**Functional Proteins and Peptides of Hen's Egg Origin**

Adham M. Abdou, Mujo Kim and Kenji Sato

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/54003

## **1. Introduction**

114 Bioactive Food Peptides in Health and Disease

International Dairy Journal 2004; 14, 889-898.

properties. Peptides 2009; 30, 1848-1853.

of Dairy Science 1996; 79, 1316-1321.

269-277.

726-731.

[280] Hernández-Ledesma B, Amigo L, Ramos M, Recio I. Release of angiotensin converting enzyme-inhibitory peptides by simulated gastrointestinal digestion of infant formulas.

[281] Quirós A, Contreras MM, Ramos M, Amigo L, Recio I. Stability to gastrointestinal enzymes and structure-activity relationship of -casein-peptides with antihypertensive

[282] Maeno M, Yamamoto N, Takano T. Identification of an antihypertensive peptide from casein hydrolyzate produced by a proteinase from *Lactobacillus helveticus* CP790. Journal

[283] Yamamoto N, Maeno M, Takano T. Purification and characterization of an antihypertensive peptide from a yogurt-like product fermented by *Lactobacillus* 

[284] Gómez-Ruiz JA, Ramos M, Recio I. Identification and formation of angiotensinconverting enzyme-inhibitory peptides in Manchego cheese by high-performance liquid chromatography-tandem mass spectrometry. Journal of Chromatography A, 2004; 1054,

[285] Miguel M, Aleixandre MA, Ramos I, López-Fandiño R. Effect of simulated gastrointestinal digestion on the antihypertensive properties of ACE-inhibitory peptides derived from ovalbumin. Journal of Agricultural and Food Chemistry 2006; 54,

*helveticus* CPN4. Journal of Dairy Science 1999; 82, 1388-1393.

Hen's egg has long history as a food. It contains a great variety of nutrients to sustain both life and growth. Egg provides an excellent, inexpensive and low calorie source of highquality proteins. Moreover, Eggs are a good source of several important nutrients including protein, total fat, monounsaturated fatty acids, polyunsaturated fatty acids, cholesterol, choline, folate, iron, calcium, phosphorus, selenium, zinc and vitamins A, B2, B6, B12, D, E and K [1]. Eggs are also a good source of the antioxidant carotenoids, lutein and zeaxanthin [2]. The high nutritional properties of eggs make them ideal for many people with special dietary requirements.

Egg proteins are nutritionally complete with a good balance of essential amino acids which are needed for building and repairing the cells in muscles and other body tissues [3]. Egg proteins are distributed in all parts of the egg, but most of them are present in the egg white and egg yolk amounting to 50% and 40%, respectively. The remaining amount of protein is in the egg shell and egg shell membranes.

In addition to excellent nutritional value, egg proteins have unique biological activities. Hyperimmunized hens could provide a convenient and economic source of specific immunoglobulin in their yolks (IgY) that have been found to be effective in preventing many bacteria and viruses infections [4]. Proteins in the egg white as lysozyme, ovotransferrin, and avidin have proven to exert numerous biological activities. Moreover, a specific protein in eggshell matrix shows unique activity; enhancement of calcium transportation in the human intestinal epithelial cells.

It is well-known that egg proteins are a source of biologically active peptides. Many researches are aiming to unlock the hidden biological functions of peptides hidden in egg proteins. These peptides are inactive within the sequence of parent proteins and can be released during gastrointestinal digestion or food processing and exerting biological

activities. Once bioactive peptides are liberated, they may act as regulatory compounds, and exhibit various activities such as anti-hypertensive, bone growth promoting, anticancer or exaggerated antimicrobial activities.

Functional Proteins and Peptides of Hen's Egg Origin 117

insoluble but under low ionic strength and acidic conditions, it becomes soluble and can become complex with various metal ions (*e.g.* Ca++, Mg++, Mn++, Co++, Fe++ and Fe+++) [5]. It could be used as a potent natural antioxidant on the basis of its potential to inhibit metalcatalyzed lipid oxidation [7]. The conjugation of egg yolk phosvitin with galactomannan produces a novel macromolecular antioxidant with significantly improved emulsifying activity and emulsion-stabilizing activity [8]. The antibacterial activity of phosvitin has been investigated against *Escherichia coli* and suggested that a significant part of the bactericidal activity of phosvitin could be attributed to the synergistic effects of the high metal-chelating ability and the high surface activity under the influence of thermal stress [9]. Phosvitin and the phosvitin-galactomannan conjugate may represent safe anti-bacterial agents for foods.

A riboflavin-binding protein exists in the egg yolk. It is a hydrophilic phosphoglycoprotein with a molecular weight of 3.6x106 Da, and it can conjugate one mole of riboflavin per mole of apoprotein. Also, biotin and cobalamin-binding proteins could be found in egg yolk.

Livetin is a water-soluble, non-lipid, globular glycoprotein, which is immunologically analogous to the plasma proteins of mammals. α-Livetin is analogous to serum albumin, βlivetin to 2-glycoprotein and γ-livetin to γ-globulin [10]. Most of the research effort has been focused on the immune proteins found in the egg yolk (IgY). Recent advances in IgY

Immunoglobulin from yolk (IgY) is the major antibody found in hen eggs. In 1893, Klemperer first described the acquisition of passive immunity in birds, by demonstrating the transfer of immunity against tetanus toxin from the hen to the chick [11]. Three immunoglobulin classes analogues to the mammalian immunoglobulin classes; IgA, IgM, and IgG, have been shown to exist in chicken. In the egg, IgA and IgM are present in the egg white, while IgG is present in the egg yolk [12]. IgG in egg yolk has been referred to as IgY

The concentration of IgY in the yolk is essentially constant (10-20 mg/mL) through the oocyte maturation. Approximately 100-400 mg IgY is packed in an egg. The concentration of IgY in the yolk is 1.23 times to the serum concentration [14]. A delay of 3 to 4 days is observed for the appearance of specific IgY in yolk after first appearance of specific IgG in

*Composition differences.* General structure of IgY molecule is the same as mammalian IgG with 2 heavy (Hv) chains with a molecular mass of 67–70 kDa each and two light (L) chains

*2.5.1. Structure and Characteristics of Avian IgY Versus Mammalian IgG* 

**2.3. Vitamin-binding protein** 

technology will be discussed.

the serum of a hen.

**2.5. Egg yolk immunoglobulin (IgY)** 

to distinguish it from its mammalian counterpart [13].

**2.4. Livetin** 

For development of bioactive peptides from parent proteins, following techniques have been conventionally used; the establishment of an assay system of biological activities, hydrolysis of proteins by digestive enzymes, isolation of peptides, determination of structures and synthesis of peptides. Recently, bioengineer technique; synthesis of peptides within egg proteins based on sequence similarities of peptides having known biological activity, has been used. The functional characteristics of either natural or modified egg proteins and the use of eggs components as "functional ingredients" are relatively new applications. A truly impressive volume of researches is now available for the egg industry to apply these new applications. Herein, some aspects concerning biologically functional egg proteins or peptides, biochemical and physiological properties as well as possible applications of egg proteins or peptides are discussed.

## **2. Egg yolk bio proteins**

The major portion of egg yolk exists as lipoproteins, which can be separated by centrifugation into a plasma fraction (which remains soluble) and a granular fraction (which precipitates). Lipovitellenin, lipovitellin, phosvitin, livetin, yolk immunoglobulins (IgY), and some minor components have been isolated and identified in egg yolk.

## **2.1. Lipoproteins**

Low density lipoprotein (LDL), which contains between 80 and 90% lipids, characterized by its emulsifying capacity. LDL is the major protein in egg yolk, accounting for 70% of yolk proteins. When LDL is treated with ether, residual fraction is referred to lipovitellenin containing 40% lipid. [5]. Shinohara et al. (1993) studied the effect of some constituents of egg yolk lipoprotein on the growth and IgM production of human-human hybridoma cells and other human-derived cells [6]. LDL-rich fractions were found to enhance the growth and IgM secretion of HB4C5 cells. The promoting activity was found in the commercial LDL.

High density lipoprotein (HDL) or lipovitellin comprise about one sixth of egg yolk solids in the granular yolk proteins. It has a molecular weight of 4X105 and composed of 80% protein and 20% lipid. HDL exists as a complex with a phosphoprotein referred to phosvitin [5]. The addition of one or two eggs a day to a healthy person's diet does not adversely affect lipoprotein levels, and can actually increase plasma HDL levels [1].

## **2.2. Phosvitin**

The name phosvitin comes from both its high phosphorus content (10%) and its source in the egg yolk. The emulsification properties of phosvitin, particularly emulsion-stabilizing activity, were found to be higher than those of other food proteins. Phosvitin is water insoluble but under low ionic strength and acidic conditions, it becomes soluble and can become complex with various metal ions (*e.g.* Ca++, Mg++, Mn++, Co++, Fe++ and Fe+++) [5]. It could be used as a potent natural antioxidant on the basis of its potential to inhibit metalcatalyzed lipid oxidation [7]. The conjugation of egg yolk phosvitin with galactomannan produces a novel macromolecular antioxidant with significantly improved emulsifying activity and emulsion-stabilizing activity [8]. The antibacterial activity of phosvitin has been investigated against *Escherichia coli* and suggested that a significant part of the bactericidal activity of phosvitin could be attributed to the synergistic effects of the high metal-chelating ability and the high surface activity under the influence of thermal stress [9]. Phosvitin and the phosvitin-galactomannan conjugate may represent safe anti-bacterial agents for foods.

### **2.3. Vitamin-binding protein**

A riboflavin-binding protein exists in the egg yolk. It is a hydrophilic phosphoglycoprotein with a molecular weight of 3.6x106 Da, and it can conjugate one mole of riboflavin per mole of apoprotein. Also, biotin and cobalamin-binding proteins could be found in egg yolk.

### **2.4. Livetin**

116 Bioactive Food Peptides in Health and Disease

exaggerated antimicrobial activities.

**2. Egg yolk bio proteins** 

**2.1. Lipoproteins** 

LDL.

**2.2. Phosvitin** 

applications of egg proteins or peptides are discussed.

some minor components have been isolated and identified in egg yolk.

lipoprotein levels, and can actually increase plasma HDL levels [1].

activities. Once bioactive peptides are liberated, they may act as regulatory compounds, and exhibit various activities such as anti-hypertensive, bone growth promoting, anticancer or

For development of bioactive peptides from parent proteins, following techniques have been conventionally used; the establishment of an assay system of biological activities, hydrolysis of proteins by digestive enzymes, isolation of peptides, determination of structures and synthesis of peptides. Recently, bioengineer technique; synthesis of peptides within egg proteins based on sequence similarities of peptides having known biological activity, has been used. The functional characteristics of either natural or modified egg proteins and the use of eggs components as "functional ingredients" are relatively new applications. A truly impressive volume of researches is now available for the egg industry to apply these new applications. Herein, some aspects concerning biologically functional egg proteins or peptides, biochemical and physiological properties as well as possible

The major portion of egg yolk exists as lipoproteins, which can be separated by centrifugation into a plasma fraction (which remains soluble) and a granular fraction (which precipitates). Lipovitellenin, lipovitellin, phosvitin, livetin, yolk immunoglobulins (IgY), and

Low density lipoprotein (LDL), which contains between 80 and 90% lipids, characterized by its emulsifying capacity. LDL is the major protein in egg yolk, accounting for 70% of yolk proteins. When LDL is treated with ether, residual fraction is referred to lipovitellenin containing 40% lipid. [5]. Shinohara et al. (1993) studied the effect of some constituents of egg yolk lipoprotein on the growth and IgM production of human-human hybridoma cells and other human-derived cells [6]. LDL-rich fractions were found to enhance the growth and IgM secretion of HB4C5 cells. The promoting activity was found in the commercial

High density lipoprotein (HDL) or lipovitellin comprise about one sixth of egg yolk solids in the granular yolk proteins. It has a molecular weight of 4X105 and composed of 80% protein and 20% lipid. HDL exists as a complex with a phosphoprotein referred to phosvitin [5]. The addition of one or two eggs a day to a healthy person's diet does not adversely affect

The name phosvitin comes from both its high phosphorus content (10%) and its source in the egg yolk. The emulsification properties of phosvitin, particularly emulsion-stabilizing activity, were found to be higher than those of other food proteins. Phosvitin is water Livetin is a water-soluble, non-lipid, globular glycoprotein, which is immunologically analogous to the plasma proteins of mammals. α-Livetin is analogous to serum albumin, βlivetin to 2-glycoprotein and γ-livetin to γ-globulin [10]. Most of the research effort has been focused on the immune proteins found in the egg yolk (IgY). Recent advances in IgY technology will be discussed.

#### **2.5. Egg yolk immunoglobulin (IgY)**

Immunoglobulin from yolk (IgY) is the major antibody found in hen eggs. In 1893, Klemperer first described the acquisition of passive immunity in birds, by demonstrating the transfer of immunity against tetanus toxin from the hen to the chick [11]. Three immunoglobulin classes analogues to the mammalian immunoglobulin classes; IgA, IgM, and IgG, have been shown to exist in chicken. In the egg, IgA and IgM are present in the egg white, while IgG is present in the egg yolk [12]. IgG in egg yolk has been referred to as IgY to distinguish it from its mammalian counterpart [13].

The concentration of IgY in the yolk is essentially constant (10-20 mg/mL) through the oocyte maturation. Approximately 100-400 mg IgY is packed in an egg. The concentration of IgY in the yolk is 1.23 times to the serum concentration [14]. A delay of 3 to 4 days is observed for the appearance of specific IgY in yolk after first appearance of specific IgG in the serum of a hen.

#### *2.5.1. Structure and Characteristics of Avian IgY Versus Mammalian IgG*

*Composition differences.* General structure of IgY molecule is the same as mammalian IgG with 2 heavy (Hv) chains with a molecular mass of 67–70 kDa each and two light (L) chains

with the molecular mass of 25 kDa each (Figure 1). The major difference is the number of constant regions (C) in H chains: IgG has 3 C regions (Cy1–Cy3), while IgY has 4 C regions (Cv1–Cv4). Due to occurrence of one additional C region with two corresponding carbohydrate chains, molecular mass of IgY (180 kDa) is larger than *mammalian* IgG (150 kDa). IgY is less flexible than mammalian IgG due to the absence of the hinge between Cy1 and Cy2. There are some regions in IgY (near the boundaries of Cv1–Cv2 and Cv2–Cv3) containing proline and glycine residues enabling only limited flexibility. IgY has isoelectric point 5.7–7.6 and is more hydrophobic than IgG [13, 15].

Functional Proteins and Peptides of Hen's Egg Origin 119

and animals. Eggs are normal dietary components and there is practically no risk of toxic side effects of IgY given orally. As mentioned above, IgY does not activate mammalian complement system nor interact with mammalian Fc-receptors that could mediate

On the basis of these facts, IgY has been used for suppression of growth of food-bone pathogens [11]. Whole egg yolks and water soluble fractions were prepared from the egg of the hens that had been immunized with pathogens such as *E. coli* O157:H7, *Salmonella enteritidis*, *Salmonella typhimurium*, *Campylobacter jejuni*, *Staphylococcus aureus*, and *Listeria monocytogenes*. It has been demonstrated that pathogen-specific IgY is bound to the surface of bacteria, resulting in structural alterations of cell wall and consequently kills bacteria. Sarker et al. (2001) performed a study for children with proven rotavirus diarrhea. The patients were treated with IgY from the eggs of chickens immunized with human rotavirus strains [22]. The treatment moderated diarrhea, which was characterized by an earlier clearance of rotavirus from the stools. Recently, IgY has been applied to cancer therapy. Hens were immunized with an antigen purified from human stomach cancer cells. The purified IgY recognized gastrointestinal cancer cells. Conjugation of antibodies and drugs

These IgY treatments have been shown to provide a safer, more efficient and less expensive method than those using conventional mammalian antibiotics for managing disease-causing pathogens. Recently, successful progresses in industrialization of IgY has been achieved in Japan, where IgY as a bioactive ingredient in food, nutraceuticals, cosmetics and other

*Helicobacter pylori*, a spiral gram-negative microaerophilic pathogen, has been shown to be a common inhabitant of the gastric and duodenal mucosa. The microorganism is recognized as one of the most prevalent human pathogens. It infects over 50% of the population worldwide [24], and is recognized as the etiologic agent of gastritis, peptic ulcer, and has been linked to the development of gastric adenocarcinoma and mucosa associated lymphoid tissue lymphoma [25, 26]. The eradication of *H. pylori* by administration of oral antimicrobials is not always successful and may be associated with adverse effects [27]. For colonization in gastric mucosa, *H. pylori* abundantly produces urease enzyme, which degrades urea into ammonia. *Helicobacter pylori* organism uses the ammonia to neutralize microenvironment in gastric mucosa. Accordingly, a novel approach in prevention and reduction of *H. pylori* infection using urease-specific IgY has been developed. It has been reported that oral administration of anti-*H. pylori* urease IgY (IgY-urease) could suppress the

*Preparation of IgY-Urease Yogurt.* Today, consumers prefer foods that promote good health and could reduce risk of diseases. Dairy products are excellent media to generate an array of products that fit into the current consumer demand for functional foods [28]. Scientific and clinical evidence is mounting to corroborate the consumer perception of health from yogurt. Designing a yogurt fortified with IgY-urease could supply passive immunization with a

inflammatory response in the gastrointestinal tract.

may be an important agent for cancer treatment [23].

*2.5.2.1. Anti-Helicobacter pylori IgY* 

bacterial colonization.

sectors is applied . The followings are the most recent applications.

**Figure 1.** Structure of IgG and IgY.

*Advantage of IgY.* Most biological effectors' functions of immunoglobulin are activated by the Fc region, where the major structural difference between IgG and IgY is located. Therefore, Fc-dependent functions of IgY are essentially different from those of mammalian IgG. First, IgY does not activate the complement system[16], second, IgY does not bind to protein-A and G [17], third, IgY is not recognized by mammalian antibodies [18] *i.e*. rheumatoid factors (RF, an autoantibody reacting with the Fc portion of IgG) or HAMA (human antimurine antibodies), and fourth, it does not bind to cell surface Fc receptor [19]. These differences in molecular interactions bring great advantages to the application of IgY antibodies. Then that were IgY has been successfully applied into a variety of methods in different areas of research, diagnostics, and medical areas. For these applications, IgY can successfully compete with antibodies (IgG) isolated from the blood of mammals [20]. The advantage of usage of the immunized hen is that it produces a large number of eggs. Approximately 40g of IgY could be collected from egg of one hen each year, compared with about 1.3g from blood of one rabbit [21]. An industrial scale production of IgY is possible because of the availability of large number of chicken farms and automation of egg breaking and processing.

#### *2.5.2. Applications and uses of IgY*

Oral administration of antibodies specific to host pathogens is an attractive approach to establish protective immunity, especially against gastrointestinal pathogens both in human and animals. Eggs are normal dietary components and there is practically no risk of toxic side effects of IgY given orally. As mentioned above, IgY does not activate mammalian complement system nor interact with mammalian Fc-receptors that could mediate inflammatory response in the gastrointestinal tract.

On the basis of these facts, IgY has been used for suppression of growth of food-bone pathogens [11]. Whole egg yolks and water soluble fractions were prepared from the egg of the hens that had been immunized with pathogens such as *E. coli* O157:H7, *Salmonella enteritidis*, *Salmonella typhimurium*, *Campylobacter jejuni*, *Staphylococcus aureus*, and *Listeria monocytogenes*. It has been demonstrated that pathogen-specific IgY is bound to the surface of bacteria, resulting in structural alterations of cell wall and consequently kills bacteria. Sarker et al. (2001) performed a study for children with proven rotavirus diarrhea. The patients were treated with IgY from the eggs of chickens immunized with human rotavirus strains [22]. The treatment moderated diarrhea, which was characterized by an earlier clearance of rotavirus from the stools. Recently, IgY has been applied to cancer therapy. Hens were immunized with an antigen purified from human stomach cancer cells. The purified IgY recognized gastrointestinal cancer cells. Conjugation of antibodies and drugs may be an important agent for cancer treatment [23].

These IgY treatments have been shown to provide a safer, more efficient and less expensive method than those using conventional mammalian antibiotics for managing disease-causing pathogens. Recently, successful progresses in industrialization of IgY has been achieved in Japan, where IgY as a bioactive ingredient in food, nutraceuticals, cosmetics and other sectors is applied . The followings are the most recent applications.

#### *2.5.2.1. Anti-Helicobacter pylori IgY*

118 Bioactive Food Peptides in Health and Disease

**Figure 1.** Structure of IgG and IgY.

and processing.

*2.5.2. Applications and uses of IgY* 

point 5.7–7.6 and is more hydrophobic than IgG [13, 15].

with the molecular mass of 25 kDa each (Figure 1). The major difference is the number of constant regions (C) in H chains: IgG has 3 C regions (Cy1–Cy3), while IgY has 4 C regions (Cv1–Cv4). Due to occurrence of one additional C region with two corresponding carbohydrate chains, molecular mass of IgY (180 kDa) is larger than *mammalian* IgG (150 kDa). IgY is less flexible than mammalian IgG due to the absence of the hinge between Cy1 and Cy2. There are some regions in IgY (near the boundaries of Cv1–Cv2 and Cv2–Cv3) containing proline and glycine residues enabling only limited flexibility. IgY has isoelectric

*Advantage of IgY.* Most biological effectors' functions of immunoglobulin are activated by the Fc region, where the major structural difference between IgG and IgY is located. Therefore, Fc-dependent functions of IgY are essentially different from those of mammalian IgG. First, IgY does not activate the complement system[16], second, IgY does not bind to protein-A and G [17], third, IgY is not recognized by mammalian antibodies [18] *i.e*. rheumatoid factors (RF, an autoantibody reacting with the Fc portion of IgG) or HAMA (human antimurine antibodies), and fourth, it does not bind to cell surface Fc receptor [19]. These differences in molecular interactions bring great advantages to the application of IgY antibodies. Then that were IgY has been successfully applied into a variety of methods in different areas of research, diagnostics, and medical areas. For these applications, IgY can successfully compete with antibodies (IgG) isolated from the blood of mammals [20]. The advantage of usage of the immunized hen is that it produces a large number of eggs. Approximately 40g of IgY could be collected from egg of one hen each year, compared with about 1.3g from blood of one rabbit [21]. An industrial scale production of IgY is possible because of the availability of large number of chicken farms and automation of egg breaking

Oral administration of antibodies specific to host pathogens is an attractive approach to establish protective immunity, especially against gastrointestinal pathogens both in human *Helicobacter pylori*, a spiral gram-negative microaerophilic pathogen, has been shown to be a common inhabitant of the gastric and duodenal mucosa. The microorganism is recognized as one of the most prevalent human pathogens. It infects over 50% of the population worldwide [24], and is recognized as the etiologic agent of gastritis, peptic ulcer, and has been linked to the development of gastric adenocarcinoma and mucosa associated lymphoid tissue lymphoma [25, 26]. The eradication of *H. pylori* by administration of oral antimicrobials is not always successful and may be associated with adverse effects [27]. For colonization in gastric mucosa, *H. pylori* abundantly produces urease enzyme, which degrades urea into ammonia. *Helicobacter pylori* organism uses the ammonia to neutralize microenvironment in gastric mucosa. Accordingly, a novel approach in prevention and reduction of *H. pylori* infection using urease-specific IgY has been developed. It has been reported that oral administration of anti-*H. pylori* urease IgY (IgY-urease) could suppress the bacterial colonization.

*Preparation of IgY-Urease Yogurt.* Today, consumers prefer foods that promote good health and could reduce risk of diseases. Dairy products are excellent media to generate an array of products that fit into the current consumer demand for functional foods [28]. Scientific and clinical evidence is mounting to corroborate the consumer perception of health from yogurt. Designing a yogurt fortified with IgY-urease could supply passive immunization with a

natural and highly specific attempt to decrease the *H. pylori* infection. In order to suppress *H. pylori* infection, a yogurt fortified with IgY-urease has been designed and developed. Three clinical studies were done to examine the efficacy of a specially designed functional yogurt containing IgY-urease on the suppression of *H. pylori* in humans. IgY-urease containing yogurt (plain and drinking) have been prepared and in markets in Japan, Korea, and Taiwan (Figure 2). IgY-urease was pasteurized and then added to yogurt mix at specific dose after all heat-treatment steps. Yogurts were cooled and stored at 4° C for up to 3 weeks. IgY-urease activity remained in the product throughout the 3 weeks of storage.

Functional Proteins and Peptides of Hen's Egg Origin 121

*Clinical studies*. Plain yogurt containing 2g IgY-urease egg yolk was produced commercially in Japan. A clinical study was conducted to determine the effect of IgYurease yogurt to decrease *H. pylori* in humans [29]. To assess presence of *H. pylori* in stomach, UBT method has been extensively used. This method based on the invasive detection of exhaled 13C-labeled carbon dioxide resulting from *H. pylori* urease activity [30]. One hundred seventy-four volunteers were screened using a 13C-urea breath test (UBT). Heavily infected volunteers (with UBT values over 30‰) were selected (16 subjects) and recruited. Each volunteer consumed 1 cups of yogurt twice daily (4 g/d egg yolk containing 40 mg IgY-urease) for 12 wk. Volunteers were tested after 4, 8 and 12 weeks. The UBT values obtained at week 8 and 12 were significantly different from those obtained at week 0 (P < 0.001), showing a 55.1% and 57.2% reduction in UBT values after 8 and 12 weeks, respectively (Figure 4). Other clinical studies using IgY-urease containing drinking yogurt were carried out in Taiwan [31] and Korea [32] showed nearly similar

The three different studies demonstrated that administration of a specially designed yogurt with highly specific antibodies from egg yolk could effectively decreases number of *H. pylori*  in humans. During the study period of the three clinical studies, the ingestion regimen was

The use of probiotics for the suppression of *H. pylori* in humans has been studied by some investigators [33, 34]. However, none of these studies were able to show a significant suppression of *H. pylori* in humans, and others showed a slight but no significant trend toward a suppressive effect of drinking yogurt containing specific lactic acid bacteria. Anti-*H. pylori* effect of yogurt containing specific lactic acid bacteria has been examined. However, no significant reduction of *H. pylori* in human stomach has been observed,

The use of IgY against a pathogenic factor of *H. pylori* would be a prudent way to suppress the infection. It was demonstrated that IgY-urease was highly specific and had a significant effectiveness against *H. pylori* because of its ability to inhibit *H. pylori* from adhering to the gastric mucosa [4, 37, 38]. Because IgY-urease binds urease only, the functional efficacy observed was presumably via capture of bacterium-associated urease within the gastric mucus layer, which resulting in bacterial aggregation and clearance via the constant washing action of the gut. By such a mechanism, consumption of IgY-urease yogurt may play a dual role in suppression and prophylaxis against *H. pylori* in humans

These findings opened new gate of applications of IgY-urease against *H. pylori* in the food industry to prevent *H. pylori*. Recently, a specially designed egg containing IgY-urease was produced in Japan. This egg has been on the Japanese market under a trade name of "stomach friendly egg". Moreover, IgY-urease was incorporated in neutraceutical formulations that launched recently in the Japanese market aiming to prevent and reduce *H.* 

well-tolerated and no adverse effects or any complications were observed.

although trend for decrease was observed [35, 36].

results.

(Figure 4).

*pylori* infection.

**Figure 2.** Different IgY-urease yogurt products in Japan, Korea, and Taiwan.

**Figure 3.** UBT Value Change of volunteers(clinical study in Japan).

*Clinical studies*. Plain yogurt containing 2g IgY-urease egg yolk was produced commercially in Japan. A clinical study was conducted to determine the effect of IgYurease yogurt to decrease *H. pylori* in humans [29]. To assess presence of *H. pylori* in stomach, UBT method has been extensively used. This method based on the invasive detection of exhaled 13C-labeled carbon dioxide resulting from *H. pylori* urease activity [30]. One hundred seventy-four volunteers were screened using a 13C-urea breath test (UBT). Heavily infected volunteers (with UBT values over 30‰) were selected (16 subjects) and recruited. Each volunteer consumed 1 cups of yogurt twice daily (4 g/d egg yolk containing 40 mg IgY-urease) for 12 wk. Volunteers were tested after 4, 8 and 12 weeks. The UBT values obtained at week 8 and 12 were significantly different from those obtained at week 0 (P < 0.001), showing a 55.1% and 57.2% reduction in UBT values after 8 and 12 weeks, respectively (Figure 4). Other clinical studies using IgY-urease containing drinking yogurt were carried out in Taiwan [31] and Korea [32] showed nearly similar results.

120 Bioactive Food Peptides in Health and Disease

natural and highly specific attempt to decrease the *H. pylori* infection. In order to suppress *H. pylori* infection, a yogurt fortified with IgY-urease has been designed and developed. Three clinical studies were done to examine the efficacy of a specially designed functional yogurt containing IgY-urease on the suppression of *H. pylori* in humans. IgY-urease containing yogurt (plain and drinking) have been prepared and in markets in Japan, Korea, and Taiwan (Figure 2). IgY-urease was pasteurized and then added to yogurt mix at specific dose after all heat-treatment steps. Yogurts were cooled and stored at 4° C for up to 3 weeks.

IgY-urease activity remained in the product throughout the 3 weeks of storage.

**Figure 2.** Different IgY-urease yogurt products in Japan, Korea, and Taiwan.

**Figure 3.** UBT Value Change of volunteers(clinical study in Japan).

The three different studies demonstrated that administration of a specially designed yogurt with highly specific antibodies from egg yolk could effectively decreases number of *H. pylori*  in humans. During the study period of the three clinical studies, the ingestion regimen was well-tolerated and no adverse effects or any complications were observed.

The use of probiotics for the suppression of *H. pylori* in humans has been studied by some investigators [33, 34]. However, none of these studies were able to show a significant suppression of *H. pylori* in humans, and others showed a slight but no significant trend toward a suppressive effect of drinking yogurt containing specific lactic acid bacteria. Anti-*H. pylori* effect of yogurt containing specific lactic acid bacteria has been examined. However, no significant reduction of *H. pylori* in human stomach has been observed, although trend for decrease was observed [35, 36].

The use of IgY against a pathogenic factor of *H. pylori* would be a prudent way to suppress the infection. It was demonstrated that IgY-urease was highly specific and had a significant effectiveness against *H. pylori* because of its ability to inhibit *H. pylori* from adhering to the gastric mucosa [4, 37, 38]. Because IgY-urease binds urease only, the functional efficacy observed was presumably via capture of bacterium-associated urease within the gastric mucus layer, which resulting in bacterial aggregation and clearance via the constant washing action of the gut. By such a mechanism, consumption of IgY-urease yogurt may play a dual role in suppression and prophylaxis against *H. pylori* in humans (Figure 4).

These findings opened new gate of applications of IgY-urease against *H. pylori* in the food industry to prevent *H. pylori*. Recently, a specially designed egg containing IgY-urease was produced in Japan. This egg has been on the Japanese market under a trade name of "stomach friendly egg". Moreover, IgY-urease was incorporated in neutraceutical formulations that launched recently in the Japanese market aiming to prevent and reduce *H. pylori* infection.

Functional Proteins and Peptides of Hen's Egg Origin 123

effectiveness of IgY with specificity to *S. mutans* prevented the colonization of mutans streptococci in the oral cavity of humans [43]. Recently, food products such as candies, chocolates and gums containing fourth- or anti-*S. mutans* IgY have been launched in the

Influenza caused by a virus is called the influenza virus. Influenza or "flu" is an infection of the respiratory tract that can affect millions of people every year. It is highly contagious and occurs mainly in the late fall, winter, or early spring. Influenza is spread from person-toperson through mists or sprays of infectious respiratory secretions caused by coughing and sneezing. Influenza affects all age groups and causes severe illness, loss of school and work,

Recently, specific anti-influenza IgY was successfully produced from hens immunized with inactivated influenza virus strain. This specific IgY significantly reacts virus in vitro [44]. Subsequently, an anti-influenza IgY biofilter which trap the influenza virus has been developed by Daikin Environment Laboratories, Japan, a research arm of Daikin Industries, in cooperation with five Japanese research institutions. It was found that 99.99% of the influenza virus sprayed over the biofilter was captured within 10 minutes. An air cleaner with anti-influenza filter is recently launched in Japan (Figure 5 and 6). Moreover, a facemask with anti-influenza IgY was developed and will be available in the Japanese

Japanese market for oral care [37].

market.

*2.5.2.3. Anti-Influenza virus IgY (Anti-influenza biofilter)* 

**Figure 5.** Diagram for the Anti-influenza IgY biofilter.

and complications such as pneumonia, hospitalization, and death.

**Figure 4.** Suppressive mechanism of anti-*H. pylori* urease IgY.

#### *2.5.2.2. Anti-Streptococcus mutans IgY*

Dental caries is still one of the most widespread diseases of mankind. Human are frequently infected with cariogenic microorganisms in early life. The cariogenic microorganisms survive in dental biofilm and can emerge under favorable environmental condition and consequently cause dental disease [39]. *Streptococcus mutans* is the main etiologic agent of dental caries and that infection is transmissible [40]. Abilities of mutants' streptococci to adhere tooth surface in the presence of sucrose and release acids by fermention play a significant role in development of dental caries [41]. Initial attachment of *S. mutans* to the saliva-coated enamel surface occurs through the surface protein of *S. mutans*. For the colonization of *S. mutans*, synthesis of water-insoluble and adherent glucan from sucrose by the glucosyl transferases (GTases) is essential. *Streptococcus mutans* produces both cellassociated (CA) and cell-free (CF) forms of GTase; the former primarily synthesizes waterinsoluble glucan, while the latter produces water soluble glucan. The combined action of these two GTases on the cell surface of *S. mutans* during its growth in the presence of sucrose is critically important in allowing firm adherence. The GTase system of *S. mutans* has therefore been considered an important virulence factor promoting caries development. Administration of IgY against *S. mutans* CA-GTase specifically inhibited insoluble glucansynthesizing CA-GTase, resulting in a significant reduction in the development of dental caries. Otake et al. (1991) reported that anti-*S. mutans* CA-GTase IgY suppressed development of dental carries in rat model [42]. Hatta et al. (1997) reported that the effectiveness of IgY with specificity to *S. mutans* prevented the colonization of mutans streptococci in the oral cavity of humans [43]. Recently, food products such as candies, chocolates and gums containing fourth- or anti-*S. mutans* IgY have been launched in the Japanese market for oral care [37].

#### *2.5.2.3. Anti-Influenza virus IgY (Anti-influenza biofilter)*

122 Bioactive Food Peptides in Health and Disease

**Figure 4.** Suppressive mechanism of anti-*H. pylori* urease IgY.

Dental caries is still one of the most widespread diseases of mankind. Human are frequently infected with cariogenic microorganisms in early life. The cariogenic microorganisms survive in dental biofilm and can emerge under favorable environmental condition and consequently cause dental disease [39]. *Streptococcus mutans* is the main etiologic agent of dental caries and that infection is transmissible [40]. Abilities of mutants' streptococci to adhere tooth surface in the presence of sucrose and release acids by fermention play a significant role in development of dental caries [41]. Initial attachment of *S. mutans* to the saliva-coated enamel surface occurs through the surface protein of *S. mutans*. For the colonization of *S. mutans*, synthesis of water-insoluble and adherent glucan from sucrose by the glucosyl transferases (GTases) is essential. *Streptococcus mutans* produces both cellassociated (CA) and cell-free (CF) forms of GTase; the former primarily synthesizes waterinsoluble glucan, while the latter produces water soluble glucan. The combined action of these two GTases on the cell surface of *S. mutans* during its growth in the presence of sucrose is critically important in allowing firm adherence. The GTase system of *S. mutans* has therefore been considered an important virulence factor promoting caries development. Administration of IgY against *S. mutans* CA-GTase specifically inhibited insoluble glucansynthesizing CA-GTase, resulting in a significant reduction in the development of dental caries. Otake et al. (1991) reported that anti-*S. mutans* CA-GTase IgY suppressed development of dental carries in rat model [42]. Hatta et al. (1997) reported that the

*2.5.2.2. Anti-Streptococcus mutans IgY* 

Influenza caused by a virus is called the influenza virus. Influenza or "flu" is an infection of the respiratory tract that can affect millions of people every year. It is highly contagious and occurs mainly in the late fall, winter, or early spring. Influenza is spread from person-toperson through mists or sprays of infectious respiratory secretions caused by coughing and sneezing. Influenza affects all age groups and causes severe illness, loss of school and work, and complications such as pneumonia, hospitalization, and death.

Recently, specific anti-influenza IgY was successfully produced from hens immunized with inactivated influenza virus strain. This specific IgY significantly reacts virus in vitro [44]. Subsequently, an anti-influenza IgY biofilter which trap the influenza virus has been developed by Daikin Environment Laboratories, Japan, a research arm of Daikin Industries, in cooperation with five Japanese research institutions. It was found that 99.99% of the influenza virus sprayed over the biofilter was captured within 10 minutes. An air cleaner with anti-influenza filter is recently launched in Japan (Figure 5 and 6). Moreover, a facemask with anti-influenza IgY was developed and will be available in the Japanese market.

