**Vigna Unguiculata as Source of Angiotensin-I Converting Enzyme Inhibitory and Antioxidant Peptides**

Maira R. Segura-Campos, Luis A. Chel-Guerrero and David A. Betancur-Ancona

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/53045

### **1. Introduction**

182 Bioactive Food Peptides in Health and Disease

chem. 57: 6618-6622.

[185] Nogata Y, Nagamine T, Sekiya K (2011) Antihypertensive effect of angiotensin Iconverting enzyme inhibitory peptides derived from wheat bran in spontaneously

[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

hypertensive rats. J. jpn. soc. food sci. technol. 58: 67-70.

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

© 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.

methods for suppressing increases in blood pressure [4]. Since discovery of ACE-I inhibitors in snake venom, extensive research has been done on synthesizing ACE-I inhibitors such as captopril, enalapril, alacepril and lisinopril, all currently in use for treatment of hypertension and heart failure in humans. However, these synthetic drugs occasionally produce side effects such as cough, taste alterations and skin rashes. Interest has consequently increased in natural ACE-I inhibitors as safer and lower cost alternatives to synthetic ones [5].

Vigna Unguiculata as Source of Angiotensin-I Converting Enzyme Inhibitory and Antioxidant Peptides 185

extract has been used as substrates for production of hydrolysates with functional and/or nutritional properties better than the original extract [10]. Bioactive peptides with ACE-I inhibitory activity has been isolated from protein hydrolysates from a number of animal and vegetable sources. Vegetable origin proteins are of particular interest, and legumes are especially promising due to their high protein content and diverse physiological activities in the human organism. Extensive hydrolysis of *V. unguiculata* protein concentrates with commercial and digestives enzymes could therefore produce a number of peptides with a myriad of potential applications; for example, as natural-source therapeutic agents in medical treatments and/or as an ingredient in functional foods. Taking this into account, the aim of the present study was to modify enzymatically protein concentrates of *V. unguiculata*, evaluate the ACE-I inhibitory and antioxidant potential of the hydrolysates and relate the

*V. unguiculata* seeds were obtained from the February 2007 harvest in Yucatan state, Mexico. Reagents were of analytical grade and purchased from J.T. Baker (Phillipsburg, NJ, USA), Sigma Chemical Co. (St. Louis, MO, USA), Merck (Darmstadt, Germany) and Bio-Rad Laboratories, Inc. (Hercules, CA, USA). Alcalase® 2.4L FG and Flavourzyme® 500MG

A single extraction was performed with 6 kg of cowpea seeds*.* Impurities and damaged seeds were removed, and sound seeds milled in a Mykros impact mill (Industrial Machinery, Monterrey, Mexico) until passing through a 20-mesh screen (0.85 mm) followed by milling in a Cyclotec 1093 (Tecator, Sweden) mill until passing through a 60-mesh screen (0.24 mm). The resulting flour was processed using the wet fractionation method of Betancur-Ancona [11]. Briefly, whole flour was suspended in distilled water at a 1:6 (w/v) ratio, pH was adjusted to 11.0 with 1 M NaOH, and the dispersion was stirred for 1 h at 0.178 x *g* with a mechanical agitator (Caframo Rz-1, Heidolph Schwabach, Germany). This suspension was wet-milled with a Kitchen-Aid® food processor, and the fiber solids were separated from the starch and protein mix by straining through 80- and 150-mesh sieves and washing the residue five times with distilled water. The protein-starch suspension was allowed to sediment for 30 min at room temperature to recover the starch and protein fractions. The pH of the separated solubilized proteins was adjusted to the isoelectric point (4.5) with 1 N HCl. The suspension was then centrifuged at 1317 x *g* for 12 min (Mistral 3000i, Curtin Matheson Sci.), the

enzymes were purchased from Novo Laboratories (Copenhagen, Denmark).

supernatants were discarded, and the precipitates were freeze-dried until use.

Hydrolysis of the protein extract was done using a totally randomized design with the treatments being the enzymatic system applied: Alcalase® 2.4L FG; Flavourzyme® 500MG; or

biological activity to their amino acid compositions.

**2. Material and methods** 

**2.1. Materials** 

**2.2. Protein isolates** 

**2.3. Enzymatic hydrolysis** 

Antioxidant deficiency also has been implicated in the occurrence of hypertension and other degenerative diseases. Reactive oxygen species (ROS) such as the superoxide anion radical (O-2), hydrogen peroxide and hydroxyl radicals (●OH) are physiological metabolites formed as result of respiration in aerobic organisms. ROS are very unstable, and react rapidly with other substances including DNA, membrane lipids and proteins. Oxidative stress is produced by an imbalance between oxidizing species and natural antioxidants in the body, and has been associated with aging, cell apoptosis and severe diseases such as cancer, Parkinson, Alzheimer, and cardiovascular disorders [6]. Epidemiological studies have demonstrated an inverse association among intake of antioxidants from fruits and vegetables, and morbidity and mortality due to coronary heart diseases and cancer. In response, researchers are searching for natural antioxidants in food that may protect the body from free radicals and retard the evolution of many chronic diseases [7].

In recent years, food proteins have gained increasing value due to the rapidly expanding knowledge about physiologically active peptides. Peptides from various dietary sources have been shown to have clearly positive effects on health by functioning as antihypertensives, antioxidants, anticarcinogens, antimicrobials and anticariogenics, among others. These properties have led to their labeling as functional or biologically-active (i.e. bioactive) peptides. Bioactive peptides may be encrypted within the amino acid sequence of a larger protein. These peptides usually consist of 3-20 amino acids and are released from the original protein after degradation [8]. The most common way to produce bioactive peptides is through enzymatic hydrolysis of whole protein molecules. After enzymatic processing, amino acid sequences that were inactive in the core of the source protein are released and can exercise special properties. Many of the known bioactive peptides have been produced using gastrointestinal enzymes, usually pepsin and trypsin. Other digestive enzymes and different enzyme combinations of proteinases-including Alcalase®, chymotrypsin, pancreatin, pepsin and thermolysin have also been utilized to generate bioactive peptides from various proteins [9].

Continued population growth worldwide, consequent food resource shortages in developing countries, and the health risks associated with excessive animal protein (and saturated fats) intake has led researchers to search for new sources of proteins from nonconventional raw materials. Legumes are cultivated worldwide and constitute an excellent protein source (protein content =20-30%). Cowpea (*Vigna unguiculata*) is a major legume crop worldwide, particularly in tropical and subtropical areas such as southeast Mexico. It serves as a major dietary protein source in both human and animal diets, and its protein content makes it a good raw material for preparation of protein extracts and hydrolysates. Protein extract has been used as substrates for production of hydrolysates with functional and/or nutritional properties better than the original extract [10]. Bioactive peptides with ACE-I inhibitory activity has been isolated from protein hydrolysates from a number of animal and vegetable sources. Vegetable origin proteins are of particular interest, and legumes are especially promising due to their high protein content and diverse physiological activities in the human organism. Extensive hydrolysis of *V. unguiculata* protein concentrates with commercial and digestives enzymes could therefore produce a number of peptides with a myriad of potential applications; for example, as natural-source therapeutic agents in medical treatments and/or as an ingredient in functional foods. Taking this into account, the aim of the present study was to modify enzymatically protein concentrates of *V. unguiculata*, evaluate the ACE-I inhibitory and antioxidant potential of the hydrolysates and relate the biological activity to their amino acid compositions.

## **2. Material and methods**

#### **2.1. Materials**

184 Bioactive Food Peptides in Health and Disease

synthetic ones [5].

(O-

methods for suppressing increases in blood pressure [4]. Since discovery of ACE-I inhibitors in snake venom, extensive research has been done on synthesizing ACE-I inhibitors such as captopril, enalapril, alacepril and lisinopril, all currently in use for treatment of hypertension and heart failure in humans. However, these synthetic drugs occasionally produce side effects such as cough, taste alterations and skin rashes. Interest has consequently increased in natural ACE-I inhibitors as safer and lower cost alternatives to

Antioxidant deficiency also has been implicated in the occurrence of hypertension and other degenerative diseases. Reactive oxygen species (ROS) such as the superoxide anion radical

In recent years, food proteins have gained increasing value due to the rapidly expanding knowledge about physiologically active peptides. Peptides from various dietary sources have been shown to have clearly positive effects on health by functioning as antihypertensives, antioxidants, anticarcinogens, antimicrobials and anticariogenics, among others. These properties have led to their labeling as functional or biologically-active (i.e. bioactive) peptides. Bioactive peptides may be encrypted within the amino acid sequence of a larger protein. These peptides usually consist of 3-20 amino acids and are released from the original protein after degradation [8]. The most common way to produce bioactive peptides is through enzymatic hydrolysis of whole protein molecules. After enzymatic processing, amino acid sequences that were inactive in the core of the source protein are released and can exercise special properties. Many of the known bioactive peptides have been produced using gastrointestinal enzymes, usually pepsin and trypsin. Other digestive enzymes and different enzyme combinations of proteinases-including Alcalase®, chymotrypsin, pancreatin, pepsin and thermolysin have also been utilized to generate

Continued population growth worldwide, consequent food resource shortages in developing countries, and the health risks associated with excessive animal protein (and saturated fats) intake has led researchers to search for new sources of proteins from nonconventional raw materials. Legumes are cultivated worldwide and constitute an excellent protein source (protein content =20-30%). Cowpea (*Vigna unguiculata*) is a major legume crop worldwide, particularly in tropical and subtropical areas such as southeast Mexico. It serves as a major dietary protein source in both human and animal diets, and its protein content makes it a good raw material for preparation of protein extracts and hydrolysates. Protein

body from free radicals and retard the evolution of many chronic diseases [7].

bioactive peptides from various proteins [9].

2), hydrogen peroxide and hydroxyl radicals (●OH) are physiological metabolites formed as result of respiration in aerobic organisms. ROS are very unstable, and react rapidly with other substances including DNA, membrane lipids and proteins. Oxidative stress is produced by an imbalance between oxidizing species and natural antioxidants in the body, and has been associated with aging, cell apoptosis and severe diseases such as cancer, Parkinson, Alzheimer, and cardiovascular disorders [6]. Epidemiological studies have demonstrated an inverse association among intake of antioxidants from fruits and vegetables, and morbidity and mortality due to coronary heart diseases and cancer. In response, researchers are searching for natural antioxidants in food that may protect the

*V. unguiculata* seeds were obtained from the February 2007 harvest in Yucatan state, Mexico. Reagents were of analytical grade and purchased from J.T. Baker (Phillipsburg, NJ, USA), Sigma Chemical Co. (St. Louis, MO, USA), Merck (Darmstadt, Germany) and Bio-Rad Laboratories, Inc. (Hercules, CA, USA). Alcalase® 2.4L FG and Flavourzyme® 500MG enzymes were purchased from Novo Laboratories (Copenhagen, Denmark).

#### **2.2. Protein isolates**

A single extraction was performed with 6 kg of cowpea seeds*.* Impurities and damaged seeds were removed, and sound seeds milled in a Mykros impact mill (Industrial Machinery, Monterrey, Mexico) until passing through a 20-mesh screen (0.85 mm) followed by milling in a Cyclotec 1093 (Tecator, Sweden) mill until passing through a 60-mesh screen (0.24 mm). The resulting flour was processed using the wet fractionation method of Betancur-Ancona [11]. Briefly, whole flour was suspended in distilled water at a 1:6 (w/v) ratio, pH was adjusted to 11.0 with 1 M NaOH, and the dispersion was stirred for 1 h at 0.178 x *g* with a mechanical agitator (Caframo Rz-1, Heidolph Schwabach, Germany). This suspension was wet-milled with a Kitchen-Aid® food processor, and the fiber solids were separated from the starch and protein mix by straining through 80- and 150-mesh sieves and washing the residue five times with distilled water. The protein-starch suspension was allowed to sediment for 30 min at room temperature to recover the starch and protein fractions. The pH of the separated solubilized proteins was adjusted to the isoelectric point (4.5) with 1 N HCl. The suspension was then centrifuged at 1317 x *g* for 12 min (Mistral 3000i, Curtin Matheson Sci.), the supernatants were discarded, and the precipitates were freeze-dried until use.

#### **2.3. Enzymatic hydrolysis**

Hydrolysis of the protein extract was done using a totally randomized design with the treatments being the enzymatic system applied: Alcalase® 2.4L FG; Flavourzyme® 500MG; or a sequential system using pepsin from porcine gastric mucosa (Sigma, P7000-100G) and pancreatin from porcine pancreas (Sigma, P3292-100G). The response variable was degree of hydrolysis (DH).

Vigna Unguiculata as Source of Angiotensin-I Converting Enzyme Inhibitory and Antioxidant Peptides 187

ACE-I inhibitory activity in the hydrolysate and its purified peptide fractions was analyzed following Hayakari et al. [17]. ACE-I hydrolyzes hippuryl-L-histidyl-L-leucine (HHL) to yield hippuric acid and His-Leu. This method relies on the colorimetric reaction of hippuric acid with 2,4,6-trichloro-s-triazine (TT) in a 0.5 mL incubation mixture containing 40 μmol potassium phosphate buffer (pH 8.3), 300 μmol sodium chloride, 40 μmol 3% HHL in potassium phosphate buffer (pH 8.3) and 100 mU/mL ACE-I. The mixture was incubated at 37ºC for 45 min and then reaction terminated by addition of TT (3% v/v) in dioxane and 3 ml of 0.2 M potassium phosphate buffer (pH 8.3). After centrifuging the reaction mixture at 10,000 x *g* for 10 min, enzymatic activity was determined in the supernatant by measuring absorbance at 382 nm. All runs were performed in triplicate. ACE-I inhibitory activity was quantified by a regression analysis of ACE-I inhibitory activity (%) versus peptide concentration and defined as an IC50 value, that is, the peptide concentration in (μg protein/mL) required to produce 50% ACE-I inhibition under the described conditions.

2,2'azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) radical cation (ABTS●+) was produced by reacting ABTS with potassium persulfate following Pukalskas et al. [18]. To prepare the stock solution, ABTS was dissolved at a 2 mM concentration in 50 mL phosphatebuffered saline (PBS) prepared from 4.0908 g NaCl, 0.1347 g KH2PO4, 0.7098 g Na2HPO4, and 0.0749 g KCl dissolved in 500 mL ultrapure water. If pH was lower than 7.4, it was adjusted with NaOH. A 70 mM K2S4O8 solution in ultrapure water was prepared. ABTS●+ radical was produced by reacting 10 mL of ABTS stock solution with 40 L K2S4O8 solution and allowing the mixture to stand in darkness at room temperature for 16-17 h before use. The radical was

stable in this form for more than 2 days when stored in darkness at room temperature.

Antioxidant compound content in the hydrolysates and their UF peptide fractions was analyzed by diluting the ABTS●+ solution with PBS to an absorbance of 0.800 ± 0.030 AU at 734 nm. After adding 990 L of diluted ABTS●+ solution (A734 nm= 0.800 ± 0.030) to 10 L antioxidant compound or Trolox standard (final concentration 0.5-3.5 mM) in PBS, absorbance was read at ambient temperature exactly 6 min after initial mixing. All analyses were run in triplicate. The percentage decrease in absorbance at 734 nm was calculated and plotted as a function of the concentration of Trolox for the standard reference data. The radical scavenging activity of the tasted samples, expressed as inhibition percentage, was

% Inhibition= [(AB-AA)/AB] x 100 Where AB was the absorbance of the blank sample (t=0), and AA was the absorbance of

Trolox equivalent antioxidant coefficient (TEAC) was quantified by a regression analysis of

TEAC = (%IM – b)/m

% Inhibition versus Trolox concentration using the following formula:

**2.6. ACE-I inhibitory activity** 

**2.7. Antioxidant activity by ABTS assay** 

calculated by the following formula:

sample with antioxidant after 6 min.

Hydrolysis was done under controlled conditions (temperature, pH and stirring) in a 1000 mL reaction vessel equipped with a stirrer, thermometer and a pH electrode. Hydrolysis with Alcalase® and Flavourzyme® was done according to Pedroche et al. [12]. Protein extracts were suspended in distilled water to produce a 4% (w/v) protein solution. This solution was equilibrated at optimum temperature and pH for each protease before adding the respective enzyme. Protease was then added to the solution at a ratio of 0.3 UA/g for Alcalase® and 50 UAPL/g for Flavourzyme®. Hydrolysis conditions were 90 min at 50°C for both enzymes, and pH 8.0 for Alcalase® and pH 7.0 for Flavourzyme®. The pH was kept constant by adding 1.0 M NaOH during hydrolysis. Hydrolysis with the sequential pepsinpancreatin system was done with a pH-stat method for 90 min: pre-digestion with pepsin for 45 min followed by incubation with pancreatin for 45 min. Hydrolysis parameters were substrate concentration 4%; enzyme/substrate ratio 1:10; pH 2 for pepsin; pH 7.5 for pancreatin; and 37°C [13, 14]. In all three treatments the reaction was stopped by heating to 80°C for 20 min, followed by centrifugation at 9,880 x *g* for 20 min to remove the insoluble portion.

## **2.4. Degree of hydrolysis**

DH was calculated by calculating free amino groups with o-phthaldialdehyde [15]: DH= h/htot \*100, where htot is the total number of peptide bonds per protein equivalent, and h is the number of hydrolyzed bonds. The htot factor is dependent on raw material amino acid composition.

## **2.5. Hydrolysate fractionation by ultrafiltration**

Following Cho et al. [16], the hydrolysate was fractionated by ultrafiltration using a high performance ultrafiltration cell (Model 2000, Millipore, Inc., Marlborough, MA, USA). Five fractions were prepared using four molecular weight cut-off membranes: 1 kDa, 3 kDa, 5 kDa and 10 kDa. Soluble fractions prepared by centrifugation were passed through the membranes stating with the largest Molecular Weight Cut off (MWCO) membrane cartridge (10 kDa). The retentate and permeate were collected separately, and the retentate recirculated into the feed until maximum permeate yield was reached, as indicated by a decreased permeate flow rate. The permeate from the 10 kDa membrane was then filtered through the 5 kDa membrane with recirculation until maximum permeate yield was reached. The 5 kDa permeate was then processed with the 3 kDa membrane and the 3 kDa permeate with the 1 kDa membrane. This process minimized contamination of the larger molecular weight fractions with smaller molecular weight fractions while producing enough retentates and permeates for the following analyses. The five ultrafiltered peptide fractions were designated as >10 kDa (10 kDa retentate); 5-10 kDa (10 kDa permeate-5 kDa retentate); 3-5 kDa (5 kDa permeate- 3 kDa retentate); 1-3 kDa (3 kDa permeate-1 kDa retentate); and <1 kDa (1 kDa permeate).

## **2.6. ACE-I inhibitory activity**

186 Bioactive Food Peptides in Health and Disease

hydrolysis (DH).

portion.

composition.

kDa (1 kDa permeate).

**2.4. Degree of hydrolysis** 

**2.5. Hydrolysate fractionation by ultrafiltration** 

a sequential system using pepsin from porcine gastric mucosa (Sigma, P7000-100G) and pancreatin from porcine pancreas (Sigma, P3292-100G). The response variable was degree of

Hydrolysis was done under controlled conditions (temperature, pH and stirring) in a 1000 mL reaction vessel equipped with a stirrer, thermometer and a pH electrode. Hydrolysis with Alcalase® and Flavourzyme® was done according to Pedroche et al. [12]. Protein extracts were suspended in distilled water to produce a 4% (w/v) protein solution. This solution was equilibrated at optimum temperature and pH for each protease before adding the respective enzyme. Protease was then added to the solution at a ratio of 0.3 UA/g for Alcalase® and 50 UAPL/g for Flavourzyme®. Hydrolysis conditions were 90 min at 50°C for both enzymes, and pH 8.0 for Alcalase® and pH 7.0 for Flavourzyme®. The pH was kept constant by adding 1.0 M NaOH during hydrolysis. Hydrolysis with the sequential pepsinpancreatin system was done with a pH-stat method for 90 min: pre-digestion with pepsin for 45 min followed by incubation with pancreatin for 45 min. Hydrolysis parameters were substrate concentration 4%; enzyme/substrate ratio 1:10; pH 2 for pepsin; pH 7.5 for pancreatin; and 37°C [13, 14]. In all three treatments the reaction was stopped by heating to 80°C for 20 min, followed by centrifugation at 9,880 x *g* for 20 min to remove the insoluble

DH was calculated by calculating free amino groups with o-phthaldialdehyde [15]: DH= h/htot \*100, where htot is the total number of peptide bonds per protein equivalent, and h is the number of hydrolyzed bonds. The htot factor is dependent on raw material amino acid

Following Cho et al. [16], the hydrolysate was fractionated by ultrafiltration using a high performance ultrafiltration cell (Model 2000, Millipore, Inc., Marlborough, MA, USA). Five fractions were prepared using four molecular weight cut-off membranes: 1 kDa, 3 kDa, 5 kDa and 10 kDa. Soluble fractions prepared by centrifugation were passed through the membranes stating with the largest Molecular Weight Cut off (MWCO) membrane cartridge (10 kDa). The retentate and permeate were collected separately, and the retentate recirculated into the feed until maximum permeate yield was reached, as indicated by a decreased permeate flow rate. The permeate from the 10 kDa membrane was then filtered through the 5 kDa membrane with recirculation until maximum permeate yield was reached. The 5 kDa permeate was then processed with the 3 kDa membrane and the 3 kDa permeate with the 1 kDa membrane. This process minimized contamination of the larger molecular weight fractions with smaller molecular weight fractions while producing enough retentates and permeates for the following analyses. The five ultrafiltered peptide fractions were designated as >10 kDa (10 kDa retentate); 5-10 kDa (10 kDa permeate-5 kDa retentate); 3-5 kDa (5 kDa permeate- 3 kDa retentate); 1-3 kDa (3 kDa permeate-1 kDa retentate); and <1 ACE-I inhibitory activity in the hydrolysate and its purified peptide fractions was analyzed following Hayakari et al. [17]. ACE-I hydrolyzes hippuryl-L-histidyl-L-leucine (HHL) to yield hippuric acid and His-Leu. This method relies on the colorimetric reaction of hippuric acid with 2,4,6-trichloro-s-triazine (TT) in a 0.5 mL incubation mixture containing 40 μmol potassium phosphate buffer (pH 8.3), 300 μmol sodium chloride, 40 μmol 3% HHL in potassium phosphate buffer (pH 8.3) and 100 mU/mL ACE-I. The mixture was incubated at 37ºC for 45 min and then reaction terminated by addition of TT (3% v/v) in dioxane and 3 ml of 0.2 M potassium phosphate buffer (pH 8.3). After centrifuging the reaction mixture at 10,000 x *g* for 10 min, enzymatic activity was determined in the supernatant by measuring absorbance at 382 nm. All runs were performed in triplicate. ACE-I inhibitory activity was quantified by a regression analysis of ACE-I inhibitory activity (%) versus peptide concentration and defined as an IC50 value, that is, the peptide concentration in (μg protein/mL) required to produce 50% ACE-I inhibition under the described conditions.

