Pursuing the Perfect Performer of Fermented Beverages: GMMs vs. Microbial Consortium

Jesús Alejandro Aldrete-Tapia, Dalia Elizabeth Miranda-Castilleja, Sofia Maria Arvizu-Medrano, Ramón Álvar Martínez-Peniche, Lourdes Soto-Muñoz and Montserrat Hernández-Iturriaga

## Abstract

Fermented beverages are widely diverse around the world and their quality is largely based on the organoleptic characteristics developed by the metabolism of the microorganisms present during fermentation. In order to achieve controllable processes in fermented beverages along with organoleptic complexity, two divergent approaches have been followed in terms of inoculum development: (1) the inoculation of multiple microorganisms, intending to promote synergism and favor organoleptic complexity derived from the metabolic diversity, and (2) the genetic modification of a single strain with the intention that it performs multiple functions. In this chapter, we discuss these divergent approaches, their achievements and perspectives.

Keywords: microbial consortium, genetic modified microorganism, biochemical changes, fermented beverages, organoleptic characteristics

## 1. Introduction

The induction of fermentation on raw materials provides new products with added nutrients and organoleptic complexity vastly appreciated by consumers. The changes in the components of the raw materials are mainly caused by the main and secondary metabolism of the microorganisms present during the fermentation processes. The microorganisms need carbon and nitrogen sources to obtain energy and structural blocks to maintain cell integrity and functions and to proliferate. However, some of the carbon and nitrogen are transformed and released to the medium as by-products of the metabolism which generate the characteristics of the fermented food. Spontaneous fermentation harbors complex evolving and diverse microbiota that provides organoleptic complexity, mainly in aromas and flavors. However, it is hard to control and usually derives in inconsistent and even defective products. This is why commercial starter cultures emerged, allowing a better control of fermentation. Nevertheless, some argue that commercial inoculation

leads to a loss of unique regional style. In these cases, flavors often considered superior are achieved, at the cost of consistency and occasional production losses. The microorganism core that causes the expected characteristics of several beverages has been studied widely, indicating the participation of multiple microorganisms through different stages of the fermentation. Two divergent approaches have been proposed to improve fermentation by the controlled inoculation of multiple microorganisms each causing different expected changes in the fermentation, or by the manipulation of the genome of single strains to perform multiple tasks by themselves. Both approaches have their strengths and weaknesses, and it seems that the next step is the combination of both strategies to provide a holistic solution.

Streptococcus thermophilus) ferment hexoses via glycolysis (the Embden-Meyerhof pathway), producing lactic acid as the major end product, whereas the heterofermentative LAB (Oenococcus oeni, Lactobacillus brevis, Lactobacillus hilgardii, and Lactobacillus buchneri) and facultative homofermentative bacteria (Lactobacillus plantarum), in contrast, ferment hexoses and pentoses via the pentose phosphate or phosphoketolase pathway to produce acid lactic, CO2, and ethanol and/or acetic

Pursuing the Perfect Performer of Fermented Beverages: GMMs vs. Microbial Consortium

Malolactic fermentation, the second important stage in winemaking, normally

Acetic fermentation, also called oxidative fermentation, is a process in which alcohol is oxidized to acetic acid by the action of a group conveniently called acetic acid bacteria (AAB). These are strict aerobic bacteria found in high-sugar, alcoholic and acidic environments, characteristics found in fermented beverage processes [13]. The AAB partially oxidate carbohydrates to generate aldehydes, ketones, and organic acids in the fermentative media [14]. AAB are evidently involved in the production of vinegar and participate in fermentation of other beverages, such as palm wine, pulque, and kombucha [15]. However, the main concern with this type of microorganisms is that they are involved in the spoilage of wine, cider, and beer,

The metabolism of microorganisms is not a straightforward pathway, and other compounds are produced in lower concentrations during the metabolization of

Higher alcohols, polyols, esters, organic acids, vicinal ketones, and aldehydes are the main secondary metabolites produced in lower concentrations, as low as ng/L, althougth human senses are able to detect them due to the low perception threshold of these compounds, providing flavor and aroma to the fermented beverages [7]. Superior alcohols, also called fusel oils, are generated as by-products of the catabolism of amino acids, specifically by transamination reaction, which yields αketo acid that enters the Ehrlich pathway, resulting in decarboxylation forming an aldehyde, and it is then oxidized to generate an alcohol [17]. Also, the aldehyde

Glycerol, the most important polyol, is formed during fermentation, as one molecule of glucose at some point is divided in two molecules of three carbons, one

Esters are formed by the reaction of an alcohol group and an acid group. The most important are the acetate esters, in which the acid group is originated from acetic acid and ethyl esters, where the alcohol group is from ethanol. Yeast produce esters to achieve the transport from cytosol to the fermenting medium as they are

Vicinal diketones are formed as intermediates of the biosynthesis of branched

where the production of acid acetic is undesired [16].

substrates, the so-called secondary metabolites.

could be released or reduced to generate an acid.

yielding glycerol and the other pyruvate [18].

able to passively diffuse the cellular membrane [19].

amino acids valine, leucine, and isoleucine [20].

89

takes place after alcoholic fermentation. The malolactic fermentation is also conducted by LAB, preferably Oenococcus oeni, which reduce acidity of the wine or cider by transforming malic acid (dicarboxylic acid) to lactic acid (monocarboxylic acid) resulting in a softer taste [7]. In addition, the malolactic fermentation also affects the final aroma and taste balance by modifying and producing aroma-active

acid [12].

compounds [12].

2.3 Acetic fermentation

DOI: http://dx.doi.org/10.5772/intechopen.81616

2.4 Secondary metabolism

### 2. Metabolism in fermented beverage processes

Fermentation is the metabolic process carried out by microorganisms to obtain energy by oxidizing carbohydrates in which the final electron acceptors are organic molecules rather than O2 [1]. The catabolism of sugars results in the production of reduced pyridine nucleotides (nicotinamide adenine dinucleotide NADH); and to regenerate it in anaerobic conditions, pyruvate acts as the electron acceptor to reoxidate NADH [2]. The different fates of pyruvate are ethanol, lactic acid, or acetate, depending on the microorganism and environmental conditions [3].

#### 2.1 Alcoholic fermentation

Alcoholic fermentation is the transformation of the sugars, mainly glucose and fructose, into ethanol and CO2. This process is carried out by yeast such as Saccharomyces cerevisiae and S. bayanus [4], as well as by some bacteria, including Zymomonas mobilis, used in Central America in the fermentation of Agave to produce pulque [5] or palm wine (Toddy) [6]. The pyruvate is decarboxylated before a final reduction by NADH, to yield ethanol. The recovery of NAD maintains the flux of glycolysis reactions [7].

In addition, other by-products of fermentation are generated, such as glycerol, acetate, succinate, higher alcohols, and esters. The production of glycerol can be considered beneficial in some cases, that is, wine production, but is undesirable in the production of distilled beverage since it represents a waste of substrate [8]. Likewise, succinate production by yeast can have an important beneficial effect on the quality of sake, while it produces a negative effect on wine favoring a salty and bitter taste [9]. Esters represent an important group of flavor-active compounds with beneficial fruity/floral flavors and aromas in fermented beverages [7].

It should be noted that alcoholic fermentation could occur in aerobic environments. For example, even in the presence of abundant oxygen, yeast cells greatly prefer fermentation to oxidative phosphorylation, as long as sugars are readily available for consumption, a phenomenon known as the Crabtree effect [10].

#### 2.2 Lactic and malolactic fermentation

Lactic acid fermentation is mainly a bacterial process that plays important roles in fermented beverages, enhancing its nutritional value and organoleptic quality. A group of morphologically and physiologically diverse bacteria has been designated the term lactic acid bacteria (LAB), due to the main production of lactic acid generated from the catabolism of carbohydrates [11]. They can be divided into two physiological groups, homo- and heterofermentative, depending on the hexose metabolic pathways used. Homofermentative LAB (Lactobacillus delbrueckii and

Pursuing the Perfect Performer of Fermented Beverages: GMMs vs. Microbial Consortium DOI: http://dx.doi.org/10.5772/intechopen.81616

Streptococcus thermophilus) ferment hexoses via glycolysis (the Embden-Meyerhof pathway), producing lactic acid as the major end product, whereas the heterofermentative LAB (Oenococcus oeni, Lactobacillus brevis, Lactobacillus hilgardii, and Lactobacillus buchneri) and facultative homofermentative bacteria (Lactobacillus plantarum), in contrast, ferment hexoses and pentoses via the pentose phosphate or phosphoketolase pathway to produce acid lactic, CO2, and ethanol and/or acetic acid [12].

Malolactic fermentation, the second important stage in winemaking, normally takes place after alcoholic fermentation. The malolactic fermentation is also conducted by LAB, preferably Oenococcus oeni, which reduce acidity of the wine or cider by transforming malic acid (dicarboxylic acid) to lactic acid (monocarboxylic acid) resulting in a softer taste [7]. In addition, the malolactic fermentation also affects the final aroma and taste balance by modifying and producing aroma-active compounds [12].

#### 2.3 Acetic fermentation

leads to a loss of unique regional style. In these cases, flavors often considered superior are achieved, at the cost of consistency and occasional production losses. The microorganism core that causes the expected characteristics of several beverages has been studied widely, indicating the participation of multiple microorganisms through different stages of the fermentation. Two divergent approaches have been proposed to improve fermentation by the controlled inoculation of multiple microorganisms each causing different expected changes in the fermentation, or by the manipulation of the genome of single strains to perform multiple tasks by themselves. Both approaches have their strengths and weaknesses, and it seems that the next step is the combination of both strategies to provide a holistic solution.

Frontiers and New Trends in the Science of Fermented Food and Beverages

Fermentation is the metabolic process carried out by microorganisms to obtain energy by oxidizing carbohydrates in which the final electron acceptors are organic molecules rather than O2 [1]. The catabolism of sugars results in the production of reduced pyridine nucleotides (nicotinamide adenine dinucleotide NADH); and to regenerate it in anaerobic conditions, pyruvate acts as the electron acceptor to reoxidate NADH [2]. The different fates of pyruvate are ethanol, lactic acid, or acetate, depending on the microorganism and environmental conditions [3].

Alcoholic fermentation is the transformation of the sugars, mainly glucose and fructose, into ethanol and CO2. This process is carried out by yeast such as Saccha-

In addition, other by-products of fermentation are generated, such as glycerol, acetate, succinate, higher alcohols, and esters. The production of glycerol can be considered beneficial in some cases, that is, wine production, but is undesirable in the production of distilled beverage since it represents a waste of substrate [8]. Likewise, succinate production by yeast can have an important beneficial effect on the quality of sake, while it produces a negative effect on wine favoring a salty and bitter taste [9]. Esters represent an important group of flavor-active compounds with beneficial fruity/floral flavors and aromas in fermented beverages [7].

It should be noted that alcoholic fermentation could occur in aerobic environments. For example, even in the presence of abundant oxygen, yeast cells greatly prefer fermentation to oxidative phosphorylation, as long as sugars are readily available for consumption, a phenomenon known as the Crabtree effect [10].

Lactic acid fermentation is mainly a bacterial process that plays important roles in fermented beverages, enhancing its nutritional value and organoleptic quality. A group of morphologically and physiologically diverse bacteria has been designated the term lactic acid bacteria (LAB), due to the main production of lactic acid generated from the catabolism of carbohydrates [11]. They can be divided into two physiological groups, homo- and heterofermentative, depending on the hexose metabolic pathways used. Homofermentative LAB (Lactobacillus delbrueckii and

romyces cerevisiae and S. bayanus [4], as well as by some bacteria, including Zymomonas mobilis, used in Central America in the fermentation of Agave to produce pulque [5] or palm wine (Toddy) [6]. The pyruvate is decarboxylated before a final reduction by NADH, to yield ethanol. The recovery of NAD maintains the flux

2. Metabolism in fermented beverage processes

2.1 Alcoholic fermentation

of glycolysis reactions [7].

2.2 Lactic and malolactic fermentation

88

Acetic fermentation, also called oxidative fermentation, is a process in which alcohol is oxidized to acetic acid by the action of a group conveniently called acetic acid bacteria (AAB). These are strict aerobic bacteria found in high-sugar, alcoholic and acidic environments, characteristics found in fermented beverage processes [13]. The AAB partially oxidate carbohydrates to generate aldehydes, ketones, and organic acids in the fermentative media [14]. AAB are evidently involved in the production of vinegar and participate in fermentation of other beverages, such as palm wine, pulque, and kombucha [15]. However, the main concern with this type of microorganisms is that they are involved in the spoilage of wine, cider, and beer, where the production of acid acetic is undesired [16].

#### 2.4 Secondary metabolism

The metabolism of microorganisms is not a straightforward pathway, and other compounds are produced in lower concentrations during the metabolization of substrates, the so-called secondary metabolites.

Higher alcohols, polyols, esters, organic acids, vicinal ketones, and aldehydes are the main secondary metabolites produced in lower concentrations, as low as ng/L, althougth human senses are able to detect them due to the low perception threshold of these compounds, providing flavor and aroma to the fermented beverages [7].

Superior alcohols, also called fusel oils, are generated as by-products of the catabolism of amino acids, specifically by transamination reaction, which yields αketo acid that enters the Ehrlich pathway, resulting in decarboxylation forming an aldehyde, and it is then oxidized to generate an alcohol [17]. Also, the aldehyde could be released or reduced to generate an acid.

Glycerol, the most important polyol, is formed during fermentation, as one molecule of glucose at some point is divided in two molecules of three carbons, one yielding glycerol and the other pyruvate [18].

Esters are formed by the reaction of an alcohol group and an acid group. The most important are the acetate esters, in which the acid group is originated from acetic acid and ethyl esters, where the alcohol group is from ethanol. Yeast produce esters to achieve the transport from cytosol to the fermenting medium as they are able to passively diffuse the cellular membrane [19].

Vicinal diketones are formed as intermediates of the biosynthesis of branched amino acids valine, leucine, and isoleucine [20].

### 2.5 Microbial stress and adaptation process during fermentation

During the fermentation process, yeast and LAB must respond to several adverse conditions, mainly low pH, increasing ethanol concentration, nutrient limitations, fluctuations of oxygen concentration, and the presence of diverse compounds with antimicrobial effects [21, 22]. One of the major stress response pathways is the global stress response, including the expression of heat shock factors [23]; this is activated by several environmental conditions, as a general non-specific cell response to adverse conditions. Likewise, specific adaptation strategies are triggered under certain circumstances. Adaptation of S. cerevisiae environmental conditions involves the activation and repression of different sets of genes during fermentation. For example, macromolecules transport and glucose signaling are repressed at initial stages of fermentation in synthetic must, while vacuolar activity is important as far as the beginning of stationary phase [24].

between microorganisms leads to stronger adaptability and stability of the consortium; (3) the participation of different microorganisms increases complexity in microbial dynamism, metabolism, transcriptomics, and interactions, that ultimately affect organoleptic characteristics of the product. Thus, along with the evolution of the medium, these microorganisms will establish relationships that will modify their individual behavior, determining temporal dominances, proportion of the participants, and thus major metabolites, which according to the substrate, will give organoleptically complex, microbiologically stable, and healthy products that con-

Pursuing the Perfect Performer of Fermented Beverages: GMMs vs. Microbial Consortium

DOI: http://dx.doi.org/10.5772/intechopen.81616

4.1 Main microorganisms present in some fermented beverages and their roles

isms and the raw materials used for their preparation.

(substrate)

Saccharomyces yeasts Fruit Fermented teas, wine, cider, perry,

Lactic acid bacteria Fruits Pulque, Taberna, tomato juice, pomegranate juice

Acetic acid bacteria Fruits Kombucha, Water kefir [33, 48] Molds Grains Sake and soy sauce [49]

Classification of some of the most common fermented foods produced worldwide according to the main groups of

fruit-fermented beverages.

Dairy Kumis, kefir [36] Grains Beer and distillates [37]

Fruits Pulque and mezcal [5] Dairies Kumis, kefir [36]

champú, Asian rice wine, among others.

Dairies Yogurt, kefir [45, 46] Grains Sourdough, Cocoa beans, Lambic beer [47]

Grains African fura, Mexican pozol, South American

Main microorganism Raw material

microorganisms and the starting substrate.

Non-Saccharomyces

yeasts

Table 1.

91

It is still unclear how much mankind has intervened in the evolution of certain groups of microorganisms in fermented foods; however, it is clear that each substrate itself exerts a different selection pressure on them. In order to determine the diversity and evolution of a microbial consortium in any type of substrates, two approaches are available nowadays. First, the traditional microbiological methods, defined as culture-dependent, which may be biased by selectivity of culture media, low populations, and the presence of viable but non-culturable cells; however, it allows to further study individual behavior of isolates. The second approach is the culture-independent or molecular methods, which nevertheless may be affected by the specificity of primers, conditions of the reaction, detection of death cells, and database availability. Culture-independent methods have allowed to obtain a more complete scene, and combining with selective flow cytometry, metabolomics, and transcriptomic studies, a further comprehensive vision of microbial biodiversity of fermented foods can be reached [32]. Some of the most important fermented beverages are presented in Table 1, according to the type of dominant microorgan-

Examples References

[33–35]

[38–41]

[5, 42–44]

sumers desire.

Yeast viability in stationary phase is fundamental to an efficient fermentation, some reactive oxygen species (ROS) could be produced and cause oxidative damage on lipids, proteins, and nucleic acids, including mitochondrial DNA. Cells respond with the production of proteins like superoxide dismutase and rhodanases [25]. Cellular accumulation of trehalose has been associated with increased resistance to oxidative stress and survival to low temperatures [22].

Assimilable nitrogen in must have a great influence over fermentation rate in wine—low nitrogen concentration leads to a low biomass yield and slow fermentation rate [26]. During nitrogen depletion different pathways are activated such as ammonium permease, nitrogen catabolic genes, post diauxic shift elements, and autophagy; all depending of target of rapamycin signaling [27].

LAB are recognized by their high acid tolerance, and indeed, malolactic fermentation is an adaptation response to reduce wine acidity, improving its survival [28]. Other strategies to respond to high acidity are citrate fermentation, amino acid degradation to produce alkaline substances, active proton pump, accumulation of trehalose and glutathione, and degradation of phenolic acids [12].

#### 3. Strategies to improve desirable characteristics

In the past, the main objective for the selection of microorganisms was that they achieve fermentation in a relatively short time, with high conversions from substrates to the metabolites of interest and without the generation of compounds detrimental to the quality of the fermented food [29]. Nowadays, the characteristics sought for in fermentation processes have increased to satisfy the needs of more customers and producers which aim to increase flavor and aroma rather than ethanol concentration [30]. The focus on the use of a single strain to perform such deeds is considered impossible. This is why two main strategies have been proposed and evaluated, the use of multiple microorganisms each carrying out a specific function and as a whole produce the desired change, or the use of single microorganisms genetically modified to perform several tasks by themselves.

#### 4. Microbial consortium

During beverage fermentations, two or more microbial groups living symbiotically define a consortium [31]. In food fermentation consortia, many aspects that are summarized as follows need to be considered: (1) different strains fulfill different and complex tasks, dividing work; (2) an adequate dynamic of the interactions

## Pursuing the Perfect Performer of Fermented Beverages: GMMs vs. Microbial Consortium DOI: http://dx.doi.org/10.5772/intechopen.81616

between microorganisms leads to stronger adaptability and stability of the consortium; (3) the participation of different microorganisms increases complexity in microbial dynamism, metabolism, transcriptomics, and interactions, that ultimately affect organoleptic characteristics of the product. Thus, along with the evolution of the medium, these microorganisms will establish relationships that will modify their individual behavior, determining temporal dominances, proportion of the participants, and thus major metabolites, which according to the substrate, will give organoleptically complex, microbiologically stable, and healthy products that consumers desire.

## 4.1 Main microorganisms present in some fermented beverages and their roles

It is still unclear how much mankind has intervened in the evolution of certain groups of microorganisms in fermented foods; however, it is clear that each substrate itself exerts a different selection pressure on them. In order to determine the diversity and evolution of a microbial consortium in any type of substrates, two approaches are available nowadays. First, the traditional microbiological methods, defined as culture-dependent, which may be biased by selectivity of culture media, low populations, and the presence of viable but non-culturable cells; however, it allows to further study individual behavior of isolates. The second approach is the culture-independent or molecular methods, which nevertheless may be affected by the specificity of primers, conditions of the reaction, detection of death cells, and database availability. Culture-independent methods have allowed to obtain a more complete scene, and combining with selective flow cytometry, metabolomics, and transcriptomic studies, a further comprehensive vision of microbial biodiversity of fermented foods can be reached [32]. Some of the most important fermented beverages are presented in Table 1, according to the type of dominant microorganisms and the raw materials used for their preparation.


#### Table 1.

Classification of some of the most common fermented foods produced worldwide according to the main groups of microorganisms and the starting substrate.

2.5 Microbial stress and adaptation process during fermentation

Frontiers and New Trends in the Science of Fermented Food and Beverages

as far as the beginning of stationary phase [24].

oxidative stress and survival to low temperatures [22].

autophagy; all depending of target of rapamycin signaling [27].

trehalose and glutathione, and degradation of phenolic acids [12].

ganisms genetically modified to perform several tasks by themselves.

