC equipment was coupled with MS detector and the compounds of interest were detected in single ion monitoring (SIM) mode and quantified by means of different internal standards properly chosen. The analytical methods described above are suitable for the analysis of the volatiles characterized by low boiling point (lower than 90°C) that is not the case for 3MH, 3MHA and 4MMP.

#### **4.2. Varietal thiols**

and the amounts of readily assimilable nitrogen in the soil [103]. Lower levels were found in must up to 100 mg/L [108] and it can be affected by exposure to oxygen, tyrosinase activity and prefermentative grape skin maceration [93, 109]. The loss of GSH in must production can negatively affect the formation of precursors of the varietal thiol compounds [73] as well as the residual content of GSH during wine aging. Glutathione was reported to be consumed by *S. cerevisiae* at the beginning of the alcoholic fermentation and then to be released by the yeast cell lysis [103, 110]. At the beginning of the alcoholic fermentation GSH almost disappears and then its concentration increases as an effect of the yeast cell synthesis and lysis. However, winemaking on an industrial scale did not preserve GSH in must and the level of this antioxidant increased during the alcoholic fermentation due to the yeast metabolism [111]. Lavigne and Dubourdieu [103] reported the GSH level becomes stable 1 month after the alcoholic fermentation is started. On the contrary, in recent researches, the highest levels of GSH were observed after the racking [111, 112]. The concentration of GSH in wine is lower than in the juice and grapes and it ranges from 3 to 35.5 mg/L [109, 111–113] and can be increased through the choice of an adequate yeast strain [15]. No GSH is released from yeast under nitrogen starvation during the alcoholic fermentation [103, 111]. The GSH concentration decreased after the racking as well as during wine aging on lees [57, 95, 111] maybe due to the adsorption of GSH on the yeast lees as occurs for other low-molecular weight

234 Grapes and Wines - Advances in Production, Processing, Analysis and Valorization

The analytical methods developed for the analysis of sulfur compounds in wine need to overcome the low concentrations and the high reactivity of these molecules as well as the complexity of the matrix. Sulfur compounds in wine are frequently divided into "light" (boiling point < 90°C) and "heavy" (boiling point > 90°C) compounds [3] indicating difficulty in using

The most common technique employed for the analysis of sulfur compounds associated with off-flavors in wine is static headspace analysis by means of solid phase micro extraction (SPME) combined with gas chromatography (GC) coupled with different detectors (**Table 3**). Methods in dynamic headspace by using Purge and Trap equipment was also described as an alternative to the static headspace. In static headspace technique, the analytes reach the equilibrium state between the liquid and gas phases and then are adsorbed in a fiber. In dynamic space, the analytes in the gas phase into the headspace and the atmosphere around the sample is constantly swept away by a flow of carrier gas, taking volatile analytes with it. Through this technique, the equilibrium state is not reached and, thus, more of the volatile dispersed in the matrix will pass into the headspace whose size results in increased sampling phase. As a consequence, the trapping stage of the analysis offers good sensitivity [43, 115]. The proper combination of sorbent and temperature may permit collection and concentration of specific analytes while venting others. Despite this, the instrumentation requires more complexity and it is more expensive than other sampling techniques, such as the static headspace. Moreover,

thiols during wine aging [114].

**4. Analytical methods**

**4.1. Sulfur off-flavors**

a relevant common sampling/enrichment technique.

Among headspace sampling techniques, only purge and trap has been used to analyze 3MH and 3MHA in wines, reaching detection limits closer to their perception threshold [121]. Despite this, the other heavy volatile sulfur aromas were not identified at perception threshold, thus leaving derivatization procedures as the most promising technique for the extraction and analysis of volatile thiols. Due to the low concentration of these compounds, in the order of magnitude of ng/L, the sample preparation has provided a liquid/liquid extraction followed by the evaporation of the organic solvent. As the varietal thiols are present in wine in low concentration as well as being highly reactive, the deuterated analogues are commonly used as internal standards compensating for the eventual loss taking place during the sample preparation [122].

volatile compounds and determined by both GC and HPLC coupled with MS detector. In the

Occurrence and Analysis of Sulfur Compounds in Wine http://dx.doi.org/10.5772/intechopen.72823 237

In the first method developed, the Cys-4MMP was detected by GC coupled with flame photometric detectors (FDP) by synthetizing it and 4MMP was then detected [69]. Tominaga and co-authors [130] quantified the cysteinylated precursors by GC-MS after their derivatization. The addition of labeled thiol precursors allowed the quantification of those contained in the must by GC-FDP and GC-MS [131]. The GC coupled either with atomic emission detection (AED) [132] or detection-capture mass spectrometry (DCMS) detector [86] were also proposed employing propyl thioacetate as internal standard and derivatizing the released volatile thiols with ethyl chloroformate, respectively. The precursors of 3MH were quantified using the

Both GC-MS and liquid chromatography techniques were used for the direct determination of thiol precursors. In the case of GC-MS, their derivatization is necessary and different derivatizing agents were proposed [133–135]. In the case of liquid chromatography, both HPLC-MS and HPLC-MS/MS were applied. The use of these types of equipment did not require a derivatization of thiol precursors, but a solid phase extraction (SPE) was applied achieving the sample purification. The quantification was carried out by means of the patterns of labeled compounds [73, 87, 136–139] as well as without them [59, 72]. The quantification by liquid

Several analytical methods have been proposed for GSH quantification in the grape, must and wine using different analytical techniques [141]. GSH was quantified after a treatment with glutathione reductase enzyme in white wine [113]. In grapevine tissues, the GSH was derivatized with 5,5′-dithiobis-2-nitrobenzoic acid (DTNB) after the enzymatic treatment and the detection was performed spectrophotometrically at 412 nm [142]. The determination of total GSH could be performed after the enzymatic treatment which allowed the breakdown of disulfide bridges. The GSH could be derivatized pre-column with *o*-phthalaldehyde (OPA) and quantified by HPLC; the detection was than conducted by fluorescence [143, 144]. The use of the fluorescence detector allowed the GSH quantification after derivatization with 2,3-naphthalenedialdehyde (NDA), as described by Marchand and de Revel [145]. The technique involved the pre-column derivatization and the separation by HPLC. The determination of oxidized glutathione was also allowed by enzymatic treatment with glutathione reductase. The fluorescence detector was employed for the GSH determination after its derivatization with monobromobimane (mBB); the reaction adduct was identified by capillary electrophoresis [110]. As described above, the mBB is photodegradable and a GSH content underestimation could occur [146]. The method proposed by Du Toit et al. [109] was based on liquid chromatography coupled with a mass spectrometry detector (LC-MS/MS). The determination of both reduced and oxidized glutathione was allowed in the same run. The ethanol present in wine had to be removed prior to the analysis and this step could cause an underestimation of the GSH content. Another analytical method for the GSH quantification was developed using the atomic adsorption spectrometry [147]. The method is based on the reactivity of the mercury toward the thiols. The liquid chromatography (both HPLC and UPLC) coupled with

second case, the precursors are quantified after their proper purification.

secondary ionization mass spectrometry (LSIMS) was also reported [140].

labeled isotopic standard d10-3MH by GC-MS.

**4.3. The reduced glutathione**

Among the derivatizing compounds, the use of *p*-hydroxymercuribenzoate (pHMB) was first proposed by Tominaga and co-authors [10]. The volatile thiols were derivatized with pHMB, isolated with the use of strong basic anion exchange column followed by the liquid/liquid extraction with dichloromethane. The sample was concentrated and analyzed by GC-MS. As a result of the analytical method improvement, varietal thiols were quantified in several wines produced with *Vitis vinifera* grape varieties [44], suggesting these sulfur compounds play a key role in the aroma of different white wines. Besides 3MH, 3MHA and 4MMP, the method allows the identification and quantification of two other sulfur-containing aromas, such as 2-furanmethanethiol [11] and benzenemethanethiol [123] in wines. The identification of these two latter molecules, together with the identification of ethyl 3-mercaptopropionate, established the role of certain volatile thiols in the bouquet of aged champagne wines [124]. Although very efficient, this procedure is time consuming. In an effort to reduce the time required for the sample preparation, a covalent chromatography was employed for the enrichment of the thiols from the wine extract. Specifically, a cross-linked agarose gel containing phenylmercuric chloride was used where the volatile thiols were trapped after their liquid/liquid extraction with dichlomethane [122]. The ability of some common solid phase extraction sorbents to retain organomercuric salts for selective concentration of thiols in wines was also proposed using styrene-divinylbenzene copolymer sorbent. Nevertheless, the organomercury salt that is formed for the detection of varietal thiols by the analytical methods described (considered as hazardous poison) still remains the key point of this method.

Other analytical approaches employ pentafluorobenzyl bromide as a derivatizing agent, which transforms thiols into their corresponding pentafluorobenzyl derivatives [125–128]. The derivatizing reaction is usually carried out in a purified extract (i.e. water) [128], organic solvent [126], in-cartridge [127], or in-fiber [125]. Another very promising derivatizing agent in the gas chromatography analysis of thiols is ethyl propiolate, which is able to derivatize thiols directly in wine matrix and is a suitable derivatizing reagent for the electron impact mass spectrometry detection system [129].

Moreover, Piano and co-authors [55] proposed an analytical method in which the varietal thiols are detected by liquid chromatography (UPLC) coupled with MS/MS. The sample preparation required several steps in order to protect the thiol aromas from oxidation and the liquid/liquid extraction in order to achieve the concentration of the analytes. The *o*-phthaldialdehyde (OPA) is used as derivatizing agent. The method allows the quantification of 3MH and 3MHA, but the derivatization of 4MMP does not occur. The formation of the OPA derivative of 4MMP was probably prevented by the hydrogen bonding between the thiol group and the carbonyl moiety within the compound itself, and its steric hindrance. Derivatization of 4MMP was an issue when other derivatizing reagents were used [127].

#### *4.2.1. Varietal thiol precursors*

The analytical methods proposed for the determination of volatile thiol precursors include both indirect and direct methods [71]. In the first case, the precursors are transformed in volatile compounds and determined by both GC and HPLC coupled with MS detector. In the second case, the precursors are quantified after their proper purification.

In the first method developed, the Cys-4MMP was detected by GC coupled with flame photometric detectors (FDP) by synthetizing it and 4MMP was then detected [69]. Tominaga and co-authors [130] quantified the cysteinylated precursors by GC-MS after their derivatization. The addition of labeled thiol precursors allowed the quantification of those contained in the must by GC-FDP and GC-MS [131]. The GC coupled either with atomic emission detection (AED) [132] or detection-capture mass spectrometry (DCMS) detector [86] were also proposed employing propyl thioacetate as internal standard and derivatizing the released volatile thiols with ethyl chloroformate, respectively. The precursors of 3MH were quantified using the labeled isotopic standard d10-3MH by GC-MS.

Both GC-MS and liquid chromatography techniques were used for the direct determination of thiol precursors. In the case of GC-MS, their derivatization is necessary and different derivatizing agents were proposed [133–135]. In the case of liquid chromatography, both HPLC-MS and HPLC-MS/MS were applied. The use of these types of equipment did not require a derivatization of thiol precursors, but a solid phase extraction (SPE) was applied achieving the sample purification. The quantification was carried out by means of the patterns of labeled compounds [73, 87, 136–139] as well as without them [59, 72]. The quantification by liquid secondary ionization mass spectrometry (LSIMS) was also reported [140].

#### **4.3. The reduced glutathione**

as being highly reactive, the deuterated analogues are commonly used as internal standards

Among the derivatizing compounds, the use of *p*-hydroxymercuribenzoate (pHMB) was first proposed by Tominaga and co-authors [10]. The volatile thiols were derivatized with pHMB, isolated with the use of strong basic anion exchange column followed by the liquid/liquid extraction with dichloromethane. The sample was concentrated and analyzed by GC-MS. As a result of the analytical method improvement, varietal thiols were quantified in several wines produced with *Vitis vinifera* grape varieties [44], suggesting these sulfur compounds play a key role in the aroma of different white wines. Besides 3MH, 3MHA and 4MMP, the method allows the identification and quantification of two other sulfur-containing aromas, such as 2-furanmethanethiol [11] and benzenemethanethiol [123] in wines. The identification of these two latter molecules, together with the identification of ethyl 3-mercaptopropionate, established the role of certain volatile thiols in the bouquet of aged champagne wines [124]. Although very efficient, this procedure is time consuming. In an effort to reduce the time required for the sample preparation, a covalent chromatography was employed for the enrichment of the thiols from the wine extract. Specifically, a cross-linked agarose gel containing phenylmercuric chloride was used where the volatile thiols were trapped after their liquid/liquid extraction with dichlomethane [122]. The ability of some common solid phase extraction sorbents to retain organomercuric salts for selective concentration of thiols in wines was also proposed using styrene-divinylbenzene copolymer sorbent. Nevertheless, the organomercury salt that is formed for the detection of varietal thiols by the analytical methods described (considered as hazardous poison) still remains the key point of this method.

Other analytical approaches employ pentafluorobenzyl bromide as a derivatizing agent, which transforms thiols into their corresponding pentafluorobenzyl derivatives [125–128]. The derivatizing reaction is usually carried out in a purified extract (i.e. water) [128], organic solvent [126], in-cartridge [127], or in-fiber [125]. Another very promising derivatizing agent in the gas chromatography analysis of thiols is ethyl propiolate, which is able to derivatize thiols directly in wine matrix and is a suitable derivatizing reagent for the electron impact

Moreover, Piano and co-authors [55] proposed an analytical method in which the varietal thiols are detected by liquid chromatography (UPLC) coupled with MS/MS. The sample preparation required several steps in order to protect the thiol aromas from oxidation and the liquid/liquid extraction in order to achieve the concentration of the analytes. The *o*-phthaldialdehyde (OPA) is used as derivatizing agent. The method allows the quantification of 3MH and 3MHA, but the derivatization of 4MMP does not occur. The formation of the OPA derivative of 4MMP was probably prevented by the hydrogen bonding between the thiol group and the carbonyl moiety within the compound itself, and its steric hindrance. Derivatization of

The analytical methods proposed for the determination of volatile thiol precursors include both indirect and direct methods [71]. In the first case, the precursors are transformed in

4MMP was an issue when other derivatizing reagents were used [127].

mass spectrometry detection system [129].

*4.2.1. Varietal thiol precursors*

compensating for the eventual loss taking place during the sample preparation [122].

236 Grapes and Wines - Advances in Production, Processing, Analysis and Valorization

Several analytical methods have been proposed for GSH quantification in the grape, must and wine using different analytical techniques [141]. GSH was quantified after a treatment with glutathione reductase enzyme in white wine [113]. In grapevine tissues, the GSH was derivatized with 5,5′-dithiobis-2-nitrobenzoic acid (DTNB) after the enzymatic treatment and the detection was performed spectrophotometrically at 412 nm [142]. The determination of total GSH could be performed after the enzymatic treatment which allowed the breakdown of disulfide bridges. The GSH could be derivatized pre-column with *o*-phthalaldehyde (OPA) and quantified by HPLC; the detection was than conducted by fluorescence [143, 144]. The use of the fluorescence detector allowed the GSH quantification after derivatization with 2,3-naphthalenedialdehyde (NDA), as described by Marchand and de Revel [145]. The technique involved the pre-column derivatization and the separation by HPLC. The determination of oxidized glutathione was also allowed by enzymatic treatment with glutathione reductase. The fluorescence detector was employed for the GSH determination after its derivatization with monobromobimane (mBB); the reaction adduct was identified by capillary electrophoresis [110]. As described above, the mBB is photodegradable and a GSH content underestimation could occur [146]. The method proposed by Du Toit et al. [109] was based on liquid chromatography coupled with a mass spectrometry detector (LC-MS/MS). The determination of both reduced and oxidized glutathione was allowed in the same run. The ethanol present in wine had to be removed prior to the analysis and this step could cause an underestimation of the GSH content. Another analytical method for the GSH quantification was developed using the atomic adsorption spectrometry [147]. The method is based on the reactivity of the mercury toward the thiols. The liquid chromatography (both HPLC and UPLC) coupled with UV detector allows the quantification of GSH after its derivatization with *p*-benzoquinone [148]. The sample preparation is not required for must and wine, while the juice needs to be obtained from grape under reductive condition prior to the derivatization [111]. Suitable preparative conditions are mandatory for monitoring GSH concentration during must extraction and clarification since such wine making steps are responsible for the chemical and enzymatic oxidations.

Microorganisms on Grapes, in Must and Wine. Heidelberg: Springer; 2008. pp. 211-233.

Occurrence and Analysis of Sulfur Compounds in Wine http://dx.doi.org/10.5772/intechopen.72823 239

[3] Mestres M, Busto O, Guasch J. Analysis of organic sulfur compounds in wine aroma. Journal of Chromatography A. 2000;**881**:569-581. DOI: 10.1016/S0021-9673(00)00220-X

[4] Maujean A, Seguin N. Contribution à l'étude des goûts de lumière dans les vins de Champagne. 3. Les réactions photochimiques responsables des goûts de lumière dans le

[5] Mattivi F, Monetti A, Vrhovšek U, Tonon D, Andrés-Lacueva C. High-performance liquid chromatographic determination of the riboflavin concentration in white wines for predicting their resistance to light. Journal of Chromatography A. 2000;**888**:121-127. DOI:

[6] Vermeulen C, Gijs L, Collin S. Sensorial contribution and formation pathways of thiols in foods: A review. Food Review International. 2005;**21**:69-137. DOI: 10.1081/FRI-200040601

[7] Swiegers JH, Bartowsky EJ, Henschke PA, Pretorius IS. Yeast and bacterial modulation of wine aroma and flavour. In: Blair RJ, Francis ME, Pretorius IS, editors. Advances in Wine Science. Glen Osmond: The Australian Wine Research Institute; 2005. pp. 159-187.

[8] Dubourdieu D, Tominaga T, Masneuf I, des Gachons CP, Murat ML. The role of yeast in grape flavour development during fermentation: The example of Sauvignon blanc.

[9] Swiegers JH, Pretorius IS.Modulation of volatile sulfur compounds by wine yeast. Applied Microbiology and Biotechnology. 2007;**74**:954-960. DOI: 10.1007/s00253-006-0828-1

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#### **5. Conclusions**

Several sulfur compounds occur in wine with a strong influence on its aromatic profile. On one hand, some of these molecules impart negative notes and their presence in wine should be counteracted and limited. The formation mechanisms are not completely clear and further researches are needed also to better understand the treatments and/or the winemaking practices potentially decreasing the appearance of these undesired compounds. On the other hand, the varietal thiols are responsible for positive characteristics of wine aroma up to a certain extent. Their protection is essential for the maintenance of the aromatic profile throughout both the winemaking and storage of wine by means of antioxidants of which reduce glutathione can represent a good natural candidate. Further studies will be necessary to investigate the fate of their precursors during winemaking on an industrial scale as well as to increase the aromatic potential of the produced wine. In the last decades, improvements of the analytical methods have been carried out in terms of sensitivity and identification of new odorants has been achieved. The goal for the researches has being the set up more sensitive and less-time consuming methods with a reduced impact on the environment in order to minimize defects and optimize the aromatic profile during winemaking.

## **Author details**

Daniela Fracassetti\* and Ileana Vigentini

\*Address all correspondence to: daniela.fracassetti@unimi.it

Department of Food, Environmental and Nutritional Sciences (DeFENS), Università degli Studi di Milano, Milan, Italy

## **References**


Microorganisms on Grapes, in Must and Wine. Heidelberg: Springer; 2008. pp. 211-233. DOI: 10.1007/978-3-540-85463-0

[3] Mestres M, Busto O, Guasch J. Analysis of organic sulfur compounds in wine aroma. Journal of Chromatography A. 2000;**881**:569-581. DOI: 10.1016/S0021-9673(00)00220-X

UV detector allows the quantification of GSH after its derivatization with *p*-benzoquinone [148]. The sample preparation is not required for must and wine, while the juice needs to be obtained from grape under reductive condition prior to the derivatization [111]. Suitable preparative conditions are mandatory for monitoring GSH concentration during must extraction and clarification since such wine making steps are responsible for the chemical and enzymatic

238 Grapes and Wines - Advances in Production, Processing, Analysis and Valorization

Several sulfur compounds occur in wine with a strong influence on its aromatic profile. On one hand, some of these molecules impart negative notes and their presence in wine should be counteracted and limited. The formation mechanisms are not completely clear and further researches are needed also to better understand the treatments and/or the winemaking practices potentially decreasing the appearance of these undesired compounds. On the other hand, the varietal thiols are responsible for positive characteristics of wine aroma up to a certain extent. Their protection is essential for the maintenance of the aromatic profile throughout both the winemaking and storage of wine by means of antioxidants of which reduce glutathione can represent a good natural candidate. Further studies will be necessary to investigate the fate of their precursors during winemaking on an industrial scale as well as to increase the aromatic potential of the produced wine. In the last decades, improvements of the analytical methods have been carried out in terms of sensitivity and identification of new odorants has been achieved. The goal for the researches has being the set up more sensitive and less-time consuming methods with a reduced impact on the environment in order to

minimize defects and optimize the aromatic profile during winemaking.

Department of Food, Environmental and Nutritional Sciences (DeFENS), Università degli

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[2] Bartowsky EJ, Pretorius IS. Microbial formation and modification of flavour and offflavour compounds in wine. In: König H, Unden G, Fröhlich J, editors. Biology of

and Biotechnology. 2008;**77**:1191-1205. DOI: 10.1007/s00253-007-1288-y

\*Address all correspondence to: daniela.fracassetti@unimi.it

oxidations.

**5. Conclusions**

**Author details**

**References**

Daniela Fracassetti\* and Ileana Vigentini

Studi di Milano, Milan, Italy


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**Chapter 12**

**Provisional chapter**

**Characterization of Red Wines from Macaronesia**

Jesús Heras-Roger, Carlos Díaz-Romero, Jacinto Darias-Martín

**Characterization of Red Wines from Macaronesia**

DOI: 10.5772/intechopen.71795

Wines from the Atlantic Islands of Macaronesia come from unusual *terroirs* due to their volcanic soils and the tropical and subtropical climatic conditions from this region. Some of these Islands produce highly appreciated fortified wines traded around the world since the sixteenth century, such as Madeira or Canary. Nowadays their distinct winemaking techniques and sweet wine traditions combine with the production of table wines. Previous studies described peculiar properties in wines from these regions, mostly related with their phenolic content and color, which are particularly important in the less produced red wines. The main purpose of this chapter is to characterize red wines produced in the Atlantic Islands of Macaronesia in terms of oenological and physicochemical properties. Wines from these islands are extremely atypical, as their climates conditions are exceptional and red grape cultivars are exclusive. Furthermore, specific viticulture techniques are applied in these latitudes to proportionate the unique characteristics outlined in this work. Original experimental data from 300 red wines produced in 8 Atlantic islands from Canary and Cape Verde archipelago and an important reviewing study for Azores and Madeira red wines are considered herein. Results are presented

**Keywords:** Macaronesia, red wine, characterization, grape varieties, Canary Islands,

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution,

© 2018 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

and reproduction in any medium, provided the original work is properly cited.

plants reached the islands via geographical dispersal but many are endemic [1].

Macaronesia is a collection of four archipelagos located in the North Atlantic Ocean, in front of the coast of Europe and Africa (see **Figure 1**). The Macaronesian archipelagos from North to South are Azores and Madeira (Portugal), Canary Islands (Spain) and Cape Verde. All the Islands of Macaronesia are volcanic and none of them were part of a continent, so their native

Jesús Heras-Roger, Carlos Díaz-Romero,

http://dx.doi.org/10.5772/intechopen.71795

according to archipelago.

Cape Verde Islands

**1. Introduction**

and Domingo Rios-Mesa

**Abstract**

Jacinto Darias-Martín and Domingo Rios-Mesa

Additional information is available at the end of the chapter

Additional information is available at the end of the chapter

**Provisional chapter**

## **Characterization of Red Wines from Macaronesia**

Jesús Heras-Roger, Carlos Díaz-Romero, Jacinto Darias-Martín

**Characterization of Red Wines from Macaronesia**

DOI: 10.5772/intechopen.71795

Jesús Heras-Roger, Carlos Díaz-Romero, Jacinto Darias-Martín and Domingo Rios-Mesa and Domingo Rios-Mesa Additional information is available at the end of the chapter

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/intechopen.71795

#### **Abstract**

Wines from the Atlantic Islands of Macaronesia come from unusual *terroirs* due to their volcanic soils and the tropical and subtropical climatic conditions from this region. Some of these Islands produce highly appreciated fortified wines traded around the world since the sixteenth century, such as Madeira or Canary. Nowadays their distinct winemaking techniques and sweet wine traditions combine with the production of table wines. Previous studies described peculiar properties in wines from these regions, mostly related with their phenolic content and color, which are particularly important in the less produced red wines. The main purpose of this chapter is to characterize red wines produced in the Atlantic Islands of Macaronesia in terms of oenological and physicochemical properties. Wines from these islands are extremely atypical, as their climates conditions are exceptional and red grape cultivars are exclusive. Furthermore, specific viticulture techniques are applied in these latitudes to proportionate the unique characteristics outlined in this work. Original experimental data from 300 red wines produced in 8 Atlantic islands from Canary and Cape Verde archipelago and an important reviewing study for Azores and Madeira red wines are considered herein. Results are presented according to archipelago.

**Keywords:** Macaronesia, red wine, characterization, grape varieties, Canary Islands, Cape Verde Islands

#### **1. Introduction**

Macaronesia is a collection of four archipelagos located in the North Atlantic Ocean, in front of the coast of Europe and Africa (see **Figure 1**). The Macaronesian archipelagos from North to South are Azores and Madeira (Portugal), Canary Islands (Spain) and Cape Verde. All the Islands of Macaronesia are volcanic and none of them were part of a continent, so their native plants reached the islands via geographical dispersal but many are endemic [1].

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2018 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Macaronesia archipelagos are similar because of their volcanic origin and oceanic influences but very different between themselves. There is also high contrast inside the archipelagos. Azores, Madeira Island or west Canary Islands (La Palma and El Hierro) are extremely mountainous, with deep valleys and steep slopes where the vines grow on little terraces rich in organic matter. Nevertheless there are also plain arid islands like Porto Santo in Madeira or the east Canary Islands (Fuerteventura and Lanzarote) where grape cultivation is highly adapted. An exceptional climatic contrast is present in Tenerife (Canary Islands), Pico (Azores) and Fogo (Cape Verde), which are the most important wine producers in their, respectively, archipelagos and also islands characterized by steep volcanoes. The fertile terraces from each of these islands volcanoes permit to cultivate quality winemaking grapes from near to the see, as in Pico or Fuerteventura, to very high altitudes with an important day/night temperature

Characterization of Red Wines from Macaronesia http://dx.doi.org/10.5772/intechopen.71795 255

Macaronesia climatic conditions combined with rugged terrain, the potential for volcanic activity, extreme winds, difficulties for mechanical harvesting and inconveniences derived from fungal attacks do not exactly add up to the most ideal winemaking environment. Moreover vines need warm and dry condition combined with regular winters to grow well, but in these areas humidity from the ocean is constant and winters are extremely mild, therefore grape cultivars have adapted and only pursued the less sensible to fungal

Red wine is produced nowadays in all Macaronesian archipelagos with several varieties. Most of the production is quality wine bottled by firms with certified origin protection figures, as *DOP* or *Vinho Regional*. Nevertheless, there are also artisanal productions for self-consumption through all the productive islands. The peculiarities from these regions in terms of volcanic soil, hours of sun, humidity and climatic conditions proportionate specific attributes to the red wines produced. This research is particularly original as the vast majority of quality winemaking regions lay between 30 and 50° latitude and there are only a few subtropical and tropical winemaking regions producing red wines. Macaronesian wines have been previ-

Macaronesian archipelagos have a long winemaking tradition associated with fortified and white wines, thus these wines are the most studied [3–10]. Furthermore the production of red wine in Macaronesia is uneven. While some islands have a long winemaking history related to fortified wines and started red winemaking just some years ago, other islands produce red wines protected by European quality figures such as DOP for decades now. Moreover some regions export red wine to international markets whereas in other areas it is only produced artisanally for self-consumption. This section briefly presents the most important conditions associated with wine production and the current red winemaking state-of-the-art in each one of the Macaronesian archipelagos from North to South. In

contrast as in Tenerife or Fogo.

ously researched, but results are rarely compared.

**2. Red wine production in Macaronesia**

diseases.

**Figure 1.** Geographical situation of Macaronesian archipelagos.

Volcanic soils are usually related with highly appreciated wines, as many traditional winemaking regions highlight their volcanic origins like Tokaj in Hungary, Struma valley in Bulgaria, Santorini in Greece or Sicilia in Italy. Nevertheless peculiarities from Macaronesia wines come not exclusively from their volcanic origin, but mostly from their specific *terroirs*. These islands present unusual geographical conditions in terms of soil and location but also atypical traditions and extremely particular microclimates. The climate from these archipelagos ranges from Mediterranean in the Azores and Madeira to arid in some islands of the Canaries (Lanzarote and Fuerteventura) and Cape Verde (Sal and Maio), subtropical in some Canary Islands (Tenerife, La Palma and El Hierro) or even tropical in the case of Fogo in Cape Verde. In general, Portuguese archipelagos enjoy a cooler climate and higher rainfall than Canary Islands and Cape Verde with warmer winters and irregular rainfalls.

Winemaking grapes were introduced in the Macaronesian archipelagos hundreds of years ago by the first Portuguese and Spanish inhabitants. Their volcanic soils are mineral rich and relatively fertile, so it is possible to naturally obtain wines with high acidity. This peculiarity makes most of these islands well suited to elaborate fortified and white wines. Moreover wines from these areas benefit from an acid/sugar balance not easily achieved in other regions. Traditional grape cultivars used in Macaronesia are characterized by a high potential alcoholic content and a valuable acidity ideal to produce fortified wines [2].

Macaronesia archipelagos are similar because of their volcanic origin and oceanic influences but very different between themselves. There is also high contrast inside the archipelagos. Azores, Madeira Island or west Canary Islands (La Palma and El Hierro) are extremely mountainous, with deep valleys and steep slopes where the vines grow on little terraces rich in organic matter. Nevertheless there are also plain arid islands like Porto Santo in Madeira or the east Canary Islands (Fuerteventura and Lanzarote) where grape cultivation is highly adapted. An exceptional climatic contrast is present in Tenerife (Canary Islands), Pico (Azores) and Fogo (Cape Verde), which are the most important wine producers in their, respectively, archipelagos and also islands characterized by steep volcanoes. The fertile terraces from each of these islands volcanoes permit to cultivate quality winemaking grapes from near to the see, as in Pico or Fuerteventura, to very high altitudes with an important day/night temperature contrast as in Tenerife or Fogo.

Macaronesia climatic conditions combined with rugged terrain, the potential for volcanic activity, extreme winds, difficulties for mechanical harvesting and inconveniences derived from fungal attacks do not exactly add up to the most ideal winemaking environment. Moreover vines need warm and dry condition combined with regular winters to grow well, but in these areas humidity from the ocean is constant and winters are extremely mild, therefore grape cultivars have adapted and only pursued the less sensible to fungal diseases.

Red wine is produced nowadays in all Macaronesian archipelagos with several varieties. Most of the production is quality wine bottled by firms with certified origin protection figures, as *DOP* or *Vinho Regional*. Nevertheless, there are also artisanal productions for self-consumption through all the productive islands. The peculiarities from these regions in terms of volcanic soil, hours of sun, humidity and climatic conditions proportionate specific attributes to the red wines produced. This research is particularly original as the vast majority of quality winemaking regions lay between 30 and 50° latitude and there are only a few subtropical and tropical winemaking regions producing red wines. Macaronesian wines have been previously researched, but results are rarely compared.

## **2. Red wine production in Macaronesia**

Volcanic soils are usually related with highly appreciated wines, as many traditional winemaking regions highlight their volcanic origins like Tokaj in Hungary, Struma valley in Bulgaria, Santorini in Greece or Sicilia in Italy. Nevertheless peculiarities from Macaronesia wines come not exclusively from their volcanic origin, but mostly from their specific *terroirs*. These islands present unusual geographical conditions in terms of soil and location but also atypical traditions and extremely particular microclimates. The climate from these archipelagos ranges from Mediterranean in the Azores and Madeira to arid in some islands of the Canaries (Lanzarote and Fuerteventura) and Cape Verde (Sal and Maio), subtropical in some Canary Islands (Tenerife, La Palma and El Hierro) or even tropical in the case of Fogo in Cape Verde. In general, Portuguese archipelagos enjoy a cooler climate and higher rainfall than Canary Islands and Cape Verde with warmer win-

Winemaking grapes were introduced in the Macaronesian archipelagos hundreds of years ago by the first Portuguese and Spanish inhabitants. Their volcanic soils are mineral rich and relatively fertile, so it is possible to naturally obtain wines with high acidity. This peculiarity makes most of these islands well suited to elaborate fortified and white wines. Moreover wines from these areas benefit from an acid/sugar balance not easily achieved in other regions. Traditional grape cultivars used in Macaronesia are characterized by a high potential alco-

holic content and a valuable acidity ideal to produce fortified wines [2].

ters and irregular rainfalls.

**Figure 1.** Geographical situation of Macaronesian archipelagos.

254 Grapes and Wines - Advances in Production, Processing, Analysis and Valorization

Macaronesian archipelagos have a long winemaking tradition associated with fortified and white wines, thus these wines are the most studied [3–10]. Furthermore the production of red wine in Macaronesia is uneven. While some islands have a long winemaking history related to fortified wines and started red winemaking just some years ago, other islands produce red wines protected by European quality figures such as DOP for decades now. Moreover some regions export red wine to international markets whereas in other areas it is only produced artisanally for self-consumption. This section briefly presents the most important conditions associated with wine production and the current red winemaking state-of-the-art in each one of the Macaronesian archipelagos from North to South. In general, these regions present relatively mild winters with irregular rainfalls, therefore viticulture has been adapted to adequately handle the sprout of the vine.

The islands of Madeira have a long winemaking history. Madeira is internationally famous for the eponymous fortified wine produced there and extensively researched [6, 13]. As in most European countries, the *phylloxera* reached the island by the nineteenth century and Madeira vines were hardly affected. Their climatic conditions also mean constant viticulture

Characterization of Red Wines from Macaronesia http://dx.doi.org/10.5772/intechopen.71795 257

Not fortified wines production in Madeira have been relatively rare, but in the last decades these volcanic and fertile lands produced red wines that break the traditional fortified wine image of the industry from this region. These wines are labeled as *Terras Madeirenses*, a Portuguese wine region for both islands classified as *Vinho Regional* or table wines for nonfortified wines. The most used red cultivar is Tinta Negra Mole (also known as Negramoll or Mulata), but Bastardo (also known as Trousseau or Maturana Tinta), Complexa and Touriga Nacional are also present in red wine blendings. Some winemakers introduce international

The archipelago of Canary Islands is an autonomous community of Spain located about 100 km west of Africa. Red wine is produced in all the islands with European figures of protection, as the archipelago counts with 10 *DOP*. The largest of the archipelago's islands, Tenerife, is the only island with more than one *DOP* and also home to the majority of the region's production, particularly for export, but most Canary table wines are present in their local markets. These islands are the largest and most populated archipelago of

These islands have a subtropical climate with long hot summers and moderately warm winters. The absence of low temperatures during winter involves problems for the sprout of the vine, as cold hours are usually not enough to obtain a regular vine cycle. Therefore grape ripening can be extremely irregular even for the same vine plant depending on the microclimate. Moreover precipitation levels and maritime influences are highly dependent on location and elevation. Thus completely fertile green areas with high humidity as well as deserts can be

Wine-growing in this fertile islands dates back to the Spanish conquest in the fifteenth century. Tenerife was the most important centre of "*Malmsey*" (*Malvasia*) production in the past with a strong reputation for fortified white wines as well as other Macaronesian regions. White sweet wines from these islands were greatly appreciated in England and the States during the sixteenth century, being known as "*Canary*". Rivalry between kingdoms restrained their commerce in the eighteenth century and red winemaking grapes were then introduced to elaborate similar products to Madeira fortified wines, being this fact the first step toward

Canary Islands grape producers' adapted viticulture to their own specific conditions. For instance, in the arid and windy Lanzarote *DOP*, they found an indigenous way of cultivating

found on the same archipelago and sometimes even in the same island.

hazards related to fungal grape diseases.

varieties such as Cabernet Sauvignon or Merlot.

the red winemaking tradition from Tenerife [14].

**2.3. Canary Islands**

Macaronesia.

#### **2.1. Azores**

The archipelago of Azores is an autonomous region of Portugal located on a line between Lisbon and New Jersey, about 1400 km west of Portugal and about 1925 km southeast of Canada. All islands have high mountains, being mount Pico, on the island of Pico, the highest point in Portugal at 2351 m. All populated islands show conditions to elaborate quality wines, but most of the production is found in the islands of Pico, Graciosa and Terceira in the center group and São Miguel in the east group.

The climate of the Azores is mild and moist all year, as it is influenced by the distance to continents and the passing Gulf Stream. Due to the marine influence, temperatures remain mild year-round being climate generally wet and cloudy. The archipelago's mid-Atlantic location means that the greatest threats to the grapes are wind and sea water, not harsh temperatures. Wind effects have been traditionally solved by building peculiar stonewalls of volcanic rocks around the vines; these walls give protection from ocean winds and radiate heat at night. Humidity and cloudy consequences are controlled continuously evaluating the risk of fungal attack [11].

Azores winemaking history starts soon after the settlement of Portuguese conquerors in the mid-fifteenth century. Winemaking traditions from this area are related to fortified wine styles, as it might be expected from a region with its oceanic mild climate. Azores fortified wine resembles Porto and Madeira wines, but its production is characterized by the vines present in the island and the distance from the continent. Azores was affected by the *phylloxera* [12].

There are no *DOP* regions in this archipelago but there are three *IPR* regions: Pico, Biscoitos and Graciosa. Pico and Biscoitos traditionally produced fortified wines but today the three regions are mostly focused on unfortified white wines. Red wine has no tradition, as the cool oceanic climate lend to produce white wines with an adequate natural acidity. Nevertheless red wines are produced as table wines classified as *Açores Vinho Regional* using European well-known cultivars as Syrah, Merlot and Cabernet Sauvignon but also Touriga Nacional, Castelão and Complexa.

#### **2.2. Madeira**

The archipelago of Madeira is an autonomous region of Portugal located about 1000 km southwest of Portugal, and about 400 km north of the Canary Islands. In Madeira and Porto Santo Islands, grapes are cultivated to elaborate quality wines. The terrain of the mountainous volcanic island of Madeira is difficult to cultivate and vineyards are planted on terraces of basaltic bedrock. Their climate is oceanic with tropical influences, such as high rainfall. The archipelago enjoys mild temperatures strongly influenced by the ocean throughout the year.

The islands of Madeira have a long winemaking history. Madeira is internationally famous for the eponymous fortified wine produced there and extensively researched [6, 13]. As in most European countries, the *phylloxera* reached the island by the nineteenth century and Madeira vines were hardly affected. Their climatic conditions also mean constant viticulture hazards related to fungal grape diseases.

Not fortified wines production in Madeira have been relatively rare, but in the last decades these volcanic and fertile lands produced red wines that break the traditional fortified wine image of the industry from this region. These wines are labeled as *Terras Madeirenses*, a Portuguese wine region for both islands classified as *Vinho Regional* or table wines for nonfortified wines. The most used red cultivar is Tinta Negra Mole (also known as Negramoll or Mulata), but Bastardo (also known as Trousseau or Maturana Tinta), Complexa and Touriga Nacional are also present in red wine blendings. Some winemakers introduce international varieties such as Cabernet Sauvignon or Merlot.

#### **2.3. Canary Islands**

general, these regions present relatively mild winters with irregular rainfalls, therefore

The archipelago of Azores is an autonomous region of Portugal located on a line between Lisbon and New Jersey, about 1400 km west of Portugal and about 1925 km southeast of Canada. All islands have high mountains, being mount Pico, on the island of Pico, the highest point in Portugal at 2351 m. All populated islands show conditions to elaborate quality wines, but most of the production is found in the islands of Pico, Graciosa and Terceira in the center

The climate of the Azores is mild and moist all year, as it is influenced by the distance to continents and the passing Gulf Stream. Due to the marine influence, temperatures remain mild year-round being climate generally wet and cloudy. The archipelago's mid-Atlantic location means that the greatest threats to the grapes are wind and sea water, not harsh temperatures. Wind effects have been traditionally solved by building peculiar stonewalls of volcanic rocks around the vines; these walls give protection from ocean winds and radiate heat at night. Humidity and cloudy consequences are controlled continuously evaluating the risk of fungal

Azores winemaking history starts soon after the settlement of Portuguese conquerors in the mid-fifteenth century. Winemaking traditions from this area are related to fortified wine styles, as it might be expected from a region with its oceanic mild climate. Azores fortified wine resembles Porto and Madeira wines, but its production is characterized by the vines present in the island and the distance from the continent. Azores was affected by the *phyl-*

There are no *DOP* regions in this archipelago but there are three *IPR* regions: Pico, Biscoitos and Graciosa. Pico and Biscoitos traditionally produced fortified wines but today the three regions are mostly focused on unfortified white wines. Red wine has no tradition, as the cool oceanic climate lend to produce white wines with an adequate natural acidity. Nevertheless red wines are produced as table wines classified as *Açores Vinho Regional* using European well-known cultivars as Syrah, Merlot and Cabernet Sauvignon but also Touriga Nacional,

The archipelago of Madeira is an autonomous region of Portugal located about 1000 km southwest of Portugal, and about 400 km north of the Canary Islands. In Madeira and Porto Santo Islands, grapes are cultivated to elaborate quality wines. The terrain of the mountainous volcanic island of Madeira is difficult to cultivate and vineyards are planted on terraces of basaltic bedrock. Their climate is oceanic with tropical influences, such as high rainfall. The archipelago enjoys mild temperatures strongly influenced by the ocean throughout the year.

viticulture has been adapted to adequately handle the sprout of the vine.

256 Grapes and Wines - Advances in Production, Processing, Analysis and Valorization

**2.1. Azores**

attack [11].

*loxera* [12].

**2.2. Madeira**

Castelão and Complexa.

group and São Miguel in the east group.

The archipelago of Canary Islands is an autonomous community of Spain located about 100 km west of Africa. Red wine is produced in all the islands with European figures of protection, as the archipelago counts with 10 *DOP*. The largest of the archipelago's islands, Tenerife, is the only island with more than one *DOP* and also home to the majority of the region's production, particularly for export, but most Canary table wines are present in their local markets. These islands are the largest and most populated archipelago of Macaronesia.

These islands have a subtropical climate with long hot summers and moderately warm winters. The absence of low temperatures during winter involves problems for the sprout of the vine, as cold hours are usually not enough to obtain a regular vine cycle. Therefore grape ripening can be extremely irregular even for the same vine plant depending on the microclimate. Moreover precipitation levels and maritime influences are highly dependent on location and elevation. Thus completely fertile green areas with high humidity as well as deserts can be found on the same archipelago and sometimes even in the same island.

Wine-growing in this fertile islands dates back to the Spanish conquest in the fifteenth century. Tenerife was the most important centre of "*Malmsey*" (*Malvasia*) production in the past with a strong reputation for fortified white wines as well as other Macaronesian regions. White sweet wines from these islands were greatly appreciated in England and the States during the sixteenth century, being known as "*Canary*". Rivalry between kingdoms restrained their commerce in the eighteenth century and red winemaking grapes were then introduced to elaborate similar products to Madeira fortified wines, being this fact the first step toward the red winemaking tradition from Tenerife [14].

Canary Islands grape producers' adapted viticulture to their own specific conditions. For instance, in the arid and windy Lanzarote *DOP*, they found an indigenous way of cultivating vines on the arid ground known as *"gerias"*. This grape production system consists on funnelshaped hollows dug into thick layers of coarse volcanic ashes with only one vine per hollow. Hollows are filled with soil and poured thick layers of volcanic ashes over it, as the porous volcanic granules known as *"picón"* retain the night humidity to feed the plants during the day and concentrates heat at night. Furthermore producers built low semicircular walls around vines to protect them from the constant winds. Another example can be found in *"La Orotava" DOP* with a unique system of vine training called *"cordón trenzado"*, literally meaning braided or plaited cord. This method of vine training evolved as a result of constraints on the areas used for cultivation as the island present limited space. Traditionally, this vine training method allowed the braids to be easily moved and thus allow the soil below them to be used for growing additional crops.

made from oak but from *pinus canariensis.* These traditional red wines share organoleptical characters with Greek retsina wines, as their introduce pine in their winemaking techniques

Characterization of Red Wines from Macaronesia http://dx.doi.org/10.5772/intechopen.71795 259

The archipelago of Cape Verde is a sovereign nation located about 650 km west of the coast of Africa and about 1300 km south of Canary Islands. Fogo is the only island producing grapes in sufficient quantity to elaborate quality wines because it registers the greatest precipitations. Fogo is characterized by its particular steep volcano covered with ash where vegetation and

As in the rest of Macaronesia, the culture of vines was started by the first settlers during the sixteenth century. Fogo wine was even exported to Guinea and Brazil during the eighteenth century. Wines from this area are mostly white as grapes cultivated there produce refreshing wines with pleasant mineral notes. There is also a strong tradition of fortified sweet wines locally known as *"Pasito",* as the homonym wine from Italy. Red wines are not the most produced in the island but they can be easily found in restaurants and local markets even though they have no origin protection figure. In fact wine tourism is taking an important role in this

The importance of latitude and altitude is one of the most relevant aspects when it comes to understanding the *terroir* from this region. Fogo is located much closer to the equator than most of the world's key wine-growing regions and its vineyards are generally more than 1500 m above sea level. These conditions temper the high temperatures and the heat associated with its latitude. Cooler nights delay grape ripening and sunlight absorbed by black soils during the day keeps the vines isolated at night. In this sense, climate cannot be strictly described as tropical. Due to its altitude and high irregular rainfall, it is possible to obtain quality wines, even with the possibility of a second harvest in the month of January given the

In Fogo, vine cultivation is not exclusive and is normally associated with other products such as corn or sweet potatoes. At the foot of the volcano is the wine-growing community of Chã das Caldeiras where most of the samples from this study come from. Chã is located at 1700 m altitude and characterized by small vineyards around the active volcano erupted several times recently (1995, 2000 and 2014). This area enjoys a microclimate with thermal amplitude, good rainfall and rich volcanic soils, being all these factors important for quality winemaking production using adequate viticulture practices [17]. Oidium is the only fungal disease

A range of European grape varieties are cultivated in the Island. The most used red cultivar is Tinta Preta (also known as Baboso Negro, Bruñal or Alfrocheiro Preto), but also other red cultivars are present such as Castelão (also known as Periquita), Bastardo (also known as

Trousseau or Maturana Tinta) and Moscatel Negro (also known as Black Hamburg).

giving them a peculiar resin aroma.

cultivation of the vine is extremely complicated.

natural vine cycle in this environment.

detected and the *phyloxera* never reached Cape Verde.

**2.4. Cape Verde**

island [15, 16].

In general vines from Canary Islands are old when compared to other winemaking regions as the *phylloxera* never reached the archipelago. Most vines are not planted on rootstocks but directly on the ground and draw water solely from the terrain as usually they are not irrigated. Mechanical harvesting is impossible given the specific methods of vine training and the small pieces of land. Vineyards are fragmented and can be found near the coast or at elevated altitudes. Most vineyards are planted on the gentle lower slopes of the mountains present where humid mists created by the winds moderate the hot maritime climate.

Red winemaking is important in the Canary Islands but some islands traditionally produce more than others. Most of these red wines are fresh and young because of the natural acidity balance of their soils but some regions are also specialized in oak-aged red wines. These red wines are usually elaborated with indigenous grapes present for centuries in the islands, but international grape varieties (Syrah, Merlot and Cabernet) can also be used. The most widespread red grape cultivar is Listán Negro (also known as Almuñeco), a variety well suited to Beaujolais-like wine style made using carbonic maceration. The workhorse grape of Madeira Tinta Negra is also present in Canary Islands known as Negramoll. The varietal richness of these islands is amazingly high, as many other red grapes can be found exclusively vinified by some winemakers, such as Tintilla, Bastardo (also known as Trousseau), Baboso (also known as Alfrocheiro or Tinta Preta in Cape Verde), Listán Prieto (also known as Mission Grape in the United States of America), Vijariego Negro or Castellana among others. Not just the grapes from ungrafted and often extremely senior vines make Canary red wines atypical. The combination of little-known grapes, high-elevation vineyards and volcanic soils made these red wines distinctive.

Red wine is produced in the 10 *DOP*. Tenerife houses half of the region's *DOP* with Abona, Tacoronte-Acentejo, Valle de Guimar, Valle de la Orotava and Ycoden-Daute-Isora. The remaining designations cover other islands in their entirety such as El Hierro, Gran Canaria, La Gomera, La Palma and Lanzarote *DOP*s. There is also a general *DOP* including all islands (also Fuerteventura). Each area has a unique microclimate and different soil composition, lending to distinctive wines with mineral notes. Some areas even present clear subzones inside the same *DOP*. For instance, only in the North area of La Palma Island a peculiar red wine called *"Vino de Tea"* is produced. These wines are sold straight from atypical barrels not made from oak but from *pinus canariensis.* These traditional red wines share organoleptical characters with Greek retsina wines, as their introduce pine in their winemaking techniques giving them a peculiar resin aroma.