#### **2.7. Antioxidant activity by ABTS assay**

2,2'azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) radical cation (ABTS●+) was produced by reacting ABTS with potassium persulfate following Pukalskas et al. [18]. To prepare the stock solution, ABTS was dissolved at a 2 mM concentration in 50 mL phosphatebuffered saline (PBS) prepared from 4.0908 g NaCl, 0.1347 g KH2PO4, 0.7098 g Na2HPO4, and 0.0749 g KCl dissolved in 500 mL ultrapure water. If pH was lower than 7.4, it was adjusted with NaOH. A 70 mM K2S4O8 solution in ultrapure water was prepared. ABTS●+ radical was produced by reacting 10 mL of ABTS stock solution with 40 L K2S4O8 solution and allowing the mixture to stand in darkness at room temperature for 16-17 h before use. The radical was stable in this form for more than 2 days when stored in darkness at room temperature.

Antioxidant compound content in the hydrolysates and their UF peptide fractions was analyzed by diluting the ABTS●+ solution with PBS to an absorbance of 0.800 ± 0.030 AU at 734 nm. After adding 990 L of diluted ABTS●+ solution (A734 nm= 0.800 ± 0.030) to 10 L antioxidant compound or Trolox standard (final concentration 0.5-3.5 mM) in PBS, absorbance was read at ambient temperature exactly 6 min after initial mixing. All analyses were run in triplicate. The percentage decrease in absorbance at 734 nm was calculated and plotted as a function of the concentration of Trolox for the standard reference data. The radical scavenging activity of the tasted samples, expressed as inhibition percentage, was calculated by the following formula:

% Inhibition= [(AB-AA)/AB] x 100

Where AB was the absorbance of the blank sample (t=0), and AA was the absorbance of sample with antioxidant after 6 min.

Trolox equivalent antioxidant coefficient (TEAC) was quantified by a regression analysis of % Inhibition versus Trolox concentration using the following formula:

$$\text{TEAC} = (\% \text{In} - \text{b}) / \text{m}$$

Where b was the intersection and m was the slope.

#### **2.8. G-50 gel filtration chromatography**

After **f**iltration through 10, 5, 3 and 1 kDa membranes in a high performance ultrafiltration cell, 10 mL of the fraction with highest ACE-I inhibitory activity was injected into a Sephadex G-50 gel filtration column (3 cm x 79 cm) at a flow rate of 25 mL/h of 50 mM ammonium bicarbonate (pH 9.1). The resulting fractions were collected for to assay ACE-I inhibitory activity. Peptide molecular masses were determined by reference to a calibration curve created by running molecular mass markers on the Sephadex G-50 under conditions identical to those used for the test samples. Molecular mass standards were thyroglobulin (670 kDa), bovine gamma globulin (158 kDa), equine myoglobin (17 kDa), vitamin B12 (1.35 kDa) and Thr-Gln (0.25 kDa). Fractions selected for further purification of peptides were pooled and lyophilized before RP-HPLC.

Vigna Unguiculata as Source of Angiotensin-I Converting Enzyme Inhibitory and Antioxidant Peptides 189

hydrolysis data and *in vitro* ACE-I inhibitory activity. A Duncan's multiple range test was used to determine differences between treatments. All analyses were performed according

Alcalase®, Flavourzyme® and pepsin-pancreatin were used to produce extensively hydrolyzed *V. unguiculata* protein extracts. Degree of hydrolysis (DH) differed (*P*<0.05) between the enzymatic systems with values of 53.0%, 58.8%, and 35.7% for Alcalase® hydrolysate (AH), Flavourzyme® hydrolysate (FH) and Pepsin-pancreatin hydrolysate

The AH had a 53.0% DH, which is lower than reported by Vioque et al. [21] for rapeseed protein hydrolysates (60% DH) produced with a mixture of Alcalase® and Flavourzyme® during 180 min. However, this DH was higher than that reported for mung bean protein hydrolysates (20.0% DH) produced with Alcalase® for 10 h[22] and for *V. unguiculata* hydrolysates (32.3% DH) produced with Alcalase® for 60 min [10].The variation in DH observed here is probably the result of protease specificity since Alcalase® is an industrial alkaline protease produced from *Bacillus licheniformis*, the main enzyme component of which (serine endopeptidase subtilisin Carlsberg) presents broad specificity and hydrolyzes most peptide bonds, with a preference for those containing aromatic amino acid residues [23]. ACE-I prefers substrates or competitive inhibitors containing hydrophobic amino acid (aromatic or branched lateral chain) residues (Hong et al., 2005). Alcalase® is therefore very suitable for production of bioactive peptides, such as those with ACE-I inhibitory activity. According to Pedroche et al. [24] the controlled liberation of biologically active peptides from protein by enzymatic hydrolysis is one of the most promising trends concerning medical applications of the protein hydrolysates with DH higher than 10% while hydrolysates with a low DH (lower than 10%) are used for the improvement of functional properties of flours or protein isolates. Therefore, the results suggest (DH=53.0%) that *V. unguiculata* protein is an appropriate substrate for producing these bioactive peptides when

Hydrolysis with Flavourzyme® produced a *V. unguiculata* hydrolysate with 58.8% DH, somewhat higher than obtained with the Alcalase® system. A similar discrepancy has been reported for chickpea protein hydrolysates (27.0% DH) produced with Flavourzyme® for 180 min [25]. DH was higher with Flavourzyme® since it is a protease complex produced by *Aspergillus orizae,* which contains endoproteinases and exopeptidases. The fungal protease complex Flavourzyme® has a broader specificity; thus, when combined with its exopeptidase activity high DH values can be achieved, perhaps as much as 50% giving

Sequential hydrolysis with pepsin-pancreatin produced cowpea protein hydrolysates with the lowest DH (35.74%) of the three studied enzymatic systems. This DH was similar to

to Montgomery [20] and processed using the Statgraphics Plus version 5.1 software.

**3. Results and discussion** 

**3.1. Protein extract hydrolysis** 

(PPH), respectively.

hydrolyzed with Alcalase®.

mostly dipeptides in the hydrolysate.

#### **2.9. HPLC C18 chromatography**

The fractions isolated with the Sephadex G-50 column were redissolved in deionized water and injected into a preparative HPLC (Agilent, Model 1110, Agilent Technologies, Inc. Santa Clara, CA, USA) reverse-phasecolumn (C18 Hi-Pore RP-318, 250 mm x10 mm, Bio-Rad). The injection volume was 100 L, and the sample concentration was 20 mg/mL. Elution was achieved by a linear gradient of acetonitrile in water (0-30% in 50 min) containing 0.1% trifluoroacetic acid at a flow rate of 4 mL/min and 30°C [13]. Elution was monitored at 215 nm, and the resulting fractions were collected for assay of ACE-I inhibitory activity as described above.

#### **2.10. Amino acid composition**

Protein amino acid composition was determined for the hydrolysate, and the peptides were purified by ultrafiltration, gel filtration chromatography and HPLC [19]. Samples (2-4 mg protein) were treated with 4 mL of 6 mol equi/L HCl, placed in hydrolysis tubes and gassed with nitrogen at 110°C for 24 h. They were then dried in a rotavapor and suspended in 1 mol/L sodium borate buffer at pH 9.0. Amino acid derivatization was performed at 50°C using diethyl ethoxymethylenemalonate. Amino acids were separated using HPLC with a reversed-phase column (300 x 3.9 mm, Nova Pack C18, 4 mm; Waters), and a binary gradient system with 25 mmol/L sodium acetate containing (A) 0.02 g/L sodium azide at pH 6.0, and (B) acetonitrile as solvent. The flow rate was 0.9 mL/min, and the elution gradient was: time 0.0–3.0 min, linear gradient A:B (91:9) to A-B (86:14); time 3.0–13.0 min, elution with A-B (86–14); time 13.0–30.0 min, linear gradient A-B (86:14) to A-B (69:31); time 30.0– 35.0 min, elution with A-B (69:31)**.** 

#### **2.11. Statistical analysis**

All results were analyzed in triplicate using descriptive statistics with a central tendency and dispersion measures. One-way ANOVAs were performed to evaluate protein isolate hydrolysis data and *in vitro* ACE-I inhibitory activity. A Duncan's multiple range test was used to determine differences between treatments. All analyses were performed according to Montgomery [20] and processed using the Statgraphics Plus version 5.1 software.

## **3. Results and discussion**

188 Bioactive Food Peptides in Health and Disease

Where b was the intersection and m was the slope.

After **f**iltration through 10, 5, 3 and 1 kDa membranes in a high performance ultrafiltration cell, 10 mL of the fraction with highest ACE-I inhibitory activity was injected into a Sephadex G-50 gel filtration column (3 cm x 79 cm) at a flow rate of 25 mL/h of 50 mM ammonium bicarbonate (pH 9.1). The resulting fractions were collected for to assay ACE-I inhibitory activity. Peptide molecular masses were determined by reference to a calibration curve created by running molecular mass markers on the Sephadex G-50 under conditions identical to those used for the test samples. Molecular mass standards were thyroglobulin (670 kDa), bovine gamma globulin (158 kDa), equine myoglobin (17 kDa), vitamin B12 (1.35 kDa) and Thr-Gln (0.25 kDa). Fractions selected for further purification of peptides were

The fractions isolated with the Sephadex G-50 column were redissolved in deionized water and injected into a preparative HPLC (Agilent, Model 1110, Agilent Technologies, Inc. Santa Clara, CA, USA) reverse-phasecolumn (C18 Hi-Pore RP-318, 250 mm x10 mm, Bio-Rad). The injection volume was 100 L, and the sample concentration was 20 mg/mL. Elution was achieved by a linear gradient of acetonitrile in water (0-30% in 50 min) containing 0.1% trifluoroacetic acid at a flow rate of 4 mL/min and 30°C [13]. Elution was monitored at 215 nm, and the resulting fractions were collected for assay of ACE-I inhibitory activity as

Protein amino acid composition was determined for the hydrolysate, and the peptides were purified by ultrafiltration, gel filtration chromatography and HPLC [19]. Samples (2-4 mg protein) were treated with 4 mL of 6 mol equi/L HCl, placed in hydrolysis tubes and gassed with nitrogen at 110°C for 24 h. They were then dried in a rotavapor and suspended in 1 mol/L sodium borate buffer at pH 9.0. Amino acid derivatization was performed at 50°C using diethyl ethoxymethylenemalonate. Amino acids were separated using HPLC with a reversed-phase column (300 x 3.9 mm, Nova Pack C18, 4 mm; Waters), and a binary gradient system with 25 mmol/L sodium acetate containing (A) 0.02 g/L sodium azide at pH 6.0, and (B) acetonitrile as solvent. The flow rate was 0.9 mL/min, and the elution gradient was: time 0.0–3.0 min, linear gradient A:B (91:9) to A-B (86:14); time 3.0–13.0 min, elution with A-B (86–14); time 13.0–30.0 min, linear gradient A-B (86:14) to A-B (69:31); time 30.0–

All results were analyzed in triplicate using descriptive statistics with a central tendency and dispersion measures. One-way ANOVAs were performed to evaluate protein isolate

**2.8. G-50 gel filtration chromatography** 

pooled and lyophilized before RP-HPLC.

**2.9. HPLC C18 chromatography** 

**2.10. Amino acid composition** 

35.0 min, elution with A-B (69:31)**.** 

**2.11. Statistical analysis** 

described above.

## **3.1. Protein extract hydrolysis**

Alcalase®, Flavourzyme® and pepsin-pancreatin were used to produce extensively hydrolyzed *V. unguiculata* protein extracts. Degree of hydrolysis (DH) differed (*P*<0.05) between the enzymatic systems with values of 53.0%, 58.8%, and 35.7% for Alcalase® hydrolysate (AH), Flavourzyme® hydrolysate (FH) and Pepsin-pancreatin hydrolysate (PPH), respectively.

The AH had a 53.0% DH, which is lower than reported by Vioque et al. [21] for rapeseed protein hydrolysates (60% DH) produced with a mixture of Alcalase® and Flavourzyme® during 180 min. However, this DH was higher than that reported for mung bean protein hydrolysates (20.0% DH) produced with Alcalase® for 10 h[22] and for *V. unguiculata* hydrolysates (32.3% DH) produced with Alcalase® for 60 min [10].The variation in DH observed here is probably the result of protease specificity since Alcalase® is an industrial alkaline protease produced from *Bacillus licheniformis*, the main enzyme component of which (serine endopeptidase subtilisin Carlsberg) presents broad specificity and hydrolyzes most peptide bonds, with a preference for those containing aromatic amino acid residues [23]. ACE-I prefers substrates or competitive inhibitors containing hydrophobic amino acid (aromatic or branched lateral chain) residues (Hong et al., 2005). Alcalase® is therefore very suitable for production of bioactive peptides, such as those with ACE-I inhibitory activity. According to Pedroche et al. [24] the controlled liberation of biologically active peptides from protein by enzymatic hydrolysis is one of the most promising trends concerning medical applications of the protein hydrolysates with DH higher than 10% while hydrolysates with a low DH (lower than 10%) are used for the improvement of functional properties of flours or protein isolates. Therefore, the results suggest (DH=53.0%) that *V. unguiculata* protein is an appropriate substrate for producing these bioactive peptides when hydrolyzed with Alcalase®.

Hydrolysis with Flavourzyme® produced a *V. unguiculata* hydrolysate with 58.8% DH, somewhat higher than obtained with the Alcalase® system. A similar discrepancy has been reported for chickpea protein hydrolysates (27.0% DH) produced with Flavourzyme® for 180 min [25]. DH was higher with Flavourzyme® since it is a protease complex produced by *Aspergillus orizae,* which contains endoproteinases and exopeptidases. The fungal protease complex Flavourzyme® has a broader specificity; thus, when combined with its exopeptidase activity high DH values can be achieved, perhaps as much as 50% giving mostly dipeptides in the hydrolysate.

Sequential hydrolysis with pepsin-pancreatin produced cowpea protein hydrolysates with the lowest DH (35.74%) of the three studied enzymatic systems. This DH was similar to 37.0% at 360 min reported for sunflower protein hydrolysates obtained with the same system [13], but higher than the reported values for soy protein hydrolysates produced with pancreatin for 60 (11.0%) and 180 min (17.0%) [26]. The *V. unguiculata* PPH represents a pool of peptides resembling those generated during digestion of *V. unguiculata* proteins in the organism. This coincides with the behavior of extensively hydrolyzed sunflower protein reported by Megías et al. [13].Pepsin is the main proteolytic enzyme generated in the stomach during food digestion, while pancreatin includes proteases such as trypsin, chymotrypsin and elastase, which are released by the pancreas in the small intestine. The resulting peptides are therefore resistant to pepsin and pancreatin, suggesting that they might be absorbed by digestive epithelial cells in the small intestine, probably might be bioavailable and exercise their biological activity.

Vigna Unguiculata as Source of Angiotensin-I Converting Enzyme Inhibitory and Antioxidant Peptides 191

In terms of ACE-I inhibitory activity, the pepsin-pancreatin system produced hydrolysate with the highest activity, while the Flavourzyme® system produced peptide fractions with the highest activity. This also confirms that *V. unguiculata* is a good protein source for

bioactive peptide extraction by gastrointestinal or commercial proteases.

**Figure 1.** IC50 values of peptide fractions obtained by ultrafiltration from *V. unguiculata* protein

Antioxidant activity of the protein hydrolysates and their corresponding UF peptide fractions was quantified and calculated as TEAC values (mM/mg protein). Antioxidant activity was not significantly different (*P*>0.05) between the three hydrolysis systems (14.7 for AH, 14.5 for FH and 14.3 mM/mg protein for PPH). Ultrafiltration improved antioxidant activity, which was dependent (*P*<0.05) on fraction molecular weight (Fig. 2).TEAC values were 303.2-1457 mM/mg protein for the AH, 357.4-10211 mM/mg protein for the FH, and 267.1-2830.4 mM/mg protein for the PPH. These values, and consequently the fraction's antioxidant activities, were higher (*P*<0.05) as fraction molecular weight decreased. According to Dávalos et al. [28] this behavior among the *V. unguiculata* peptide fractions may reflect the enhanced accessibility of small peptides to the redox reaction system, for the

Antioxidant activity in the *V. unguiculata* protein hydrolysates and their UF peptide fractions was measured with an ABTS assay, which quantifies an antioxidant's (i.e. hydrogen or electron donor) suppression of the radical cation ABTS●+ based on singleelectron reduction of the relatively stable radical cation ABTS●+ formed previously by an oxidation reaction. When added to PBS medium (pH 7.2) containing ABTS●+, the proteins in the hydrolysates and peptide fractions very probably acted as electron donors, transforming

hydrolysates.

**3.3. Antioxidant activity by ABTS assay** 

prescence of critical amino acid residues.

#### **3.2. ACE-I inhibitory activity**

ACE-I inhibitory activity of AH, FH and PPH was measured and calculated as IC50. Biological activity was highest in the PPH, as indicated by a lower IC50 value (1397.9 g/ml) compared to the AH (2564.7 g/ml) and FH (2634.4 g/ml). In other words, more ACE-I inhibitory active peptides were produced using the pepsin-pancreatin treatment.

ACE-I inhibitory activity has been reported for enzymatic hydrolysates from different protein sources with IC50 values ranging from 0.2 to 246.7 g/mL [22]. Many of these hydrolysates have been shown to have antihypertensive activities in spontaneously hypertensive rats (SHR). In the present study, the IC50 value for the hydrolysate prepared with pepsin-pancreatin at 90 min incubation is within the concentration range likely to mediate an antihypertensive effect. Therefore, it is to be expected that *V. unguiculata* protein derived ACE-I inhibitory peptides would have antihypertensive activity. However, further investigations are necessary to examine whether the peptide mixture may exert antihypertensive activity *in vivo* because the inhibitory potencies of the peptides on ACE-I activity do not always correlate with their antihypertensive activities found in SHR[22].

Ultrafiltration of AH, FH and PPH produced peptide fractions with increased biological activity (Fig. 1). ACE-I inhibitory activity of peptide fractions ranged from 24.3-123 g/ml in the AH, from 0.04 to 170.6 g/ml in the FH and from 44.7 to 112 g/ml in the PPH. ACE-I inhibitory activity was significantly (*P*<0.05) dependent on peptide fraction molecular weight, with the lowest activity being in the >10kDa fractions and the highest in the <1kDa fractions for all hydrolysates. Similar ACE-I inhibitory activity behavior was reported by Je et al.[5]for five peptide fractions from pepsin-hydrolyzed Alaska pollack frame protein run through an ultrafiltration membrane-bioreactor system with MWCOs of 30, 10, 5, 3 and 1 kDa. Higher ACE-I inhibitory activity (%) in lower molecular weight fractions was also reported by Xue-Ying et al. [27] for yak casein hydrolysate fractions separated using 6 and 10 kDa MWCOs: >10kDa (23.1%), 6-10kDa (29.2%) and <6 kDa (85.4%).

Of the three hydrolysates tested in the present study, FH, which had the highest DH, also exhibited the highest ACE-I inhibitory activity in the <1kDa fraction. The biological activity of this fraction provides it potential commercial applications as a 'health-enhancing ingredient' in functional food production.

In terms of ACE-I inhibitory activity, the pepsin-pancreatin system produced hydrolysate with the highest activity, while the Flavourzyme® system produced peptide fractions with the highest activity. This also confirms that *V. unguiculata* is a good protein source for bioactive peptide extraction by gastrointestinal or commercial proteases.

**Figure 1.** IC50 values of peptide fractions obtained by ultrafiltration from *V. unguiculata* protein hydrolysates.

## **3.3. Antioxidant activity by ABTS assay**

190 Bioactive Food Peptides in Health and Disease

**3.2. ACE-I inhibitory activity** 

bioavailable and exercise their biological activity.