4. Microbial consortium

90

3. Strategies to improve desirable characteristics

During the fermentation process, yeast and LAB must respond to several adverse conditions, mainly low pH, increasing ethanol concentration, nutrient limitations, fluctuations of oxygen concentration, and the presence of diverse compounds with antimicrobial effects [21, 22]. One of the major stress response pathways is the global stress response, including the expression of heat shock factors [23]; this is activated by several environmental conditions, as a general non-specific cell response to adverse conditions. Likewise, specific adaptation strategies are triggered under certain circumstances. Adaptation of S. cerevisiae environmental conditions involves the activation and repression of different sets of genes during fermentation. For example, macromolecules transport and glucose signaling are repressed at initial stages of fermentation in synthetic must, while vacuolar activity is important

Yeast viability in stationary phase is fundamental to an efficient fermentation, some reactive oxygen species (ROS) could be produced and cause oxidative damage on lipids, proteins, and nucleic acids, including mitochondrial DNA. Cells respond with the production of proteins like superoxide dismutase and rhodanases [25]. Cellular accumulation of trehalose has been associated with increased resistance to

Assimilable nitrogen in must have a great influence over fermentation rate in wine—low nitrogen concentration leads to a low biomass yield and slow fermentation rate [26]. During nitrogen depletion different pathways are activated such as ammonium permease, nitrogen catabolic genes, post diauxic shift elements, and

LAB are recognized by their high acid tolerance, and indeed, malolactic fermentation is an adaptation response to reduce wine acidity, improving its survival [28]. Other strategies to respond to high acidity are citrate fermentation, amino acid degradation to produce alkaline substances, active proton pump, accumulation of

In the past, the main objective for the selection of microorganisms was that they achieve fermentation in a relatively short time, with high conversions from substrates to the metabolites of interest and without the generation of compounds detrimental to the quality of the fermented food [29]. Nowadays, the characteristics sought for in fermentation processes have increased to satisfy the needs of more customers and producers which aim to increase flavor and aroma rather than ethanol concentration [30]. The focus on the use of a single strain to perform such deeds is considered impossible. This is why two main strategies have been proposed and evaluated, the use of multiple microorganisms each carrying out a specific function and as a whole produce the desired change, or the use of single microor-

During beverage fermentations, two or more microbial groups living symbiotically define a consortium [31]. In food fermentation consortia, many aspects that are summarized as follows need to be considered: (1) different strains fulfill different and complex tasks, dividing work; (2) an adequate dynamic of the interactions

## 4.2 Interaction between microorganisms in mixed cultures

In order to survive in an environment, a group of different type of microorganisms need to adapt and specialize through the time they spend in it. Microbial relationships are needed to establish and maintain the microbial consortium; the type of interaction that can emerge may be positive as mutualism or synergism, in which both parts benefit from being together. However, the relationships can also be negative or antagonistic, when one microorganism inhibits another, for instance by nutrients or space competition; or by producing a metabolite that harms the other; or by presenting parasitism, in which one microorganism benefits at the expense of other, damaging and even killing it [50, 51]. Any type of interaction starts by recognizing the environment, then transferring the information to others. The phenomenon is regulated by mechanisms such as quorum sensing, which consists in a stimuliresponse system that regulates gene expression in response to population density [51].

In the particular environment of beverage fermentations, as exhaustively reviewed by [50], microorganisms manifest a variety of interactions. During fermentation, the environment generated maintains most of human pathogenic or food spoilage microorganisms. This role is achieved through competition and antagonism, through the fast consumption of nutrients and production of inhibitory compounds, mainly ethanol and organic acids, usually acting together with medium, short-chain fatty acids and proteinaceous toxins such as yeast's killer toxin in wine. On the other hand, throughout the evolution of the original substrate, the limiting factors change and the dominant microorganisms also change along with them. This succession of species has been reported in almost every fermented food studied. Positive interactions determine largely the succession of microbes in a particular substrate, for instance in sake production, where the saccharification of starch by Aspergillus flavus var. oryzae is first required in order to let S. cerevisiae conduct the alcoholic fermentation [52].

In a different context, regarding a particular metabolic interest or synergism, a study carried out in tequila fermentation is briefly presented. An important safety issue in the consumption of tequila (and in general, distillates) to take into account is the elevated concentration of ethyl carbamate generated by the reaction between urea and ethanol driven by the elevated temperatures occurring during the distillation stage. While ethanol is the desired metabolite in this process, urea is the byproduct of nitrogen metabolism of S. cerevisiae and thus its production cannot be totally eliminated. On the other side, bacteria are capable of consuming urea as nitrogen source [59]. Taking advantage of the usual symbiosis across yeast, LAB, and AAB, an alternative approach that has been explored to reduce ethyl carbamate production is the use of mixed cultures, combining a selected S. cerevisiae strain and bacteria strains isolated from spontaneously fermenting agave juice (Figure 2).

Urea concentration produced by S. cerevisiae strain Teq-199 individually (C-) or in combination with seven

DOiwithoutyeast h i. Strain 450®

Compatibility of four native LAB grown into the medium produced by five native S. cerevisiae strains (N42, SR25, N05), measured as relative optical density increase ODi <sup>¼</sup> ODiafteryeast�ODiwithoutyeast

Pursuing the Perfect Performer of Fermented Beverages: GMMs vs. Microbial Consortium

(O. oeni) and K1-V1116® (K1) were used as commercial references.

DOI: http://dx.doi.org/10.5772/intechopen.81616

Figure 1.

Figure 2.

93

native bacteria species (data not published).

Besides the simply descriptive craving to know the diversity and roles that each microorganism plays, by understanding the types of interactions and how they emerge, a more controllable process can be achieved and the quality of the products can be improved. Finding the combination of microorganisms (species and strains) that will give desired characteristics is a strategy vastly explored in wine [53, 54], and also in cachaça [55], prickly pear wine [56] where mixed populations of Saccharomyces, non-Saccharomyces yeast, and even LAB have been explored.

One important aspect to consider when a proper combination of microorganisms is sought is to investigate their compatibility, that is, not negative type of interaction, as well as to determine if the intended promoting role actually occurs during the fermentation process. For instance, regarding compatibility, a study was conducted to observe synergism, antagonism, or no apparent interaction between selected native yeasts and LAB strains for the production of wine in the region of Queretaro, Mexico [57]. For this, yeast strains were grown in a medium resembling must, after 12 h yeast biomass was removed and the resulting broth was used to incubate the different strains of LAB and to observe their growth by means of optical density (OD) (Figure 1).

Positive values indicate a growth promotion from yeast to LAB observed in different extent, showing synergism superior in the combinations of native yeast strains compared with the growth promotion given by the commercial yeast (K1-V1116). It is also observable that the behaviors were strain-combination dependent, an aspect cited by other authors [58]. This test allowed to foresee and select compatible strains in order to further analyze their performance in a traditional winemaking process, where LAB strain is inoculated after the alcoholic fermentation performed by the yeast strain.

Pursuing the Perfect Performer of Fermented Beverages: GMMs vs. Microbial Consortium DOI: http://dx.doi.org/10.5772/intechopen.81616

#### Figure 1.

4.2 Interaction between microorganisms in mixed cultures

Frontiers and New Trends in the Science of Fermented Food and Beverages

In order to survive in an environment, a group of different type of microorgan-

isms need to adapt and specialize through the time they spend in it. Microbial relationships are needed to establish and maintain the microbial consortium; the type of interaction that can emerge may be positive as mutualism or synergism, in which both parts benefit from being together. However, the relationships can also be negative or antagonistic, when one microorganism inhibits another, for instance by nutrients or space competition; or by producing a metabolite that harms the other; or by presenting parasitism, in which one microorganism benefits at the expense of other, damaging and even killing it [50, 51]. Any type of interaction starts by recognizing the environment, then transferring the information to others. The phenomenon is regulated by mechanisms such as quorum sensing, which consists in a stimuliresponse system that regulates gene expression in response to population density [51]. In the particular environment of beverage fermentations, as exhaustively reviewed by [50], microorganisms manifest a variety of interactions. During fermentation, the environment generated maintains most of human pathogenic or food spoilage microorganisms. This role is achieved through competition and antagonism, through the fast consumption of nutrients and production of inhibitory

compounds, mainly ethanol and organic acids, usually acting together with

charomyces, non-Saccharomyces yeast, and even LAB have been explored.

conduct the alcoholic fermentation [52].

optical density (OD) (Figure 1).

mentation performed by the yeast strain.

92

medium, short-chain fatty acids and proteinaceous toxins such as yeast's killer toxin in wine. On the other hand, throughout the evolution of the original substrate, the limiting factors change and the dominant microorganisms also change along with them. This succession of species has been reported in almost every fermented food studied. Positive interactions determine largely the succession of microbes in a particular substrate, for instance in sake production, where the saccharification of starch by Aspergillus flavus var. oryzae is first required in order to let S. cerevisiae

Besides the simply descriptive craving to know the diversity and roles that each microorganism plays, by understanding the types of interactions and how they emerge, a more controllable process can be achieved and the quality of the products can be improved. Finding the combination of microorganisms (species and strains) that will give desired characteristics is a strategy vastly explored in wine [53, 54], and also in cachaça [55], prickly pear wine [56] where mixed populations of Sac-

One important aspect to consider when a proper combination of microorganisms is sought is to investigate their compatibility, that is, not negative type of interaction, as well as to determine if the intended promoting role actually occurs during the fermentation process. For instance, regarding compatibility, a study was conducted to observe synergism, antagonism, or no apparent interaction between selected native yeasts and LAB strains for the production of wine in the region of Queretaro, Mexico [57]. For this, yeast strains were grown in a medium resembling must, after 12 h yeast biomass was removed and the resulting broth was used to incubate the different strains of LAB and to observe their growth by means of

Positive values indicate a growth promotion from yeast to LAB observed in different extent, showing synergism superior in the combinations of native yeast strains compared with the growth promotion given by the commercial yeast (K1-V1116). It is also observable that the behaviors were strain-combination dependent, an aspect cited by other authors [58]. This test allowed to foresee and select compatible strains in order to further analyze their performance in a traditional winemaking process, where LAB strain is inoculated after the alcoholic fer-

Compatibility of four native LAB grown into the medium produced by five native S. cerevisiae strains (N42, SR25, N05), measured as relative optical density increase ODi <sup>¼</sup> ODiafteryeast�ODiwithoutyeast DOiwithoutyeast h i. Strain 450® (O. oeni) and K1-V1116® (K1) were used as commercial references.

In a different context, regarding a particular metabolic interest or synergism, a study carried out in tequila fermentation is briefly presented. An important safety issue in the consumption of tequila (and in general, distillates) to take into account is the elevated concentration of ethyl carbamate generated by the reaction between urea and ethanol driven by the elevated temperatures occurring during the distillation stage. While ethanol is the desired metabolite in this process, urea is the byproduct of nitrogen metabolism of S. cerevisiae and thus its production cannot be totally eliminated. On the other side, bacteria are capable of consuming urea as nitrogen source [59]. Taking advantage of the usual symbiosis across yeast, LAB, and AAB, an alternative approach that has been explored to reduce ethyl carbamate production is the use of mixed cultures, combining a selected S. cerevisiae strain and bacteria strains isolated from spontaneously fermenting agave juice (Figure 2).

#### Figure 2.

Urea concentration produced by S. cerevisiae strain Teq-199 individually (C-) or in combination with seven native bacteria species (data not published).

Compared with fermentation individually carried out by S. cerevisiae strain, a clear tendency to decrease urea concentration of approximately 0.2 mg/L was observed when Weissella confusa and Pediococcus acidilactici were co-inoculated with the S. cerevisiae strain. Conversely, a moderate increase was obtained with the rest of the bacterial strain, especially with Weissella paramesenteriodes, with an increase of about 0.4 mg/L compared with the control. These changes are respectively associated with a consumption and production of the metabolite in question, depending on the species used.

These cases exemplify some of the strategies that have been followed in order to choose or validate the use of mixed cultures, seeking to achieve particular objectives and trying to ensure the success of combining certain strains.

## 5. Genetically modified microorganisms

Natural genetic differences are shown in strains of the same species. This variability can be replicated under laboratory conditions intended to improve characteristics of microorganisms [60]. These traits could be modified by directed or by "natural" methodologies. Even though both approaches result in genetically modified microorganisms (GMMs), the laws that dictate the feasibility on food production depend on the strategy used [61].

It is necessary to consider that the strains to be modified for food fermentation must be labeled as generally recognized as safe (GRAS) or qualified presumption of safety (QPS), not related with pathogens; so, they should be taxonomically identified, as well as being genetically stable under industrial processes [62]. Under these considerations the most investigated eukaryotic microorganism is S. cerevisiae, used for several centuries for food and alcoholic production; thus, their metabolic pathways and gene-related regulation are well known. Furthermore, the genome of this species has been completely sequenced, providing the basis for applications of genetic engineering [63]. Meanwhile, technological improvement investigation has been carried out mainly on LAB (Table 2).

5.2 Natural genetic modifications

Acetic acid bacterium

Modification technique

Adaptive evolution

Random mutagenesis

Natural conjugation

Genome shuffling

Table 2.

95

Yeast (species not identified)

S. cerevisiae S. bayanus

DOI: http://dx.doi.org/10.5772/intechopen.81616

S. cerevisiae S. bayanus

S. cerevisiae S. paradoxus S. pastorianus

5.2.1 Adaptive evolution

pressure will be able to grow [82].

To obtain microorganisms with desired genetic characteristics using natural techniques, growth conditions are guided in the laboratory to improve the probability of inducing the desired genome modifications. All these natural techniques target the whole genome of the strain, generating several different genotypic changes and, thus, generating the need to further select the strains with the pheno-

Examples of genetic modifications applied to microorganisms for fermented beverages improvement.

S. cerevisiae Determine gene implicated in nitrogen requirements

S. cerevisiae Improve fermentation performance, affected

Species Modified trait Reference

S. cerevisiae Flocculation in the surface [64] S. cerevisiae Ethanol reduction and flavor increase [65] S. cerevisiae Ethanol reduction [66]

Pursuing the Perfect Performer of Fermented Beverages: GMMs vs. Microbial Consortium

L. lactis Domestication from plant to milk fermentation [67]

O. oeni Malolactic efficiency and sensory properties [69]

S. cerevisiae Acid- and thermo-tolerance [73]

negatively the flocculation capacity

S. cerevisiae Improve fermentation performance [75] S. cerevisiae Improve fermentation performance [76] Candida krusei Improve acetic acid tolerance [77]

Reduction of acetic acid [68]

Fermentation at low temperature [70]

Stress resistance and fermentation performance [71]

Aroma production [60]

Improve tolerance of ethanol [78]

[72]

[74]

In this methodology, strains are grown in a medium exerting an increasing selective pressure to allow the most adapted generations to become dominant. During the replication of DNA, mutations could accumulate in the offspring without causing an evident modification. However, in a selective condition, only strains with the genetic pool needed to maintain the homeostasis of the cell under the stress

typic variation desired. These methodologies are "allowed," or at least not prohibited by the law as they do not enter in the legal definition of GMM [30]. Among other strategies, some of the most important are described below.

#### 5.1 Directed genetic modifications

The directed modification is carried out by genetic engineering causing a punctual manipulation in a known region in the genome that in turn will improve a characteristic of interest or the repression of a negative trait. The changes usually involve the promoter region to induce or repress gene translation, or the deletion or insertion of new genes from other microorganisms. This approach presents several drawbacks in food industry. First, it requires the global knowledge of metabolic pathways, genes involved, and their regulation [79]. Second, a single gene modification cannot produce the expected result, since some pathways are regulated by several genes, making a complex process to obtain the desirable trait [61]. And third, the use of microorganisms modified this way is prohibited in foods by law in the European Union, USA, and other countries [80].

The only permitted directed genetically engineered strain used in USA is a S. cerevisiae strain able to fully carry out a malo-alcoholic fermentation. This strain was generated by the integration of a malate permease gene from Schizosaccharomyces pombe and malic enzyme from O. oeni to the constitutive promoter of the 3-phosphoglycerate kinase of S. cerevisiae [81].


Pursuing the Perfect Performer of Fermented Beverages: GMMs vs. Microbial Consortium DOI: http://dx.doi.org/10.5772/intechopen.81616

#### Table 2.

Compared with fermentation individually carried out by S. cerevisiae strain, a clear tendency to decrease urea concentration of approximately 0.2 mg/L was observed when Weissella confusa and Pediococcus acidilactici were co-inoculated with the S. cerevisiae strain. Conversely, a moderate increase was obtained with the rest of the bacterial strain, especially with Weissella paramesenteriodes, with an increase of about 0.4 mg/L compared with the control. These changes are respectively associated with a consumption and production of the metabolite in question,

These cases exemplify some of the strategies that have been followed in order to choose or validate the use of mixed cultures, seeking to achieve particular objectives

Natural genetic differences are shown in strains of the same species. This variability can be replicated under laboratory conditions intended to improve characteristics of microorganisms [60]. These traits could be modified by directed or by "natural" methodologies. Even though both approaches result in genetically modified microorganisms (GMMs), the laws that dictate the feasibility on food

It is necessary to consider that the strains to be modified for food fermentation must be labeled as generally recognized as safe (GRAS) or qualified presumption of safety (QPS), not related with pathogens; so, they should be taxonomically identified, as well as being genetically stable under industrial processes [62]. Under these considerations the most investigated eukaryotic microorganism is S. cerevisiae, used for several centuries for food and alcoholic production; thus, their metabolic pathways and gene-related regulation are well known. Furthermore, the genome of this species has been completely sequenced, providing the basis for applications of genetic engineering [63]. Meanwhile, technological improvement investigation has

The directed modification is carried out by genetic engineering causing a punctual manipulation in a known region in the genome that in turn will improve a characteristic of interest or the repression of a negative trait. The changes usually involve the promoter region to induce or repress gene translation, or the deletion or insertion of new genes from other microorganisms. This approach presents several drawbacks in food industry. First, it requires the global knowledge of metabolic pathways, genes involved, and their regulation [79]. Second, a single gene modification cannot produce the expected result, since some pathways are regulated by several genes, making a complex process to obtain the desirable trait [61]. And third, the use of microorganisms modified this way is prohibited in foods by law in the European Union, USA, and other

The only permitted directed genetically engineered strain used in USA is a S. cerevisiae strain able to fully carry out a malo-alcoholic fermentation. This strain was generated by the integration of a malate permease gene from Schizosaccharomyces pombe and malic enzyme from O. oeni to the constitutive promoter of the

and trying to ensure the success of combining certain strains.

Frontiers and New Trends in the Science of Fermented Food and Beverages

5. Genetically modified microorganisms

production depend on the strategy used [61].

been carried out mainly on LAB (Table 2).

3-phosphoglycerate kinase of S. cerevisiae [81].

5.1 Directed genetic modifications

countries [80].

94

depending on the species used.

Examples of genetic modifications applied to microorganisms for fermented beverages improvement.

#### 5.2 Natural genetic modifications

To obtain microorganisms with desired genetic characteristics using natural techniques, growth conditions are guided in the laboratory to improve the probability of inducing the desired genome modifications. All these natural techniques target the whole genome of the strain, generating several different genotypic changes and, thus, generating the need to further select the strains with the phenotypic variation desired. These methodologies are "allowed," or at least not prohibited by the law as they do not enter in the legal definition of GMM [30]. Among other strategies, some of the most important are described below.

#### 5.2.1 Adaptive evolution

In this methodology, strains are grown in a medium exerting an increasing selective pressure to allow the most adapted generations to become dominant. During the replication of DNA, mutations could accumulate in the offspring without causing an evident modification. However, in a selective condition, only strains with the genetic pool needed to maintain the homeostasis of the cell under the stress pressure will be able to grow [82].

Adaptive evolution has been applied to divert ethanol to glycerol production, then reducing ethanol graduation in wine. It was achieved by increasing osmotic stress with salts in growth media. Glycerol is produced and accumulated in the interior of the yeast cell to counteract the osmotic pressure in the environment [66]. interest [75]. As this methodology is relatively new, their evaluation at industrial

Pursuing the Perfect Performer of Fermented Beverages: GMMs vs. Microbial Consortium

In nature, horizontal gene transfer occurs in fungi and bacteria kingdoms, it involves the insertion of sequence elements, conjugation, transformation, and transduction from one microorganism to another [90]. These transferences could happen in non-taxonomically related microorganisms. In yeast, this mechanism is not well known; however, it has provided important features such as the identified in a S. cerevisiae strain by whole genome sequencing, in which a total of 34 genes were found to be transferred from non-Saccharomyces and Zygosaccharomyces bailii,

Regarding bacteria, mating process involves close physical contact between a strain that donates its genetic material, mainly a plasmid, to a recipient. The vast majority of the plasmids transferred do not contain any technological use [91]. In LAB, important plasmids naturally present provide the ability to ferment lactose, gain resistance to bacteriophages, and produce bacteriocins [92]. Plasmids could also encode for antibiotic resistance and further transferring could occur to other

During the last years, there has been an increase in the demand of natural, artisanal, and organic-labeled products, leading to a rise in the request for autochthonous starters, which reflect the biodiversity of a particular area, supported by

An alternative to the use of single-strain starter cultures, which leads to very standardized products, is the use of autochthonous mixed starters (consortia), able to mimic the natural biodiversity, increasing organoleptic properties, but still

On the other hand, considering the fact that mixed populations can perform functions that are difficult or even impossible for individual strains or species to do, nowadays the theoretical support to successfully obtain synthetic microbial consortium exists and presents a wider application potential than single synthetic cells. Taking into consideration the knowledge acquired on naturally occurring microbial interactions, the application of such technology seems feasible and attractive for many industries. This approach would make it possible to efficiently complete many tasks and to acquire a specific product profile compartmentalizing molecular components of each pathway, transcriptional regulators, and chemical intermediates in each different microbial individual. Nevertheless, the use of this technology would face many drawbacks until it is approved to be used in fermented foods, in

spite of being the focus of several studies in other similar fields [94-96].

ages maintaining control of the process and quality of the products. Both

The genetic modification of strains and the development of mixed starter cultures aim for similar objectives, to improve the characteristics of fermented bever-

approaches possess strengths and weaknesses. While some advocate that changes in the genome open a vast opportunity to achieve all the desired characteristics in

level to provide certainty of the results is still needed.

DOI: http://dx.doi.org/10.5772/intechopen.81616

providing important fructose fermentation capability [30].

species of importance to pathogenic bacteria [93].