#### **2.4. Cape Verde**

vines on the arid ground known as *"gerias"*. This grape production system consists on funnelshaped hollows dug into thick layers of coarse volcanic ashes with only one vine per hollow. Hollows are filled with soil and poured thick layers of volcanic ashes over it, as the porous volcanic granules known as *"picón"* retain the night humidity to feed the plants during the day and concentrates heat at night. Furthermore producers built low semicircular walls around vines to protect them from the constant winds. Another example can be found in *"La Orotava" DOP* with a unique system of vine training called *"cordón trenzado"*, literally meaning braided or plaited cord. This method of vine training evolved as a result of constraints on the areas used for cultivation as the island present limited space. Traditionally, this vine training method allowed the braids to be easily moved and thus allow the soil below them to be used for grow-

258 Grapes and Wines - Advances in Production, Processing, Analysis and Valorization

In general vines from Canary Islands are old when compared to other winemaking regions as the *phylloxera* never reached the archipelago. Most vines are not planted on rootstocks but directly on the ground and draw water solely from the terrain as usually they are not irrigated. Mechanical harvesting is impossible given the specific methods of vine training and the small pieces of land. Vineyards are fragmented and can be found near the coast or at elevated altitudes. Most vineyards are planted on the gentle lower slopes of the mountains present

Red winemaking is important in the Canary Islands but some islands traditionally produce more than others. Most of these red wines are fresh and young because of the natural acidity balance of their soils but some regions are also specialized in oak-aged red wines. These red wines are usually elaborated with indigenous grapes present for centuries in the islands, but international grape varieties (Syrah, Merlot and Cabernet) can also be used. The most widespread red grape cultivar is Listán Negro (also known as Almuñeco), a variety well suited to Beaujolais-like wine style made using carbonic maceration. The workhorse grape of Madeira Tinta Negra is also present in Canary Islands known as Negramoll. The varietal richness of these islands is amazingly high, as many other red grapes can be found exclusively vinified by some winemakers, such as Tintilla, Bastardo (also known as Trousseau), Baboso (also known as Alfrocheiro or Tinta Preta in Cape Verde), Listán Prieto (also known as Mission Grape in the United States of America), Vijariego Negro or Castellana among others. Not just the grapes from ungrafted and often extremely senior vines make Canary red wines atypical. The combination of little-known grapes, high-elevation vineyards and volcanic soils made

Red wine is produced in the 10 *DOP*. Tenerife houses half of the region's *DOP* with Abona, Tacoronte-Acentejo, Valle de Guimar, Valle de la Orotava and Ycoden-Daute-Isora. The remaining designations cover other islands in their entirety such as El Hierro, Gran Canaria, La Gomera, La Palma and Lanzarote *DOP*s. There is also a general *DOP* including all islands (also Fuerteventura). Each area has a unique microclimate and different soil composition, lending to distinctive wines with mineral notes. Some areas even present clear subzones inside the same *DOP*. For instance, only in the North area of La Palma Island a peculiar red wine called *"Vino de Tea"* is produced. These wines are sold straight from atypical barrels not

where humid mists created by the winds moderate the hot maritime climate.

ing additional crops.

these red wines distinctive.

The archipelago of Cape Verde is a sovereign nation located about 650 km west of the coast of Africa and about 1300 km south of Canary Islands. Fogo is the only island producing grapes in sufficient quantity to elaborate quality wines because it registers the greatest precipitations. Fogo is characterized by its particular steep volcano covered with ash where vegetation and cultivation of the vine is extremely complicated.

As in the rest of Macaronesia, the culture of vines was started by the first settlers during the sixteenth century. Fogo wine was even exported to Guinea and Brazil during the eighteenth century. Wines from this area are mostly white as grapes cultivated there produce refreshing wines with pleasant mineral notes. There is also a strong tradition of fortified sweet wines locally known as *"Pasito",* as the homonym wine from Italy. Red wines are not the most produced in the island but they can be easily found in restaurants and local markets even though they have no origin protection figure. In fact wine tourism is taking an important role in this island [15, 16].

The importance of latitude and altitude is one of the most relevant aspects when it comes to understanding the *terroir* from this region. Fogo is located much closer to the equator than most of the world's key wine-growing regions and its vineyards are generally more than 1500 m above sea level. These conditions temper the high temperatures and the heat associated with its latitude. Cooler nights delay grape ripening and sunlight absorbed by black soils during the day keeps the vines isolated at night. In this sense, climate cannot be strictly described as tropical. Due to its altitude and high irregular rainfall, it is possible to obtain quality wines, even with the possibility of a second harvest in the month of January given the natural vine cycle in this environment.

In Fogo, vine cultivation is not exclusive and is normally associated with other products such as corn or sweet potatoes. At the foot of the volcano is the wine-growing community of Chã das Caldeiras where most of the samples from this study come from. Chã is located at 1700 m altitude and characterized by small vineyards around the active volcano erupted several times recently (1995, 2000 and 2014). This area enjoys a microclimate with thermal amplitude, good rainfall and rich volcanic soils, being all these factors important for quality winemaking production using adequate viticulture practices [17]. Oidium is the only fungal disease detected and the *phyloxera* never reached Cape Verde.

A range of European grape varieties are cultivated in the Island. The most used red cultivar is Tinta Preta (also known as Baboso Negro, Bruñal or Alfrocheiro Preto), but also other red cultivars are present such as Castelão (also known as Periquita), Bastardo (also known as Trousseau or Maturana Tinta) and Moscatel Negro (also known as Black Hamburg).

Most of the samples from this study are red wines from the vineyards located around the caldera and produced by Chã das Caldeiras wine cooperative. These wines are mainly elaborated Tinta Preta and destined to early consumption without periods of guard. In addition to the bottled quality wines from Chã our study also consider sweet red wines produced artisanally from other vineyards situated on the volcanic soils inside the caldera. These wines are less representative, produced in small volumes and mostly destined for self-consumption soon after the fermentation. These traditional homemade wines are known as *"manecom". "Manecoms"* can be both white or red but sweet red wines are the most popular and the only ones considered in our study. Although homemade, some of these wines are intended for both personal consumption and to be marketed in bulk.

In addition to Chã Caldeiras winery whose vineyards are in the caldera, there are other wineries in the island like Monte Barro or Achada Grand producing bottled red wines. Most probably these wines differ in the physico-chemical characteristics due to the differences in the vine cultivation area, related to both altitude (1700 m vs. 700 m) and rainfall.

## **3. Macaronesian red wine characterization**

#### **3.1. Material and methods**

Experimental results from 300 red wines produced in Canary Islands and Fogo (Cape Verde) are described herein together with scientific literature from red wines produced in Azores and Madeira. The 250 samples from Canary Islands and 40 red wines from Fogo were bottled as quality wine destined to the regional market. The remaining 10 samples of red wine from Fogo were *"manecom"*; this means red wine elaborated for selfconsumption.

Samples come from different harvest (2003–2014). Most analysis was performed using International Organisation of Vine and Wine (OIV) reference methods [18]. A phenolic and metallic profile was also obtained. Color characteristics using CIELab and magnitude of co-pigmentation are important in red wines and thus were analytically quantified. **Table 1** presents the methods applied with the uncertainty associated to each parameter. All data have been grouped and compared according to the archipelago of origin introducing similar data from published scientific works when available. Significant differences between islands from the same archipelago or even between regions from the same island have been previously detailed in the literature due to particular microclimates influences. Nevertheless differences inside archipelagos are not considered in the present chapter as they are not part of the aim of this study.

islands. Most red wines considered were produced to be consumed in the short term, so they have typical young wines values in terms of phenolics. Nevertheless some samples present

All samples conformed to the established legal wine standards as far as analytical parameters are concerned. Canary Islands results are within the usual ranges and comparable to those published for the same geographic origin [19, 20], although pH is slightly higher than usual [21]. This can be due to their characteristic soils and warm climate conditions, as it have been previously demonstrated that wines produced in warm areas trend to present higher pHs than those produced in colder regions [22]. Moreover the tannin content is also higher than

high potential for aging, as they are distinguished by high total phenol index (IPT).

**Wine characteristics Canary (bottled with DOP) Cape Verde/Fogo**

13.72 ± 1.20 (11.00–19.54)

(0.9893–1.0513)

(3.23–4.54)

5.16 ± 0.72 (3.93–7.59)

1.90 ± 6.90 (n.d.–59.00)

(11.0–184.0)

38.0 ± 29.0 (n.d.–207.0)

(14.6–103.1)

(0.20–4.40)

296.1 ± 178.8 (29.2–798.4)

Results expressed as Χ ± σ (Min.–Max. value if available). n.d. = not detected.

**Table 1.** Conventional red wine parameters from Canary and Cape Verde islands.

(mg/l) 78.0 ± 34.0

IPT DO 280 nm (UA) 52.7 ± 12.9

Total tannins (g/l) 2.00 ± 0.80

) 0.9941 ± 0.006

pH 3.74 ± 0.18

Alcoholic content

Titrable acidity (g.

Glucose + fructose

Nitrogen ammonia

Total anthocyanins

(mg/l)

Density (g/cm3

(%v/v)

tart./l)

(g/l)

(g/l)

Free SO<sup>2</sup>

Total SO<sup>2</sup>

**(n = 250) (n = 60) [19, 20] Chã Caldeiras bottled** 

Ethanal (mg/l) 16 ± 39 (n.d.–246) 30 ± 24 30 ± 45 (n.d.–150) 50 ± 65 (15–310)

Glycerol (g/l) 11.0 ± 2.9 (0.4–20.2) – 7.6±0.3 (7.4–7.8) 8.0 ± 0.1 (7.8–8.2)

Folin–Ciocalteu Index 50 ± 11 (27–83) – 62 ± 15 (32–93) 32 ± 25 (15–76)

(mg/l) 16.0 ± 8.0 (5.0–48.0) 16.0 ± 9.0 24.4 ± 13.8 (7.0–47.0) 1.5 ± 0.71 (1.0–2.0)

12.28 ± 0.60 15.58 ± 0.61

0.9922 ± 0.001 0.9896 ± 0.0116

**(n = 40)**

(14.70–17.66)

(0.9370–0.9944)

3.55 ± 0.17 3.99 ± 0.18 (3.46–4.23) 3.94 ± 0.02 (3.93–3.95)

5.10 ± 0.54 4.95 ± 0.71 (4.15–6.62) 7.89 ± 1.61 (6.75–9.03)

1.95 ± 0.95 1.70 ± 2.21 (n.d.–5.9) 43.05 ± 31.75 (20.60–65.5)

57.0 ± 32.0 83.4 ± 27.6 (26.0–129.0) 12.5 ± 3.5 (10.0–15.0)

– 27.6 ± 19.1 (10.3–59.8) 53.3 ± 20.4 (20.1–60.8)

35.0 ± 5.6 68.3 ± 8.7 (56.7–89.3) 70.6 ± 16.2 (59.1–82.0)

1.30 ± 0.40 4.78 ± 0.61 (3.97–6.25) 4.94 ± 1.13 (4.14–5.74)

108.2 ± 55.2 350 ± 202 (33.7–815.3) 123.1 ± 198.9 (12.2–489.5)

**"Manecom" (n = 10)**

Characterization of Red Wines from Macaronesia http://dx.doi.org/10.5772/intechopen.71795

> 1.0130 ± 0.0096 (1.0062–1.0198)

16.67 ± 0.87 (16.06–17.29)

261

#### **3.2. Oenological parameters**

Results for the most important oenological parameters in red wines are summarized in **Table 1**. Canary wines are characterized by a great variability and heterogeneity, as these wines come from different harvest and are elaborated with different varietals in various


Results expressed as Χ ± σ (Min.–Max. value if available). n.d. = not detected.

Most of the samples from this study are red wines from the vineyards located around the caldera and produced by Chã das Caldeiras wine cooperative. These wines are mainly elaborated Tinta Preta and destined to early consumption without periods of guard. In addition to the bottled quality wines from Chã our study also consider sweet red wines produced artisanally from other vineyards situated on the volcanic soils inside the caldera. These wines are less representative, produced in small volumes and mostly destined for self-consumption soon after the fermentation. These traditional homemade wines are known as *"manecom". "Manecoms"* can be both white or red but sweet red wines are the most popular and the only ones considered in our study. Although homemade, some of these wines are intended for both personal

In addition to Chã Caldeiras winery whose vineyards are in the caldera, there are other wineries in the island like Monte Barro or Achada Grand producing bottled red wines. Most probably these wines differ in the physico-chemical characteristics due to the differences in the

Experimental results from 300 red wines produced in Canary Islands and Fogo (Cape Verde) are described herein together with scientific literature from red wines produced in Azores and Madeira. The 250 samples from Canary Islands and 40 red wines from Fogo were bottled as quality wine destined to the regional market. The remaining 10 samples of red wine from Fogo were *"manecom"*; this means red wine elaborated for self-

Samples come from different harvest (2003–2014). Most analysis was performed using International Organisation of Vine and Wine (OIV) reference methods [18]. A phenolic and metallic profile was also obtained. Color characteristics using CIELab and magnitude of co-pigmentation are important in red wines and thus were analytically quantified. **Table 1** presents the methods applied with the uncertainty associated to each parameter. All data have been grouped and compared according to the archipelago of origin introducing similar data from published scientific works when available. Significant differences between islands from the same archipelago or even between regions from the same island have been previously detailed in the literature due to particular microclimates influences. Nevertheless differences inside archipelagos

are not considered in the present chapter as they are not part of the aim of this study.

Results for the most important oenological parameters in red wines are summarized in **Table 1**. Canary wines are characterized by a great variability and heterogeneity, as these wines come from different harvest and are elaborated with different varietals in various

vine cultivation area, related to both altitude (1700 m vs. 700 m) and rainfall.

consumption and to be marketed in bulk.

**3.1. Material and methods**

**3.2. Oenological parameters**

consumption.

**3. Macaronesian red wine characterization**

260 Grapes and Wines - Advances in Production, Processing, Analysis and Valorization

**Table 1.** Conventional red wine parameters from Canary and Cape Verde islands.

islands. Most red wines considered were produced to be consumed in the short term, so they have typical young wines values in terms of phenolics. Nevertheless some samples present high potential for aging, as they are distinguished by high total phenol index (IPT).

All samples conformed to the established legal wine standards as far as analytical parameters are concerned. Canary Islands results are within the usual ranges and comparable to those published for the same geographic origin [19, 20], although pH is slightly higher than usual [21]. This can be due to their characteristic soils and warm climate conditions, as it have been previously demonstrated that wines produced in warm areas trend to present higher pHs than those produced in colder regions [22]. Moreover the tannin content is also higher than the values described for French vines [21], although this can be due to an over-quantification, since the method counts with limited specificity.

Most wines from Cape Verde present high ethanol content, probably due to the natural conditions affecting this archipelago and to late grape picking. The lowest ethanol content in Fogo attained 14.7%vol, higher than the average in Canary. *"Manecoms"* show an alcoholic graduation even higher than bottled wines, probably because Fogo inhabitants are used to wines with high alcoholic content.

Fogo wines also present unusual high pHs. These values lead to color losses in red wines due to anthocyanins chemical form and their greater oxidation [23]. High pH also reduces the protective action from SO<sup>2</sup> . pH depends on various factors from soil, climate or grape ripening to winemaking techniques and the values observed might be due to regional characteristics, such as soil potassium content, climate and skins macerations. In this sense Fogo winemakers could diminish wine pH by reducing maceration periods, with earlier harvest or introducing specific rootstocks. The relationship between high pH in these wines and potassium content are further discussed in the metallic profile section.

The residual nitrogen ammonia for most *"manecoms"* samples is high in relation with conventional levels [21]. This nitrogen bioavailability leads to a greater ease of wine diseases increased by their high pH and low SO<sup>2</sup> protection. Nevertheless Fogo wines avoid many microbiological problems due to their high alcoholic content. In general, these wines contain low levels of ethanol, as most samples are not subjected to micro oxygenation. Their high pHs discourage winemakers to elaborate oak-aged wines, as color stability would be hardly affected by time [24]. Red wines from both archipelagos were also characterized by high glycerol values in agreement with their high alcohol content.

grapes fungal attacks are common in Canary Islands according to our results. Madeira's citric acid content is unusually high and extremely low in Fogo. In Canary Islands, the citric acid

**Canary (bottled with DOP) Cape Verde/Fogo Madeira (n <sup>=</sup> 12) [25] (n <sup>=</sup> 250) (n <sup>=</sup> 60) [19] Chã Bottled (n = 40)**

2.08 ± 0.42 1.60 ± 0.61

– 2.40 ± 0.26

0.50 ± 0.14 0.81 ± 0.14

0.24 ± 0.34 0.56 ± 1.11

– 0.05 ± 0.05

– 70 ± 42

Results expressed as Χ ± σ (Min. – Max. value if available). n.d. = not detected.

**Table 2.** Red wine acid profile from canary, Cape Verde and Madeira islands.

*"Manecom***" (n = 10)**

(2.20–2.70) 1.35 ± 1.39 (0.10–2.70) –

(0.53–1.19) 2.09 ± 1.56 (0.99–3.20) –

(0.10–3.72) 0.27 ± 0.31 (0.05–0.50) –

(n.d.–0.1) 0.02 ± 0.04 (n.d.–0.10) –

(n.d.–150) <sup>30</sup> <sup>±</sup> 12 (n.d.–50) 445 ± 220

(1.20–2.30) 1.95 ± 0.92 (1.30–2.60) 1.97 ± 0.33

Characterization of Red Wines from Macaronesia http://dx.doi.org/10.5772/intechopen.71795

(1.50–2.73)

263

(141–707)

Some scientist previously addressed wine metallic differentiation depending on the Canary Island of origin [26–28] and studied the content from Azores and Madeira wines [29]. Differences between *DOP* and islands are in some cases highly significant. The cited references are useful to study these specific profiles but this is not our aim, as it introduces other considerations. **Table 3** resumes experimental data and average results from

The metallic content conforms to commercial standards though potassium concentration is slightly higher than values previously reported [30]. Its source in wine is diverse; on one hand the addition of potassium during winemaking can increase it, but soil content due to other crops enrichment or fertilizers is also relevant. However, soil characteristics considered alone cannot explain its high content. It can be also increased by winemaking techniques, such as excessive macerations with grape skins for instance. Most probably its concentration is related to the grapevine needs, as in warm, dry and windy climates vine plants need more water as it is constantly evaporating from their leaves. Therefore grapevines breathe intensively in order to absorb soil water in a greater extent than those plants located in cold or humid climates. Greater water absorption contributes to a greater mineral absorption. Thus in warm climates plants absorb more water and by addition more potassium, concentrating

content highly varies probably due to the different elaborations considered.

**3.4. Metallic content**

literature.

Tartaric acid (g/l)

Lactic acid (g/l)

Acetic acid (g/l)

L-malic acid (g/l)

Gluconic acid (g/l)

Citric acid (mg/l)

2.50 ± 1.00 (1.10–4.90)

1.78 ± 1.00 (0.07–5.53)

0.60 ± 0.24 (0.15–1.53)

0.38 ± 0.71 (n.d.–4.21)

0.36 ± 0.42 (n.d.–2.29)

140 ± 102 (n.d.–620)

#### **3.3. Acid profile**

Wine acids are classified in two groups. The first one includes those whose concentrations evolve during grape ripening, such as tartaric, malic and citric acid. In the second group are those derived from fermentations such as lactic, acetic and succinic acid. Gluconic acid is out of this classification because of its fungal origin. These acids content is detailed on **Table 2**.

None bottled wine exceeds the legislated 1.2 g/l value for volatile acidity. Those presenting greater values correspond to sweet wines with greater limits. "*Manecoms*" show extremely high values due to their artisan winemaking. The initial malic acid content in wine depends on grape ripening, but its concentration drastically reduces after malolactic fermentation. This fermentation was completed in most red wines, even though some samples present high values above 4 g/l. These wines acid content is within the values considered normal [21], although some Canary wines present lactic acid contents above the average.

The very low gluconic acid in Fogo wines reveals none important fungal incidences, probably due to the extremely dry climatic conditions derived from latitude and altitude. Nevertheless


**Table 2.** Red wine acid profile from canary, Cape Verde and Madeira islands.

grapes fungal attacks are common in Canary Islands according to our results. Madeira's citric acid content is unusually high and extremely low in Fogo. In Canary Islands, the citric acid content highly varies probably due to the different elaborations considered.

#### **3.4. Metallic content**

the values described for French vines [21], although this can be due to an over-quantification,

Most wines from Cape Verde present high ethanol content, probably due to the natural conditions affecting this archipelago and to late grape picking. The lowest ethanol content in Fogo attained 14.7%vol, higher than the average in Canary. *"Manecoms"* show an alcoholic graduation even higher than bottled wines, probably because Fogo inhabitants are used to wines

Fogo wines also present unusual high pHs. These values lead to color losses in red wines due to anthocyanins chemical form and their greater oxidation [23]. High pH also reduces the pro-

to winemaking techniques and the values observed might be due to regional characteristics, such as soil potassium content, climate and skins macerations. In this sense Fogo winemakers could diminish wine pH by reducing maceration periods, with earlier harvest or introducing specific rootstocks. The relationship between high pH in these wines and potassium content

The residual nitrogen ammonia for most *"manecoms"* samples is high in relation with conventional levels [21]. This nitrogen bioavailability leads to a greater ease of wine diseases

microbiological problems due to their high alcoholic content. In general, these wines contain low levels of ethanol, as most samples are not subjected to micro oxygenation. Their high pHs discourage winemakers to elaborate oak-aged wines, as color stability would be hardly affected by time [24]. Red wines from both archipelagos were also characterized by high glyc-

Wine acids are classified in two groups. The first one includes those whose concentrations evolve during grape ripening, such as tartaric, malic and citric acid. In the second group are those derived from fermentations such as lactic, acetic and succinic acid. Gluconic acid is out of this classification because of its fungal origin. These acids content is detailed

None bottled wine exceeds the legislated 1.2 g/l value for volatile acidity. Those presenting greater values correspond to sweet wines with greater limits. "*Manecoms*" show extremely high values due to their artisan winemaking. The initial malic acid content in wine depends on grape ripening, but its concentration drastically reduces after malolactic fermentation. This fermentation was completed in most red wines, even though some samples present high values above 4 g/l. These wines acid content is within the values considered normal [21], although some Canary wines present lactic acid contents above

The very low gluconic acid in Fogo wines reveals none important fungal incidences, probably due to the extremely dry climatic conditions derived from latitude and altitude. Nevertheless

. pH depends on various factors from soil, climate or grape ripening

protection. Nevertheless Fogo wines avoid many

since the method counts with limited specificity.

262 Grapes and Wines - Advances in Production, Processing, Analysis and Valorization

are further discussed in the metallic profile section.

erol values in agreement with their high alcohol content.

increased by their high pH and low SO<sup>2</sup>

with high alcoholic content.

tective action from SO<sup>2</sup>

**3.3. Acid profile**

on **Table 2**.

the average.

Some scientist previously addressed wine metallic differentiation depending on the Canary Island of origin [26–28] and studied the content from Azores and Madeira wines [29]. Differences between *DOP* and islands are in some cases highly significant. The cited references are useful to study these specific profiles but this is not our aim, as it introduces other considerations. **Table 3** resumes experimental data and average results from literature.

The metallic content conforms to commercial standards though potassium concentration is slightly higher than values previously reported [30]. Its source in wine is diverse; on one hand the addition of potassium during winemaking can increase it, but soil content due to other crops enrichment or fertilizers is also relevant. However, soil characteristics considered alone cannot explain its high content. It can be also increased by winemaking techniques, such as excessive macerations with grape skins for instance. Most probably its concentration is related to the grapevine needs, as in warm, dry and windy climates vine plants need more water as it is constantly evaporating from their leaves. Therefore grapevines breathe intensively in order to absorb soil water in a greater extent than those plants located in cold or humid climates. Greater water absorption contributes to a greater mineral absorption. Thus in warm climates plants absorb more water and by addition more potassium, concentrating


The content of iron is noteworthy in all the archipelagos probably because of their volcanic soils, as it is difficult to suggest any other cause affecting all islands. Copper concentration is unusually variable between regions, relatively high in Madeira but extremely low in Fogo. Similarly, zinc and manganese content are low in Fogo wines when compared to other Macaronesia regions. Calcium and magnesium content are similar between archipelagos. The different concentration of all these minerals might be an interesting source for wine characterization according to geographical area using them as potential origin

Characterization of Red Wines from Macaronesia http://dx.doi.org/10.5772/intechopen.71795 265

Wines from Madeira are mostly characterized by 2-ethylhexan-1-ol, 3,5,5-trimethylhexan-1-ol, ethyl 2-methylbutanoate, ethyl DL-2-hydroxycaproate, decanoic acid and 2-ethoxythiazole, whereas wines from Azores and Canary Islands are mainly characterized by 3-ethoxypropan-1-ol, 1-octen-3-ol, (Z)-3-hexenyl butanoate, 2,3-dihydrobenzofuran and 4-(methylthio)-1-butanol [32]. This latter compound is particularly important in Fogo, where red wines present an unusual high concentration of heavy sulfur volatiles in its aroma [33]. These concentrations might be due to the active volcano where grapevines are located. Fogo wines also present the highest concentration in the minor volatiles fatty acids. Results for the most common volatile

Higher alcohols followed by ethyl esters and fatty acids are the most predominant chemical groups among the volatiles from red wines elaborated in Macaronesia. In fact hexanolderived compounds with decaonic acids and whiskey lactones discriminate Madeira red wines from the rest of Macaronesian archipelagos [32] and a characteristic profile for alcohols, ethyl esters and fatty acids has been obtained for these wines [13]. Similarly, the concentration of esters in Azores wines revealed to be significantly lower than in red wines from the other Macaronesian archipelagos. The unique exception is the ethyl hexanoate compound, which

Canary wines studies conclude that 3-metyl-1-butanol, 2,2-butanodiona and ethyl butirate were the main odorants [37, 38]. Moreover specific studies have been conducted to analyze

Phenolic compounds are responsible for the most important sensory attributes of red wines. Phenolic content depends on the winemaking, which are influenced by factors such as variety, ripening, cultivation techniques and climatic conditions among other considerations [40]. Thus the phenolic composition of Macaronesian red wines is potentially different from other regions because of their specific *"terroir".* Gallic and caftaric acids are the most concentrated hydroxybenzoic and hydroxycinnamic acid respectively in Canary wines [41] but there is high heterogeneity. Previous studies for the same region confirmed that these wines polyphenolic composition is highly heterogenic and depends on various factors such as local climate

markers.

**3.5. Volatile compounds**

**3.6. Phenolic content**

compounds in red wines are summarized in **Table 4**.

seems to be present in a greater extent than in other islands [33–36].

the volatiles from red wines aged in pine casks from La Palma Island [39].

**Table 3.** Metallic content (mg/l) from canary, Cape Verde, Madeira and Azores islands.

this metal in grapes and latter in wine. The greater or lesser concentration might depend on the climatic conditions of each harvest, on the grapevine situation as greater winds or dryness enhances it, and even on the vine itself, as roots absorb potassium differently depending on the cultivar. This climate influence might explain the potassium increase in Canary Islands wine, as the literature cited is almost 15 years old and today vines are exposed to warmer conditions due to the temperature increase from the last decade.

Potassium concentrations correlate with high wine pHs, a peculiar characteristic already observed in the previous section for Fogo wines. Since high pHs lead to a greater wine sensitivity to microbiological diseases, it is advisable to control potassium content. Obviously potassium is not the only factor conditioning the final wine pH, although it is one of the most important according to our experimental results.

The sodium content for all Macaronesian regions except Fogo is higher than those from other origins [31], probably due to oceanic winds and the marine aerosol. Fogo samples contain significantly less than most Canary, Azorean and Madeiran red wines because of the high altitude of Chã vineyards. In fact Fogo sodium content is similar to continental wines, where the marine aerosol is non-existent and its origin is essentially due to soil and agronomic practices. Some Canary red wines also presented low sodium content when compared with most samples from the same region, coming these wines from Tenerife vineyards located at high altitudes, which are presumably less influenced by oceanic winds.

The content of iron is noteworthy in all the archipelagos probably because of their volcanic soils, as it is difficult to suggest any other cause affecting all islands. Copper concentration is unusually variable between regions, relatively high in Madeira but extremely low in Fogo. Similarly, zinc and manganese content are low in Fogo wines when compared to other Macaronesia regions. Calcium and magnesium content are similar between archipelagos. The different concentration of all these minerals might be an interesting source for wine characterization according to geographical area using them as potential origin markers.

#### **3.5. Volatile compounds**

Wines from Madeira are mostly characterized by 2-ethylhexan-1-ol, 3,5,5-trimethylhexan-1-ol, ethyl 2-methylbutanoate, ethyl DL-2-hydroxycaproate, decanoic acid and 2-ethoxythiazole, whereas wines from Azores and Canary Islands are mainly characterized by 3-ethoxypropan-1-ol, 1-octen-3-ol, (Z)-3-hexenyl butanoate, 2,3-dihydrobenzofuran and 4-(methylthio)-1-butanol [32]. This latter compound is particularly important in Fogo, where red wines present an unusual high concentration of heavy sulfur volatiles in its aroma [33]. These concentrations might be due to the active volcano where grapevines are located. Fogo wines also present the highest concentration in the minor volatiles fatty acids. Results for the most common volatile compounds in red wines are summarized in **Table 4**.

Higher alcohols followed by ethyl esters and fatty acids are the most predominant chemical groups among the volatiles from red wines elaborated in Macaronesia. In fact hexanolderived compounds with decaonic acids and whiskey lactones discriminate Madeira red wines from the rest of Macaronesian archipelagos [32] and a characteristic profile for alcohols, ethyl esters and fatty acids has been obtained for these wines [13]. Similarly, the concentration of esters in Azores wines revealed to be significantly lower than in red wines from the other Macaronesian archipelagos. The unique exception is the ethyl hexanoate compound, which seems to be present in a greater extent than in other islands [33–36].

Canary wines studies conclude that 3-metyl-1-butanol, 2,2-butanodiona and ethyl butirate were the main odorants [37, 38]. Moreover specific studies have been conducted to analyze the volatiles from red wines aged in pine casks from La Palma Island [39].

#### **3.6. Phenolic content**

this metal in grapes and latter in wine. The greater or lesser concentration might depend on the climatic conditions of each harvest, on the grapevine situation as greater winds or dryness enhances it, and even on the vine itself, as roots absorb potassium differently depending on the cultivar. This climate influence might explain the potassium increase in Canary Islands wine, as the literature cited is almost 15 years old and today vines are exposed to warmer

**Canary (bottled with DOP) Fogo Chã (n = 40) Madeira (n = 36) [29] Azores (n = 28) [29]**

(531–3727) <sup>835</sup> <sup>±</sup> <sup>333</sup> <sup>1270</sup> <sup>±</sup> 247 (988–1849) <sup>936</sup> <sup>±</sup> <sup>215</sup> <sup>923</sup> <sup>±</sup> <sup>217</sup>

(19.0–351.2) 92.0 <sup>±</sup> 38.2 23.6 <sup>±</sup> 3.6 (17.1–29.0) 51.0 <sup>±</sup> 30.1 57.7 <sup>±</sup> 30.5

(24.1–83.2) 68.7 <sup>±</sup> 15.0 50.5 <sup>±</sup> 35.4 (30.5–90.3) 73.1 <sup>±</sup> 23.8 80.5 <sup>±</sup> 15.7

(65.0–263.1) 84.1 <sup>±</sup> 15.4 115.0 <sup>±</sup> 40.0 (75.0–125.6) 101.2 <sup>±</sup> 13.9 112.5 <sup>±</sup> 41.8

(0.3–7.4) 2.6 <sup>±</sup> 1.1 2.6 <sup>±</sup> 1.9 (0.9–7.3) 2.3 <sup>±</sup> 1.4 1.5 <sup>±</sup> 1.0

(n.d.–6.70) 0.17 <sup>±</sup> 0.20 0.02 <sup>±</sup> 0.01 (n.d.–0.1) 0.63 <sup>±</sup> 0.66 0.22 <sup>±</sup> 0.41

(n.d.–0.53) 0.56 <sup>±</sup> 0.31 0.18 <sup>±</sup> 0.10 (0.10–0.20) 1.02 <sup>±</sup> 0.60 0.63 <sup>±</sup> 0.55

(n.d.–5.1) 1.0 <sup>±</sup> 0.4 0.4 <sup>±</sup> 0.1 (0.3–0.5) 1.9 <sup>±</sup> 0.8 0.7 <sup>±</sup> 0.2

Potassium concentrations correlate with high wine pHs, a peculiar characteristic already observed in the previous section for Fogo wines. Since high pHs lead to a greater wine sensitivity to microbiological diseases, it is advisable to control potassium content. Obviously potassium is not the only factor conditioning the final wine pH, although it is one of the most

The sodium content for all Macaronesian regions except Fogo is higher than those from other origins [31], probably due to oceanic winds and the marine aerosol. Fogo samples contain significantly less than most Canary, Azorean and Madeiran red wines because of the high altitude of Chã vineyards. In fact Fogo sodium content is similar to continental wines, where the marine aerosol is non-existent and its origin is essentially due to soil and agronomic practices. Some Canary red wines also presented low sodium content when compared with most samples from the same region, coming these wines from Tenerife vineyards located at high

conditions due to the temperature increase from the last decade.

Results expressed as Χ ± σ (Min. – Max. value if available). n.d. = not detected.

**Table 3.** Metallic content (mg/l) from canary, Cape Verde, Madeira and Azores islands.

altitudes, which are presumably less influenced by oceanic winds.

important according to our experimental results.

**(n = 250) (n = 249) [30]**

264 Grapes and Wines - Advances in Production, Processing, Analysis and Valorization

K 1428 ± 459

Na 98 ± 57

Ca 69.3 ± 22.1

Mg 129.0 ± 27.0

Fe 1.7 ± 1.0

Cu 0.20 ± 0.70

Zn 0.26 ± 0.15

Mn 1.3 ± 0.7

Phenolic compounds are responsible for the most important sensory attributes of red wines. Phenolic content depends on the winemaking, which are influenced by factors such as variety, ripening, cultivation techniques and climatic conditions among other considerations [40]. Thus the phenolic composition of Macaronesian red wines is potentially different from other regions because of their specific *"terroir".* Gallic and caftaric acids are the most concentrated hydroxybenzoic and hydroxycinnamic acid respectively in Canary wines [41] but there is high heterogeneity. Previous studies for the same region confirmed that these wines polyphenolic composition is highly heterogenic and depends on various factors such as local climate


**Table 4.** Volatiles compounds from Canary, Cape Verde, Madeira and Azores islands.

and vinification conditions [42]. Results for the most common phenolic compounds in red

Quercetin is the most concentrated flavonol and the content of this compound group, that is flavonols, in Azores [36], Madeira [44], Fogo [33] and Canary [41] is unusually higher than in other winemaking areas [45, 46]. Stilbenes content in Macaronesia red wines has been also described as higher than in continental wines [25, 36, 43] but similar to those from Greek red wines [47]. These differences may be due to the sunny climate of Macaronesia. Flavonol and stilbene content depends on sun exposure because these phenolics protect against solar radiation, thus Macaronesian vines might increase their synthesis to combat UV radiation [48]. Anthocyanins content is also in the upper quartile when compared with other winemaking regions probably because of these solar radiation influences, which are more important in the most southern islands. In fact it has been considered that the content of catechins and proanthocyanidins in Canary wines is higher

Phenolic reactions are responsible for the colorimetric changes observed while wine aging [24]. **Table 6** summarizes the main colorimetric indexes for Canary and Fogo red wines. Wines from both archipelagos present high chromacity, as the average color intensity for Canary is 9 Units of Absorbance (U.A.) and *"manecom"* even achieves 17.7 U.A. Hue color values lead to

wines are summarized in **Table 5** for all Macaronesian archipelagos.

**Table 5.** Phenolic compounds (mg/l) from Canary, Fogo, Madeira and Azores islands.

**Canary wines Fogo (n = 4)** 

21.1 ± 11.5 (5.6–44.7)

2.0 ± 0.8 (0.9–4.0)

2.1 ± 2.3 (0.1–6.8)

20.2 ± 8.5 (9.4–38.4)

3.3 ± 1.1 (0.2–5.7)

Results expressed as Χ ± σ (Min. – Max. value if available). n.d. = not detected.

17.5 ± 11.48 (1.9–49.8)

Gallic acid 41.8 ± 24.0

Syringic acid 7.9 ± 2.8

Coumaric acid 9.7 ± 9.8

Catechin 85.9 ± 29.9

Resveratrol 5.1 ± 3.0

Quercetin 2.8 ± 2.5

Malv.3gluc. 92.7 ± 79.4

(3.6–125.8)

(2.1–20.4)

(<0.9–67.8)

(6.6–199.7)

(<0.7–13.3)

(n.d.–13.7)

(1.5–371.2)

**[33]**

10.8 ± 3.4 (6–13.7)

10.9 ± 5.5 (7.4–19.1)

6.9 ± 2.5 (3.8–10.0)

4.1 ± 0.5 (3.4–4.5)

(19.6–116.0)

– 68.5 ± 39.6

– 0.3 ± 0.1

**[36] (n <sup>=</sup> 250) [41] (n <sup>=</sup> 55) [43] (n <sup>=</sup> 12) [25] (n <sup>=</sup> 5) [44]**

– – 398.1 ± 34.5

0.7 ± 0.1 (0.5–0.9)

(0.2–0.5)

– 18.6 ± 8.8

– 55.7 ± 18.0

**Madeira Azores (n = 3)**

Characterization of Red Wines from Macaronesia http://dx.doi.org/10.5772/intechopen.71795

– –

–

267

–

4.2 ± 2.1 (2.8–5.7)

5.6 ± 4.1 (3.0–12.0)

(341.2–429.0)

(4.8–28.6)

24.5 ± 25.4 (4.5–57.7)

(35.9–79.1)

– – –

– 8.1 ± 5.4 (4.5–16.1) –

than in Madeira red wines [44].

low perceptions of oxidation.

**3.7. Color**


**Table 5.** Phenolic compounds (mg/l) from Canary, Fogo, Madeira and Azores islands.

and vinification conditions [42]. Results for the most common phenolic compounds in red wines are summarized in **Table 5** for all Macaronesian archipelagos.

Quercetin is the most concentrated flavonol and the content of this compound group, that is flavonols, in Azores [36], Madeira [44], Fogo [33] and Canary [41] is unusually higher than in other winemaking areas [45, 46]. Stilbenes content in Macaronesia red wines has been also described as higher than in continental wines [25, 36, 43] but similar to those from Greek red wines [47]. These differences may be due to the sunny climate of Macaronesia. Flavonol and stilbene content depends on sun exposure because these phenolics protect against solar radiation, thus Macaronesian vines might increase their synthesis to combat UV radiation [48]. Anthocyanins content is also in the upper quartile when compared with other winemaking regions probably because of these solar radiation influences, which are more important in the most southern islands. In fact it has been considered that the content of catechins and proanthocyanidins in Canary wines is higher than in Madeira red wines [44].

#### **3.7. Color**

**Compound group**

Alcohols (μg/l) (50–60%)

Hexanol Benzyl alcohol

Phenylethanol

2-(Methylthio)

0.139 ±

0.012 (0.091–0.175)

54.333 ±

10.204 (25.500–74.500)

 0.002 ±

0.001 (0.001–0.005)

–

ethanol

Linalool

Esters (mg/l) (30–40%)

Ethyl lactate Ethyl hexanoate

Diethyl succinate

Ethyl octanoate

Phenylethyl acetate

Ethyl decanoate

Isoamyl acetate

Fatty acids (μg/l) (5%)

Butanoic acid Decanoic acid Hexanoic acid

Results expressed as Χ

**Table 4.**

 ±

σ (Min. – Max. value if available). n.d.

Volatiles compounds from Canary, Cape Verde, Madeira and Azores islands.

 =

not detected.

9.674 ±

0.546 (8.486–11.056)

 n.d.

0.194 ±

0.088 (0.098–0.286)

2.750 ±

0.781 (2.500 – 3.000)

0.694 ± 0.080 (0.098–1.607)

1.537 ±

0.208 (1.486–1.625)

0.123 ±

0.145 (0.030–0.290)

0.065 ±

0.154 (0.026–0.073)

 –

–

1.184 ±

0.751 (0.561–1.905)

3.985 ± 2.048 ±

0.091 (1.350–2.660)

0.542 ± 0.026 ±

0.004 (0.019–0.040)

–

0.100 ±

0.127 (0.010–0.190)

0.343 (0.491–0.654)

0.038 ±

0.019 (0.022–0.059)

0.152 (3.300–4.730)

0.248 ±

0.014 (0.181–0.380)

0.197 ±

0.025 (0.176–0.224)

0.945 ±

0.451 (0.316–1.641)

1.565 ±

0.104 (1.160–1.770)

3.584 ±

0.113 (2.993–5.090)

–

1.223 ±

1.014 (0.848–2.351)

2.530 ±

0.133 (2.150–2.930)

34.506 ±

6.598 (20.069–54.823)

 0.186 ±

0.119 (0.066–0.304)

–

3.348 ±

0.288 (2.751–4.560)

2.383 ± 1.479 ±

0.088 (0.957–2.180)

0.566 ±

0.041 (0.184–1.049)

0.005 ±

0.002 (0.003–0.007)

0.155 (2.050–2.760)

1.788 ±

0.404 (1.544–2.271)

4.402 ±

1.615 (2.55–5.516)

266 Grapes and Wines - Advances in Production, Processing, Analysis and Valorization

–

2.522 ±

0.678 (0.151–5.021)

3.664 ± 4.138 ±

0.710 (2.250–6.370)

5.300 ±

0.175 (3.088–7.206)

0.004 ±

0.001 (0.003–0.005)

0.461 (0.475–6.301)

–

0.147 ±

0.031 (0.120–0.180)

1.086 ±

0.122 (0.961–1.313)

7.320 ±

0.739 (6.250–8.300)

12.348 ±

1.011 (9.706–18.005)

0.745 ±

0.181 (0.574–0.935)

–

–

**Volatile compound**

**Canary (n = 6) [34]}**

**Fogo (n = 4) [33]**

0.214 ±

0.131 ±

0.01 (0.032–0.184)

0.137 ±

0.007 (0.106–0.224)

0.113 ±

0.129 (0.022–0.204)

0.011 (0.167–0.256)

8.359 ±

0.469 (7.604–8.779)

0.076 ±

0.03 (0.042–0.1)

**Madeira (n =**

**48) [35]}**

**Azores (n = 3) [36]**

> Phenolic reactions are responsible for the colorimetric changes observed while wine aging [24]. **Table 6** summarizes the main colorimetric indexes for Canary and Fogo red wines. Wines from both archipelagos present high chromacity, as the average color intensity for Canary is 9 Units of Absorbance (U.A.) and *"manecom"* even achieves 17.7 U.A. Hue color values lead to low perceptions of oxidation.


**4. Conclusion**

during the coming years.

**Author details**

Jesús Heras-Roger<sup>1</sup>

**References**

j.aca.2005.10.031

\*, Carlos Díaz-Romero<sup>2</sup>

1 Excelentísimo Cabildo Insular de Tenerife, Tenerife, Spain

Tecnología de Alimentos, Universidad de La Laguna, La Laguna, Spain

\*Address all correspondence to: jherasr@gmail.com

Macaronesian red wines present peculiar characteristics and wide diversity. Their detailed study is still a current research issue with a promising future as these winemaking regions are mostly known because of fortified and white wines. Their atypical *terroir* combined with traditions and exclusive grape cultivars makes them a prospective red winemaking region. Red wines from Macaronesia can be grouped according to archipelago of origin but are also highly heterogeneous due to microclimates. Significant differences have been obtained in terms of oenological properties, acids, minerals, volatiles, phenolics and color influencing their organoleptic peculiarities. These wines are highly affected by the unusual circumstances of their regions making them unique. For instance, their volcanic soil might increase the iron concentration, whereas oceanic winds modify their sodium content. Moreover the warm and dry climates from these islands determine higher water needs from grapevines, which carry out a greater potassium absorption and thus a greater wine content increasing pH. Furthermore solar radiation involves a higher flavonols and stilbenes biosynthesis in grapes. In summary, Macaronesian red wines need further research but most probably their valorization as atypical food products from an exclusive environment would be enhanced

, Jacinto Darias-Martín<sup>2</sup>

2 Departamento de Ingeniería Química y Tecnología Farmacéutica, Área de Ciencia y

[1] Petit J. Chapter 5. Macaronesia. In: Petit J, Prudent G, editors. Climate Change and Biodiversity in the European Union Overseas Entities. Switzerland. IUCN; 2010. pp. 122-

[2] Marques JC. Vinhos da Macaronésia: conhecer e diferenciar para competir. Seminário Vitivinicultura Atlântica. Available from: https://vitiviniculturaatlantic.0a.picowines.net

[3] Câmara JS, Alves MA, Marques JC. Changes in volatile composition of Madeira wines during their oxidative ageing. Analytica Chimica Acta. 2006;**563**(1):188-197. DOI: 10.1016/

135. https://portals.iucn.org/library/sites/library/files/documents/2010-064.pdf

and Domingo Rios-Mesa<sup>1</sup>

Characterization of Red Wines from Macaronesia http://dx.doi.org/10.5772/intechopen.71795 269

**Table 6.** Colorimetric red wines parameters from canary and Cape Verde islands.

The minimum values for coordinates a\* and b\* in CIELab Units (C.U.) are lower than those obtained in wines from the mainland of Spain [49]. Chroma (C\*) and saturation (S\*) reveal great heterogeneity. Fogo red wine lightness (L\*) is significantly lower than Canary red wines giving a darker color, probably due to a higher anthocyanin extraction derived from the winemaking techniques applied to maximize color and alcoholic content.

One of the most important pigment interactions is co-pigmentation, which occurs when anthocyanic pigments associates with itself or with another substance known as co-pigment, these compounds can be very variable, from other colorless anthocyanin to phenolic acids or flavonols depending on the red wine considered [40]. The percentage of co-pigmentation in parts per unit provides an estimation of co-pigments and their influence in color. The average co-pigmentation in Canary wines is 18 ± 11% of color, ranging from 0.5 to 40%. Co-pigmentation in Fogo wines is lower in disagreement with studies where warm regions produced wines with significantly higher co-pigmentation than cold areas [50]. This low co-pigmenation is related with the high amount of polymeric pigment present in Fogo red wines.

The percentage of polymeric pigments in the young Fogo wines is similar to the percentage quantified for Canary wines where also oak-aged red wines are considered. Short-aged red wines do not normally present so many polymeric pigments. Its content in Fogo wines might be related to late harvest, excessive grape ripening and peculiar winemaking techniques which maximize oxidation and thus increase phenolic polymerizations during winemaking and storage.

## **4. Conclusion**

Macaronesian red wines present peculiar characteristics and wide diversity. Their detailed study is still a current research issue with a promising future as these winemaking regions are mostly known because of fortified and white wines. Their atypical *terroir* combined with traditions and exclusive grape cultivars makes them a prospective red winemaking region. Red wines from Macaronesia can be grouped according to archipelago of origin but are also highly heterogeneous due to microclimates. Significant differences have been obtained in terms of oenological properties, acids, minerals, volatiles, phenolics and color influencing their organoleptic peculiarities. These wines are highly affected by the unusual circumstances of their regions making them unique. For instance, their volcanic soil might increase the iron concentration, whereas oceanic winds modify their sodium content. Moreover the warm and dry climates from these islands determine higher water needs from grapevines, which carry out a greater potassium absorption and thus a greater wine content increasing pH. Furthermore solar radiation involves a higher flavonols and stilbenes biosynthesis in grapes. In summary, Macaronesian red wines need further research but most probably their valorization as atypical food products from an exclusive environment would be enhanced during the coming years.

## **Author details**

The minimum values for coordinates a\* and b\* in CIELab Units (C.U.) are lower than those obtained in wines from the mainland of Spain [49]. Chroma (C\*) and saturation (S\*) reveal great heterogeneity. Fogo red wine lightness (L\*) is significantly lower than Canary red wines giving a darker color, probably due to a higher anthocyanin extraction derived from the wine-

Color intensity A420 <sup>+</sup> A520 <sup>+</sup> A620 (U.A.) 9.00 <sup>±</sup> 3.60 (1.41–24.15) 10.73 ± 4.00 (2.10–18.00) 17.67 ± 0.80

Chroma, C\* (C.U.) 53.71 ± 11.12 (9.91–70.60) 36.15 ± 6.68

268 Grapes and Wines - Advances in Production, Processing, Analysis and Valorization

Hue angle, h\* (C.U.) 28.91 ± 6.51 (13.72–44.03) 18.56 ± 3.95

Redness-greenness axis a\* (C.U.) 41.61 ± 12.83 (5.40–60.43) 34.09 ± 5.58

**Table 6.** Colorimetric red wines parameters from canary and Cape Verde islands.

Results expressed as Χ ± σ (Min.–Max. value if available).

