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## **Meet the editor**

Editor, Dr. Naofumi Shiomi studied recombinant yeast as a researcher at the Laboratory of Production Technology of Kanena Corporation for 15 years until 1998 and earned his PhD degree in Engineering from Kyoto University. He now works as a professor at the School of Human Sciences of Kobe College in Japan, where he teaches biotechnology and life science in his

"Applied Life Science" laboratory. He has studied bioremediation and biomedical science for 20 years at Kobe College and has published more than 40 papers and several book chapters. His recent research has focused on the prevention of obesity and aging.

Co-Editor, Dr. Viduranga Waisundara obtained her PhD from the Department of Chemistry, National University of Singapore in Food Science and Technology in 2010. She was a lecturer at Temasek Polytechnic, Singapore, from July 2009 to March 2013. Following this, she relocated to her motherland Sri Lanka and spearheaded the Functional Food Product Development Project at

the National Institute of Fundamental Studies from April 2013 to October 2016. She is currently pursuing independent writing projects in Kandy, Sri Lanka. Dr. Waisundara is a prolific writer with many research publications and articles in newspapers and magazines. She has also been an invited speaker in international conferences and participated in local school events in Sri Lanka to spread awareness on functional food and dietary habits.

## Contents

## **Preface XI**


## Preface

Chapter 9 **Protective Effects of Curcumin on Gastric Inflammation and**

Chapter 10 **Glutamine: A Conditionally Essential Amino Acid with Multiple**

Chapter 11 **The Effect of Dietary Intake of Omega-3 Polyunsaturated Fatty Acids on Cardiovascular Health: Revealing Potentials of**

Ines Drenjančević, Gordana Kralik, Zlata Kralik, Martina Mihalj, Ana

Roberto Carlos Burini, Caroline das Neves Mendes Nunes and Franz

Alberto Leguina-Ruzzi and Marcial Cariqueo

Stupin, Sanja Novak and Manuela Grčević

Chapter 12 **Evolution and Therapy of Brain by Foods Containing**

**Unsaturated Fatty Acids 233**

**Liver Disease 173**

**VI** Contents

Duangporn Werawatganon

**Biological Functions 187**

**Functional Food 207**

Homero Paganini Burini

Based on the concept of "medicine and food are the same," which means that people who eat suitable foods will become healthy, many foods that are effective for improving health have been discovered and investigated in Japan. However, the effects of these foods were not always sufficient to treat or prevent disease. To improve these effects, artificial foods have been created in which effective compounds have been added. Such foods are referred to as "functional food." In Japan, many products containing purified dietary fiber, polyphe‐ nols, calcium, etc., are commercially used as functional foods to maintain health.

The use of natural food containing large amounts of effective compounds is another way to improve the insufficient effects. Such foods are called "superfoods"—famous superfoods in‐ clude fruits and vegetables, such as acai, coconut, and hemp. The idea of superfoods was discussed by doctors and researchers studying diet therapy in Canada and the USA. The roles of superfoods in medicine have been reported in many countries; thus, there is great interest in the potential for healthy food to protect against diseases.

Nowadays, the market has increased to more than several hundred billion dollars, and new sources and functions of superfoods have been developed.

This book and the topic-related book *Superfood and Functional Food- An Overview of Their Processing and Utilization* introduce recent advances in the fields of superfood and functional food.

I recommend looking at the book *Superfood and Functional Food- An Overview of Their Process‐ ing and Utilization* in addition to this book because it provides an overview of the many kinds of superfoods and functional foods and provides the readers with important informa‐ tion about the characteristics of these two types of food.

This book focuses on "the production of superfoods and their role as medicine." In the early chapters, prominent researchers introduce the role and production of spirulina or microal‐ gae, the production of functional fruits through metabolic engineering, and the use of food waste. Moreover, effective methods for preparing and consuming superfoods and functional foods are introduced. These chapters will provide improvement to the readers' understand‐ ing on the development of superfood. In the latter chapters, other prominent researchers introduce the medical effects of superfoods and functional foods. The roles of a Mediterra‐ nean diet in health are introduced first. In the subsequent chapters, the protective effects of isoflavones, anthocyanins, curcumin, glutamine, and unsaturated fatty acids—which are contained in superfoods and functional foods—against several diseases and their therapeu‐ tic use are discussed. These chapters will suggest that superfoods and functional foods are very effective in the prevention and treatment of many diseases. I believe that this book will be useful for researchers and students who are studying or developing new functional foods and superfoods.

Finally, I would like to thank Ms. Romina Rovan and the publishing process managers of InTech Publisher for their great support and assistance throughout the writing and publica‐ tion process of this book.

> **Naofumi Shiomi** Kobe College, Japan

#### **A Prominent Superfood:** *Spirulina platensis* **A Prominent Superfood:** *Spirulina platensis*

Nilay Seyidoglu, Sevda Inan and Cenk Aydin Nilay Seyidoglu, Sevda Inan and Cenk Aydin

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

http://dx.doi.org/10.5772/66118

#### **Abstract**

be useful for researchers and students who are studying or developing new functional foods

Finally, I would like to thank Ms. Romina Rovan and the publishing process managers of InTech Publisher for their great support and assistance throughout the writing and publica‐

> **Naofumi Shiomi** Kobe College,

> > Japan

and superfoods.

VIII Preface

tion process of this book.

Our planet's resources have been declining, as you know. The life qualities of humans have also changed a little because of their economy, nutrition, sports, and family life. Therefore, more alternative resources are being sought after by humans. Also, in the food supply for animals, scientists have been researching different and alternative supplements for growth performance, immunity, reproduction, and metabolism. *Spirulina platensis* and its contents have been linked to a nutritional component in both human and animal health and welfare. Growth and immunomodulation properties of this supplement have been widely studied in animals and humans, recently. Nowadays, nutritional specifics of *S. platensis* are a main focus for researchers. *S. platensis* is a singlecell protein due to its rich components, such as protein, essential amino acids, fatty acids, antioxidant pigments, carotenoids, beta-carotene, and phycocyanin. Today, researchers study the nutritional quality and investigate the effects of *S. platensis* on growth, immunity, antioxidant, antitoxicologic, anticancerogenic, cholesterol and glucose metabolism, and fertility. For these reasons, *S. platensis* may be acceptable as an alternative and/or superfood for the next generation. So, we review this information regarding *S. platensis* using historical background, literature reviews, qualitative studies, and microscopic appearances in this chapter.

**Keywords:** super food, *Spirulina platensis*, microalgae

## **1. Introduction**

Population growth, depletion of food resources, and balanced diets require the usage of new food sources. For many years, there have been antibiotics, hormones, or drugs used for improving health and immunity, and to fight against disease. Today, antibiotic resistance has become a reality, and using a more natural approach to additives in both humans and animals has become a more acceptable alternative. The natural additives are using a protein source to

© 2017 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2017 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

replace the use of the antibiotics, hormones, and drugs. Natural additives are contained in a big scientific family, but mostly they come from plant derivatives and extracts. The nutritional status of these supplements is important for use as a food additive. Among these additives, microalgae are prevalent throughout history. The utilization of these algae as a protein source has been observed by researchers for many years. There are several types of microalgae, but especially Spirulina, namely *S. platensis*, has been studied more than others due to its rich components, positive effects, and being a supplement that is nontoxic.

*S. platensis* is a filamentous cyanobacterium known as blue-green algae, which is often used as a single cell protein. This microalgae contain essential amino acids, proteins, fatty acids, antioxidant pigments, carotenoids, beta-carotene, and phycocyanin. It has been designated as a health food by the World Health Organization (WHO), and it has the potential to become one of the best alternative treatments in the twenty-first century. Also, according to the National Institutes of Health (NIH), *S. platensis* can be used as a treatment for the nervous system and metabolism, including weight loss, diabetes, and high cholesterol. And today, well-known scientific sources say that Spirulina is a "superfood" and a "miracle from the sea."

## **1.1. Classification**

*S. platensis* is a member of Phormidiaceae family. It is a filamentous and multicellular cyanobacterium which is figured like a cylindrical filament [1]. Also, it is a photosynthetic bacterium, and according to Bergey's Manual of Determinative Bacteriology (1974) it is considered to be in eukaryotic organisms [2]. Actually, there have only been one more algae in this family, named Arthrospira, which was confirmed by Gomont in 1989 [3]. He explained that Spirulina and Arthrospira are different due to their features such as helix type, cell wall, visibility under microscopy, diameters, and filaments (**Figure 1**). According to Botanics, the name of *S. platensis* was called *Arthrospira platensis* [4] at first because of its oxygenic photosynthetic feature, but today, the worldwide researchers use the term "Spirulina" for this microalgae.

## **1.2. Historical perspective**

*S. platensis* was first isolated from Lake Texcoco by the Aztecs in the sixteenth century and they called it "tecuitlatl" [5]. Later, Dangeard happened upon the Kanembu tribe which had been harvesting these excellent microalgae from Lake Chad in Africa [6]. He then coined the name "dihe" for *S. platensis* which had been used for bread, meals, and cakes in the 1940s. *S. platensis* was analyzed chemically and it quickly prompted research in 1964 [7]. During that year, studies began on this microalgae by botanists, microbiologists, and scientists, and also reviewed by some researchers [8, 9].

Early in the 1990s, NASA studied the cultivation of *S. platensis* as a food source for long-term outer-space programs. They modified the growth process using environmental factors and suggested that this microalgae could be used as palatable diet [10]. Also, in 1967, *S. platensis* was touted as a "wonderful future food source" by the International Association of Applied Microbiology [11].

The World Health Organization reported that *S. platensis* has no risk and is a good food supplement for health [12]. Included in this issue, in 2003, the Intergovernmental Institution studied this microalgae for malnutrition (IIMSAM) and developed a charter with the United Nations Economic and Social Council (UNECOSOC). They agreed that Spirulina should be used against malnutrition for humans, especially in developing countries.

replace the use of the antibiotics, hormones, and drugs. Natural additives are contained in a big scientific family, but mostly they come from plant derivatives and extracts. The nutritional status of these supplements is important for use as a food additive. Among these additives, microalgae are prevalent throughout history. The utilization of these algae as a protein source has been observed by researchers for many years. There are several types of microalgae, but especially Spirulina, namely *S. platensis*, has been studied more than others due to its rich

*S. platensis* is a filamentous cyanobacterium known as blue-green algae, which is often used as a single cell protein. This microalgae contain essential amino acids, proteins, fatty acids, antioxidant pigments, carotenoids, beta-carotene, and phycocyanin. It has been designated as a health food by the World Health Organization (WHO), and it has the potential to become one of the best alternative treatments in the twenty-first century. Also, according to the National Institutes of Health (NIH), *S. platensis* can be used as a treatment for the nervous system and metabolism, including weight loss, diabetes, and high cholesterol. And today, well-known

*S. platensis* is a member of Phormidiaceae family. It is a filamentous and multicellular cyanobacterium which is figured like a cylindrical filament [1]. Also, it is a photosynthetic bacterium, and according to Bergey's Manual of Determinative Bacteriology (1974) it is considered to be in eukaryotic organisms [2]. Actually, there have only been one more algae in this family, named Arthrospira, which was confirmed by Gomont in 1989 [3]. He explained that Spirulina and Arthrospira are different due to their features such as helix type, cell wall, visibility under microscopy, diameters, and filaments (**Figure 1**). According to Botanics, the name of *S. platensis* was called *Arthrospira platensis* [4] at first because of its oxygenic photosynthetic feature, but

*S. platensis* was first isolated from Lake Texcoco by the Aztecs in the sixteenth century and they called it "tecuitlatl" [5]. Later, Dangeard happened upon the Kanembu tribe which had been harvesting these excellent microalgae from Lake Chad in Africa [6]. He then coined the name "dihe" for *S. platensis* which had been used for bread, meals, and cakes in the 1940s. *S. platensis* was analyzed chemically and it quickly prompted research in 1964 [7]. During that year, studies began on this microalgae by botanists, microbiologists, and scientists, and also

Early in the 1990s, NASA studied the cultivation of *S. platensis* as a food source for long-term outer-space programs. They modified the growth process using environmental factors and suggested that this microalgae could be used as palatable diet [10]. Also, in 1967, *S. platensis* was touted as a "wonderful future food source" by the International Association of Applied

scientific sources say that Spirulina is a "superfood" and a "miracle from the sea."

today, the worldwide researchers use the term "Spirulina" for this microalgae.

components, positive effects, and being a supplement that is nontoxic.

2 Superfood and Functional Food - The Development of Superfoods and Their Roles as Medicine

**1.1. Classification**

**1.2. Historical perspective**

reviewed by some researchers [8, 9].

Microbiology [11].

In 2011, the National Institutes of Health proposed that *S. platensis* could be used in human research, but they requested further studies on the effects of Spirulina [13]. *S. platensis* was suggested as a safe dietary supplement by The Food and Drug Administration (FDA) in 2012 [14]. They recommended a 3–10-g daily dose of this microalgae for human health. Notably, according to the European Food Safety Authority (EFSA), *S. platensis* also helps to control the blood sugar level for glycemic health [15].

**Figure 1.** (A) Microscopic view of microalgae *Spirulina platensis* and (B) scanning electron micrograph of *Spirulina platensis*. Photograph by N. Seyidoglu.

#### **1.3. Nutritional composition**

The superfood *S. platensis* includes bioactive components such as proteins, amino acids, minerals, vitamins, pigments, nucleic acids, carbohydrates, and lipids, shown in **Tables 1**–**6**.


**Table 1.** Quantity of *Spirulina platensis* proteins and other foods [33].


National Nutrient Database for Standard Reference, Release 28 slightly revised May, 2016. Available from: https:// ndb.nal.usda.gov/ndb/foods/show/3306?

fgcd=&manu=&lfacet=&format=Full&count=&max=50&offset=&sort=default&order=asc&qlookup=11667&ds

**Table 2.** Protein and amino acids in *Spirulina platensis* powder (nutritional value per 100 g).


**Table 3.** Vitamins in *Spirulina platensis* powder [133].

**1.3. Nutritional composition**

The superfood *S. platensis* includes bioactive components such as proteins, amino acids, minerals, vitamins, pigments, nucleic acids, carbohydrates, and lipids, shown in **Tables 1**–**6**.

**Food protein origin Protein (%)** Spirulina powder 60–70 Whole dried egg 47 Beer yeast 45 Skimmed powdered milk 36 Whole soybean flour 36 Parmesan cheese 36 Wheat germ 27 Peanuts 26 Chicken 19–24 Fish 19.2–20.6 Beef meat 17.4

4 Superfood and Functional Food - The Development of Superfoods and Their Roles as Medicine

**Protein and amino acids g/100 g** Protein 57.47 Tryptophan 0.929 Threonine 2.97 Isoleucine 3.209 Leucine 4.947 Lysine 3.025 Methionine 1.149 Cystine 0.662 Phenylalanine 2.777 Tyrosine 2.584 Valine 3.512 Arginine 4.147 Histidine 1.085 Alanine 4.515 Aspartic acid 5.793 Glutamic acid 8.386 Glycine 3.099 Proline 2.382 Serine 2.998

National Nutrient Database for Standard Reference, Release 28 slightly revised May, 2016. Available from: https://

fgcd=&manu=&lfacet=&format=Full&count=&max=50&offset=&sort=default&order=asc&qlookup=11667&ds

**Table 2.** Protein and amino acids in *Spirulina platensis* powder (nutritional value per 100 g).

**Table 1.** Quantity of *Spirulina platensis* proteins and other foods [33].

ndb.nal.usda.gov/ndb/foods/show/3306?


**Table 4.** Fatty acid composition of *Spirulina platensis* powder [134].


**Table 5.** Minerals in *Spirulina platensis* powder [133].

#### 6 Superfood and Functional Food - The Development of Superfoods and Their Roles as Medicine


**Table 6.** Pigments in *Spirulina platensis* powder [133].

## *1.3.1. Protein and amino acids*

*S. platensis* is the most useful microalgae for nutrition due to its components, especially protein. The nutritional level of protein is almost 70% of its dry weight and also has a high quantity and quality belonging to amino acids [1]. *S. platensis* contains all of the essential amino acids, as shown in **Table 2**. Researchers reported that although methionine and cysteine are found in a lower value, albumin and casein are found in a higher value, of animal proteins, respectively, in eggs and milk [8, 16]. *S. platensis* contains biliproteins, especially C-phycocyanin which is 20% of all protein fractions. C-Phycocyanin molecule has an antioxidant feature, which regulates immunity and protects the organism against diseases [17].

#### *1.3.2. Vitamins*

*S. platensis* has the richest vitamin source of vitamin A (beta-carotene), vitamin E, thiamin (vitamin B1), biotin (vitamin B7), and inositol (vitamin B8) in food. Beta-carotene is in a biotransformed state which can be absorbed by humans, and is also important for antioxidant processes in organisms [18]. On the other hand, there is a conflict of cobalamin (vitamin B12) content in *S. platensis*. Some researchers reported that *S. platensis* has no reliable vitamin B12. They explain that it is a pseudovitamin B12 which is inactive and in a form that the human organism cannot uptake at a cellular level [19, 20]. However, other researchers claimed that *S. platensis* has a great amount of B12 compared to other sea algae and they indicated that vitamin B12 in this microalgae is important for vegetable nutrition, especially for humans who do not eat meat [21, 22].

## *1.3.3. Minerals*

*S. platensis* contains many minerals such as potassium, calcium, chromium, copper, iron, magnesium, manganese, phosphorus, selenium, sodium, and zinc. This microalgae is a good component due to its iron, calcium, and phosphorus contents. The ferrous component in this microalgae can be easily digested and bioactive in an organism easily which is important for pregnant adult females [23]. The utilization of calcium and phosphorus contents of *S. platensis* has an important impact on bone calcification and improves bone health [24].

#### *1.3.4. Lipids*

Lipid contents of *S. platensis* are only 4–7%, but it has important essential fatty acids for humans: gamma-linolenic acid and linolenic acid. These components are also mediators of immune and cardiovascular system due to their precursor effects of prostaglandins and leukotrienes [25]. The *S. platensis*' other lipids are stearidonic acid, eicosapentaenoic acid, docosahexaenoic acid, and arachidonic acid, respectively.

#### *1.3.5. Carbohydrates*

**Pigments mg/100g** Carotenoids 370 Chlorophyll a 1000 C-Phycocyanin 14,000

6 Superfood and Functional Food - The Development of Superfoods and Their Roles as Medicine

*S. platensis* is the most useful microalgae for nutrition due to its components, especially protein. The nutritional level of protein is almost 70% of its dry weight and also has a high quantity and quality belonging to amino acids [1]. *S. platensis* contains all of the essential amino acids, as shown in **Table 2**. Researchers reported that although methionine and cysteine are found in a lower value, albumin and casein are found in a higher value, of animal proteins, respectively, in eggs and milk [8, 16]. *S. platensis* contains biliproteins, especially C-phycocyanin which is 20% of all protein fractions. C-Phycocyanin molecule has an antioxidant feature,

*S. platensis* has the richest vitamin source of vitamin A (beta-carotene), vitamin E, thiamin (vitamin B1), biotin (vitamin B7), and inositol (vitamin B8) in food. Beta-carotene is in a biotransformed state which can be absorbed by humans, and is also important for antioxidant processes in organisms [18]. On the other hand, there is a conflict of cobalamin (vitamin B12) content in *S. platensis*. Some researchers reported that *S. platensis* has no reliable vitamin B12. They explain that it is a pseudovitamin B12 which is inactive and in a form that the human organism cannot uptake at a cellular level [19, 20]. However, other researchers claimed that *S. platensis* has a great amount of B12 compared to other sea algae and they indicated that vitamin B12 in this microalgae is important for vegetable nutrition, especially for humans who do not

*S. platensis* contains many minerals such as potassium, calcium, chromium, copper, iron, magnesium, manganese, phosphorus, selenium, sodium, and zinc. This microalgae is a good component due to its iron, calcium, and phosphorus contents. The ferrous component in this microalgae can be easily digested and bioactive in an organism easily which is important for pregnant adult females [23]. The utilization of calcium and phosphorus contents of *S. platensis*

Lipid contents of *S. platensis* are only 4–7%, but it has important essential fatty acids for humans: gamma-linolenic acid and linolenic acid. These components are also mediators of immune and cardiovascular system due to their precursor effects of prostaglandins and leukotrienes [25].

has an important impact on bone calcification and improves bone health [24].

which regulates immunity and protects the organism against diseases [17].

**Table 6.** Pigments in *Spirulina platensis* powder [133].

*1.3.1. Protein and amino acids*

*1.3.2. Vitamins*

eat meat [21, 22].

*1.3.3. Minerals*

*1.3.4. Lipids*

*S. platensis* contains 13.6% carbohydrates, which are glucose, mannose, galactose, and xylose. Nevertheless, it does not contain cellulose, which cannot be absorbed by humans and thereby this feature makes *S. platensis* easily digestible and a safe nutrient for human consumption. It is significant for people who have intestinal malabsorption and for the elderly [24]. Likewise, there is also a polysaccharide molecule, isolated from *S. platensis*, which has a huge molecular weight. This polysaccharide has an immunomodulator effect called "immulina" by scientific authorities [26, 27].

#### *1.3.6. Nucleic acids*

Nucleic acids play a role in uric acid metabolism. They catabolize the uric acid to adenine and guanine which causes gout and cardiovascular diseases [28]. So, the World Health Organization recommends about 80-g daily dosage of *S. platensis*.

## *1.3.7. Pigments*

*S. platensis* has some natural pigments which color this microalgae, such as c-phycocyanin, chlorophyll, xanthophyle, beta-carotene, zeaxanthin, and allophycocyanin. The most important are phycocyanin, chlorophyll, and beta-carotene. C-Phycocyanin is the most important pigment, which includes iron, and contains 14% of its dry weight. Also, *S. platensis* is one of the best nutrients that contains the highest chlorophyll value (1%). Chlorophyll is known as a detoxifier and purifier phyto-nutrient. It improves the carbohydrate, protein, and lipid metabolism and affects reproduction positively. Carotenes constitute half of this microalgae, especially beta-carotene. The carotenes and xanthophyle in *S. platensis* are demonstrated in different metabolism pathways in the body, and also better influence the function of vitamins and minerals in an organism [29]. Nowadays, diets rich in carotenes are found to be important for human health due to its effects in reducing the risk of diseases [30, 31] (**Table 6**).

## **2. Utilization of** *S. platensis* **worldwide**

## **2.1. Usage as food**

Plants and plant extracts have been the focus for improved health in recent years. *S. platensis* is one of the most sought after natural alternatives for nutrition in both human and animal. *S. platensis* is a microalgae that has been consumed for centuries due to its high nutritional value and supposed health benefits. Apart from its easy production, *S. platensis* has a high nutritional ability. Its affects on growth, antioxidants and antiviral features, immunomodulator activity, and hypocholesterolemic influence have been proposed by researchers over the years. Likewise, it is indicated as a nontoxic supplement, and the World Health Organization has supported it as a health nutrient [32]. *S. platensis* is used in many countries, such as Mexico, United States, Japan, Taiwan, India, Singapore, Germany, Spain, Switzerland, Holland, and many others. It is added in food marketing such as candies, chewing gums, appetizers, sports tablets, and bread. As well as its many uses in food, it is a component in some cosmetics such as creams, masks, tonics, and shampoos [33].

Natural additives have also been added to animal feed for healthy animal growth in recent years. At the same time, in the farming sector, it is preferred as it is a natural and economical product, as well as healthy, and it is shown to have rapid growth performance. *S. platensis* is one of the most sought after ingredients for animal feed as compared to other nutrients due to its high protein contents and nourishing features. Its growth, antiviral, antidiaretic, antioxidant, probiotic, hypocholesterolemic, antiallergic, analgesic, anthelmintic, anticarcinogenic, antiparasitic, immune system activator, and cardiovascular protective effects for animals have been reported by researchers [34–39].

*S. platensis* grows naturally in shallow bodies of water and in the presence of an alkaline medium of high salinity [40, 41]. The primary component for growing this microalgae is sodium bicarbonate. The production systems for this microalgae are found in Thailand, United States, Africa, China, and Chile, mostly where the Pacific Ocean, fresh water, and deep oceans exist. On the other hand, in Turkey and Bulgaria, *S. platensis* has been cultivated experimentally and recently.

Clinical and experimental trials have shown that *S. platensis* can be utilized for both human and animal safety. There have also been many studies that can help explain the benefits of this interesting microalgae. Its high biological components are an interest for scientists in recent centuries. Although it has been reported as a nontoxic supplement, current studies have continued to test its safety.

*S. platensis* can be used for immune enhancement, growth, as a nutritional food source, protector of metabolism, and many other important benefits for both humans and animals. It is amazing that all of these different features exist in this one specific microalgae. This is why scientific evidences call this microalgae a "super food." Nevertheless, in that respect there is always a need for continued studies regarding natural additives such as *S. platensis* to explain the study of their effects on humans and animals.

## **2.2. Effect of** *S. platensis* **on the growth of bacteria and animals**

*S. platensis* does not contain cellulose on its cell wall. Therefore, this microalgae can be absorbed in the intestinal mucosa and improve the intestinal function and mucosal digestion. Although *S. platensis* can repress the harmful microorganism such as Candida, it can help to increase the good microorganism such as *Lactobacillus* and *Bifidobacteria*. So, this increase of *Lactobacillus* population helps the absorption and digestion of food [42–44]. At the same time, the biological components in *S. platensis*, such as phycocyanin, polysaccharide, and gamma-linolenic acid, have an important role for improving overall body function. The Scientific Committee on Food (SCF) and the European Food Safety Authority (EFSA) also recommend 10 g of *S. platensis* as a supplement for daily intake in order to protect the health of humans, and research indicates that there is no risk with this microalgae use as a food [14].

The focus on *S. platensis* is due to its protein bioavailability, and that is the reason for this important microalgae to be compared to others. Its high protein content can improve growth performances of both humans and animals. The application of *S. platensis* for protein malnutrition has resulted in good weight gain, hematological responses, and positive nitrogen balance in metabolism with no side effects. Foods containing high protein are especially useful for malnutrition in humans, as malnutrition is a global problem. Studies, which estimate the effects, were performed in Africa, where malnutrition is prevalent, especially in children. The children and older people were separated according to their protein malnutrition first, and then rehabilitated with *S. platensis* for these studies [45–49]. The studies resulted in positive weight gain, normalized blood values, and optimized the health of human immunodeficiency virus (HIV)-negative children. The study of Simpore et al. [47] compared HIV-negative and positive children, and showed a positive weight gain between 15 and 25 g/day with children given *S. platensis*. They reported that *S. platensis* is a good food source for malnutrition. On the other hand, Azabji Kenfazk et al. [49] studied HIV-infected and malnourished adults, using *S. platensis* for 12 weeks. At the end of that study, positive improvement in body composition and body weight was concluded.

There are many different studies that point out the growth performance of *S. platensis* in animals [50–57]. For example, Moreira et al. [50] studied the Wistar rat using *S. platensis* as an added nutrient at 8.8, 17.6, and 26.4% doses of forage. They reported that there was a significant increase in weight in the 17.6% group. Heidarpour [35] used 0-, 2-, 6-, and 25-g *S. platensis* for cattle, and noted weight gain every 15 days. He observed no statistical differences in growth performances when comparing all groups. On the other hand, although some researchers found positive effects of *S. platensis* as a supplement with fish [52], some of them reported no significant changes in growth performances in fish [53, 54]. Seyidoglu and Galip [51] tried to elucidate the effects of *S. platensis* on growth performance in rabbits. They indicated that there was a positive effect of supplementing *S. platensis* on growth performance due to dose, animals, and environmental changes.

When comparing all these studies, there were different results about the supplementing dose and effects of the *S. platensis*. So, there continues to be more studies which are necessary to determine dietary concentration and the effects of this interesting microalgae.

## **3. Utilization of** *S. platensis* **for health**

## **3.1. Immune system and allergy**

supported it as a health nutrient [32]. *S. platensis* is used in many countries, such as Mexico, United States, Japan, Taiwan, India, Singapore, Germany, Spain, Switzerland, Holland, and many others. It is added in food marketing such as candies, chewing gums, appetizers, sports tablets, and bread. As well as its many uses in food, it is a component in some cosmetics such

8 Superfood and Functional Food - The Development of Superfoods and Their Roles as Medicine

Natural additives have also been added to animal feed for healthy animal growth in recent years. At the same time, in the farming sector, it is preferred as it is a natural and economical product, as well as healthy, and it is shown to have rapid growth performance. *S. platensis* is one of the most sought after ingredients for animal feed as compared to other nutrients due to its high protein contents and nourishing features. Its growth, antiviral, antidiaretic, antioxidant, probiotic, hypocholesterolemic, antiallergic, analgesic, anthelmintic, anticarcinogenic, antiparasitic, immune system activator, and cardiovascular protective effects for animals have

*S. platensis* grows naturally in shallow bodies of water and in the presence of an alkaline medium of high salinity [40, 41]. The primary component for growing this microalgae is sodium bicarbonate. The production systems for this microalgae are found in Thailand, United States, Africa, China, and Chile, mostly where the Pacific Ocean, fresh water, and deep oceans exist. On the other hand, in Turkey and Bulgaria, *S. platensis* has been cultivated experimentally

Clinical and experimental trials have shown that *S. platensis* can be utilized for both human and animal safety. There have also been many studies that can help explain the benefits of this interesting microalgae. Its high biological components are an interest for scientists in recent centuries. Although it has been reported as a nontoxic supplement, current studies have

*S. platensis* can be used for immune enhancement, growth, as a nutritional food source, protector of metabolism, and many other important benefits for both humans and animals. It is amazing that all of these different features exist in this one specific microalgae. This is why scientific evidences call this microalgae a "super food." Nevertheless, in that respect there is always a need for continued studies regarding natural additives such as *S. platensis* to explain

*S. platensis* does not contain cellulose on its cell wall. Therefore, this microalgae can be absorbed in the intestinal mucosa and improve the intestinal function and mucosal digestion. Although *S. platensis* can repress the harmful microorganism such as Candida, it can help to increase the good microorganism such as *Lactobacillus* and *Bifidobacteria*. So, this increase of *Lactobacillus* population helps the absorption and digestion of food [42–44]. At the same time, the biological components in *S. platensis*, such as phycocyanin, polysaccharide, and gamma-linolenic acid, have an important role for improving overall body function. The Scientific Committee on Food (SCF) and the European Food Safety Authority (EFSA) also recommend 10 g of *S. platensis* as

as creams, masks, tonics, and shampoos [33].

been reported by researchers [34–39].

and recently.

continued to test its safety.

the study of their effects on humans and animals.

**2.2. Effect of** *S. platensis* **on the growth of bacteria and animals**

Hematopoietic system is important for repairing tissues, generating important body cells, and protecting healthy regulation. The immune system is one of the most important systems within the hematopoietic system. Together, they are all responsible for protecting the host. The immune system of the organism is classified as an innate immune system and adaptive immune system. The innate immune system is the first barrier to protect the organism against infections. This system includes macrophages, neutrophiles, natural killer cells, and lectins. On the other hand, providing a more specialized and active defense against diseases is called an adaptive immune system, in which there are antibodies, lymphocytes, and cytokines. These two immune systems are in a sensitive balance with each other.

*S. platensis* can produce high protein, amino acids, vitamins, beta-carotene, pigments, and polysaccharides as a bioactive agent. All these components have an enhanced effect on the production of antibodies and cytokines. Especially polysaccharide, in this microalgae, has an effect on macrophages and T- and B-cell proliferations, and so it is said that *S. platensis* can improve the resistance of the organism. However, the effects of *S. platensis* on the immune system have not yet been precisely determined. The first experimental study was performed on mice in 1994, and it investigated at the effects of supporting antibody production [58]. In that study, it was reported that C-phycocyanin and polysaccharide in *S. platensis* activated the proliferation of monocytes, erythrocytes, granulocytes, and fibroblastosis in the bone marrow, and thereby the hematopoietic and immune systems were activated. In the University of Mississippi, a polysaccharide that is called "Immulina" was extracted from *S. platensis* by researchers [26]. They measured the immunostimulatory activity on human monocyte cells in vitro, and reported positive monocyte activation due to the effect of polysaccharide. Some researchers demonstrated that *S. platensis* plays an important role in the balance of immune system cells [59–65]. All these researchers reported that polysaccharides and phycocyanin have a positive role in erythropoietin activity, which is based on improved T-lymphocytes, and triggered leukocytes and bone marrow growth. Moreover, Løbner et al. [60] observed the increased CD4+ cell proliferation in humans using Immulina. There are two studies which also used *S. platensis* supplement (Immulina) with healthy humans. They reported that hemoglobin levels, natural killer cell activity, and monocytes were increased [27, 61]. Although some of the studies did showed the immune stimulatory effect of this microalgae on adaptive immune system [62–64], some of the studies [65] found no effect on the immune system, which can be explained by mutation in protocols and strains, and also the ratio of *S. platensis*.

An allergic response is a reaction of the immune system against a harmless substance such as pollen, nutrition, house mites, or other substances. Today, it is an increasing problem in the world. The protection and treatment process of allergies is aided by natural foods, especially *S. platensis*. According to researchers [66], *S. platensis* can regulate T-helper cells (Th) in allergic rhinitis. In that study, which was the first human study investigating at allergies, the role of Thelper 2 cells (Th2) and IL-4, which induced the production of IgE, was inhibited by this microalgae. According to the results, *S. platensis* supplements had a positive effect on allergic patients. In another study about food allergies, the researchers investigated the immunoglobulins role (IgA, E, G1) in the protective effects of *S. platensis*. They suggested that *S. platensis* may enhance the IgA antibody, which worked as a blocking antibody toward IgE, and thereby had protective effects against allergic reactions [67].

The supplementing of *S. platensis* was also used for adolescent animals, which have an immature immune system, which has been shown to improve the immune system and living ratio [62, 68]. Some researchers studied this concept with poultry and reported that there was a positive immunomodulator effect of *S. platensis* through the decreasing of the nutrients in macrophages [69]. According to other studies in animals, there have been increases in hemoglobin, erythrocytes, natural killer value, T–lymphocytes, and cytokine activity with this microalgae [70–72]. Prompya and Chitmanat [53] studied fish over a 60-day duration using this microalgae and found a statistically significant increase in white and red blood cells. There was another research which studied newborn pigs, and the results found a significant increase in cytokines and interleukins [72].

For many years, *S. platensis* has been used as a food additive for both humans and animals. According to scientific findings, the components are sufficient for healthy nutrition, the protective activity of the body and disease therapies. Also, according to the Food and Drug Administration, *S. platensis* has been designated as a "safe food" [14] due to its natural properties for health therapies.

## **3.2. Anemia**

immune system. The innate immune system is the first barrier to protect the organism against infections. This system includes macrophages, neutrophiles, natural killer cells, and lectins. On the other hand, providing a more specialized and active defense against diseases is called an adaptive immune system, in which there are antibodies, lymphocytes, and cytokines. These

*S. platensis* can produce high protein, amino acids, vitamins, beta-carotene, pigments, and polysaccharides as a bioactive agent. All these components have an enhanced effect on the production of antibodies and cytokines. Especially polysaccharide, in this microalgae, has an effect on macrophages and T- and B-cell proliferations, and so it is said that *S. platensis* can improve the resistance of the organism. However, the effects of *S. platensis* on the immune system have not yet been precisely determined. The first experimental study was performed on mice in 1994, and it investigated at the effects of supporting antibody production [58]. In that study, it was reported that C-phycocyanin and polysaccharide in *S. platensis* activated the proliferation of monocytes, erythrocytes, granulocytes, and fibroblastosis in the bone marrow, and thereby the hematopoietic and immune systems were activated. In the University of Mississippi, a polysaccharide that is called "Immulina" was extracted from *S. platensis* by researchers [26]. They measured the immunostimulatory activity on human monocyte cells in vitro, and reported positive monocyte activation due to the effect of polysaccharide. Some researchers demonstrated that *S. platensis* plays an important role in the balance of immune system cells [59–65]. All these researchers reported that polysaccharides and phycocyanin have a positive role in erythropoietin activity, which is based on improved T-lymphocytes, and triggered leukocytes and bone marrow growth. Moreover, Løbner et al. [60] observed the increased CD4+ cell proliferation in humans using Immulina. There are two studies which also used *S. platensis* supplement (Immulina) with healthy humans. They reported that hemoglobin levels, natural killer cell activity, and monocytes were increased [27, 61]. Although some of the studies did showed the immune stimulatory effect of this microalgae on adaptive immune system [62–64], some of the studies [65] found no effect on the immune system, which can be explained by mutation in protocols and strains, and also the ratio of *S. platensis*.

An allergic response is a reaction of the immune system against a harmless substance such as pollen, nutrition, house mites, or other substances. Today, it is an increasing problem in the world. The protection and treatment process of allergies is aided by natural foods, especially *S. platensis*. According to researchers [66], *S. platensis* can regulate T-helper cells (Th) in allergic rhinitis. In that study, which was the first human study investigating at allergies, the role of Thelper 2 cells (Th2) and IL-4, which induced the production of IgE, was inhibited by this microalgae. According to the results, *S. platensis* supplements had a positive effect on allergic patients. In another study about food allergies, the researchers investigated the immunoglobulins role (IgA, E, G1) in the protective effects of *S. platensis*. They suggested that *S. platensis* may enhance the IgA antibody, which worked as a blocking antibody toward IgE, and thereby

The supplementing of *S. platensis* was also used for adolescent animals, which have an immature immune system, which has been shown to improve the immune system and living ratio [62, 68]. Some researchers studied this concept with poultry and reported that there was

had protective effects against allergic reactions [67].

two immune systems are in a sensitive balance with each other.

10 Superfood and Functional Food - The Development of Superfoods and Their Roles as Medicine

Anemia refers to a decreased number of circulating red blood cells and is the most common blood disorder. Insufficient nutritional intake, toxic metals, and environmental contamination cause there to be a disruption in the red blood cell production pathways, and thereby anemia is the result. Also, iron deficiency is the most common cause of anemia in pregnant women, older people, and children [61]. In literature reviews, several studies have shown that several types of anemia have been treated by *S. platensis* due to its phycocyanin content [73–75]. The mechanism of C-phycocyanin is explained through the stimulation of the hematopoiesis and the endogenous erythropoietin (Epo). The Epo is known as an indicator for the proliferation and differentiation of erythrocytes. Along with this result, some research have also demonstrated that *S. platensis* has a positive impact on different types of anemia due to its rich components such as essential amino acids, folic acids, vitamin B12, and high iron which have an important role in erythropoiesis [48, 76, 77]. There are also some animal studies regarding anemia that have shown the beneficial effects of *S. platensis* on hemoglobin and serum iron levels [47, 86, 88].

#### **3.3. Obesity**

*S. platensis* has a hypocholesterolemic effect due to its C-phycocyanin component. It was reported that C-phycocyanin inhibits the reabsorption of bile acids in the ileum and also cholesterol in the jejunum [78–80]. In some studies, humans using *S. platensis* supplements showed lower results in cholesterol and triacylglycerol levels, and an increase in high-density lipoprotein levels. All of these effects indirectly reduced both diastolic and systolic blood pressure and gave a protective effect on the cardiovascular system [51, 81–83]. In another study [84], researchers treated hyperlipidemia nephrotic syndrome with *S. platensis* by applying 1-g *S. platensis* per day for 2 months and observed whether *S. platensis* decreased essential fatty acids and cholesterol values or not. They concluded that *S. platensis* consumption decreases lipid profile and helps to reduce the hyperlipidemia nephrotic syndrome. Also, all these researchers suggested that *S. platensis* is important to maintain a healthy cardiovascular system including blood lipid profile as well as treating precardiovascular disease. In vascular lesions such as coronary artery disease, the proteoglycan metabolism protecting cardiovascular cells is associated with exogen polysaccharides that are present in *S. platensis*. This pathway was studied by Sato et al. [85] and has been found to be an important element in coronary artery disease.

Cardiovascular diseases, obesity, and diabetes are linked with each other. The risk of cancer development is enhanced by these diseases in both humans and animals. On that point, some researchers point out the effects of *S. platensis* on obesity and diabetes [86–89]. During a 4-week study, *S. platensis* supplement (2.8 g) was taken by obese people, and the total body weight and biochemical values were determined. A reduction in body weight and lower cholesterol levels in obese humans was observed, in the lower significant level. Also, the other researchers observed the positive effects on diabetics using supplements of *S. platensis* [86, 89]. In these studies, obese humans with high blood sugar and lipid profiles were studied to determine the antidiabetic mechanism of this microalgae and have suggested that the gamma-linolenic acid in *S. platensis* may be attributed to the reduction in hyperglycemia.

*S. platensis* has been applied to animal feed and it has been reported that *S. platensis* plays a substantial part in lipid metabolism in animals, such as a decreased effect on total cholesterol, lipid profile, and glucose [5, 35, 90, 91]. They suggested that *S. platensis* could reduce serum cholesterol, and thereby have positive effects on lipid metabolism. In fact, cholesterol metabolism is significant in these creatures, especially in the milk production during lactation. The fatty acid profile of this microalgae is a prominent source and may stimulate milk production. The application of *S. platensis* to both humans and animals has been reviewed by The Dietary Supplements Information Expert Committee (DSI-EC) with experimental researches of animals, human clinical, and animal studies, and has reported that *S. platensis* does not have any risk for nutritional consumption. However, as there are quite limited studies in animals, especially in ruminants shown by researchers, more animal studies will be necessary to study this functional microalgae.

#### **3.4. Healing and antibiotic effects**

Wound healing is a process of repairing skin or tissue, and this process is also important for regulating hemostasis. During the healing process, bacteria and other pathogens are present at damaged areas where the pyretic situation occurs as a result of the inflammation. Natural pharmaceutical compounds are generally used to heal such wound areas. In addition, *S. platensis* or its extracts have been widely used in creams, solutions, raw juices, and ointments for skin health in recent times. Collagen fibrils, which is the plant constituent contained in microalgae, have attributes that have positive effects on wound closure during the healing process [92]. Rabadiya et al. [93] suggested that the antibiotic effects of *S. platensis* had inhibitive effects of bacteria and promoted skin healing, during the scarring process. Also, another study suggested that aqueous extract of *S. platensis* has a healing activity and it is an economical method for promoting skin, especially for diabetic wounds [94].