6. Trends and perspectives

the idea of microbial "terroir."

7. Conclusions

97

maintaining controllable processes [52, 53].

5.2.5 Horizontal gene transfer

#### 5.2.2 Random mutagenesis

The exposure of microorganisms to physical factors such as UV light, or chemical mutagens as alkylating agent, allows increasing the rate of mistakes in the replication. The offspring then are screened to select colonies with improved characteristics. The randomness of the mutations causes a big drawback, and the modification of regions other than the target of interest could impact negatively on the performance [83]. Also, as the genes occur in more than one copy in the genome, the mutation should be present in all the copies to obtain a strain with changed phenotype [84].

#### 5.2.3 Natural conjugation

This methodology mainly has been applied to yeast, in which two strains, both having an interesting characteristic are crossed using their sexual cycle, thus also receiving the name of direct mating [60]. The resulting hybrid strain contains half genes from each parental strain, meaning that it will obtain some characteristics and lose others [85]. To discriminate the new hybrids from the parental strains, the latter must be differentiated, usually using respiratory-deficient and auxotrophic strains, which in turn only hybrids with prototrophy and respiratory proficiency would be able to grow in a selective media [60].

The most famous yeast strain generated by natural hybridization is the lager beer S. pastorius, having characteristics of S. cerevisiae and cryotolerance of S. eubayanus, which gave the desired fermentative proficiency at low temperatures [37]. Laboratory hybridization of S. cerevisiae x S. mikatae has also generated strains with improved and diverse volatile compounds that provide complexity to wines [86]. In addition, a hybrid of S. cerevisiae and S. kudriavzevii accumulated more glycerol, providing more cryotolerance, osmotolerance, and ethanol tolerance [87].

The major drawback of the sexual reproduction in yeast is that industrial strains poorly sporulate [61]. Rare mating is applied in these cases, switching the mating type of diploid or polyploid cells, and then being able to hybridize with the contrary mating type, to generate a new hybrid [88].

#### 5.2.4 Cell fusion

In this methodology, the cell wall is disrupted generating spheroplasts that will spontaneously fuse to other cells, integrating their DNA into a single cell and, then, recombination occurs. The insertion of genetic material could be done even from microorganisms of other kingdoms [89].

Genome shuffling is based on protoplast fusion and nowadays several methodologies are integrated to provide complex phenotypes. It involves the induction of mutagenesis in a population of a specific strain, and then this new genetically diverse population could be screened by the evaluation of individual isolates or by applying a selective pressure to the media containing the mutants. The resulting exceptional mutants are hybridized by protoplast fusion or by mating. The resulting combinations could be further hybridized repeatedly to improve characteristics of

interest [75]. As this methodology is relatively new, their evaluation at industrial level to provide certainty of the results is still needed.

## 5.2.5 Horizontal gene transfer

Adaptive evolution has been applied to divert ethanol to glycerol production, then reducing ethanol graduation in wine. It was achieved by increasing osmotic stress with salts in growth media. Glycerol is produced and accumulated in the interior of the yeast cell to counteract the osmotic pressure in the environment [66].

Frontiers and New Trends in the Science of Fermented Food and Beverages

The exposure of microorganisms to physical factors such as UV light, or chem-

This methodology mainly has been applied to yeast, in which two strains, both having an interesting characteristic are crossed using their sexual cycle, thus also receiving the name of direct mating [60]. The resulting hybrid strain contains half genes from each parental strain, meaning that it will obtain some characteristics and lose others [85]. To discriminate the new hybrids from the parental strains, the latter must be differentiated, usually using respiratory-deficient and auxotrophic strains, which in turn only hybrids with prototrophy and respiratory proficiency

The most famous yeast strain generated by natural hybridization is the lager beer S. pastorius, having characteristics of S. cerevisiae and cryotolerance of S. eubayanus, which gave the desired fermentative proficiency at low temperatures [37]. Laboratory hybridization of S. cerevisiae x S. mikatae has also generated strains with improved and diverse volatile compounds that provide complexity to wines [86]. In addition, a hybrid of S. cerevisiae and S. kudriavzevii accumulated more glycerol, providing more cryotolerance, osmotolerance, and ethanol toler-

The major drawback of the sexual reproduction in yeast is that industrial strains poorly sporulate [61]. Rare mating is applied in these cases, switching the mating type of diploid or polyploid cells, and then being able to hybridize with the contrary

In this methodology, the cell wall is disrupted generating spheroplasts that will spontaneously fuse to other cells, integrating their DNA into a single cell and, then, recombination occurs. The insertion of genetic material could be done even from

Genome shuffling is based on protoplast fusion and nowadays several methodologies are integrated to provide complex phenotypes. It involves the induction of mutagenesis in a population of a specific strain, and then this new genetically diverse population could be screened by the evaluation of individual isolates or by applying a selective pressure to the media containing the mutants. The resulting exceptional mutants are hybridized by protoplast fusion or by mating. The resulting combinations could be further hybridized repeatedly to improve characteristics of

ical mutagens as alkylating agent, allows increasing the rate of mistakes in the replication. The offspring then are screened to select colonies with improved characteristics. The randomness of the mutations causes a big drawback, and the modification of regions other than the target of interest could impact negatively on the performance [83]. Also, as the genes occur in more than one copy in the genome, the mutation should be present in all the copies to obtain a strain with changed

5.2.2 Random mutagenesis

phenotype [84].

ance [87].

5.2.4 Cell fusion

96

5.2.3 Natural conjugation

would be able to grow in a selective media [60].

mating type, to generate a new hybrid [88].

microorganisms of other kingdoms [89].

In nature, horizontal gene transfer occurs in fungi and bacteria kingdoms, it involves the insertion of sequence elements, conjugation, transformation, and transduction from one microorganism to another [90]. These transferences could happen in non-taxonomically related microorganisms. In yeast, this mechanism is not well known; however, it has provided important features such as the identified in a S. cerevisiae strain by whole genome sequencing, in which a total of 34 genes were found to be transferred from non-Saccharomyces and Zygosaccharomyces bailii, providing important fructose fermentation capability [30].

Regarding bacteria, mating process involves close physical contact between a strain that donates its genetic material, mainly a plasmid, to a recipient. The vast majority of the plasmids transferred do not contain any technological use [91]. In LAB, important plasmids naturally present provide the ability to ferment lactose, gain resistance to bacteriophages, and produce bacteriocins [92]. Plasmids could also encode for antibiotic resistance and further transferring could occur to other species of importance to pathogenic bacteria [93].

## 6. Trends and perspectives

During the last years, there has been an increase in the demand of natural, artisanal, and organic-labeled products, leading to a rise in the request for autochthonous starters, which reflect the biodiversity of a particular area, supported by the idea of microbial "terroir."

An alternative to the use of single-strain starter cultures, which leads to very standardized products, is the use of autochthonous mixed starters (consortia), able to mimic the natural biodiversity, increasing organoleptic properties, but still maintaining controllable processes [52, 53].

On the other hand, considering the fact that mixed populations can perform functions that are difficult or even impossible for individual strains or species to do, nowadays the theoretical support to successfully obtain synthetic microbial consortium exists and presents a wider application potential than single synthetic cells. Taking into consideration the knowledge acquired on naturally occurring microbial interactions, the application of such technology seems feasible and attractive for many industries. This approach would make it possible to efficiently complete many tasks and to acquire a specific product profile compartmentalizing molecular components of each pathway, transcriptional regulators, and chemical intermediates in each different microbial individual. Nevertheless, the use of this technology would face many drawbacks until it is approved to be used in fermented foods, in spite of being the focus of several studies in other similar fields [94-96].

## 7. Conclusions

The genetic modification of strains and the development of mixed starter cultures aim for similar objectives, to improve the characteristics of fermented beverages maintaining control of the process and quality of the products. Both approaches possess strengths and weaknesses. While some advocate that changes in the genome open a vast opportunity to achieve all the desired characteristics in

fermented beverages, the other groups remark that only natural diversity and traditional methods could generate best products with typicity. Furthermore, the application of genetic modifications is badly perceived by consumers and legally prohibited in some cases. It seems that the next step in the improvement agenda is the combination of both approaches, the incorporation of mixtures of natural, genetically modified microorganisms and native strains to provide a holistic solution to the existing difficulties in fermentation.

References

[1] El-Mansi E, Bryce C, Hartley B, Demain A. Fermentation microbiology and biotechnology: An historical perspective. In: El-Mansi E, Bryce C, Dahhou B, Sanchez S, Demain A, Allman A, editors. Fermentation Microbiology and Biotechnology. Boca Raton: CRC Press; 2012. pp. 2-4

DOI: http://dx.doi.org/10.5772/intechopen.81616

Pursuing the Perfect Performer of Fermented Beverages: GMMs vs. Microbial Consortium

[9] Swiegers J, Saerens S, Pretorius I. The development of yeast strains as tools for adjusting the flavor of fermented beverages to market specifications. In: Havkin-Frenkel D, Dudai N, editors. Biotechnology in Flavor Production. Chichester: John Wiley & Sons; 2008. pp. 6-7. DOI: 10.1002/9781444302493.ch1

[10] Hagman A, Säll T, Compagno C, Piskur J. Yeast "make-accumulateconsume" life strategy evolved as a multi-step process that predates the whole genome duplication. PLoS One. 2013;8:e68734. DOI: 10.1371/journal.

[11] Muñoz R, Moreno-Arribas M, de las

[12] Liu S. Malolactic fermentation in wine—Beyond deacidification. Journal of Applied Microbiology. 2002;92: 589-601. DOI: 10.1046/j.1365-2672.

[13] Gullo M, Giudici P. Acetic acid bacteria in traditional balsamic vinegar: Phenotypic traits relevant for starter cultures selection. International Journal of Food Microbiology. 2008;125:46-53. DOI: 10.1016/j.ijfoodmicro.2007.11.076

[14] Sengun I, Karabiyikli S. Importance of acetic acid bacteria in food industry. Food Control. 2011;22:647-656. DOI: 10.1016/j.foocont.2010.11.008

[15] Kersters K, Lisdiyanti P, Komagata K, Swings J. The family Acetobacteraceae: The genera Acetobacter, Acidomonas, Asaia, Gluconacetobacter, Gluconobacter and Kozakia. In: Dworkin M, Falkow S, Rosenberg E, Schleifer K, Stackebrandt E, editors. The Prokaryotes. 3. New York: Springer; 2006. pp. 163-200. DOI:

10.1007/0-387-30745-1\_9

Rivas B. Lactic acid bacteria. In: Carrascosa AV, Muñoz R, González R, editors. Molecular Wine Microbiology.

1st ed. 2011. pp. 191-226

pone.0068734

2002.01589.x

[2] Stanbury P, Whitaker A, Hall S. Principles of Fermentation Technology. Butterworth-Heinemann; 2016. pp. 1-20

[3] Kamal-Eldin A, Mehta B, Iwanski R.

properties. Boca Raton: CRC Press; 2012.

[4] Hui Y. Fermented plant products and their manufacturing. In: Hui Y, Evranuz E, editors. Handbook of Plant-based Fermented Food and Beverage Technology. Burlington: CRC Press;

Fermentation: Effects on food

[5] Escalante A, Giles-Gómez M, Hernández G, Córdova-Aguilar M, López-Munguía A, Gosset G, et al. Analysis of bacterial community during the fermentation of pulque, a traditional Mexican alcoholic beverage, using a polyphasic approach. International Journal of Food Microbiology. 2008;124:

126-134. DOI: 10.1016/j. ijfoodmicro.2008.03.003

[6] Weir P. The ecology of Zymomonas: A review. Folia Microbiologica. 2016;61: 385-392. DOI: 10.1007/s12223-016-

[7] Walker G, Stewart G. Saccharomyces cerevisiae in the production of fermented beverages. Beverages. 2016;2:30. DOI:

[8] Nicol D. Rum. In: Lea A, Piggott J,

10.3390/beverages2040030

editors. Fermented Beverage Production. Boston: Springer; 2003. p. 265. DOI: 10.1007/978-1-4615-0187-9

pp. 8-44

2012. pp. 1-3

0447-x

99

## Acknowledgements

The authors thank the Universidad Autónoma de Querétaro for their financial support, and CONACYT for the scholarships given to Dalia Miranda-Castilleja and Alejandro Aldrete-Tapia.

## Conflict of interest

The authors state that there is not conflict of interest.

## Author details

Jesús Alejandro Aldrete-Tapia, Dalia Elizabeth Miranda-Castilleja, Sofia Maria Arvizu-Medrano, Ramón Álvar Martínez-Peniche, Lourdes Soto-Muñoz and Montserrat Hernández-Iturriaga\* Universidad Autónoma de Querétaro, Queretaro, Mexico

\*Address all correspondence to: montshi@uaq.mx

© 2018 The Author(s). Licensee IntechOpen. 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.

Pursuing the Perfect Performer of Fermented Beverages: GMMs vs. Microbial Consortium DOI: http://dx.doi.org/10.5772/intechopen.81616

## References

fermented beverages, the other groups remark that only natural diversity and traditional methods could generate best products with typicity. Furthermore, the application of genetic modifications is badly perceived by consumers and legally prohibited in some cases. It seems that the next step in the improvement agenda is the combination of both approaches, the incorporation of mixtures of natural, genetically modified microorganisms and native strains to provide a holistic solu-

Frontiers and New Trends in the Science of Fermented Food and Beverages

The authors thank the Universidad Autónoma de Querétaro for their financial support, and CONACYT for the scholarships given to Dalia Miranda-Castilleja and

tion to the existing difficulties in fermentation.

The authors state that there is not conflict of interest.

Jesús Alejandro Aldrete-Tapia, Dalia Elizabeth Miranda-Castilleja,

Universidad Autónoma de Querétaro, Queretaro, Mexico

\*Address all correspondence to: montshi@uaq.mx

Sofia Maria Arvizu-Medrano, Ramón Álvar Martínez-Peniche, Lourdes Soto-Muñoz

© 2018 The Author(s). Licensee IntechOpen. 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,

Acknowledgements

Alejandro Aldrete-Tapia.

Conflict of interest

Author details

98

and Montserrat Hernández-Iturriaga\*

provided the original work is properly cited.

[1] El-Mansi E, Bryce C, Hartley B, Demain A. Fermentation microbiology and biotechnology: An historical perspective. In: El-Mansi E, Bryce C, Dahhou B, Sanchez S, Demain A, Allman A, editors. Fermentation Microbiology and Biotechnology. Boca Raton: CRC Press; 2012. pp. 2-4

[2] Stanbury P, Whitaker A, Hall S. Principles of Fermentation Technology. Butterworth-Heinemann; 2016. pp. 1-20

[3] Kamal-Eldin A, Mehta B, Iwanski R. Fermentation: Effects on food properties. Boca Raton: CRC Press; 2012. pp. 8-44

[4] Hui Y. Fermented plant products and their manufacturing. In: Hui Y, Evranuz E, editors. Handbook of Plant-based Fermented Food and Beverage Technology. Burlington: CRC Press; 2012. pp. 1-3

[5] Escalante A, Giles-Gómez M, Hernández G, Córdova-Aguilar M, López-Munguía A, Gosset G, et al. Analysis of bacterial community during the fermentation of pulque, a traditional Mexican alcoholic beverage, using a polyphasic approach. International Journal of Food Microbiology. 2008;124: 126-134. DOI: 10.1016/j. ijfoodmicro.2008.03.003

[6] Weir P. The ecology of Zymomonas: A review. Folia Microbiologica. 2016;61: 385-392. DOI: 10.1007/s12223-016- 0447-x

[7] Walker G, Stewart G. Saccharomyces cerevisiae in the production of fermented beverages. Beverages. 2016;2:30. DOI: 10.3390/beverages2040030

[8] Nicol D. Rum. In: Lea A, Piggott J, editors. Fermented Beverage Production. Boston: Springer; 2003. p. 265. DOI: 10.1007/978-1-4615-0187-9 [9] Swiegers J, Saerens S, Pretorius I. The development of yeast strains as tools for adjusting the flavor of fermented beverages to market specifications. In: Havkin-Frenkel D, Dudai N, editors. Biotechnology in Flavor Production. Chichester: John Wiley & Sons; 2008. pp. 6-7. DOI: 10.1002/9781444302493.ch1

[10] Hagman A, Säll T, Compagno C, Piskur J. Yeast "make-accumulateconsume" life strategy evolved as a multi-step process that predates the whole genome duplication. PLoS One. 2013;8:e68734. DOI: 10.1371/journal. pone.0068734

[11] Muñoz R, Moreno-Arribas M, de las Rivas B. Lactic acid bacteria. In: Carrascosa AV, Muñoz R, González R, editors. Molecular Wine Microbiology. 1st ed. 2011. pp. 191-226

[12] Liu S. Malolactic fermentation in wine—Beyond deacidification. Journal of Applied Microbiology. 2002;92: 589-601. DOI: 10.1046/j.1365-2672. 2002.01589.x

[13] Gullo M, Giudici P. Acetic acid bacteria in traditional balsamic vinegar: Phenotypic traits relevant for starter cultures selection. International Journal of Food Microbiology. 2008;125:46-53. DOI: 10.1016/j.ijfoodmicro.2007.11.076

[14] Sengun I, Karabiyikli S. Importance of acetic acid bacteria in food industry. Food Control. 2011;22:647-656. DOI: 10.1016/j.foocont.2010.11.008

[15] Kersters K, Lisdiyanti P, Komagata K, Swings J. The family Acetobacteraceae: The genera Acetobacter, Acidomonas, Asaia, Gluconacetobacter, Gluconobacter and Kozakia. In: Dworkin M, Falkow S, Rosenberg E, Schleifer K, Stackebrandt E, editors. The Prokaryotes. 3. New York: Springer; 2006. pp. 163-200. DOI: 10.1007/0-387-30745-1\_9

[16] Bartowsky E, Henschke P. Acetic acid bacteria spoilage of bottled red wine—A review. International Journal of Food Microbiology. 2008; 125:60-70. DOI: 10.1016/j. ijfoodmicro.2007.10.016

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535-569. DOI: 10.1111/ j.1574-6976.2007.00076.x

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[69] Li N, Duan J, Gao D, Luo J, Zheng R, Bian Y, et al. Mutation and selection of Oenococcus oeni for controlling wine malolactic fermentation. European Food Research and Technology. 2015;240: 93-100. DOI: 10.1007/s00217-014-2310-0

[70] Sato M, Kishimoto M, Watari J, Takashio M. Breeding of brewer's yeast by hybridization between top fermenting yeast Saccharomyces cerevisiae and cryophilic yeast Saccharomyces bayanus. Journal of Bioscience and Bioengineering. 2002;93:509-511. DOI: 10.1016/ S1389-1723(02)80101-3

[71] Garcia R, Solodovnikova N, Wendland J. Breeding of lager yeast with Saccharomyces cerevisiae improves resistance and fermentation performance. Yeast. 2012;29:343-355. DOI: 10.1002/yea.2914

[72] Brice C, Sanchez I, Bigey F, Legras J, Blongin B. A genetic approach of wine yeast fermentation capacity in nitrogenstarvation reveals the key role of nitrogen signaling. BMC Genomics. 2014;15:495. DOI: 10.1186/1471-2164- 15-495

[73] Mitsumasu K, Liu Z, Tang Y, Akamatsu T, Taguchi H, Kida K. Development of industrial yeast strain with improved acid- and thermotolerance through evolution under continuous fermentation conditions followed by haploidization and mating. Journal of Bioscience and Bioengineering. 2014;118:689-695. DOI: 10.1016/j.jbiosc.2014.05.012

[74] Mukai N, Nishimori C, Wilson I, Mizuno A, Takahashi T, Sato K. Beer brewing using fusant between a sake yeast and a brewer's yeast. Journal of Bioscience and Bioengineering. 2001;91: 482-486. DOI: 10.1016/S1389-1723(01) 80277-2

[75] Hou L. Improved production of ethanol by novel genome shuffling in Saccharomyces cerevisiae. Applied Biochemistry and Biotechnology. 2010; 160:1084-1093. DOI: 10.1007/ s12010-009-8552-9

[76] Wang H, Hou L. Genome shuffling to improve fermentation properties of top-fermenting yeast by the improvement of stress tolerance. Food Science and Biotechnology. 2010;19: 145-150. DOI: 10.1007/s10068-010- 0020-3

[77] Wei P, Li Z, He P, Lin Y, Jiang N. Genome shuffling in the ethanologenic yeast Candida krusei to improve acetic acid tolerance. Biotechnology and Applied Biochemistry. 2010;49:113-120. DOI: 10.1042/BA20070072

[78] Wei K, Cao X, Li X, Wang C, Hou L. Genome shuffling to improve fermentation properties of acetic acid bacterium by the improvement of ethanol tolerance. International Journal of Food Science and Technology. 2012; 47:2184-2189. DOI: 10.1111/j.1365-2621. 2012.03086.x

Letters. 2007;29:191-200. DOI: 10.1007/

DOI: http://dx.doi.org/10.5772/intechopen.81616

Pursuing the Perfect Performer of Fermented Beverages: GMMs vs. Microbial Consortium

[92] Shea E, Cotter P, Ross R, Hill C. Strategies to improve the bacteriocin protection provided by lactic acid bacteria. Current Opinion in Biotechnology. 2013;24:130-134

[93] Verraes C, Van Boxsael S, Van Meervenee E, Van Coillie, Butaye P, Catry B, et al. Antimicrobial resistance

International Journal of Environmental Research and Public Health. 2013;10:

in the food chain: A review.