**Figure 5.** Diagram for the Anti-influenza IgY biofilter.

Functional Proteins and Peptides of Hen's Egg Origin 125

Phosvitin phosphopeptides are new functional bioactive peptides derived from egg yolk with molecular masses of 1-3 kDa prepared from tryptic hydrolysate by partial dephosphorylation [46]. The phosvitin phosphopeptides were shown to be effective for enhancing the calcium binding capacity and inhibiting the formation of insoluble calcium phosphate. The results suggest a potential application of phosvitin peptides as novel

Hen egg turns into a full skeleton chick within 3 weeks; based on this fact, some investigations were carried out to explore the biologically active substances in hen egg that would initiate and enhance bone growth. It has been found that a specific yolk water-soluble protein (YSP) has a bone growth promotion activity *in vitro* [47] and *in vivo* [48]. These findings encouraged to search the functional yolk peptides that would promote bone growth. Different enzymes were used to hydrolyze YSP and the effect of the peptide preparations on osteoblast MC3T3-E1 cell proliferation was investigated. A novel peptides preparation (Bonepep®) with bone growth promotion activity was obtained. An *in vitro* study showed that Bonepep enhances the osteoblast MC3T3-E1 cell proliferation (Figure 7). Furthermore, an *in vivo* study showed that it promotes the elongation of rat tibia bone [49]. Compared with control and YSP fed rats; rats fed on Bonepep® showed marked and significant increase of chondrocytes proliferation and the bone formation in the tibia and

**3.2. Phosvitin peptide** 

functional peptides for the prevention of osteoporosis.

consequently significantly increased elongation of their bone.

(A: Bonepep, B~H: peptides by other enzymes, YSP: Yolk Soluble Protein).

**Figure 7.** Promotion of osteoblast MC3T3-E1 cell proliferation by Bonepep®.

**3.3. Egg yolk bone peptides (Bonepep®)** 

**Figure 6.** An air cleaner with anti-Influenza virus biofilter available in Japanese market.

#### *2.5.2.4. Future prospects of IgY applications*

Many research activities and proposals are going on in order to provide new applications for IgY technology. An anti-*Bacteriodes gengivalis* is under development for improving the oral health. For cosmetics sector, IgY against *Propionibacterium acnes* and its lipases has been developed to prevent and treat acne that is the most common skin disease [45]. For the medical sector, many researchers are working on using transgenic chicken to produce human antibodies in the transgenic hen's eggs in the form of IgY to help for treating human diseases.

## **3. Egg yolk bio peptides**

Recently, it has become clear that proteins are a source for biologically active peptides. These peptides are inactive within the sequence of parent protein and can be released during gastrointestinal digestion or food processing. Egg yolk proteins could be an important source of bioactive peptides. The resultant peptides could show biologically new function with improved stability and/or solubility. In this section, some biologically functional egg yolk derived peptides are introduced and their underlying mechanisms are discussed.

#### **3.1. Lipovitellenin peptide**

Vitellenin is the apoprotein of lipovitellenin. Digestion of vitellenin with pronase gives two glycopeptides. Glycopeptide A has high content of sialic acid and glycopeptide B contains most of the carbohydrates of vitellenin but devoid of sialic acid (N-Acetylneuraminic acid). Sialic acid is naturally occurring carbohydrate with numerous biological functions, including blood protein half-life regulation, variety of toxin neutralization, regulation of cellular adhesion and glycoprotein lytic protection.

#### **3.2. Phosvitin peptide**

124 Bioactive Food Peptides in Health and Disease

*2.5.2.4. Future prospects of IgY applications* 

diseases.

discussed.

**3. Egg yolk bio peptides** 

**3.1. Lipovitellenin peptide** 

cellular adhesion and glycoprotein lytic protection.

**Figure 6.** An air cleaner with anti-Influenza virus biofilter available in Japanese market.

Many research activities and proposals are going on in order to provide new applications for IgY technology. An anti-*Bacteriodes gengivalis* is under development for improving the oral health. For cosmetics sector, IgY against *Propionibacterium acnes* and its lipases has been developed to prevent and treat acne that is the most common skin disease [45]. For the medical sector, many researchers are working on using transgenic chicken to produce human antibodies in the transgenic hen's eggs in the form of IgY to help for treating human

Recently, it has become clear that proteins are a source for biologically active peptides. These peptides are inactive within the sequence of parent protein and can be released during gastrointestinal digestion or food processing. Egg yolk proteins could be an important source of bioactive peptides. The resultant peptides could show biologically new function with improved stability and/or solubility. In this section, some biologically functional egg yolk derived peptides are introduced and their underlying mechanisms are

Vitellenin is the apoprotein of lipovitellenin. Digestion of vitellenin with pronase gives two glycopeptides. Glycopeptide A has high content of sialic acid and glycopeptide B contains most of the carbohydrates of vitellenin but devoid of sialic acid (N-Acetylneuraminic acid). Sialic acid is naturally occurring carbohydrate with numerous biological functions, including blood protein half-life regulation, variety of toxin neutralization, regulation of

Phosvitin phosphopeptides are new functional bioactive peptides derived from egg yolk with molecular masses of 1-3 kDa prepared from tryptic hydrolysate by partial dephosphorylation [46]. The phosvitin phosphopeptides were shown to be effective for enhancing the calcium binding capacity and inhibiting the formation of insoluble calcium phosphate. The results suggest a potential application of phosvitin peptides as novel functional peptides for the prevention of osteoporosis.

#### **3.3. Egg yolk bone peptides (Bonepep®)**

Hen egg turns into a full skeleton chick within 3 weeks; based on this fact, some investigations were carried out to explore the biologically active substances in hen egg that would initiate and enhance bone growth. It has been found that a specific yolk water-soluble protein (YSP) has a bone growth promotion activity *in vitro* [47] and *in vivo* [48]. These findings encouraged to search the functional yolk peptides that would promote bone growth. Different enzymes were used to hydrolyze YSP and the effect of the peptide preparations on osteoblast MC3T3-E1 cell proliferation was investigated. A novel peptides preparation (Bonepep®) with bone growth promotion activity was obtained. An *in vitro* study showed that Bonepep enhances the osteoblast MC3T3-E1 cell proliferation (Figure 7). Furthermore, an *in vivo* study showed that it promotes the elongation of rat tibia bone [49]. Compared with control and YSP fed rats; rats fed on Bonepep® showed marked and significant increase of chondrocytes proliferation and the bone formation in the tibia and consequently significantly increased elongation of their bone.

(A: Bonepep, B~H: peptides by other enzymes, YSP: Yolk Soluble Protein).

**Figure 7.** Promotion of osteoblast MC3T3-E1 cell proliferation by Bonepep®.

## **4. Egg white bio proteins**

Well-known biological functions of egg white proteins are the prevention of microorganisms' penetration into the yolk and supply of nutrients to the embryo during the late stages of development. Most of the egg white proteins appear to possess antimicrobial properties or certain physiological functions to interfere with the growth and spread of invading microorganisms.

Functional Proteins and Peptides of Hen's Egg Origin 127

implicated in the transport of iron in a soluble form to the target cells. The recognition of transferrin molecules by the target cells is mediated by membrane-bound transferrin receptors [56]. The significant structural similarities between lactoferrin and ovotransferrin justify the similarity of their biological roles. Ovotransferrin can be used as a nutritional ingredient in iron fortified products such as iron supplements, iron-fortified mixes for

There is also extensive evidence of an antibacterial effect of ovotransferrin based on iron deprivation, iron being an essential growth factor for most microorganisms. The high affinity of transferrins for iron means that, in the presence of unsaturated transferrin (apotransferrin), iron will be sequestered and rendered unavailable for the growth of microorganisms. *In vivo*, ovotransferrin has been shown to have therapeutic properties

The name lysozyme was originally used to describe an enzyme which had lytic action against bacterial cells. Lysozyme is one of the oldest egg components to be utilized commercially after it was discovered by Alexander Fleming in 1922. It is a bacteriolytic enzyme commonly found in nature and is present in almost all secreted body fluids and tissues of humans and animals. It has also been isolated from some plants, bacteria and

The lysozyme content of a laying hen's blood is 10-fold higher than in mammals because it is being transferred to the egg white. Lysozyme constitutes approximately 3.5% of hen egg white [50]. Egg white lysozyme consists of 129 amino acid residues with a molecular weight of 14.4 kDa. Because of its basic character, lysozyme binds to ovomucin, transferrin or ovalbumin in egg white [58]. In nature, lysozyme is found mainly as a monomer but it has been reported to also exist as a reversible dimer, which can be evoked by pH, concentration

It has long been believed that lysozyme's antimicrobial action could only be attributed to its catalytic effect on certain Gram-positive bacteria, by splitting the bond between Nacetylmuramic acid and N-acetyl-glucosamine of peptidoglycan in the bacterial cell wall [59]. Beside this well-known inactivation mechanism, a non enzymatic antibacterial mode of action of lysozyme was achieved by denatured form of lysozyme without enzymatic action.

Lysozyme demonstrates antimicrobial activity against a limited spectrum of bacteria and fungi [60]. However, the antimicrobial activity of lysozyme is greater for certain Grampositive bacteria. On the other hand, Gram-negative bacteria are less susceptible to the bacteriolytic action of the enzyme [61]. The cell walls of different bacteria show varying degrees of susceptibility to digestion with hen egg white lysozyme. The walls of *Micrococcus lysodeikticus* were the most sensitive and the walls of *Staphylococci* were the less sensitive to the bacteriolytic action of lysozyme. Among Gram-negative bacteria, the walls of *Salmonella*  and *Shigella* were the most sensitive whereas those of *E.coli*, *Vibrio* and *Proteus* were much

bacteriophages. Avian egg white is a rich and easily available source of lysozyme.

and/or temperature-dependent phase transition of the molecule.

instant drinks, sport bars, protein supplements and iron-fortified beverages.

against acute enteritis in infants [57].

**4.3. Lysozyme** 

Most of egg white proteins are soluble and can easily be isolated. Egg white contains approximately 40 different proteins. Egg white proteins possess unique functional properties, such as antimicrobial, enzymatic and anti-enzymatic, cell growth stimulatory, metal binding, vitamin binding, and immunological activities [50].

## **4.1. Ovalbumin**

Ovalbumin is a predominant protein contributing to the functional properties of egg white [51]. Ovalbumin is a monomeric phosphoglycoprotein with a molecular weight of 44.5 kDa and an isoelectric point of 4.5. Ovalbumin is a key reference protein in biochemistry. As a carrier, stabilizer, blocking agent or standard, highly purified ovalbumin has served the fundamentalists as well as the food industry. It has long been the subject of physical and chemical studies as a convenient protein model.

It is believed that ovalbumin, especially its unphosphorylated form, serves as a source of amino acids for the developing embryo. Despite the intensive investigations undertaken on ovalbumin, its function remains largely unknown. Ovalbumin is the only egg white protein which contains free sulfhydryl groups. The complete amino acid sequence of hen ovalbumin (which comprises 385 residues) and its crystal structure have been reported [52]. The unexpected finding that this protein belongs to the serpin superfamily has stimulated new interest in the structure and function of ovalbumin. The serpins are a family of more than 300 homologous proteins with diverse functions found in animals, plants, insects and viruses, but not in prokaryotes [53]. They include the major serine protease inhibitors of human plasma that control enzymes of the coagulation, fibrinolytic, complement and kinin cascades, as well as proteins without any known inhibitory properties such as hormone binding globulins, angiotensinogen and ovalbumin [54].

## **4.2. Ovotransferrin**

Ovotransferrin (also known as conalbumin) has been identified as the iron-binding protein from avian egg white. Ovotransferrin, which constitutes 12% of the egg white protein, has a molecular weight of 77.7 kDa and a pI of about 6.1. It contains 686 amino acid residues and has 15 disulfide bridges [55]. It is glycosylated and contains a single glycan chain (composed of mannose and N-acetylglucosamine residues) in the C-terminal domain. Ovotransferrin is a neutral glycoprotein synthesized in the hen oviduct and deposited in the albumen fraction of eggs. Furthermore, it has two similar domains in N and C terminal regions, each one binding one atom of transition metal (Fe+++, Cu++, Al+++) very tightly and specifically [55]. It is implicated in the transport of iron in a soluble form to the target cells. The recognition of transferrin molecules by the target cells is mediated by membrane-bound transferrin receptors [56]. The significant structural similarities between lactoferrin and ovotransferrin justify the similarity of their biological roles. Ovotransferrin can be used as a nutritional ingredient in iron fortified products such as iron supplements, iron-fortified mixes for instant drinks, sport bars, protein supplements and iron-fortified beverages.

There is also extensive evidence of an antibacterial effect of ovotransferrin based on iron deprivation, iron being an essential growth factor for most microorganisms. The high affinity of transferrins for iron means that, in the presence of unsaturated transferrin (apotransferrin), iron will be sequestered and rendered unavailable for the growth of microorganisms. *In vivo*, ovotransferrin has been shown to have therapeutic properties against acute enteritis in infants [57].

#### **4.3. Lysozyme**

126 Bioactive Food Peptides in Health and Disease

**4. Egg white bio proteins** 

invading microorganisms.

**4.1. Ovalbumin** 

**4.2. Ovotransferrin** 

Well-known biological functions of egg white proteins are the prevention of microorganisms' penetration into the yolk and supply of nutrients to the embryo during the late stages of development. Most of the egg white proteins appear to possess antimicrobial properties or certain physiological functions to interfere with the growth and spread of

Most of egg white proteins are soluble and can easily be isolated. Egg white contains approximately 40 different proteins. Egg white proteins possess unique functional properties, such as antimicrobial, enzymatic and anti-enzymatic, cell growth stimulatory,

Ovalbumin is a predominant protein contributing to the functional properties of egg white [51]. Ovalbumin is a monomeric phosphoglycoprotein with a molecular weight of 44.5 kDa and an isoelectric point of 4.5. Ovalbumin is a key reference protein in biochemistry. As a carrier, stabilizer, blocking agent or standard, highly purified ovalbumin has served the fundamentalists as well as the food industry. It has long been the subject of physical and

It is believed that ovalbumin, especially its unphosphorylated form, serves as a source of amino acids for the developing embryo. Despite the intensive investigations undertaken on ovalbumin, its function remains largely unknown. Ovalbumin is the only egg white protein which contains free sulfhydryl groups. The complete amino acid sequence of hen ovalbumin (which comprises 385 residues) and its crystal structure have been reported [52]. The unexpected finding that this protein belongs to the serpin superfamily has stimulated new interest in the structure and function of ovalbumin. The serpins are a family of more than 300 homologous proteins with diverse functions found in animals, plants, insects and viruses, but not in prokaryotes [53]. They include the major serine protease inhibitors of human plasma that control enzymes of the coagulation, fibrinolytic, complement and kinin cascades, as well as proteins without any known inhibitory properties such as hormone

Ovotransferrin (also known as conalbumin) has been identified as the iron-binding protein from avian egg white. Ovotransferrin, which constitutes 12% of the egg white protein, has a molecular weight of 77.7 kDa and a pI of about 6.1. It contains 686 amino acid residues and has 15 disulfide bridges [55]. It is glycosylated and contains a single glycan chain (composed of mannose and N-acetylglucosamine residues) in the C-terminal domain. Ovotransferrin is a neutral glycoprotein synthesized in the hen oviduct and deposited in the albumen fraction of eggs. Furthermore, it has two similar domains in N and C terminal regions, each one binding one atom of transition metal (Fe+++, Cu++, Al+++) very tightly and specifically [55]. It is

metal binding, vitamin binding, and immunological activities [50].

chemical studies as a convenient protein model.

binding globulins, angiotensinogen and ovalbumin [54].

The name lysozyme was originally used to describe an enzyme which had lytic action against bacterial cells. Lysozyme is one of the oldest egg components to be utilized commercially after it was discovered by Alexander Fleming in 1922. It is a bacteriolytic enzyme commonly found in nature and is present in almost all secreted body fluids and tissues of humans and animals. It has also been isolated from some plants, bacteria and bacteriophages. Avian egg white is a rich and easily available source of lysozyme.

The lysozyme content of a laying hen's blood is 10-fold higher than in mammals because it is being transferred to the egg white. Lysozyme constitutes approximately 3.5% of hen egg white [50]. Egg white lysozyme consists of 129 amino acid residues with a molecular weight of 14.4 kDa. Because of its basic character, lysozyme binds to ovomucin, transferrin or ovalbumin in egg white [58]. In nature, lysozyme is found mainly as a monomer but it has been reported to also exist as a reversible dimer, which can be evoked by pH, concentration and/or temperature-dependent phase transition of the molecule.

It has long been believed that lysozyme's antimicrobial action could only be attributed to its catalytic effect on certain Gram-positive bacteria, by splitting the bond between Nacetylmuramic acid and N-acetyl-glucosamine of peptidoglycan in the bacterial cell wall [59]. Beside this well-known inactivation mechanism, a non enzymatic antibacterial mode of action of lysozyme was achieved by denatured form of lysozyme without enzymatic action.

Lysozyme demonstrates antimicrobial activity against a limited spectrum of bacteria and fungi [60]. However, the antimicrobial activity of lysozyme is greater for certain Grampositive bacteria. On the other hand, Gram-negative bacteria are less susceptible to the bacteriolytic action of the enzyme [61]. The cell walls of different bacteria show varying degrees of susceptibility to digestion with hen egg white lysozyme. The walls of *Micrococcus lysodeikticus* were the most sensitive and the walls of *Staphylococci* were the less sensitive to the bacteriolytic action of lysozyme. Among Gram-negative bacteria, the walls of *Salmonella*  and *Shigella* were the most sensitive whereas those of *E.coli*, *Vibrio* and *Proteus* were much

less sensitive [59]. The susceptibility differences are believed to be due to the complex envelope structure of Gram-negative bacteria such as *E.coli* or *Salmonella typhimurium*. The outer membrane serves to reduce the access of lysozyme to its site of action (peptidoglycan layer).

Functional Proteins and Peptides of Hen's Egg Origin 129

was safe to be used in food [69]. The enzyme shows a number of properties important for food application. It is a heat stable protein, active in a broad range of temperatures (from 1oC to nearly 100oC), withstands boiling for 1-2 min, and stable in freeze-drying and thermal drying. Moreover, lysozyme is not inactivated by solvents and it maintains its activity when re-dissolved in water. It has optimum activity at pH 5.3 to 6.4 (*i.e.* typical for low-acidic

In cheese making, lysozyme has been used to prevent growth of *Clostridium tyrobutyricum,*  which causes off-flavors and late blowing in some cheeses [70]. Another application of lysozyme may be the possible acceleration of cheese ripening, because lysis of starter bacteria would cause release of cytoplasmic enzymes which play a key role in proteolysis during cheese ripening [71]. Moreover, egg white lysozyme was used as an antimicrobial

*Pharmaceuticals.* In the pharmaceutical industry, avian egg white lysozyme can protect the body against bacterial, viral or inflammatory diseases [73]. It has been used in aerosols for the treatment of broncho pulmonary diseases, prophylactically for dental caries, for nasal tissue protection and is incorporated into various therapeutic creams for the protection and topical preparation of certain dystrophic and inflammatory lesions of the skin and soft

Regardless of the direct bacteriolytic action, many other biological functions of lysozyme have recently been reported. These include anti-viral action by forming an insoluble complex with acidic viruses, enhanced antibiotic effects, anti-inflammatory and anti-

Ovomucoid is a glycoprotein with heat stable trypsin inhibitor activity. Ovomucoid, which constitutes about 11% of the egg white protein, has a molecular weight of approximately 28 kDa and a pI of 4.1. It has nine disulfides and no free sulfydryl groups. The molecule consists of three tandem domains, each of which is homologous to pancreatic secretory trypsin inhibitor (Kazal-type). It has a putative reactive site for the inhibition of serine proteases. A large proportion of the carbohydrate present in this glycoprotein (about 25%*)* is joined to the polypeptide chain through asparginyl residues [78]. Ovomucoid can be heated at 100o C under acidic conditions for long periods without any apparent changes in its physical or chemical properties. Ovomucoid may play a more important role in the

Ovomucin comprises 1.5-3.5% of the total egg white solids. It is a highly viscous glycoprotein with an extremely large molecular weight (8300-23000 kDa). The specific jellying property of egg white is attributed to ovomucin. It consists of two subunits; αsubunit and a β-subunit which are bound by disulfide bonds. The biological function of

histaminic actions, direct activation of immune cells and anti-tumor action [74-77].

pathogenesis of allergic reactions to egg white than other egg white proteins [79].

agent to control lactic acid bacteria in some fermented beverages [72].

tissues (*e.g.* burns and viral diseases).

**4.4. Ovomucoid** 

**4.5. Ovomucin** 

food).

#### *4.3.1. Molecular modification for functional improvement*

*Lipophilization*. A number of chemical modifications of lysozyme have been undertaken to increase its efficacy as an antimicrobial agent. The effect of lipophilization with long chain fatty acids (palmitic or stearic acid) and shorter chain saturated fatty acids (caproic, capric or myristic acid) on the bactericidal action of lysozyme was investigated [62]. Lipophilization broadened the bactericidal action of lysozyme to Gram-negative bacteria with little loss of enzymatic activity [63].

*Glycosylation.* It is one of the most promising techniques, involves the attachment of carbohydrate chains to lysozyme. Glycosylation produces more stable proteins with improved conformational stability, protease resistance, modulated charge effects and waterbinding capacity [64]. Conjugation of lysozyme with dextran by Maillard reaction increases antimicrobial activity. In addition, emulsifying activity of the conjugate was approximately 30 times that of native lysozyme [65]. Extending the function of lysozyme by conjugation with food compounds gives a novel and potentially useful bi-functional food additive. Similarly, hen egg lysozyme conjugated with xyloglucan hydrolysates; totally conserved enzymatic activity of lysozyme and increased the emulsifying properties 5 times higher than that of the native protein [66]. An antibacterial emulsifier was prepared by conjugating a fatty acylated saccharide with lysozyme through the Maillard reaction; the conjugate exhibited considerable resistance to proteolysis and much enhanced emulsifying activity and emulsion stability. The conjugate maintained approximately 70% of the bactericidal activity of native hen egg lysozyme without significant conformational changes of the protein [67].

*Combination of lipophilization and glycosylation.* An egg white lysozyme, which had been modified using the Maillard-type glycosylation method prior to lipophilization with palmitic acid, was prepared [68]. The yield of lipophilized lysozyme was increased significantly by pre-glycosylation of the protein and showed strong antimicrobial activity against *Escherichia coli*. Lipophilization of lysozyme combined with glycosylation is a promising method for potential industrial applications of lysozyme due to its enhanced antimicrobial activity towards Gram-negative bacteria and improved yield.

#### *4.3.2. Applications*

The bacteriostatic and bactericidal properties of lysozyme have been used to preserve various food items, as well as in pharmacy, medicine and veterinary medicine.

*Natural food preservative.* Lysozyme has been used as an antimicrobial agent in various foods. In 1992, the Joint FAO/WHO Expert Committee on Food Additives declared that lysozyme was safe to be used in food [69]. The enzyme shows a number of properties important for food application. It is a heat stable protein, active in a broad range of temperatures (from 1oC to nearly 100oC), withstands boiling for 1-2 min, and stable in freeze-drying and thermal drying. Moreover, lysozyme is not inactivated by solvents and it maintains its activity when re-dissolved in water. It has optimum activity at pH 5.3 to 6.4 (*i.e.* typical for low-acidic food).

In cheese making, lysozyme has been used to prevent growth of *Clostridium tyrobutyricum,*  which causes off-flavors and late blowing in some cheeses [70]. Another application of lysozyme may be the possible acceleration of cheese ripening, because lysis of starter bacteria would cause release of cytoplasmic enzymes which play a key role in proteolysis during cheese ripening [71]. Moreover, egg white lysozyme was used as an antimicrobial agent to control lactic acid bacteria in some fermented beverages [72].

*Pharmaceuticals.* In the pharmaceutical industry, avian egg white lysozyme can protect the body against bacterial, viral or inflammatory diseases [73]. It has been used in aerosols for the treatment of broncho pulmonary diseases, prophylactically for dental caries, for nasal tissue protection and is incorporated into various therapeutic creams for the protection and topical preparation of certain dystrophic and inflammatory lesions of the skin and soft tissues (*e.g.* burns and viral diseases).

Regardless of the direct bacteriolytic action, many other biological functions of lysozyme have recently been reported. These include anti-viral action by forming an insoluble complex with acidic viruses, enhanced antibiotic effects, anti-inflammatory and antihistaminic actions, direct activation of immune cells and anti-tumor action [74-77].

## **4.4. Ovomucoid**

128 Bioactive Food Peptides in Health and Disease

enzymatic activity [63].

protein [67].

*4.3.2. Applications* 

*4.3.1. Molecular modification for functional improvement* 

layer).

less sensitive [59]. The susceptibility differences are believed to be due to the complex envelope structure of Gram-negative bacteria such as *E.coli* or *Salmonella typhimurium*. The outer membrane serves to reduce the access of lysozyme to its site of action (peptidoglycan

*Lipophilization*. A number of chemical modifications of lysozyme have been undertaken to increase its efficacy as an antimicrobial agent. The effect of lipophilization with long chain fatty acids (palmitic or stearic acid) and shorter chain saturated fatty acids (caproic, capric or myristic acid) on the bactericidal action of lysozyme was investigated [62]. Lipophilization broadened the bactericidal action of lysozyme to Gram-negative bacteria with little loss of

*Glycosylation.* It is one of the most promising techniques, involves the attachment of carbohydrate chains to lysozyme. Glycosylation produces more stable proteins with improved conformational stability, protease resistance, modulated charge effects and waterbinding capacity [64]. Conjugation of lysozyme with dextran by Maillard reaction increases antimicrobial activity. In addition, emulsifying activity of the conjugate was approximately 30 times that of native lysozyme [65]. Extending the function of lysozyme by conjugation with food compounds gives a novel and potentially useful bi-functional food additive. Similarly, hen egg lysozyme conjugated with xyloglucan hydrolysates; totally conserved enzymatic activity of lysozyme and increased the emulsifying properties 5 times higher than that of the native protein [66]. An antibacterial emulsifier was prepared by conjugating a fatty acylated saccharide with lysozyme through the Maillard reaction; the conjugate exhibited considerable resistance to proteolysis and much enhanced emulsifying activity and emulsion stability. The conjugate maintained approximately 70% of the bactericidal activity of native hen egg lysozyme without significant conformational changes of the

*Combination of lipophilization and glycosylation.* An egg white lysozyme, which had been modified using the Maillard-type glycosylation method prior to lipophilization with palmitic acid, was prepared [68]. The yield of lipophilized lysozyme was increased significantly by pre-glycosylation of the protein and showed strong antimicrobial activity against *Escherichia coli*. Lipophilization of lysozyme combined with glycosylation is a promising method for potential industrial applications of lysozyme due to its enhanced

The bacteriostatic and bactericidal properties of lysozyme have been used to preserve

*Natural food preservative.* Lysozyme has been used as an antimicrobial agent in various foods. In 1992, the Joint FAO/WHO Expert Committee on Food Additives declared that lysozyme

antimicrobial activity towards Gram-negative bacteria and improved yield.

various food items, as well as in pharmacy, medicine and veterinary medicine.

Ovomucoid is a glycoprotein with heat stable trypsin inhibitor activity. Ovomucoid, which constitutes about 11% of the egg white protein, has a molecular weight of approximately 28 kDa and a pI of 4.1. It has nine disulfides and no free sulfydryl groups. The molecule consists of three tandem domains, each of which is homologous to pancreatic secretory trypsin inhibitor (Kazal-type). It has a putative reactive site for the inhibition of serine proteases. A large proportion of the carbohydrate present in this glycoprotein (about 25%*)* is joined to the polypeptide chain through asparginyl residues [78]. Ovomucoid can be heated at 100o C under acidic conditions for long periods without any apparent changes in its physical or chemical properties. Ovomucoid may play a more important role in the pathogenesis of allergic reactions to egg white than other egg white proteins [79].

#### **4.5. Ovomucin**

Ovomucin comprises 1.5-3.5% of the total egg white solids. It is a highly viscous glycoprotein with an extremely large molecular weight (8300-23000 kDa). The specific jellying property of egg white is attributed to ovomucin. It consists of two subunits; αsubunit and a β-subunit which are bound by disulfide bonds. The biological function of

ovomucin is shown to inhibition of haemagglutination by viruses. Its affinity with viruses such as bovine rotavirus, hen Newcastle disease virus and human influenza virus was already proved [80]. Moreover, the β-subunit from ovomucin was shown to have a cytotoxic effect on the cultured tumor cells [81].

Functional Proteins and Peptides of Hen's Egg Origin 131

This trypsin inhibitor was discovered by Matsushima in 1958. While it is a Kazal-type inhibitor (like ovomucoid), ovoinhibitor functions as a multi-headed inhibitor and inhibits bacterial serine proteinase, fungal serine proteinase and mammalian chymotrypsin [5].

It is the third proteinase inhibitor in egg white (also called ficin-papain inhibitor). In contrast to ovomucin, cystatin is a small molecule (12.7 kDa) and it has no carbohydrates and a high thermal stability. The potential of their broad application in medical treatments has been reported in the literature, which includes antimicrobial and antiviral activities [83], the

Ovoglycoprotein is an acidic glycoprotein with a molecular weight of 24.4 kDa. This protein contains hexoses 13.6%, glucosamine 13.8%, and N-acetylneuraminic acid 3%. The biological

Ovokinin, a vasorelaxing octapeptide derived from pepsin digest of ovalbumin, has been shown to significantly lower the systolic blood pressure of spontaneously hypertensive rats [86]. Oral vailability of ovokinin is improved after emulsification. When we eat whole egg, ovalbumin peptides will be released by the action of pepsin in the stomach, and the peptide will be instantly emulsified with egg yolk and effectively absorbed from the intestines.

A vasorelaxing peptide – ovokinin (2-7) – was isolated from chymotryptic digest of ovalbumin [87]. However, the mechanisms for the relaxation were different from ovokinin. More anti-hypertensive peptide was obtained by modifying the amino acid residues of ovokinin (2-7). The minimum effective dose of [Pro2, Phe3]-ovokinin (2-7) was about onethirtieth of that of ovokinin (2-7). [Pro2, Phe3]-ovokinin (2-7) proved to be a potent antihypertensive peptide with little effect on normal blood pressure when administered orally

The ovotransferrin antimicrobial peptide (OTAP-92) is a 92 amino acid cationic fragment of hen ovotransferrin located (109-299) in the N-lobe of ovotransferrin. The peptide OTAP-92 showed strong bactericidal activity against both Gram-positive *S.aureus* and Gram-negative *E.coli* strains [89]. OTAP-92 has also been shown to possess a unique structural motif similar to the insect defensins. Furthermore, this cationic antimicrobial peptide is capable of killing

prevention of cerebral hemorrhage [84] and control of cancer cell metastasis [85].

**4.10. Ovoinhibitor** 

**4.11. Cystatin** 

**4.12. Ovoglycoprotein** 

**5. Egg white bio peptides** 

**5.2. Ovotransferrin peptides** 

[88].

functions of ovoglycoprotein are still unclear [5].

**5.1. Ovoalbumin peptide (Ovokinin)** 

### **4.6. Avidin**

Avidin is a strongly basic glycoprotein synthesized in the hen oviduct and deposited in the albumen fraction of eggs. Avidin is a tetrameric protein, composed of subunits of identical amino acid composition and sequence (15.6 kDa and 128 amino acids each). Avidin is a trace component (0.05%) of egg white, but it has been well studied because of its ability to tightly and specifically bind biotin, one of Vitamin B group. Each subunit of avidin binds to a molecule of biotin. The high affinity of avidin for biotin has been widely used as a biochemical tool in molecular biology, affinity chromatography, molecular recognition and labeling, Enzyme Linked Immuno Sorbent Assay (ELISA), histochemistry and cytochemistry [82].

#### **4.7. Ovoglobulin**

In early studies, six globulin fractions were thought to be present in egg white. They are macroglobulin, ovoglobulins G1, G2 and G3 and two other globulins. However, the two globulins were later classified as ovoinhibitors and ovoglobulin G1 was identified as lysozyme. Currently, the name ovoglobulin is given only to ovoglobulins G2 and G3, which have molecular weights of 36 and 45 kDa, respectively. The biological function of these proteins has not been clearly elucidated, but they appear to be important in the foaming capacity of egg white [5].

#### **4.8. Ovomacroglobulin**

Ovomacroglobulin is the second largest egg glycoprotein after ovomucin and its molecular weight is 760-900 kDa. Ovomacroglobulin, like ovomucin, has the ability to inhibit hemagglutination [5].

### **4.9. Ovoflavoprotein**

Ovoflavoprotein is acidic protein with a molecular weight of 32-36 kDa, and contains a carbohydrate moiety (14%) made up of mannose, galactose and glucosamines, 7-8 phosphate groups and 8 disulfide bonds. After being transported from the blood to the egg white, most of the riboflavin (Vitamin B2) is stored in the egg white bound to an apoprotein called flavoprotein. One mole of apoprotein binds one mole of riboflavin, but this binding ability is lost when the protein is exposed to a pH below its isoelectric pH 4.2 [5]. It has antimicrobial properties due to depriving the microorganisms from its riboflavin content [50].

### **4.10. Ovoinhibitor**

130 Bioactive Food Peptides in Health and Disease

effect on the cultured tumor cells [81].

**4.6. Avidin** 

cytochemistry [82].

**4.7. Ovoglobulin** 

capacity of egg white [5].

**4.8. Ovomacroglobulin** 

hemagglutination [5].

**4.9. Ovoflavoprotein** 

riboflavin content [50].

ovomucin is shown to inhibition of haemagglutination by viruses. Its affinity with viruses such as bovine rotavirus, hen Newcastle disease virus and human influenza virus was already proved [80]. Moreover, the β-subunit from ovomucin was shown to have a cytotoxic

Avidin is a strongly basic glycoprotein synthesized in the hen oviduct and deposited in the albumen fraction of eggs. Avidin is a tetrameric protein, composed of subunits of identical amino acid composition and sequence (15.6 kDa and 128 amino acids each). Avidin is a trace component (0.05%) of egg white, but it has been well studied because of its ability to tightly and specifically bind biotin, one of Vitamin B group. Each subunit of avidin binds to a molecule of biotin. The high affinity of avidin for biotin has been widely used as a biochemical tool in molecular biology, affinity chromatography, molecular recognition and labeling, Enzyme Linked Immuno Sorbent Assay (ELISA), histochemistry and

In early studies, six globulin fractions were thought to be present in egg white. They are macroglobulin, ovoglobulins G1, G2 and G3 and two other globulins. However, the two globulins were later classified as ovoinhibitors and ovoglobulin G1 was identified as lysozyme. Currently, the name ovoglobulin is given only to ovoglobulins G2 and G3, which have molecular weights of 36 and 45 kDa, respectively. The biological function of these proteins has not been clearly elucidated, but they appear to be important in the foaming

Ovomacroglobulin is the second largest egg glycoprotein after ovomucin and its molecular weight is 760-900 kDa. Ovomacroglobulin, like ovomucin, has the ability to inhibit

Ovoflavoprotein is acidic protein with a molecular weight of 32-36 kDa, and contains a carbohydrate moiety (14%) made up of mannose, galactose and glucosamines, 7-8 phosphate groups and 8 disulfide bonds. After being transported from the blood to the egg white, most of the riboflavin (Vitamin B2) is stored in the egg white bound to an apoprotein called flavoprotein. One mole of apoprotein binds one mole of riboflavin, but this binding ability is lost when the protein is exposed to a pH below its isoelectric pH 4.2 [5]. It has antimicrobial properties due to depriving the microorganisms from its This trypsin inhibitor was discovered by Matsushima in 1958. While it is a Kazal-type inhibitor (like ovomucoid), ovoinhibitor functions as a multi-headed inhibitor and inhibits bacterial serine proteinase, fungal serine proteinase and mammalian chymotrypsin [5].

### **4.11. Cystatin**

It is the third proteinase inhibitor in egg white (also called ficin-papain inhibitor). In contrast to ovomucin, cystatin is a small molecule (12.7 kDa) and it has no carbohydrates and a high thermal stability. The potential of their broad application in medical treatments has been reported in the literature, which includes antimicrobial and antiviral activities [83], the prevention of cerebral hemorrhage [84] and control of cancer cell metastasis [85].

## **4.12. Ovoglycoprotein**

Ovoglycoprotein is an acidic glycoprotein with a molecular weight of 24.4 kDa. This protein contains hexoses 13.6%, glucosamine 13.8%, and N-acetylneuraminic acid 3%. The biological functions of ovoglycoprotein are still unclear [5].

## **5. Egg white bio peptides**

#### **5.1. Ovoalbumin peptide (Ovokinin)**

Ovokinin, a vasorelaxing octapeptide derived from pepsin digest of ovalbumin, has been shown to significantly lower the systolic blood pressure of spontaneously hypertensive rats [86]. Oral vailability of ovokinin is improved after emulsification. When we eat whole egg, ovalbumin peptides will be released by the action of pepsin in the stomach, and the peptide will be instantly emulsified with egg yolk and effectively absorbed from the intestines.

A vasorelaxing peptide – ovokinin (2-7) – was isolated from chymotryptic digest of ovalbumin [87]. However, the mechanisms for the relaxation were different from ovokinin. More anti-hypertensive peptide was obtained by modifying the amino acid residues of ovokinin (2-7). The minimum effective dose of [Pro2, Phe3]-ovokinin (2-7) was about onethirtieth of that of ovokinin (2-7). [Pro2, Phe3]-ovokinin (2-7) proved to be a potent antihypertensive peptide with little effect on normal blood pressure when administered orally [88].