The anti-inflammatory effect of *S. platensis* is explained as an inhibitive effect of gammalinolenic acid [95–97]. Gamma-linolenic acid is important to control inflammation and cell proliferation. The high value of gamma-linolenic acid inhibits the work of prostaglandin and

the progression of inflammation. On the other hand, some researchers reported that *S. platensis* and its extract C-phycocyanin, can regulate the cytotoxicity and inflammation-associated factors such as ions, COX-2, tumor necrosis factor (TNF)-α, and IL-6 with BV-2 microglial cell during the inflammatory process [98].

Antibacterial activity of *S. platensis* is also caused by the activation of phagocytosis in mononuclear cells and this bacterial clearance is associated with liver health. The increase in T-cell and mononuclear phagocytes in liver by *S. platensis* has been reported [99].

*S. platensis* and its extracts, especially calcium, do not allow the viruses to attack and infect the cells. On that point, there are some written reports about the inhibition effect of viral replication and natural defenses [100]. Referring to the animal studies, *S. platensis* has been shown to be beneficial as an antiviral agent and lead to a limitation of foot and mouth disease [101]. The researchers studied the calcium extract of this microalgae in vitro, and indicated that the replication of viruses, such as herpes, measles, or mumps, was interfered by this extract. In some other studies, aqueous extracts of *S. platensis* diminished the HIV-1 virus and enterovirus replication in T-cells, Langerhans, and peripheral blood mononuclear cells due to the polysaccharides activity of this microalgae [102, 103].

Helminth infections contribute to diseases such as anemia, eosinophilia, and malnutrition. Studies about marine natural products, which are used for anthelmintic situation, were reviewed by Mayer et al. [104]; however, sufficient anthelmintic effect by *S. platensis* on the parasites was not observed.

## **3.5. Fertility**

lesions such as coronary artery disease, the proteoglycan metabolism protecting cardiovascular cells is associated with exogen polysaccharides that are present in *S. platensis*. This pathway was studied by Sato et al. [85] and has been found to be an important element in coronary

Cardiovascular diseases, obesity, and diabetes are linked with each other. The risk of cancer development is enhanced by these diseases in both humans and animals. On that point, some researchers point out the effects of *S. platensis* on obesity and diabetes [86–89]. During a 4-week study, *S. platensis* supplement (2.8 g) was taken by obese people, and the total body weight and biochemical values were determined. A reduction in body weight and lower cholesterol levels in obese humans was observed, in the lower significant level. Also, the other researchers observed the positive effects on diabetics using supplements of *S. platensis* [86, 89]. In these studies, obese humans with high blood sugar and lipid profiles were studied to determine the antidiabetic mechanism of this microalgae and have suggested that the gamma-linolenic

*S. platensis* has been applied to animal feed and it has been reported that *S. platensis* plays a substantial part in lipid metabolism in animals, such as a decreased effect on total cholesterol, lipid profile, and glucose [5, 35, 90, 91]. They suggested that *S. platensis* could reduce serum cholesterol, and thereby have positive effects on lipid metabolism. In fact, cholesterol metabolism is significant in these creatures, especially in the milk production during lactation. The fatty acid profile of this microalgae is a prominent source and may stimulate milk production. The application of *S. platensis* to both humans and animals has been reviewed by The Dietary Supplements Information Expert Committee (DSI-EC) with experimental researches of animals, human clinical, and animal studies, and has reported that *S. platensis* does not have any risk for nutritional consumption. However, as there are quite limited studies in animals, especially in ruminants shown by researchers, more animal studies will be necessary to study

Wound healing is a process of repairing skin or tissue, and this process is also important for regulating hemostasis. During the healing process, bacteria and other pathogens are present at damaged areas where the pyretic situation occurs as a result of the inflammation. Natural pharmaceutical compounds are generally used to heal such wound areas. In addition, *S. platensis* or its extracts have been widely used in creams, solutions, raw juices, and ointments for skin health in recent times. Collagen fibrils, which is the plant constituent contained in microalgae, have attributes that have positive effects on wound closure during the healing process [92]. Rabadiya et al. [93] suggested that the antibiotic effects of *S. platensis* had inhibitive effects of bacteria and promoted skin healing, during the scarring process. Also, another study suggested that aqueous extract of *S. platensis* has a healing activity and

it is an economical method for promoting skin, especially for diabetic wounds [94].

The anti-inflammatory effect of *S. platensis* is explained as an inhibitive effect of gammalinolenic acid [95–97]. Gamma-linolenic acid is important to control inflammation and cell proliferation. The high value of gamma-linolenic acid inhibits the work of prostaglandin and

acid in *S. platensis* may be attributed to the reduction in hyperglycemia.

12 Superfood and Functional Food - The Development of Superfoods and Their Roles as Medicine

artery disease.

this functional microalgae.

**3.4. Healing and antibiotic effects**

There are many factors that affect infertility in female humans and animals such as age, size and physical condition, reproductive history, and nutrition [105]. *S. platensis* is an amazing food for supporting fertility and pregnancy due to its contents. It was reported that high protein and essential amino acid components of *S. platensis* may have improved fertility by enhancing the gonad weight and gonadosomatic index, and thereby had positive effects on reproductive function [106]. Granaci et al. [107] studied with boars and found that *S. platensis* can increase the fertilizing ability of sperms. Some researchers suggested that *S. platensis* improves the sperm motility and tone due to lactate dehydrogenase (LDH) in spermatozoa, which is increased by this microalgae [108, 109]. Also, it is known that thyroid hormones (T3 and T4) are associated with increased testosterone stimulation [110], which in turn helps spermatogenesis, which were studied in rats supplemented with *S. platensis*. It was also described that these thyroid hormones regulated by this microalgae can show an improvement in rats, which have a testicular injury and dysfunction, due to its antioxidant components [111, 112].

## **3.6. Antioxidant, anticancer and antitoxicity effects**

The natural antioxidants are vitamins (B1, B5, B6, and E6), minerals (zinc, manganese, and copper), amino acid (methionine), beta-carotene, and trace elements (selenium). *S. platensis*, which contains phenolic acids, beta-carotene, and tocopherols, is a very important natural source for the intake of antioxidants. The antioxidant effect has been examined in vivo and in vitro [113, 114]. *S. platensis* has antioxidant and immunomodulatory properties which appear in the mechanism of tumor destruction and also in cancer prevention [115]. Some researchers studied liver cancer and reported that lymphocyte activity and survival rate in cancer-stricken organisms can be increased by the supply of *S. platensis* [17] through C-phycocyanin activating the immune system and playing an important role to prevent the progress of local and oral cancer [116].

Beta-carotene contained in *S. platensis* at a high value protects the free radicals and tumors induced by chemicals and enhances the immunologic resistance of the body, also decreasing lung cancer [117, 118]. The inhibitory effects of *S. platensis* and its extracts on carcinogenesis for both humans and animals were reported in some studies [119–121]. Grawish et al. [119] showed the inhibition of dysplastic tumoral changes in cheek pouch mucosa in hamsters. In another study, the protective phyto-antioxidant functions of liver tumors were determined, by an increase of the Bax/Bcl-2 ratio, which is associated in the apoptosis mechanism of hepatocellular carcinoma cell line HepG2 [120]. Additionally, *S. platensis* and its contents have protective effects against drugs, chemicals, and xenobiotics on liver tissue [120, 122, 123]. Abdel Daim et al. [124] reported that the protective mechanism of *S. platensis* against Deltamethrin induced oxidative stress through the inhibition of lipid peroxidation and releasing of free radicals or enhancing of the activity superoxide dismutase. Related to all these studies, it has been suggested that *S. platensis* may have a positive effect on anticancerogenic and oxidative situations.

*S. platensis* consists of proteins, lipids, carbohydrates, elements, and vitamins such as βcarotene, riboflavin, cyanocobalamin, α-tocopherol, and α-lipoic acid [125]. As discussed, with all these substances, *S. platensis* has beneficial effects against nephrotoxicity and cardiotoxicity [125–127]. Mohan et al. [126] showed that *S. platensis* may protect against cisplatin-induced nephrotoxicity in rats. Also, Khan et al. [127] described the protective effect of *S. platensis* against doxorubicin-induced cardiotoxicity. In the world, there are some threats which are spreading dangerously such as arsenic and radiation in the water. The millions of people living in Bangladesh, India, Taiwan, and Chile are consuming high concentrations of arseniccontaminated drinking water and thousands of them are exposed to chronic arsenic poisoning [128]. Specific treatment for this situation is unavailable. Misbahuddin et al. [128] showed that *S. platensis* extract plus zinc could be beneficial for the treatment of chronic arsenic poisoning with melanosis and keratosis. Likewise, in another study it was determined that *S. platensis* could protect the testes against mercury chloride-induced testicular damage by its rich antioxidants and antitoxicity activity [129]. An important example of radiation and *S. platensis* effects is the Chernobyl disaster. In Ukraine and Belarus, people live with radiation, which is in contaminated water, land, and nutrients. Due to this effect, poisoning, leukemia, cancer, birth defects, anemia, and thyroid disease have appeared. On that point, there is some unpublished work which talks about the effects of *S. platensis* on these symptoms and diseases [130]. Also, the protective effects of this microalgae and its extract polysaccharides and phycocyanin were shown by Belookaya et al., Wu et al., and Qishen et al [130–132]. They reported that *S. platensis* and its extracts decrease the radioactivities, and improve the bone marrow reproduction and immune system.

## **4. Conclusions**

vitro [113, 114]. *S. platensis* has antioxidant and immunomodulatory properties which appear in the mechanism of tumor destruction and also in cancer prevention [115]. Some researchers studied liver cancer and reported that lymphocyte activity and survival rate in cancer-stricken organisms can be increased by the supply of *S. platensis* [17] through C-phycocyanin activating the immune system and playing an important role to prevent the progress of local and oral

14 Superfood and Functional Food - The Development of Superfoods and Their Roles as Medicine

Beta-carotene contained in *S. platensis* at a high value protects the free radicals and tumors induced by chemicals and enhances the immunologic resistance of the body, also decreasing lung cancer [117, 118]. The inhibitory effects of *S. platensis* and its extracts on carcinogenesis for both humans and animals were reported in some studies [119–121]. Grawish et al. [119] showed the inhibition of dysplastic tumoral changes in cheek pouch mucosa in hamsters. In another study, the protective phyto-antioxidant functions of liver tumors were determined, by an increase of the Bax/Bcl-2 ratio, which is associated in the apoptosis mechanism of hepatocellular carcinoma cell line HepG2 [120]. Additionally, *S. platensis* and its contents have protective effects against drugs, chemicals, and xenobiotics on liver tissue [120, 122, 123]. Abdel Daim et al. [124] reported that the protective mechanism of *S. platensis* against Deltamethrin induced oxidative stress through the inhibition of lipid peroxidation and releasing of free radicals or enhancing of the activity superoxide dismutase. Related to all these studies, it has been suggested that *S. platensis* may have a positive effect on anticancerogenic and oxidative

*S. platensis* consists of proteins, lipids, carbohydrates, elements, and vitamins such as βcarotene, riboflavin, cyanocobalamin, α-tocopherol, and α-lipoic acid [125]. As discussed, with all these substances, *S. platensis* has beneficial effects against nephrotoxicity and cardiotoxicity [125–127]. Mohan et al. [126] showed that *S. platensis* may protect against cisplatin-induced nephrotoxicity in rats. Also, Khan et al. [127] described the protective effect of *S. platensis* against doxorubicin-induced cardiotoxicity. In the world, there are some threats which are spreading dangerously such as arsenic and radiation in the water. The millions of people living in Bangladesh, India, Taiwan, and Chile are consuming high concentrations of arseniccontaminated drinking water and thousands of them are exposed to chronic arsenic poisoning [128]. Specific treatment for this situation is unavailable. Misbahuddin et al. [128] showed that *S. platensis* extract plus zinc could be beneficial for the treatment of chronic arsenic poisoning with melanosis and keratosis. Likewise, in another study it was determined that *S. platensis* could protect the testes against mercury chloride-induced testicular damage by its rich antioxidants and antitoxicity activity [129]. An important example of radiation and *S. platensis* effects is the Chernobyl disaster. In Ukraine and Belarus, people live with radiation, which is in contaminated water, land, and nutrients. Due to this effect, poisoning, leukemia, cancer, birth defects, anemia, and thyroid disease have appeared. On that point, there is some unpublished work which talks about the effects of *S. platensis* on these symptoms and diseases [130]. Also, the protective effects of this microalgae and its extract polysaccharides and phycocyanin were shown by Belookaya et al., Wu et al., and Qishen et al [130–132]. They reported that *S. platensis* and its extracts decrease the radioactivities, and improve the bone

cancer [116].

situations.

marrow reproduction and immune system.

A prominent super food, *S. platensis*, has been known for its importance for health instead of medicine for centuries. Many studies have been performed on the effects of this interesting microalgae on both humans and animals. Today, studies observe at the nutritional quality and investigate the medicinal aspects of *S. platensis* on growth, hematopoietic system, immune system, allergy, anemia, cholesterol, obesity, diabetes, wound healing, fertility, viral and bacterial diseases, parasites, and helminth diseases. Besides these effects, anti-inflammatory, antibiotic, antipyretic, antioxidant, anticancer, and antitoxicity effects have also been determined by researchers. The potential effects have been addressed with in vivo and in vitro experiments, and contribute to the literature.

## **Acknowledgements**

Special thanks are to Susan Korucubasi who assisted our chapter in proofreading.

## **Author details**

Nilay Seyidoglu1\*, Sevda Inan2 and Cenk Aydin3

\*Address all correspondence to: nseyidoglu@nku.edu.tr


## **References**


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## **Quality Assessment of Microalgae Exposed to Trace Metals Using Flow Cytometry Quality Assessment of Microalgae Exposed to Trace Metals Using Flow Cytometry**

Toshiyuki Takahashi Toshiyuki Takahashi

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

http://dx.doi.org/10.5772/65516

#### **Abstract**

Seaweed has long been an important kitchen ingredient and a functional food material. Microalgae have attracted the same attention as seaweed from food, pharmaceutical and cosmetic companies because several algae contain unique functional materials. Industry application of algae requires the selection of useful algal species, evaluation of their features and monitoring of their quality in culture. Taking *Chlorella* for example, this chapter presents a method using flow cytometry (FCM) to assess not only the number of algae but also algal quality. First, *Chlorella* was cultured in media containing eluate from steel slag as an experimental factor and trace metals. After the treatment of algae with eluate, the number and physiological features of algae were evaluated, respectively, using hemocytometry and FCM. Results show that eluate from slag induced neither lethality nor growth inhibition. Coupled with hemocytometry, FCM was used to estimate vigorous and aberrant algal status. Consequently, the eluate did not give rise to algae stresses. Interestingly, the addition of slag eluate increased the amounts of the carbonate species. The increase in the carbonate species actually triggered the potential increase in aqueous CO2 for photosynthesis, eventually inducing algal proliferation. These analyses can support evaluation of algal features and maintenance of their quality for industry application.

**Keywords:** food science, *Chlorella*, Chlorophyll, flow cytometry, fluorescence spectro‐ scopy, trace elements, steel slag

## **1. Introduction**

Aqueous photosynthetic organisms such as algae are the foundation of aquatic ecosystems. The quantities of aqueous photosynthetic organisms as producers in the aqueous ecosystem

© 2017 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2017 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

support yields of both fish and shellfish. In addition to its role as a producer in aqueous ecosystems, seaweed has long been an important kitchen ingredient as a functional food material, providing functional nutrients such as carotenoids and anti‐oxidative fucoxanthin. A biorefinery that can take advantage of biofunctions presents an alternative concept to that of conventional refineries that manufacture materials using fossil fuels. Especially, autotrophic algal biorefineries present great advantages over conventional microbial biorefineries such as those using fermentation. Some microalgae species such as *Chlorella* spp., *Dunaliella salina* and *Haematococcus pluvialis* are appreciated respectively as sources of β‐1, 3‐glucan, β‐carotene and astaxanthin [1]. They have attracted attention equal to that devoted to seaweed products from several pharmaceutical, vitamin supplement, cosmetic and food companies [1, 2] because these algae have functional materials that are rare among land plants. Moreover, other microalgae have attracted attention for use as biofuel materials [2, 3] and bioremediation materials for environmental biodegradation [4, 5].

The industrial application of algae demands the selection of useful algal *sp.,* the evaluation of algal features and the assessment of their qualities in culture. Open pond culture systems, rather than closed systems, are the main type of culture system for the commercial scale culture of microalgae because of their relative low cost [6]. As commonly known, human activities have major impacts on the global and regional cycles of most of the trace elements including toxic heavy metals [7]. Atmospheric transport and deposition are potentially important processes for delivering a wide range of anthropogenic contaminations to aquatic environ‐ ments [7, 8]. Microalgae are very sensitive to changes in their environment [9]. Their overall metabolisms are greatly affected by even trace amounts of various organic and inorganic pollutants including heavy metals [9]. Such fear factors might pose a threat to open culture systems of algae. Therefore, it is especially important to routinely control and manage algal qualities in culture.

Taking green algae *Chlorella* spp*.,* for example, this chapter presents a method to assess algal quality using flow cytometry (FCM). *Chlorella* was cultured in media containing eluate from steel slag as an experimental factor and trace metals. After treatment of algae with eluate, the number and physiological features of algae were evaluated respectively using hemocytometry and FCM. These analyses are expected to contribute to the evaluation of algal features and to the maintenance of their quality for industry applications.

## **2. Algal characteristics using FCM**

Over the last few decades, FCM has become widely used as a powerful and valuable tool for studies of cell biology, microbiology, protein engineering and healthcare. Several functions of FCM include several procedures such as cell counting, biomarker detection and cell sorting through assessment of cell optical information. **Figure 1** presents an outline of a flow cyto‐ metric instrument used for this study. This flow cytometer, which detects several optical properties, is equipped with a green laser operating at 532 nm. Forward scatter (FSC) signals were collected to ascertain the cell size. Red fluorescence is detected in the red fluorescence

channel through a 680/30 nm band pass filter. Simultaneously, a yellow fluorescence channel through a 576/28 nm band pass filter is used [10–13]. Each fluorescence is converted into an electrical pulse. The electrical intensity is then quantified for each level of fluorescence intensity.

support yields of both fish and shellfish. In addition to its role as a producer in aqueous ecosystems, seaweed has long been an important kitchen ingredient as a functional food material, providing functional nutrients such as carotenoids and anti‐oxidative fucoxanthin. A biorefinery that can take advantage of biofunctions presents an alternative concept to that of conventional refineries that manufacture materials using fossil fuels. Especially, autotrophic algal biorefineries present great advantages over conventional microbial biorefineries such as those using fermentation. Some microalgae species such as *Chlorella* spp., *Dunaliella salina* and *Haematococcus pluvialis* are appreciated respectively as sources of β‐1, 3‐glucan, β‐carotene and astaxanthin [1]. They have attracted attention equal to that devoted to seaweed products from several pharmaceutical, vitamin supplement, cosmetic and food companies [1, 2] because these algae have functional materials that are rare among land plants. Moreover, other microalgae have attracted attention for use as biofuel materials [2, 3] and bioremediation materials for

30 Superfood and Functional Food - The Development of Superfoods and Their Roles as Medicine

The industrial application of algae demands the selection of useful algal *sp.,* the evaluation of algal features and the assessment of their qualities in culture. Open pond culture systems, rather than closed systems, are the main type of culture system for the commercial scale culture of microalgae because of their relative low cost [6]. As commonly known, human activities have major impacts on the global and regional cycles of most of the trace elements including toxic heavy metals [7]. Atmospheric transport and deposition are potentially important processes for delivering a wide range of anthropogenic contaminations to aquatic environ‐ ments [7, 8]. Microalgae are very sensitive to changes in their environment [9]. Their overall metabolisms are greatly affected by even trace amounts of various organic and inorganic pollutants including heavy metals [9]. Such fear factors might pose a threat to open culture systems of algae. Therefore, it is especially important to routinely control and manage algal

Taking green algae *Chlorella* spp*.,* for example, this chapter presents a method to assess algal quality using flow cytometry (FCM). *Chlorella* was cultured in media containing eluate from steel slag as an experimental factor and trace metals. After treatment of algae with eluate, the number and physiological features of algae were evaluated respectively using hemocytometry and FCM. These analyses are expected to contribute to the evaluation of algal features and to

Over the last few decades, FCM has become widely used as a powerful and valuable tool for studies of cell biology, microbiology, protein engineering and healthcare. Several functions of FCM include several procedures such as cell counting, biomarker detection and cell sorting through assessment of cell optical information. **Figure 1** presents an outline of a flow cyto‐ metric instrument used for this study. This flow cytometer, which detects several optical properties, is equipped with a green laser operating at 532 nm. Forward scatter (FSC) signals were collected to ascertain the cell size. Red fluorescence is detected in the red fluorescence

the maintenance of their quality for industry applications.

**2. Algal characteristics using FCM**

environmental biodegradation [4, 5].

qualities in culture.

**Figure 1.** Overview of the flow cytometric system used for this study. Algae that had passed through a capillary were analysed. In addition to the red and yellow fluorescence derived from algae, FSC signals of algae were collected simul‐ taneously as shown.

When heterotrophic cells, such as animal cells, are targeted for FCM measurement, fluores‐ cence‐labelling antibodies against certain biomolecules are used to detect and quantify the biomolecules. When using phototrophic cells, such as phytoplankton and plant cells, a photosynthetic pigment, chlorophyll, can also function as a biomarker similar to a fluorescence labelling antibody. When exposed to appropriate excitation light, chlorophyll in each cell irradiates red fluorescence (**Figure 2A** and **B**) [12]. **Figure 2C** depicts emission spectra of *Chlorella*‐like algae [14, 16]. The wavelength of the maximal fluorescence of algal chlorophyll is approximately 680 nm (green curve in **Figure 2C**). Consequently, chlorophyll fluorescence is mainly detectable using the red fluorescence channel of the instrument used for this study. The cell size and chlorophyll content of algae are correlated strongly with the algal cell cycle [14–16].

**Figure 2.** Fluorescence characteristics of algae and microphotographs of *Chlorella*‐like algae isolated from protozoa *Par‐ amecium bursaria*. Algal images obtained using bright field (A) and fluorescence microscopy (B) were referred from the literature [12]. Panel C presents fluorescence characteristics of algae obtained using fluorescence spectroscopy referred from the literature [10]. Emission spectra of algae are shown with (black line, heated algae) or without (green line, con‐ trol algae). Yellow and pink areas, respectively, show detection ranges of yellow and red fluorescence channels used for FCM in this study.

**Figure 3.** Excitation spectra of *Chlorella*‐like algae with or without heat treatment referred from the literature [10]. The fluorescence intensities at 575 nm were measured to produce excitation spectra. Two vertical lines signifying 488 nm (blue) and 532 nm (green) are shown in the graph.

Chlorophyll is sensitive to physiological factors such as heat and acid. These physical factors eventually cause inactive chlorophyll because of degradation [11–13]. In fact, a previous study using *Chlorella*‐like algae [10] demonstrated that algae without stress irradiated only red fluorescence derived from chlorophyll (green curve in **Figure 2C**). In contrast, dead algae, subjected to extraneous stress, tended to have less red fluorescence and more yellow fluores‐ cence because of the biodegradation of chlorophyll (black curve in **Figure 2C**). Moreover, this instrument presents benefits for evaluation of algal status because the excitation efficiency of the green laser at 532 nm in the yellow fluorescence is higher than that of a conventionally used blue laser at 488 nm (**Figure 3**) [10]. It suggests that the red and yellow fluorescence intensities are regarded respectively as indices of vigorous algae and of variant algae when the green laser irradiates algae moving through the capillary of this flow cytometer [11, 13].

## **3. Features of steel slag used for this study**

**Figure 2.** Fluorescence characteristics of algae and microphotographs of *Chlorella*‐like algae isolated from protozoa *Par‐ amecium bursaria*. Algal images obtained using bright field (A) and fluorescence microscopy (B) were referred from the literature [12]. Panel C presents fluorescence characteristics of algae obtained using fluorescence spectroscopy referred from the literature [10]. Emission spectra of algae are shown with (black line, heated algae) or without (green line, con‐ trol algae). Yellow and pink areas, respectively, show detection ranges of yellow and red fluorescence channels used

32 Superfood and Functional Food - The Development of Superfoods and Their Roles as Medicine

**Figure 3.** Excitation spectra of *Chlorella*‐like algae with or without heat treatment referred from the literature [10]. The fluorescence intensities at 575 nm were measured to produce excitation spectra. Two vertical lines signifying 488 nm

for FCM in this study.

(blue) and 532 nm (green) are shown in the graph.

Iron and steel slags from blast furnace slag and steel making slag including converter slag and electric arc furnace (EAF) slag are produced as steel industrial by‐products. All blast furnace slag can be recycled completely for the use of steel making slag base and cement as soil aggregate [17, 18], although several volumes of steel making slag, particularly EAF slag, ultimately end up in landfill sites [19]. New applications of slag, such as depurative and sand capping materials in aquatic environments [20] have been regarded both as decreasing the amounts of discarded slag and as reducing high costs of discarding slag. Several environmental pollution laws, however, have restricted slag use in aquatic environments because steel slag contains environmentally hazardous substances. The toxicity of eluate from EAF slag for aquatic organisms remains poorly understood [11, 13, 21], but the physiochemical properties and effects of converter slag on organisms have been documented often [22–27].

This study specifically examined stainless steel slag (designated as slag A) and common steel slag (slag B), exhausted respectively from oxidation processes of stainless and common steelmaking in EAF processes [11, 13]. **Table 1** presents compositions of EAF slags used for this study [11, 13, 21, 28, 29]. In brief, slag A contains more SiO2, CaO, and Cr2O3 than slag B does, whereas slag A contains less FeO than slag B. All Fe and Cr compounds are described respectively as FeO or Cr2O3 because it is generally difficult to distinguish FeO and Cr2O3 formed form Fe and Cr in a suspended metal solution after alkali fusion of stainless steel slag [11, 13].


**Table 1.** Chemical compositions of EAF steel slags used for this study (mass %).

## **4. Research methods**

The author used *Chlorella* as the model organism representing algae in this study. Several methods used to examine algal behaviours have been established using *Chlorella* spp. The author used *Chlorella kessleri* (C‐531 strain) which was obtained from the Institute of Applied Microbiology (IAM) culture collection at The University of Tokyo. The scientific name of *C. kessleri* was recently changed to *Parachlorella kessleri* because the taxonomy of *Chlorella* has been re‐validated using multidisciplinary approaches based on combining classical and modern methods including molecular phylogeny and bioinformatics [30]. Before experiments, algae on the CA agar plates [31] were scratched with an inoculating needle and were suspended in CA liquid medium.

Steel slag was subjected to a leaching test based on JIS K0058‐1: 2005 (Method for chemicals in slags Part 1: Leaching test) to elute metal components of slag with HCl [11, 13, 21, 28, 29, 32]. After elution, the solution was filtrated with a 0.45 μm pore filter to eliminate slag particles as described in previous reports. The filtrated eluate from the slag (designated respectively as eluate A and eluate B) was used for bioassay with *Chlorella* as a test solution including trace metals.

To assess the eluate effects on algal growth, *Chlorella* was cultured in CA medium containing an eluate from steel slag as an experimental factor and sources of trace metals [11, 13]. Compared with general culture media for algae, slag eluates used for this study contained insufficient nutrients for algal growth. To supplement nutrients for algal growth, the following assessments of algal growth were conducted with CA liquid medium at pH 7.2 [11, 13]. Nutrient amounts of CA medium containing eluates were the same as those of CA medium alone, but the concentrations of chemicals derived from each eluate differed from those of CA medium without eluate. Here, CA medium without eluate was designated as "control".

Algae (initial density of 1.0 × 104 cells/ml adjusted using hemocytometry) were cultured with CA medium containing eluate from each slag for 1 week in a plastic tube under an LD cycle (12 h light/12 h dark) at approximately 1100 lux of natural white fluorescent light and 23 ± 2°C as described in previous reports [11, 13]. After treatment of algae with eluate, the number and physiological features of algae were quantified respectively using hemocytometry and FCM. The algal proliferation ratio (average ± standard error) was expressed as a proportion of the number of algae treated with eluate to that of control without eluate [11, 13].

To investigate algal status using FCM, the algal status was analysed and estimated based on the corresponding fluorescence. In brief, the stress of each alga is portrayed as a two‐dimen‐ sional graph (2D map) of red fluorescence intensity (665–695 nm) as the index of vigorous algae and yellow fluorescence intensity (562–590 nm) as the index of variant algae [11, 13]. To facilitate comparison of vigorous algae with stressed and dying algae, a reference standard of algae subjected to stress was prepared by treatment of algae with heat for 5 min at 100°C (designated as heated algae) [11, 13]. For FCM analyses, FSC signals detected only in the culture medium were removed as technical noise from FCM measurements as described in previous reports [11, 13]. The remaining signals were re‐analysed as algal signals.

Aquatic CO2 (CO2(aq)) concentrations are related directly with photosynthesis and algal proliferation. CO2(aq), HCO3 − and CO3 2− are present as the carbonate species in solution, as presented in Eqs. (1)–(3) [11, 13].

**4. Research methods**

CA liquid medium.

Algae (initial density of 1.0 × 104

metals.

The author used *Chlorella* as the model organism representing algae in this study. Several methods used to examine algal behaviours have been established using *Chlorella* spp. The author used *Chlorella kessleri* (C‐531 strain) which was obtained from the Institute of Applied Microbiology (IAM) culture collection at The University of Tokyo. The scientific name of *C. kessleri* was recently changed to *Parachlorella kessleri* because the taxonomy of *Chlorella* has been re‐validated using multidisciplinary approaches based on combining classical and modern methods including molecular phylogeny and bioinformatics [30]. Before experiments, algae on the CA agar plates [31] were scratched with an inoculating needle and were suspended in

34 Superfood and Functional Food - The Development of Superfoods and Their Roles as Medicine

Steel slag was subjected to a leaching test based on JIS K0058‐1: 2005 (Method for chemicals in slags Part 1: Leaching test) to elute metal components of slag with HCl [11, 13, 21, 28, 29, 32]. After elution, the solution was filtrated with a 0.45 μm pore filter to eliminate slag particles as described in previous reports. The filtrated eluate from the slag (designated respectively as eluate A and eluate B) was used for bioassay with *Chlorella* as a test solution including trace

To assess the eluate effects on algal growth, *Chlorella* was cultured in CA medium containing an eluate from steel slag as an experimental factor and sources of trace metals [11, 13]. Compared with general culture media for algae, slag eluates used for this study contained insufficient nutrients for algal growth. To supplement nutrients for algal growth, the following assessments of algal growth were conducted with CA liquid medium at pH 7.2 [11, 13]. Nutrient amounts of CA medium containing eluates were the same as those of CA medium alone, but the concentrations of chemicals derived from each eluate differed from those of CA medium without eluate. Here, CA medium without eluate was designated as "control".

CA medium containing eluate from each slag for 1 week in a plastic tube under an LD cycle (12 h light/12 h dark) at approximately 1100 lux of natural white fluorescent light and 23 ± 2°C as described in previous reports [11, 13]. After treatment of algae with eluate, the number and physiological features of algae were quantified respectively using hemocytometry and FCM. The algal proliferation ratio (average ± standard error) was expressed as a proportion of the

To investigate algal status using FCM, the algal status was analysed and estimated based on the corresponding fluorescence. In brief, the stress of each alga is portrayed as a two‐dimen‐ sional graph (2D map) of red fluorescence intensity (665–695 nm) as the index of vigorous algae and yellow fluorescence intensity (562–590 nm) as the index of variant algae [11, 13]. To facilitate comparison of vigorous algae with stressed and dying algae, a reference standard of algae subjected to stress was prepared by treatment of algae with heat for 5 min at 100°C (designated as heated algae) [11, 13]. For FCM analyses, FSC signals detected only in the culture medium were removed as technical noise from FCM measurements as described in previous

number of algae treated with eluate to that of control without eluate [11, 13].

reports [11, 13]. The remaining signals were re‐analysed as algal signals.

cells/ml adjusted using hemocytometry) were cultured with

$$\text{CO}\_2\text{(gas)} \leftrightarrow \text{CO}\_2\text{(aq)}\tag{1}$$

$$\rm H\_2O + CO\_2(aq) \leftrightarrow H\_2CO\_3(aq) \leftrightarrow HCO\_3^- + H^+ \tag{2}$$

$$\text{HCO}\_3^- \leftrightarrow \text{CO}\_3^{2-} + \text{H}^+ \tag{3}$$

For aqueous photosynthetic organisms, CO2(aq) of these carbonate species is particularly necessary to support photosynthesis. We ascertained the concentration of CO2(aq) in slag eluate using potentiometry with a diaphragm‐type electrode to measure the CO2(aq) concen‐ trations [11, 13]. Both HCO3 − and CO3 2‐ can be estimated as CO2(aq) in acidic conditions (≤ pH 4.0) as portrayed in **Figure 4** resulting from the following Henderson–Hasselbalch Eqs. (4) and (5) [11, 13].

$$\text{pH} = \text{p} \text{K}\_1 + \log(\text{[HCO}\_3^-] \text{[} \text{CO}\_2(\text{aq})]) \tag{4}$$

$$\text{pH} = \text{p} \text{K}\_2 + \log(\text{[CO}\_3^{2-}\text{]} \text{[HCO}\_3^{-}\text{]}) \tag{5}$$

The respective pK values of pK1 = 6.35 and pK2 = 10.33 [33] were used for this study. The carbonate species aside from CO2(aq) were converted into CO2(aq) by adding a pH‐adjustable solution. Then they were estimated as the amounts of total carbonate species. Each concen‐ tration of CO2(aq), HCO3 − , and CO3 2‐ was calculated from the amounts of total carbonate species and pH values using Eqs. (4) and (5) above. Here, the [H2CO3(aq)] given by Eq. (2) was expressed as [CO2(aq)] in Eq. (4) because it was difficult to distinguish CO2(aq) from H2CO3(aq) in solution, as described in previous reports [11, 13]. The result was expressed as the concen‐ tration of CO2(aq) (average ± standard error) under each condition [11, 13].

In general, the amounts of Ca2+ and Mg2+ are related to the water hardness and are highly reactive with carbonate species. To examine whether these elements contribute to the concen‐ tration of CO2(aq) in solution, the amounts of Ca2+ and Mg2+ were measured before and after treatment of algae with CA medium containing slag eluate [11, 13]. After treatment of algae with CA medium containing eluate, the culture tube including the algae was centrifuged. The supernatant, which no longer included algae, was collected and subjected to elemental analysis. Several organic compounds, such as biomolecules reportedly interfere with these measurements [34]. Therefore, this study applied colorimetric determination using specific chelate reagents to elucidate the amounts of Ca2+ and Mg2+ in the culture supernatant. In practice, the chlorophosphonazo‐III method [35] and the xylidyl blue‐I method [36] were used for the evaluation of the concentration of Ca2+ and that of Mg2+. Moreover, elemental concen‐ trations before treatment of algae with eluate were compared statistically with those after treatment using *t*‐tests.

**Figure 4.** Concentrations of CO2(aq), HCO3 − , and CO3 2‐ for each pH [mol%] referred from the literature [11, 13].

## **5. Results and discussion**

Before evaluating the effects of slag eluates on algae, the concentrations of elements in each slag eluate were analysed and discussed (**Table 2**). In addition to the results of leaching tests for slag, the environmental quality standards (EQSs) for soil pollution and for marine and water pollution are shown as reference values in **Table 2** [13]. In brief, concentrations of Ca, Mg and Si eluted from the slag samples were high because these slags contained large amounts of those materials (**Table 1**). In contrast to the elements above, the eluted concentrations of Al and Fe were quite low in spite of their high concentrations in the slag particles. An earlier report [32] explained this contradictory phenomenon as a difference of these elements in terms of solubility. Leaching tests revealed that concentrations of components eluted from two slags used for this study were almost all lower than the respective EQSs, except for selenium (Se) in eluate from the slag A (eluate A), as described in earlier reports [11–13].

This study examined the effects of slag eluates as an experimentally stress factor on algal growth, particularly that of *Chlorella* spp. [11–13]. **Figure 5** shows the relation between the *Chlorella* proliferation ratio and concentrations of the slag eluate in the test solution. Here, all nutrient amounts derived from CA medium, other than elements derived from each slag eluate, were constant with each experimental condition. A detailed account of the results showed that the number of algae increased according to the concentration of each eluate up to 30 vol%. Subsequent comparison of the algal proliferation ratios in eluate A and eluate B showed that these ratios were almost equal at concentrations lower than 50 vol% of the respective eluates. However, the 70 vol% of eluate B showed a slightly more algal proliferation than that of eluate A, as described in previous reports [11–13]. After explaining the results from FCM analysis, we subsequently discussed the difference in the algal proliferation ratio between eluate A and eluate B.

practice, the chlorophosphonazo‐III method [35] and the xylidyl blue‐I method [36] were used for the evaluation of the concentration of Ca2+ and that of Mg2+. Moreover, elemental concen‐ trations before treatment of algae with eluate were compared statistically with those after

36 Superfood and Functional Food - The Development of Superfoods and Their Roles as Medicine

treatment using *t*‐tests.

**Figure 4.** Concentrations of CO2(aq), HCO3

**5. Results and discussion**

− , and CO3

eluate from the slag A (eluate A), as described in earlier reports [11–13].

Before evaluating the effects of slag eluates on algae, the concentrations of elements in each slag eluate were analysed and discussed (**Table 2**). In addition to the results of leaching tests for slag, the environmental quality standards (EQSs) for soil pollution and for marine and water pollution are shown as reference values in **Table 2** [13]. In brief, concentrations of Ca, Mg and Si eluted from the slag samples were high because these slags contained large amounts of those materials (**Table 1**). In contrast to the elements above, the eluted concentrations of Al and Fe were quite low in spite of their high concentrations in the slag particles. An earlier report [32] explained this contradictory phenomenon as a difference of these elements in terms of solubility. Leaching tests revealed that concentrations of components eluted from two slags used for this study were almost all lower than the respective EQSs, except for selenium (Se) in

This study examined the effects of slag eluates as an experimentally stress factor on algal growth, particularly that of *Chlorella* spp. [11–13]. **Figure 5** shows the relation between the *Chlorella* proliferation ratio and concentrations of the slag eluate in the test solution. Here, all nutrient amounts derived from CA medium, other than elements derived from each slag eluate, were constant with each experimental condition. A detailed account of the results

2‐ for each pH [mol%] referred from the literature [11, 13].



1 Not detected.

2 Reportable detection limit.

3 These data from a previous study reported by Takahashi et al. [17].

4 Standard value is not applied to coastal waters.

5 Standard value is applied to coastal waters.

6 The Cd value has changed from 0.1 to 0.03 mg/L since December 2014.

7 Habitable river or lake for aquatic life.

8 Habitable coastal water for aquatic life.

9 Habitable coastal water that requires conservation in particular for nidus and nursery ground.

10Total concentrations of both calcium and magnesium are limited for water hardness.

11Habitable lake for aquatic life.

12Total N contents derived from nitrite nitrogen.

13Total N contents derived from both nitrite nitrogen and nitrate nitrogen.

**Table 2.** Environmental quality standards regarding pollutions and others for effluent and drinking water, and concentrations of elements of each eluate (mg/L) quoted with permission from Ref. [9].

**Figure 5.** Effects of respective eluates on algal growth referred from the literature [11, 13].

**Origin of slag Eluate of EAF**

**Substances out of regulation**

1

2

3

4

5

6

7

8

9

Not detected.

Reportable detection limit.

**stainless steel oxidation slag (Slag A)**

Total Al ND 1.8

Total Si 1.8 1.9

These data from a previous study reported by Takahashi et al. [17].

The Cd value has changed from 0.1 to 0.03 mg/L since December 2014.

13Total N contents derived from both nitrite nitrogen and nitrate nitrogen.

Standard value is not applied to coastal waters.

Standard value is applied to coastal waters.

Habitable river or lake for aquatic life.

Habitable coastal water for aquatic life.

12Total N contents derived from nitrite nitrogen.

11Habitable lake for aquatic life.

**Eluate of EAF normal steel oxidation slag (Slag B)**

38 Superfood and Functional Food - The Development of Superfoods and Their Roles as Medicine

**Soil pollution**

Total Ca 9.3 10.1 30010 Total Fe ND 0.23 10 0.3 Total Mg 0.9 1.1 30010 Total Mn 0.028 ND 10 0.05

Total N 0.4 0.3623 0.1–111

Total P ND (RDL: 0.1) ND3 0.005–111

Habitable coastal water that requires conservation in particular for nidus and nursery ground.

**Table 2.** Environmental quality standards regarding pollutions and others for effluent and drinking water, and

10Total concentrations of both calcium and magnesium are limited for water hardness.

concentrations of elements of each eluate (mg/L) quoted with permission from Ref. [9].

**Environmental quality standards**

**Marine pollution** **Water pollutant**

0.2–18

0.02–0.098

16

**Effluent standard** **Drinking water standard**

100 0.0412, 1013

In addition to algal population estimation using hemocytometry, estimation of the cellular status of algae using FCM was conducted. The results are presented two‐dimensionally in **Figure 6** [11–13]. Here, each single dot on the 2D map represents optical information of a single alga. To compare the status of vigorous algae with that of stressed and dying algae, algae treated with heat (heated sample) were prepared as a reference standard of algae subjected to stress [11–13]. Results show that the 2D map of the red versus the yellow fluorescence intensity for control algae differs clearly from that for the heated algae (**Figure 6**). The 2D map of the red versus yellow fluorescence intensity for control algae showed respectively 102 –103 on the red channel and 101 –102 on the yellow, whereas that for the heated algae did 101 –102 on the red channel and 101 –103 on the yellow. It is particularly interesting that the dot distribution of algae treated with each slag eluate closely resembled that of control, although that with each eluate shifted slightly upward relative to that of control algae [11–13].