2643-2669. DOI: 10.3390/

[94] Brenner K, Joe L, Arnold F. Engineering microbial consortia: a new frontier in synthetic biology. Journal of Trends in Biotechnology. 2008;26:483- 489. DOI: 10.1016/j.tibtech.2018.05.004

[95] Hays S, Patrick W, Ziesack M, Oxman N, Silver P. Better together: engineering and application of microbial

[96] Jia X, Liu C, Song H, Ding M, Du J, Ma Q, Yuan Y. Design, analysis and application of synthetic microbial consortia. Synthetic and Systems Biotechnology. 2016;1:109-117. DOI: 10.1016/j.synbio. 2016.02.001

symbioses. Current Opinion in Biotechnology. 2015;36:40-49. DOI: 10.1016/j.copbio.2015.08.008

ijerph10072643

[86] Bellon J, Schmid F, Capone D, Dunn B, Chambers P. Introducing a new breed

hybridization between a commercial Saccharomyces cerevisiae wine yeast and Saccharomyces mikatae. PLoS One. 2013;

[87] González S, Gallo L, Climent M, Barrio E, Querol A. Ecological

[88] Bellon J, Eglington J, Siebert T, Pollnitz A, Rose L, de Barros M, et al. Newly generated interspecific wine yeast hybrids introduce flavour and aroma diversity to wines. Applied Microbiology and Biotechnology. 2011; 91:603-612. DOI: 10.1007/s00253-011-

[89] Zhao D, Wu B, Zhang Y, Jia H, Zhang X, Cheng S. Identification of protoplast fusion strain Fhhh by randomly amplified polymorphic DNA.

Biotechnology. 2009;25:1181-1188. DOI:

[90] Rossi F, Rizzotti L, Felis G, Torriani S. Horizontal gene transfer among microorganisms in food: Current knowledge and future perspectives. Food Microbiology. 2014;42:232-243. DOI: 10.1016/j.fm.2014.04.004

[91] Frost L, Koraimann G. Regulation of

bacterial conjugation: Balancing opportunity with adversity. Future Microbiology. 2010;5:1057-1071

Journal of Microbiology and

10.1007/s-11274-009-9999-0

characterization of natural hybrids from

8:e62053. DOI: 10.1371/journal.

Saccharomyces cerevisiae and S. kudriavzevii. International Journal of Food Microbiology. 2007;116:11-18. DOI: 10.1016/j.ijfoodmicro.2006.10.047

pone.0062053

3294-3

105

of wine yeast: Interspecific

[85] Haber J. Mating-type genes and MAT switching in Saccharomyces cerevisiae. Genetics. 2012;191:33-64. DOI: 10.1534/genetics.111.134577

s10529-006-9236-y

[79] Dai Z, Nielsen J. Advancing metabolic engineering through systems biology of industrial microorganisms. Current Opinion in Biotechnology. 2015;36:8-15. DOI: 10.1016/j. copbio.2015.08.006

[80] Sybesma W, Hugenholt J, de-Vos W, Smid E. Safe use of genetically modified lactic acid bacteria in food: Bringing the gap between consumers, green groups, and industry. Electronic Journal of Biotechnology. 2006;9: 424-448. DOI: 10.2225/vol9-issue4 fulltext-12

[81] Husnik J, Volschenk H, Bauer J, Colavizza D, Luo Z, van Vuuren H. Metabolic engineering of malolactic wine yeast. Metabolic Engineering. 2006;8:315-323. DOI: 10.1016/j. ymben.2006.02.003

[82] Dragosits M, Mattanovich D. Adaptive laboratory evolution— Principles and applications for biotechnology. Microbial Cell Factories. 2013;12:64. DOI: 10.1186/1475-2859- 12-64

[83] Derkx P, Janzen T, Sorensen K, Christensen J, Stuer-Lauridsen B, Johansen E. The art of strain improvement of industrial lactic acid bacteria without the use of recombinant DNA technology. Microbial Cell Factories. 2014;13:S5. DOI: 10.1186/ 1475-2859-13-S1-S5

[84] Cebollero E, Gonzalez-Ramos D, Tabera L, Gonzalez R. Transgenic wine yeast technology comes to age: Is it time for trangenic wine? Biotechnology

Pursuing the Perfect Performer of Fermented Beverages: GMMs vs. Microbial Consortium DOI: http://dx.doi.org/10.5772/intechopen.81616

Letters. 2007;29:191-200. DOI: 10.1007/ s10529-006-9236-y

with Saccharomyces cerevisiae improves

Frontiers and New Trends in the Science of Fermented Food and Beverages

[78] Wei K, Cao X, Li X, Wang C, Hou L.

fermentation properties of acetic acid bacterium by the improvement of ethanol tolerance. International Journal of Food Science and Technology. 2012; 47:2184-2189. DOI: 10.1111/j.1365-2621.

Genome shuffling to improve

[79] Dai Z, Nielsen J. Advancing

metabolic engineering through systems biology of industrial microorganisms. Current Opinion in Biotechnology. 2015;36:8-15. DOI: 10.1016/j.

[80] Sybesma W, Hugenholt J, de-Vos W, Smid E. Safe use of genetically modified lactic acid bacteria in food: Bringing the gap between consumers, green groups, and industry. Electronic Journal of Biotechnology. 2006;9: 424-448. DOI: 10.2225/vol9-issue4-

[81] Husnik J, Volschenk H, Bauer J, Colavizza D, Luo Z, van Vuuren H. Metabolic engineering of malolactic wine yeast. Metabolic Engineering. 2006;8:315-323. DOI: 10.1016/j.

[82] Dragosits M, Mattanovich D. Adaptive laboratory evolution— Principles and applications for

biotechnology. Microbial Cell Factories. 2013;12:64. DOI: 10.1186/1475-2859-

[83] Derkx P, Janzen T, Sorensen K, Christensen J, Stuer-Lauridsen B, Johansen E. The art of strain

improvement of industrial lactic acid bacteria without the use of recombinant

[84] Cebollero E, Gonzalez-Ramos D, Tabera L, Gonzalez R. Transgenic wine yeast technology comes to age: Is it time for trangenic wine? Biotechnology

DNA technology. Microbial Cell Factories. 2014;13:S5. DOI: 10.1186/

1475-2859-13-S1-S5

2012.03086.x

copbio.2015.08.006

fulltext-12

12-64

ymben.2006.02.003

performance. Yeast. 2012;29:343-355.

[72] Brice C, Sanchez I, Bigey F, Legras J, Blongin B. A genetic approach of wine yeast fermentation capacity in nitrogen-

starvation reveals the key role of nitrogen signaling. BMC Genomics. 2014;15:495. DOI: 10.1186/1471-2164-

[73] Mitsumasu K, Liu Z, Tang Y, Akamatsu T, Taguchi H, Kida K. Development of industrial yeast strain with improved acid- and thermotolerance through evolution under continuous fermentation conditions followed by haploidization and mating.

Bioengineering. 2014;118:689-695. DOI:

[74] Mukai N, Nishimori C, Wilson I, Mizuno A, Takahashi T, Sato K. Beer brewing using fusant between a sake yeast and a brewer's yeast. Journal of Bioscience and Bioengineering. 2001;91: 482-486. DOI: 10.1016/S1389-1723(01)

[75] Hou L. Improved production of ethanol by novel genome shuffling in Saccharomyces cerevisiae. Applied Biochemistry and Biotechnology. 2010;

[76] Wang H, Hou L. Genome shuffling to improve fermentation properties of

improvement of stress tolerance. Food Science and Biotechnology. 2010;19: 145-150. DOI: 10.1007/s10068-010-

[77] Wei P, Li Z, He P, Lin Y, Jiang N. Genome shuffling in the ethanologenic yeast Candida krusei to improve acetic acid tolerance. Biotechnology and Applied Biochemistry. 2010;49:113-120.

160:1084-1093. DOI: 10.1007/

top-fermenting yeast by the

DOI: 10.1042/BA20070072

s12010-009-8552-9

Journal of Bioscience and

10.1016/j.jbiosc.2014.05.012

resistance and fermentation

DOI: 10.1002/yea.2914

15-495

80277-2

0020-3

104

[85] Haber J. Mating-type genes and MAT switching in Saccharomyces cerevisiae. Genetics. 2012;191:33-64. DOI: 10.1534/genetics.111.134577

[86] Bellon J, Schmid F, Capone D, Dunn B, Chambers P. Introducing a new breed of wine yeast: Interspecific hybridization between a commercial Saccharomyces cerevisiae wine yeast and Saccharomyces mikatae. PLoS One. 2013; 8:e62053. DOI: 10.1371/journal. pone.0062053

[87] González S, Gallo L, Climent M, Barrio E, Querol A. Ecological characterization of natural hybrids from Saccharomyces cerevisiae and S. kudriavzevii. International Journal of Food Microbiology. 2007;116:11-18. DOI: 10.1016/j.ijfoodmicro.2006.10.047

[88] Bellon J, Eglington J, Siebert T, Pollnitz A, Rose L, de Barros M, et al. Newly generated interspecific wine yeast hybrids introduce flavour and aroma diversity to wines. Applied Microbiology and Biotechnology. 2011; 91:603-612. DOI: 10.1007/s00253-011- 3294-3

[89] Zhao D, Wu B, Zhang Y, Jia H, Zhang X, Cheng S. Identification of protoplast fusion strain Fhhh by randomly amplified polymorphic DNA. Journal of Microbiology and Biotechnology. 2009;25:1181-1188. DOI: 10.1007/s-11274-009-9999-0

[90] Rossi F, Rizzotti L, Felis G, Torriani S. Horizontal gene transfer among microorganisms in food: Current knowledge and future perspectives. Food Microbiology. 2014;42:232-243. DOI: 10.1016/j.fm.2014.04.004

[91] Frost L, Koraimann G. Regulation of bacterial conjugation: Balancing opportunity with adversity. Future Microbiology. 2010;5:1057-1071

[92] Shea E, Cotter P, Ross R, Hill C. Strategies to improve the bacteriocin protection provided by lactic acid bacteria. Current Opinion in Biotechnology. 2013;24:130-134

[93] Verraes C, Van Boxsael S, Van Meervenee E, Van Coillie, Butaye P, Catry B, et al. Antimicrobial resistance in the food chain: A review. International Journal of Environmental Research and Public Health. 2013;10: 2643-2669. DOI: 10.3390/ ijerph10072643

[94] Brenner K, Joe L, Arnold F. Engineering microbial consortia: a new frontier in synthetic biology. Journal of Trends in Biotechnology. 2008;26:483- 489. DOI: 10.1016/j.tibtech.2018.05.004

[95] Hays S, Patrick W, Ziesack M, Oxman N, Silver P. Better together: engineering and application of microbial symbioses. Current Opinion in Biotechnology. 2015;36:40-49. DOI: 10.1016/j.copbio.2015.08.008

[96] Jia X, Liu C, Song H, Ding M, Du J, Ma Q, Yuan Y. Design, analysis and application of synthetic microbial consortia. Synthetic and Systems Biotechnology. 2016;1:109-117. DOI: 10.1016/j.synbio. 2016.02.001

Chapter 7

Abstract

1. Introduction

107

Perspectives and Uses of

Fermented Beverages

Waldir Desiderio Estela Escalante

Non-Saccharomyces Yeasts in

Fermented beverages such as wine, cider and beer are normally fermented with Saccharomyces yeasts due to their well-known fermentative behavior. These yeasts have been extensively investigated and are used in commercial processes. On the other hand, non-Saccharomyces yeasts were always considered contaminants in winemaking and brewing. Most researchers in the past argued that these yeasts produce several compounds that may alter the sensory quality of wine and beers. However, recent studies have demonstrated that their fermentative metabolism can be regulated and addressed to the production of compounds of sensory importance. Currently, some non-Saccharomyces yeasts belonging to the genera Kloeckera, Candida, Hanseniaspora are getting importance due to their high potentiality to be used in the production of fermented beverages such as special wines and craft beers. The emergence of new consumption patterns and market niches demanding products with new sensory characteristics has catapulted the exploitation of these yeasts.

Keywords: non-Saccharomyces yeasts, fermented beverages, wine, craft beers

Fermentation of wines, beers and ciders is traditionally carried out with Saccharomyces cerevisiae strains, the most common and commercially available yeast. They are well known for their fermentative behavior and technological characteristics which allow obtaining products of uniform and standard quality. Saccharomyces cerevisiae is the most used yeast in fermentative processes. In wine fermentation, strains with specific characteristics are needed, for instance, highly producers of ethanol to reach values of 11–13% v/v, typically found in this beverage. On the other hand, beers and ciders contain less amounts of ethanol with a balanced and distinctive sensory profile characteristic of each one. In recent years, new consuming trends and requirements for new and innovative products have emerged. This situation led to rethink about the existing fermented beverages and to meet the demands of consumers. Yeasts are largely responsible for the complexity and sensory quality of fermented beverages. Based on this, current studies are mainly focused on the search of new type of yeasts with technological application. Non-Saccharomyces yeasts have always been considered contaminants in the manufacture of wine and beer. Therefore, procedures for eliminating them are routinely utilized such as must pasteurization, addition of sulfite and sanitization of equipment and

## Chapter 7

## Perspectives and Uses of Non-Saccharomyces Yeasts in Fermented Beverages

Waldir Desiderio Estela Escalante

## Abstract

Fermented beverages such as wine, cider and beer are normally fermented with Saccharomyces yeasts due to their well-known fermentative behavior. These yeasts have been extensively investigated and are used in commercial processes. On the other hand, non-Saccharomyces yeasts were always considered contaminants in winemaking and brewing. Most researchers in the past argued that these yeasts produce several compounds that may alter the sensory quality of wine and beers. However, recent studies have demonstrated that their fermentative metabolism can be regulated and addressed to the production of compounds of sensory importance. Currently, some non-Saccharomyces yeasts belonging to the genera Kloeckera, Candida, Hanseniaspora are getting importance due to their high potentiality to be used in the production of fermented beverages such as special wines and craft beers. The emergence of new consumption patterns and market niches demanding products with new sensory characteristics has catapulted the exploitation of these yeasts.

Keywords: non-Saccharomyces yeasts, fermented beverages, wine, craft beers

## 1. Introduction

Fermentation of wines, beers and ciders is traditionally carried out with Saccharomyces cerevisiae strains, the most common and commercially available yeast. They are well known for their fermentative behavior and technological characteristics which allow obtaining products of uniform and standard quality. Saccharomyces cerevisiae is the most used yeast in fermentative processes. In wine fermentation, strains with specific characteristics are needed, for instance, highly producers of ethanol to reach values of 11–13% v/v, typically found in this beverage. On the other hand, beers and ciders contain less amounts of ethanol with a balanced and distinctive sensory profile characteristic of each one. In recent years, new consuming trends and requirements for new and innovative products have emerged. This situation led to rethink about the existing fermented beverages and to meet the demands of consumers. Yeasts are largely responsible for the complexity and sensory quality of fermented beverages. Based on this, current studies are mainly focused on the search of new type of yeasts with technological application. Non-Saccharomyces yeasts have always been considered contaminants in the manufacture of wine and beer. Therefore, procedures for eliminating them are routinely utilized such as must pasteurization, addition of sulfite and sanitization of equipment and

processing halls. In recent years, the negative perception about non-Saccharomyces yeasts has been changing due to the fact that several studies have shown that during spontaneous fermentations of wine, these yeasts play an important role in the definition of the sensory quality of the final product. Based on this evidence, the fermentative behavior of some non-Saccharomyces yeasts is being studied in deep with the purpose of finding the most adequate conditions and the most suitable strain to be utilized in the production of fermented beverages.

fermentation processes, asexual reproduction of yeasts is preferable to ensure the conservation of the genotype and to maintain their fermentative behavior over time. Regarding their metabolism, yeasts are usually characterized by fermenting a broad spectrum of sugars, among them, glucose, fructose, sucrose, maltose and maltotriose, which are found in ripen fruits and processed cereals. In addition, yeasts tolerate acidic environments with pH values around 3.5 or even less. According to technological convenience, yeasts are divided into two large groups namely Saccharomyces and non-Saccharomyces. Morphologically, Saccharomyces yeasts can be round or ellipsoidal in shape depending on the growth phase and cultivation conditions. S. cerevisiae is the most studied species and the most utilized in the fermentation of wines and beers due to its excellent fermentative capacity, rapid growth and easy adaptation. They tolerate concentrations of SO2 that normally most non-Saccharomyces yeasts do not survive. However, despite these advantages, it is possible to find in the nature representatives of S. cerevisiae that do not necessarily present these features.

Perspectives and Uses of Non-Saccharomyces Yeasts in Fermented Beverages

DOI: http://dx.doi.org/10.5772/intechopen.81868

Non-Saccharomyces yeasts are a group of microorganisms genetically diverse with specific metabolic characteristics and high potential for using in fermentation processes. In the past, many of them have been considered contaminants due to the production of compounds that alters the sensory quality of wines [4, 5]. With the purpose of eliminating them and avoiding their fermentative activity, for instance, in wine processing, disinfection of fermentation tanks and containers with sulfite is commonly performed. However, over time, the importance of non-Saccharomyces yeasts in spontaneous fermentation has been demonstrated since they contribute positively to the definition of the sensory quality of wines. These yeasts predominate at the initial stage of the spontaneous fermentation [6–8] until certain concentration of ethanol is reached (usually between 4 and 5% v/v), which are then inhibited due to the effect of the ethanol and the depletion of dissolved oxygen [9, 10]. At the end of the process, Saccharomyces yeasts, the most resistant to ethanol, predominate and complete the fermentation. It has been reported that some non-Saccharomyces yeasts are able to survive toward the end of the spontaneous fermentation and exert their metabolic activity, thus contributing positively to the sensory quality of wines. Based on this evidence, in recent years, many researchers have focused their studies in understanding the nature and fermentative activity of the non-Saccharomyces yeasts [8, 11–21]. The findings demonstrated the enormous potential of these yeasts for use in the fermentation of traditional and nontraditional beverages. Despite the fact that most non-Saccharomyces yeasts show some technological disadvantages compared to Saccharomyces cerevisiae such as lower fermentative power and production of ethanol, non-Saccharomyces yeasts possess characteristics that in S. cerevisiae are absent, for instance, production of high levels of aromatic compounds such as esters, higher alcohols and fatty acids [22, 23]. In addition, it has been reported that the fermentative activity of these yeasts is manifested in the presence of small amounts of oxygen which leads to an increase in cell biomass and the decrease in ethanol yield, a strategy that can be used to reduce the ethanol content of wines produced in coculture with S cerevisiae [24–26]. With the aim of exploiting the positive characteristics of non-Saccharomyces yeasts and reducing their negative impact, fermentations with mixed and sequential cultures with S. cerevisiae can be performed to produce

fermented beverages with different sensory profiles [27–29]. The most important fact is related to the potential for producing a broad variety of compounds of sensory importance necessary to improve the organoleptic quality of wines and beers. The findings reported so far in literature have led to rethink the role of these yeasts in

3. Non-Saccharomyces yeasts

109

## 2. Yeasts

Yeasts are eukaryotic microorganisms that inhabit a variety of ecological niches such as water, soil, air and the surface of plants and fruits. Commonly, they are present during the decomposition of ripen fruits and participate in the fermentation process. In this natural environment, the yeasts find nutrients and substrates necessary for their metabolism and fermentative activity [1, 2]. Yeasts are not nutritionally demanding compared to other microorganisms such as lactic acid bacteria. For supporting their growth, they need common compounds such as fermentable sugars, amino acids, vitamins, minerals and also oxygen. Morphologically the yeasts are very diverse, being the round, ellipsoidal and oval shapes mostly predominant. During the identification, the microscopic evaluation is the first resource followed by microbiological and biochemical tests; subsequently, assays of sugar fermentation and assimilation of amino acids are necessary [3]. The production and tolerance to ethanol, organic acids and SO2 are also important tools to differentiate among species. The reproduction of yeasts is mainly by budding, which results in a new and genetically identical cell. Budding is the most common type of asexual reproduction, although cell fission is a characteristic of yeasts belonging to the genus Schizosaccharomyces (Figure 1). Cultivation conditions leading to the starvation of nutrients such as the lack of amino acids induce sporulation, which is a mechanism used by yeasts to survive under unfavorable conditions. As a consequence of the sporulation, yeast cells undergo genetic variability. In industrial

#### Figure 1.

Asexual reproduction of yeasts. (a) Budding, typically observed in Saccharomyces cerevisiae, Candida, Kloeckera, Brettanomyces and (b) fission, typically observed in Schizosaccharomyces pombe.

Perspectives and Uses of Non-Saccharomyces Yeasts in Fermented Beverages DOI: http://dx.doi.org/10.5772/intechopen.81868

fermentation processes, asexual reproduction of yeasts is preferable to ensure the conservation of the genotype and to maintain their fermentative behavior over time. Regarding their metabolism, yeasts are usually characterized by fermenting a broad spectrum of sugars, among them, glucose, fructose, sucrose, maltose and maltotriose, which are found in ripen fruits and processed cereals. In addition, yeasts tolerate acidic environments with pH values around 3.5 or even less. According to technological convenience, yeasts are divided into two large groups namely Saccharomyces and non-Saccharomyces. Morphologically, Saccharomyces yeasts can be round or ellipsoidal in shape depending on the growth phase and cultivation conditions. S. cerevisiae is the most studied species and the most utilized in the fermentation of wines and beers due to its excellent fermentative capacity, rapid growth and easy adaptation. They tolerate concentrations of SO2 that normally most non-Saccharomyces yeasts do not survive. However, despite these advantages, it is possible to find in the nature representatives of S. cerevisiae that do not necessarily present these features.