Hue color (A420/A520) 0.75 ± 0.16 (0.51–1.28) 0.69 ± 0.15 (0.41–0.99) 0.73 ± 0.11 (0.48–0.105) Lightness, L\* (C.U.) 19.90 ± 10.12 (1.42–68.02 6.87 ± 2.58 (3.87–10.14) 1.07 ± 1.10 (0.29–1.85)

yellowness-blueness axis b\* (C.U.) 29.81 ± 11.83 (2.52–56.01) 11.82 ± 4.42 (6.68–17.39) 1.84 ± 1.90 (0.50–3.18) Saturation, S (C.U.) 3.52 ± 1.22 (0.61–7.33) 5.61 ± 1.16 (4.28–7.01) 6.91 ± 0.07 (6.86–6.96) Co-pigmentation (parts per unit) 0.18 ± 0.11 (0.05–0.41) 0.11±0.09 (0.02–0.35) 0.09 ± 0.08 (0.02–0.25) Polymeric pigments (parts per unit) 0.41 ± 0.15 (0.06–0.80). 0.40 ± 0.14 (0.10–0.60) 0.49 ± 0.12 (0.19–0.72)

**Canary (n = 250) Fogo Chã (n = 40)** *Manecom* **(n = 10)**

(14.24– 23.62)

(17.10–18.23)

14.45 ± 0.15 (14.35–14.56)

(27.14–43.39) 7.41 ± 7.69 (1.98–12.85)

(26.31–39.75) 7.18 ± 7.45 (1.92–12.45)

One of the most important pigment interactions is co-pigmentation, which occurs when anthocyanic pigments associates with itself or with another substance known as co-pigment, these compounds can be very variable, from other colorless anthocyanin to phenolic acids or flavonols depending on the red wine considered [40]. The percentage of co-pigmentation in parts per unit provides an estimation of co-pigments and their influence in color. The average co-pigmentation in Canary wines is 18 ± 11% of color, ranging from 0.5 to 40%. Co-pigmentation in Fogo wines is lower in disagreement with studies where warm regions produced wines with significantly higher co-pigmentation than cold areas [50]. This low co-pigmenation is related with the high amount of polymeric pigment present in Fogo red

The percentage of polymeric pigments in the young Fogo wines is similar to the percentage quantified for Canary wines where also oak-aged red wines are considered. Short-aged red wines do not normally present so many polymeric pigments. Its content in Fogo wines might be related to late harvest, excessive grape ripening and peculiar winemaking techniques which maximize oxidation and thus increase phenolic polymerizations during winemaking

making techniques applied to maximize color and alcoholic content.

wines.

and storage.

Jesús Heras-Roger<sup>1</sup> \*, Carlos Díaz-Romero<sup>2</sup> , Jacinto Darias-Martín<sup>2</sup> and Domingo Rios-Mesa<sup>1</sup>

\*Address all correspondence to: jherasr@gmail.com

1 Excelentísimo Cabildo Insular de Tenerife, Tenerife, Spain

2 Departamento de Ingeniería Química y Tecnología Farmacéutica, Área de Ciencia y Tecnología de Alimentos, Universidad de La Laguna, La Laguna, Spain

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**Chapter 13**

Provisional chapter

**Novel Memory-Based Sensory Approach to Assess**

Novel Memory-Based Sensory Approach to Assess

**Red Wines**

Red Wines

Anibal Jose-Coutinho, Patricia Avila and

Anibal Jose-Coutinho, Patricia Avila and

Additional information is available at the end of the chapter

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/intechopen.73039

Jorge M. Ricardo-da-Silva

Jorge M. Ricardo-da-Silva

Abstract

**Large-Scale Typicality: The Case of Mainland Portugal**

DOI: 10.5772/intechopen.73039

Large-Scale Typicality: The Case of Mainland Portugal

Twenty professional wine experts were asked to describe their prototypical construct of a representative young red wine from each of the 12 Protected Geographical Indications (PGI) of mainland Portugal. No samples were assessed; the experiment was based on memory alone by completion of 12 extended self-reported sensory questionnaires. Four large-scale areas were differentiated, the typicality being statistically validated and described from a sensory standpoint. Alcohol, acidity, bitterness, and astringency were cross-linked; the respective variations were correlated with published literature and expressed as key factors for the regional macroscale area differentiation. Bitterness and astringency were found to be sensory different and related on a geographical scale, as bitterness was primarily affected by inland/coastal influence; while astringency confirmed its customary north/south dependence that finding is to be considered a new understanding. Moreover, with the proposed methodology, it was possible to achieve a novel nationwide sensory characterization of PGIs, overcoming present day limitations on macroscale sensory research and sample representativeness. Results by uncalibrated prototypical memory assessment of single PGI Beira Atlântico were compared with the outcome of calibrated wine sampling assessment by local experts, using the same sensory questionnaire, and were found significantly correlated. The need for a calibration stage was found

> © 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and eproduction in any medium, provided the original work is properly cited.

> © 2018 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

uneven regarding the overall group of scrutinized wine descriptors.

Keywords: red wine, sensory, typicality, astringency, bitterness

Provisional chapter

## **Novel Memory-Based Sensory Approach to Assess Large-Scale Typicality: The Case of Mainland Portugal Red Wines** Novel Memory-Based Sensory Approach to Assess Large-Scale Typicality: The Case of Mainland Portugal Red Wines

DOI: 10.5772/intechopen.73039

Anibal Jose-Coutinho, Patricia Avila and Anibal Jose-Coutinho, Patricia Avila and

Jorge M. Ricardo-da-Silva Jorge M. Ricardo-da-Silva

Additional information is available at the end of the chapter Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/intechopen.73039

#### Abstract

Twenty professional wine experts were asked to describe their prototypical construct of a representative young red wine from each of the 12 Protected Geographical Indications (PGI) of mainland Portugal. No samples were assessed; the experiment was based on memory alone by completion of 12 extended self-reported sensory questionnaires. Four large-scale areas were differentiated, the typicality being statistically validated and described from a sensory standpoint. Alcohol, acidity, bitterness, and astringency were cross-linked; the respective variations were correlated with published literature and expressed as key factors for the regional macroscale area differentiation. Bitterness and astringency were found to be sensory different and related on a geographical scale, as bitterness was primarily affected by inland/coastal influence; while astringency confirmed its customary north/south dependence that finding is to be considered a new understanding. Moreover, with the proposed methodology, it was possible to achieve a novel nationwide sensory characterization of PGIs, overcoming present day limitations on macroscale sensory research and sample representativeness. Results by uncalibrated prototypical memory assessment of single PGI Beira Atlântico were compared with the outcome of calibrated wine sampling assessment by local experts, using the same sensory questionnaire, and were found significantly correlated. The need for a calibration stage was found uneven regarding the overall group of scrutinized wine descriptors.

Keywords: red wine, sensory, typicality, astringency, bitterness

© 2018 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and eproduction in any medium, provided the original work is properly cited.

## 1. Introduction

The geographical origin of wine as well as its price has a strong impact on consumers. Wine is a product in which the concept of typicality is prevalent, especially in Europe [1]. Protected geographical indications (PGI) and protected designation of origin (PDO) products are the base of European artisan production, maintaining sensory characteristics, assuring consumer confidence, and preserving market position [2].

perceptual one, and if all of the perceptually based techniques have a persistent low wine sampling hazard due to the logistical and/or time-consuming factors, then why are conceptual techniques, based on the long-term memory from professional wine experts and keepers of

Novel Memory-Based Sensory Approach to Assess Large-Scale Typicality: The Case of Mainland Portugal Red Wines

http://dx.doi.org/10.5772/intechopen.73039

277

Currently, there are 12 Protected Geographical Wine Indications (PGI) in mainland Portugal (Figure 1). For such a small country, does the actual range of 12 PGI's offer 12 single and

Figure 1. Protected geographical indications (and respective inner protected designations of origin) in mainland Portugal.

memory [1], not accepted as a sensory methodology?

A large number of sensory methodologies are based on the use of professional tasters to show the sensory properties of a wine category (wine variety or PDO). Scientists have tested different sensory approaches, but the perceptual sample-tasting methods may be considered the most commonly used. These approaches work correctly, if the samples are grouped in wellseparated regions; however, in more complex distributions, their classification power becomes poor [3]. Concerns regarding the scarcity and representation of samples that are assessed have always undermined typicality research. A recent study stated that a sample-tasting panel did not discriminate among the wines for astringency and bitterness, probably because the samples were inexpensive wines with very similar phenolic contents [4].

The role of expertise, as well as the importance of experience and long-term memory (LTM) for extraordinary performance, is widely documented. Wine professionals (wine-makers, wine journalists, etc.) are often nonavailable for testing [5]; hence, our experts were not formally trained, as usually done in quantitative descriptive analysis (DA). Due to their high level of long-term professional expertise, we hypothesized that the abstraction of sensory prototypes is memorized and could be considered as a synthesis of all previous wine tasting experiences from the category. Descriptions stored in LTM can be used to generate images of objects and scenes [6]. Recognition of meaningful stimuli such as words or objects rapidly activates conceptual information and leads to the retrieval of additional relevant information from LTM [7]. Neuroscience research stated that new information can come from different sources, including sensory stimuli (SS) or LTM; however, no (brain) regions were found to be more active during updating from SS than updating from LTM [8]. In the case of sensory analysis, flavor may be processed by tasters as a psychological construct, the data being more consistent with a perceptual/cognitive process rather than a consequence of rating strategies [9].

Our nationwide study, a novel macroscale approach on wine sensory typicality, was built on the case that senior experts have common mental representations of typical wines from each PGI, resulting in a feasible assessment of predetermined attributes and respective lexicon [10] which can be used to describe the sensory characteristics of a typical exemplar of the category or prototype. Clear agreement between experts concerning typicality scores [11] and existence of shared cognitive constructs of typicality [12] has been demonstrated. The literature does not always clearly delineate what constitutes training and what is experience. Experience relates to a familiarity with a product class resulting from long-term exposure to a wide variety of members representing that class [13]. Qualitative methods can be used to enhance quantitative studies, and with time, labor or financial limitations, may be considered a valid substitute for quantitative methods with heavy training/calibration requirements. Sensory methods are constantly evolving, becoming simpler, faster, and highly reviewed [14–20]. If neuroscience and sensory science results suggest that expertise may be more a cognitive skill rather than a perceptual one, and if all of the perceptually based techniques have a persistent low wine sampling hazard due to the logistical and/or time-consuming factors, then why are conceptual techniques, based on the long-term memory from professional wine experts and keepers of memory [1], not accepted as a sensory methodology?

1. Introduction

confidence, and preserving market position [2].

276 Grapes and Wines - Advances in Production, Processing, Analysis and Valorization

ples were inexpensive wines with very similar phenolic contents [4].

perceptual/cognitive process rather than a consequence of rating strategies [9].

Our nationwide study, a novel macroscale approach on wine sensory typicality, was built on the case that senior experts have common mental representations of typical wines from each PGI, resulting in a feasible assessment of predetermined attributes and respective lexicon [10] which can be used to describe the sensory characteristics of a typical exemplar of the category or prototype. Clear agreement between experts concerning typicality scores [11] and existence of shared cognitive constructs of typicality [12] has been demonstrated. The literature does not always clearly delineate what constitutes training and what is experience. Experience relates to a familiarity with a product class resulting from long-term exposure to a wide variety of members representing that class [13]. Qualitative methods can be used to enhance quantitative studies, and with time, labor or financial limitations, may be considered a valid substitute for quantitative methods with heavy training/calibration requirements. Sensory methods are constantly evolving, becoming simpler, faster, and highly reviewed [14–20]. If neuroscience and sensory science results suggest that expertise may be more a cognitive skill rather than a

The geographical origin of wine as well as its price has a strong impact on consumers. Wine is a product in which the concept of typicality is prevalent, especially in Europe [1]. Protected geographical indications (PGI) and protected designation of origin (PDO) products are the base of European artisan production, maintaining sensory characteristics, assuring consumer

A large number of sensory methodologies are based on the use of professional tasters to show the sensory properties of a wine category (wine variety or PDO). Scientists have tested different sensory approaches, but the perceptual sample-tasting methods may be considered the most commonly used. These approaches work correctly, if the samples are grouped in wellseparated regions; however, in more complex distributions, their classification power becomes poor [3]. Concerns regarding the scarcity and representation of samples that are assessed have always undermined typicality research. A recent study stated that a sample-tasting panel did not discriminate among the wines for astringency and bitterness, probably because the sam-

The role of expertise, as well as the importance of experience and long-term memory (LTM) for extraordinary performance, is widely documented. Wine professionals (wine-makers, wine journalists, etc.) are often nonavailable for testing [5]; hence, our experts were not formally trained, as usually done in quantitative descriptive analysis (DA). Due to their high level of long-term professional expertise, we hypothesized that the abstraction of sensory prototypes is memorized and could be considered as a synthesis of all previous wine tasting experiences from the category. Descriptions stored in LTM can be used to generate images of objects and scenes [6]. Recognition of meaningful stimuli such as words or objects rapidly activates conceptual information and leads to the retrieval of additional relevant information from LTM [7]. Neuroscience research stated that new information can come from different sources, including sensory stimuli (SS) or LTM; however, no (brain) regions were found to be more active during updating from SS than updating from LTM [8]. In the case of sensory analysis, flavor may be processed by tasters as a psychological construct, the data being more consistent with a Currently, there are 12 Protected Geographical Wine Indications (PGI) in mainland Portugal (Figure 1). For such a small country, does the actual range of 12 PGI's offer 12 single and

Figure 1. Protected geographical indications (and respective inner protected designations of origin) in mainland Portugal.

typical wine sensory profiles, so different from one another that typicality governs consumers on their purchasing decisions?

2.2. Questionnaire

2.3. Procedure

"extreme intensity of the attribute" (score 10).

red wine of each of the 12 Portuguese wine regions.

2.4. Statistical data analysis

The questionnaire was designed according to the classical three-tier sensory method of assessment: visual, aromatic, and gustatory/tactile descriptors (full questionnaire in supporting information). With the exception of hue, the questionnaire used an 11-integer point rating scale. On the left was written: "no trace of the attribute" (score 0) and on the right was written:

Novel Memory-Based Sensory Approach to Assess Large-Scale Typicality: The Case of Mainland Portugal Red Wines

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279

The color was assessed in its intensity and hue. Results from this last descriptor were standardized to match the same scale because the previously reported research justified the use of a 4 integer point scale with 1—Violet-Purple, 2—Purple-Ruby, 3—Ruby-Garnet, and 4—Garnet-Brick [32]. The aroma was evaluated with 1 overall intensity measure and 18 aromatic categories that summarize a significant amount of scientific research [33], much of which has already been used in the form of aroma wheels [34]. The taste of red wines was evaluated using 14 measures, including essential tastes (sour, sweet, salty, and bitter), mouthfeel categories resulting from the various types of astringency [35], prickling, weight, persistence, and mouthcoat sensation [10].

Respondents were invited and briefed by the same experimenter. They received oral instructions on how to fill the questionnaire. The experts were told to quantify their prototypical memory activated by the following question: "How would you sensory assess a typical young commercial red wine (released during the first year following harvest) from this particular PGI?" Despite some reasonable doubts on similar cognitive construct within the panel, no training phase or leveling pre-stage was performed. No wine samples were assessed. All experts independently completed individual self-reported questionnaires by scoring several sensory attributes that characterized their own expert construct for what is a typical PGI young

Descriptive statistics (central tendency and dispersion) were computed for each sensory attribute, using IBM SPSS Statistics 20.0. Using the same software, bivariate statistics such as oneway ANOVA were performed in order to analyze the relation between the sensory attributes and 12 PGIs. Moreover, this tool reveals whether or not significant differences are found and also the strength of the association. This was followed by the completion of principal components analysis (PCA) with a Varimax rotation which sought to explore the possibility of reducing the initial amount of sensory variables into fewer dimensions—the principal components (PC). PCA of sensory data permitted the differentiation among geographic areas [28]. The number of retained components was based on the Kaiser criterion—according to which the components with an eigenvalue greater than or equal to 1 are retained—and occasionally, the next principal component was also retained. Latent variables encompassing different initial variables found to be highly correlated on each PC were estimated, named accordingly, and

This study builds on the suggestion, which is commonly expressed in Portuguese and Anglophone wine press, that typicality assessment of PGI-certified still red wines of mainland Portugal does not match 12 exclusive sensory profiles, 1 for each Geographical Indication [21]. The supporting evidence of typicality similitude among certain PGIs can be found in bioclimatic [22–24] and biogeographical zoning studies [25]. Moreover, the phenolic composition [26, 27] and aroma [28] exhibited zonal differences.

Following our recently published white wine nationwide study [29], encompassing a novel memory-based DA, each member of our panel of professional experts was asked to mentally retrieve and assess his prototypical sensory profile of each of the 12 mainland Portugal PGIcertified young red wines. To our knowledge, this is the first macroscale sensory study covering a whole wine-producing country.

Finally, the outcome of our sample-free and uncalibrated DA methodology was compared with the results of a classic sampling DA single PGI study [30] assessed by a calibrated panel of PGI Beira Atlântico local experts, both studies sharing the same sensory questionnaire.

## 2. Materials and methods

#### 2.1. Subjects

When compared with previous studies [31], rigorous selection criteria were applied, as the definition of a wine expert considered the following categories:


As this method is mostly dependent on indisputable knowledge brought by well-known senior Portuguese professionals, graduate wine students, wine consumers and enthusiasts, even with a long-term wine involvement, were not eligible for group of wine experts. The sensory raw data of this work was generated by 20 renowned wine-makers, referenced scholars and professional opinion leaders, wine buyers, and distributors from the Portuguese wine industry. Recent literature showed that the number of panelists is well above average to ensure a statistically robust outcome [4].

#### 2.2. Questionnaire

typical wine sensory profiles, so different from one another that typicality governs consumers

This study builds on the suggestion, which is commonly expressed in Portuguese and Anglophone wine press, that typicality assessment of PGI-certified still red wines of mainland Portugal does not match 12 exclusive sensory profiles, 1 for each Geographical Indication [21]. The supporting evidence of typicality similitude among certain PGIs can be found in bioclimatic [22–24] and biogeographical zoning studies [25]. Moreover, the phenolic composi-

Following our recently published white wine nationwide study [29], encompassing a novel memory-based DA, each member of our panel of professional experts was asked to mentally retrieve and assess his prototypical sensory profile of each of the 12 mainland Portugal PGIcertified young red wines. To our knowledge, this is the first macroscale sensory study cover-

Finally, the outcome of our sample-free and uncalibrated DA methodology was compared with the results of a classic sampling DA single PGI study [30] assessed by a calibrated panel of PGI Beira Atlântico local experts, both studies sharing the same sensory questionnaire.

When compared with previous studies [31], rigorous selection criteria were applied, as the

• Renowned wine-makers with tasting activity of >4 times per week in more than one

• Oenology researchers and scholars who were regularly involved in wine-making and/or

As this method is mostly dependent on indisputable knowledge brought by well-known senior Portuguese professionals, graduate wine students, wine consumers and enthusiasts, even with a long-term wine involvement, were not eligible for group of wine experts. The sensory raw data of this work was generated by 20 renowned wine-makers, referenced scholars and professional opinion leaders, wine buyers, and distributors from the Portuguese wine industry. Recent literature showed that the number of panelists is well above average to

• Wine professionals (e.g., Master of Wine, wine judges, wine writers, wine buyers); • Wine experts with an extensive (more than 15 years) history of wine involvement.

on their purchasing decisions?

ing a whole wine-producing country.

2. Materials and methods

Portuguese wine region;

ensure a statistically robust outcome [4].

wine evaluation;

2.1. Subjects

tion [26, 27] and aroma [28] exhibited zonal differences.

278 Grapes and Wines - Advances in Production, Processing, Analysis and Valorization

definition of a wine expert considered the following categories:

The questionnaire was designed according to the classical three-tier sensory method of assessment: visual, aromatic, and gustatory/tactile descriptors (full questionnaire in supporting information). With the exception of hue, the questionnaire used an 11-integer point rating scale. On the left was written: "no trace of the attribute" (score 0) and on the right was written: "extreme intensity of the attribute" (score 10).

The color was assessed in its intensity and hue. Results from this last descriptor were standardized to match the same scale because the previously reported research justified the use of a 4 integer point scale with 1—Violet-Purple, 2—Purple-Ruby, 3—Ruby-Garnet, and 4—Garnet-Brick [32]. The aroma was evaluated with 1 overall intensity measure and 18 aromatic categories that summarize a significant amount of scientific research [33], much of which has already been used in the form of aroma wheels [34]. The taste of red wines was evaluated using 14 measures, including essential tastes (sour, sweet, salty, and bitter), mouthfeel categories resulting from the various types of astringency [35], prickling, weight, persistence, and mouthcoat sensation [10].

#### 2.3. Procedure

Respondents were invited and briefed by the same experimenter. They received oral instructions on how to fill the questionnaire. The experts were told to quantify their prototypical memory activated by the following question: "How would you sensory assess a typical young commercial red wine (released during the first year following harvest) from this particular PGI?" Despite some reasonable doubts on similar cognitive construct within the panel, no training phase or leveling pre-stage was performed. No wine samples were assessed. All experts independently completed individual self-reported questionnaires by scoring several sensory attributes that characterized their own expert construct for what is a typical PGI young red wine of each of the 12 Portuguese wine regions.

#### 2.4. Statistical data analysis

Descriptive statistics (central tendency and dispersion) were computed for each sensory attribute, using IBM SPSS Statistics 20.0. Using the same software, bivariate statistics such as oneway ANOVA were performed in order to analyze the relation between the sensory attributes and 12 PGIs. Moreover, this tool reveals whether or not significant differences are found and also the strength of the association. This was followed by the completion of principal components analysis (PCA) with a Varimax rotation which sought to explore the possibility of reducing the initial amount of sensory variables into fewer dimensions—the principal components (PC). PCA of sensory data permitted the differentiation among geographic areas [28]. The number of retained components was based on the Kaiser criterion—according to which the components with an eigenvalue greater than or equal to 1 are retained—and occasionally, the next principal component was also retained. Latent variables encompassing different initial variables found to be highly correlated on each PC were estimated, named accordingly, and new descriptive statistics (mean and standard deviation) were computed on the basis of such aggregate variables (three initial variables were kept isolated: color intensity, color tonality (hue), and aromatic intensity).

Given that the central hypothesis of this study anchored on similarity of several PGIs typical wine sensory profiles, global research of this proximity was assessed by hierarchical cluster analysis (HCA), which is an exploratory data analysis tool that aims at sorting different objects (the 12 PGIs) into groups in a way that the similarity between two objects is maximal if they belong to the same group and minimal otherwise. The squared Euclidean distance was used as measure of proximity, and the complete linkage method algorithm was applied to group the PGIs. Raw data from the above-mentioned three initial sensory variables excluded from the PCA, as well the means of the latent sensory variables that emerged from the PCA outcome, were lined up as the HCA input.

## 3. Results and discussion

Twelve Protected Geographical Indications, memory assessed by the typicality of their respective young commercial red wines, were sensory validated, since all PGIs showed differences with significant statistical meaning (at least in 1 attribute) which was verified by the ANOVA method, considering one fixed factor: the Geographical Indication. Relative standard deviation, also known as coefficient of variation [36], showed a lower percentage for the majority of the sensory attributes, which indicates low variability in the data set means (Table 1).

#### 3.1. Representation of aroma and taste

Except for the aromatic intensity assessment, the 18 initial aroma attributes were grouped into 5 principal components (Table 2) that were named according to the previously reported literature on aromatic series: PC1 Aredfruitwoody, PC2 Aripefruit, PC3 Agreenchemical, PC4 Aoverripeness and PC5 Aflorcitrusmineral.

It was possible to find an aggregate sensory measurement that explained 66.7% of the total variance. The simplification procedure that was based on the expert panel assessments may be optimal for macroscale area profiling purposes in contrast to the previously reported use of highly specific, isolated, and less obvious sensory descriptors.

The application of PCA to all tactile/textural and mouthfeel categories simplified the characterization of the taste of the PGI young red wines.

Hue Color

Aroma

(PC1)

(PC2)

(PC3)

(PC4)

(PC5) Aflor

(PC1)

(PC2) T

(PC3) T

(PC4) T

Tdryastringent

sweetviscous

bittersalty

fullpersistent

intensity

Algarve

 Mean 3.00 6.19 SD 0.37 0.98

Alentejano

 Mean 2.70 7.30 SD 0.47 0.73

Beira

Mean 2.00 5.65

Atlântico

Duriense

 Mean 1.65 7.55 SD 0.59 1.00

Lisboa

 Mean 1.82 6.35 SD 0.64 1.46

Minho

 Mean 1.11 8.58 SD 0.32 1.64

Península

Mean 2.76 6.24

Setúbal

Tejo

Mean 2.84 6.32 SD 0.37 0.58

> Terras da

Mean 2.32 6.16

Beira

Terras de

Mean 2.60 5.10

Cister

Terras do Dão Mean 2.00 6.60 SD 0.46 1.10

Transmontano

 Mean 2.38 6.25 SD 0.50 0.86

> Total

Mean 2.23 6.60 SD 0.73 1.38

%

33% 21%

 20%

 33%

RSD

Table 1. Mean and standard deviation of scores on all final sensory attributes by PGI, after PCA. Includes relative standard deviation.

SD 0.52 0.99

SD 0.67 1.17

SD 0.44 0.90

SD 0.46 1.39

 6.13

 1.02

 7.15

 0.88

 5.30

 1.08

 6.95

 0.89

 5.35

 0.79

 5.84

 1.71

 6.12

 0.86

 6.05

 0.91

 5.74

 0.93

 5.20

 1.03

 6.90

 1.02

 5.38

 0.81

 6.07

 1.20

 1.43

 4.29

 1.45

 3.98

 1.47

 4.57

 1.19

 3.74

 1.22

 4.13

 1.35

 4.73

 1.38

 4.84

 1.19

 2.93

 1.36

 4.25

 1.34

 4.71

 1.13

 4.07

 1.45

 4.79

 1.69

 4.50

5.77

1.52

6.95

1.10

3.28

1.22

6.04

1.15

4.13

1.17

3.24

1.39

5.46

1.44

5.09

0.92

4.21

1.13

3.45

1.14

4.19

1.56

4.91

1.19

4.77

1.68

35%

 38%

 1.52

 4.02

 1.42

 3.91

 1.30

 3.94

 1.08

 3.73

 1.57

 3.96

 1.48

 3.95

 1.53

 3.87

 1.73

 5.26

 1.25

 4.31

 1.48

 3.81

 1.51

 4.93

 1.31

 3.48

 1.18

 2.80

2.84 1.61 3.85 1.90 3.33 1.40 3.48 1.35 2.91 1.28 1.89 1.14 3.56 1.69 2.95 1.61 3.08 1.56 2.70 0.98 3.63 1.64 3.66 1.71 3.18 1.58 50%

43%

33%

30%

62%

18%

281

1.64

1.57

1.44

1.36

1.07

3.79

4.71

4.82

2.19

5.84

1.50

1.20

0.77

1.06

0.89

http://dx.doi.org/10.5772/intechopen.73039

3.83

4.63

4.71

1.88

6.00

1.62

1.11

0.73

1.18

0.83

5.23

5.03

4.52

1.92

6.70

1.24

1.42

0.67

1.04

0.76

3.77

5.03

3.94

2.20

5.55

1.42

1.28

0.76

1.22

0.69

4.39

5.07

4.52

1.95

6.08

1.68

1.64

0.62

1.25

0.75

3.32

4.26

5.37

1.79

5.47

Novel Memory-Based Sensory Approach to Assess Large-Scale Typicality: The Case of Mainland Portugal Red Wines

1.73

1.33

0.69

1.49

0.85

3.47

3.66

5.93

2.31

5.50

1.81

1.43

0.87

1.45

1.52

3.35

6.43

2.43

3.79

5.03

1.58

1.12

0.89

1.05

0.70

3.61

5.18

4.18

2.63

5.35

1.63

1.26

0.81

1.09

0.81

4.77

4.61

5.66

1.58

7.05

1.25

1.34

0.63

1.43

0.95

3.80

5.73

3.24

3.12

5.60

1.06

1.05

0.72

0.97

1.04

2.87

3.43

6.68

1.57

6.00

1.35

1.58

0.56

0.98

0.85

2.77

3.31

6.48

1.54

5.38

intensity

Aredfruitwoody

Aripefruit

Agreenchemical

Aoverripeness

citrusmineral

The 14 initial taste attributes were grouped into 4 principal components (Table 3) that were named according to the classical tastes and contributing research on mouthfeel perceptions, reported above: PC1 Tdryastringent, PC2 Tsweetviscous, PC3 Tbittersalty and PC4 Tfullpersistent.

We found an aggregate sensory measurement that explained 75% of the total variance, again supporting the role of the simplified procedure on the tannins tactile range of descriptors [35].


Table 1. Mean and standard deviation of scores on all final sensory attributes by PGI, after PCA. Includes relative standard deviation.

new descriptive statistics (mean and standard deviation) were computed on the basis of such aggregate variables (three initial variables were kept isolated: color intensity, color tonality

Given that the central hypothesis of this study anchored on similarity of several PGIs typical wine sensory profiles, global research of this proximity was assessed by hierarchical cluster analysis (HCA), which is an exploratory data analysis tool that aims at sorting different objects (the 12 PGIs) into groups in a way that the similarity between two objects is maximal if they belong to the same group and minimal otherwise. The squared Euclidean distance was used as measure of proximity, and the complete linkage method algorithm was applied to group the PGIs. Raw data from the above-mentioned three initial sensory variables excluded from the PCA, as well the means of the latent sensory variables that emerged from the PCA outcome,

Twelve Protected Geographical Indications, memory assessed by the typicality of their respective young commercial red wines, were sensory validated, since all PGIs showed differences with significant statistical meaning (at least in 1 attribute) which was verified by the ANOVA method, considering one fixed factor: the Geographical Indication. Relative standard deviation, also known as coefficient of variation [36], showed a lower percentage for the majority of

Except for the aromatic intensity assessment, the 18 initial aroma attributes were grouped into 5 principal components (Table 2) that were named according to the previously reported literature on aromatic series: PC1 Aredfruitwoody, PC2 Aripefruit, PC3 Agreenchemical, PC4

It was possible to find an aggregate sensory measurement that explained 66.7% of the total variance. The simplification procedure that was based on the expert panel assessments may be optimal for macroscale area profiling purposes in contrast to the previously reported use of

The application of PCA to all tactile/textural and mouthfeel categories simplified the charac-

The 14 initial taste attributes were grouped into 4 principal components (Table 3) that were named according to the classical tastes and contributing research on mouthfeel perceptions, reported above: PC1 Tdryastringent, PC2 Tsweetviscous, PC3 Tbittersalty and PC4

We found an aggregate sensory measurement that explained 75% of the total variance, again supporting the role of the simplified procedure on the tannins tactile range of descriptors [35].

the sensory attributes, which indicates low variability in the data set means (Table 1).

(hue), and aromatic intensity).

280 Grapes and Wines - Advances in Production, Processing, Analysis and Valorization

were lined up as the HCA input.

3. Results and discussion

3.1. Representation of aroma and taste

Aoverripeness and PC5 Aflorcitrusmineral.

highly specific, isolated, and less obvious sensory descriptors.

terization of the taste of the PGI young red wines.

Tfullpersistent.


Table 2. Loadings of 18 aromatic attributes in the first 5 principal components named PC1 Aredfruitwoody, PC2 Aripefruit, PC3 Agreenchemical, PC4 Aoverripeness and PC5 Aflorcitrusmineral.

proposed. The experts' typicality construct for the PGI red wines from mainland Portugal led to a sensory aggregation around four clusters (Figures 2 and 3) that were named according to their respective geography and a combination of geomorphological and bioclimatic character-

Table 3. Loadings of 14 tactile/textural and mouthfeel descriptors in the first 4 principal components named PC1

Grain (Mouthfeel) 0.879 �0.073 0.071 0.122 Rough (Mouthfeel) 0.862 �0.216 0.154 0.075 Astringent (Global) 0.812 �0.341 0.186 0.165 Dry (Mouthfeel) 0.787 �0.045 0.247 �0.186 Sweet �0.071 0.886 0.071 �0.092 Alcohol �0.299 0.799 �0.113 0.168 Smooth (Mouthfeel) �0.163 0.787 �0.045 0.206 Oily (Mouthcoat) 0.044 0.622 �0.166 0.583 Acid (Sour) 0.492 �0.621 0.363 0.187 Salty 0.084 0.025 0.864 0.009 Bitter 0.393 �0.011 0.715 0.053 Bubbly 0.167 �0.395 0.572 �0.209 Length 0.188 �0.107 0.077 0.862 Full body �0.078 0.466 �0.1 0.714 Variance explained (cumulative) 24.2 (24.2) 24.1 (48.3) 13.6 (61.9) 13.1 (75.0)

Novel Memory-Based Sensory Approach to Assess Large-Scale Typicality: The Case of Mainland Portugal Red Wines

PC1 PC2 PC3 PC4

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283

• Cluster #1: SOUTHERN (PGI Pen. Setubal (PS) + PGI Tejo (T) + PGI Alentejano (AL) + PGI

• Cluster #3: CENTRAL COAST and PERIPHERAL VALLEYS (PGI Beira Atlântico (BA) + PGI Lisboa (L)) and (PGI Terras Beira (TB) + PGI Transmontano (TM) + PGI Terras

Clustered groups were, finally, sensory described on the basis of mean scores comparison as the ANOVA results confirmed the statistical robustness of our design (Table 4). A radar chart of such means is presented, such representation being straightforward and relatively effortless

• Cluster #2: CENTRAL VALLEYS (PGI Duriense (D) + PGI Terras do Dão (TD))

istics [22]:

Algarve (AG))

Cister (TC))

to analyze (Figure 4).

• Cluster#4: (Single) PGI MINHO (M)

Major loadings on each PC are highlighted in bold.

Tdryastringent, PC2 Tsweetviscous, PC3 Tbittersalty and PC4 Tfullpersistent.

Taste Principal component (PC)

Wine phenolic compounds may either taste astringent, bitter or both [37]. Furthermore, mutual suppression is a fundamental property among all tastes. However, this does not occur with astringency [38] because this tactile sensation plays a strong role as a key sensory profiler. Despite the close relationship between bitterness and astringent [39], our panel of professional experts placed major factor loadings for each one in different principal components, hence endorsing the greater sensitivity and lingual acuity of our super tasters [40]. Although the significant grouping of the originally segmented terms may lead to doubt about their usefulness for providing quality data regarding the astringency of the wines [35], our results suggest that the variation in the PC1 Tdryastringent that was elicited by the red wines can be differentiated and rated.

#### 3.2. Sensory differentiation

Multivariate statistical methods showed that evidence of sensory aggregations between PGIs and extended geographical clusters or macroscale areas with similar sensory profile was


Table 3. Loadings of 14 tactile/textural and mouthfeel descriptors in the first 4 principal components named PC1 Tdryastringent, PC2 Tsweetviscous, PC3 Tbittersalty and PC4 Tfullpersistent.

proposed. The experts' typicality construct for the PGI red wines from mainland Portugal led to a sensory aggregation around four clusters (Figures 2 and 3) that were named according to their respective geography and a combination of geomorphological and bioclimatic characteristics [22]:


Wine phenolic compounds may either taste astringent, bitter or both [37]. Furthermore, mutual suppression is a fundamental property among all tastes. However, this does not occur with astringency [38] because this tactile sensation plays a strong role as a key sensory profiler. Despite the close relationship between bitterness and astringent [39], our panel of professional experts placed major factor loadings for each one in different principal components, hence endorsing the greater sensitivity and lingual acuity of our super tasters [40]. Although the significant grouping of the originally segmented terms may lead to doubt about their usefulness for providing quality data regarding the astringency of the wines [35], our results suggest that the variation in the PC1

Table 2. Loadings of 18 aromatic attributes in the first 5 principal components named PC1 Aredfruitwoody, PC2 Aripefruit,

PC1 PC2 PC3 PC4 PC5

Woody 0.814 0.054 0.085 0.151 0.111 Spicy 0.674 0.02 0.236 0.154 0.206 Red fruit 0.644 0.139 0.088 �0.253 0.346 Bread and pastry 0.595 0.185 0.012 0.426 0.04 Caramelized 0.492 0.442 0.083 0.433 �0.219 Black fruit 0.058 0.846 �0.131 �0.14 0.249 Stone fruit �0.027 0.809 0.054 0.167 0.048 Dried fruit 0.251 0.717 �0.092 0.351 �0.23 Jam and jelly fruit 0.513 0.65 �0.2 0.203 �0.086 Chemical �0.036 0.064 0.757 �0.026 �0.095 Vegetable 0.292 �0.192 0.742 �0.157 0.177 Animal �0.012 �0.024 0.733 0.307 0.109 Herbal 0.231 �0.16 0.709 0.094 0.243 Dry flowers 0.157 0.058 0.189 0.763 0.234 Nut fruit 0.118 0.206 �0.009 0.691 0.216 Floral 0.304 0.046 �0.079 0.077 0.784 Mineral 0.11 �0.091 0.218 0.208 0.697 Citrus fruit �0.001 0.177 0.381 0.29 0.574 Variance explained (cumulative) 15.2 (15.2) 15.1 (30.3) 14.1 (44.5) 11.2 (55.6) 11.1 (66.7)

Multivariate statistical methods showed that evidence of sensory aggregations between PGIs and extended geographical clusters or macroscale areas with similar sensory profile was

Tdryastringent that was elicited by the red wines can be differentiated and rated.

AROMA Principal component (PC)

282 Grapes and Wines - Advances in Production, Processing, Analysis and Valorization

3.2. Sensory differentiation

Major loadings on each PC are highlighted in bold.

PC3 Agreenchemical, PC4 Aoverripeness and PC5 Aflorcitrusmineral.

Clustered groups were, finally, sensory described on the basis of mean scores comparison as the ANOVA results confirmed the statistical robustness of our design (Table 4). A radar chart of such means is presented, such representation being straightforward and relatively effortless to analyze (Figure 4).

Figure 2. Dendogram showing the distance between PGIs (PS, peninsula de Setubal; T, Tejo; AG, Algarve; AL, Alentejano; D, Duriense; TD, Terras do Dão; BA, Beira Atlântico; L, Lisboa; TB, Terras da Beira; TM, Transmontano; TC, Terras de Cister; M, Minho).

the hemiketal (colorless), the quinoidal (blue) and the flavylium cation (red) anthocyanin structures [43, 44]. One study showed that raisining decreased the lightness and increased the color saturation and hue [45]. These findings may explain the ruby-garnet dominant hue and the low color intensity of typical young red wines from the SOUTHERN macroscale area.

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Figure 3. Four large-scale clusters in mainland Portugal originated from experimental design on sensory typicality.

For the aroma, the lowest assessments were given to PC3 Agreenchemical and PC5 Aflorcitrusmineral. Inversely, PC1 Aredfruitwoody and PC2 Aripefruit received higher scores (Table 4 and Figure 4). Published results indicate that a masking effect of vegetative aromas by fruit aromas may occur [46]. The aroma profile showed an intense raisiny and jammy character with dominant black and stone fruit; however, the experts noted that the aromas were also woody, spicy, and of young yeasty red fruits. Red wine fruitiness may correlate directly with the ethanol enhancement because studies on red wine aroma have confirmed that when ethanol is enhanced, the intensity of the fruity odor increases [47]. Additionally, the existence of inodorous constituents in red grape skins, which are extractible by ethanol and transformed by yeasts, produced a

#### 3.3. Typical wine profiles of the new-found macroscale areas

#### 3.3.1. Typical young red wine profile of the SOUTHERN macroscale area

The final sensory attributes reveal some characteristics that sustain the fast clustering dynamics that encompass the four SOUTHERN PGIs (PGI Peninsula de Setubal, PGI Tejo, PGI Alentejano and PGI Algarve), thus enabling the creation of a typical sensory profile for this macroscale area (Figure 4). In agreement with our results, similar geoviticultural (temperate hot climate, temperate warm nights and a moderately strong drought) and sensory analysis (high alcohol content and intense aromatic ripe fruity wines) groupings were reported for these same four PGIs [41].

The color hue got the highest assessment (Table 4 and Figure 4), indicating a Ruby-Garnet hue, and color intensity rated second lowest. Temperatures of 30C and higher might lead to lower anthocyanin synthesis [42]. Lower acidity levels are usually correlated with higher grape pH, although the relation is affected by potassium accumulation, which is itself temperature dependent. At a higher pH, the chalcone form (slightly yellow) increases progressively over Novel Memory-Based Sensory Approach to Assess Large-Scale Typicality: The Case of Mainland Portugal Red Wines http://dx.doi.org/10.5772/intechopen.73039 285

Figure 3. Four large-scale clusters in mainland Portugal originated from experimental design on sensory typicality.

3.3. Typical wine profiles of the new-found macroscale areas

284 Grapes and Wines - Advances in Production, Processing, Analysis and Valorization

these same four PGIs [41].

Terras de Cister; M, Minho).

3.3.1. Typical young red wine profile of the SOUTHERN macroscale area

The final sensory attributes reveal some characteristics that sustain the fast clustering dynamics that encompass the four SOUTHERN PGIs (PGI Peninsula de Setubal, PGI Tejo, PGI Alentejano and PGI Algarve), thus enabling the creation of a typical sensory profile for this macroscale area (Figure 4). In agreement with our results, similar geoviticultural (temperate hot climate, temperate warm nights and a moderately strong drought) and sensory analysis (high alcohol content and intense aromatic ripe fruity wines) groupings were reported for

Figure 2. Dendogram showing the distance between PGIs (PS, peninsula de Setubal; T, Tejo; AG, Algarve; AL, Alentejano; D, Duriense; TD, Terras do Dão; BA, Beira Atlântico; L, Lisboa; TB, Terras da Beira; TM, Transmontano; TC,

The color hue got the highest assessment (Table 4 and Figure 4), indicating a Ruby-Garnet hue, and color intensity rated second lowest. Temperatures of 30C and higher might lead to lower anthocyanin synthesis [42]. Lower acidity levels are usually correlated with higher grape pH, although the relation is affected by potassium accumulation, which is itself temperature dependent. At a higher pH, the chalcone form (slightly yellow) increases progressively over the hemiketal (colorless), the quinoidal (blue) and the flavylium cation (red) anthocyanin structures [43, 44]. One study showed that raisining decreased the lightness and increased the color saturation and hue [45]. These findings may explain the ruby-garnet dominant hue and the low color intensity of typical young red wines from the SOUTHERN macroscale area.

For the aroma, the lowest assessments were given to PC3 Agreenchemical and PC5 Aflorcitrusmineral. Inversely, PC1 Aredfruitwoody and PC2 Aripefruit received higher scores (Table 4 and Figure 4). Published results indicate that a masking effect of vegetative aromas by fruit aromas may occur [46].

The aroma profile showed an intense raisiny and jammy character with dominant black and stone fruit; however, the experts noted that the aromas were also woody, spicy, and of young yeasty red fruits. Red wine fruitiness may correlate directly with the ethanol enhancement because studies on red wine aroma have confirmed that when ethanol is enhanced, the intensity of the fruity odor increases [47]. Additionally, the existence of inodorous constituents in red grape skins, which are extractible by ethanol and transformed by yeasts, produced a


Table 4. Mean scores of final sensory attributes regarding the four clustered zonings of mainland Portugal.

specific aroma of red-berry and black-berry fruit in the finished wines [48]. The SOUTHERN macroscale area ranked second on the aromatic intensity scale and received the lowest scores for mineral, floral, and citrus fruit. Chemical and vegetable notes were almost nonexistent.

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Figure 4. Radar chart of the red wine sensory aggregate attribute means per immediate cluster.

For the taste, the lowest assessment was given to PC1 Tdryastringent, and the highest assessment was given to PC2 Tsweetviscous (Table 4 and Figure 4). The taste characterization demonstrated a clear typicality for the sweet and oily intensive textures, which are higher alcohol-driven tactile sensations, with a weakness in the fresh acidic taste and tannin smoothness. Lower levels of dry, astringent, rough, and grain tannin-related mouthfeels are frequent in these medium-bodied immediate southern red wines. An oily viscosity enhancement results in a smoother palate texture [49] and reduces the astringency [38]. Wines with high levels of polysaccharides tend to decrease the impact of the bitterness and astringency [50]. The phenolic composition may be due to macroscale area differentiation because the amount of epigallocatechin (EGC) was negatively correlated with the perceived astringency [51] and could decrease the coarse perception [52]. PC2 Tsweetviscous encompassed alcohol, sweet taste, smooth mouthfeel, and oily mouthcoat sensations as positively correlated; however, other published results indicate that ethanol and glycerol do not significantly

3.3.2. Typical young red wine profile of the CENTRAL VALLEYS macroscale area

their centered geography on the mountainous continental plateau (Figure 4).

A portion of inner center-northern Portugal was sensory-zoned. PGI Terras do Dão and PGI Terras do Douro were immediately clustered and named CENTRAL VALLEYS because of

contribute to viscous mouthfeel [53].

Summary of ANOVA results.

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Figure 4. Radar chart of the red wine sensory aggregate attribute means per immediate cluster.

specific aroma of red-berry and black-berry fruit in the finished wines [48]. The SOUTHERN macroscale area ranked second on the aromatic intensity scale and received the lowest scores for mineral, floral, and citrus fruit. Chemical and vegetable notes were almost nonexistent.

For the taste, the lowest assessment was given to PC1 Tdryastringent, and the highest assessment was given to PC2 Tsweetviscous (Table 4 and Figure 4). The taste characterization demonstrated a clear typicality for the sweet and oily intensive textures, which are higher alcohol-driven tactile sensations, with a weakness in the fresh acidic taste and tannin smoothness. Lower levels of dry, astringent, rough, and grain tannin-related mouthfeels are frequent in these medium-bodied immediate southern red wines. An oily viscosity enhancement results in a smoother palate texture [49] and reduces the astringency [38]. Wines with high levels of polysaccharides tend to decrease the impact of the bitterness and astringency [50]. The phenolic composition may be due to macroscale area differentiation because the amount of epigallocatechin (EGC) was negatively correlated with the perceived astringency [51] and could decrease the coarse perception [52]. PC2 Tsweetviscous encompassed alcohol, sweet taste, smooth mouthfeel, and oily mouthcoat sensations as positively correlated; however, other published results indicate that ethanol and glycerol do not significantly contribute to viscous mouthfeel [53].

#### 3.3.2. Typical young red wine profile of the CENTRAL VALLEYS macroscale area

Final clustered zonings vs. final

Hue\* Color

Aroma

(PC1)

(PC2)

(PC3)

(PC4)

(PC5) Aflor

(PC1)

(PC2)

(PC3)

(PC4)

intensity

intensity

Aredfruitwoody

Aripefruit

Agreenchemical

Aoverripeness

citrusmineral

Tdryastringent

Tsweetviscous

Tbittersalty

Tfullpersistent

sensory attributes

Southern CENTRAL VALLEYS

Central coast and peripheral valleys

PGI MINHO Univariate ANOVA

Sigma

Eta

0.806 0.641

 0.730

 0.910

Squared

\*Hue was standardized to encompass the 0–10 points integer scale.

Minimal scores in italic; maximal scores in bold.

Summary of ANOVA results.

Table 4. Mean scores of final sensory attributes regarding the four clustered zonings of mainland Portugal.

 0.001 0.010

 0.003

 0.000

> results (By Attribute)

Mean

0.35 8.58

5.84

2.93

3.24 0.031

0.518

 0.459

 0.049

5.26

1.89 0.047 0.466

0.810

 0.818

0.770

0.599

 0.754

286 Grapes and Wines - Advances in Production, Processing, Analysis and Valorization

0.001

 0.001

0.002

0.016

 0.002

3.35

6.43

2.43

3.79

5.03

 Mean Mean

 4.08 5.90

 5.39

4.03

3.99

 4.17

 2.75 7.08

Mean

6.09 6.51

 6.36 6.93

4.64

5.11

 3.88

4.71

5.82

3.52

3.30 3.55 3.13

3.88

5.12

4.12

2.35

 5.72

5.00

4.82

5.09

1.75

6.88

3.11

3.67

6.11

1.80

 5.59

> A portion of inner center-northern Portugal was sensory-zoned. PGI Terras do Dão and PGI Terras do Douro were immediately clustered and named CENTRAL VALLEYS because of their centered geography on the mountainous continental plateau (Figure 4).

The CENTRAL VALLEYS macroscale area was presented as an immediate clustering output. According to the cluster analysis method and based on the related isolated and aggregate means, the sensory profile has the following characterization:

The hue of the red wines was rated from Purple/Ruby to Ruby/Garnet halfway between typical fresh and warm region hues. Indeed, this peripheral setting includes a vast macroscale area border that may explain the wider values observed in our results. The color intensity received

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289

The aromatic intensity of PERIPHERAL VALLEYS and CENTRAL COAST red wines was the lowest among all of the macroscale areas (Table 4 and Figure 4), which is another difference with the neighboring Central Valleys and is a similarity with the Northern-coastal

The taste characterization showed long mouthfeel persistence, and it ranked second highest for the assessment of full bodied taste, a feature that is similar to the neighboring Central Valleys. The levels of salty, bitter, and tactile bubbly were slightly increased, suggesting a similarity with the Northern-coastal PGI Minho and some typicality in the acidic-fresh sensory profile

The northern-coastal PGI MINHO is a wine region, where the world-renowned PDO Vinho Verde is located. This region presented the highest primary typicality assessments according to the stand-alone cluster analysis results (Figure 2). This result was strongly supported by 10 of the 12 analyzed attributes that had extreme scores (Table 4 and Figure 4). Similar observations were previously reported by other authors [41], which were based on their geoviticultural multicriteria climatic classification system [61]. The sensory attributes revealed characteristics

For the color, the highest assessment was for C Intensity, and the lowest assessment was for C Hue, indicating a Violet-Purple hue. The high intensity is related to the inky local red cultivar, Vinhão, which has been shown to have anthocyanin levels that are 24 times higher than other cultivars [62]. A correlation between the anthocyanic content and the bitterness that is elicited was recently published [63]. Moreover, the lowest assessment for PC2 Tsweetviscous correlates with the highest acidity levels, and it thus correlates with the lowest pH, explaining the highest content of the colored anthocyanins [43]. The hue assessment that indicated a violet-purple hue is aligned with the first-year consumption of extremely young reds [32], which is typical of PGI

The aroma was considered to be typical as the experts scored one highest assessment for PC3 Agreenchemical and three lowest assessments for PC1 Aredfruitwoody, PC2 Aripefruit, and PC4

The aromatic profile is built on several extreme assessments. The high levels of green and chemical notes and the low levels of ripe fruit may relate to the cool climate and the low ripening conditions [64]. An average intensity was found for the PGI MINHO red wines with

an extremely low presence of woody, spicy, and young yeasty red fruit notes.