37.0% at 360 min reported for sunflower protein hydrolysates obtained with the same system [13], but higher than the reported values for soy protein hydrolysates produced with pancreatin for 60 (11.0%) and 180 min (17.0%) [26]. The *V. unguiculata* PPH represents a pool of peptides resembling those generated during digestion of *V. unguiculata* proteins in the organism. This coincides with the behavior of extensively hydrolyzed sunflower protein reported by Megías et al. [13].Pepsin is the main proteolytic enzyme generated in the stomach during food digestion, while pancreatin includes proteases such as trypsin, chymotrypsin and elastase, which are released by the pancreas in the small intestine. The resulting peptides are therefore resistant to pepsin and pancreatin, suggesting that they might be absorbed by digestive epithelial cells in the small intestine, probably might be

ACE-I inhibitory activity of AH, FH and PPH was measured and calculated as IC50. Biological activity was highest in the PPH, as indicated by a lower IC50 value (1397.9 g/ml) compared to the AH (2564.7 g/ml) and FH (2634.4 g/ml). In other words, more ACE-I

ACE-I inhibitory activity has been reported for enzymatic hydrolysates from different protein sources with IC50 values ranging from 0.2 to 246.7 g/mL [22]. Many of these hydrolysates have been shown to have antihypertensive activities in spontaneously hypertensive rats (SHR). In the present study, the IC50 value for the hydrolysate prepared with pepsin-pancreatin at 90 min incubation is within the concentration range likely to mediate an antihypertensive effect. Therefore, it is to be expected that *V. unguiculata* protein derived ACE-I inhibitory peptides would have antihypertensive activity. However, further investigations are necessary to examine whether the peptide mixture may exert antihypertensive activity *in vivo* because the inhibitory potencies of the peptides on ACE-I activity do not always correlate with their antihypertensive activities found in SHR[22].

Ultrafiltration of AH, FH and PPH produced peptide fractions with increased biological activity (Fig. 1). ACE-I inhibitory activity of peptide fractions ranged from 24.3-123 g/ml in the AH, from 0.04 to 170.6 g/ml in the FH and from 44.7 to 112 g/ml in the PPH. ACE-I inhibitory activity was significantly (*P*<0.05) dependent on peptide fraction molecular weight, with the lowest activity being in the >10kDa fractions and the highest in the <1kDa fractions for all hydrolysates. Similar ACE-I inhibitory activity behavior was reported by Je et al.[5]for five peptide fractions from pepsin-hydrolyzed Alaska pollack frame protein run through an ultrafiltration membrane-bioreactor system with MWCOs of 30, 10, 5, 3 and 1 kDa. Higher ACE-I inhibitory activity (%) in lower molecular weight fractions was also reported by Xue-Ying et al. [27] for yak casein hydrolysate fractions separated using 6 and

Of the three hydrolysates tested in the present study, FH, which had the highest DH, also exhibited the highest ACE-I inhibitory activity in the <1kDa fraction. The biological activity of this fraction provides it potential commercial applications as a 'health-enhancing

10 kDa MWCOs: >10kDa (23.1%), 6-10kDa (29.2%) and <6 kDa (85.4%).

ingredient' in functional food production.

inhibitory active peptides were produced using the pepsin-pancreatin treatment.

Antioxidant activity of the protein hydrolysates and their corresponding UF peptide fractions was quantified and calculated as TEAC values (mM/mg protein). Antioxidant activity was not significantly different (*P*>0.05) between the three hydrolysis systems (14.7 for AH, 14.5 for FH and 14.3 mM/mg protein for PPH). Ultrafiltration improved antioxidant activity, which was dependent (*P*<0.05) on fraction molecular weight (Fig. 2).TEAC values were 303.2-1457 mM/mg protein for the AH, 357.4-10211 mM/mg protein for the FH, and 267.1-2830.4 mM/mg protein for the PPH. These values, and consequently the fraction's antioxidant activities, were higher (*P*<0.05) as fraction molecular weight decreased. According to Dávalos et al. [28] this behavior among the *V. unguiculata* peptide fractions may reflect the enhanced accessibility of small peptides to the redox reaction system, for the prescence of critical amino acid residues.

Antioxidant activity in the *V. unguiculata* protein hydrolysates and their UF peptide fractions was measured with an ABTS assay, which quantifies an antioxidant's (i.e. hydrogen or electron donor) suppression of the radical cation ABTS●+ based on singleelectron reduction of the relatively stable radical cation ABTS●+ formed previously by an oxidation reaction. When added to PBS medium (pH 7.2) containing ABTS●+, the proteins in the hydrolysates and peptide fractions very probably acted as electron donors, transforming this radical cation (maximum absorbance at 734nm) into the non-radical ABTS. The higher antioxidant activity of the UF peptide fractions versus their source hydrolysates is related to unique properties provided by their amino acid composition. The fractions' increased ability to decrease free radical reactivity is linked to the greater exposure of their amino acids, which leads to increased peptide/free radical reactions.

Vigna Unguiculata as Source of Angiotensin-I Converting Enzyme Inhibitory and Antioxidant Peptides 193

**Figure 3.** Elution profile of the <1 kDa ultrafiltration fraction of the cowpea *V. unguiculata* protein

The profile was typical of a protein hydrolysate formed by a pool of peptides, with gradually decreasing molecular masses. Elution volumes between 406 and 518 mL included free amino acids and peptides with molecular masses ranging from 3.6 to 0.4 kDa. This range was fractionated into eleven fractions (1 to 11) and ACE-I inhibitory activity determined for each. Fractions with elution volumes smaller than 406 mL and greater than 518 mL were not analyzed because they largely included peptides with high molecular weights, as well as free amino acids. ACE-I inhibitory activity (%) in the eleven fractions ranged from 5.29 to 47.43% and differed (*P*<0.05) between fractions (Table 1). The highest ACE-I inhibitory activity was observed in fractions F4 (47.43%; 437.5-444.5 mL elution volume) and F5 (45.14%, 448-455 mL elution volume), which were not statistically different (*P*<0.05). Their molecular masses were approximately 1.8 kDa (indicative of 7 amino acid residues) and 1.5 kDa (indicative of 10 amino acid residues), respectively. The IC50 value for F4 (14.19 g/mL) was similar than those of *Fagopirum esculentum* peptide fractions purified by Sephadex LH-20 gel filtration (15.1 g/mL) [31], but lower than those of gel filtration (Sephadex G-25) peptide fractions from tuna broth hydrolysate (210 to 25,260 g/mL) [32], from *Fagopyrum esculentum* Moench (Sephadex C-25 = 25,715.1 g/mL; Sephadex G-10 = 21,315.1 g/mL), and from the peptic hydrolysate of *Acetes chinensis* (Sephadex C-15 = 770 – 1590 g/mL) [33]. A similar behavior pattern was observed with Konjac peptides purified by series connection of Sephadex G-25 and Sephadex G-15 columns that resulted in purified peptides with molecular weights of 1500 and 1000 Da and IC50 values of 120 g/mL and 88 g/mL, respectively [34]. Ji-Eun et al. [35] reported similar results to separate peptide fractions below 3 kDa through size exclusion chromatography (IC50= 500 g/mL) from

hydrolysate with Flavorzyme® purified in a Sephadex G-50 gel filtration column.

**Figure 2.** Antioxidant activity of peptide fractions obtained by ultrafiltration from *V. unguiculata* protein hydrolysates.

Overall, the <1 kDa peptide fraction from the FH had the highest TEAC values and was shown to undergo single-electron transfer reactions in the ABTS●+ reduction assay, demonstrating its antioxidant capacity. This is an extremely attractive property since oxidants are known to be involved in many human diseases and aging processes. Oxidants are associated with the chronic damage of ageing, and destructive oxidants and oxygen-free radicals can be extremely toxic to tissues by promoting tissue necrosis and cell damage. Some authors claim that proteins possess antioxidant properties; for instance, [29] reported that protein insufficiency aggravates enhanced lipid peroxidation and reduces antioxidative enzyme activities in rats, while Larson et al. [30] observed that proteins affect lipid metabolism in laboratory animals. The biological effect exhibited by the FH<1kDa fraction apparently reinforces the claim that proteins possess antioxidant properties. This makes the FH<1kDa fraction a potential "antioxidant" ingredient in functional food production.

#### **3.4. Gel filtration chromatography**

Because it exhibited the highest ACE-I inhibitory (IC50 value of 0.04 g/mL) and antioxidant activity (TEAC value of 10211 mM/mg protein), the <1 kDa fraction from FH was selected for further fractionation. Gel filtration chromatography was used to generate a molecular weight profile of this fraction (Fig. 3).

protein hydrolysates.

**3.4. Gel filtration chromatography** 

weight profile of this fraction (Fig. 3).

which leads to increased peptide/free radical reactions.

this radical cation (maximum absorbance at 734nm) into the non-radical ABTS. The higher antioxidant activity of the UF peptide fractions versus their source hydrolysates is related to unique properties provided by their amino acid composition. The fractions' increased ability to decrease free radical reactivity is linked to the greater exposure of their amino acids,

**Figure 2.** Antioxidant activity of peptide fractions obtained by ultrafiltration from *V. unguiculata*

FH<1kDa fraction a potential "antioxidant" ingredient in functional food production.

Because it exhibited the highest ACE-I inhibitory (IC50 value of 0.04 g/mL) and antioxidant activity (TEAC value of 10211 mM/mg protein), the <1 kDa fraction from FH was selected for further fractionation. Gel filtration chromatography was used to generate a molecular

Overall, the <1 kDa peptide fraction from the FH had the highest TEAC values and was shown to undergo single-electron transfer reactions in the ABTS●+ reduction assay, demonstrating its antioxidant capacity. This is an extremely attractive property since oxidants are known to be involved in many human diseases and aging processes. Oxidants are associated with the chronic damage of ageing, and destructive oxidants and oxygen-free radicals can be extremely toxic to tissues by promoting tissue necrosis and cell damage. Some authors claim that proteins possess antioxidant properties; for instance, [29] reported that protein insufficiency aggravates enhanced lipid peroxidation and reduces antioxidative enzyme activities in rats, while Larson et al. [30] observed that proteins affect lipid metabolism in laboratory animals. The biological effect exhibited by the FH<1kDa fraction apparently reinforces the claim that proteins possess antioxidant properties. This makes the

**Figure 3.** Elution profile of the <1 kDa ultrafiltration fraction of the cowpea *V. unguiculata* protein hydrolysate with Flavorzyme® purified in a Sephadex G-50 gel filtration column.

The profile was typical of a protein hydrolysate formed by a pool of peptides, with gradually decreasing molecular masses. Elution volumes between 406 and 518 mL included free amino acids and peptides with molecular masses ranging from 3.6 to 0.4 kDa. This range was fractionated into eleven fractions (1 to 11) and ACE-I inhibitory activity determined for each. Fractions with elution volumes smaller than 406 mL and greater than 518 mL were not analyzed because they largely included peptides with high molecular weights, as well as free amino acids. ACE-I inhibitory activity (%) in the eleven fractions ranged from 5.29 to 47.43% and differed (*P*<0.05) between fractions (Table 1). The highest ACE-I inhibitory activity was observed in fractions F4 (47.43%; 437.5-444.5 mL elution volume) and F5 (45.14%, 448-455 mL elution volume), which were not statistically different (*P*<0.05). Their molecular masses were approximately 1.8 kDa (indicative of 7 amino acid residues) and 1.5 kDa (indicative of 10 amino acid residues), respectively. The IC50 value for F4 (14.19 g/mL) was similar than those of *Fagopirum esculentum* peptide fractions purified by Sephadex LH-20 gel filtration (15.1 g/mL) [31], but lower than those of gel filtration (Sephadex G-25) peptide fractions from tuna broth hydrolysate (210 to 25,260 g/mL) [32], from *Fagopyrum esculentum* Moench (Sephadex C-25 = 25,715.1 g/mL; Sephadex G-10 = 21,315.1 g/mL), and from the peptic hydrolysate of *Acetes chinensis* (Sephadex C-15 = 770 – 1590 g/mL) [33]. A similar behavior pattern was observed with Konjac peptides purified by series connection of Sephadex G-25 and Sephadex G-15 columns that resulted in purified peptides with molecular weights of 1500 and 1000 Da and IC50 values of 120 g/mL and 88 g/mL, respectively [34]. Ji-Eun et al. [35] reported similar results to separate peptide fractions below 3 kDa through size exclusion chromatography (IC50= 500 g/mL) from textured and fermented vegetable protein (IC50 = 2190 g/mL). They purified peptide fraction with a molecular weight range of 500-999 Da with IC50 values of 94 g/mL and that represented peptides of approximately 7 amino acids residues.

Vigna Unguiculata as Source of Angiotensin-I Converting Enzyme Inhibitory and Antioxidant Peptides 195

RP-HPLC fractions obtained of *Mustelus mustelus* intestines with alkaline proteases (130- 783 g/ml) [37] and with peptide fractions obtained from the peptic hydrolysate of the

**Figure 4.** ACE inhibition percentage of RP-HPLC fractions obtained from fractions F4 and F5 produced after hydrolysis of *V. unguiculata* with Flavourzyme®.a-cDifferent letters in the same gel filtration chromatography fraction indicate statistical difference (*P<*0.05). Data are the mean of three replicates.

Fractions with the highest ACE-I inhibitory activity were analyzed to produce an amino acid profile. During hydrolysis, asparagine and glutamine partially converted to aspartic acid and glutamic acid, respectively; the data for asparagine and/or aspartic acid were therefore reported as Asx while those for glutamine and/or glutamic acid were reported as Glx. The higher ACE-I inhibitory activity exhibited by the <1 kDa fraction (IC50=0.04 g/mL) compared to FH (IC50=2634.4 g/mL) was probably due to its higher concentration of neutral amino acids, such as Ser (3.03%) and Thr (11.36%), hydrophilics such as His (12.5%) or hydrophobics such as Ala (7.84%), Pro (23.80%), Val (10%), Met (66.66%), Ile (10%), Leu

Compared to the <1 kDa fraction amino acid profile, the G-50 gel filtration chromatography fractions had higher Asx, Glx and Arg concentrations. Hydrophobic amino acid content decreased by 20.27% in F4 (34.56g/100g) and 21.19% in F5 (34.16g/100g) compared to the <1 kDa fraction (43.35 g/100g), while hydrophilic residues increased by 19.25% (48 g/100g) in F4 and 22.48% (50 g/100g) in F5. The ACE-I inhibitory activity observed in F4 (47.43%) and F5 (45.14%) could therefore be the result of their higher Arg, Asx or Asp concentrations. These residues are known to play an important role in the antihypertensive activity of peptides from white and red wines and from French flor-sherry wine [38]. The F4 and F5 fractions may also have the added benefit of low bitterness. Many of the ACE-I-inhibitory peptides

freshwater rotifer *Brachionus calyciflonus* (40.01 g/ml) [38].

**3.6. Amino acid composition** 

(21.6%), Phe (18.6%) and Trp (16.6%) (Table 2).


**Table 1.** ACE-I inhibition percentage of peptide fractions purified in a Sephadex G-50 gel filtration column.a-eDifferent superscripts letters indicate statistical difference (*P <*0.05). Data are the mean of three replicates.

#### **3.5. Reverse-phase HPLC chromatography of pooled fractions**

Fractions F4 and F5 from the gel filtration chromatography treatment were pooled and analyzed using RP-HPLC to produce a chromatographic profile from mass-transfer between stationary and mobile phases. Mixture components were separated by dissolving fractions F4 and F5 in acetonitrile and forcing them through a chromatographic column under high pressure. The mixture resolved into its components in the column, separating F4 and F5 based on differences in hydrophobicity. In this process, the components of both fractions passed over stationary-phase particles containing pores large enough for them to enter, and in which interactions with the hydrophobic surface removed them from the flowing mobilephase stream. The strength and nature of the interaction between the sample particles and stationary phase depended on hydrophobic and polar interactions. As the eluent organic solvent concentration increased, it reached a critical value for each analyte and desorbed it from the hydrophobic stationary-phase surface to allowing it to elute from the column into the flowing mobile phase. Because this elution depended on the precise distribution of hydrophobic residues in each specie, each analyte eluted from the column at a characteristic time and the resulting peaks were used to qualitatively analyze both fractions' components. Within each gel filtration fraction, the eluates were divided into four major fractions: F4-1, F4-2, F4-3, F4-4; F5-1, F5-2, F5-3, F5-4. The peptides were relatively pure, although a small shoulder still appeared behind the peaks in the chromatogram.

Enough material from each fraction was collected in successive analyses to determine ACE-I inhibitory activity (Fig. 4). Fraction F4 had a larger (*P*<0.05) ACE-I inhibitory activity range (33.83 to 75.42%) than F5 (32.31 to 49.71%). Overall, F4-2 (75.42%) had the highest ACE-I inhibitory activity (IC50 = 0.4704 g/mL). This value is lower than the 6.3 g/ml reported for a peptide from *F. esculentum* Moench purified by RP-HPLC [31] and within the 8.1 to 91.6 g/mL range reported for fractions from caprine kefir water-soluble extract purified by preparative RP-HPLC [36]. The same behavior was observed when comparing the results of RP-HPLC fractions obtained of *Mustelus mustelus* intestines with alkaline proteases (130- 783 g/ml) [37] and with peptide fractions obtained from the peptic hydrolysate of the freshwater rotifer *Brachionus calyciflonus* (40.01 g/ml) [38].

**Figure 4.** ACE inhibition percentage of RP-HPLC fractions obtained from fractions F4 and F5 produced after hydrolysis of *V. unguiculata* with Flavourzyme®.a-cDifferent letters in the same gel filtration chromatography fraction indicate statistical difference (*P<*0.05). Data are the mean of three replicates.

#### **3.6. Amino acid composition**

194 Bioactive Food Peptides in Health and Disease

three replicates.

textured and fermented vegetable protein (IC50 = 2190 g/mL). They purified peptide fraction with a molecular weight range of 500-999 Da with IC50 values of 94 g/mL and that

> F1 28.47C F7 37.61d F2 10.67a F8 29.9c F3 25.61bc F9 22.01b F4 47.43e F10 20.92b

**Table 1.** ACE-I inhibition percentage of peptide fractions purified in a Sephadex G-50 gel filtration column.a-eDifferent superscripts letters indicate statistical difference (*P <*0.05). Data are the mean of

Fractions F4 and F5 from the gel filtration chromatography treatment were pooled and analyzed using RP-HPLC to produce a chromatographic profile from mass-transfer between stationary and mobile phases. Mixture components were separated by dissolving fractions F4 and F5 in acetonitrile and forcing them through a chromatographic column under high pressure. The mixture resolved into its components in the column, separating F4 and F5 based on differences in hydrophobicity. In this process, the components of both fractions passed over stationary-phase particles containing pores large enough for them to enter, and in which interactions with the hydrophobic surface removed them from the flowing mobilephase stream. The strength and nature of the interaction between the sample particles and stationary phase depended on hydrophobic and polar interactions. As the eluent organic solvent concentration increased, it reached a critical value for each analyte and desorbed it from the hydrophobic stationary-phase surface to allowing it to elute from the column into the flowing mobile phase. Because this elution depended on the precise distribution of hydrophobic residues in each specie, each analyte eluted from the column at a characteristic time and the resulting peaks were used to qualitatively analyze both fractions' components. Within each gel filtration fraction, the eluates were divided into four major fractions: F4-1, F4-2, F4-3, F4-4; F5-1, F5-2, F5-3, F5-4. The peptides were relatively pure, although a small

Enough material from each fraction was collected in successive analyses to determine ACE-I inhibitory activity (Fig. 4). Fraction F4 had a larger (*P*<0.05) ACE-I inhibitory activity range (33.83 to 75.42%) than F5 (32.31 to 49.71%). Overall, F4-2 (75.42%) had the highest ACE-I inhibitory activity (IC50 = 0.4704 g/mL). This value is lower than the 6.3 g/ml reported for a peptide from *F. esculentum* Moench purified by RP-HPLC [31] and within the 8.1 to 91.6 g/mL range reported for fractions from caprine kefir water-soluble extract purified by preparative RP-HPLC [36]. The same behavior was observed when comparing the results of

**3.5. Reverse-phase HPLC chromatography of pooled fractions** 

shoulder still appeared behind the peaks in the chromatogram.

**activity (%) Fraction ACE-I inhibitory** 

F11 5.29a

**activity (%)** 

represented peptides of approximately 7 amino acids residues.

**Fraction ACE-I inhibitory** 

F5 45.14e

F6 37.58d

Fractions with the highest ACE-I inhibitory activity were analyzed to produce an amino acid profile. During hydrolysis, asparagine and glutamine partially converted to aspartic acid and glutamic acid, respectively; the data for asparagine and/or aspartic acid were therefore reported as Asx while those for glutamine and/or glutamic acid were reported as Glx. The higher ACE-I inhibitory activity exhibited by the <1 kDa fraction (IC50=0.04 g/mL) compared to FH (IC50=2634.4 g/mL) was probably due to its higher concentration of neutral amino acids, such as Ser (3.03%) and Thr (11.36%), hydrophilics such as His (12.5%) or hydrophobics such as Ala (7.84%), Pro (23.80%), Val (10%), Met (66.66%), Ile (10%), Leu (21.6%), Phe (18.6%) and Trp (16.6%) (Table 2).

Compared to the <1 kDa fraction amino acid profile, the G-50 gel filtration chromatography fractions had higher Asx, Glx and Arg concentrations. Hydrophobic amino acid content decreased by 20.27% in F4 (34.56g/100g) and 21.19% in F5 (34.16g/100g) compared to the <1 kDa fraction (43.35 g/100g), while hydrophilic residues increased by 19.25% (48 g/100g) in F4 and 22.48% (50 g/100g) in F5. The ACE-I inhibitory activity observed in F4 (47.43%) and F5 (45.14%) could therefore be the result of their higher Arg, Asx or Asp concentrations. These residues are known to play an important role in the antihypertensive activity of peptides from white and red wines and from French flor-sherry wine [38]. The F4 and F5 fractions may also have the added benefit of low bitterness. Many of the ACE-I-inhibitory peptides isolated from food sources are composed of multiple food components and hydrophobic and/or aromatic amino acid residues. However, practical use of these food protein hydrolysates is complicated by formation of peptides, which impart a bitter taste, the result of the formation of low molecular weight peptides containing mostly hydrophobic amino acids. To address this problem, Kim et al. [40] recommended use of Flavourzyme®, a fungal endoprotease and exo-protease complex that produces hydrolysates or peptides with ACE-I inhibitory activity and low bitterness. Fractions F4 and F5 are promising prospects for use in new product development because they had clear ACE-I inhibitory activity and low hydrophobic amino acid content, which may ensure that they have low bitterness.