#### 3. Non-Saccharomyces yeasts

processing halls. In recent years, the negative perception about non-Saccharomyces yeasts has been changing due to the fact that several studies have shown that during spontaneous fermentations of wine, these yeasts play an important role in the definition of the sensory quality of the final product. Based on this evidence, the fermentative behavior of some non-Saccharomyces yeasts is being studied in deep with the purpose of finding the most adequate conditions and the most suitable

Yeasts are eukaryotic microorganisms that inhabit a variety of ecological niches such as water, soil, air and the surface of plants and fruits. Commonly, they are present during the decomposition of ripen fruits and participate in the fermentation process. In this natural environment, the yeasts find nutrients and substrates necessary for their metabolism and fermentative activity [1, 2]. Yeasts are not nutritionally demanding compared to other microorganisms such as lactic acid bacteria. For supporting their growth, they need common compounds such as fermentable sugars, amino acids, vitamins, minerals and also oxygen. Morphologically the yeasts are very diverse, being the round, ellipsoidal and oval shapes mostly predominant. During the identification, the microscopic evaluation is the first resource followed by microbiological and biochemical tests; subsequently, assays of sugar fermentation and assimilation of amino acids are necessary [3]. The production and tolerance to ethanol, organic acids and SO2 are also important tools to differentiate among species. The reproduction of yeasts is mainly by budding, which results in a new and genetically identical cell. Budding is the most common type of asexual reproduction, although cell fission is a characteristic of yeasts belonging to the genus

Schizosaccharomyces (Figure 1). Cultivation conditions leading to the starvation of nutrients such as the lack of amino acids induce sporulation, which is a mechanism used by yeasts to survive under unfavorable conditions. As a consequence of the sporulation, yeast cells undergo genetic variability. In industrial

Asexual reproduction of yeasts. (a) Budding, typically observed in Saccharomyces cerevisiae, Candida, Kloeckera, Brettanomyces and (b) fission, typically observed in Schizosaccharomyces pombe.

strain to be utilized in the production of fermented beverages.

Frontiers and New Trends in the Science of Fermented Food and Beverages

2. Yeasts

Figure 1.

108

Non-Saccharomyces yeasts are a group of microorganisms genetically diverse with specific metabolic characteristics and high potential for using in fermentation processes. In the past, many of them have been considered contaminants due to the production of compounds that alters the sensory quality of wines [4, 5]. With the purpose of eliminating them and avoiding their fermentative activity, for instance, in wine processing, disinfection of fermentation tanks and containers with sulfite is commonly performed. However, over time, the importance of non-Saccharomyces yeasts in spontaneous fermentation has been demonstrated since they contribute positively to the definition of the sensory quality of wines. These yeasts predominate at the initial stage of the spontaneous fermentation [6–8] until certain concentration of ethanol is reached (usually between 4 and 5% v/v), which are then inhibited due to the effect of the ethanol and the depletion of dissolved oxygen [9, 10]. At the end of the process, Saccharomyces yeasts, the most resistant to ethanol, predominate and complete the fermentation. It has been reported that some non-Saccharomyces yeasts are able to survive toward the end of the spontaneous fermentation and exert their metabolic activity, thus contributing positively to the sensory quality of wines. Based on this evidence, in recent years, many researchers have focused their studies in understanding the nature and fermentative activity of the non-Saccharomyces yeasts [8, 11–21]. The findings demonstrated the enormous potential of these yeasts for use in the fermentation of traditional and nontraditional beverages. Despite the fact that most non-Saccharomyces yeasts show some technological disadvantages compared to Saccharomyces cerevisiae such as lower fermentative power and production of ethanol, non-Saccharomyces yeasts possess characteristics that in S. cerevisiae are absent, for instance, production of high levels of aromatic compounds such as esters, higher alcohols and fatty acids [22, 23]. In addition, it has been reported that the fermentative activity of these yeasts is manifested in the presence of small amounts of oxygen which leads to an increase in cell biomass and the decrease in ethanol yield, a strategy that can be used to reduce the ethanol content of wines produced in coculture with S cerevisiae [24–26]. With the aim of exploiting the positive characteristics of non-Saccharomyces yeasts and reducing their negative impact, fermentations with mixed and sequential cultures with S. cerevisiae can be performed to produce fermented beverages with different sensory profiles [27–29]. The most important fact is related to the potential for producing a broad variety of compounds of sensory importance necessary to improve the organoleptic quality of wines and beers. The findings reported so far in literature have led to rethink the role of these yeasts in

fermentative processes and to evaluate their use in the development of new products. Among the most studied non-Saccharomyces yeasts that reached special importance for researchers include Candida, Kloeckera, Hanseniaspora, Brettanomyces, Pichia, Lanchacea and Kluyveromyces, among others.

generation of energy is greater since glucose undergoes a complete oxidation, and as a result 36 net moles of ATP per mole of glucose are generated. The low-energy yield obtained by yeasts under anaerobic conditions forces the cell to increase the flow of glucose consumption in order to obtain a higher amount of energy in the form of ATP. As consequence, the ethanol accumulates in the fermentation medium and exerts its inhibitory effect, thus stopping the fermentative activity of the yeasts [31]. The low amount of energy generated under anaerobic conditions is used by the yeast cells in requirements for maintenance and growth. Glucose is easily transported and metabolized inside the cell; however, disaccharides such as sucrose, maltose or lactose must be first hydrolyzed to their simple forms (hexoses) which are then catabolized in the glycolysis pathway. Sucrose is hydrolyzed to fructose and glucose, maltose to two glucose units and lactose to glucose and galactose. The disaccharides are preferably hydrolyzed in the periplasmic space before entering the cytosol. Under anaerobic conditions besides ethanol, glycerol is also produced, thus contributing to restore the redox balance inside the cell. The production of glycerol increases in fermentations with musts of high specific gravity as a response to the osmotic stress [32]. It has been found that yeasts unable of metabolizing dihydroxyacetone (Figure 2) are not capable of producing glycerol, and as a consequence dihydroxyacetone accumulates and inhibits the fermentation. Moreover, glucose apart of being metabolized via glycolysis, it is also broken by complementary pathways that are not necessarily related to the generation of energy. The hexose monophosphate pathway (HMP) also known as the pentose phosphate cycle usually accompanies the glycolytic pathway [33]. In addition, yeasts during fermentation produce small amounts of acetic acid either from acetaldehyde or acetyl-CoA (Figure 2). Acetic acid is the main organic acid produced by yeasts during the fermentation of glucose, and it is responsible for the acidification and the decrease of pH of the medium. Ethanol is the most important fermentation by-product, and from the technological point of view, the production capacity of yeasts is an important parameter that determines their usability in fermentative processes. Gay Lussac defined a stoichiometric theoretical relationship to explain

Perspectives and Uses of Non-Saccharomyces Yeasts in Fermented Beverages

DOI: http://dx.doi.org/10.5772/intechopen.81868

the production of ethanol by Saccharomyces cerevisiae yeasts which is:

higher alcohols, esters, aldehydes and organic acids, among others.

! 2 C2H5OH ethanol

According to this relationship, from 180.0 grams of glucose, 92.0 grams of ethanol and 88.0 grams of carbon dioxide are produced, which results in a theoretical yield of 0.511 g ethanol/g glucose. However, in practice, besides ethanol and CO2, the production of biomass, glycerol and other minority compounds also hap-

C6H12O6 þ nitrogen ! C2H5OH þ CO2 þ glycerol þ biomass þ minority compounds

At industrial scale, a yield of 0.45 g ethanol/g glucose is acceptable [34]. In the case of fermentations with non-Saccharomyces yeasts, lower yields are commonly observed. Regarding to glycerol, in the case of S. cerevisiae, its production represents approximately 3% of the utilized sugar. Minor compounds are represented by

Oxygen is an important element during the complete oxidation of glucose since it serves as final acceptor of electrons under aerobic conditions. It is also essential for other metabolic processes such as the synthesis of structural components of the

þ 2 CO2 carbon dioxide (1)

(2)

1 C6H12O6 glucose

pens, that is:

111

3.1.1 Importance of oxygen

### 3.1 Fermentative metabolism of sugars

Either non-Saccharomyces or Saccharomyces yeasts share common pathways for the central metabolism of carbon; thus, both groups metabolize glucose through glycolysis. However, the mechanisms involved in the regulation of respirefermentative metabolism can differ significantly among them [30]. The glycolysis operates indistinctly under aerobic and anaerobic conditions, and through it, the glucose is metabolized to pyruvate by means of a series of biochemical reactions (Figure 2). Under anaerobic or oxygen-limited conditions, pyruvate is converted to acetaldehyde and then to ethanol, and as a result, two net moles of ATP are generated. Under fully aerobic conditions and in the absence of any repression effect, the

#### Figure 2.

Fermentative metabolism of glucose by yeasts: Glycolysis (black lines) and ethanol and glycerol production (blue lines). Enzymes: 1, hexokinase; 2, phosphoglucose isomerase; 3, phosphofructokinase; 4, fructose 1,6 bisphosphate aldolase; 5, triosephosphate isomerase; 6, glyceraldehyde 3-phosphate dehydrogenase; 7, phosphoglycerate kinase; 8, phosphoglycerate mutase; 9, enolase; 10, pyruvate kinase; 11, pyruvate decarboxylase; 12, alcohol dehydrogenase; 13, aldehyde dehydrogenase; 14, acetyl-CoA hydrolase; 15, acetyl-CoA synthetase; 16, pyruvate dehydrogenase; 17, glycerol 3-P dehydrogenase; 18, glycerol 3-phosphatase.

Perspectives and Uses of Non-Saccharomyces Yeasts in Fermented Beverages DOI: http://dx.doi.org/10.5772/intechopen.81868

generation of energy is greater since glucose undergoes a complete oxidation, and as a result 36 net moles of ATP per mole of glucose are generated. The low-energy yield obtained by yeasts under anaerobic conditions forces the cell to increase the flow of glucose consumption in order to obtain a higher amount of energy in the form of ATP. As consequence, the ethanol accumulates in the fermentation medium and exerts its inhibitory effect, thus stopping the fermentative activity of the yeasts [31]. The low amount of energy generated under anaerobic conditions is used by the yeast cells in requirements for maintenance and growth. Glucose is easily transported and metabolized inside the cell; however, disaccharides such as sucrose, maltose or lactose must be first hydrolyzed to their simple forms (hexoses) which are then catabolized in the glycolysis pathway. Sucrose is hydrolyzed to fructose and glucose, maltose to two glucose units and lactose to glucose and galactose. The disaccharides are preferably hydrolyzed in the periplasmic space before entering the cytosol. Under anaerobic conditions besides ethanol, glycerol is also produced, thus contributing to restore the redox balance inside the cell. The production of glycerol increases in fermentations with musts of high specific gravity as a response to the osmotic stress [32]. It has been found that yeasts unable of metabolizing dihydroxyacetone (Figure 2) are not capable of producing glycerol, and as a consequence dihydroxyacetone accumulates and inhibits the fermentation. Moreover, glucose apart of being metabolized via glycolysis, it is also broken by complementary pathways that are not necessarily related to the generation of energy. The hexose monophosphate pathway (HMP) also known as the pentose phosphate cycle usually accompanies the glycolytic pathway [33]. In addition, yeasts during fermentation produce small amounts of acetic acid either from acetaldehyde or acetyl-CoA (Figure 2). Acetic acid is the main organic acid produced by yeasts during the fermentation of glucose, and it is responsible for the acidification and the decrease of pH of the medium. Ethanol is the most important fermentation by-product, and from the technological point of view, the production capacity of yeasts is an important parameter that determines their usability in fermentative processes. Gay Lussac defined a stoichiometric theoretical relationship to explain the production of ethanol by Saccharomyces cerevisiae yeasts which is:

$$\underset{\text{glucose}}{1\,\text{C}\_6\text{H}\_{12}\text{O}\_6} \to 2\,\text{C}\_2\text{H}\_5\text{OH} + 2\,\text{CO}\_2\tag{1}$$

According to this relationship, from 180.0 grams of glucose, 92.0 grams of ethanol and 88.0 grams of carbon dioxide are produced, which results in a theoretical yield of 0.511 g ethanol/g glucose. However, in practice, besides ethanol and CO2, the production of biomass, glycerol and other minority compounds also happens, that is:

$$\text{C}\_6\text{H}\_{12}\text{O}\_6 + \text{nitrogen} \rightarrow \text{C}\_2\text{H}\_5\text{OH} + \text{CO}\_2 + \text{glycine} + \text{biomass} + \text{minority compounds} \tag{2}$$

At industrial scale, a yield of 0.45 g ethanol/g glucose is acceptable [34]. In the case of fermentations with non-Saccharomyces yeasts, lower yields are commonly observed. Regarding to glycerol, in the case of S. cerevisiae, its production represents approximately 3% of the utilized sugar. Minor compounds are represented by higher alcohols, esters, aldehydes and organic acids, among others.

#### 3.1.1 Importance of oxygen

Oxygen is an important element during the complete oxidation of glucose since it serves as final acceptor of electrons under aerobic conditions. It is also essential for other metabolic processes such as the synthesis of structural components of the

fermentative processes and to evaluate their use in the development of new products. Among the most studied non-Saccharomyces yeasts that reached special importance for researchers include Candida, Kloeckera, Hanseniaspora, Brettanomyces, Pichia,

Frontiers and New Trends in the Science of Fermented Food and Beverages

Either non-Saccharomyces or Saccharomyces yeasts share common pathways for the central metabolism of carbon; thus, both groups metabolize glucose through glycolysis. However, the mechanisms involved in the regulation of respirefermentative metabolism can differ significantly among them [30]. The glycolysis operates indistinctly under aerobic and anaerobic conditions, and through it, the glucose is metabolized to pyruvate by means of a series of biochemical reactions (Figure 2). Under anaerobic or oxygen-limited conditions, pyruvate is converted to acetaldehyde and then to ethanol, and as a result, two net moles of ATP are generated. Under fully aerobic conditions and in the absence of any repression effect, the

Fermentative metabolism of glucose by yeasts: Glycolysis (black lines) and ethanol and glycerol production (blue lines). Enzymes: 1, hexokinase; 2, phosphoglucose isomerase; 3, phosphofructokinase; 4, fructose 1,6 bisphosphate aldolase; 5, triosephosphate isomerase; 6, glyceraldehyde 3-phosphate dehydrogenase; 7, phosphoglycerate kinase; 8, phosphoglycerate mutase; 9, enolase; 10, pyruvate kinase; 11, pyruvate decarboxylase; 12, alcohol dehydrogenase; 13, aldehyde dehydrogenase; 14, acetyl-CoA hydrolase; 15, acetyl-CoA synthetase; 16, pyruvate dehydrogenase; 17, glycerol 3-P dehydrogenase; 18, glycerol 3-phosphatase.

Lanchacea and Kluyveromyces, among others.

3.1 Fermentative metabolism of sugars

Figure 2.

110

cytoplasmic membrane of yeasts. During alcoholic fermentation, as ethanol accumulates, it exerts a detrimental effect on the integrity and stability of the cytoplasmic membrane [31]. Under this condition, the supply of small amounts of oxygen to the medium through aeration promotes the synthesis of unsaturated fatty acids and sterols (mainly ergosterol) which are important components of the yeast cell membrane. Thus, the produced compounds can be used to replace the damaged fraction caused by the effect of ethanol that acts as a solvent [35, 36]. The replacement of unsaturated fatty acids and sterols is important to maintain the cell viability and allow the yeasts to complete successfully the fermentation. From the technological point of view, the supply of small amounts of oxygen is recommended in fermentations with musts of high specific gravity in order to avoid some drawbacks such as sluggish fermentation. It is also necessary for promoting the fermentative metabolism of non-Saccharomyces yeasts which are unable to ferment under fully anaerobic conditions [37]. The optimization of the aeration rate is very important to ensure the predominance of the fermentative metabolism and to reach the highest ethanol yield. In Crabtree-negative yeasts, as the concentration of oxygen in the medium increases above a certain value, the metabolism may become predominantly oxidative; thus, the ethanol yield decreases and the production of biomass increases. The highest ethanol yield is possible to achieve, adjusting properly the aeration rate of the fermentation medium. Aeration also affects the production of glycerol by yeasts; thus, as the concentration of oxygen increases, the production of glycerol decreases. From the technological point of view, aeration of the fermentation medium is an interesting tool to control the metabolic activity of non-Saccharomyces yeasts during fermentation, for instance, wines and beers [38, 39]. In addition, aeration can be also used in winemaking to improve the quality of wines since it provokes the transformation of phenols, which reduces the astringency.

## 3.2 Production of higher alcohols

During alcoholic fermentation, either non-Saccharomyces or Saccharomyces yeasts produce diverse volatile compounds of sensory importance such as higher alcohols, aldehydes, fatty acids and esters in different concentrations depending on the species of yeasts and the fermentation conditions. The harmonic balance of the compounds determines the sensory quality of the fermented beverage. Higher alcohols are a group of compounds that mostly confer unpleasant organoleptic character when present at high concentrations [40, 41]. In adequate concentrations, they contribute positively in defining the organoleptic quality of alcoholic beverage such as wines, beers and ciders. They are produced in the cytosol and then exported outside the yeast cell where it accumulates. Higher alcohols result from the decarboxylation of ketoacids that leads to the formation of the respective aldehydes, which are then reduced to form the corresponding higher alcohols (Figure 3). Ketoacids can be originated either from the metabolism of glucose or the catabolism of amino acids [42, 43], which are taken by the yeast cell from the fermentation medium. The synthesis of higher alcohols involves the participation of at least three enzymes: a transaminase, a carboxylase and an alcohol dehydrogenase. Factors that increase the metabolism of sugar and amino acids promote the synthesis of higher alcohols. The factors include temperature of fermentation, amino acid concentration and composition of the fermentation medium.

action of specific enzymes that catalyze the reaction between an alcohol and a volatile fatty acid (Figure 4). The synthesis of esters by yeast initially involves the activation of fatty acids to acyl coenzyme A mediated by energy and the subsequent condensation of the active compound with an alcohol present in the medium to form the corresponding ester [44]. From the sensory point of view, acetate esters are the most important compounds present in fermented beverages, which include ethyl acetate, butyl acetate, propyl acetate, phenyl ethyl acetate and amyl acetate, among others. The esters produced by S. cerevisiae involve the activity of at least three acetyltransferases (AAT, EC 2.3.1.84): an alcohol acetyltransferase, an ethanol acetyltransferase and an isoamyl alcohol acetyltransferase [45, 46]. Other enzymes such as ester synthase were also reported to participate in the synthesis of esters.

Production of higher alcohols by yeast. Ehrlich's pathway and glucose catabolism.

Perspectives and Uses of Non-Saccharomyces Yeasts in Fermented Beverages

DOI: http://dx.doi.org/10.5772/intechopen.81868

Figure 3.

Figure 4.

113

Mechanisms for the production of esters by yeasts.

#### 3.3 Production of esters

Esters are a group of compounds that mostly impart positive sensory characteristics to fermented beverages such as wines, beer and ciders. They are formed by the Perspectives and Uses of Non-Saccharomyces Yeasts in Fermented Beverages DOI: http://dx.doi.org/10.5772/intechopen.81868

Figure 4. Mechanisms for the production of esters by yeasts.

action of specific enzymes that catalyze the reaction between an alcohol and a volatile fatty acid (Figure 4). The synthesis of esters by yeast initially involves the activation of fatty acids to acyl coenzyme A mediated by energy and the subsequent condensation of the active compound with an alcohol present in the medium to form the corresponding ester [44]. From the sensory point of view, acetate esters are the most important compounds present in fermented beverages, which include ethyl acetate, butyl acetate, propyl acetate, phenyl ethyl acetate and amyl acetate, among others. The esters produced by S. cerevisiae involve the activity of at least three acetyltransferases (AAT, EC 2.3.1.84): an alcohol acetyltransferase, an ethanol acetyltransferase and an isoamyl alcohol acetyltransferase [45, 46]. Other enzymes such as ester synthase were also reported to participate in the synthesis of esters.

cytoplasmic membrane of yeasts. During alcoholic fermentation, as ethanol accumulates, it exerts a detrimental effect on the integrity and stability of the cytoplasmic membrane [31]. Under this condition, the supply of small amounts of oxygen to the medium through aeration promotes the synthesis of unsaturated fatty acids and sterols (mainly ergosterol) which are important components of the yeast cell membrane. Thus, the produced compounds can be used to replace the damaged fraction caused by the effect of ethanol that acts as a solvent [35, 36]. The replacement of unsaturated fatty acids and sterols is important to maintain the cell viability and allow the yeasts to complete successfully the fermentation. From the technological point of view, the supply of small amounts of oxygen is recommended in fermentations with musts of high specific gravity in order to avoid some drawbacks such as sluggish fermentation. It is also necessary for promoting the fermentative metabolism of non-Saccharomyces yeasts which are unable to ferment under fully anaerobic conditions [37]. The optimization of the aeration rate is very important to ensure the predominance of the fermentative metabolism and to reach the highest ethanol yield. In Crabtree-negative yeasts, as the concentration of oxygen in the medium increases above a certain value, the metabolism may become predominantly oxidative; thus, the ethanol yield decreases and the production of biomass increases. The highest ethanol yield is possible to achieve, adjusting properly the aeration rate of the fermentation medium. Aeration also affects the production of glycerol by yeasts; thus, as the concentration of oxygen increases, the production of glycerol decreases. From the technological point of view, aeration of the fermentation medium is an interesting tool to control the metabolic activity of non-Saccharomyces yeasts during fermentation, for instance, wines and beers [38, 39]. In addition, aeration can be also used in winemaking to improve the quality of wines since it

Frontiers and New Trends in the Science of Fermented Food and Beverages

provokes the transformation of phenols, which reduces the astringency.

amino acid concentration and composition of the fermentation medium.