3.3.4. Typical young red wine profile of the single-clustered PGI MINHO

that allowed clustering dynamics and helped establish a typical sensory profile:

the lowest score (Table 4 and Figure 4).

Minho and its inner PDO Vinho Verde.

Aoverripeness.

PGI Minho.

(Table 4 and Figure 4).

The color got the second lowest assessment for its hue, indicating a Purple-Ruby hue, and the second highest assessment was for C Intensity (Table 4 and Figure 4). These results are characteristic of fresh/cool continental regions with extreme thermal amplitudes, and this explains the high-color intensities [54].

For the aroma, there was a strong typicality with the three highest assessments for Aintensity, Aoverripeness, and Aflorcitrusmineral (Table 4 and Figure 4). The red wine aromatic intensity (Aintensity) was the highest for the CENTRAL VALLEYS macroscale area. Additionally, the floral and citrus fruit notes (PC5 Aflorcitrusmineral) may be related to the Touriga Nacional cultivar (representative of PGI Terras do Dão and PGI Duriense) because they exhibited maximum scores [55]. The highest mineral assessment [56] and the exhibited essence of dried fruits and flowers from PC4 Aoverripeness are attributes that may result from the heterogeneous mountain that is known to have significant gaps in altitude and semi-arid locations, especially near the Spanish border [23]. This may explain the observed proximity with the Southern regions, as shown in the clustering dendogram (Figure 2).

For the taste, the lowest assessment was for PC3 Tbittersalty, and the highest assessment was for PC4 Tfullpersistent (Table 4 and Figure 4). The taste characterization exhibited a clear typicality based on the extremely long persistence, which relates with the highest assessment of fullness. This feature may relate to a higher phenolic content of such high density wines which has shown to enhance flavors and mouthfeel persistence [57, 58]. Both the main yeast polysaccharides (mannoproteins) and the principal grape polysaccharides (arabinogalactanproteins and rhamnogalacturonans) increase the perception of body [52]. The evaluation of wine finish duration by trained panelists indicated the finish of the high ethanol wines lasted longer than the ones with low ethanol [59]. Similar to the Southern reds, wines from the CENTRAL VALLEYS exhibited the lowest salty, bitter, and bubbly sensation levels, suggesting the partial proximity. The bitterness intensities elicited by ethanol-sugar mixtures are lower than those elicited by unmixed ethanol solutions [60], these findings in perfect alignment with our results.

#### 3.3.3. Typical young red wine profile of the CENTRAL COAST and PERIPHERAL VALLEYS macroscale area

For the remaining continental PGIs, which are listed under the PERIPHERAL VALLEYS cluster, they were sensory assessed as similar to the CENTRAL COAST PGIs, and the data were combined (Figure 3). Interestingly, the late PGI Beiras, which encompass the administrative merging of coastal and inner wine regions (PGI Terras da Beira (TB), PGI Terras de Cister (TC), and PGI Beira Atlântico (BA)), was officially extinct after the harvest of 2011.

The hue of the red wines was rated from Purple/Ruby to Ruby/Garnet halfway between typical fresh and warm region hues. Indeed, this peripheral setting includes a vast macroscale area border that may explain the wider values observed in our results. The color intensity received the lowest score (Table 4 and Figure 4).

The aromatic intensity of PERIPHERAL VALLEYS and CENTRAL COAST red wines was the lowest among all of the macroscale areas (Table 4 and Figure 4), which is another difference with the neighboring Central Valleys and is a similarity with the Northern-coastal PGI Minho.

The taste characterization showed long mouthfeel persistence, and it ranked second highest for the assessment of full bodied taste, a feature that is similar to the neighboring Central Valleys. The levels of salty, bitter, and tactile bubbly were slightly increased, suggesting a similarity with the Northern-coastal PGI Minho and some typicality in the acidic-fresh sensory profile (Table 4 and Figure 4).

#### 3.3.4. Typical young red wine profile of the single-clustered PGI MINHO

The CENTRAL VALLEYS macroscale area was presented as an immediate clustering output. According to the cluster analysis method and based on the related isolated and aggregate

The color got the second lowest assessment for its hue, indicating a Purple-Ruby hue, and the second highest assessment was for C Intensity (Table 4 and Figure 4). These results are characteristic of fresh/cool continental regions with extreme thermal amplitudes, and this

For the aroma, there was a strong typicality with the three highest assessments for Aintensity, Aoverripeness, and Aflorcitrusmineral (Table 4 and Figure 4). The red wine aromatic intensity (Aintensity) was the highest for the CENTRAL VALLEYS macroscale area. Additionally, the floral and citrus fruit notes (PC5 Aflorcitrusmineral) may be related to the Touriga Nacional cultivar (representative of PGI Terras do Dão and PGI Duriense) because they exhibited maximum scores [55]. The highest mineral assessment [56] and the exhibited essence of dried fruits and flowers from PC4 Aoverripeness are attributes that may result from the heterogeneous mountain that is known to have significant gaps in altitude and semi-arid locations, especially near the Spanish border [23]. This may explain the observed proximity with the Southern regions, as shown in the clustering dendogram

For the taste, the lowest assessment was for PC3 Tbittersalty, and the highest assessment was for PC4 Tfullpersistent (Table 4 and Figure 4). The taste characterization exhibited a clear typicality based on the extremely long persistence, which relates with the highest assessment of fullness. This feature may relate to a higher phenolic content of such high density wines which has shown to enhance flavors and mouthfeel persistence [57, 58]. Both the main yeast polysaccharides (mannoproteins) and the principal grape polysaccharides (arabinogalactanproteins and rhamnogalacturonans) increase the perception of body [52]. The evaluation of wine finish duration by trained panelists indicated the finish of the high ethanol wines lasted longer than the ones with low ethanol [59]. Similar to the Southern reds, wines from the CENTRAL VALLEYS exhibited the lowest salty, bitter, and bubbly sensation levels, suggesting the partial proximity. The bitterness intensities elicited by ethanol-sugar mixtures are lower than those elicited by unmixed ethanol solutions [60], these findings in perfect alignment with

3.3.3. Typical young red wine profile of the CENTRAL COAST and PERIPHERAL VALLEYS

(TC), and PGI Beira Atlântico (BA)), was officially extinct after the harvest of 2011.

For the remaining continental PGIs, which are listed under the PERIPHERAL VALLEYS cluster, they were sensory assessed as similar to the CENTRAL COAST PGIs, and the data were combined (Figure 3). Interestingly, the late PGI Beiras, which encompass the administrative merging of coastal and inner wine regions (PGI Terras da Beira (TB), PGI Terras de Cister

means, the sensory profile has the following characterization:

288 Grapes and Wines - Advances in Production, Processing, Analysis and Valorization

explains the high-color intensities [54].

(Figure 2).

our results.

macroscale area

The northern-coastal PGI MINHO is a wine region, where the world-renowned PDO Vinho Verde is located. This region presented the highest primary typicality assessments according to the stand-alone cluster analysis results (Figure 2). This result was strongly supported by 10 of the 12 analyzed attributes that had extreme scores (Table 4 and Figure 4). Similar observations were previously reported by other authors [41], which were based on their geoviticultural multicriteria climatic classification system [61]. The sensory attributes revealed characteristics that allowed clustering dynamics and helped establish a typical sensory profile:

For the color, the highest assessment was for C Intensity, and the lowest assessment was for C Hue, indicating a Violet-Purple hue. The high intensity is related to the inky local red cultivar, Vinhão, which has been shown to have anthocyanin levels that are 24 times higher than other cultivars [62]. A correlation between the anthocyanic content and the bitterness that is elicited was recently published [63]. Moreover, the lowest assessment for PC2 Tsweetviscous correlates with the highest acidity levels, and it thus correlates with the lowest pH, explaining the highest content of the colored anthocyanins [43]. The hue assessment that indicated a violet-purple hue is aligned with the first-year consumption of extremely young reds [32], which is typical of PGI Minho and its inner PDO Vinho Verde.

The aroma was considered to be typical as the experts scored one highest assessment for PC3 Agreenchemical and three lowest assessments for PC1 Aredfruitwoody, PC2 Aripefruit, and PC4 Aoverripeness.

The aromatic profile is built on several extreme assessments. The high levels of green and chemical notes and the low levels of ripe fruit may relate to the cool climate and the low ripening conditions [64]. An average intensity was found for the PGI MINHO red wines with an extremely low presence of woody, spicy, and young yeasty red fruit notes.

The taste was considered to be typical as the experts scored the highest assessment for PC1 Tdryastringent and PC3 Tbittersalty and the lowest assessment for PC2 Tsweetviscous and PC4 Tfullpersistent.

Located on the extreme northwest corner of mainland Portugal, the PGI MINHO sensory profile exhibited typical gustatory characteristics, such as the highest intensity of dryness, astringency, roughness, and grain tannin mouthfeel, which are related to the greener ripeness [26]. Polyphenols are recognized as substances that provide astringency sensation. However, other substances such as organic acids, sugars, and ethanol can also influence this sensation [65]. The fresh profile was amplified to its peak by the highest presence of salty, bitter, and bubbly sensations. The bubbly sensations were due to typical carbonic gas addition. Inversely, these red wines were assessed as light bodied with a fair persistence, low alcohol-driven tactile sensations, and reduced tannin smoothness. The lowest assessments were for sweet and oily textures. The highest assessment of astringency occurred for the low alcoholic wines [66]. The maximal rate of fresh acidic taste was found for the PC2 Tsweetviscous, and this is also typical of the PGI MINHO profile (Figure 4). Furthermore, the lack of ethanol and proanthocyanidins greatly increased the perceived acidity [67]. The combination of the effect of excess acidity and astringency was termed Green by a panel of experienced wine tasters [35]. Acidity reduces the perception of body [68]. The results of this study contradict the reports of enhanced bitterness by alcohol [69], although some authors suggest that alcohol may suppress the bitter taste when held in the mouth [70].

#### 3.4. Astringency vs. bitterness: A novel macroscale orthogonal approach

The fact that four PCs—PC1 Tdryastringent, PC2 Tsweetviscous, PC3 Tbittersalty and PC4 Tfullpersistent—explained 75% of the total variance of our experiment, justified further


research on potential correlation with an orthogonal mapping of the leading sensory attributes of each PC, envisaging red wine PGI typicality. Pearson Correlation was computed between those four PCs (Table 5), and results showed expected significant correlation between PC1

Figure 5. Standardized score bi-plots of the four aggregate tactile descriptors over 12 PGIs (PS, peninsula de Setubal; T, Tejo; AG, Algarve; AL, Alentejano; D, Duriense; TD, Terras do Dão; BA, Beira Atlântico; L, Lisboa; TB, Terras da Beira;

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The coefficient of �0.957 (close to maximum �1) between PC1 Tdryastringent and PC2 Tsweetviscous reflected a strong linear mapping of mainland Portugal PGIs, enabling a clear sensory profiling statement, virtually with a North-South reading, as shown in

Astringency was assessed as drier and rougher in the north and smoother in the South. The bi-plot in Figure 5a also showed a clear northward decrease in alcohol content. Conversely, acidity was enhanced northward. The sensory measure PC3 Tbittersalty included bitterness,

and PC2.

TM, Transmontano; TC, Terras de Cister; M, Minho).

Figure 5a.

Correlation is significant at the 0.05 level.

\*\*Correlation is significant at the 0.01 level.

Table 5. Pearson correlation matrix of four aggregate tactile attributes.

The taste was considered to be typical as the experts scored the highest assessment for PC1 Tdryastringent and PC3 Tbittersalty and the lowest assessment for PC2 Tsweetviscous and PC4

290 Grapes and Wines - Advances in Production, Processing, Analysis and Valorization

Located on the extreme northwest corner of mainland Portugal, the PGI MINHO sensory profile exhibited typical gustatory characteristics, such as the highest intensity of dryness, astringency, roughness, and grain tannin mouthfeel, which are related to the greener ripeness [26]. Polyphenols are recognized as substances that provide astringency sensation. However, other substances such as organic acids, sugars, and ethanol can also influence this sensation [65]. The fresh profile was amplified to its peak by the highest presence of salty, bitter, and bubbly sensations. The bubbly sensations were due to typical carbonic gas addition. Inversely, these red wines were assessed as light bodied with a fair persistence, low alcohol-driven tactile sensations, and reduced tannin smoothness. The lowest assessments were for sweet and oily textures. The highest assessment of astringency occurred for the low alcoholic wines [66]. The maximal rate of fresh acidic taste was found for the PC2 Tsweetviscous, and this is also typical of the PGI MINHO profile (Figure 4). Furthermore, the lack of ethanol and proanthocyanidins greatly increased the perceived acidity [67]. The combination of the effect of excess acidity and astringency was termed Green by a panel of experienced wine tasters [35]. Acidity reduces the perception of body [68]. The results of this study contradict the reports of enhanced bitterness by alcohol [69], although some

authors suggest that alcohol may suppress the bitter taste when held in the mouth [70].

(PC1) Tdryastringent 1 �0.957\*\* 0.434 0.154 (PC2) Tsweetviscous �0.957\*\* <sup>1</sup> �0.592\* �0.017 (PC3) Tbittersalty 0.434 �0.592\* <sup>1</sup> �0.502 (PC4) Tfullpersistent 0.154 �0.017 �0.502 1 N 12 12 12 12

The fact that four PCs—PC1 Tdryastringent, PC2 Tsweetviscous, PC3 Tbittersalty and PC4 Tfullpersistent—explained 75% of the total variance of our experiment, justified further

(PC1) Tdryastringent (PC2) Tsweetviscous (PC3) Tbittersalty (PC4) Tfullpersistent

3.4. Astringency vs. bitterness: A novel macroscale orthogonal approach

Tfullpersistent.

Correlations

\*

Correlation is significant at the 0.05 level. \*\*Correlation is significant at the 0.01 level.

Table 5. Pearson correlation matrix of four aggregate tactile attributes.

Figure 5. Standardized score bi-plots of the four aggregate tactile descriptors over 12 PGIs (PS, peninsula de Setubal; T, Tejo; AG, Algarve; AL, Alentejano; D, Duriense; TD, Terras do Dão; BA, Beira Atlântico; L, Lisboa; TB, Terras da Beira; TM, Transmontano; TC, Terras de Cister; M, Minho).

research on potential correlation with an orthogonal mapping of the leading sensory attributes of each PC, envisaging red wine PGI typicality. Pearson Correlation was computed between those four PCs (Table 5), and results showed expected significant correlation between PC1 and PC2.

The coefficient of �0.957 (close to maximum �1) between PC1 Tdryastringent and PC2 Tsweetviscous reflected a strong linear mapping of mainland Portugal PGIs, enabling a clear sensory profiling statement, virtually with a North-South reading, as shown in Figure 5a.

Astringency was assessed as drier and rougher in the north and smoother in the South. The bi-plot in Figure 5a also showed a clear northward decrease in alcohol content. Conversely, acidity was enhanced northward. The sensory measure PC3 Tbittersalty included bitterness, saltiness, and fizziness (natural or added CO2) perceptions as its leading scores. The interpretation must be merely indicative, as the explained variance was weaker than PC1 and PC2 levels; however, those three aggregate perceptions seemed to act as key profilers for sensory differentiation, as showed in Figure 5b and c. This finding is statistically significant, the negative correlation of PC3 Tbittersalty with PC2 Tsweetviscous suggesting an inland to coastal increase in bitterness, leading to a well-defined cluster including super humid coastal PGI Minho and temperate coastal PGI Beira Atlântico and PGI Lisboa, as well as a surprising stand-alone southern coastal region (PGI Peninsula de Setubal), which was replaced, in its customary cluster, by a sweet, lowest bitter-salty, north-continental PGI Duriense (Figure 5c). The nationwide differentiation of astringency and bitterness introduced an orthogonal reading (N north-S south vs. E inland-W coastal, respectively) rather than a linear one, these findings add novelty to sensory research. The weakest contributing PC4 Tfullpersistent presented several inverse scores when compared to PC3 bittersalty (Figure 5d), and this finding, lacking statistical significance, may justify new and comprehensive studies to confirm the following orthogonal sensory mapping PC1 (northward) – PC2 (southward) – PC3 (coastal westward) – PC4 (inland eastward), the geographical reading showed in Figure 6.

#### 3.5. Classic sampling vs. prototypical memory

Among the scientific community, our novel, nationwide, sample-free, noncalibrated, expert memory-based method was accepted [29, 71], as it was challenged on the basis that the data relied on respondents' perceptions rather than direct experience. In this new research, we compare the results mentioned before in this study—signaled as Experiment 1 (LTM) with the completion of the same questionnaire (Supporting Information) by 19 local experts from the single PGI Beira Atlântico—Experiment 2 (WS)—as a result of a blind tasting of 15 young red wine samples, representing the leading supermarket brands of this wine region.

Figure 6. Mean differences between long term memory (LTM) and wine sampling (WS) methods. ABS scores represent

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Descriptor (PD) Descriptor Long-term memory Wine sampling Δ (LTM-WS)

Experiment 1 Experiment 2

Primary Aggregate (PD Means) (PD Means) ABS ()

Aroma vegetable Aroma-greenchemical 6.25 2.36 3.89 Taste grain astringency Taste-dryastringent 5.00 2.19 2.81 Aroma herbal Aroma-greenchemical 5.95 3.21 2.74 Aroma red fruit Aroma-redfruitwoody 5.55 2.85 2.70 Taste acid Taste-sweetviscous (inverted) 7.00 4.46 2.54 Aroma mineral Aroma-citrusmineral 5.15 2.66 2.49 Taste salty Taste-bittersalty 3.65 1.27 2.38 Taste rough astringency Taste-dryastringent 5.60 3.24 2.36 Aroma animal Aroma-greenchemical 4.25 1.91 2.34 Taste bitter Taste-bittersalty 4.60 2.30 2.30

absolute differences between both methods.

Table 6. Top 10 underperfoming primary descriptors.

#### 3.5.1. Experiment 2

Results of the wine tasting assessment by local PGI Beira Atlântico wine experts were organized in order to fully compare with extracted results of experiment 1 regarding the same PGI (Figure 6). The panel is widely calibrated, as these professionals belong to the certification panel which must approve typicality and quality of all submitted PGI Beira Atlântico wines. This panel complies with an annual program of inner and inter-laboratorial calibration on aromatic thresholds, triangular tests, and tastant solutions (control standards of sucrose, NaCl, citric acid, quinine, and others).

The assessment of the same sensory questionnaire by the nationwide professional uncalibrated panel and the local professional PGI Atlântico calibrated panel showed a significant Pearson positive correlation of 0.669, this to be considered promising and a green flag for newcoming comparative studies regarding other PGIs.

saltiness, and fizziness (natural or added CO2) perceptions as its leading scores. The interpretation must be merely indicative, as the explained variance was weaker than PC1 and PC2 levels; however, those three aggregate perceptions seemed to act as key profilers for sensory differentiation, as showed in Figure 5b and c. This finding is statistically significant, the negative correlation of PC3 Tbittersalty with PC2 Tsweetviscous suggesting an inland to coastal increase in bitterness, leading to a well-defined cluster including super humid coastal PGI Minho and temperate coastal PGI Beira Atlântico and PGI Lisboa, as well as a surprising stand-alone southern coastal region (PGI Peninsula de Setubal), which was replaced, in its customary cluster, by a sweet, lowest bitter-salty, north-continental PGI Duriense (Figure 5c). The nationwide differentiation of astringency and bitterness introduced an orthogonal reading (N north-S south vs. E inland-W coastal, respectively) rather than a linear one, these findings add novelty to sensory research. The weakest contributing PC4 Tfullpersistent presented several inverse scores when compared to PC3 bittersalty (Figure 5d), and this finding, lacking statistical significance, may justify new and comprehensive studies to confirm the following orthogonal sensory mapping PC1 (northward) – PC2 (southward) – PC3 (coastal westward) – PC4 (inland eastward), the geographical

292 Grapes and Wines - Advances in Production, Processing, Analysis and Valorization

Among the scientific community, our novel, nationwide, sample-free, noncalibrated, expert memory-based method was accepted [29, 71], as it was challenged on the basis that the data relied on respondents' perceptions rather than direct experience. In this new research, we compare the results mentioned before in this study—signaled as Experiment 1 (LTM) with the completion of the same questionnaire (Supporting Information) by 19 local experts from the single PGI Beira Atlântico—Experiment 2 (WS)—as a result of a blind tasting of 15 young red wine samples, representing the leading supermarket brands of this wine

Results of the wine tasting assessment by local PGI Beira Atlântico wine experts were organized in order to fully compare with extracted results of experiment 1 regarding the same PGI (Figure 6). The panel is widely calibrated, as these professionals belong to the certification panel which must approve typicality and quality of all submitted PGI Beira Atlântico wines. This panel complies with an annual program of inner and inter-laboratorial calibration on aromatic thresholds, triangular tests, and tastant solutions (control standards of sucrose, NaCl,

The assessment of the same sensory questionnaire by the nationwide professional uncalibrated panel and the local professional PGI Atlântico calibrated panel showed a significant Pearson positive correlation of 0.669, this to be considered promising and a green flag for newcoming

reading showed in Figure 6.

region.

3.5.1. Experiment 2

citric acid, quinine, and others).

comparative studies regarding other PGIs.

3.5. Classic sampling vs. prototypical memory

Figure 6. Mean differences between long term memory (LTM) and wine sampling (WS) methods. ABS scores represent absolute differences between both methods.


Table 6. Top 10 underperfoming primary descriptors.

Focusing on the major mean differences, results show that the local experts tend to evaluate with higher appreciation their local wines, regarding negative sensory descriptors such as PC3 Agreenchemical and PC1 Tdryastringent.

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#### 3.5.2. Uneven calibration needs

There is an ongoing line of research focusing on cost-benefit of calibration stages [72]. Our findings emphasize uneven calibration needs. In Table 6, we have highlighted the initial sensory descriptors which could gain accuracy with a calibration stage, such as mineral aroma [56], as opposed to other attributes that, according to our results, need no calibration among wine experts, hence, saving time and money [73].

## 4. Conclusions

In this sensory study on wine typicality, four large-scale areas were identified. PGI MINHO was found to be the most typical of all PGIs, with several extreme rates for the Color, Aroma and Taste. Encompassed by the four Mediterranean PGIs (PGI Peninsula de Setubal, PGI Tejo, PGI Alentejano and PGI Algarve), the SOUTHERN cluster presented several extreme sensory assessments that were essentially opposed to the single-clustered PGI MINHO's profile. Alcohol, acidity, bitterness and astringency were cross-linked; the respective variations were correlated with published literature and expressed as key factors for the regional macroscale differentiation. Bitterness and astringency were found to be sensory different and related on a geographical scale, as bitterness was primarily affected by inland/coastal influence, while astringency confirmed its customary north/south dependence, this finding to be considered new. Moreover, with the proposed methodology, it was possible to achieve a novel nationwide sensory characterization of PGIs, overcoming present day limitations on macroscale sensory research and sample representativeness.

Results by memory of (1 out of 12) extracted PGI Beira Atlântico were compared with the outcome of wine sampling assessment by local experts, using the same sensory questionnaire, and were found significantly correlated. Major differences between results by memory and by sampling were found mostly on unpleasant (negative-prone) sensory descriptors, the local experts showing greater sympathy for local wines. The need for a calibration stage was found uneven regarding the overall group of scrutinized wine descriptors, envisaging potential accuracy on final results only with a selective calibration phase on fewer descriptors.

These findings may lead to condensed information on typicality, which may contribute to a feasible macroscale regulation of small-sized wine regions and allow Portuguese red wines to be readily understood and sorted by a larger group of consumers.

#### Supporting information

Sensory Questionnaire (Table 7).

294 Grapes and Wines - Advances in Production, Processing, Analysis and Valorization Novel Memory-Based Sensory Approach to Assess Large-Scale Typicality: The Case of Mainland Portugal Red Wines http://dx.doi.org/10.5772/intechopen.73039 295


Focusing on the major mean differences, results show that the local experts tend to evaluate with higher appreciation their local wines, regarding negative sensory descriptors such as PC3

There is an ongoing line of research focusing on cost-benefit of calibration stages [72]. Our findings emphasize uneven calibration needs. In Table 6, we have highlighted the initial sensory descriptors which could gain accuracy with a calibration stage, such as mineral aroma [56], as opposed to other attributes that, according to our results, need no calibration among

In this sensory study on wine typicality, four large-scale areas were identified. PGI MINHO was found to be the most typical of all PGIs, with several extreme rates for the Color, Aroma and Taste. Encompassed by the four Mediterranean PGIs (PGI Peninsula de Setubal, PGI Tejo, PGI Alentejano and PGI Algarve), the SOUTHERN cluster presented several extreme sensory assessments that were essentially opposed to the single-clustered PGI MINHO's profile. Alcohol, acidity, bitterness and astringency were cross-linked; the respective variations were correlated with published literature and expressed as key factors for the regional macroscale differentiation. Bitterness and astringency were found to be sensory different and related on a geographical scale, as bitterness was primarily affected by inland/coastal influence, while astringency confirmed its customary north/south dependence, this finding to be considered new. Moreover, with the proposed methodology, it was possible to achieve a novel nationwide sensory characterization of PGIs, overcoming present day limitations on macroscale sensory research and sample representativeness. Results by memory of (1 out of 12) extracted PGI Beira Atlântico were compared with the outcome of wine sampling assessment by local experts, using the same sensory questionnaire, and were found significantly correlated. Major differences between results by memory and by sampling were found mostly on unpleasant (negative-prone) sensory descriptors, the local experts showing greater sympathy for local wines. The need for a calibration stage was found uneven regarding the overall group of scrutinized wine descriptors, envisaging potential

accuracy on final results only with a selective calibration phase on fewer descriptors.

be readily understood and sorted by a larger group of consumers.

These findings may lead to condensed information on typicality, which may contribute to a feasible macroscale regulation of small-sized wine regions and allow Portuguese red wines to

Agreenchemical and PC1 Tdryastringent.

wine experts, hence, saving time and money [73].

3.5.2. Uneven calibration needs

4. Conclusions

Supporting information

Sensory Questionnaire (Table 7).


Table 7. Sensory Questionnaire.

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Table 7. Sensory Questionnaire.

## Author details

Anibal Jose-Coutinho<sup>1</sup> \*, Patricia Avila<sup>2</sup> and Jorge M. Ricardo-da-Silva1

\*Address all correspondence to: ajosecoutinho@gmail.com

1 Laboratory Ferreira Lapa, Instituto Superior de Agronomia, Linking Landscape Environment Agriculture and Food (LEAF), University of Lisbon, Lisbon, Portugal

2 Department of Social Methods Research, University Institute of Lisbon (ISCTE-IUL), Lisbon, Portugal

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Author details

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[72] Lesschaeve I. Sensory evaluation of wine and commercial realities: Review of current practices and perspectives. American Journal of Enology and Viticulture. 2007;58(2):252-258

[73] Findlay CJ, Castura JC, Schlich P, Lesschaeve I. Use of feedback calibration to reduce the training time for wine panels. Food Quality and Preference. 2006;17(3):266-276. DOI:

cultural and Food Chemistry. 2013;61:8861-8870. DOI: 10.1021/jf401041q

10.1016/j.agrformet.2003.06.001

10.1016/j.talanta.2008.01.012

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DOI: 10.1016/B978-0-12-381468-5.00001-4

901-908. DOI: 10.1016/j.foodqual.2007.02.004

69-83. DOI: doi.org/10.1051/ctv/20153002069

10.1016/j.foodqual.2005.07.005


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[49] Jones PR, Gawel R, Francis IL, Waters EJ. The influence of interactions between major white wine components on the aroma, flavour and texture of model white wine. Food

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[54] Mateus N, Machado JM, de Freitas V. Development changes of anthocyanins in Vitis vinifera grapes grown in the Douro Valley and concentration in respective wines. Journal of the Science of Food and Agriculture. 2002;82(14):1689-1695. DOI: 10.1002/jsfa.1237

[55] Guedes-de-Pinho P, Falqué E, Castro M, E Silva HO, Machado B, Ferreira ACS. Further insights into the floral character of Touriga Nacional wines. Journal of Food Science. 2007

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[57] Kontoudakis N, Esteruelas M, Fort F, Canals JM, De Freitas V, Zamora F. Influence of the heterogeneity of grape phenolic maturity on wine composition and quality. Food Chem-

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302 Grapes and Wines - Advances in Production, Processing, Analysis and Valorization

jf061835h

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j.1745-459X.2010.00322.x


**Section 3**

**Economic Analysis and Valorization of Wine**

**Products**

**Economic Analysis and Valorization of Wine Products**

**Chapter 14**

Provisional chapter

**Wine Firm's Size and Economic Performance: Evidence**

DOI: 10.5772/intechopen.71320

Wine Firm's Size and Economic Performance: Evidence

The wine market is an excellent example of monopolistic competition, demonstrating both vertical and horizontal product differentiation. The propensity toward monopolistic competition and the asymmetric preferences of wine consumption can justify the coexistence of different sized wineries. The main goal of this chapter is to analyze the economic performance of wineries, using indicators widely employed in economic and business literature, and to assess the potential influence of size on firm's performance. To achieve this goal, different statistical tests are applied to firms´ data collected from two representative Portuguese demarcated wine regions (Douro and Vinho Verde). The results confirm that the wine firm's performance is a multidimensional construct, exhibiting similarities and differences, according to the index used. The size of firms increases their export performance but exhibits a nonstatistical effect on the financial performance. The indices of productivity and productive efficiency show that there is no standard behavior. The results of this study can be useful both for managers and public decision-makers, given the importance of controlling economic performance in order to guarantee the industry's competitiveness in an increasingly globalized wine business.

Keywords: competitiveness, demarcated wine regions, economic performance, size,

Over recent decades, the wine industry has been under an intense process of globalization, with an impressive increase in competition as a consequence of the new suppliers entering the market: new world wine producing countries (such as Australia, New Zealand, South Africa,

> © The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and eproduction in any medium, provided the original work is properly cited.

© 2018 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

**from Traditional Portuguese Wine Regions**

from Traditional Portuguese Wine Regions

João Rebelo, Sofia Gouveia, Lina Lourenço-Gomes and Ana Alexandra Marta-Costa

João Rebelo, Sofia Gouveia, Lina Lourenço-Gomes and Ana Alexandra Marta-Costa

Abstract

1. Introduction

Additional information is available at the end of the chapter

Additional information is available at the end of the chapter

wine industry, monopolistic competition

http://dx.doi.org/10.5772/intechopen.71320

Provisional chapter

## **Wine Firm's Size and Economic Performance: Evidence from Traditional Portuguese Wine Regions** Wine Firm's Size and Economic Performance: Evidence

DOI: 10.5772/intechopen.71320

from Traditional Portuguese Wine Regions

João Rebelo, Sofia Gouveia, Lina Lourenço-Gomes and Ana Alexandra Marta-Costa João Rebelo, Sofia Gouveia, Lina Lourenço-Gomes and

Additional information is available at the end of the chapter Ana Alexandra Marta-Costa Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/intechopen.71320

#### Abstract

The wine market is an excellent example of monopolistic competition, demonstrating both vertical and horizontal product differentiation. The propensity toward monopolistic competition and the asymmetric preferences of wine consumption can justify the coexistence of different sized wineries. The main goal of this chapter is to analyze the economic performance of wineries, using indicators widely employed in economic and business literature, and to assess the potential influence of size on firm's performance. To achieve this goal, different statistical tests are applied to firms´ data collected from two representative Portuguese demarcated wine regions (Douro and Vinho Verde). The results confirm that the wine firm's performance is a multidimensional construct, exhibiting similarities and differences, according to the index used. The size of firms increases their export performance but exhibits a nonstatistical effect on the financial performance. The indices of productivity and productive efficiency show that there is no standard behavior. The results of this study can be useful both for managers and public decision-makers, given the importance of controlling economic performance in order to guarantee the industry's competitiveness in an increasingly globalized wine business.

Keywords: competitiveness, demarcated wine regions, economic performance, size, wine industry, monopolistic competition

## 1. Introduction

Over recent decades, the wine industry has been under an intense process of globalization, with an impressive increase in competition as a consequence of the new suppliers entering the market: new world wine producing countries (such as Australia, New Zealand, South Africa,

© 2018 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

© The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and eproduction in any medium, provided the original work is properly cited.

Argentina, Chile, and more recently China), and at the same time, the decreasing consumption of the old world traditional wine-producing countries (namely, Italy, France, Spain, and Portugal). The opening up and globalization of the wine markets raise both challenges and opportunities to the wine regions and consequently to the respective wine firms, which must adopt strategies that address the consumer's demand in an economic and sustainable way.

In 2015, there were 39,727 farms of grape vine cultivation that represented a planted area of 201,445 ha [2], the fourth largest in European Union (EU). The total production of 2015 was 6,205,756 hl [2] (fifth in the EU, after France, Italy, Spain, and Germany and 11th in the world wine production). From the total wine production (77.76% protected Designation of Origin), 70.5% is red wine, 29.5% white wine, 13.2% fortified wine, namely Port wine. The domestic consumption reached 4.94 million hectoliters (79.6% of production), and 2.8 million hectoliters were exported (45.11% of production), meaning that Portugal is both an importer and exporter [2]. The average price of exporting was 2.63 Euro/Liter, against the 0.55 Euro/Liter for

Wine Firm's Size and Economic Performance: Evidence from Traditional Portuguese Wine Regions

http://dx.doi.org/10.5772/intechopen.71320

309

Due to the soil, climatic, social, and economic factors, Portugal is a heterogeneous wine country, with very specific conditions as testified by one of the currently most recognized Portuguese winemakers (Dirk Nieeport in [3]): Portugal has one thing that only few countries nowadays have traditions, history, old vines, old and diversified varieties, and personalized methods of vinification. Due to its diversity, the country has long since been divided into various wine regions, there currently being fourteen demarcated wine regions, each one with the own terroir characteristics. The Douro and Vinho Verde demarcated regions are representatives of the Portuguese wine terroir model, located in the Northeast and Northwest of Portugal, respectively. In 2015, the Douro region had an area of 43,659 ha (21.7% of the Portugal area) and produced 1,407,006 hl (22.7% of the total Portuguese production), shared by Port wine (almost 50%) and still wines. The Vinho Verde region extends along an area of 15,821 ha (7.9% of the Portuguese area) with a production

In a microeconomic level, the Portuguese wine supply is characterized by the presence of a diversified typology of economic agents [2]: warehouse, distiller, bottler, exporter/importer, producer of vinegar and wine, merchant without establishment, preparer, producer, winemaker, and winemaker bottler. Considering the classification of economic activities in the EU (NACE) in the code 11021, production of still and liquors wines, in 2015, there were 1246 active

Similarly to other traditional wine countries [5], it is known that Portuguese wine firms, including the ones located in the Douro and Vinho Verde regions, embrace different marketing strategies that range from the low cost or mass market types to those based on product differentiation or a market niche. In general, the first strategy is adopted by the largest firms, which prefer to offer standardized wines and to cater to large distribution channels. On the other hand, small firms prefer to satisfy the needs of narrow markets, emphasizing the importance of differentiation based on the terroir, appellation, and geographic identity in order to enhance perceived wine quality, which endorses the monopolistic competition model. Given this multifaceted market structure, the importance of monitoring and controlling economic performance becomes clear, as such entrepreneurial strategies may guarantee survival in the long term. The assessment of a wine firm's performance is a critical managerial issue [6], despite its measure is a multidimensional construct, whose indicators to be used depend on

Researchers in economics and management strategy showed a long interest in understanding the main determinants of the firm performance in different market structures. Two theoretical

importing, meaning that Portugal exports wine with a higher added value [2].

of 693,026 hl (6.38% of the Portuguese production), only still wines [2].

the stakeholder perspective and on the outcomes to be achieved.

firms, according to the database racius [4].

The wine market reveals itself to be an excellent example of monopolistic competition, wherein the market is determined by significant variety, differentiated by growing regions, and consumer preference/demand for taste, price, and advertising. There is not only one market for wine, but many different ones depending on price, tastes, and brands, with consumers selecting within red or white wines, or even within grape varieties. This is why "wine" is a bundle of monopolistic and competitive markets and not a single competitive or monopoly undifferentiated market.

Following Church and Ware [1], the assumption of symmetry and consumers´ taste for variety supports the essential feature of monopolistic competition, wherein every brand is in competition with each other. Consequently, the focus of the models of monopolistic competition is not typically on strategic decisions regarding to product specification or design, since the products are assumed to be equally differentiated. Instead, the model focuses on the issue of the extent of variety: the number of products available in the market. Two conditions must be satisfied to achieve equilibrium: profit maximization and free-entry condition. In the process of profit maximization, it is expected that the group of larger firms has a behavior referred to as true or perfect monopolistic competition and the group of small firms as oligopolistic, in which the equilibrium is attained whether the firms compete over price or quantity. The free-entry condition implies that the profit must be an incentive to enter or exit. The profit of an additional entrant must be nonpositive and the profit of a firm in the industry non-negative. Ignoring the integer constraint, these two considerations imply that the profit of firms in the industry must be zero, at equilibrium. Moreover, in this market structure, the number of firms depends on product elasticity substitution and on the extent of scale economies. As elasticity substitution increases, products become less differentiated, and as result, the demand becomes more elastic, reducing the market power of the firm and the equilibrium prices tend toward a state of perfect competition. This squeezes price-cost margins, reducing profitability. The opposite occurs if the elasticity substitution decreases. Otherwise, the greater the extent of economies of scale, the smaller the number of firms that will be in the industry and hence varieties, leading to an increase in prices and in the market share at which firm's breakeven. Conversely, reduction in the extent of scale economies reduces the minimum price markup and the market share for a firm to break even, resulting in an increase in the number of firms where equilibrium occurs and in the variety of products.

Theoretically, the model of monopolistic competition justifies the coexistence of wineries of different sizes and production technologies, charging different prices for distinct wines, according to the consumer's willingness to pay. In this scenario, the profitability could be independent of the firm's size.

Portugal seems to be a good example of a traditional wine producing country, with a strong presence in the international market and a market structure of monopolistic competition. In 2015, there were 39,727 farms of grape vine cultivation that represented a planted area of 201,445 ha [2], the fourth largest in European Union (EU). The total production of 2015 was 6,205,756 hl [2] (fifth in the EU, after France, Italy, Spain, and Germany and 11th in the world wine production). From the total wine production (77.76% protected Designation of Origin), 70.5% is red wine, 29.5% white wine, 13.2% fortified wine, namely Port wine. The domestic consumption reached 4.94 million hectoliters (79.6% of production), and 2.8 million hectoliters were exported (45.11% of production), meaning that Portugal is both an importer and exporter [2]. The average price of exporting was 2.63 Euro/Liter, against the 0.55 Euro/Liter for importing, meaning that Portugal exports wine with a higher added value [2].

Argentina, Chile, and more recently China), and at the same time, the decreasing consumption of the old world traditional wine-producing countries (namely, Italy, France, Spain, and Portugal). The opening up and globalization of the wine markets raise both challenges and opportunities to the wine regions and consequently to the respective wine firms, which must adopt

The wine market reveals itself to be an excellent example of monopolistic competition, wherein the market is determined by significant variety, differentiated by growing regions, and consumer preference/demand for taste, price, and advertising. There is not only one market for wine, but many different ones depending on price, tastes, and brands, with consumers selecting within red or white wines, or even within grape varieties. This is why "wine" is a bundle of monopolistic and competitive markets and not a single competitive or monopoly

Following Church and Ware [1], the assumption of symmetry and consumers´ taste for variety supports the essential feature of monopolistic competition, wherein every brand is in competition with each other. Consequently, the focus of the models of monopolistic competition is not typically on strategic decisions regarding to product specification or design, since the products are assumed to be equally differentiated. Instead, the model focuses on the issue of the extent of variety: the number of products available in the market. Two conditions must be satisfied to achieve equilibrium: profit maximization and free-entry condition. In the process of profit maximization, it is expected that the group of larger firms has a behavior referred to as true or perfect monopolistic competition and the group of small firms as oligopolistic, in which the equilibrium is attained whether the firms compete over price or quantity. The free-entry condition implies that the profit must be an incentive to enter or exit. The profit of an additional entrant must be nonpositive and the profit of a firm in the industry non-negative. Ignoring the integer constraint, these two considerations imply that the profit of firms in the industry must be zero, at equilibrium. Moreover, in this market structure, the number of firms depends on product elasticity substitution and on the extent of scale economies. As elasticity substitution increases, products become less differentiated, and as result, the demand becomes more elastic, reducing the market power of the firm and the equilibrium prices tend toward a state of perfect competition. This squeezes price-cost margins, reducing profitability. The opposite occurs if the elasticity substitution decreases. Otherwise, the greater the extent of economies of scale, the smaller the number of firms that will be in the industry and hence varieties, leading to an increase in prices and in the market share at which firm's breakeven. Conversely, reduction in the extent of scale economies reduces the minimum price markup and the market share for a firm to break even, resulting in an increase in the number of firms where

Theoretically, the model of monopolistic competition justifies the coexistence of wineries of different sizes and production technologies, charging different prices for distinct wines, according to the consumer's willingness to pay. In this scenario, the profitability could be

Portugal seems to be a good example of a traditional wine producing country, with a strong presence in the international market and a market structure of monopolistic competition.

strategies that address the consumer's demand in an economic and sustainable way.

308 Grapes and Wines - Advances in Production, Processing, Analysis and Valorization

undifferentiated market.

equilibrium occurs and in the variety of products.

independent of the firm's size.

Due to the soil, climatic, social, and economic factors, Portugal is a heterogeneous wine country, with very specific conditions as testified by one of the currently most recognized Portuguese winemakers (Dirk Nieeport in [3]): Portugal has one thing that only few countries nowadays have traditions, history, old vines, old and diversified varieties, and personalized methods of vinification. Due to its diversity, the country has long since been divided into various wine regions, there currently being fourteen demarcated wine regions, each one with the own terroir characteristics. The Douro and Vinho Verde demarcated regions are representatives of the Portuguese wine terroir model, located in the Northeast and Northwest of Portugal, respectively. In 2015, the Douro region had an area of 43,659 ha (21.7% of the Portugal area) and produced 1,407,006 hl (22.7% of the total Portuguese production), shared by Port wine (almost 50%) and still wines. The Vinho Verde region extends along an area of 15,821 ha (7.9% of the Portuguese area) with a production of 693,026 hl (6.38% of the Portuguese production), only still wines [2].

In a microeconomic level, the Portuguese wine supply is characterized by the presence of a diversified typology of economic agents [2]: warehouse, distiller, bottler, exporter/importer, producer of vinegar and wine, merchant without establishment, preparer, producer, winemaker, and winemaker bottler. Considering the classification of economic activities in the EU (NACE) in the code 11021, production of still and liquors wines, in 2015, there were 1246 active firms, according to the database racius [4].

Similarly to other traditional wine countries [5], it is known that Portuguese wine firms, including the ones located in the Douro and Vinho Verde regions, embrace different marketing strategies that range from the low cost or mass market types to those based on product differentiation or a market niche. In general, the first strategy is adopted by the largest firms, which prefer to offer standardized wines and to cater to large distribution channels. On the other hand, small firms prefer to satisfy the needs of narrow markets, emphasizing the importance of differentiation based on the terroir, appellation, and geographic identity in order to enhance perceived wine quality, which endorses the monopolistic competition model. Given this multifaceted market structure, the importance of monitoring and controlling economic performance becomes clear, as such entrepreneurial strategies may guarantee survival in the long term. The assessment of a wine firm's performance is a critical managerial issue [6], despite its measure is a multidimensional construct, whose indicators to be used depend on the stakeholder perspective and on the outcomes to be achieved.

Researchers in economics and management strategy showed a long interest in understanding the main determinants of the firm performance in different market structures. Two theoretical

alternatives approaches have been used [7] to analyze the importance of the industry and the business unit factors in the firm performance: the paradigm structure-conduct-performance (SCP) of the industrial organization [8, 9], suggesting that the industry structure is the main determinant of performance differences across firms, and the business strategy [10, 11], assuming that the resources and capabilities are the main determinants of differences of profitability across firms.

Profitability indices measure the extent to which a business generates a profit from the resources applied to the business. Traditionally, to compute profitability, indices such as ROA (ratio between total earnings before interests and taxes and total assets), ROE (ratio between net income and total equity), and ROI (ratio between total earnings before interests and taxes and the sum of plants, equipment, and stocks) are used. In a market structure, such as monopolistic competition, where firms of different sizes coexist and survive, it is expected that there should be

Wine Firm's Size and Economic Performance: Evidence from Traditional Portuguese Wine Regions

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311

Complementary to the profitability indices, the solvency ratios gauge the firms´ ability to pay all financial obligations if all assets are sold or to continue operations after financial adversity. Two ratios can be used: the leverage or debt to equity ratio (Lev) and financial autonomy or equity to asset ratio (FA). Since microfirms have difficulties in accessing market capital and/or

To estimate productivity, two measures specifically relating to labor are used: turnover per employee (T/E) and added value per Employee (AV/E). In the profit maximization strategy, it is expected that managers try to optimize the use of labor, independently of size. Conse-

The indices of economic (productive) efficiency allow us to get information about the relative performance of a firm in comparison with the optimum (frontier) that it is possible to achieve. Based on the firms included in a technologically homogeneous sample, the frontier and, consequently, productive efficiency can be computed through parametric or nonparametric approaches. In our case, productive efficiency is computed using the data envelopment analysis (DEA), a nonparametric approach initially proposed by several authors [15, 16]. This approach has the advantage of not needing to assume a functional form to the production (cost or profit) function, since the frontier surface is constructed through linear programming. The main disadvantage is that any deviation between observed and estimated value is considered as (in)efficiency, or even as a consequence of random factors. A firm is efficient if no other firm is able to produce a higher level of output from the same input (output oriented), or a firm is efficient if it produces the same output from less input (input oriented). Following the methodology described by Sellers and Alampi-Sottini [6], we apply an input-oriented model, since the inputs are under the control of firms more than the output. Three controllable inputs

the output data on annual turnover have been considered. Assuming j = 1, …, n firms, each one using vector of m inputs, Xj = (x1j, x2j, …, xmj), to produce vector of outputs, Yj = (y1j, y2j, …, ysj) and linear variable returns to scale (VRS). The programming model is given by Eq. (1):

> Xs r¼1 s þ <sup>r</sup> þ ε

This variable is included because access to loans and the correspondent cost is a crucial issue in the firm performance.

Xm i¼1 s � i

) have been chosen. Moreover,

no strong statistical relationships between profitability and size, namely on the ROE.

to bank loans, it is expected that these ratios change according to size.

quently, the indices of productivity may or may not be related to size.

(cost of raw materials, number of employees, and value of debts<sup>1</sup>

subject to:

1

Max z<sup>0</sup> ¼ θ þ ε

Some studies on the agrifood sector conclude that there is a direct relation between firm size and financial performance in the food industry [e.g., 7, 12]. The findings suggest a positive relationship between firm size with return on assets (ROA) [7, 12], return on equity (ROE), and return on investment (ROI) [12] and based in arguments like greater resources and market opportunities of large firms, which enables to be more efficient in the use of inputs, to benefit from economies of scale and to a stronger market power. However, other studies [13] reported a negative relation, showing that smaller firms can obtain a better financial performance. Thus, the empirical evidence is inconclusive on the effect of firm size on the financial performance of agrifood firms, suggesting the need of additional empirical research, that takes into account both the market structure and different indexes of firm performance.

Based on information collected from the Douro and Vinho Verde wine regions, the main goal of this chapter is to analyze the economic performance of wineries, using indicators widely employed in economic and management literature and to assess the potential influence of size on economic performance. To achieve this goal, different nonparametric and parametric tests are applied to indices related to export performance, profitability, productivity, productive efficiency, and solvency.

## 2. Methodology and sampling

#### 2.1. Methodology

In line with the aims of the chapter in the first step, based on information included in firm's annual financial reports, indices are computed related to export performance, profitability, solvency ratios, labor productivity, and economic efficiency.

The index of export performance (EP), measured by the ratio between firm exports and turnover, reflects the firm's ability to be competitive in international markets. Size is an important factor in shaping the firm's potential for internationalization. In free markets, the size of a firm can reveal its potential market power, capability, and scale economies: all essential elements in gaining an international market. Vivas and Sousa [14] identify internationalization strategies of firms in the wine industry in Portugal, as well as the option to export to a wide range of countries, pointing out a lack of size of the production units, coupled with poor investment in the commercial area. This framework constraints the ability to compete with larger producers, more experienced in international markets and holding a more consistent commercial attitude. Therefore, it is expected that EP will be positively correlated with size.

Profitability indices measure the extent to which a business generates a profit from the resources applied to the business. Traditionally, to compute profitability, indices such as ROA (ratio between total earnings before interests and taxes and total assets), ROE (ratio between net income and total equity), and ROI (ratio between total earnings before interests and taxes and the sum of plants, equipment, and stocks) are used. In a market structure, such as monopolistic competition, where firms of different sizes coexist and survive, it is expected that there should be no strong statistical relationships between profitability and size, namely on the ROE.

Complementary to the profitability indices, the solvency ratios gauge the firms´ ability to pay all financial obligations if all assets are sold or to continue operations after financial adversity. Two ratios can be used: the leverage or debt to equity ratio (Lev) and financial autonomy or equity to asset ratio (FA). Since microfirms have difficulties in accessing market capital and/or to bank loans, it is expected that these ratios change according to size.