Quantitative analysis of algal distribution patterns (**Table 3**) was conducted along with qualitative analysis of those patterns (**Figure 6**). Each graph in **Figure 6** is divisible into four subareas (regions I–IV) based on algal viability [11–13]: region I represents an area for vigorous algae; region II includes dead and variant algae such as heated algae; region III includes algae with low red fluorescence intensity; and region IV includes data from which algae are virtually absent. Algal distribution patterning revealed clear differences between the algal distribution in control samples and those in heated algae. The signals of algae treated with eluate were also distributed almost entirely to region I. The ratio was 96.81 ± 2.60% in control, 98.15 ± 0.31% in eluate A, and 98.13 ± 0.24% in eluate B. Ratio analysis shows that the percentages of algae treated with slag eluates were slightly higher than those of control algae. Components dissolved from slags did not apparently give rise to algae stress because the quantities of algae in media containing the respective eluates were equal to or greater than those in media with no eluate (**Figure 5**). The tested slags contain metals such as copper, zinc and aluminium (**Table 2**). Aluminium, which is also not contained in the CA medium, has been particularly reported as inhibiting plant growth [37, 38]. Although the culture medium containing aluminium and other metal elements was predicted to affect algal growth and status, they caused no effect on algal growth directly. The data demonstrated that components eluted from slag showed no marked toxicity to algae. This assessment system using FCM, which estimates chlorophyll fluorescence of photosynthetic pigments, might be applicable to other algae, other phytoplankton and aquatic plants with chlorophyll, although this report presents data only for *Chlorella* spp. as a model organism. This technique can contribute to evaluation of algal features and monitoring their qualities in culture for industry application of algae.

**Figure 6.** Distribution of *Chlorella* using FCM referred from the literature [11–13]. The red fluorescence intensity of al‐ gae is shown versus the yellow fluorescence intensity. The heated sample is the heat treatment sample of algae. Eluate A and eluate B denote solutions with respective concentrations of eluate A and eluate B of 50 vol%.


**Table 3.** Distribution of untreated *Chlorella* and treated with heat or eluate from slag referred from earlier reports [7, 9].

It remains unclear why algae in media containing eluate proliferated more than algae in media without eluate (**Figure 5**). In general, the growth and proliferation of photosynthetic organ‐ isms, such as land plants and algae, depend strongly on photosynthetic efficiency. Photosyn‐ thesis is divisible mainly into two metabolizing systems: light‐dependent reactions, which harvest light energy from sunlight and which perform electron transport; and the Calvin cycle, which performs CO2 fixation to synthesize glucose. This study particularly examined the concentrations of CO2, which are related to the Calvin cycle, because all experiments in this study were conducted under constant light conditions [11, 13]. This CO2(aq) can be detected as infrared absorption near 2350 cm‐1 using FT‐IR [11, 13], which is identical to the infrared absorption attributable to anti‐symmetric stretching of CO2 [39]. To examine the relation between algal proliferation and the concentration of CO2(aq) under treatment of algae with slag eluate, this study directly evaluated CO2(aq) in medium containing eluate using FT‐IR [11, 13]. The result shows that both eluates had higher infrared absorption identical to CO2 than that of controls without slag eluate [11, 13]. Next, concentrations of CO2(aq) under each test condition were quantified from the amounts of total carbonate species using a diaphragm‐type electrode to measure CO2(aq) and from calculation using the Henderson–Hasselbalch equa‐ tions (**Figure 7**) [11, 13]. The result also showed that concentrations of CO2(aq) in media containing eluate were higher than those of control samples. Moreover, the concentration in the medium containing eluate B had higher concentrations than that in eluate A. Speculating based on these obtained data, the addition of slag eluate appears to improve aqueous envi‐ ronments for photosynthetic organisms. It might facilitate algal photosynthesis more than CA medium alone. Consequently, increasing concentrations of CO2(aq) by adding slag eluates induced greater algal proliferation than that in the control sample (**Figure 5**). **Figure 5** shows that this study also stumbled on the fact that the addition of eluate B to the culture medium induced greater proliferation of algae than that of eluate A. Accounting for the different concentrations of CO2(aq) between eluate A and eluate B, the greater effects of eluate B than those of eluate A on algal proliferation might also be explained.

caused no effect on algal growth directly. The data demonstrated that components eluted from slag showed no marked toxicity to algae. This assessment system using FCM, which estimates chlorophyll fluorescence of photosynthetic pigments, might be applicable to other algae, other phytoplankton and aquatic plants with chlorophyll, although this report presents data only for *Chlorella* spp. as a model organism. This technique can contribute to evaluation of algal

**Figure 6.** Distribution of *Chlorella* using FCM referred from the literature [11–13]. The red fluorescence intensity of al‐ gae is shown versus the yellow fluorescence intensity. The heated sample is the heat treatment sample of algae. Eluate

**Table 3.** Distribution of untreated *Chlorella* and treated with heat or eluate from slag referred from earlier reports [7, 9].

It remains unclear why algae in media containing eluate proliferated more than algae in media without eluate (**Figure 5**). In general, the growth and proliferation of photosynthetic organ‐ isms, such as land plants and algae, depend strongly on photosynthetic efficiency. Photosyn‐ thesis is divisible mainly into two metabolizing systems: light‐dependent reactions, which harvest light energy from sunlight and which perform electron transport; and the Calvin cycle, which performs CO2 fixation to synthesize glucose. This study particularly examined the concentrations of CO2, which are related to the Calvin cycle, because all experiments in this study were conducted under constant light conditions [11, 13]. This CO2(aq) can be detected as infrared absorption near 2350 cm‐1 using FT‐IR [11, 13], which is identical to the infrared

**I II III IV**

A and eluate B denote solutions with respective concentrations of eluate A and eluate B of 50 vol%.

Control 96.81 ± 2.60 2.40 ± 2.91 0.73 ± 0.31 0.07 ± 0.08 Heated sample 0.28 ± 0.36 97.27 ± 0.81 2.59 ± 0.42 0.02 ± 0.03 Eluate A 98.15 ± 0.31 0.17 ± 0.08 1.67 ± 0.21 0.02 ± 0.03 Eluate B 98.13 ± 0.24 0.52 ± 0.51 1.29 ± 0.33 0.05 ± 0.05

features and monitoring their qualities in culture for industry application of algae.

40 Superfood and Functional Food - The Development of Superfoods and Their Roles as Medicine

**Figure 7.** Concentrations of CO2(aq) in each solution modified from the literature [11, 13]. Eluate A and eluate B, re‐ spectively, denote solutions in which concentrations of eluate A and eluate B were 70 vol%.

Formation of the greater concentrations of CO2(aq) by addition of slag eluate than the control condition has been discussed in the literature [11, 13]. Ca2+ and Mg2+ are highly reactive substrates with the carbonate species. These elements conveniently existed in higher contents of eluates than other elements (see **Table 2**). As portrayed in **Figure 4**, the fraction of HCO3 − is the highest content of the carbonate species at pH 7.2, which were experimental conditions used for this study. Therefore, Ca2+ in eluate, for instance, might be presumably reacted with HCO3 − as described in the following Eq. (6).

$$\rm Ca^{2+} + 2HCO\_3^- \rightarrow \rm Ca(HCO\_3)\_2(aq) \tag{6}$$

Ca(HCO3)2 can interfere in the chemical equilibrium of the carbonate species as HCO3 − because Ca(HCO3)2 is ionized completely. Here, the ratios of concentrations of the respective carbonate species must be constant in solution. Consequently, the increase in HCO3 − concentrations in solution prompts Eq. (2) to proceed leftward, resulting in increasing concentrations of CO2(aq). Increased CO2(aq) might be consumed by algae as a raw material of photosynthesis. Assess‐ ment of the transitional change of concentrations of Ca2+ and Mg2+ revealed no significant difference between the concentrations of these elements before and after incubation of algae with eluates, even at 7 days after incubation (**Table 4**). These obtained data support the hypothesis that the addition of slag eluate, particularly Ca2+ in eluate, increases the amounts of the total carbonate species and that the increase in the total amounts of the carbonate species by adding slag eluates triggers the potential increase of CO2(aq), eventually inducing algal proliferation.


**Table 4.** Concentration of alkarin earth elements before and after incubation of *Chlorella* with each eluate, referred from references [7, 9].

The biochemical importance of CO2(aq) increased by slag eluates was discussed as described in earlier reports [11, 13]. In general, the increase of CO2(aq) can promote carbon dioxide assimilation by photosynthesis on an algal cellular level. However, the present concentrations of CO2(gas) in air determine the concentrations of CO2(aq) that can be dissolved in water. Consequently, the concentrations of CO2(gas) are regarded as a rate‐determining factor of photosynthesis [40]. Its action as the rate‐determining factor of CO2(gas) is common not only to land plants using CO2(gas) directly but also to aquatic organisms such as phytoplankton using CO2(aq) dissolved into water. This study indicates that slag components in solution did not cause toxicity to *Chlorella* and that the eluates were able to increase concentrations of CO2(aq), which functions as the rate‐determining factor of photosynthetic organisms in the aqueous environment. This feature of slags in aqueous environment is regarded as beneficial for aqueous photosynthetic organisms including algae. This study was performed using *Chlorella* spp. as the model organism of algae and aqueous photosynthetic organisms. To present the usefulness of slag eluate and their components in algal culture more precisely, additional experiments must be done using photosynthetic organisms other than *Chlorella*.

## **6. Conclusion**

( )( ) <sup>2</sup>

Ca(HCO3)2 is ionized completely. Here, the ratios of concentrations of the respective carbonate

solution prompts Eq. (2) to proceed leftward, resulting in increasing concentrations of CO2(aq). Increased CO2(aq) might be consumed by algae as a raw material of photosynthesis. Assess‐ ment of the transitional change of concentrations of Ca2+ and Mg2+ revealed no significant difference between the concentrations of these elements before and after incubation of algae with eluates, even at 7 days after incubation (**Table 4**). These obtained data support the hypothesis that the addition of slag eluate, particularly Ca2+ in eluate, increases the amounts of the total carbonate species and that the increase in the total amounts of the carbonate species by adding slag eluates triggers the potential increase of CO2(aq), eventually inducing algal

**Before incubation After incubation** *p* **value (%)**

**Table 4.** Concentration of alkarin earth elements before and after incubation of *Chlorella* with each eluate, referred from

The biochemical importance of CO2(aq) increased by slag eluates was discussed as described in earlier reports [11, 13]. In general, the increase of CO2(aq) can promote carbon dioxide assimilation by photosynthesis on an algal cellular level. However, the present concentrations of CO2(gas) in air determine the concentrations of CO2(aq) that can be dissolved in water. Consequently, the concentrations of CO2(gas) are regarded as a rate‐determining factor of photosynthesis [40]. Its action as the rate‐determining factor of CO2(gas) is common not only to land plants using CO2(gas) directly but also to aquatic organisms such as phytoplankton using CO2(aq) dissolved into water. This study indicates that slag components in solution did not cause toxicity to *Chlorella* and that the eluates were able to increase concentrations of CO2(aq), which functions as the rate‐determining factor of photosynthetic organisms in the aqueous environment. This feature of slags in aqueous environment is regarded as beneficial for aqueous photosynthetic organisms including algae. This study was performed using *Chlorella* spp. as the model organism of algae and aqueous photosynthetic organisms. To present the usefulness of slag eluate and their components in algal culture more precisely, additional experiments must be done using photosynthetic organisms other than *Chlorella*.

Eluate A 9.904 8.945 ± 0.917 *p* > 0.05 Eluate B 10.464 9.763 ± 1.056 *p* > 0.05

Eluate A 2.602 3.362 ± 0.381 *p* > 0.05 Eluate B 2.742 2.931 ± 0.075 *p* > 0.05

Ca(HCO3)2 can interfere in the chemical equilibrium of the carbonate species as HCO3

species must be constant in solution. Consequently, the increase in HCO3

42 Superfood and Functional Food - The Development of Superfoods and Their Roles as Medicine

proliferation.

references [7, 9].

**Concentration of Ca2+ (mg/L)**

**Concentration of Mg2+ (mg/L)**

3 3 <sup>2</sup> Ca 2HCO Ca HCO aq + - + ® (6)

− because

concentrations in

−

For autotrophic algal biorefineries, biofuel materials, and bioremediation materials, it is important to evaluate features of interesting algae and their qualities in culture. In this study, *Chlorella* was cultured with CA medium containing an eluate from steel slag as an experimental factor and sources of trace metals. Results obtained from this study can be summarized as the following: (1) Slag eluates used for this study met the EQSs for soil pollution, effluent and drinking water, except for Se in eluate A. (2) Analyses of algae treated with the eluate revealed that the eluate from used slag induced neither lethality nor growth inhibition. (3) In addition to cell counting using hemocytometry, bioassay using FCM was able to estimate vigorous and aberrant algal growth simultaneously and graphically. (4) In contrast to comparison of control algae with the heat stress, the distribution of algae treated with the eluate was appropriately similar to that of control, suggesting that the eluate from slags did not give rise to algae stresses. (5) The addition of eluates to the medium increased the concentrations of CO2(aq). The increased CO2(aq), which was found to be related to the presence of Ca2+ in eluates, improved the rates of photosynthesis and algal proliferation.

## **Acknowledgements**

This research was mainly supported by a Grant for Young Scientists from the Iron and Steel Institute of Japan and partly by a Grant‐in‐Aid for Young Scientists (B) (Grant No. 26870820).

## **Author details**

Toshiyuki Takahashi

Address all correspondence to: mttaka@cc.miyakonojo‐nct.ac.jp

Department of Chemical Science and Engineering, National Institute of Technology, Miyakonojo College, Miyakonojo, Miyazaki, Japan

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## **Functional Fruits Through Metabolic Engineering Functional Fruits Through Metabolic Engineering**

Luis Quiroz-Iturra, Carolina Rosas-Saavedra and Claudia Stange Klein Luis Quiroz-Iturra, Carolina Rosas-Saavedra and Claudia Stange Klein

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

http://dx.doi.org/10.5772/67219

#### **Abstract**

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46 Superfood and Functional Food - The Development of Superfoods and Their Roles as Medicine

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Metabolic engineering is a main focus of many plant biotechnology programs that look for the production of novel plant varieties with improved human health benefits. Among the most interesting goals are those that are focused in the production of functional fruits. A fruit can be considered as functional if it produces additional benefits to human health and well-being, beyond nutrition. Fruits that present higher levels of beneficial compounds such as essential vitamins, antioxidants, and phytochemicals can be considered as functional as those compounds have long-term benefits in reducing the occurrence of certain diseases. Through the expression, silencing, or mutagenesis strategies, many functional fruit crops have been produced during the last 40 years. Novel plants produce higher amount of carotenoids, antocyanins, and folic acid in their fruits, as well as higher color, sweetness, flavor, and aroma. The improvement of postharvest and resistance to biotic and abiotic stress in commercial plants has been also enhanced as it can led to a better fruit production. Taken together, this chapter will present a revision of the main fruits that have been improved by means of metabolic engineering within the framework of functional foods and super foods.

**Keywords:** biotechnology, functional food, fruits, metabolic engineering

## **1. Introduction**

Today, the quality of life is becoming one of the pivotal reasons for people to be concerned about its health. Also, the steady increase in life expectancy accompanied by the growing cost of health care and the increasing rate of metabolic disorders (heart disease, obesity, diabetes, and arthritis) are the factors to consider in terms of life quality. On the other hand, vast scientific evidence determines a pivotal link between diet and human health, showing the crucial

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

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, © 2017 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

role of nutrition in the prevention of chronic diseases, such as coronary heart diseases, cancer, neurodegenerative, and respiratory disease, along with aging. Therefore, this scenario had led to the development of functional foods as a recognized category of foods to be part of an international strategy to overcome diseases related to human diet and life style.

The first conceptual approach developed by the European Commission Concerted Action on Functional Food Science in Europe (FUFOSE), coordinated by the International Life Sciences Institute (ILSI) Europe established that "A food can be regarded as 'functional' if it is satisfactorily demonstrated to affect beneficially one or more target functions in the body, beyond adequate nutritional effects, in a way that is relevant to either an improved state of health and well-being and/or reduction of risk of disease. Functional foods must remain foods and they must demonstrate their effects in amounts that can normally be expected to be consumed in the diet: they are not pills or capsules but part of a normal food pattern" [1, 2]. Today, an universal accepted definition for the term functional food is not accorded yet [1, 3]. In fact, this concept has been defined several times [4], although in most countries there is not a legislative definition [3].

From a practical point of view, a whole fruit that contains sufficient quantities of beneficial components represents the simplest example of functional food and therefore may be considered as functional fruit. The main characteristic of functional fruits is their high content of bioactive compounds, which promote a state of health and well-being and/or reduce the risk of some diseases and may even be used to cure some illnesses. However, there is a slight difference between conventional and functional fruits, even for experts such as nutritionists, because many if not most fruits contain natural components that provide benefits beyond basic nutrition, such as lycopene in tomatoes [5] or anthocyanins in pomegranate [6]. Moreover, not all fruits can be considered as functional because the health benefits strongly depend on the absorption and transformation of nutrients (bioaccesibility) during gastrointestinal digestion [7]. The bioaccesibility of these compounds permits to have a clear idea of their potential bioavailability, term that involves the biological activity of these compounds.

To meet future demand for functional foods, the food industry must address critical challenges such as developing strategies to increase the yield of healthy compounds in fruits or to add nutritional value with new components to improve the quality standards aiming to maintain the well-being. The beneficial components of functional fruits can be enhanced through special growing conditions [8–10], through breeding techniques [11] or through metabolic engineering for delivering truly unique health benefits [12]. In this context, metabolic engineering plays a key role in developing functional fruits, in terms of being defined as "the direct improvement of production, formation, or cellular properties through the modification of specific biochemical reactions or the introduction of new ones with the use of recombinant DNA technology" [13, 14]. In plants, metabolic engineering can be used to develop functional foods and super foods by handling the flow of primary and secondary metabolic pathways, allowing the redirection of carbon flow toward products of interest or the synthesis of new products.

Nowadays, metabolic engineering has been used to produce new plant varieties with higher levels of valuable compounds such as pro-vitamin A, antocyanins, folic acid, antioxidants, as well as higher color, sweetness, flavor, and aroma. Many novel functional fruit crops have been generated during the last 40 years by using molecular strategies such as the over-expression, silencing, or mutagenesis of specific genes. The improvement of postharvest and resistance to biotic and abiotic stress in commercial plants has been also enhanced as it can lead to a better fruit production.

Taken together, in the following sections, we will review the progress in biotechnological approaches in developing functional fruits by describing strategies employed in metabolic engineering, and the characteristics that have been improved in several agronomic traits to insert novel functional fruits into the market.

## **2. Metabolic engineering strategies**

role of nutrition in the prevention of chronic diseases, such as coronary heart diseases, cancer, neurodegenerative, and respiratory disease, along with aging. Therefore, this scenario had led to the development of functional foods as a recognized category of foods to be part of an

The first conceptual approach developed by the European Commission Concerted Action on Functional Food Science in Europe (FUFOSE), coordinated by the International Life Sciences Institute (ILSI) Europe established that "A food can be regarded as 'functional' if it is satisfactorily demonstrated to affect beneficially one or more target functions in the body, beyond adequate nutritional effects, in a way that is relevant to either an improved state of health and well-being and/or reduction of risk of disease. Functional foods must remain foods and they must demonstrate their effects in amounts that can normally be expected to be consumed in the diet: they are not pills or capsules but part of a normal food pattern" [1, 2]. Today, an universal accepted definition for the term functional food is not accorded yet [1, 3]. In fact, this concept has been defined several times [4], although in most countries there is not a legislative definition [3]. From a practical point of view, a whole fruit that contains sufficient quantities of beneficial components represents the simplest example of functional food and therefore may be considered as functional fruit. The main characteristic of functional fruits is their high content of bioactive compounds, which promote a state of health and well-being and/or reduce the risk of some diseases and may even be used to cure some illnesses. However, there is a slight difference between conventional and functional fruits, even for experts such as nutritionists, because many if not most fruits contain natural components that provide benefits beyond basic nutrition, such as lycopene in tomatoes [5] or anthocyanins in pomegranate [6]. Moreover, not all fruits can be considered as functional because the health benefits strongly depend on the absorption and transformation of nutrients (bioaccesibility) during gastrointestinal digestion [7]. The bioaccesibility of these compounds permits to have a clear idea of their potential bioavailability, term that involves the biological activity of these compounds. To meet future demand for functional foods, the food industry must address critical challenges such as developing strategies to increase the yield of healthy compounds in fruits or to add nutritional value with new components to improve the quality standards aiming to maintain the well-being. The beneficial components of functional fruits can be enhanced through special growing conditions [8–10], through breeding techniques [11] or through metabolic engineering for delivering truly unique health benefits [12]. In this context, metabolic engineering plays a key role in developing functional fruits, in terms of being defined as "the direct improvement of production, formation, or cellular properties through the modification of specific biochemical reactions or the introduction of new ones with the use of recombinant DNA technology" [13, 14]. In plants, metabolic engineering can be used to develop functional foods and super foods by handling the flow of primary and secondary metabolic pathways, allowing the redirection of carbon flow toward products of interest or the synthesis of new

Nowadays, metabolic engineering has been used to produce new plant varieties with higher levels of valuable compounds such as pro-vitamin A, antocyanins, folic acid, antioxidants, as well as higher color, sweetness, flavor, and aroma. Many novel functional fruit crops have been

international strategy to overcome diseases related to human diet and life style.

48 Superfood and Functional Food - The Development of Superfoods and Their Roles as Medicine

products.

In plants, metabolic engineering has been developed through different approaches. Within them, the most widely used are the gain-of-function by transgenesis (including cisgenia and intragenia) and the loss-of-function carried out through gene silencing and mutagenesis. These strategies are achieved by *Agrobacterium tumefaciens* or biobalistics transformation systems.

## **2.1. Production of transgenic plants that express a functional gene**

Transgenesis is the process by which one (or more) exogenous gene (called transgene) is inserted into a living organism, giving them a new feature that has to be stable over next generations. Cisgenia and intragenia terms are used when the exogenous genes belong to the same or related plants, respectively [15]. The functionality of the transgene in the new plant is accomplished due to the fact that the genetic code of all living organisms is exactly the same. This means that a specific DNA sequence may encode the same protein in any living organism. Transgenesis itself is a process that occurs in nature without human intervention. The better example is taken from plants that are infected by the bacteria *A. tumefaciens*. This bacteria produces the disease known as "grown call" in which *Agrobacterium* induces the formation of a tumor in the stem of more than 140 species of Eudicotyledoneae. The symptoms are caused by inserting a DNA segment in a semirandom manner in the plant genome. This DNA segment (known as T-DNA, 'transfer DNA') codifies for plant hormones that induce the generation of tumors when produced together in high levels in plant cells [16]. This feature has allowed scientists to use a modified strain of *A. tumefaciens* for inserting genes of interest instead of the phytohormone-inducing-tumor genes [17]. For instances, several commercial transgenic plant varieties produced through Agrobacterium transformation are cultivated and consumed in around 25 countries in the world. Most of them are transgenic varieties of maize, soybean, cotton, and canola that are tolerant to herbicides and resistant to insects, among others. Actually, fruits have also been improved through this technique and will be described later.

## **2.2. Gene silencing of specific genes in plants**

In the case of plant gene silencing, also termed RNA interference (RNAi) or post-transcriptional gene silencing (PTGS), a small fragment of 100–400 pb of the gene in antisense orientation is introduced into the plant, causing the degradation of the target RNA, diminishing thus the amount of mRNA and of the protein from 40 to 99%. The process for gene silencing has been described extensively and is stated through the degradation of a double-stranded RNA molecule (dsRNA) in the cell coming from the hybridization of the antisense RNA and the endogenous sense RNA of the target gene, which triggers the RNAi pathway [18, 19]. The fragments generated include small interfering RNAs (siRNA) of about 21–23 nucleotides in length that through the host RNA-induced silencing complex (RISC) allows the systemic degradation of the target mRNA. It is believed that siRNA system has evolved as a cellular defense mechanism against RNA viruses or to combat the proliferation of transposons within the cell [19]. Currently, siRNAs are now widely used to suppress the expression of specific genes and to assess gene function. Gene silencing is considered a mechanism of gene knockdown, where the expression of a gene is reduced at least 99% but not completely [20].

#### **2.3. Plant mutagenesis**

Several standardized procedures that induce mutations have been used for the production of new crops varieties of commercial interest [21]. This technique uses physical or chemical mutagenic agents. The chemical procedure, by using EMS (ethyl-methane sulfonate), is simpler to achieve and has demonstrated to be one of the most reliable inducers of mutagenesis [21]. EMS tends to generate random changes in nucleotides, generating single-nucleotide polymorphisms (SNPs) or deletions (indel) that affect the functionality of some gene(s) in the genome [22]. This is translated in the modification of phenotypes and/or physiological characters. Mutations are inherit events in any alive species and naturally and randomly happen around 50,000 times per year which genetically change from normal cells to mutated cells every day [23]. Many of those alterations are repaired, but when not, a mutation persists being a key piece in the evolution of the species. Therefore, chemical mutagenesis has been used for more than 60 years in breeding programs in the world. Plants generated by mutagenesis do not require long evaluation processes as transgenic or silenced plants and are accepted and introduced to the market more efficiently. Approximately 2965 induced mutagenesis cultivars such as crops that include wheat, barley, and rice, and among others have been generated and released during the last 40 years. Novel varieties of fruits, which include Kiwifruits, produced through EMS mutagenesis, have also been approved for commercialization [21–24].

One of the most innovative system, which has gained great impact few years ago in the field of metabolic engineering, is the genome editing system CRISPR/Cas. This is a versatile and effective tool for editing genomes in a site-specific manner [25, 26]. The CRISPR/Cas system is a natural defense mechanism in eubacteria and Aequeas against plasmids and viruses [27, 28]. For metabolic engineering, a chimeric guide RNA (gRNA) that contains 20 nucleotides must specifically bind to their target sequence in the DNA. The target sequence must also contain the protospacer adjacent motif (PAM) sequence that is recognized by Cas9, cutting 3–4 nucleotides upstream of the PAM sequence [25]. The most common editing events are small Indels (insertion or deletion) of 1–10 nt [29]. The CRISPR/Cas9 system has been effectively used as a tool for editing the genome of numerous plants including *Arabidopsis thaliana, Nicotiana tabacum, Oryza sativa, Zea mays, Glycine max, Triticum aestivum,* and citrus [29].

## **3. Metabolic engineering for fruit-trees improvement**

introduced into the plant, causing the degradation of the target RNA, diminishing thus the amount of mRNA and of the protein from 40 to 99%. The process for gene silencing has been described extensively and is stated through the degradation of a double-stranded RNA molecule (dsRNA) in the cell coming from the hybridization of the antisense RNA and the endogenous sense RNA of the target gene, which triggers the RNAi pathway [18, 19]. The fragments generated include small interfering RNAs (siRNA) of about 21–23 nucleotides in length that through the host RNA-induced silencing complex (RISC) allows the systemic degradation of the target mRNA. It is believed that siRNA system has evolved as a cellular defense mechanism against RNA viruses or to combat the proliferation of transposons within the cell [19]. Currently, siRNAs are now widely used to suppress the expression of specific genes and to assess gene function. Gene silencing is considered a mechanism of gene knockdown, where the

Several standardized procedures that induce mutations have been used for the production of new crops varieties of commercial interest [21]. This technique uses physical or chemical mutagenic agents. The chemical procedure, by using EMS (ethyl-methane sulfonate), is simpler to achieve and has demonstrated to be one of the most reliable inducers of mutagenesis [21]. EMS tends to generate random changes in nucleotides, generating single-nucleotide polymorphisms (SNPs) or deletions (indel) that affect the functionality of some gene(s) in the genome [22]. This is translated in the modification of phenotypes and/or physiological characters. Mutations are inherit events in any alive species and naturally and randomly happen around 50,000 times per year which genetically change from normal cells to mutated cells every day [23]. Many of those alterations are repaired, but when not, a mutation persists being a key piece in the evolution of the species. Therefore, chemical mutagenesis has been used for more than 60 years in breeding programs in the world. Plants generated by mutagenesis do not require long evaluation processes as transgenic or silenced plants and are accepted and introduced to the market more efficiently. Approximately 2965 induced mutagenesis cultivars such as crops that include wheat, barley, and rice, and among others have been generated and released during the last 40 years. Novel varieties of fruits, which include Kiwifruits, produced

through EMS mutagenesis, have also been approved for commercialization [21–24].

One of the most innovative system, which has gained great impact few years ago in the field of metabolic engineering, is the genome editing system CRISPR/Cas. This is a versatile and effective tool for editing genomes in a site-specific manner [25, 26]. The CRISPR/Cas system is a natural defense mechanism in eubacteria and Aequeas against plasmids and viruses [27, 28]. For metabolic engineering, a chimeric guide RNA (gRNA) that contains 20 nucleotides must specifically bind to their target sequence in the DNA. The target sequence must also contain the protospacer adjacent motif (PAM) sequence that is recognized by Cas9, cutting 3–4 nucleotides upstream of the PAM sequence [25]. The most common editing events are small Indels (insertion or deletion) of 1–10 nt [29]. The CRISPR/Cas9 system has been effectively used as a tool for editing the genome of numerous plants including *Arabidopsis thaliana, Nicotiana tabacum, Oryza sativa, Zea mays, Glycine max, Triticum aestivum,* and citrus [29].

expression of a gene is reduced at least 99% but not completely [20].

50 Superfood and Functional Food - The Development of Superfoods and Their Roles as Medicine

**2.3. Plant mutagenesis**

There are several strategies for metabolic engineering of plants. Nevertheless, the nutritive fruits are normally produced from trees or from "recalcitrant" plants that are difficult to regenerate *in vitro* and to transform [30]. Usually, the success of genetic transformation depends on the success of the regeneration process for each plant species. This is influenced by several factors, such as the genotype of the variety, the source of explant, and the degree of tissue differentiation. Therefore, the tissue culture conditions must be optimized for each range of each crop independently [31]. Generally, two methods of tissue culture have been used for regeneration of transgenic plants: organogenesis and somatic embryogenesis. Organogenesis is the process in which the regeneration of a new seedling occurs directly from the explants while in somatic embryogenesis, the formation of embryos from somatic cells that are present in the explant tissue is produced [32]. Somatic embryogenesis has many advantages over organogenesis, including its potentially high rates of multiplication, the genetic uniformity among embryo clones, the potential for expansion in bioreactors, and cryopreserved through synthetic seeds [33]. Despite the above, somatic embryogenesis has not been standardized for most fruit species, and therefore, somatic organogenesis is normally carried out for transgenic plant regeneration.

## **3.1. Disease control in plants to improve fruit crops**

Owing to the economic importance that represents fruit production in various countries, there have been many efforts to generate plants with increased resistance against diseases and pathogens, whether fungi, bacteria, or viruses. Today, only virus resistance is used commercially, which only represents a small proportion of all transgenic plants grown in the world. Most of the plant-infecting viruses are of single-stranded RNA+ type, which codify for a capsid protein, movement protein, and replicases, among others. Within the first cases of success in the generation of virus-resistant plants through metabolic engineering, there are those using viral capsid protein (CP) as the transgene [34]. In this case, plants expressing the CP gene confer viral resistance mediated by RNAi gene silencing when plants are infected by the specific virus.

In 2004, the first example of a genetically engineered horticultural crop that has made it to market was produced in Hawaii. The genetically engineered Rainbow and SunUp Papaya, which are resistant to the papaya ringspot virus (PRSV) were successfully commercialized and adopted by farmers in the Puna area of Hawaii [35]. In addition, after 20 years of testing and risk assessment in the laboratory, in greenhouse and field, plum 'HoneySweet' is now used as a GM crop resistant to Sharka disease caused by the plum pox virus (PPV), which has been validated for US cultivation [36]. PPV protection is based on RNAi, and resistance has been shown to be highly effective, stable, durable, and heritable as a dominant trait. Extensive testing has also demonstrated on the safety and the ability of the RNAi technology for fruit production [36]. Chandrasekaran et al. [37] developed a virus-resistant variety of cucumber (*Cucumis sativus L*.) by CRISPR/Cas9 technology. The Ipomovirus infects cucumber and produces a vein yellowing of the leaves. During the infection, the virus requires the plant eIF4E gene (eukaryotic initiation factor of translation 4E) to carry out the recognition and transcription of their genes [38]. With the CRISPR/Cas9 tool, mutations in the eIF4E gene were introduced in cucumber, and the transgenic plants present small deletions or single-nucleotide polymorphisms (SNPs) in the mutated region of eIF4E. By the culture of the plant to next generations, homozygous mutant progeny was selected. The obtained one showed immunity to the Ipomovirus Cucumber vein yellowing infection and increased resistance to Zucchini yellow mosaic virus and papaya ring spot mosaic virus-W. A system for cucumber virus resistance was generated for the first time, without the need to produce a transgenic organism [37].

Regarding the generation of plants resistant to fungal and bacterial diseases, the main strategies are the expression of resistant genes coding for resistance receptors (R) and those involved in the defense mechanisms such as pathogenic-related proteins (PR proteins), peptides, and antimicrobial metabolites, and genes involved in detoxification mechanisms [39]. The plant defense response is triggered by the recognition of pathogen avirulence factors (avr) by the resistance receptors (R) equipped in the host plant [40]. The avr and R interaction activates one or more signal transduction pathways and eventually triggers a local response termed hypersensitive response (HR) and a systemic acquired resistance (SAR). Both of them induced by the signal-molecule salicylic acid (SA) which permits the accumulation of PR proteins through the activation of a signal transduction machinery [41]. Arabidopsis NPR1 gene (PR gene nonexpresser) is well recognized as a key regulator of signal transduction by SA leading to SAR. The NPR1 overexpression in citrus showed a positive response to the increased citrus canker resistance, caused by the bacterial pathogen *Xanthomonas citri* subsp. *Citri* [42]. In apple (*Malus domestica*), transgenic plants overexpressing the MdNPR1 gene exhibited increased resistance to two important fungal pathogens of apple, *Venturia inaequalis* and *Gymnosporangium juniperi-virginianae* [43]. The expression of the R gene was achieved in apples to also overcome the infection by the fungus *V. inaequalis,* which causes Scabies disease which is one of the most serious diseases that hinder the apple crop production. The gene that confers resistance to this disease is termed Rvi6/Vf scab and is present originally in the *Malus floribunda* 821 wild species. This gene has been incorporated in different commercial apple cultivars by classical breeding. However, as *M. floribunda* 821 has not an edible and attractive fruit, the new breeded species that have the resistance gene Rvi6/Vf produce low-quality fruits that do not reach the market [44]. In order to obtain high-resistant species, the Rvi6 gene (formerly HcrVf2) was inserted in susceptible apple cultivar 'Gala', thereby obtaining a commercially attractive variety that is resistant to Scab disease [45]. Unlike other crops, this variety is considered a cisgenic line, as the Rvi6 gene was taken from another variety of apple.

The hydrolytic enzymes chitinase and glucanase, the best characterized class of PR proteins, are able to degrade the cell wall of pathogenic fungi invaders. PR proteins are important components of the response of plant defense against fungal and bacterial pathogens [46, 47]. Transgenic plants expressing genes encoding for chitinase and glucanase showed increased resistance to fungal diseases in many fruit plants [34]. Furthermore, the use of antimicrobial peptides expressed constitutively in plant tissues has been recommended for genetic engineering of plants to increase disease resistance against fungal and bacterial pathogens [39]. Defensins, one of the classic examples of small antimicrobial peptides, play an important role in the response of plant defense against fungi. Defensins produce the permeabilization of the membrane inhibiting the growth of the fungi through the interaction with membrane components of the fungus [48]. Two defensins genes derived from petunia (PhDef1 and PhDef2) were expressed in banana. *In vitro* and *ex vivo* assays clearly suggested that transgenic banana plants were resistant against the pathogenic fungus *Fusarium oxysporum* sp. cubense [49]. Moreover, antimicrobial proteins from other organisms, such as insects or animals, have been used to increase the resistance to pathogens. Some of these nonplant antimicrobial proteins, such as attacin or cecropin from *Hyalophora cecropia* and magainin from *Xenopus laevis*, showed antimicrobial activity and increased resistance to pathogens in transgenic fruit, such as apple, papaya, pear, potato, sugarcane, and grape [39, 50]. Another alternative is to enhance the production of phytoalexins, antimicrobial metabolites that contribute significantly to the resistance against pathogens [51]. Even so, the production of antimicrobial metabolites generally requires coordinated action of a number of biosynthetic enzymes, which means that many genes are required. This feature makes very difficult to increment antimicrobial metabolites to generate plant resistance varieties [39].

Usually, necrotrophic pathogens produce toxins and enzymes that degrade the cell wall to invade the plant cell for a successful infection. Detoxification and degradation of these phytotoxins by the generation of transgenic plants could provide an opportunity to improve resistance to several diseases [52]. However, the strategy to develop disease-resistant plants is not accessible because some phytotoxins are harmful to mammals, and the product of a detoxification reaction could remain toxic in the plant [39].

## **3.2. Abiotic stress tolerance in fruit crops**

gene (eukaryotic initiation factor of translation 4E) to carry out the recognition and transcription of their genes [38]. With the CRISPR/Cas9 tool, mutations in the eIF4E gene were introduced in cucumber, and the transgenic plants present small deletions or single-nucleotide polymorphisms (SNPs) in the mutated region of eIF4E. By the culture of the plant to next generations, homozygous mutant progeny was selected. The obtained one showed immunity to the Ipomovirus Cucumber vein yellowing infection and increased resistance to Zucchini yellow mosaic virus and papaya ring spot mosaic virus-W. A system for cucumber virus resistance was generated for the first time, without the need to produce a transgenic organism [37].

52 Superfood and Functional Food - The Development of Superfoods and Their Roles as Medicine

Regarding the generation of plants resistant to fungal and bacterial diseases, the main strategies are the expression of resistant genes coding for resistance receptors (R) and those involved in the defense mechanisms such as pathogenic-related proteins (PR proteins), peptides, and antimicrobial metabolites, and genes involved in detoxification mechanisms [39]. The plant defense response is triggered by the recognition of pathogen avirulence factors (avr) by the resistance receptors (R) equipped in the host plant [40]. The avr and R interaction activates one or more signal transduction pathways and eventually triggers a local response termed hypersensitive response (HR) and a systemic acquired resistance (SAR). Both of them induced by the signal-molecule salicylic acid (SA) which permits the accumulation of PR proteins through the activation of a signal transduction machinery [41]. Arabidopsis NPR1 gene (PR gene nonexpresser) is well recognized as a key regulator of signal transduction by SA leading to SAR. The NPR1 overexpression in citrus showed a positive response to the increased citrus canker resistance, caused by the bacterial pathogen *Xanthomonas citri* subsp. *Citri* [42]. In apple (*Malus domestica*), transgenic plants overexpressing the MdNPR1 gene exhibited increased resistance to two important fungal pathogens of apple, *Venturia inaequalis* and *Gymnosporangium juniperi-virginianae* [43]. The expression of the R gene was achieved in apples to also overcome the infection by the fungus *V. inaequalis,* which causes Scabies disease which is one of the most serious diseases that hinder the apple crop production. The gene that confers resistance to this disease is termed Rvi6/Vf scab and is present originally in the *Malus floribunda* 821 wild species. This gene has been incorporated in different commercial apple cultivars by classical breeding. However, as *M. floribunda* 821 has not an edible and attractive fruit, the new breeded species that have the resistance gene Rvi6/Vf produce low-quality fruits that do not reach the market [44]. In order to obtain high-resistant species, the Rvi6 gene (formerly HcrVf2) was inserted in susceptible apple cultivar 'Gala', thereby obtaining a commercially attractive variety that is resistant to Scab disease [45]. Unlike other crops, this variety is considered a cisgenic line,

The hydrolytic enzymes chitinase and glucanase, the best characterized class of PR proteins, are able to degrade the cell wall of pathogenic fungi invaders. PR proteins are important components of the response of plant defense against fungal and bacterial pathogens [46, 47]. Transgenic plants expressing genes encoding for chitinase and glucanase showed increased resistance to fungal diseases in many fruit plants [34]. Furthermore, the use of antimicrobial peptides expressed constitutively in plant tissues has been recommended for genetic engineering of plants to increase disease resistance against fungal and bacterial pathogens [39].

as the Rvi6 gene was taken from another variety of apple.

Resistance to abiotic stresses is a challenging goal to develop biotechnological fruit cultivars and varieties because many plants face rough conditions of drought, salinity, cold, and heat, among others. These environmental factors are significant plant stressors, and their effects on plant development and productivity are reflected in serious agricultural yield losses [11].

Survival of plants under adverse environmental conditions is realized by structural and metabolic changes into endogenous developmental programs. Therefore, methods for agronomic processes and crop improvement are required to enhance these adaptive responses. Efforts have been made to introduce traits with improved drought tolerance, but in many cases, the strategies involved the insertion of a wide range of genes into plants [53].