Esters are a group of compounds that mostly impart positive sensory characteristics to fermented beverages such as wines, beer and ciders. They are formed by the

During alcoholic fermentation, either non-Saccharomyces or Saccharomyces yeasts produce diverse volatile compounds of sensory importance such as higher alcohols, aldehydes, fatty acids and esters in different concentrations depending on the species of yeasts and the fermentation conditions. The harmonic balance of the compounds determines the sensory quality of the fermented beverage. Higher alcohols are a group of compounds that mostly confer unpleasant organoleptic character when present at high concentrations [40, 41]. In adequate concentrations, they contribute positively in defining the organoleptic quality of alcoholic beverage such as wines, beers and ciders. They are produced in the cytosol and then exported outside the yeast cell where it accumulates. Higher alcohols result from the decarboxylation of ketoacids that leads to the formation of the respective aldehydes, which are then reduced to form the corresponding higher alcohols (Figure 3). Ketoacids can be originated either from the metabolism of glucose or the catabolism of amino acids [42, 43], which are taken by the yeast cell from the fermentation medium. The synthesis of higher alcohols involves the participation of at least three enzymes: a transaminase, a carboxylase and an alcohol dehydrogenase. Factors that increase the metabolism of sugar and amino acids promote the synthesis of higher alcohols. The factors include temperature of fermentation,

3.2 Production of higher alcohols

3.3 Production of esters

112

However, the relevance attributed to the activity of this enzyme is quite limited. Ethyl acetate is the most abundant ester present in wines and largely responsible for the sensory character. Studies carried out with non-Saccharomyces yeasts related to the ability of producing esters allowed to select species of Hanseniaspora and Pichia able to promote esterification of various alcohols such as ethanol, isoamyl alcohol and 2-phenyl ethanol to produce the corresponding esters [47].

and K. corticis were isolated from a variety of niches including the spontaneous fermentation of grape must and ciders [6, 8, 49, 59]. Most representatives present a lemon shape (apiculate yeasts) and asexual reproduction with bipolar budding. It was reported that these yeasts participate positively in the early stage of the spontaneous fermentation of wine [59, 60], strains of Kloeckera apiculata being the most dominant [19, 49, 51, 52]. During spontaneous fermentation, as the ethanol concentration increases, the fermentative activity of these yeasts slows down and stops toward the end of fermentation by the effect of the ethanol [61]. These yeasts are characterized by producing amounts of ethanol around 4–5% v/v, values typically found in commercial beers. It was reported that the control of aeration during fermentation has effect on the production of ethanol and compounds of sensory importance such as esters, higher alcohols and organic acids [14]. Based on the information available in literature, these yeasts are promissory for being used in brewing; however, before defining a strategy of exploitation, it is necessary to carry out more in-depth studies on the effect of temperature, wort composition and inoculation rate in the fermentative activity of these yeasts. In addition, it is also necessary to carry out studies on the behavior of these yeasts in fermentations with mixed and sequential cultures with Saccharomyces cerevisiae and the production of compounds of sensory importance. Studies carried out with pure cultures of Kloeckera corticis showed that these yeasts are capable of producing acetic acid, acetaldehyde, ethyl acetate and acetoin at high concentrations [62]. In addition, it has been reported that strains of Kloeckera apiculata are capable of producing higher concentrations of ethyl and isoamyl acetate than other non-Saccharomyces yeasts [14, 63]. From the technological point of view, techniques of cell immobilization can be an additional strategy to improve the fermentative behavior and the production of compounds of sensory importance. The ability of these yeasts to produce a variety of aromatic compounds with positive impact on the sensory quality makes

Perspectives and Uses of Non-Saccharomyces Yeasts in Fermented Beverages

DOI: http://dx.doi.org/10.5772/intechopen.81868

them attractive and potentially exploitable in fermentation processes.

Few studies have been conducted regarding the potential use of yeasts belonging to the genus Hanseniaspora (apiculate yeasts) in the production of fermented beverages. The studied yeasts were isolated from the spontaneous fermentation of grape musts [6, 8, 59] and include species of Hanseniaspora uvarum, H. osmophila and H. guilliermondii, among others. It has been shown that these yeasts play an important role during the early stage of spontaneous fermentation of wine and strains of Hanseniaspora uvarum (also called Kloeckera apiculata) are dominant [19, 51, 52]. They are characterized by tolerating and producing low amounts of ethanol that do not exceed the values of 5.0% v/v [61]. This limitation explains why these yeasts do not participate actively toward the end of spontaneous fermentation of wines where the ethanol content reaches values even higher than 10%v/v. However, the fermentative capability of these yeasts is enough to produce beers of standard ethanol content similarly to those found in the market (4.5–5%v/v). In addition, they are able to ferment a wide range of sugars including maltose, which is an important feature needed for the production of beers. Regarding the production of compounds of sensory importance, studies have reported that strains of Hanseniaspora osmophila are characterized by producing high concentrations of acetic acid, acetaldehyde and ethyl acetate [62]. Additionally, it was also found that strains of Hanseniaspora uvarum are able to produce a variety of esters that confer fruitiness to fermented beverages [11, 62, 64]. However, other studies reported that mixed cultures of H. uvarum with S. cerevisiae produce higher amounts of higher alcohols than monocultures with S. cerevisiae

4.3 Hanseniaspora yeasts

115

## 4. Most important non-Saccharomyces yeasts

#### 4.1 Candida yeasts

In the last years, the fermentative behavior of some Candida yeasts has been studied with respect to the production of wines and beers. The most studied species include Candida stellata, C. zemplinina and C. pulcherrima, among others [16, 20, 21, 48–50]. Representatives of Candida yeasts have been isolated from the early stages of spontaneous fermentation of different types of wines [8, 19, 51, 52]. The isolated species were characterized by being round in shape and smaller than S. cerevisiae. These yeasts are able to sediment toward the end of fermentation in a similar manner as S. cerevisiae [20]. Currently, the most important characteristics reported include the production of considerable amounts of ethanol and glycerol and a balanced production of volatile compounds of sensory importance, for instance, esters, fatty acids, aldehydes and higher alcohols. The production of ethanol is an important feature to define the use of yeasts in the production of fermented beverages with high ethanol contents such as wines. It has been reported that C. zemplinina strains are capable of producing ethanol up to 11.0% v/v [53], amount normally reached during the fermentation of sweet and semidry wines with S. cerevisiae. In addition, it has been demonstrated that Candida yeasts are capable of producing high amounts (up to 25.0 g/L) of glycerol [53–56], compound that contributes positively to the sensory quality of wines, beers and other beverages. The fermentative behavior of these yeasts was also evaluated as mixed cultures with S. cerevisiae [57]. The results were promising and interesting for being scaled-up to pilot fermentations. For instance, fermentation experiments of mixed cultures of C. stellata with S. cerevisiae produced higher levels of esters and fatty acids than monocultures of S. cerevisiae [19, 57]. Fermentations with mixed and even sequential cultures of yeasts are an interesting field of research to evaluate the potential use of non-Saccharomyces yeasts to produce sensory differentiated beverages. In addition, individual fermentations with C. stellata and C. zemplinina strains using immobilized systems have been also performed [53, 58]. The results showed the improvement of some technological properties such as the fermentation rate, ethanol production and the reusability of the strains in successive fermentations. Currently, studies to evaluate the usability of C. zemplinina strains in beer fermentation have been carried out using malt wort of 14 and 20°P, typically used in beer fermentation processes [21, 22]. The yeast strains showed a suitable fermentative behavior for the production of lager and ale beers. One interesting feature is that Candida zemplinina is unable to ferment maltose, the main fermentable sugar of the malt wort. This characteristic is of special importance since it would enable the production of beers with low ethanol content and particular sensory profiles.

#### 4.2 Kloeckera yeasts

Yeasts species belonging to this genus have recently become of interest for the production of fermented beverages. Species such as Kloeckera apiculata, K. javanica Perspectives and Uses of Non-Saccharomyces Yeasts in Fermented Beverages DOI: http://dx.doi.org/10.5772/intechopen.81868

and K. corticis were isolated from a variety of niches including the spontaneous fermentation of grape must and ciders [6, 8, 49, 59]. Most representatives present a lemon shape (apiculate yeasts) and asexual reproduction with bipolar budding. It was reported that these yeasts participate positively in the early stage of the spontaneous fermentation of wine [59, 60], strains of Kloeckera apiculata being the most dominant [19, 49, 51, 52]. During spontaneous fermentation, as the ethanol concentration increases, the fermentative activity of these yeasts slows down and stops toward the end of fermentation by the effect of the ethanol [61]. These yeasts are characterized by producing amounts of ethanol around 4–5% v/v, values typically found in commercial beers. It was reported that the control of aeration during fermentation has effect on the production of ethanol and compounds of sensory importance such as esters, higher alcohols and organic acids [14]. Based on the information available in literature, these yeasts are promissory for being used in brewing; however, before defining a strategy of exploitation, it is necessary to carry out more in-depth studies on the effect of temperature, wort composition and inoculation rate in the fermentative activity of these yeasts. In addition, it is also necessary to carry out studies on the behavior of these yeasts in fermentations with mixed and sequential cultures with Saccharomyces cerevisiae and the production of compounds of sensory importance. Studies carried out with pure cultures of Kloeckera corticis showed that these yeasts are capable of producing acetic acid, acetaldehyde, ethyl acetate and acetoin at high concentrations [62]. In addition, it has been reported that strains of Kloeckera apiculata are capable of producing higher concentrations of ethyl and isoamyl acetate than other non-Saccharomyces yeasts [14, 63]. From the technological point of view, techniques of cell immobilization can be an additional strategy to improve the fermentative behavior and the production of compounds of sensory importance. The ability of these yeasts to produce a variety of aromatic compounds with positive impact on the sensory quality makes them attractive and potentially exploitable in fermentation processes.

#### 4.3 Hanseniaspora yeasts

However, the relevance attributed to the activity of this enzyme is quite limited. Ethyl acetate is the most abundant ester present in wines and largely responsible for the sensory character. Studies carried out with non-Saccharomyces yeasts related to the ability of producing esters allowed to select species of Hanseniaspora and Pichia able to promote esterification of various alcohols such as ethanol, isoamyl alcohol

In the last years, the fermentative behavior of some Candida yeasts has been studied with respect to the production of wines and beers. The most studied species include Candida stellata, C. zemplinina and C. pulcherrima, among others [16, 20, 21, 48–50]. Representatives of Candida yeasts have been isolated from the early stages of spontaneous fermentation of different types of wines [8, 19, 51, 52]. The isolated species were characterized by being round in shape and smaller than S. cerevisiae. These yeasts are able to sediment toward the end of fermentation in a similar manner as S. cerevisiae [20]. Currently, the most important characteristics reported include the production of considerable amounts of ethanol and glycerol and a balanced production of volatile compounds of sensory importance, for instance, esters, fatty acids, aldehydes and higher alcohols. The production of etha-

nol is an important feature to define the use of yeasts in the production of

fermented beverages with high ethanol contents such as wines. It has been reported that C. zemplinina strains are capable of producing ethanol up to 11.0% v/v [53], amount normally reached during the fermentation of sweet and semidry wines with S. cerevisiae. In addition, it has been demonstrated that Candida yeasts are capable of producing high amounts (up to 25.0 g/L) of glycerol [53–56], compound that contributes positively to the sensory quality of wines, beers and other beverages. The fermentative behavior of these yeasts was also evaluated as mixed cultures with S. cerevisiae [57]. The results were promising and interesting for being scaled-up to pilot fermentations. For instance, fermentation experiments of mixed cultures of C. stellata with S. cerevisiae produced higher levels of esters and fatty acids than monocultures of S. cerevisiae [19, 57]. Fermentations with mixed and even sequential cultures of yeasts are an interesting field of research to evaluate the potential use of non-Saccharomyces yeasts to produce sensory differentiated beverages. In addition, individual fermentations with C. stellata and C. zemplinina strains using immobilized systems have been also performed [53, 58]. The results showed the improvement of some technological properties such as the fermentation rate, ethanol production and the reusability of the strains in successive fermentations. Currently, studies to evaluate the usability of C. zemplinina strains in beer fermentation have been carried out using malt wort of 14 and 20°P, typically used in beer fermentation processes [21, 22]. The yeast strains showed a suitable fermentative behavior for the production of lager and ale beers. One interesting feature is that Candida zemplinina is unable to ferment maltose, the main fermentable sugar of the malt wort. This characteristic is of special importance since it would enable the production of beers with low ethanol content and particular sensory profiles.

Yeasts species belonging to this genus have recently become of interest for the production of fermented beverages. Species such as Kloeckera apiculata, K. javanica

and 2-phenyl ethanol to produce the corresponding esters [47].

Frontiers and New Trends in the Science of Fermented Food and Beverages

4. Most important non-Saccharomyces yeasts

4.1 Candida yeasts

4.2 Kloeckera yeasts

114

Few studies have been conducted regarding the potential use of yeasts belonging to the genus Hanseniaspora (apiculate yeasts) in the production of fermented beverages. The studied yeasts were isolated from the spontaneous fermentation of grape musts [6, 8, 59] and include species of Hanseniaspora uvarum, H. osmophila and H. guilliermondii, among others. It has been shown that these yeasts play an important role during the early stage of spontaneous fermentation of wine and strains of Hanseniaspora uvarum (also called Kloeckera apiculata) are dominant [19, 51, 52]. They are characterized by tolerating and producing low amounts of ethanol that do not exceed the values of 5.0% v/v [61]. This limitation explains why these yeasts do not participate actively toward the end of spontaneous fermentation of wines where the ethanol content reaches values even higher than 10%v/v. However, the fermentative capability of these yeasts is enough to produce beers of standard ethanol content similarly to those found in the market (4.5–5%v/v). In addition, they are able to ferment a wide range of sugars including maltose, which is an important feature needed for the production of beers. Regarding the production of compounds of sensory importance, studies have reported that strains of Hanseniaspora osmophila are characterized by producing high concentrations of acetic acid, acetaldehyde and ethyl acetate [62]. Additionally, it was also found that strains of Hanseniaspora uvarum are able to produce a variety of esters that confer fruitiness to fermented beverages [11, 62, 64]. However, other studies reported that mixed cultures of H. uvarum with S. cerevisiae produce higher amounts of higher alcohols than monocultures with S. cerevisiae

[4, 19]. Regarding fermentation parameters, the control of aeration and temperature exerts an important effect on the dynamics and activity of Hanseniaspora yeasts. Both parameters are important to control the production of compounds of sensory importance, which influence the quality of fermented beverages [11, 65]. However, in view of the scarce information on the fermentative behavior of Hanseniaspora yeasts, particularly referring to the production of fermented beverages, additional studies are needed to perform in order to find the adequate conditions for their usage, for instance, in the production of beers with new sensory profiles.

5. Production of special wines

DOI: http://dx.doi.org/10.5772/intechopen.81868

sensory qualities.

117

6. Production of craft beers

It is of common agreement that non-Saccharomyces yeasts contribute beneficially to the sensory quality of spontaneously fermented wines, an evidence that served as a starting point to pay attention to particular yeast species that could be exploited in fermentations of commercial and noncommercial fermented beverages. Non-Saccharomyces species are characterized by producing a greater

diversity of compounds of sensory importance than S. cerevisiae yeasts. Although these yeasts show a low fermentation power, some species possess important

Hanseniaspora yeasts produce a variety of compounds of sensory impact, particularly esters at concentrations even higher than S. cerevisiae. On the other hand, Candida zemplinina, a fructofilic yeast, has been shown to produce glycerol in higher concentrations than S. cerevisiae. It is also capable of producing ethanol in concentrations high enough to produce different types of wines. In view of the complementary characteristics of both groups of yeasts (Saccharomyces and non-Saccharomyces), the use of non-Saccharomyces yeasts can be proposed in fermentations with mixed or sequential cultures with S. cerevisiae as an important strategy to improve sensory complexity and mouthfeel of wines [19, 73]. The fermentative versatility of non-Saccharomyces yeasts would enable the production of special wines with different and innovative sensory characteristics. In addition, among the techniques that can be implemented for enabling their practical exploitation include the selection of new strains, the development of fermentation strategies (mixed or sequential cultures with two or more yeast strains), the ratio of both strains in the inoculum (non-Saccharomyces/Saccharomyces cerevisiae) and the inoculation rate at the beginning of fermentation [57, 76]. Finally, some technological characteristics of non-Saccharomyces yeasts can be also modified by using cultivation techniques in bioreactors with the aim of improving, for instance, the fermentation rate. The possibility of commercializing as starter cultures is an attractive opportunity for the production of different types of wines with special

In the last 10 years, the market of craft beers has increased in the USA, Latin America and some countries of Europe [77, 78]. This phenomenon is related to the expectation of consumers for discovering in these beers sensory characteristics different from those routinely found in commercial beers [74]. Current consumers are curious and interested in sensing new flavors and aromas that can satisfy their preferences. As consequence, new market segments have emerged in response to the broad possibility of offering new types of beers produced using different methods and techniques of fermentation. The production of craft beer is generally

processing methods. Craft beers are not usually filtered; due to this, their shelf life is relatively short, and therefore, their consumption must be within few days after bottling. There are a variety of innovative alternatives to produce different types of craft beers which include the use of new types of adjuncts either amylaceous (cereal grains) or non-amylaceous (fruit pulps or juices) and selected strains of non-Saccharomyces yeasts which have an enormous exploitation potential. Although most non-Saccharomyces yeasts produce low concentrations of ethanol, the fermentative capacity of some representatives of Kloeckera and Hanseniaspora yeasts is adequate to produce beers with an ethanol content typically found in the market

carried out in small-scale breweries and involves the use of non-technified

fermentative features, for instance, representatives of Kloeckera and

Perspectives and Uses of Non-Saccharomyces Yeasts in Fermented Beverages

### 4.4 Brettanomyces yeasts

Yeasts of this genus do not have a good reputation in fermentation processes such as in winemaking. For instance, representatives of Brettanomyces bruxellensis are considered detrimental due to the production of compounds such as 4 ethylguaiacol, 4-ethylphenol and 4-ethylcatechol which impart unpleasant sensory character to wines known as "Bretty" [5, 66]. These compounds result from the activity of a decarboxylase that acts on hydroxycinnamic acids followed by a reduction reaction [67]. The hydroxycinnamic acids are phenolic compounds naturally present in the skin and seeds of grapes. The common representatives of this genus were isolated from the spontaneous fermentation of wine, beer, cider and even kombucha [68–70]. It was also isolated from equipment and utensils utilized in fermentation processes, which are difficult to sanitize. The commonly isolated species include Brettanomyces bruxellensis, B. lambicus, B. intermedius and B. anomalus, among others [68, 69]. Particularly, strains of B. bruxellensis are able to ferment only in the presence of oxygen (positive Crabtree effect), a broad spectrum of sugars and even maltooligosaccharides which are not fermentable by S. cerevisiae [71]. Under anaerobic conditions, these yeasts are unable to ferment and produce ethanol; thus, at low concentration of sugar in the medium, the fermentation of glucose to ethanol is blocked. On the contrary, the fermentation is stimulated in the presence of oxygen, an effect known as Custer or negative Pasteur [72]. Apart from producing ethanol in the presence of oxygen, Brettanomyces bruxellensis also produces high concentrations of acetic acid, which acidifies and lowers the pH of the medium. However, yeasts of this genus are not entirely undesirable; some representatives participate, for instance, during the fermentation of certain beers known as "Lambic" and "Gueuze" consumed commonly in Belgium and "Coolship Ales" in North America. The fermentation of "Lambic" beer is a spontaneous process which goes through a complex succession of microorganisms where Brettanomyces bruxellensis participates during the final stage acidifying the product [73]. The participation of these yeasts gives the beer its characteristic acidity and dryness and additionally is responsible for the production of compounds such as ethyl phenol, ethyl acetate, ethyl caprylate, ethyl decanoate and ethyl lactate, which synergistically confer their typical aroma character [18, 74]. It has been shown that esters soften the sour taste and add fruity notes to this kind of beers [75]. Based on these findings, it was demonstrated that these yeasts and particularly B. bruxellensis contribute positively to defining the floral and fruity character of "Lambic" beers [18]. Beyond the contribution of Brettanomyces yeasts in spontaneous fermentation processes, in recent years, their use in controlled fermentations has been investigated, both in pure and in coculture with S. cerevisiae [15, 17]. Interesting findings were reported, indicating that the control of aeration during fermentation is a critical point to guide the fermentative metabolism toward the production of important volatile compounds that may contribute to the organoleptic character of fermented beverages.

## 5. Production of special wines

[4, 19]. Regarding fermentation parameters, the control of aeration and temperature exerts an important effect on the dynamics and activity of Hanseniaspora yeasts. Both parameters are important to control the production of compounds of sensory importance, which influence the quality of fermented beverages [11, 65]. However, in view of the scarce information on the fermentative behavior of Hanseniaspora yeasts, particularly referring to the production of fermented beverages, additional studies are needed to perform in order to find the adequate conditions for their usage, for instance, in the production of beers with new

Frontiers and New Trends in the Science of Fermented Food and Beverages

Yeasts of this genus do not have a good reputation in fermentation processes such as in winemaking. For instance, representatives of Brettanomyces bruxellensis are considered detrimental due to the production of compounds such as 4-

ethylguaiacol, 4-ethylphenol and 4-ethylcatechol which impart unpleasant sensory character to wines known as "Bretty" [5, 66]. These compounds result from the activity of a decarboxylase that acts on hydroxycinnamic acids followed by a reduction reaction [67]. The hydroxycinnamic acids are phenolic compounds naturally present in the skin and seeds of grapes. The common representatives of this genus were isolated from the spontaneous fermentation of wine, beer, cider and even kombucha [68–70]. It was also isolated from equipment and utensils utilized in fermentation processes, which are difficult to sanitize. The commonly isolated

species include Brettanomyces bruxellensis, B. lambicus, B. intermedius and

B. anomalus, among others [68, 69]. Particularly, strains of B. bruxellensis are able to ferment only in the presence of oxygen (positive Crabtree effect), a broad spectrum of sugars and even maltooligosaccharides which are not fermentable by S. cerevisiae [71]. Under anaerobic conditions, these yeasts are unable to ferment and produce ethanol; thus, at low concentration of sugar in the medium, the fermentation of glucose to ethanol is blocked. On the contrary, the fermentation is stimulated in the presence of oxygen, an effect known as Custer or negative Pasteur [72]. Apart from producing ethanol in the presence of oxygen, Brettanomyces bruxellensis also produces high concentrations of acetic acid, which acidifies and lowers the pH of the medium. However, yeasts of this genus are not entirely undesirable; some representatives participate, for instance, during the fermentation of certain beers known as "Lambic" and "Gueuze" consumed commonly in Belgium and "Coolship Ales" in North America. The fermentation of "Lambic" beer is a spontaneous process which goes through a complex succession of microorganisms where Brettanomyces bruxellensis participates during the final stage acidifying the product [73]. The participation of these yeasts gives the beer its characteristic acidity and dryness and additionally is responsible for the production of compounds such as ethyl phenol, ethyl acetate, ethyl caprylate, ethyl decanoate and ethyl lactate, which synergistically confer their typical aroma character [18, 74]. It has been shown that esters soften the sour taste and add fruity notes to this kind of beers [75]. Based on these findings, it was demonstrated that these yeasts and particularly B. bruxellensis contribute positively to defining the floral and fruity character of "Lambic" beers [18]. Beyond the contribution of Brettanomyces yeasts in spontaneous fermentation processes, in recent years, their use in controlled fermentations has been investigated, both in pure and in coculture with S. cerevisiae [15, 17]. Interesting findings were reported, indicating that the control of aeration during fermentation is a critical point to guide the fermentative metabolism toward the production of important volatile compounds that may contribute to the organoleptic character of

sensory profiles.