#### **5.2. Ovotransferrin peptides**

The ovotransferrin antimicrobial peptide (OTAP-92) is a 92 amino acid cationic fragment of hen ovotransferrin located (109-299) in the N-lobe of ovotransferrin. The peptide OTAP-92 showed strong bactericidal activity against both Gram-positive *S.aureus* and Gram-negative *E.coli* strains [89]. OTAP-92 has also been shown to possess a unique structural motif similar to the insect defensins. Furthermore, this cationic antimicrobial peptide is capable of killing

Gram-negative bacteria by crossing the outer membrane by a self-promoted uptake pathway and damaging the cytoplasmic membrane by channel formation. Knowledge of the structure-function relationship may allow combinations of antimicrobial agents with different mechanisms to be designed for pharmaceutical applications. OTAP-92 may represent a novel antimicrobial agent for the food and pharmaceutical industries [55].

Functional Proteins and Peptides of Hen's Egg Origin 133

*In vitro*, it showed dose-dependent vasorelaxant activity of both aortic and mesenteric vascular smooth muscle*. In vivo,* Runpep® exhibited dose-dependent hypotensive effect on both systolic and diastolic blood pressures with more pronounced effect on the systolic one by ingestion. Runpep® has been proved to have anti-hypertensive effect and has been shown to significantly lower the systolic blood pressure (Figure 9) of spontaneously

The hydrolysate of egg white with pepsin was found to exhibit a strong angiotensin I– converting enzyme (ACE) inhibitory activity in vitro [95]. Other work reports the antioxidant activity of peptides produced by pepsin hydrolysis of egg white; four peptides included in the protein sequence of ovalbumin possessed radical scavenging activity higher than that of Trolox. The combined antioxidant and ACE inhibition properties make it a very useful multifunctional preparation for the control of cardiovascular diseases, particularly hypertension. No correlation was found between antioxidant and ACE inhibitory activities

**Figure 9.** Antihypertensive activity of Runpep® after oral administration to SHR rats using cuff method

SHR control 1 2 3 4

Time after 300 mg RunPep oral administration (hour)

**Systolic blood pressure Mean blood pressure Diastolic blood pressure**

Lysozyme is known to have a variety of folding topologies around the active site cleft [97]. Enzymatic hydrolysis of lysozyme is a novel technology that uses proteolytic enzymes for

hypertensive rats [94].

(Sphygmomanometer).

**5.5. Lysozyme peptides** 

of pepsin digest of egg white [96].

Blood pressure (mm/Hg)

//

#### **5.3. Ovomucoid and ovomucin peptides**

A pepsin-digest of egg white ovomucoid was prepared to enhance digestibility and lower allergenicity [90]. A highly glycosylated peptide fragments (220 and 120 kDa) was separated from pronase digest of avian egg white ovomucin; It was derived from the β-subunit. Both fragments inhibited the growth of tumors [91].

#### **5.4. Egg white peptides (Runpep®)**

Egg white hydrolysate (Runpep®) has been produced by standardized technology to provide a highly nutritive source of peptides. Runpep® contains all essential amino acids with amino acid score of 100 [5, 50]. It is rich in branched chain amino acids (BCAA); these are the essential amino acids leucine, isoleucine, and valine. BCAA's are of special importance for athletes because they are metabolized in the muscle, rather than in the liver. Moreover, it is a rich source of sulpher containing amino acids as methionine and cysteine. It is in the form of small peptides (less than 3000 Da) and contains more than 91% protein providing easily absorbable amino acids source (Figure 8). An *in vitro* study showed that Runpep® has anticoagulant activity (reduce the formation of coagulum in blood vessels, reduce the risk of embolism) and helps in lowering the blood platelets aggregations [92, 93].

**Figure 8.** Runpep® profile by GPC.

*In vitro*, it showed dose-dependent vasorelaxant activity of both aortic and mesenteric vascular smooth muscle*. In vivo,* Runpep® exhibited dose-dependent hypotensive effect on both systolic and diastolic blood pressures with more pronounced effect on the systolic one by ingestion. Runpep® has been proved to have anti-hypertensive effect and has been shown to significantly lower the systolic blood pressure (Figure 9) of spontaneously hypertensive rats [94].

The hydrolysate of egg white with pepsin was found to exhibit a strong angiotensin I– converting enzyme (ACE) inhibitory activity in vitro [95]. Other work reports the antioxidant activity of peptides produced by pepsin hydrolysis of egg white; four peptides included in the protein sequence of ovalbumin possessed radical scavenging activity higher than that of Trolox. The combined antioxidant and ACE inhibition properties make it a very useful multifunctional preparation for the control of cardiovascular diseases, particularly hypertension. No correlation was found between antioxidant and ACE inhibitory activities of pepsin digest of egg white [96].

#### **5.5. Lysozyme peptides**

132 Bioactive Food Peptides in Health and Disease

**5.3. Ovomucoid and ovomucin peptides** 

fragments inhibited the growth of tumors [91].

Column: YMC-Pack Diol-60 (500×8.0mmI.D.) Mobile phase: 0.1M KH2PO4-K2HPO4 containing 0.2MNaCl/Acetonitrile

**5.4. Egg white peptides (Runpep®)** 

**Figure 8.** Runpep® profile by GPC.


10000

30000

50000

70000

90000

110000

(70/30)

Flow rate: 0.7mL/min Temperature: ambient Ditection: UV215nm (1.0AUFS) Injection volume: 20μL Sample conc : 0 1%

130000

Gram-negative bacteria by crossing the outer membrane by a self-promoted uptake pathway and damaging the cytoplasmic membrane by channel formation. Knowledge of the structure-function relationship may allow combinations of antimicrobial agents with different mechanisms to be designed for pharmaceutical applications. OTAP-92 may

A pepsin-digest of egg white ovomucoid was prepared to enhance digestibility and lower allergenicity [90]. A highly glycosylated peptide fragments (220 and 120 kDa) was separated from pronase digest of avian egg white ovomucin; It was derived from the β-subunit. Both

Egg white hydrolysate (Runpep®) has been produced by standardized technology to provide a highly nutritive source of peptides. Runpep® contains all essential amino acids with amino acid score of 100 [5, 50]. It is rich in branched chain amino acids (BCAA); these are the essential amino acids leucine, isoleucine, and valine. BCAA's are of special importance for athletes because they are metabolized in the muscle, rather than in the liver. Moreover, it is a rich source of sulpher containing amino acids as methionine and cysteine. It is in the form of small peptides (less than 3000 Da) and contains more than 91% protein providing easily absorbable amino acids source (Figure 8). An *in vitro* study showed that Runpep® has anticoagulant activity (reduce the formation of coagulum in blood vessels, reduce the risk of embolism) and helps in lowering the blood platelets aggregations [92, 93].

分子

10000

5000

3000

1000

500

100

量

<sup>10</sup> <sup>15</sup> <sup>20</sup> <sup>25</sup> <sup>30</sup> <sup>35</sup> Retention Time (min)

Amino acid Runpep

represent a novel antimicrobial agent for the food and pharmaceutical industries [55].

Lysozyme is known to have a variety of folding topologies around the active site cleft [97]. Enzymatic hydrolysis of lysozyme is a novel technology that uses proteolytic enzymes for

hydrolyzing native lysozyme to produce potent antimicrobial peptides that hidden within its folds. Lysozyme was digested by different proteolytic enzymes, such as clostripain [58, 60], pepsin, and trypsin [97, 98, 99]. All these researchers proved that the resulting peptides lost the enzymatic activities of lysozyme, but exhibited strong bactericidal activities against both Gram-negative (*E.coli*, *Salmonella*, *Pseudomonas*, and *Aeromonas*) and Gram-positive bacteria (*Listeria monocytogenes*, *Staph aureus, Bacillus* spp., and *Leuconostic*s spp.) as well as yeasts (*Saccharomyces*). Scanning electron microscopy clearly demonstrated that cell membrane of both Gram-negative and –positive bacteria was damaged by these peptides. Thus these peptides probably have a different mechanism of action than native lysozyme [58, 99]. Mine et al (2004) could isolation, purification, and characterization of novel antimicrobial peptides from chicken egg white lysozyme hydrolysate, obtained by peptic digestion and subsequent tryptic digestion. The hydrolysate was composed of over 20 small peptides of less than 1000 Da, and had no enzymatic activity. The water-soluble peptide mixture showed bacteriostatic activity against Gram-positive bacteria (Staphylococcus aureus 23-394) and Gram-negative bacteria (*E. coli* K-12). Two bacteriostatic peptides were purified and sequenced. One peptide, with the sequence Ile-Val-Ser-Asp-Gly-Asp-Gly-Met-Asn-Ala-Trp, inhibited Gram-negative bacteria *E. coli* K-12 and corresponded to amino acid residues 98-108, which are located in the middle part of the helix-loop-helix. Another novel antimicrobial peptide inhibited *S. aureus* and was identified as His-Gly-Leu-Asp-Asn-Tyr-Arg, corresponding to amino acid residues 15-21 of lysozyme. These peptides have broadened the antimicrobial activity of lysozyme to Gram-negative bacteria. The results obtained in this study indicate that lysozyme possesses nonenzymatic bacteriostatic domains in its primary sequence and they are released by proteolytic hydrolysis [99].

Functional Proteins and Peptides of Hen's Egg Origin 135

egg membrane. The effects of this egg shell membrane protein on cell growth have been studied. The growth of normal human skin fibroblasts on egg membrane protein-coated tissue cultures increased in relation to increasing egg membrane protein concentration.

The egg shell membranes contain several bacteriolytic enzymes (*e.g.* lysozyme and Nacetylglucosaminidase) and other membrane components which may alter the thermal resistance of Gram-positive and Gram-negative bacterial pathogens (*Salmonella Enteritidis,* 

The presence of hydroxyproline in hydrolysates of the egg shell membrane layers has suggested that the membrane layers contain collagen [103]. This has been confirmed using biochemical and immunological tests. It has been established that about 10% of the total proteinaceous content of the membrane structure of an egg shell is collagen. The outer shell membrane contains predominately Type I collagen and the inner shell membrane contains Types I and V collagen. In addition, Type X collagen has been found in both the inner and outer shell membranes using immunohistochemical analysis. It is important to recognize the

Different Non-collagenous proteins have been identified in the organic matrix of the hen's egg shell. Ovocleidin-17 is a soluble matrix protein component and distributed in palisade

Ovocalyxin-32 has been identified as a novel 32-kDa protein. It is expressed at high levels in the uterine and isthmus regions of the oviduct, and concentrated in the eggshell. In the eggshell, ovocalyxin-32 localizes to the outer palisade layer, the vertical crystal layer, and the cuticle of the eggshell, in agreement with its demonstration by Western blotting at high levels in the uterine fluid during the termination phase of eggshell formation. Ovocalyxin-32 is therefore identified as a novel protein synthesized in the distal oviduct where hen eggshell

Osteopontin, a phosphorylated bone glycoprotein involved in formation and remodeling of the mineralised tissue, has also been demonstrated in the hen egg shell. Gene expression for

Ovalbumin, lysozyme and ovotransferrin, as egg white proteins, have been identified in hen's egg shell. The organic matrix also contains several proteoglycan molecules [107].

Chicken eggshell powder has been proposed as an attractive source of calcium for human health to increase bone mineral density in an elderly population with osteoporosis. However, factors affecting calcium transport of eggshell calcium have not yet been evaluated. Chicken eggshell contains about 1.0% (w/w) matrix proteins in addition to a major form of calcium carbonate (95%, w/w). It was found that soluble eggshell matrix proteins remarkably enhance calcium transport using in vitro Caco-2 cell monolayers grown on a permeable support. The total calcium transport across Caco-2 monolayers showed an

this protein has been shown to be higher during the period of calcification [106].

*Escherichia coli* 0157:H7, *Listeria monocytogenes* and *Staphylococcus aureus*).

presence of collagen in egg shell membranes because of its potential value.

**7. Egg shell bio proteins** 

and mammillary layers [104].

formation occurs [105].

Consumers are increasingly demanding food that is free from pathogens, but with less preservatives and additives. As a response to these conflicting demands, current trends in the food industry include the investigation of alternative natural preservative in foods. Six Gram-negative bacteria (*Escherichia coli*, *Salmonella enteritidis* NBRC 3313, *Salmonella typhimurium*, *Pseudomonas fluorescens*, *Pseudomonas aeruginosa*, *Aeromonas hydrophila*) were checked for sensitivity to native hen egg white lysozyme and hydrolysate preparation of lysozyme derived peptides. Generally, lysozyme peptides preparation acts on the tested organisms and on different strains of *Bacillus* spp. with much more potency comparing to native lysozyme [100, 101]. The resulting peptides lost the enzymatic activities of lysozyme, but exhibited strong bactericidal activities against both Gram-negative and Gram-positive bacteria [97, 102]. Being natural antimicrobial, lysozyme peptides preparation will find its way as a safe shelf-life extender in the food industry.

#### **6. Egg shell membrane bio proteins**

The egg shell membrane has been thought to be beneficial in the treatment of some injuries. For example, in Japan, when Sumo wrestlers get flesh abrasions, they will often peel the egg membrane from the egg shell and cover their injuries. They believe that it facilitates their recovery. Peptides product which are stable in water have been prepared from hydrolyzed egg membrane. The effects of this egg shell membrane protein on cell growth have been studied. The growth of normal human skin fibroblasts on egg membrane protein-coated tissue cultures increased in relation to increasing egg membrane protein concentration.

The egg shell membranes contain several bacteriolytic enzymes (*e.g.* lysozyme and Nacetylglucosaminidase) and other membrane components which may alter the thermal resistance of Gram-positive and Gram-negative bacterial pathogens (*Salmonella Enteritidis, Escherichia coli* 0157:H7, *Listeria monocytogenes* and *Staphylococcus aureus*).

The presence of hydroxyproline in hydrolysates of the egg shell membrane layers has suggested that the membrane layers contain collagen [103]. This has been confirmed using biochemical and immunological tests. It has been established that about 10% of the total proteinaceous content of the membrane structure of an egg shell is collagen. The outer shell membrane contains predominately Type I collagen and the inner shell membrane contains Types I and V collagen. In addition, Type X collagen has been found in both the inner and outer shell membranes using immunohistochemical analysis. It is important to recognize the presence of collagen in egg shell membranes because of its potential value.

## **7. Egg shell bio proteins**

134 Bioactive Food Peptides in Health and Disease

hydrolyzing native lysozyme to produce potent antimicrobial peptides that hidden within its folds. Lysozyme was digested by different proteolytic enzymes, such as clostripain [58, 60], pepsin, and trypsin [97, 98, 99]. All these researchers proved that the resulting peptides lost the enzymatic activities of lysozyme, but exhibited strong bactericidal activities against both Gram-negative (*E.coli*, *Salmonella*, *Pseudomonas*, and *Aeromonas*) and Gram-positive bacteria (*Listeria monocytogenes*, *Staph aureus, Bacillus* spp., and *Leuconostic*s spp.) as well as yeasts (*Saccharomyces*). Scanning electron microscopy clearly demonstrated that cell membrane of both Gram-negative and –positive bacteria was damaged by these peptides. Thus these peptides probably have a different mechanism of action than native lysozyme [58, 99]. Mine et al (2004) could isolation, purification, and characterization of novel antimicrobial peptides from chicken egg white lysozyme hydrolysate, obtained by peptic digestion and subsequent tryptic digestion. The hydrolysate was composed of over 20 small peptides of less than 1000 Da, and had no enzymatic activity. The water-soluble peptide mixture showed bacteriostatic activity against Gram-positive bacteria (Staphylococcus aureus 23-394) and Gram-negative bacteria (*E. coli* K-12). Two bacteriostatic peptides were purified and sequenced. One peptide, with the sequence Ile-Val-Ser-Asp-Gly-Asp-Gly-Met-Asn-Ala-Trp, inhibited Gram-negative bacteria *E. coli* K-12 and corresponded to amino acid residues 98-108, which are located in the middle part of the helix-loop-helix. Another novel antimicrobial peptide inhibited *S. aureus* and was identified as His-Gly-Leu-Asp-Asn-Tyr-Arg, corresponding to amino acid residues 15-21 of lysozyme. These peptides have broadened the antimicrobial activity of lysozyme to Gram-negative bacteria. The results obtained in this study indicate that lysozyme possesses nonenzymatic bacteriostatic

domains in its primary sequence and they are released by proteolytic hydrolysis [99].

way as a safe shelf-life extender in the food industry.

**6. Egg shell membrane bio proteins** 

Consumers are increasingly demanding food that is free from pathogens, but with less preservatives and additives. As a response to these conflicting demands, current trends in the food industry include the investigation of alternative natural preservative in foods. Six Gram-negative bacteria (*Escherichia coli*, *Salmonella enteritidis* NBRC 3313, *Salmonella typhimurium*, *Pseudomonas fluorescens*, *Pseudomonas aeruginosa*, *Aeromonas hydrophila*) were checked for sensitivity to native hen egg white lysozyme and hydrolysate preparation of lysozyme derived peptides. Generally, lysozyme peptides preparation acts on the tested organisms and on different strains of *Bacillus* spp. with much more potency comparing to native lysozyme [100, 101]. The resulting peptides lost the enzymatic activities of lysozyme, but exhibited strong bactericidal activities against both Gram-negative and Gram-positive bacteria [97, 102]. Being natural antimicrobial, lysozyme peptides preparation will find its

The egg shell membrane has been thought to be beneficial in the treatment of some injuries. For example, in Japan, when Sumo wrestlers get flesh abrasions, they will often peel the egg membrane from the egg shell and cover their injuries. They believe that it facilitates their recovery. Peptides product which are stable in water have been prepared from hydrolyzed Different Non-collagenous proteins have been identified in the organic matrix of the hen's egg shell. Ovocleidin-17 is a soluble matrix protein component and distributed in palisade and mammillary layers [104].

Ovocalyxin-32 has been identified as a novel 32-kDa protein. It is expressed at high levels in the uterine and isthmus regions of the oviduct, and concentrated in the eggshell. In the eggshell, ovocalyxin-32 localizes to the outer palisade layer, the vertical crystal layer, and the cuticle of the eggshell, in agreement with its demonstration by Western blotting at high levels in the uterine fluid during the termination phase of eggshell formation. Ovocalyxin-32 is therefore identified as a novel protein synthesized in the distal oviduct where hen eggshell formation occurs [105].

Osteopontin, a phosphorylated bone glycoprotein involved in formation and remodeling of the mineralised tissue, has also been demonstrated in the hen egg shell. Gene expression for this protein has been shown to be higher during the period of calcification [106].

Ovalbumin, lysozyme and ovotransferrin, as egg white proteins, have been identified in hen's egg shell. The organic matrix also contains several proteoglycan molecules [107].

Chicken eggshell powder has been proposed as an attractive source of calcium for human health to increase bone mineral density in an elderly population with osteoporosis. However, factors affecting calcium transport of eggshell calcium have not yet been evaluated. Chicken eggshell contains about 1.0% (w/w) matrix proteins in addition to a major form of calcium carbonate (95%, w/w). It was found that soluble eggshell matrix proteins remarkably enhance calcium transport using in vitro Caco-2 cell monolayers grown on a permeable support. The total calcium transport across Caco-2 monolayers showed an increase of 64% in the presence of 100 microg/well soluble eggshell matrix proteins. The active enhancer with a molecular mass of 21 kDa was isolated by reversed phase highperformance liquid chromatography and did not match to any previously identified protein. The N-terminal sequence was determined to be Met-Ala-Val-Pro-Gln-Thr-Met-Val-Gln. The possible mechanisms of eggshell matrix protein-mediated increase in calcium transport and the potential significance of eggshell calcium as a nutraceutical are discussed [108].

Functional Proteins and Peptides of Hen's Egg Origin 137

[5] Sugino H, Nitoda T, Juneja LR. General chemical composition of hen eggs. In: Yammamoti T, Juneja LR, Hatta H, Kim M, editors. Hen eggs: Their basic and applied

[6] Shinohara K, Fukushima T, Suzuki M, Tsutsumi M, Kobori M, Kong ZL. Effect of some constituents of chicken egg yolk lipoprotein on the growth and IgM production of human-human hybridoma cells and other human-derived cells. Cytotechnology 1993;

[7] Hegenauer J, Saltmann P, Nace G. Iron (III)-phosphoprotein charts: Stoichiometricequilibrium constant for interaction of iron (III) and phosphoserine

[8] Nakamura S, Ogawa M, Nakai S, Kato A, Kitts DD. Antioxidant activity of a Maillardtype phosvitin-galactomannan conjugate with emulsifying properties and heat stability.

[9] Sattar Khan MA, Nakamura S, Ogawa M, Akita E, Azakami H, Kato A. Bactericidal action of egg yolk phosvitin against *Escherichia coli* under thermal stress. Journal of

[10] Williams T. Serum proteins and the livetins of hen's egg yolk. Biochemistry Journal

[11] Sunwoo HH, Sim JS. IgY technology for human health: Antimicrobial activities of IgY preparations against foodborne pathogens. The 225th ACS National Meeting, New

[12] Hatta H, Tsuda K, Akachi S, Kim M, Yamamoto T. Productivity and some properties of egg yolk antibody (IgY) against human rotavirus compared with rabbit IgG. Bioscience

[13] Sun S, Mo W, Ji Y, Lius S. Preparation and mass spectrometric study of egg yolk antibody (IgY) against rabies virus, Rapid Communications in Mass Spectrometry 2001;

[14] Woolley JA, Landon J. Comparison of antibody production to human interlukin-6 (IL-6) by sheep and chickens. Journal of Immunological Methods 1995; 187: 253-265. [15] Shimizu M, Nagashima H, Sano K, Hashimoto K, Ozeki M, Tsuda K. Molecular stability of chicken and rabbit immunoglobulin G. Bioscience Biotechnology and Biochemistry

[16] Campbell RD, Dodds AW, Porter RR. The binding of human complement component

[17] Fischer M, Hlinak A. The lack of binding ability of staphylococcal protein A and streptococcal protein G to egg yolk immunoglobulins of different fowl species. Berl

[18] Larsson A, Karlsson-Parra A, Sjoquist J. Use of chicken antibodies in enzyme immunoassays to avoid interferences by rheumatoid factors. Clinical Biochemistry 1991;

C4 to antibody-antigen aggregates. Biochemistry Journal 1980; 189: 67-80.

residues of phosvitin and casein. Biochemistry 1979; 18: 3865-3879.

Journal of Agriculture Food Chemistry 1998; 46: 3958-3963.

Agriculture Food Chemistry 2000; 48: 1503-1506.

Biotechnology and Biochemistry 1993; 57: 450-454.

Munch Tierarztl Wochenschr 2000; 113: 94-96.

science. Boca Raton: CRC Press. 1997; pp. 13-24.

11: 149-154.

1962; 83: 346-355.

Orleans, LA.: 2003.

15: 708-712.

1992; 56: 270-274.

37: 411-414.

Experimental and clinical studies performed to date have shown a number of positive properties of eggshell powder, such as antirachitic effects in rats and humans. A positive effect was observed on bone density in animal models of postmenopausal osteoporosis in ovariectomized female rats. In vitro eggshell powder stimulates chondrocyte differentiation and cartilage growth. Clinical studies in postmenopausal women and women with senile osteoporosis showed that eggshell powder reduces pain and osteoresorption and increases mobility and bone density or arrests its loss. The bioavailability of calcium from this source, as tested in piglets, was similar or better than that of food grade purified calcium carbonate. Clinical and experimental studies showed that eggshell powder has positive effects on bone and cartilage and that it is suitable in the prevention and treatment of osteoporosis [109].

## **Author details**

Adham M. Abdou *Food Control Department, Benha University, Moshtohor, Kaliobiya, Egypt* 

Mujo Kim *Research Department, Pharma Foods International Co., Ltd., Kyoto, Japan* 

Kenji Sato *Division of Applied Life Sciences, Kyoto Prefectural University, Kyoto, Japan* 

## **8. References**


[5] Sugino H, Nitoda T, Juneja LR. General chemical composition of hen eggs. In: Yammamoti T, Juneja LR, Hatta H, Kim M, editors. Hen eggs: Their basic and applied science. Boca Raton: CRC Press. 1997; pp. 13-24.

136 Bioactive Food Peptides in Health and Disease

**Author details** 

Adham M. Abdou

Mujo Kim

Kenji Sato

**8. References** 

1061-1066.

Publishing Co. 2002; pp. 9-18.

Nutrition 2000; 19: 499S-506S.

increase of 64% in the presence of 100 microg/well soluble eggshell matrix proteins. The active enhancer with a molecular mass of 21 kDa was isolated by reversed phase highperformance liquid chromatography and did not match to any previously identified protein. The N-terminal sequence was determined to be Met-Ala-Val-Pro-Gln-Thr-Met-Val-Gln. The possible mechanisms of eggshell matrix protein-mediated increase in calcium transport and

Experimental and clinical studies performed to date have shown a number of positive properties of eggshell powder, such as antirachitic effects in rats and humans. A positive effect was observed on bone density in animal models of postmenopausal osteoporosis in ovariectomized female rats. In vitro eggshell powder stimulates chondrocyte differentiation and cartilage growth. Clinical studies in postmenopausal women and women with senile osteoporosis showed that eggshell powder reduces pain and osteoresorption and increases mobility and bone density or arrests its loss. The bioavailability of calcium from this source, as tested in piglets, was similar or better than that of food grade purified calcium carbonate. Clinical and experimental studies showed that eggshell powder has positive effects on bone and cartilage and that it is suitable in the prevention and treatment of osteoporosis [109].

the potential significance of eggshell calcium as a nutraceutical are discussed [108].

*Food Control Department, Benha University, Moshtohor, Kaliobiya, Egypt* 

*Research Department, Pharma Foods International Co., Ltd., Kyoto, Japan* 

*Division of Applied Life Sciences, Kyoto Prefectural University, Kyoto, Japan* 

[1] Kerver JM, Park Y, Song WO. The role of eggs in American diets: health implications and benefits. In: Watson R., editor. Eggs and health promotion. Iowa: Blackwell

[2] Hasler CM. The changing face of functional foods. Journal American College of

[3] Watkins BA. The nutritive value of the egg. In: Stadelman WJ, Cotterill OJ, editors. Egg science and technology. New York: The Haworth Press Inc. 1995; pp. 177-194. [4] Shin JH, Yang M, Nam SW, Kim JT, Myung NH, Bang WG, Roe IH. Use of egg yolkderived immunoglobulin as an alternative to antibiotic treatment for control of *Helicobacter pylori* infection. Clinical Diagnostic Laboratory of Immunology 2002; 9:


[19] Rubinstein E, Kouns WC, Jennings LK, Boucheix C, Carroll RC. Interaction of two GPIIb/IIa monoclonal antibodies with platlet Fc receptor (Fc gamma RII). British Journal of Haematology 1991; 87: 80-86.

Functional Proteins and Peptides of Hen's Egg Origin 139

[34] Wendakoon CN, Thomson ABR, Ozimek L. Lack of therapeutic effect of a specially designed yogurt for the suppression of *Helicobacter pylori* infection. Digestion 2002; 65:

[35] Sakamoto I, Igarashi M, Kimura K, Takagi A, Miwa T, Koga Y. Suppressive effect of *Lactobacillus gasseri* OLL2716 (LG21) on *Helicobacter pylori* infection in humans.

[36] Cats A, Kuipers EJ, Bosschaert MAR, Pot RGJ, Vandenbroucke-Grauls CMJE, Kusters JG. Effect of frequent consumption of a Lactobacillus casei-containing milk drink in Helicobacter pylori-colonized subjects. Alimentary Pharmacology and Therapeutics

[37] Kim M, Higashiguchi S, Iwamoto Y, Yang HC, Cho HY, Hatta H. Egg yolk antibody

[38] Shin JH, Roe IH, Kim HG. Production of anti- Helicobacter pylori urease-specific immunoglobulin in egg yolk using an antigenic epitope of *H. pylori* urease. Journal of

[39] Smith DJ. Dental caries vaccines: Prospects and concerns. Critical Reviews in Oral

[40] Loesche WJ. Role of *Streptococcus mutans* in human dental decay. Microbiological

[41] Hamada S, Koga T, Ooshima T. Virulence factors of *Streptococcus mutans* and dental

[42] Otake S, Nishihara Y, Makimura M, Hatta H, Kim M, Yamamoto T, Hirasawa M. Protection of rats against dental carries by passive immunization with hen-egg-yolk

[43] Hatta H, Tsuda K, Ozeki M, Kim M, Yamamoto T, Otake S, Hirasawa M, Katz J, Childers NK., Michalek SM. Passive immunization against dental plaque formation in humans: Effect of a mouth rinse containing egg yolk antibodies (IgY) specific to

[44] Nakamura T, Umeda K, Kusonoki S, Arai J, Namiki H. Advances in IgY applications: characteristics of specific egg yolk immunoglobulin against influenza virus, Page 72 in Proc. 16thAnnual meeting Japanese Society of Bioscience Biotechnology and

[45] Fathy G, Abdel-Raheem SM, Abdou AM, Horie M, Kim M, Suzuki H. Suppressive effect of Topical Specific Egg Yolk Immunoglobulin (IgY) on *Propionibacterium acnes* in

[46] Jiang B, Mine M. Preparation of novel functional oligophoshopeptides from hen egg

[47] Leem KH, Kim MG, Kim HK, Oi Y, Kim M. Effect of egg yolk proteins on the longitudinal bone growth in rat, Page 200 in Proc. 16thAnnual meeting Jpn. Soc. Biosci.

yolk phosvitin. Journal of Agricultural and Food Chemistry 2000; 48: 990.

caries prevention. Journal of Dental Research 1984; 63: 407.

antibody (IgY). Journal of Dental Research 1991; 70: 162.

*Streptococcus mutans*. Caries Research 1997; 31: 268.

Agrochemistry (in Japanese), Hiroshima, Japan: 2004.

Acne Vulgaris. Sci. J. Al-Azhar Med. Fac. (girls). 2007; 28: 2177.

Biotechnol. & Agrochem. (in Japanese), Hiroshima, Japan: 2004.

and its application. Biotechnology and Bioprocess Engineering 2000; 5: 75.

Antimicrobial Chemotherapy 2001; 47: 709.

Medical Microbiology 2004; 53: 31.

Biology and Medicine 2002; 13: 335.

Reviews 1989; 50: 353.

16.

2003; 17: 429.


[34] Wendakoon CN, Thomson ABR, Ozimek L. Lack of therapeutic effect of a specially designed yogurt for the suppression of *Helicobacter pylori* infection. Digestion 2002; 65: 16.

138 Bioactive Food Peptides in Health and Disease

2003; 41: 259-267.

2001; 32: 19-25.

10: 720-741.

128.

2003; 41: 408.

Science 2004; 87: 4073.

Digestion 1999; 60: 203.

Science 1999; 82: 2245-2256.

of Haematology 1991; 87: 80-86.

[19] Rubinstein E, Kouns WC, Jennings LK, Boucheix C, Carroll RC. Interaction of two GPIIb/IIa monoclonal antibodies with platlet Fc receptor (Fc gamma RII). British Journal

[20] Narat M. Production of antibodies in chickens. Food Technology and Biotechnology

[23] Yang H, Jin Z, Yu Q, Yang T, Wang H, Liu L. The selective recognition of IgY for

[24] Dunn BE, Cohen H, Blaser M J. *Helicobacter pylori*. Clinical Microbiology Reviews 1997;

[25] Blaser M J. *Helicobacter pylori* and the pathogenesis of gastroduodenal inflammation.

[26] Parsonnet J, Hansen S, Rodriguez L, Gleb AB, Warnke RA, Jellum E, Orentreich N, Vogelman JH, Friedman GD. *Helicobacter pylori* infection and gastric lymphoma. The

[27] Marshall BJ. *Helicobacter pylori*. American Journal of Gastroenterolology 1994; 89: 116-

[28] Chandan RC. Enhancing market value of milk by adding cultures. Journal of Dairy

[29] Yamane T, Saito Y, Takizawa S, Goshima H, Kodama Y, Horie N, Kim M. Development of anti-Helicobacter pylori urease IgY and its application for food product. Food

[30] Chang Y, Min S, Kim K, Han Y, Lee J. Delta [sup 13] C-urea breath test value is a useful indicator for *Helicobacter pylori* eradication in patients with functional dyspepsia.

[31] Chen JP, Chang MC. Effect of anti-Helicobacter pylori urease antibody (IgY) as a food ingredient on the decrease of H. pylori in the stomach of humans infected with *H. pylori*. Taiwanese Journal of Agricultural Chemistry and Food Science (in Taiwanese)

[32] Horie K, Horie N, Abdou AM, Yang JO, Yun SS, Chun HN, Park CK, Kim M, Hatta H. Egg Yolk Immunoglobulin (IgY) on *Helicobacter pylori* in Humans. Journal of Dairy

[33] Michetti P, Dorta G, Wiesel PH, Brassart D, Verdu E, Herranz M, Felley C, Porta N, Rouvet M, Blum AL, Corthésy-Theulaz I. Effect of whey-based culture supernatant of *Lactobacillus acidophilus* (johnsonii) La1 on *Helicobacter pylori* infection in humans.

digestive cancers. Chinese Journal of Biotechnology 1997; 13: 85-90.

The Journal of Infectious Diseases 1990; 161: 626-633.

New England Journal of Medicine 1994; 330: 1267-1271.

Processing and Ingredients (in Japanese) 2003; 38: 70.

Journal of Gastroenterology and Hepatology 2003; 18: 726.

[21] Zeidler G. Eggs vital to human and animal medicine. World Poultry 1998; 14: 33-34. [22] Sarker SA, Casswall TH, Juneja LR, Hoq E, Hossain I, Fuchs GJ. Randomized, placebocontrolled, clinical trial of hyperimmunized chicken egg yolk immunoglobulin in children with rotavirus diarrhea. Journal of Pediatric Gastroenterology and Nutrition


[48] Leem KH, Kim MG, Kim HM, Kim M, Lee YJ, Kim HK. Effect of egg yolk proteins on the longitudinal bone growth of adolescent male rat, Bioscience Biotechnology and Biochemistry 2004; 68: 2388.

Functional Proteins and Peptides of Hen's Egg Origin 141

[63] Liu ST, Sugimoto T, Azakami H, Kato A. Lipophilization of lysozyme by short and middle chain fatty acids. Journal of Agricultural and Food Chemistry 2000; 48: 265. [64] Kato A, Ibrahim HR, Watanabe H, Honma K, Kobayashi K. New approach to improve the gelling and surface functional properties of dried egg white by heating in dry state.

[65] Nakamura NK, Furukawa N, Matsuoka M, Takahashi T, Yamanaka Y. Enzyme activity of lysozyme-dextran complex prepared by high-pressure treatment. Food Science and

[66] Nakamura S, Saito M, Goto T, Saeki H, Ogawa M, Gotoh M, Gohya Y, Hwang JK. Rapid formation of biologically active neoglycoprotein from lysozyme and xyloglucan hydroysates throughnaturally occurring Maillard reaction. Journal of Food Science and

[67] Takahashi K, Lou XF, Ishii Y, Hattori M. Lysozyme-glucose stearic acid monoester conjugate formed through the Maillard reaction as an antibacterial emulsifier. Journal of

[68] Liu ST, Azakami H, Kato A. Improvement in the yield of lipophilized lysozyme by the

[69] Kijowski J, Lesnierowski G. Separation, polymer formation and antibacterial activity of

[70] Stadhouders J, Stegink H, Van den Berg G, Van Ginkel W. The use of lysozyme to prevent butyric fermentation in Gouda cheese. Voedingsmiddelen technlogie 1987; 25:

[71] de Roos AL, Walstra P, Geurts TJ. The association of lysozyme with casein.

[72] Daeschel MA, Bruslind L, Clawson J. Application of the enzyme lysozyme in brewing.

[73] Lacono VJ, Mackay BJ, Dirienzo S, Pollock JJ. Selective antibacterial properties of

[74] El-Nimr A, Hardee GE, Perrin JH. A fluorimetric investigation of the binding of drugs

[75] Siwicki AK, Klein P, Morand M, Kiczka W, Studnicka M. Immunostimulatory effects of dimerized lysozyme (KLP-602) on the nonspecific defense mechanisms and protection against furunculosis in salmonids. Veterinary Immunology and Immunopathology

[76] Sugahara T, Murakami F, Yamada Y, Sasaki T. The mode of action of lysozyme as an immunoglobulin production stimulation factor. Biochimica and Biophysica Acta 2000;

[77] Sava G. Reduction of B16 melanoma metastases by oral administration of egg-white

[78] Yamashita K, Tachibana Y, Hitoi A. Sialic acid-containing sugar chains of hen

Master Brewers. Association of American Technology Quart. 1999; 36: 219.

lysozyme for oral microorganisms. Infection and Immunity 1980; 29: 523.

to lysozyme. Journal of Pharmacy and Pharmacology 1981; 33: 117.

lysozyme. Cancer Chemotherapy Pharmacology 1989; 25: 221.

ovalbumin and ovomucoid. Carbohydrate Research 1984; 130: 271.

combination with Maillard-type glycosylation. Nahrung 2000; 44: s407.

lysozyme. Polish Journal of Food and Nutrition Sciences 1999; 8/49: 3.

Journal of Agricultural and Food Chemistry 1989; 37: 433.

Technology International 1997; 3: 235.

Agricultural and Food Chemistry 2000; 48: 2044.

International Dairy Journal 1998; 8: 319.

Nutrition 2000; 5: 65.

17.

1998; 61: 369.

1475: 27.


[63] Liu ST, Sugimoto T, Azakami H, Kato A. Lipophilization of lysozyme by short and middle chain fatty acids. Journal of Agricultural and Food Chemistry 2000; 48: 265.

140 Bioactive Food Peptides in Health and Disease

Biochemistry 2004; 68: 2388.

LandersRG. Austin, TX: 1996.

Chromatography B 2001; 756: 189.

New York: CRS Press, Inc. 2000.

Journal 1996; 319: 361.

Microbiology 1997; 82: 372.