Drought, salinity, extreme temperatures, and oxidative stress are interconnected environmental stresses [54] that often activate similar cell signaling pathways [55, 56] and cellular responses, such as the accumulation of compatible solutes, production of stress proteins, and the up-regulation of anti-oxidants [57, 58]. Therefore, plant modification for abiotic-enhanced tolerance is mostly based on the manipulation of one or several genes that are either involved in signaling and regulatory pathways [59, 60] or that encode enzymes present in pathways leading to the synthesis of functional and structural protectants, such as osmolytes and antioxidants [61, 62] or that encode stress-tolerance-conferring proteins [63]. For example, the overexpression of SK3-type DHN gene (ShDHN) in transgenic tomato, which codes for a type of dehydrin (DHN), increased tolerance to drought and cold stresses and improved seedling growth under salt and osmotic stresses [63]. DHN is also known as Group 2 LEA (late embryogenesis abundant) proteins [64], and the overexpression of ShDHN in tomato accumulated more proline, maintained higher enzymatic activities of superoxide dismutase and catalase, and suffered less membrane damage under cold and drought stresses.

Another interesting example in abiotic tolerance is the transformation effect of an important rootstock for lemon, *Citrus macrophylla* W. that constitutively expresses the CBF3/DREB1A transcription factor from *Arabidopsis*. CBF3/DREB1A is a member of transcription factors induced on abiotic stress conditions [65]. Transgenic lemon lines showed normal development and, under salt stress, showed greater growth, better stomatal conductance, and similar accumulation of chloride and sodium in the leaves, in comparison with wild-type plants [66].

The adaptation to stress often affects metabolic and energy requirements that sometimes result in deleterious collateral effects such as yield penalty, which mask and limit its benefit to agriculture. In consequence, some authors have succeeded to enhance the abiotic stress tolerance of agricultural species by combining traditional and molecular breeding [67, 68] with the transformation of specific genes [54] such as those reported in this section. Therefore, these strategies have been applied to other species such as soybean, corn, cotton, and canola, which are currently on the market, although additional research is still necessary to evaluate stress resistance of fruit trees varieties in field trials under real stress conditions [68]. Even more, the problems of high costs on the development and releasing processes and safety requirements in regulatory demands must be solved in this kind of fruit crops.

## **4. Metabolic engineering for functional fruits development**

## **4.1. Nutritional improvement in fruits**

Fruits and their processed derivatives are important nutritional sources, not only for carbohydrates, but also for a wide range of secondary metabolites. That are beneficial to human health. Most important metabolites are carotenoids, flavonoids, and anthocyanins, which are widely known to be powerful antioxidants and anticancer agents. Therefore, since many years ago, a great effort has been done to increase the content of these metabolites in plants to improve the nutritional value of fruits.

#### **4.2. Functional fruits with improved carotenoid content**

Carotenoids are the second most abundant pigments found in nature, with more than 750 structurally different compounds responsible for yellow, orange, and red colors [69]. Carotenoids are metabolites synthesized in plants, algae, fungi, and yeasts, and some bacteria where they have photosynthetic, antioxidants, and/or photoprotectant functions [70]. In vertebrates, carotenoids are precursors of vitamin A, and they are also involved in the formation and maintenance of bones and retina. Mammals such as human are not able to synthesize vitamin A, and therefore, they have to include carotenoids in the diet [71]. From a pharmaceutical point of view, these pigments are used as nutritional supplements and antioxidants, highlighting by their protection against UV damage, anticarcinogenic properties, prevention of cardiovascular diseases, cataracts, and macular degeneration [70–73]. Owing to all these important features for human health, the improvement of enhancing carotenoids content in plants and fruits has been carried out since many years, and the production of new varieties of fruits with enhanced amount of carotenoids has been succeeded a few years ago.

The most recognized fruit produced by metabolic engineering is the "super banana," which is part of an Australian project [74]. The aim of this project is to increase the levels of pro-vitamin A in the pulp of commercial banana using metabolic engineering. The overexpression of Psy gene (Apsy2a) from wild banana Asupina, which accumulates carotenoid in the pulp, showed the highest increase in β-carotene levels in the transgenic super banana variety (**Figure 1A**) [75]. Psy gene encodes for the phytoene synthase (PSY), which is the first and the key step in the biosynthetic pathway of carotenoids. Currently, the super banana is in the human-feeding trial stage in the United States. The aim is to transfer the technology to East Africa, where bananas are one of the major basic foods and the levels of vitamin A deficiency are high [76]. Another fruit, which has been modified to improve carotenoid concentration by metabolic engineering, is the kiwifruit (*Actinidia deliciosa*). Transgenic kiwi lines with improved carotenoid content were generated by overexpression of geranylgeranyl diphosphate synthase gene (GGPS), which is part of the metabolic precursors route of carotenoids and Psy of *Citrus unshiu* [77]. Pons et al. showed that it is possible to increase the content of β-carotene in orange fruit (*Citrus sinensis*) through gene silencing of β-carotene hydroxylase gene (CsB-CHX) [78]. This gene is involved in the conversion of β-carotene into xanthophylls. Transgenic fruits showed a dark yellow (golden) phenotype (**Figure 1C**). The levels of β-carotene in transgenic fruit pulp were 36 times higher. Besides, *in vivo* studies performed in *Caenorhabditis elegans* suggested that antioxidant effect in golden fruit was 20% higher than that in conventional fruits [78]. Overexpression of key genes for enhancement of β-carotene synthesis has been extended to other fruits such as tomato (*Solanum lycopersicum*) [79, 80] or the Hong Kong kumquat (*Fortunella hindsii*) [81].

#### **4.3. Functional fruits with improved antocyanin content**

overexpression of SK3-type DHN gene (ShDHN) in transgenic tomato, which codes for a type of dehydrin (DHN), increased tolerance to drought and cold stresses and improved seedling growth under salt and osmotic stresses [63]. DHN is also known as Group 2 LEA (late embryogenesis abundant) proteins [64], and the overexpression of ShDHN in tomato accumulated more proline, maintained higher enzymatic activities of superoxide dismutase

Another interesting example in abiotic tolerance is the transformation effect of an important rootstock for lemon, *Citrus macrophylla* W. that constitutively expresses the CBF3/DREB1A transcription factor from *Arabidopsis*. CBF3/DREB1A is a member of transcription factors induced on abiotic stress conditions [65]. Transgenic lemon lines showed normal development and, under salt stress, showed greater growth, better stomatal conductance, and similar accumulation of chloride and sodium in the leaves, in comparison with wild-type plants [66].

The adaptation to stress often affects metabolic and energy requirements that sometimes result in deleterious collateral effects such as yield penalty, which mask and limit its benefit to agriculture. In consequence, some authors have succeeded to enhance the abiotic stress tolerance of agricultural species by combining traditional and molecular breeding [67, 68] with the transformation of specific genes [54] such as those reported in this section. Therefore, these strategies have been applied to other species such as soybean, corn, cotton, and canola, which are currently on the market, although additional research is still necessary to evaluate stress resistance of fruit trees varieties in field trials under real stress conditions [68]. Even more, the problems of high costs on the development and releasing processes and safety requirements in regulatory demands must be solved in this kind of fruit crops.

Fruits and their processed derivatives are important nutritional sources, not only for carbohydrates, but also for a wide range of secondary metabolites. That are beneficial to human health. Most important metabolites are carotenoids, flavonoids, and anthocyanins, which are widely known to be powerful antioxidants and anticancer agents. Therefore, since many years ago, a great effort has been done to increase the content of these metabolites in plants to

Carotenoids are the second most abundant pigments found in nature, with more than 750 structurally different compounds responsible for yellow, orange, and red colors [69]. Carotenoids are metabolites synthesized in plants, algae, fungi, and yeasts, and some bacteria where they have photosynthetic, antioxidants, and/or photoprotectant functions [70]. In vertebrates, carotenoids are precursors of vitamin A, and they are also involved in the

**4. Metabolic engineering for functional fruits development**

**4.1. Nutritional improvement in fruits**

improve the nutritional value of fruits.

**4.2. Functional fruits with improved carotenoid content**

and catalase, and suffered less membrane damage under cold and drought stresses.

54 Superfood and Functional Food - The Development of Superfoods and Their Roles as Medicine

Anthocyanins are one of the most important water-soluble plant pigments. They are synthesized by the flavonoid branch in the phenylpropanoid pathway. In plants, anthocyanins are secondary metabolites involved in multiple processes, such as attracting pollinators, protection against damage from UV light, seed dispersal, and pathogen attack [84]. For humans, anthocyanins have taken great importance due to its antioxidant and anti-inflammatory effects [85]. Tomato, which is a red fruit, owes its color to the accumulation of carotenoids in the pulp and peel (mainly lycopene) [86]. However, the content of anthocyanins in tomato is very low. Metabolic engineering has been used to enrich the anthocyanin content in tomatoes. For instance, the expression of Delila (DEL) and Rosea1 (ROS1) transcription factors, from *Antirrhinum majus*, in transgenic tomato allowed the increase of anthocyanin content in the fruit [83]. It was observed that DEL and ROS1 transcription factors were able to activate multiple genes related to anthocyanin biosynthesis, such as phenylalanine ammonia lyase (PAL) and flavonoid 3′5′ hydroxylase (F3′5′H). Thus, transgenic tomatoes exhibit intense purple coloration in the pulp and fruit peel as shown in **Figure 1E**, because they contain high concentration of anthocyanin [86]. Several studies on the regulation of the biosynthetic pathway of anthocyanins showed that the family of MYB transcription factors regulates the expression of genes involved in this pathway [87]. Espley et al. reported that greater accumulation of anthocyanins in apple fruits (**Figure 1B**) was obtained by transforming with the transcription factor *MdMYB10* [82]. Moreover, the expression of *FaMYB10* in strawberry generated an increase in anthocyanins in the root, leaf, and strawberry fruit [88].

**Figure 1.** Examples of transgenic fruits. (A) Fruits of wild banana (*left*) and transgenic golden banana fruits with higher levels of β-carotene (*right*) [75]. (B) Wild apple fruit (*left*) and transgenic fruit that accumulate higher levels of anthocyanins in the pulp (*right*) [82]. (C) Wild apple fruit which presents pulp oxidation (*left*) and arctic apple which has a higher resistance to oxidation of the pulp (*right*). (D) Wild orange fruit (*bottom*) and golden orange fruits with higher levels of β-carotene [78]. (E) Wild tomato fruits (*top*) and transgenic tomato fruits that accumulate higher levels of anthocyanins in the pulp [83].

#### **4.4. Functional fruits with improved folic acid content**

Folic acid, also known as vitamin B9, is part of the water-soluble vitamin B complex, which is necessary for the formation of structural proteins and hemoglobin [89]. Folate deficiency is considered as a worldwide problem because it is mainly caused by poor nutrition in poor countries. Folate deficiency during pregnancy causes premature births and babies with low weight and possible defects in neural tube development. In adults, the clearest sign of folate deficiency is anemia while children can also slow growth [89]. Folates are synthesized from pteridine, p-aminobenzoate (PABA) and glutamate precursor [90]. Diaz de la Garza et al. [90] developed transgenic tomatoes, which overexpressed the genes involved in the first steps in the biosynthesis of pteridine and PABA proteins: GTP cyclohydrolase I and amynodeoxychorismate synthase, respectively, in a fruit-specific manner [90]. The amount of folic acid contained in ripe tomato fruits was 25 times higher than in nontransgenic one, and the amount responds to the daily requirement for adult consumers with less than a standard serving.

## **5. Functional fruits as oral vaccines**

*Antirrhinum majus*, in transgenic tomato allowed the increase of anthocyanin content in the fruit [83]. It was observed that DEL and ROS1 transcription factors were able to activate multiple genes related to anthocyanin biosynthesis, such as phenylalanine ammonia lyase (PAL) and flavonoid 3′5′ hydroxylase (F3′5′H). Thus, transgenic tomatoes exhibit intense purple coloration in the pulp and fruit peel as shown in **Figure 1E**, because they contain high concentration of anthocyanin [86]. Several studies on the regulation of the biosynthetic pathway of anthocyanins showed that the family of MYB transcription factors regulates the expression of genes involved in this pathway [87]. Espley et al. reported that greater accumulation of anthocyanins in apple fruits (**Figure 1B**) was obtained by transforming with the transcription factor *MdMYB10* [82]. Moreover, the expression of *FaMYB10* in strawberry generated an increase in anthocyanins in

56 Superfood and Functional Food - The Development of Superfoods and Their Roles as Medicine

the root, leaf, and strawberry fruit [88].

**4.4. Functional fruits with improved folic acid content**

of anthocyanins in the pulp [83].

Folic acid, also known as vitamin B9, is part of the water-soluble vitamin B complex, which is necessary for the formation of structural proteins and hemoglobin [89]. Folate deficiency is considered as a worldwide problem because it is mainly caused by poor nutrition in poor

**Figure 1.** Examples of transgenic fruits. (A) Fruits of wild banana (*left*) and transgenic golden banana fruits with higher levels of β-carotene (*right*) [75]. (B) Wild apple fruit (*left*) and transgenic fruit that accumulate higher levels of anthocyanins in the pulp (*right*) [82]. (C) Wild apple fruit which presents pulp oxidation (*left*) and arctic apple which has a higher resistance to oxidation of the pulp (*right*). (D) Wild orange fruit (*bottom*) and golden orange fruits with higher levels of β-carotene [78]. (E) Wild tomato fruits (*top*) and transgenic tomato fruits that accumulate higher levels Metabolic engineering has been used not only to increase its content of healthy secondary metabolites but also for generating edible oral vaccines. The strategy of oral vaccines offers several advantages over the classical system of injection, as oral vaccine is a cheaper strategy to produce immunization and is a more practical system to be implemented in universal vaccination programs [91]. An example is the strategy to immunize against the enterovirus 71 (EV71), which is known to cause seasonal epidemics of hand, foot, and mouth and can reach fatal neurological complications in young children. Tomato plants expressing the VP1 epitope and the coat protein of the enterovirus 71 (EV71) were produced to induce immunization when eaten [92]. Tomato fruit–expressing VP1 protein was firstly tested in mice, which presented an increment of specific IgA and IgG immunoglobulins against VP1. Besides, the serum from mice fed with transgenic tomato was able to neutralize EV71 infection in rhabdomyosarcoma cell culture and the proliferation of spleen cells in orally immunized mice was also enhanced by VP1 tomatoes, which activated both humoral and cellular immunity. The results of this study not only demonstrated the feasibility of using transgenic tomato as an oral vaccine to generate protective immunity against EV71 in mice but also the likelihood of vaccine development against other kinds of enterovirus [92].

Another example of oral vaccine aims to immunize against the enterobacteria *Yersinia pestis*, which is a gram-negative bacilli, anaerobic facultative and pathogenic to humans that causes pneumonic and bubonic pest. This pathogen affects mainly people in Africa, Asia, and Latin America. Owing to the increasing reports of the emergence of antibiotic-resistant *Y. pestis* strains, the need for a safer and cheaper vaccination system increases. Among all *Y*. *pestis* antigens, only the F1 and V antigens have generated immunogenicity in conventional vaccines. Alvarez et al. reported the expression of a fusion protein F1-V in tomato plants. The immunogenicity of transgenic tomatoes F1-V was tested in mice, and the immune response of mice vaccinated with antigens of bacterial origin (conventional system) and oral transgenic vaccine was compared. The results showed a similar level of immunization with both strategies [93].

## **6. Enrichment of organoleptic properties in fruits**

Despite the best efforts in metabolic engineering of fruits which have been focused on increasing the nutritional value and defense against biotic and abiotic stress, this technology is also applied in handling the organoleptic properties of fruits such as color, texture, flavor, and aroma to get new varieties more attractive and pleasant to the consumer.

#### **6.1. Color improvement in fruits**

The color is a key feature of the quality of fruits and flowers and is often associated with carotenoids, flavonoids, and anthocyanins. As we described in the previous section (nutritional improvement), many fruits have been modified in their metabolism to increase these beneficial molecules for health, but most of them are also responsible for giving color to several organs of the plant. Taken the last example of the "super banana" and tomato with increased content in β-carotene, it is important to note that both fruits have an orange color in the pulp. On the other hand, fruits of tomato, apple, and strawberry modified for increasing anthocyanins accumulation have a more bluish (tomato) [83, 86] and red (apple and strawberry) [82, 88] pulp and peel (**Figure 1**). Thus, the organoleptic property of color can be modified by creating more striking and novel varieties.

#### **6.2. Sweetness increment in fruits**

Sweetness is one of the major determinants of the quality of fruits and generally depends on two factors, the composition and content of sugars. In plants, sugar also works as substrates in carbon metabolism and energy [94]. ADP-glucose pyrophosphorylase (AGPase), a key enzyme in the metabolic pathway from sucrose to starch, catalyzes the rate-limiting step of the biosynthesis of starch by generating the ADP glucose and inorganic pyrophosphate from glucose 1-phosphate and ATP. Transgenic plants of strawberry were developed by gene silencing using an antisense sequence for *FaGPS* gene, which codes for AGPase. A decrease in starch content and an increase in total soluble sugar content of 16–37% were obtained in transgenic fruits [95].

Currently, there are many alternative sweeteners that have been approved by the European Union regulators. Some of these are aspartame, saccharin, cyclamate, neohesperidin DC, acesulfame-K, and thaumatin. The first five compounds are low-molecular-weight molecules and are obtained by technology of traditional organic synthesis, although it should be noted that aspartame is an unnatural peptide. In contrast, thaumatin, is a natural protein that is produced normally in the *Thaumatococcus benth* plant, which is native to West Africa. In addition to thaumatin, there are several other sweet proteins in nature. Some of them have been isolated, purified, and characterized. Their genes have been cloned, and in some cases, recombinant versions of the natural protein have been obtained. Consequently, many of these sweet proteins can be used for the development of transgenic plants to enhance sweetness and fruit quality [96]. The tridimensional structure of thaumatin was determined at a high resolution and shows a marked homology with the PR-5 proteins (pathogenesis-related protein-5), which are involved in biotic stress defense [97, 98]. Despite their structural similarity, it has not been reported that any PR-5 proteins produce a sweet taste [99]. Thaumatin is about 100,000 times sweeter than sucrose on a molar basis (about 1600 times on a weight basis). Thus, the threshold sweetness value of thaumatin is about 50 nM [100]. The thaumatin II gene was expressed in cucumber plants. The transgenic fruits accumulating thaumatin II showed a sweet phenotype and a positive correlation between the levels of accumulation of thaumatin and the intensity of sweet taste [101]. In addition, the concentration of E,Z-2,6-nonadienal, which is the main molecular odorant in cucumber, was enhanced in the transgenic cucumber fruits. Thus, transgenic expression of the thaumatin II gene resulted not only in a sweeter taste of fruits compared to control but also in a greater aroma intensity [102]. Tomato, pear, and strawberry were also transformed with the thaumatin II gene, and they also showed a direct correlation between amount of protein expression and the increase in sweetness [103–105]. As a particular case, the strawberry expressing thaumatin II showed a significantly higher resistance to gray mold caused by *Botrytis cinerea* [105].

#### **6.3. Aroma as an important feature in fruits**

**6. Enrichment of organoleptic properties in fruits**

58 Superfood and Functional Food - The Development of Superfoods and Their Roles as Medicine

**6.1. Color improvement in fruits**

creating more striking and novel varieties.

**6.2. Sweetness increment in fruits**

transgenic fruits [95].

aroma to get new varieties more attractive and pleasant to the consumer.

Despite the best efforts in metabolic engineering of fruits which have been focused on increasing the nutritional value and defense against biotic and abiotic stress, this technology is also applied in handling the organoleptic properties of fruits such as color, texture, flavor, and

The color is a key feature of the quality of fruits and flowers and is often associated with carotenoids, flavonoids, and anthocyanins. As we described in the previous section (nutritional improvement), many fruits have been modified in their metabolism to increase these beneficial molecules for health, but most of them are also responsible for giving color to several organs of the plant. Taken the last example of the "super banana" and tomato with increased content in β-carotene, it is important to note that both fruits have an orange color in the pulp. On the other hand, fruits of tomato, apple, and strawberry modified for increasing anthocyanins accumulation have a more bluish (tomato) [83, 86] and red (apple and strawberry) [82, 88] pulp and peel (**Figure 1**). Thus, the organoleptic property of color can be modified by

Sweetness is one of the major determinants of the quality of fruits and generally depends on two factors, the composition and content of sugars. In plants, sugar also works as substrates in carbon metabolism and energy [94]. ADP-glucose pyrophosphorylase (AGPase), a key enzyme in the metabolic pathway from sucrose to starch, catalyzes the rate-limiting step of the biosynthesis of starch by generating the ADP glucose and inorganic pyrophosphate from glucose 1-phosphate and ATP. Transgenic plants of strawberry were developed by gene silencing using an antisense sequence for *FaGPS* gene, which codes for AGPase. A decrease in starch content and an increase in total soluble sugar content of 16–37% were obtained in

Currently, there are many alternative sweeteners that have been approved by the European Union regulators. Some of these are aspartame, saccharin, cyclamate, neohesperidin DC, acesulfame-K, and thaumatin. The first five compounds are low-molecular-weight molecules and are obtained by technology of traditional organic synthesis, although it should be noted that aspartame is an unnatural peptide. In contrast, thaumatin, is a natural protein that is produced normally in the *Thaumatococcus benth* plant, which is native to West Africa. In addition to thaumatin, there are several other sweet proteins in nature. Some of them have been isolated, purified, and characterized. Their genes have been cloned, and in some cases, recombinant versions of the natural protein have been obtained. Consequently, many of these sweet proteins can be used for the development of transgenic plants to enhance sweetness and fruit quality [96]. The tridimensional structure of thaumatin was determined at a high Aroma has an important influence at the moment of choosing foods. The threshold for human perception of a volatile molecule can be low as 0.007 μg/L in water [106]. Thus, both the unique combination of volatile compound and the specific proportions of each of the volatile components, determine the properties of flavor in fruits and other foods. Additionally, plant volatiles greatly influence pollination and fruit defense responses and are therefore critical for breeders. Thus, the aroma is presented as a complex mixture of a large number of volatile compounds, whose composition is species specific and often for the particular variety of a fruit [107]. Although different fruits share many aromatic characteristics, each fruit has a characteristic aroma, which depends on the combination of volatile compounds, concentration, and perception of volatiles. The most important aromatic compounds are derived from amino acids, lipids, phenols, and mono and sesquiterpenes [107]. Lewinsohn et al. showed that transgenic tomatoes that express the gene for S-linalool synthase (LIS), under the control of the fruit-specific promoter E8, are able to synthesize and accumulate linalool S-terpenoid and 8-hydroxylinalool compounds. No other phenotypic alterations were observed, including levels of other terpenoids such as γ- and α-tocopherols, lycopene, β-carotene, and lutein, and the results show that it is possible to improve levels of monoterpenes in fruit ripening by metabolic engineering [108]. Transgenic tomato with higher levels of other terpenoids was developed by overexpression of geraniol synthase (*GES*) gene from *Ocimum basilicum*, which catalyzed the synthesis of geraniol from geranyl diphosphate, under the direction of the polygalacturonase gene (PG) fruit-specific promoter, it was possible to increase the content of geraniol in tomato fruits, although pigments were decreased. This would indicate that geraniol accumulation occurs at the expense of the accumulation of lycopene probably because geranyl diphosphate is a common precursor for the synthesis of both metabolites. The aroma of transgenic tomatoes was stronger compared to nontransgenic ones. Aroma test was performed to clarify the matter by several panelists, showing that they preferred the aroma of transgenic tomatoes [109].

## **7. Improvement of the characteristics in post-harvest**

A critical decision for fruit growers is the time to harvest a crop. The time depends on factors such as the time required to reach the market and the management in route. Therefore, harvest is carried out when it has reached "harvest maturity" in any type of fresh fruits.

All fruits continue their metabolic processes after harvest. Therefore, the maintenance of the postharvest life is important to avoid that the product become inedible. In other words, many efforts are focused on preserve certain fruit traits, including nutritional value, processing qualities, flavor, and shelf life until the development of the ideal condition for consumption. Otherwise, the time lag of the postharvest life has the risk to expose serious losses in an evergrowing market. Indeed, the postharvest losses of fruits and vegetables, including roots and tubers, reach almost 50% of the production in developing countries, and the ratio of wastage is highest among food products [110, 111]. Also more than 40% of the food products losses occur at postharvest and processing levels in developing countries [112].

Within the postharvest life, fruit ripening occurs through physiological and biochemical reactions that alter visual appearance, flavor, aroma, texture, and fruit firmness [113, 114]. Therefore, many breeders and researchers have studied the complexity of fruit ripening and the development of engineered plants with high quality levels of fruit production in terms of flavor, color, and aroma.

#### **7.1. Inhibition of ripening**

The inhibition of fruit ripening has been achieved by reducing ethylene production [115]. Inhibition of ethylene production can be carried out by downregulating genes encoding key enzymes in the biosynthetic pathway of ethylene [116, 117] or by diverging the metabolic flux away from ethylene synthesis through the overexpression of enzymes degrading its immediate precursor, the 1-aminocyclopropane-1-carboxylic acid (ACC) [118, 119]. Even though most tomatoes have successfully lower levels of ethylene and an extended shelf life, most of them also compromised fruit-quality traits. An interesting work employed RNAi technology in which three homologs of 1-aminocyclopropane-1-carboxylate (ACC) synthase (ACS) gene were silenced during the course of ripening. Engineered fruits exhibited delayed ripening, prolonged shelf life for ~45 days, and improved juice quality. Indeed, total soluble solids (TSS) recorded in RNAi-ACS tomatoes increased up to ~40–45% compared to control [120]. In melon and papaya (*Carica papaya*), inhibition of fruit ripening and shelf life extension have also been achieved by silencing of genes coding for the enzymes or regulators of the ethylene biosynthesis pathway [121, 122].

Through Targeting Induced Local Lesions in Genomes (TILLING) approach [123–126], a melon mutant was isolated, which showed delayed fruit ripening and rind yellowing and an increase of fruit firmness and shelf life. The missense mutation G194D occurred in a highly conserved amino acid position of the ethylene biosynthetic enzyme, ACC oxidase 1 (CmACO1), and was predicted to affect the enzymatic activity of CmACO1.

By using RNAseq analysis, it was recently reported that the Polycomb-group (PcG) protein multicopy suppressor of IRA1 (MSI1) negatively regulates a large set of fruit-ripening genes along with the MADS-box protein RIN (ripening inhibitor) and its regulons. In fact, the genetic manipulation of SlMSI1 and RIN transcription factor successfully prolonged the fruit shelf life in tomato [127]. This may be an optimal approach to improve the post-harvest life of functional fruits by employing high-throughput techniques and addressing multiple metabolic pathways such as light-signaling pathways, which have modulatory components to adjust pigmentation during ripening and could be a selective advantage for primeval fleshy fruited plants [128].

## **7.2. Reduction of softening in ripe fruits**

**7. Improvement of the characteristics in post-harvest**

60 Superfood and Functional Food - The Development of Superfoods and Their Roles as Medicine

occur at postharvest and processing levels in developing countries [112].

flavor, color, and aroma.

**7.1. Inhibition of ripening**

biosynthesis pathway [121, 122].

A critical decision for fruit growers is the time to harvest a crop. The time depends on factors such as the time required to reach the market and the management in route. Therefore, har-

All fruits continue their metabolic processes after harvest. Therefore, the maintenance of the postharvest life is important to avoid that the product become inedible. In other words, many efforts are focused on preserve certain fruit traits, including nutritional value, processing qualities, flavor, and shelf life until the development of the ideal condition for consumption. Otherwise, the time lag of the postharvest life has the risk to expose serious losses in an evergrowing market. Indeed, the postharvest losses of fruits and vegetables, including roots and tubers, reach almost 50% of the production in developing countries, and the ratio of wastage is highest among food products [110, 111]. Also more than 40% of the food products losses

Within the postharvest life, fruit ripening occurs through physiological and biochemical reactions that alter visual appearance, flavor, aroma, texture, and fruit firmness [113, 114]. Therefore, many breeders and researchers have studied the complexity of fruit ripening and the development of engineered plants with high quality levels of fruit production in terms of

The inhibition of fruit ripening has been achieved by reducing ethylene production [115]. Inhibition of ethylene production can be carried out by downregulating genes encoding key enzymes in the biosynthetic pathway of ethylene [116, 117] or by diverging the metabolic flux away from ethylene synthesis through the overexpression of enzymes degrading its immediate precursor, the 1-aminocyclopropane-1-carboxylic acid (ACC) [118, 119]. Even though most tomatoes have successfully lower levels of ethylene and an extended shelf life, most of them also compromised fruit-quality traits. An interesting work employed RNAi technology in which three homologs of 1-aminocyclopropane-1-carboxylate (ACC) synthase (ACS) gene were silenced during the course of ripening. Engineered fruits exhibited delayed ripening, prolonged shelf life for ~45 days, and improved juice quality. Indeed, total soluble solids (TSS) recorded in RNAi-ACS tomatoes increased up to ~40–45% compared to control [120]. In melon and papaya (*Carica papaya*), inhibition of fruit ripening and shelf life extension have also been achieved by silencing of genes coding for the enzymes or regulators of the ethylene

Through Targeting Induced Local Lesions in Genomes (TILLING) approach [123–126], a melon mutant was isolated, which showed delayed fruit ripening and rind yellowing and an increase of fruit firmness and shelf life. The missense mutation G194D occurred in a highly conserved amino acid position of the ethylene biosynthetic enzyme, ACC oxidase 1

(CmACO1), and was predicted to affect the enzymatic activity of CmACO1.

vest is carried out when it has reached "harvest maturity" in any type of fresh fruits.

Reduction in fruit firmness due to softening that accompanies ripening plays a major role in determining the cost factor because exacerbates damage during handling and shipping processes having also a pivotal effect on shelf life, palatability, consumer acceptance, and postharvest resistance to pathogens [129–131]. The excessive softening causes losses around 35–40% of fruits and vegetables produced by India known as the second largest producer of these crops in the world [110]. It is suggested that the cell wall modifications are the major determinant of fruit softening induced as a consequence of the increased levels of cell wall–degrading enzymes [132]. Some strategies to control fruit softening include the manipulation of genes coding cell wall–degrading enzymes such as polygalacturonase or β-galactosidase [133–136]. This is the case of the first genetically modified tomato with reduced levels of expression of polygalacturonase gene through PTGS that was marketed as Flavr Savr™ to remark the potentially positive effect on the flavor [137, 138]. Although this strategy was developed to improve the flavor traits, delayed ripening with an expanded shelf life also occurred in the transgenic tomatoes by delaying cell wall softening.

In nonclimateric fruits such as strawberry and capsicum, efforts to control fruit ripening based on slowing down the rate of fruit softening has been successfully achieved by targeting genes involved in cell wall modification. In strawberry (*Fragaria* × *ananassa* Duch.), the down expression of pectate lyase or the fruit-specific polygalacturonase (FaPG1) genes by antisense technology resulted in extended fruit firmness and postharvest shelf life [139–143].

However, the other studies on the suppression of the expression of cell wall–degrading enzymes have not enough impact in prevent softening of genetically engineer fruits [133, 144]. This may be due to the redundant functionality of components taking part of a complex metabolic process [114, 145]. Therefore, the improvement of fruit shelf life constitutes a strong challenge for the identification of new targets to achieve this goal.

## **7.3. Browning reduction in fruits**

The postharvest storage and quality of fresh fruits are also affected by the enzymatic browning having negative effects on color, flavor, taste, and nutritional value. This reaction may be responsible for up to 50% of total losses of fruits and vegetables production [146]. Browning is triggered by the oxidation of phenolic compounds to quinones catalyzed by the polyphenol oxidase (PPO) enzyme [147, 148]. The subsequent nonenzymatic polymerization of the quinones results in the brown pigments formation that induces the postharvest deterioration [149]. This is particularly easy to appear in apples, which are highly susceptible to enzymatic browning and contain high levels of polyphenols [150, 151]. In order to reduce postharvest browning, the silencing of PPO gene in an apple was accomplished. The apples produced less PPO activity, and 50% of browning was inhibited compared to wild type control in golden delicious (GD) and granny Smith (GS) [152, 153]. Transgenic apples can keep the original color of the apple flesh when they are subjected to mechanical damage, such as bruising or slicing. This "nonbrowning" phenotype minimizes shrinkage caused by harvest and postharvest damage and also decreases the need for antibrowning compounds on cut fruit (**Figure 1C**). Similar approaches have been performed to reduce grape berry darkening [149], blackheart in pineapple [154], and the browning process in fruits of Yali pear [61].

## **8. Conclusion**

Metabolic engineering generally involves the redirection of cellular metabolism by modifying the expression of genes and enzymes affecting to the regulatory functions within the cell. For a successful metabolic engineering, rate-limiting step is the target for the increase of specific molecules or newly introduced molecules.

Traditional strategies for modifying gene expression such as using *A. tumefaciens* for overexpressing and silencing genes will be continue to be used but the strategies to manipulate the gene expression have been gradually refined. For instance, the selection markers have been removed, and only the transgene or mutagenic effect remains in the plant. In case of using CRISPR/Cas vectors, this methodology induces site-specific mutations and can produce thereby specific knock-out plants with the absence of external DNAs requirements. Since the modified plants do not contain a transgene, they are not included in the category of GMO.

Even more sophisticated metabolomic tools based on biochemical, genetic, environmental, and developmental parameters will offer the possibility to study the production of metabolites through the improvement of primary and secondary metabolic pathways in fruits. The increasing number of plant genomes sequenced, and the availability of many molecular markers can be used to track candidate genes that are associated with the desired trait and feature. However, traditional plant breeding together with genetic engineering provides greater opportunities to develop fruit crops with the desired amount and/or composition of specific metabolites. The most relevant characteristics for genetic improvement of plants that bear economic interest were discussed in this chapter. These characteristics included higher resistance to biotic and abiotic stress and improvement of pre-harvest and post-harvest features to face those problems that affect the vast cultivars and cause a high degree of economic losses. On the other hand, nutritional and organoleptic improvement of functional fruits are in direct benefit for end consumers.

Despite the benefits of metabolic engineering, the development and marketing of genetically modified fruit plants are hampered by many regulatory and social barriers. From the biosafety and consumers point of view, the presence of selectable marker genes, which are essential for the initial selection of transgenic plants, is undesirable. Therefore, the production of transgenic fruit plants without markers is now an essential requirement for commercial exploitation. The techniques such as RNAi in rootstocks for virus silencing, cisgenesis, or intragenesis show great potential and greater acceptance when generating genetically modified organisms. Additionally, selection marker free plants may improve the confidence and bring the benefits of genetically modified products to consumers.

## **Acknowledgements**

The Chilean Regular Fondecyt 1130245 and Fondef VIU 140049.

## **Author details**

is triggered by the oxidation of phenolic compounds to quinones catalyzed by the polyphenol oxidase (PPO) enzyme [147, 148]. The subsequent nonenzymatic polymerization of the quinones results in the brown pigments formation that induces the postharvest deterioration [149]. This is particularly easy to appear in apples, which are highly susceptible to enzymatic browning and contain high levels of polyphenols [150, 151]. In order to reduce postharvest browning, the silencing of PPO gene in an apple was accomplished. The apples produced less PPO activity, and 50% of browning was inhibited compared to wild type control in golden delicious (GD) and granny Smith (GS) [152, 153]. Transgenic apples can keep the original color of the apple flesh when they are subjected to mechanical damage, such as bruising or slicing. This "nonbrowning" phenotype minimizes shrinkage caused by harvest and postharvest damage and also decreases the need for antibrowning compounds on cut fruit (**Figure 1C**). Similar approaches have been performed to reduce grape berry darkening [149], blackheart

Metabolic engineering generally involves the redirection of cellular metabolism by modifying the expression of genes and enzymes affecting to the regulatory functions within the cell. For a successful metabolic engineering, rate-limiting step is the target for the increase of specific

Traditional strategies for modifying gene expression such as using *A. tumefaciens* for overexpressing and silencing genes will be continue to be used but the strategies to manipulate the gene expression have been gradually refined. For instance, the selection markers have been removed, and only the transgene or mutagenic effect remains in the plant. In case of using CRISPR/Cas vectors, this methodology induces site-specific mutations and can produce thereby specific knock-out plants with the absence of external DNAs requirements. Since the modified plants do not contain a transgene, they are not included in the category

Even more sophisticated metabolomic tools based on biochemical, genetic, environmental, and developmental parameters will offer the possibility to study the production of metabolites through the improvement of primary and secondary metabolic pathways in fruits. The increasing number of plant genomes sequenced, and the availability of many molecular markers can be used to track candidate genes that are associated with the desired trait and feature. However, traditional plant breeding together with genetic engineering provides greater opportunities to develop fruit crops with the desired amount and/or composition of specific metabolites. The most relevant characteristics for genetic improvement of plants that bear economic interest were discussed in this chapter. These characteristics included higher resistance to biotic and abiotic stress and improvement of pre-harvest and post-harvest features to face those problems that affect the vast cultivars and cause a high degree of economic losses. On the other hand, nutritional and organoleptic improvement of functional fruits are

in pineapple [154], and the browning process in fruits of Yali pear [61].

62 Superfood and Functional Food - The Development of Superfoods and Their Roles as Medicine

**8. Conclusion**

of GMO.

molecules or newly introduced molecules.

in direct benefit for end consumers.

Luis Quiroz-Iturra, Carolina Rosas-Saavedra and Claudia Stange Klein\*

\*Address all correspondence to: cstange@uchile.cl

Department of Biology, Faculty of Science, Centre of Plant Molecular Biology, University of Chile, Santiago, Chile

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## **Food Wastes as Valuable Sources of Bioactive Molecules Food Wastes as Valuable Sources of Bioactive Molecules**

Sonia A. Socaci, Anca C. Fărcaş, Dan C. Vodnar and Maria Tofană Maria Tofană Additional information is available at the end of the chapter

Sonia A. Socaci, Anca C. Fărcaş, Dan C. Vodnar and

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/66115

#### **Abstract**

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Food industry produces worldwide millions of tons of plant‐derived wastes which can be exploited as sources of high‐value components: proteins, fibres, polysaccharides, flavour compounds or different phytochemicals. These bioactive compounds can be valorised as functional ingredients in food, pharmaceutical, health care, cosmetic and other products. Using the recovered bioactive molecules as functional ingredients represents a sustainable alternative of food wastes exploitation as inexpensive source of valuable compounds, while developing innovative food and non‐food products with health‐promoting benefits and at the same time contributing to an efficient waste reduction management. This chapter gives an overview of the main classes of bioactive compounds recovered from food wastes and their potential applications as functional chemicals, without being exhaustive.

**Keywords:** bioactive compounds, functional ingredients, food waste exploitation, renewable resources, recovered biomolecules

## **1. Introduction**

Large amounts of wastes are generated annually by the food industry, their efficient management and valorisation representing one of the main objectives of European Union (EU) actions against food waste and towards sustainable development [1, 2]. The Waste Framework Directive [3] emphasised the importance of prevention of waste generation and the exploitation of wastes by reuse and recycling. Thus, in the 'bioeconomy' concept, the possibilities of conversion of renewable biological resources into economically viable products are addressed. In 2014, the European Commission provided the definition for the term 'food waste' as '*food (including inedible parts) lost from the food supply chain, not including food diverted to material uses such as bio‐based*

© 2017 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2017 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

*products, animal feed, or sent for re‐distribution'* [4]. The processing by‐products are also included among food waste, if these are not used for other high‐value functions (e.g. animal feed and industrial uses). In this chapter, we address only the exploitation of plant‐derived by‐products as sources of bioactive compounds.

Until few decades ago, food wastes, if not discarded into environment, were mainly used as animal feed. Nowadays, this attitude towards wastes changed, especially due to the growing interest in protecting the environment but also due to the increasing awareness of the benefits deriving from their exploitation. The by‐products resulted from the processing of raw vegetables contain sometimes appreciable amounts of bioactive compounds such as proteins, dietary fibres, polysaccharides, fatty acids, flavour compounds and phytochemicals (e.g. polyphenols) that can be extracted, purified, concentrated and reused as functional ingredients in food industry or other related sectors (e.g. pharmaceuticals, cosmetics and health‐care products) [5, 6].

## **2. Bioactive compounds recovered from plant‐derived wastes and their potential applications**

#### **2.1. General overview**

The wastes generated from the food industry can be separated into two main categories: plant‐ derived wastes and animal‐derived wastes. The animal‐derived wastes can be divided in three subcategories: (i) meat products, (ii) fish and seafood and (iii) dairy products, whereas the plant‐derived wastes can be classified into four subcategories: (i) cereals (e.g. rice bran, wheat bran and brewers' spent grain), (ii) root and tubers (e.g. potato peel, sugar beet and molasses), (iii) oil crops and pulses (e.g. sunflower seeds, soybean seed and olive pomace) and (iv) fruit and vegetables (e.g. orange peel, grape pomace, apple pomace, tomato skin and pomace) [5, 7]. We further focus only on the plant‐derived wastes chemical characterisation in terms of composition and content in functional compounds. The plant‐derived by‐products and especially those from fruits, vegetables and oil crops processing are generated in large amounts, some of them being produced in millions of tons annually worldwide [5, 8–10]. Disposal of such quantities of waste represents a challenge and an environmental problem. Apart from being used as animal feeds or fertilisers, the research conducted in the last decades clearly showed that the by‐products resulted from processing of plant materials contained valuable nutrients which could be exploited in the development and production of new functional ingredients [11–15].