4.4 Brettanomyces yeasts

fermented beverages.

116

It is of common agreement that non-Saccharomyces yeasts contribute beneficially to the sensory quality of spontaneously fermented wines, an evidence that served as a starting point to pay attention to particular yeast species that could be exploited in fermentations of commercial and noncommercial fermented beverages. Non-Saccharomyces species are characterized by producing a greater diversity of compounds of sensory importance than S. cerevisiae yeasts. Although these yeasts show a low fermentation power, some species possess important fermentative features, for instance, representatives of Kloeckera and Hanseniaspora yeasts produce a variety of compounds of sensory impact, particularly esters at concentrations even higher than S. cerevisiae. On the other hand, Candida zemplinina, a fructofilic yeast, has been shown to produce glycerol in higher concentrations than S. cerevisiae. It is also capable of producing ethanol in concentrations high enough to produce different types of wines. In view of the complementary characteristics of both groups of yeasts (Saccharomyces and non-Saccharomyces), the use of non-Saccharomyces yeasts can be proposed in fermentations with mixed or sequential cultures with S. cerevisiae as an important strategy to improve sensory complexity and mouthfeel of wines [19, 73]. The fermentative versatility of non-Saccharomyces yeasts would enable the production of special wines with different and innovative sensory characteristics. In addition, among the techniques that can be implemented for enabling their practical exploitation include the selection of new strains, the development of fermentation strategies (mixed or sequential cultures with two or more yeast strains), the ratio of both strains in the inoculum (non-Saccharomyces/Saccharomyces cerevisiae) and the inoculation rate at the beginning of fermentation [57, 76]. Finally, some technological characteristics of non-Saccharomyces yeasts can be also modified by using cultivation techniques in bioreactors with the aim of improving, for instance, the fermentation rate. The possibility of commercializing as starter cultures is an attractive opportunity for the production of different types of wines with special sensory qualities.

## 6. Production of craft beers

In the last 10 years, the market of craft beers has increased in the USA, Latin America and some countries of Europe [77, 78]. This phenomenon is related to the expectation of consumers for discovering in these beers sensory characteristics different from those routinely found in commercial beers [74]. Current consumers are curious and interested in sensing new flavors and aromas that can satisfy their preferences. As consequence, new market segments have emerged in response to the broad possibility of offering new types of beers produced using different methods and techniques of fermentation. The production of craft beer is generally carried out in small-scale breweries and involves the use of non-technified processing methods. Craft beers are not usually filtered; due to this, their shelf life is relatively short, and therefore, their consumption must be within few days after bottling. There are a variety of innovative alternatives to produce different types of craft beers which include the use of new types of adjuncts either amylaceous (cereal grains) or non-amylaceous (fruit pulps or juices) and selected strains of non-Saccharomyces yeasts which have an enormous exploitation potential. Although most non-Saccharomyces yeasts produce low concentrations of ethanol, the fermentative capacity of some representatives of Kloeckera and Hanseniaspora yeasts is adequate to produce beers with an ethanol content typically found in the market

(4.5–5%v/v). Among non-Saccharomyces yeasts considered important in beer fermentation, Brettanomyces lambicus is the most representative which is involved in the production of "Lambic" and "Gueuze" beers. Currently, some studies with Candida zemplinina strains were performed in fermentations with pure malt wort and with different adjuncts (grape or apple juice) at different temperatures and specific gravities. The findings were promissory and showed the capability of these yeasts to ferment at low temperatures (14°C) and in medium with high specific gravity (16°P), which demonstrates the possibility for being exploited in the production of craft beers. In addition, it was also proposed that these yeasts can be used for the production of beers with low ethanol content since they are not able to ferment maltose, the main and most abundant sugar present in the wort [20, 21]. Additionally, other non-Saccharomyces yeasts such as Dekkera anomala, Naumovozyma dairenensis and Debaryomyces spp. have been also reported with a high potential for being used in the fermentation of beers. In view of the different fermentative behavior of non-Saccharomyces yeasts and the variety of compounds of sensory importance that they can produce during fermentation, their use in controlled fermentations has aroused the interest of brewers for producing beers with distinctive sensory features [23, 79].

## 7. Conclusion

Non-Saccharomyces yeasts show a great potential to be used in the production of fermented beverages mainly wines and beers. These yeasts show a variety of fermentative patterns, and depending on the fermentation conditions, they produce a wide range of volatile compounds of sensory importance. For their practical application in a particular fermentative process, it is necessary knowing the parameters that directly influence on the fermentative activity and the production of desirable volatile compounds. Among the non-Saccharomyces yeasts that have attracted interest of researchers due to their fermentative qualities include strains of Candida stellata, C. zemplinina, Kloeckera apiculata and Hanseniaspora uvarum. Particularly, strains of Candida stellata and C. zemplinina have become very attractive for using in fermentations of different types of wines and beers. These yeasts are capable of producing significant concentrations of glycerol, an important compound that imparts a positive impact on the sensory quality of wines and beers. Candida yeasts, especially C. zemplinina, also produce high concentrations of ethanol, high enough to drive fermentation processes of wines. On the other hand, species of Kloeckera and Hanseniaspora yeasts are characterized by producing considerable amounts of acetate esters, valuable compounds that contribute positively to the sensory character of beers. Based on this, if a fermentation process that involves the use of non-Saccharomyces yeasts is going to be implemented, it is necessary to select the best representatives and then define the appropriate fermentation conditions for the production of fermented beverages with the desired sensory qualities.

Author details

119

Waldir Desiderio Estela Escalante

provided the original work is properly cited.

Laboratory of Bioprocessing and Technology of Fermentation, Faculty of Chemistry and Chemical Engineering, Universidad Nacional Mayor de San Marcos, Lima, Peru

© 2018 The Author(s). Licensee IntechOpen. 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,

\*Address all correspondence to: waldir.estela@unmsm.edu.pe

Perspectives and Uses of Non-Saccharomyces Yeasts in Fermented Beverages

DOI: http://dx.doi.org/10.5772/intechopen.81868

## Conflict of interest

The author certifies that he has no affiliation with or involvement in any organization or entity with any financial interest or non-financial interest in the subject matter or materials discussed in this manuscript.

Perspectives and Uses of Non-Saccharomyces Yeasts in Fermented Beverages DOI: http://dx.doi.org/10.5772/intechopen.81868

## Author details

(4.5–5%v/v). Among non-Saccharomyces yeasts considered important in beer fermentation, Brettanomyces lambicus is the most representative which is involved in the production of "Lambic" and "Gueuze" beers. Currently, some studies with Candida zemplinina strains were performed in fermentations with pure malt wort and with different adjuncts (grape or apple juice) at different temperatures and specific gravities. The findings were promissory and showed the capability of these yeasts to ferment at low temperatures (14°C) and in medium with high specific gravity (16°P), which demonstrates the possibility for being exploited in the production of craft beers. In addition, it was also proposed that these yeasts can be used for the production of beers with low ethanol content since they are not able to ferment maltose, the main and most abundant sugar present in the wort [20, 21]. Additionally, other non-

Frontiers and New Trends in the Science of Fermented Food and Beverages

Saccharomyces yeasts such as Dekkera anomala, Naumovozyma dairenensis and Debaryomyces spp. have been also reported with a high potential for being used in the fermentation of beers. In view of the different fermentative behavior of non-Saccharomyces yeasts and the variety of compounds of sensory importance that they can produce during fermentation, their use in controlled fermentations has aroused the interest of brewers for producing beers with distinctive sensory features

Non-Saccharomyces yeasts show a great potential to be used in the production of fermented beverages mainly wines and beers. These yeasts show a variety of fermentative patterns, and depending on the fermentation conditions, they produce a wide range of volatile compounds of sensory importance. For their practical application in a particular fermentative process, it is necessary knowing the parameters that directly influence on the fermentative activity and the production of desirable volatile compounds. Among the non-Saccharomyces yeasts that have attracted interest of researchers due to their fermentative qualities include strains of Candida stellata, C. zemplinina, Kloeckera apiculata and Hanseniaspora uvarum. Particularly, strains of Candida stellata and C. zemplinina have become very attractive for using in fermentations of different types of wines and beers. These yeasts are capable of producing significant concentrations of glycerol, an important compound that imparts a positive impact on the sensory quality of wines and beers. Candida yeasts, especially C. zemplinina, also produce high concentrations of ethanol, high enough to drive fermentation processes of wines. On the other hand, species of Kloeckera and Hanseniaspora yeasts are characterized by producing considerable amounts of acetate esters, valuable compounds that contribute

positively to the sensory character of beers. Based on this, if a fermentation process that involves the use of non-Saccharomyces yeasts is going to be implemented, it is necessary to select the best representatives and then define the appropriate fermentation conditions for the production of fermented beverages with the desired

The author certifies that he has no affiliation with or involvement in any organization or entity with any financial interest or non-financial interest in the subject

[23, 79].

7. Conclusion

sensory qualities.

118

Conflict of interest

matter or materials discussed in this manuscript.

Waldir Desiderio Estela Escalante Laboratory of Bioprocessing and Technology of Fermentation, Faculty of Chemistry and Chemical Engineering, Universidad Nacional Mayor de San Marcos, Lima, Peru

\*Address all correspondence to: waldir.estela@unmsm.edu.pe

© 2018 The Author(s). Licensee IntechOpen. 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.

## References

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[2] Clavijo A, Calderón IL, Paneque P. Diversity of Saccharomyces and non-Saccharomyces yeasts in three red grape varieties cultured in the Serranía de Ronda (Spain) vine-growing region. International Journal of Food Microbiology. 2010;143:241-245

[3] Barnett JA, Payne RW, Yarrow D. Yeasts: Characteristics and Identification. 2nd ed. Cambridge, UK: Cambridge University Press; 1990

[4] Ciani M, Maccarelli F. Oenological properties of non-Saccharomyces yeasts associated with wine-making. World Journal of Microbiology and Biotechnology. 1998;14:199-203

[5] Loureiro V, Malfeito-Ferreira M. Spoilage activities of Dekkera/ Brettanomyces spp. In: Blackburn C, editor. Food Spoilage Microorganisms. Cambridge: Woodhead Publishers; 2006. pp. 354-398

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[9] Pardo I, Garcia MJ, Zuñiga M, Uruburu E. Dynamics of microbial populations during fermentation of wines from the Utiel-Requena region of Spain. Applied and Environmental Microbiology. 1989;55:539-541

[10] Tamang JP, Fleet GH. Yeasts diversity in fermented foods and beverages. In: Satyanarayana T, Kunze G, editors. Yeast Biotechnology. Netherlands: Diversity and Applications. Springer; 2009. pp. 169-198

[11] Estela Escalante W, Rychtera M, Melzoch K, Hatta-Sakoda B, et al. Actividad fermentativa de Hanseniaspora uvarum y su importancia en la producción de bebidas fermentadas. Revista de la Sociedad Venezolana de Microbiología. 2011;31:57-63

[12] Estela Escalante W, Rychtera M, Melzoch K, Quillama Polo E, Hatta Sakoda B. Study of the fermentative activity of Hansenula anomala and production of chemical compounds of sensory importance. Revista Peruana de Biología. 2011;18(3):325-334

[13] Estela-Escalante W, Rychtera M, Melzoch K, Hatta-Sakoda B, et al. Actividad fermentativa de Saccharomycodes ludwigii y evaluación de la síntesis de compuestos de importancia sensorial durante la fermentación de jugo de manzana. TIP Revista Especializada en Ciencias Químico-Biológicas. 2011;14(1):12-23

[14] Estela-Escalante WD, Rychtera M, Melzoch K, Guerrero-Ochoa MR. Influence of aeration in the fermentative activity of Kloeckera apiculata during fermentation of apple juice. Acta Biológica Colombiana. 2012; 17(2):309-321

Perspectives and Uses of Non-Saccharomyces Yeasts in Fermented Beverages DOI: http://dx.doi.org/10.5772/intechopen.81868

[15] Estela-Escalante WD, Rychtera M, Melzoch K, et al. Efecto de la aireación en la producción de compuestos volátiles por cultivo mixto de Brettanomyces intermedius y Saccharomyces cerevisiae durante la fermentación de sidra. TIP Revista Especializada en Ciencias Químico-Biológicas. 2014;17(1):5-14

References

2007;24:403-412

[1] Mercado L, Dalcero A, Masuelli R, Combina M. Diversity of Saccharomyces strains on grapes and winery surfaces: Analysis of their contribution of

Frontiers and New Trends in the Science of Fermented Food and Beverages

grape must. Journal of Microbiological

[9] Pardo I, Garcia MJ, Zuñiga M, Uruburu E. Dynamics of microbial populations during fermentation of wines from the Utiel-Requena region of Spain. Applied and Environmental Microbiology. 1989;55:539-541

[10] Tamang JP, Fleet GH. Yeasts diversity in fermented foods and beverages. In: Satyanarayana T, Kunze G, editors. Yeast Biotechnology.

Springer; 2009. pp. 169-198

uvarum y su importancia en la producción de bebidas fermentadas. Revista de la Sociedad Venezolana de

Microbiología. 2011;31:57-63

Biología. 2011;18(3):325-334

Netherlands: Diversity and Applications.

[11] Estela Escalante W, Rychtera M, Melzoch K, Hatta-Sakoda B, et al. Actividad fermentativa de Hanseniaspora

[12] Estela Escalante W, Rychtera M, Melzoch K, Quillama Polo E, Hatta Sakoda B. Study of the fermentative activity of Hansenula anomala and production of chemical compounds of sensory importance. Revista Peruana de

[13] Estela-Escalante W, Rychtera M, Melzoch K, Hatta-Sakoda B, et al. Actividad fermentativa de

Saccharomycodes ludwigii y evaluación de la síntesis de compuestos de importancia sensorial durante la fermentación de jugo de manzana. TIP Revista Especializada en Ciencias Químico-Biológicas. 2011;14(1):12-23

[14] Estela-Escalante WD, Rychtera M, Melzoch K, Guerrero-Ochoa MR. Influence of aeration in the fermentative activity of Kloeckera apiculata during fermentation of apple juice. Acta Biológica Colombiana. 2012;

17(2):309-321

Methods. 2016;121:50-58

fermentative flora of Malbec wine from Mendoza (Argentina) during two consecutive years. Food Microbiology.

[2] Clavijo A, Calderón IL, Paneque P. Diversity of Saccharomyces and non-Saccharomyces yeasts in three red grape varieties cultured in the Serranía de Ronda (Spain) vine-growing region. International Journal of Food Microbiology. 2010;143:241-245

[3] Barnett JA, Payne RW, Yarrow D.

Identification. 2nd ed. Cambridge, UK: Cambridge University Press; 1990

[4] Ciani M, Maccarelli F. Oenological properties of non-Saccharomyces yeasts associated with wine-making. World

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[24] Quiros M, Rojas V, Gonzalez R, Morales P. Selection of non-Saccharomyces yeast strains for reducing alcohol levels in wine by sugar respiration. International Journal of Food Microbiology. 2014;181:85-91

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[26] Ciani M, Capece A, Comitini F, Canonico L, Siesto G, Romano P. Yeast interactions in inoculated wine fermentation. Frontiers in Microbiology. 2016;7(555)

[27] Gobbi M, Comitini F, Domizio P, Romani C, Lencioni L, Mannazzu I, et al. Lachancea thermotolerans and Saccharomyces cerevisiae in simultaneous and sequential co-fermentation: A strategy to enhance acidity and improve the overall quality of wine. Food Microbiology. 2013;33:271-281. DOI: 10.1016/j.fm.2012.10.004

[28] Loira I, Vejarano R, Banuelos MA, Morata A, Tesfaye W, Uthurry C, et al. Influence of sequential fermentation with Torulaspora delbrueckii and Saccharomyces cerevisiae on wine quality. LWT- Food Science and Technology. 2014;59:915-922

[29] Canonico L, Agarbati A, Comitini F, Ciani M. Torulaspora delbrueckii in the brewing process: A new approach to enhance bioflavour and to reduce ethanol content. Food Microbiology. 2016;56:45-51. DOI: 10.1016/j.fm. 2015.12.005

[30] Flores CL, Rodríguez C, Petit T, Gancedo C. Carbohydrate and energyyielding metabolism in non-conventional yeasts. FEMS Microbiology Reviews. 2000;24:507-529

[31] Puligundla P, Smogrovicova D, Obulam VSR, Ko S. Very high gravity (VHG) ethanolic brewing and fermentation: A research update. Journal of Industrial Microbiology & Biotechnology. 2011;38:1133-1144. DOI: 10.1007/s10295-011-0999-3

[32] Prior BA, Hohmann S. Glycerol production and osmoregulation. In: Zimmermann FK, Entian KD, editors. Yeast sugar metabolism. Lancaster: Technomic Publishing; 1997. pp. 313-337

[33] Steel CC, Grbin PR, Nichol AW. The pentose phosphate pathway in the yeasts Saccharomyces cerevisiae and Kloeckera apiculata, an exercise in comparative metabolism for food and wine science students. Biochemistry and Molecular Biology Education. 2001;29: 245-249

[34] Boudarel MJ. Contribution ál étude de la fermentation alcoolique á partir de jus de Betteraves avec. Saccharomyces cerevisiae [Thèse de Doctorat]. Université de Dijon, Francia; 1984

[35] Buttke TM, Jones SD, Bloch K. Effect of sterol side chains on growth and membrane fatty acid composition of Saccharomyces cerevisiae. Journal of Bacteriology. 1980;144(1):124-130

[44] Nordstrom K. Possible control of volatile ester formation in brewing. In: Proc. Eur. Brew. Conv. 10th, Stockholm.

DOI: http://dx.doi.org/10.5772/intechopen.81868

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[52] Beltran G, Torija MJ, Novo M, Ferrer N, Poblet M, Guillamon JM, et al. Analysis of yeast populations during alcoholic fermentation: A six year follow-up study. Systematic and Applied Microbiology. 2002;25(2):

[53] Englezos V, Rantsiou K, Torchio F, Rolle L, Gerbi V, Cocolin L. Exploitation

Starmerella bacillaris (synonym Candida zemplinina) in wine fermentation: Physiological and molecular

characterizations. International Journal of Food Microbiology. 2015;199:33-40

[54] Zara G, Mannazzu I, Del Caro A, Budroni M, Pinna MB, Murru M, et al. Wine quality improvement through the combined utilisation of yeast hulls and Candida zemplinina/Saccharomyces cerevisiae mixed starter cultures. Australian Journal of Grape and Wine

[55] Mestre MV, Maturano YP, Combina M, Mercado LA, Toro ME, Vazquez F. Selection of non-Saccharomyces yeasts to be used in grape musts with high alcoholic potential: A strategy to obtain wines with reduced ethanol content. FEMS Yeast Research. 2017;17(2):1-10.

[56] Rantsiou K, Englezos V, Torchio F, Risse PA, Cravero F, Gerbi F, et al. Modeling of the fermentation behavior of Starmerella bacillaris. American Journal of Enology and Viticulture.

[57] Englezos V, Torchio F, Cravero F, Marengo F, Giacosa S, Gerbi V, et al. Aroma profile and composition of Barbera wines obtained by mixed fermentations of Starmerella bacillaris (synonym Candida zemplinina) and Saccharomyces cerevisiae. LWT- Food Science and Technology. 2016;73:567-575

[58] Balli D, Flari V, Sakellaraki E, Schoina V, Iconomopoulou M,

Research. 2014;20:199-207

DOI: 10.1093/femsyr/fox010

2017;68:378-385

of the non-Saccharomyces yeast

287-293

[45] Yoshioka K, Hashimoto N. Ester formation by alcohol acetyltransferase from brewers' yeast. Agricultural and Biological Chemistry. 1981;45(10):

[46] Lilly M, Lambrechts MG, Pretorius IS. Effect of increased yeast alcohol acetyltransferase activity on flavor profiles of wine and distillates. Applied and Environmental Microbiology. 2000;

[47] Rojas V, Gil JV, Manzanares P, Gavara R, Piñaga F, Flors A.

[48] Soden A, Francis IL, Oakey H, Henschke PA. Effects of co-

Research. 2000;6:21-30

2002;38:319-324

2001;79(3):345-352

123

fermentation with Candida stellata and Saccharomyces cerevisiae on the aroma and composition of chardonnay wine. Australian Journal of Grape and Wine

[49] Zohre DE, Erten H. The influence of

Kloeckera apiculata and Candida pulcherrima yeasts on wine

fermentation. Process Biochemistry.

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[51] Torija MJ, Rozes N, Poblet M, Guillamon JM, Mas A. Yeast population dynamics in spontaneous fermentations comparison between two different wine-producing areas over a period of three years. Antonie Van Leeuwenhoek.