Food Chemistry 1991; 39: 2077.

2002: 8: 671.

Agrochemistry (in Japanese), Hokkaido, Japan: 2005.

Journal of Agricultural and Food Chemistry 1991; 39: 443.

enteritis in the infant. Journal Tissue Reactions 1983; 1: 117.

Microscopy Research and Technique 1997; 39: 297.

[48] Leem KH, Kim MG, Kim HM, Kim M, Lee YJ, Kim HK. Effect of egg yolk proteins on the longitudinal bone growth of adolescent male rat, Bioscience Biotechnology and

[49] Oi Y, Ji MY, Leem KH, Cho SW, Kim M. The effect of hen egg yolk peptide on bone growth, 17thAnnual meeting Japanese Society of Bioscience Biotechnology and

[50] Ibrahim HR. Insight into the structure-function relationships of ovalbumin, ovotransferrin, and lysozyme. In: Yamamoto T, Juneja LR, Hatta H, Kim M, editors.

[51] Mine Y, Noutomi T, Haga N. Emulsifying and structural properties of ovalbumin.

[52] Nisbet AD, Saundry RH, Moir AJ, Fothergill LA, Fothergill JE. The complete amino-acid sequence of hen ovalbumin, European Journal of Biochemistry/FEBS 1981; 115: 335. [53] Gettins PGW, Patston PA, Olson ST. Serpins: Structure, Function and Biology,

[54] Huntington JA, Stein PE. Structure and properties of ovalbumin. Journal of

[55] Ibrahim HR. Ovotransferrin. In: Naidu AS, editor. Natural food antimicrobial systems.

[56] Mason AB, Woodworth RC, Oliver RW, Green BN, Lin LN, Brandts JF, Savage KJ, Tam BM, MacGillivrays TA. Association of the two lobes of ovotransferrin is a prerequisite for receptor recognition: Studies with recombinant ovotransferrins. Biochemistry

[57] Corda R, Biddau P, Corrias A, Puxeddu E. Conalbumin in the treatment of acute

[58] Ibrahim HR, Aoki T, Pellegrini A. Strategies for new antimicrobial proteins and peptides: lysozyme and aprotinin as model molecules. Current Pharmacological Design

[59] Masschalck B, Michiels CW. Antimicrobial properties of lysozyme in relation to foodborne vegetative bacteria. Critical Reviews in Microbiology 2003; 29: 191. [60] Pellegrini A, Bramaz TN, Klauser S, Hunziker P, von Fellenberg R. Identification and isolation of bactericidal domain in chicken egg white lysozyme. Journal of Applied

[61] Wild P, Gabrieli A, Schraner EM, Pellegrini A, Thomas U, Frederik PM, Stuart MC, Vonfellenberg R. Reevaluation of the effect of lysozyme on Escherichia coli employing ultrarapid freezing followed by cryoelectron microscopy or freeze substitution.

[62] Ibrahim HR, Kato A, Kobayashi K. Antimicrobial effects of lysozyme against Gramnegative bacteria due to covalent binding of palmitic acid. Journal of Agricultural and

Hen Eggs: Their basic and applied science. New York: CRC press, Inc. 1997.


[79] Urisu A, Ando H, Morita Y, Wada E, Yasaki T, Yamada K, Komada K, Torii S, Goto M, Wakamatsu T. Allergenic activity of heated and ovomucoid-depleted egg white. Journal of Allergy and Clinical Immunology 1997; 100: 171.

Functional Proteins and Peptides of Hen's Egg Origin 143

[93] Cho HJ, Kittaka R, Abdou AM, Kim M, Kim HS, Lee DH, Park HJ. Inhibitory effects of oligopeptides from hen egg white on both human platelet aggregation and blood

[94] El-Mahmoudy A, Kittaka R, Okamoto K, Shimizu Y, Shiina T, Takewaki T, Kim M. Pharmacologically active peptide derived from egg white with circulatory beneficial effects. 17thAnnual meeting Japanese Society of Bioscience Biotechnology and

[95] Miguel M, Recio JA, Gómez-Ruiz M, Ramos M, Amos M, López-Fandiňo R. Angiotensin I–converting enzyme inhibitory activity of peptides derived from egg white proteins by enzymatic hydrolysis. Journal of Food Protection 2004; 67: 1914. [96] Dávalos A, Miguel M, Bartolomé B, López-Fandiňo R. Antioxidant activity of peptides derived from egg white proteins by enzymatic hydrolysis. Journal of Food Protection

[97] Ibrahim HR, Inazaki D, Abdou AM, Aoki T, Kim M. Processing of Lysozyme at distinct loops by pepsin: A novel action for generating multiple antimicrobial peptide motifs in

[99] Mine Y, Lauriau S, Ma F. Antimicrobial peptides released by enzymatic hydrolysis of hen egg white lysozyme. Journal of Agricultural and Food Chemistry 2004; 52: 1088. [100] Abdou AM, Higashiguchi S, Aboueleinin A, Kim M, Ibrahim HR. Lysopep: a lysozyme peptide preparation has an exaggerated antimicrobial activity against Gramnegative bacteria, Page 226 in Proc. 16thAnnual meeting Japanese Society of Bioscience

[101] Abdou AM, Higashiguchi S, Aboueleinin AM, Kim M, Ibrahim HR. Antimicrobial peptides derived from hen egg lysozyme with inhibitory effect against Bacillus species.

[102] Abdou AM, Higashiguchi S, Kim M, Ibrahim HR. A marvelous inhibitory effect of lysozyme peptides preparation against Bacillus species. Page 221 in Proc. 15thAnnual meeting Japanese Society of Bioscience Biotechnology and Agrochemistry, Yokohama,

[103] Nakano T, Ikawa NI, Ozimek L. Chemical composition of chicken eggshell and shell

[104] Hincke MT, Tsang CPW, Courtney M, Hill V, Narbaitz R. Purification and immunochemistry of a soluble matrix protein of the chicken eggshell (ovocleidin-17).

[105] Gautron J, Hincke MT, Mann K, Panhéleux M, Bain M, McKee MD, Solomon SE, Nys, Y. Ovocalyxin-32, a novel chicken eggshell matrix protein. Journal of Biological

the newborn stomach. Biochimica et Biophysica Acta (BBA) 2005; 1726 (1): 102. [98] Ibrahim HR, Thomas U, Pellegrini A. A helix-loop-helix peptide at the upper lip of the active site cleft of lysozyme confers potent antimicrobial activity with membrane

permeabilization action. Journal of Biological Chemistry 2001; 276: 43767.

Biotechnology and Agrochemistry, Hiroshima, Japan. 2004.

Food Control 2007; 18 (2): 173.

Chemistry 2001; 276: 39243.

membranes. Poultry Science 2003; 82: 510.

Calcified Tissue International 1995; 56: 578.

Japan. 2003.

coagulation. Archives in Pharmacology Research 2009; 32(6): 945.

Agrochemistry, Hokkaido, Japan. 2005.

2004; 67: 1939.


[93] Cho HJ, Kittaka R, Abdou AM, Kim M, Kim HS, Lee DH, Park HJ. Inhibitory effects of oligopeptides from hen egg white on both human platelet aggregation and blood coagulation. Archives in Pharmacology Research 2009; 32(6): 945.

142 Bioactive Food Peptides in Health and Disease

of Allergy and Clinical Immunology 1997; 100: 171.

Medicine and Biology 1993; 126: 19.

mesenteric artery. FEBS Letters 1999; 452: 181.

Bioscience Biotechnology and Biochemistry 2001; 65: 736.

14.

Plenum Press. 1993.

1995; 59: 2344.

289.

Chemistry 2000; 48: 6261.

Hiroshima, Japan. 2004.

Bioscience Biotechnology and Biochemistry 1996; 60: 1503.

[82] Green NM. Avidin. Advances in Protein Chemistry 1975; 29: 85.

[79] Urisu A, Ando H, Morita Y, Wada E, Yasaki T, Yamada K, Komada K, Torii S, Goto M, Wakamatsu T. Allergenic activity of heated and ovomucoid-depleted egg white. Journal

[80] Tsuge Y, Shimoyamada M, Watanabe K. Binding of egg white proteins to viruses.

[81] Ohami H, Ohishi H, Yokota T, Mori T, Watanabe K. Cytotoxic effect of sialoglycoprotein derived from avian egg white ovomucin on the cultured tumor cell.

[83] Ebina T, Tsukada K. Protease inhibitors prevent the development of human rotavirus induced diarrhea in suckling mice. Microbiology and Immunology 1991; 35: 583. [84] Abrahamson M, Dalboge H, Olafsson I, Carlsen S, Grubb A. Efficient production of native, biologically active human cystatin C by Escherichia coli. FEBS Letters 1988; 236:

[85] Kennedy AR. Anti-carcinogenic activity of protease inhibitors. In: Troll W, Kennedy AR, editors. Protease Inhibitors as Cancer Chemopreventive Agents. New York :

[86] Fujita H, Sasaki R, Yoshikawa M. Potentiation of the anthypertensive activity of orally administered ovokinin, a vasorelaxing peptide derived from ovalbumin, by emulsification in egg phosphatidylcholine. Bioscience Biotechnology and Biochemistry

[87] Matoba N, Usui H, Fujita H, Yoshikawa M. A novel anti-hypertensive peptide derived from ovalbumin induced nitric oxide-mediated vasorelaxation in an isolated SHR

[88] Matoba N, Yamada Y, Usui H, Fujita H, Nakagiri R, Yoshikawa M. Designing potent derivatives of ovokinin (2-7), an anti-hypertensive peptide derived from ovalbumin.

[89] Ibrahim HR, Iwamori E, Sugimoto Y, Aoki T. Identification of a distinct antibacterial domain within the N-lobe of ovotransferrin. Biochimica et Biophysica Acta 1998; 1401:

[90] Kovacs-Nolan J, Zhang JW, Hayakawa S, Mine Y. Immunochemical and structural analysis of pepsin-digested egg white ovomucoid. Journal of Agricultural and Food

[91] Watanabe K, Tsuge Y, Shimoyamada M, Ogama N, Ebina T. Antitumor effects of pronase treated fragments, glycopeptides, from ovomucin in hen egg white in a double grafted tumor system. Journal of Agricultural and Food Chemistry 1998; 46: 3033. [92] Kittaka R, Cho HJ, Choi SH, Kang HC, Choi SA, Jung YJ, Lee TK, Kim CR, Park HJ, Kim M. Anti-thrombotic action of egg white peptides. Page 198 in Proc. 16thAnnual meeting Japanese Society of Bioscience Biotechnology and Agrochemistry (in Japanese),


[106] Pines M, Knopov V, Bar A. Involvement of osteopontin in egg shell formation in the layingchicken. Matrix Biology 1994; 14: 765.

**Chapter 6** 

© 2013 Pihlanto and Mäkinen, licensee InTech. This is an open access chapter 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.

© 2013 Pihlanto and Mäkinen, licensee InTech. This is a paper 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.

**Antihypertensive Properties of Plant Protein** 

Cardiovascular diseases (CVD) are a major health problem in the industrialized countries, representing the main cause of death in the world. It is estimated that 17 million people globally die of CVD every year and these diseases are responsible for more than half of all deaths in Europe [1]. Therefore, primary prevention is becoming an increasing part of public health strategies aimed at reducing societal burden due to CVD-related morbidity and mortality worldwide. There are several behavioural factors such as tobacco use, ethanol consumption, unhealthy diet and physical inactivity that can lead to hypertension, hyperlipidemia, diabetes, overweight and obesity, and thereby contribute to CVD development. The World Health Organisation emphasises the importance of improved nutrition as means of controlling the expected rise in global CVD incidence over the next

Angiotensin I-converting enzyme (ACE: EC 3.4.15.1) is a peptidyldipeptide hydrolase that plays an important physiological role in both the regulation of blood pressure and cardiovascular function [2] through two different reactions. First, ACE catalyzes the hydrolysis of angiotensin I, an inactive decapeptide, to angiotensin II, a powerful vasoconstrictor and salt-retaining octapeptide. Thus, ACE-inhibition has a hypotensive effect. Secondly, ACE catalyzes the inactivation of the vasodilator bradykinin that regulates different biological processes including vascular endothelial nitric oxide (NO) release [3].

Oxidative stress has a well documented role in CVD development. Oxidative stress is defined as the situation characterized by increased generation of free radicals (reactive oxygen species, ROS), resulting in increased oxidative damage of biological structures. Within the cell, physiologic levels of some ROS are involved as key intermediates in signalling pathways to maintain basal cellular functions. In contrast, when ROS are

**Derived Peptides** 

http://dx.doi.org/10.5772/54565

**1. Introduction** 

decades.

Anne Pihlanto and Sari Mäkinen

Additional information is available at the end of the chapter


## **Antihypertensive Properties of Plant Protein Derived Peptides**

Anne Pihlanto and Sari Mäkinen

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/54565

## **1. Introduction**

144 Bioactive Food Peptides in Health and Disease

Pharmacology 2003; 23: 83.

layingchicken. Matrix Biology 1994; 14: 765.

[106] Pines M, Knopov V, Bar A. Involvement of osteopontin in egg shell formation in the

[107] Ferandez MS, Araya M, Arias JL. Eggshells are shaped by a precise spatio-temperal arrangement of sequentially deposited macromolecules. Matrix Biology 1997; 16: 13. [108] Daengprok W, Garnjanagoonchorn W, Naivikul O, Pornsinlpatip P, Issigonis K, Mine Y. Chicken eggshell matrix proteins enhance calcium transport in the human intestinal epithelial cells, Caco-2. Journal of Agricultural and Food Chemistry 2003; 51: 6056. [109] Rovensky J, Stancikova M, Masaryk P, Svik K, Istok R. Eggshell calcium in the prevention and treatment of osteoporosis. International Journal of Clinical

> Cardiovascular diseases (CVD) are a major health problem in the industrialized countries, representing the main cause of death in the world. It is estimated that 17 million people globally die of CVD every year and these diseases are responsible for more than half of all deaths in Europe [1]. Therefore, primary prevention is becoming an increasing part of public health strategies aimed at reducing societal burden due to CVD-related morbidity and mortality worldwide. There are several behavioural factors such as tobacco use, ethanol consumption, unhealthy diet and physical inactivity that can lead to hypertension, hyperlipidemia, diabetes, overweight and obesity, and thereby contribute to CVD development. The World Health Organisation emphasises the importance of improved nutrition as means of controlling the expected rise in global CVD incidence over the next decades.

> Angiotensin I-converting enzyme (ACE: EC 3.4.15.1) is a peptidyldipeptide hydrolase that plays an important physiological role in both the regulation of blood pressure and cardiovascular function [2] through two different reactions. First, ACE catalyzes the hydrolysis of angiotensin I, an inactive decapeptide, to angiotensin II, a powerful vasoconstrictor and salt-retaining octapeptide. Thus, ACE-inhibition has a hypotensive effect. Secondly, ACE catalyzes the inactivation of the vasodilator bradykinin that regulates different biological processes including vascular endothelial nitric oxide (NO) release [3].

> Oxidative stress has a well documented role in CVD development. Oxidative stress is defined as the situation characterized by increased generation of free radicals (reactive oxygen species, ROS), resulting in increased oxidative damage of biological structures. Within the cell, physiologic levels of some ROS are involved as key intermediates in signalling pathways to maintain basal cellular functions. In contrast, when ROS are

© 2013 Pihlanto and Mäkinen, licensee InTech. This is an open access chapter 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. © 2013 Pihlanto and Mäkinen, licensee InTech. This is a paper 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.

generated in the absence of a physiological stimulus, small-molecule antioxidants are depleted, or antioxidant enzymatic systems are overwhelmed. This leads to a net increase in biologically active ROS and oxidant stress ensues. In blood vessels, oxidant stress has deleterious consequences for basal vascular function. Then, the cellular mechanisms that result in vascular redox imbalance leading to an increase in oxidant stress are implicated in the pathogenesis of vascular disease [4].

Antihypertensive Properties of Plant Protein Derived Peptides 147

were identified. Potatoes may have a role in controlling appetite and therefore weight gain, by contributing to satiety. Gastrointestinal hormones such as cholecystokinin (CCK) are key factors in the regulation of food intake and maintaining energy homeostasis. Hill and colleagues [15] reported reduced energy intake and increased CCK release, when protease

The protein content of defatted rapeseed meal is high, approximately 32%, making it a potential food ingredient. Rapeseed protein has recently been demonstrated to be of high nutritional value in human subjects and substituting Cys-rich rapeseed protein, for milk protein prevented the early onset of insulin resistance, similar to those achieved by manipulating dietary fat and carbohydrates in a rat model [16, 17]. Flaxseed and its defatted meal contain high amounts of proteins, which are comparable in amino acid composition to food proteins like soy, with a preponderance of basic and branch-chain amino acids. The high Cys and Met content can boost the body's antioxidant levels, potentially stabilising DNA during cell division and reducing risk of certain forms of colon cancer [18]. Yet, there are only few studies examining rapeseed and flaxseed meals as source of bioactive peptides

Legumes could represent valuable tools to prevent CVD, in addition to constitute an important source of dietary proteins (18-40 %), dietary fibre, minerals and vitamins. Epidemiological studies have provided consistent evidence of the inverse relationship between legume consumption and the incidence of CVD. The majority of studies that have evaluated the hypocholesterolemic effects of legume consumption examined soybeans [19]. In addition, the meta-analysis showed that diet rich in legumes, such as a variety of beans, peas, and some seeds other than soy decreases total and low-density lipoprotein (LDL) cholesterol [20]. Different legumes have been identified as sources of ACE-inhibitory and

Cereal grains contain relatively little protein compared to legume seeds, with an average of about 10–12% of dry weight. Storage proteins account for about 50% of the total protein in mature cereal grains and have important impacts on their nutritional quality for humans and livestock and on their functional properties in food processing. Proteins can be separated into albumin, globulin, prolamin (hordein) and gluten fractions as described by [29]. The prolamins are characterized by their high content of Pro and Glu and low content of Lys and Trp. Alcohol-soluble endosperm proteins (prolamins) from some cereals (e.g. wheat, barley, and rye) give origin upon proteolytic digestion to biologically active antinutritional peptides able to adversely affect *in vivo* the intestinal mucosa of coeliac patients, whereas prolamins from other cereals (e.g. maize and rice) do not [30]. A large deal of cereal proteins originates from by-products following production of starch, malting or brewing industry. For example, -zein protein, derived from corn starch production, is rich in Pro and hydrolysis by thermolysin liberates ACE-inhibitors [31]. Wheat proteins are also source of opioid peptides [32] and tryptic hydrolysate of rice proteins yields the

inhibitors extracted from potatoes were tested in 11 lean subjects.

to enhance the value of these rapeseed and flaxseed industry by-products.

antioxidative peptides, mainly soybean [21-24], chickpea and pea [25-28].

immunomodulatory peptide, oryzatensin [33].

The search for dietary compounds that prevent the development of CVD is deemed crucial to tackle this major health problem worldwide, and recent observational studies and clinical trials have suggested that increased protein consumption, particularly from plant sources, might reduce blood pressure and prevent CVD. Recently, interest has been emerging to identify and characterize bioactive peptides from plant and animal sources. Bioactive peptides are considered specific protein fragments that are inactive within the sequence of the parent protein. After they are released they may exert various physiological functions. The type of bioactive peptides generated from a particular protein is dependent on two factors: (a) the primary sequence of the source protein and (b) the specificity of the enzyme(s) used to generate such peptides. The hydrolysis of plant proteins has led to the production of a variety of biologically active peptides, such as opioid, antihypertensive, antioxidative, immunomodulatory or antimicrobial peptides [5, 6]. Bioactivity of peptides depend on the structure, however, the structure activity relationship is not yet fully understood for all biological activities described. This present paper focuses on the peptides beneficial to CVD derived from plant proteins.

## **2. Biological activities of plant proteins**

Potato tuber proteins are classified into three major groups: patatins, protease inhibitors, and other proteins. Patatin is the major storage protein and an allergen for some people. The second major potato tuber storage protein is a diverse group of low molecular weight protease inhibitors [7]. The majority of the patatin and proteinase inhibitor isoforms possess enzymatic and inhibitory activities, respectively, which might be of physiological relevance. Activities are associated with the defense mechanisms of potato against pathogens and they inhibit a variety of proteases and some other enzymes, for example invertase [8]. Low molecular weight antimicrobial potato peptides from potato tubers have recently been reported to exhibit antibiotic/antifungal activity also against human pathogenic fungal and microbial strains [9]. The broad phospholipase activity of patatin has been characterized and documented rather extensively [10, 11]. The results indicate that the patatin-related enzymes are, in addition to fat metabolism, involved in the stress responses and signal transduction in the potato tubers. Patatin has also been shown to possess antioxidant or antiradical activity. Liu and colleagues [12] found that purified patatin exert antioxidant or antiradical activity in various *in vitro* tests, such as radical, scavenging activity assay and protection against hydroxyl radical-induced calf thymus DNA damage. Potato protein hydrolysates showed antioxidant activity [13] and enhanced oxidative stability of soybean oil emulsions [14]. Two peptides derived from metallocarboxypeptidase inhibitor and lipoxygenase 1 were identified. Potatoes may have a role in controlling appetite and therefore weight gain, by contributing to satiety. Gastrointestinal hormones such as cholecystokinin (CCK) are key factors in the regulation of food intake and maintaining energy homeostasis. Hill and colleagues [15] reported reduced energy intake and increased CCK release, when protease inhibitors extracted from potatoes were tested in 11 lean subjects.

146 Bioactive Food Peptides in Health and Disease

the pathogenesis of vascular disease [4].

beneficial to CVD derived from plant proteins.

**2. Biological activities of plant proteins** 

generated in the absence of a physiological stimulus, small-molecule antioxidants are depleted, or antioxidant enzymatic systems are overwhelmed. This leads to a net increase in biologically active ROS and oxidant stress ensues. In blood vessels, oxidant stress has deleterious consequences for basal vascular function. Then, the cellular mechanisms that result in vascular redox imbalance leading to an increase in oxidant stress are implicated in

The search for dietary compounds that prevent the development of CVD is deemed crucial to tackle this major health problem worldwide, and recent observational studies and clinical trials have suggested that increased protein consumption, particularly from plant sources, might reduce blood pressure and prevent CVD. Recently, interest has been emerging to identify and characterize bioactive peptides from plant and animal sources. Bioactive peptides are considered specific protein fragments that are inactive within the sequence of the parent protein. After they are released they may exert various physiological functions. The type of bioactive peptides generated from a particular protein is dependent on two factors: (a) the primary sequence of the source protein and (b) the specificity of the enzyme(s) used to generate such peptides. The hydrolysis of plant proteins has led to the production of a variety of biologically active peptides, such as opioid, antihypertensive, antioxidative, immunomodulatory or antimicrobial peptides [5, 6]. Bioactivity of peptides depend on the structure, however, the structure activity relationship is not yet fully understood for all biological activities described. This present paper focuses on the peptides

Potato tuber proteins are classified into three major groups: patatins, protease inhibitors, and other proteins. Patatin is the major storage protein and an allergen for some people. The second major potato tuber storage protein is a diverse group of low molecular weight protease inhibitors [7]. The majority of the patatin and proteinase inhibitor isoforms possess enzymatic and inhibitory activities, respectively, which might be of physiological relevance. Activities are associated with the defense mechanisms of potato against pathogens and they inhibit a variety of proteases and some other enzymes, for example invertase [8]. Low molecular weight antimicrobial potato peptides from potato tubers have recently been reported to exhibit antibiotic/antifungal activity also against human pathogenic fungal and microbial strains [9]. The broad phospholipase activity of patatin has been characterized and documented rather extensively [10, 11]. The results indicate that the patatin-related enzymes are, in addition to fat metabolism, involved in the stress responses and signal transduction in the potato tubers. Patatin has also been shown to possess antioxidant or antiradical activity. Liu and colleagues [12] found that purified patatin exert antioxidant or antiradical activity in various *in vitro* tests, such as radical, scavenging activity assay and protection against hydroxyl radical-induced calf thymus DNA damage. Potato protein hydrolysates showed antioxidant activity [13] and enhanced oxidative stability of soybean oil emulsions [14]. Two peptides derived from metallocarboxypeptidase inhibitor and lipoxygenase 1 The protein content of defatted rapeseed meal is high, approximately 32%, making it a potential food ingredient. Rapeseed protein has recently been demonstrated to be of high nutritional value in human subjects and substituting Cys-rich rapeseed protein, for milk protein prevented the early onset of insulin resistance, similar to those achieved by manipulating dietary fat and carbohydrates in a rat model [16, 17]. Flaxseed and its defatted meal contain high amounts of proteins, which are comparable in amino acid composition to food proteins like soy, with a preponderance of basic and branch-chain amino acids. The high Cys and Met content can boost the body's antioxidant levels, potentially stabilising DNA during cell division and reducing risk of certain forms of colon cancer [18]. Yet, there are only few studies examining rapeseed and flaxseed meals as source of bioactive peptides to enhance the value of these rapeseed and flaxseed industry by-products.

Legumes could represent valuable tools to prevent CVD, in addition to constitute an important source of dietary proteins (18-40 %), dietary fibre, minerals and vitamins. Epidemiological studies have provided consistent evidence of the inverse relationship between legume consumption and the incidence of CVD. The majority of studies that have evaluated the hypocholesterolemic effects of legume consumption examined soybeans [19]. In addition, the meta-analysis showed that diet rich in legumes, such as a variety of beans, peas, and some seeds other than soy decreases total and low-density lipoprotein (LDL) cholesterol [20]. Different legumes have been identified as sources of ACE-inhibitory and antioxidative peptides, mainly soybean [21-24], chickpea and pea [25-28].

Cereal grains contain relatively little protein compared to legume seeds, with an average of about 10–12% of dry weight. Storage proteins account for about 50% of the total protein in mature cereal grains and have important impacts on their nutritional quality for humans and livestock and on their functional properties in food processing. Proteins can be separated into albumin, globulin, prolamin (hordein) and gluten fractions as described by [29]. The prolamins are characterized by their high content of Pro and Glu and low content of Lys and Trp. Alcohol-soluble endosperm proteins (prolamins) from some cereals (e.g. wheat, barley, and rye) give origin upon proteolytic digestion to biologically active antinutritional peptides able to adversely affect *in vivo* the intestinal mucosa of coeliac patients, whereas prolamins from other cereals (e.g. maize and rice) do not [30]. A large deal of cereal proteins originates from by-products following production of starch, malting or brewing industry. For example, -zein protein, derived from corn starch production, is rich in Pro and hydrolysis by thermolysin liberates ACE-inhibitors [31]. Wheat proteins are also source of opioid peptides [32] and tryptic hydrolysate of rice proteins yields the immunomodulatory peptide, oryzatensin [33].

## **2.1. Production of peptides**

ACE-inhibitory peptides have been produced by enzymatic hydrolysis and microbial fermentation of food proteins. In addition, solvent extraction has been used to isolate ACEinhibitory activity from plant materials, such as mushrooms, broccoli and buckwheat [6]. The ACE-inhibitory potency is expressed as the IC50 value (inhibitor concentration leading to 50% inhibition), being used to estimate the effectiveness of different hydrolysates and peptides. The most common way to produce bioactive peptides is through enzymatic hydrolysis of whole protein molecules. The specificity of enzyme and process conditions influence the peptide composition of hydrolysates and thus their activities.

Antihypertensive Properties of Plant Protein Derived Peptides 149

**Response (Δ SBP mmHg)** 

[118]

[119]

[120]


[123]

[86] [175]

[122]

[122]






**Identified peptides** *In vivo* **response Ref** 

**Dose administratio n model** 

500 mg/kg hydrolysate, oral, SHR

oral, SHR

154 mg/kg oral, SHR 154 mg/kg oral, SHR

140 mg daily oral, human

40 mg/kg oral, SHR

40 mg/kg oral, SHR

**Sequences IC50 value (µM)**

> 9.5 80.8 52.3 300.4

94.25 265.43 84.12 61.67 1.31 75.93

1.73 1.14 0.42 1.03

276.2 746.4 228.3 208.6 2.3 275.8 392.2 141.56 523.5 849.7

ND 60 mg/kg

**Source protein** 

Sweetpotato tuber protein isolate

Sweetpotato tuber defensin

Sweet potato tuber Thioredoxin

Sweetpotato tuber Trypsin inhibitor

Yam tuber Powdered yam product, alcoholinsoluble-solids Water extract (15% protein) Heat treated (90°C) water extract Dioscorin, lyophilized yam powder Storage protein ion-exhange chromatography Pepsin hydrolysate

h

**Enzyme or other process conditions** 

 **IC50**

Thermoase PC 10F, Protease S & Proleather FG-F

**ACE inhibition** 

**(mg/ml)** 

Trypsin 0.190 GFR

Trypsin 0.152 EVPK

Pepsin 0.188 HDHM

0.018 ITP

IIP GQY STYQT

FK IMVAEAR GPCSR CFCTKPC MCESASSK

VVGAK FTDVDFIK MMEPMVK

LR SNIP VRL TYCQ GTEKC RF VKAGE AH KIEL

Generally, enzymatic hydrolysis is widely applied to upgrade functional features (such as emulsifying properties of hydrolysed protein) and nutritional properties of proteins [34-36]. It has been reported that additional advantage of hydrolysis can be the development of hydrophobicity since proteolysis unfolds the protein chains. The cleavage of peptide bonds enhances levels of free amino and carboxyl groups resulting in enhanced solubility. Therefore, hydrolysis can increase or decrease the hydrophobicity, which mostly depends on the nature of the precursor protein and molecular weight of the generated peptides [34]. Moreover, hydrolysis leads to production of small bioactive peptides [35] and bitterness of peptides of below 1000 Da is much less than fractions with a higher molecular mass [36]. However, it has been reported that extensive hydrolysis could adversely affect functional properties of peptides [37]. Some factors to consider in producing bioactive peptides include hydrolysis time, degree of hydrolysis of the proteins, enzyme–substrate ratio, and pretreatment of the protein prior to hydrolysis. For example, thermal treatment of proteins can enhance enzymatic hydrolysis [38] possibly by increasing enzyme–protein interactions due to thermal-induced unfolding of the proteins.

According to the literature, enzymatic hydrolysis has been the main process for producing ACE-inhibitory and antioxidative peptides from food proteins. Use of exogenous enzymes is preferred in most cases over the autolytic process (i.e., use of endogenous enzymes present in the food source itself), due to the shorter time required to obtain similar degree of hydrolysis as well as better control of the hydrolysis to obtain more consistent molecular weight profiles and peptide composition [39-45]. Industrial food-grade proteinases such as Alcalase, Flavourzyme, and Protamex derived from microorganisms, as well as enzymes from plant (e.g. Papain) and animal sources (e.g., pepsin and trypsin), have been widely used in producing ACE-inhibitory and antioxidative peptides. The serine type endoprotease Alcalase, has produced the highest ACE-inhibitory activities *in vitro* in case of several plant proteins (Table 1).

Alcalase digests of rapeseed, canola, flaxseed, sunflower seed protein, legumes as well as and mung and chick beans showed high potency for ACE inhibition [41, 43-48]. Moreover, Alcalase digestion increased the ACE inhibition of the protein-rich by-product fraction from potato starch industry, potato tuber liquid fraction [13]. The IC50 -values were ranged between 0.020 and 0.64 mg protein/ml, which are similar to those reported for milk whey and casein hydrolysates [5, 41, 45-47,49]. Mäkinen and colleagues [50] reported that


to thermal-induced unfolding of the proteins.

several plant proteins (Table 1).

ACE-inhibitory peptides have been produced by enzymatic hydrolysis and microbial fermentation of food proteins. In addition, solvent extraction has been used to isolate ACEinhibitory activity from plant materials, such as mushrooms, broccoli and buckwheat [6]. The ACE-inhibitory potency is expressed as the IC50 value (inhibitor concentration leading to 50% inhibition), being used to estimate the effectiveness of different hydrolysates and peptides. The most common way to produce bioactive peptides is through enzymatic hydrolysis of whole protein molecules. The specificity of enzyme and process conditions

Generally, enzymatic hydrolysis is widely applied to upgrade functional features (such as emulsifying properties of hydrolysed protein) and nutritional properties of proteins [34-36]. It has been reported that additional advantage of hydrolysis can be the development of hydrophobicity since proteolysis unfolds the protein chains. The cleavage of peptide bonds enhances levels of free amino and carboxyl groups resulting in enhanced solubility. Therefore, hydrolysis can increase or decrease the hydrophobicity, which mostly depends on the nature of the precursor protein and molecular weight of the generated peptides [34]. Moreover, hydrolysis leads to production of small bioactive peptides [35] and bitterness of peptides of below 1000 Da is much less than fractions with a higher molecular mass [36]. However, it has been reported that extensive hydrolysis could adversely affect functional properties of peptides [37]. Some factors to consider in producing bioactive peptides include hydrolysis time, degree of hydrolysis of the proteins, enzyme–substrate ratio, and pretreatment of the protein prior to hydrolysis. For example, thermal treatment of proteins can enhance enzymatic hydrolysis [38] possibly by increasing enzyme–protein interactions due

According to the literature, enzymatic hydrolysis has been the main process for producing ACE-inhibitory and antioxidative peptides from food proteins. Use of exogenous enzymes is preferred in most cases over the autolytic process (i.e., use of endogenous enzymes present in the food source itself), due to the shorter time required to obtain similar degree of hydrolysis as well as better control of the hydrolysis to obtain more consistent molecular weight profiles and peptide composition [39-45]. Industrial food-grade proteinases such as Alcalase, Flavourzyme, and Protamex derived from microorganisms, as well as enzymes from plant (e.g. Papain) and animal sources (e.g., pepsin and trypsin), have been widely used in producing ACE-inhibitory and antioxidative peptides. The serine type endoprotease Alcalase, has produced the highest ACE-inhibitory activities *in vitro* in case of

Alcalase digests of rapeseed, canola, flaxseed, sunflower seed protein, legumes as well as and mung and chick beans showed high potency for ACE inhibition [41, 43-48]. Moreover, Alcalase digestion increased the ACE inhibition of the protein-rich by-product fraction from potato starch industry, potato tuber liquid fraction [13]. The IC50 -values were ranged between 0.020 and 0.64 mg protein/ml, which are similar to those reported for milk whey and casein hydrolysates [5, 41, 45-47,49]. Mäkinen and colleagues [50] reported that

influence the peptide composition of hydrolysates and thus their activities.