There is a wide range of extraction techniques used for the isolation and purification of the bioactive compounds from plant‐derived wastes, some of them being based on new emerging techniques. The development of new extraction methods as well as the optimisation of existing ones, in order to increase, for example, the extraction yield or the selectivity for a certain compound, or to improve the production of a natural bioactive compound from a waste, has seen a real upsurge in the last decade [16]. Nevertheless, there is no universal extraction


*products, animal feed, or sent for re‐distribution'* [4]. The processing by‐products are also included among food waste, if these are not used for other high‐value functions (e.g. animal feed and industrial uses). In this chapter, we address only the exploitation of plant‐derived by‐products

76 Superfood and Functional Food - The Development of Superfoods and Their Roles as Medicine

Until few decades ago, food wastes, if not discarded into environment, were mainly used as animal feed. Nowadays, this attitude towards wastes changed, especially due to the growing interest in protecting the environment but also due to the increasing awareness of the benefits deriving from their exploitation. The by‐products resulted from the processing of raw vegetables contain sometimes appreciable amounts of bioactive compounds such as proteins, dietary fibres, polysaccharides, fatty acids, flavour compounds and phytochemicals (e.g. polyphenols) that can be extracted, purified, concentrated and reused as functional ingredients in food industry or other related sectors (e.g. pharmaceuticals, cosmetics and health‐care

**2. Bioactive compounds recovered from plant‐derived wastes and their**

The wastes generated from the food industry can be separated into two main categories: plant‐ derived wastes and animal‐derived wastes. The animal‐derived wastes can be divided in three subcategories: (i) meat products, (ii) fish and seafood and (iii) dairy products, whereas the plant‐derived wastes can be classified into four subcategories: (i) cereals (e.g. rice bran, wheat bran and brewers' spent grain), (ii) root and tubers (e.g. potato peel, sugar beet and molasses), (iii) oil crops and pulses (e.g. sunflower seeds, soybean seed and olive pomace) and (iv) fruit and vegetables (e.g. orange peel, grape pomace, apple pomace, tomato skin and pomace) [5, 7]. We further focus only on the plant‐derived wastes chemical characterisation in terms of composition and content in functional compounds. The plant‐derived by‐products and especially those from fruits, vegetables and oil crops processing are generated in large amounts, some of them being produced in millions of tons annually worldwide [5, 8–10]. Disposal of such quantities of waste represents a challenge and an environmental problem. Apart from being used as animal feeds or fertilisers, the research conducted in the last decades clearly showed that the by‐products resulted from processing of plant materials contained valuable nutrients which could be exploited in the development and production of new

There is a wide range of extraction techniques used for the isolation and purification of the bioactive compounds from plant‐derived wastes, some of them being based on new emerging techniques. The development of new extraction methods as well as the optimisation of existing ones, in order to increase, for example, the extraction yield or the selectivity for a certain compound, or to improve the production of a natural bioactive compound from a waste, has seen a real upsurge in the last decade [16]. Nevertheless, there is no universal extraction

as sources of bioactive compounds.

products) [5, 6].

**potential applications**

functional ingredients [11–15].

**2.1. General overview**

**Table 1.** Examples of bioactive compounds from plant‐derived wastes and the employed extraction techniques.

technique for the bioactive compounds. When an extraction technique is chosen, several criteria have to be considered, such as waste composition, aggregation state, homogeneity, and so on. Also, plant‐derived waste is prone to microbial degradation, so an appropriate way of preservation is necessary for its storage and further exploitation. One of the most common and economically feasible methods used for preservation is the drying of the waste and thus reducing the water content and lowering the microbiological activity [11].

In **Table 1**, examples of some of the most common extraction technique for the main classes of high‐value compounds and their sources are given.

## **2.2. Proteins**

Proteins are macronutrients with an important role in human nutrition, having high nutritional value. Nowadays, the consumers are more concerned about their health and are starting to realise the tight correlation between health and diet. The trend is towards vegetarianism, and thus finding new plant sources of protein is crucial for the food industry. For a by‐product to be considered as a source of protein, it has to fulfil major requirements: to have high protein content and this protein to be quality protein (well‐balanced essential amino acid composi‐ tion) [12]. Also, the allergic or toxic substances that may be present in the by‐product must be removed prior to its utilisation as source of protein.

The main wastes with a relatively high content of protein are the defatted meals obtained from oil industry, including sunflower, canola, rapeseed, but also palm and peanuts. The defatted by‐products generated from oil refineries (oil cake, stem and grain husk) are not only good sources of proteins but are also available in large quantities and at a low cost.

Sunflower proteins have been extensively evaluated as food ingredients. Sunflower seeds content in proteins ranges between 10% and 27.1% (dry weight (DW) basis), thus making the sunflower oil cake a good source of quality protein. The sunflower protein isolate's or concen‐ trate's characteristic is the relatively high content in phenolics, compounds that may alter the proteins' functional properties and their shelf life [49]. However, the current tendency is not to obtain protein isolates free of phenolics, but to keep these compounds into the isolates due to the antioxidant activity they exert. The protein concentrates containing different concen‐ trations of phenolics were studied and the results showed that they have high water solubility, moderate water‐holding capacity, emulsifying, foaming and gelation capacity similar to commercial isolates [21].

Another source of plant protein is the canola seeds. These seeds contain two main types of storage proteins: salt‐soluble (cruciferin) and water‐soluble (napin), the total protein content in the defatted canola meal being around 32% [24]. The concentration of proteins in canola protein isolates, when conventional direct alkaline extraction is used, ranged between 66% and 76% [23, 24], while using salt precipitation method may increase the concentration of proteins in isolates up to 93% [24]. There are new emerging non‐invasive methods, such as electro‐ activated solutions, that can be used for the extraction of proteins from canola meals with better extraction yields by solubilising the proteins without damaging their native conformations and maintaining their functional properties [25].

Rapeseed stem, the residual biomass remaining after the extraction of oil, represents roughly 30% of the plant and may also be considered to be used for proteins' recovery. The protein concentration in the rapeseed stem extract, using a green solvent (water) in an enhanced ultrasound extraction, was up to 0.03 g BSA/100 g DW. The ultrasound‐assisted extraction showed an increase in extractability and at the same offering the possibility of scaling up [20].

technique for the bioactive compounds. When an extraction technique is chosen, several criteria have to be considered, such as waste composition, aggregation state, homogeneity, and so on. Also, plant‐derived waste is prone to microbial degradation, so an appropriate way of preservation is necessary for its storage and further exploitation. One of the most common and economically feasible methods used for preservation is the drying of the waste and thus

In **Table 1**, examples of some of the most common extraction technique for the main classes of

Proteins are macronutrients with an important role in human nutrition, having high nutritional value. Nowadays, the consumers are more concerned about their health and are starting to realise the tight correlation between health and diet. The trend is towards vegetarianism, and thus finding new plant sources of protein is crucial for the food industry. For a by‐product to be considered as a source of protein, it has to fulfil major requirements: to have high protein content and this protein to be quality protein (well‐balanced essential amino acid composi‐ tion) [12]. Also, the allergic or toxic substances that may be present in the by‐product must be

The main wastes with a relatively high content of protein are the defatted meals obtained from oil industry, including sunflower, canola, rapeseed, but also palm and peanuts. The defatted by‐products generated from oil refineries (oil cake, stem and grain husk) are not only good

Sunflower proteins have been extensively evaluated as food ingredients. Sunflower seeds content in proteins ranges between 10% and 27.1% (dry weight (DW) basis), thus making the sunflower oil cake a good source of quality protein. The sunflower protein isolate's or concen‐ trate's characteristic is the relatively high content in phenolics, compounds that may alter the proteins' functional properties and their shelf life [49]. However, the current tendency is not to obtain protein isolates free of phenolics, but to keep these compounds into the isolates due to the antioxidant activity they exert. The protein concentrates containing different concen‐ trations of phenolics were studied and the results showed that they have high water solubility, moderate water‐holding capacity, emulsifying, foaming and gelation capacity similar to

Another source of plant protein is the canola seeds. These seeds contain two main types of storage proteins: salt‐soluble (cruciferin) and water‐soluble (napin), the total protein content in the defatted canola meal being around 32% [24]. The concentration of proteins in canola protein isolates, when conventional direct alkaline extraction is used, ranged between 66% and 76% [23, 24], while using salt precipitation method may increase the concentration of proteins in isolates up to 93% [24]. There are new emerging non‐invasive methods, such as electro‐ activated solutions, that can be used for the extraction of proteins from canola meals with better extraction yields by solubilising the proteins without damaging their native conformations

sources of proteins but are also available in large quantities and at a low cost.

reducing the water content and lowering the microbiological activity [11].

78 Superfood and Functional Food - The Development of Superfoods and Their Roles as Medicine

high‐value compounds and their sources are given.

removed prior to its utilisation as source of protein.

and maintaining their functional properties [25].

**2.2. Proteins**

commercial isolates [21].

Functional proteins can also be extracted from hazelnut cake (contains up to 54.4% proteins). The isolated hazelnut meal protein was found to exert good antioxidant activity (158–461 mmol Trolox/kg), iron chelation (60.7–126.7 mmol EDTA/kg), antiproliferative activity on colon cancer cells (IC50: 3.0–4.6 mg/ml) and good oil absorption (7.4–9.4 g/g) [22].

In the palm oil‐producing countries (e.g. Indonesia and Malaysia), the palm kernel cake is one of the main by‐products generated by food industry [26]. Palm kernel cake contains in average 15–21% crude protein, but it is deficient in lysine, methionine and tryptophan, and thus has a poor utility being usually used as feed for ruminants [50, 51]. Nevertheless, palm kernel cake is still a potential source of plant protein. The extracted protein isolates have a 68.50% protein concentration when alkaline extraction was used. Attempts in optimisation of extraction technology were carried out in order to transform the extracted protein into a bioactive plant protein (e.g. by enzymatic hydrolysis) by adding functional properties such as antioxidant function [26, 52].

Cereal origin wastes represent another potential source of bioactive molecules, including plant proteins. Brewers' spent grain is the main insoluble residue generated by the brewing industry. This by‐product results after the production of wort and it mainly consists in barley grain husks with minor fractions of pericarp and endosperm [53]. Its chemical composition is dependent on several intrinsic and extrinsic factors (barley cultivar, harvest time, type of malt used in the brewing process, mashing conditions, etc.) [54], but regardless of these factors it contains appreciable amounts of valuable compounds (proteins, lipids, carbohydrates, polyphenols and minerals) that remain unexploited in the brewing process. Brewers' spent grain has a high content (18–35.4%, w/w) [18, 55, 56] of quality protein, with lysine accounting for 14.3% of total protein content [55]. The extraction of protein from brewers' spent grain may be performed by classical alkaline extraction, but recently new integrated processes are developed for a more efficient exploitation of this by‐product. For example, simultaneous extraction of proteins and arabinoxylans by use of alkaline reagents directly from brewers' spent grain without any pre‐ treatment [18] has a great potential to be scaled up being an innovative environmental friendly process that allows the recycling of the reagents and at the same time saving 93% in costs [57]. The incorporation of chitosan into the brewers' spent grain protein had as result a composite film with antimicrobial and antioxidant activities which can be used in packaging materials for foods [58].

The apricot kernel press cake, the waste remaining after the oil extraction, contains 34.5% crude protein which may be valorised by as protein isolates. In this case, before the alkaline extraction of proteins, a pre‐step of detoxification is required in order to remove the HCN present in the kernel cake. The obtained isolates had a protein concentration of 68.8% and fairly good functional properties, especially water and oil absorption capacity and foaming properties [27]. The proteins recovered from plant‐derived wastes have several functional properties when incorporated in food products: emulsifying agents, film‐forming properties, flavour binding, viscosity increase by binding the water and gelation properties. The recovered proteins are successfully used for food fortification, especially in meat and milk products, infant formulae, bakery products and pasta products [20, 22, 27, 59].

#### **2.3. Polysaccharides**

Polysaccharides are widely distributed in nature, with about 99% being located in plants and vegetables, the representative ones including starch, cellulose, hemicelluloses, pectin and inulin [60]. These compounds are also referred to as dietary fibre and can be divided into two categories based on their water solubility [61]:


In plants, polysaccharides have important functional roles: maintaining the living cell struc‐ ture, and water binding or energy suppliers. These properties are exploited by the food industry and other related fields in the development of new food additives, functional ingredients or materials for bioactive molecules delivery and controlled release. Their suita‐ bility for pharmaceutical or medicinal uses is due to their innocuousness, biocompatibility, biodegradability and water solubility. Thus, there is an increasing and constant interest in finding new sources of plant‐derived polysaccharides—the bioagro‐waste streams being very promising in this sense [60, 62].

The fruit‐ and vegetable‐processing sector produces wastes (peels, pulp and seeds) that are rich, low cost and sustainable sources of polysaccharides. After isolation and purification, the recovered polysaccharides may have manifold applications.

Pectin is a polysaccharide with a heterogeneous structure that depends on the plant origin, the part of the plant where it is located (peels, pulp, seed, etc.) and how it is extracted. The 'building block' is the uronic acid residue link through α‐1‐4‐glycosidic bonds, forming a galacturonyl polymer backbone. The structural diversity of pectin provides a wide range of physico‐ chemical and functional properties (gelling, emulsifier, thickening agents, film‐forming, water‐ holding, prebiotic activities, etc.) essential for food industry. According to the Joint Food and Agriculture Organization/World Health Organization (FAO/WHO) Expert Committee on Food Additives and the European Commission, a pectic polysaccharide must have a content of minimum 65% in galacturonic acid [60–63]. Wastes such as orange peels or apple pomace are well‐known sources of pectins, but there are also other waste streams that can be exploited in this sense. The pectins from 26 vegetable wastes were characterised in a very complex study, in the framework of EU project NOSHAN, including orange peel, onion hulls, parsley, endive roots and leaves, leek leaves, fresh cabbage, pea pod, sugar beet flakes, berries, apple pomace, sea buckthorn pulp, hop, olive pomace, tomato skin, grape pomace, whole pear and shabal. The results showed that the structure of the pectin extracted from wastes is similar to that from the raw matrices, although the methylation and acetylation degrees are lower due to the processing and/or enzymatic actions. The collected data also emphasise the potential of the recovered pectin to be used either as food additives or other applications (if the minimum concentration in galacturonic acid is not reached) [63].

The most important sources of soluble dietary fibres are the wastes derived from citrus fruits processing. The pectin content differs considerably among citrus varieties, but it generally ranges between 20% and 30% of citrus peel dry weight. Cellulose and hemicellulose can also be recovered from citrus waste as it comprises approximately 50–60% of citrus peel weight. The dietary fibres are not only present in high amount in citrus peels but also have important features due to the presence of associated bioactive constituents (flavonoids and vitamin C) with antioxidant properties, which may provide additional health‐promoting effects [64, 65]. For example, the pectin extracted from citrus peel and apple pomace by subcritical water extraction (with maximum yields of 22 and 17%, respectively) showed a high antioxidative and anti‐tumour activity [30]. Soluble dietary fibres also reduce the intestinal absorption of blood cholesterol, whereas insoluble dietary fibre associates to water absorption and intestinal regulation apart from the well‐known probiotic and health benefits [66].

As previously mentioned, brewers' spent grain besides being a source of quality plant protein is also a good source of carbohydrates, their level being up to 50% of the by‐product weight [28]. The main carbohydrates in brewers' spent grain are cellulose (∼17% dw) [13, 18, 32] and hemicelluloses, mainly arabinoxylan (25–28% dw) [13, 18]. The vegetable matrices being rich in hemicelluloses can be hydrolysed (e.g. with diluted acid) in order to release the monosac‐ charides (xylose and arabinose) which can be further subjected to a fermentation process to generate valuable products (e.g. xylitol, a sweetener used in food industry) [29]. Arabinoxylans are considered dietary fibres with a broad range of potential uses as functional ingredients in food products. Their extraction from brewers' spent grain may be performed under strong alkali conditions and also by using an innovative fully integrated process that sequentially extracts the proteins and arabinoxylans [18].

## **2.4. Phenolics**

The proteins recovered from plant‐derived wastes have several functional properties when incorporated in food products: emulsifying agents, film‐forming properties, flavour binding, viscosity increase by binding the water and gelation properties. The recovered proteins are successfully used for food fortification, especially in meat and milk products, infant formulae,

80 Superfood and Functional Food - The Development of Superfoods and Their Roles as Medicine

Polysaccharides are widely distributed in nature, with about 99% being located in plants and vegetables, the representative ones including starch, cellulose, hemicelluloses, pectin and inulin [60]. These compounds are also referred to as dietary fibre and can be divided into two

**1.** insoluble dietary fibre—are insoluble in water and resistant to hydrolysis by digestive tract enzymes (cellulose, hemicelluloses, lignin—non‐carbohydrate compounds); **2.** soluble dietary fibres are soluble in water and well fermented by digestive tract enzymes

In plants, polysaccharides have important functional roles: maintaining the living cell struc‐ ture, and water binding or energy suppliers. These properties are exploited by the food industry and other related fields in the development of new food additives, functional ingredients or materials for bioactive molecules delivery and controlled release. Their suita‐ bility for pharmaceutical or medicinal uses is due to their innocuousness, biocompatibility, biodegradability and water solubility. Thus, there is an increasing and constant interest in finding new sources of plant‐derived polysaccharides—the bioagro‐waste streams being very

The fruit‐ and vegetable‐processing sector produces wastes (peels, pulp and seeds) that are rich, low cost and sustainable sources of polysaccharides. After isolation and purification, the

Pectin is a polysaccharide with a heterogeneous structure that depends on the plant origin, the part of the plant where it is located (peels, pulp, seed, etc.) and how it is extracted. The 'building block' is the uronic acid residue link through α‐1‐4‐glycosidic bonds, forming a galacturonyl polymer backbone. The structural diversity of pectin provides a wide range of physico‐ chemical and functional properties (gelling, emulsifier, thickening agents, film‐forming, water‐ holding, prebiotic activities, etc.) essential for food industry. According to the Joint Food and Agriculture Organization/World Health Organization (FAO/WHO) Expert Committee on Food Additives and the European Commission, a pectic polysaccharide must have a content of minimum 65% in galacturonic acid [60–63]. Wastes such as orange peels or apple pomace are well‐known sources of pectins, but there are also other waste streams that can be exploited in this sense. The pectins from 26 vegetable wastes were characterised in a very complex study, in the framework of EU project NOSHAN, including orange peel, onion hulls, parsley, endive roots and leaves, leek leaves, fresh cabbage, pea pod, sugar beet flakes, berries, apple pomace, sea buckthorn pulp, hop, olive pomace, tomato skin, grape pomace, whole pear and shabal. The results showed that the structure of the pectin extracted from wastes is similar to that from

bakery products and pasta products [20, 22, 27, 59].

categories based on their water solubility [61]:

(pectin, inulin, gums and mucilages).

recovered polysaccharides may have manifold applications.

promising in this sense [60, 62].

**2.3. Polysaccharides**

Phenolics are among the most studied phytochemicals in the last decades. The interest showed by the scientific community in finding new and unconventional sources of phenolic com‐ pounds is due to the many studies that suggested that there is an association between the consumption of diets rich in phenolic compounds and a reduced risk of cardiovascular and neurodegenerative diseases [37, 66, 67]. Also, the recovery of phenolic compounds from food processing by‐products and their use as functional ingredients sustain the increasing efforts for a sustainable food production.

During fruit processing, the beverage industry leaves between 25 and 35% mass of the raw material called fruit pomace. Unfortunately, some part of pomace in the fruit industry still goes to landfill, and causes environmental pollution and huge losses of valuable materials which could be exploited as a great variety of natural additives and many health‐promoting ingre‐ dients (phenolic compounds, vitamins, carotenoids and dietary fibre) [68, 69]. Phenolic compounds of different plant sources such as grape and apple pomace are known as potent antioxidants and radical scavengers. The wine‐making industries produce millions of tons of residues (grape pomace), which represents a management issue from both ecological and economical point of view [70]. Grape pomace is a phenolic‐rich dietary fibre matrix that combines the benefits of both fibre and antioxidants in the prevention of cancer and cardio‐ vascular diseases [66]. Moreover, the grape seeds are considered to be a disposable waste material by the majority of wineries. They are usually discarded, burned or used as animal feed [45]. The oil extracted from the grape seed offers a wide range of benefits for human health, due to its high content of unsaturated fatty acids and antioxidant compounds such as mono‐ meric flavan‐3‐ols, phenolic acids and oligomeric proanthocyanidins, which is the reason why the valorisation of this by‐product is of great interest. Crude grape seed oil consists mainly of linoleic and oleic unsaturated fatty acids and also of palmitic and stearic saturated fatty acids [33, 34]. A study regarding the chemical characterisation of the grape seed extracts obtained by supercritical CO2 extraction showed that their content in trans‐resveratrol was similar to the contents reported in the literature for red wines. This demonstrates that a considerable amount of trans‐resveratrol remains unexploited in grape seeds after the fermentation process [33]. An alternative of reuse of grape seeds is as flour incorporated in food products. For example, formulations of frankfurters with grape seed flour showed a decrease in oxidation processes (due to the strong antioxidant activity of the flour), increased total dietary fibre content and water‐holding capacity of the final product [59], while the addition of apple pomace extract in meat products reduces the number of synthetic antioxidants needed to be added, and increases the health‐promoting properties of the finished product [68].

Besides being a serious environmental problem, olive by‐products can also represent a precious resource of potentially valuable molecules. It is worth mentioning that 98% of olive fruit phenols are lost during oil extraction. These compounds are distributed between the olive mill wastewaters (OMWs) phase (approximately 53%) and the solid phase—the 'pomace' (approximately 45%). Consequently, only a 2% fraction of the phenolic classes remains the oil phase depending on the extraction system and olive variety [71]. The evidence relating to decreased prevalence of chronic heart diseases, atherosclerosis or other diseases caused by oxidative stress, through a Mediterranean diet, has oriented scientific research towards the best use of olive‐processing by‐products (olive leaves and olive mill wastewaters) in order to produce purified natural antioxidants or high antioxidant‐rich preparations that could be incorporated in foods, cosmetics and pharmaceuticals [37, 67]. The studies on chemical constituents of olive leaves revealed that phenolic compounds stand out as predominant micronutrients, hydroxytyrosol and oleuropein considered as majority [72]. For example, the hydroxytyrosol‐rich olive leaf extract had an inhibitory activity against breast cancer cell proliferation [37]. Also, phenolic‐rich extract from OMW and hydroxytyrosol and oleuropein extracts from olive leaves had very pronounced hypocholesterolaemic effects, hypoglycaemic effect, protective action against lipid peroxidation and enhanced antioxidant defence system [73, 74].

Sunflower seeds contain high amounts of polyphenols such as caffeoylquinic and caffeic acids, accounting up to 4% dw. Among all, 5‐*O*‐caffeoylquinic acid (chlorogenic acid) is the predom‐ inant compound. To achieve sustainability of sunflower processing and complete utilisation of by‐products arising from sunflower oil production, polyphenols co‐extracted during sunflower protein recovery from the expeller were recovered by adsorption technology. In addition, an integrated process was optimised in order to enhance the recovery of polyphe‐ nolics as by‐products of protein production from sunflower press cake [38, 75].

Other unconventional source of phenolic compounds is the potato peels. Phenolic acids are the most abundant phenolic compounds in potatoes peels, the main representative being the chlorogenic acid (up to 95–98% of phenolic compounds) [39, 76]. It is present in the form of three main isomers: chlorogenic acid (5‐*O*‐caffeoylquinic acid), neochlorogenic acid (3‐*O*‐ caffeoylquinic acid) and cryptochlorogenic acid (4‐*O*‐caffeoylquinic acid) [76]. Its extraction from potato peels may be performed by conventional solvent extraction [40], ultrasound‐ assisted extraction [41] or using an optimised solvent extraction using pressurised liquid extractor [39]. The optimisation of an extraction method is a crucial step for researchers to accurately quantify the content in phenolic compounds and also to be able to estimate their potential health benefits when incorporated in food as functional ingredients. The extracted quantity of phenolic acids from potato peels depends not only on the method parameters but also on genetic factors. While the total phenolics content varies between cultivars and geo‐ graphical regions, the most abounding isomer of chlorogenic acid was in all cases the 5‐*O*‐ caffeoylquinic acid [39, 40].

## **2.5. Carotenoids**

antioxidants and radical scavengers. The wine‐making industries produce millions of tons of residues (grape pomace), which represents a management issue from both ecological and economical point of view [70]. Grape pomace is a phenolic‐rich dietary fibre matrix that combines the benefits of both fibre and antioxidants in the prevention of cancer and cardio‐ vascular diseases [66]. Moreover, the grape seeds are considered to be a disposable waste material by the majority of wineries. They are usually discarded, burned or used as animal feed [45]. The oil extracted from the grape seed offers a wide range of benefits for human health, due to its high content of unsaturated fatty acids and antioxidant compounds such as mono‐ meric flavan‐3‐ols, phenolic acids and oligomeric proanthocyanidins, which is the reason why the valorisation of this by‐product is of great interest. Crude grape seed oil consists mainly of linoleic and oleic unsaturated fatty acids and also of palmitic and stearic saturated fatty acids [33, 34]. A study regarding the chemical characterisation of the grape seed extracts obtained by supercritical CO2 extraction showed that their content in trans‐resveratrol was similar to the contents reported in the literature for red wines. This demonstrates that a considerable amount of trans‐resveratrol remains unexploited in grape seeds after the fermentation process [33]. An alternative of reuse of grape seeds is as flour incorporated in food products. For example, formulations of frankfurters with grape seed flour showed a decrease in oxidation processes (due to the strong antioxidant activity of the flour), increased total dietary fibre content and water‐holding capacity of the final product [59], while the addition of apple pomace extract in meat products reduces the number of synthetic antioxidants needed to be

82 Superfood and Functional Food - The Development of Superfoods and Their Roles as Medicine

added, and increases the health‐promoting properties of the finished product [68].

[73, 74].

Besides being a serious environmental problem, olive by‐products can also represent a precious resource of potentially valuable molecules. It is worth mentioning that 98% of olive fruit phenols are lost during oil extraction. These compounds are distributed between the olive mill wastewaters (OMWs) phase (approximately 53%) and the solid phase—the 'pomace' (approximately 45%). Consequently, only a 2% fraction of the phenolic classes remains the oil phase depending on the extraction system and olive variety [71]. The evidence relating to decreased prevalence of chronic heart diseases, atherosclerosis or other diseases caused by oxidative stress, through a Mediterranean diet, has oriented scientific research towards the best use of olive‐processing by‐products (olive leaves and olive mill wastewaters) in order to produce purified natural antioxidants or high antioxidant‐rich preparations that could be incorporated in foods, cosmetics and pharmaceuticals [37, 67]. The studies on chemical constituents of olive leaves revealed that phenolic compounds stand out as predominant micronutrients, hydroxytyrosol and oleuropein considered as majority [72]. For example, the hydroxytyrosol‐rich olive leaf extract had an inhibitory activity against breast cancer cell proliferation [37]. Also, phenolic‐rich extract from OMW and hydroxytyrosol and oleuropein extracts from olive leaves had very pronounced hypocholesterolaemic effects, hypoglycaemic effect, protective action against lipid peroxidation and enhanced antioxidant defence system

Sunflower seeds contain high amounts of polyphenols such as caffeoylquinic and caffeic acids, accounting up to 4% dw. Among all, 5‐*O*‐caffeoylquinic acid (chlorogenic acid) is the predom‐ inant compound. To achieve sustainability of sunflower processing and complete utilisation Carotenoid compounds are known for their health‐promoting effects, especially due to their high free radical‐scavenging activity. Being powerful antioxidants, when ingested they protect the human body from the damaging actions of the reactive oxygen species and thus lowering the risk of several chronic diseases (cardiovascular diseases, diabetes and cancer). They are fat‐ soluble pigments which are responsible for the bright‐yellow colour of many fruits and vegetables [77].

Lycopene is the main carotenoid found in tomatoes. Some studies suggested that a direct correlation may be established between the consumption of foods rich in lycopene and a low risk of prostate cancer [78].

Tomato (*Solanum lycopersicum* L.) is the second‐most consumed vegetable in the world [79]. The solid by‐products resulted from its processing into food products such as tomato juice, paste, puree, ketchup and sauce reaching up to 50,000 tons per year [16]. Their exploitation as a source of carotenoids (mainly lycopene) may provide economic benefits. Several techniques are used for the extraction of lycopene from tomato by‐products of which enzymatic‐assisted process is a promising one. When enzymatic method is used, the tomato by‐products are pre‐ treated by crude enzyme extracts with pectinolytic, cellulolytic and cutinolytic activities prior to their conventional solvent extraction. The results showed an enhancement in the extraction of lycopene from tomato by‐products (2.7 mg/100 g) and also a higher overall antioxidant activity for the enzymatic extract (even higher than that of BHA) compared to the one obtained by conventional ethanol extraction [16].

In general, bioaccessibility of carotenoids is low. However, in some fruits, such as mango and papaya, they are present in oil droplet in an esterified form with fatty acids. This kind of structure enhances their extraction and bioavailability during digestion [80]. Poor postharvest technology is one of the major inconveniences in mango annual production, accounting for nearly 60–80% of losses. Therefore, processing mango into flour represents a viable alternative for its use as a functional ingredient and to reduce wastage. The carotenoid content of mango flours ranged from 56.46 to 160.64 μg/g and was found to be higher in ripe mango flours than in green mango flours. In addition, the flour processed from the mango peel has been found to contain significant superior qualities than that from mango pulp in terms of total phenolic, anthocyanins, flavonoids and vitamin C contents and antioxidant activities [81].

Citrus waste is voluminous, heterogeneous, chemically complex and highly biodegradable; therefore, it cannot be disposed of in a landfill without a previous valorisation, in order to avoid both economic loss and environmental pollution issues. About 40–50% of the quantity of this fruit is processed for juice and marmalade production and approximately 50–60% w/w of the processed fruit becomes waste. This by‐product contains a wide range of bioactive compounds, such as essential oils, carotenoids, fibre, hesperidin and limonin, which have many applications in food, cosmetic and pharmaceutical industry. After the production of orange juice, the remaining outer layer called flavedo contains considerable amounts of the natural carotenoids. These bioactive compounds comprise approximately 0.1–0.5% of citrus peel dry weight. The major carotenoids available in citrus are α‐ and β‐carotene, lutein, zeaxanthin and β‐cryptoxanthin, which are known to be responsible for a wide range of functional properties, mainly offering protection against the reactive oxygen species damaging actions at the cellular level [64, 82–84].

#### **2.6. Other compounds**

The wastes from fruits and vegetables can be exploited by microbial processing in order to obtain valuable enzymes such as amylolytic enzymes from banana waste, mango kernels; pectinolytic enzymes from orange peel, lemon peel; tannase from grape seeds; protease from mango peel, potato peel; lipase from coconut cake, lemon peel; and invertase from orange peel, banana peel. The microbial treatment can also be used for the production of organic acids, including lactic acid, citric acid, succinic acid and acetic acid from wastes of potatoes, banana, mango, apple, pineapple and many others [85]. These valuable chemicals can be further exploited as raw materials for other processes or as functional ingredients for newly developed food products and so on [86]. Another example of valuable products recovered from fruit wastes, more exactly, from citrus fruits peels (orange, mandarin, lime, lemons, etc.), is the essential oils. Citrus essential oils extracted from the peels discarded after the fruits processing can be valorised: as flavouring agents in different food products (e.g. soft drinks and confec‐ tioneries), perfumes, personal care products, household products; in food preservation enhancing the product's shelf life due to their antioxidant and antimicrobial properties, and thus representing an attractive alternative to synthetic antioxidants and preservatives; and as functional chemicals in agriculture as insects repellent and other more uses [48, 87–89].

## **3. Conclusion**

In general, bioaccessibility of carotenoids is low. However, in some fruits, such as mango and papaya, they are present in oil droplet in an esterified form with fatty acids. This kind of structure enhances their extraction and bioavailability during digestion [80]. Poor postharvest technology is one of the major inconveniences in mango annual production, accounting for nearly 60–80% of losses. Therefore, processing mango into flour represents a viable alternative for its use as a functional ingredient and to reduce wastage. The carotenoid content of mango flours ranged from 56.46 to 160.64 μg/g and was found to be higher in ripe mango flours than in green mango flours. In addition, the flour processed from the mango peel has been found to contain significant superior qualities than that from mango pulp in terms of total phenolic,

Citrus waste is voluminous, heterogeneous, chemically complex and highly biodegradable; therefore, it cannot be disposed of in a landfill without a previous valorisation, in order to avoid both economic loss and environmental pollution issues. About 40–50% of the quantity of this fruit is processed for juice and marmalade production and approximately 50–60% w/w of the processed fruit becomes waste. This by‐product contains a wide range of bioactive compounds, such as essential oils, carotenoids, fibre, hesperidin and limonin, which have many applications in food, cosmetic and pharmaceutical industry. After the production of orange juice, the remaining outer layer called flavedo contains considerable amounts of the natural carotenoids. These bioactive compounds comprise approximately 0.1–0.5% of citrus peel dry weight. The major carotenoids available in citrus are α‐ and β‐carotene, lutein, zeaxanthin and β‐cryptoxanthin, which are known to be responsible for a wide range of functional properties, mainly offering protection against the reactive oxygen species damaging

The wastes from fruits and vegetables can be exploited by microbial processing in order to obtain valuable enzymes such as amylolytic enzymes from banana waste, mango kernels; pectinolytic enzymes from orange peel, lemon peel; tannase from grape seeds; protease from mango peel, potato peel; lipase from coconut cake, lemon peel; and invertase from orange peel, banana peel. The microbial treatment can also be used for the production of organic acids, including lactic acid, citric acid, succinic acid and acetic acid from wastes of potatoes, banana, mango, apple, pineapple and many others [85]. These valuable chemicals can be further exploited as raw materials for other processes or as functional ingredients for newly developed food products and so on [86]. Another example of valuable products recovered from fruit wastes, more exactly, from citrus fruits peels (orange, mandarin, lime, lemons, etc.), is the essential oils. Citrus essential oils extracted from the peels discarded after the fruits processing can be valorised: as flavouring agents in different food products (e.g. soft drinks and confec‐ tioneries), perfumes, personal care products, household products; in food preservation enhancing the product's shelf life due to their antioxidant and antimicrobial properties, and thus representing an attractive alternative to synthetic antioxidants and preservatives; and as functional chemicals in agriculture as insects repellent and other more uses [48, 87–89].

anthocyanins, flavonoids and vitamin C contents and antioxidant activities [81].

84 Superfood and Functional Food - The Development of Superfoods and Their Roles as Medicine

actions at the cellular level [64, 82–84].

**2.6. Other compounds**

Food wastes are renewable resources of high‐value extractable or convertible chemicals which can be exploited for the development of new functional ingredients, respectively, for the generation of bio‐fuels. The scientific research is focused on finding new ways of valorisation of food industry by‐products by identifying or optimising the most appropriate extraction methods for the recovery of the biomolecules, as well as by strengthening the cooperation with food industry partners in implementing adequate solutions for a sustainable development and increased competitiveness.

The 'zero‐waste' desiderate can be reached by reusing the high‐value compounds from by‐ products in innovative and unconventional ways which may generate profits in a sustainable food production system. The recovered biomolecules are also of great interest for pharma‐ ceutical industry (e.g. carrier agents and controlled release), cosmetics, agriculture, chemical industry and so on.

## **Acknowledgements**

This work was supported by a grant of the Romanian National Authority for Scientific Research, CNCS–UEFISCDI, project number PN‐II‐RU‐TE‐2014‐4‐0842. Authors S.A. Socaci and A.C. Fărcaş contributed equally to this work.

## **Author details**

Sonia A. Socaci\* , Anca C. Fărcaş, Dan C. Vodnar and Maria Tofană

\*Address all correspondence to: sonia.socaci@usamvcluj.ro

University of Agricultural Sciences and Veterinary Medicine, Cluj‐Napoca, Romania

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## **Selected Superfoods and Their Derived Superdiets Selected Superfoods and Their Derived Superdiets**

Beatrice Nakhauka Ekesa Beatrice Nakhauka Ekesa

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

http://dx.doi.org/10.5772/67239

#### **Abstract**

Despite the reported decline in undernourishment in developing regions from 23.3% to 12.9% within 25 years, sub-Saharan Africa is the most malnourished region in the world, and the situation could get even worse depending on how the continent's love affairs with the few popular foods play out. The irony is that there are millions of nutrient-rich edible plants, insects and animals within tropical Africa, but due to modernization, only 3% of these foods are utilized within diets. Through a comprehensive literature review, this chapter will explore eight of the most feasible superfoods with an objective of using a systems approach to further look into their derived superdiets. Superfoods are naturally occurring plant or animal-based foods dense in nutrients, antioxidants and healthy fats, whilst superdiets are defined as feasible dishes prepared based on selected superfoods, incorporating other food ingredients and using appropriate processing and cooking techniques. The selected superfoods will include amaranth, teff, fonio, moringa leaves, baobab fruit, tamarind and hibiscus leaves. With the dense vitamins, minerals, healthy fats and antioxidants, these superfoods and more importantly their derived dishes have great potential in boosting the immune system, reducing risk of chronic diseases and promoting a healthy and productive population.

**Keywords:** superfoods, superdiets, moringa, hibiscus, tamarind, amaranth, teff, fonio, baobab

## **1. Introduction**

The number of hungry people in the world has dropped to 795 million from 1 billion in 1990/1992 to the latest state of food insecurity in the world as reported in 2015 [1]. In addition, in the developing regions, the prevalence of undernourishment declined to 12.9% of the population, down from 23.3% a quarter century ago [1]. Despite the progress, sub-Saharan Africa is the region with the highest prevalence of undernourishment in the world, and the

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

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, © 2017 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

situation could get better—or worse—depending on how the continent's love affairs with some of its increasingly popular foods play out [2]. The irony is that there are millions of edible plants, insects and animals, and just like the Amazon, tropical Africa is still hiding most of the food items considered to be superfoods. Although superfood is a term originally used just as an advertisement and marketing tool, a superfood can be defined as a nutrientdense, antioxidant-rich, natural-food product that is minimally processed and bioavailable in numerous, potent nutritive constituents. Consumption of superfoods increases energy and vitality, regulates cholesterol and blood pressure and may help to prevent or fight cancer and other diseases [3]. Superfoods are generally beneficial for health and well-being. Basing on the above definitions, there is a big range of foods considered to be superfoods, but for this chapter, focus will be on those mainly available within Africa and with higher potential of integration within existing food and diet systems. The objective is to downplay on the 'superfood' and emphasize more on 'super diets', where the emphasis is on a healthy balanced diet. The foods that will be explored include moringa leaves, hibiscus, amaranth, baobab fruit, tamarind, teff and fonio.

## **2. Selected superfoods**

#### **2.1. Amaranth**

Botanically referred to as *Amaranthus*, this crop was cultivated by the Aztecs 8000 years ago and is still a native crop in Peru. The ancient history of amaranth can be traced to Mexico and the Yucatan Peninsula. The name for amaranth comes from a Greek word *amarantos* meaning 'one that does not wither' or 'never fades'; this is true as amaranth's bushy flowers retain their vibrancy even after harvesting and drying. In addition, some varieties of ornamental amaranth do not produce the fancy flowers but produce flashy foliage, sprouting leaves [4]. *Amaranthus* are now grown in Africa, India, China, Russia, throughout South America and North America. Amaranth is tall about 6 feet, has broad leaves with colours ranging from deep blood red to light green with purple veins and has around 60 different species [4], several of which are cultivated as leaf vegetables, grains or ornamental plants [5]. It is commonly known as pigweed (English), hanekam (Afrikaans), thepe (Sesotho), imbuya (isiZulu), mchicha (Swahili), terere (Gikuyu, Meru and Embu of Kenya), doodo (Luganda), shoko (Yoruba) [5] and lengalenga (Democratic Republic of Congo and Burundi) [6]. Both the amaranth leaves and seeds are useful in terms of human health [5]. Whether you choose to consume amaranth as a leaf vegetable, a cereal grain or grain flour, considering the versatility and high concentration of antioxidants and nutrients, amaranth is one of the most of valuable health foods that you may have never heard of [5].

#### *2.1.1. Vegetable/leafy amaranth*

Vegetable amaranths are probably the most widely eaten boiled or steamed greens throughout Africa's humid lowlands. They secure the food supply for millions. The leaves and stems make excellent boiled or steamed vegetables as stew or sauce; they have a soft texture, mild flavour and no trace of bitterness [7]. As already indicated, raw vegetables have higher nutrient levels than cooked vegetables, but it is also obvious that not all vegetables can be consumed raw. Amaranth leaves are one of those vegetables that have to be cooked. It is therefore important that when cooking the amaranth, the cooking time should not exceed 5 min, and in the case water is used, it should be used in minimal quantities and not discarded as most nutrients leach into the water. If the amaranth is being cooked together with other food items that require longer cooking time such as legumes and meats, it should be added to the food just a few minutes before the meal is ready. Therefore, following appropriate cooking methods, amaranth leaves have great nutrition value.

Cooked amaranth leaves are packed with antioxidants and an excellent source of several nutrients especially vitamins and minerals [8]. According to the FAO West African food composition table, 100 g of boiled leafy amaranth contains 4.6 g protein, 380 mg calcium, 4.9 mg iron, 58 mg magnesium, 54 mg potassium, 42 mcg folate, 19 mg vitamin C, 0.25 mg vitamin E and 228 mcg RAE of vitamin A [9]. There are very few leafy vegetables with high levels of calcium, and therefore amaranth is an absolute superfood in terms of boosting bone strength and preventing osteoporosis, thus extending your 'active life' well into old age. The significant level of carotenoids and vitamin A in amaranth leaves is a major boost for eye health, as these antioxidants can prevent macular degeneration and slow or stop the development of cataracts [10]. By lowering oxidative stress in the ocular system, amaranth keeps your vision healthy and strong. In addition, vitamin A plays a major role in boosting the immune system, thus reducing the likelihood of contracting infections and the severity if contracted. The type of vitamin E in this leafy vegetable is tocotrienols, a type which helps in reducing bad cholesterol (low density lipoprotein-LDL) levels in the body and prevents coronary heart diseases [10]. The high levels of potassium and magnesium are crucial for maintaining proper electrolyte balance in the body, and the presence of significant amounts of dietary fibre aids in the management and prevention of hypertension [10]. The antioxidant property of vitamin E, vitamin C and lysine in addition to other essential nutrients makes it possible for these leaves to fight against harmful free radicals and prevent the formation of malignant cells responsible for cancer [10]. Basing on the important role that folate plays especially in preventing neural tube defects in newborns, including amaranth vegetables to your diet would help protect your newest addition to the family.