Measurement of alcohol acetyltransferase and ester hydrolase activities in yeast extracts. Enzyme and Microbial Technology. 2002;30(2):224-230. DOI: 10.1016/S0141-0229(01)00483-5

1965. pp. 195-208

2183-2190

66(2):744-753

[36] Gibson BR. 125th anniversary review: Improvement of higher gravity brewery fermentation via wort enrichment and supplementation. Journal of the Institute of Brewing. 2011;117(3):268-284. DOI: 10.1002/ j.2050-0416.2011.tb00472.x

[37] Visser W, Scheffers WA, Batenburg-Van Der Vegte WH, Van Dijken JP. Oxygen requirements of yeasts. Applied and Environmental Microbiology. 1990;56(12):3785-3792

[38] González R, Quiros M, Morales P. Yeast respiration of sugars by non-Saccharomyces yeast species: A promising and barely explored approach to lowering alcohol content of wines. Trends in Food Science and Technology. 2013;29(1):55-61. DOI: 10.1016/j.tifs. 2012.06.015

[39] Rodrigues AJ, Raimbourg T, Gonzalez R, Morales P. Environmental factors influencing the efficacy of different yeast strains for alcohol level reduction in wine by respiration. LWT-Food Science and Technology. 2016;65: 1038-1043

[40] Romano P, Suzzi G, Comi G, Zironi R. Higher alcohol and acetic acid production by apiculate wine yeasts. The Journal of Applied Bacteriology. 1992;73:126-130

[41] Vidrih R, Hribar J. Synthesis of higher alcohols during cider processing. Food Chemistry. 1999;67:287-294

[42] Ehrlich F. Uber eine Methode zur spaltung racemischer aminosauren mittels Hefe. Biochemische Zeitschrift. 1906;1:8

[43] Webb AD, Ingraha JL. Fusel oil. Advances in Applied Microbiology. 1963;5:317-353

Perspectives and Uses of Non-Saccharomyces Yeasts in Fermented Beverages DOI: http://dx.doi.org/10.5772/intechopen.81868

[44] Nordstrom K. Possible control of volatile ester formation in brewing. In: Proc. Eur. Brew. Conv. 10th, Stockholm. 1965. pp. 195-208

Influence of sequential fermentation with Torulaspora delbrueckii and

2014;59:915-922

2015.12.005

2000;24:507-529

pp. 313-337

245-249

122

Saccharomyces cerevisiae on wine quality. LWT- Food Science and Technology.

Frontiers and New Trends in the Science of Fermented Food and Beverages

Saccharomyces cerevisiae. Journal of Bacteriology. 1980;144(1):124-130

[36] Gibson BR. 125th anniversary review: Improvement of higher gravity

brewery fermentation via wort enrichment and supplementation. Journal of the Institute of Brewing. 2011;117(3):268-284. DOI: 10.1002/

j.2050-0416.2011.tb00472.x

[37] Visser W, Scheffers WA, Batenburg-Van Der Vegte WH,

2012.06.015

1038-1043

1992;73:126-130

1906;1:8

1963;5:317-353

Van Dijken JP. Oxygen requirements of yeasts. Applied and Environmental Microbiology. 1990;56(12):3785-3792

[38] González R, Quiros M, Morales P. Yeast respiration of sugars by non-Saccharomyces yeast species: A

promising and barely explored approach to lowering alcohol content of wines. Trends in Food Science and Technology. 2013;29(1):55-61. DOI: 10.1016/j.tifs.

[40] Romano P, Suzzi G, Comi G, Zironi R. Higher alcohol and acetic acid production by apiculate wine yeasts. The Journal of Applied Bacteriology.

[41] Vidrih R, Hribar J. Synthesis of higher alcohols during cider processing. Food Chemistry. 1999;67:287-294

[42] Ehrlich F. Uber eine Methode zur spaltung racemischer aminosauren mittels Hefe. Biochemische Zeitschrift.

[43] Webb AD, Ingraha JL. Fusel oil. Advances in Applied Microbiology.

[39] Rodrigues AJ, Raimbourg T, Gonzalez R, Morales P. Environmental factors influencing the efficacy of different yeast strains for alcohol level reduction in wine by respiration. LWT-Food Science and Technology. 2016;65:

[29] Canonico L, Agarbati A, Comitini F, Ciani M. Torulaspora delbrueckii in the brewing process: A new approach to enhance bioflavour and to reduce ethanol content. Food Microbiology. 2016;56:45-51. DOI: 10.1016/j.fm.

[30] Flores CL, Rodríguez C, Petit T, Gancedo C. Carbohydrate and energyyielding metabolism in non-conventional yeasts. FEMS Microbiology Reviews.

[31] Puligundla P, Smogrovicova D, Obulam VSR, Ko S. Very high gravity

[32] Prior BA, Hohmann S. Glycerol production and osmoregulation. In: Zimmermann FK, Entian KD, editors. Yeast sugar metabolism. Lancaster: Technomic Publishing; 1997.

[33] Steel CC, Grbin PR, Nichol AW. The pentose phosphate pathway in the yeasts Saccharomyces cerevisiae and Kloeckera apiculata, an exercise in comparative metabolism for food and wine science students. Biochemistry and Molecular Biology Education. 2001;29:

[34] Boudarel MJ. Contribution ál étude de la fermentation alcoolique á partir de jus de Betteraves avec. Saccharomyces cerevisiae [Thèse de Doctorat]. Université de Dijon, Francia; 1984

[35] Buttke TM, Jones SD, Bloch K. Effect of sterol side chains on growth and membrane fatty acid composition of

(VHG) ethanolic brewing and fermentation: A research update. Journal of Industrial Microbiology & Biotechnology. 2011;38:1133-1144. DOI:

10.1007/s10295-011-0999-3

[45] Yoshioka K, Hashimoto N. Ester formation by alcohol acetyltransferase from brewers' yeast. Agricultural and Biological Chemistry. 1981;45(10): 2183-2190

[46] Lilly M, Lambrechts MG, Pretorius IS. Effect of increased yeast alcohol acetyltransferase activity on flavor profiles of wine and distillates. Applied and Environmental Microbiology. 2000; 66(2):744-753

[47] Rojas V, Gil JV, Manzanares P, Gavara R, Piñaga F, Flors A. Measurement of alcohol acetyltransferase and ester hydrolase activities in yeast extracts. Enzyme and Microbial Technology. 2002;30(2):224-230. DOI: 10.1016/S0141-0229(01)00483-5

[48] Soden A, Francis IL, Oakey H, Henschke PA. Effects of cofermentation with Candida stellata and Saccharomyces cerevisiae on the aroma and composition of chardonnay wine. Australian Journal of Grape and Wine Research. 2000;6:21-30

[49] Zohre DE, Erten H. The influence of Kloeckera apiculata and Candida pulcherrima yeasts on wine fermentation. Process Biochemistry. 2002;38:319-324

[50] Tofalo R, Schirone M, Torriani S, Rantsiou K, Cocolin L, Perpetuini G, et al. Diversity of Candida zemplinina strains from grapes and Italian wines. Food Microbiology. 2012;29:18-26

[51] Torija MJ, Rozes N, Poblet M, Guillamon JM, Mas A. Yeast population dynamics in spontaneous fermentations comparison between two different wine-producing areas over a period of three years. Antonie Van Leeuwenhoek. 2001;79(3):345-352

[52] Beltran G, Torija MJ, Novo M, Ferrer N, Poblet M, Guillamon JM, et al. Analysis of yeast populations during alcoholic fermentation: A six year follow-up study. Systematic and Applied Microbiology. 2002;25(2): 287-293

[53] Englezos V, Rantsiou K, Torchio F, Rolle L, Gerbi V, Cocolin L. Exploitation of the non-Saccharomyces yeast Starmerella bacillaris (synonym Candida zemplinina) in wine fermentation: Physiological and molecular characterizations. International Journal of Food Microbiology. 2015;199:33-40

[54] Zara G, Mannazzu I, Del Caro A, Budroni M, Pinna MB, Murru M, et al. Wine quality improvement through the combined utilisation of yeast hulls and Candida zemplinina/Saccharomyces cerevisiae mixed starter cultures. Australian Journal of Grape and Wine Research. 2014;20:199-207

[55] Mestre MV, Maturano YP, Combina M, Mercado LA, Toro ME, Vazquez F. Selection of non-Saccharomyces yeasts to be used in grape musts with high alcoholic potential: A strategy to obtain wines with reduced ethanol content. FEMS Yeast Research. 2017;17(2):1-10. DOI: 10.1093/femsyr/fox010

[56] Rantsiou K, Englezos V, Torchio F, Risse PA, Cravero F, Gerbi F, et al. Modeling of the fermentation behavior of Starmerella bacillaris. American Journal of Enology and Viticulture. 2017;68:378-385

[57] Englezos V, Torchio F, Cravero F, Marengo F, Giacosa S, Gerbi V, et al. Aroma profile and composition of Barbera wines obtained by mixed fermentations of Starmerella bacillaris (synonym Candida zemplinina) and Saccharomyces cerevisiae. LWT- Food Science and Technology. 2016;73:567-575

[58] Balli D, Flari V, Sakellaraki E, Schoina V, Iconomopoulou M,

Bekatorou A, et al. Effect of yeast cell immobilization and temperature on glycerol content in alcoholic fermentation with respect to wine making. Process Biochemistry. 2003;39: 499-506

[59] Suarez-Valles B, Pando Bedrinana R, Ferandez Tasco N, Querol Simon A, Rodriguez Madrera R. Yeast species associated with the spontaneous fermentation of cider. Food Microbiology. 2007;24:25-31

[60] Zott K, Miot-Sertier C, Claisse O, Lonvaud-Funel A, Masneufpomarede I. Dynamics and diversity of non-Saccharomyces yeasts during the early stages in winemaking. International Journal of Food Microbiology. 2008;125: 197-203

[61] Di Maro E, Ercolini D, Coppola S. Yeast dynamics during spontaneous wine fermentation of the Catalanesca grape. International Journal of Food Microbiology. 2007;117(2):201-210. DOI: 10.1016/j.ijfoodmicro.2007.04.007

[62] Granchi L, Ganucci D, Messini A, Vincenzini M. Oenological properties of Hanseniaspora osmophila and Kloeckera corticis from wines produced by spontaneous fermentations of normal and dried grapes. FEMS Yeast Research. 2002;(2):403-407

[63] Plata C, Millan C, Mauricio JC, Ortega JM. Formation of ethyl acetate and isoamyl acetate by various species of wine yeasts. Food Microbiology. 2003;20:217-224. DOI: 10.1016/ S0740-0020(02)00101-6

[64] Caridi A, Tini V. Caratteristiche enologiche di Hanseniaspora guilliermondii. Vini d'Italia. 1991;23: 51-57

[65] Maturano YP, Mestre MV, Esteve-Zarzoso B, Nally MC, Lerena MC, Toro ME, et al. Yeast population dynamics during prefermentative cold soak of

cabernet sauvignon and Malbec wines. International Journal of Food Microbiology. 2015;199:23-32. DOI: 10.1016/j.ijfoodmicro.2015.01.005

traditional spontaneously fermented lambic beer. PLoS One. 2014;9(4):1-13. DOI: 10.1371/journal.pone.0095384

DOI: http://dx.doi.org/10.5772/intechopen.81868

Perspectives and Uses of Non-Saccharomyces Yeasts in Fermented Beverages

[74] Vanderhaegen B, Neven H, Coghe S, Verstrepen KJ, Derdelinckx G, Verachtert H. Bioflavoring and beer refermentation. Applied Microbiology and Biotechnology. 2003;62:140-150

[75] Verstrepen KJ, Derdelinckx G, Dufour JP, Winderickx J, Thevelein JM, Pretorius IS, et al. Flavor-active esters: Adding fruitiness to beer. Journal of Bioscience and Bioengineering. 2003;96: 110-118. DOI: 10.1263/jbb.96.110

[76] Comitini F, Gobbi M, Domizio P, Romani C, Lencioni L, Mannazzu I, et al. Selected non-Saccharomyces wine yeasts in controlled multistarter fermentations with Saccharomyces cerevisiae. Food Microbiology. 2011;28:

[77] Kell J. What You Didn't Know About The Boom In Craft Beer. 2016. Available from: http://fortune.com/ 2016/03/22/craft-beer-sales-rise-2015/.

[78] Brewers-Association. National beer sales and production data. The New Brewer. 2017. Available from: https://

statistics/national-beer-sales production

[79] Johnson E. Biotechnology of non-Saccharomyces yeasts-the ascomycetes.

Biotechnology. 2013;97(2):503-517. DOI: 10.1007/s00253-012-4497-y

[Accessed: 2018-07-20]

www.brewersassociation.org/

data/. [Accessed: 2018-07-18]

Applied Microbiology and

125

873-882

[66] Romano A, Perello MC, Lonvaud-Funel A, Sicard G, de Revel G. Sensory and analytical re-evaluation of "Brett character". Food Chemistry. 2009;114: 15-19

[67] Suarez R, Suárez-Lepe JA, Morata A, Calderón F. The production of ethylphenols in wine by yeasts of the genera Brettanomyces and Dekkera: A review. Food Chemistry. 2007;102:10-21

[68] Martens H, Iserentant D, Verachtert H. Microbiological aspects of a mixed yeast-bacterial fermentation in the production of a special Belgian acidic ale. Journal of the Institute of Brewing. 1997;103:85-91

[69] Morrissey WF, Davenport B, Querol A, Dobson ADW. The role of indigenous yeasts in traditional Irish cider fermentations. Journal of Applied Microbiology. 2004;97:647-655

[70] Teoh AL, Heard G, Cox J. Yeast ecology of Kombucha fermentation. International Journal of Food Microbiology. 2004;95:119-126

[71] Verachtert H, Derdelinckx G. Belgian acidic beers: Daily reminiscences of the past. Cerevisia. 2014;38(4):121-128. DOI: 10.1016/j. cervis.2014.04.002

[72] Wijsman MR, van Dijken JP, van Kleeff BH, Scheffers WA. Inhibition of fermentation and growth in batch cultures of the yeast Brettanomyces intermedius upon a shift from aerobic to anaerobic conditions (Custers effect). Antonie Van Leeuwenhoek. 1984;50: 183-192

[73] Spitaels F, Wieme AD, Janssens M, Aerts M, Daniel HM, Van Landschoot A, et al. The microbial diversity of

Perspectives and Uses of Non-Saccharomyces Yeasts in Fermented Beverages DOI: http://dx.doi.org/10.5772/intechopen.81868

traditional spontaneously fermented lambic beer. PLoS One. 2014;9(4):1-13. DOI: 10.1371/journal.pone.0095384

Bekatorou A, et al. Effect of yeast cell immobilization and temperature on

Frontiers and New Trends in the Science of Fermented Food and Beverages

cabernet sauvignon and Malbec wines.

[66] Romano A, Perello MC, Lonvaud-Funel A, Sicard G, de Revel G. Sensory and analytical re-evaluation of "Brett character". Food Chemistry. 2009;114:

[67] Suarez R, Suárez-Lepe JA, Morata A, Calderón F. The production of ethylphenols in wine by yeasts of the genera Brettanomyces and Dekkera: A review. Food Chemistry. 2007;102:10-21

[68] Martens H, Iserentant D, Verachtert H. Microbiological aspects of a mixed yeast-bacterial fermentation in the production of a special Belgian acidic ale. Journal of the Institute of Brewing.

[69] Morrissey WF, Davenport B, Querol A, Dobson ADW. The role of indigenous

yeasts in traditional Irish cider fermentations. Journal of Applied Microbiology. 2004;97:647-655

[70] Teoh AL, Heard G, Cox J. Yeast ecology of Kombucha fermentation. International Journal of Food Microbiology. 2004;95:119-126

[71] Verachtert H, Derdelinckx G. Belgian acidic beers: Daily

cervis.2014.04.002

183-192

reminiscences of the past. Cerevisia. 2014;38(4):121-128. DOI: 10.1016/j.

[72] Wijsman MR, van Dijken JP, van Kleeff BH, Scheffers WA. Inhibition of fermentation and growth in batch cultures of the yeast Brettanomyces intermedius upon a shift from aerobic to anaerobic conditions (Custers effect). Antonie Van Leeuwenhoek. 1984;50:

[73] Spitaels F, Wieme AD, Janssens M, Aerts M, Daniel HM, Van Landschoot A,

et al. The microbial diversity of

International Journal of Food Microbiology. 2015;199:23-32. DOI: 10.1016/j.ijfoodmicro.2015.01.005

15-19

1997;103:85-91

[59] Suarez-Valles B, Pando Bedrinana R, Ferandez Tasco N, Querol Simon A, Rodriguez Madrera R. Yeast species associated with the spontaneous fermentation of cider. Food Microbiology. 2007;24:25-31

[60] Zott K, Miot-Sertier C, Claisse O, Lonvaud-Funel A, Masneufpomarede I.

[61] Di Maro E, Ercolini D, Coppola S. Yeast dynamics during spontaneous wine fermentation of the Catalanesca grape. International Journal of Food Microbiology. 2007;117(2):201-210. DOI: 10.1016/j.ijfoodmicro.2007.04.007

[62] Granchi L, Ganucci D, Messini A, Vincenzini M. Oenological properties of Hanseniaspora osmophila and Kloeckera corticis from wines produced by spontaneous fermentations of normal and dried grapes. FEMS Yeast Research.

[63] Plata C, Millan C, Mauricio JC, Ortega JM. Formation of ethyl acetate and isoamyl acetate by various species of wine yeasts. Food Microbiology. 2003;20:217-224. DOI: 10.1016/ S0740-0020(02)00101-6

[64] Caridi A, Tini V. Caratteristiche

guilliermondii. Vini d'Italia. 1991;23:

[65] Maturano YP, Mestre MV, Esteve-Zarzoso B, Nally MC, Lerena MC, Toro ME, et al. Yeast population dynamics during prefermentative cold soak of

enologiche di Hanseniaspora

51-57

124

Dynamics and diversity of non-Saccharomyces yeasts during the early stages in winemaking. International Journal of Food Microbiology. 2008;125:

glycerol content in alcoholic fermentation with respect to wine making. Process Biochemistry. 2003;39:

499-506

197-203

2002;(2):403-407

[74] Vanderhaegen B, Neven H, Coghe S, Verstrepen KJ, Derdelinckx G, Verachtert H. Bioflavoring and beer refermentation. Applied Microbiology and Biotechnology. 2003;62:140-150

[75] Verstrepen KJ, Derdelinckx G, Dufour JP, Winderickx J, Thevelein JM, Pretorius IS, et al. Flavor-active esters: Adding fruitiness to beer. Journal of Bioscience and Bioengineering. 2003;96: 110-118. DOI: 10.1263/jbb.96.110

[76] Comitini F, Gobbi M, Domizio P, Romani C, Lencioni L, Mannazzu I, et al. Selected non-Saccharomyces wine yeasts in controlled multistarter fermentations with Saccharomyces cerevisiae. Food Microbiology. 2011;28: 873-882

[77] Kell J. What You Didn't Know About The Boom In Craft Beer. 2016. Available from: http://fortune.com/ 2016/03/22/craft-beer-sales-rise-2015/. [Accessed: 2018-07-20]

[78] Brewers-Association. National beer sales and production data. The New Brewer. 2017. Available from: https:// www.brewersassociation.org/ statistics/national-beer-sales production data/. [Accessed: 2018-07-18]

[79] Johnson E. Biotechnology of non-Saccharomyces yeasts-the ascomycetes. Applied Microbiology and Biotechnology. 2013;97(2):503-517. DOI: 10.1007/s00253-012-4497-y

**127**

**Chapter 8**

**Abstract**

*Torulaspora delbrueckii*

product differ greatly between recipes.

**1. Introduction**

*Torulaspora delbrueckii*: Towards

Innovating in the Legendary

*Antonio Garcia-Triana and Rosa Lidia Solís-Oviedo*

Baking and brewing are among the oldest bioprocesses refined by human societ-

**Keywords:** alcoholic beverages, beer production, baking industry, brewing industry,

Bread and beer are among the oldest foods in the human history. The consumption of both of these fermented products has been rooted as a basic human food, and at the present time, these are still among the most consumed foods around the world. The ubiquity of their production allowed a diversification and development of refined, artisan techniques, which currently comprises innumerable recipes [1, 2]. All the recipes include essentially the same basic ingredients such as cereals, yeast, and water. However, the organoleptic properties (aroma, flavours, etc.) of the final

Since early times, both cereals and water were identified as fundamental ingredients for the preparation of beer or bread. Despite the fact that these ingredients have been recognised as essentials for centuries, the experimental approaches developed by Pasteur during the mid-nineteenth century revealed the existence of a third element much more essential to the fermentation process: yeast. The fermentation performed by yeast is undoubtedly the oldest and the largest biotechnology

ies. Both fermentative processes have successfully used domesticated strains of *Saccharomyces cerevisiae* in their process as the biocatalyst throughout their evolution. However, the dominance of *S. cerevisiae* has limited the capability for diversification of many organoleptic properties of the final product, such as aroma and flavours. The use of non-*Saccharomyces* yeasts can be an enormous source of opportunities for innovation in both fermentative processes. *Torulaspora delbrueckii* is a ubiquitous yeast species, and numerous strains have been isolated from many different bioprocesses. The strains of *T. delbrueckii*, once considered microbial contamination, have recently shown several advantages over *S. cerevisiae* strains, including higher ethanol tolerance; better capabilities to consume wort sugars; higher resistance to hop/pH/osmotic stress; and freeze-thaw resistance, among others. This chapter aims to present a comprehensive review of frontier research on *T. delbrueckii* regarding its

*Ángel De La Cruz Pech-Canul, David Ortega,* 

potential and prospects for the baking and brewing industries.

Baking and Brewing Industries

## **Chapter 8**