Vigna Unguiculata as Source of Angiotensin-I Converting Enzyme Inhibitory and Antioxidant Peptides 197

inhibitory activity of peptides. For instance, similar observations have been made for an orientase hydrolysate with notable ACE-I inhibition attributed to its high basic and aromatic amino acids contents [33]. Cheung et al. [43] reported a peptide with strong, competitive ACE-I inhibition in which aromatic amino acid residues at its C-terminal and basic or hydrophobic ones at its N-terminal played an essential role. Based on the above, the higher ACE-I inhibitory potential of the <1 kDa fraction from the cowpea hydrolysate can be attributed to the steric properties of its aromatic amino acids and the lipophilicity and

The most active fraction among those purified from F4 by preparative RP-HPLC was F4-2, which had much higher neutral amino acid content (80.6 g/100g) than F4-1 (46.6 g/100g), F4- 3(6.1 g/100g) and F4-4 (28.6 g/100g) (Table 3). Of the neutral amino acids, Tyr was higher in F4-2 (71.3 g/100g) compared to F4-3 (1.5 g/100g), F4-4 (3.9 g/100g) and F4-1 (3.9 g/100g). This supports the importance of aromatic residues in a peptides' biological potential, probably due to their high steric properties and low lipophilicity. The Tyr amino acid has been reported in peptides from milk (e.g., Tyr-Pro-Tyr-Tyr) isolated by a combination of lactic acid bacteria fermentation and Flavourzyme® hydrolysis. These peptides are bioavailable and exhibit *in vitro* (90.9 M) and *in vivo* ACE-I inhibitory activity, the latter in the form of

reduced hypertension in SHR (15.9 mmHg reduction in systolic blood pressure) [43].

angiotensin system and induces a prolonged reduction in blood pressure [45].

Of the fractions purified from F5 by preparative RP-HPLC chromatography (Table 3), F5-2, F5-3 and F5-4 exhibited ACE-I inhibition that was not different among them (*P*<0.05), but was greater than that of F5-1. The amino acids Arg, Tyr, Met, Ile, Leu, Phe and Lys very probably played a key role in providing greater activity to the first three fractions. As mentioned above, residues such as Leu and Ile are preferred at the amino terminus of

F4-2 was also unique in containing Met (0.5 g/100g) and Trp (0.2 g/100g), as well as higher concentrations of Ile (3.2 g/100g) and Leu (10.2 g/100g) than the other fractions eluted from F4, all of which could have significantly increased its relative ACE-I inhibition activity. This would coincide with previous reports of inhibitory activity in peptides with these amino acids. The residues Val, Leu and Ile are preferred in the amino terminal position in active tripeptides for their low lipophilicity and steric properties or side chain bulk/ molecular size [40]. Clearly, the structure of ACE-I inhibitory peptides influences their activity, as shown by Cheung et al. [42], who reported that peptides with Tyr at the C-terminus and Ile at the N-terminus exhibit highly potent inhibitory activity. In a randomized, double-blind, placebo-controlled human study, a significant depressor effect was observed in mild essential hypertensive volunteers and Val-Tyr was shown to be one of the predominant ACE-I inhibitory peptides involved in this effect [44]. In addition to its high Tyr content, F4- 2 may also be absorbed intact into the human circulatory system and induce a reduction in blood pressure. For instance, intravenous and oral administration of Val-Tyr in SHR have shown that this di-peptide caused a long-lasting depressor effect. Val-Tyr is known to be absorbed intact into the human circulatory system, and studies using cross-mated transgenic mice carrying the human renin gene and the human angiotensinogen gene have shown that, as a natural ACE-I inhibitory dipeptide, Val-Tyr regulates the enhanced human renin-

electronic properties of its cyclic amino acids.


\*Data are the mean of three replicates

**Table 2.** Amino acid contents of the cowpea *V. unguiculata* protein concentrate (PC), Flavourzyme hydrolysate (FH), <1 kDa ultrafiltered fraction and F4 and F5 gel filtration chromatography fractions.

Comparison of amino acid composition and properties between the cowpea *V. unguiculata*  hydrolysate and its peptides isolated by ultrafiltration and G-50 gel chromatography showed the most active fraction to be the <1 kDa fraction, which had abundant aromatic (e.g., Phe) and cyclic amino acids (e.g., Pro). Amino acids such as Phe, with large bulky chains and hydrophobic side chains, are preferred in both positions of a dipeptide for their high steric properties and low lipophilicity, while amino acids such as Pro are preferred in the carboxyl terminus of active tripeptides for their low lipophilicity, and high steric and electronic properties [41]. Aromatic and basic amino acids are important to the ACE-I inhibitory activity of peptides. For instance, similar observations have been made for an orientase hydrolysate with notable ACE-I inhibition attributed to its high basic and aromatic amino acids contents [33]. Cheung et al. [43] reported a peptide with strong, competitive ACE-I inhibition in which aromatic amino acid residues at its C-terminal and basic or hydrophobic ones at its N-terminal played an essential role. Based on the above, the higher ACE-I inhibitory potential of the <1 kDa fraction from the cowpea hydrolysate can be attributed to the steric properties of its aromatic amino acids and the lipophilicity and electronic properties of its cyclic amino acids.

196 Bioactive Food Peptides in Health and Disease

\*Data are the mean of three replicates

isolated from food sources are composed of multiple food components and hydrophobic and/or aromatic amino acid residues. However, practical use of these food protein hydrolysates is complicated by formation of peptides, which impart a bitter taste, the result of the formation of low molecular weight peptides containing mostly hydrophobic amino acids. To address this problem, Kim et al. [40] recommended use of Flavourzyme®, a fungal endoprotease and exo-protease complex that produces hydrolysates or peptides with ACE-I inhibitory activity and low bitterness. Fractions F4 and F5 are promising prospects for use in new product development because they had clear ACE-I inhibitory activity and low

**PC FH F<1 kDa F4 F5** 

**Asx** 10.8 ± 0.003 9.6 ± 0.387 9.4 ± 0.448 14 ± 0.274 16.7 ± 0.152 **Glx** 19 ± 0.016 18.6 ± 0.013 14.9 ± 0.171 17.9 ± 0.182 18.3 ± 0.17 **Ser** 6.5 ± 0.041 6.4 ± 0.016 6.6 ± 0.046 5.8 ± 0.302 5.0 ± 0.126 **His** 2.9 ± 0.028 2.8 ± 0.001 3.2 ± 0.024 2.9 ± 0.008 2.7 ± 0.061 **Gly** 4.4 ± 0.005 4.5 ± 0.006 4.1 ± 0.035 4.3 ± 0.011 3.0 ± 0.177 **Thr** 4.3 ± 0.009 3.9 ± 0.009 4.4 ± 0.032 4.0 ± 0.040 3.4 ± 0.036 **Arg** 7.8 ± 0.067 7.9 ± 0.025 6.0 ± 0.073 7.5 ± 0.026 6.9 ± 0.039 **Ala** 4.4 ± 0.008 4.7 ± 0.002 5.1 ± 0.106 4.3 ± 0.016 3.6 ± 0.625 **Pro** 2.7 ± 0.078 1.6 ± 0.038 2.1 ± 0.01 0 ± 0 0 ± 0 **Tyr** 2.6 ± 0.098 2.7 ± 0.006 2.6 ± 0.026 2.2 ± 0.018 1.2 ± 0.078 **Val** 5.4 ± 0.052 6.3 ± 0.015 7.0 ± 0.029 5.3 ± 0.004 4.2 ± 0.211 **Met** 0.1 ± 0.109 0.3 ± 0.001 0.9 ± 0.013 0.7 ± 0.006 2.2 ± 0.292 **Cys** 0.4 ± 0.002 0.4 ± 0.006 0.2 ± 0.002 1.2 ± 0.01 3.2 ± 0.1 **Ile** 4.4 ± 0.019 5.4 ± 0.207 6.0 ± 0.073 5.2 ± 0.015 4.7 ± 0.236 **Leu** 9.2 ± 0.115 9.8 ± 0.175 12.5 ± 0.029 11.6 ± 0.092 12.6 ± 0.229 **Phe** 6.9 ± 0.008 7.0 ± 0.004 8.6 ± 0.056 6.7 ± 0.046 5.8 ± 0.043 **Lys** 7.3 ± 0.089 7.1 ± 0 5.3 ± 0.072 5.7 ± 0.042 5.3 ± 0.263 **Trp** 0.7 ± 0.064 1.0 ± 0.038 1.2 ± 0.041 0.8 ± 0.241 1.0 ± 0.535

**Table 2.** Amino acid contents of the cowpea *V. unguiculata* protein concentrate (PC), Flavourzyme hydrolysate (FH), <1 kDa ultrafiltered fraction and F4 and F5 gel filtration chromatography fractions.

Comparison of amino acid composition and properties between the cowpea *V. unguiculata*  hydrolysate and its peptides isolated by ultrafiltration and G-50 gel chromatography showed the most active fraction to be the <1 kDa fraction, which had abundant aromatic (e.g., Phe) and cyclic amino acids (e.g., Pro). Amino acids such as Phe, with large bulky chains and hydrophobic side chains, are preferred in both positions of a dipeptide for their high steric properties and low lipophilicity, while amino acids such as Pro are preferred in the carboxyl terminus of active tripeptides for their low lipophilicity, and high steric and electronic properties [41]. Aromatic and basic amino acids are important to the ACE-I

hydrophobic amino acid content, which may ensure that they have low bitterness.

Amino acid Composition (g/100g)\*

The most active fraction among those purified from F4 by preparative RP-HPLC was F4-2, which had much higher neutral amino acid content (80.6 g/100g) than F4-1 (46.6 g/100g), F4- 3(6.1 g/100g) and F4-4 (28.6 g/100g) (Table 3). Of the neutral amino acids, Tyr was higher in F4-2 (71.3 g/100g) compared to F4-3 (1.5 g/100g), F4-4 (3.9 g/100g) and F4-1 (3.9 g/100g). This supports the importance of aromatic residues in a peptides' biological potential, probably due to their high steric properties and low lipophilicity. The Tyr amino acid has been reported in peptides from milk (e.g., Tyr-Pro-Tyr-Tyr) isolated by a combination of lactic acid bacteria fermentation and Flavourzyme® hydrolysis. These peptides are bioavailable and exhibit *in vitro* (90.9 M) and *in vivo* ACE-I inhibitory activity, the latter in the form of reduced hypertension in SHR (15.9 mmHg reduction in systolic blood pressure) [43].

F4-2 was also unique in containing Met (0.5 g/100g) and Trp (0.2 g/100g), as well as higher concentrations of Ile (3.2 g/100g) and Leu (10.2 g/100g) than the other fractions eluted from F4, all of which could have significantly increased its relative ACE-I inhibition activity. This would coincide with previous reports of inhibitory activity in peptides with these amino acids. The residues Val, Leu and Ile are preferred in the amino terminal position in active tripeptides for their low lipophilicity and steric properties or side chain bulk/ molecular size [40]. Clearly, the structure of ACE-I inhibitory peptides influences their activity, as shown by Cheung et al. [42], who reported that peptides with Tyr at the C-terminus and Ile at the N-terminus exhibit highly potent inhibitory activity. In a randomized, double-blind, placebo-controlled human study, a significant depressor effect was observed in mild essential hypertensive volunteers and Val-Tyr was shown to be one of the predominant ACE-I inhibitory peptides involved in this effect [44]. In addition to its high Tyr content, F4- 2 may also be absorbed intact into the human circulatory system and induce a reduction in blood pressure. For instance, intravenous and oral administration of Val-Tyr in SHR have shown that this di-peptide caused a long-lasting depressor effect. Val-Tyr is known to be absorbed intact into the human circulatory system, and studies using cross-mated transgenic mice carrying the human renin gene and the human angiotensinogen gene have shown that, as a natural ACE-I inhibitory dipeptide, Val-Tyr regulates the enhanced human reninangiotensin system and induces a prolonged reduction in blood pressure [45].

Of the fractions purified from F5 by preparative RP-HPLC chromatography (Table 3), F5-2, F5-3 and F5-4 exhibited ACE-I inhibition that was not different among them (*P*<0.05), but was greater than that of F5-1. The amino acids Arg, Tyr, Met, Ile, Leu, Phe and Lys very probably played a key role in providing greater activity to the first three fractions. As mentioned above, residues such as Leu and Ile are preferred at the amino terminus of tripeptides with ACE-I inhibitory activity due to their low lipophilicity and steric properties or side chain bulk/molecular size values. In addition, Lys and Arg are expected in positions adjacent to the amino terminus due to their low electronic properties and high lipophilicity and steric property values, while residues such as Phe are preferred at the carboxyl terminus due to their low lipophilicity and high steric and electronic property values [41].

Vigna Unguiculata as Source of Angiotensin-I Converting Enzyme Inhibitory and Antioxidant Peptides 199

Content (g/100g)\* F4-1 F4-2 F4-3 F4-4 F5-1 F5-2 F5-3 F5-4

> 2.1 ± 0.492

0.329 0 ± 0 1.5 ±

30.5 ± 0.834

16.3 ± 0.194

13.3 ± 0.159

5.8 ± 0.069

0.151 0 ± 0 0.2 ±

1.1 ± 1.536

0.109 0 ± 0 74.7 ±

31 ± 0.369

0.059 0 ± 0 1.6 ±

0.024 0 ± 0 3.1 ±

0.068 0 ± 0 0.2 ±

1.2 ± 0.005

0.046

0.9 ± 0.014

0.3 ± 0.049

2.9 ± 0.017

0.3 ± 0.048

0.236

0.3 ± 0.048

0.280

0.071

8.8 ± 0.098

0.035

0.066

0.765

0.009

0 ± 0 3.5 ±

2 ± 0.004

2 ± 0.071

1 ± 0.014

0.4 ± 0.017

2 ± 0.016

0.4 ± 0.024

0.6 ± 0.028

0.5 ± 0.012

0.7 ± 0.109

0.1 ± 0.096

0.9 ± 0.019

0.5 ± 0.026

0.8 ± 0.093

87.9 ± 0.234

0.3 ± 0.002 10.6 ± 0.731

14.2 ± 0.762

8.4 ± 0.009

2.7 ± 0.957

10 ± 0.057

2.7 ± 0.957

3 ± 0.139

3.4 ± 0.089

4.2 ± 0.289

0.5 ± 0.039

6.8 ± 0.070

1.8 ± 0.032

6.1 ± 0.896

22.4 ± 0.304

2.4 ± 0.073

12.5 ± 0.242

12.6 ±

8.4 ± 0.177

4.1 ± 0.133

9.2 ± 0.226

3.5 ± 0.066

7.1 ±

3.8 ± 0.046

3.9 ±

3.6 ± 0.288

2.8 ±

4.7 ±

20 ±

0.778

3.8 ±

Trp 0 ± 0 0.2 ± 0.040 0 ± 0 0 ± 0 0 ± 0 0 ± 0 0 ± 0 0 ± 0 **Table 3.** Amino acid contents of fractions from the F4 and F5 gel filtration chromatography fraction

Pro 0 ± 0 0 ± 0 0 ± 0 0 ± 0 0 ± 0 0 ± 0 0 ± 0 0 ± 0

Val 0 ± 0 0 ± 0 0 ± 0 0 ± 0 0 ± 0 0 ± 0 0 ± 0 0 ± 0

intestine to the blood stream (in the case of oral administration) and resist plasma peptidase degradation (in the case of oral and intravenous administration) to reach their target sites and exert an antihypertensive effect *in vivo*. Therefore, *in vivo* research is needed to determine to what extent any of the studied ACE-I inhibitory peptides can exercise their

0.001

0.006

1.2 ± 0.021

0.009

0.031

0.018

0.01

0.005

1.5 ± 0.026

Met 0 ± 0 0.5 ± 0.013 0 ± 0 0 ± 0 0 ± 0 0.6 ±

0.085

0.020

0.3 ± 0.008

0.230

0.008

antihypertensive activity *in vivo*.

Ser 22.7 ± 0.291 1 ±

Asx 9.9 ± 0.063 1.2 ± 0.009 0.8 ±

Glx 5.1 ± 0.214 1.4 ± 0.048 1.1 ±

His 8.2 ± 0.677 0.4 ± 0.015 0.3 ±

Gly 19.6 ± 0.983 1.4 ± 0.008 1.7 ±

Thr 4.3 ± 0.987 0.3 ± 0.005 0.4 ±

Arg 3.2 ± 0.065 0.8 ± 0.002 0.8 ±

Ala 14.3 ± 0.620 0.4 ± 0.001 0.3 ±

Cys 0 ± 0 6.6 ± 0.001 1.3 ±

Ile 0.4 ± 0.555 3.2 ± 0.041 0.2 ±

Phe 0 ± 0 0.8 ± 0.003 89.6 ±

0.012

0.006

0.532 0.3 ± 0.007 0.3 ±

purified by preparative RP-HPLC C-18 chromatography.

Tyr 0 ± 0 71.3 ±

Leu 9.3 ± 0.217 10.2 ±

Lys 3±

0.001

Amino acid

Although the precise substrate specificity is not fully understood, ACE-I appears to prefer substrates containing hydrophobic amino acid residues at the three C-terminal positions, suggesting that the higher hydrophobic amino acid content in F5-2 (9.1 g/100g), F5-3 (89.8 g/100g) and F5-4 (34.2 g/100g) versus F5-1 (1.1 g/100g) probably made a substantial contribution to their inhibitory potency. This agrees with Wu et al. [40], who state that aromatic, positively-charged and hydrophobic amino acids are preferred in active tripeptides. Due to substrate specificity differences between the two ACE-I catalytic sites, ACE-I inhibitors may inhibit only one site. Moskowitz [46] proposed a model explaining the clinical superiority of hydrophobic ACE-I inhibitory drugs relative to hydrophilic ones: all ACE-I inhibitors bind to the C-terminal catalytic site, but only hydrophobic ones bind to the occluded N-terminal catalytic site and are therefore better at blocking Ang II production. This would also explain why hydrophobic ACE-I inhibitors have specific local benefits such as organ damage prevention, in addition to reducing blood pressure [46]. The high hydrophobic amino acid (particularly aromatic side-chains) content in the F5 may therefore make a substantial contribution to its fractions' ACE inhibitory activity by blocking Ang II production.

Overall, the highest *in vitro* ACE-I inhibitory activity (IC50) among the cowpea hydrolysate and its derivative fractions was present in the ultrafiltered <1 kDa fraction (0.04 g/mL), followed by the RP-HPLC F4-2 fraction (0.4704 g/mL), the gel filtration chromatography fraction F4 (14.195 g/mL) and finally the hydrolysate (2634.4 g/mL). Although the IC50 values for the hydrolysate, F4 and F4-2 fractions were substantially higher than that of the F<1 kDa fraction, they are still in the same order of magnitude as values reported for many other natural ACE-I inhibitory peptides. Nevertheless, the IC50 values for all the studied *V. unguiculata* derivatives are far higher than that of the synthetic ACE-I inhibitor Captopril® (0.0013 g/mL) [32]. The biological potential in the peptides purified from *V. unguiculata*, and the high ACE-I inhibitory activity in the F<1 kDa fraction, reinforce the need for ACE-I inhibitory peptides to be rich in hydrophobic amino acids (aromatic or branched chains) and peptides rich in Pro. Pro is well-documented as the most favorable amino acid for ACE-I binding (most commercial inhibitors include this residue), but it was not present in the peptides with potential biological activity that had been purified by G-50 gel filtration chromatography and RP-HPLC.

The studied cowpea *V. unguiculata* protein hydrolysate has potential applications in the development of physiologically functional foods aimed at preventing and/or treating hypertension. An added benefit is the balanced amino acid profile of the protein hydrolysate and its peptide fractions, which makes them an appropriate protein source in human nutrition. It should be considered, however, that the *in vitro* ACE-I inhibitory potencies of peptides do not always correlate with their *in vivo* antihypertensive activities as quantified in SHR. This is because they must be absorbed and transported intact from the intestine to the blood stream (in the case of oral administration) and resist plasma peptidase degradation (in the case of oral and intravenous administration) to reach their target sites and exert an antihypertensive effect *in vivo*. Therefore, *in vivo* research is needed to determine to what extent any of the studied ACE-I inhibitory peptides can exercise their antihypertensive activity *in vivo*.

198 Bioactive Food Peptides in Health and Disease

chromatography and RP-HPLC.

tripeptides with ACE-I inhibitory activity due to their low lipophilicity and steric properties or side chain bulk/molecular size values. In addition, Lys and Arg are expected in positions adjacent to the amino terminus due to their low electronic properties and high lipophilicity and steric property values, while residues such as Phe are preferred at the carboxyl terminus

Although the precise substrate specificity is not fully understood, ACE-I appears to prefer substrates containing hydrophobic amino acid residues at the three C-terminal positions, suggesting that the higher hydrophobic amino acid content in F5-2 (9.1 g/100g), F5-3 (89.8 g/100g) and F5-4 (34.2 g/100g) versus F5-1 (1.1 g/100g) probably made a substantial contribution to their inhibitory potency. This agrees with Wu et al. [40], who state that aromatic, positively-charged and hydrophobic amino acids are preferred in active tripeptides. Due to substrate specificity differences between the two ACE-I catalytic sites, ACE-I inhibitors may inhibit only one site. Moskowitz [46] proposed a model explaining the clinical superiority of hydrophobic ACE-I inhibitory drugs relative to hydrophilic ones: all ACE-I inhibitors bind to the C-terminal catalytic site, but only hydrophobic ones bind to the occluded N-terminal catalytic site and are therefore better at blocking Ang II production. This would also explain why hydrophobic ACE-I inhibitors have specific local benefits such as organ damage prevention, in addition to reducing blood pressure [46]. The high hydrophobic amino acid (particularly aromatic side-chains) content in the F5 may therefore make a substantial

due to their low lipophilicity and high steric and electronic property values [41].

contribution to its fractions' ACE inhibitory activity by blocking Ang II production.