To estimate productivity, two measures specifically relating to labor are used: turnover per employee (T/E) and added value per Employee (AV/E). In the profit maximization strategy, it is expected that managers try to optimize the use of labor, independently of size. Consequently, the indices of productivity may or may not be related to size.

The indices of economic (productive) efficiency allow us to get information about the relative performance of a firm in comparison with the optimum (frontier) that it is possible to achieve. Based on the firms included in a technologically homogeneous sample, the frontier and, consequently, productive efficiency can be computed through parametric or nonparametric approaches. In our case, productive efficiency is computed using the data envelopment analysis (DEA), a nonparametric approach initially proposed by several authors [15, 16]. This approach has the advantage of not needing to assume a functional form to the production (cost or profit) function, since the frontier surface is constructed through linear programming. The main disadvantage is that any deviation between observed and estimated value is considered as (in)efficiency, or even as a consequence of random factors. A firm is efficient if no other firm is able to produce a higher level of output from the same input (output oriented), or a firm is efficient if it produces the same output from less input (input oriented). Following the methodology described by Sellers and Alampi-Sottini [6], we apply an input-oriented model, since the inputs are under the control of firms more than the output. Three controllable inputs (cost of raw materials, number of employees, and value of debts<sup>1</sup> ) have been chosen. Moreover, the output data on annual turnover have been considered. Assuming j = 1, …, n firms, each one using vector of m inputs, Xj = (x1j, x2j, …, xmj), to produce vector of outputs, Yj = (y1j, y2j, …, ysj) and linear variable returns to scale (VRS). The programming model is given by Eq. (1):

$$\mathbf{Max}\,z\_0 = \theta + \varepsilon \sum\_{r=1}^{s} s\_r^+ + \varepsilon \sum\_{i=1}^{m} s\_i^- $$

subject to:

alternatives approaches have been used [7] to analyze the importance of the industry and the business unit factors in the firm performance: the paradigm structure-conduct-performance (SCP) of the industrial organization [8, 9], suggesting that the industry structure is the main determinant of performance differences across firms, and the business strategy [10, 11], assuming that the resources and capabilities are the main determinants of differences of profitability across firms. Some studies on the agrifood sector conclude that there is a direct relation between firm size and financial performance in the food industry [e.g., 7, 12]. The findings suggest a positive relationship between firm size with return on assets (ROA) [7, 12], return on equity (ROE), and return on investment (ROI) [12] and based in arguments like greater resources and market opportunities of large firms, which enables to be more efficient in the use of inputs, to benefit from economies of scale and to a stronger market power. However, other studies [13] reported a negative relation, showing that smaller firms can obtain a better financial performance. Thus, the empirical evidence is inconclusive on the effect of firm size on the financial performance of agrifood firms, suggesting the need of additional empirical research, that takes into account

Based on information collected from the Douro and Vinho Verde wine regions, the main goal of this chapter is to analyze the economic performance of wineries, using indicators widely employed in economic and management literature and to assess the potential influence of size on economic performance. To achieve this goal, different nonparametric and parametric tests are applied to indices related to export performance, profitability, productivity, productive

In line with the aims of the chapter in the first step, based on information included in firm's annual financial reports, indices are computed related to export performance, profitability,

The index of export performance (EP), measured by the ratio between firm exports and turnover, reflects the firm's ability to be competitive in international markets. Size is an important factor in shaping the firm's potential for internationalization. In free markets, the size of a firm can reveal its potential market power, capability, and scale economies: all essential elements in gaining an international market. Vivas and Sousa [14] identify internationalization strategies of firms in the wine industry in Portugal, as well as the option to export to a wide range of countries, pointing out a lack of size of the production units, coupled with poor investment in the commercial area. This framework constraints the ability to compete with larger producers, more experienced in international markets and holding a more consistent commercial attitude. Therefore, it is expected that EP will be positively correlated with

both the market structure and different indexes of firm performance.

310 Grapes and Wines - Advances in Production, Processing, Analysis and Valorization

solvency ratios, labor productivity, and economic efficiency.

efficiency, and solvency.

2.1. Methodology

size.

2. Methodology and sampling

<sup>1</sup> This variable is included because access to loans and the correspondent cost is a crucial issue in the firm performance.

$$\sum\_{j=1}^{n} \mathbf{x}\_{ij}\boldsymbol{\lambda}\_{j} + \mathbf{s}\_{i}^{-} = \mathbf{x}\_{r0}$$

$$\sum\_{j=1}^{n} \mathbf{y}\_{rj}\boldsymbol{\lambda}\_{j} - \mathbf{s}\_{r}^{+} = \theta \mathbf{y}\_{i0}$$

$$\sum\_{j=1}^{n} \boldsymbol{\lambda}\_{j} = 1$$

$$\boldsymbol{\lambda}\_{j}, \mathbf{s}\_{r}^{+}, \mathbf{s}\_{i}^{-} \ge 0$$

$$\mathbf{j} = \mathbf{1}, \dots, \mathbf{n}, \mathbf{r} = \mathbf{1}, \dots \text{ s}; \mathbf{i} = \mathbf{1}, \dots, \mathbf{m}. \tag{1}$$

This means that this study is carried out with a pooled sample data of firms and years, and the databases are obtained from the Financial and Economic Entrepreneurial Data Base provided by Informa Dun & Bradstreet (D&B) and reporting wine firms included in the 11,021 NACE-2009 code (production of still and liquors wines). Considering that not all the financial reports include the information required for this research, we eventually got a sample database that includes: for the Douro region, 434 pooled observations, 218 for 2014 and 216 for 20152

Wine Firm's Size and Economic Performance: Evidence from Traditional Portuguese Wine Regions

204 wine firms observed for both years and 26 for only 1 year, and for the Vinho Verde region, the sample includes a pool of 299 observations, 154 of 2014 and 145 of 2015, in a total of 162

Table 1 shows the main descriptive statistics of the variables used, for both wine regions. The range between the minimum and the maximum, together with the comparison between the standard deviation (SD) and mean, allows us to conclude that each sample reflects a heterogeneous market structure, typical of monopolistic competition. According to the value of the mean of the variables, the firms of the Douro region are bigger than those of the Vinho Verde

In order to build a detailed basis of information about the market structure of each wine region, we have classified firms according their size, adopting the European Commission

firm, the number of employees, the turnover, or total assets on the balance sheet (Table 2). Consequently, these criteria should be used to analyze any industry, including in the wine business. These criteria also suggest the use of financial and productive indicators to assess the

In this section, performance indices (export performance, profitability, solvency, productivity, and efficiency) are presented, both for the Douro and Vinho Verde regions (Table 3). As can been seen, the statistical descriptive measures show a highly heterogeneous industry in and for

For the Douro region, the average of the index of EP is 18%. The profitability ratios are ROA = 0.9%, ROE = 20.5%, and ROI = 68.1%. Regarding solvency, the mean of leverage (Lev) is 2.25 and the equity/asset (AF) ratio is 28%. The productivity indices show that on average, the amount sold per employee (T/E) is 121.5 thousand Euro and the added value per employee (AV/E) is 31.5 thousand Euro. Comparing the standard deviation (SD) with the mean, we conclude that there is a high relative dispersion between observations, which is confirmed through the values of the weighted means of the indices: EP = 58%, ROA = 3.9%, ROE = 4.4%,

Considering that the total national number of firms active in 2015 is 1246, the sample of Douro represents 17.33% and

The classification of firms by the EU, according their size, aims to improve the consistency and effectiveness of policies targeting small and medium enterprises and would, to this end, homogenize the definition of the size of the firm between

member states, to limit the risk of distortion of competition resulting from different levels of public support.

. This recommendation is used to obtain criteria to define the size of the

wine firms, 137 observed for both years and 25 for only 1 year.

region (Table 1).

3. Results

2

3

both wine regions.

Vinho Verde 11.64%.

recommendation [18]<sup>3</sup>

performance of the industry.

, with

313

http://dx.doi.org/10.5772/intechopen.71320

where θ is the measure of efficiency for each unit. A firm is efficient if θ<sup>∗</sup> =1 and all the slacks are equal to zero, and ε > 0 is a non-Archimedean element that is defined to be smaller than any positive real number. Since the indices of productive efficiency summarize the relative management performance in the inputs allocation, they may or not be related to size, although a positive relation stands out.

In the second step, the data are analyzed using descriptive statistics and both nonparametric and parametric tests, in order to test if there is an association between indices and size. More specifically, since we have different groups of observations for each variable, the Kruskal-Wallis equality-of-population rank test is used. To test if there are differences between means of groups in the same variable, the Hotelling's T2 is used. Furthermore, to check the robustness of the nonparametric tests, ordinary least squares (OLS) regression models are estimated, where the dependent variables are the indices and the explanatory variables are dummies according to size.

#### 2.2. Sampling

In accordance with the aim of the study, as well as to assure the technological homogeneity of the sample, two demarcated wine regions are considered: the Douro region and the Vinho Verde region, both in the North of Portugal. The Douro is one of the oldest world wine regions, fitting well the terroir model [17]. It is characterized by the production of two different products: the fortified Port wine and still wines. The Vinho Verde region only produces still wines. In both regions, there are a very large number of wine brands that are sold in different markets at different prices.

Given the economic and technological differences between the two regions, it is recommended that each one of them is analyzed as a separate sample. Therefore, in order to define the respective sample of each region, we start by selecting the firms that are registered as a winery in the regulator of Port and/or still Douro wines (Port and Douro Wines Institute: www.ivdp. pt) and in the regulator of Vinho Verde (Commission of Viticulture from Vinhos Verdes region: www.vinhoverde.pt). Moreover, to overcome any possible effects of seasonality, we decided to collect economic and financial data that are available for the last 2 years, 2014 and 2015. This means that this study is carried out with a pooled sample data of firms and years, and the databases are obtained from the Financial and Economic Entrepreneurial Data Base provided by Informa Dun & Bradstreet (D&B) and reporting wine firms included in the 11,021 NACE-2009 code (production of still and liquors wines). Considering that not all the financial reports include the information required for this research, we eventually got a sample database that includes: for the Douro region, 434 pooled observations, 218 for 2014 and 216 for 20152 , with 204 wine firms observed for both years and 26 for only 1 year, and for the Vinho Verde region, the sample includes a pool of 299 observations, 154 of 2014 and 145 of 2015, in a total of 162 wine firms, 137 observed for both years and 25 for only 1 year.

Table 1 shows the main descriptive statistics of the variables used, for both wine regions. The range between the minimum and the maximum, together with the comparison between the standard deviation (SD) and mean, allows us to conclude that each sample reflects a heterogeneous market structure, typical of monopolistic competition. According to the value of the mean of the variables, the firms of the Douro region are bigger than those of the Vinho Verde region (Table 1).

In order to build a detailed basis of information about the market structure of each wine region, we have classified firms according their size, adopting the European Commission recommendation [18]<sup>3</sup> . This recommendation is used to obtain criteria to define the size of the firm, the number of employees, the turnover, or total assets on the balance sheet (Table 2). Consequently, these criteria should be used to analyze any industry, including in the wine business. These criteria also suggest the use of financial and productive indicators to assess the performance of the industry.

## 3. Results

Xn j¼1

312 Grapes and Wines - Advances in Production, Processing, Analysis and Valorization

Xn j¼1

where θ is the measure of efficiency for each unit. A firm is efficient if θ<sup>∗</sup>

a positive relation stands out.

according to size.

at different prices.

2.2. Sampling

xijλ<sup>j</sup> þ s � <sup>i</sup> ¼ xr<sup>0</sup>

yrjλ<sup>j</sup> � s<sup>þ</sup>

λ<sup>j</sup> ¼ 1

Xn j¼1

λj, s<sup>þ</sup> <sup>r</sup> , s� <sup>i</sup> ≥ 0

are equal to zero, and ε > 0 is a non-Archimedean element that is defined to be smaller than any positive real number. Since the indices of productive efficiency summarize the relative management performance in the inputs allocation, they may or not be related to size, although

In the second step, the data are analyzed using descriptive statistics and both nonparametric and parametric tests, in order to test if there is an association between indices and size. More specifically, since we have different groups of observations for each variable, the Kruskal-Wallis equality-of-population rank test is used. To test if there are differences between means of groups in the same variable, the Hotelling's T2 is used. Furthermore, to check the robustness of the nonparametric tests, ordinary least squares (OLS) regression models are estimated, where the dependent variables are the indices and the explanatory variables are dummies

In accordance with the aim of the study, as well as to assure the technological homogeneity of the sample, two demarcated wine regions are considered: the Douro region and the Vinho Verde region, both in the North of Portugal. The Douro is one of the oldest world wine regions, fitting well the terroir model [17]. It is characterized by the production of two different products: the fortified Port wine and still wines. The Vinho Verde region only produces still wines. In both regions, there are a very large number of wine brands that are sold in different markets

Given the economic and technological differences between the two regions, it is recommended that each one of them is analyzed as a separate sample. Therefore, in order to define the respective sample of each region, we start by selecting the firms that are registered as a winery in the regulator of Port and/or still Douro wines (Port and Douro Wines Institute: www.ivdp. pt) and in the regulator of Vinho Verde (Commission of Viticulture from Vinhos Verdes region: www.vinhoverde.pt). Moreover, to overcome any possible effects of seasonality, we decided to collect economic and financial data that are available for the last 2 years, 2014 and 2015.

<sup>r</sup> ¼ θyi0

j ¼ 1, …, n,r ¼ 1,… s; i ¼ 1, …, m: (1)

=1 and all the slacks

In this section, performance indices (export performance, profitability, solvency, productivity, and efficiency) are presented, both for the Douro and Vinho Verde regions (Table 3). As can been seen, the statistical descriptive measures show a highly heterogeneous industry in and for both wine regions.

For the Douro region, the average of the index of EP is 18%. The profitability ratios are ROA = 0.9%, ROE = 20.5%, and ROI = 68.1%. Regarding solvency, the mean of leverage (Lev) is 2.25 and the equity/asset (AF) ratio is 28%. The productivity indices show that on average, the amount sold per employee (T/E) is 121.5 thousand Euro and the added value per employee (AV/E) is 31.5 thousand Euro. Comparing the standard deviation (SD) with the mean, we conclude that there is a high relative dispersion between observations, which is confirmed through the values of the weighted means of the indices: EP = 58%, ROA = 3.9%, ROE = 4.4%,

<sup>2</sup> Considering that the total national number of firms active in 2015 is 1246, the sample of Douro represents 17.33% and Vinho Verde 11.64%.

<sup>3</sup> The classification of firms by the EU, according their size, aims to improve the consistency and effectiveness of policies targeting small and medium enterprises and would, to this end, homogenize the definition of the size of the firm between member states, to limit the risk of distortion of competition resulting from different levels of public support.


Table 1. Descriptive statistics of the variables used for Douro (434 observations) and Vinhos Verdes (299 observations) regions (years 2014 and 2015).


Table 2. Criteria to identify size of the firms using European Commission recommendation [18].

ROI = 4.94%, Lev = 0.85, FA = 0.54, T/E = 181.8 thousand Euro, AV/E = 46.4 thousand Euro, a picture that is different of that one given by the arithmetic mean and calls for a group analysis. The average of economic efficiency (EF) is 0.454 meaning that the Douro wine industry could use 55.6% fewer inputs to obtain the same level of output, if all the firms adopted the observed best productive practice. The averages of technical efficiency (TE) and scale efficiency (SE) are 0.692 and 0.692, respectively, suggesting that the deviation from the production frontier is due to a similar extent to the inefficient use of inputs (technical efficiency) and to firms not operating at an optimum size (scale efficiency).

Regarding the Vinho Verde region, the arithmetic means of the sample are: EP = 11.8%, ROA = �0.5%, ROE = �8.2%, ROI = �0.07%, Lev = 7.52, FA = 0.25, T/E = 101.3 thousand Euro, AV/E = 24.3 thousand Euro. However, as with the Douro region, the simple means compares poorly with the weighted means, which have the following values: EP = 30.7%, ROA = 4.4%, ROE = 5.3%, ROI = 5.9%, Lev = 1.21, FA =0.45, T/E = 134.4 thousand Euro, and AV/E = 35.7 thousand Euro. The average of EF is 0.417, meaning that Vinho Verde firms could use 59.3% fewer inputs if all of them were on the production frontier. The averages of TE and SE are 0.678

Table 3. Indices of performance of Douro (434 observations) and Vinho Verde regions (years 2014 and 2015).

Indices Minimum Maximum Mean SD

EP 0.000 0.930 0.180 0.250

1.060 41.170 165.840

Wine Firm's Size and Economic Performance: Evidence from Traditional Portuguese Wine Regions

204.720 0.970

2799.500 305.200

1.000 1.000 1.000

0.899 8.634 4.511

897.680 0.998

3053.000 410.333

1.000 1.000 1.000

Legend: SD – Standard Deviation; EP - Export Performance; ROA - Return on Assets; ROE - Return On Equity; ROI - Return on Investment; Lev - Leverage; FA - Financial Autonomy; T/E - Turnover per Employee; AV/E - Added Value per

EP 0.000 0.993 0.118 0.203

0.009 0.205 0.681

http://dx.doi.org/10.5772/intechopen.71320

2.250 0.280

121.500 31.500

0.454 0.692 0.693

�0.005 �0.082 �0.073

7.517 0.252

101.337 24.287

0.417 0.678 0.675 0.430 3.180 8.920 315

20.890 0.940

0.250 0.282 0.267

0.163 1.800 0.904

57.255 0.706

263.676 38.608

0.227 0.285 0.283

227,917.000 37,534.000

�8.415 �34.850 �10.020

�258.000 �14.490

80.000 �120.706

0.000 0.083 0.000

�1.116 �21.665 �10.295

�18.731 �5.165

0.398 �83.004

0.008 0.084 0.008

Employee; EF - Economic Efficiency; TE - Technical Efficiency; SE - Scale Efficiency.

Douro region

Profitability ROA ROE ROI

Solvency Lev FA

Productivity T/E (10<sup>3</sup> Euro) AV/E (10<sup>3</sup> Euro)

Efficiency EF TE SE

Profitability ROA ROE ROI

Solvency Lev FA

Productivity T/E (10<sup>3</sup> Euro) AV/E (10<sup>3</sup> Euro)

Efficiency EF TE SE

Vinhos Verdes region

Wine Firm's Size and Economic Performance: Evidence from Traditional Portuguese Wine Regions http://dx.doi.org/10.5772/intechopen.71320 315


Legend: SD – Standard Deviation; EP - Export Performance; ROA - Return on Assets; ROE - Return On Equity; ROI - Return on Investment; Lev - Leverage; FA - Financial Autonomy; T/E - Turnover per Employee; AV/E - Added Value per Employee; EF - Economic Efficiency; TE - Technical Efficiency; SE - Scale Efficiency.

Table 3. Indices of performance of Douro (434 observations) and Vinho Verde regions (years 2014 and 2015).

ROI = 4.94%, Lev = 0.85, FA = 0.54, T/E = 181.8 thousand Euro, AV/E = 46.4 thousand Euro, a picture that is different of that one given by the arithmetic mean and calls for a group analysis. The average of economic efficiency (EF) is 0.454 meaning that the Douro wine industry could use 55.6% fewer inputs to obtain the same level of output, if all the firms adopted the observed best productive practice. The averages of technical efficiency (TE) and scale efficiency (SE) are 0.692 and 0.692, respectively, suggesting that the deviation from the production frontier is due to a similar extent to the inefficient use of inputs (technical efficiency) and to firms not

Table 2. Criteria to identify size of the firms using European Commission recommendation [18].

Variables Minimum Maximum Mean SD

314 Grapes and Wines - Advances in Production, Processing, Analysis and Valorization

Assets (10<sup>3</sup> euro) 3.000 257,010.000 7,886.000 29,006.000 Equity (10<sup>3</sup> euro) �2060.000 184,547.000 4249.000 18,806.000 Investments (103 euro) 0.100 220,150.000 6231.000 23,708.000 Turnover (10<sup>3</sup> euro) 0.100 132,503.000 2881.000 12,199.000 Added value (10<sup>3</sup> euro) �1,012.000 46,618.000 735.000 3299.000 Employees (#) 1 593 16.000 53.000

Assets (10<sup>3</sup> euro) 6.900 50,727.000 2137.000 5167.000 Equity (10<sup>3</sup> euro) �1,277.000 31,492.000 966.000 2898.000 Investments (103 euro) 0.100 41,583.000 1594.000 3973.000 Turnover (10<sup>3</sup> euro) 0.400 31,999.000 996.000 3130.000 Added value (10<sup>3</sup> euro) �660.000 13,401.000 265.000 1112.000 Employees (#) 1 148 7.000 14.000

Table 1. Descriptive statistics of the variables used for Douro (434 observations) and Vinhos Verdes (299 observations)

Micro Employment: < 10 workers; and turnover or assets: < 2 million Euro Small 10 ≤ workers < 50; and 2 ≤ turnover or assets < 50 million Euro Medium 50 ≤ workers < 250; and or 50 ≤ turnover or assets < 250 million Euro Big Workers ≥ 250; and or turnover or assets ≥ 250 million Euro

Douro region

Vinhos verdes region

Legend: SD – Standard Deviation.

regions (years 2014 and 2015).

Regarding the Vinho Verde region, the arithmetic means of the sample are: EP = 11.8%, ROA = �0.5%, ROE = �8.2%, ROI = �0.07%, Lev = 7.52, FA = 0.25, T/E = 101.3 thousand Euro, AV/E = 24.3 thousand Euro. However, as with the Douro region, the simple means compares

operating at an optimum size (scale efficiency).

Size Criteria

poorly with the weighted means, which have the following values: EP = 30.7%, ROA = 4.4%, ROE = 5.3%, ROI = 5.9%, Lev = 1.21, FA =0.45, T/E = 134.4 thousand Euro, and AV/E = 35.7 thousand Euro. The average of EF is 0.417, meaning that Vinho Verde firms could use 59.3% fewer inputs if all of them were on the production frontier. The averages of TE and SE are 0.678


\*, \*\*, \*\*\* statistically significant at 1%, 5% and 10%, respectively.

Legend: EP - Export Performance; ROA - Return on Assets; ROE - Return On Equity; ROI - Return on Investment; Lev - Leverage; FA - Financial Autonomy; T/E - Turnover per Employee; AV/E - Added Value per Employee; EF - Economic Efficiency; TE - Technical Efficiency; SE - Scale Efficiency.

Table 4. Pearson correlation coefficients of indices of performance of Douro region (434 observations, years 2014 and 2015).

and 0.675, respectively, indicating that the causes of productive inefficiency are due to a similar extent to the inefficient use of inputs and to firms not operating at an optimum size.

SE; the FA is positively correlated with EP, ROA, ROI, EF, and SE; the T/E is positively correlated with ROA, AV/E, EF, TE, and SE; the AV/E is positively correlated with EP, ROA, ROI, T/E, and with the indices of efficiency; the EF is positively correlated with EP, ROA, ROI, FA, T/E, AV/E, TE, and SE and negatively with leverage; the TE is positively correlated with ROA, Lev, TE/E, AV/EEF, and negatively with SE; and the SE is positively correlated with EP, ROA, ROI, FA, T/E, AV/E, and EF and negatively with Lev and TE. For this region, from the 55 linear correlation coefficients computed, 45.5% are statistically significant, with 5 of them being negative (ROE-ROI, ROE-Lev, Lev-EF, Lev-SE, and SE-TE). For instance, the negative correlation Lev and EF implies that the more indebted the firm is, the less efficient it is, both in terms of technical efficiency and scale efficiency. Moreover, the negative signal of the correlation coefficient between SE and TE shows that the firms´ optimum size does not lead to the better

Table 5. Pearson correlation coefficients of indices of performance Vinho Verde (299 observations, years 2014 and 2015).

EP ROA ROE ROI Lev FA T/E AV/E EF TE SE

Wine Firm's Size and Economic Performance: Evidence from Traditional Portuguese Wine Regions

http://dx.doi.org/10.5772/intechopen.71320

317

EP 1

ROA 0.165\* 1

ROE 0.018 0.006 1

ROI 0.083 0.602\* �0.108\*\*\* 1

Lev �0.045 0.002 �0.779\* 0.019 1

FA 0.127\*\* 0.437\* 0.003 0.378\* �0.029 1

\*, \*\*, \*\*\* statistically significant at 1%, 5% and 10%, respectively.

Efficiency; TE - Technical Eficiency; SE - Scale Efficiency.

T/E 0.063 0.135\*\* 0.032 0.078 �0.029 0.055 1

AV/E 0.179\* 0.358\* 0.055 0.170\* �0.042 0.213 0.474\* 1

EF 0.177\* 0.374\* 0.079 0.116\*\* �0.127\*\* 0.312\* 0.404\* 0.534\* 1

TE 0.006 0.265\* 0.090 0.039 0.046\* 0.017 0.182\* 0.236\* 0.401\* 1

SE 0.182\* 0.158\* 0.017 0.108\*\*\* �0.172\* 0.314\* 0.186\* 0.258\* 0.544\* �0.502\* 1

Legend: EP - Export Performance; ROA - Return on Assets; ROE - Return On Equity; ROI - Return on Investment; Lev - Leverage; FA - Financial Autonomy; T/E - Turnover per Employee; AV/E - Added Value per Employee; EF - Economic

Summarizing, the statistical significance of the Pearson correlation coefficients confirms the assumption that the performance of the company should not be assessed on one index, highlighting the importance computing, and analyzing alternative performance indices, in order to reinforce the results. Moreover, both in terms of signals and statistical significance, there are differences between the correlation coefficients of the Douro and Vinho Verde

Therefore, in order to ascertain whether there are differences in the performance of the wine firms, according to their size, each sample was divided into different groups according to the referred criteria of Table 2, which are the number of employees, total turnover, and total assets. Since the results are very similar, we only report the results ordered according to the number of employees.

use of inputs, as has also been demonstrated to be the case in Douro region.

regions, giving yet another clear reason to analyze them as separate samples.

In order to analyze the linear relation between the indices of performance, coefficients of Person correlation are computed for both regions (Tables 4 and 5) and special attention is given to the signal and statistical significance of these coefficients. For the Douro region (Table 4), the EP shows a highly positive correlated with FA, AV/E, EF, and SE. The ROA is positively correlated with ROE, ROI, FA, AV/E, EF, and SE. The ROE is positively correlated only with ROA, AV/E, and EF. The ROI is only positively related to ROA. The Lev is only positively related to SE. The FA is positively related to EP, ROA, EF, and SE. The T/E is positively correlated with ROA, AV/E, EF, TE, and SE. The AV/E is positively correlated with EP, ROA, ROI, T/E, and with the three indices of efficiency. The EF is positively correlated with the other indices of efficiency, and with EP, ROA, ROE, ROE, FA, T/E, and AV/E. The TE is positively correlated with T/E, AV/E, EF, and negatively with SE. The SE is positively correlated with EP, ROA, Lev, FA, T/E, FA, and negatively with TE. This last result means that firms being close to the optimum size do not mean they are efficient in the use of inputs. From the 55 linear correlation coefficients computed, 43.6% are statistically significant and only the correlation between TE and SE is negative. ROI and Lev are the indexes that present more linear independence to the other indices.

Regarding to the Vinho Verde region (Table 5), the EP is positively correlated with ROA, FA, AV/E, EF, and SE; the ROA is positively correlated ROI, FA, T/E AV/E, EF; the ROE is negatively correlated with ROI and Lev; the ROI is positively correlated with ROA, AV/E, EF, and SE and negatively correlated with ROE; the Lev is negatively correlated with ROE, EF, and


\*, \*\*, \*\*\* statistically significant at 1%, 5% and 10%, respectively.

and 0.675, respectively, indicating that the causes of productive inefficiency are due to a similar

Table 4. Pearson correlation coefficients of indices of performance of Douro region (434 observations, years 2014 and 2015).

EP ROA ROE ROI Lev FA T/E AV/E EF TE SE

In order to analyze the linear relation between the indices of performance, coefficients of Person correlation are computed for both regions (Tables 4 and 5) and special attention is given to the signal and statistical significance of these coefficients. For the Douro region (Table 4), the EP shows a highly positive correlated with FA, AV/E, EF, and SE. The ROA is positively correlated with ROE, ROI, FA, AV/E, EF, and SE. The ROE is positively correlated only with ROA, AV/E, and EF. The ROI is only positively related to ROA. The Lev is only positively related to SE. The FA is positively related to EP, ROA, EF, and SE. The T/E is positively correlated with ROA, AV/E, EF, TE, and SE. The AV/E is positively correlated with EP, ROA, ROI, T/E, and with the three indices of efficiency. The EF is positively correlated with the other indices of efficiency, and with EP, ROA, ROE, ROE, FA, T/E, and AV/E. The TE is positively correlated with T/E, AV/E, EF, and negatively with SE. The SE is positively correlated with EP, ROA, Lev, FA, T/E, FA, and negatively with TE. This last result means that firms being close to the optimum size do not mean they are efficient in the use of inputs. From the 55 linear correlation coefficients computed, 43.6% are statistically significant and only the correlation between TE and SE is negative. ROI and Lev are the indexes that present more linear

Regarding to the Vinho Verde region (Table 5), the EP is positively correlated with ROA, FA, AV/E, EF, and SE; the ROA is positively correlated ROI, FA, T/E AV/E, EF; the ROE is negatively correlated with ROI and Lev; the ROI is positively correlated with ROA, AV/E, EF, and SE and negatively correlated with ROE; the Lev is negatively correlated with ROE, EF, and

extent to the inefficient use of inputs and to firms not operating at an optimum size.

independence to the other indices.

EP 1

ROA 0.036 1

ROE �0.052 0.085\*\*\* 1

ROI �0.051 0.134\* 0.022 1

Lev 0.022 0.029 �0.009 �0.007 1

FA 0.143\* 0.549\* �0.022 �0.044 0.004 1

\*, \*\*, \*\*\* statistically significant at 1%, 5% and 10%, respectively.

Efficiency; TE - Technical Efficiency; SE - Scale Efficiency.

T/E 0.057 0.058 0.039 �0.015 �0.003 �0.052 1

316 Grapes and Wines - Advances in Production, Processing, Analysis and Valorization

AV/E 0.244\* 0.303\* 0.111\* 0.026 0.019 0.004 0.632\* 1

EF 0.177\* 0.199\* 0.086\*\*\* 0.045 0.017 0.235\* 0.538\* 0.619\* 1

TE 0.038 �0.015 0.057 0.078 �0.065 0.026 0.258\* 0.238\* 0.499\* 1

SE 0.164\* 0.200\* 0.016 �0.021 0.092\*\* 0.254\* 0.319\* 0.412\* 0.601\* �0.346\* 1

Legend: EP - Export Performance; ROA - Return on Assets; ROE - Return On Equity; ROI - Return on Investment; Lev - Leverage; FA - Financial Autonomy; T/E - Turnover per Employee; AV/E - Added Value per Employee; EF - Economic Legend: EP - Export Performance; ROA - Return on Assets; ROE - Return On Equity; ROI - Return on Investment; Lev - Leverage; FA - Financial Autonomy; T/E - Turnover per Employee; AV/E - Added Value per Employee; EF - Economic Efficiency; TE - Technical Eficiency; SE - Scale Efficiency.

Table 5. Pearson correlation coefficients of indices of performance Vinho Verde (299 observations, years 2014 and 2015).

SE; the FA is positively correlated with EP, ROA, ROI, EF, and SE; the T/E is positively correlated with ROA, AV/E, EF, TE, and SE; the AV/E is positively correlated with EP, ROA, ROI, T/E, and with the indices of efficiency; the EF is positively correlated with EP, ROA, ROI, FA, T/E, AV/E, TE, and SE and negatively with leverage; the TE is positively correlated with ROA, Lev, TE/E, AV/EEF, and negatively with SE; and the SE is positively correlated with EP, ROA, ROI, FA, T/E, AV/E, and EF and negatively with Lev and TE. For this region, from the 55 linear correlation coefficients computed, 45.5% are statistically significant, with 5 of them being negative (ROE-ROI, ROE-Lev, Lev-EF, Lev-SE, and SE-TE). For instance, the negative correlation Lev and EF implies that the more indebted the firm is, the less efficient it is, both in terms of technical efficiency and scale efficiency. Moreover, the negative signal of the correlation coefficient between SE and TE shows that the firms´ optimum size does not lead to the better use of inputs, as has also been demonstrated to be the case in Douro region.

Summarizing, the statistical significance of the Pearson correlation coefficients confirms the assumption that the performance of the company should not be assessed on one index, highlighting the importance computing, and analyzing alternative performance indices, in order to reinforce the results. Moreover, both in terms of signals and statistical significance, there are differences between the correlation coefficients of the Douro and Vinho Verde regions, giving yet another clear reason to analyze them as separate samples.

Therefore, in order to ascertain whether there are differences in the performance of the wine firms, according to their size, each sample was divided into different groups according to the referred criteria of Table 2, which are the number of employees, total turnover, and total assets. Since the results are very similar, we only report the results ordered according to the number of employees.


In order to check the robustness of these results, the OLS regressions, whose dependent variable is the respective index of performance, are estimated. The explanatory variables are dummy variables of the size, with "big" being omitted. The results of these models for the Douro region are also included in Table 6. The signal of the coefficients and, namely, the global significance (values of F) confirm that there are differences in the performance of firms dependent on their respective size. Specifically, it is noted that export performance, financial autonomy, productivity, and efficiency are lower in micro and small wine enterprises. However, this difference seems not to have consequences on the profitability and leverage indices, since the

Wine Firm's Size and Economic Performance: Evidence from Traditional Portuguese Wine Regions

Concerning the Vinho Verde region (Table 7), according to the number of employees, there are no big wine firms, with 77.6% being micro, 21.4% small, and 1% medium, confirming the dominance of very small companies. The Kruskal-Wallis test allows us to infer that there is only a positive relationship between size and the indices of EP, T/E, AV/E, and TE and that there are no statistically significant relationships with the other indices. The Hotelling F test

(10<sup>3</sup> €)

93.541 292.740

126.066 114.987

216.533 26.386

65.340 (0.090)

23.650 (0.000)

216.533 (0.000) 122.992 (0.010) �92.933 (0.130) 3.260 (0.040)

22.251 40.684

29.720 28.139

69.279 43.879

10.420 (0.210)

15.570 (0.000)

69.279 (0.230) 47.027 (0.419) 39.720 (0.490) 1.020 (0.360)

AV/E (103 €) EF TE SE

http://dx.doi.org/10.5772/intechopen.71320

0.414 0.239

0.427 0.178

0.551 0.131

28.260 (0.130)

3.240 (0.190)

0.551 (0.000) �0.137 (0.180) �0.129 (0.210) 0.920 (0.400)

0.702 0.283

0.587 0.273

0.880 0.183

55.230 (0.090)

10.770 (0.000)

0.880 (0.000) �0.178 (0.090) �0.300 (0.010) 8.430 (0.000)

0.650 0.310 319

0.765 0.134

0.623 0.029

639.950 (0.030)

2.270 (0.320)

0.623 (0.000) 0.027 (0.370) 0.144 (0.000) 25.540 (0.000)

respective regressions are not statistically significant.

Micro (n = 232)

Small (n = 64) Mean SD

Medium (n = 3)

0.102 0.202

0.157 0.183

0.533 0.193

44.230 (0.110)

20.970 (0.000)

0.533 (0.000) �0.432 (0.010) �0.376 (0.020) 5.080 (0.010)

test; OLS - Ordinary Least Squares.

�0.011 0.179

0.010 0.083

0.130 0.113

0.710 (0.640)

3.890 (0.140)

0.128 (0.470) �0.139 (0.440) �0.119 (0.500) 0.890 (0.410)

�0.096 2.033

�0.043 0.424

0.155 0.159

0.620 (0.670)

0.920 (0.630)

0.155 (0.800) �0.251 (0.690) �0.193 (0.760) 0.120 (0.880)

Mean SD

Mean SD

KW (Sig.)

Statistical F (Sig.)

OLS regressions Constant (Sig.) Micro (Sig.) Small (Sig) Statistical F (Sig.)

employees.

EP ROA ROE ROI Lev FA T/E

�0.100 1.022

0.015 0.139

0.160 0.139

0.700 (0.650)

2.950 (0.230)

0.159 (0.670) �0.260 (0.490) �0.147 (0.690) 1.470 (0.230)

8.875 64.888

2.973 6.121

1.359 1.139

0.620 (0.670)

2.710 (0.260)

1.359 (0.680) 7.516 (0.140) 1.521 (0.680) 1.240 (0.290) 0.226 0.764

0.349 0.439

0.483 0.185

3.880 (0.340)

0.990 (0.620)

0.483 (0.060) �0.256 (0.330) �0.147 (0.579) 1.260 (0.280)

Legend: EP - Export Performance; ROA - Return on Assets; ROE - Return On Equity; ROI - Return on Investment; Lev - Leverage; FA - Financial Autonomy; T/E - Turnover per Employee; AV/E - Added Value per Employee; EF - Economic Efficiency; TE - Technical Efficiency; SE - Scale Efficiency; SD – Standard Deviation; Sig - significance; KW - Kruskal-Wallis

Table 7. Indices of performance of Vinho Verde (299 observations, years 2014 and 2015) ordered according the number of

Legend: EP - Export Performance; ROA - Return on Assets; ROE - Return On Equity; ROI - Return on Investment; Lev - Leverage; FA - Financial Autonomy; T/E - Turnover per Employee; AV/E - Added Value per Employee; EF - Economic Efficiency; TE - Technical Efficiency; SE - Scale Efficiency; SD – Standard Deviation; Sig - significance; KW - Kruskal-Wallis test; OLS - Ordinary Least Squares.

Table 6. Indices of performance of Douro region (434 observations, years 2014 and 2015) ordered according to the number of employees.

Table 6 includes the results of the Douro region. Taking into account the number of observations for each size, we observe that almost 72% of the firms are micro, 23% are small, 3.9% are medium, and only 1.1% are big, confirming the hypothesis of a heterogeneous market structure that underlies the monopolistic competition approach. Assuming the value and significance of the Kruskal-Wallis test, since the null hypothesis of equality of population rank test is rejected, the results indicate a positive relationship between size and the indices of EP, Lev, FA, T/E, AV/E, EF, and TE. This is not the case for the profitability indicators (ROA, ROE, and ROI) and SE. However, the Hotelling statistical F is applied to examine that the means of the four sizes are the same. The results only reject this hypothesis for the EP, T/E, and EF (at a 5% level of significance) and TE (at 10% of level of significance).

In order to check the robustness of these results, the OLS regressions, whose dependent variable is the respective index of performance, are estimated. The explanatory variables are dummy variables of the size, with "big" being omitted. The results of these models for the Douro region are also included in Table 6. The signal of the coefficients and, namely, the global significance (values of F) confirm that there are differences in the performance of firms dependent on their respective size. Specifically, it is noted that export performance, financial autonomy, productivity, and efficiency are lower in micro and small wine enterprises. However, this difference seems not to have consequences on the profitability and leverage indices, since the respective regressions are not statistically significant.

Concerning the Vinho Verde region (Table 7), according to the number of employees, there are no big wine firms, with 77.6% being micro, 21.4% small, and 1% medium, confirming the dominance of very small companies. The Kruskal-Wallis test allows us to infer that there is only a positive relationship between size and the indices of EP, T/E, AV/E, and TE and that there are no statistically significant relationships with the other indices. The Hotelling F test


Legend: EP - Export Performance; ROA - Return on Assets; ROE - Return On Equity; ROI - Return on Investment; Lev - Leverage; FA - Financial Autonomy; T/E - Turnover per Employee; AV/E - Added Value per Employee; EF - Economic Efficiency; TE - Technical Efficiency; SE - Scale Efficiency; SD – Standard Deviation; Sig - significance; KW - Kruskal-Wallis test; OLS - Ordinary Least Squares.

Table 6 includes the results of the Douro region. Taking into account the number of observations for each size, we observe that almost 72% of the firms are micro, 23% are small, 3.9% are medium, and only 1.1% are big, confirming the hypothesis of a heterogeneous market structure that underlies the monopolistic competition approach. Assuming the value and significance of the Kruskal-Wallis test, since the null hypothesis of equality of population rank test is rejected, the results indicate a positive relationship between size and the indices of EP, Lev, FA, T/E, AV/E, EF, and TE. This is not the case for the profitability indicators (ROA, ROE, and ROI) and SE. However, the Hotelling statistical F is applied to examine that the means of the four sizes are the same. The results only reject this hypothesis for the EP, T/E, and EF (at a 5% level

Table 6. Indices of performance of Douro region (434 observations, years 2014 and 2015) ordered according to the

Legend: EP - Export Performance; ROA - Return on Assets; ROE - Return On Equity; ROI - Return on Investment; Lev - Leverage; FA - Financial Autonomy; T/E - Turnover per Employee; AV/E - Added Value per Employee; EF - Economic Efficiency; TE - Technical Efficiency; SE - Scale Efficiency; SD – Standard Deviation; Sig - significance; KW - Kruskal-Wallis

EP ROA ROE ROI Lev FA T/E

0.930 10.515

0.038 0.096

0.059 0.031

0.052 0.022

0.670 0.647

2.280 0.517

0.052 (0.560) 0.878 (0.090) �0.013 (0.890) 0.009 (0.930) 1.040 (0.370)

2.363 24.462

2.223 5.454

0.758 0.472

0.592 0.024

1.500 0.423

9.490 0.023

0.689 (0.000) 1.673 (0.180) 1.533 (0.120) 0.067 (0.890) 1.380 (0.250) 0.223 1.099

0.399 0.208

0.608 0.160

0.592 0.024

6.040 0.145

19.210 0.000

0.592 (0.000) �0.369 (0.000) �0.193 (0.000) 0.016 (0.900) 12.560 (0.000)

Micro (n = 312)

Small (n = 100)

Medium (n = 17)

0.107 0.189

0.306 0.281

0.636 0.163

0.750 0.134

48.870 0.020

92.610 0.000

0.750 (0.000) �0.643 (0.000) �0.443 (0.000) �0.115 (0.190) 97.030 (0.000)

test; OLS - Ordinary Least Squares.

number of employees.

0.002 0.506

0.024 0.063

0.051 0.024

0.044 0.019

2.680 0.283

4.720 0.192

0.043 (0.620) �0.041 (0.640) �0.019 (0.830) 0.007 (0.930) 0.420 (0.740)

0.278 3.734

318 Grapes and Wines - Advances in Production, Processing, Analysis and Valorization

0.014 0.600

0.055 0.037

0.044 0.029

0.700 0.633

3.280 0.350

0.044 (0.750) 0.232 (0.360) �0.031 (0.840) 0.010 (0.940) 0.520 (0.670)

Mean SD

Mean SD

Mean SD

Big (n = 5) Medium SD

Statistical F (Sig.)

OLS regressions Constant (Sig.) Micro (Sig.) Small (Sig) Medium (Sig) Statistical F (Sig.)

KW (Sig.) (10<sup>3</sup> €)

99.247 144.965

171.342 386.509

214.696 189.556

193.381 63.845

239.800 0.004

33.050 0.000

193.381 (0.000) �94.113 (0.060) �22.039 (0.690) 21.315 (0.810) 3.750 (0.010)

AV/E (10<sup>3</sup> €)

27.298 35.737

37.889 37.780

68.480 46.094

39.880 32.928

2.750 0.278

31.480 0.000

39.880 (0.160) �12.581 (0.660) 1.990 (0.940) 28.600 (0.380) 4.320 (0.010)

0.437 0.260

0.465 0.213

0.631 0.158

0.714 0.197

49.200 0.020

19.080 0.000

0.712 (0.000) �0.275 (0.000) �0.247 (0.000) �0.008 (0.340) 10.590 (0.000)

EF TE SE

0.709 0.296

0.610 0.239

0.808 0.150

0.866 0.155

10.520 0.088

15.310 0.002

0.866 (0.000) �0.156 (0.000) �0.266 (0.000) �0.058 (0.290) 17.850 (0.000)

0.666 0.299

0.759 0.144

0.779 0.109

0.808 0.098

3.050 0.257

1.160 0.763

0.808 (0.000) �0.142 (0.000) �0.049 (0.160) �0.029 (0.550) 7.940 (0.000)

of significance) and TE (at 10% of level of significance).

Table 7. Indices of performance of Vinho Verde (299 observations, years 2014 and 2015) ordered according the number of employees.

only permits the rejection of the null hypothesis of equality means of the three groups, for the variables T/E, TE (both at 10%), and SE (at 5%). The results of the OLS regressions show that the indices EP, T/E, and TE increase positively with the size, but negatively with SE. This last result implies that the larger firms tend to deviate from the optimal size, although they are more technical efficient in the use of inputs.

competition model. The indicators of productivity show that there is no standard result as these differ by region, demonstrating that the productivity of labor is higher in the bigger firms of the Douro region, but not in the Vinho Verde region where the turnover per employee is

Wine Firm's Size and Economic Performance: Evidence from Traditional Portuguese Wine Regions

http://dx.doi.org/10.5772/intechopen.71320

321

The indices of productive efficiency of Douro firms show that the bigger firms are empowered to adopt management strategies that improve the use of economic resources. This is a consequence of a better use of inputs and the adoption of an appropriate size. In opposite, the firms of the Vinho Verde region show a different result, in which the economic efficiency is independent of the firms' size. This could be a consequence of the better use of inputs by bigger

Summing up, the results do not show in a clear way that bigger wine firms outperform small ones, depending on the conclusions drawn from the indices that are used. The results also highlight the diversity among wine firms and the need to understand why they are different. Consequently, future research will be directed toward understanding which specific resources constitute a source of competitive advantage, expressed in variables that are not considered in

Regarding policy implications, mainly in the areas of public support and regulation, this research shows that micro and small wineries are the most common types in the Portuguese wine industry and that, in the short run, this situation will not change. Moreover, assuming that globalization and international competition continue to increase in the wine industry, the search for new international markets should be aware of the importance of scale economies. This is an aim that can be achieved through organic growth or strategic alliances, either along

This work is supported by the project NORTE -01-0145-FEDER-000038 (INNOVINE & WINE: Innovation Platform of Vine & Wine) and by European and Structural and Investment Funds in the FEDER component, through the Operational Competitiveness and Internationalization Program (COMPETE 2020) [Project No 006971 (UIC/SOC/04011)]; and national funds are received through the FCT: Portuguese Foundation for Science and Technology under the

the whole production and distribution chain or exclusively in the area of marketing.

João Rebelo\*, Sofia Gouveia, Lina Lourenço-Gomes and Ana Alexandra Marta-Costa

Department of Economics, Sociology and Management (DESG), Centre for Transdisciplinary Development Studies (CETRAD), University of Trás-os-Montes and Alto Douro (UTAD), Vila

higher for microfirms, there being no statistical differences between groups.

wineries to be annulled by the deviation from the optimal size.

Acknowledgements

UID/SOC/04011/2013.

\*Address all correspondence to: jrebelo@utad.pt

Author details

Real, Portugal

this study and that are only available through the use of firm's enquires.

Generally speaking, we can summarize that Douro wine firms are more heterogeneous than their Vinho Verde counterparts, which can be explained by comparing the technologies and market structures of the two markets. In both cases, the relationship between size and performance depends on the index of performance used, and it becomes clear that organizational performance is a multidimensional construct that should take into account the different dimensions of economic performance and even the respective regions<sup>4</sup> (spatial differentiation) in which they are located.

## 4. Conclusions

The wine industry neatly illustrates the market structure of monopolistic competition, exhibiting as it does varying degrees of product differentiation and free entry into the industry, with competition between groups of firm's size. Some of them search for market niches acting as oligopolies and others for mass markets acting as in perfect competition. In this context of horizontal product differentiation, notwithstanding the globalization of the wine market, a better understanding of the performance of the wine industry requires a microeconomic analysis based on measures at both the level of the firm and that of the wine region.

Based on data for the Douro and Vinho Verde regions, two representative Portuguese wine regions that fit well into the terroir model, and where horizontal product differentiation is clearly present, the main goal of this research has been to estimate the economic performance of wineries using different approaches and to assess the influence of the respective sizes of firms on performance indices, including ratios of traditional profitability, solvency, labor productivity and export performance, and productive efficiency. The results show that the wine firm's performance is a multidimensional construct and there are expected differences between wine regions, which are well predicted by the monopolistic competition model specifically regarding horizontal product differentiation.

Nonetheless, in both regions, export performance increases with size, which can be explained by the fact that big firms have at their disposal greater marketing resources and the ability to make substantial investment in the search for new international markets. Relative to the profitability indices, in both regions, there is no statistically significant relationship to size, and there coexist different typologies of firms in the market, according to the monopolistic

<sup>4</sup> In our case (results not reported in the chapter but provided by the authors) in order to test if there are differences between the two wine regions, the estimation of OLS regression, where the only explanatory dummy variable is the region, indicates that EP, ROI, AV/T, and EF are higher for the Douro region and not statistically different for the other variables.

competition model. The indicators of productivity show that there is no standard result as these differ by region, demonstrating that the productivity of labor is higher in the bigger firms of the Douro region, but not in the Vinho Verde region where the turnover per employee is higher for microfirms, there being no statistical differences between groups.

The indices of productive efficiency of Douro firms show that the bigger firms are empowered to adopt management strategies that improve the use of economic resources. This is a consequence of a better use of inputs and the adoption of an appropriate size. In opposite, the firms of the Vinho Verde region show a different result, in which the economic efficiency is independent of the firms' size. This could be a consequence of the better use of inputs by bigger wineries to be annulled by the deviation from the optimal size.

Summing up, the results do not show in a clear way that bigger wine firms outperform small ones, depending on the conclusions drawn from the indices that are used. The results also highlight the diversity among wine firms and the need to understand why they are different. Consequently, future research will be directed toward understanding which specific resources constitute a source of competitive advantage, expressed in variables that are not considered in this study and that are only available through the use of firm's enquires.