## *2.1.1.1. Amaranth-based diets*

situation could get better—or worse—depending on how the continent's love affairs with some of its increasingly popular foods play out [2]. The irony is that there are millions of edible plants, insects and animals, and just like the Amazon, tropical Africa is still hiding most of the food items considered to be superfoods. Although superfood is a term originally used just as an advertisement and marketing tool, a superfood can be defined as a nutrientdense, antioxidant-rich, natural-food product that is minimally processed and bioavailable in numerous, potent nutritive constituents. Consumption of superfoods increases energy and vitality, regulates cholesterol and blood pressure and may help to prevent or fight cancer and other diseases [3]. Superfoods are generally beneficial for health and well-being. Basing on the above definitions, there is a big range of foods considered to be superfoods, but for this chapter, focus will be on those mainly available within Africa and with higher potential of integration within existing food and diet systems. The objective is to downplay on the 'superfood' and emphasize more on 'super diets', where the emphasis is on a healthy balanced diet. The foods that will be explored include moringa leaves, hibiscus, amaranth, baobab fruit,

96 Superfood and Functional Food - The Development of Superfoods and Their Roles as Medicine

Botanically referred to as *Amaranthus*, this crop was cultivated by the Aztecs 8000 years ago and is still a native crop in Peru. The ancient history of amaranth can be traced to Mexico and the Yucatan Peninsula. The name for amaranth comes from a Greek word *amarantos* meaning 'one that does not wither' or 'never fades'; this is true as amaranth's bushy flowers retain their vibrancy even after harvesting and drying. In addition, some varieties of ornamental amaranth do not produce the fancy flowers but produce flashy foliage, sprouting leaves [4]. *Amaranthus* are now grown in Africa, India, China, Russia, throughout South America and North America. Amaranth is tall about 6 feet, has broad leaves with colours ranging from deep blood red to light green with purple veins and has around 60 different species [4], several of which are cultivated as leaf vegetables, grains or ornamental plants [5]. It is commonly known as pigweed (English), hanekam (Afrikaans), thepe (Sesotho), imbuya (isiZulu), mchicha (Swahili), terere (Gikuyu, Meru and Embu of Kenya), doodo (Luganda), shoko (Yoruba) [5] and lengalenga (Democratic Republic of Congo and Burundi) [6]. Both the amaranth leaves and seeds are useful in terms of human health [5]. Whether you choose to consume amaranth as a leaf vegetable, a cereal grain or grain flour, considering the versatility and high concentration of antioxidants and nutrients, amaranth is one of the most of valuable health

Vegetable amaranths are probably the most widely eaten boiled or steamed greens throughout Africa's humid lowlands. They secure the food supply for millions. The leaves and stems make excellent boiled or steamed vegetables as stew or sauce; they have a soft texture, mild

tamarind, teff and fonio.

**2.1. Amaranth**

**2. Selected superfoods**

foods that you may have never heard of [5].

*2.1.1. Vegetable/leafy amaranth*

Amaranth leaves come in different colour shades ranging from dark green and reddish green to deep red and purple, but the most popular variety in Africa especially East Africa is the dark green leafy type. Although there is no standard recipe of cooking amaranth, the basic ingredients include amaranth leaves and small parts of the stem, cooking oil, tomatoes, onions and salt. Depending on the culture and economic ability, other ingredients that could be added include meat, small fish, groundnuts, African eggplant, green pepper, garlic or red beans.

The most popular use of amaranth vegetable is as a vegetable sauce accompanying starchy staples such as steamed/boiled/stewed banana; 'ugali' (African polenta or cornmeal mush) also called sima, sembe, kaunga, dona, banku, kenkey pup, posho, fufu; rice; and potatoes or sweet potatoes. As detailed in **Table 1**, it can also be cooked together with other food items to form a complete meal. In Burundi, amaranth is cooked together with beans and bananas and sometimes small fish locally called 'dagala' added. This provides a very nutritious meal able to meet a good range of both macro- and micronutrient needs.


**Table 1.** Selected amaranth leaf-based dishes.

## *2.1.2. Grain amaranth*

also called sima, sembe, kaunga, dona, banku, kenkey pup, posho, fufu; rice; and potatoes or sweet potatoes. As detailed in **Table 1**, it can also be cooked together with other food items to form a complete meal. In Burundi, amaranth is cooked together with beans and bananas and sometimes small fish locally called 'dagala' added. This provides a very nutritious meal able

Add oil to a hot pan and immediately add the onions. Saute

Serve hot with starch accompaniment such as banana, 'ugali'








NB: The amaranth can be cooked with beans excluding the bananas and sometimes with bananas excluding the beans. All the options can be served alone or with rice, ugali or any


Add the chopped tomatoes and cook covered for 1 min

until translucent; do not let them turn brown Add the leaves and stir to prevent them from burning

until translucent; do not let them turn brown -Add the leaves and stir to prevent them from burning


stir into a smooth paste and keep aside

Cook covered on low heat for 3 min

Add seasoning of your choice

potatoes, rice, etc.

covered for 1 min

for 5 more minutes

until translucent

covered for 2 more minutes

other starchy staple

constantly.

tender



to meet a good range of both macro- and micronutrient needs.

98 Superfood and Functional Food - The Development of Superfoods and Their Roles as Medicine

**Dish name Ingredients Cooking procedure**



chopped


and drained

**Table 1.** Selected amaranth leaf-based dishes.





NB: one can either use flour made from grinding uncooked groundnuts or paste made from slightly roasted groundnuts)


Basic amaranth sauce

Amaranth in groundnut sauce

Beans, cooking banana and amaranth leaves Amaranth grain is reported to have been domesticated between 6000 and 8000 years ago, and it has a long and colourful history in Mexico [11]. When ground, the amaranth flour is generally a pale ivory shade, but the red 'buds' can be ground as well for a red-tinged and very healthful grain. One of the most important aspects of this tiny grain is that it is gluten-free [11, 12], thus providing a viable wheat alternative for millions of people suffering from celiac disease or gluten intolerance.

At about 13–14%, grain amaranth easily trumps the protein content of most other grains, and you may hear the protein in amaranth referred to as 'complete' because it contains lysine, an amino acid missing or negligible in many grains [12]. It also contains other primary proteins called albumin and globulins, which, in comparison with the prolamins in wheat, are more soluble and digestible [12]. A 100 g of raw amaranth contains 14 g of protein, 15 mg of iron, 159 mg of calcium, 4 mg of vitamin C and 18 mg of fibre [9]. The high fibre level results in smooth digestion of food and facilitates an efficient uptake of minerals level. At 105% of the daily value per serving, the manganese in amaranth is off the charts, yet it contains fewer carbohydrates [4]. Amaranth contains 6–10% oil, predominantly unsaturated, or around of which 77% are unsaturated fatty acids, including linoleic acid, acid that is required for optimum nutrition [4]. With all the above nutrients, amaranth grain is a true powerhouse, likely to prevent a number of chronic health conditions such as diabetes, heart disease, cancer, and stroke.

## *2.1.2.1. Grain amaranth-based dishes*

Grain amaranth has been used for food by humans in a number of ways. Being extremely dense, amaranth is too heavy to be used by itself. Although it can be popped and eaten like popcorn or flaked like oatmeal, it is best used with other grains for a lighter texture. The ground grain is used as an enrichment to staple-based diets such as porridge, soups, ugali, etc., thus supplying more nutrients to vulnerable population groups. For instance, amaranth grain porridge (1 cup) combined together with moringa leaf powder (1 tbsp) from moringa leaves not only provides an excellent nutritional food for individuals with compromised immune system (HIV/AIDs), but also those consuming the amaranth/moringa combination are able to take anti-retroviral drugs with no complications [13].

The ground grain is also mixed with wheat flour and used in making more nutritious breads, noodles, pancakes, cereals, cookies and other flour-based products. There are more than 40 products containing amaranth and that can be used by consumers of different social economic/cultural backgrounds. Amaranth grain flour is also used as an exceptional thickener for sauces, soups, stews and even jellies; the four can be made from freshly ground grains of by sprouting/germinating the grain, drying it and milling/grinding it into flour. Eaten as a snack, amaranth can have a light, nutty or peppery-crunchy texture and flavour.

Cooking amaranth grain alone is comparable to cooking pasta or rice: boil plenty of water (six cups of water per one cup of amaranth), measure the grain into it, cook and stir for 15–20 min, drain, rinse, and its ready to eat. **Table 2** gives details of selected dishes based on amaranth grain.



**Table 2.** Examples of amaranth grain-based dishes.

#### **2.2. Teff**

**Dish name Ingredients Cooking procedure and serving recommendations**

15 min

more)

thick

2 min

bowl

brown)

taste hard or gritty)

in more boiling water -Serve in small bowls

medium-sized saucepan

cool whilst stirring, and serve

you begin to smell burning corn

other vegetable or/ and any protein dish




water; keep stirring
















hot water for 10 min to soften

any grit on the bottom of the cup


onions, and cook till soft and translucent -Stir in the amaranth; add the soaked chopped mushrooms with the soaking liquid, taking care to leave




100 Superfood and Functional Food - The Development of Superfoods and Their Roles as Medicine


Enriched porridge -One cup maize meal flour/any

Enriched 'ugali' -One cup of maize meal flour/any

Chapati -Wheat flour (1400 g)

(600 g) -Water (1000 ml) -Grated carrots (156 g) -Cooking oil (35 ml) -Salt to taste (15 g)

other common porridge flour -½ cup amaranth flour -2 ½ cups water -2 ½ cups milk (optional) -Two tbsp. margarine (optional)

other common ugali flour (cassava flour/ cassava and millet flours) -One cup of amaranth flour -Three cups of water


Amaranth polenta with mushrooms

> Teff is a tiny fine ivory, red/brown or mixed (ivory, red/brown) grain. Red/brown teff has a subtle hazelnut, almost chocolate-like flavour and a moist texture similar to millet (but more exotic). Ivory teff has a milder flavour than the brown. This grain is the national pride of Ethiopia, where it has been consumed for more than 1000 years (way BC). Teff is scientifically known as Eragrostis teff. 'Teffa', the Amharic name for 'lost', is so named because of 'teff's' small size; it is the smallest grain in the world and often is lost in the harvesting and threshing process [14]. It is now starting to get global attention which is good news for all of us especially because it is a durable crop that can grow in almost every climate.

> With its subtle nutty flavour, the same flexibility holds also in the kitchen. Teff leads all the known grains by a large margin. Its small size means that the germ and the bran—the most nutrient-dense layers—make up a large proportion of the overall seed as the grain cannot be separated into bran, germ and endosperm. Apart from its gluten-free nature which makes it a delicious wheat alternative, the teff grain is also known for its superior amino acid profile, being high in lysine, a protein essential for muscle repair [15]. Teff is the primary carbohydrate source for most Ethiopians. It has an estimated 20–40% resistant starch and high fibre; these particular components are important in dealing with diabetes and assisting with blood sugar control [15, 16]. 100 g of edible portion of raw teff has 13 g protein, 8 g fibre, 180 mg Calcium and 8 mg iron [9]. Just a cup of cooked teff contains 123 mg of calcium, about the same as half a cup of spinach [2, 15, 16]. It is also high in iron, calcium and vitamin C. It is also packed full of B vitamins, which makes it great for energy. Last but not least, teff packs a little something that the others do not 'vitamin K', a fat soluble vitamin which is required for blood clotting and also bone health [17].

#### *2.2.1. Teff-based dishes*

As shown in **Table 3**, Teff has the versatility of corn meal and millet. Delicious in porridge, stews, stuffing and pilaf, teff can be cooked alone or in combination with other grains and vegetables. Alone, teff's cooking time is 20 min, and for each cup of grain, you need three cups of water. All you need to do is combine teff and water in a pot and bring to a boil. Reduce heat, cover and simmer for 20 min, until water is absorbed. You may stir occasionally towards the end of cooking.


**Table 3.** Examples of teff-based dishes.

As the preferred staple in the Ethiopian and Eritrean dishes, teff flour is used in making engera/injera (pronounced en-jer-a and sometimes spelled injera), a flat sour-like fermented pancake that is used with 'wot', a stew made with spices, meats and pulses, such as lentils, beans and split peas.

In combination with other ingredients which is a better option as enhances nutrient-nutrient interaction, teff grain and teff flour are wonderful alternatives to wheat, barley and rye for those on a gluten-free diet. Teff flour will expand food choices beyond potato, corn and rice flour!.

## **2.3. Fonio**

There are two types, white fonio (*Digitaria exilis*) and black fonio (*Digitaria iburua*), but both are actually a type of millet grain. White fonio is grown in the Sahel area that borders the Sahara Desert, and it grows well in dry and grassy savannah as well as in richer climates. Black fonio is found in Benin, Niger, Nigeria and Togo and is generally less common (and even more nutritious). Although fonio is found all over West Africa, it is especially prized in the Fouta Djallon region of Guinea and Senegal and the Akposso region of Togo and Central Nigeria [18].

Like teff, fonio matures quickly, producing grain in just 6–8 weeks, which makes it the world's fasted maturing cereal. It can therefore be relied upon in semiarid areas with poor soil and unreliable rainfall [2, 18]. After they are mature, fonio's tiny grains must be dried and removed from their husk before they are ready to cook. Before machines did the dehusking, the fonio was dehusked in a mortar and pestle, where the grains were pounded with sand. Fonio could also be slowly toasted in a large pan until it popped out of its husk and then pounded to separate the grain from its covering [18].

A staple in African cuisine and diets, it is prepared steamed as the anchor in many meals and is also milled into flour to be used in baking. It is called the 'seed of the universe' in Malian mythology [19]. It provides 3.6 calories per gramme of grain that is similar to other cereals [20]. Just as teff and amaranth grain, it is also gluten-free making it another great wheat replacement. Fonio is simpler to digest making it suitable for children and older people [20]. Fonio is consumed as a whole grain; the barn and germ of fonio are full of nutrients. A 100 g of boiled whole-grain black fonio has 3.7 g protein, 21 mg calcium, 4.1 mg iron, 9 mg folate, 181 mg magnesium and 3.1 g of fibre [9]. In addition, fonio has essential amino acids methionine and cysteine which jointly help liver function and help in detoxing process. The high fibre content makes it necessary to keep the digestive system smooth. It helps in bowel motions and helps prevent constipation; in certain parts of Africa, fonio is provided as food to individuals struggling with stomach problems [20]. Fonio has got lower glycemic index. It really is absorbed in body gradually and therefore effect on blood sugar increment is gradual. The presence of essential amino acids helps in preventing liver damage and colon cancer and is also useful in drug removal symptoms, whilst the high levels of folic acid as well as iron play an important role in iron metabolism [20]. It is good in avoiding anaemia. This particular nourishing food is typically suitable for pregnant as well as lactating women in Africa [20]. Moreover, because of its insulinsecreting properties, fonio products have found that diabetics are their key customers.

#### *2.3.1. Fonio-based dishes*

As the preferred staple in the Ethiopian and Eritrean dishes, teff flour is used in making engera/injera (pronounced en-jer-a and sometimes spelled injera), a flat sour-like fermented pancake that is used with 'wot', a stew made with spices, meats and pulses, such as lentils,

**Dish name Ingredients Cooking procedure and serving recommendations**

102 Superfood and Functional Food - The Development of Superfoods and Their Roles as Medicine














cinnamon, and stir in banana the milk mixture




Ground-up flax seeds easily take the place of egg

one heaping tablespoon for each pancake



sit for 1 day/24 hrs without any stirring or agitation

barely detect the saltiness -Stir in the baking powder

the pan or the injera will crack -Do not flip or brown its underside

paper until you use up all the batter

berbere spice

blend well

3 min

to taste

more minutes

on one tsp of oil

bubbles and begins to dry

Injera -1 1/2 cups of teff flour

Teff banana pancakes

Teff grain/ flour porridge




(optional)

**Table 3.** Examples of teff-based dishes.






In combination with other ingredients which is a better option as enhances nutrient-nutrient interaction, teff grain and teff flour are wonderful alternatives to wheat, barley and rye

beans and split peas.

The small grains are beloved in Burkina Faso, Guinea, Mali and Nigeria, Senegal and Togo, where fonio is a staple part of most people's diets. Fonio is a favourite in salads, stews and porridges [18]. In Togo, fonio is cooked with black-eyed peas and, in other places, mixed with nutrient-dense sesame seeds to add even more vegetarian nutrition [18].

Fonio grains can be cooked whole, or it can be ground into a gluten-free flour and used as a substitute for wheat flour [18]. **Table 4** provides details of selected fonio-based dishes.




**Table 4.** Examples of fonio-based dishes.

## **2.4. Moringa**

Fonio grains can be cooked whole, or it can be ground into a gluten-free flour and used as a substitute for wheat flour [18]. **Table 4** provides details of selected fonio-based dishes.

> -Bring the water to a boil in a pot -Add the salt and the fonio, and cover

fonio has absorbed all its water

shorted/rub in using finger tips

consistency of your liking -Beat in the egg

dish, serve with vegetables, stew or sauce


bananas and a glass of milk or juice

simmer for some time (10–15 min)

let simmering for additional time -Put the cooked vegetables aside

margarine and mix properly


yeast is well incorporated -Fill the cake tins with the dough

with a spatula, up until paste is smooth

onions are translucent

additional 5 min -Serve the complete dish



















**Dish name Ingredients Cooking procedure and serving recommendations**

104 Superfood and Functional Food - The Development of Superfoods and Their Roles as Medicine

Simple fonio -Two cups of fonio grains

Paleo pancakes using fonio flour -Two cups of water -Salt to taste -(Serves four)








Fonio Pilaf -Two cups of fonio rinsed and drained

> (optional) -Ten cups of water -(Serves six to eight)

> -1/2 kg wheat flour -650 g sugar -Three eggs -1/2 Cup dried milk -1/2 margarine -10 g yeast -½ litre water -(Serves six to eight)

Fonio cake -1/2 kg fonio flour

The *Moringa oleifera* tree is a small tree that is native to the Himalayas of India and was being used in Indian medicine around 5000 years ago. There are also accounts of it being utilized by the ancient Greeks, Romans and Egyptians [21]. Although there are technically 13 different species of moringa tree [21], for simplicity, this chapter is in reference to the *Moringa oleifera* tree and using the common name 'moringa'. This tree was, and still is, considered a panacea and is referred to as the 'The Wonder Tree', 'The Divine Tree' and 'The Miracle Tree' amongst many others. It is priced as a multipurpose tree with all parts usable either as a raw or cooked nutriment, medicine or as a water purification additive. It is also known for its long twisted pods, from which it derives its name. 'Murungai' means 'twisted pod' in the Tamil language [21].

Moringa is beneficial for both food and medicine because of its ability to grow in virtually all countries. Currently, its growth is most prevalent in Africa, Central and South America and Asia. But its effects are being felt around the world [21].

The leaves typically the most common part of the plant are especially high in protein and considered a multivitamin-mineral complex. A 100 g of mature moringa leaves contain 5.7 g protein, 15 mg β-carotene, 459 mg vitamin C, 25 mg vitamin E, 9.2 mg iron and 638 mg calcium [22]. The moringa leaves also contain 18 amino acids, including the 9 essentials: histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan and valine [23].

The leaves are harvested and steamed as a green vegetable but are also dried and ground into a powder used in sauces, soups and cooked grains. In these different forms, moringa is used as a vitamin-mineral supplement that is extremely effective at balancing nutritional uptake needed for greater dietary balance. With the high nutrition content, moringa leaves are gaining popularity as a remedy for malnutrition in Africa, especially amongst infants, children and nursing mothers [2]. Moringa could also be a great nutrient safety net because the tree is in full leaf at the end of the dry season when other foods are typically scarce [2]. The seeds and pods can also be eaten just as you would green beans, and the flowers and buds make a nice tea although they contain a laxative effect [24].

#### *2.4.1. Moringa-based foods*

Whilst the leaves can be steamed or boiled and eaten as a green leafy vegetable with a slight 'bite' taste to them, the moringa leave powder has a mild, somewhat spinach-like taste and works well in smoothies, green juices and soups or sprinkled over most anything. **Table 5** provides details on how moringa can be used as leaves and as powder in selected recipes.


**Table 5.** Examples of moringa-based dishes.

## **2.5. Baobab fruit**

*2.4.1. Moringa-based foods*

Moringa sprinkles -Moringa powder

Moringa, lemon, honey, mint leaves, ginger and ice blend

Stir fried moringa

Scrambled eggs with moringa leaves

Moringa leaves with maize/corn and beans

leaves







Two cups of fresh beans from the pods -One onion sliced -Two tomatoes sliced -Cooking oil

mint leaves -Three tbsp honey

onion -Salt to taste


off the cob

**Table 5.** Examples of moringa-based dishes.

leaves -Cooking oil -Salt to taste -Minced fresh garlic cloves or garlic salt (optional)

Whilst the leaves can be steamed or boiled and eaten as a green leafy vegetable with a slight 'bite' taste to them, the moringa leave powder has a mild, somewhat spinach-like taste and works well in smoothies, green juices and soups or sprinkled over most anything. **Table 5** provides details on how moringa can be used as leaves and as powder in selected recipes.














need to sweeten the drink depends on your preference -Experiment with the ratios, but know these ingredients all play


**Dish name Ingredients Cooking procedure and serving recommendations**

106 Superfood and Functional Food - The Development of Superfoods and Their Roles as Medicine

a sandwich


serving with plenty of ice


ugali, matooke, etc.

your dish is ready


starts to brown on the edges a bit

they do not stick at the bottom or clump

from the leaves during cooking is evaporated





nice together

browned

spoon

cooked

The iconic baobab is a common tree in eastern and southern Africa's savannahs [2]. It is one of the most nutrient-dense wholefoods on the planet. It is a 100% organic, raw superfruit that dries naturally on the branch [25]. In Africa, the baobab fruit has been used medicinally for centuries to treat everything from fevers, malaria and gastrointestinal problems to vitamin C deficiency [26].

The unusually high levels of vitamin C in moringa fruit are what contribute to the great potential health benefits of moringa fruit powder and its fresh counterpart [26]. A 100 g of baobab fruit pulp contains 247 mg vitamin C (nearly four times of the daily requirements); therefore, a single serving of baobab powder (10 g or two to three tsp) will have about 24.7 mg of vitamin C providing about 40% of your daily Vitamin C requirement making baobab fruit one of the best sources of vitamin C in the world [25].

Vitamin C plays a crucial role in our bodies. It contributes to normal collagen formation supporting healthy gums, teeth, skin, bones, cartilage and blood vessels; energy release, energyyielding metabolism and reduction of fatigue; immune function; functioning of the nervous system and psychological function; and protection of cells from oxidative stress [25].

There are two types of fibre that your body needs: soluble and insoluble and baobab being 50% fibre contains equal quantities of both. The soluble fibre in baobab helps to slow down the release of sugar into the bloodstream, thus reducing energy spikes. Fibre also helps maintain a healthy digestive system including bowel regularity, and the fact that it helps you feel fuller for longer, it can be helpful for weight management [25].

Baobab powder has twice the antioxidants gramme per gramme of goji berries and more than blueberries and pomegranates combined, thus having the highest antioxidant content amongst all fruits [25]. Antioxidants are essential for protecting, repairing and preventing cell damage; supporting the ageing process of the skin particularly over the long term; and neutralizing free radicals, unstable atoms and molecules that can cause damage to your body at the cellular level, increasing the risk of degenerative diseases and other signs of ageing, including wrinkles and fine lines on the skin. Antioxidants counteract oxidative stress and the effects of free radicals (unstable molecules that damage collagen causing skin dryness, fine lines, wrinkles and premature ageing). When fresh baobab pulp is used in cooking or concentrated baobab fruit powder added to dishes, it boosts the supply of beneficial minerals including calcium, copper, iron, magnesium, potassium and zinc. These minerals act both individually and synergistically to perform hundreds of tasks in the human body. A 100 g of fresh baobab pulp contains 295 mg calcium, 1.6 mg copper, 9.3 mg iron, 90 mg magnesium, 1240 mg potassium, 27.9 mg sodium and 1.8 mg zinc [26].

#### *2.5.1. Baobab-based dishes*

Baobab fruit is very dry so it keeps almost indefinitely, and it is used to make juice by soaking the fruit and straining out the pulp and seeds [2]. The fruit powder (or fresh baobab fruit if you can get) can be added to your diet (liquid or solid) to enhance your body's fat-burning capacities, especially if you are working on losing weight and your current diet is not rich in vitamin C. Absorption of iron and vitamin C actually increases your body's absorption of iron, which is why vitamin C-rich baobab and iron-rich moringa work so well together. See **Table 6** for selected Baobab-based recipes.


**Table 6.** Examples of baobab-based dishes.

#### **2.6. Hibiscus**

The hibiscus plant (*Hibiscus sabdariffa*) is thought to originate from the areas surrounding central Africa presumably Angola. It is also widely cultivated throughout many tropical and subtropical regions, particularly Mexico, India, Thailand and China [27, 28]. Hibiscus is recognized for its large, colourful flowers that are often used as decorative pieces in gardens and homes. When the petals of the hibiscus flower begin to detach from the main plant, underneath they reveal flower bud-like structures known as calyces. These deep red buds are subsequently used to produce hibiscus tea and hibiscus extract. Whilst the tea is popular with health-conscious consumers, the extract is more versatile. It can be used in a number of different food and beverages and still maintaining its health-promoting properties [27].

Hibiscus is a rich source of vitamins and microelements including 13 organic acids. A 100 g of hibiscus tea will provide approximately 6, 31 and 48% of the daily values of vitamin A, vitamin C and iron [28]. These nutrients and microelements boost your immune system. It makes your blood vessels stronger, lowers the blood pressure and the level of 'bad' cholesterol and even has an antibacterial effect; this makes it prevent and reduce symptoms of metabolic syndrome (a combination of diabetes, obesity and high blood pressure), thus reducing the risk of developing heart disease and stroke [29]. It is also good for the pancreas and liver [28]. Hibiscus erases post-effects of alcohol intoxication and contains antioxidant elements similar to those of red wine; this helps the extract act as an anti-solar agent, by absorbing skin-damaging ultraviolet radiation from the sun. The antioxidants also minimize cell damage from free radicals, which may help to slow down the natural ageing process and reduce the risk of developing a number of age-related diseases [28, 29].

Hibiscus extract is also thought to promote a healthy digestive system. It has antibacterial properties, which may help to maintain a favourable gut flora. Hibiscus can also act as a mild laxative, which may help with symptoms of constipation and indigestion. One study also found that the extract demonstrates anti-urolithiatic activity, meaning that it can help reduce the formation of kidney stones. All these properties explain why the Arabs call carcade 'the remedy from all illnesses'. It is said that three cups of red tea per day is enough to get the most of it. On the other hand, like any remedy, it is not good for everyone. People with low blood pressure, ulcer or gastritis should be very careful with it.

## *2.6.1. Hibiscus-based dishes*

capacities, especially if you are working on losing weight and your current diet is not rich in vitamin C. Absorption of iron and vitamin C actually increases your body's absorption of iron, which is why vitamin C-rich baobab and iron-rich moringa work so well together. See

**Dish name Ingredients Cooking procedure and serving** 




powder

108 Superfood and Functional Food - The Development of Superfoods and Their Roles as Medicine

(optional)

**recommendations**

and shake or stir -It is ready to drink





and blend until smooth

for a thinner texture

The hibiscus plant (*Hibiscus sabdariffa*) is thought to originate from the areas surrounding central Africa presumably Angola. It is also widely cultivated throughout many tropical and subtropical regions, particularly Mexico, India, Thailand and China [27, 28]. Hibiscus is recognized for its large, colourful flowers that are often used as decorative pieces in gardens and homes. When the petals of the hibiscus flower begin to detach from the main plant, underneath they reveal flower bud-like structures known as calyces. These deep red buds are subsequently used to produce hibiscus tea and hibiscus extract. Whilst the tea is popular with health-conscious consumers, the extract is more versatile. It can be used in a number of differ-

Hibiscus is a rich source of vitamins and microelements including 13 organic acids. A 100 g of hibiscus tea will provide approximately 6, 31 and 48% of the daily values of vitamin A, vitamin C and iron [28]. These nutrients and microelements boost your immune system. It makes your blood vessels stronger, lowers the blood pressure and the level of 'bad' cholesterol and even has an antibacterial effect; this makes it prevent and reduce symptoms of metabolic syndrome (a combination of diabetes, obesity and high blood pressure), thus reducing the risk of developing heart disease and stroke [29]. It is also good for the pancreas and liver [28]. Hibiscus erases post-effects of alcohol intoxication and contains antioxidant elements similar to those of red wine; this helps the extract act as an anti-solar agent, by absorbing skin-damaging ultraviolet radiation from the sun. The antioxidants also minimize cell damage from free radicals, which may help to slow down the natural ageing process and reduce the risk of

ent food and beverages and still maintaining its health-promoting properties [27].

developing a number of age-related diseases [28, 29].

**Table 6** for selected Baobab-based recipes.

Baobab nutri-shake -One glass of water

Tropical baobab-papaya smoothie -One tsp baobab powder

**2.6. Hibiscus**

**Table 6.** Examples of baobab-based dishes.

When dried hibiscus flowers are steeped in hot water, the dark red hibiscus tea is called karkadeh/karkady in Arabic and is popular in North Africa, particularly Egypt and Sudan where it is used to not only maintain normal body temperature, support heart health and encourage fluid balance [2, 29] but also at wedding celebrations as a toasting drink [2]. In West Africa, it is known as bissap, tsoborodo or wonjo; bissap is called the 'national drink of Senegal'. It is either served hot (it loses a bit of its characteristic sour) or can also be served chilled with ice [2].

The recipes in **Table 7** show that hibiscus powder is added to hot or cold water to make a simple, slightly tart-tasting tea, but it is also increasingly used as a functional ingredient in many applications, from sorbets to confectionary. It can also be used as a colouring and flavouring agent in jams, relishes, sauces and baked goods. In addition, hibiscus extract has been applied as a colourant and antioxidant in the skin and hair care applications.


**Table 7.** Examples of hibiscus-based foods.

## **2.7. Tamarind**

Tamarind trees are native to tropical Africa but found in tropical regions throughout the world [2]. The tree produces an abundance of long, curved, brown pods filled with small brown seeds, surrounded by a sticky pulp that dehydrates naturally to a sticky paste. The pods look a bit like huge, brown, overly mature green beans [30].

Just as the other ancient foods do, tamarind has a long history of being used as a medical remedy. It has been known to ease stomach discomfort, aid digestion and act as a laxative [30]. Tamarind preparations are used to relieve fevers, sore throat, rheumatism, inflammation and sunstroke. Dried or boiled tamarind leaves and flowers are made into compresses used for swollen joints, sprains, boils, haemorrhoids and conjunctivitis. Similar to the natural gums and pectins found in other foods, the tamarind sticky pulp contains non-starch polysaccharides, which contribute to its high dietary fibre content (5.1 g/100 g fruit pulp). They bind with bile to help flush waste through the colon, decreasing the chances of it sticking around, thus reducing chances of colon cancer. Prized for its sweet-and-sour flavour, tamarind (also known as ukwaju in Swahili) is used to make juice and is rich in vitamins, minerals and antioxidants [2]. 100 g of tamarind contain 36% of the thiamin, 35% of the iron, 23% of magnesium and 16% of the phosphorus recommended for a day's worth of nutrition [30]. Other prominent nutrients include niacin, calcium, vitamin C, copper and pyridoxine. Tamarinds also contain high levels of tartaric acid, just as citrus fruits contain citric acid, providing not just a zing to the taste buds but evidence of powerful antioxidant action against harmful free radicals floating through your system [30].

Other phytochemicals found in tamarinds include limonene, safrole (a natural oil also found in sassafras), geraniol (a natural antioxidant with a rose-like scent), methyl salicylate (a plant essence with counterirritant properties), cinnamic acid, pyrazine and alkyl-thiazoles (natural flavours and fragrances derived from plants and vegetables). Each of these phytochemicals brings their own healing property and flavour to the fruit's overall make-up [30]. In addition, due to its ability to restore electrolyte imbalance during dehydration, many East African coastal communities will serve a glass of ukwaju (tamarind juice) to a guest coming in from a hot day or as a hangover remedy [2].

#### *2.7.1. Tamarind-based dishes*

In addition to being used alone as a drink/juice/tea, **Table 8** shows that the pulp from tamarind fruit is also used as a spice and souring agent in sauces, marinades, salads, stir fries, even sorbets and cool refreshing summer drinks. The English word 'tamarind' is taken from the Arabic tamar-hindi, meaning 'Indian date', and it is popular in equatorial cuisines, such as Indian, Mexican and Thai. Also known as imli, tamarind is used as a souring agent in many cuisines, especially those of South and Southeast Asia. There, you will find it in curries, stirred into drinks, made into relishes and sauces and even cooked down into a sweet-and-spicy dessert paste. The pulp can be pressed to form a 'cake' or processed to make a paste. When used in marinades as indicated earlier, besides adding flavour, the fruit's natural acidity helps to tenderize tougher cuts of beef by breaking down the fibres in the meat. Marinated overnight in a tamarind-tinged liquid, beef becomes succulent and tender. But it is important to note that when marinating fish or chicken, if left in the marinade too long, the tamarind will begin to chemically 'cook' it.


**Table 8.** Examples of tamarind-based dishes.

## **3. Conclusions**

**2.7. Tamarind**

Tamarind trees are native to tropical Africa but found in tropical regions throughout the world [2]. The tree produces an abundance of long, curved, brown pods filled with small brown seeds, surrounded by a sticky pulp that dehydrates naturally to a sticky paste. The

Just as the other ancient foods do, tamarind has a long history of being used as a medical remedy. It has been known to ease stomach discomfort, aid digestion and act as a laxative [30]. Tamarind preparations are used to relieve fevers, sore throat, rheumatism, inflammation and sunstroke. Dried or boiled tamarind leaves and flowers are made into compresses used for swollen joints, sprains, boils, haemorrhoids and conjunctivitis. Similar to the natural gums and pectins found in other foods, the tamarind sticky pulp contains non-starch polysaccharides, which contribute to its high dietary fibre content (5.1 g/100 g fruit pulp). They bind with bile to help flush waste through the colon, decreasing the chances of it sticking around, thus reducing chances of colon cancer. Prized for its sweet-and-sour flavour, tamarind (also known as ukwaju in Swahili) is used to make juice and is rich in vitamins, minerals and antioxidants [2]. 100 g of tamarind contain 36% of the thiamin, 35% of the iron, 23% of magnesium and 16% of the phosphorus recommended for a day's worth of nutrition [30]. Other prominent nutrients include niacin, calcium, vitamin C, copper and pyridoxine. Tamarinds also contain high levels of tartaric acid, just as citrus fruits contain citric acid, providing not just a zing to the taste buds but evidence of powerful antioxidant action against harmful free radicals floating through your system [30]. Other phytochemicals found in tamarinds include limonene, safrole (a natural oil also found in sassafras), geraniol (a natural antioxidant with a rose-like scent), methyl salicylate (a plant essence with counterirritant properties), cinnamic acid, pyrazine and alkyl-thiazoles (natural flavours and fragrances derived from plants and vegetables). Each of these phytochemicals brings their own healing property and flavour to the fruit's overall make-up [30]. In addition, due to its ability to restore electrolyte imbalance during dehydration, many East African coastal communities will serve a glass of ukwaju (tamarind juice) to a guest coming in from a

In addition to being used alone as a drink/juice/tea, **Table 8** shows that the pulp from tamarind fruit is also used as a spice and souring agent in sauces, marinades, salads, stir fries, even sorbets and cool refreshing summer drinks. The English word 'tamarind' is taken from the Arabic tamar-hindi, meaning 'Indian date', and it is popular in equatorial cuisines, such as Indian, Mexican and Thai. Also known as imli, tamarind is used as a souring agent in many cuisines, especially those of South and Southeast Asia. There, you will find it in curries, stirred into drinks, made into relishes and sauces and even cooked down into a sweet-and-spicy dessert paste. The pulp can be pressed to form a 'cake' or processed to make a paste. When used in marinades as indicated earlier, besides adding flavour, the fruit's natural acidity helps to tenderize tougher cuts of beef by breaking down the fibres in the meat. Marinated overnight in a tamarind-tinged liquid, beef becomes succulent and tender. But it is important to note that when marinating fish or chicken, if left in the marinade too long, the tamarind will begin to chemically 'cook' it.

pods look a bit like huge, brown, overly mature green beans [30].

110 Superfood and Functional Food - The Development of Superfoods and Their Roles as Medicine

hot day or as a hangover remedy [2].

*2.7.1. Tamarind-based dishes*

Just as in the Amazon, there are millions of edible plants, insects and animals within tropical Africa that are not only nutrient rich but also contain essential elements beneficial in preventing and/or managing a range of health conditions that are of great public concern. Amaranth, teff, fonio, moringa leaves, baobab fruit, tamarind and hibiscus leaves just to mention a few are some of the superfoods that can be used alone but more importantly transformed into health superdiets to provide simple, acceptable and sustainable remedies that not only address malnutrition but also play a major role in the battle against non-communicable diseases like cardiovascular diseases (heart attacks and stroke), cancers, chronic respiratory diseases and diabetes.

## **Acknowledgements**

The author first acknowledges the authors of all articles cited in this chapter. Secondly, Bioversity International www.bioversityinternational.org is thanked for the staff time spent during the writing of this chapter. Last but not least, the author acknowledges her family for bearing with her as she spent some family time in putting this chapter together.

## **Author details**

Beatrice Nakhauka Ekesa Address all correspondence to: b.ekesa@cgiar.org Bioversity International, Kampala, Uganda

## **References**


[12] Cholorexa. Amaranth (Kiwicha) [Internet]. Available from http://colorexa.com/products/amaranth-kiwicha/ [Accessed: 13th August 2016]

**Author details**

**References**

Beatrice Nakhauka Ekesa

Address all correspondence to: b.ekesa@cgiar.org

112 Superfood and Functional Food - The Development of Superfoods and Their Roles as Medicine

[1] FAO, IFAD and WFP. The State of Food Insecurity in the World 2015. Meeting the 2015 International Hunger Targets: Taking Stock of Uneven Progress. 2015. Rome, FAO.

[2] Christine M. The Secret is Out: 10 African Super Foods you Need to Eat and Drink Right Now [Internet]. 1st June 2015. Available from: http://mgafrica.com/article/2015-05-30

[3] What is a Superfood. Global Health Centre. 3rd June 2014.[Internet] Available from http://www.globalhealingcenter.com/natural-health/what-is-a-superfood/ [Accessed:

[4] Joseph M, Food Facts, What is Amaranth Good for? 2016. [Internet] Available from http://foodfacts.mercola.com/amaranth.html [Accessed 29th September 2016]

[5] Republic of South Africa (2010). Amaranthus Production Guideline. Department of Agriculture, Forestry and Fisheries, South Africa. 2010. Available from http://www.nda.

[6] DianaBuja's Blog. Amaranth Greens (Lenga-Lenga)—Politically Correct, Easy to Grow, and Delicious. 16th November 2012 [Internet]. Available from https://dianabuja.word-

[7] National Academy of Sciences. Amaranth, Lost Crops of Africa, Volume II: Vegetables. 2016. Available from https://www.nap.edu/read/11763/chapter/3#45 [Accessed: 10th

[8] Innovateus. The 12 Health Benefits of Eating Amaranth Leaves. Available from http:// www.innovateus.net/health/12-health-benefits-eating-amaranth-leaves [Accessed: 10th

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[10] Organic Facts. Health Benefits of Amaranth. [Internet] Available from https://www. organicfacts.net/health-benefits/vegetable/amaranth.html Accessed: 10th August 2016] [11] Whole Grains Council. Amaranth-May Grain of the Month. May 2016. Available from http://wholegrainscouncil.org/whole-grains-101/easy-ways-enjoy-whole-grains/grain-

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**Provisional chapter**

## **The Mediterranean Diet in the Prevention of Degenerative Chronic Diseases The Mediterranean Diet in the Prevention of**

Elisabetta Della Valle, Francesco Cacciatore,

**Degenerative Chronic Diseases**

Eduardo Farinaro, Francesco Salvatore, Elisabetta Della Valle, Francesco Cacciatore, Eduardo Farinaro, Francesco Salvatore,

Roberto Marcantonio, Saverio Stranges and Roberto Marcantonio, Saverio Stranges and

Maurizio Trevisan Maurizio Trevisan

[24] One Green planet. All About Moringa: The Uber Nutritious Superfood that could Combat Malnutrition.[Internet] 13th October 2014. Available from http://www. onegreenplanet.org/natural-health/all-aboutmoringa-the-uber-nutritious-super-

[25] Aduna. Baobab Superfruit: Health and Beauty Benefits. Available from https://aduna.

[26] Heal With Food. Health Benefits of Baobab Fruit Powder (A Superfood Packed with Vitamin C). Available from http://www.healwithfood.org/health-benefits/baobab-fruit-

[27] Superfoods Ingredients (SFI). Wholesale Superfood Ingredients in the Spotlight: High Antioxidant Hibiscus Extract. Available from http://www.sfisuperfoods.com/superfood-

[28] Jon J. 10 Surprising Health Benefits of Hibiscus Tea, Foods and Nutrition. Doctors Health Press. 2nd August 2015. Available from http://www.doctorshealthpress.com/food-and-

[29] Hudson T The Surprising Health Benefits of Hibiscus, Gaia Herbs. 9th July, 2013. Available from http://www.gaiaherbs.com/articles/detail/42/The-Surprising-Health-

[30] Joseph M, Food Facts, What Is Tamarind Good For? 2016. [Internet] Available from http://foodfacts.mercola.com/tamarind.html [Accessed: 12th September 2016]

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114 Superfood and Functional Food - The Development of Superfoods and Their Roles as Medicine

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

http://dx.doi.org/10.5772/67119

#### **Abstract**

Degenerative chronic diseases are a problem related to the aging phenomenon of industrialized countries due to the increase of risk factors and related comorbidity such as overweight, obesity, metabolic syndrome, diabetes, hypertension and hyperlipidemia with a consequent increased risk of cardiovascular disease (CVD) and cancer. Moreover, the significant reduction of physical activity in daily life and the huge growth in food availability have considerably increased the risk of such diseases. Particular attention should be paid to primary prevention by means of health strategies based on improvement in lifestyle intervention such as implementation of Mediterranean diet and promotion of physical activity programs. In this chapter, the protective effect of Mediterranean diet and the role of certain foods and/or their constituents are analyzed; the possible mechanisms by which Mediterranean diet is effective in the prevention of cardiovascular and other chronic diseases are presented, in particular the effects exerted by antioxidants, polyphenols, fibers, unsaturated fatty acids, and alcohol. The genetic revolution in the past decades has produced new fields of study where the interaction between foods, nutrients, and our genetic makeup is investigated. The relationship between nutrigenetics and nutrigenomics and the Mediterranean diet are the future area that research should discover.