Overall, the highest *in vitro* ACE-I inhibitory activity (IC50) among the cowpea hydrolysate and its derivative fractions was present in the ultrafiltered <1 kDa fraction (0.04 g/mL), followed by the RP-HPLC F4-2 fraction (0.4704 g/mL), the gel filtration chromatography fraction F4 (14.195 g/mL) and finally the hydrolysate (2634.4 g/mL). Although the IC50 values for the hydrolysate, F4 and F4-2 fractions were substantially higher than that of the F<1 kDa fraction, they are still in the same order of magnitude as values reported for many other natural ACE-I inhibitory peptides. Nevertheless, the IC50 values for all the studied *V. unguiculata* derivatives are far higher than that of the synthetic ACE-I inhibitor Captopril® (0.0013 g/mL) [32]. The biological potential in the peptides purified from *V. unguiculata*, and the high ACE-I inhibitory activity in the F<1 kDa fraction, reinforce the need for ACE-I inhibitory peptides to be rich in hydrophobic amino acids (aromatic or branched chains) and peptides rich in Pro. Pro is well-documented as the most favorable amino acid for ACE-I binding (most commercial inhibitors include this residue), but it was not present in the peptides with potential biological activity that had been purified by G-50 gel filtration

The studied cowpea *V. unguiculata* protein hydrolysate has potential applications in the development of physiologically functional foods aimed at preventing and/or treating hypertension. An added benefit is the balanced amino acid profile of the protein hydrolysate and its peptide fractions, which makes them an appropriate protein source in human nutrition. It should be considered, however, that the *in vitro* ACE-I inhibitory potencies of peptides do not always correlate with their *in vivo* antihypertensive activities as quantified in SHR. This is because they must be absorbed and transported intact from the


**Table 3.** Amino acid contents of fractions from the F4 and F5 gel filtration chromatography fraction purified by preparative RP-HPLC C-18 chromatography.

## **4. Conclusions**

After modification by Alcalase®, Flavourzyme® and pepsin-pancreatin cowpea *V. unguiculata* proteins proved to be a source of bioactive peptides with ACE-I inhibitory and antioxidant activity. Fractionation of *V. unguiculata* enzymatic hydrolysates by ultrafiltration enhanced their ACE-I inhibitory and antioxidant activity in all the resulting peptides, although the <1 kDa fraction of the Flavourzyme hydrolysate had the highest overall biological activity. Further purification of this fraction by gel filtration chromatography and RP-HPLC produced fractions with different activities, all of which were much higher than the source hydrolysate. Separation of protein hydrolysates by molecular weight and hydrophobicity clearly enhanced peptide ACE-I inhibitory activity, particularly purification by ultrafiltration and chromatography. The highest biological potential among the purified peptides was observed in the ultrafiltered <1 kDa fraction, which supports the importance of high hydrophobic amino acid and proline content in ACE-I inhibitory peptides. These results highlight the promise of controlled protein hydrolysis with a fungal protease complex to isolate bioactive peptides from cowpea *V. unguiculata* proteins, which can then be further purified and/or used as an ingredient in functional foods designed for specific diets.

Vigna Unguiculata as Source of Angiotensin-I Converting Enzyme Inhibitory and Antioxidant Peptides 201

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[8] Moller NP, Scholz-Ahrens KE, Schrezenmeir NR. Bioactive peptides and protein from foods: indication for health effects. European Journal of Nutrition 2008; 47: 171-182. [9] Korhonen H, Pihlanto A. Bioactive peptides: Production and functionality. International

[10] Araujo-González F, Chel-Guerrero L, Betancur-Ancona D. Functional properties of hydrolysates from cowpea (*V. unguiculata*) seeds. Advances in Food Science and Food

[11] Betancur-Ancona D, Gallegos-Tintoré S, Chel-Guerrero L. Wet fractionation of *Phaseolus lunatus* seeds: partial characterization of starch and protein. Journal of the Science of

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[13] Megías C, Yust MM, Pedroche J, Lquari H, Girón-Calle J, Alaiz M, Millán F, Vioque J. Purification of an ACE inhibitory peptide after hydrolysis of sunflower (*Helianthus annuus* L.) protein isolates. Journal of Agricultural and Food Chemistry 2004; 52: 1928-

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rubisco. Journal of Agricultural and Food Chemistry 2003; 51: 4897-4902.

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5901.

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## **Author details**

Maira R. Segura-Campos, Luis A. Chel-Guerrero and David A. Betancur- Ancona\* *Facultad de Ingeniería Química, Campus de Ciencias Exactas e Ingenierías, Universidad Autónoma de Yucatán, Periférico Nte, Tablaje Catastral 13615, Col. Chuburná de Hidalgo Inn, Mérida, Yucatán, México* 

## **Acknowledgement**

This research was supported by the Consejo Nacional de Ciencia y Tecnología (CONACYT) through doctoral scholarship 164186. This research forms part of Project 153012 "Actividad biológica de fracciones peptídicas derivadas de la hidrólisis enzimática de proteínas de frijoles lima (*Phaseolus lunatus*) y caupí (*Vigna unguiculata*)", financed by the CONACYT.

#### **5. References**


<sup>\*</sup> Corresponding Authors

[3] Tzakos AG, Galanis AS, Spyroulias GA, Cordopatis P, Manessi-Zoupa E, Gerothanassis IP. Structure-function discrimination of the N and C-catalytic domains of human angiotensin-converting enzyme: implications for Cl-activation and peptide hydrolysis mechanisms. Protein Engineering 2003; 16: 993-1003.

200 Bioactive Food Peptides in Health and Disease

functional foods designed for specific diets.

*Col. Chuburná de Hidalgo Inn, Mérida, Yucatán, México* 

After modification by Alcalase®, Flavourzyme® and pepsin-pancreatin cowpea *V. unguiculata* proteins proved to be a source of bioactive peptides with ACE-I inhibitory and antioxidant activity. Fractionation of *V. unguiculata* enzymatic hydrolysates by ultrafiltration enhanced their ACE-I inhibitory and antioxidant activity in all the resulting peptides, although the <1 kDa fraction of the Flavourzyme hydrolysate had the highest overall biological activity. Further purification of this fraction by gel filtration chromatography and RP-HPLC produced fractions with different activities, all of which were much higher than the source hydrolysate. Separation of protein hydrolysates by molecular weight and hydrophobicity clearly enhanced peptide ACE-I inhibitory activity, particularly purification by ultrafiltration and chromatography. The highest biological potential among the purified peptides was observed in the ultrafiltered <1 kDa fraction, which supports the importance of high hydrophobic amino acid and proline content in ACE-I inhibitory peptides. These results highlight the promise of controlled protein hydrolysis with a fungal protease complex to isolate bioactive peptides from cowpea *V. unguiculata* proteins, which can then be further purified and/or used as an ingredient in

Maira R. Segura-Campos, Luis A. Chel-Guerrero and David A. Betancur- Ancona\*

This research was supported by the Consejo Nacional de Ciencia y Tecnología (CONACYT) through doctoral scholarship 164186. This research forms part of Project 153012 "Actividad biológica de fracciones peptídicas derivadas de la hidrólisis enzimática de proteínas de frijoles lima (*Phaseolus lunatus*) y caupí (*Vigna unguiculata*)", financed by the CONACYT.

[1] Miguel M, Alonso M, Salaices M, Aleixandre A, López-Fandiño R. Antihypertensive, ACE inhibitory and vasodilator properties of an egg white hydrolysate: Effect of a

[2] Hooper NM, Turner AJ. An ACE structure. Nature Structural & Molecular Biology

simulated intestinal digestion. Food Chemistry 2011; 104: 163-168.

*Facultad de Ingeniería Química, Campus de Ciencias Exactas e Ingenierías, Universidad Autónoma de Yucatán, Periférico Nte, Tablaje Catastral 13615,* 

**4. Conclusions** 

**Author details** 

**Acknowledgement** 

**5. References** 

 \*

2003; 10: 155-157.

Corresponding Authors


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Vigna Unguiculata as Source of Angiotensin-I Converting Enzyme Inhibitory and Antioxidant Peptides 203

[32] Hwang JS, Ko WC. Angiotensin I-converting enzyme inhibitory activity of protein hydrolysates from tuna broth. Journal of Food and Drug Analysis 2004; 12(3): 232-237. [33] Cao W, Zhang C, Hong P, Ji H, Hao J. Purification and identification of an ACE inhibitory peptide from the peptic hydrolysates of *Acetes chinensis* and its antihypertensive effects in spontaneously hypertensive rats. International Journal of

[34] Wang L, Xu H, He X, Wang X. Studies on the preparation of ACE inhibitory peptides from konjac fly powder by alkaline protease enzymolysis. Journal of Chinese Institute

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[45] Matsui T, Hayashi A, Tamaya K, Matsumoto K, Kawasaki T, Murakami K, Kimoto K. Depressor effect induced by dipeptide, Val-Tyr, in part, to the suppression of human circulating renin-angiotensin system. Clinical and Experimental Pharmacology 2003; 30: 262-265.

**Chapter 8** 

© 2013 Hsu 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 Hsu 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.

**Dipeptidyl Peptidase-IV Inhibitory Activity of** 

**Peptides in Porcine Skin Gelatin Hydrolysates** 

During a meal, two incretin hormones, glucagon-like peptide 1 (GLP-1) and glucosedependent insulinotropic polypeptide (GIP), are released from the small intestine into the vasculature and augment glucose-induced insulin secretion from the islet β-cells [1]. It has been estimated that 50-60% of the total insulin secreted during a meal results from the incretin response, mainly the combined effects of GIP and GLP-1 [2]. Previous studies have shown both GIP and GLP-1 stimulate β-cell proliferation, differentiation, and prevent apoptosis [3-6]. GLP-1 also has some actions such as inhibition of glucagon secretion and food intake, glucose homeostasis and slowing the gastric emptying [7-9]. However, GLP-1 has a short half-life of 1- 2 min following secretion in response to the nutrients ingestion because of its inactivation by

DPP-IV (CD26; E.C. 3.4.14.5) is a 110-kDa plasma membrane glycoprotein ectopeptidase that belongs to the prolyl oligopeptidase family [11]. It acts as a cleaving enzyme with the specificity for removing X-Pro or X-Ala dipeptides from the N terminus of polypeptides and proteins. It has a strong preference for Pro > Ala > Ser as the penultimate amino acid residue [10-12]. This enzyme is also capable of cleavage of N-terminal dipeptides with hydroxyproline (Hyp), dehydroproline, Gly, Val, Thr or Leu [10-14]. GLP-1 has Ala as the N-terminal penultimate amino acid residue, and therefore it is the substrate of DPP-IV. This finding that over 95% of the degradation of GLP-1 is attributed to the action of DPP-IV led to an elevated interest in inhibition of this enzyme for the treatment of type 2 diabetes [15]. Some previous studies have shown that specific DPP-IV inhibition increased the half-life of total circulating GLP-1, decreased plasma glucose, and improved impaired glucose

There are several chemical compounds used *in vitro* and in animal models to inhibit DPP-IV activity, such as Val-pyrrolidide [17], NVP-DPP728 [18], Lys[Z(NO2)]-thiazolidide and

dipeptidyl peptidase-IV (DPP-IV) [10], resulting in loss of insulinotropic activities.

Kuo-Chiang Hsu, Yu-Shan Tung, Shih-Li Huang and Chia-Ling Jao

Additional information is available at the end of the chapter

tolerance in animal and human experiments [16-18].

http://dx.doi.org/10.5772/51264

**1. Introduction** 

[46] Moskowitz DW. Is 'somatic' angiotensin I-converting enzyme a mechanosensor? Diabetes Technology and Therapeutics 2003; 4: 841-858.

## **Dipeptidyl Peptidase-IV Inhibitory Activity of Peptides in Porcine Skin Gelatin Hydrolysates**

Kuo-Chiang Hsu, Yu-Shan Tung, Shih-Li Huang and Chia-Ling Jao

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/51264

## **1. Introduction**

204 Bioactive Food Peptides in Health and Disease

262-265.

[45] Matsui T, Hayashi A, Tamaya K, Matsumoto K, Kawasaki T, Murakami K, Kimoto K. Depressor effect induced by dipeptide, Val-Tyr, in part, to the suppression of human circulating renin-angiotensin system. Clinical and Experimental Pharmacology 2003; 30:

[46] Moskowitz DW. Is 'somatic' angiotensin I-converting enzyme a mechanosensor?

Diabetes Technology and Therapeutics 2003; 4: 841-858.

During a meal, two incretin hormones, glucagon-like peptide 1 (GLP-1) and glucosedependent insulinotropic polypeptide (GIP), are released from the small intestine into the vasculature and augment glucose-induced insulin secretion from the islet β-cells [1]. It has been estimated that 50-60% of the total insulin secreted during a meal results from the incretin response, mainly the combined effects of GIP and GLP-1 [2]. Previous studies have shown both GIP and GLP-1 stimulate β-cell proliferation, differentiation, and prevent apoptosis [3-6]. GLP-1 also has some actions such as inhibition of glucagon secretion and food intake, glucose homeostasis and slowing the gastric emptying [7-9]. However, GLP-1 has a short half-life of 1- 2 min following secretion in response to the nutrients ingestion because of its inactivation by dipeptidyl peptidase-IV (DPP-IV) [10], resulting in loss of insulinotropic activities.

DPP-IV (CD26; E.C. 3.4.14.5) is a 110-kDa plasma membrane glycoprotein ectopeptidase that belongs to the prolyl oligopeptidase family [11]. It acts as a cleaving enzyme with the specificity for removing X-Pro or X-Ala dipeptides from the N terminus of polypeptides and proteins. It has a strong preference for Pro > Ala > Ser as the penultimate amino acid residue [10-12]. This enzyme is also capable of cleavage of N-terminal dipeptides with hydroxyproline (Hyp), dehydroproline, Gly, Val, Thr or Leu [10-14]. GLP-1 has Ala as the N-terminal penultimate amino acid residue, and therefore it is the substrate of DPP-IV. This finding that over 95% of the degradation of GLP-1 is attributed to the action of DPP-IV led to an elevated interest in inhibition of this enzyme for the treatment of type 2 diabetes [15]. Some previous studies have shown that specific DPP-IV inhibition increased the half-life of total circulating GLP-1, decreased plasma glucose, and improved impaired glucose tolerance in animal and human experiments [16-18].

There are several chemical compounds used *in vitro* and in animal models to inhibit DPP-IV activity, such as Val-pyrrolidide [17], NVP-DPP728 [18], Lys[Z(NO2)]-thiazolidide and

© 2013 Hsu 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 Hsu 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.

Lys[Z(NO2)]-pyrrolidide [19]. However, such chemical compounds, which often have to be administered by injection, may result in side effects as chemical drugs often do. Thus, to develop safe and natural DPP-IV inhibitors as the therapeutic agents of type 2 diabetes is necessary.

Dipeptidyl Peptidase-IV Inhibitory Activity of Peptides in Porcine Skin Gelatin Hydrolysates 207

zymes North America Inc. (Salem, NC, Canada). DPP-IV (D7052, from porcine kidney), Gly-Pro-*p*-nitroanilide hydrochloride, trichloroacetic acid (TCA), L-Leu and Diprotin A were purchased from Sigma-Aldrich. Trinitrobenzenesulfonic acid (TNBS) was from Fluka Biochemika (Oakville, ON, Canada). Other chemicals and reagents used were analytical

One gram of the gelatin added with 50 mL ddH2O was incubated at 50°C for 10 min prior to the enzymatic hydrolysis. ALA in liquid form were weighed 10, 30, 50 mg and mixed with 1 mL ddH2O. The hydrolysis reaction was started by the addition of enzymes at various enzyme/substrate ratios (E/S: 1%, 3%, and 5%). The reaction with ALA was conducted at pH 8.0, respectively, and 50°C for up to 6 h. After hydrolysis, the hydrolysates were heated in boiling water for 15 min to inactivate enzymes and then cooled in cold water at room temperature for 20 min. Hydrolysates were adjusted their pH to 7.0 with 1 M NaOH and centrifuged (Du Pont Sorvall Centrifuge RC 5B, Mandel Scientific Co. Ltd, Guelph, ON, Canada) at 12,000*g* and room temperature for 15 min. The supernatant was lyophilized and

Immediately prior to termination of hydrolysis, 4 mL of the hydrolysate were mixed with an equal volume of 24% TCA solution and centrifuged at 12200*g* for 5 min. The supernatant (0.2 mL) was added to 2.0 mL of 0.05 M sodium tetraborate buffer (pH 9.2) and 1 mL of 4.0 mM TNBS and incubated at room temperature for 30 min in the dark. Then the mixture was added with 1.0 mL of 2.0 M NaH2PO4 containing 18 mM Na2SO3, and the absorbance was measured at 420 nm using a spectrophotometer (Cary 50 Bio UV-vis spectrophotometer, Varian, Inc., Santa Clara, CA, USA) [35,36]. Degree of hydrolysis (DH) was calculated as % DH=(*h*/*h*tot) × 100, where DH=percent ratio of the number of peptide bonds broken (*h*) to the total number bonds per unit weight (*h*tot) and *h*tot=11.1 mequiv/g of gelatin [35]. L-Leu was

DPP-IV activity determination in this study was performed in 96-well microplates measuring the increase in absorbance at 405 nm using Gly-Pro-p-nitroanilide as DPP-IV substrate [37]. The lyophilized hydrolysates were dissolved in 100 mM Tris buffer (pH 8.0) to the concentration of 10 mg/mL and then serially diluted. The hydrolysates (25 μL) were added with 25 μL of 1.59 mM Gly-Pro-p-nitroanilide (in 100 mM Tris buffer, pH 8.0). The mixture was incubated at 37°C °C for 10 min, followed by the addition of 50 μL of DPP-IV (diluted with the same Tris buffer to 0.01 Unit/mL). The reaction mixture was incubated at 37°C for 60 min, and the reaction was stopped by adding 100 μL of 1 M sodium acetate buffer (pH 4.0). The absorbance of the resulting solution was measured at 405 nm with a microplate reader (iEMS reader MF; Labsystems, Helsinki, Finland). Under the conditions of

grade and commercially available.

**2.3. Measurement of degree of hydrolysis** 

used for drawing a standard curve.

**2.4. Determination of DPP-IV inhibitory activity** 

**2.2. Enzymatic hydrolysis** 

stored at -25°C .

Proteins are well known as precursors of bioactive peptides. In recent years, peptides have been identified to possess physiological functions, such as immunomodulatory [20], antimicrobial [21], antihypertensive [22], anticancer [23], antioxidative [24] and cholesterollowering activities [25]. These bioactive peptides are mostly derived from milk, wheat, soybean, egg and fish proteins by enzymatic hydrolysis or fermentation [26]. Food protein hydrolysates are well-used and natural food ingredients, and therefore they are believed to be safe for consumers when they are served as functional foods. Some studies have reported that bioactive peptides possessed DPP-IV inhibitory activity. Diprotins A and B, isolated from culture filtrates of *Bacillus cereus* BMF673-RF1, are bioactive peptides found to exhibit the DPP-IV inhibitory activity with IC50 values of 1.1 and 5.5 μg/mL; they were elucidated to be Ile-Pro-Ile and Val-Pro-Leu, respectively [27]. Two bioactive peptides, Ile-Pro-Ala and Val-Ala-Gly-Thr-Trp-Tyr, derived from β-lactoglobulin hydrolysed by proteinase K and trypsin, showed IC50 values of 49 and 174 μM, respectively, against DPP-IV *in vitro* [28,29]. Two patents, WO 2006/068480 and WO 2009/128713 have shown that peptides derived from casein and lysozyme hydrolysates, respectively display DPP-IV inhibitory activity, and the peptides show in particular the presence of at least one Pro within the sequence and mostly as the penultimate N-terminal residue [30,31].

It is well-known that the dominant amino acid in gelatin is Gly, while the imino acids (Pro and Hyp) come second in abundance [32]. The amino acid composition of gelatin is characterized by a repeating sequence of Gly-X-Y triplets, where X is mostly Pro and Y is mostly Hyp. Inside gelatin molecule, Gly constitutes approximately 27% of the total amino acid pool [33]. The total amount of the imino acids is higher in mammalian (20-24%) than in fish (16-20%). In our previous study, we successfully isolated two peptides, Gly-Pro-Ala-Glu and Gly-Pro-Gly-Ala from Atlantic salmon skin gelatin, that showed dose-dependent inhibitory effects on DPP-IV with IC50 values of 49.6 and 41.9 μM, respectively [34]. According to the report of previous studies, DPP-IV inhibitory peptides consisted of at least one Pro and mostly as the penultimate N-terminal residue [32]. Therefore, the aim of this study was to examine the DPP-IV inhibitory activity of peptides derived from porcine skin gelatin, which constitutes higher content of imino acids than skin gelatin of Atlantic salmon, a kind of cold-water fish. This is expected to give insight into the possible utilization of porcine skin as a potential source of DPP-IV inhibitors that may be used in the treatment of type 2 diabetes to lower the risk of side effects.