Regarding policy implications, mainly in the areas of public support and regulation, this research shows that micro and small wineries are the most common types in the Portuguese wine industry and that, in the short run, this situation will not change. Moreover, assuming that globalization and international competition continue to increase in the wine industry, the search for new international markets should be aware of the importance of scale economies. This is an aim that can be achieved through organic growth or strategic alliances, either along the whole production and distribution chain or exclusively in the area of marketing.

## Acknowledgements

only permits the rejection of the null hypothesis of equality means of the three groups, for the variables T/E, TE (both at 10%), and SE (at 5%). The results of the OLS regressions show that the indices EP, T/E, and TE increase positively with the size, but negatively with SE. This last result implies that the larger firms tend to deviate from the optimal size, although they are

Generally speaking, we can summarize that Douro wine firms are more heterogeneous than their Vinho Verde counterparts, which can be explained by comparing the technologies and market structures of the two markets. In both cases, the relationship between size and performance depends on the index of performance used, and it becomes clear that organizational performance is a multidimensional construct that should take into account the different dimensions of economic performance and even the respective regions<sup>4</sup> (spatial differentiation)

The wine industry neatly illustrates the market structure of monopolistic competition, exhibiting as it does varying degrees of product differentiation and free entry into the industry, with competition between groups of firm's size. Some of them search for market niches acting as oligopolies and others for mass markets acting as in perfect competition. In this context of horizontal product differentiation, notwithstanding the globalization of the wine market, a better understanding of the performance of the wine industry requires a microeconomic

Based on data for the Douro and Vinho Verde regions, two representative Portuguese wine regions that fit well into the terroir model, and where horizontal product differentiation is clearly present, the main goal of this research has been to estimate the economic performance of wineries using different approaches and to assess the influence of the respective sizes of firms on performance indices, including ratios of traditional profitability, solvency, labor productivity and export performance, and productive efficiency. The results show that the wine firm's performance is a multidimensional construct and there are expected differences between wine regions, which are well predicted by the monopolistic competition model specifically

Nonetheless, in both regions, export performance increases with size, which can be explained by the fact that big firms have at their disposal greater marketing resources and the ability to make substantial investment in the search for new international markets. Relative to the profitability indices, in both regions, there is no statistically significant relationship to size, and there coexist different typologies of firms in the market, according to the monopolistic

In our case (results not reported in the chapter but provided by the authors) in order to test if there are differences between the two wine regions, the estimation of OLS regression, where the only explanatory dummy variable is the region, indicates that EP, ROI, AV/T, and EF are higher for the Douro region and not statistically different for the other

analysis based on measures at both the level of the firm and that of the wine region.

more technical efficient in the use of inputs.

320 Grapes and Wines - Advances in Production, Processing, Analysis and Valorization

regarding horizontal product differentiation.

in which they are located.

4. Conclusions

4

variables.

This work is supported by the project NORTE -01-0145-FEDER-000038 (INNOVINE & WINE: Innovation Platform of Vine & Wine) and by European and Structural and Investment Funds in the FEDER component, through the Operational Competitiveness and Internationalization Program (COMPETE 2020) [Project No 006971 (UIC/SOC/04011)]; and national funds are received through the FCT: Portuguese Foundation for Science and Technology under the UID/SOC/04011/2013.

## Author details

João Rebelo\*, Sofia Gouveia, Lina Lourenço-Gomes and Ana Alexandra Marta-Costa

\*Address all correspondence to: jrebelo@utad.pt

Department of Economics, Sociology and Management (DESG), Centre for Transdisciplinary Development Studies (CETRAD), University of Trás-os-Montes and Alto Douro (UTAD), Vila Real, Portugal

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Wine Firm's Size and Economic Performance: Evidence from Traditional Portuguese Wine Regions

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**Chapter 15**

**Provisional chapter**

**Potential for Use of the Residues of the Wine Industry**

**Potential for Use of the Residues of the Wine Industry** 

The use of underutilized resources, with the aim of increasing productivity and creating wealth, will increasingly deserve the attention of the wine sector. The treatment of agricultural by-products will increasingly enter the priority agenda of the agribusiness sector, with a view to its use, the environment's re-cleanliness and, in many cases, whenever possible, for both purposes. Solid waste from the process of grape industrialization, when not adequately disposed, is aggressive to the environment. Such residues release significant amounts of liquid effluents when disposed in the soil, and this liquid contains high content of nutrients, organic matter, and other elements that can change the environment, especially of streams and sources, causing the death of aquatic beings. However, if properly used, they can be used as raw materials for other purposes. The solid residues of industrially processed grapes, which may have some potential economic interest, are pomace, seeds, liquid (lees), and other materials. In this context, this chapter presents the

**Keywords:** *Vitis* spp., by-products, pomace, winery, flour, polyphenols, antioxidants,

Approximately 75,100 million tons of grapes are produced annually in the world and are destined mostly to wine production or in natura consumption, with Italy, France, Spain, the United States, Australia, China, Chile, South Africa, Argentina, and Germany being the main producers. These countries produce about 275.7 million hectoliters of wine.

> © 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

© 2018 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

DOI: 10.5772/intechopen.73132

**in Human Nutrition and as Agricultural Input**

**in Human Nutrition and as Agricultural Input**

Renato Vasconcelos Botelho, Gabriela Datsch Bennemann, Yohandra Reyes Torres and Alessandro Jefferson Sato

Renato Vasconcelos Botelho, Gabriela Datsch Bennemann,

Additional information is available at the end of the chapter

Additional information is available at the end of the chapter

Yohandra Reyes Torres and Alessandro Jefferson Sato

description of these by-products and their potential for use.

http://dx.doi.org/10.5772/intechopen.73132

**Abstract**

seed oil

**1. Introduction**

#### **Potential for Use of the Residues of the Wine Industry in Human Nutrition and as Agricultural Input Potential for Use of the Residues of the Wine Industry in Human Nutrition and as Agricultural Input**

DOI: 10.5772/intechopen.73132

Renato Vasconcelos Botelho, Gabriela Datsch Bennemann, Yohandra Reyes Torres and Alessandro Jefferson Sato Renato Vasconcelos Botelho, Gabriela Datsch Bennemann, Yohandra Reyes Torres and Alessandro Jefferson Sato Additional information is available at the end of the chapter

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/intechopen.73132

#### **Abstract**

The use of underutilized resources, with the aim of increasing productivity and creating wealth, will increasingly deserve the attention of the wine sector. The treatment of agricultural by-products will increasingly enter the priority agenda of the agribusiness sector, with a view to its use, the environment's re-cleanliness and, in many cases, whenever possible, for both purposes. Solid waste from the process of grape industrialization, when not adequately disposed, is aggressive to the environment. Such residues release significant amounts of liquid effluents when disposed in the soil, and this liquid contains high content of nutrients, organic matter, and other elements that can change the environment, especially of streams and sources, causing the death of aquatic beings. However, if properly used, they can be used as raw materials for other purposes. The solid residues of industrially processed grapes, which may have some potential economic interest, are pomace, seeds, liquid (lees), and other materials. In this context, this chapter presents the description of these by-products and their potential for use.

**Keywords:** *Vitis* spp., by-products, pomace, winery, flour, polyphenols, antioxidants, seed oil

## **1. Introduction**

Approximately 75,100 million tons of grapes are produced annually in the world and are destined mostly to wine production or in natura consumption, with Italy, France, Spain, the United States, Australia, China, Chile, South Africa, Argentina, and Germany being the main producers. These countries produce about 275.7 million hectoliters of wine.

This results in approximately 13 million tons of residues, which are normally used as fertilizer or simply discarded [1].

In this context, the use of winery residues is indicated as an important perspective in the management of grapevine, once the action of its components, such as phenolic acids and flavonoids, have already been reported as efficient against microorganisms [12] and also weeds [13]. The isolated effect of some compounds present in this residue has already been characterized on some insects, such as the flavonoids, which caused inhibited development of *Phyllotreta* 

Potential for Use of the Residues of the Wine Industry in Human Nutrition and as Agricultural…

http://dx.doi.org/10.5772/intechopen.73132

327

The main by-products of winemaking are separated during the crushing and pressing stages of the grapes, and only small quantities of these residues are availed. Recovery of compounds from the continuous waste produced by the wine industry could represent a significant step forward in maintaining the balance of the environment, as the large quantities of waste generated in the wineries present serious problems of storage, processing, or disposal in ecological and economic terms. This situation explains the growing interest in exploring the by-products of winemaking. Solid residues from the grape industrialization process, when disposed outdoor, are aggressive to the environment, releasing significant amounts of liquid effluents, which contain high nutrient content, organic matter, and other elements that, in contact with the soil and sources of water, can cause death of living organisms. However, adopting some technologies, they can be used as raw material for other purposes. Solid residues of industrially processed grapes, which may have some potential economic interest, are stalks, pomace, seeds, liquid (lees), and other materials. Although many polyphenols are transferred from the grapes to the wine during the maceration process, the residual seeds have been the focus of studies that relate them

The pomace consists of the skin, the seeds, and the remains of the pulp of the grape, resulting from the crushing of the berries through a process of separation of the juice or must. Under

Other solid wastes are lees and tartar. The sludge originates in the bottom of the kites, is dense, and comes from the processes of purification of the wine stored. The tartar is solid and deposits on the walls of the containers (pipes) used to age the wine. Liquid wastes result from washes and spills of raw material. These materials should be submitted to the treatment of effluents in canteens. Regulation (EC) No 1493/99 defines wine sludge as the residue which is deposited in receptacles containing wine after fermentation, or at the time of storage, or after authorized treatment, as well as the residue obtained by the filtration and/or centrifugation of this wine product [12]. Also considered as wine lees is waste that is deposited in containers containing grape must. The amount of lees obtained annually depends on several factors, namely those inherent to the constitution of the grape varieties, maturation stage and phytosanitary state of the berries, climatic factors, and the winemaking techniques adopted. In general, these represent about 5% of the wine volume. A quantity of 140 kg of grapes produces

Pomace is the main by-product of winemaking, not only because of its alcoholic and tartaric richness but also because of the economic interest of some of its physical components. Pomace,

normal conditions, the pomace is equivalent to 15% of the weight of the berries.

approximately 1 hL of wine, giving 5.5 kg of liquid lees with 4.5% alcohol.

*cruciferae* (Coleoptera: Chrysomelidae) [14].

as good sources of phenolic compounds [7].

**2. Wine production and waste generation**

Although not widely used, the residues from the wine industry have high fiber content and antioxidant substances that could be beneficial for use in food, but there are few reports about this potential. One of the best applications for grape residues, especially the pomace, could be to obtain flour, which can be used in the preparation of biscuits, breads, cereal bars, homemade pasta, and juices [2, 3].

The phenols are defined as the largest group of natural antioxidants, with about 8000 different compounds. They are distributed in several plant foods, being secondary metabolites of these and considered fundamental for the proper development of the plant, defense against environmental injuries, and infectious processes. Among the different vegetables, grapes are considered one of the major sources of phenolic compounds; however, it is known that there is considerable diversity among cultivars, and this results in grapes with different characteristics, such as flavor and color, which is certainly associated with the content and profile of polyphenols [4].

Grape seeds usually contain 8–20% of oil, which represent about 5% of the fruit weight, with about 3 million tons of grape seeds discarded annually in the world [5]. The seeds constitute about 20% of the gross weight of the fruit and calculated on dry matter, representing between 40 and 60% [6]. The complete use of grapes, including seeds, is considered an important economic and sustainability factor, since the oil has a pleasant and neutral taste and has a high concentration of linoleic acid and natural vitamin E, which provides considerable oxidative stability to the product. Grape seeds also have a considerable content of phenolic compounds (about 60–70% of their content), with smaller percentages found in other parts of the fruit such as 28–35% in the skin and approximately 10% in the pulp [6].

These compounds present in grapes are recognized for their role in modulating the expression of antioxidant enzymes [7], protection against oxidative damage in rat brain cells [8], and some anti-inflammatory effects. Some studies have shown anticholinergic effects of grape seed oil, with a proven reduction of low-density lipoprotein (LDL) and increases in highdensity lipoprotein (HDL) levels, characteristics related to cardioprotective effects [9].

Some phenolic compounds were identified as the monomers, gallic acid, (+)-catechin, (+)-epicatechin, and epicatechin-3-*O*-gallate, and a large variety of procyanidins oligomers in skin and grape seeds [10, 11]. These high contents of bioactive compounds in both flour and grape seed oil characterize them as a functional food, widely disseminated in current nutritional practices.

Due to its abundance and its richness in compounds with bioactive properties, the study of the use of grape residues as an agricultural input, aiming at the management of plant cover, pests, and diseases, represents a great potential, especially considering the issues related to sustainability, impacts on the environment, and production costs.

In viticulture, intensive management practices and the use of agrochemicals are adopted on a large scale to control pests, diseases, and weeds. These practices tend to have significant impacts on agroecosystems, since they alter the characteristics of the habitat and the composition of the trophic chain. Thus, in recent years, there has been a growing demand for alternative management methods that reduce impacts on nontarget organisms and are satisfactorily effective against target organisms.

In this context, the use of winery residues is indicated as an important perspective in the management of grapevine, once the action of its components, such as phenolic acids and flavonoids, have already been reported as efficient against microorganisms [12] and also weeds [13]. The isolated effect of some compounds present in this residue has already been characterized on some insects, such as the flavonoids, which caused inhibited development of *Phyllotreta cruciferae* (Coleoptera: Chrysomelidae) [14].

## **2. Wine production and waste generation**

This results in approximately 13 million tons of residues, which are normally used as fertil-

Although not widely used, the residues from the wine industry have high fiber content and antioxidant substances that could be beneficial for use in food, but there are few reports about this potential. One of the best applications for grape residues, especially the pomace, could be to obtain flour, which can be used in the preparation of biscuits, breads, cereal bars, home-

The phenols are defined as the largest group of natural antioxidants, with about 8000 different compounds. They are distributed in several plant foods, being secondary metabolites of these and considered fundamental for the proper development of the plant, defense against environmental injuries, and infectious processes. Among the different vegetables, grapes are considered one of the major sources of phenolic compounds; however, it is known that there is considerable diversity among cultivars, and this results in grapes with different characteristics, such as flavor

Grape seeds usually contain 8–20% of oil, which represent about 5% of the fruit weight, with about 3 million tons of grape seeds discarded annually in the world [5]. The seeds constitute about 20% of the gross weight of the fruit and calculated on dry matter, representing between 40 and 60% [6]. The complete use of grapes, including seeds, is considered an important economic and sustainability factor, since the oil has a pleasant and neutral taste and has a high concentration of linoleic acid and natural vitamin E, which provides considerable oxidative stability to the product. Grape seeds also have a considerable content of phenolic compounds (about 60–70% of their content), with smaller percentages found in other parts of the fruit

These compounds present in grapes are recognized for their role in modulating the expression of antioxidant enzymes [7], protection against oxidative damage in rat brain cells [8], and some anti-inflammatory effects. Some studies have shown anticholinergic effects of grape seed oil, with a proven reduction of low-density lipoprotein (LDL) and increases in high-

Some phenolic compounds were identified as the monomers, gallic acid, (+)-catechin, (+)-epicatechin, and epicatechin-3-*O*-gallate, and a large variety of procyanidins oligomers in skin and grape seeds [10, 11]. These high contents of bioactive compounds in both flour and grape seed oil characterize them as a functional food, widely disseminated in current nutritional practices. Due to its abundance and its richness in compounds with bioactive properties, the study of the use of grape residues as an agricultural input, aiming at the management of plant cover, pests, and diseases, represents a great potential, especially considering the issues related to

In viticulture, intensive management practices and the use of agrochemicals are adopted on a large scale to control pests, diseases, and weeds. These practices tend to have significant impacts on agroecosystems, since they alter the characteristics of the habitat and the composition of the trophic chain. Thus, in recent years, there has been a growing demand for alternative management methods that reduce impacts on nontarget organisms and are satisfactorily

density lipoprotein (HDL) levels, characteristics related to cardioprotective effects [9].

and color, which is certainly associated with the content and profile of polyphenols [4].

such as 28–35% in the skin and approximately 10% in the pulp [6].

sustainability, impacts on the environment, and production costs.

effective against target organisms.

izer or simply discarded [1].

326 Grapes and Wines - Advances in Production, Processing, Analysis and Valorization

made pasta, and juices [2, 3].

The main by-products of winemaking are separated during the crushing and pressing stages of the grapes, and only small quantities of these residues are availed. Recovery of compounds from the continuous waste produced by the wine industry could represent a significant step forward in maintaining the balance of the environment, as the large quantities of waste generated in the wineries present serious problems of storage, processing, or disposal in ecological and economic terms. This situation explains the growing interest in exploring the by-products of winemaking.

Solid residues from the grape industrialization process, when disposed outdoor, are aggressive to the environment, releasing significant amounts of liquid effluents, which contain high nutrient content, organic matter, and other elements that, in contact with the soil and sources of water, can cause death of living organisms. However, adopting some technologies, they can be used as raw material for other purposes. Solid residues of industrially processed grapes, which may have some potential economic interest, are stalks, pomace, seeds, liquid (lees), and other materials. Although many polyphenols are transferred from the grapes to the wine during the maceration process, the residual seeds have been the focus of studies that relate them as good sources of phenolic compounds [7].

The pomace consists of the skin, the seeds, and the remains of the pulp of the grape, resulting from the crushing of the berries through a process of separation of the juice or must. Under normal conditions, the pomace is equivalent to 15% of the weight of the berries.

Other solid wastes are lees and tartar. The sludge originates in the bottom of the kites, is dense, and comes from the processes of purification of the wine stored. The tartar is solid and deposits on the walls of the containers (pipes) used to age the wine. Liquid wastes result from washes and spills of raw material. These materials should be submitted to the treatment of effluents in canteens. Regulation (EC) No 1493/99 defines wine sludge as the residue which is deposited in receptacles containing wine after fermentation, or at the time of storage, or after authorized treatment, as well as the residue obtained by the filtration and/or centrifugation of this wine product [12]. Also considered as wine lees is waste that is deposited in containers containing grape must. The amount of lees obtained annually depends on several factors, namely those inherent to the constitution of the grape varieties, maturation stage and phytosanitary state of the berries, climatic factors, and the winemaking techniques adopted. In general, these represent about 5% of the wine volume. A quantity of 140 kg of grapes produces approximately 1 hL of wine, giving 5.5 kg of liquid lees with 4.5% alcohol.

Pomace is the main by-product of winemaking, not only because of its alcoholic and tartaric richness but also because of the economic interest of some of its physical components. Pomace, as is well known, is the product resulting from the pressing of the wine masses, consisting of the solid parts of the grapes and the must or the wort/wine assembly that soaks them. Regulation (EC) No 1493/1999 defines it as the residue of pressing of fresh grapes, fermented or not. The pomace consists mainly of water, wines, and lees—these being dependent on the pressing; alcohols, especially ethanol, and also methanol and glycerol; aldehydes, esters, volatile acids, polyphenols, proteins, cellulose, pectins, mineral salts, and sugar residues [12].

## **3. Composition of grapes pomace and its potential for use**

Botanically, the grape can be classified as a fruit of the vine, belonging to the family Vitaceae and to the genus *Vitis*, with the following main species: *Vitis vinifera*, *Vitis labrusca*, *Vitis rupestris*, *Vitis aestivalis*, *Vitis riparia*, *Vitis cinerea*, and *Vitis rotundifolia* [15]. In this sense, the existence of numerous grape varieties causes differences in their chemical composition, which allows to select the most suitable cultivars for both industrialization and in natura consumption.

The composition of the grape berry is generally formed by 6–12% of skin, 2–5% of seed, and 85–92% of pulp. The pulp, which constitutes the main part of the grape, is composed of the following constituents: 65–85% water, 12–25% reducing sugars, 0.6–1.4% organic acids, 0.25– 0.5% of mineral substances, and 0.05–0.1% of nitrogenous compounds, besides several watersoluble and fat-soluble vitamins [16].

Like the other nutrients presented, the mineral composition of the grape may vary according to the conditions provided for growth, such as the composition of the soil and the use of fertilizers and herbicides. The nutritional relevance of the juice is mainly due to the high content of potassium, calcium, iron, magnesium and phosphorus, and low levels of sodium. Thus, fresh grape juice presents from 2.5 to 3.5 g L−1 of mineral substances [17].

Pomace (a mixture of grapes skin and seed) accounts for about 16% of the total processed grape and is one of the most abundant residues in the wine industry, and this material is known to be rich in many compounds such as phenolic acids, flavonoids, tannins, and saponins and, therefore, has a great utility potential for a variety of purposes, including pests, diseases, and weed control in agricultural crops, and the use of grape extract as a corrosion inhibitor by the metallurgical industry due to its high antioxidant capacity. The pomace contains another coproduct with high added value: the seed, which generates vegetable oil [4, 5].

In a research carried out in Brazil [17], it was analyzed for the mineral content (N, P, K, S, Ca, Fe, Mg, Mn, Fe, and Zn), anthocyanins, and phenolic compounds in flours produced from residues of different grape cultivars from different wineries (**Tables 1** and **2**). Mineral analysis showed a significant difference for all grape cultivar, with the exception in phosphorus content. Residues from cv. Seibel showed higher levels of N, Cu, and Mg. The cultivars Ancelotta, Tannat, and Ives present higher contents of K, Zn, Mn, Fe, and Ca. For the concentration of anthocyanins, cultivars Cabernet Sauvignon (114.7 mg/100 g), Tannat (88.5 mg/100 g), and Ancelotta (33.8 mg/100 g) had the highest concentrations. The cultivars Pinot Noir (7.0 g AGE/100 g), Tannat (4.3 g AGE/100 g),

**Grape variety**

**x ± DP** Cabernet Sauvignon

Tannat Ancelotta Pinot Noir

Malbec Merlot

Seibel

Ives Cabernet Franc

**Variety**

**x ± DP** Cabernet Sauvignon

Tannat Ancelotta Pinot Noir

Malbec Merlot

Seibel

Ives Cabernet Franc

**Table 1.**

Mineral content in grape pomace flour in southern Brazil [18].

42.64 ± 1.64abc

86.52 ± 11.8ad 63.97 ± 19.18ac

89.94 ± 0.71ad 52.95 ± 1.44abc 125.06 ± 2.57d

9.25 ± 0.22b 296.42 ± 44.61e

36.38 ± 1.11bc

24.87 ± 5.3abc

36.64 ± 0.26d

32.27 ± 1.25bd 25.84 ± 1.37abc

19.91 ± 3.15c

9.02 ± 1.25e 30 ± 6.58abd 29.26 ± 2.32abd

21.09 ± 0.78ac Means followed by different letters on the same column are significantly different according to NSK at *P*

62.21 ± 1.96b 104.32 ± 3.03a

86.93 ± 1.75ac 105.51 ± 3.62a

75.49 ± 1.95c 58.73 ± 2.94b 154.7 ± 20.42d 102.87 ± 5.70a 101.36 ± 1.73a

209.78 ± 7.02ab

286.5 ± 6.05c

212.89 ± 8.48ab

164.84 ± 4.71a 250.06 ± 18.54bc

169.26 ± 11.66a 166.36 ± 10.37a 241.54 ± 19.81bc

155.3 ± 9.05a

> <

0.05. NS, not significant.

329

284.22 ± 3.59b

429.5 ± 4.74d

Potential for Use of the Residues of the Wine Industry in Human Nutrition and as Agricultural…

357.02 ± 4.2c 362.84 ± 1.67c 312.13 ± 1.21b 158.04 ± 4.63a 356.21 ± 13.68c

http://dx.doi.org/10.5772/intechopen.73132

308.8 ± 3.96b

296.21 ± 7.84b

45.14 ± 5.3 **Copper (mcg/100**

 **g)**

**Manganese (mg/100**

 **g)**

**Magnesium (mg/100**

 **g)**

**Iron (mg/100 g)**

**Calcium (mg/100**

 **g)**

39.59 ± 1.3n.s.

33.05 ± 0.99 43.88 ± 1.33 49.76 ± 9.81 42.43 ± 2.01 39.96 ± 3.42

44.1 ± 1.77 35.65 ± 0.86

21.83 ± 0.99a 22.09 ± 1.31a 22.09 ± 1.06a 22.45 ± 0.59a 26.17 ± 0.87b

3.92 ± 0.72c 30.93 ± 1.45e

25.05 ± 0.91b

17.77 ± 0.74d

52.27 ± 1.64ab

38.53 ± 2.5b 85.4 ± 4.32c 56.49 ± 2.69ab 53.94 ± 3.07ab 54.92 ± 1.46ab

67.47 ± 0.58ab

87.88 ± 0.98c

63.12 ± 1.25ab

13.77 ± 2.02a 23.52 ± 3.14b 25.82 ± 0.01b 13.44 ± 1.49a

19 ± 0.94c 10.84 ± 0.56a

5.96 ± 1.12d

25.15 ± 0.69b 18.66 ± 0.56c

NS

16.35 ± 0.44d 33.59 ± 1.66e

6.7 ± 0.50b 9.84 ± 0.81c 0.08 ± 0.02a

6.29 ± 0.35b

17.47 ± 0.24d

NS

**Phosphor (mg/100**

 **g)**

**Nitrogen (mg/100**

 **g)**

**Sulfur (mg/100**

 **g)**

**Potassium (mg/100**

 **g)**

**Zinc (mg/100 g)**


as is well known, is the product resulting from the pressing of the wine masses, consisting of the solid parts of the grapes and the must or the wort/wine assembly that soaks them. Regulation (EC) No 1493/1999 defines it as the residue of pressing of fresh grapes, fermented or not. The pomace consists mainly of water, wines, and lees—these being dependent on the pressing; alcohols, especially ethanol, and also methanol and glycerol; aldehydes, esters, volatile acids, polyphenols, proteins, cellulose, pectins, mineral salts, and sugar residues [12].

Botanically, the grape can be classified as a fruit of the vine, belonging to the family Vitaceae and to the genus *Vitis*, with the following main species: *Vitis vinifera*, *Vitis labrusca*, *Vitis rupestris*, *Vitis aestivalis*, *Vitis riparia*, *Vitis cinerea*, and *Vitis rotundifolia* [15]. In this sense, the existence of numerous grape varieties causes differences in their chemical composition, which allows to select the most suitable cultivars for both industrialization and in natura consumption.

The composition of the grape berry is generally formed by 6–12% of skin, 2–5% of seed, and 85–92% of pulp. The pulp, which constitutes the main part of the grape, is composed of the following constituents: 65–85% water, 12–25% reducing sugars, 0.6–1.4% organic acids, 0.25– 0.5% of mineral substances, and 0.05–0.1% of nitrogenous compounds, besides several water-

Like the other nutrients presented, the mineral composition of the grape may vary according to the conditions provided for growth, such as the composition of the soil and the use of fertilizers and herbicides. The nutritional relevance of the juice is mainly due to the high content of potassium, calcium, iron, magnesium and phosphorus, and low levels of sodium. Thus, fresh

Pomace (a mixture of grapes skin and seed) accounts for about 16% of the total processed grape and is one of the most abundant residues in the wine industry, and this material is known to be rich in many compounds such as phenolic acids, flavonoids, tannins, and saponins and, therefore, has a great utility potential for a variety of purposes, including pests, diseases, and weed control in agricultural crops, and the use of grape extract as a corrosion inhibitor by the metallurgical industry due to its high antioxidant capacity. The pomace contains another coproduct with high added value: the seed, which generates veg-

In a research carried out in Brazil [17], it was analyzed for the mineral content (N, P, K, S, Ca, Fe, Mg, Mn, Fe, and Zn), anthocyanins, and phenolic compounds in flours produced from residues of different grape cultivars from different wineries (**Tables 1** and **2**). Mineral analysis showed a significant difference for all grape cultivar, with the exception in phosphorus content. Residues from cv. Seibel showed higher levels of N, Cu, and Mg. The cultivars Ancelotta, Tannat, and Ives present higher contents of K, Zn, Mn, Fe, and Ca. For the concentration of anthocyanins, cultivars Cabernet Sauvignon (114.7 mg/100 g), Tannat (88.5 mg/100 g), and Ancelotta (33.8 mg/100 g) had the highest concentrations. The cultivars Pinot Noir (7.0 g AGE/100 g), Tannat (4.3 g AGE/100 g),

**3. Composition of grapes pomace and its potential for use**

328 Grapes and Wines - Advances in Production, Processing, Analysis and Valorization

grape juice presents from 2.5 to 3.5 g L−1 of mineral substances [17].

soluble and fat-soluble vitamins [16].

etable oil [4, 5].

Potential for Use of the Residues of the Wine Industry in Human Nutrition and as Agricultural… http://dx.doi.org/10.5772/intechopen.73132 329

**Table 1.** Mineral content in grape pomace flour in southern Brazil [18].


A study with grape pomace of the cv. Marselan (*V. vinifera*) was carried out to evaluate the effect of its inclusion in extruded snack with respect to nutritional, technological, and sensory parameters [22]. The microbiological determinations of coliforms at 45°C and *Salmonella* in the flours were also performed. The centesimal composition presented fiber (58.01%), carbohydrates (17.62%), and ashes (12.46%) as the main constituents. Resveratrol (6.14 mg g−1), luteolin (5.16 mg g−1), and kaempferol (3.01 mg g−1) were the phenolic compounds detected in greater quantity in the flour. The fiber formulation containing 9% (5% fiber) of flour presented better acceptance results with regard to color, aroma, and texture attributes compared to the standard snack formulation. According to these authors [22], for the nutritional enrichment (fibers and phytochemicals) and for adding value to the agro-industrial residue discarded by the wineries, the addition of pomace flour in extruded snacks is viable and quite interesting. Grape pomace is known to be rich in many compounds such as phenolic acids, flavonoids, tannins, and saponins presenting a high potential for pests, diseases, and weed control in agricultural crops, including vineyards. The antimicrobial effect of 'Pinot Noir' grape pomace

Potential for Use of the Residues of the Wine Industry in Human Nutrition and as Agricultural…

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331

extracts against *Staphylococcus aureus* and *Candida albicans* was already reported [18].

accumulation at the 3% dose.

**4. Composition of grape seeds oil**

discarded annually worldwide [26].

organic system.

Considering the composition of the grape pomace, one of the possibilities is the induction of resistance of plants to pathogens. In this context, a study was carried out to evaluate the effect of autoclaved grape pomace extract (GPE) in the induction of phytoalexin deoxyanthocyanidine in sorghum [23]. To obtain the extract, ground dry pomace was macerated in water at 70°C for approximately 12 h in the dark. The GPE was sterilized by autoclaving for 20 min at 120°C under a pressure of 1.1 kgf cm−2, followed by fractionation in the tested doses. These authors reported the effect of GBE on the deoxythyanocyanidine synthesis, with a higher

The downy mildew (*Plasmopara viticola*) is responsible for qualitative and quantitative losses in grapevines production. In this context, a study was carried out aiming to verify the action of grape pomace extract (GPE) on sporangia germination of *P. viticola* and the severity of mildew on grapevines cv. Carmen [24]. These authors verified reduction of germination of the pathogen and incidence of mildew in grapevines. The highest dose of the extract (12%) controlled mildew severity at 36%, demonstrating the potential of the residue extract for the

Pomace represents approximately 16% of the grapes, of which about 20–26% are seeds [25]. Although winemakers have traditionally considered these wastes an economic and environmental problem, they are gradually being considered as a potential to add value to the products. Grape seeds usually contain 8–20% oil, with about 3 million tons of grape seeds being

The complete use of grapes, including seeds, is considered an important economic and sustainability factor, since the oil has a pleasant and neutral taste and has a high concentration of linoleic acid and natural vitamin E, which provides oxidative stability to the product.

Means followed by different letters on the same column are significantly different according to NSK at *P* < 0.05. NS, not significant.

**Table 2.** Anthocyanin content and phenolic compounds in grape pomace flour in southern Brazil [18].

and Ancelotta (3.9 g AGE/100 g) had the highest content of phenolic compounds. Considering these results, the potential of using the residue of winemaking to produce flour for human consumption became evident, highlighting the grapes 'Tannat' and 'Ancelotta'.

The main phenolic compounds presented in the pomace of 'Pinot Noir' grapes were (1) flavonoids: flavan-3-ols (catechin, epicatechin, epicatechin gallate, procyanidins A and B), flavonols (quercetin and quercetin methyl glucoside), and anthocyanins (delphinidin-3-glucoside, 3-glucoside cyanidin-3-glucoside, petunidin-3-glucoside, peonidin-3-glucoside, malvidin-3-glucoside, vitisin) and (2) non-flavonoids: hydroxybenzoate (gallic acid) [5].

Grape pomace also showed a great wealth of biologically active substances in the study that were developed to determine the chemical composition of seeds and skin of grape pomace of different cultivars of *V. vinifera* grapes both white and red in Italy and in California [19]. These authors verified that in the seeds and in the skin, the Italian samples presented higher content of organic matter—lignin and copper. In addition, the contents of K, Fe, and Zn were higher in grapes from California. The Italian white grapes had a higher content of saponins in the pell and tended to have a higher phenolic content in both the skin and the seeds.

A study with grape pomace extracts (GPEs) of the grapevines 'Niagara' and 'Isabella' (*V. labrusca*) was carried out to evaluate their effects on oxidative stability and quality of chicken meat. The use of the grape pomace extract was efficient to maintain the lipid stability of the chicken meat, presenting results compatible with those exhibited by the synthetic antioxidant butyl hydroxytoluene (BHT) [14].

Due to the richness of bioactive components, the use of grape pomace flour has been tested in different food product such as biscuits [3], muffins [20], and cookies [21]. These studies showed that, in general, the addition of flour does not negatively affect the preference for food. Moreover, the presence of bioactive compounds was verified, indicating the possibility of adding nutritional value to the food with the supplementation of flour of grape pomace.

A study with grape pomace of the cv. Marselan (*V. vinifera*) was carried out to evaluate the effect of its inclusion in extruded snack with respect to nutritional, technological, and sensory parameters [22]. The microbiological determinations of coliforms at 45°C and *Salmonella* in the flours were also performed. The centesimal composition presented fiber (58.01%), carbohydrates (17.62%), and ashes (12.46%) as the main constituents. Resveratrol (6.14 mg g−1), luteolin (5.16 mg g−1), and kaempferol (3.01 mg g−1) were the phenolic compounds detected in greater quantity in the flour. The fiber formulation containing 9% (5% fiber) of flour presented better acceptance results with regard to color, aroma, and texture attributes compared to the standard snack formulation. According to these authors [22], for the nutritional enrichment (fibers and phytochemicals) and for adding value to the agro-industrial residue discarded by the wineries, the addition of pomace flour in extruded snacks is viable and quite interesting.

Grape pomace is known to be rich in many compounds such as phenolic acids, flavonoids, tannins, and saponins presenting a high potential for pests, diseases, and weed control in agricultural crops, including vineyards. The antimicrobial effect of 'Pinot Noir' grape pomace extracts against *Staphylococcus aureus* and *Candida albicans* was already reported [18].

Considering the composition of the grape pomace, one of the possibilities is the induction of resistance of plants to pathogens. In this context, a study was carried out to evaluate the effect of autoclaved grape pomace extract (GPE) in the induction of phytoalexin deoxyanthocyanidine in sorghum [23]. To obtain the extract, ground dry pomace was macerated in water at 70°C for approximately 12 h in the dark. The GPE was sterilized by autoclaving for 20 min at 120°C under a pressure of 1.1 kgf cm−2, followed by fractionation in the tested doses. These authors reported the effect of GBE on the deoxythyanocyanidine synthesis, with a higher accumulation at the 3% dose.

The downy mildew (*Plasmopara viticola*) is responsible for qualitative and quantitative losses in grapevines production. In this context, a study was carried out aiming to verify the action of grape pomace extract (GPE) on sporangia germination of *P. viticola* and the severity of mildew on grapevines cv. Carmen [24]. These authors verified reduction of germination of the pathogen and incidence of mildew in grapevines. The highest dose of the extract (12%) controlled mildew severity at 36%, demonstrating the potential of the residue extract for the organic system.

## **4. Composition of grape seeds oil**

**Grape variety Phenolic compounds (g AGE/100 g) Anthocyanins (mg/100 g)**

Means followed by different letters on the same column are significantly different according to NSK at *P* < 0.05. NS, not

and Ancelotta (3.9 g AGE/100 g) had the highest content of phenolic compounds. Considering these results, the potential of using the residue of winemaking to produce flour for human con-

The main phenolic compounds presented in the pomace of 'Pinot Noir' grapes were (1) flavonoids: flavan-3-ols (catechin, epicatechin, epicatechin gallate, procyanidins A and B), flavonols (quercetin and quercetin methyl glucoside), and anthocyanins (delphinidin-3-glucoside, 3-glucoside cyanidin-3-glucoside, petunidin-3-glucoside, peonidin-3-glucoside, malvidin-

Grape pomace also showed a great wealth of biologically active substances in the study that were developed to determine the chemical composition of seeds and skin of grape pomace of different cultivars of *V. vinifera* grapes both white and red in Italy and in California [19]. These authors verified that in the seeds and in the skin, the Italian samples presented higher content of organic matter—lignin and copper. In addition, the contents of K, Fe, and Zn were higher in grapes from California. The Italian white grapes had a higher content of saponins in the pell

A study with grape pomace extracts (GPEs) of the grapevines 'Niagara' and 'Isabella' (*V. labrusca*) was carried out to evaluate their effects on oxidative stability and quality of chicken meat. The use of the grape pomace extract was efficient to maintain the lipid stability of the chicken meat, presenting results compatible with those exhibited by the synthetic

Due to the richness of bioactive components, the use of grape pomace flour has been tested in different food product such as biscuits [3], muffins [20], and cookies [21]. These studies showed that, in general, the addition of flour does not negatively affect the preference for food. Moreover, the presence of bioactive compounds was verified, indicating the possibility of adding nutritional value to the food with the supplementation of flour of grape pomace.

**Table 2.** Anthocyanin content and phenolic compounds in grape pomace flour in southern Brazil [18].

sumption became evident, highlighting the grapes 'Tannat' and 'Ancelotta'.

3-glucoside, vitisin) and (2) non-flavonoids: hydroxybenzoate (gallic acid) [5].

and tended to have a higher phenolic content in both the skin and the seeds.

antioxidant butyl hydroxytoluene (BHT) [14].

Cabernet Sauvignon 1.7 ± 0.29<sup>a</sup> 114.7 ± 0.15<sup>f</sup> Tannat 4.3 ± 0.18<sup>e</sup> 88.5 ± 2.02<sup>e</sup> Ancelotta 3.8 ± 0.04<sup>d</sup> 33.8 ± 2.4<sup>d</sup> Pinot Noir 7.0 ± 0.17<sup>f</sup> 3.48 ± 0.35bc Malbec 2.3 ± 0.31<sup>b</sup> 12.89 ± 0.51ac Merlot 1.7 ± 0.02<sup>a</sup> 15.78 ± 0.38<sup>a</sup> Seibel 5.9 ± 0.39<sup>c</sup> 18.35 ± 2.1<sup>a</sup> Ives 2.4 ± 0.10<sup>b</sup> 19.86 ± 6.46<sup>a</sup> Cabernet Franc 1.5 ± 0.12<sup>a</sup> 0.9 ± 0.96<sup>b</sup>

330 Grapes and Wines - Advances in Production, Processing, Analysis and Valorization

significant.

Pomace represents approximately 16% of the grapes, of which about 20–26% are seeds [25]. Although winemakers have traditionally considered these wastes an economic and environmental problem, they are gradually being considered as a potential to add value to the products. Grape seeds usually contain 8–20% oil, with about 3 million tons of grape seeds being discarded annually worldwide [26].

The complete use of grapes, including seeds, is considered an important economic and sustainability factor, since the oil has a pleasant and neutral taste and has a high concentration of linoleic acid and natural vitamin E, which provides oxidative stability to the product. These properties are related to the reduction of low-density lipoprotein (LDL) and increases in high-density lipoprotein (HDL) levels—characteristics related to cardioprotective effects [26].

**5. Conclusions**

**Author details**

Renato Vasconcelos Botelho1

\*Address all correspondence to: rbotelho@unicentro.br

4 Federal University of Paraná (UFPR), Palotina, Paraná, Brazil

http://www.oiv.org/uk/accueil/ndex.php [Accessed: 2017/04/20]

Technology. 2015;**18**(2):128-136. DOI: 10.1590/1981-6723.6014

2013;**16**(1):68-72. DOI: 10.1590/S1981-67232013005000007

Alessandro Jefferson Sato<sup>4</sup>

Guarapuava, Paraná, Brazil

Guarapuava, Paraná, Brazil

Guarapuava, Paraná, Brazil

**References**

Considering the aspects presented in this chapter, it was evident to the innumerable nutritional and pharmaceutical properties of the residues of the wine industry. Both pomace and grape seed are materials that are considered as industrial waste but are rich in vitamins, polyphenols, unsaturated fatty acids, and other important components for nutrition and with positive effects on human health. Considering the increasing need for sustainability of agricultural and industrial activities, the use of these wine by-products may represent an advance

Potential for Use of the Residues of the Wine Industry in Human Nutrition and as Agricultural…

in the production chain from an economic, social, and environmental point of view.

\*, Gabriela Datsch Bennemann2

1 Department of Agronomy, State University of Midwestern of Paraná (Unicentro),

2 Department of Nutrition, State University of Midwestern of Paraná (Unicentro),

3 Department of Chemistry, State University of Midwestern of Paraná (Unicentro),

[1] OIV. Organisation International de la Vigne et du Vin [Internet]. 2016. Available from:

[2] Paz MF, Marques RV, Schumann C, Corrêa LB, Corrêa ÉK. Características tecnológicas de pães elaborados com farelo de arroz desengordurado. Brazilian. Journal of Food

[3] Piovesana A, Bueno MM, Klajn VM. Elaboração e aceitabilidade de biscoitos enriquecidos com aveia e farinha de bagaço de uva. Brazilian Journal of Food Technology.

[4] Poudel PR, Tamura H, Kataoka I, Mochioka R. Phenolic compounds and antioxidant activities of skins and seeds of five wild grapes an two hybrids native to Japan. Journal of Food Composition and Analysis. 2008;**21**:622-625. DOI: 10.1016/j.jfca.2008.07.003

, Yohandra Reyes Torres3

http://dx.doi.org/10.5772/intechopen.73132

and

333

The oils present in grape seeds have numerous health benefits, especially vitamin E and essential fatty acids. These fatty acids are considered as protectors of cardiovascular diseases, while vitamin E has antitumor and neuroprotective properties, is able to lower cholesterol levels, and has antioxidant activity [26]. Additionally, in the extraction of the oil, part of it still remains in the residue and could be used for animal nutrition.

Seeds are rich sources of polyphenol components and have been widely studied by several groups [27–29]. Oils that are not refined may contain tocopherol and other active compounds with antioxidant properties [10].

The mineral content of grape seeds prior to the winemaking process and wine manufacturing residues has been shown to be an important source of nutrients and essential elements. In some grape seeds collected from different locations in Turkey, the mineral contents (Al, B, Ca, Co, Mo, Cr, Fe, K, Mg, Mn, Na, P, S, Se, and Zn) were determined [28]. Ca, K, Mg, Na, and P were the main minerals contained in grape seeds. While the contents of vitamins A and B1 and of the minerals Fe, Mn, and Zn were elevated in all seeds, Cu, Mo, and Cr contents were very low.

A trial was carried out to study the composition of fatty acids, oxidative stability, and antioxidant and antiproliferative activity of cold pressed oils and flours extracted from the pomace of grape cultivars Chardonnay, Concord, and Rubi [29]. In the seeds, the phenolic profiles were also measured. The most abundant fatty acid in the oils was linoleic acid, which ranged from 66.0 g 100 g−1 of total fatty acids, in cv. Rubi; to 75.3 g 100 g−1 in 'Concord' grape seed oil. The oils also had high levels of oleic acid and low levels of saturated fat. Seed oil of cv. Rubi had the highest oxidative stability index. The total phenolic content was up to 100 times lower in oils than in flours. Lutein, zeaxanthin, cryptoxanthin, β-carotene, and α-tocopherol levels were also measured. The antioxidant activity evaluated by the 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical elimination capacity ranged from 0.07 to 2.22 mmol Trolox (TE) g−1 oil equivalents and 11.8 to 15.0 mmol TE g−1 flour. In this study, the antiproliferative activity against HT-29 colon cancer cells was also tested. Seed meal and grape seed oil recorded significant (*p* < 0.05) inhibition of cancer cell growth, demonstrating the potential for the development of value-added applications for these seed oils and flours as dietary sources of natural antioxidants and anti-proliferative agents for health.

Studies on vitamin E extractions with oil of seeds of different grape varieties showed variations in concentrations. In the study with grape cultivars Barbera, Malbec, Gamay, Cabernet Sauvignon, Pinot Noir, Merlot, Cabernet Franc, and Syrah extracted with supercritical fluid, CO2, and petroleum ether, the authors found values between 3.58 mg 100 g−1 and 30.9 mg 100 g−1 of vitamin E [29]. The result of the analysis of grapes grown in Brazil presented as results the oil of the 'Isabella' grape seed containing values lower than 1 mg of tocopherol in 100 g of oil. The best results were obtained for the cvs. Cabernet Sauvignon and Merlot (5.67 and 7.03 mg 100 g−1) [30].

## **5. Conclusions**

These properties are related to the reduction of low-density lipoprotein (LDL) and increases in high-density lipoprotein (HDL) levels—characteristics related to cardioprotective effects

The oils present in grape seeds have numerous health benefits, especially vitamin E and essential fatty acids. These fatty acids are considered as protectors of cardiovascular diseases, while vitamin E has antitumor and neuroprotective properties, is able to lower cholesterol levels, and has antioxidant activity [26]. Additionally, in the extraction of the oil, part of it still

Seeds are rich sources of polyphenol components and have been widely studied by several groups [27–29]. Oils that are not refined may contain tocopherol and other active compounds

The mineral content of grape seeds prior to the winemaking process and wine manufacturing residues has been shown to be an important source of nutrients and essential elements. In some grape seeds collected from different locations in Turkey, the mineral contents (Al, B, Ca, Co, Mo, Cr, Fe, K, Mg, Mn, Na, P, S, Se, and Zn) were determined [28]. Ca, K, Mg, Na, and P were the main minerals contained in grape seeds. While the contents of vitamins A and B1 and of the minerals Fe, Mn, and Zn were elevated in all seeds, Cu, Mo, and Cr contents were

A trial was carried out to study the composition of fatty acids, oxidative stability, and antioxidant and antiproliferative activity of cold pressed oils and flours extracted from the pomace of grape cultivars Chardonnay, Concord, and Rubi [29]. In the seeds, the phenolic profiles were also measured. The most abundant fatty acid in the oils was linoleic acid, which ranged from 66.0 g 100 g−1 of total fatty acids, in cv. Rubi; to 75.3 g 100 g−1 in 'Concord' grape seed oil. The oils also had high levels of oleic acid and low levels of saturated fat. Seed oil of cv. Rubi had the highest oxidative stability index. The total phenolic content was up to 100 times lower in oils than in flours. Lutein, zeaxanthin, cryptoxanthin, β-carotene, and α-tocopherol levels were also measured. The antioxidant activity evaluated by the 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical elimination capacity ranged from 0.07 to 2.22 mmol Trolox (TE) g−1 oil equivalents and 11.8 to 15.0 mmol TE g−1 flour. In this study, the antiproliferative activity against HT-29 colon cancer cells was also tested. Seed meal and grape seed oil recorded significant (*p* < 0.05) inhibition of cancer cell growth, demonstrating the potential for the development of value-added applications for these seed oils and flours as dietary sources of natural antioxi-

Studies on vitamin E extractions with oil of seeds of different grape varieties showed variations in concentrations. In the study with grape cultivars Barbera, Malbec, Gamay, Cabernet Sauvignon, Pinot Noir, Merlot, Cabernet Franc, and Syrah extracted with supercritical fluid, CO2, and petroleum ether, the authors found values between 3.58 mg 100 g−1 and 30.9 mg 100 g−1 of vitamin E [29]. The result of the analysis of grapes grown in Brazil presented as results the oil of the 'Isabella' grape seed containing values lower than 1 mg of tocopherol in 100 g of oil. The best results were obtained for the cvs. Cabernet Sauvignon and Merlot (5.67

remains in the residue and could be used for animal nutrition.

332 Grapes and Wines - Advances in Production, Processing, Analysis and Valorization

with antioxidant properties [10].

dants and anti-proliferative agents for health.

and 7.03 mg 100 g−1) [30].

[26].

very low.

Considering the aspects presented in this chapter, it was evident to the innumerable nutritional and pharmaceutical properties of the residues of the wine industry. Both pomace and grape seed are materials that are considered as industrial waste but are rich in vitamins, polyphenols, unsaturated fatty acids, and other important components for nutrition and with positive effects on human health. Considering the increasing need for sustainability of agricultural and industrial activities, the use of these wine by-products may represent an advance in the production chain from an economic, social, and environmental point of view.