**2.1. Production of peptides** 


Antihypertensive Properties of Plant Protein Derived Peptides 151

[61]

intravenous, SHR

oral, SHR 25-30 g/day oral,

[131]

600 mg/kg oral, SHR

intragastric, SHR

rat 3g/day oral, Human, consumption

Han:SPDR-cy

**Response (Δ SBP mmHg)** 





[41]

[43]



[39]

[48]

[142]

[144]

[143]

**Identified peptides** *In vivo* **response Ref** 

**Dose administratio n model** 

**Sequences IC50 value (µM)**

0.070 50 mg/kg

ND 200 mg/kg

0.103- 0.117 mg/ml

0.011-0.021 mg/ml

0.053- 0.190 [64]

600 mg/kg

26.5 82.4 13.4

4.8 12.3

fractions

peaks

**Source protein** 

Sunflower protein

Pea protein isolate

Chick pea protein

Chick pea legumin

Common beans Pinto beans Green lentils

Red and green lentil protein

Mung bean protein

Mung bean sprout

**Enzyme or other process conditions** 

 **IC50**

Alcalase Peptide fraction from affinity purification

digest *in vitro*

Thermolysin, 3kDa MWCO permeate

Heat treatment and *in vitro* gastro-intestinal digestion

*In vitro* gastrointestinal digestion

Alcalase, 6kDa MWCO permeate

Raw sprout extract Dried sprout extract Pepsin, trypsin

chymotrypsin

Soy protein Alcalase 0.065 DLP

and

Pea protein Gastrointestinal

**ACE inhibition** 

**(mg/ml)** 

0.062 1.18x10-3

Alcalase Peptide

Alcalase Peptide

0.78-0.83 0.15-0.69 0.008-0.89

0.64 KDYRL

VTPALR KLPAGTLF

DG


**Enzyme or other process conditions** 

 **IC50**

protein extract Alcalase digest of potato tuber liguid fraction

Water extract (rich in proline)

Pepsin Subtilisin

Alcalase Peptide fraction from affinity purification Alcalase

Heat treatment and Alcalase

Gastrointestinal digest *in vitro*, <1kDa permeate fraction

Trypsin & Pronase cationic peptide fraction

Potato tuber Autolysed

**ACE inhibition** 

**(mg/ml)** 

0.36

0.018

0.16 0.16

0.038 0.25x10-3

0.020

0.027 VSV

0.4 QGR

FL

RW SVR GQMRQPI QQQG ASVRT DYLRSC ARDLPGQ RDLPG RGLERA TCRGLERA

0.040 WNI/LNA

NI/LDTDI/L

**Identified peptides** *In vivo* **response Ref** 

**Dose administratio n model** 

[50]

oral, SHR

500 mg/kg hydrolysate, 12.5 mg/kg 7.5 mg/kg 7.5 mg/kg 7.5 mg/kg oral, SHR

[45]

**Response (Δ SBP mmHg)** 




[13] 

[124]

[125]

[47]

[46]

[44]

**Sequences IC50 value (µM)**

127 200 mg/kg

30 1.6 3.7 28

0.15 1.33

200 mg/kg

hydrolysate, oral, SHR

[127]

VWIS VW IY RIY

**Source protein** 

*Apios Americana Medikus* tuber

Rapeseed protein

Rapeseed protein

Canola meal, defatted

Flaxseed protein

Flaxseed protein


Antihypertensive Properties of Plant Protein Derived Peptides 153

reported that peptides produced by Alcalase have diverse biological activities, including antioxidant activity [55]. In comparison to other proteases, it provided higher yields of antioxidative peptides and develops shorter peptides. Udenigwe and colleagues [55] observed that release of radical scavenging peptides depends on the specificity of protease used in hydrolysis. In another study, flaxseed protein was treated with thermolysin followed by pronase to produce antioxidant peptides [42]. Moreover, pepsin, pancreatin, neutrase and esperase have been used to produce antioxidative hydrolysates and peptides

Simulated gastrointestinal enzymatic process has also been used to mimic normal human digestion of proteins to evaluate the possibility of releasing potent bioactive peptides after normal consumption of food proteins. The combination of pepsin-trypsin-chymotrypsin or pepsin-pancreatin has been used to simulate the gastrointestinal degradation of proteins in humans [58]. Pepsin treatment alone cannot effectively elicit ACE-inhibitory peptides from buckwheat protein, while this enzyme followed by chymotrypsin and trypsin lead to a significant increase in ACE-inhibitory activity [59]. In some studies, plant protein hydrolysates generated during pepsin digestion have potent ACE-inhibitory peptides. Lower ACE-inhibitory activity was found after subsequent digestion with pancreatin, suggesting that the active components were hydrolyzed [60, 61]. For the pea proteins, high ACE-inhibitory activity is reached at the early stage of pepsin hydrolysis and the level is maintained during the small intestine phase using trypsin-chymotrypsin treatment [62]. While digestion of red lentils with trypsin showed moderate ACE inhibition (IC50 value of 0.44 mg/ml), addition of pepsin and chymotrypsin clearly improved it (IC50 of 0.09 mg/ml)

Careful choice of suitable enzymes and digestion conditions such as optimal temperature, degree of hydrolysis and enzyme-substrate ratio, as well as the control of hydrolysis time, are crucial for obtaining protein hydrolysates with desirable functional and bioactive properties. Hydrolysis can be performed by conventional batch hydrolysis or by continuous hydrolysis using ultrafiltration membranes. The traditional batch method has several disadvantages, such as the relatively high cost of the enzymes and their inefficiency compared to a continuous process, as noted in numerous studies [65, 66]. The hydrolysis process is feasible to scale-up production of peptides from laboratory scale to pilot and industrial plant scales with conserved peptide profiles and bioactivity of the resulting products [67]. The crude protein hydrolysate may be further processed, for example by passage through ultrafiltration membranes, in order to obtain a more uniform product with the desired range of molecular mass [68]. Alcalase hydrolysate from soy isolate was ultrafiltrated with 1-30 kDa membranes and ACE-inhibitory activities were analysed. The IC50-values for 1 kDa and 10 kDa permeates were almost the same, 0.080 and 0.078 mg/ml, respectively, but recovery yield of 10 kDa permeate was much higher than that of 1 kDa permeate [69]. Ultrafiltration membrane reactors have been shown to improve the efficiency of enzyme-catalysed bioconversion, to increase product yields, and to be easily scaled up. Furthermore, ultrafiltration membrane reactors yield a consistently uniform product with desired molecular mass characteristics [65, 68]. Low molecular mass cut-off membranes are useful for concentrating bioactive peptides from the higher molecular mass components

[13, 42, 51, 56, 57].

[63, 64].

**Table 1.** ACE-inhibitory activities *in vitro* and antihypertensive effect *in vivo* of plant protein–derived hydrolysates and peptides

autolysis of protein isolates from the potato tuber tissue enhances ACE-inhibition which may be due to the native proteolytic activity of potato tuber proteins.

Peña-Ramos and Xiong [51] used different enzymes to produce hydrolysates from native and heated soy protein isolates. They reported that using different enzymes resulted in the formation of a mixture of peptides with different degrees of hydrolysis and accordingly different ranges of antioxidant activity. It has been found that antioxidant activity of Alcalase derived hydrolysates is higher than that of other hydrolysates [52-54]. It is also reported that peptides produced by Alcalase have diverse biological activities, including antioxidant activity [55]. In comparison to other proteases, it provided higher yields of antioxidative peptides and develops shorter peptides. Udenigwe and colleagues [55] observed that release of radical scavenging peptides depends on the specificity of protease used in hydrolysis. In another study, flaxseed protein was treated with thermolysin followed by pronase to produce antioxidant peptides [42]. Moreover, pepsin, pancreatin, neutrase and esperase have been used to produce antioxidative hydrolysates and peptides [13, 42, 51, 56, 57].

152 Bioactive Food Peptides in Health and Disease

**Enzyme or other process conditions** 

 **IC50**

Rye Sourdough

hydrolysates and peptides

Maize, αzein

*Lactobacillus reuteri* TMW 1.106 and added protease

Soybean Fermentation 0.45 AW

**ACE inhibition** 

**(mg/ml)** 

**Identified peptides** *In vivo* **response Ref** 

**Dose administratio n model** 

Diet contained 10% v/w of fermented soy product oral, SHR

hydrolysate 30 mg peptide/kg SHR, oral

Peptide (LRP) 30 mg/kg SHR, intravenous

 10 mg/ml oral, SHR

**Response (Δ SBP mmHg)** 


[149]





[184]

[153]

[31]

[186]

**Sequences IC50 value (µM)**

> µg/ml 0.03 0.05 0.07 0.10 0.19 0.48 0.69 1.1

0.29 1.7 2.0

peptidefraction 0.14 mg/ml

GW AY SY GY AF VP AI VG

IPP LQP LLP

LSP LQP

IQP LRP VY IY TF

may be due to the native proteolytic activity of potato tuber proteins.

**Table 1.** ACE-inhibitory activities *in vitro* and antihypertensive effect *in vivo* of plant protein–derived

autolysis of protein isolates from the potato tuber tissue enhances ACE-inhibition which

Peña-Ramos and Xiong [51] used different enzymes to produce hydrolysates from native and heated soy protein isolates. They reported that using different enzymes resulted in the formation of a mixture of peptides with different degrees of hydrolysis and accordingly different ranges of antioxidant activity. It has been found that antioxidant activity of Alcalase derived hydrolysates is higher than that of other hydrolysates [52-54]. It is also

VPP

Rice Alcalase 0.14 TQVY 18.2 600 mg/kg

Thermolysin LRP

Wheat bran Autolysis 0.08 LQP

**Source protein** 

> Simulated gastrointestinal enzymatic process has also been used to mimic normal human digestion of proteins to evaluate the possibility of releasing potent bioactive peptides after normal consumption of food proteins. The combination of pepsin-trypsin-chymotrypsin or pepsin-pancreatin has been used to simulate the gastrointestinal degradation of proteins in humans [58]. Pepsin treatment alone cannot effectively elicit ACE-inhibitory peptides from buckwheat protein, while this enzyme followed by chymotrypsin and trypsin lead to a significant increase in ACE-inhibitory activity [59]. In some studies, plant protein hydrolysates generated during pepsin digestion have potent ACE-inhibitory peptides. Lower ACE-inhibitory activity was found after subsequent digestion with pancreatin, suggesting that the active components were hydrolyzed [60, 61]. For the pea proteins, high ACE-inhibitory activity is reached at the early stage of pepsin hydrolysis and the level is maintained during the small intestine phase using trypsin-chymotrypsin treatment [62]. While digestion of red lentils with trypsin showed moderate ACE inhibition (IC50 value of 0.44 mg/ml), addition of pepsin and chymotrypsin clearly improved it (IC50 of 0.09 mg/ml) [63, 64].

> Careful choice of suitable enzymes and digestion conditions such as optimal temperature, degree of hydrolysis and enzyme-substrate ratio, as well as the control of hydrolysis time, are crucial for obtaining protein hydrolysates with desirable functional and bioactive properties. Hydrolysis can be performed by conventional batch hydrolysis or by continuous hydrolysis using ultrafiltration membranes. The traditional batch method has several disadvantages, such as the relatively high cost of the enzymes and their inefficiency compared to a continuous process, as noted in numerous studies [65, 66]. The hydrolysis process is feasible to scale-up production of peptides from laboratory scale to pilot and industrial plant scales with conserved peptide profiles and bioactivity of the resulting products [67]. The crude protein hydrolysate may be further processed, for example by passage through ultrafiltration membranes, in order to obtain a more uniform product with the desired range of molecular mass [68]. Alcalase hydrolysate from soy isolate was ultrafiltrated with 1-30 kDa membranes and ACE-inhibitory activities were analysed. The IC50-values for 1 kDa and 10 kDa permeates were almost the same, 0.080 and 0.078 mg/ml, respectively, but recovery yield of 10 kDa permeate was much higher than that of 1 kDa permeate [69]. Ultrafiltration membrane reactors have been shown to improve the efficiency of enzyme-catalysed bioconversion, to increase product yields, and to be easily scaled up. Furthermore, ultrafiltration membrane reactors yield a consistently uniform product with desired molecular mass characteristics [65, 68]. Low molecular mass cut-off membranes are useful for concentrating bioactive peptides from the higher molecular mass components

remaining, including undigested polypeptide chains and enzymes. Other techniques such as nanofiltration, ion-exchange membranes, or column chromatographic methods can be used in further concentration and purification of the peptides [70].

Antihypertensive Properties of Plant Protein Derived Peptides 155

statistical computer models potentially capable of identifying ACE-inhibitory peptides based on structure–activity data [85]. A relationship was found between hydrophobicity and positively charged amino acid in C-terminal position, size of amino acid next to C-terminal position and ACE inhibition of peptides up to six amino acids in length. Moreover, no

The exact mechanism underlying the antioxidant activity of peptides has not fully been understood, as various studies have displayed that they are inhibitors of lipid peroxidation, scavengers of free radicals and chelators of transition metal ions [86, 87]. In addition, it has been reported that antioxidative peptides keep cells safe from damage by ROS through the induction of genes [88]. Antioxidative properties of the peptides are more related to their composition, structure, and hydrophobicity. Tyr, Trp, Met, Lys, Cys, and His are examples of amino acids that cause antioxidant activity. Amino acids with aromatic residues can donate protons to electron deficient radicals. This property improves the radical scavenging properties of the amino acid residues [89, 90]. It is proposed that the antioxidative activity of His containing peptides is in relation with the hydrogen donating, lipid peroxyl radical trapping and/or the metal ion chelating ability of the imidazole group [87, 91]. On the other hand, SH group in Cys has an independently crucial antioxidant action due to its direct interaction with radicals [92]. In addition to the amino acid composition, their correct position in peptide sequence plays an important role in antioxidative properties of peptides. Chen and colleagues [93] designed 28 synthetic peptides following the structure of an antioxidative peptide (Leu-Leu-Pro-His-His) from digestion of soybean protein conglycinin. According to the results, Pro-His-His sequence displayed the greatest antioxidative activity among all tested peptides. The antioxidant activity of a peptide was more dependent on His-His segment in the Leu-Leu-Pro-His-His-domain and its activity was decreased by removing a His residue from the C-terminus. Moreover, substitution of L-His by D-His in a peptide leads reduction of activity [93]. They concluded that the correct position of imidazole group is the key factor influencing the antioxidant activity. Saito and co-workers [94] also studied antioxidative activity of peptides created in two tripeptide libraries. According to their results, for the 114 peptides containing either His or Tyr residues, tripeptides containing two Tyr residues showed higher activity in the linoleic acid peroxidation system than tripeptides containing two His residues. Further, Tyr-His-Tyr showed strong synergistic effects with phenolic antioxidants. It has been shown that certain amino acids can exert higher antioxidative properties when they are incorporated in dipeptides [95] and some peptide bond or its structural conformation can reduce the

The search for *in vitro* ACE-inhibitors is the most common strategy followed in the selection of potential antihypertensive peptides derived from food proteins. *In vitro* ACE-inhibitory activity is generally measured by monitoring the conversion of an appropriate substrate by ACE in the presence and absence of inhibitors. There are several methods, and those based on spectrophotometric [97-99] and high-performance liquid chromatography (HPLC) assays

relationship between N-terminal structure and inhibition activity was found.

antioxidant activity of the constituent amino acids [96].

**2.3.** *In vitro* **and** *in vivo* **activity** 

A number of studies have shown that antihypertensive peptides are liberated during fermentation of milk. Fermented milk products prepared using different strains of lactic acid bacteria have been found to exert antihypertensive and antioxidative activities [71, 72]. Moreover, several antioxidative and ACE-inhibitory peptide sequences have been identified from fermented milk [73]. Only few experimental investigations to produce these compounds by fermentation of plant proteins have been reported. Fermentation of rapeseed and flaxseed proteins with *Lactobacillus helveticus* and *Bacillus subtilis* yields to products containing compounds with ACE-inhibitory and inhibition of lipid peroxidation capacities [74]. Fermented soybean products such as natto, tempeh, and douche also contain antioxidative and ACE-inhibitory peptides due to the action of fungal proteases. The results have indicated that the processing techniques have an impact on the ACE-inhibitory activities of soy products. Different fermented soybean foods showed IC50 values of 0.51 mg/ml for tempeh, 1.77 mg/ml for tofuyo, 3.44 and 0.71-17.80 mg/ml for soy sauce, 5.35 and 1.27 mg/ml for miso paste, and 0.16, 0.19 and 0.27 mg/ml for natto [24,75, 76]. Commercial Chinese style soy paste exhibited ACE-inhibitory activities with the lowest and the highest IC50 values of 0.012 and 3.241 mg/ml, respectively [77]. Tsai and colleagues [78] fermented soy milk with lactic acid bacteria (*Lb. casei, Lb., acidophilus, Lb. bulcaricus, Streptococcus thermophilus* and *Bifidobacterium longum*) and IC50-value was 2.89 mg/ml after 30 h fermentation. When a protease (Prozyme 6) was added after 5 h fermentation, much lower IC50-value (0.66 mg/ml) was obtained. Natto has shown to have radical scavenging activity and inhibitory effect on the oxidation of rat plasma LDL *in vitro* [79]. The aqueous extracts of Douchi showed radical scavenging activities and chelating ability of ferrous ions. The radical scavenging activities were higher than that of Trolox, an analogue of vitamin E used as a standard [80].

#### **2.2. Structure-activity relationship**

ACE-inhibitory peptides are generally short sequences often carrying polar amino acid residues like Pro. This is in agreement with the results of Natesh and co-workers [81] who showed that the active site of ACE cannot accommodate large peptide molecules. The Cterminal tripeptide strongly influences the binding of substrate or a competitive inhibitor to ACE. Many ACE-inhibitory peptides identified from different food sources, structure– activity studies indicated that C-terminal tripeptide residues play a predominant role in competitive binding to the active site of ACE. It has been reported that this enzyme prefers substrates or inhibitors containing hydrophobic (aromatic or branched side chains) amino acid residues at each of the three C-terminal positions. The most effective ACE-inhibitory peptides identified contain Tyr, Phe, Trp, and/or Pro at the C-terminal. In addition, structure–activity data suggests that the positive charge of Lys (εamino group) and Arg (guanidine group) as the C-terminal residue may contribute to the inhibitory potency [82- 84]. Quantitative structure–activity relationship (QSAR) modelling was used to develop statistical computer models potentially capable of identifying ACE-inhibitory peptides based on structure–activity data [85]. A relationship was found between hydrophobicity and positively charged amino acid in C-terminal position, size of amino acid next to C-terminal position and ACE inhibition of peptides up to six amino acids in length. Moreover, no relationship between N-terminal structure and inhibition activity was found.

The exact mechanism underlying the antioxidant activity of peptides has not fully been understood, as various studies have displayed that they are inhibitors of lipid peroxidation, scavengers of free radicals and chelators of transition metal ions [86, 87]. In addition, it has been reported that antioxidative peptides keep cells safe from damage by ROS through the induction of genes [88]. Antioxidative properties of the peptides are more related to their composition, structure, and hydrophobicity. Tyr, Trp, Met, Lys, Cys, and His are examples of amino acids that cause antioxidant activity. Amino acids with aromatic residues can donate protons to electron deficient radicals. This property improves the radical scavenging properties of the amino acid residues [89, 90]. It is proposed that the antioxidative activity of His containing peptides is in relation with the hydrogen donating, lipid peroxyl radical trapping and/or the metal ion chelating ability of the imidazole group [87, 91]. On the other hand, SH group in Cys has an independently crucial antioxidant action due to its direct interaction with radicals [92]. In addition to the amino acid composition, their correct position in peptide sequence plays an important role in antioxidative properties of peptides. Chen and colleagues [93] designed 28 synthetic peptides following the structure of an antioxidative peptide (Leu-Leu-Pro-His-His) from digestion of soybean protein conglycinin. According to the results, Pro-His-His sequence displayed the greatest antioxidative activity among all tested peptides. The antioxidant activity of a peptide was more dependent on His-His segment in the Leu-Leu-Pro-His-His-domain and its activity was decreased by removing a His residue from the C-terminus. Moreover, substitution of L-His by D-His in a peptide leads reduction of activity [93]. They concluded that the correct position of imidazole group is the key factor influencing the antioxidant activity. Saito and co-workers [94] also studied antioxidative activity of peptides created in two tripeptide libraries. According to their results, for the 114 peptides containing either His or Tyr residues, tripeptides containing two Tyr residues showed higher activity in the linoleic acid peroxidation system than tripeptides containing two His residues. Further, Tyr-His-Tyr showed strong synergistic effects with phenolic antioxidants. It has been shown that certain amino acids can exert higher antioxidative properties when they are incorporated in dipeptides [95] and some peptide bond or its structural conformation can reduce the antioxidant activity of the constituent amino acids [96].

#### **2.3.** *In vitro* **and** *in vivo* **activity**

154 Bioactive Food Peptides in Health and Disease

as a standard [80].

**2.2. Structure-activity relationship** 

in further concentration and purification of the peptides [70].

remaining, including undigested polypeptide chains and enzymes. Other techniques such as nanofiltration, ion-exchange membranes, or column chromatographic methods can be used

A number of studies have shown that antihypertensive peptides are liberated during fermentation of milk. Fermented milk products prepared using different strains of lactic acid bacteria have been found to exert antihypertensive and antioxidative activities [71, 72]. Moreover, several antioxidative and ACE-inhibitory peptide sequences have been identified from fermented milk [73]. Only few experimental investigations to produce these compounds by fermentation of plant proteins have been reported. Fermentation of rapeseed and flaxseed proteins with *Lactobacillus helveticus* and *Bacillus subtilis* yields to products containing compounds with ACE-inhibitory and inhibition of lipid peroxidation capacities [74]. Fermented soybean products such as natto, tempeh, and douche also contain antioxidative and ACE-inhibitory peptides due to the action of fungal proteases. The results have indicated that the processing techniques have an impact on the ACE-inhibitory activities of soy products. Different fermented soybean foods showed IC50 values of 0.51 mg/ml for tempeh, 1.77 mg/ml for tofuyo, 3.44 and 0.71-17.80 mg/ml for soy sauce, 5.35 and 1.27 mg/ml for miso paste, and 0.16, 0.19 and 0.27 mg/ml for natto [24,75, 76]. Commercial Chinese style soy paste exhibited ACE-inhibitory activities with the lowest and the highest IC50 values of 0.012 and 3.241 mg/ml, respectively [77]. Tsai and colleagues [78] fermented soy milk with lactic acid bacteria (*Lb. casei, Lb., acidophilus, Lb. bulcaricus, Streptococcus thermophilus* and *Bifidobacterium longum*) and IC50-value was 2.89 mg/ml after 30 h fermentation. When a protease (Prozyme 6) was added after 5 h fermentation, much lower IC50-value (0.66 mg/ml) was obtained. Natto has shown to have radical scavenging activity and inhibitory effect on the oxidation of rat plasma LDL *in vitro* [79]. The aqueous extracts of Douchi showed radical scavenging activities and chelating ability of ferrous ions. The radical scavenging activities were higher than that of Trolox, an analogue of vitamin E used

ACE-inhibitory peptides are generally short sequences often carrying polar amino acid residues like Pro. This is in agreement with the results of Natesh and co-workers [81] who showed that the active site of ACE cannot accommodate large peptide molecules. The Cterminal tripeptide strongly influences the binding of substrate or a competitive inhibitor to ACE. Many ACE-inhibitory peptides identified from different food sources, structure– activity studies indicated that C-terminal tripeptide residues play a predominant role in competitive binding to the active site of ACE. It has been reported that this enzyme prefers substrates or inhibitors containing hydrophobic (aromatic or branched side chains) amino acid residues at each of the three C-terminal positions. The most effective ACE-inhibitory peptides identified contain Tyr, Phe, Trp, and/or Pro at the C-terminal. In addition, structure–activity data suggests that the positive charge of Lys (εamino group) and Arg (guanidine group) as the C-terminal residue may contribute to the inhibitory potency [82- 84]. Quantitative structure–activity relationship (QSAR) modelling was used to develop

The search for *in vitro* ACE-inhibitors is the most common strategy followed in the selection of potential antihypertensive peptides derived from food proteins. *In vitro* ACE-inhibitory activity is generally measured by monitoring the conversion of an appropriate substrate by ACE in the presence and absence of inhibitors. There are several methods, and those based on spectrophotometric [97-99] and high-performance liquid chromatography (HPLC) assays

are most commonly utilized [100-102]. Hippuryl-His-Leu (HHL) is one of the oldest and most used methods for determining the ACE activity or inhibition [97, 99-101]. The broadly used spectrophotometric method of Cushman and Cheung [97] is based on the hydrolysis of HHL by ACE to hippuric acid and His-Leu, and the extent of hippuric acid released is measured after its extraction with ethyl acetate. The liberated hippuric acid can also be measured by chromatographic assays avoiding the extraction step [102]. The method based on the substrate 2-furanacryloyl-phenylalanylglycyl-glycine (FAPGG) was developed by Holmquist and colleagues [103] and can be applied in microtiter plates [98]. Substrates such as o-aminobenzoylglycyl-p-nitrophenylalanylproline are designed to perform in fluorometric assays [104, 105].

Antihypertensive Properties of Plant Protein Derived Peptides 157

The antihypertensive effects can be assessed by *in vivo* experiments using spontaneously hypertensive rats (SHR) that constitute an accepted animal model to study human essential hypertension [88]. A great number of studies have addressed the effects of both short-term and long-term administration of potential antihypertensive milk protein-derived peptides using this animal model [88, 112]. *In vitro* measurements of antioxidant capacity of compounds in interest cannot be directly related to their capacity *in vivo*. Biomarkers of lipid and protein peroxides as well as DNA damage can be assessed to monitor changes in oxidative stress *in vivo*. Only few studies so far have been done on plant derived peptides in

During the last decade the *in vitro* capacity of tuber protein -derived peptides to inhibit ACE have gained increasing interest (Table 1). Hsu and colleagues [113] reported that yam (*Dioscorea alata*) tuber dioscorin possess high ACE-inhibitory capacity and the digestion with pepsin increased the efficacy further. Moderate ACE-inhibition *in vitro* has been reported for purified yam (*Dioscorea batatas*) tuber mucilage [114] and for an enzymatic digest as well as for an autolysate of yam (*Dioscorea opposita*) tuber extract [115, 116]. However, the potential impact of other compounds, such as phenolic compounds and sugars, on the observed ACE inhibition should be taken into consideration. It has been shown that naturally occurring phenolic compounds, such as flavonoids and proanthocyanidins, have inhibition activity

Sweet potato proteins defensin and thioredoxin h2 which were overproduced in *Esherichia coli* showed moderate ACE-inhibitory capacity (IC50 of 0.190 and 0.152 mg/ml, respectively) and both proteins showed mixed type inhibitor against ACE using FAPGG as substrate. Hydrolysis with trypsin increased the capacity. Several peptides contained in the hydrolysate with IC50 values from 1.31 to 265.43 µM were analyzed [118, 119]. Trypsin inhibitor from the root storage protein of sweet potato, inhibited ACE in a dose-dependent manner (50-200 µg/ml, with 31.9-53.2% inhibition), and the IC50 value was 187.96 µg/ml. After digestion with pepsin the ACE-inhibition increased and peptides were designed by simulating the pepsin cutting sites of sporamin A. Finally, ten new ACE-inhibitory peptides showed IC50-values from 2.3 to 849.7 µM [120]. Sweet potato protein isolate digested with 16 different proteases showed variability in digestibility from 44.7 to 97.3% and IC50 values from 0.16 to 1.08 mg/ml. Based on these results four most potent enzymes (Thermoase PC 10F, Protease S, Proleather FG-F and Orientase 22BF) were selected and combined effect of enzymes were tested. Combination of Thermoase PC 10F, Protease S and Proleather FG-F produced potent ACE-inhibition (IC50 of 0.137 mg/ml). Finally, four different peptides derived from sweet potato storage protein, sporamin, were identified with IC50 values from 9.5 to 300.4 µM [121]. The lowest IC50 values were obtained for synthetic tripeptides, Ile-Thr-

animal models or human clinical trials.

*2.4.1. Potato and other root crops* 

towards ACE [117].

**2.4. ACE-inhibitory and antioxidant peptides** 

Pro (9.5 µM), Gly-Gln-Tyr (52.3 µM) and Ile-Ile-Pro (80.8 µM).

Specific assays have not yet been developed or standardized to measure the antioxidative capacity of peptides or peptide mixtures. Therefore, assays that are commonly used for measuring antioxidative capacity of non-peptidic antioxidants have been used in the literature to measure the antioxidative capacity of peptides as well. Due to the complexity of oxidative processes occurring in food or biological systems as well as the different antioxidative mechanisms by which various compounds may act, finding one method that can characterize the overall antioxidative potential of food is not an easy task. Nevertheless, methods such as the Trolox equivalent antioxidant capacity (TEAC) assay, oxygen radical absorbance capacity (ORAC) assay, and the total radical-trapping antioxidant parameter (TRAP) assay have been widely reported in the literature for measuring antioxidative capacity of food and biological samples [106, 107]. Commonly used assays include measuring the inhibition of lipid peroxidation in a linoleic acid model system and the capacity to scavenge the 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid)/2,2- Diphenyl-1-picrylhydrazyl (ABTS/DPPH) radicals.

*In vitro* cultured cell model systems allow for rapid, inexpensive screening of compounds for their bioavailability, metabolism, as well as bioactivity, compared to expensive and timeconsuming animal studies and human clinical trials. Endothelial cells are currently used as *in vitro* model systems for various physiological and pathological processes, especially in angiogenesis research. Endothelial dysfunction, an initiator of atherosclerosis, is manifested by altered NO and endothelin-1 (ET-1) homeostasis. ET-1 is one of the most potent endogenous vasoconstrictors and impaired NO release precedes the development of atherosclerosis [108]. NO is produced by the enzyme endothelial NO synthase (eNOS) which converts L-Arg in the presence of O2 and NADPH into L-citruline and NO. There are several assays available to analyze NO, ET-1, or expression of eNOS after exposure to compounds of interest. Use of cell culture models for antioxidant research is particularly important since the studies to date have demonstrated that the mechanism of the action of antioxidants in human health promotion go beyond the antioxidant activity of scavenging free radicals [109]. During experiments, intracellular oxidation of cells can be induced by using a peroxy radical generator or by using hydrogen peroxide (H2O2) [110]. The 20,70 dichlorofluorescein diacetate (DCFH-DA) probe can be used to measure the extent of intracellular radical formation with and without added antioxidative compound in order to assess the cellular antioxidant activity (CAA) [111].

The antihypertensive effects can be assessed by *in vivo* experiments using spontaneously hypertensive rats (SHR) that constitute an accepted animal model to study human essential hypertension [88]. A great number of studies have addressed the effects of both short-term and long-term administration of potential antihypertensive milk protein-derived peptides using this animal model [88, 112]. *In vitro* measurements of antioxidant capacity of compounds in interest cannot be directly related to their capacity *in vivo*. Biomarkers of lipid and protein peroxides as well as DNA damage can be assessed to monitor changes in oxidative stress *in vivo*. Only few studies so far have been done on plant derived peptides in animal models or human clinical trials.

#### **2.4. ACE-inhibitory and antioxidant peptides**

#### *2.4.1. Potato and other root crops*

156 Bioactive Food Peptides in Health and Disease

fluorometric assays [104, 105].

Diphenyl-1-picrylhydrazyl (ABTS/DPPH) radicals.

assess the cellular antioxidant activity (CAA) [111].

are most commonly utilized [100-102]. Hippuryl-His-Leu (HHL) is one of the oldest and most used methods for determining the ACE activity or inhibition [97, 99-101]. The broadly used spectrophotometric method of Cushman and Cheung [97] is based on the hydrolysis of HHL by ACE to hippuric acid and His-Leu, and the extent of hippuric acid released is measured after its extraction with ethyl acetate. The liberated hippuric acid can also be measured by chromatographic assays avoiding the extraction step [102]. The method based on the substrate 2-furanacryloyl-phenylalanylglycyl-glycine (FAPGG) was developed by Holmquist and colleagues [103] and can be applied in microtiter plates [98]. Substrates such as o-aminobenzoylglycyl-p-nitrophenylalanylproline are designed to perform in

Specific assays have not yet been developed or standardized to measure the antioxidative capacity of peptides or peptide mixtures. Therefore, assays that are commonly used for measuring antioxidative capacity of non-peptidic antioxidants have been used in the literature to measure the antioxidative capacity of peptides as well. Due to the complexity of oxidative processes occurring in food or biological systems as well as the different antioxidative mechanisms by which various compounds may act, finding one method that can characterize the overall antioxidative potential of food is not an easy task. Nevertheless, methods such as the Trolox equivalent antioxidant capacity (TEAC) assay, oxygen radical absorbance capacity (ORAC) assay, and the total radical-trapping antioxidant parameter (TRAP) assay have been widely reported in the literature for measuring antioxidative capacity of food and biological samples [106, 107]. Commonly used assays include measuring the inhibition of lipid peroxidation in a linoleic acid model system and the capacity to scavenge the 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid)/2,2-

*In vitro* cultured cell model systems allow for rapid, inexpensive screening of compounds for their bioavailability, metabolism, as well as bioactivity, compared to expensive and timeconsuming animal studies and human clinical trials. Endothelial cells are currently used as *in vitro* model systems for various physiological and pathological processes, especially in angiogenesis research. Endothelial dysfunction, an initiator of atherosclerosis, is manifested by altered NO and endothelin-1 (ET-1) homeostasis. ET-1 is one of the most potent endogenous vasoconstrictors and impaired NO release precedes the development of atherosclerosis [108]. NO is produced by the enzyme endothelial NO synthase (eNOS) which converts L-Arg in the presence of O2 and NADPH into L-citruline and NO. There are several assays available to analyze NO, ET-1, or expression of eNOS after exposure to compounds of interest. Use of cell culture models for antioxidant research is particularly important since the studies to date have demonstrated that the mechanism of the action of antioxidants in human health promotion go beyond the antioxidant activity of scavenging free radicals [109]. During experiments, intracellular oxidation of cells can be induced by using a peroxy radical generator or by using hydrogen peroxide (H2O2) [110]. The 20,70 dichlorofluorescein diacetate (DCFH-DA) probe can be used to measure the extent of intracellular radical formation with and without added antioxidative compound in order to During the last decade the *in vitro* capacity of tuber protein -derived peptides to inhibit ACE have gained increasing interest (Table 1). Hsu and colleagues [113] reported that yam (*Dioscorea alata*) tuber dioscorin possess high ACE-inhibitory capacity and the digestion with pepsin increased the efficacy further. Moderate ACE-inhibition *in vitro* has been reported for purified yam (*Dioscorea batatas*) tuber mucilage [114] and for an enzymatic digest as well as for an autolysate of yam (*Dioscorea opposita*) tuber extract [115, 116]. However, the potential impact of other compounds, such as phenolic compounds and sugars, on the observed ACE inhibition should be taken into consideration. It has been shown that naturally occurring phenolic compounds, such as flavonoids and proanthocyanidins, have inhibition activity towards ACE [117].

Sweet potato proteins defensin and thioredoxin h2 which were overproduced in *Esherichia coli* showed moderate ACE-inhibitory capacity (IC50 of 0.190 and 0.152 mg/ml, respectively) and both proteins showed mixed type inhibitor against ACE using FAPGG as substrate. Hydrolysis with trypsin increased the capacity. Several peptides contained in the hydrolysate with IC50 values from 1.31 to 265.43 µM were analyzed [118, 119]. Trypsin inhibitor from the root storage protein of sweet potato, inhibited ACE in a dose-dependent manner (50-200 µg/ml, with 31.9-53.2% inhibition), and the IC50 value was 187.96 µg/ml. After digestion with pepsin the ACE-inhibition increased and peptides were designed by simulating the pepsin cutting sites of sporamin A. Finally, ten new ACE-inhibitory peptides showed IC50-values from 2.3 to 849.7 µM [120]. Sweet potato protein isolate digested with 16 different proteases showed variability in digestibility from 44.7 to 97.3% and IC50 values from 0.16 to 1.08 mg/ml. Based on these results four most potent enzymes (Thermoase PC 10F, Protease S, Proleather FG-F and Orientase 22BF) were selected and combined effect of enzymes were tested. Combination of Thermoase PC 10F, Protease S and Proleather FG-F produced potent ACE-inhibition (IC50 of 0.137 mg/ml). Finally, four different peptides derived from sweet potato storage protein, sporamin, were identified with IC50 values from 9.5 to 300.4 µM [121]. The lowest IC50 values were obtained for synthetic tripeptides, Ile-Thr-Pro (9.5 µM), Gly-Gln-Tyr (52.3 µM) and Ile-Ile-Pro (80.8 µM).

The protein-rich by-product fraction from potato (*Solanum tuberosum*) starch industry, potato tuber liquid, has been found to be a valuable source of ACE-inhibitory peptides [13]. The ACE-inhibitory activity of potato tuber liquid was moderate and enzymatic digestion was needed to enhance the activity to high level. Alcalase digest showed the highest ACEinhibitory activity and the digest was chromatographically separated to highly active peptide fractions. The potato liquid Alcalase hydrolysate produced the highest radical scavenging potency even though no statistically significant differences were found among hydrolysates produced by Alcalase, Neutrase and Esperase. Mäkinen and colleagues [47] reported that autolysis of protein isolates from the potato tuber tissue enhances ACE inhibition which may be due to the native proteolytic activity of potato tuber proteins. The results indicated a relevant role of potato tuber storage proteins in the production of ACEinhibitory peptides during the autolysis. Enrichment of recombinant potato tuber protein to the autolysis enhanced the production of activity significantly, which suggests possibility to enhance potato tuber ACE-inhibitory potential by means of biotechnological tools. Anyhow, more research is needed to characterize and identify the ACE-inhibitory potato peptides and to evaluate the *in vivo* antihypertensive potential.

Antihypertensive Properties of Plant Protein Derived Peptides 159

According to the published data, enzymatic hydrolysis is required to release antihypertensive peptides from oil seed plant proteins. Only few peptide sequences have been identified from oil seed plants. Four ACE-inhibitory peptides were isolated from the rapeseed subtilisin digest of which Ile-Tyr and Arg-Ile-Tyr can be found in the primary structure of napin and Val-Trp and Val-Trp-Ile-Ser exist in the primary structure of cruciferin and ribosomal protein, respectively. Among the peptides isolated, Ile-Tyr and Val-Trp can be considered true ACE-inhibitors because IC50 values for these peptides before and after pre-incubation with ACE were found to be the same. Val-Trp-Ile-Ser is a pro-drug type ACE-inhibitor, as pre-incubation with ACE of Val-Trp-Ile-Ser intensified inhibitory activity of this peptide [125]. In addition, to these rapeseed peptides, two peptide sequences with high ACE-inhibitory capacity were identified from canola meal hydrolysed with Alcalase, Val-Ser-Val and Phe-Leu, located in the primary structure of canola napin and cruciferin proteins [46]. Low-molecular weight cationic peptide fractions from flaxseed protein hydrolysed by Alcalase or thermolysin showed concentration dependent ACEinhibition (IC50 0.0275-0.151 mg/ml) [126]. The Alcalase cationic peptide and thermolysin hydrolysate showed mixed type inhibition of ACE activity. Several peptides were detected in a cationic peptide fraction of a trypsin & Pronase digest of flaxseed, which showed moderate ACE inhibition *in vitro* and antihypertensive effects in SHR [44]. A pentapeptide Trp-Asn-Ile-Leu-Asn-Ile-Leu and a hexapeptide Asn-Ile-Leu-Asp-Thr-Asp-Ile-Leu were identified from flaxseed protein digested with an *in vitro* digestion model [127]. Anyhow, ACE-inhibitory activity of these flaxseed derived peptides has not been evaluated individually and thus, the ACE-inhibitory capacity of these peptides in a pure form is not

Despite the high *in vitro* ACE-inhibitory potency of Alcalase digests of oil seed proteins, their antihypertensive effects *in vivo* have not been evaluated yet. Subtilisin digest of rapeseed and tryptic digest of flaxseed have shown antihypertensive properties in SHR. Marczak and co-workers [125] studied the Subtilisin and pepsin digests of rapeseed in SHR. Subtilisin digest of rapeseed protein showed dose dependent antihypertensive effect after oral administration to SHR and its effect was significant even at a single dose of 0.15 g/kg. The Subtilisin digest was subjected to hydrolysis with different proteases to simulate gastrointestinal digestion *in vitro* and the ACE-inhibitory activity was changed only slightly indicating that ACE-inhibitory peptides present in the Subtilisin digest are relatively resistant. The antihypertensive activities of Val-Trp, Val-Trp-Ile-Ser, Ile-Tyr and Arg-Ile-Tyr were tested following oral administration to SHR. The maximum hypotensive activity of Val-Trp, Val-Trp-Ile-Ser and Ile-Tyr occurred 2 h after administration, whereas Arg-Ile-Tyr (rapakinin) had the maximum effect 4 h after administration. All peptides displayed dosedependent antihypertensive effect. Hypotensive activity of the peptides was compared after oral administration to young (19-20 weeks) and old (28-30 weeks) SHR. Usually the hypotensive effect of ACE inhibitors in old SHR is lower than in young SHR. The hypotensive effects of Val-Trp, Val-Trp-Ile-Ser and Ile-Tyr were lower in old rats, but in the case of rapakinin the effect was similar in old and young rats. The authors suggested that

*2.4.2. Oil seed plants-derived peptides* 

clear [44, 126, 127].