**Keywords:** functional foods, Mediterranean diet, cardiovascular diseases, chronic diseases, prevention

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

© 2017 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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

## **1. Introduction**

The progressive improvement of socioeconomic conditions, which occurred in the second half of the last century in industrialized countries, has produced a major change in lifestyle with a significant reduction of physical activity, due to the mechanization of work activities and transport systems, and thus of total energy expenditure, and a contemporary huge increase in food availability. Eating habits have changed substantially and have acquired two characteristics: excess and inadequacy. The biggest change is the adoption of a high-calorie diet, rich in animal fats, cholesterol, refined sugars, salt, and alcohol, with a low ratio of nutrients to calories. These aspects of modern life have favored an increase in overweight and obesity and consequently also the frequency of diabetes, hypertension, and hyperlipidemia. The genetic constitution, meanwhile, has remained the same as that of primitive man as the human genome has not had the time to adapt to the new environment that instead has changed rapidly. The natural selection has favored the appropriate mechanisms to address the deficiency of food rather than those to limit weight gain; consequently, most of the current diseases are the result of a precarious genetic adaptation to the new environment created by man [1]. In addition, improved sanitary conditions, together with the introduction of antibiotic therapy and vaccination, have led to an increase in lifespan: humanity in general has aged and presents all the issues related to aging. There has been a gradual, steady increase of chronic degenerative diseases and in some countries there has been a real epidemic of these diseases and in particular of cardiovascular diseases (CVDs). The social impact of these diseases is considerable, especially for costs, both direct and indirect. The first type of cost is related to medical interventions necessary to the care (hospitalization, drugs, and rehabilitation), while the indirect costs are related to the loss of productivity and the need to replace the person affected by the disease in a period of his/her life characterized by a high professional qualification in its business work. CVDs are still the leading cause of death and disability in many industrialized countries. Recently, also in developing countries or economies in transition, we are observing a continuous and rapid increase in CVD. Smoking is considerably widespread, especially among women, and among the youngest which has further increased the risk of CVD.

These are already the first cause of death worldwide with the exception of Sub-Saharan Africa [2]. They represent a crucial issue in public health and, consequently, their prevention, especially at primary level, is an essential point in the choice of health strategies developed by the governments of several countries.

The importance of environmental factors in the pathogenesis of CVD is recognized as certain and among the most important environmental factors is lifestyle, defined as eating habits and physical activity.

The genetic revolution in the past decades has produced new fields of study focused on the interaction between foods, nutrients, and human genetic make-up to investigate our predisposition and ability to prevent or treat CVD, cancer, diabetes, obesity, cognitive decline and dementia, and inflammatory bowel disease [3–5]. Experimental data demonstrated that environment and foods could regulate gene expression and structure [6]. Food constituents and nutrients may induce the change of structure and function of genes and may be able to prevent or cause specific diseases; these new areas of study are called nutrigenomics and nutrigenetics [7]. Nutrigenomics aims at relating in the population the effects of certain foods on human health on the basis of genetic predisposition. It will therefore be possible, in the next few years, to identify the best strategy for the prevention of many chronic degenerative diseases, and with specific tests, it will be possible to understand which foods are the most suitable and which ones need to be avoided. Nutrigenetics specifically investigates the modifying effects of inheritance in nutrition-related genes on micronutrient uptake and metabolism as well as dietary effects on health. In this way, it is possible to hypothesize a diet tailored to the patient, based on his/her genotype, the quality, and quantity of the daily required nutrients to his/her body, with determination of minimum and maximum amounts needed to obtain the most benefits. These two branches of science can combine genetics with nutrition trying to play an active preventive role in defense of the organism; this is the new pathway for genetics applied to nutrition. A new frontier has been opened and has created a new scientific approach to prevention based on genetics. Proper and targeted feeding combined with the genotypic diversity of each individual will allow us to clarify the guidelines for the prevention of a large number of diseases and will allow the development of new experimental therapies, aimed at the treatment of complex pathologies such as metabolic diseases. A proper and balanced diet is essential for a long and healthy life, but it is not the same for everyone; modern genetic testing allows us to determine the best suited diet to each individual. This systemic approach, based on genetics, once fully operational, will provide results that can be functional to the improvement of human well-being. The typing of biomolecules with enhanced nutritional properties will be reflected on the dietary recommendation with a more accurate and effective action of prevention and population health protection.

Actually, nutrigenomics and nutrigenetics are still in a beginning phase, without definite scientific evidence that the effects observed in experimental and small clinical studies have real clinical implications. We have to rely on existing scientific evidence, suggesting nutritional models known to be effective on the health of individuals and communities. One of the most widespread diets is the "*Mediterranean diet (MD)*."

## **2. Mediterranean diet**

**1. Introduction**

governments of several countries.

physical activity.

The progressive improvement of socioeconomic conditions, which occurred in the second half of the last century in industrialized countries, has produced a major change in lifestyle with a significant reduction of physical activity, due to the mechanization of work activities and transport systems, and thus of total energy expenditure, and a contemporary huge increase in food availability. Eating habits have changed substantially and have acquired two characteristics: excess and inadequacy. The biggest change is the adoption of a high-calorie diet, rich in animal fats, cholesterol, refined sugars, salt, and alcohol, with a low ratio of nutrients to calories. These aspects of modern life have favored an increase in overweight and obesity and consequently also the frequency of diabetes, hypertension, and hyperlipidemia. The genetic constitution, meanwhile, has remained the same as that of primitive man as the human genome has not had the time to adapt to the new environment that instead has changed rapidly. The natural selection has favored the appropriate mechanisms to address the deficiency of food rather than those to limit weight gain; consequently, most of the current diseases are the result of a precarious genetic adaptation to the new environment created by man [1]. In addition, improved sanitary conditions, together with the introduction of antibiotic therapy and vaccination, have led to an increase in lifespan: humanity in general has aged and presents all the issues related to aging. There has been a gradual, steady increase of chronic degenerative diseases and in some countries there has been a real epidemic of these diseases and in particular of cardiovascular diseases (CVDs). The social impact of these diseases is considerable, especially for costs, both direct and indirect. The first type of cost is related to medical interventions necessary to the care (hospitalization, drugs, and rehabilitation), while the indirect costs are related to the loss of productivity and the need to replace the person affected by the disease in a period of his/her life characterized by a high professional qualification in its business work. CVDs are still the leading cause of death and disability in many industrialized countries. Recently, also in developing countries or economies in transition, we are observing a continuous and rapid increase in CVD. Smoking is considerably widespread, especially

116 Superfood and Functional Food - The Development of Superfoods and Their Roles as Medicine

among women, and among the youngest which has further increased the risk of CVD.

These are already the first cause of death worldwide with the exception of Sub-Saharan Africa [2]. They represent a crucial issue in public health and, consequently, their prevention, especially at primary level, is an essential point in the choice of health strategies developed by the

The importance of environmental factors in the pathogenesis of CVD is recognized as certain and among the most important environmental factors is lifestyle, defined as eating habits and

The genetic revolution in the past decades has produced new fields of study focused on the interaction between foods, nutrients, and human genetic make-up to investigate our predisposition and ability to prevent or treat CVD, cancer, diabetes, obesity, cognitive decline and dementia, and inflammatory bowel disease [3–5]. Experimental data demonstrated that environment and foods could regulate gene expression and structure [6]. Food constituents and nutrients may induce the change of structure and function of genes and may be able to prevent The term Mediterranean diet has become a synonymous of a healthful and tasteful pattern of eating. The MD is a way to "enjoy food" while ensuring a long and healthy life. The increasing interest is the consequence of numerous studies conducted around the world in the last 50 years, when the famous nutritionist Ancel Keys launched and organized the Seven Countries Study, an epidemiological study that analyzed the role of diet and other cardiovascular risk factors on cardiovascular disease and death [8–10]. The study originated from the observations that in nations such as Greece, Italy, and Japan the cases of myocardial infarction (at least those in the hospitals) were much lower than those he had observed in Minnesota, in Netherlands, and in Finland.

The observation was conducted in 16 cohorts enrolled in Finland, Greece, Italy, Japan, the Netherlands, USA, and former Yugoslavia. The Seven Countries Study was one of the first examples of international collaboration in medical research and has represented, over the years, the groundbreaking evidence on the effect of diet on health and in cardiovascular and chronic disease epidemiology. The main findings of the study were the demonstration of a significant association between coronary heart disease (CHD) and diet, particularly positive correlation between the consumption of saturated fatty acids and CHD and relevant inverse relationship between the consumption of monounsaturated fat and CHD. The 15-year mortality follow-up demonstrated an inverse association between coronary deaths and the ratio of the dietary monounsaturated/saturated fats [8–10]. Olive oil has been considered one of the principal components of the MD. Wine, garlic, fish, vegetables, legumes, almonds, and other nuts, other constituents of this dietary pattern, have also been identified to have beneficial effects on health [11, 12]. The data of 15-year follow-up of the Seven Countries Study have been followed up by numerous evidences showing important inverse relationships between the MD, and/or its elements, and either CVD or its risk factors [13–16].

More recent studies have demonstrated that MD and its components may be a powerful aid against certain conditions, such as diabetes, stroke, dementia, colorectal cancer (CRC), and mortality. The greater part of the findings up to now comes from epidemiological studies, even if the cause-effect relationship is not so clear. A recent clinical trial study, based on randomized population, showed the positive outcomes of the MD in the prevention of CVD in individual at a high risk for this disease [17]. A recent study aimed to evaluate the effects of adherence to MD on survival on a large sample of 71,333 Swedish men and women, followed up for 14 years, demonstrated a linear dose-response association between the MD score average and the length of life with the higher score associated with longer survival. The difference in the average length of life between subjects with extremes scores (0 vs. 8) of MD was up to 2 years [18]. The PREDIMED trial was performed using an energy-unrestricted MD, enriched with nuts or extra-virgin oil; the relative risk of cardiovascular events was the reduction of approximately 30% in people who were free of CVD at the beginning of the study, reinforcing the evidence of the MD in the primary prevention of CVD with relevant risk reduction [17]. MD is also effective in reducing the rate of cardiovascular complications after myocardial infarction in the secondary prevention as demonstrated in the Lyon Diet Heart Study where a large reduction in rates of coronary heart disease events was observed with a modified MD enriched with alpha-linolenic acid (a key constituent of walnuts) [19]. More recently, the ATTICA study carried out between 2001 and 2002 on 3024 prevalently male individuals between 20 and 89 years living in the province of Attica (Greece) demonstrated on individuals free of CVD or chronic viral infections that higher the level of adherence to the traditional MD pattern lower the risk of left ventricular systolic dysfunction in patients affected by acute coronary syndrome [20, 21].

#### **3. Effective components contained in the Mediterranean diet**

The apparent ability of the traditional MD to reduce the risk of CVD, cancer, and degenerative diseases development and progression has been attributed, at least in part, to the content of micronutrients and compounds with antithrombotic and antioxidant capacity.

#### **3.1. Antioxidants**

examples of international collaboration in medical research and has represented, over the years, the groundbreaking evidence on the effect of diet on health and in cardiovascular and chronic disease epidemiology. The main findings of the study were the demonstration of a significant association between coronary heart disease (CHD) and diet, particularly positive correlation between the consumption of saturated fatty acids and CHD and relevant inverse relationship between the consumption of monounsaturated fat and CHD. The 15-year mortality follow-up demonstrated an inverse association between coronary deaths and the ratio of the dietary monounsaturated/saturated fats [8–10]. Olive oil has been considered one of the principal components of the MD. Wine, garlic, fish, vegetables, legumes, almonds, and other nuts, other constituents of this dietary pattern, have also been identified to have beneficial effects on health [11, 12]. The data of 15-year follow-up of the Seven Countries Study have been followed up by numerous evidences showing important inverse relationships between

More recent studies have demonstrated that MD and its components may be a powerful aid against certain conditions, such as diabetes, stroke, dementia, colorectal cancer (CRC), and mortality. The greater part of the findings up to now comes from epidemiological studies, even if the cause-effect relationship is not so clear. A recent clinical trial study, based on randomized population, showed the positive outcomes of the MD in the prevention of CVD in individual at a high risk for this disease [17]. A recent study aimed to evaluate the effects of adherence to MD on survival on a large sample of 71,333 Swedish men and women, followed up for 14 years, demonstrated a linear dose-response association between the MD score average and the length of life with the higher score associated with longer survival. The difference in the average length of life between subjects with extremes scores (0 vs. 8) of MD was up to 2 years [18]. The PREDIMED trial was performed using an energy-unrestricted MD, enriched with nuts or extra-virgin oil; the relative risk of cardiovascular events was the reduction of approximately 30% in people who were free of CVD at the beginning of the study, reinforcing the evidence of the MD in the primary prevention of CVD with relevant risk reduction [17]. MD is also effective in reducing the rate of cardiovascular complications after myocardial infarction in the secondary prevention as demonstrated in the Lyon Diet Heart Study where a large reduction in rates of coronary heart disease events was observed with a modified MD enriched with alpha-linolenic acid (a key constituent of walnuts) [19]. More recently, the ATTICA study carried out between 2001 and 2002 on 3024 prevalently male individuals between 20 and 89 years living in the province of Attica (Greece) demonstrated on individuals free of CVD or chronic viral infections that higher the level of adherence to the traditional MD pattern lower the risk of left ventricular systolic dysfunction in patients affected by acute

the MD, and/or its elements, and either CVD or its risk factors [13–16].

118 Superfood and Functional Food - The Development of Superfoods and Their Roles as Medicine

**3. Effective components contained in the Mediterranean diet**

micronutrients and compounds with antithrombotic and antioxidant capacity.

The apparent ability of the traditional MD to reduce the risk of CVD, cancer, and degenerative diseases development and progression has been attributed, at least in part, to the content of

coronary syndrome [20, 21].

It is conceivable that the protective effect of the MD, which guarantees a regular intake of substances with antioxidant activity (ascorbic acid, α-tocopherol, retinol, and β-carotene), estimated that 10–100 mg per day is to be ascribed essentially to its ability to maintain constantly high antioxidant capacity in the blood [22, 23]. The abundance of fruits and vegetables, along with extra-virgin olive oil, red wine, aromatic herbs (oregano, parsley, and rosemary), garlic, onion, and pepper (ingredients generously used in Mediterranean cuisine), offers a number of phenolic compounds with a strong antioxidant action that is hardly possible to achieve with other types of diet. Examples are allyl sulfides, which are present in garlic and raw onions, give cardiovascular benefits, improve cognitive ability, and have chemopreventive activity; it was shown that certain isothiocyanates (degradation products of glucosinolates, compounds present in caper berries) can affect the cell cycle and induce apoptosis in HT-29 human colon cancer cells and other isothiocyanates, present in high concentration in cruciferous vegetables (cabbage and broccoli) [24, 25], have the capacity to modulate the metabolism of carcinogens; kaemferolo and flavonoids quercetin and hydrocinnamic acids from capers have well-known anti-inflammatory and antioxidant effects and chives also rich in phenolic compounds with diuretic, antihypertensive, anti-inflammatory, and antioxidant substances [26–28]; catechins fruit (e.g., apple skin and grape) antioxidant molecules prevent the production of reactive oxygen species generated by oxidative stress; the anthocyanins, plant pigments, give the red or blue color to fruits and vegetables (berries, eggplant, black grapes, and red beet), are antioxidants, photoprotective, and are able to inhibit angiogenesis. One other major constituent of MD is vitamin E, which contains a group of eight isomers: four tocopherols (α, β, γ, δ-tocopherol) and four tocotrienols (α, β, γ, δ-tocotrienol). There are several studies demonstrating healthful effects of α-tocopherol, while little is known on γ-tocopherol, the main form of vitamin E in food. In the last 20 years, much of the supposed beneficial effects of antioxidant vitamins were not confirmed in controlled clinical trials [29–31]. However, it is hard to believe that such vitamins may adverse the development of CVD events when administered in patients with advanced stages of the disease, while a protective effect could be supposed in population in which this nutrient is present throughout the life.

Lycopene, a carotenoid present in tomatoes and tomato products, is a dietary antioxidant that has received great consideration. Epidemiological studies have demonstrated a lower incidence of CVD in those with higher consumption of tomatoes and lycopene, confirmed also by lycopene levels in serum and adipose tissue [32–34]. A protective effect on acute myocardial infarction (AMI) with an odds ratio of 0.75 was found in one of the earlier studies that investigated the serum antioxidant status and lycopene [35]. The most remarkable population-based evidence from a multicenter case-control study (EURAMIC) [36] indicated lycopene levels, and not β-carotene, to be protective against myocardial infarction with an odds ratio of 0.52 comparing the 10th to the 90th percentiles. In the Malaga region, the component of EURAMIC study adipose tissue lycopene levels showed an odds ratio of 0.39 [37]. In Atherosclerosis Risk in Communities (ARIC) case-control study, fasting serum antioxidant levels were inversely related to the intima-media thickness with an odds ratio of 0.81 [38]. Although these epidemiological studies provide convincing evidence for the role of lycopene in CVD prevention, they can only suggest but not prove a causal relationship between lycopene intake and the risk of CVD. Such a proof can be obtained only by performing controlled clinical dietary intervention studies where both the biomarkers of the status of oxidative stress and the disease are measured.

#### **3.2. Polyphenols**

Polyphenols are the most abundant antioxidants in the diet, present in fruits and plant-derived beverages such as fruit juices, tea, coffee, red wine, cereals, chocolates, and dry legumes. The total dietary intake could be as high as 1 g/d; this is 10 times higher than the intake of vitamin C and 100 times higher than the intakes of vitamin E and carotenoids [39].

Despite the wide distribution in plants, the effects of polyphenols on health have come to the consideration of nutritionists only in recent times. Polyphenols and other antioxidants were considered to protect cell constituents against oxidative damage, through scavenging of free radicals for many years. Nowadays, this theory is drastically changed; polyphenols give signals principally through the receptors or enzymes related to signal transduction and the signal may lead to modification of the redox status of the cell, and may activate a series of redox-dependent responses [40]. Many evidences on the prevention of diseases exerted by polyphenols derives from in vitro or animal experiments, which are often done with higher doses than those humans exposed with a regular diet [41, 42].

Epidemiological studies are necessary to establish the effects of polyphenol consumption on CVD [43]. Moreover, it was shown that short- and long-term black tea consumption increases plasma flavonoids and reverses endothelial dysfunction in CVD patients [44].

All these observations suggest that polyphenols can protect vascular damage via antioxidant effects and nitric oxide restoration. However, clinical trials using different antioxidants have failed to demonstrate preventive effects on major CVD events. One imaginable explanation for this discrepancy is that experimental studies are not comparable to real life in humans, although very useful to understand pathophysiological mechanisms [45]. Moreover, antioxidants quantity used in studies conducted in humans may not have been appropriate, and/or the state of disease too severe to evaluate the protective effect that could probably be existent in a preclinical state.

## **3.3. Dietary fiber**

Dietary fiber (DF) has been widely studied and numerous evidences support the health benefits of its consumption. Several prospective studies have demonstrated the inverse association between DF intake and cardiovascular risk. An important pooled analysis of 10 cohort studies demonstrated that DF consumption was inversely related to coronary heart disease. Thence, the introduction of functional foods enriched in DF—alone or in combination with other bioactive compounds—in the diet may represent a useful strategy to improve the cardio-metabolic profile in high-risk subjects preventing cardio-metabolic diseases. The promotion in use of both natural and functional foods might facilitate adherence to a healthy diet with a higher fiber intake compared with the common nutritional conducts of western populations.

Cohort studies have found a consistent protective effect of dietary fibers on glucose control and serum lipoproteins in diabetic patients [46] and in turn on CVD [47].

However, the biologic mechanisms of fibers on the cardiovascular system have yet to be fully elucidated. In the Nurses Health Study, women in the highest quintile of fiber intake had an age-adjusted relative risk for major coronary events that was 47% lower than women in the lowest quintile [48].

Practical recommendations for CVD prevention include food-based approach favoring an increased intake of whole-grain and dietary fiber (especially soluble fiber), fruits, and vegetables providing a mixture of different types of fibers [49].

## **3.4. Unsaturated fatty acids**

risk of CVD. Such a proof can be obtained only by performing controlled clinical dietary intervention studies where both the biomarkers of the status of oxidative stress and the disease

Polyphenols are the most abundant antioxidants in the diet, present in fruits and plant-derived beverages such as fruit juices, tea, coffee, red wine, cereals, chocolates, and dry legumes. The total dietary intake could be as high as 1 g/d; this is 10 times higher than the intake of vitamin

Despite the wide distribution in plants, the effects of polyphenols on health have come to the consideration of nutritionists only in recent times. Polyphenols and other antioxidants were considered to protect cell constituents against oxidative damage, through scavenging of free radicals for many years. Nowadays, this theory is drastically changed; polyphenols give signals principally through the receptors or enzymes related to signal transduction and the signal may lead to modification of the redox status of the cell, and may activate a series of redox-dependent responses [40]. Many evidences on the prevention of diseases exerted by polyphenols derives from in vitro or animal experiments, which are often done with higher

Epidemiological studies are necessary to establish the effects of polyphenol consumption on CVD [43]. Moreover, it was shown that short- and long-term black tea consumption increases

All these observations suggest that polyphenols can protect vascular damage via antioxidant effects and nitric oxide restoration. However, clinical trials using different antioxidants have failed to demonstrate preventive effects on major CVD events. One imaginable explanation for this discrepancy is that experimental studies are not comparable to real life in humans, although very useful to understand pathophysiological mechanisms [45]. Moreover, antioxidants quantity used in studies conducted in humans may not have been appropriate, and/or the state of disease too severe to evaluate the protective effect that could probably be existent

Dietary fiber (DF) has been widely studied and numerous evidences support the health benefits of its consumption. Several prospective studies have demonstrated the inverse association between DF intake and cardiovascular risk. An important pooled analysis of 10 cohort studies demonstrated that DF consumption was inversely related to coronary heart disease. Thence, the introduction of functional foods enriched in DF—alone or in combination with other bioactive compounds—in the diet may represent a useful strategy to improve the cardio-metabolic profile in high-risk subjects preventing cardio-metabolic diseases. The promotion in use of both natural and functional foods might facilitate adherence to a healthy diet with a higher fiber intake compared with the common nutritional conducts of western

plasma flavonoids and reverses endothelial dysfunction in CVD patients [44].

C and 100 times higher than the intakes of vitamin E and carotenoids [39].

120 Superfood and Functional Food - The Development of Superfoods and Their Roles as Medicine

doses than those humans exposed with a regular diet [41, 42].

are measured.

**3.2. Polyphenols**

in a preclinical state.

**3.3. Dietary fiber**

populations.

Dietary sources of n-3 polyunsaturated fatty acids (PUFAs) include fish oils, rich in eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), along with plants rich in a-linolenic acid. Regular consumption of fish, characteristic of the MD, allows satisfying the need for omega-3 fatty acids. PUFAs contained in fish regulate effectively hemostatic factors, cancer, and hypertension, and play a crucial role in the maintenance of neural functions in humans and in the prevention of certain psychiatric disorders; evidence from epidemiological and clinical secondary prevention trials suggests a significant role of fish consumption on long-term (20 year) mortality from coronary heart disease and n-3 PUFA in the prevention of CHD and arrhythmias [50, 51]. Prospective randomized trials show a favorable impact on CV health of both fish and plant sources of n-3 PUFAs. The omega-6 is present mainly in vegetable oils (sunflower and corn oils which, however, should not be cooked since these oils are thermolabile). Among them, the linoleic acid content in nuts, grains, legumes, corn and sunflower oil, synthesized, comes from the gammalinoleic acid (or GLA) [52]. Randomized secondary prevention clinical trials with fish oils and a-linolenic acid have demonstrated a reduction in risk that compare favorably to those seen in landmark secondary prevention trials with lipidlowering drugs. A meta-analysis of randomized trials involving patients with cardiac disease showed that supplementation with the marine n-3 fatty acids EPA and DHA reduced the rate of death from coronary heart disease by 20% [53].

The beneficial effects of olive oil on cardiovascular disease risk factors are now recognized and often attributed only to the high levels of monounsaturated fatty acids (MUFAs). The olive oil is a functional food. Secondary components of olive oil, which constitute only 1–2% of the total virgin olive oil content, are classified into two types: the unsaponifiable fraction, defined as the fraction of the oil extracted after saponification through the use of solvents, and the soluble fraction which includes the phenolic residual. The unsaponifiable fractions of the components are hydrocarbons (squalene), tocopherols, fatty alcohols, triterpene alcohol, 4-methylsterols, sterols, terpene, and other polar compound pigments (chlorophyll and pheophytins). The accumulation of scientific evidence suggests that flavoring and seasoning foods with olive oil bring great health benefits including reducing the risk of coronary heart disease and preventing various cancers (by inhibiting proliferation, inducing apoptosis, and minimizing DNA damage). Also, it appears to have a role in bone mineralization reducing the risk of osteopenia and osteoporosis [54].

## **3.5. Alcohol**

Although the excessive consumption of alcohol must be discouraged due to the significant health damage to individuals and societies [55, 56], increasing evidence shows that moderate consumption of alcoholic beverages may decrease CVD [57]. A dose-response relation between wine intake and vascular risk resulted in a J-shaped curve, with a significant risk reduction at moderate (one to two drinks) consumption (trend analysis *p* = 0.032) [58].

Data derived from PREDIMED demonstrated that moderate red-wine consumption is associated with a lower prevalence of the metabolic syndrome in an elderly Mediterranean population at a high cardiovascular risk [59].

The protective effects of alcohol have been primarily explained by an action on blood lipids (increase in high-density lipoprotein (HDL) levels) and platelets (decreased aggregation) resulting in a reduced rate of coronary artery obstruction [58]. Moderate drinking may improve the early outcomes after AMI and prevent sudden cardiac death, suggesting a direct effect of ethanol on the ischemic myocardium that has been referred to as "ethanol preconditioning" [60].

Moreover, a protective effect of moderate alcohol intake is demonstrated by the Italian Longitudinal Study on Aging (ILSA). In this study, participants with moderate cognitive alterations who consumed approximately 15 g of alcohol a day (moderate drinkers) experienced a decreased rate of progression toward dementia compared to non-drinkers. In the same study, alcohol consumption in older age is associated with healthier hematological values of fibrinogen, HDL cholesterol, Apo A-I lipoprotein, and insulin [61].

## **4. Effects of the Mediterranean diet on health**

#### **4.1. Effects of the Mediterranean diet on cancer and other degenerative diseases**

As life expectancy increases, there are an increased number of elderly individuals suffering from cardiovascular disease, dementia, and cancer.

In sedentary people eating Western-type diets, aging is associated with several chronic diseases, including type 2 diabetes mellitus, cancer, and cardiovascular diseases. About 80% of elderly (over 65 years of age) have at least one chronic disease, and 50% have at least two chronic diseases, with an increase in disability related to comorbidity [62]. Data from epidemiological studies and clinical trials indicate that many age-associated chronic diseases can be prevented, and even reversed, by the implementation of healthy lifestyle interventions [63]. Recent data demonstrate that higher Mediterranean-type diet adherence and higher physical activity were independently associated with a reduced risk for Alzheimer disease [64].

Epidemiological burden of cancer in Mediterranean countries is lower when compared to other states, such as the UK and the USA. There is increasing evidence that Mediterranean dietary adherence reduces the risk of several cancer types and cancer mortality. Particularly, high consumption of fruits and vegetables, whole grains, and little assumption of processed meat, characteristic aspects of the Mediterranean diet, is inversely related to the risk of tumor pathogenesis at different cancer sites. Observational studies provide new evidence suggesting that high adherence to a MD is associated with a reduced risk of overall cancer mortality as well as a reduced risk of incidence of cancers of the colorectum, aerodigestive tract, breast, stomach, pancreas, prostate, liver, and head and neck [65]. A recent review and meta-analysis of 23 observational studies with an overall population of 1,784,404 demonstrated that the highest adherence to MD was significantly associated with a 13% lower risk of all-cause cancer mortality, 17% colorectal cancer, 7% breast cancer, 27% gastric cancer, 4% prostate cancer, 42% liver cancer, 60% head and neck cancer, 52% pancreatic cancer, and 90% respiratory cancer. The meta-analyses confirm a prominent and consistent inverse association provided by adherence to MD in relation to cancer mortality and the risk of several cancer types [66].

**3.5. Alcohol**

tioning" [60].

tion at a high cardiovascular risk [59].

Although the excessive consumption of alcohol must be discouraged due to the significant health damage to individuals and societies [55, 56], increasing evidence shows that moderate consumption of alcoholic beverages may decrease CVD [57]. A dose-response relation between wine intake and vascular risk resulted in a J-shaped curve, with a significant risk

Data derived from PREDIMED demonstrated that moderate red-wine consumption is associated with a lower prevalence of the metabolic syndrome in an elderly Mediterranean popula-

The protective effects of alcohol have been primarily explained by an action on blood lipids (increase in high-density lipoprotein (HDL) levels) and platelets (decreased aggregation) resulting in a reduced rate of coronary artery obstruction [58]. Moderate drinking may improve the early outcomes after AMI and prevent sudden cardiac death, suggesting a direct effect of ethanol on the ischemic myocardium that has been referred to as "ethanol precondi-

Moreover, a protective effect of moderate alcohol intake is demonstrated by the Italian Longitudinal Study on Aging (ILSA). In this study, participants with moderate cognitive alterations who consumed approximately 15 g of alcohol a day (moderate drinkers) experienced a decreased rate of progression toward dementia compared to non-drinkers. In the same study, alcohol consumption in older age is associated with healthier hematological val-

ues of fibrinogen, HDL cholesterol, Apo A-I lipoprotein, and insulin [61].

**4.1. Effects of the Mediterranean diet on cancer and other degenerative diseases**

As life expectancy increases, there are an increased number of elderly individuals suffering

In sedentary people eating Western-type diets, aging is associated with several chronic diseases, including type 2 diabetes mellitus, cancer, and cardiovascular diseases. About 80% of elderly (over 65 years of age) have at least one chronic disease, and 50% have at least two chronic diseases, with an increase in disability related to comorbidity [62]. Data from epidemiological studies and clinical trials indicate that many age-associated chronic diseases can be prevented, and even reversed, by the implementation of healthy lifestyle interventions [63]. Recent data demonstrate that higher Mediterranean-type diet adherence and higher physical activity were independently associated with a reduced risk for Alzheimer disease [64].

Epidemiological burden of cancer in Mediterranean countries is lower when compared to other states, such as the UK and the USA. There is increasing evidence that Mediterranean dietary adherence reduces the risk of several cancer types and cancer mortality. Particularly, high consumption of fruits and vegetables, whole grains, and little assumption of processed

**4. Effects of the Mediterranean diet on health**

from cardiovascular disease, dementia, and cancer.

reduction at moderate (one to two drinks) consumption (trend analysis *p* = 0.032) [58].

122 Superfood and Functional Food - The Development of Superfoods and Their Roles as Medicine

The Healthy Ageing: a Longitudinal Study in Europe (HALE) Study, which evaluated 3496 participants in 10 European countries, reported that individuals between 70 and 90 years who follow up an MD experienced more than 50% reduction in all-cause mortality [67]. The EPIC study designed to clarify the relationship between diet, environmental factors, lifestyles, and the incidence of cancer and other chronic diseases demonstrated in the Spanish cohort that a lower incidence of cancer (12% reduction) is observed in those with a greater adherence to MD after an 8-year follow-up. The EPIC study has also shown that the contemporary consumption of more components of the diet has a greater effect than the single-component assumption [68].

In an epidemiological study investigating the role of both Dietary Inflammatory Index (DII) and Mediterranean Diet Score (MDS), the DII was positively associated with a risk of lung cancer in current smokers while the MDS was inversely associated with lung cancer risk overall (hazard ratio (HR) = 0.64) and for current smokers (HR = 0.38), demonstrating a protective effect even more evident in high-risk patients [69].

The Women's Health Initiative Observational Study assessed the association between diet quality index scores on Healthy Eating Index 2010 (HEI-2010), Alternative HEI-2010, alternative Mediterranean Diet Index, and the Dietary Approaches to Stop Hypertension (DASH and colorectal cancer (1993–2012)), a US study of postmenopausal women. During an average of 12.4 years of follow-up, there were 938 cases of CRC and 238 CRC-specific deaths. Closer adherence to HEI-2010 and DASH dietary recommendations was inversely associated with a risk of CRC in this large cohort of postmenopausal women [70].

Data from PREDIMED find an effect of a long-term dietary intervention on breast cancer incidence, suggesting a beneficial effect of an MD supplemented with extra-virgin olive oil in the primary prevention of breast cancer. The multivariate-adjusted hazard ratios were 0.32 for the MD with extra-virgin olive oil group and 0.59 for the MD with nuts group. In analyses with yearly cumulative updated dietary exposures, the hazard ratio for each additional 5% of calories from extra-virgin olive oil was 0.72 [71]. Mediterranean diet appeared to exert a protective effect also on hip fracture in two Swedish cohort studies consisting of 37,903 men and 33,403 women (total *n* = 71,333, mean age 60 years) free of previous cardiovascular disease and cancer who answered a medical and a food-frequency questionnaire in 1997. One unit increase in modified Mediterranean diet score (mMED; range 0–8 points) was associated with 6% lower hip fracture. Comparing the highest quintile of adherence to the mMED (6–8 points) with the lowest (0–2 points) conferred an adjusted HR of hip fracture of 0.78 [72].

## **4.2. Reduction in caloric intake in lowering incidence of degenerative disease**

A more drastic nutritional interventions and implementation of physical activity programs may have additional beneficial effects on several metabolic and hormonal factors, implicated in the etiology of degenerative diseases and aging [73–75].

The traditional MD means also a diet with a reduced caloric intake, at least referred to the past century; caloric restriction (CR) can be defined as the reduction of all dietary nutrients, except vitamins and minerals (to avoid malnutrition), and has recently emerged as the most promising pro-longevity/anti-aging candidate measure); in fact, it is a highly robust phenomenon capable of slowing aging [76]. Moderate CR can prevent or reverse the damaging effects of visceral obesity, insulin resistance, type 2 diabetes, high blood pressure, dyslipidemia, and inflammation. Energy deficits induced by CR and physical activity in overweight and obese subjects are accompanied by similar improvements in glucose tolerance and insulin action, and similar reductions in several major CHD risk factors, with a loss after 2 years of intervention of 14 kg [77].

CR improves metabolic status also in normal-weight individuals. Data from a series of studies conducted in a group of self-imposed CR (approximately 30% reduction in daily calories) show that a prolonged CR determines sustained beneficial effects on lipid profile, blood pressure, and carotid artery intima-media thickness [78].

Finally, weight loss obtained with an energy deficit of 500–750 kcal per day from their daily energy requirement and exercise was effective in improving the score of physical functioning in obese elderly with frailty, improving, body composition, bone mineral density, physical functions, and quality of life [79].

## **4.3. Dietary supplements**

Dietary supplementation has increased significantly in the last years because of the perception that antioxidant vitamins and minerals may reduce the risk of CVD, cancer, and other chronic diseases. However, no clear evidence in chronic disease prevention is demonstrated for dietary supplements, at least among healthy individuals in the general population [80]. Nevertheless, from a public health perspective, it is extremely important to understand the effects of nutrients on health. At the moment, the use of selenium as supplement in the diet to prevent cardio-metabolic disease is not justified and thus not to be encouraged.

Longitudinal epidemiological studies have led to the identification of recognized functional foods and dietary patterns as beneficial in the primary prevention of cardio-metabolic diseases. The mechanisms by which these foods exert their protective effects are complex and probably related to the macro- and micronutrient contents of the food [81]. The benefits may depend on the clinical status due to risk factors, and state of diseases, may be dose dependent, and may be affected by the food preparation. The benefits of functional food have been reproduced using isolated components of foods as supplements. At the moment, randomized, double-blind, placebo-controlled trials of clinical end points are necessary to establish the efficacy in modifying cardiovascular risk profile in humans.

## **5. Future directions of nutrigenetics and nutrigenomics in the Mediterranean diet**

Increasing evidence enhances the idea that functional foods may improve health status by means of physiologically active components [81]. This area of research is now developing and additional studies are necessary to demonstrate the potential benefit of those foods for which the diet-health relationships are not yet scientifically validated.

A personalized diet based on specific nutrition strategies exerts a pivotal role in the treatment of phenylketonuria, galactosemia, and fructose intolerance, diseases known as "singlegene autosomal recessive disorders." More than 6000 human monogenic disorders have been identified, including over hundreds of protein-based metabolic disorders. Some are rare and complex dietary diseases, namely fatty-acid oxidation disorders, organic acid metabolism disorders, urea cycle defects, and glycogen storage disease. Patients may reduce their intake of the dietary substrates or metabolites that accumulate in these conditions and nutrigenetics will improve prevention and treatment by identifying specific mutations or haplotype combinations that modulate the dietary response in affected patients [81]. In multifactorial pathologies such as CVD, obesity, type 2 diabetes mellitus, cancer, and so on, nutrigenomic studies have shown that dietary intervention may modulate the onset and progression of the disease.

Recently, there has been notable progress in gene-environment interaction evaluation; this field is now accessible to patients to help them to improve their health. Therefore, the current challenge for nutritional genomics is to clarify the role of food and the human microbiota in human health, to better understand the relationship between them and to use this knowledge to promote and preserve a healthy status [82, 83].

## **6. Conclusions**

hip fracture. Comparing the highest quintile of adherence to the mMED (6–8 points) with the

A more drastic nutritional interventions and implementation of physical activity programs may have additional beneficial effects on several metabolic and hormonal factors, implicated

The traditional MD means also a diet with a reduced caloric intake, at least referred to the past century; caloric restriction (CR) can be defined as the reduction of all dietary nutrients, except vitamins and minerals (to avoid malnutrition), and has recently emerged as the most promising pro-longevity/anti-aging candidate measure); in fact, it is a highly robust phenomenon capable of slowing aging [76]. Moderate CR can prevent or reverse the damaging effects of visceral obesity, insulin resistance, type 2 diabetes, high blood pressure, dyslipidemia, and inflammation. Energy deficits induced by CR and physical activity in overweight and obese subjects are accompanied by similar improvements in glucose tolerance and insulin action, and similar reductions in several major CHD risk factors, with a loss after 2 years of interven-

CR improves metabolic status also in normal-weight individuals. Data from a series of studies conducted in a group of self-imposed CR (approximately 30% reduction in daily calories) show that a prolonged CR determines sustained beneficial effects on lipid profile, blood pres-

Finally, weight loss obtained with an energy deficit of 500–750 kcal per day from their daily energy requirement and exercise was effective in improving the score of physical functioning in obese elderly with frailty, improving, body composition, bone mineral density, physical

Dietary supplementation has increased significantly in the last years because of the perception that antioxidant vitamins and minerals may reduce the risk of CVD, cancer, and other chronic diseases. However, no clear evidence in chronic disease prevention is demonstrated for dietary supplements, at least among healthy individuals in the general population [80]. Nevertheless, from a public health perspective, it is extremely important to understand the effects of nutrients on health. At the moment, the use of selenium as supplement in the diet to

Longitudinal epidemiological studies have led to the identification of recognized functional foods and dietary patterns as beneficial in the primary prevention of cardio-metabolic diseases. The mechanisms by which these foods exert their protective effects are complex and probably related to the macro- and micronutrient contents of the food [81]. The benefits may depend on the clinical status due to risk factors, and state of diseases, may be dose dependent, and may be affected by the food preparation. The benefits of functional food have been reproduced using isolated components of foods as supplements. At the moment, randomized,

prevent cardio-metabolic disease is not justified and thus not to be encouraged.

lowest (0–2 points) conferred an adjusted HR of hip fracture of 0.78 [72].

124 Superfood and Functional Food - The Development of Superfoods and Their Roles as Medicine

in the etiology of degenerative diseases and aging [73–75].

sure, and carotid artery intima-media thickness [78].

functions, and quality of life [79].

**4.3. Dietary supplements**

tion of 14 kg [77].

**4.2. Reduction in caloric intake in lowering incidence of degenerative disease**

Scientific research and the wider dissemination of its results made aware the industrialized countries population of the strong connection between nutrition and health, and the role of certain foods and/or their constituents in maintaining this balance. This helps to clarify the role of diet in the prevention and control of morbidity and premature mortality caused by non-communicable diseases. Adaptations to the diet can not only influence today's health but also act in determining whether a person will develop or not, in the course of his/her life, diseases such as cancer, cardiovascular diseases, or diabetes. A healthy diet based on the balance between nutrients represents the first preventive intervention to protect the health and physical harmony. As a result, today, nutrition has new meanings. The concept of food has undergone a radical modification to the point of attributing to each food, in addition to its intrinsic nutritional and sensory properties, an important role in maintaining health and the psycho-physical well-being. Improving eating habits and increasing physical activity levels will reduce the risk of death and disability related to chronic diseases. The practical implications of these recommendations should lead to increased consumption of fruits, vegetables, and fish, and to change the quality of fats and oils, as well as the amount of sugar and starch, by acting as much as possible to match the Mediterranean diet dictates that is found in the Hippocrates famous quote "Let food be thy medicine and let thy medicine be food," the needed action for the twenty-first century population.