## **Author details**

Renato Vasconcelos Botelho1 \*, Gabriela Datsch Bennemann2 , Yohandra Reyes Torres3 and Alessandro Jefferson Sato<sup>4</sup>

\*Address all correspondence to: rbotelho@unicentro.br

1 Department of Agronomy, State University of Midwestern of Paraná (Unicentro), Guarapuava, Paraná, Brazil

2 Department of Nutrition, State University of Midwestern of Paraná (Unicentro), Guarapuava, Paraná, Brazil

3 Department of Chemistry, State University of Midwestern of Paraná (Unicentro), Guarapuava, Paraná, Brazil

4 Federal University of Paraná (UFPR), Palotina, Paraná, Brazil

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[21] Karnopp AR, Figueroa AM, Los PR, Teles JC, Simões DRS, Barana AC, Kubiaki FT, Oliveira JGB, Granato D. Effects of whole-wheat flour and bordeaux grape pomace (*Vitis labrusca* L.) on the sensory, physicochemical and functional properties of cookies. Food

[22] Bender ABB, Luvielmo MM, Loureiro BB, Speroni CS, Boligon AA, Silva LP. Obtenção e caracterização de farinha de casca de uva e sua utilização em snack extrusado. Brazilian

[23] Horst MV, Leite CD, Bortuli D, Santos I, Botelho RV, Faria CMDR. Induction of deoxiantocianidine in sorghum with the use of grape pomace autoclaved extract. In: Anais do VII Reunião sobre Indução de Resistência de plantas a patógenos. 2014; Maringá, Paraná

[24] Leite CD, Horst MV, Maia, AJ, Santos I, Botelho RV, Faria CMDR. Extato de bagaço de

[25] Rice AC. Solid-waste generation and by-product recovery pontential from winery resi-

[26] Choi Y, Lee J. Antioxidant and antiproliferative properties of a tocotrienol-rich fraction.

[27] Fuleki T, Ricardo-da-Silva JM. Catechin and procyanidin composition of seeds from grape cultivars grown in Ontario. Journal of Agricultural and Food Chemistry. 1997;**45**:

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[30] Freitas LS, Jacques RA, Richter MF, Silva AL, Caramão EB. Pressurized liquid extraction of vitamin E from Brazilian grape seed oil. Journal of Chromatography. 2008;**120**:80-83

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**Chapter 16**

**Provisional chapter**

**Revalorization of Grape Seed Oil for Innovative Non-**

Grape processing produces a substantial amount of residues that are highly polluting and expensive to treat, being grape seed one of the main by-products with high commercial interest. During the extraction process of grape seed oil, most of the nutraceutical compounds remain on the solid cake. This book chapter resumes the potential utilization of grape seed oil for producing biobased materials through environmentally friendly processes that could substitute petroleum-derived products. Special attention is given to transesterification and epoxidation processes. The transesterification of grape seed oil in presence of methanol drives to the production of a biodiesel with excellent low-temperature properties. According to EN 14214, grape seed oil-based biodiesel presents a slightly lower cetane number than the specified limit. In addition, this biodiesel presents a low oxidation stability which can be improved by the incorporation of oxidation stabilizer. Attending to the epoxidation of grape seed oil, short reaction times and high temperatures are advised. Epoxidized grape seed oil can be used for the synthesis of biobased polyols and its further

**Keywords:** biodiesel, biopolyol, epoxidation, grape seed oil, polyurethane foam,

**Revalorization of Grape Seed Oil for Innovative** 

DOI: 10.5772/intechopen.71710

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution,

© 2018 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

and reproduction in any medium, provided the original work is properly cited.

Grapes and grape-related products are among one of the most important horticultural products and therefore they are of high commercial interest. According to the Food and Agriculture Organization of the United Nations (FAO), the grape farming occupies 7.12 million hectares and approximately 74.49 million tonnes were produced in 2014. The top five countries

**Food Applications**

**Non-Food Applications**

Juan Carlos de Haro, Juan Francisco Rodríguez,

Juan Carlos de Haro, Juan Francisco Rodríguez,

Additional information is available at the end of the chapter

application on the synthesis of polyurethane compounds.

Additional information is available at the end of the chapter

Manuel Carmona and Ángel Pérez

Manuel Carmona and Ángel Pérez

http://dx.doi.org/10.5772/intechopen.71710

**Abstract**

transesterification

**1. Introduction**

## **Revalorization of Grape Seed Oil for Innovative Non-Food Applications Revalorization of Grape Seed Oil for Innovative Non-Food Applications**

DOI: 10.5772/intechopen.71710

Juan Carlos de Haro, Juan Francisco Rodríguez, Manuel Carmona and Ángel Pérez Manuel Carmona and Ángel Pérez Additional information is available at the end of the chapter

Juan Carlos de Haro, Juan Francisco Rodríguez,

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/intechopen.71710

#### **Abstract**

Grape processing produces a substantial amount of residues that are highly polluting and expensive to treat, being grape seed one of the main by-products with high commercial interest. During the extraction process of grape seed oil, most of the nutraceutical compounds remain on the solid cake. This book chapter resumes the potential utilization of grape seed oil for producing biobased materials through environmentally friendly processes that could substitute petroleum-derived products. Special attention is given to transesterification and epoxidation processes. The transesterification of grape seed oil in presence of methanol drives to the production of a biodiesel with excellent low-temperature properties. According to EN 14214, grape seed oil-based biodiesel presents a slightly lower cetane number than the specified limit. In addition, this biodiesel presents a low oxidation stability which can be improved by the incorporation of oxidation stabilizer. Attending to the epoxidation of grape seed oil, short reaction times and high temperatures are advised. Epoxidized grape seed oil can be used for the synthesis of biobased polyols and its further application on the synthesis of polyurethane compounds.

**Keywords:** biodiesel, biopolyol, epoxidation, grape seed oil, polyurethane foam, transesterification

## **1. Introduction**

Grapes and grape-related products are among one of the most important horticultural products and therefore they are of high commercial interest. According to the Food and Agriculture Organization of the United Nations (FAO), the grape farming occupies 7.12 million hectares and approximately 74.49 million tonnes were produced in 2014. The top five countries

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2018 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

producer of grapes are China (12.6 MTon), USA (7.15 MTon), Italy (6.93 MTon), Spain (6.22 MTon), and France (6.17 MTon) [1]. Despite grapes can be commercialized as fresh fruit, dried (raisins) and in the form of jellies and jams, beverages (wine and juice) are the most important derived products. As an estimation performed by the International Organization of Vine and Wine (OIV), 276 million hL were globally produced in 2015.

fermentation, during storage or after authorized treatments, as well as the residue obtained by the filtration or centrifugation of this product. The lees consists of a solid phase, mainly composed by microorganisms (yeast and bacteria), insoluble carbohydrates from the cellulosic and hemicellulosic fractions, phenolic compounds, lignin, proteins and tartrates; and

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The legislative situation in Europe governing the by-products produced by the wine industries only indicates that wine lees have to be withdrawn once they have been denatured to make their use in winemaking impossible. Producers with a capacity of less than 25 hL/year of wine may be exempted by the Member State from this obligation. However, most of the Member States have specified the rules for the withdrawal and legal destinations of winery

As it was previously commented, grape seed is one of the main by-products of winery industries. Even some investigations have focused on the use of grape seeds as a fuel via pyrolysis [15, 16] or gasification [17] processes, the most interesting activity from an economic point of view consists on the extraction of the interesting compounds prior to the thermal exploitation. Grape seed oil composition has been studied by different authors, focusing mainly on the fatty acid profile, the phytochemical composition, and antioxidant properties. **Table 1** shows the fatty acid profile for grape seed oil found on a previous study [18]. These values agree with the ones found in literature. For example, Beveridge et al. [19] compared 8 different grape seed oils obtained by two different extraction methods, finding a linoleic acid content between 66.76 and 73.23%. They also reported oleic acid, palmitic acid, and stearic acids as main fatty acids in the range of 12.63–18.95%, 6.28–8.62%, 3.60–5.26%, respectively. However, slight differences on the fatty acid profile can be found due to the use of different grapes vari-

a liquid phase rich in ethanol, lactic acid and acetic acid [9].

industries by-products [14].

eties and/or extraction methods [20–23].

**Table 1.** Fatty acid composition (wt.%) of grape seed oil.

**Fatty acid Percentage (wt.%)**

Palmitic C16:0 6.9 Palmitoleic C16:1 0.1 Stearic C18:0 4.0 Oleic C18:1 19.0 Linoleic C18:2 69.1 Arachidic C20:0 0.3 Gadoleic C20:1 0.3 Others 0.3

**1.1. Grape seed oil**

The socioeconomic activity of grape pressing for producing grape juice and wine imply the generation of a considerable amount of solid wastes, since approximately 30% (w/w) of the material used become a waste. There are several environmental problems associated with wineries, including water pollution, soil degradation, damage to natural vegetation, odors, and air emissions [2].

The main solid by-products and residues produced during wine production are vine shoots, grape marc or pomace (composed by skins, seeds, and stems), and wine lees.

	- Grape skins can represent up to 20% of grape pomace. This winery by-product was mainly used as compost or fuel as unusable waste, overall on the small wineries. However, grape skin represents nowadays a valuable source of biologically active phytochemicals due to the high amount of phenolic compounds that contents [11].
	- Grape stems are the structure of grape bunch and represent around 3% of grape marc mass. These are composed mainly of cellulose (30.3%), hemicelluloses (21.0%), lignin (17.4%), tannins (15.9%), and proteins (6.1%). However, the compounds that can be found in grape stems depend on several factors, such as geographic origin, time of harvest, and climatic conditions [12].
	- Grape seeds are one of the main by-products from grape processing industries. An individual grape berry typically contains two/three seeds and it constitutes around 4% of grape marc. Grape seeds contain 13–19% oil, which is rich in essential fatty acids, about 11% protein, 60–70% of non-digestible carbohydrates, non-phenolic antioxidants, such as tocopherols and beta-carotene [10], and also phenolic compounds with antioxidant capacity [13].

fermentation, during storage or after authorized treatments, as well as the residue obtained by the filtration or centrifugation of this product. The lees consists of a solid phase, mainly composed by microorganisms (yeast and bacteria), insoluble carbohydrates from the cellulosic and hemicellulosic fractions, phenolic compounds, lignin, proteins and tartrates; and a liquid phase rich in ethanol, lactic acid and acetic acid [9].

The legislative situation in Europe governing the by-products produced by the wine industries only indicates that wine lees have to be withdrawn once they have been denatured to make their use in winemaking impossible. Producers with a capacity of less than 25 hL/year of wine may be exempted by the Member State from this obligation. However, most of the Member States have specified the rules for the withdrawal and legal destinations of winery industries by-products [14].

#### **1.1. Grape seed oil**

producer of grapes are China (12.6 MTon), USA (7.15 MTon), Italy (6.93 MTon), Spain (6.22 MTon), and France (6.17 MTon) [1]. Despite grapes can be commercialized as fresh fruit, dried (raisins) and in the form of jellies and jams, beverages (wine and juice) are the most important derived products. As an estimation performed by the International Organization of Vine and

The socioeconomic activity of grape pressing for producing grape juice and wine imply the generation of a considerable amount of solid wastes, since approximately 30% (w/w) of the material used become a waste. There are several environmental problems associated with wineries, including water pollution, soil degradation, damage to natural vegetation, odors,

The main solid by-products and residues produced during wine production are vine shoots,

• Vine shoots are a non-wood lignocellulosic residue generated during the vine pruning. Manzone et al. [3] quantified the pruning residue between 1850 and 5360 kg/ha, depending on the weather conditions during the year and the structure of plantation. This residue, which is mainly composed by cellulose (34%), hemicelluloses (19%), and lignin (27%), have been used for the extraction of phenolic acids [4], lactic acid [5], and also for production of

• Grape marc is the main by-product of the wine industry. It is composed by stems, skins, and seeds that remains after pressing the grapes. Taking into account that the marc represents 15–20% of grapes' weight [9], around 13.03 Mt of grape marc are produced annually. The main chemical components of grape marc are phenols, which confer a high antioxidant

○ Grape skins can represent up to 20% of grape pomace. This winery by-product was mainly used as compost or fuel as unusable waste, overall on the small wineries. However, grape skin represents nowadays a valuable source of biologically active phyto-

○ Grape stems are the structure of grape bunch and represent around 3% of grape marc mass. These are composed mainly of cellulose (30.3%), hemicelluloses (21.0%), lignin (17.4%), tannins (15.9%), and proteins (6.1%). However, the compounds that can be found in grape stems depend on several factors, such as geographic origin, time of har-

○ Grape seeds are one of the main by-products from grape processing industries. An individual grape berry typically contains two/three seeds and it constitutes around 4% of grape marc. Grape seeds contain 13–19% oil, which is rich in essential fatty acids, about 11% protein, 60–70% of non-digestible carbohydrates, non-phenolic antioxidants, such as tocopherols and beta-carotene [10], and also phenolic compounds with antioxidant capacity [13].

• Wine lees, which are generated during the fermentation and aging processes of the wine, are defined as the residue formed at the bottom of recipients containing wine, after

chemicals due to the high amount of phenolic compounds that contents [11].

grape marc or pomace (composed by skins, seeds, and stems), and wine lees.

Wine (OIV), 276 million hL were globally produced in 2015.

338 Grapes and Wines - Advances in Production, Processing, Analysis and Valorization

and air emissions [2].

biosurfactants [6] and energy [7, 8].

vest, and climatic conditions [12].

capacity, peptic substances, cellulose, and lignin [10].

As it was previously commented, grape seed is one of the main by-products of winery industries. Even some investigations have focused on the use of grape seeds as a fuel via pyrolysis [15, 16] or gasification [17] processes, the most interesting activity from an economic point of view consists on the extraction of the interesting compounds prior to the thermal exploitation.

Grape seed oil composition has been studied by different authors, focusing mainly on the fatty acid profile, the phytochemical composition, and antioxidant properties. **Table 1** shows the fatty acid profile for grape seed oil found on a previous study [18]. These values agree with the ones found in literature. For example, Beveridge et al. [19] compared 8 different grape seed oils obtained by two different extraction methods, finding a linoleic acid content between 66.76 and 73.23%. They also reported oleic acid, palmitic acid, and stearic acids as main fatty acids in the range of 12.63–18.95%, 6.28–8.62%, 3.60–5.26%, respectively. However, slight differences on the fatty acid profile can be found due to the use of different grapes varieties and/or extraction methods [20–23].


**Table 1.** Fatty acid composition (wt.%) of grape seed oil.

With respect to vitamin E active compounds, most of the vegetable oils only contain a significant content of tocopherols, meanwhile tocotrienols are seldom. Virgin grape seed oil contains up to 10 mg α-tocopherol/100 g and different tocotrienols with a total amount of around 35 mg/100 g [24]. Crews et al. [25] investigated the total content of tocopherols and tocotrienols of 30 samples of grape seed oil, finding a total content between 63 and 1208 mg/kg. This range is much wider than the values given at the Codex Alimentarius (240–410 mg/kg). Hassanein and Abedel-Razek [26] reported a value of 380 mg/kg of total tocopherol content. These values are relatively low compared with other vegetable oils, indicating that most of vitamin E active compounds remain on the solid phase of the seed (grape seed flour).

transport of seeds into, through and out of the high-pressure extractor. Smaller amounts of seeds can be extracted in semi-batch mode, with the supercritical fluid flowing through a fixed bed of material [30]. Molero Gómez et al. [31] compared the supercritical extraction

on oil yield by working at supercritical conditions. Jokić et al. [32] determined that the optimal conditions for obtaining the highest oil yield (14.49%) and antioxidant activity

break-even value of grape seed oil obtained at 550 bar and 60°C of 5.9 €/kg using the same technology. Freitas et al. [23] pointed that supercritical propane is a more suitable solvent for grape seed oil extraction since a higher extraction yield and faster kinetic was observed. However, most of authors conclude that the grape variety, the harvesting conditions, and the cultivation structure have a major influence on the grape seed oil yield, composition

As a result of the fatty acid profile of grape seed oil with a high content of linoleic acid and the low amount of nutraceutical compounds compared with other vegetable oils, grape seed oil is not as important as olive or rapeseed oil. Moreover, phenolic compounds, whose concentration is very high in the grape seed, are only extracted into the oil only to a very small extent [24]. From this, it can be drawn that grape seed oil is not as an interesting vegetable oil as others from a nutritional point of view. Hence, in this chapter, the major non-food applications of grape seed oil are discussed, mainly focusing on the transesterification to produce biodiesel

and the epoxidation and further ring-opening reactions to produce biobased polyols.

Biodiesel is an alternative fuel for diesel engines which is made from renewable resources, such as vegetable oils and animal fats. It is biodegradable, nontoxic, sulfur- and benzene-free and, therefore, is environmentally advantageous. The recent concern about the increase in the crude oil price, the limited resources of fossil oil, and the environmental concerns have

Different alternatives have been proposed on literature to increase the content of vegetable oil-based compounds on diesel fuels. The first approach consisted on directly using vegetable oils without any chemical transformation as direct fuel. Thus, soybean oil [35], sunflower oil [36], canola oil [37] or rapeseed oil [38] have been studied. However, the direct use of vegetable oils or the use of blends with traditional petroleum-based diesel has not been considered a suitable solution due to the high viscosity, the gum formation due to oxidation processes and the deposition of carbon on piston and head of the engines [39]. As an alternative, some authors proposed the pyrolysis of rapeseed oil [40], soybean oil [41] and safflower oil [42] to yield small molecules. Nevertheless, the required equipment is expensive and the obtained results do not justify the use of this technology. In addition, the pyrolysis of vegetable oils implies the remove of oxygen from biofuel, which nowadays is a legal requirement in biobased fuels [43]. Therefore, the most interesting route to produce biodiesel from vegetable

**2. Transesterification of grape seed oil for biodiesel production**

increased the interest on vegetable oils to make biodiesel [34].

oils is the transesterification.

as extraction agent, showing a great increment

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341

Revalorization of Grape Seed Oil for Innovative Non-Food Applications

as extracting agent were 400 bar and 41°C. Fiori [33] determined a

and the non-supercritical one using CO2

and antioxidant components concentration [19].

using supercritical CO2

Phytosterols are natural sterols which occur in plants and vegetable oils. A content of phytosterols ranged from 2580 to 11,250 mg/kg phytosterols have been reported in literature. However, independently of the author, β-sitosterol is the main phytosterol found in grape seed oil (67–70%) [25, 26].

Different techniques have been proposed for grape seed oil extraction. The traditional method consists of pressing the whole seeds in a hydraulic press or the milled and heated seeds in screw press. Cold-pressing is a method of oil extraction that involves no heat or chemical treatment, and hence might retain more health beneficial components, such as natural antioxidants. The cold-pressed oils may be a better source of beneficial components, such as antioxidative phenolic compounds, as well as other health-beneficial phytochemicals. Although the yield is usually lower than that with conventional solvent extraction, there is no concern about solvent residues in the oil, making for a safer and more consumer-desired product [27]. Using this technique, Lutterodt et al. [20] determined no significant differences on the fatty acid profile of cold-pressed grape seed oil, but an increment on the oxidative stability index, indicating a higher content of antioxidant compounds.

Solvent extraction of grape seed oil is a diffusion process achieved by immersing the seed in solvent or by percolating solvent through a bed of seeds. Solvent is recovered from the oil-solvent mixture (known as micella) by an evaporator and recycled to the process. In order to determine the influence of the solvent, Fernández et al. [28] compared hexane, acetone, pentane, acetonitrile, diethyl ether, methanol, and ethanol. Among them, diethyl ether showed the highest extraction yield (20.8%). However, its use was rejected due to its high flammability and hexane was chosen as an extraction agent. It was also found that the use of polar solvents enhanced the oxidation stability of the extracted grape seed oil, indicating a higher antioxidant content in those cases. In order to combine both effects, solvents mixtures were tested and it was observed a maximum in oil yield (18.5%) and oxidation stability (16.3 h) by using a mixture hexane/acetone 1:1 (v/v). Moreover, Soxhlet and Soxtherm extration technologies were compared, without observing a significant diference on the extraction yield nor on the oxidation stability of the oil. Luque-Rodríguez et al. [29] compared superheated hexane extraction with conventional Soxhlet system, finding that similar yields can be obtained in shorter extraction times.

Supercritical fluid extraction of vegetable oils has been intensively studied in the last decades and the results of the research are protected by several patents. However, this procedure is not applied in Iarge-scale plants because of the difficulties with continuous transport of seeds into, through and out of the high-pressure extractor. Smaller amounts of seeds can be extracted in semi-batch mode, with the supercritical fluid flowing through a fixed bed of material [30]. Molero Gómez et al. [31] compared the supercritical extraction and the non-supercritical one using CO2 as extraction agent, showing a great increment on oil yield by working at supercritical conditions. Jokić et al. [32] determined that the optimal conditions for obtaining the highest oil yield (14.49%) and antioxidant activity using supercritical CO2 as extracting agent were 400 bar and 41°C. Fiori [33] determined a break-even value of grape seed oil obtained at 550 bar and 60°C of 5.9 €/kg using the same technology. Freitas et al. [23] pointed that supercritical propane is a more suitable solvent for grape seed oil extraction since a higher extraction yield and faster kinetic was observed. However, most of authors conclude that the grape variety, the harvesting conditions, and the cultivation structure have a major influence on the grape seed oil yield, composition and antioxidant components concentration [19].

With respect to vitamin E active compounds, most of the vegetable oils only contain a significant content of tocopherols, meanwhile tocotrienols are seldom. Virgin grape seed oil contains up to 10 mg α-tocopherol/100 g and different tocotrienols with a total amount of around 35 mg/100 g [24]. Crews et al. [25] investigated the total content of tocopherols and tocotrienols of 30 samples of grape seed oil, finding a total content between 63 and 1208 mg/kg. This range is much wider than the values given at the Codex Alimentarius (240–410 mg/kg). Hassanein and Abedel-Razek [26] reported a value of 380 mg/kg of total tocopherol content. These values are relatively low compared with other vegetable oils, indicating that most of vitamin E active

Phytosterols are natural sterols which occur in plants and vegetable oils. A content of phytosterols ranged from 2580 to 11,250 mg/kg phytosterols have been reported in literature. However, independently of the author, β-sitosterol is the main phytosterol found in grape

Different techniques have been proposed for grape seed oil extraction. The traditional method consists of pressing the whole seeds in a hydraulic press or the milled and heated seeds in screw press. Cold-pressing is a method of oil extraction that involves no heat or chemical treatment, and hence might retain more health beneficial components, such as natural antioxidants. The cold-pressed oils may be a better source of beneficial components, such as antioxidative phenolic compounds, as well as other health-beneficial phytochemicals. Although the yield is usually lower than that with conventional solvent extraction, there is no concern about solvent residues in the oil, making for a safer and more consumer-desired product [27]. Using this technique, Lutterodt et al. [20] determined no significant differences on the fatty acid profile of cold-pressed grape seed oil, but an increment on the oxidative stability index,

Solvent extraction of grape seed oil is a diffusion process achieved by immersing the seed in solvent or by percolating solvent through a bed of seeds. Solvent is recovered from the oil-solvent mixture (known as micella) by an evaporator and recycled to the process. In order to determine the influence of the solvent, Fernández et al. [28] compared hexane, acetone, pentane, acetonitrile, diethyl ether, methanol, and ethanol. Among them, diethyl ether showed the highest extraction yield (20.8%). However, its use was rejected due to its high flammability and hexane was chosen as an extraction agent. It was also found that the use of polar solvents enhanced the oxidation stability of the extracted grape seed oil, indicating a higher antioxidant content in those cases. In order to combine both effects, solvents mixtures were tested and it was observed a maximum in oil yield (18.5%) and oxidation stability (16.3 h) by using a mixture hexane/acetone 1:1 (v/v). Moreover, Soxhlet and Soxtherm extration technologies were compared, without observing a significant diference on the extraction yield nor on the oxidation stability of the oil. Luque-Rodríguez et al. [29] compared superheated hexane extraction with conventional Soxhlet system, finding that

Supercritical fluid extraction of vegetable oils has been intensively studied in the last decades and the results of the research are protected by several patents. However, this procedure is not applied in Iarge-scale plants because of the difficulties with continuous

compounds remain on the solid phase of the seed (grape seed flour).

340 Grapes and Wines - Advances in Production, Processing, Analysis and Valorization

indicating a higher content of antioxidant compounds.

similar yields can be obtained in shorter extraction times.

seed oil (67–70%) [25, 26].

As a result of the fatty acid profile of grape seed oil with a high content of linoleic acid and the low amount of nutraceutical compounds compared with other vegetable oils, grape seed oil is not as important as olive or rapeseed oil. Moreover, phenolic compounds, whose concentration is very high in the grape seed, are only extracted into the oil only to a very small extent [24]. From this, it can be drawn that grape seed oil is not as an interesting vegetable oil as others from a nutritional point of view. Hence, in this chapter, the major non-food applications of grape seed oil are discussed, mainly focusing on the transesterification to produce biodiesel and the epoxidation and further ring-opening reactions to produce biobased polyols.

## **2. Transesterification of grape seed oil for biodiesel production**

Biodiesel is an alternative fuel for diesel engines which is made from renewable resources, such as vegetable oils and animal fats. It is biodegradable, nontoxic, sulfur- and benzene-free and, therefore, is environmentally advantageous. The recent concern about the increase in the crude oil price, the limited resources of fossil oil, and the environmental concerns have increased the interest on vegetable oils to make biodiesel [34].

Different alternatives have been proposed on literature to increase the content of vegetable oil-based compounds on diesel fuels. The first approach consisted on directly using vegetable oils without any chemical transformation as direct fuel. Thus, soybean oil [35], sunflower oil [36], canola oil [37] or rapeseed oil [38] have been studied. However, the direct use of vegetable oils or the use of blends with traditional petroleum-based diesel has not been considered a suitable solution due to the high viscosity, the gum formation due to oxidation processes and the deposition of carbon on piston and head of the engines [39]. As an alternative, some authors proposed the pyrolysis of rapeseed oil [40], soybean oil [41] and safflower oil [42] to yield small molecules. Nevertheless, the required equipment is expensive and the obtained results do not justify the use of this technology. In addition, the pyrolysis of vegetable oils implies the remove of oxygen from biofuel, which nowadays is a legal requirement in biobased fuels [43]. Therefore, the most interesting route to produce biodiesel from vegetable oils is the transesterification.

Transesterification reactions consists of the alcoholysis of a triglyceride to form esters and glycerol. To improve the reaction rate and yield, a catalyst is usually used. As the reaction is limited by an equilibrium, excess of alcohol is used to enhance the production of esters. Among the alcohols that can be used during the transesterification process, methanol and ethanol are the most efficient and commonly used ones. With respect to catalysts, alkalis (NaOH, KOH, carbonates, and alkoxides), acids (sulfuric acid, sulfonic acid, and hydrochloric acid), and enzymes (lipases) have been proposed in literature [44]. However, alkali-catalyzed transesterification is much faster and economically viable and, therefore, is the most often used industrial system for producing biodiesel from vegetable oils [34]. However, for alkali-catalyzed transesterification, the glycerides and alcohol must be substantially anhydrous, because water causes a partial reaction change to saponification which produces soaps. Soaps consume the catalyst and reduce the catalytic efficiency, as well as causing an increase in viscosity, the formation of gels and difficulty in achieving separation of glycerol at the end of the reaction [44].

**EU (EN 14214:2012) USA (ASTM D6751-15ce1)**

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— —

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>93 ASTM D93

>3.0 EN 15751

<0.02 ASTM D6584

<15 ASTM D5453

— —

EN ISO 12185

/s 3.50–5.00 EN ISO 3104 1.9–6.0 ASTM D445

EN ISO 3679

°C — — <360 ASTM D1160

EN 15751

EN 16300

EN 14106

EN ISO 20884 EN ISO 13032

**Parameter Units Limit Method Limit Method** Ester content % (m/m) >96.5 EN 14103 — —

Cetane number — >51.0 EN ISO 5165 >47 ASTM D613 Copper band corrosion Classification 1 EN ISO 2160 3 ASTM D130

Acid value mg KOH/g <0.50 EN 14104 <0.50 ASTM D664

Cloud point °C \* EN 23015 Report ASTM D2500

Methanol content % (m/m) <0.2 EN 14110 <0.2 EN 14110 Monoglyceride content % (m/m) <0.70 EN 14105 <0.40 ASTM D6584

Total glycerol % (m/m) <0.25 EN 14105 ≤0.24 ASTM D6584

Carbon residue % (m/m) — — <0.05 ASTM D4530 Water and sediment % vol. — — <0.05 ASTM D2709

Sulphated ash content % (m/m) <0.02 ISO 3987 <0.02 ASTM D874

CFFP °C \* EN 116 — — Methyl linoleate % (m/m) <12.0 EN 14103 — — Polyunsaturated methyl esters % (m/m) <1.00 EN 15779 — —

Diglyceride content % (m/m) <0.20 EN 14105 — — Triglyceride content % (m/m) <0.20 EN 14105 — —

Water content mg/kg <500 EN ISO 12937 — —

Total contamination mg/kg <24 EN 12662 — —

Density at 15°C kg/m3 860–900 EN ISO 3675

Flash point °C >101 EN ISO 2719

Oxidation stability h >8.0 EN 14112

Iodine value g I/100 g <120 EN 14111

Free glycerol % (m/m) <0.02 EN 14105

Sulfur content mg/kg <10.0 EN ISO 20846

Viscosity at 40°C mm2

Distillation temperature (90% recovered)

In respect to the transesterification mechanism, it consists of three consecutive reversible reactions where the triglyceride is converted into diglyceride, monoglyceride, and finally, glycerol, liberating a mole of esters at each step (**Figure 1**). After the transesterification reaction, a mixture of esters, glycerol, alcohol, catalyst, and unreacted tri-, di-, and monoglycerides is obtained.

The obtained transesterification product should be purified and characterized to quantify the global biodiesel quality. **Table 2** shows the requirements of the most important regional standards and the analytic methods.

**Figure 1.** The transesterification reaction of triglycerides with alcohol.


Transesterification reactions consists of the alcoholysis of a triglyceride to form esters and glycerol. To improve the reaction rate and yield, a catalyst is usually used. As the reaction is limited by an equilibrium, excess of alcohol is used to enhance the production of esters. Among the alcohols that can be used during the transesterification process, methanol and ethanol are the most efficient and commonly used ones. With respect to catalysts, alkalis (NaOH, KOH, carbonates, and alkoxides), acids (sulfuric acid, sulfonic acid, and hydrochloric acid), and enzymes (lipases) have been proposed in literature [44]. However, alkali-catalyzed transesterification is much faster and economically viable and, therefore, is the most often used industrial system for producing biodiesel from vegetable oils [34]. However, for alkali-catalyzed transesterification, the glycerides and alcohol must be substantially anhydrous, because water causes a partial reaction change to saponification which produces soaps. Soaps consume the catalyst and reduce the catalytic efficiency, as well as causing an increase in viscosity, the formation of gels and difficulty in achieving separation of glycerol

342 Grapes and Wines - Advances in Production, Processing, Analysis and Valorization

In respect to the transesterification mechanism, it consists of three consecutive reversible reactions where the triglyceride is converted into diglyceride, monoglyceride, and finally, glycerol, liberating a mole of esters at each step (**Figure 1**). After the transesterification reaction, a mixture of esters, glycerol, alcohol, catalyst, and unreacted tri-, di-, and monoglycerides is

The obtained transesterification product should be purified and characterized to quantify the global biodiesel quality. **Table 2** shows the requirements of the most important regional stan-

at the end of the reaction [44].

dards and the analytic methods.

**Figure 1.** The transesterification reaction of triglycerides with alcohol.

obtained.


\* Differences exist between the national versions of the EN 14214 standard. These differences relate to cold weather requirements and are detailed in the national annex of each standard.

**Table 2.** Comparison of the European and American biodiesel quality standards.

Among the general parameters for biodiesel, the viscosity controls the characteristics of the injection into the engine. The viscosity of fatty acid methyl esters can reach very high values when the vegetable oil used as raw material is highly unsaturated and, therefore, it should be controlled. The flash point of a fuel is the temperature at which it will ignite when exposed to a flame or a spark. The flash point of biodiesel is higher than the petroleum-based diesel, which is safe for transport purposes. Cetane number measures how easily ignition occurs and the smoothness of combustion. Cetane number is a critical variable because it affects important performance parameters such us combustion, stability, production of white smokes, noise, and emissions of CO and hydrocarbons. The presence of mono-, di-, and triglycerides, and other non-interesting products (alcohol, glycerol, phosphorylated and sulfated compounds, etc.) cause engine problems, such as fuel filter plugging, carbon deposition, and hydrolytic and oxidative reactions of fatty acid methyl esters [34].

oxidation stability is one of the major issues affecting the use of biodiesel because it is very difficult to meet the 6 h of stability required by most of the main standards, even for many common raw materials. Therefore, since linoleic acid (C18:2) is the majoritarian fatty acid on grape seed oil composition, cetane number, iodine value, and oxidation stability require-

**Table 3.** Properties of biodiesel obtained from different vegetable oils via transesterification.

**Parameter Grape Palm Olive Rape Soybean Sunflower** Ester content (% mass) 97.8 97.7 99.0 99.5 96.9 97.2

Flash point (°C) 175 176 178 170 171 177 Cetane number 48 61 57 55 49 50 Oxidative stability at 110°C (h) 0.5 4.0 3.3 2.0 1.3 0.8 Acid value (mg KOH/g) 0.27 0.12 0.13 0.16 0.14 0.15 Iodine value (g I/100 g) 138 57 84 109 128 132 Linoleic acid content (% mass) 0.4 0.2 0.6 7.9 6.3 0.2 CFPP (°C) −6 10 −6 −10 −5 −3 Methanol content (% mass) 0.00 0.00 0.00 0.00 0.00 0.00 Monoglyceride content (% mass) 0.28 0.17 0.67 0.41 0.21 0.37 Diglyceride content (% mass) 0.08 0.06 0.09 0.08 0.10 0.07 Triglyceride content (% mass) 0.03 0.04 0.03 0.03 0.07 0.04 Free glycerol (% mass) 0.00 0.01 0.00 0.01 0.07 0.00 Total glycerol (% mass) 0.09 0.06 0.19 0.09 0.00 0.11

/s) 4.1 4.5 4.5 4.4 4.2 4.2

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345

On the other hand, biodiesel obtained from grape seed oil showed some excellent properties, such us a very low cold filter plugging point (CFPP). Certain types of biodiesel present an operability problem based on the wax settling and plugging of filters and fuel lines when overnight temperatures approach. Low-temperature properties depend mostly on the saturated ester content. In this sense, grape seed oil barely contains a 11% of saturated fatty acid

To improve the performance of grape seed oil as biodiesel feedstock, different approaches have been discussed in literature. One of the most common solutions consists of the biodiesel-biodiesel blending from different feedstocks. Fernández et al. [28] studied the feasibility of blending jatropha, grape, rape and palm biodiesel, finding good properties at specific mixing portions. Atabani et al. [48] improved the kinematic viscosity of *Sterculia foetida* methyl esters by blending it with soybean-based biodiesel. Also, biodiesel has been blended with petroleum-based diesel and other fuels to meet the standards requirements

ments cannot be easily accomplished.

Viscosity at 40°C (mm<sup>2</sup>

[49, 50].

methyl esters, so its excellent CFPP is justified [46, 47].

In their previous work, Ramos et al. [18] studied the influence of vegetable oils properties and composition on the quality of biodiesel synthesized from different vegetable oils. Among them, grape seed oil was also used as raw material. As a resume, **Table 3** shows a comparison of properties of biodiesel obtained using different vegetable oils.

As can be observed, grape seed oil-based biodiesel shows similar values of ester content, viscosity, flash point, and acid value to those obtained from traditional vegetable oils (soybean oil, sunflower oil, etc.). However, iodine number, cetane number, and oxidative stability exclude the direct use of this biodiesel because their values are out of the range indicated by the major biodiesel standards.

Iodine number is an important parameter on biodiesel quality, since heating highly unsaturated fatty acid methyl esters results in polymerization. This can lead to the formation of deposits or to deterioration of the lubricant properties of biodiesel. It is well known that biodiesel cetane number depends on the feedstock used for its production. The longer and the more saturated the fatty acid carbon chains, the higher the cetane number [45]. Also,


**Table 3.** Properties of biodiesel obtained from different vegetable oils via transesterification.

Among the general parameters for biodiesel, the viscosity controls the characteristics of the injection into the engine. The viscosity of fatty acid methyl esters can reach very high values when the vegetable oil used as raw material is highly unsaturated and, therefore, it should be controlled. The flash point of a fuel is the temperature at which it will ignite when exposed to a flame or a spark. The flash point of biodiesel is higher than the petroleum-based diesel, which is safe for transport purposes. Cetane number measures how easily ignition occurs and the smoothness of combustion. Cetane number is a critical variable because it affects important performance parameters such us combustion, stability, production of white smokes, noise, and emissions of CO and hydrocarbons. The presence of mono-, di-, and triglycerides, and other non-interesting products (alcohol, glycerol, phosphorylated and sulfated compounds, etc.) cause engine problems, such as fuel filter plugging, carbon deposition, and hydrolytic

Differences exist between the national versions of the EN 14214 standard. These differences relate to cold weather

**Parameter Units Limit Method Limit Method** Cold soak filterability test s — — <200 ASTM 7501

Group II metals (Ca + Mg) mg/kg <5.0 EN 14538 <5.0 EN 14538

Group I metals (Na + K) mg/kg <5.0 EN 14108

344 Grapes and Wines - Advances in Production, Processing, Analysis and Valorization

Phosphorous content mg/kg <4.0 EN 14107

**Table 2.** Comparison of the European and American biodiesel quality standards.

requirements and are detailed in the national annex of each standard.

**EU (EN 14214:2012) USA (ASTM D6751-15ce1)**

<5.0 EN 14538

<10 ASTM D4951

EN 14109 EN 14538

EN 16294

In their previous work, Ramos et al. [18] studied the influence of vegetable oils properties and composition on the quality of biodiesel synthesized from different vegetable oils. Among them, grape seed oil was also used as raw material. As a resume, **Table 3** shows a comparison

As can be observed, grape seed oil-based biodiesel shows similar values of ester content, viscosity, flash point, and acid value to those obtained from traditional vegetable oils (soybean oil, sunflower oil, etc.). However, iodine number, cetane number, and oxidative stability exclude the direct use of this biodiesel because their values are out of the range indicated by

Iodine number is an important parameter on biodiesel quality, since heating highly unsaturated fatty acid methyl esters results in polymerization. This can lead to the formation of deposits or to deterioration of the lubricant properties of biodiesel. It is well known that biodiesel cetane number depends on the feedstock used for its production. The longer and the more saturated the fatty acid carbon chains, the higher the cetane number [45]. Also,

and oxidative reactions of fatty acid methyl esters [34].

the major biodiesel standards.

\*

of properties of biodiesel obtained using different vegetable oils.

oxidation stability is one of the major issues affecting the use of biodiesel because it is very difficult to meet the 6 h of stability required by most of the main standards, even for many common raw materials. Therefore, since linoleic acid (C18:2) is the majoritarian fatty acid on grape seed oil composition, cetane number, iodine value, and oxidation stability requirements cannot be easily accomplished.

On the other hand, biodiesel obtained from grape seed oil showed some excellent properties, such us a very low cold filter plugging point (CFPP). Certain types of biodiesel present an operability problem based on the wax settling and plugging of filters and fuel lines when overnight temperatures approach. Low-temperature properties depend mostly on the saturated ester content. In this sense, grape seed oil barely contains a 11% of saturated fatty acid methyl esters, so its excellent CFPP is justified [46, 47].

To improve the performance of grape seed oil as biodiesel feedstock, different approaches have been discussed in literature. One of the most common solutions consists of the biodiesel-biodiesel blending from different feedstocks. Fernández et al. [28] studied the feasibility of blending jatropha, grape, rape and palm biodiesel, finding good properties at specific mixing portions. Atabani et al. [48] improved the kinematic viscosity of *Sterculia foetida* methyl esters by blending it with soybean-based biodiesel. Also, biodiesel has been blended with petroleum-based diesel and other fuels to meet the standards requirements [49, 50].

The issue of oxidative stability affects biodiesel mainly during extended storage. Generally, factors such as presence of air, elevated temperatures or presence of metals facilitate oxidation. Several approaches have been discussed in literature to either prevent or decelerate oxidation rate [51, 52]. The most obvious solution consists on preventing contact of the biodiesel with atmospheric air. Another solution is to prevent contact with prooxidative substances and avoid elevated temperatures and light. However, these solutions are not always viable and the use of antioxidants is of significant interest. Different natural and synthetic antioxidants had been used on biodiesel obtained from different vegetable oils. The most interesting result, apart from the obvious increment on oxidative stability, was that properties such us viscosity, CFPP, density, carbon residue and sulphated ash remained constant [53]. Different antioxidants, such as vitamin E [54], tocopherols [55], and synthetic ones (butylated hydroxytoluene, tert-butyl hydroquinone, and pyrogallol) [56], had been studied over biodiesel obtained from different feedstocks.

However, none of these vegetable oils are commercially available at a competitive cost. Therefore, the only remaining solution is the chemical transformation of unsaturated veg-

Revalorization of Grape Seed Oil for Innovative Non-Food Applications

http://dx.doi.org/10.5772/intechopen.71710

347

A wide variety of methods have been proposed for epoxidation of vegetable oils and related products (e.g., unsaturated free fatty acids). The most important ones are listed below [74]:

• Chlorohydrin process: this indirect epoxidation process is based on the reaction of the unsaturation with HOCl, forming the chlorohydrin which subsequently yields the epoxide on

• Halcon reaction: epoxides can also be prepared by treating the unsaturations with tertbutyl hydroperoxide using vanadium, titanium of molybdenum complexes as catalysts. • Epoxidation with dioxirane: the enantioselective epoxidation in a neutral medium is made possible by using dioxirane and an optically active manganese (III) salt as catalyst.

• Epoxidation with molecular oxygen: this process is catalyzed by silver and is the cheapest and greenest route to epoxidize low molecular weight molecules (e.g., ethylene and butadiene). However, this process is not efficient to be applied to vegetable oils, but its use is

However, the selectivity and workup of these processes are not satisfactory for industrial applications, and currently, the industrial processes are based on the use of peracids. The Prilezhaev reaction consists on the epoxidation of alkenes with peracids, typically performic or peracetic acid, formed *in situ* from hydrogen peroxide and formic or acetic acid, respectively. Among them, peracetic acid is the most used one due to its low price, higher epoxidation efficiency, and safety issues at ordinary temperatures. Acidic catalysts, either strong acids or acidic ion exchange resins, are required on this process. Dinda et al. [75] found that the order of catalyst effectiveness for the vegetable oil epoxidation reaction based on mineral

PO4

During the *in situ* epoxidation process, different reactions take place. Firstly, the peracid must be formed from hydrogen peroxide and the carboxylic acid. Secondly, the previously formed peracid attacks the alkene group, generating an oxirane group and the carboxylic acid. Hence, the carboxylic acid is not consumed during the epoxidation reaction. However, two side reaction might take place during the epoxidation process. Since oxirane rings are formed in aqueous acidic media, they can be hydrolysed into hydroxyl groups. Consequently, reactions between hydroxyl groups form different triglycerides led to the formation of oligomers linked

Grape seed oil was epoxidized using peracetic acid formed *in situ* from acetic acid and hydro-

and 100°C [73]. To evaluate the conversion and selectivity of the process, the concentration of

, HNO<sup>3</sup>

, and HCl.

as catalyst. The epoxidation reactions were carried out at 50, 60, 90,

etable oils, such us soybean [72] or grape seed oil [73], on epoxidized vegetable oils.

alkaline treatment. However, this method is highly unfriendly environmentally.

mostly restricted to low molecular weight substrates.

SO4

SO4

different functional groups was determined.

, followed by H<sup>3</sup>

**3.1. A case study: influence of temperature on grape seed oil epoxidation**

acids was headed by H2

by ether groups.

gen peroxide using H2

Nevertheless, grape seed oil-based biodiesel has been used for different applications. For example, Karthikeyan [57] proposed its use as a biofuel in marine engines by blending it with traditional petroleum-based fuel. It was found that flash point, cloud point and pour point where improved and NOx emissions were significantly reduced. Bazooyar et al. [58] compared the behavior of biodiesels based on various vegetable oils in a semi-industrial boiler. Grape seed oil biodiesel showed the lowest CO and CO2 emissions. Hence, it can be concluded that grape seed oil can be used.

## **3. Epoxidation of grape seed oil**

Epoxidation reaction consists of the addition of an oxygen atom to a carbon-carbon double bond. This reaction has been established as one of the most important methods for the formation of carbon-oxygen bonds [59]. Epoxides are highly reactive and versatile functional groups which are commonly used as precursors for alcohols, glycols, carbonyls, alkanolamines, substituted olefins, polyester polyurethanes, and epoxy resins [60, 61]. Increasing environmental problems related to fossil sources overuse have made plant oils an attractive alternative for the production of epoxy-based materials.

In the industry, epoxidized vegetable oil, and more specifically soybean and linseed oil, are currently used as plasticizers for PVC and related resins, reaching an annual production of about 200,000 tons [62]. Indeed, the epoxides change the solubility and flexibility of the PVC resins and react with hydrochloric acid liberated from the PVC resins under the prolonged action of light and heat. Epoxidized vegetable oils are also used as lubricants [63, 64] and as prepolymer in coating formulations [65–67]. The applicability of an epoxidized oil depends on its purity, oxirane number, and iodine number.

There are three different potential sources of vegetable oils containing epoxy groups. Firstly, there is a variety of natural occurring epoxy vegetable oil, mainly *Vernonia galamensis* [68] and *Euphorbia lagascae* oil [69], which contain up to 70% of vernolic acid (12S,13R-epoxy-9 cis-octadecenoic acid). The second option consists of the production of oil from genetically modified seeds which already contains vernolic acid in order to increase its content [70, 71]. However, none of these vegetable oils are commercially available at a competitive cost. Therefore, the only remaining solution is the chemical transformation of unsaturated vegetable oils, such us soybean [72] or grape seed oil [73], on epoxidized vegetable oils.

The issue of oxidative stability affects biodiesel mainly during extended storage. Generally, factors such as presence of air, elevated temperatures or presence of metals facilitate oxidation. Several approaches have been discussed in literature to either prevent or decelerate oxidation rate [51, 52]. The most obvious solution consists on preventing contact of the biodiesel with atmospheric air. Another solution is to prevent contact with prooxidative substances and avoid elevated temperatures and light. However, these solutions are not always viable and the use of antioxidants is of significant interest. Different natural and synthetic antioxidants had been used on biodiesel obtained from different vegetable oils. The most interesting result, apart from the obvious increment on oxidative stability, was that properties such us viscosity, CFPP, density, carbon residue and sulphated ash remained constant [53]. Different antioxidants, such as vitamin E [54], tocopherols [55], and synthetic ones (butylated hydroxytoluene, tert-butyl hydroquinone, and pyrogallol) [56], had been studied over biodiesel obtained from different feedstocks.

Nevertheless, grape seed oil-based biodiesel has been used for different applications. For example, Karthikeyan [57] proposed its use as a biofuel in marine engines by blending it with traditional petroleum-based fuel. It was found that flash point, cloud point and pour point where improved and NOx emissions were significantly reduced. Bazooyar et al. [58] compared the behavior of biodiesels based on various vegetable oils in a semi-industrial boiler.

Epoxidation reaction consists of the addition of an oxygen atom to a carbon-carbon double bond. This reaction has been established as one of the most important methods for the formation of carbon-oxygen bonds [59]. Epoxides are highly reactive and versatile functional groups which are commonly used as precursors for alcohols, glycols, carbonyls, alkanolamines, substituted olefins, polyester polyurethanes, and epoxy resins [60, 61]. Increasing environmental problems related to fossil sources overuse have made plant oils an attractive alternative for

In the industry, epoxidized vegetable oil, and more specifically soybean and linseed oil, are currently used as plasticizers for PVC and related resins, reaching an annual production of about 200,000 tons [62]. Indeed, the epoxides change the solubility and flexibility of the PVC resins and react with hydrochloric acid liberated from the PVC resins under the prolonged action of light and heat. Epoxidized vegetable oils are also used as lubricants [63, 64] and as prepolymer in coating formulations [65–67]. The applicability of an epoxidized oil depends

There are three different potential sources of vegetable oils containing epoxy groups. Firstly, there is a variety of natural occurring epoxy vegetable oil, mainly *Vernonia galamensis* [68] and *Euphorbia lagascae* oil [69], which contain up to 70% of vernolic acid (12S,13R-epoxy-9 cis-octadecenoic acid). The second option consists of the production of oil from genetically modified seeds which already contains vernolic acid in order to increase its content [70, 71].

emissions. Hence, it can be con-

Grape seed oil biodiesel showed the lowest CO and CO2

346 Grapes and Wines - Advances in Production, Processing, Analysis and Valorization

cluded that grape seed oil can be used.

**3. Epoxidation of grape seed oil**

the production of epoxy-based materials.

on its purity, oxirane number, and iodine number.

A wide variety of methods have been proposed for epoxidation of vegetable oils and related products (e.g., unsaturated free fatty acids). The most important ones are listed below [74]:


However, the selectivity and workup of these processes are not satisfactory for industrial applications, and currently, the industrial processes are based on the use of peracids. The Prilezhaev reaction consists on the epoxidation of alkenes with peracids, typically performic or peracetic acid, formed *in situ* from hydrogen peroxide and formic or acetic acid, respectively. Among them, peracetic acid is the most used one due to its low price, higher epoxidation efficiency, and safety issues at ordinary temperatures. Acidic catalysts, either strong acids or acidic ion exchange resins, are required on this process. Dinda et al. [75] found that the order of catalyst effectiveness for the vegetable oil epoxidation reaction based on mineral acids was headed by H2 SO4 , followed by H<sup>3</sup> PO4 , HNO<sup>3</sup> , and HCl.