Recently, antihypertensive effects of some tuber plant –derived protein digests have been evaluated *in vivo* using SHR animal model, although no tuber protein –derived peptides in pure form have been reported. The proteins tested in the *in vivo* trials have concerned the antihypertensive effects of the main storage proteins of the tubers and peptides derived from these proteins. Among the tuber proteins, the *in vivo* antihypertensive effects of yam (*Dioscorea alata*) tuber proteins are the most studied. Lin and co-workers [122] purified storage proteins, dioscorins from yam tubers, that were digested with pepsin and evaluated for their antihypertensive effects in SHR. The maximum effect after single oral administration was observed after 4 h with the dioscorin isolate and after 8 h with the peptic hydrolysate, while the antihypertensive effect of the peptic digest was more pronounced (- 33.7 mmHg Mean arterial pressure, MAP) and less transient than that of the dioscorin isolate (-21.5 mmHg MAP). The long-term antihypertensive effect of the dioscorin isolate was tested for 25 days with daily oral administration and the greatest reductions in systolic blood pressure (SBP) and diastolic blood pressure (DBP) were observed on the ninth day. Liu and co-workers [123] tested the antihypertensive effects of different yam tuber products on SHR. The yam tuber alcohol-insoluble solids and water extract before and after heat treatment were observed to decrease the blood pressure after single oral administration. The most pronounced effect with the lowest dose was found with the alcohol-insoluble-solids product, which contained the yam tuber dioscorins. Iwai and Matsue [124] reported moderate antihypertensive effects of an edible tuber *Apios Americana* Medikus in SHR. The animals ingested water extract of the tubers that was rich in Pro. The antihypertensive effect was suggested to be due to Pro-rich peptides, which were released during digestion. Sweet potato protein digest made with combination of three proteases (Thermoase PC10F, Protease S and Proleather FG-F) showed a dose dependent decrease in SBP after single oral administration in SHR [121].

#### *2.4.2. Oil seed plants-derived peptides*

158 Bioactive Food Peptides in Health and Disease

to evaluate the *in vivo* antihypertensive potential.

administration in SHR [121].

The protein-rich by-product fraction from potato (*Solanum tuberosum*) starch industry, potato tuber liquid, has been found to be a valuable source of ACE-inhibitory peptides [13]. The ACE-inhibitory activity of potato tuber liquid was moderate and enzymatic digestion was needed to enhance the activity to high level. Alcalase digest showed the highest ACEinhibitory activity and the digest was chromatographically separated to highly active peptide fractions. The potato liquid Alcalase hydrolysate produced the highest radical scavenging potency even though no statistically significant differences were found among hydrolysates produced by Alcalase, Neutrase and Esperase. Mäkinen and colleagues [47] reported that autolysis of protein isolates from the potato tuber tissue enhances ACE inhibition which may be due to the native proteolytic activity of potato tuber proteins. The results indicated a relevant role of potato tuber storage proteins in the production of ACEinhibitory peptides during the autolysis. Enrichment of recombinant potato tuber protein to the autolysis enhanced the production of activity significantly, which suggests possibility to enhance potato tuber ACE-inhibitory potential by means of biotechnological tools. Anyhow, more research is needed to characterize and identify the ACE-inhibitory potato peptides and

Recently, antihypertensive effects of some tuber plant –derived protein digests have been evaluated *in vivo* using SHR animal model, although no tuber protein –derived peptides in pure form have been reported. The proteins tested in the *in vivo* trials have concerned the antihypertensive effects of the main storage proteins of the tubers and peptides derived from these proteins. Among the tuber proteins, the *in vivo* antihypertensive effects of yam (*Dioscorea alata*) tuber proteins are the most studied. Lin and co-workers [122] purified storage proteins, dioscorins from yam tubers, that were digested with pepsin and evaluated for their antihypertensive effects in SHR. The maximum effect after single oral administration was observed after 4 h with the dioscorin isolate and after 8 h with the peptic hydrolysate, while the antihypertensive effect of the peptic digest was more pronounced (- 33.7 mmHg Mean arterial pressure, MAP) and less transient than that of the dioscorin isolate (-21.5 mmHg MAP). The long-term antihypertensive effect of the dioscorin isolate was tested for 25 days with daily oral administration and the greatest reductions in systolic blood pressure (SBP) and diastolic blood pressure (DBP) were observed on the ninth day. Liu and co-workers [123] tested the antihypertensive effects of different yam tuber products on SHR. The yam tuber alcohol-insoluble solids and water extract before and after heat treatment were observed to decrease the blood pressure after single oral administration. The most pronounced effect with the lowest dose was found with the alcohol-insoluble-solids product, which contained the yam tuber dioscorins. Iwai and Matsue [124] reported moderate antihypertensive effects of an edible tuber *Apios Americana* Medikus in SHR. The animals ingested water extract of the tubers that was rich in Pro. The antihypertensive effect was suggested to be due to Pro-rich peptides, which were released during digestion. Sweet potato protein digest made with combination of three proteases (Thermoase PC10F, Protease S and Proleather FG-F) showed a dose dependent decrease in SBP after single oral According to the published data, enzymatic hydrolysis is required to release antihypertensive peptides from oil seed plant proteins. Only few peptide sequences have been identified from oil seed plants. Four ACE-inhibitory peptides were isolated from the rapeseed subtilisin digest of which Ile-Tyr and Arg-Ile-Tyr can be found in the primary structure of napin and Val-Trp and Val-Trp-Ile-Ser exist in the primary structure of cruciferin and ribosomal protein, respectively. Among the peptides isolated, Ile-Tyr and Val-Trp can be considered true ACE-inhibitors because IC50 values for these peptides before and after pre-incubation with ACE were found to be the same. Val-Trp-Ile-Ser is a pro-drug type ACE-inhibitor, as pre-incubation with ACE of Val-Trp-Ile-Ser intensified inhibitory activity of this peptide [125]. In addition, to these rapeseed peptides, two peptide sequences with high ACE-inhibitory capacity were identified from canola meal hydrolysed with Alcalase, Val-Ser-Val and Phe-Leu, located in the primary structure of canola napin and cruciferin proteins [46]. Low-molecular weight cationic peptide fractions from flaxseed protein hydrolysed by Alcalase or thermolysin showed concentration dependent ACEinhibition (IC50 0.0275-0.151 mg/ml) [126]. The Alcalase cationic peptide and thermolysin hydrolysate showed mixed type inhibition of ACE activity. Several peptides were detected in a cationic peptide fraction of a trypsin & Pronase digest of flaxseed, which showed moderate ACE inhibition *in vitro* and antihypertensive effects in SHR [44]. A pentapeptide Trp-Asn-Ile-Leu-Asn-Ile-Leu and a hexapeptide Asn-Ile-Leu-Asp-Thr-Asp-Ile-Leu were identified from flaxseed protein digested with an *in vitro* digestion model [127]. Anyhow, ACE-inhibitory activity of these flaxseed derived peptides has not been evaluated individually and thus, the ACE-inhibitory capacity of these peptides in a pure form is not clear [44, 126, 127].

Despite the high *in vitro* ACE-inhibitory potency of Alcalase digests of oil seed proteins, their antihypertensive effects *in vivo* have not been evaluated yet. Subtilisin digest of rapeseed and tryptic digest of flaxseed have shown antihypertensive properties in SHR. Marczak and co-workers [125] studied the Subtilisin and pepsin digests of rapeseed in SHR. Subtilisin digest of rapeseed protein showed dose dependent antihypertensive effect after oral administration to SHR and its effect was significant even at a single dose of 0.15 g/kg. The Subtilisin digest was subjected to hydrolysis with different proteases to simulate gastrointestinal digestion *in vitro* and the ACE-inhibitory activity was changed only slightly indicating that ACE-inhibitory peptides present in the Subtilisin digest are relatively resistant. The antihypertensive activities of Val-Trp, Val-Trp-Ile-Ser, Ile-Tyr and Arg-Ile-Tyr were tested following oral administration to SHR. The maximum hypotensive activity of Val-Trp, Val-Trp-Ile-Ser and Ile-Tyr occurred 2 h after administration, whereas Arg-Ile-Tyr (rapakinin) had the maximum effect 4 h after administration. All peptides displayed dosedependent antihypertensive effect. Hypotensive activity of the peptides was compared after oral administration to young (19-20 weeks) and old (28-30 weeks) SHR. Usually the hypotensive effect of ACE inhibitors in old SHR is lower than in young SHR. The hypotensive effects of Val-Trp, Val-Trp-Ile-Ser and Ile-Tyr were lower in old rats, but in the case of rapakinin the effect was similar in old and young rats. The authors suggested that another mechanism besides ACE inhibition may be involved in hypotensive effect of rapakinin. Recently, the hypotensive effect of rapakinin was found to be mediated mainly by the prostaglandin IP (PGI2-IP) receptor followed by CCK1 receptor-dependent vasorelaxation [128]. In addition to hypotensive effects, the napin-derived peptide rapakinin has been reported to possess multifunctional properties. Rapakinin dose-dependently decreased food intake and gastric emptying after oral administration at a dose of 150 mg/kg in mice [129] and recently, Yamada and colleagues [130] reported anti-opioid activity related to the hypotensive effects for rapakinin.

Antihypertensive Properties of Plant Protein Derived Peptides 161

inhibitory properties with an IC50 value of 0.190 mg/ml. Four peptide-fractions with average molecular weight of 900 Da, representing peptides with six to eight amino acid residues were isolated. The IC50 values were 0.103-0.117 mg/ml and two of the peptides showed competitive and two showed uncompetitive mechanism [41]. In addition, six ACEinhibitory peptides with IC50 values ranging from 0.011 to 0.012 mg/ml have been isolated from chickpea hydrolysed by Alcalase. All these peptides contained Met and were rich in other hydrophobic amino acids [43]. Two lentil varieties were hydrolysed with different enzymes and IC50 values ranged between 0.053 and 0.190 mg/ml. Furthermore, the inhibition mechanism investigated using Lineweaver–Burk plots revealed a non-competitive inhibition of ACE with inhibitor constants (Ki) between 0.16 and 0.46 mg/ml [40]. Three dipeptides, Ile-Arg, Lys-Phe and Glu-Phe were isolated and identified from Alcalase hydrolysate of pea protein isolate. The peptides showed strong inhibitions (IC50 values <25

Enzymatic hydrolysate of soy protein showed a moderate ACE-inhibitory activity (0.034 mg/ml) [39] as compared with values reported ranging from 0.021 to 1.73 mg/ml [5, 6, 49, 70]. Lo and co-workers [134] applied the dynamic model for the *in vitro* digestion of isolated soy proteins. They concluded that ACE-inhibitory activity was dependent on the digestion time and the heat treatment of soy protein. Pepsin hydrolysis of isolated soy protein produced peptides with a higher ACE-inhibitory activity due to the increased digestion time. Hydrolysis by pancreatin produced soy peptides with higher ACE‑inhibitory activity as compared to pepsin hydrolysates, but decresed inhibitory activity appeared after longer digestion time. These results suggest that at the longer digestion time pancreatin may have hydrolysed the peptides from pepsin digestion that had strong ACE-inhibitory activity, and

Pea protein hydrolysates by thermolysin contained low molecular weight (<3 kDa) peptides with various antioxidant activities that were dependent on the amounts of hydrophobic and aromatic amino acid constituents [135] (Table 2). Peptide fractions with the least cationic property had significantly stronger scavenging activity against DPPH and H2O2. Generally, the scavenging of O2− and H2O2 was negatively related with the cationic property of the peptide fractions [136]. Chick pea hydrolysate with antioxidant activities was prepared from chickpea protein isolates by Alcalase. This hydrolysate was separated with Sephadex G-25. Four fractions were obtained, and fraction IV had the highest antioxidant activities assayed by free radical scavenging effects [137]. The active peptide was identified as Asn-Arg-Tyr-His-Glu. This peptide quenched the free radical sources DPPH, hydroxyl, and superoxide free radicals. Furthermore, the inhibition of the peptide on lipid peroxidation was greater

Different hydrolysis conditions of soy protein isolates have resulted in peptide mixtures with different antioxidant properties. Native and heated soy protein isolate hydrolysed with different enzymes resulted in different degree of hydrolysis ranging from 1.7-20.6 with antioxidant activity ranging from 28% to 65% [138]. Zhang and coworkers [139] used three microbial proteases to produce hydrolysates with degree of hydrolysis values from 13.4% to 26.1% and with different oxygen radical absorbance capacity (ORAC), DPPH-radical

then turned them into peptides with lower ACE-inhibitory activity.

mM) of ACE and renin [133].

than that of -tocopherol [57].

Peptide fraction from the enzymatic digest of flaxseed protein was recently reported to possess hypotensive activity in SHR [44]. An Arg-rich peptide fraction was produced from flaxseed protein using trypsin and pronase and by subsequent concentration with combined electrodialysis and ultrafiltration. The hypotensive effect of the flaxseed derived Arg-rich peptide fraction was tested on SHR and the effects were compared to the effects of inherent flaxseed protein isolate and amino acid form of Arg. The maximum hypotensive effect of the cationic peptide fraction was observed already 2 h after oral administration while the amino acid form of Arg showed lower hypotensive activity, -10.3 mmHg. On the other hand, the flaxseed protein isolate exhibited a slow-acting hypotensive effect with maximum of -18.4 mmHg (SBP) at 6 h after the administration. The hypotensive effect of the Arg-rich peptide fraction was longer-lasting when compared to the free amino acid form of Arg and the authors suggested that this might be related to more efficient absorption of peptides and the ability of peptides to translocate directly into the cells, obviating the need for transporters. The observed hypotensive activity of flaxseed protein and peptide fraction could be due to vasodilatory activity of NO synthesized from the Arg through the L-Arg-NO pathway in the vascular endothelium, or ACE- and renin-inhibition observed *in vitro* by the cationic peptide fraction.

#### *2.4.3. Legume derived peptides*

Several enzymes have been used to produce pulse protein hydrolysates having bioactive properties. It has been suggested that hydrolysates of chickpea legume and mung bean obtained by Alcalase treatments are good sources of ACE-inhibitory peptides [43, 48]. Potential ACE-inhibitory potencies of common dry beans, dry pinto beans and green lentils increased during *in vitro* gastrointestinal digestion have been reported, with IC50 values of 0.78–0.83, 0.15–0.69 and 0.008–0.89 mg protein/ml, respectively [131]. In addition, 15 min heat treatment effectively increased the ACE-inhibitory activity of the stomach digest [43, 131]. Digestion simulating the physiological conditions of pea proteins sufficed to achieve the highest ACE-inhibitory activity with IC50 value of 0.076 mg/ml [132]. Furthermore, it has been suggested that red lentil protein hydrolysates have ACE-inhibitory properties. The ACE-inhibitory property of the tryptic hydrolysates varied as a function of the protein fraction with the total lentil protein hydrolysate having the lowest IC50 (0.440 ± 0.004 mg/ml). This indicates that lentil varieties having higher amounts of legumin and albumin proteins may have higher ACE-inhibitory properties [63]. Pedroche and co-workers [41] hydrolysed chickpea protein isolate with Alcalase to produce a bioactive hydrolysate having ACE-

inhibitory properties with an IC50 value of 0.190 mg/ml. Four peptide-fractions with average molecular weight of 900 Da, representing peptides with six to eight amino acid residues were isolated. The IC50 values were 0.103-0.117 mg/ml and two of the peptides showed competitive and two showed uncompetitive mechanism [41]. In addition, six ACEinhibitory peptides with IC50 values ranging from 0.011 to 0.012 mg/ml have been isolated from chickpea hydrolysed by Alcalase. All these peptides contained Met and were rich in other hydrophobic amino acids [43]. Two lentil varieties were hydrolysed with different enzymes and IC50 values ranged between 0.053 and 0.190 mg/ml. Furthermore, the inhibition mechanism investigated using Lineweaver–Burk plots revealed a non-competitive inhibition of ACE with inhibitor constants (Ki) between 0.16 and 0.46 mg/ml [40]. Three dipeptides, Ile-Arg, Lys-Phe and Glu-Phe were isolated and identified from Alcalase hydrolysate of pea protein isolate. The peptides showed strong inhibitions (IC50 values <25 mM) of ACE and renin [133].

160 Bioactive Food Peptides in Health and Disease

to the hypotensive effects for rapakinin.

fraction.

*2.4.3. Legume derived peptides* 

another mechanism besides ACE inhibition may be involved in hypotensive effect of rapakinin. Recently, the hypotensive effect of rapakinin was found to be mediated mainly by the prostaglandin IP (PGI2-IP) receptor followed by CCK1 receptor-dependent vasorelaxation [128]. In addition to hypotensive effects, the napin-derived peptide rapakinin has been reported to possess multifunctional properties. Rapakinin dose-dependently decreased food intake and gastric emptying after oral administration at a dose of 150 mg/kg in mice [129] and recently, Yamada and colleagues [130] reported anti-opioid activity related

Peptide fraction from the enzymatic digest of flaxseed protein was recently reported to possess hypotensive activity in SHR [44]. An Arg-rich peptide fraction was produced from flaxseed protein using trypsin and pronase and by subsequent concentration with combined electrodialysis and ultrafiltration. The hypotensive effect of the flaxseed derived Arg-rich peptide fraction was tested on SHR and the effects were compared to the effects of inherent flaxseed protein isolate and amino acid form of Arg. The maximum hypotensive effect of the cationic peptide fraction was observed already 2 h after oral administration while the amino acid form of Arg showed lower hypotensive activity, -10.3 mmHg. On the other hand, the flaxseed protein isolate exhibited a slow-acting hypotensive effect with maximum of -18.4 mmHg (SBP) at 6 h after the administration. The hypotensive effect of the Arg-rich peptide fraction was longer-lasting when compared to the free amino acid form of Arg and the authors suggested that this might be related to more efficient absorption of peptides and the ability of peptides to translocate directly into the cells, obviating the need for transporters. The observed hypotensive activity of flaxseed protein and peptide fraction could be due to vasodilatory activity of NO synthesized from the Arg through the L-Arg-NO pathway in the vascular endothelium, or ACE- and renin-inhibition observed *in vitro* by the cationic peptide

Several enzymes have been used to produce pulse protein hydrolysates having bioactive properties. It has been suggested that hydrolysates of chickpea legume and mung bean obtained by Alcalase treatments are good sources of ACE-inhibitory peptides [43, 48]. Potential ACE-inhibitory potencies of common dry beans, dry pinto beans and green lentils increased during *in vitro* gastrointestinal digestion have been reported, with IC50 values of 0.78–0.83, 0.15–0.69 and 0.008–0.89 mg protein/ml, respectively [131]. In addition, 15 min heat treatment effectively increased the ACE-inhibitory activity of the stomach digest [43, 131]. Digestion simulating the physiological conditions of pea proteins sufficed to achieve the highest ACE-inhibitory activity with IC50 value of 0.076 mg/ml [132]. Furthermore, it has been suggested that red lentil protein hydrolysates have ACE-inhibitory properties. The ACE-inhibitory property of the tryptic hydrolysates varied as a function of the protein fraction with the total lentil protein hydrolysate having the lowest IC50 (0.440 ± 0.004 mg/ml). This indicates that lentil varieties having higher amounts of legumin and albumin proteins may have higher ACE-inhibitory properties [63]. Pedroche and co-workers [41] hydrolysed chickpea protein isolate with Alcalase to produce a bioactive hydrolysate having ACE-

Enzymatic hydrolysate of soy protein showed a moderate ACE-inhibitory activity (0.034 mg/ml) [39] as compared with values reported ranging from 0.021 to 1.73 mg/ml [5, 6, 49, 70]. Lo and co-workers [134] applied the dynamic model for the *in vitro* digestion of isolated soy proteins. They concluded that ACE-inhibitory activity was dependent on the digestion time and the heat treatment of soy protein. Pepsin hydrolysis of isolated soy protein produced peptides with a higher ACE-inhibitory activity due to the increased digestion time. Hydrolysis by pancreatin produced soy peptides with higher ACE‑inhibitory activity as compared to pepsin hydrolysates, but decresed inhibitory activity appeared after longer digestion time. These results suggest that at the longer digestion time pancreatin may have hydrolysed the peptides from pepsin digestion that had strong ACE-inhibitory activity, and then turned them into peptides with lower ACE-inhibitory activity.

Pea protein hydrolysates by thermolysin contained low molecular weight (<3 kDa) peptides with various antioxidant activities that were dependent on the amounts of hydrophobic and aromatic amino acid constituents [135] (Table 2). Peptide fractions with the least cationic property had significantly stronger scavenging activity against DPPH and H2O2. Generally, the scavenging of O2− and H2O2 was negatively related with the cationic property of the peptide fractions [136]. Chick pea hydrolysate with antioxidant activities was prepared from chickpea protein isolates by Alcalase. This hydrolysate was separated with Sephadex G-25. Four fractions were obtained, and fraction IV had the highest antioxidant activities assayed by free radical scavenging effects [137]. The active peptide was identified as Asn-Arg-Tyr-His-Glu. This peptide quenched the free radical sources DPPH, hydroxyl, and superoxide free radicals. Furthermore, the inhibition of the peptide on lipid peroxidation was greater than that of -tocopherol [57].

Different hydrolysis conditions of soy protein isolates have resulted in peptide mixtures with different antioxidant properties. Native and heated soy protein isolate hydrolysed with different enzymes resulted in different degree of hydrolysis ranging from 1.7-20.6 with antioxidant activity ranging from 28% to 65% [138]. Zhang and coworkers [139] used three microbial proteases to produce hydrolysates with degree of hydrolysis values from 13.4% to 26.1% and with different oxygen radical absorbance capacity (ORAC), DPPH-radical


Antihypertensive Properties of Plant Protein Derived Peptides 163

Alcalase. Purification of peptide by ultrafiltration and chromatographic techniques enhanced the specific activity 67.9-fold compared to hydrolysate. The final potent antioxidant peptide contained hydrophobic amino acids and among them, Phe was

Pea and mung bean protein digests have been reported to possess antihypertensive activity in SHR [48, 142]. Mung bean protein hydrolysate prepared with alcalase decreased significantly SBP (-30.8 mmHg) of SHR 6 h after single oral administration at a dose of 600 mg/ml. The blood pressure-lowering effect continued for at least 8 h, and the blood pressure returned to initial levels at 12 h after administration [48]. Single administration of mung bean raw sprout extract (at dose of 600 mg/kg) reduced significantly SBP (-40 mmHg) 6 h after administration. Plasma ACE activities in the treated rats also decreased (0.007 Unit/ml). Long-term intervention (1 month) test showed that blood pressure in the treated animals fluctuated according to the treatments. While raw sprout extract showed effective results after 1 week of intervention, dried sprout extracts did not have significant effects until 2 weeks [142]. Pea protein hydrolysate was made by thermolysin action followed by membrane filtration. Oral administration of the pea protein hydrolysate, containing <3 kDa peptides, to SHR at doses of 100 and 200 mg/kg body weight led to a lowering of SBP, with a maximum reduction of 19 mmHg at 4 h. In contrast, orally administered unhydrolysed pea protein isolate had no blood pressure reducing effect in SHR, suggesting that thermolysin hydrolysis may have been responsible for releasing bioactive peptides from the native protein [143]. Pea protein peptides from *in vitro* gastrointestinal digestion were observed to absorb poorly with *in vitro* model and the hypotensive effect was tested with intravenous

The seed storage proteins of wheat, barley, rye, and oats contain known ACE-inhibitory diand tripeptides in their primary structures. Barley and barley by-products extract possesses a biological activity such as free radical scavenging activity, tyrosinase, xanthin oxidase, and ACE-inhibition effect [145]. Hydrolysates of barley prolamin fraction exhibited the highest antioxidant and ACE-inhibitory activity compared to other protein fractions and protein isolate. Moreover, positive correlations were obtained between antioxidant and ACEinhibitory activity and the degree of hydrolysis of hydrolysed protein fractions and protein

The computer analysis of amino acid sequences of wheat gliadins made by means of BIOPEP database [147] showed the presence of fragments that are homological with the sequences regarded as antihypertensive peptides. They were: Leu-Gln-Pro (α-, β- and γgliadins), Pro-Tyr-Pro (α-, β- and γ-gliadins), Ile-Pro-Pro (α-and β-gliadins), Leu-Pro-Pro (γgliadins) and Leu-Val-Leu (γ-gliadins). Bioinformatic analysis of cereal proteins sequences revealed that particularly four tripeptides with known ACE-inhibitory activity, Leu-Gln-Pro, Val-Pro-Pro, Ile-Pro-Pro, and Leu-Leu-Pro, are frequently encrypted in the primary structure of rye secalin, wheat gluten, and barley hordein. Sourdoughs fermented with different strains showed different concentrations of Leu-Gln-Pro and Leu-Leu-Pro. These

especially abundant [141].

administration [144].

*2.4.4. Cereals* 

isolate [146].

1 The enzyme indicated in bold is the most effective of the enzymes to produce antioxidative activity/peptides

**Table 2.** Antioxidative capacity of plant protein-derived hydrolysates and peptides

scavenging activities as well transition metal chelating activities. Chen and coworkers [140] isolated 6 antioxidative peptide fragments from the digests of -conglycinin, a main soybean protein component, by using protease S from *Bacillus* sp. The antioxidant activity of the soybean hydrolysates, based on a linoleic acid oxidation system study, was attributed to the Leu-Leu-Pro-His-His peptide sequence [89, 93]. A potent antioxidant peptide, with inhibitory activity of lipid peroxidation, was isolated from soy protein isolate hydrolysed by Alcalase. Purification of peptide by ultrafiltration and chromatographic techniques enhanced the specific activity 67.9-fold compared to hydrolysate. The final potent antioxidant peptide contained hydrophobic amino acids and among them, Phe was especially abundant [141].

Pea and mung bean protein digests have been reported to possess antihypertensive activity in SHR [48, 142]. Mung bean protein hydrolysate prepared with alcalase decreased significantly SBP (-30.8 mmHg) of SHR 6 h after single oral administration at a dose of 600 mg/ml. The blood pressure-lowering effect continued for at least 8 h, and the blood pressure returned to initial levels at 12 h after administration [48]. Single administration of mung bean raw sprout extract (at dose of 600 mg/kg) reduced significantly SBP (-40 mmHg) 6 h after administration. Plasma ACE activities in the treated rats also decreased (0.007 Unit/ml). Long-term intervention (1 month) test showed that blood pressure in the treated animals fluctuated according to the treatments. While raw sprout extract showed effective results after 1 week of intervention, dried sprout extracts did not have significant effects until 2 weeks [142]. Pea protein hydrolysate was made by thermolysin action followed by membrane filtration. Oral administration of the pea protein hydrolysate, containing <3 kDa peptides, to SHR at doses of 100 and 200 mg/kg body weight led to a lowering of SBP, with a maximum reduction of 19 mmHg at 4 h. In contrast, orally administered unhydrolysed pea protein isolate had no blood pressure reducing effect in SHR, suggesting that thermolysin hydrolysis may have been responsible for releasing bioactive peptides from the native protein [143]. Pea protein peptides from *in vitro* gastrointestinal digestion were observed to absorb poorly with *in vitro* model and the hypotensive effect was tested with intravenous administration [144].

#### *2.4.4. Cereals*

162 Bioactive Food Peptides in Health and Disease

Soybean proteins Liposome

*In vitro* **methods used in measuring antioxidant capacity** 

ABTS+-- scavenging Emulsion oxidative

oxidizing system

Radical (DPPH, O2- , H2O2) scavenging and inhibition of linoleic acid oxidation

capacity (DPPH/O2-

), Fe2+ chelating effect and reducing power

peroxidation system

, OH and DPPH

Barley glutelin Radical scavenging

Wheat gluten Linoleic acid

/OH-

O2-

radical sacavenging capacity, Linoleic acid peroxidation

system

Linoleic acid peroxidation system

stability

**Enzymes or other process conditions** 

Esperase, Neutrase

**Chymotrypsin**, Pepsin, Papain, **Flavourzyme,**  Alcalase, Protamex

Protease M, Protease N, Protease P, **Protease S**

Alcalase, Chymotrypsin, **Neutrase,** Papain, Flavorase

1 The enzyme indicated in bold is the most effective of the enzymes to produce antioxidative activity/peptides

scavenging activities as well transition metal chelating activities. Chen and coworkers [140] isolated 6 antioxidative peptide fragments from the digests of -conglycinin, a main soybean protein component, by using protease S from *Bacillus* sp. The antioxidant activity of the soybean hydrolysates, based on a linoleic acid oxidation system study, was attributed to the Leu-Leu-Pro-His-His peptide sequence [89, 93]. A potent antioxidant peptide, with inhibitory activity of lipid peroxidation, was isolated from soy protein isolate hydrolysed by

**Table 2.** Antioxidative capacity of plant protein-derived hydrolysates and peptides

Alcalase SSEFTY

**Antioxidative peptides** 

Not specified [13]

Not specified [51]

**Ref** 

[14]

[141]

[54]

[56]

[151]

**identified** 

IYLGQ

VNPHDHQN LVNPHDQN LLPHH LLPHHADADY VIPAGYP

Thermolysin NRYHE [135]

PQIPEEF

AQIPQQ

FRDEHKK KHNRGDEF

LRTLPMSVNVPL

Alcalase QKPFPQQPPF

Pepsin LQPGQGQQG

LQSGDALRVPSGTTYY

**used** 

ABTS+- scavenging **Alcalase**1,

**Source of proteins or hydrolysates** 

Potato liquid fraction

Potato protein concentration

Soybean protein b-conglycin

Yellow pea seed

Rice endosperm

protein

protein

The seed storage proteins of wheat, barley, rye, and oats contain known ACE-inhibitory diand tripeptides in their primary structures. Barley and barley by-products extract possesses a biological activity such as free radical scavenging activity, tyrosinase, xanthin oxidase, and ACE-inhibition effect [145]. Hydrolysates of barley prolamin fraction exhibited the highest antioxidant and ACE-inhibitory activity compared to other protein fractions and protein isolate. Moreover, positive correlations were obtained between antioxidant and ACEinhibitory activity and the degree of hydrolysis of hydrolysed protein fractions and protein isolate [146].

The computer analysis of amino acid sequences of wheat gliadins made by means of BIOPEP database [147] showed the presence of fragments that are homological with the sequences regarded as antihypertensive peptides. They were: Leu-Gln-Pro (α-, β- and γgliadins), Pro-Tyr-Pro (α-, β- and γ-gliadins), Ile-Pro-Pro (α-and β-gliadins), Leu-Pro-Pro (γgliadins) and Leu-Val-Leu (γ-gliadins). Bioinformatic analysis of cereal proteins sequences revealed that particularly four tripeptides with known ACE-inhibitory activity, Leu-Gln-Pro, Val-Pro-Pro, Ile-Pro-Pro, and Leu-Leu-Pro, are frequently encrypted in the primary structure of rye secalin, wheat gluten, and barley hordein. Sourdoughs fermented with different strains showed different concentrations of Leu-Gln-Pro and Leu-Leu-Pro. These differences corresponded to strain-specific differences in endopeptidase (PepO) and aminopeptidase (PepN) activities. The highest levels of peptides Val-Pro-Pro, Ile-Pro-Pro, Leu-Gln-Pro, and Leu-Leu-Pro, 0.23, 0.71, 1.09, and 0.09 mmol/ kg dry matter (DM), respectively, were observed in rye malt: gluten sourdoughs fermented with *Lactobacillus reuteri* TMW 1.106 and added protease [148]. Several clinical trials with hypertensive humans show a moderate but relatively consistent reduction of blood pressure upon consumption of the fermented milk products containing Val-Pro-Pro and Ile-Pro-Pro [88, 112]. Cheung and co-workers [149] used *in silico* approach to evaluate the potential of using oats as a protein source for generation of ACE-inhibitory peptides, and to screen for candidate enzymes to hydrolyse the oat protein for this purpose. It was found that thermolysin under high enzyme to substrate ratio (3%) and short time (20 min) conditions produced strong and stable ACE-inhibitory activity.

Antihypertensive Properties of Plant Protein Derived Peptides 165

take place after oral administration of a bioactive peptide and need to be considered on the final activity. It's highly likely that antihypertensive reported peptide sequences are subjected to alteration before the final activity *in vivo* after the various steps, such as attack of gastrointestinal enzymes and brush border peptidases, absorption through the intestinal barrier, attack of intracellular peptidases in the transcellular absorption and plasma enzymes after the peptides have entered the circulation [58, 154]. Therefore, the different aspects of bioavailability of antihypertensive peptide sequences have attracted a growing interest in the last years. The possibility of modification or breakdown of peptides during the gastrointestinal digestion is one of the most important factors to be considered when evaluating potential food-derived peptides for promotion of human health. Various models have been implemented to simulate gastrointestinal digestion; static and dynamic models which both differ in enzymes applied and reaction conditions, such as agitation and duration. For instance, authors in references [155] and [47] utilized human digestive liquids to model digestion *in vitro* whereas several reports have concerned implementation of porcine enzyme mixtures [e.g. 60, 127, 132]. In addition to studying the resistance of antihypertensive peptide sequences against the digestive enzymes, the models have been utilized in order to produce bioactive peptides, plant derived ACE-inhibitory peptides among them. For instance pea, lentil, bean and chickpea proteins have been reported to release ACE-inhibitory peptides during *in vitro* digestion [40, 127, 132]. The digestive characteristics of commercial proteases mixtures are known to differ from those of human origin [155]. Zhu and co-workers [156] reported that the antioxidative activity of a zein hydrolysate, which had previously shown antioxidant activity in aqueous solutions and in food systems, was either decreased or improved during the course of *in vitro* digestion, depending on the enzymes encountered and the duration of hydrolysis. Thus, direct comparison of the results between the different models is difficult. A consensus concerning the basic parameters would be relevant in order to harmonize the various *in vitro* digestion

Study of intestinal absorption *in vitro* is another common aim when elucidating the bioavailability. It has been indicated that a small portion of bioactive peptides can pass the intestine barrier and although it is usually too small to be considered nutritionally important, it can present the biological effects in tissue level [157, 158]. Molecular size and structural properties, such as hydrophobicity, affect the major transport route for peptides [158]. Research findings indicate that peptides with 2–6 amino acids are absorbed more readily in comparison to protein and free amino acids. As the molecular weight of peptides increases, their chance to pass the intestinal barrier decreases. Peptides are transported by active transcellular transport or by passive process [159]. The absorption studies are commonly performed with the monolayer of intestinal cell lines, such as Caco-2 cells, simulating intestinal epithelium, and analysis of peptides and metabolites in serum after *in vivo* and clinical studies. Foltz *et al*. [160] investigated the transport of Ile-Pro-Pro and Val-Pro-Pro by using three different absorption models and demonstrated that these tri-peptides are transported in small amounts intact across the barrier of the intestinal epithelium. In another study, the absolute bioavailability of the tri-peptides in pigs was below 0.1%, with an extremely short elimination half-life ranging from 5 to 20 min [161]. In humans, maximal

models.

Barley glutelin possess high hydrophobic amino acid content and enzymatic release by Alcalase produced peptides that had antioxidant capacity. Large size peptides possessed stronger DPPH scavenging activity and reducing power, whereas small-sized peptides were more effective in Fe2+ and hydroxyl radical scavenging activity [54]. Pepsin hydrolysis of byproduct of the wheat starch industry has shown antioxidant properties. Especially ultrafiltration produced fraction showed strong inhibition of the autoxidation of linoleic acid and scavenging activity of DPPH, superoxide and hydroxyl free radicals. The molecular weight distribution ranged from 0.1-1.7 kDa and high content of total hydrophobic amino acid was found in the active fraction [150]. Rice endosperm protein was, respectively, digested by five different protease treatments (Alcalase, chymotrypsin, Neutrase, Papain and Flavorase), and Neutrase produced the most desirable quality of antioxidant peptides. Two different peptides showing strong antioxidant activities were isolated from the hydrolysate using consecutive chromatographic methods. Especially, Phe-Arg-Asp-Glu-His-Lys-Lys significantly inhibited lipid peroxidation in a linoleic acid emulsion system more effectively than α-tocopherol [151].

The Alcalase-generated rice hydrolysate showed ACE-inhibitory activity with an IC50 value of 0.14 mg/ml. A potent ACE-inhibitory peptide with the amino acid sequence of Thr-Gln-Val-Tyr (IC50 of 18.2 µM) was isolated and identified from the hydrolysate. Single oral administration of the hydrolysate (600 mg/kg) and Thr-Gln-Val-Tyr (30 mg/kg) showed significantly decreased blood pressure in SHR, -25.6 and -40 mmHg SBP, respectively, after 6h [152]. Three strong ACE-inhibitors with the Leu-Arg-Pro, Leu-Ser-Pro and Leu-Gln-Pro sequences were isolated from maize α-zein hydrolysed with thermolysin. After intravenous administration of these peptides (30 mg/kg body weight), SBP was found to decrease up to a maximum of 15 mmHg [31]. Moreover, a tripeptide (Ile-Val-Tyr) isolated from wheat germ hydrolysate reduced MAP of 19.2 mmHg at dose of 5 mg/ml in SHR [153].