## **2. Materials and methods**

#### **2.1. Materials and reagents**

Porcine skin gelatin (G-2500) was purchased from Sigma-Aldrich (St. Louis, MO, USA). Alcalase® 2.4 L FG (from *Bacillus licheniformis*, 2.4 AU/g) (ALA) was the product from Novozymes North America Inc. (Salem, NC, Canada). DPP-IV (D7052, from porcine kidney), Gly-Pro-*p*-nitroanilide hydrochloride, trichloroacetic acid (TCA), L-Leu and Diprotin A were purchased from Sigma-Aldrich. Trinitrobenzenesulfonic acid (TNBS) was from Fluka Biochemika (Oakville, ON, Canada). Other chemicals and reagents used were analytical grade and commercially available.

#### **2.2. Enzymatic hydrolysis**

206 Bioactive Food Peptides in Health and Disease

as the penultimate N-terminal residue [30,31].

type 2 diabetes to lower the risk of side effects.

**2. Materials and methods** 

**2.1. Materials and reagents** 

necessary.

Lys[Z(NO2)]-pyrrolidide [19]. However, such chemical compounds, which often have to be administered by injection, may result in side effects as chemical drugs often do. Thus, to develop safe and natural DPP-IV inhibitors as the therapeutic agents of type 2 diabetes is

Proteins are well known as precursors of bioactive peptides. In recent years, peptides have been identified to possess physiological functions, such as immunomodulatory [20], antimicrobial [21], antihypertensive [22], anticancer [23], antioxidative [24] and cholesterollowering activities [25]. These bioactive peptides are mostly derived from milk, wheat, soybean, egg and fish proteins by enzymatic hydrolysis or fermentation [26]. Food protein hydrolysates are well-used and natural food ingredients, and therefore they are believed to be safe for consumers when they are served as functional foods. Some studies have reported that bioactive peptides possessed DPP-IV inhibitory activity. Diprotins A and B, isolated from culture filtrates of *Bacillus cereus* BMF673-RF1, are bioactive peptides found to exhibit the DPP-IV inhibitory activity with IC50 values of 1.1 and 5.5 μg/mL; they were elucidated to be Ile-Pro-Ile and Val-Pro-Leu, respectively [27]. Two bioactive peptides, Ile-Pro-Ala and Val-Ala-Gly-Thr-Trp-Tyr, derived from β-lactoglobulin hydrolysed by proteinase K and trypsin, showed IC50 values of 49 and 174 μM, respectively, against DPP-IV *in vitro* [28,29]. Two patents, WO 2006/068480 and WO 2009/128713 have shown that peptides derived from casein and lysozyme hydrolysates, respectively display DPP-IV inhibitory activity, and the peptides show in particular the presence of at least one Pro within the sequence and mostly

It is well-known that the dominant amino acid in gelatin is Gly, while the imino acids (Pro and Hyp) come second in abundance [32]. The amino acid composition of gelatin is characterized by a repeating sequence of Gly-X-Y triplets, where X is mostly Pro and Y is mostly Hyp. Inside gelatin molecule, Gly constitutes approximately 27% of the total amino acid pool [33]. The total amount of the imino acids is higher in mammalian (20-24%) than in fish (16-20%). In our previous study, we successfully isolated two peptides, Gly-Pro-Ala-Glu and Gly-Pro-Gly-Ala from Atlantic salmon skin gelatin, that showed dose-dependent inhibitory effects on DPP-IV with IC50 values of 49.6 and 41.9 μM, respectively [34]. According to the report of previous studies, DPP-IV inhibitory peptides consisted of at least one Pro and mostly as the penultimate N-terminal residue [32]. Therefore, the aim of this study was to examine the DPP-IV inhibitory activity of peptides derived from porcine skin gelatin, which constitutes higher content of imino acids than skin gelatin of Atlantic salmon, a kind of cold-water fish. This is expected to give insight into the possible utilization of porcine skin as a potential source of DPP-IV inhibitors that may be used in the treatment of

Porcine skin gelatin (G-2500) was purchased from Sigma-Aldrich (St. Louis, MO, USA). Alcalase® 2.4 L FG (from *Bacillus licheniformis*, 2.4 AU/g) (ALA) was the product from NovoOne gram of the gelatin added with 50 mL ddH2O was incubated at 50°C for 10 min prior to the enzymatic hydrolysis. ALA in liquid form were weighed 10, 30, 50 mg and mixed with 1 mL ddH2O. The hydrolysis reaction was started by the addition of enzymes at various enzyme/substrate ratios (E/S: 1%, 3%, and 5%). The reaction with ALA was conducted at pH 8.0, respectively, and 50°C for up to 6 h. After hydrolysis, the hydrolysates were heated in boiling water for 15 min to inactivate enzymes and then cooled in cold water at room temperature for 20 min. Hydrolysates were adjusted their pH to 7.0 with 1 M NaOH and centrifuged (Du Pont Sorvall Centrifuge RC 5B, Mandel Scientific Co. Ltd, Guelph, ON, Canada) at 12,000*g* and room temperature for 15 min. The supernatant was lyophilized and stored at -25°C .

#### **2.3. Measurement of degree of hydrolysis**

Immediately prior to termination of hydrolysis, 4 mL of the hydrolysate were mixed with an equal volume of 24% TCA solution and centrifuged at 12200*g* for 5 min. The supernatant (0.2 mL) was added to 2.0 mL of 0.05 M sodium tetraborate buffer (pH 9.2) and 1 mL of 4.0 mM TNBS and incubated at room temperature for 30 min in the dark. Then the mixture was added with 1.0 mL of 2.0 M NaH2PO4 containing 18 mM Na2SO3, and the absorbance was measured at 420 nm using a spectrophotometer (Cary 50 Bio UV-vis spectrophotometer, Varian, Inc., Santa Clara, CA, USA) [35,36]. Degree of hydrolysis (DH) was calculated as % DH=(*h*/*h*tot) × 100, where DH=percent ratio of the number of peptide bonds broken (*h*) to the total number bonds per unit weight (*h*tot) and *h*tot=11.1 mequiv/g of gelatin [35]. L-Leu was used for drawing a standard curve.

#### **2.4. Determination of DPP-IV inhibitory activity**

DPP-IV activity determination in this study was performed in 96-well microplates measuring the increase in absorbance at 405 nm using Gly-Pro-p-nitroanilide as DPP-IV substrate [37]. The lyophilized hydrolysates were dissolved in 100 mM Tris buffer (pH 8.0) to the concentration of 10 mg/mL and then serially diluted. The hydrolysates (25 μL) were added with 25 μL of 1.59 mM Gly-Pro-p-nitroanilide (in 100 mM Tris buffer, pH 8.0). The mixture was incubated at 37°C °C for 10 min, followed by the addition of 50 μL of DPP-IV (diluted with the same Tris buffer to 0.01 Unit/mL). The reaction mixture was incubated at 37°C for 60 min, and the reaction was stopped by adding 100 μL of 1 M sodium acetate buffer (pH 4.0). The absorbance of the resulting solution was measured at 405 nm with a microplate reader (iEMS reader MF; Labsystems, Helsinki, Finland). Under the conditions of

the assay, IC50 values were determined by assaying appropriately diluted samples and plotting the DPP-IV inhibition rate as a function of the hydrolysate concentration.

Dipeptidyl Peptidase-IV Inhibitory Activity of Peptides in Porcine Skin Gelatin Hydrolysates 209

The DH of porcine skin gelatin hydrolyzed with ALA increased dramatically during the initial 1 h, and then increased gradually thereafter (Fig. 1). Also the highest DH was obtained with the highest E/S ratio. The highest DH (%) for ALA was 16.7% and obtained at

**Hydrolysis Time (h)**

**Figure 1.** Degree of hydrolysis of porcine skin gelatin hydrolyzed with ALA at various E/S ratio.

The DPP-IV inhibitory activity of porcine skin gelatin hydrolysates at the concentration of 10 mg/mL was shown in Fig. 2. The gelatin sample without hydrolysis (0 h) showed 9.2% inhibitory rate on DPP-IV. The DPP-IV inhibitory activity of the gelatin hydrolysates increased with E/S ratio and hydrolysis time. The DPP-IV inhibition rates of the 1-h hydrolysates with the E/S ratio of 1, 3 and 5% were 27.2, 44.3 and 48.8%, respectively; while those of 6-h hydrolysates increased to 52.0, 59.7 and 60.0%. The hydrolysates with the E/S

**3.2. DPP-IV inhibitory activity of hydrolysates** 

**01234567**

**E/S: 1% E/S: 3% E/S: 5%**

**3. Results and discussion** 

the E/S ratio of 5% and 6-h hydrolysis.

**3.1. Degree of hydrolysis** 

**Degree of Hydrolysis (%)**

**0**

**5**

**10**

**15**

**20**

**25**

## **2.5. Ultrafiltration**

Hydrolysates were fractionated by ultrafiltration (UF; Model ABL085, Lian Sheng Tech. Co., Taichung, Taiwan) with spiral wound membranes having molecular mass cutoffs of 2.5 and 1 kDa. The fractions were collected as follows: >2.5 kDa, peptides retained without passing through 2.5 kDa membrane; 1-2.5 kDa, peptides permeating through the 2.5 kDa membrane but not the 1 kDa membrane; <1 kDa, peptides permeating through the 1 kDa membrane. All collected fractions were lyophilized and stored in a desiccator until use.

## **2.6. High Performance Liquid Chromatography (HPLC)**

The fractionated hydrolysates by ultrafiltration exhibiting DPP-IV inhibitory activity were further purified using high performance liquid chromatography (Model L-2130 HPLC, Hitachi Ltd., Katsuda, Japan). The lyophilized hydrolysate fraction (100 μg) by gel filtration was dissolved in 1 mL of 0.1% trifluoroacetic acid (TFA) and 90 μL of the mixture, was then injected into a column (ZORBAX Eclipse Plus C18, 4.6 × 250 mm, Agilent Tech. Inc., CA, USA) using a linear gradient of acetonitrile (5 to 15% in 20 min) in 0.1% TFA under a flow rate of 0.7 mL/min. The peptides were detected at 215 nm. Each collected fraction was lyophilized and stored in a desiccator until use.

### **2.7. Identification of amino acid sequence**

An accurate molecular mass and amino acid sequence of the purified peptides was determined using a Q-TOF mass spectrometer (Micromass, Altrincham, UK) coupled with an electrospray (ESI) source. The purified peptides were separately infused into the ESI source after being dissolved in methanol/water (1:1, v/v), and the molecular mass was determined by the doubly charged (M+2H)+2 state in the mass spectrum. Automated Edman sequencing was performed by standard procedures using a 477-A protein sequencer chromatogram (Applied Biosystems, Foster, CA, USA).

#### **2.8. Peptide synthesis**

Peptides were prepared by the conventional Fmoc solid-phase synthesis method with an automatic peptide synthesizer (Model CS 136, CS Bio Co. San Carlos, CA, USA), and their purity was verified by analytical RP-HPLC-MS/MS.

## **2.9. Statistical analysis**

Each data represents the mean of three samples was subjected to analysis of variance (ANOVA) followed by Tukey's studentized range test, and the significance level of *P*<0.05 was employed.

#### **3. Results and discussion**

#### **3.1. Degree of hydrolysis**

208 Bioactive Food Peptides in Health and Disease

**2.5. Ultrafiltration** 

the assay, IC50 values were determined by assaying appropriately diluted samples and

Hydrolysates were fractionated by ultrafiltration (UF; Model ABL085, Lian Sheng Tech. Co., Taichung, Taiwan) with spiral wound membranes having molecular mass cutoffs of 2.5 and 1 kDa. The fractions were collected as follows: >2.5 kDa, peptides retained without passing through 2.5 kDa membrane; 1-2.5 kDa, peptides permeating through the 2.5 kDa membrane but not the 1 kDa membrane; <1 kDa, peptides permeating through the 1 kDa membrane.

The fractionated hydrolysates by ultrafiltration exhibiting DPP-IV inhibitory activity were further purified using high performance liquid chromatography (Model L-2130 HPLC, Hitachi Ltd., Katsuda, Japan). The lyophilized hydrolysate fraction (100 μg) by gel filtration was dissolved in 1 mL of 0.1% trifluoroacetic acid (TFA) and 90 μL of the mixture, was then injected into a column (ZORBAX Eclipse Plus C18, 4.6 × 250 mm, Agilent Tech. Inc., CA, USA) using a linear gradient of acetonitrile (5 to 15% in 20 min) in 0.1% TFA under a flow rate of 0.7 mL/min. The peptides were detected at 215 nm. Each collected fraction was

An accurate molecular mass and amino acid sequence of the purified peptides was determined using a Q-TOF mass spectrometer (Micromass, Altrincham, UK) coupled with an electrospray (ESI) source. The purified peptides were separately infused into the ESI source after being dissolved in methanol/water (1:1, v/v), and the molecular mass was determined by the doubly charged (M+2H)+2 state in the mass spectrum. Automated Edman sequencing was performed by standard procedures using a 477-A protein sequencer

Peptides were prepared by the conventional Fmoc solid-phase synthesis method with an automatic peptide synthesizer (Model CS 136, CS Bio Co. San Carlos, CA, USA), and their

Each data represents the mean of three samples was subjected to analysis of variance (ANOVA) followed by Tukey's studentized range test, and the significance level of *P*<0.05

plotting the DPP-IV inhibition rate as a function of the hydrolysate concentration.

All collected fractions were lyophilized and stored in a desiccator until use.

**2.6. High Performance Liquid Chromatography (HPLC)** 

lyophilized and stored in a desiccator until use.

**2.7. Identification of amino acid sequence** 

chromatogram (Applied Biosystems, Foster, CA, USA).

purity was verified by analytical RP-HPLC-MS/MS.

**2.8. Peptide synthesis** 

**2.9. Statistical analysis** 

was employed.

The DH of porcine skin gelatin hydrolyzed with ALA increased dramatically during the initial 1 h, and then increased gradually thereafter (Fig. 1). Also the highest DH was obtained with the highest E/S ratio. The highest DH (%) for ALA was 16.7% and obtained at the E/S ratio of 5% and 6-h hydrolysis.

**Figure 1.** Degree of hydrolysis of porcine skin gelatin hydrolyzed with ALA at various E/S ratio.

#### **3.2. DPP-IV inhibitory activity of hydrolysates**

The DPP-IV inhibitory activity of porcine skin gelatin hydrolysates at the concentration of 10 mg/mL was shown in Fig. 2. The gelatin sample without hydrolysis (0 h) showed 9.2% inhibitory rate on DPP-IV. The DPP-IV inhibitory activity of the gelatin hydrolysates increased with E/S ratio and hydrolysis time. The DPP-IV inhibition rates of the 1-h hydrolysates with the E/S ratio of 1, 3 and 5% were 27.2, 44.3 and 48.8%, respectively; while those of 6-h hydrolysates increased to 52.0, 59.7 and 60.0%. The hydrolysates with the E/S ratio of 3% and 5%, and the hydrolysis time of 4 h and 6 h showed the highest DPP-IV inhibition rates between 57.4 to 60.0% among all the samples (*P*<0.05); in the meanwhile, the inhibition rates between the four samples were not significantly different (*P*>0.05). The results showed that the hydrolysates with the smaller size of peptides due to the higher DH possessed greater DPP-IV inhibitory activity. Patent WO 2006/068480 has demonstrated that the hydrolysates possessed great DPP-IV inhibitory activities referred to a mixture of peptides derived from hydrolysis of proteins with the percentage of hydrolysed peptide bonds of most preferably 20 to 40% [30]. As the economic concern for saving time and enzyme used, the hydrolysate with E/S ratio of 3% and 4-h hydrolysis was adopted for furher purification and analysis.

Dipeptidyl Peptidase-IV Inhibitory Activity of Peptides in Porcine Skin Gelatin Hydrolysates 211

b

**y = 38.9423 + 27.3496 ln (x)**

DPP-IV inhibition rates of 30.6% and 30.7%, respectively (*P*<0.05), than that within the > 2.5 kDa fraction displaying an inhibition rate of 28.2%. < 1-kDa fraction was selected for further analysis on the basis of the small size peptides may pass through the digestive tract without degradation. The IC50 value of the < 1 kDa fraction was determined and found as 1.50 mg/mL (Fig. 3B). The result in this study is in agreement with the former studies using various protein sources that reported the preferable DPP-IV inhibitory peptides derived from food protein consisted of 2-8 amino acid residues [30,31], and their molecular weights

> **Molecular mass cut-off >2.5 kDa 1-2.5 kDa <1 kD**

**Concentration (mg/mL) 0123456**

**Figure 3.** (A) DPP-IV inhibition rate of porcine skin gelatin hydrolysate fractionated by UF at the concentration of 1 mg/mL. (B) DPP-IV inhibition rate of the < 1 kDa UF fraction at various

concentrations. Different letters indicate the significant differences (*P*<0.05).

**R2**

 **= 0.9754**

**IC50 = 1.50 mg/mL**

b

were supposed between 200 to 1000 Da.

**A**

**Inhibition Rate (%)**

**Inhibition Rate (%)**

**0**

**20**

**40**

**60**

**80**

**100**

**B**

**0**

**10**

**20**

**30**

a

**40**

**Figure 2.** DPP-IV inhibitory rate of porcine skin gelatin hydrolysates. Different letters indicate the significant differences (*P*<0.05).

#### **3.3. DPP-IV inhibitory activtiy of hydrolysates fractionated by UF**

The DPP-IV inhibitory activity of hydrolysates with the E/S ratio of 3% and 4-h hydrolysis at the concentration of 1 mg/mL fractionated by UF was shown in Fig. 3A. The result showed the UF fractions of 1-2.5 kDa and < 1 kDa had insignificantly different (*P*>0.05) and higher DPP-IV inhibition rates of 30.6% and 30.7%, respectively (*P*<0.05), than that within the > 2.5 kDa fraction displaying an inhibition rate of 28.2%. < 1-kDa fraction was selected for further analysis on the basis of the small size peptides may pass through the digestive tract without degradation. The IC50 value of the < 1 kDa fraction was determined and found as 1.50 mg/mL (Fig. 3B). The result in this study is in agreement with the former studies using various protein sources that reported the preferable DPP-IV inhibitory peptides derived from food protein consisted of 2-8 amino acid residues [30,31], and their molecular weights were supposed between 200 to 1000 Da.

210 Bioactive Food Peptides in Health and Disease

furher purification and analysis.

**Gelatin without hydrolysis**

b

d

d

**E/S: 1% E/S: 3% E/S: 5%**

a

**Inhibition Rate (%)**

**0**

significant differences (*P*<0.05).

**10**

**20**

**30**

**40**

**50**

**60**

**70**

ratio of 3% and 5%, and the hydrolysis time of 4 h and 6 h showed the highest DPP-IV inhibition rates between 57.4 to 60.0% among all the samples (*P*<0.05); in the meanwhile, the inhibition rates between the four samples were not significantly different (*P*>0.05). The results showed that the hydrolysates with the smaller size of peptides due to the higher DH possessed greater DPP-IV inhibitory activity. Patent WO 2006/068480 has demonstrated that the hydrolysates possessed great DPP-IV inhibitory activities referred to a mixture of peptides derived from hydrolysis of proteins with the percentage of hydrolysed peptide bonds of most preferably 20 to 40% [30]. As the economic concern for saving time and enzyme used, the hydrolysate with E/S ratio of 3% and 4-h hydrolysis was adopted for

**Hydrolysis Time (h)**

The DPP-IV inhibitory activity of hydrolysates with the E/S ratio of 3% and 4-h hydrolysis at the concentration of 1 mg/mL fractionated by UF was shown in Fig. 3A. The result showed the UF fractions of 1-2.5 kDa and < 1 kDa had insignificantly different (*P*>0.05) and higher

**Figure 2.** DPP-IV inhibitory rate of porcine skin gelatin hydrolysates. Different letters indicate the

**3.3. DPP-IV inhibitory activtiy of hydrolysates fractionated by UF** 

**01246**

c

fg

ef <sup>g</sup> fg h

<sup>h</sup> <sup>h</sup> <sup>h</sup>

**Figure 3.** (A) DPP-IV inhibition rate of porcine skin gelatin hydrolysate fractionated by UF at the concentration of 1 mg/mL. (B) DPP-IV inhibition rate of the < 1 kDa UF fraction at various concentrations. Different letters indicate the significant differences (*P*<0.05).

#### **3.4. Purification of DPP-IV-inhibitory peptides by HPLC**

The elution profile and DPP-IV inhibitory activity of the peptide fractions from the < 1 kDa UF fraction separated by HPLC were shown in Fig. 4A and B. To obtain a sufficient amount of purified peptide, chromatographic separations were performed repeatedly. Five fractions (F-1 to F-5) were obtained upon HPLC separation of the < 1 kDa UF fraction (Fig. 4A), and they were lyophilized and then used to determine their DPP-IV inhibitory activities at the concentration of 100 μg/mL. The result showed that the fraction F-3 had the highest DPP-IV inhibition rate of 64.6% (*P*<0.05), as compared to the others which showed the inhibition rates between 26.7 to 45.6%, and its IC50 value was also determined as 62.9 μg/mL (Fig. 4C). Therefore, the fraction F-3 was used to identify the amino acid sequences of the peptides.