## **Author details**

Elisabetta Della Valle<sup>1</sup> , Francesco Cacciatore2 , Eduardo Farinaro1 \*, Francesco Salvatore4 , Roberto Marcantonio1 , Saverio Stranges3 and Maurizio Trevisan<sup>5</sup>

\*Address all correspondence to: elisabetta.dellavalle@unina.it

1 Department of Public Health, Federico II University, Naples, Italy

2 U.O. of Cardiac Rehabilitation, Salvatore Maugeri Foundation, IRCCS, Telese Terme Institute, Benevento, Italy

3 Department of Epidemiology and Biostatistics, Schulich School of Medicine & Dentistry, University of Western Ontario, London, Canada

4 CEINGE-Advanced Biotechnologies, Naples, Italy

5 CUNY School of Medicine, City College of New York, New York, USA

## **References**


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## **Isoflavones: Vegetable Sources, Biological Activity, and Analytical Methods for Their Assessment Isoflavones: Vegetable Sources, Biological Activity, and Analytical Methods for Their Assessment**

Daniela-Saveta Popa and Marius Emil Rusu Daniela-Saveta Popa and Marius Emil Rusu Additional information is available at the end of the chapter

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/66531

#### **Abstract**

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Phytoestrogens are natural compounds found in various plant species and they have the ability to bind to the estrogenic receptors, exerting agonist and/or antagonist effects. The main classes of phytoestrogens are isoflavones, lignans, and coumestranes. Isoflavones are plant bioactive nonsteroidal polyphenolic metabolites with antioxidant properties. They have a very close structure with 17*β*-estradiol and possess estrogenic/antiestrogenic effects. The main dietary source of isoflavones is soy (*Glycine max* L.). Other legumes, such as red clover (*Trifolium pratense* L.), alfalfa (*Medicago sativa* L.), and *Genista* species, have important content in isoflavones, showing nutritional or phytotherapeutic interest. In plants, isoflavones can be found mainly as non-active glycosides which are converted after ingestion, in the corresponding aglycones (e.g., genistein, daidzein) that have pharmacological activity. Many studies have demonstrated the benefits of dietary isoflavones in menopause and multiple chronic pathologies, including cardiovascular diseases, osteoporosis, and hormonal cancers. Dietary intake of isoflavones is widespread, mainly due to the consumption of soybean products. Analytical methods applied for the quantification of isoflavones allow both assessment of dietary intake of isoflavones and highlighting natural sources with phytotherapeutic potential. Health benefits of isoflavones justify the interest for this class of functional food; therefore, further clinical and epidemiological studies are required.

**Keywords:** nutraceuticals, phytoestrogens, isoflavones, vegetables, analysis

## **1. Introduction**

Phytoestrogens are natural nonsteroidal compounds able to bind to estrogenic receptors and have both estrogenic and antiestrogenic activities. They are widespread in the plant kingdom being considered ubiquitous. The main classes of phytoestrogens are isoflavones, coumestans, and lignans.

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2017 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Isoflavones are plant-derived secondary metabolites with a polyphenolic structure and antioxidant properties [1]. They pertain to the flavonoid class and are found mostly in plants belonging to Fabaceae family. Soy (*Glycine max* L.) is the major natural source of isoflavones, and the benefits associated with a soy diet occur mostly because of these phytochemicals. Other natural sources of isoflavones are red clover (*Trifolium pratense* L.), alfalfa (*Medicago sativa* L.), and species of the genus *Genista*. All of these plants present phytotherapeutic and nutraceutical significance, and their by-products, herbal teas, and food supplements are often used.

Several epidemiological studies have demonstrated the benefits of dietary isoflavones in menopause and multiple chronic pathologies, including cardiovascular diseases, osteoporosis, and hormonal cancers. The main mechanisms of action of isoflavones, their benefits to human health, and the factors involved in the modulation of their bioactivity are shown in this chapter. Moreover, the analytical methods used for their quantification in plant and food samples are introduced. These are very important methods to evaluate the human exposure to isoflavones and also to assess the optimum intake for human well-being.

## **2. Characteristics of isoflavones**

## **2.1. Chemistry and metabolism of isoflavones**

Isoflavones (IFs) are yellow pigments derived from 3-phenyl-benzopyrone (3-phenyl-chromone) structure. They are found in plants mostly as biologically inactive glycosides: 7-*O*-β-D-glycosides, 6″-*O*-acetyl-7-*O*-β-D-glucosides, and 6″-*O*-malonyl-7-*O*-β-D-glycosides [1, 2]. After ingestion, glycosides are not bioavailable to be absorbed through enterocytes [3]. They are hydrolyzed into bioactive aglycones by both intestinal mucosa and bacterial *β*-glucosidases from the gut microbiota. Only these forms are absorbed into systemic circulation directly or after subsequent metabolism in the bowel by intestinal bacteria [3]. Soybeans incorporate predominantly genistin, daidzin, and glycitin as inactive glycosides, which are hydrolyzed into their corresponding biologically active aglycones: genistein, daidzein, and glycitein. Other isoflavones observed in legumes are ononin and sissotrin, with their aglycones, formononetin, and biochanin A, respectively (**Figure 1**).

The absorption of aglycones is fast and efficient. Plasmatic isoflavone levels increase up to micromolar-level values after the consumption of soy-based foods, compared to the nanomolar (≤40 nm) levels found in diets without soy [4]. First pharmacokinetic study on isolated and purified isoflavones was performed, when a single dose of 50 mg of aglycone or the equivalent dose of *β*-glycoside, respectively, was given to healthy adult volunteers. The plasmatic peak values (Cmax) were 341 ± 74 ng/mL for genistein and 194 ± 30.6 ng/mL for daidzein. The times when the values reached the peaks were 5.2 and 6.6 hours (tmax) in the case of direct aglycones ingestion and 9.3 and 9.0 h in the case of the ingestion of *β*-glycosides, genistin, and daidzin, due to the time required for their hydrolysation. The bioavailability of genistein and daidzein (based on the area under the curve in plasma concentration *versus* time graph) was higher after consumption of *β*-glycosides [5].

Formononetin and biochanin A can be transformed to daidzein and genistein, respectively, through 4′-*O*-demethylation by the gut microflora or in the liver [6]. Aglycones can be further metabolized through several steps: reduction, deoxygenation, hydroxylation, and C-ring Isoflavones: Vegetable Sources, Biological Activity, and Analytical Methods for Their Assessment http://dx.doi.org/10.5772/66531 135


**Figure 1.** Chemical structure of main isoflavones.

Isoflavones are plant-derived secondary metabolites with a polyphenolic structure and antioxidant properties [1]. They pertain to the flavonoid class and are found mostly in plants belonging to Fabaceae family. Soy (*Glycine max* L.) is the major natural source of isoflavones, and the benefits associated with a soy diet occur mostly because of these phytochemicals. Other natural sources of isoflavones are red clover (*Trifolium pratense* L.), alfalfa (*Medicago sativa* L.), and species of the genus *Genista*. All of these plants present phytotherapeutic and nutraceutical significance, and their by-products, herbal teas, and food supplements are often used.

Several epidemiological studies have demonstrated the benefits of dietary isoflavones in menopause and multiple chronic pathologies, including cardiovascular diseases, osteoporosis, and hormonal cancers. The main mechanisms of action of isoflavones, their benefits to human health, and the factors involved in the modulation of their bioactivity are shown in this chapter. Moreover, the analytical methods used for their quantification in plant and food samples are introduced. These are very important methods to evaluate the human exposure

Isoflavones (IFs) are yellow pigments derived from 3-phenyl-benzopyrone (3-phenyl-chromone) structure. They are found in plants mostly as biologically inactive glycosides: 7-*O*-β-D-glycosides, 6″-*O*-acetyl-7-*O*-β-D-glucosides, and 6″-*O*-malonyl-7-*O*-β-D-glycosides [1, 2]. After ingestion, glycosides are not bioavailable to be absorbed through enterocytes [3]. They are hydrolyzed into bioactive aglycones by both intestinal mucosa and bacterial *β*-glucosidases from the gut microbiota. Only these forms are absorbed into systemic circulation directly or after subsequent metabolism in the bowel by intestinal bacteria [3]. Soybeans incorporate predominantly genistin, daidzin, and glycitin as inactive glycosides, which are hydrolyzed into their corresponding biologically active aglycones: genistein, daidzein, and glycitein. Other isoflavones observed in legumes are ononin and sissotrin, with their aglycones, formonone-

The absorption of aglycones is fast and efficient. Plasmatic isoflavone levels increase up to micromolar-level values after the consumption of soy-based foods, compared to the nanomolar (≤40 nm) levels found in diets without soy [4]. First pharmacokinetic study on isolated and purified isoflavones was performed, when a single dose of 50 mg of aglycone or the equivalent dose of *β*-glycoside, respectively, was given to healthy adult volunteers. The plasmatic peak values (Cmax) were 341 ± 74 ng/mL for genistein and 194 ± 30.6 ng/mL for daidzein. The times when the values reached the peaks were 5.2 and 6.6 hours (tmax) in the case of direct aglycones ingestion and 9.3 and 9.0 h in the case of the ingestion of *β*-glycosides, genistin, and daidzin, due to the time required for their hydrolysation. The bioavailability of genistein and daidzein (based on the area under the curve in plasma concentration *versus* time graph) was

Formononetin and biochanin A can be transformed to daidzein and genistein, respectively, through 4′-*O*-demethylation by the gut microflora or in the liver [6]. Aglycones can be further metabolized through several steps: reduction, deoxygenation, hydroxylation, and C-ring

to isoflavones and also to assess the optimum intake for human well-being.

134 Superfood and Functional Food - The Development of Superfoods and Their Roles as Medicine

**2. Characteristics of isoflavones**

**2.1. Chemistry and metabolism of isoflavones**

tin, and biochanin A, respectively (**Figure 1**).

higher after consumption of *β*-glycosides [5].

cleavage. Daidzein forms *S*-(−)equol and *O*-desmethylangolensin (O-DMA) via dihydrodaidzein (**Figure 2**). Similarly, genistein is metabolized first as dihydrogenistein and then as 5′-hydroxy-equol and p-ethyl phenol (**Figure 2**). Another possible minor pathway is the hydroxylation of isoflavone rings at different positions, catalyzed by hepatic cytochrome P450 isoenzymes [2]. Metabolites with phenolic or polyphenolic structures are conjugated to *O*-glucuronides and sulfate esters during and after absorption through the gut barrier and more intense in the liver. The conjugated metabolites are urinary or biliary excreted and have enterohepatic circulation [4, 7].

Gut microbiota play a very important role in the isoflavone metabolism. The positive effects of a soy-rich diet derive from the existence of microorganisms in the gut capable of intense metabolization of isoflavones. It is the so-called equol producer phenotype, responsible for metabolizing daidzein to equol and identified through the equol/daidzein ratio in the 24-hour urine. Asian people (Japanese, Korean, or Chinese) and Western adult vegetarians are 50–60% equol producers, but equol producers are only 25–30% in Western population. This phenotype is rather stable and cannot be modulated through prebiotic or probiotic nutritional interventions [8]. Otherwise, there are differences between human and animal metabolism, and therefore in vivo results are not relevant to humans [9]. All tested animals had equol in urine after the ingestion of soy or clover [8]. Notably in rodents, equol constitutes 70–90% from the serum isoflavones, compared to humans where only 30% of the daidzein absorbed is metabolized as equol [4].

#### **2.2. Isoflavone content in different sources**

Isoflavones can be found in legumes [10–12], nuts, and some fruits, such as currants and raisins [13], coffee [14], and cereals [15], but the most important dietary sources are soybeans and

**Figure 2.** Metabolic pathways of daidzein and genistein.

their by-products [10, 12]. The content of isoflavones in several plants and foods is presented in **Tables 1** and **2**. Soy can be ingested as textured soy protein, as soy milk or drink, added to many fortified foods (e.g., energized bars, cereals, baby formula), or consumed as fermented soybean products, such as miso, natto, and tempeh (**Table 3**) [12]. Also, many food supplements containing soy isoflavones are on the market [16].

Isoflavone content in plants can vary greatly (up to threefold) for the same variety by growth conditions, geographical areas, years, biotic stress factors (e.g., pests), and abiotic stress factors, such as temperature, nutritional status, or drought [4]. Dietary culture has an especially


**Table 1.** Isoflavone content in selected legumes (mg/100 g, edible portion—the mean value derived from multiple experiments) [12].


their by-products [10, 12]. The content of isoflavones in several plants and foods is presented in **Tables 1** and **2**. Soy can be ingested as textured soy protein, as soy milk or drink, added to many fortified foods (e.g., energized bars, cereals, baby formula), or consumed as fermented soybean products, such as miso, natto, and tempeh (**Table 3**) [12]. Also, many food supple-

Isoflavone content in plants can vary greatly (up to threefold) for the same variety by growth conditions, geographical areas, years, biotic stress factors (e.g., pests), and abiotic stress factors, such as temperature, nutritional status, or drought [4]. Dietary culture has an especially

ments containing soy isoflavones are on the market [16].

136 Superfood and Functional Food - The Development of Superfoods and Their Roles as Medicine

**Figure 2.** Metabolic pathways of daidzein and genistein.

**Table 2.** Coumestrol, Formononetin, and Biochanin A in selected foods (mg/100 g, edible portion—the mean value derived from multiple experiments) [12].


**Table 3.** Isoflavone content in soy foods (mg/100 g, edible portion—the mean value derived from multiple experiments) [12].

big influence on isoflavone content in the diet. Asian and vegetarian diets provide 20–50 mg isoflavones/day, in some cases reaching 100 mg/day, while the Western diet contributes only 0.2–1.5 mg isoflavones/day [2]. Based on recent report of European Food Safety Authority (EFSA), in Europe the dietary isoflavone intake is usually under 1 mg/day, despite an increase in the soy food consumption [17]. The differences between the types of diets refer to the amount of isoflavone in foods, as well as the type of food consumed. In the Western diet, solid processed soy products (such as tofu) and soymilk dominate the diet, and they contain both glycosides (genistin and daidzin which are stable during processing) and aglycones. In the Asian diet, most soy products are obtained through fermentation and have higher amounts of aglycones [3]. Miso, fermented soybean paste (Japan); doenjang, fermented soybean paste (Korea); douchi, fermented soybeans (China); and tempeh, fermented soybean cake (Indonesia) are staple foods in some Asian countries. Simultaneously, health benefit probiotics are formed in these foods during the fermentation processes [18].

Besides soy, other plants in the Fabaceae family have a high content of isoflavones: species of clover, mainly red clover (*Trifolium pratense* L.), alfalfa (*Medicago sativa* L.), and hop clover (*Medicago lupulina* L.), form important part of animal feed. These plants are used in phytotherapy, as medicinal teas or as food supplements. Red clover (*Trifolium pratense* L.) incorporates mainly genistein, daidzein and formononetin, and their respective *β*-glycosides [19–22]. It also contains important quantities of coumestrol, a phytoestrogen part of coumestan class [19, 20], and antioxidant compounds [23, 24]. Data from scientific literature show that red clover extracts can be used as replacement for conventional hormonal therapy in menopause or hormone-dependent diseases [25]. Alfalfa (*Medicago sativa* L.) contains isoflavones (genistein, daidzein, formononetin, biochanin A) in addition to other phytoestrogens (coumestrol) and many nutrients. It is used in phytotherapy for its antianemic, antihemorrhagic, and remineralization properties [26] and for its hypocholesterolemic, antimicrobial, hypolipidemic, antioxidant, antiulcer, neuroprotective, and estrogenic properties [27]. Species of *Genista* (*G. tinctoria* L., *G*. *sagittalis* L.) contain essentially genistin and genistein [19, 20, 28, 29]. They are known for their hypoglycemic [30], anti-inflammatory, antiulcer, spasmolytic, antioxidant, and estrogenic properties [31]. Among these plants, *Genista tinctoria* L. show antioxidant and antitoxic activities [32, 33], protective effect against ultraviolet (UV) radiation, and in vitro melanoma cell proliferation [31].

#### **2.3. Mechanism of estrogen-like action of isoflavones**

According to the xenohormesis theory, plants synthesize phytochemicals to withstand and adapt under stress. Indeed, isoflavone biosynthesis depends on the environmental conditions in which the plant grows and is stimulated by stress. The stress-induced plant compounds have the ability to upregulate stress adaptive pathways in animals and humans. In the body, the biological effects of isoflavones are exercised by modulating pathways mediated by estrogen receptors (ERs) or various key enzymes involved in cellular signaling or metabolism and antioxidant potential [4].

## **3. The estrogenic/antiestrogenic effects**

big influence on isoflavone content in the diet. Asian and vegetarian diets provide 20–50 mg isoflavones/day, in some cases reaching 100 mg/day, while the Western diet contributes only 0.2–1.5 mg isoflavones/day [2]. Based on recent report of European Food Safety Authority (EFSA), in Europe the dietary isoflavone intake is usually under 1 mg/day, despite an increase in the soy food consumption [17]. The differences between the types of diets refer to the amount of isoflavone in foods, as well as the type of food consumed. In the Western diet, solid processed soy products (such as tofu) and soymilk dominate the diet, and they contain both glycosides (genistin and daidzin which are stable during processing) and aglycones. In the Asian diet, most soy products are obtained through fermentation and have higher amounts of aglycones [3]. Miso, fermented soybean paste (Japan); doenjang, fermented soybean paste (Korea); douchi, fermented soybeans (China); and tempeh, fermented soybean cake (Indonesia) are staple foods in some Asian countries. Simultaneously, health benefit pro-

**Table 3.** Isoflavone content in soy foods (mg/100 g, edible portion—the mean value derived from multiple

Soy cheese, unspecified 5.79 11.14 - 25.72 Soy drink 2.75 5.10 - 7.85 Soy flour (textured) 67.69 89.42 20.02 172.55 Soy meal, defatted, raw 80.77 114.71 16.12 209.58 Soy protein drink 27.98 42.91 10.76 81.65 Soy protein isolate 30.81 57.28 8.54 91.05 Soy yogurt 13.77 16.59 2.80 33.17

**Food description Daidzein Genistein Glycitein Total IFs** Miso 16.43 23.24 3.00 41.45 Natto 33.22 37.66 10.55 82.29 Tempeh 22.66 36.15 3.82 60.61

138 Superfood and Functional Food - The Development of Superfoods and Their Roles as Medicine

8.56 12.99 1.98 22.73

20.34 22.57 7.57 48.95

7.41 7.06 4.60 17.92

12.86 18.77 2.88 34.39

40.07 62.18 10.90 109.51

Tofu, raw, regular, prepared with calcium

Soybeans, green, raw (includes edamame)

Soybeans, green, cooked, boiled, drained, without salt (includes edamame)

Soybeans, mature seeds, sprouted, raw

Instant beverage, soy, powder, not reconstituted

experiments) [12].

sulfate

biotics are formed in these foods during the fermentation processes [18].

Isoflavones produce both estrogenic and antiestrogenic effects through several ways. Due to their structure similar to that of 17*β*-estradiol, they have the ability to bind to the nuclear ERs, but their affinity for these receptors is rather weak. Only genistein shows stronger affinity for ER*β* to which it binds preferentially. Its relative affinity (0.87) is closer to that of the reference hormone, 17*β*-estradiol. Daidzein affinity for these receptors is 0.005, but equol, its metabolite, has a 5.7 times stronger affinity, thus increasing its estrogenic potential. The affinity for ERα decreases as follows: genistein > equol > daidzein, with the values of 0.04, 0.005, and 0.001, respectively. The affinities of other isoflavones are less than 0.0001 [2, 4].

Isoflavones induce agonist/antagonist effects depending on the level of the endogenous estrogen. For people with high levels of estrogen, (women premenopause, especially in the follicular phase of the menstrual cycle), the isoflavones bind to the estrogen receptors. Because of their weak estrogen potency, isoflavones exert an antagonist effect. They block the action of endogenous estrogens on their receptors. In case of low concentration of endogenous estrogens (women in menopause, after ovariectomy, or males), the estrogenic action of isoflavones becomes evident, showing additive agonist effect [34]. This is the reason why isoflavones can be used as a long-term complementary or alternative hormone therapy [35].

Isoflavones and their active metabolites can bind to the membrane ERs and induce rapid non-genomic effects by which they modulate cellular metabolism. Thus, they can change the protein kinase and lipid kinase cell signaling pathways [1]. It is believed that the activation of these signaling pathways by isoflavones causes some beneficial effects, in particular in the tissues that are not specific targets for the estrogens. At the circulatory system, the isoflavones induce vasodilation by increasing the production of nitric oxide (NO) after the activation of the endothelial NO− synthase. At the central nervous system, they improve the cognitive function by affecting cell membrane permeability and altering the neuronal excitability. In the skeletal system, the isoflavones inhibit the tyrosine kinase causing changes in the alkaline phosphatase activity. On the other hand, they induce the apoptosis of the osteoclasts, suppress the formation of osteoclasts [34], and prevent the bone demineralization [35].

Also, isoflavones influence the activity of some of the enzymes involved in the metabolism of the sex steroid hormones. In this way they inhibit 5α-reductase (the enzyme responsible for the conversion of testosterone to 5α-dihydrotestosterone) and aromatase (involved in the conversion of testosterone to estradiol) in low concentrations, but they increase the aromatase activity at high concentrations. Isoflavones have an affinity for sex hormone-binding globulin (SHBG) and they induce its expression. Therefore, they affect the free-steroid hormone level in the systemic circulation. But these outcomes depend on many factors, including species, gender, and the hormonal status [35].

Xenoestrogens can modulate the enzyme activity of aromatase. Thus, they induce alterations in the metabolism of fats and carbohydrates through effects on ERα. The decrease of endogenous estrogen levels on ERα, aromatase inhibition or the existence of mutations affecting the enzyme activity has been correlated with visceral obesity or truncate, hyperlipidemia, glucose intolerance and insulin resistance, low physical activity, and reduced energy expenditure. Isoflavones compensate for the deficit of estrogens and have the ability to prevent the associated negative effects. Asian diets, rich in isoflavones, are correlated with low incidence of obesity and metabolic syndrome, favorable plasma profile, and a reduced body mass index in postmenopausal women [4].

## **4. Health benefits of isoflavones**

## **4.1. Isoflavones and their effects on diseases**

Numerous epidemiological and clinical studies have demonstrated the protective role of dietary isoflavones against development of specific menopause symptoms [36–38] and several chronic diseases, including cardiovascular diseases [39, 40], osteoporosis [38], cognitive impairment [37], and hormone-dependent cancers [41–43]. Based on human health benefits of soy diet, the Food and Drug Administration (FDA) approved the use of the following health claim on the labels: "25 grams of soy protein a day, as part of a low in saturated fat and cholesterol, may reduce the risk of heart disease" [44].

Isoflavones, as all polyphenols, have a strong antioxidant activity. They can neutralize free radicals and prevent the lipid peroxydation by stopping the chain reactions. Also, isoflavones induce the antioxidant enzymes (glutathione peroxidase, catalase, and superoxide dismutase) and inhibit the expression of some enzymes, such as xanthine oxidase [1]. The antioxidant protective action of isoflavones from soy or plant extracts, such as *Trifolium pratense* L. or *Genista tinctoria* L., was proven in clinical studies [45, 46], as well as in animal models [32, 47].

## **4.2. Anticarcinogenic activity of isoflavones**

endogenous estrogens on their receptors. In case of low concentration of endogenous estrogens (women in menopause, after ovariectomy, or males), the estrogenic action of isoflavones becomes evident, showing additive agonist effect [34]. This is the reason why isoflavones can

Isoflavones and their active metabolites can bind to the membrane ERs and induce rapid non-genomic effects by which they modulate cellular metabolism. Thus, they can change the protein kinase and lipid kinase cell signaling pathways [1]. It is believed that the activation of these signaling pathways by isoflavones causes some beneficial effects, in particular in the tissues that are not specific targets for the estrogens. At the circulatory system, the isoflavones induce vasodilation by increasing the production of nitric oxide (NO) after the activation of

tion by affecting cell membrane permeability and altering the neuronal excitability. In the skeletal system, the isoflavones inhibit the tyrosine kinase causing changes in the alkaline phosphatase activity. On the other hand, they induce the apoptosis of the osteoclasts, sup-

Also, isoflavones influence the activity of some of the enzymes involved in the metabolism of the sex steroid hormones. In this way they inhibit 5α-reductase (the enzyme responsible for the conversion of testosterone to 5α-dihydrotestosterone) and aromatase (involved in the conversion of testosterone to estradiol) in low concentrations, but they increase the aromatase activity at high concentrations. Isoflavones have an affinity for sex hormone-binding globulin (SHBG) and they induce its expression. Therefore, they affect the free-steroid hormone level in the systemic circulation. But these outcomes depend on many factors, including species,

Xenoestrogens can modulate the enzyme activity of aromatase. Thus, they induce alterations in the metabolism of fats and carbohydrates through effects on ERα. The decrease of endogenous estrogen levels on ERα, aromatase inhibition or the existence of mutations affecting the enzyme activity has been correlated with visceral obesity or truncate, hyperlipidemia, glucose intolerance and insulin resistance, low physical activity, and reduced energy expenditure. Isoflavones compensate for the deficit of estrogens and have the ability to prevent the associated negative effects. Asian diets, rich in isoflavones, are correlated with low incidence of obesity and metabolic syndrome, favorable plasma profile, and a reduced body mass index

Numerous epidemiological and clinical studies have demonstrated the protective role of dietary isoflavones against development of specific menopause symptoms [36–38] and several chronic diseases, including cardiovascular diseases [39, 40], osteoporosis [38], cognitive impairment [37], and hormone-dependent cancers [41–43]. Based on human health benefits of soy diet, the Food and Drug Administration (FDA) approved the use of the following health

press the formation of osteoclasts [34], and prevent the bone demineralization [35].

synthase. At the central nervous system, they improve the cognitive func-

be used as a long-term complementary or alternative hormone therapy [35].

140 Superfood and Functional Food - The Development of Superfoods and Their Roles as Medicine

the endothelial NO−

gender, and the hormonal status [35].

in postmenopausal women [4].

**4. Health benefits of isoflavones**

**4.1. Isoflavones and their effects on diseases**

The anticarcinogenic potential of isoflavones is based on multiple actions: binding to estrogen receptors (ERs), changing of cell signaling pathways, and inhibition of the key enzymes involved in the metabolism of sex hormones. Also, the anticarcinogenic potential of isoflavones has positive effects through independent mechanisms which do not involve ERs, such as antioxidant activity, reduction in the bioactivation of carcinogens, and stimulation of detoxification [2, 48].

Anticarcinogenic activity of genistein has been assessed more thoroughly among isoflavones. Genistein initiates apoptosis, alters cell proliferation and angiogenesis, and inhibits metastasis in many types of cancer cells [49]. It is a tyrosine kinase inhibitor. Therefore, in breast cancer cells, it slows down tumorigenesis; in the circulatory system, it prevents tumor vascularization; in the nervous system, it induces neuroprotective effects. In addition, genistein affects tumorigenesis by inhibiting DNA topoisomerases I and II [50], alteration of epigenetic regulations (both histone methylation and DNA methylation), and activating tumor suppressor genes [51]. As a polyphenol, genistein has antioxidant [1] and anti-inflammatory potential [52]. Another possible action pathway for genistein is the competitive inhibition of estrone metabolism through cytochrome P450 isoenzymes by altering the 2-hydroxy-estrone (2-OH-E1 )/16α-hydroxy-estrone (16α-OH-E1 ) ratio, as noticed in vitro [53]. While 2-OH-E1 is a weak estrogen, 16α-OH-E1 has an important role in carcinogenesis, showing a strong estrogen effect and genotoxic properties [54]. 16α-OH-E1 covalently binds to the estrogenic receptors and thus stimulates cell proliferation [55]. The ratio 2-OH-E1 /16α-OH-E1 has been proposed and studied as a biomarker of breast cancer risk [55–59], but now its significance is controversial. In high concentrations, genistein decreases the hydroxylation of estrone in position 2 in favor of hydroxylation in position 16α [55]. Other studies show that genistein has no mutagenic or clastogenic activity in vivo. But in high concentration of genistein, it has clastogenic potential in vitro, explained by the topoisomerase inhibitory effect, which is known to cause chromosome damage above a certain threshold dose [60].

Anti-proliferative effects of high concentrations of genistein were demonstrated in all breast cancer cells, both ER positive and ER negative. However, there are several studies showing that genistein shows both anti-proliferative and proliferative effects, depending on the concentration, type of tumor, level of endogenous estrogens present in the tissue, or development stage. At low physiological concentrations, genistein stimulates tumorigenesis and cancels the effects of tamoxifen in ER-positive breast cancer cells [50]. Similar dual effects were observed in the case of tamoxifen and other selective estrogen receptor modulators (SERMs) [16].

In fermented soybean products (e.g., natto, miso, tempeh), aglycons can suffer changes under the effect of enzymes produced by the microorganisms involved in the fermentation process. Thus, *ortho*-hydroxygenistein (6-OHG, 8-OHG, 3′-OHG) and *ortho*-hydroxydaidzein (6-OHD, 8-OHD, 3′-OHD) were identified. These compounds are not synthesized by the plants. The hydroxylation reaction that occurs in the *ortho* position gives molecules a high antioxidant potential and a free radical scavenging activity. Moreover, several of their abilities have been proven: to suppress cell proliferation and to inhibit tyrosinase (anti-melanogenesis properties) and antimutagenic, anti-inflammatory, and hepatoprotective properties [18].

Equol has a higher estrogenic potential than daidzein, its precursor, and a preferential affinity for ER*β*, as it has already been stated. This detail is of high interest for its beneficial effect in the treatment of prostate cancer, since both isomers, *S*-(−)equol and *R*-(+)equol, can bind in vivo dihydrotestosterone without having an affinity for the androgen receptor. Therefore, equol prevents the endogenous hormone to exert its stimulating effect on prostate growth. In addition, equol possesses the highest antioxidant capacity of all isoflavones tested. It causes blood vessel relaxation and modifies the inflammatory response in activated macrophages and has beneficial effects in cardiovascular and inflammatory diseases [52].

#### **4.3. Effects of isoflavones on hormone-dependent cancers**

Clinical studies show contradictory results of the efficacy of isoflavones in the treatment of breast cancer. The effects depend on a number of factors such as age, gender, hormonal status, type of isoflavones consumed (soy proteins or isolated isoflavones), dose, diet (type of food), and extent of consumption [2].

A recent meta-analysis of 35 studies shows that soy isoflavones lower the risk of breast cancer in both premenopausal and post-menopausal women. The effect is more evident in Asian women than in those living in Western countries, probably due to differences in quality (traditionally fermented foods) and quantity of the isoflavone products ingested [41]. In Asian women, a diet rich in soy food lowers breast cancer risk with 30% [61]. A higher prevalence of equol-producer phenotype in Asian population can be an essential factor. Equol-producer phenotype is associated with a substantial reduction in the risk of breast cancer. Several specific biomarkers are favorable modified, such as sex hormone-binding globulin (SHBG) and steroid hormone levels in plasma, a higher urinary 2-hydroxy-estrone/16α-hydroxy-estrone ratio, and a lower mammographic breast density [2]. However, because several studies have provided mixed or contradictory results, the general recommendation for patients diagnosed with estrogen-dependent breast cancer is to avoid consuming high quantities of products containing isoflavone. Indeed, isoflavones are selective estrogen receptor modulators (SERMs), and their effects would depend on multiple factors.

Another meta-analysis of five cohort studies that included more than 11,000 female patients diagnosed with breast cancer focused on the post-diagnostic relationship between consumption of soy foods and mortality or cancer recurrence. The study concluded that the ingestion of soy foods reduced mortality and recurrence in all types of breast cancer, especially in the ER-negative, ER-positive/PR-positive, and postmenopausal patients [42]. In women diagnosed with breast cancer under tamoxifen treatment, the consumption of plants containing isoflavones did not alter plasma levels of the drug and its metabolites [62]. Moreover, a recent study shows that a moderate intake of soy isoflavones (5–10 g soy protein/day) would have an optimal effect on tamoxifen treatment on these patients [63].

In some studies [64], excessive consumption of soy was associated with a negative impact on male fertility and reproductive hormones and the disruption of the thyroid gland function. In other studies these effects were inconsistent [65].

Isoflavones can modulate the toxicity of other xenoestrogens, but the interactions are complex and difficult to predict relying only on in vitro steroid receptor affinities [66]. In these kinds of interactions, multiple mechanisms are involved, both estrogen and non-estrogen type, such as oxidative stress [32, 47, 53]. European Food Safety Authority (EFSA) has recently conducted a systematic study of published medical literature, focusing on the correlation between the intake of soy isoflavones and the induced effects on the breast (mammographic density, proliferative marker Ki67 expression), uterus (endometrial thickness, histopathology changes), and thyroid (the thyroid hormone). Results showed that the intake of 35–150 mg isoflavones/ day does not affect these organs in peri- and postmenopausal women [17]. Isoflavones have demonstrated prostate cancer efficacy in several studies: in vitro, on prostate cancer cell lines, in vivo, and in numerous clinical trials [43, 67, 68]. Conclusion of a recent meta-analysis suggests that phytoestrogen intake, mostly genistein and daidzein, can be correlated with a decreased risk of prostate cancer [69].

## **5. Recent advances in analytical methods of isoflavones**

## **5.1. Isolation of isoflavones in foods and vegetable materials**

effects of tamoxifen in ER-positive breast cancer cells [50]. Similar dual effects were observed in the case of tamoxifen and other selective estrogen receptor modulators (SERMs) [16].

In fermented soybean products (e.g., natto, miso, tempeh), aglycons can suffer changes under the effect of enzymes produced by the microorganisms involved in the fermentation process. Thus, *ortho*-hydroxygenistein (6-OHG, 8-OHG, 3′-OHG) and *ortho*-hydroxydaidzein (6-OHD, 8-OHD, 3′-OHD) were identified. These compounds are not synthesized by the plants. The hydroxylation reaction that occurs in the *ortho* position gives molecules a high antioxidant potential and a free radical scavenging activity. Moreover, several of their abilities have been proven: to suppress cell proliferation and to inhibit tyrosinase (anti-melanogenesis proper-

Equol has a higher estrogenic potential than daidzein, its precursor, and a preferential affinity for ER*β*, as it has already been stated. This detail is of high interest for its beneficial effect in the treatment of prostate cancer, since both isomers, *S*-(−)equol and *R*-(+)equol, can bind in vivo dihydrotestosterone without having an affinity for the androgen receptor. Therefore, equol prevents the endogenous hormone to exert its stimulating effect on prostate growth. In addition, equol possesses the highest antioxidant capacity of all isoflavones tested. It causes blood vessel relaxation and modifies the inflammatory response in activated macrophages

Clinical studies show contradictory results of the efficacy of isoflavones in the treatment of breast cancer. The effects depend on a number of factors such as age, gender, hormonal status, type of isoflavones consumed (soy proteins or isolated isoflavones), dose, diet (type of food),

A recent meta-analysis of 35 studies shows that soy isoflavones lower the risk of breast cancer in both premenopausal and post-menopausal women. The effect is more evident in Asian women than in those living in Western countries, probably due to differences in quality (traditionally fermented foods) and quantity of the isoflavone products ingested [41]. In Asian women, a diet rich in soy food lowers breast cancer risk with 30% [61]. A higher prevalence of equol-producer phenotype in Asian population can be an essential factor. Equol-producer phenotype is associated with a substantial reduction in the risk of breast cancer. Several specific biomarkers are favorable modified, such as sex hormone-binding globulin (SHBG) and steroid hormone levels in plasma, a higher urinary 2-hydroxy-estrone/16α-hydroxy-estrone ratio, and a lower mammographic breast density [2]. However, because several studies have provided mixed or contradictory results, the general recommendation for patients diagnosed with estrogen-dependent breast cancer is to avoid consuming high quantities of products containing isoflavone. Indeed, isoflavones are selective estrogen receptor modulators (SERMs),

Another meta-analysis of five cohort studies that included more than 11,000 female patients diagnosed with breast cancer focused on the post-diagnostic relationship between consumption of soy foods and mortality or cancer recurrence. The study concluded that the ingestion

ties) and antimutagenic, anti-inflammatory, and hepatoprotective properties [18].

142 Superfood and Functional Food - The Development of Superfoods and Their Roles as Medicine

and has beneficial effects in cardiovascular and inflammatory diseases [52].

**4.3. Effects of isoflavones on hormone-dependent cancers**

and their effects would depend on multiple factors.

and extent of consumption [2].

In recent years, due to the health benefits provided by isoflavones, higher attention has been paid to the analytical methods that allow identification and quantification of isoflavones from different types of samples: (a) food, for dietary intake assessing [15, 70]; (b) food supplements, for standardization of nutraceuticals [5, 71]; (c) vegetable products, for phytotherapeutic evaluation [19, 20, 28]; and (d) human biological samples (plasma, urine) [5]. These analytical methods are commonly used for isoflavone bioavailability assessing and in pharmacokinetic or pharmacological studies.

Isoflavones are solubilized from food or vegetable material by refluxing or maceration, shaking, and stirring [72]. The isolation of isoflavones from the mixture can be achieved either by conventional methods, liquid-liquid extraction [11, 15, 19] or Soxhlet, or by modern ones supercritical fluid extraction, ultrasound-assisted extraction [19, 71], pressurized fluid extraction, microwave-assisted extraction, and solid-phase extraction [5, 73] (**Table 4**).

The methods used to isolate isoflavones from food are selected function of the nature of the food, the type of the isoflavones analyzed (the total of aglycones or aglycones and


**Table 4.** HPLC and UPLC methods applied for analysis of isoflavones in different samples.

glycosides), and the instrumental method used for identification and quantification. Several examples are presented below.

Liggins et al. isolated isoflavones from cereals and derivatives after a prior sonication in a polar solvent (methanol/water 4:1, v/v), in order to break apart the cellular material, followed by filtration and evaporation of the solvent under nitrogen. In order to determine the total aglycones, glycosides were hydrolyzed in an acid medium (0.1 M acetate buffer, pH 5) by overnight incubation at 37 °C in the presence of cellulase (enzyme used for hydrolytic removal of the hydrolysis resulted carbohydrates). Aglycones were extracted into ethyl acetate and were derivatized and analyzed using GC-MS [15]. Otieno et al. analyzed isoflavones from fermented and unfermented soy milk. For the solubilization of analytes, the freeze-dried sample was refluxed in methanol for 1 hour and filtered, and after adding the internal standard, the solvent has been evaporated to dryness under nitrogen. The residue has been suspended into a buffer (10 mm ammonium acetate containing 0.1% trifluoroacetic acid) and centrifuged, and the supernatant was filtered and analyzed using high-performance liquid chromatography (HPLC) [74].

Extraction and analysis of isoflavones in soybeans can be realized through maceration of the powdered beans with 70% ethanol at room temperature, for 24 hours under constant stirring. After centrifugation and filtering, the supernatant is analyzed directly by HPLC [70]. Also, analysis of isoflavones contained in food supplements requires a simple preparation of the samples: fine powdering of tablets, refluxing in 80% methanol for 1 hour, filtering, and injection into the HPLC system [5].

Hydroalcoholic extracts or tinctures can be prepared from either fresh or dry and pulverized vegetable materials. The hydroalcoholic extracts can be made in 70% ethanol or methanol, by refluxing and filtration; by cold maceration, pressing, and filtration [20]; by percolation [28]; or using modern methods, such as ultrasound-assisted extraction in 50% ethanol [19]. The extracts can be analyzed directly by LC-MS/MS, after an adequate dilution [20], or they can be subjected to an acid hydrolysis [19] in order to release aglycones. Further, the aglycones can be assessed directly or after liquid-liquid extraction, for a concentration of the analytes [19].

In biological samples (e.g., plasma and human urine) isoflavones can be found in different forms: as aglycones (active metabolites), aglycone derivatives (with or without bioactivity), or conjugated metabolites (*β*-glucuronides and sulfate esters). Isoflavone analysis can focus on individual quantification of aglycones and their metabolites or quantification of aglycones after the hydrolysis of conjugated forms. Hydrolysis of conjugated metabolites is achieved by incubation at 37 °C with a mixture of *β*-glucuronidase/sulfatase in the presence of a buffer (0.5 M acetate) at pH 4.5 for several hours or overnight. Isolation of free forms and/or of those freed after hydrolysis can be done by liquid-liquid extraction or solid-phase extraction [5].

## **5.2. Quantification of isoflavones in foods and vegetable materials**

glycosides), and the instrumental method used for identification and quantification. Several

Liggins et al. isolated isoflavones from cereals and derivatives after a prior sonication in a polar solvent (methanol/water 4:1, v/v), in order to break apart the cellular material, followed by filtration and evaporation of the solvent under nitrogen. In order to determine the total aglycones, glycosides were hydrolyzed in an acid medium (0.1 M acetate buffer, pH 5) by overnight incubation at 37 °C in the presence of cellulase (enzyme used for hydrolytic removal of the hydrolysis resulted carbohydrates). Aglycones were extracted into ethyl acetate and were derivatized and analyzed using GC-MS [15]. Otieno et al. analyzed isoflavones from fermented and unfermented soy milk. For the solubilization of analytes, the freeze-dried sample was refluxed in methanol for 1 hour and filtered, and after adding the internal standard, the solvent has been evaporated to dryness under nitrogen. The residue has been suspended into a buffer (10 mm ammonium acetate containing 0.1% trifluoroacetic acid) and centrifuged, and the supernatant was filtered and analyzed using high-performance liquid chromatography (HPLC) [74].

Extraction and analysis of isoflavones in soybeans can be realized through maceration of the powdered beans with 70% ethanol at room temperature, for 24 hours under constant stirring.

examples are presented below.

\*\* UAE, ultrasound-assisted extraction.

\* Cou, coumestrol.