## **3. Bioavailability**

Bioavailability is a major issue when establishing correspondence between *in vitro* and *in vivo* activities of bioactive peptides. The capacity to reach target organ in an active conformation determines the physiological effect of bioactive peptides. Various processes take place after oral administration of a bioactive peptide and need to be considered on the final activity. It's highly likely that antihypertensive reported peptide sequences are subjected to alteration before the final activity *in vivo* after the various steps, such as attack of gastrointestinal enzymes and brush border peptidases, absorption through the intestinal barrier, attack of intracellular peptidases in the transcellular absorption and plasma enzymes after the peptides have entered the circulation [58, 154]. Therefore, the different aspects of bioavailability of antihypertensive peptide sequences have attracted a growing interest in the last years. The possibility of modification or breakdown of peptides during the gastrointestinal digestion is one of the most important factors to be considered when evaluating potential food-derived peptides for promotion of human health. Various models have been implemented to simulate gastrointestinal digestion; static and dynamic models which both differ in enzymes applied and reaction conditions, such as agitation and duration. For instance, authors in references [155] and [47] utilized human digestive liquids to model digestion *in vitro* whereas several reports have concerned implementation of porcine enzyme mixtures [e.g. 60, 127, 132]. In addition to studying the resistance of antihypertensive peptide sequences against the digestive enzymes, the models have been utilized in order to produce bioactive peptides, plant derived ACE-inhibitory peptides among them. For instance pea, lentil, bean and chickpea proteins have been reported to release ACE-inhibitory peptides during *in vitro* digestion [40, 127, 132]. The digestive characteristics of commercial proteases mixtures are known to differ from those of human origin [155]. Zhu and co-workers [156] reported that the antioxidative activity of a zein hydrolysate, which had previously shown antioxidant activity in aqueous solutions and in food systems, was either decreased or improved during the course of *in vitro* digestion, depending on the enzymes encountered and the duration of hydrolysis. Thus, direct comparison of the results between the different models is difficult. A consensus concerning the basic parameters would be relevant in order to harmonize the various *in vitro* digestion models.

164 Bioactive Food Peptides in Health and Disease

produced strong and stable ACE-inhibitory activity.

effectively than α-tocopherol [151].

**3. Bioavailability** 

differences corresponded to strain-specific differences in endopeptidase (PepO) and aminopeptidase (PepN) activities. The highest levels of peptides Val-Pro-Pro, Ile-Pro-Pro, Leu-Gln-Pro, and Leu-Leu-Pro, 0.23, 0.71, 1.09, and 0.09 mmol/ kg dry matter (DM), respectively, were observed in rye malt: gluten sourdoughs fermented with *Lactobacillus reuteri* TMW 1.106 and added protease [148]. Several clinical trials with hypertensive humans show a moderate but relatively consistent reduction of blood pressure upon consumption of the fermented milk products containing Val-Pro-Pro and Ile-Pro-Pro [88, 112]. Cheung and co-workers [149] used *in silico* approach to evaluate the potential of using oats as a protein source for generation of ACE-inhibitory peptides, and to screen for candidate enzymes to hydrolyse the oat protein for this purpose. It was found that thermolysin under high enzyme to substrate ratio (3%) and short time (20 min) conditions

Barley glutelin possess high hydrophobic amino acid content and enzymatic release by Alcalase produced peptides that had antioxidant capacity. Large size peptides possessed stronger DPPH scavenging activity and reducing power, whereas small-sized peptides were more effective in Fe2+ and hydroxyl radical scavenging activity [54]. Pepsin hydrolysis of byproduct of the wheat starch industry has shown antioxidant properties. Especially ultrafiltration produced fraction showed strong inhibition of the autoxidation of linoleic acid and scavenging activity of DPPH, superoxide and hydroxyl free radicals. The molecular weight distribution ranged from 0.1-1.7 kDa and high content of total hydrophobic amino acid was found in the active fraction [150]. Rice endosperm protein was, respectively, digested by five different protease treatments (Alcalase, chymotrypsin, Neutrase, Papain and Flavorase), and Neutrase produced the most desirable quality of antioxidant peptides. Two different peptides showing strong antioxidant activities were isolated from the hydrolysate using consecutive chromatographic methods. Especially, Phe-Arg-Asp-Glu-His-Lys-Lys significantly inhibited lipid peroxidation in a linoleic acid emulsion system more

The Alcalase-generated rice hydrolysate showed ACE-inhibitory activity with an IC50 value of 0.14 mg/ml. A potent ACE-inhibitory peptide with the amino acid sequence of Thr-Gln-Val-Tyr (IC50 of 18.2 µM) was isolated and identified from the hydrolysate. Single oral administration of the hydrolysate (600 mg/kg) and Thr-Gln-Val-Tyr (30 mg/kg) showed significantly decreased blood pressure in SHR, -25.6 and -40 mmHg SBP, respectively, after 6h [152]. Three strong ACE-inhibitors with the Leu-Arg-Pro, Leu-Ser-Pro and Leu-Gln-Pro sequences were isolated from maize α-zein hydrolysed with thermolysin. After intravenous administration of these peptides (30 mg/kg body weight), SBP was found to decrease up to a maximum of 15 mmHg [31]. Moreover, a tripeptide (Ile-Val-Tyr) isolated from wheat germ

Bioavailability is a major issue when establishing correspondence between *in vitro* and *in vivo* activities of bioactive peptides. The capacity to reach target organ in an active conformation determines the physiological effect of bioactive peptides. Various processes

hydrolysate reduced MAP of 19.2 mmHg at dose of 5 mg/ml in SHR [153].

Study of intestinal absorption *in vitro* is another common aim when elucidating the bioavailability. It has been indicated that a small portion of bioactive peptides can pass the intestine barrier and although it is usually too small to be considered nutritionally important, it can present the biological effects in tissue level [157, 158]. Molecular size and structural properties, such as hydrophobicity, affect the major transport route for peptides [158]. Research findings indicate that peptides with 2–6 amino acids are absorbed more readily in comparison to protein and free amino acids. As the molecular weight of peptides increases, their chance to pass the intestinal barrier decreases. Peptides are transported by active transcellular transport or by passive process [159]. The absorption studies are commonly performed with the monolayer of intestinal cell lines, such as Caco-2 cells, simulating intestinal epithelium, and analysis of peptides and metabolites in serum after *in vivo* and clinical studies. Foltz *et al*. [160] investigated the transport of Ile-Pro-Pro and Val-Pro-Pro by using three different absorption models and demonstrated that these tri-peptides are transported in small amounts intact across the barrier of the intestinal epithelium. In another study, the absolute bioavailability of the tri-peptides in pigs was below 0.1%, with an extremely short elimination half-life ranging from 5 to 20 min [161]. In humans, maximal plasma concentration did not exceed picomolar concentration [162]. Studies concerning absorption of plant derived peptides are rare, but milk-derived peptide Leu-His-Leu-Pro-Leu-Pro is an interesting example of a peptide with evaluation of bioavailability. This peptide resisted gastrointestinal simulation, but cellular peptidases digested the peptide to His-Leu-Pro-Leu-Pro before crossing Caco-2 cell monolayer [163, 164]. The degradation product, His-Leu-Pro-Leu-Pro, has been demonstrated to absorb in human intestine as it has been identified in human plasma after oral administration [165].

Antihypertensive Properties of Plant Protein Derived Peptides 167

case must be studied. Many strategies are currently demonstrated for enhancing bioavailability [171], among them microencapsulation for controlled release of the active compounds, stabilization of the active molecules to improve transportation through the intestinal barrier and provide resistance against degradation, and development of highly

ACE-inhibitory peptides have been studied extensively in the past two decades and ACEinhibition is the main mechanism concerning bioactive peptides with proven antihypertensive effects. ACE is a constituent enzyme of the Renin-Angiotensin-Aldosterone System (RAAS), which is a crucial regulator in human physiology. It controls blood pressure, fluid and electrolyte balance and affects the heart, vasculature and kidney [2]. Among the food-derived ACE-inhibitory peptides milk-derived peptides are the most extensively studied. The relevance of vegetable proteins as a source of antihypertensive peptides is increasing and several *in vivo* studies performed in SHR have demonstrated that plant-derived ACE-inhibitory protein hydrolysates and peptides significantly reduce blood pressure, either after oral or intravenous administration. For instance, a clinical randomized, placebo-controlled crossover study was performed in order to elucidate further the antihypertensive potential of yam tuber dioscorins [175]. The dioscorin meal or placebo was intervened as a morning drink daily for five weeks, followed by a washout stage for one week and the trial was then crossed over for five weeks. The SBP and DBP values were decreased after the five weeks of dioscorin meal intervention. The clinical trial as well as the animal trials with dioscorin intervention suggests that the gastrointestinal digestion may

Furthermore, related to the RAAS, plant derived ACE-inhibitory peptides have been reported to possess inhibition activity against renin, the first and rate-determining enzyme in RAAS [2]. The inhibition of renin is being suggested as a major alternative in hypertension prevention. The first direct renin-inhibitor, aliskiren, is currently under phase III trials to evaluate its potential as an antihypertensive drug [176]. Thermolysin digest of pea protein decreased remarkably the renal expression of renin mRNA levels *in vivo* and lowered plasma levels of angiotensin II, thus the reduction in blood pressure in SHR and human subjects was likely due to the effects on the renal angiotensin system [143]. Peaderived peptides Ile-Arg, Lys-Phe and Glu-Phe showed strong inhibitions *in vitro* studies of ACE and renin [133] as well as ACE-inhibitory peptide fractions from flaxseed protein

Opioid receptors are involved in various physiological phenomenons, e.g. in the regulation of blood pressure and circulation, and these receptors are related to the antihypertensive properties of some food derived peptides. Other vasodilatory substances, such as ET-1, have also been suggested to be involved in the antihypertensive effects of food-derived peptides [173, 178, 179]. However, peptide sequences derived specifically from plant proteins inducing endothelial NO liberation have not been reported this far. A cationic Arg-rich

produce antihypertensive peptides from the yam tuber dioscorins.

hydrolysates possessed inhibition also against renin [126, 177, 178].

stabile peptide analogues [172-174].

**4. Health benefits** 

Fujita and colleagues [166] established a bioavailability factor in relation to antihypertensive activity and ACE inhibition mechanism. The classification is based on inhibitor type and substrate type, the possible conversion of peptides by ACE into peptides with weaker activity and pro-drug type inhibitors, or possible conversion of peptides into true inhibitors by ACE or gastrointestinal proteases. A delayed antihypertensive effect is characteristic for pro-drug type peptides as they need to degrade further to reach the final active form [167, 168]. For instance, flaxseed protein showed pro-drug type characteristics compared to hydrolysed cationic peptide fraction [126]. The protein fraction showed a delayed hypotensive effect in SHR comparable to captopril (3 mg/kg body weight) and the effect was more sustained than the effect of the digested peptide fraction. The slow-acting character of the protein fraction was expected since the digestion of the proteins. Anyhow, more research is needed to identify the active peptide sequences released in the digestive tract and to evaluate the bioavailability of these peptides.

It can be deduced due to the incomplete bioavailability of peptide following oral ingestion, a peptide with pronounced antioxidant activity *in vitro* may exert little or no activity *in vivo*. However, bypass routes which increase the chance of peptide absorption can diminish the problem and it is possible that *in vivo* antioxidant activity can be higher than *in vitro* activity. In such cases, bioactive peptides may display their biological functions by mechanisms other than what is applied in experiment. In addition, it has been suggested that the strong *in vivo* activity can be due to increased activity of peptides following their breakdown by gastrointestinal proteases [88].

The improvement and optimization of bioavailability of antihypertensive peptides have gained a great interest during the last decade. The improvement of limited absorption and stability of peptides has been a goal when evaluating their effectiveness. For example, some carriers interact with the peptide molecule to create an insoluble entity at low pH, which later dissolves and facilitates intestinal uptake, by enhancing peptide transport over the non-polar biological membrane [169]. Bioavailability of bioactive tri-peptides (Val-Pro-Pro, Ile-Pro-Pro, Leu-Pro-Pro) was improved by administering them with a meal containing fibre, as compared to a meal containing no fibre. High methylated citrus pectin was used as a fibre [170]. Among drug delivery systems, emulsions have been used to enhance oral bioavailability or promoting absorption through mucosal surfaces of peptides and proteins [169]. Individually, various components of emulsions have been considered as candidates for improving bioavailability of peptides. Anyhow, it seems that no general strategy for improving bioavailability of antihypertensive peptides exists and due to the number of processes involved and different characteristics of peptides depending on the sequence each case must be studied. Many strategies are currently demonstrated for enhancing bioavailability [171], among them microencapsulation for controlled release of the active compounds, stabilization of the active molecules to improve transportation through the intestinal barrier and provide resistance against degradation, and development of highly stabile peptide analogues [172-174].

## **4. Health benefits**

166 Bioactive Food Peptides in Health and Disease

plasma concentration did not exceed picomolar concentration [162]. Studies concerning absorption of plant derived peptides are rare, but milk-derived peptide Leu-His-Leu-Pro-Leu-Pro is an interesting example of a peptide with evaluation of bioavailability. This peptide resisted gastrointestinal simulation, but cellular peptidases digested the peptide to His-Leu-Pro-Leu-Pro before crossing Caco-2 cell monolayer [163, 164]. The degradation product, His-Leu-Pro-Leu-Pro, has been demonstrated to absorb in human intestine as it has

Fujita and colleagues [166] established a bioavailability factor in relation to antihypertensive activity and ACE inhibition mechanism. The classification is based on inhibitor type and substrate type, the possible conversion of peptides by ACE into peptides with weaker activity and pro-drug type inhibitors, or possible conversion of peptides into true inhibitors by ACE or gastrointestinal proteases. A delayed antihypertensive effect is characteristic for pro-drug type peptides as they need to degrade further to reach the final active form [167, 168]. For instance, flaxseed protein showed pro-drug type characteristics compared to hydrolysed cationic peptide fraction [126]. The protein fraction showed a delayed hypotensive effect in SHR comparable to captopril (3 mg/kg body weight) and the effect was more sustained than the effect of the digested peptide fraction. The slow-acting character of the protein fraction was expected since the digestion of the proteins. Anyhow, more research is needed to identify the active peptide sequences released in the digestive tract

It can be deduced due to the incomplete bioavailability of peptide following oral ingestion, a peptide with pronounced antioxidant activity *in vitro* may exert little or no activity *in vivo*. However, bypass routes which increase the chance of peptide absorption can diminish the problem and it is possible that *in vivo* antioxidant activity can be higher than *in vitro* activity. In such cases, bioactive peptides may display their biological functions by mechanisms other than what is applied in experiment. In addition, it has been suggested that the strong *in vivo* activity can be due to increased activity of peptides following their breakdown by

The improvement and optimization of bioavailability of antihypertensive peptides have gained a great interest during the last decade. The improvement of limited absorption and stability of peptides has been a goal when evaluating their effectiveness. For example, some carriers interact with the peptide molecule to create an insoluble entity at low pH, which later dissolves and facilitates intestinal uptake, by enhancing peptide transport over the non-polar biological membrane [169]. Bioavailability of bioactive tri-peptides (Val-Pro-Pro, Ile-Pro-Pro, Leu-Pro-Pro) was improved by administering them with a meal containing fibre, as compared to a meal containing no fibre. High methylated citrus pectin was used as a fibre [170]. Among drug delivery systems, emulsions have been used to enhance oral bioavailability or promoting absorption through mucosal surfaces of peptides and proteins [169]. Individually, various components of emulsions have been considered as candidates for improving bioavailability of peptides. Anyhow, it seems that no general strategy for improving bioavailability of antihypertensive peptides exists and due to the number of processes involved and different characteristics of peptides depending on the sequence each

been identified in human plasma after oral administration [165].

and to evaluate the bioavailability of these peptides.

gastrointestinal proteases [88].

ACE-inhibitory peptides have been studied extensively in the past two decades and ACEinhibition is the main mechanism concerning bioactive peptides with proven antihypertensive effects. ACE is a constituent enzyme of the Renin-Angiotensin-Aldosterone System (RAAS), which is a crucial regulator in human physiology. It controls blood pressure, fluid and electrolyte balance and affects the heart, vasculature and kidney [2]. Among the food-derived ACE-inhibitory peptides milk-derived peptides are the most extensively studied. The relevance of vegetable proteins as a source of antihypertensive peptides is increasing and several *in vivo* studies performed in SHR have demonstrated that plant-derived ACE-inhibitory protein hydrolysates and peptides significantly reduce blood pressure, either after oral or intravenous administration. For instance, a clinical randomized, placebo-controlled crossover study was performed in order to elucidate further the antihypertensive potential of yam tuber dioscorins [175]. The dioscorin meal or placebo was intervened as a morning drink daily for five weeks, followed by a washout stage for one week and the trial was then crossed over for five weeks. The SBP and DBP values were decreased after the five weeks of dioscorin meal intervention. The clinical trial as well as the animal trials with dioscorin intervention suggests that the gastrointestinal digestion may produce antihypertensive peptides from the yam tuber dioscorins.

Furthermore, related to the RAAS, plant derived ACE-inhibitory peptides have been reported to possess inhibition activity against renin, the first and rate-determining enzyme in RAAS [2]. The inhibition of renin is being suggested as a major alternative in hypertension prevention. The first direct renin-inhibitor, aliskiren, is currently under phase III trials to evaluate its potential as an antihypertensive drug [176]. Thermolysin digest of pea protein decreased remarkably the renal expression of renin mRNA levels *in vivo* and lowered plasma levels of angiotensin II, thus the reduction in blood pressure in SHR and human subjects was likely due to the effects on the renal angiotensin system [143]. Peaderived peptides Ile-Arg, Lys-Phe and Glu-Phe showed strong inhibitions *in vitro* studies of ACE and renin [133] as well as ACE-inhibitory peptide fractions from flaxseed protein hydrolysates possessed inhibition also against renin [126, 177, 178].

Opioid receptors are involved in various physiological phenomenons, e.g. in the regulation of blood pressure and circulation, and these receptors are related to the antihypertensive properties of some food derived peptides. Other vasodilatory substances, such as ET-1, have also been suggested to be involved in the antihypertensive effects of food-derived peptides [173, 178, 179]. However, peptide sequences derived specifically from plant proteins inducing endothelial NO liberation have not been reported this far. A cationic Arg-rich peptide fraction from flaxseed, which possessed hypotensive effects in SHR, was suggested to mediate blood pressure through vasodilatory activity of NO synthesized from Arg. The observed effect might also be due to ACE- and renin-inhibition and in-depth research is needed to measure the renin and ACE protein levels and activities in SHR tissues and plasma and to specify the prior mechanism of antihypertensive action [44].

Antihypertensive Properties of Plant Protein Derived Peptides 169

activity of these hydrolysates and peptides in animal or in humans. These findings open up an interesting field aiming to revaluation of plant derived protein-rich by-products formed

Certain aspects, such as identification of the active form of the peptides in the organism and the different mechanisms of action that contribute in the antihypertensive effect still need to be further investigated. Recent advances on specific analytical techniques enable to follow small amounts of the peptides or derivatives in complex matrices and biological fluids. This will allow performing the kinetic studies in model animals and humans. Similarly, identifying novel and more complex biomarkers of exposure and activity by advances in new disciplines such as nutrigenomic and nutrigenetic will open new ways to follow bioactivity in the organism. There is still poor knowledge on the resistance of peptides to gastric degradation, and low bioavailability of peptides has been observed. This reinforces

More emphasis has been put on the legal regulation of the health claims attached to the products. Systematic approaches for review and assessment of scientific data have been developed by authorities around the world. The scientific evidence on the beneficial effects of the product should be enough detailed, extensive and conclusive for the use of a health claim in the functional food product labeling and marketing. First, it is necessary to identify and quantify the active sequences in the product. It is mandatory to monitor the hydrolytic or fermentative industrial production process as the antihypertensive peptides are only minor constituents in highly complex food matrices. Second, the antihypertensive effect in humans as well as the minimal dose needed to show the effect has to be proven in extensive investigations to fulfill the requirements of the legislation concerning functional foods. Besides being based on generally accepted scientific evidence, the claims should be well understood by the average consumer. Japan is the pioneer in the area of regulation of the health claims concerning food products. The concept of Foods for Specified Health Use (FOSHU) was established in 1991. In EU, the European Regulation on nutrition and health claims was established in January 2007 and the regulations are governed by European Food

[1] World Health Organisation (2011) Cardiovascular Diseases (CVD's). Fact sheet N°317. [2] Lavoie JL, Sigmund CD (2003) Minireview: Overview of the renin-angiotensin system -

[3] Landmesser U, Spiekermann S, Dikalov S, Tatge H, Wilke R, Kohler C, Harrison DG, Hornig B, Drexler H (2002) Vascular oxidative stress and endothelial dysfunction in

An endocrine and paracrine system. Endocrinol. 144: 2179-2189.

the need of various strategies to improve the oral bioavailability of peptides.

in food industry processes in remarkable amounts.

Safety Authority (EFSA).

Anne Pihlanto and Sari Mäkinen

*MTT, Biotechnology and Food Research, Jokioinen, Finland* 

**Author details** 

**6. References** 

Calmodulin (CaM) –dependent cyclic nucleotide phosphodiesterase (CaMPDE) regulates a large variety of cellular functions and excessive levels of CaM and CaMPDE play important roles in many physiological conditions, symptoms of cardiovascular disease among them. Recently, food derived peptides capable to inhibit CaMPDE have been reported, flaxseed and pea protein derived peptides among them [125, 133, 178]. Oxidative stress is a crucial causative factor for the initiation and progression of hypertension and CVD. Increased production of ROS, such as H2O2 and superoxide anion, reduced NO synthesis, and decreased bioavailability of antioxidants have been demonstrated in experimental and human hypertension. Diet rich in antioxidants can reduce blood pressure and thus, antioxidant properties of food-derived peptides may also affect on blood pressure regulation [180, 181]. Several food derived peptides have been reported to possess dual (ACE-inhibition and antioxidant) activity, among them plant protein derived hydrolysates of flaxseed [44, 55,182], rapeseed [47], potato [13] and yam [115, 116].

New mechanisms of action of antihypertensive peptides have been demonstrated in the recent years. The antihypertensive effect of a rapeseed derived tri-peptide rapakinin, was suggested to be mediated through other mechanism than ACE-inhibition [125]. Later on, different mechanisms were considered and the vasorelaxing activity of rapakinin was not blocked by eNOS inhibitor, while antagonists of IP and CCK1 receptor blocked the vasorelaxing effect of rapakinin significantly [128]. The results demonstrated that rapakinin relaxes the mesenteric artery of SHR through the PGI2-IP receptor followed by CCK pathway and the antihypertensive activity is mediated mainly by the PGI2-IP CCK-CCK1 receptor-dependent vasorelaxation. Moreover, inhibition of platelet-activating factor acetylhydrolase (PAF-AH) is suggested to play a crucial role in the hypertension prevention. PAF-AH is a circulatory enzyme secreted by inflammatory cells and it is involved in atherosclerosis. The discovery and application of natural PAF-AH into health promoting foods open up considerable potential [183].

## **5. General conclusions**

The interest on foods possessing health-promoting or disease-preventing properties has been increasing. So far most of the studies on antihypertensive peptides have been done on milk protein-derived peptides. In fact, much work has been done with dietary antihypertensive peptides and evidence of their effect in animal and clinical studies. However, it has been highlighted that there is a huge potential for obtaining antihypertensive peptides from protein sources other than milk. Much work has been done on plant protein hydrolysates and their activity *in vitro*. So far only limited number of peptides has been identified from plant proteins. In addition very little is known on the activity of these hydrolysates and peptides in animal or in humans. These findings open up an interesting field aiming to revaluation of plant derived protein-rich by-products formed in food industry processes in remarkable amounts.

Certain aspects, such as identification of the active form of the peptides in the organism and the different mechanisms of action that contribute in the antihypertensive effect still need to be further investigated. Recent advances on specific analytical techniques enable to follow small amounts of the peptides or derivatives in complex matrices and biological fluids. This will allow performing the kinetic studies in model animals and humans. Similarly, identifying novel and more complex biomarkers of exposure and activity by advances in new disciplines such as nutrigenomic and nutrigenetic will open new ways to follow bioactivity in the organism. There is still poor knowledge on the resistance of peptides to gastric degradation, and low bioavailability of peptides has been observed. This reinforces the need of various strategies to improve the oral bioavailability of peptides.

More emphasis has been put on the legal regulation of the health claims attached to the products. Systematic approaches for review and assessment of scientific data have been developed by authorities around the world. The scientific evidence on the beneficial effects of the product should be enough detailed, extensive and conclusive for the use of a health claim in the functional food product labeling and marketing. First, it is necessary to identify and quantify the active sequences in the product. It is mandatory to monitor the hydrolytic or fermentative industrial production process as the antihypertensive peptides are only minor constituents in highly complex food matrices. Second, the antihypertensive effect in humans as well as the minimal dose needed to show the effect has to be proven in extensive investigations to fulfill the requirements of the legislation concerning functional foods. Besides being based on generally accepted scientific evidence, the claims should be well understood by the average consumer. Japan is the pioneer in the area of regulation of the health claims concerning food products. The concept of Foods for Specified Health Use (FOSHU) was established in 1991. In EU, the European Regulation on nutrition and health claims was established in January 2007 and the regulations are governed by European Food Safety Authority (EFSA).

## **Author details**

168 Bioactive Food Peptides in Health and Disease

peptide fraction from flaxseed, which possessed hypotensive effects in SHR, was suggested to mediate blood pressure through vasodilatory activity of NO synthesized from Arg. The observed effect might also be due to ACE- and renin-inhibition and in-depth research is needed to measure the renin and ACE protein levels and activities in SHR tissues and

Calmodulin (CaM) –dependent cyclic nucleotide phosphodiesterase (CaMPDE) regulates a large variety of cellular functions and excessive levels of CaM and CaMPDE play important roles in many physiological conditions, symptoms of cardiovascular disease among them. Recently, food derived peptides capable to inhibit CaMPDE have been reported, flaxseed and pea protein derived peptides among them [125, 133, 178]. Oxidative stress is a crucial causative factor for the initiation and progression of hypertension and CVD. Increased production of ROS, such as H2O2 and superoxide anion, reduced NO synthesis, and decreased bioavailability of antioxidants have been demonstrated in experimental and human hypertension. Diet rich in antioxidants can reduce blood pressure and thus, antioxidant properties of food-derived peptides may also affect on blood pressure regulation [180, 181]. Several food derived peptides have been reported to possess dual (ACE-inhibition and antioxidant) activity, among them plant protein derived hydrolysates

New mechanisms of action of antihypertensive peptides have been demonstrated in the recent years. The antihypertensive effect of a rapeseed derived tri-peptide rapakinin, was suggested to be mediated through other mechanism than ACE-inhibition [125]. Later on, different mechanisms were considered and the vasorelaxing activity of rapakinin was not blocked by eNOS inhibitor, while antagonists of IP and CCK1 receptor blocked the vasorelaxing effect of rapakinin significantly [128]. The results demonstrated that rapakinin relaxes the mesenteric artery of SHR through the PGI2-IP receptor followed by CCK pathway and the antihypertensive activity is mediated mainly by the PGI2-IP CCK-CCK1 receptor-dependent vasorelaxation. Moreover, inhibition of platelet-activating factor acetylhydrolase (PAF-AH) is suggested to play a crucial role in the hypertension prevention. PAF-AH is a circulatory enzyme secreted by inflammatory cells and it is involved in atherosclerosis. The discovery and application of natural PAF-AH into health promoting

The interest on foods possessing health-promoting or disease-preventing properties has been increasing. So far most of the studies on antihypertensive peptides have been done on milk protein-derived peptides. In fact, much work has been done with dietary antihypertensive peptides and evidence of their effect in animal and clinical studies. However, it has been highlighted that there is a huge potential for obtaining antihypertensive peptides from protein sources other than milk. Much work has been done on plant protein hydrolysates and their activity *in vitro*. So far only limited number of peptides has been identified from plant proteins. In addition very little is known on the

plasma and to specify the prior mechanism of antihypertensive action [44].

of flaxseed [44, 55,182], rapeseed [47], potato [13] and yam [115, 116].

foods open up considerable potential [183].

**5. General conclusions** 

Anne Pihlanto and Sari Mäkinen *MTT, Biotechnology and Food Research, Jokioinen, Finland* 

## **6. References**


patients with chronic heart failure - Role of xanthine-oxidase and extracellular superoxide dismutase. Circulation 106: 3073–3078.

Antihypertensive Properties of Plant Protein Derived Peptides 171

[20] Reynolds K, Chin A, Lees KA, Nguyen A, Bujnowski D, He J (2006) A meta-analysis of the effect of soy protein supplementation on serum lipids. Am j. cardiol. 98: 633-640. [21] Gibbs BF, Zougman A, Masse R, Mulligan C (2004) Production and characterization of bioactive peptides from soy hydrolysate and soy-fermented. Food res. int. 37: 123-131. [22] Zhang JH, Tatsumi E, Ding CH, Li LT (2006) Angiotensin I-converting enzyme inhibitory peptides in Douchi, a Chinese traditional fermented soybean product. Food

[23] Fan J, Hu X, Tan S, Zhang Y, Tatsumi E, Li L (2009) Isolation and characterisation of a novel angiotensin I-converting enzyme-inhibitory peptide derived from douchi, a

[24] Ibe S, Yoshida K, Kumada K, Tsurushin S, Furusho T, Otobe K (2009) Antihypertensive effects of natto, a traditional Japanese fermented food, in spontaneously hypertensive

[25] Roy F, Boye JI, Simpson BK (2010) Bioactive proteins and peptides in pulse crops: Pea,

[26] Aluko RE (2008) Determination of nutritional and bioactive properties of peptides in enzymatic pea, chickpea, and mung bean protein hydrolysates. J. AOAC int. 91: 947-

[27] Ndiaye F, Tri Vuong, Duarte J, Aluko RE, Matar C (2012) Anti-oxidant, antiinflammatory and immunomodulating properties of an enzymatic protein hydrolysate

[28] Arcan I, Yemenicioglu A (2010) Effects of controlled pepsin hydrolysis on antioxidant potential and fractional changes of chickpea proteins. Food res. int. 43: 140-147. [29] Shewry PR, Halford NG (2002) Cereal seed storage proteins: structures, properties and

[30] Silano M, De Vincenzi M (1999) Bioactive antinutritional peptides derived from cereal

[31] Miyoshi S, Ishikawa H, Kaneko T, Fukui F, Tanaka H, Maruyama S (1991) Structures and activity of angiotensin-converting enzyme inhibitors in an alpha-zein hydrolysate.

[32] Kong X, Zhou H, Hua Y, Qian H (2008). Preparation of wheat gluten hydrolysates with

[33] Takahashi M, Moriguchi S, Yoshikawa M, Sakasaki R (1994) Isolation and characterization of oryzatensin - a novel bioactive peptide with ileum-contracting and immunomodulating activities derived from rice albumin. Biochem. mol. biol. int. 33:

[34] Calderón de la Barca AM, RuizSalazar RA, JaraMarini ME (2000) Enzymatic hydrolysis of soy protein to improve its amino acid composition and functional properties. J. food

[35] Moure A, Domínguez H, Parajó JC (2005) Fractionation and enzymatic hydrolysis of soluble protein present in waste liquors from soy processing. J. agric. food chem. 53:

traditional Chinese fermented soybean food. J. sci. food agric. 89: 603-608.

chem. 98: 551-557.

956.

rats. Food sci. technol. res. 15: 199-202.

chickpea and lentil. Food res. int. 43: 432-442.

from yellow field pea seeds. Eur. j. nutr. 51: 29-37.

role in grain utilization. J. exp. bot. 53: 947–958.

high opioid activity. Eur. food res. technol. 227: 511-517.

prolamins: a review. Nahrung 43:175-184.

Agric. biol. chem. 55: 1313-1318.

1151-1158.

7600–7608.

sci. 65: 246–253.


[20] Reynolds K, Chin A, Lees KA, Nguyen A, Bujnowski D, He J (2006) A meta-analysis of the effect of soy protein supplementation on serum lipids. Am j. cardiol. 98: 633-640.

170 Bioactive Food Peptides in Health and Disease

276.

15: 693-700.

51: 4389-4393.

Kuras. FEBS J. 273: 3569-3584.

superoxide dismutase. Circulation 106: 3073–3078.

peptides. J. agric. food chem. 57: 5113-5120.

cardiovascular diseases. Curr. pharm. biotechnol. 7: 101-108.

(Solanum tuberosum L.). Mol. gen. genet. 269: 535-541.

of potato (Solanum tuberosum). Food chem. 109: 104-112.

high metabolic utilization in humans. J. nutr. 137: 594-600.

soybean oil emulsions. J. food sci.75: C760-C765.

high-sucrose diet in rats. Br. j. nutr. 100: 984-991.

intern. med. 161: 2573-2578.

patients with chronic heart failure - Role of xanthine-oxidase and extracellular

[4] Yao EH, Yu Y, Fukuda N (2006) Oxidative stress on progenitor and stem cells in

[5] Pihlanto A, Korhonen H (2003) Bioactive peptides and proteins. Adv. food res. 47: 175-

[6] Guang C, Phillips RD (2009) Plant food-derived angiotensin I converting enzyme

[7] Pihlanto A, Mäkinen S, Mattila P (2012) Potential health-promoting properties of potato-derived proteins, peptides and phenolic Compounds. In: Caprara C, editor. Potatoes: Production, Consumption and Health Benefits. Nova Publishers pp. 173-194. [8] Heibges A, Salamini F, Gebhardt C (2003) Functional comparison of homologous members of three groups of Kunitz-type enzyme inhibitors from potato tubers

[9] Kim J-Y, Park C-S, Hwang I, Cheong H, Nah J-W, Hahm K-S, Park Y (2009) Protease inhibitors from plants with antimicrobial activity. Int. j. mol. sci. 10: 2860–2872. [10] Scherer GFE, Ryu SB, Wang XM, Matos AR, Heitz T (2010) Patatin-related phospholipase A: nomenclature, subfamilies and functions in plants. Trends plant sci.

[11] Bauw G, Nielsen HV, Emmersen J, Nielsen KL, Jorgensen M, Welinder KG (2006) Patatins, Kunitz protease inhibitors and other major proteins in tuber of potato cv.

[12] Liu YW, Han CH, Lee MH, Hsu FL, Hou WC (2003) Patatin, the tuber storage protein of potato (*Solanum tuberosum L.*), exhibits antioxidant activity *in vitro*. J. agric. food chem.

[13] Pihlanto A, Akkanen S, Korhonen HJ (2008). ACE-inhibitory and antioxidant properties

[14] Cheng Y, Xiong YL, Chen J (2010) Fractionation, separation, and identification of antioxidative peptides in potato protein hydrolysate that enhance oxidative stability of

[15] Hill AJ, Peikin SR, Ryan CA, Blundell JE (1990) Oral administration of proteinase inhibitor II from potatoes reduces energy intake in man. Physiol. behave. 48: 241-246. [16] Bos C, Airinei G, Mariotti F, Benamouzig R, Bérot S, Evrard J, Fénart E, Tomé D, Gaudichon C (2007) The poor digestibility of rapeseed protein is balanced by its very

[17] Mariotti F, Hermier D, Sarrat C, Magné J, Fenart E, Evrard J, Tome D, Huneau JF (2008) Rapeseed protein inhibits the initiation of insulin resistance by a high-saturated fat,

[18] Ooman DB (2001) Flaxseed as a functional food source. J. sci. food agric. 81: 889-894. [19] Bazzano LA, He J, Ogden LG, Loria C, Vupputuri S, Myers L, Whelton PK (2001) Legume consumption and risk of coronary heart disease in US men and women. Arch.

	- [36] Yim MH, Lee JH (2000) Functional properties of fractionated soy protein isolates by proteases from Meju. Food sci. biotechnol. 9: 253–257.

Antihypertensive Properties of Plant Protein Derived Peptides 173

proteins by enzymatic activities confined to different parts of the potato tuber. J. agric.

[51] Pêna-Ramos EA, Xiong YL (2002) Antioxidant activity of soy protein hydrolyzates in a

[52] Li Y, Jiang B, Zhang T, Mu W, Liu J (2008) Antioxidant and free radical scavenging

[53] Dryakova A, Pihlanto A, Marnila P, Curda L, Korhonen HJT (2010) Antioxidant properties of whey protein hydrolysates as measured by three methods. Eur. food res.

[54] Xia Y, Bamdad F, Gänzle M, Chen L (2012) Fractionation and characterization of antioxidant peptides derived from barley glutelin by enzymatic hydrolysis. Food chem.

[55] Udenigwe CC, Lu Y, Han C, Hou W, Aluko RE (2009) Flaxseed protein-derived peptide fractions: Antioxidant properties and inhibition of lipopolysaccharide-induced nitric

[56] Suetsuna K, Chen JR (2002) Isolation and characterization of peptides with antioxidant

[57] Zhang T, Li Y, Miao M, Jiang B (2011) Purification and characterisation of a new antioxidant peptide from chickpea (Cicer arietium L.) protein hydrolysates. Food chem.