Dipeptidyl Peptidase-IV Inhibitory Activity of Peptides in Porcine Skin Gelatin Hydrolysates 213

Two peptides were identified in fraction F-3, and their amino acid sequences were Gly-Pro-Hyp (285.3 Da) and Gly-Pro-Ala-Gly (300.4 Da). Patent WO 2006/068480 has reported that 21 peptides which were capable of inhibiting DPP-IV activity showed a hydrophobic character, had a length varying from 3-7 amino acid residues and in particular the presence of Pro residue within the sequence [30]. The Pro residue was located as the first, second, third or fourth N-terminal residue, but mostly as the second N-terminal residue. Besides, the Pro residue was flanked by Leu, Val, Phe, Ala and Gly. In the present study, both peptides comprised Pro as the second N-terminal residue, and the Pro residue was flanked by Ala and Gly. Moreover, the peptides were composed of mostly hydrophobic amino acid residues, such as Ala, Gly and Pro, and one peptide comprised a charged amino acid, Glu, as the C-terminal residue. The present results therefore are consistent with the hypothesis demonstrated in the previous study

The DPP-IV inhibitory activity of the two synthetic peptides and Diprotin A at various concentrations was determined (Fig. 5). The IC50 was calculated for each of the peptides. Diprotin A is well-known as the peptide with the greatest DPP-IV inhibitory activity, and its IC50 value was found as 24.7 μM in the present study (Fig. 4C). The IC50 values of the two synthetic peptides, Gly-Pro-Hyp and Gly-Pro-Ala-Gly, were 49.6 and 41.9 μM, respectively (Fig. 4A, B). In the previous study, the IC50 values against DPP-IV of Diprotin A and Diprotin B isolated from culture filtrates of *B. cereus* BMF673-RF1 were 3.2 and 16.8 μM, respectively [27]. Moreover, Ile-Pro-Ala and Val-Ala-Gly-Thr-Trp-Tyr, both prepared from β-lactoglobulin showed IC50 values of 49 and 174 μM against DPP-IV, respectively [28,29]. Patent WO 2006/068480 reported that Diprotin A showed the IC50 value of about 5 μM against DPP-IV, and five peptides, His-Pro-Ile-Lys, Leu-Pro-Leu-Pro, Leu-Pro-Val-Pro, Met-Pro-Leu-Trp and Gly-Pro-Phe-Pro, comprised 4 amino acids with Pro as the penultimate N-terminal residue displayed their IC50 values between 76 to 120 μM [30]. The results showed that the two peptides obtained in this study showed lower DPP-IV inhibitory activity than only Diprotin A and B, which were composed with 3 amino acid residues. However, they had similar inhibition effect to Ile-Pro-Ala but greater than other peptides comprised 4 or more amino acid residues. It is interesting that the ultimate N-terminal residues of the peptides mentioned above are all hydrophobic amino acids, and therefore we assumed that DPP-IV inhibitory activity of bioactive peptides may be determined by the amino acid length and the two N-terminal amino acid sequence of X-Pro, where X is the hydrophobic amino acid and preferably smaller in size. In conclusion, we found two peptides, Gly-Pro-Hyp and Gly-Pro-Ala-Gly, isolated from porcine skin gelatin hydrolysates having the inhibitory activity

**3.5. Amino acid sequence of DPP-IV inhibitory peptides** 

**3.6. DPP-IV-inhibitory activity of the synthetic peptides** 

[30].

against DPP-IV.

**Figure 4.** (A) Elution profile and (B) DPP-IV inhibition rate of the peptide fractions from the < 1 kDa UF fraction separated by HPLC. (C) DPP-IV inhibition rate of fraction F-3 at various concentrations. The DPP-IV inhibition rate was determined with each HPLC fraction at the concentration of 100 μg/mL.

#### **3.5. Amino acid sequence of DPP-IV inhibitory peptides**

212 Bioactive Food Peptides in Health and Disease

peptides.

**3.4. Purification of DPP-IV-inhibitory peptides by HPLC** 

The elution profile and DPP-IV inhibitory activity of the peptide fractions from the < 1 kDa UF fraction separated by HPLC were shown in Fig. 4A and B. To obtain a sufficient amount of purified peptide, chromatographic separations were performed repeatedly. Five fractions (F-1 to F-5) were obtained upon HPLC separation of the < 1 kDa UF fraction (Fig. 4A), and they were lyophilized and then used to determine their DPP-IV inhibitory activities at the concentration of 100 μg/mL. The result showed that the fraction F-3 had the highest DPP-IV inhibition rate of 64.6% (*P*<0.05), as compared to the others which showed the inhibition rates between 26.7 to 45.6%, and its IC50 value was also determined as 62.9 μg/mL (Fig. 4C). Therefore, the fraction F-3 was used to identify the amino acid sequences of the

**Figure 4.** (A) Elution profile and (B) DPP-IV inhibition rate of the peptide fractions from the < 1 kDa UF fraction separated by HPLC. (C) DPP-IV inhibition rate of fraction F-3 at various concentrations. The DPP-IV inhibition rate was determined with each HPLC fraction at the concentration of 100 μg/mL.

Two peptides were identified in fraction F-3, and their amino acid sequences were Gly-Pro-Hyp (285.3 Da) and Gly-Pro-Ala-Gly (300.4 Da). Patent WO 2006/068480 has reported that 21 peptides which were capable of inhibiting DPP-IV activity showed a hydrophobic character, had a length varying from 3-7 amino acid residues and in particular the presence of Pro residue within the sequence [30]. The Pro residue was located as the first, second, third or fourth N-terminal residue, but mostly as the second N-terminal residue. Besides, the Pro residue was flanked by Leu, Val, Phe, Ala and Gly. In the present study, both peptides comprised Pro as the second N-terminal residue, and the Pro residue was flanked by Ala and Gly. Moreover, the peptides were composed of mostly hydrophobic amino acid residues, such as Ala, Gly and Pro, and one peptide comprised a charged amino acid, Glu, as the C-terminal residue. The present results therefore are consistent with the hypothesis demonstrated in the previous study [30].

#### **3.6. DPP-IV-inhibitory activity of the synthetic peptides**

The DPP-IV inhibitory activity of the two synthetic peptides and Diprotin A at various concentrations was determined (Fig. 5). The IC50 was calculated for each of the peptides. Diprotin A is well-known as the peptide with the greatest DPP-IV inhibitory activity, and its IC50 value was found as 24.7 μM in the present study (Fig. 4C). The IC50 values of the two synthetic peptides, Gly-Pro-Hyp and Gly-Pro-Ala-Gly, were 49.6 and 41.9 μM, respectively (Fig. 4A, B). In the previous study, the IC50 values against DPP-IV of Diprotin A and Diprotin B isolated from culture filtrates of *B. cereus* BMF673-RF1 were 3.2 and 16.8 μM, respectively [27]. Moreover, Ile-Pro-Ala and Val-Ala-Gly-Thr-Trp-Tyr, both prepared from β-lactoglobulin showed IC50 values of 49 and 174 μM against DPP-IV, respectively [28,29]. Patent WO 2006/068480 reported that Diprotin A showed the IC50 value of about 5 μM against DPP-IV, and five peptides, His-Pro-Ile-Lys, Leu-Pro-Leu-Pro, Leu-Pro-Val-Pro, Met-Pro-Leu-Trp and Gly-Pro-Phe-Pro, comprised 4 amino acids with Pro as the penultimate N-terminal residue displayed their IC50 values between 76 to 120 μM [30]. The results showed that the two peptides obtained in this study showed lower DPP-IV inhibitory activity than only Diprotin A and B, which were composed with 3 amino acid residues. However, they had similar inhibition effect to Ile-Pro-Ala but greater than other peptides comprised 4 or more amino acid residues. It is interesting that the ultimate N-terminal residues of the peptides mentioned above are all hydrophobic amino acids, and therefore we assumed that DPP-IV inhibitory activity of bioactive peptides may be determined by the amino acid length and the two N-terminal amino acid sequence of X-Pro, where X is the hydrophobic amino acid and preferably smaller in size. In conclusion, we found two peptides, Gly-Pro-Hyp and Gly-Pro-Ala-Gly, isolated from porcine skin gelatin hydrolysates having the inhibitory activity against DPP-IV.

Dipeptidyl Peptidase-IV Inhibitory Activity of Peptides in Porcine Skin Gelatin Hydrolysates 215

This study has clearly demonstrated that porcine skin gelatin could be a good protein source to produce DPP-IV inhibitory peptides by hydrolysis with ALA. The two peptides identified in this study may have the potential for the therapy or prevention of type 2 diabetes. Further studies using *in vitro* simulated gastrointestinal digestion and Caco-2 cell permeate analysis and *in vivo* animal model systems are therefore necessary to ascertain the required standards of evidence for DPP-IV inhibitory potential of bioactive peptides derived from porcine skin

*Department of Nutrition, China Medical University, Taiwan, Republic of China* 

*National Kaohsiung University of Hospitality and Tourism, Taiwan, Republic of China* 

[1] Creutzfeldt W. The Entero-Insular Axis in Type 2 Diabetes-Incretins as Therapeutic Agents. Experimental and Clinical Endocrinology & Diabetes 2001;109 288-300. [2] Creutzfeldt W, Nauck MA. Gut Hormones and Diabetes Mellitus. Diabetes/Metabolism

[3] Drucker DJ. Enhancing Incretin Action for the Treatment of Type 2 Diabetes. Diabetes

[4] Ehses JA, Casilla V, Doty T, Pospisilik JA, Demuth HU, Pederson RA. Glucose-Dependent Insulinotropic Polypeptide Pormotes β-(INS-1) Cell Survival via Cyclic Adenosine Monophosphate-Mediated Caspase-3 Inhibition and Regulation of p38

[5] Trümper A, Trümper K, Trusheim H, Arnold R, Göke B, Hörsch D. Glucose-Dependent Insulinotropic Polypeptide is a Growth Factor for β-(INS-1) Cells by Pleiotropic

[6] Drucker DJ. Glucagon-Like Peptides: Regulators of Cell Proliferation, Differentiation

[7] Ahren B. GLP-1 and Extra-Islet Effects. Hormone and Metabolic Research 2004;36 842-

Mitogen-Activated Protein Kinase. Endocrinology 2003;144 4433-4455.

Signaling. Molucular Endocrinology 2001;15 1559-1570.

and Apoptosis. Molecular Endocrinology 2003;17 161-171.

**4. Conclusion** 

**Author details** 

Shih-Li Huang

Chia-Ling Jao

**5. References** 

845.

Kuo-Chiang Hsu and Yu-Shan Tung

*Department of Baking Technology and Management,* 

*Department of Food and Beverage Management,* 

Research and Reviews 1992;8 565-573.

Care 2003;26 2929-2940.

*Tung Fang Design University, Taiwan, Republic of China* 

gelatin.

**Figure 5.** DPP-IV inhibition rates and IC50 values of the synthetic peptides and diprotin A.

## **4. Conclusion**

214 Bioactive Food Peptides in Health and Disease

**Figure 5.** DPP-IV inhibition rates and IC50 values of the synthetic peptides and diprotin A.

This study has clearly demonstrated that porcine skin gelatin could be a good protein source to produce DPP-IV inhibitory peptides by hydrolysis with ALA. The two peptides identified in this study may have the potential for the therapy or prevention of type 2 diabetes. Further studies using *in vitro* simulated gastrointestinal digestion and Caco-2 cell permeate analysis and *in vivo* animal model systems are therefore necessary to ascertain the required standards of evidence for DPP-IV inhibitory potential of bioactive peptides derived from porcine skin gelatin.

## **Author details**

Kuo-Chiang Hsu and Yu-Shan Tung *Department of Nutrition, China Medical University, Taiwan, Republic of China* 

Shih-Li Huang *Department of Baking Technology and Management, National Kaohsiung University of Hospitality and Tourism, Taiwan, Republic of China* 

Chia-Ling Jao *Department of Food and Beverage Management, Tung Fang Design University, Taiwan, Republic of China* 

## **5. References**


[8] De León DD, Crutchlow MF, Ham JYN, Stoffers AS. Role of Glucagon-Like Peptide-1 in the Pathogenesis and Treatment of Diabetes Mellitus. The International Journal of Biochemistry & Cell Biology 2006;38 845-859.

Dipeptidyl Peptidase-IV Inhibitory Activity of Peptides in Porcine Skin Gelatin Hydrolysates 217

[21] Galvez A, Abriouel H, Lopez RL, Ben Omar N. Bacteriocin-Based Strategies for Food

[22] Hsu KC, Cheng ML, Hwang JS. Hydrolysates from Tuna Cooking Juice as An Anti-

[23] Kim SE, Kim HH, Kim JY, Kang YI, Woo HJ, Lee HJ. Anticancer Activity of

[24] Hsu KC, Lu GH, Jao CL. (2009). Antioxidative Properties of Peptides Prepared from Tuna Cooking Juice Hydrolysates with Orientase (*Bacillus subtilis*). Food Research

[25] Fukui K, Tachibana N, Wanezaki S, Tsuzaki S, Takamatsu K, Yamamoto T, Hashimoto Y, Shimoda T. (2002). Isoflavone-Free Soy Protein Prepared by Column Chromatography Reduces Plasma Cholesterol in Rats. Journal of Agricultural and Food

[26] Wang W, de Mejia EG. A New Frontier in Soy Bioactive Peptides That May Prevent Age-Related Chronic Diseases. Comprehensive Reviews in Food Science and Food

[27] Umezawa H, Aoyagi T, Ogawa K, Naganawa H, Hamada M, Takeuchi T. Diprotins A and B, Inhibitors of Dipeptidyl Aminopeptidase IV, Produced by Bacteria. The Journal

[28] Tulipano G, Sibilia V, Caroli AM, Cocchi D. Whey Proteins as Source of Dipeptidyl Dipeptidase IV (Dipeptidyl Peptidase-4) Inhibitors. Peptides 2011;32 835-

[29] Uchida M, Ohshiba Y, Orie, Mogami. Novel Dipeptidyl Peptidase-4-Inhibiting Peptide Derived from β-lactoglobulin. Journal of Pharmacological Sciences 2011;117 63-

[30] Pieter BJW. WO 2006/068480 200, Protein Hydrolysate Enriched in Peptides Inhibiting

[31] Aart VA, Catharina MJ, Zeeland-Wolbers V, Maria LA, Gilst V, Hendrikus W, Nelissen

[32] Tabata Y, Ikada Y. Protein Release from Gelatin Matrices. Advanced Drug Delivery

[33] Eastoe JE, Leach AA. Hemical Constitution of Gelatin. In The Science and Technology of Gelatin. Ward AG, Courts A, Eds. Academic Press: New York, NY; 1977. p73-107. [34] Li-Chan ECY, Huang SL, Jao CL, Ho KP, Hsu KC. Peptides Derived from Atlantic Salmon Skin Gelatin as Dipeptidyl-Peptidase IV Inhibitors. Journal of Agricultural and

[35] Alder-Nissen J. Enzymic Hydrolysis of Food Proteins. Elsevier Applied Science

[36] Lo WMY, Li-Chan ECY. Angiotensin I Converting Enzyme Inhibitory Peptides from in vitro Pepsin-Pancreatin Digestion of Soy Protein. Journal of Agricultural and Food

BJH, Maria JWP. WO 2009/128713, Egg Protein Hydrolysates. 2009.

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[8] De León DD, Crutchlow MF, Ham JYN, Stoffers AS. Role of Glucagon-Like Peptide-1 in the Pathogenesis and Treatment of Diabetes Mellitus. The International Journal of

[9] Hansotia T, Drucker DJ. GIP and GLP-1 as Incretin Hormones: Lessons from Single and Double Incretin Receptor Knockout Mice. Regulatory Peptides 2005;128 125-134. [10] Mentlein R, Gallwitz B, Schmidt WE. Dipeptidyl-Peptidase IV Hydrolyses Gastric Inhibitory Polypeptide, Glucagon-Like Peptide-1 (7-36) Amide, and Peptide Histidine Methionine and is Responsible for Their Degradation in Human Serum. European

[11] De Meester I, Lambeir AM, Proost P, Scharpé S. Dipeptidyl Peptidase IV Substrates.

[12] Lambeir AM, Durinx C, Scharpé S, De Meester I. Dipeptidyl-Peptidase IV from Bench to Bedside: An Update on Structural Properties, Functions and Clinical Aspects of the Enzyme DPP IV. Critical Reviews in Clinical Laboratory Science 2003;40

[13] Mentlein R. Dipeptidyl-Peptidase IV (CD-26)-Role in the Inactivation of Regulatory

[14] Augustyns K, Bal G, Thonus G, Belyaev A, Zhang XM, Bollaert W. The Unique Properties of Dipeptidyl Peptidase IV Inhibitors. Current Medicinal Chemistry 1999;6

[15] McIntosh CHS, Demuth HU, Pospisilik JA, Pederson R. Dipeptidyl Peptidase IV Inhibitors: How do They Work as New Antidiabetic Agents. Regulatory Peptides

[16] Deacon CF, Hughes TE, Holst JJ. Dipeptidyl Peptidase IV Inhibition Potentiates the Insulinotropic Effect of Glucagon-Like Peptide 1 in the Anesthetized Pig. Diabetes

[17] Deacon CF, Nauck MA, Meier J, Hücking K, Holst JJ. Degradation of Endogenous and Exogenous Gastric Inhibitory Polypeptide in Healthy and in Type 2 Diabetes Subjects as Revealed Using a New Assay for the Intact Peptide. The Journal of Clinical

[18] Mitani H, Takimoto M, Hughes TE, Kimura M. Dipeptidyl Peptidase IV Inhibition Improves Impaired Glucose Tolerance in High-Fat Diet-Fed Rats: Study Using a Fischer 344 Rat Substrain Deficient in Its Enzyme Activity. The Japanese Journal of

[19] Reinhold D, Vetterb RW, Mnich K, Bühling F, Lendeckel U, Born I, Faust J, Neubert K, Gollnick H, Ansorge S. Dipeptidyl Peptidase IV (DP IV, CD26) is Involved in Regulation of DNA Synthesis in Human Keratinocytes. FEBS Letters 1998;428 100-

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**Chapter 9** 

© 2013 Sarray 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 Sarray 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.

**Snake Venom Peptides:** 

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/51263

structures known as focal adhesions [4].

**1. Introduction** 

**Promising Molecules with Anti-Tumor Effects** 

Tumorigenesis and metastasis are two processes with inter-related mechanisms. These include tumor growth and angiogenesis, detachment of tumor cells from the primary tumor, followed by migration through the local connective tissue and penetration into the circulation (intravasation). Once in the blood stream, tumor cells interact with circulating blood cells, arrest in the microvasculature of target organs, then extravasate and secondary proliferate. During each of these steps, integrin-mediated adhesion, migration, proliferation

Integrins are a family of heterodimeric transmembrane receptors that mediate cell-cell and cell-extracellular matrix (ECM) interactions. These cell adhesion molecules are composed by non covalent association of α and β subunits. Although 18 α and 8 β subunits have been described, only 24 different combinations have been identified to date [3]. Specific integrin heterodimers preferentially bind distinct ECM proteins. The repertory of integrins present on a given cell dictates the extent to which that cell will adhere to and migrate on different matrices. Several integrins, among others αv and α5β1, recognize the RGD sequence on their respective ligands. Other adhesive sequences in ECM proteins have also been observed, including the EILDV and REDV sequences that are recognized by integrin α4β1 in an alternatively spliced form of fibronectin [3]. On ligation to the ECM, integrins cluster in the plane of the membrane and recruit various signalling and adaptor proteins to form

Integrin expression can also vary considerably between normal and tumor tissue. Most notably, integrins αvβ3, α5β1 and αvβ6 are usually expressed at low or undetectable levels in most adult epithelia but can be highly up-regulated in some tumors. Expression levels of some integrins, such as α2β1, decrease in tumor cells; potentially increasing tumor cell dissemination [5]. The integrin αvβ3 is particularly important for tumor growth and

and survival of tumor cells and angiogenic endothelial cells play crucial roles [1,2].

Sameh Sarray, Jose Luis, Mohamed El Ayeb and Naziha Marrakchi

## **Snake Venom Peptides: Promising Molecules with Anti-Tumor Effects**

Sameh Sarray, Jose Luis, Mohamed El Ayeb and Naziha Marrakchi

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/51263

## **1. Introduction**

218 Bioactive Food Peptides in Health and Disease

[37] Kojima K, Ham T, Kato T. Rapid Chromatographic Purification of Dipeptidyl Peptidase IV in Human Submaxiallry Gland. Journal of Chromatography A 1980;189 233-240.

> Tumorigenesis and metastasis are two processes with inter-related mechanisms. These include tumor growth and angiogenesis, detachment of tumor cells from the primary tumor, followed by migration through the local connective tissue and penetration into the circulation (intravasation). Once in the blood stream, tumor cells interact with circulating blood cells, arrest in the microvasculature of target organs, then extravasate and secondary proliferate. During each of these steps, integrin-mediated adhesion, migration, proliferation and survival of tumor cells and angiogenic endothelial cells play crucial roles [1,2].

> Integrins are a family of heterodimeric transmembrane receptors that mediate cell-cell and cell-extracellular matrix (ECM) interactions. These cell adhesion molecules are composed by non covalent association of α and β subunits. Although 18 α and 8 β subunits have been described, only 24 different combinations have been identified to date [3]. Specific integrin heterodimers preferentially bind distinct ECM proteins. The repertory of integrins present on a given cell dictates the extent to which that cell will adhere to and migrate on different matrices. Several integrins, among others αv and α5β1, recognize the RGD sequence on their respective ligands. Other adhesive sequences in ECM proteins have also been observed, including the EILDV and REDV sequences that are recognized by integrin α4β1 in an alternatively spliced form of fibronectin [3]. On ligation to the ECM, integrins cluster in the plane of the membrane and recruit various signalling and adaptor proteins to form structures known as focal adhesions [4].