During the *in situ* epoxidation process, different reactions take place. Firstly, the peracid must be formed from hydrogen peroxide and the carboxylic acid. Secondly, the previously formed peracid attacks the alkene group, generating an oxirane group and the carboxylic acid. Hence, the carboxylic acid is not consumed during the epoxidation reaction. However, two side reaction might take place during the epoxidation process. Since oxirane rings are formed in aqueous acidic media, they can be hydrolysed into hydroxyl groups. Consequently, reactions between hydroxyl groups form different triglycerides led to the formation of oligomers linked by ether groups.

#### **3.1. A case study: influence of temperature on grape seed oil epoxidation**

Grape seed oil was epoxidized using peracetic acid formed *in situ* from acetic acid and hydrogen peroxide using H2 SO4 as catalyst. The epoxidation reactions were carried out at 50, 60, 90, and 100°C [73]. To evaluate the conversion and selectivity of the process, the concentration of different functional groups was determined.

**Figure 2** shows the evolution of double bonds, epoxy groups, and hydroxyl groups along the reaction time at the previously commented reaction temperatures.

The concentration of oligomers (dimers and trimers) could be calculated through the mass balance presented in Eq. (1). This mass balance indicates that the initial content of double

(TRIM) was quantified using the area ratio of these compounds from the chromatograms. In this estimation, the same response factor was assumed for both dimers and trimers. The

When the epoxidation reaction was performed at low temperature (50 or 60°C), oligomer formation was almost neglected. However, the oligomer concentration profiles obtained at high temperatures (90 and 100°C) indicate that the formation of dimers (**Figure 3A**) is required to form a trimer (**Figure 3B**) and that a unique reaction step is not taken place. Moreover, a change in the trend can be observed at a reaction time of 1.5 and 2.5 h for the reactions performed at 90 and 100°C, respectively. This result could be explained by the lack of oxirane ring groups in the reactor at this time, suggesting that the formation of oligomers requires the

Janković and Sinadinović-Fišer [76] have already postulated a kinetic model for the *in situ* formation of peracetic acid from acetic acid and hydrogen peroxide in the presence of a homogeneous acid catalyst. The mechanism can be summarized in the following steps: (i) formation of peracetic acid in the presence of the catalyst and (ii) reaction between peracetic acid and the double bonds to produce oxirane rings, releasing acetic acid. Moreover, different secondary reactions were observed during the epoxidation of grape seed oil: the hydrolysis of oxirane

**Figure 3.** Concentration of dimmers and trimmers along the reaction time as a function of different temperature used.

*OHt*

), the oxirane rings (ORt

Revalorization of Grape Seed Oil for Innovative Non-Food Applications

). The portion of dimers (DIM) and trimers

http://dx.doi.org/10.5772/intechopen.71710

<sup>2</sup> + *OLIGt* (1)

), half of the

349

) is equal to the unreacted double bonds (DBt

), and the oligomers (OLIG<sup>t</sup>

bonds (DBt <sup>=</sup> <sup>0</sup>

hydroxyl groups (OHt

obtained values are presented in **Figure 3**.

*DBt*=0 <sup>=</sup> *DBt* <sup>+</sup> *ORt* <sup>+</sup> \_\_\_\_

presence of both oxirane rings and hydroxyl groups.

**3.2. Kinetic model for the** *in situ* **epoxidation of grape seed oil**

As can be observed, a gap exists in the double bond conversion (**Figure 2A**), depending on the reaction temperature, with increased conversion at greater temperature. If the temperature is higher than 90°C, conversions greater than 90% can be obtained in less than 1.5 h. Complete conversion of double bonds was only achieved when the reaction temperature and time were 100°C and 4 h, respectively. However, the double bonds conversion decreased much slower when the reaction temperature was 60°C or lower, requiring reaction times longer than 6 h for complete double bond conversion. Nevertheless, high double bond conversion rate cannot be the desired reaction conditions because the presence of secondary reactions can be promoted.

In respect to the oxirane ring formation (**Figure 2B**), the obtained data also indicated an important influence of reaction temperature. The reactions performed at high temperature (90 and 100°C) showed a maximum concentration of epoxide groups at short reaction times, between 1 and 1.5 h. After this, oxirane rings were completely consumed when the reaction time was 6 h. However, when the reaction was performed at lower temperatures (50 and 60°C), the epoxide group concentration increased constantly during the reaction, without observing any depletion.

In the same way, the hydroxyl group concentration (**Figure 2C**) increased abruptly at 100°C, reaching its maximum value in 4 h. At this point, the hydroxyl group concentration decreased in the reaction media, indicating the presence of another secondary reaction. On the other hand, when the reaction was performed at lower temperatures, the hydroxyl group formation was almost negligible.

The diminishing in the concentration of hydroxyl groups at 100°C indicates the presence of another secondary reaction, which mainly takes place when oxirane rings exist. The most common reaction in these cases reported in literature consists of the oligomerization of triglycerides. The generation of oligomers in the reaction bulk was confirmed by Gel Permeation Chromatography (GPC). All chromatograms presented three peaks, indicating the presence of dimers and trimers in addition to the main product.

**Figure 2.** Concentration of different functional groups along the reaction time as function of different temperature used.

The concentration of oligomers (dimers and trimers) could be calculated through the mass balance presented in Eq. (1). This mass balance indicates that the initial content of double bonds (DBt <sup>=</sup> <sup>0</sup> ) is equal to the unreacted double bonds (DBt ), the oxirane rings (ORt ), half of the hydroxyl groups (OHt ), and the oligomers (OLIG<sup>t</sup> ). The portion of dimers (DIM) and trimers (TRIM) was quantified using the area ratio of these compounds from the chromatograms. In this estimation, the same response factor was assumed for both dimers and trimers. The obtained values are presented in **Figure 3**.

$$DB\_{t=0} = DB\_t + OR\_t + \frac{OH\_t}{2} + OLG\_t \tag{1}$$

When the epoxidation reaction was performed at low temperature (50 or 60°C), oligomer formation was almost neglected. However, the oligomer concentration profiles obtained at high temperatures (90 and 100°C) indicate that the formation of dimers (**Figure 3A**) is required to form a trimer (**Figure 3B**) and that a unique reaction step is not taken place. Moreover, a change in the trend can be observed at a reaction time of 1.5 and 2.5 h for the reactions performed at 90 and 100°C, respectively. This result could be explained by the lack of oxirane ring groups in the reactor at this time, suggesting that the formation of oligomers requires the presence of both oxirane rings and hydroxyl groups.

#### **3.2. Kinetic model for the** *in situ* **epoxidation of grape seed oil**

**Figure 2** shows the evolution of double bonds, epoxy groups, and hydroxyl groups along the

As can be observed, a gap exists in the double bond conversion (**Figure 2A**), depending on the reaction temperature, with increased conversion at greater temperature. If the temperature is higher than 90°C, conversions greater than 90% can be obtained in less than 1.5 h. Complete conversion of double bonds was only achieved when the reaction temperature and time were 100°C and 4 h, respectively. However, the double bonds conversion decreased much slower when the reaction temperature was 60°C or lower, requiring reaction times longer than 6 h for complete double bond conversion. Nevertheless, high double bond conversion rate cannot be the desired reaction conditions because the presence of

In respect to the oxirane ring formation (**Figure 2B**), the obtained data also indicated an important influence of reaction temperature. The reactions performed at high temperature (90 and 100°C) showed a maximum concentration of epoxide groups at short reaction times, between 1 and 1.5 h. After this, oxirane rings were completely consumed when the reaction time was 6 h. However, when the reaction was performed at lower temperatures (50 and 60°C), the epoxide group concentration increased constantly during the reaction, without

In the same way, the hydroxyl group concentration (**Figure 2C**) increased abruptly at 100°C, reaching its maximum value in 4 h. At this point, the hydroxyl group concentration decreased in the reaction media, indicating the presence of another secondary reaction. On the other hand, when the reaction was performed at lower temperatures, the hydroxyl group formation

The diminishing in the concentration of hydroxyl groups at 100°C indicates the presence of another secondary reaction, which mainly takes place when oxirane rings exist. The most common reaction in these cases reported in literature consists of the oligomerization of triglycerides. The generation of oligomers in the reaction bulk was confirmed by Gel Permeation Chromatography (GPC). All chromatograms presented three peaks, indicating the presence

**Figure 2.** Concentration of different functional groups along the reaction time as function of different temperature used.

reaction time at the previously commented reaction temperatures.

348 Grapes and Wines - Advances in Production, Processing, Analysis and Valorization

secondary reactions can be promoted.

of dimers and trimers in addition to the main product.

observing any depletion.

was almost negligible.

Janković and Sinadinović-Fišer [76] have already postulated a kinetic model for the *in situ* formation of peracetic acid from acetic acid and hydrogen peroxide in the presence of a homogeneous acid catalyst. The mechanism can be summarized in the following steps: (i) formation of peracetic acid in the presence of the catalyst and (ii) reaction between peracetic acid and the double bonds to produce oxirane rings, releasing acetic acid. Moreover, different secondary reactions were observed during the epoxidation of grape seed oil: the hydrolysis of oxirane

**Figure 3.** Concentration of dimmers and trimmers along the reaction time as a function of different temperature used.

rings (iii) and the formation and dimers (iv) and trimers (v). Finally, it is important to take into account the degradation of hydrogen peroxide in water (vi) [77].

From the above mechanism, the following reaction system (Eqs. (2)–(7)) can be proposed:

$$AA \star H\_2O\_2 \xleftarrow{k\_1} PAA \star H\_2O \tag{2}$$

*<sup>d</sup>*[*OR*] \_\_\_\_\_

*dt* <sup>=</sup> *<sup>k</sup>*<sup>2</sup>

*<sup>d</sup>*[*TRIM*] \_\_\_\_\_\_\_

Kutta method. Hence, all the kinetic constants (k<sup>i</sup>

*<sup>d</sup>*[*OH*] \_\_\_\_\_\_

*<sup>d</sup>*[*DIM*] \_\_\_\_\_\_

[*PAA*][*DB*] − *k*<sup>3</sup>

*dt* <sup>=</sup> <sup>2</sup> *<sup>k</sup>*<sup>3</sup>

*dt* <sup>=</sup> *<sup>k</sup>*<sup>4</sup>

[*OR*]

[*OR*]

*dt* <sup>=</sup> *<sup>k</sup>*<sup>5</sup>

concentrations of the species to those predicted from the equations Eqs. (8)–(16).

[*H*<sup>2</sup> *O*] − *k*<sup>4</sup>

[*H*<sup>2</sup> *O*] − *k*<sup>4</sup>

[*OR*][*OH*] − *k*<sup>5</sup>

The differential equation system was solved numerically by using the fourth-order Runge-

A comparison between the experimental values and the theoretical trends for the concentration of DB, OR, OH, DIMS, and TRIMS is shown at **Figure 4**. As can be observed, a good agreement exists among the experimental and predicted values, with an error lower than

**Figure 4.** Theoretical and experiential profiles of functional groups along the reaction time for different temperatures.

[*OR*][*OH*] − *k*<sup>5</sup>

Revalorization of Grape Seed Oil for Innovative Non-Food Applications

[*OR*][*DIM*] (13)

351

[*OR*][*OH*] (14)

http://dx.doi.org/10.5772/intechopen.71710

[*OR*][*DIM*] (15)

[*OR*][*DIM*] (16)

) were calculated by fitting the experimental

$$\text{PAA} \star \text{DB} \xrightarrow{\bar{\bar{k}}\_2} \text{OR} \star \text{AA} \tag{3}$$

$$\text{OR} \star \text{H}\_2\text{O} \xrightarrow{\text{k}\_3} \text{2OH} \tag{4}$$

$$\text{OR} \star \text{OH} \xrightleftharpoons[]{k\_{\underline{\quad}}} \text{DIM} \tag{5}$$

$$\text{OR} + \text{DIM} \overset{k\_{\sharp}}{\rightleftharpoons} \text{TRIM} \tag{6}$$

$$H\_2O\_2 \xrightarrow{k\_6} H\_2O + 0.5 \text{ O}\_2 \tag{7}$$

where AA, PAA, DB, OR, OH, DIM, and TRIM represent the acetic acid, peracetic acid, double bond, oxirane ring, hydroxyl groups, dimers, and trimers, respectively.

The kinetic model was simplified assuming that the epoxidation reaction takes place in a unique phase (i.e., pseudo-homogeneous system). This assumption avoids the use of distribution constants, which would be necessary to quantify the concentration of the species in the organic and aqueous phases. To minimize the error caused by this assumption on the kinetic model, a high agitation rate was used. This high agitation rate favors the formation of small oil drops in the aqueous phase, increasing the external surface for mass transfer and minimizing limitations to the mass transfer [73].

Therefore, the corresponding differential equation system (Eqs. (8)–(16)) can be drawn from the proposed reaction set:

$$\frac{d[AA]}{dt} = -k\_0[AA][H\_2O\_2] + k\_1[PAA][H\_2O] + k\_2[PAA][DB] \tag{8}$$

$$\frac{d[H\_2O\_2]}{dt} = -k\_0[AA][H\_2O\_2] + k\_1[PAA][H\_2O] - k\_b[H\_2O\_2] \tag{9}$$

$$\frac{d[\text{PAA}]}{dt} = k\_y[AA] \left[H\_2O\_2\right] - k\_y[\text{PAA}] \left[H\_2O\right] - k\_2'[PAA] \left[DB\right] \tag{10}$$

$$\frac{d[H\_2O]}{dt} = k\_v[AA] \left[ H\_2O\_2 \right] - k\_v[PAA] \left[ H\_2O \right] - k\_s[OR] \left[ H\_2O \right] + k\_s[H\_2O\_2] \tag{11}$$

$$\frac{d[DB]}{dt} = -k\_2[PAA][DB] \tag{12}$$

$$\frac{d[OR]}{dt} = k\_\natural [PAA][DB] - k\_\natural [OR][H\_\natural O] - k\_\natural [OR][OH] - k\_\natural [OR][DIM] \tag{13}$$

$$\frac{d[OH]}{dt} = 2\,k\_3[OR][H\_2O] - k\_4[OR][OH] \tag{14}$$

$$\frac{d[DIM]}{dt} = k\_4[OR][OH] - k\_5[OR][DIM] \tag{15}$$

$$\frac{d[\text{TRIM}]}{dt} = k\_s[\text{OR}][\text{DIM}] \tag{16}$$

The differential equation system was solved numerically by using the fourth-order Runge-Kutta method. Hence, all the kinetic constants (k<sup>i</sup> ) were calculated by fitting the experimental concentrations of the species to those predicted from the equations Eqs. (8)–(16).

rings (iii) and the formation and dimers (iv) and trimers (v). Finally, it is important to take into

*k* ⟶2

> *k* ⟶3

*k* ⟶4

*k* ⟶5

where AA, PAA, DB, OR, OH, DIM, and TRIM represent the acetic acid, peracetic acid, double

The kinetic model was simplified assuming that the epoxidation reaction takes place in a unique phase (i.e., pseudo-homogeneous system). This assumption avoids the use of distribution constants, which would be necessary to quantify the concentration of the species in the organic and aqueous phases. To minimize the error caused by this assumption on the kinetic model, a high agitation rate was used. This high agitation rate favors the formation of small oil drops in the aqueous phase, increasing the external surface for mass transfer and minimiz-

Therefore, the corresponding differential equation system (Eqs. (8)–(16)) can be drawn from

[*PAA*]

[*PAA*]

[*PAA*]

[*H*<sup>2</sup> *O*] − *k*<sup>3</sup>

[*H*<sup>2</sup> *O*] + *k*<sup>2</sup>

[*H*<sup>2</sup> *O*] − *k*<sup>2</sup>

[*OR*]

[*H*<sup>2</sup> *O*2] + *k*<sup>1</sup>

[*H*<sup>2</sup> *O*2] − *k*<sup>1</sup>

[*H*<sup>2</sup> *O*2] + *k*<sup>1</sup>

[*PAA*]

*dt* <sup>=</sup> <sup>−</sup>*k*<sup>2</sup>

*k* ⟶6

bond, oxirane ring, hydroxyl groups, dimers, and trimers, respectively.

*PAA* + *H*<sup>2</sup>

*O* (2)

*OR* + *AA* (3)

2*OH* (4)

*DIM* (5)

*TRIM* (6)

[*PAA*][*DB*] (8)

[*PAA*][*DB*] (10)

[*H*<sup>2</sup> *O*] + *k*6[*H*<sup>2</sup> *O*2] (11)

[*H*<sup>2</sup> *O*] − *k*6[*H*<sup>2</sup> *O*2] (9)

[*PAA*][*DB*] (12)

*H*<sup>2</sup> *O* + 0.5 *O*<sup>2</sup> (7)

From the above mechanism, the following reaction system (Eqs. (2)–(7)) can be proposed:

*O*2 *k*1 ⟷ *k*0

account the degradation of hydrogen peroxide in water (vi) [77].

350 Grapes and Wines - Advances in Production, Processing, Analysis and Valorization

*AA* + *H*<sup>2</sup>

*PAA* + *DB*

*OR* + *H*<sup>2</sup> *O*

*OR* + *OH*

*OR* + *DIM*

*H*<sup>2</sup> *O*<sup>2</sup>

ing limitations to the mass transfer [73].

*dt* <sup>=</sup> <sup>−</sup>*k*<sup>0</sup>

*dt* <sup>=</sup> *<sup>k</sup>*<sup>0</sup>

[*AA*]

*dt* <sup>=</sup> <sup>−</sup>*k*<sup>0</sup>

*d*[*H*<sup>2</sup> *O*2]

[*AA*]

[*AA*]

[*AA*]

[*H*<sup>2</sup> *O*2] − *k*<sup>1</sup>

the proposed reaction set:

*<sup>d</sup>*[*AA*] \_\_\_\_\_\_

\_\_\_\_\_\_\_

*<sup>d</sup>*[*PAA*] \_\_\_\_\_\_\_

*d*[*H*<sup>2</sup> *O*] *dt* <sup>=</sup> *<sup>k</sup>*<sup>0</sup>

*<sup>d</sup>*[*DB*] \_\_\_\_\_

\_\_\_\_\_\_

A comparison between the experimental values and the theoretical trends for the concentration of DB, OR, OH, DIMS, and TRIMS is shown at **Figure 4**. As can be observed, a good agreement exists among the experimental and predicted values, with an error lower than

**Figure 4.** Theoretical and experiential profiles of functional groups along the reaction time for different temperatures.


**Table 4.** Kinetic constants for the proposed model.

0.5% in all cases. Student's *t*-test was performed, observing in all cases *t* values lower than the critical one (2.56) [73].

**Table 4** shows the obtained values for the kinetic constants. All the kinetic constants presented the same order of magnitude, excepting k<sup>3</sup> , which was the lowest. This lower value could be related to the high concentration of water that was considered in this model by neglecting the presence of a secondary aqueous phase. Therefore, it is expected that this value would increase in a two-phase model. Respecting to the values of k0 and k1 , it indicates that the peracetic formation is not favorable at high temperatures. Moreover, the kinetic constants k4 and k5 indicate that the formation of trimers is favored over dimers one.

One of the most traditional representations of kinetic constants is the Arrhenius equation (Eq. (17)). To obtain the pre-exponential factor (A) and the activation energy (EA), all obtained constants for the chemical reactions were adjusted by non-linear fitting.

$$k = A \cdot e^{\frac{-E\_s}{kT}} \tag{17}$$

a strong influence on the activation energies. Moreover, EA values are affected by the simplifications and assumptions considered along the proposed model. Therefore, slight differences in

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The uncertainty of petroleum price and its availability, combined with the global and institutional tendencies toward the principles of green chemistry, have forced the chemical industry to explore the use of renewable resources. Polyurethanes (PUs), with a global production of 12.28 Mt in 2010, are the sixth most widely used polymer [80]. This is due to the high versatility of use in mostly all the fields of polymer applications: foams, elastomers, adhesives, coatings, sealants, fibers, etc. PUs are obtained by the reaction of a polyol and a polyisocyanate, being both of these raw materials typically obtained from petroleum. However, the chemical

industry is paying a great interest to the production of biobased polyols (biopolyols).

The most interesting alternative to produce biopolyols consists of the chemical transformation of unsaturated vegetable oils through different alternatives to produce hydroxyl groups, which enable the reaction with polyisocyanates to get PUs. Some of the proposed routes in literature are the ozonolysis-hydrogenation process [81], hydroformylation [82], dimerization of fatty acids [83], thiol-ene coupling [84], and formaldehyde addition [85]. However, the most used one consists of the epoxidation of double bonds and its further epoxide ring opening.

To obtain desired biopolyols, the oxirane rings can be opened with compounds containing active hydrogen atoms, such as monoalcohols [86], amines [87, 88] or carboxylic acids [89],

In addition, if diols or triols are used as nucleophile for epoxide ring opening, then both primary and secondary hydroxyl functions could be inserted on each epoxide group. Thus, although the production of polyols from different vegetable oils has been reported, there is a lack of information about the yield of this reaction toward hydroxyl groups and the viability

Therefore, in this work, the ring-opening reaction was carried out using glycerol as ring-

The epoxidized grape seed oil previously produced, with an oxirane oxygen concentration of 5.87%, a hydroxyl value of 31.98 mg KOH/g, and an average molecular weight of 1151.75, was stirred at 900 rpm during 2 h at 80°C. **Figure 6** shows the infrared spectrum of the grape seed oil, the epoxidized oil, and the green-polyol obtained after ring-opening reaction with glycerol. It can be observed a drop in the intensity of the bands at 3009 and 1660 cm−1 related to unsaturations during the epoxidation reaction. Moreover, a big increase in the intensity area at the stretching band of 3500 cm−1 is observed, which confirms the rupture of the oxirane ring and the incorporation of a large number of terminal hydroxyl groups from the glycerol molecule.

opening agent and double metal cyanide (DMC) complex as catalyst (**Figure 5**) [90].

the activation energies values are expected.

**4.1. Ring-opening reaction with glycerol**

of using grape seed oil as unsaturated vegetable oil.

among others.

**4. Synthesis of biopolyols**

The EA, A, and coefficient of determination values (R<sup>2</sup> ) are summarized in **Table 5**. A good fit between the experimental kinetic constants and the Arrhenius equation was observed for all reactions, except for the reverse reaction of peracetic acid synthesis (k1 ).

Different authors have determined the EA value of different *in situ* epoxidation system using different vegetable oils as raw materials. For example, Cai et al. [78] and Mungroo et al. [79] found a value of 10.3 and 10.7 kcal/mol, using soybean and canola oil as raw material, respectively. There is a small difference among these values and our determined one (7.3 kcal/mol). Janković and Sinadinović-Fišer [76] concluded that the number and the position of the double bonds, their position with respect to the carboxylic group, and the presence of *cis-* or *trans-*isomers have


**Table 5.** Arrhenius equation parameters.

a strong influence on the activation energies. Moreover, EA values are affected by the simplifications and assumptions considered along the proposed model. Therefore, slight differences in the activation energies values are expected.

## **4. Synthesis of biopolyols**

0.5% in all cases. Student's *t*-test was performed, observing in all cases *t* values lower than the

**k3 (L/mol min)**

 1.35 × 10−2 6.56 × 10−2 0.129 0.247 × 10−4 0.106 × 10−3 1.04 × 10−2 5.85 × 10−3 1.20 × 10−2 4.02 × 10−2 0.178 0.288 × 10−4 5.94 × 10−3 1.05 × 10−2 7.10 × 10−3 1.01 × 10−2 0.102 × 10−2 0.485 1.38 × 10−4 3.81 × 10−3 1.50 × 10−2 0.14 × 10−3 0.990 × 10−2 0.573 × 10−2 0.560 1.75 × 10−4 3.07 × 10−3 1.70 × 10−2 6.30 × 10−3

**k4 (L/mol min)**

**Table 4** shows the obtained values for the kinetic constants. All the kinetic constants pre-

could be related to the high concentration of water that was considered in this model by neglecting the presence of a secondary aqueous phase. Therefore, it is expected that this value

the peracetic formation is not favorable at high temperatures. Moreover, the kinetic constants

One of the most traditional representations of kinetic constants is the Arrhenius equation (Eq. (17)). To obtain the pre-exponential factor (A) and the activation energy (EA), all obtained

between the experimental kinetic constants and the Arrhenius equation was observed for all

Different authors have determined the EA value of different *in situ* epoxidation system using different vegetable oils as raw materials. For example, Cai et al. [78] and Mungroo et al. [79] found a value of 10.3 and 10.7 kcal/mol, using soybean and canola oil as raw material, respectively. There is a small difference among these values and our determined one (7.3 kcal/mol). Janković and Sinadinović-Fišer [76] concluded that the number and the position of the double bonds, their position with respect to the carboxylic group, and the presence of *cis-* or *trans-*isomers have

**Kinetic constant k0 k1 k2 k3 k4 k5 k6** EA (kcal/mol) −1.46 −16.51 7.30 10.19 −5.33 2.47 5.33 A 1.35 × 10−3 4.35 × 10−13 1.12 × 10<sup>4</sup> 171 2.27 × 10−6 0.468 23.18 R2 0.972 0.738 0.994 0.979 0.937 0.966 0.996

−*E*\_\_\_\_*<sup>A</sup>*

indicate that the formation of trimers is favored over dimers one.

, which was the lowest. This lower value

**k5 (L/mol min)**

**k6 (L/min)**

and k1

*<sup>R</sup>*·*<sup>T</sup>* (17)

).

) are summarized in **Table 5**. A good fit

, it indicates that

critical one (2.56) [73].

 **(L/mol min)**

**k1 (L/mol min)**

**Table 4.** Kinetic constants for the proposed model.

**k2 (L/mol min)**

352 Grapes and Wines - Advances in Production, Processing, Analysis and Valorization

**T (°C) k0**

k4 and k5

sented the same order of magnitude, excepting k<sup>3</sup>

*k* = *A* · *e*

**Table 5.** Arrhenius equation parameters.

The EA, A, and coefficient of determination values (R<sup>2</sup>

would increase in a two-phase model. Respecting to the values of k0

constants for the chemical reactions were adjusted by non-linear fitting.

reactions, except for the reverse reaction of peracetic acid synthesis (k1

The uncertainty of petroleum price and its availability, combined with the global and institutional tendencies toward the principles of green chemistry, have forced the chemical industry to explore the use of renewable resources. Polyurethanes (PUs), with a global production of 12.28 Mt in 2010, are the sixth most widely used polymer [80]. This is due to the high versatility of use in mostly all the fields of polymer applications: foams, elastomers, adhesives, coatings, sealants, fibers, etc. PUs are obtained by the reaction of a polyol and a polyisocyanate, being both of these raw materials typically obtained from petroleum. However, the chemical industry is paying a great interest to the production of biobased polyols (biopolyols).

The most interesting alternative to produce biopolyols consists of the chemical transformation of unsaturated vegetable oils through different alternatives to produce hydroxyl groups, which enable the reaction with polyisocyanates to get PUs. Some of the proposed routes in literature are the ozonolysis-hydrogenation process [81], hydroformylation [82], dimerization of fatty acids [83], thiol-ene coupling [84], and formaldehyde addition [85]. However, the most used one consists of the epoxidation of double bonds and its further epoxide ring opening.

#### **4.1. Ring-opening reaction with glycerol**

To obtain desired biopolyols, the oxirane rings can be opened with compounds containing active hydrogen atoms, such as monoalcohols [86], amines [87, 88] or carboxylic acids [89], among others.

In addition, if diols or triols are used as nucleophile for epoxide ring opening, then both primary and secondary hydroxyl functions could be inserted on each epoxide group. Thus, although the production of polyols from different vegetable oils has been reported, there is a lack of information about the yield of this reaction toward hydroxyl groups and the viability of using grape seed oil as unsaturated vegetable oil.

Therefore, in this work, the ring-opening reaction was carried out using glycerol as ringopening agent and double metal cyanide (DMC) complex as catalyst (**Figure 5**) [90].

The epoxidized grape seed oil previously produced, with an oxirane oxygen concentration of 5.87%, a hydroxyl value of 31.98 mg KOH/g, and an average molecular weight of 1151.75, was stirred at 900 rpm during 2 h at 80°C. **Figure 6** shows the infrared spectrum of the grape seed oil, the epoxidized oil, and the green-polyol obtained after ring-opening reaction with glycerol. It can be observed a drop in the intensity of the bands at 3009 and 1660 cm−1 related to unsaturations during the epoxidation reaction. Moreover, a big increase in the intensity area at the stretching band of 3500 cm−1 is observed, which confirms the rupture of the oxirane ring and the incorporation of a large number of terminal hydroxyl groups from the glycerol molecule.

**Figure 5.** Reaction scheme of the ring-opening process in presence of glycerol.

The developed renewable green-polyol presents the following characteristics: an oxirane oxygen concentration of 1.06%, a hydroxyl value of 80.84 mg KOH/g, and an average molecular weight of 1800.94 g/mol. These values indicate that 82% of the oxirane rings were opened by the glycerol.

Moreover, the azide group is one of the two main parts for performing the 1,3-dipolar cycloaddition reaction among azides and alkynes in the field of click chemistry [95]. The latter one is of the most actively investigated approaches for preparing tailor-made bioactive substances

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The azidification reaction was performed at three different temperatures (50, 60, and 70°C) for 24 h. Samples were extracted at four points: 1, 2, 4, and 24 h [92]. The hydroxyl value and

As can be observed, high reaction times and temperatures favor the transformation of oxirane rings into hydroxyl groups. The influence of temperature was determined considering that the reactions follow a pseudo-first-order kinetics. Hence, the observed kinetic constant (kobs) can be determined from the slope of the representation ln(Cepox) vs. reaction time. The high correlation coefficient

 = 0.990) indicated a good fit between the experimental and theoretical values. Moreover, it is also remarkable that longer reaction times than 24 h are required to achieve full conversion of epoxide groups. However, the polyol obtained after 24 h at 70°C was used for the subse-

oxirane content of the synthesized azidified biopolyols are presented in **Figure 8**.

**Figure 6.** Infrared spectrum of the grape seed oil, the epoxidized oil, and the vegetable biopolyol.

at this time.

(R2

quent foaming process.

## **4.2. Ring-opening reaction with NaN3**

As it was previously commented, biopolyols synthetized from epoxidized vegetable oils can be functionalized by different groups, when the epoxide group is hydrolysed. Among the possible pathways, the ring-opening reaction with sodium azide [91] is one of the most interesting alternatives for obtaining very interesting compounds (**Figure 7**) [92].

The presence of nitrogen atoms in the structure of the biopolyol resulting from the incorporation of azide groups enables the polyol to act as thermal stabilizer compound in polyurethane foams [93]. Additionally, the azide group is highly polar, and when it is linked to a hydrocarbon chain, the hydrophobic character is modified, favoring its use as a surfactant [94].

**Figure 6.** Infrared spectrum of the grape seed oil, the epoxidized oil, and the vegetable biopolyol.

The developed renewable green-polyol presents the following characteristics: an oxirane oxygen concentration of 1.06%, a hydroxyl value of 80.84 mg KOH/g, and an average molecular weight of 1800.94 g/mol. These values indicate that 82% of the oxirane rings were opened by the glycerol.

As it was previously commented, biopolyols synthetized from epoxidized vegetable oils can be functionalized by different groups, when the epoxide group is hydrolysed. Among the possible pathways, the ring-opening reaction with sodium azide [91] is one of the most inter-

The presence of nitrogen atoms in the structure of the biopolyol resulting from the incorporation of azide groups enables the polyol to act as thermal stabilizer compound in polyurethane foams [93]. Additionally, the azide group is highly polar, and when it is linked to a hydrocarbon chain, the hydrophobic character is modified, favoring its use as a surfactant [94].

esting alternatives for obtaining very interesting compounds (**Figure 7**) [92].

**Figure 5.** Reaction scheme of the ring-opening process in presence of glycerol.

354 Grapes and Wines - Advances in Production, Processing, Analysis and Valorization

**4.2. Ring-opening reaction with NaN3**

Moreover, the azide group is one of the two main parts for performing the 1,3-dipolar cycloaddition reaction among azides and alkynes in the field of click chemistry [95]. The latter one is of the most actively investigated approaches for preparing tailor-made bioactive substances at this time.

The azidification reaction was performed at three different temperatures (50, 60, and 70°C) for 24 h. Samples were extracted at four points: 1, 2, 4, and 24 h [92]. The hydroxyl value and oxirane content of the synthesized azidified biopolyols are presented in **Figure 8**.

As can be observed, high reaction times and temperatures favor the transformation of oxirane rings into hydroxyl groups. The influence of temperature was determined considering that the reactions follow a pseudo-first-order kinetics. Hence, the observed kinetic constant (kobs) can be determined from the slope of the representation ln(Cepox) vs. reaction time. The high correlation coefficient (R2 = 0.990) indicated a good fit between the experimental and theoretical values. Moreover, it is also remarkable that longer reaction times than 24 h are required to achieve full conversion of epoxide groups. However, the polyol obtained after 24 h at 70°C was used for the subsequent foaming process.

**Figure 7.** Reaction scheme of the ring-opening process in presence of sodium azide.

Kinetic constant values of 2.70 × 10−2, 5.05 × 10−2, and 7.18 × 10−2 s−1 were obtained at 50, 60, and 70°C, respectively. As expected, higher reaction temperatures favored the ring-opening reaction. Arrhenius equation (Eq. (17)) was used to determine the EA and A for the proposed reaction system. Also in this case, a good correlation (R2 = 0.987) was observed. EA and A was calculated from the slope and y-intercept of its linearized form, obtaining values of EA = 0.341 J/ mol and A = 0.121.

#### **4.3. Rigid polyurethane foam synthesis**

As was stated previously, polyurethanes constitute a large family of polymers which is characterized for their versatility. Depending on the characteristics and structures of polyols and polyisocyanates, polyurethanes can be obtained as thermoplastics, adhesives, coatings, foams, etc. Rigid polyurethane foams are widely used in different applications, such as thermal [96] and acoustic [97] insulation.

The versatility of these products and the great consumption of polyurethanes (PUs) in the new emerging economies, which are believed to increase in incoming years, are forcing researchers to find alternative raw materials to avoid the petroleum dependence. Related to this, use and disposal of petroleum-based PUs is to be taken into account. Therefore, since polyol components usually are around half the weight of the final product, the use of greener polyols in PU synthesis has attracted much interest during the last decade [98].

In this section, different rigid PU foams were synthesized from the previously synthesized grape-seed-oil-based polyols. The commercial polyol Alcupol R4520 was used as a reference material to compare the internal structure and properties of the final product. **Table 6** shows the formulations used to synthesize the PU foams. The required quantities of these reactants were calculated based on the hydroxyl number of the polyol used to synthesize the rigid PU

**Figure 8.** Oxirane content and hydroxyl values for the azidified biopolyol obtained at different reaction temperatures

Type of polyol R4520 Vegetable biopolyol Azidified biopolyol

Polyol (wt.%) 47.75 48.43 47.75 Water (wt.%) 0.75 1.21 0.75 Tegostab B8404 (wt.%) 3 0.73 3 Tegoamin 33 (wt.%) 0.37 0.37 0.37 Tegoamin BDE (wt.%) 0.37 1.20 0.37 MDI (wt.%) 47.76 48.06 47.76

**Table 6.** Weight percentages of raw materials for the synthesis of rigid PU foams.

**Foam A Foam B Foam C**

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foam, as indicated elsewhere [99].

during 24 h.

Revalorization of Grape Seed Oil for Innovative Non-Food Applications http://dx.doi.org/10.5772/intechopen.71710 357

**Figure 8.** Oxirane content and hydroxyl values for the azidified biopolyol obtained at different reaction temperatures during 24 h.


**Table 6.** Weight percentages of raw materials for the synthesis of rigid PU foams.

Kinetic constant values of 2.70 × 10−2, 5.05 × 10−2, and 7.18 × 10−2 s−1 were obtained at 50, 60, and 70°C, respectively. As expected, higher reaction temperatures favored the ring-opening reaction. Arrhenius equation (Eq. (17)) was used to determine the EA and A for the proposed

calculated from the slope and y-intercept of its linearized form, obtaining values of EA = 0.341 J/

As was stated previously, polyurethanes constitute a large family of polymers which is characterized for their versatility. Depending on the characteristics and structures of polyols and polyisocyanates, polyurethanes can be obtained as thermoplastics, adhesives, coatings, foams, etc. Rigid polyurethane foams are widely used in different applications, such as thermal [96]

The versatility of these products and the great consumption of polyurethanes (PUs) in the new emerging economies, which are believed to increase in incoming years, are forcing researchers to find alternative raw materials to avoid the petroleum dependence. Related to this, use and disposal of petroleum-based PUs is to be taken into account. Therefore, since polyol components usually are around half the weight of the final product, the use of greener polyols in PU synthesis has attracted much interest during the last

In this section, different rigid PU foams were synthesized from the previously synthesized grape-seed-oil-based polyols. The commercial polyol Alcupol R4520 was used as a reference

= 0.987) was observed. EA and A was

reaction system. Also in this case, a good correlation (R2

**Figure 7.** Reaction scheme of the ring-opening process in presence of sodium azide.

356 Grapes and Wines - Advances in Production, Processing, Analysis and Valorization

**4.3. Rigid polyurethane foam synthesis**

mol and A = 0.121.

and acoustic [97] insulation.

decade [98].

material to compare the internal structure and properties of the final product. **Table 6** shows the formulations used to synthesize the PU foams. The required quantities of these reactants were calculated based on the hydroxyl number of the polyol used to synthesize the rigid PU foam, as indicated elsewhere [99].

No differences during the foaming process (growth start time, growth rate, and final aspect of the foam) were observed between the two polyols. The only appreciable difference that was observed between the three PU foams was that the azidified foam was slightly darker than the commercial one due to the higher coloration of the azidified biopolyol [91].

This increase can primarily be attributed to two effects: the different volumes of pendant groups and the unadjusted foam formulation, which modify the nucleation and foaming rate. Finally, thermogravimetric analyses (TGA) were used to investigate the effect of the incorporation of azide groups on the thermal stability of the biopolyols. **Figure 10** compares the TGA

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Two degradation steps can be observed for both polyurethane foams. The first one is assigned to the thermal degradation of the crosslinked polymer, which can be divided into hard and soft segments. The second step is attributed to the decomposition of the hydrocarbon chains formed by the main components of the foam (polyol and isocyanate) [100, 101]. As can be observed in the thermogravimetric curve, the incorporation of 0.09 g of nitrogen per gram of Foam C increased the maximum degradation temperature from 330 to 450°C. Moreover, the residue yield observed at 650°C improved from 6.28 to 8.11, indicating that Foam C is more thermally stable than Foam A. These results confirm the enhancement in the thermal stability of the PU foams because of incorporating nitrogen groups into their chemical structures by

Cosmetics are substances or products used to enhance or modify the aspect or smell of the body. During the last years, a wide sector of cosmetic consumers is expressing their interest in natural products due to the appearance of allergies and skin irritations due to the synthetic preservatives (mainly parabens), colorants, stabilizers, etc. [102]. Grape seed oil has been presented by Fiume et al. [103], as a safe alternative to these synthetic compounds, which are to be incorporated in cosmetics. Mbah [104] compared different vegetable oils with grape seed to act as lipophilic substance, indicating that it can be a potential dermal permeation enhancer. Attending to micro- and nano-structured cosmetic systems, Contri et al. [105] determined the viability of preparing grape seed oil nanocapsules for cutaneous applications, which enhanced the antioxidant activity. Moreover, Glampedaki and Dutschk [102] studied the preparation of oil-in-water emulsions using wine and grape seed oil, observing a good stability, when using glycerol monostearate as

Grape seed oil was compared with soybean and rapeseed oil for producing renewable crosslinked polyester resins by Clark et al. [106]. They concluded that grape seed oil presented a lower crosslink density due to its higher linoleic acid content but a higher thermal stability, being this product a viable substitute of traditional polyester resins in low stress or high-

Grape seed oil is one of the main by-products of the winery industry. The fatty acid profile of this oil showed that it is mainly composed by linoleic and oleic acid, but the total amount

and DTG curves for Foam A and Foam C under an air atmosphere.

using azidified biopolyols from grape seed oil as raw materials.

**5. Other non-food applications**

emulsifying agent.

**6. Conclusions**

demanding thermal applications.

The internal morphology of all the rigid PU foams was observed using a scanning electron microscope (SEM) (**Figure 9**). In all three foams, a polyhedral cell structure can be observed. A slight increase in the cell size of Foams B and C with respect to that of Foam A can be observed.

**Figure 9.** SEM images of (a) Foam A (synthesized from commercial polyol) (b) Foam B (synthesized from the biopolyol obtained using glycol as ring-opening agent) and (c) Foam C (synthesized from the azidified biopolyol).

**Figure 10.** Comparison between thermogravimetric curves of Foam A (commercial foam) and Foam C (azidified foam).

This increase can primarily be attributed to two effects: the different volumes of pendant groups and the unadjusted foam formulation, which modify the nucleation and foaming rate.

Finally, thermogravimetric analyses (TGA) were used to investigate the effect of the incorporation of azide groups on the thermal stability of the biopolyols. **Figure 10** compares the TGA and DTG curves for Foam A and Foam C under an air atmosphere.

Two degradation steps can be observed for both polyurethane foams. The first one is assigned to the thermal degradation of the crosslinked polymer, which can be divided into hard and soft segments. The second step is attributed to the decomposition of the hydrocarbon chains formed by the main components of the foam (polyol and isocyanate) [100, 101]. As can be observed in the thermogravimetric curve, the incorporation of 0.09 g of nitrogen per gram of Foam C increased the maximum degradation temperature from 330 to 450°C. Moreover, the residue yield observed at 650°C improved from 6.28 to 8.11, indicating that Foam C is more thermally stable than Foam A. These results confirm the enhancement in the thermal stability of the PU foams because of incorporating nitrogen groups into their chemical structures by using azidified biopolyols from grape seed oil as raw materials.

## **5. Other non-food applications**

No differences during the foaming process (growth start time, growth rate, and final aspect of the foam) were observed between the two polyols. The only appreciable difference that was observed between the three PU foams was that the azidified foam was slightly darker than the

The internal morphology of all the rigid PU foams was observed using a scanning electron microscope (SEM) (**Figure 9**). In all three foams, a polyhedral cell structure can be observed. A slight increase in the cell size of Foams B and C with respect to that of Foam A can be observed.

**Figure 9.** SEM images of (a) Foam A (synthesized from commercial polyol) (b) Foam B (synthesized from the biopolyol

**Figure 10.** Comparison between thermogravimetric curves of Foam A (commercial foam) and Foam C (azidified foam).

obtained using glycol as ring-opening agent) and (c) Foam C (synthesized from the azidified biopolyol).

commercial one due to the higher coloration of the azidified biopolyol [91].

358 Grapes and Wines - Advances in Production, Processing, Analysis and Valorization

Cosmetics are substances or products used to enhance or modify the aspect or smell of the body. During the last years, a wide sector of cosmetic consumers is expressing their interest in natural products due to the appearance of allergies and skin irritations due to the synthetic preservatives (mainly parabens), colorants, stabilizers, etc. [102]. Grape seed oil has been presented by Fiume et al. [103], as a safe alternative to these synthetic compounds, which are to be incorporated in cosmetics. Mbah [104] compared different vegetable oils with grape seed to act as lipophilic substance, indicating that it can be a potential dermal permeation enhancer. Attending to micro- and nano-structured cosmetic systems, Contri et al. [105] determined the viability of preparing grape seed oil nanocapsules for cutaneous applications, which enhanced the antioxidant activity. Moreover, Glampedaki and Dutschk [102] studied the preparation of oil-in-water emulsions using wine and grape seed oil, observing a good stability, when using glycerol monostearate as emulsifying agent.

Grape seed oil was compared with soybean and rapeseed oil for producing renewable crosslinked polyester resins by Clark et al. [106]. They concluded that grape seed oil presented a lower crosslink density due to its higher linoleic acid content but a higher thermal stability, being this product a viable substitute of traditional polyester resins in low stress or highdemanding thermal applications.

## **6. Conclusions**

Grape seed oil is one of the main by-products of the winery industry. The fatty acid profile of this oil showed that it is mainly composed by linoleic and oleic acid, but the total amount and proportion of this depend on the grape variety, the weather, and the extraction method. However, grape seed oil has a limited application from a nutritional point of view, since most nutraceutical compounds (vitamin E active compounds, phytosterols, etc.) remain on other by-products, mainly on the grape skin and grape seed flour. Hence, different nonfood applications have been proposed and discussed along with this book chapter.

[2] Jin B, Kelly JM. Wine industry residues. In: Biotechnology for Agro-Industrial Residues Utilisation: Utilisation of Agro-Residues. Dordrecht, Netherlands: Springer; 2009.

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jf903441d

bp049603v

10.1016/j.indcrop.2015.11.003

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The transesterification of grape seed oil leads to the production of a biodiesel with excellent low-temperature properties. On the other hand, other quality requirements, such as cetane number and oxidation stability, cannot be easily accomplished using grape seed oil as raw material due to its high content of polyunsaturated fatty acids. However, this can be easily solved by blending the grape seed oil based biodiesel with other fuels.

The epoxidation of grape seed oil using peracetic acid formed *in situ* from acetic acid and hydrogen peroxide is a feasible process for obtaining a highly epoxidated vegetable oil. The negative effect of the secondary reactions of this process, namely the hydrolysis and further oligomerization, can be diminished by working at high temperature (90°C) and short reaction times (1 h).

Epoxidized vegetable oils, such as grape seed oil, are of high interest for producing biobased materials that nowadays are produced from petroleum. So, biopolyols can be produced from epoxidized grape seed oil using different ring-opening agents such us glycerol and sodium azide. Different rigid polyurethane foams were synthesized from these biopolyols, using a commercial petroleum-based polyol as reference material to compare the internal structure and their properties. No internal differences were observed between the foams. However, it was observed an increase on the thermal stability of the foam which incorporated N<sup>3</sup> groups.

## **Acknowledgements**

Authors gratefully acknowledge the financial support from Spanish Ministry of Education, Culture and Sport for the fellowship for PhD studies FPU014/00009.

## **Author details**

Juan Carlos de Haro\*, Juan Francisco Rodríguez, Manuel Carmona and Ángel Pérez

\*Address all correspondence to: jcarlos.haro@uclm.es

Department of Chemical Engineering, Institute of Chemical and Environmental Technology, University of Castilla-La Mancha, Ciudad Real, Spain

## **References**

[1] FAOSTAT [Internet]. 2014. Available from: http://www.fao.org/faostat [Accessed: 2017]

[2] Jin B, Kelly JM. Wine industry residues. In: Biotechnology for Agro-Industrial Residues Utilisation: Utilisation of Agro-Residues. Dordrecht, Netherlands: Springer; 2009. pp. 293-311. DOI: 10.1007/978-1-4020-9942-7\_15

and proportion of this depend on the grape variety, the weather, and the extraction method. However, grape seed oil has a limited application from a nutritional point of view, since most nutraceutical compounds (vitamin E active compounds, phytosterols, etc.) remain on other by-products, mainly on the grape skin and grape seed flour. Hence, different non-

The transesterification of grape seed oil leads to the production of a biodiesel with excellent low-temperature properties. On the other hand, other quality requirements, such as cetane number and oxidation stability, cannot be easily accomplished using grape seed oil as raw material due to its high content of polyunsaturated fatty acids. However, this can be easily

The epoxidation of grape seed oil using peracetic acid formed *in situ* from acetic acid and hydrogen peroxide is a feasible process for obtaining a highly epoxidated vegetable oil. The negative effect of the secondary reactions of this process, namely the hydrolysis and further oligomerization, can be diminished by working at high temperature (90°C) and short reaction times (1 h). Epoxidized vegetable oils, such as grape seed oil, are of high interest for producing biobased materials that nowadays are produced from petroleum. So, biopolyols can be produced from epoxidized grape seed oil using different ring-opening agents such us glycerol and sodium azide. Different rigid polyurethane foams were synthesized from these biopolyols, using a commercial petroleum-based polyol as reference material to compare the internal structure and their properties. No internal differences were observed between the foams. However, it

was observed an increase on the thermal stability of the foam which incorporated N<sup>3</sup>

Juan Carlos de Haro\*, Juan Francisco Rodríguez, Manuel Carmona and Ángel Pérez

Culture and Sport for the fellowship for PhD studies FPU014/00009.

\*Address all correspondence to: jcarlos.haro@uclm.es

University of Castilla-La Mancha, Ciudad Real, Spain

Authors gratefully acknowledge the financial support from Spanish Ministry of Education,

Department of Chemical Engineering, Institute of Chemical and Environmental Technology,

[1] FAOSTAT [Internet]. 2014. Available from: http://www.fao.org/faostat [Accessed: 2017]

**Acknowledgements**

**Author details**

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## *Edited by António Manuel Jordão and Fernanda Cosme*

The book "*Grapes and Wines: Advances in Production, Processing, Analysis, and Valorization*" intends to provide to the reader a comprehensive overview of the current state-of-the-art and different perspectives regarding the most recent knowledge related to grape and wine production. Thus, this book is composed of three different general sections: (1) Viticulture and Environmental Conditions, (2) Wine Production and Characterization, and (3) Economic Analysis and Valorization of Wine Products. Inside these 3 general sections, 16 different chapters provide current research on different topics of recent advances on production, processing, analysis, and valorization of grapes and wines. All chapters are written by a group of international researchers, in order to provide up-to-date reviews, overviews, and summaries of current research on the different dimensions of grape and wine production. This book is not only intended for technicians actively engaged in the field but also for students attending technical schools and/or universities and other professionals that might be interested in reading and learning about some fascinating areas of grape and wine research.

Grapes and Wines - Advances in Production, Processing, Analysis and Valorization

Grapes and Wines

Advances in Production, Processing,

Analysis and Valorization

*Edited by António Manuel Jordão* 

*and Fernanda Cosme*

Photo by khuang54 / iStock