[58] Vermeirssen V, Van Camp J, Verstraete A, Verstraete W (2004) Bioavailability of

[59] Li C, Matsui T, Matsumoto K, Yamasaki R, Kawasaki T (2002) Latent production of angiotensin I-converting enzyme inhibitors from buckwheat protein. J. pept. sci. 8: 267–

[60] Lo WMY, Li-Chan ECY (2005) Angiotensin I converting enzyme inhibitory peptides from *in vitro* pepsin-pancreatin digestion of soy protein. J. agric. food chem. 53: 3369-

[61] Megías C, Yust MM, Pedroche J, Lquari H, Girón-Calle J, Alaiz M, Millán F, Vioque J (2004) Purification of an ACE inhibitory peptide after hydrolysis of sunflower

[62] Vermeirssen V, van der Bent A, Van Camp J, van Amerongen A, Verstraete W (2004) A quantitative *in silico* analysis calculates the angiotensin I converting enzyme (ACE)

[63] Boye JI, Roufik S, Pesta N, Barbana C (2010) Angiotensin I-converting enzyme inhibitory properties and SDS-PAGE of red lentil protein hydrolysates. LWT – Food sci.

[64] Barbana C, Boye JI (2011) Angiotensin I-converting enzyme inhibitory properties of lentil protein hydrolysates: Determination of the kinetics of inhibition. Food chem. 127:

[65] Mannheim A, Cheryan M (1990) Continuous hydrolysis of milk protein in a membrane

activities of chickpea protein hydrolysate (CPH). Food chem. 106: 444–450.

oxide production in murine macrophages. Food chem. 116: 277-284.

activity derived from wheat gluten. Food sci. tehnol. res. 8: 227–230.

angiotensin I converting enzyme inhibitory peptides. Br. j. nutr. 92: 357.

(*Helianthus annuus* L.) protein isolates. J. agric. food chem. 52: 1928–1932.

inhibitory activity in pea and whey protein digests. Biochimie 86: 231-239.

food chem. 56: 9875-9883.

technol. 230: 865-874.

134: 1509-1518.

128: 28-33.

274.

3376.

technol. 43: 987-991.

reactor. J. food sci. 55: 381-385.

94-101.

liposomial system. J. food sci. 67: 2952–2956


proteins by enzymatic activities confined to different parts of the potato tuber. J. agric. food chem. 56: 9875-9883.

[51] Pêna-Ramos EA, Xiong YL (2002) Antioxidant activity of soy protein hydrolyzates in a liposomial system. J. food sci. 67: 2952–2956

172 Bioactive Food Peptides in Health and Disease

chem. 48: 657–666.

agric. food chem. 58: 4762-4768.

flaxseed protein. Food chem. 132: 468-475.

converting enzyme. Br. j. nutr. 84: S33-S37.

immobilized ACE. J. agric. food chem. 54: 7120-7124.

81: 363–369.

chem. 111: 942-950.

funct. foods 4: 575-583.

733–736.

23.

[36] Yim MH, Lee JH (2000) Functional properties of fractionated soy protein isolates by

[37] Kristinsson HG, Rasco BA (2000) Biochemical and functional properties of Atlantic salmon (Salmo salar) muscle hydrolyzed with various alkaline proteases. J. agric. food

[38] Inouye K, Nakano K, Asaoka K, Yasukawa K (2009) Effects of thermal treatment on the coagulation of soy proteins induced by subtilisin Carlsberg. J agric. food chem. 57:717–

[39] Wu J, Ding X (2002) Characterization of inhibition and stability of soy-protein-derived angiotensin I-converting enzyme inhibitory peptides. Food res. int. 35: 367-375. [40] Barbana C, Boye JI (2010) Angiotensin I-converting enzyme inhibitory activity of

[41] Pedroche J, Yust MM, Giron-Calle J, Alaiz M, Millan F, Vioque J (2002) Utilisation of chickpea protein isolates for production of peptides with angiotensin I-converting

[42] Udenigwe CC, Aluko RE (2010) Antioxidant and angiotensin converting enzymeinhibitory properties of a flaxseed protein-derived high fischer ratio peptide mixture. J.

[43] Yust MM, Pedroche J, Giron-Calle J, Alaiz M, Francisco M, Vioque J (2003) Production of ace inhibitory peptides by digestion of chickpea legumin with alcalase. Food chem.

[44] Udenigwe CC, Adebiyi AP, Doyen A, Li H, Bazinet L, Aluko RE (2012) Low molecular weight flaxseed protein-derived arginine-containing peptides reduced blood pressure of spontaneously hypertensive rats faster than amino acid form of arginine and native

[45] Megias C, Pedroche J, Yust MDM, Alaiz M, Giron-Calle J, Millan F, Vioque J (2006) Affinity purification of angiotensin converting enzyme inhibitory peptides using

[46] Wu J, Aluko RE, Muir AD (2008) Purification of angiotensin I-converting enzymeinhibitory peptides from the enzymatic hydrolysate of defatted canola meal. Food

[47] Mäkinen S, Johansson T, Vegarud G, Pihlava J-M, Pihlanto A (2012) Angiotensin Iconverting enzyme inhibitory and antioxidant properties of rapeseed hydrolysates. J.

[48] Li G, Shi Y, Liu H, Le G (2006) Antihypertensive effect of alcalase generated mung bean protein hydrolysates in spontaneously hypertensive rats. Eur. food res. technol. 222:

[49] FitzGerald R, Meisel H (2000) Milk protein-derived peptide inhibitors of angiotensin-I-

[50] Makinen S, Kelloniemi J, Pihlanto A, Makinen K, Korhonen H, Hopia A, Valkonen JPT (2008) Inhibition of angiotensin converting enzyme I caused by autolysis of potato

chickpea and pea protein hydrolysates. Food res. int. 43: 1642–1649.

enzyme (ACE)-inhibitory activity. J. sci. food. agric. 82: 960-965.

proteases from Meju. Food sci. biotechnol. 9: 253–257.


[66] Wang Y-K, He H-L, Wang G-F, Wu H, Zhou B-C, Chen X-L, Zhang YZ (2010) Oyster (Crassostrea gigas) hydrolysates produced on a plant scale have antitumor activity and immunostimulating effects in BALB/c mice. Mar. drugs 8:255–68.

Antihypertensive Properties of Plant Protein Derived Peptides 175

[81] Natesh R, Schwager SLU, Sturrock ED, Acharya KR (2003) Crystal structure of the human angiotensin-converting enzyme lisonopril complex. Nature 421: 551–554. [82] Ondetti MA, Rubin B, Cushman DW (1977) Design of specific inhibitors of angiotensinconverting enzyme: new class of orally active antihypertensive agents. Science 196: 441-

[83] Cheung HS, Wang FL, Ondetti MA, Sabo EF, Cushman DW (1980) Binding of peptide substrates and inhibitors of angiotensin-converting enzyme: importance of the COOH-

[84] Ariyoshi Y (1993) Angiotensin-converting enzyme inhibitors derived from food

[85] Pripp AH, Isaksson T, Stepaniak L, Sorhaug T (2004) Quantitative structure–activity relationship modelling of ACE-inhibitory peptides derived from milk proteins. Eur.

[86] Moure A, Domínguez H, Parajó JC (2006) Antioxidant properties of ultrafiltration recovered soy protein fractions from industrial effluents and their hydrolysates. Process

[87] Rajapakse N, Mendis E, Jung WK, Je JY, Kim SK (2005) Purification of a radical scavenging peptide from fermented mussel sauce and its antioxidant properties. Food

[88] Erdmann K, Cheung BWY, Schröder H (2008) The possible roles of food derived bioactive peptides in reducing the risk of cardiovascular disease. J. nutr. biol. 19: 643–

[89] Chen HM, Muramoto K, Yamauchi F, Fujimoto K, Nokihara K (1998) Antioxidative properties of histidine-containing peptides designed from peptide fragments found in

[90] Pihlanto A (2006) Antioxidative peptides derived from milk proteins. Int. dairy j. 16:

[91] Chan KM, Decker EA (1994) Endogenous muscle antioxidants. Crit. rev. food. sci. 34:

[92] Qian ZJ, Jung WK, Kim SK (2008) Free radical scavenging activity of a novel antioxidative peptide purified from hydrolysate of bullfrog skin, Rana catesbeiana

[93] Chen HM, Muramoto K, Yamauchi F, Nokihara K (1996) Antioxidant activity of designed peptides based on the antioxidative peptide isolated from digests of a soybean

[94] Saito K, Jin DH, Ogawa T, Muramoto K, Hatakeyama E, Yasuhara T, Nokihara K (2003) Antioxidative properties of tripeptide libraries prepared by the combinatorial

[95] Nagasawa T, Yonekura T, Nishizawa N, Kitts DD (2001) *In vitro* and *in vivo* inhibition of muscle lipid and protein oxidation by carnosine. Mol. cell. biochem. 225: 29–34.

the digests of a soybean protein. J. agric. food chem. 46: 49–53.

Shaw. bioresource technol. 99: 1690–1698.

protein. J. agric. food chem. 44: 2619–2623.

chemistry. J. agric. food chem. 51: 3668–3674.

terminal dipeptide sequence. J biol. chem. 255: 401-407.

proteins. Trends food sci. technol. 4:139-144.

food res. technol. 219: 579-583.

biochem. 41: 447–456.

res. int. 38: 175–182.

654.

1306–1314.

403–426.

444.


[81] Natesh R, Schwager SLU, Sturrock ED, Acharya KR (2003) Crystal structure of the human angiotensin-converting enzyme lisonopril complex. Nature 421: 551–554.

174 Bioactive Food Peptides in Health and Disease

Publishing Limited pp. 107–143.

reactor. Food chem. 98: 725–732.

DOI: 10.1002/elsc.201100137.

biotech. biochem. 67: 1278-1283.

soypaste. JARQ 44:167-172.

biochem. 41: 1282-1288.

102: 106–115.

1048.

[66] Wang Y-K, He H-L, Wang G-F, Wu H, Zhou B-C, Chen X-L, Zhang YZ (2010) Oyster (Crassostrea gigas) hydrolysates produced on a plant scale have antitumor activity and

[67] Chiang WE, Cordle CT, Thomas RL (1995) Casein hydrolysate produced using a

[68] Guérard F (2007). Enzymatic methods for marine by-products recovery. In: Shahidi F editor. Maximizing the value of marine by-products. Campridge: Woodward

[69] Chiang WD, Tsou MJ, Tsai ZY, Tsai TC (2006) Angiotensin I-converting enzyme inhibitor derived from soy protein hydrolysate and produced by using membrane

[70] Korhonen H, Pihlanto A (2003) Food-derived bioactive peptides - Opportunities for

[71] Virtanen T, Pihlanto A, Akkanen S, Korhonen H (2007) Development of antioxidant activity in milk whey during fermentation with lactic acid bacteria. J. appl. microbiol.

[72] Pihlanto A, Virtanen T, Korhonen H (2010) Angiotensin I converting enzyme (ACE) inhibitory activity and antihypertensive effect of fermented milk. Int. dairy j. 20: 3-10. [73] Hernández-Ledesma B, Miralles B, Amigo L, Ramos M, Recio, I (2005) Identification of antioxidant and ACE-inhibitory peptides in fermented milk. J. sci. food agric. 85: 1041-

[74] Pihlanto A, Johansson T, Mäkinen S (2012) Inhibition of angiotensin I-converting enzyme and lipid peroxidation by fermented rapeseed and flaxseed meal. Eng. life sci.

[75] Okamoto A, hanagata H, Matsumoto E, kawamura Y, Koizumi Y, Yanagida F (1995) Angiotensin I-converting enzyme inhibitory peptides isolated from tofuyo fermented

[76] Kuba M, Tanaka K, Tawata S, Takeda Y, Yasuda M (2003) Angiotensin I-converting enzyme inhibitory peptides isolated from tofuyo fermented soybean food. Biosci.

[77] Li F-J, Yin L-J, Cheng Y-Q, Saito M, Yamaki K, Li L-T (2010) Angiotensin I-converting enzyme inhibitory activities of extracts from commercial chinese style fermented

[78] Tsai JS, Lin YS, Pan BS, Chen TJ (2006) Antihypertensive peptides and -aminobutyric acid from prozyme 6 facilitated lactic acid bacteria fermentation of soymilk. Process

[79] Iwai K, Nakaya N, Kawasaki Y, Matsue H (2002) Inhibitory effect of natto, a kind of fermented soybeans, on LDL oxidation in vitro. J. agric. food chem. 50: 3592-3596. [80] Wang D, Wang L, Zhu F, Zhu J, Chen XD, Zou L, Saito, M, Li L (2008) In vitro and in vivo studies on the antioxidant activities of the aqueous extracts of Douchi (a traditional

Chinese salt-fermented soybean food). Food chem. 107:1421-1428.

immunostimulating effects in BALB/c mice. Mar. drugs 8:255–68.

formed-in-place membrane reactor. J. food sci. 60: 1349-1352.

designing future foods. Curr. pharm. design 9: 1297-1308.

soybean food. Biosci. biotech. biochem 59: 1147-1149.


[96] Hernández-Ledesma B, Davalos A, Bartolome B, Amigo L (2005) Preparation of antioxidant enzymatic hydrolysates from a-lactalbumin and b-lactoglobulin. Identification of active peptides by HPLC–MS/MS. J. agric. food chem. 53: 588–593.

Antihypertensive Properties of Plant Protein Derived Peptides 177

[113] Hsu F, Lin Y, Lee M, Lin C, Hou W (2002) Both dioscorin, the tuber storage protein of yam (Dioscorea alata cv. Tainong No. 1), and its peptic hydrolysates exhibited angiotensin converting enzyme inhibitory activities. J. agric. food chem. 50: 6109-

[114] Lee M, Lin Y, Lin Y, Hsu F, Hou W (2003) The mucilage of yam (Dioscorea batatas Decne) tuber exhibited angiotensin converting enzyme inhibitory activities. Bot. bull.

[115] Nagai T, Suzuki N, Nagashima T (2007) Autolysate and enzymatic hydrolysates from yam (Dioscorea opposita Thunb.) tuber mucilage tororo have antioxidant and angiotensin I-converting enzyme inhibitory activities. J. food agric. environ. 5: 39-43. [116] Nagai T, Suzuki N, Tanoue Y, Kai N, Nagashima T (2007) Antioxidant and antihypertensive activities of autolysate and enzymatic hydrolysates from yam

[117] Lacaille-Dubois MA, Franck U, Wagner H (2001) Search for potential angiotensin

[118] Huang G, Lu T, Chiu C, Chen H, Wu C, Lin Y, Hsieh W, Liao J, Sheu M, Lin Y (2011) Sweet potato storage root defensin and its tryptic hydrolysates exhibited angiotensin

[119] Huang G, Chen H, Susumu K, Wu J, Hou W, Wu C, Sheu M, Huang S, Lin Y (2011) Sweet potato storage root thioredoxin h2 and their peptic hydrolysates exhibited

[121] Ishiguro K, Sameshima Y, Kume T, Ikeda K, Matsumoto J, Yoshimoto M (2012) Hypotensive effect of a sweet potato protein digest in spontaneously hypertensive rats and purification of angiotensin I-converting enzyme inhibitory peptides. Food chem.

[122] Lin C, Lin S, Lin Y, Hou W (2006) Effects of tuber storage protein of yam (Dioscorea alata cv. Tainong No. 1) and its peptic hydrolyzates on spontaneously hypertensive

[123] Liu Y, Lin Y, Liu D, Han C, Chen C, Fan M, Hou W (2009) Effects of different types of yam (Dioscorea alata) products on the blood pressure of spontaneously hypertensive

[124] Iwai K, Matsue H (2007) Ingestion of Apios americana Medikus tuber suppresses blood pressure and improves plasma lipids in spontaneously hypertensive rats. Nutr.

[125] Marczak E, Usui H, Fujita H, Yang Y, Yokoo M, Lipkowski A, Yoshikawa M (2003) New antihypertensive peptides isolated from rapeseed. Peptides 24: 791-798. [126] Udenigwe CC, Lin YS, Hou WC, Aluko RE (2009) Kinetics of the inhibition of renin and angiotensin I-converting enzyme by flaxseed protein hydrolysate fractions. J. funct.

angiotensin converting enzyme inhibitory activity *in vitro*. Bot. stud. 52: 15-22 [120] Huang G, Ho Y, Chen H, Chang Y, Huang S, Hung H, Lin Y (2008) Sweet potato storage root trypsin inhibitor and their peptic hydrolysates exhibited angiotensin

(Dioscorea opposita Thunb.) ichyoimo tubers. J. food agric. environ. 5: 64-68.

converting enzyme (ACE)-inhibitors from plants. Phytomedicine 8: 47–52.

converting enzyme inhibitory activity *in vitro*. Bot. stud. 52: 257-264.

converting enzyme inhibitory activity *in vitro*. Bot. stud. 49: 101-108.

6113.

acad. sinica 44: 267-273.

131: 774-779.

res. 27: 218-224.

foods 1: 199-207.

rats. J. sci. food agric. 86: 1489-1494.

Rats. Biosci. biotech. biochem. 73: 1371-1376.


[113] Hsu F, Lin Y, Lee M, Lin C, Hou W (2002) Both dioscorin, the tuber storage protein of yam (Dioscorea alata cv. Tainong No. 1), and its peptic hydrolysates exhibited angiotensin converting enzyme inhibitory activities. J. agric. food chem. 50: 6109- 6113.

176 Bioactive Food Peptides in Health and Disease

J. biochem. biophys. methods 51: 75.

pharm. biomed. anal. 37: 219.

J. chrom. biomed. appl. 613: 145.

carboxypeptidase. Eur. j. biochem. 87: 265.

Free radical bio. med. 26: 1231–1237.

food chem. 53: 4311–4314.

J. nutr. 134: 980-988.

178.

[96] Hernández-Ledesma B, Davalos A, Bartolome B, Amigo L (2005) Preparation of antioxidant enzymatic hydrolysates from a-lactalbumin and b-lactoglobulin. Identification of active peptides by HPLC–MS/MS. J. agric. food chem. 53: 588–593. [97] Cushman DW, Cheung HS (1971) Spectrophotometric assay and properties of the angiotensin converting enzyme of rabbit lung. Biochem. pharmacol. 20: 1637. [98] Vermeirssen V, Van Camp J, Verstraete W (2002) Optimisation and validation of an angiotensin-converting enzyme inhibition assay for the screening of bioactive peptides.

[99] Li GH, Liu H, Shi Y-H, Le GW (2005) Direct spectrophotometric measurement of angiotensin I-converting enzyme inhibitory activity for screening bioactive peptides. J.

[100] Shalaby SM, Zakora M, Otte J (2006) Performance of two commonly used angiotensinconverting enzyme inhibition assays using synthetic peptide substrates. J. dairy res. 73:

[101] Doig MT, Smiley JW (1993) Direct injection assay of angiotensin-converting enzyme by high-performance liquid-chromatography using a shielded hydrophobic phase column.

[102] Hyun C, Shin H (2000) Utilization of bovine blood plasma proteins for the production of angiotensin I converting enzyme inhibitory peptides, Process biochem. 36: 65-71. [103] Holmquist B, Bunning P, Riordan J (1979) Continuous spectrophotometric assay for

[104] Carmel A, Yaron A (1978) An intramolecularly quenched fuorescent tripeptide as a fuorogenic substrate of angiotensin-I-converting enzyme and of bacterial dipeptidyl

[105] Sentandreu MA, Toldrá F (2006) A rapid, simple and sensitive fuorescence method for

[106] Cao G, Prior RL (1998) Comparison of different analytical methods for assessing total

[107] Re R, Pellegrini N, Proteggente A, Pannala A, Yang M, Rice- Evans C (1999) Antioxidant activity applying an improved ABTS radical cation decolorization assay.

[108] Verma S, Buchanan MR, Anderson TJ (2003) Endothelial function testing as a

[109] Liu RH, Finley J (2005) Potential cell culture models for antioxidant research. J. agric.

[110] Elisia I, Kitts DD (2008) Anthocyanins inhibit peroxy radical-induced apoptosis in

[111] Wolfe KL, Liu RH (2007) Cellular antioxidant activity (CAA) assay for assessing antioxidants, foods, and dietary supplements. J. agric. food chem.. 55: 8896–8907. [112] Fitzgerald RJ, Murray BA, Walsh DJ (2004) Hypotensive peptides from milk proteins.

angiotensin converting enzyme. Anal. biochem. 95: 540-548.

the assay of angiotensin-I converting enzyme. Food chem. 97: 546.

antioxidant capacity of human serum. Clin. chem. 44: 1309–1315.

biomarker of vascular disease. Circulation 108: 2054-9.

Caco-2 cells. Mol. cell. biochem. 312: 139–145.


[127] Marambe HK, Shand PJ, Wanasundara, JPD (2011) Release of angiotensin I-converting enzyme inhibitory peptides from flaxseed (Linum usitatissimum L.) protein under simulated gastrointestinal digestion. J. agric. food chem. 59: 9596-9604.

Antihypertensive Properties of Plant Protein Derived Peptides 179

[141] Park SY, Lee JS, Baek HH, Lee HG (2010) Purification and characterization of antioxidant peptides from soy protein hydrolysate. J. food biochem.34:120-134 [142] Hsu GW, Lu Y, Chang S, Hsu S (2011) Antihypertensive effect of mung bean sprout

[143] Li H, Prairie N, Udenigwe CC, Adebiyi AP, Tappia PS, Aukema HM, Jones PJH, Aluko RE (2011) Blood pressure lowering effect of a pea protein hydrolysate in

[144] Vermeirssen V, Augustijns P, Morel N, Van Camp J, Opsomer A, Verstraete W (2005) *In vitro* intestinal transport and antihypertensive activity of ACE inhibitory pea and

[145] Lee NY, Kim Y, Choi I, Cho S, Hyun J, Choi J, Park K, Kim K, Lee M (2010) Biological activity of barley (Hordeum vulgare L.) and barley byproduct extracts. Food sci.

[146] Aludatt MH, Ereifej K, Abou-zaitoun A, Al-Rababah M, Almajwal A, Rababeh T, Yang W (2012) Oxidant, anti-diabetic and anti-hypertensive effects of extracted phenolics and

[148] Ying Hu, Stromeck A, Loponen J, Lopes-Lutz D, Schieber A, Gänzle MG (2011) LC-MS/MS Quantification of bioactive angiotensin I-converting enzyme inhibitory peptides

[149] Cheung IWY, Nakayama S, Hsu MNK, Samaranayaka AGP, Li-Chan ECY (2009) Angiotensin-I converting enzyme inhibitory activity of hydrolysates from oat (Avena sativa) proteins by *in silico* and *in vitro* analyses. J agric. food chem. 57: 9234–9242. [150] Kong X, Zhou H, Hua Y (2008) Preparation and antioxidant activity of wheat gluten hydrolysates (WGHs) using ultrafiltration membranes. J sci. food agric. 88: 920–926. [151] Zhang J, Zhang H, Wang L, Guo X, Wang X, Yao H (2010) Isolation and identification of antioxidative peptides from rice endosperm protein enzymatic hydrolysate by consecutive chromatography and MALDI-TOF/TOF MS/MS. Food

[152] Li GH, Qu MR, Wan JZ, You JM (2007) Antihypertensive effect of rice protein hydrolysate with *in vitro* angiotensin I-converting enzyme inhibitory activity in

[153] Matsui T, Li CH, Tanaka T, Maki T, Osajima Y, Matsumoto K (2000) Depressor effect of wheat germ hydrolysate and its novel angiotensin I–converting enzyme inhibitory peptide, Ile-Val-Tyr, and the metabolism in rat and human plasma. Biol. pharm. bull.

[154] De Leo F, Panarese S, Gallerani R, Ceci LR (2009) Angiotensin converting enzyme (ACE) inhibitory peptides: production and implementation of functional food. Curr.

spontaneously hypertensive rats. Asia pa. j. clin. nutr. 16: 275-280.

hydrolyzed peptides from barley protein fraction. Int. j. food prop. 15: 781-795. [147] Iwaniak A, Dziuba B (2009) Motifs with potential physiological activity in food proteins –Biopep database. Acta Scientiarum Polonorum: Technologia Alimentaria 8:

extracts in spontaneously hypertensive rats. J. food biochem. 35: 278-288.

hypertensive rats and humans. J. agric. food chem. 59: 9854-9860.

in rye malt sourdoughs. *J. agric. food chem. 59:* 11983–11989.

whey digests. Int. j. food sci. nutr. 56: 415-430.

biotechnol. 19: 785-791.

chem. 119: 226-234.

23: 427–431.

pharm. design 15: 3622-3643.

59-85.


[141] Park SY, Lee JS, Baek HH, Lee HG (2010) Purification and characterization of antioxidant peptides from soy protein hydrolysate. J. food biochem.34:120-134

178 Bioactive Food Peptides in Health and Disease

281-285.

438.

1074.

food res. technol. 229: 915–921.

agric. food chem. 58: 4712–4718.

[127] Marambe HK, Shand PJ, Wanasundara, JPD (2011) Release of angiotensin I-converting enzyme inhibitory peptides from flaxseed (Linum usitatissimum L.) protein under

[128] Yamada Y, Iwasaki M, Usui H, Ohinata K, Marczak ED, Lipkowski, AW, Yoshikawa M (2010) Rapakinin, an anti-hypertensive peptide derived from rapeseed protein, dilates mesenteric artery of spontaneously hypertensive rats via the prostaglandin IP

[129] Marczak, E. D., Ohinata, K., Lipkowski, A. W., & Yoshikawa, M. (2006). Arg-Ile-Tyr (RIY) derived from rapeseed protein decreases food intake and gastric emptying after

[130] Yamada Y, Ohinata K, Lipkowski AW, Yoshikawa M (2011) Rapakinin, Arg-Ile-Tyr, derived from rapeseed napin, shows anti-opioid activity via the prostaglandin IP receptor followed by the cholecystokinin CCK2 receptor in mice. Peptides 32:

[131] Aklloglu HG, Karakaya, S (2009) Effects of heat treatment and *in vitro* digestion on the angiotensin converting enzyme inhibitory activity of some legume species. Eur.

[132] Vermeirssen V, Van Camp J, Decroos K, Van Wijmelbeke L, Verstraete W (2003) The impact of fermentation and *in vitro* digestion on the formation of angiotensin-Iconverting enzyme Inhibitory activity from pea and whey protein. J. dairy. sci. 86: 429-

[133] Li H, Aluko RF (2010) Identification and inhibitory properties of multifunctional

[134] Lo WMY, Farnworth ER, Li-Chan ECY (2006) Angiotensin I-converting enzyme inhibitory activity of soy protein digests in a dynamic model system simulating the

[135] Pownall TL, Udenigwe CC, Aluko RE (2010) Amino acid composition and antioxidant properties of pea seed (Pisum sativum L.) enzymatic protein hydrolysate fractions. J.

[136] Pownall TL, Udenigwe CC, Aluko RE (2011) Effects of cationic property on the *in vitro* antioxidant activities of pea protein hydrolysate fractions. Food res. int. 44: 1069-

[137] Li Y, Jiang B, Zhang T, Mu W, Liu J (2008) Antioxidant and free radical-scavenging

[138] Liu JR, Chen MJ, Lin CW (2005) Antimutagenic and antioxidant properties of milk-

[139] Zhang L, Lia J, Zhoub K (2010) Chelating and radical scavenging activities of soy protein hydrolysates prepared from microbial proteases and their effect on meat lipid

[140] Chen HM, Muramoto K, Yamauchi F. 1995. Structural analysis of antioxidant peptides

activities of chickpea protein hydrolysate (CPH). Food chem. 106: 444-450.

peptides from pea protein hydrolysate. J. agric. food chem. 58: 11471-11476.

simulated gastrointestinal digestion. J. agric. food chem. 59: 9596-9604.

receptor followed by CCK1 receptor. Peptides 31: 909-914.

oral administration in mice. Peptides 27: 2065-2068.

upper gastrointestinal tract. J. food. sci. 71: S231–S237.

kefir and soymilk-kefir. J. agric. food chem 53: 2467-2474.

peroxidation. Bioresource technology, 101: 2084-2089.

from soybean b-conglycinin. J Agric Food Chem 43:574-578.


[155] Eriksen EK, Holm H, Jensen E, Aaboe R, Devold TG, Jacobsen M, Vegarud GE (2010) Different digestion of caprine whey proteins by human and porcine gastrointestinal enzymes. Br. j. nutr. 104: 374-381.

Antihypertensive Properties of Plant Protein Derived Peptides 181

[170] Kies AK, Van Der Pijl P (2012) Peptide availability. USA Patent Application

[171] Nestor JJ Jr. (2009) The medicinal chemistry of peptides. Curr. med. chem. 16: 4399-

[172] Yamada Y, Matoba N, Usui H, Onishi K, Yoshikawa M (2002) Design of a highly potent anti-hypertensive peptide based on ovokinin(2-7). Biosci. biotechn. biochem. 66:

[173] Matoba N, Yamada Y, Usui H, Nakagiri R, Yoshikawa H (2001) Designing potent derivatives of ovokinin(2-7), an anti-hypertensive peptide derived from ovalbumin.

[174] Gomez-Guillen MC, Gimenez B, Lopez-Caballero ME, Montero MP (2011) Functional and bioactive properties of collagen and gelatin from alternative sources: A review.

[175] Liu D, Liang H, Han C, Lin S, Chen C, Fan M, Hou W (2009) Feeding trial of instant food containing lyophilised yam powder in hypertensive subjects. J. sci. food agric. 89:

[176] Peach MJ (1997) Renin-angiotensin system: biochemistry and mechanism of action.

[177] Gradman A, Schmieder R, Lins R, Nussberger J, Chiang Y, Bedigian, M (2005) Aliskiren, a novel orally effective renin inhibitor, provides dose-dependent antihypertensive efficacy and placebo-like tolerability in hypertensive patients.

[178] Udenigwe CC, Aluko RE (2012) Multifunctional cationic peptide fractions from

[179] Erdmann K, Grosser N, Schipporeit K, Schroeder H (2006) The ACE inhibitory dipeptide Met-Tyr diminishes free radical formation in human endothelial cells via

[180] Touyz R (2004) Reactive oxygen species, vascular oxidative stress, and redox signaling

[181] Akpaffiong M, Taylor A (1998) Antihypertensive and vasodilator actions of

[182] Marambe PWMLHK, Shand PJ, Wanasundara JPD (2008) An in-vitro investigation of selected biological activities of hydrolysed flaxseed (Linum usitatissimum L.) proteins.

[183] Fitzgerald C, Gallagher E, Tasdemir D, Hayes M (2011) Heart health peptides from macroalgae and their potential use in functional foods. J. agric. food chem. 59: 6829-

[184] Nakahara T, Sano A, Yamaguchi H, Sugimoto K, Chikata H, Kinoshita E, Uchida R (2010) Antihypertensive effect of peptide-enriched soy sauce-like seasoning and identification of its angiotensin I-converting enzyme inhibitory substances. J. agric. food

in hypertension - What is the clinical significance? Hypertension 44: 248-252.

antioxidants in spontaneously hypertensive rats. Am. j. hyp. 11: 1450-1460.

flaxseed protein hydrolysates. Plant food hum. nutr. 67: 1-9.

induction of heme oxygenase-1 and ferritin. J. nutr. 136: 2148-2152.

20120040895.

1213-1217.

138-143.

6836.

chem.58: 821-827.

Biosci. biotechn. biochem. 65: 736-739.

Food hydrocolloids 25: 1813-1827.

Physiol. rev. 57: 313-370.

Circulation 111: 1012-1018.

J. am. oil chem. soc. 85: 1155-1164.

4418.


[170] Kies AK, Van Der Pijl P (2012) Peptide availability. USA Patent Application 20120040895.

180 Bioactive Food Peptides in Health and Disease

2714-2721.

329–350.

286.

enzymes. Br. j. nutr. 104: 374-381.

targeting. London: Portland Press Ltd.

human intestinal cell, Caco2. Peptides 18: 681–687.

relevance of absorption models. Peptides 29: 1312–1320.

with antihypertensive properties. Peptides 30: 1848-1853.

mass spectrometry. J. chromatogr. B 830: 151-157.

proteins. J. food sci. 65: 564-569.

sci. 70: 269-277.

[155] Eriksen EK, Holm H, Jensen E, Aaboe R, Devold TG, Jacobsen M, Vegarud GE (2010) Different digestion of caprine whey proteins by human and porcine gastrointestinal

[156] Zhu L, Chen J, Tang X, Xiong YL (2008) Reducing, radical scavenging, and chelation properties of *in vitro* digests of alcalase-treated zein hydrolysate. J. agric. food chem. 56:

[157] Gardner MLG (1988) Gastrointestinal absorption of intact proteins. Annu. rev. nutr. 8:

[158] Gardner MLG (1998) Transmucosal passage on intact peptides in mammalian metabolism. In: Grimble GK, Backwell FRG, editors. Tissue utilization and clinical

[159] Shimizu M, Tsunogai M, Arai S (1997) Transepithelial transport of oligopeptides in the

[160] Foltz M, Cerstiaens A, van Meensel A, Mols R, van der Pijl PC, Duchateau GSMJE, Augustijns P (2008) The angiotensin converting enzyme inhibitory tripeptides Ile-Pro-Pro and Val-Pro-Pro show increasing permeabilities with increasing physiological

[161] van der Pijl PC, Kies AK, Ten Have GA, Duchateau GS, Deutz NE (2008) Pharmacokinetics of proline-rich tripeptides in the pig. Peptides 29: 2196–2202. [162] Foltz M, Meynen EE, Bianco V, van Platerink C, Koning TMMG, Kloek J (2007) Angiotensin converting enzyme inhibitory peptides from a lactotripeptide-enriched

[163] Quiros A, Davalos A, Lasuncion MA, Ramos M, Recio I (2008) Bioavailability of the antihypertensive peptide LHLPLP: Transepithelial flux of HLPLP. Int. dairy j. 18: 279-

[164] Quiros A, del Mar Contreras M, Ramos M, Amigo L, Recio I (2009) Stability to gastrointestinal enzymes and structure-activity relationship of beta-casein-peptides

[165] van Platerink C, Janssen H, Horsten R, Haverkamp J (2006) Quantification of ACE inhibiting peptides in human plasma using high performance liquid chromatography-

[166] Fujita H, Yokoyama K, Yoshikawa M (2000) Classification and antihypertensive activity of angiotensin I-converting enzyme inhibitory peptides derived from food

[167] Zhao Y, Li B, Dong S, Liu Z, Zhao X, Wang J, Zeng M (2009) A novel ACE inhibitory peptide isolated from Acaudina molpadioidea hydrolysate. Peptides 30: 1028-1033. [168] Muguruma M, Ahhmed AM, Katayama K, Kawahara S, Maruyama M, Nakamura T (2009) Identification of pro-drug type ACE inhibitory peptide sourced from porcine myosin B: Evaluation of its antihypertensive effects *in vivo*. Food chem. 114: 516-522. [169] Shaji J, Patole V (2008) Protein and peptide drug delivery: Oral approaches. J. pharm.

milk beverage are absorbed intact into the circulation. J. nutr. 137: 953–958.


[185] Nogata Y, Nagamine T, Sekiya K (2011) Antihypertensive effect of angiotensin Iconverting enzyme inhibitory peptides derived from wheat bran in spontaneously hypertensive rats. J. jpn. soc. food sci. technol. 58: 67-70.

**Chapter 7** 

© 2013 Betancur-Ancona et al., licensee InTech. This is an open access chapter 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.

© 2013 Betancur-Ancona et al., licensee InTech. This is a paper 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.

**Vigna Unguiculata as Source of** 

**Angiotensin-I Converting Enzyme** 

Maira R. Segura-Campos, Luis A. Chel-Guerrero and

Additional information is available at the end of the chapter

David A. Betancur-Ancona

http://dx.doi.org/10.5772/53045

**1. Introduction** 

**Inhibitory and Antioxidant Peptides** 

The frequency of lifestyle-related diseases is steadily increasing, particularly of hypertension, a risk factor for cardiovascular diseases such as coronary heart disease, peripheral arterial disease and stroke. Indeed, cardiovascular diseases are the primary cause of morbidity and mortality in Western countries, with hypertension affecting about 20% of the world's adult population [1]. Blood pressure is controlled by various regulatory factors in the body, including angiotensin I-converting enzyme (ACE-I). ACE-I (peptidyldipeptidaseA, kininase II, EC 3.4.15.1) is a zinc dipeptidylcarboxypeptidase. This membrane-bound exopeptidase is found on the plasma membranes of various cell types, including vascular endothelial cells, microvillar brush border epithelial cells and neuroepithelial cells. It is thought to be physiologically important. The primary activity of ACE-I is to cleave broad specificity free carboxyl group oligopeptides. Substrates containing Pro at the P1' position and Asp or Glu at P2' are resistant to ACE-I. However, on certain substrates ACE-I can also function as an endopeptidase or a tripeptidylcarboxypeptidase. With ACE-I, endopeptidase activity is observed on substrates having amidated carboxyl groups where the enzyme can cleave a C-terminal dipeptide amide and/or a C-terminal tripeptide amide [2]. ACE-I is responsible for converting angiotensin I (Ang I) to the powerful vasoconstrictor angiotensin II (Ang II) and inactivating the vasodilator peptide bradykinin (BK) by removal of C-terminal dipeptides [3]. In a functional sense, therefore, the enzymatic actions of ACE-I potentially cause increased vasoconstriction and decreased vasodilation. ACE-I has attracted interest for development of orally-active ACE-I inhibitors to treat hypertension due to its central role in vasoactive peptide metabolism. Inhibition of ACE-I prevents conversion of Ang I into Ang II, making it becomes one of the most effective

[186] Nogata Y, Nagamine T, Yanaka M, Ohta, H (2009) Angiotensin I converting enzyme inhibitory peptides produced by autolysis reactions from wheat bran. J. agric. food chem. 57: 6618-6622.

**Chapter 7** 