> Integrin expression can also vary considerably between normal and tumor tissue. Most notably, integrins αvβ3, α5β1 and αvβ6 are usually expressed at low or undetectable levels in most adult epithelia but can be highly up-regulated in some tumors. Expression levels of some integrins, such as α2β1, decrease in tumor cells; potentially increasing tumor cell dissemination [5]. The integrin αvβ3 is particularly important for tumor growth and

© 2013 Sarray 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 Sarray 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.

invasiveness [6]. The receptor plays a major role in neo-vessels formation, its expression being strongly up-regulated in endothelial cells and specifically required during angiogenesis stimulated by basic fibroblast growth factor (bFGF) and tumor necrosis factorα [7,8]. αvβ3 is functionally involved in the malignant spread of various tumor cell types such as breast carcinoma, prostate carcinoma and melanoma, and supports tumor cell adhesion and migration through endothelium [9] and matrix proteins [10,1]. Blocking αvβ3 is therefore expected to have a broad impact in cancer therapy and diagnosis. In the last decade, several clinical trials evaluating the efficacy of αvβ3 blockers have led to encouraging results. Thus, MEDI-522 (Vitaxin), a humanized antibody derived from the mouse LM609 monoclonal antibody, was recently reported to give positive results in a phase II trial enrolling patients with stage IV metastatic melanoma [11]. Cilengitide is an inhibitor of both αvβ3 and αvβ5 integrins; it is currently being tested in phase II trials in patients with lung and prostate cancers [12] and in phase II and Phase III trials studying their role against glioblastoma are currently underway.

Snake Venom Peptides: Promising Molecules with Anti-Tumor Effects 221

Most of the functional activities of SVMPs are associated with hemorrhage or the disruption of the hemostatic system, which are primarily mediated by the proteolytic activity of the M domain. SVMPs cause hemorrhage by disturbing the interactions between endothelial cells and the basement membrane through the degradation of endothelial cell membrane proteins (e.g., integrin, cadherin) and basement membrane components (e.g., fibronectin, laminin, nidogen, type IV collagen) [17]. Blood coagulation proteins (e.g., fibrinogen, factor X,

*Echis carinatus* venom contains the specific prothrombin activators, ecarin [18,19] and carinactivase [20]. Adamalysin II, a non-hemorrhagic P-I SVMP isolated from *Crotalus adamantus* venom, cleaves and inactivates serum proteinase inhibitors including antithrombin III [21]. Kaouthiagin, isolated from the venom of *Naja kaouthia* specifically binds and cleaves von Willebrand factor (vWF), resulting in loss of both the ristocetininduced platelet aggregation and collagen-binding activity of vWF [22]. Additionally, a large number of the P-III SVMPs can inhibit platelet aggregation, thus enhancing the hemorrhagic state [23]. The hemorrhagic P-III SVMP jararhagin from the venom of *Bothrops jararaca* has been shown to degrade platelet collagen receptor α2β1 integrin in addition to fibrinogen and vWF, resulting in the inhibition of platelet aggregation [24]. Other platelet receptors are also degraded by SVMPs. GPIbα is cleaved by kistomin; mocarhagin and crotalin [25-27], and GPVI is degraded by alborhagin, crotarhagin and kistomin [28,29].

In the other side, it was reported that several SVMPs inhibited integrin-mediated adhesion of cancer cells on ECM proteins (table 1). BaG, a dimeric PIII class of SVMP from *Bothrops alternatus* with inactivated enzymatic domain but intact D/C domain, has been reported to

**Proteins Snake Integrins Effects References** 

*flavoviridis* - Inhibits adhesion of HUVEC

*gramineus* α1β1;α5β1 Inhibits proliferation and

*stejnegeri* - Inhibits cell proliferation and

<sup>α</sup>3,α6,β1 Induce apoptosis of HUVEC [31,36]

Induces apoptosis of HUVEC

Induces apoptosis of HUVEC

Induces apoptosis of HUVEC

*alternatus* αβ Inhibits adhesion of K562 cells [30]

induces transient cell morphologic changes of endothelial cells.

[32]

[33]

[34]

[35]

[113]

and induces apoptosis

inhibit fibronectin-mediated K562 cell adhesion *via* α5β1 integrin [30].

Halysase *Gloydius halys* <sup>α</sup>1β1;α5β1 Inhibits proliferation and

VLAIPs *Vipera lebetina* - Inhibits proliferation and

VAP1, VAP2 *Crotalus atrox*

Graminelysin *Trimeresurus* 

BaG *Bothrops* 

TSV-DM *Trimeresurus* 

**Table 1.** SVMP affecting tumor cells

HV1 *Trimeruserus* 

prothrombin) are also targets of their proteolytic activities.

In addition to their role in tumor cells, integrins are also important for the host cellular response against cancer. Endothelial cells, fibroblasts, pericytes, bone marrow-derived cells, inflammatory cells and platelets all use integrins for various functions, including angiogenesis, desmoplasia and immune response.

Nature has been a source of medicinal products for thousands of years among which snake venoms form a rich source of bioactive molecules such as peptides, proteins and enzymes with important pharmacological activities. International research and development in this area, based on multidisciplinary approaches including molecular screening, proteomics, genomics and pharmacological *in vitro*, *ex vivo* and *in vivo* assays, allow the identification and characterization of highly specific molecules from snake venom that can potently inhibit integrin functions. These anti-adhesive snake venom proteins belong to different families (phospholipases, disintegrins, C-type lectins and metalloproteinases). By targeting integrins, they exhibit various pharmacological activities such as anti-tumor, anti-angiogenic and/or pro-apoptotic effects.

## **2. Snake venom protein families**

#### **2.1. The Snake Venom Metalloproteinases (SVMP)**

Metalloproteinases are among the most abundant toxins in many *Viperidae* venoms. SVMPs are monozinc endopeptidases varying in size from 20 to 100 kDa. They are phylogenetically most closely related to the mammalian disintegrin and metalloproteinase (ADAM) family of proteins. SVMPs are grouped into several subclasses according to their domain organization [13, 14, 15]. P-I SVMPs are the simplest class of enzymes that contain only a metalloproteinase (M) domain. P-II SVMPs contain a M domain followed by a disintegrin (D) domain. P-III SVMPs contain M, disintegrin-like (D) and cysteine-rich (C) domains. Formally called P-IV, the heterotrimeric class of SVMPs that contain an additional snake C-type lectin-like (snaclec) domain [16] is now included in the P-III group as a subclass (P-IIId).

Most of the functional activities of SVMPs are associated with hemorrhage or the disruption of the hemostatic system, which are primarily mediated by the proteolytic activity of the M domain. SVMPs cause hemorrhage by disturbing the interactions between endothelial cells and the basement membrane through the degradation of endothelial cell membrane proteins (e.g., integrin, cadherin) and basement membrane components (e.g., fibronectin, laminin, nidogen, type IV collagen) [17]. Blood coagulation proteins (e.g., fibrinogen, factor X, prothrombin) are also targets of their proteolytic activities.

*Echis carinatus* venom contains the specific prothrombin activators, ecarin [18,19] and carinactivase [20]. Adamalysin II, a non-hemorrhagic P-I SVMP isolated from *Crotalus adamantus* venom, cleaves and inactivates serum proteinase inhibitors including antithrombin III [21]. Kaouthiagin, isolated from the venom of *Naja kaouthia* specifically binds and cleaves von Willebrand factor (vWF), resulting in loss of both the ristocetininduced platelet aggregation and collagen-binding activity of vWF [22]. Additionally, a large number of the P-III SVMPs can inhibit platelet aggregation, thus enhancing the hemorrhagic state [23]. The hemorrhagic P-III SVMP jararhagin from the venom of *Bothrops jararaca* has been shown to degrade platelet collagen receptor α2β1 integrin in addition to fibrinogen and vWF, resulting in the inhibition of platelet aggregation [24]. Other platelet receptors are also degraded by SVMPs. GPIbα is cleaved by kistomin; mocarhagin and crotalin [25-27], and GPVI is degraded by alborhagin, crotarhagin and kistomin [28,29].


In the other side, it was reported that several SVMPs inhibited integrin-mediated adhesion of cancer cells on ECM proteins (table 1). BaG, a dimeric PIII class of SVMP from *Bothrops alternatus* with inactivated enzymatic domain but intact D/C domain, has been reported to inhibit fibronectin-mediated K562 cell adhesion *via* α5β1 integrin [30].

**Table 1.** SVMP affecting tumor cells

220 Bioactive Food Peptides in Health and Disease

their role against glioblastoma are currently underway.

angiogenesis, desmoplasia and immune response.

pro-apoptotic effects.

**2. Snake venom protein families** 

**2.1. The Snake Venom Metalloproteinases (SVMP)** 

domain [16] is now included in the P-III group as a subclass (P-IIId).

invasiveness [6]. The receptor plays a major role in neo-vessels formation, its expression being strongly up-regulated in endothelial cells and specifically required during angiogenesis stimulated by basic fibroblast growth factor (bFGF) and tumor necrosis factorα [7,8]. αvβ3 is functionally involved in the malignant spread of various tumor cell types such as breast carcinoma, prostate carcinoma and melanoma, and supports tumor cell adhesion and migration through endothelium [9] and matrix proteins [10,1]. Blocking αvβ3 is therefore expected to have a broad impact in cancer therapy and diagnosis. In the last decade, several clinical trials evaluating the efficacy of αvβ3 blockers have led to encouraging results. Thus, MEDI-522 (Vitaxin), a humanized antibody derived from the mouse LM609 monoclonal antibody, was recently reported to give positive results in a phase II trial enrolling patients with stage IV metastatic melanoma [11]. Cilengitide is an inhibitor of both αvβ3 and αvβ5 integrins; it is currently being tested in phase II trials in patients with lung and prostate cancers [12] and in phase II and Phase III trials studying

In addition to their role in tumor cells, integrins are also important for the host cellular response against cancer. Endothelial cells, fibroblasts, pericytes, bone marrow-derived cells, inflammatory cells and platelets all use integrins for various functions, including

Nature has been a source of medicinal products for thousands of years among which snake venoms form a rich source of bioactive molecules such as peptides, proteins and enzymes with important pharmacological activities. International research and development in this area, based on multidisciplinary approaches including molecular screening, proteomics, genomics and pharmacological *in vitro*, *ex vivo* and *in vivo* assays, allow the identification and characterization of highly specific molecules from snake venom that can potently inhibit integrin functions. These anti-adhesive snake venom proteins belong to different families (phospholipases, disintegrins, C-type lectins and metalloproteinases). By targeting integrins, they exhibit various pharmacological activities such as anti-tumor, anti-angiogenic and/or

Metalloproteinases are among the most abundant toxins in many *Viperidae* venoms. SVMPs are monozinc endopeptidases varying in size from 20 to 100 kDa. They are phylogenetically most closely related to the mammalian disintegrin and metalloproteinase (ADAM) family of proteins. SVMPs are grouped into several subclasses according to their domain organization [13, 14, 15]. P-I SVMPs are the simplest class of enzymes that contain only a metalloproteinase (M) domain. P-II SVMPs contain a M domain followed by a disintegrin (D) domain. P-III SVMPs contain M, disintegrin-like (D) and cysteine-rich (C) domains. Formally called P-IV, the heterotrimeric class of SVMPs that contain an additional snake C-type lectin-like (snaclec) Several apoptosis-inducing proteins have been purified from hemorrhagic snake venom, such as VAP1 and VAP2 (*Crotalus atrox*), HV1 (*Trimeresurus flavoviridis*), halysase (*Gloydius halys*), and VLAIPs (*Vipera lebetina*) [31-34], graminelysin [35]. They are members of the SVMP and ADAM family and induce apoptosis of human umbilical vein endothelial cells (HUVECs) [31,36]. The detachment of endothelial cells and resulting apoptosis could be an additional mechanism for the disruption of normal hemostasis by SVMPs. TSV-DM a basic metalloproteinase from *Trimeresurus stejnegeri* venom inhibits cell proliferation and induces cell morphologic changes transiently of ECV304 cells. However, DNA fragmentation and DNA content analysis demonstrated that this metalloproteinase could not induce ECV304 cells apoptosis.

Snake Venom Peptides: Promising Molecules with Anti-Tumor Effects 223

[46]. Triflavin strongly inhibited cell migration toward vitronectin and fibronectin nearly thirty orders of magnitude greater than anti-αvβ3 monoclonal antibodies [46]. Triflavin was also more effective in inhibiting TNF-α-induced angiogenesis in the chicken chorioallantoic membrane (CAM) assay. Similar results were obtained with another RGD-disintegrin, rhodostomin, from *Agkistrodon rhodostoma* venom, which inhibits endothelial cell migration, invasion and tube formation induced by bFGF in MatrigelTM both *in vitro* and *in vivo* [47]. Rhodostomin effects were inhibited by anti-αvβ3 but not by anti-αvβ5 antibodies, thus supporting the hypothesis that the effects of RGD-disintegrins are mediated by blockade of

**Proteins Snake Integrins Effects References** 

Inhibits adhesion of tumor cells to matrix proteins, cell migration and angiogenesis

invasion of endothelial cells; inhibits angiogenesis *in vivo*

Blocks adhesion, migration invasion of different type of

invasion of endothelial cells; anti-angiogenic activity *in vitro* and *in vivo*; elicites

Inhibits cell motility; no effect on cell proliferation or [46]

[47]

[48]

[56]

[58]

[59,60]

[62]

[114]

[115]

*in vitro* and *in vivo*

αvβ3,αvβ5 Inhibits cell migration,

and *in vitro*

tumor cells

α1β1 Inhibits migration and angiogenesis

αvβ3 Prevents migration and

angiogenesis

αvβ3 Anti-angiogenic and antimetastatic effect on melanoma cells

αvβ3 Inhibits cell adhesion of

apoptosis

melanoma tumor cells

αvβ3 Inhibits angiogenesis *in vitro* and *in vivo*; induces

anoïkis

α5β1,αvβ3, α3β1

αvβ3,α5β1, αvβ5, αIIββ3

α4β1,other integrin not yet determined

the vitronectin receptor.

Triflavin *Trimeresurus* 

Rhodostomin *Agikistrodon* 

Contortrostatin *Agkistrodon* 

Lebestatin *Macrovipera* 

Accurhagin-C *Agkistrodon* 

Eristostatin *Eritocophis* 

DisBa-01 *Bothrops* 

Leberagin-C *Macrovipera* 

Accutin *Agkistrodon* 

*flavoviridis* 

*rhodostoma* 

*contortrix contortrix* 

*lebetina* 

*acutus* 

*macmahoni* 

*alternatus* 

*lebetina* 

*acutus* 

**Table 2.** Effects of disintegrins on cancerous cells

## **2.2. The disintegrins**

Disintegrins are a family of non-enzymatic and low molecular weight proteins derived from viper venom [37-39]. They are able to inhibit platelet aggregation and interact with adhesion molecules in particular integrins in a dose-dependent manner. They have a K / RTS sequence which is known as the RGD adhesive loop [37-39]. Their primary structure shows a strong conservation in the arrangement of cysteines [38]. Most disintegrins represent the C-terminal domain of metalloproteinases PIIa, d and e classes and are released into the venom by proteolytic cleavage [40,37,38]. A minority of these proteins exist as D / C domains from the class of SVMPs PIIIb.

Disintegrins can be conveniently divided into five different groups according to their length and the number of disulfide bridges [41]. The first group includes short disintegrins, single polypeptide composed of 49 - 51 amino acids with four disulfide bridges. The second group comprises medium disintegrins containing about 70 amino acids and six disulfide bridges. The third group includes long disintegrins of 83 residues linked by seven disulfide bridges. The disintegrin domains of PIII snake-venom metalloproteinases, containing approx. hundred amino acids with 16 Cysteine residues involved in the formation of eight disulfide bonds, constitute the fourth subgroup of the disintegrin family. Unlike short-, medium- and long-sized disintegrins, which are single-chain molecules**,** the fifth subgroup is composed of homo and heterodimers. The dimeric disintegrins subunits contain about 67 residues with four disulfide intra-chain bridges and two interchain bridges [42,43].

Although disintegrins are highly homologous, significant differences exist in their affinity and selectivity for integrins, which explains the multitude of effects of these molecules (Table 2).

Disintegrins were first identified as inhibitors of platelet aggregation and were subsequently shown to antagonize fibrinogen binding to platelet integrin αIIbβ3 [44,45]. After that, studies on disintegrins have revealed new uses in the diagnosis of cardiovascular diseases and the design of therapeutic agents in arterial thrombosis, osteoporosis, and angiogenesisrelated tumor growth and metastasis (table 2). Triflavin from *Trimeresurus flavoviridis* venom was one of the first RGD-disintegrins shown to inhibit angiogenesis both *in vitro* and *in vivo*  [46]. Triflavin strongly inhibited cell migration toward vitronectin and fibronectin nearly thirty orders of magnitude greater than anti-αvβ3 monoclonal antibodies [46]. Triflavin was also more effective in inhibiting TNF-α-induced angiogenesis in the chicken chorioallantoic membrane (CAM) assay. Similar results were obtained with another RGD-disintegrin, rhodostomin, from *Agkistrodon rhodostoma* venom, which inhibits endothelial cell migration, invasion and tube formation induced by bFGF in MatrigelTM both *in vitro* and *in vivo* [47]. Rhodostomin effects were inhibited by anti-αvβ3 but not by anti-αvβ5 antibodies, thus supporting the hypothesis that the effects of RGD-disintegrins are mediated by blockade of the vitronectin receptor.


**Table 2.** Effects of disintegrins on cancerous cells

222 Bioactive Food Peptides in Health and Disease

cells apoptosis.

(Table 2).

**2.2. The disintegrins** 

domains from the class of SVMPs PIIIb.

Several apoptosis-inducing proteins have been purified from hemorrhagic snake venom, such as VAP1 and VAP2 (*Crotalus atrox*), HV1 (*Trimeresurus flavoviridis*), halysase (*Gloydius halys*), and VLAIPs (*Vipera lebetina*) [31-34], graminelysin [35]. They are members of the SVMP and ADAM family and induce apoptosis of human umbilical vein endothelial cells (HUVECs) [31,36]. The detachment of endothelial cells and resulting apoptosis could be an additional mechanism for the disruption of normal hemostasis by SVMPs. TSV-DM a basic metalloproteinase from *Trimeresurus stejnegeri* venom inhibits cell proliferation and induces cell morphologic changes transiently of ECV304 cells. However, DNA fragmentation and DNA content analysis demonstrated that this metalloproteinase could not induce ECV304

Disintegrins are a family of non-enzymatic and low molecular weight proteins derived from viper venom [37-39]. They are able to inhibit platelet aggregation and interact with adhesion molecules in particular integrins in a dose-dependent manner. They have a K / RTS sequence which is known as the RGD adhesive loop [37-39]. Their primary structure shows a strong conservation in the arrangement of cysteines [38]. Most disintegrins represent the C-terminal domain of metalloproteinases PIIa, d and e classes and are released into the venom by proteolytic cleavage [40,37,38]. A minority of these proteins exist as D / C

Disintegrins can be conveniently divided into five different groups according to their length and the number of disulfide bridges [41]. The first group includes short disintegrins, single polypeptide composed of 49 - 51 amino acids with four disulfide bridges. The second group comprises medium disintegrins containing about 70 amino acids and six disulfide bridges. The third group includes long disintegrins of 83 residues linked by seven disulfide bridges. The disintegrin domains of PIII snake-venom metalloproteinases, containing approx. hundred amino acids with 16 Cysteine residues involved in the formation of eight disulfide bonds, constitute the fourth subgroup of the disintegrin family. Unlike short-, medium- and long-sized disintegrins, which are single-chain molecules**,** the fifth subgroup is composed of homo and heterodimers. The dimeric disintegrins subunits contain about 67 residues with

Although disintegrins are highly homologous, significant differences exist in their affinity and selectivity for integrins, which explains the multitude of effects of these molecules

Disintegrins were first identified as inhibitors of platelet aggregation and were subsequently shown to antagonize fibrinogen binding to platelet integrin αIIbβ3 [44,45]. After that, studies on disintegrins have revealed new uses in the diagnosis of cardiovascular diseases and the design of therapeutic agents in arterial thrombosis, osteoporosis, and angiogenesisrelated tumor growth and metastasis (table 2). Triflavin from *Trimeresurus flavoviridis* venom was one of the first RGD-disintegrins shown to inhibit angiogenesis both *in vitro* and *in vivo* 

four disulfide intra-chain bridges and two interchain bridges [42,43].

Contortrostatin, a disintegrin isolated from the venom of the southern copperhead snake, exhibits anti-cancer activity in a variety of tumor cells [48-50]. It does not display cytotoxic activity *in vitro* nor animals upon injection. Contortrostatin inhibits adhesion, migration, invasion, metastatic and angiogenesis of tumor and endothelial cells mediated by αvβ3,α5β1 and αvβ5 [48,50-54]. Recently, contortrostatin showed an additive inhibitory effect in combination with docetaxel on the growth of xenograft tumors derived from prostate cancer cells [55].

Snake Venom Peptides: Promising Molecules with Anti-Tumor Effects 225

[69,70]. Snake venom sPLA2 are secreted enzymes belonging to only two groups that are based on their primary structure and disulfide bridge pattern [68,71,72]. Those of group I are similar to pancreatic sPLA2 present in mammals, were found in venom of *Elapidae* snakes, while group II PLA2s belong to the *Viperidae* and are similar to mammals nonpancreatic, inflammatory sPLA2s [73,74]. The group II can be subdivided mainly in two subgroups, depending on the residue at position 49 in the primary structure: Aspartic acid-49 PLA2s are enzymatically active, while Lysine 49 present low or no enzymatic activity [75]. There are other subgroups, such as Asparagine-49, Serine-49, Glutamine-49 and Arginine -49 [76-83]. Studies have found that catalytic activity is reduced or even abolished

Despite a high identity of their amino acid sequences, sPLA2 exhibit a wide variety of pharmacological properties such as anticoagulant, haemolytic, neurotoxic, myotoxic, oedema-inducing, hemorrhagic, cytolytic, cardiotoxic, muscarinic inhibitor and antiplatelet

Recently, PLA2s have been shown to possess anti-tumor and anti-angiogenic properties (Table 3). CC-PLA2-1 and CC-PLA2-2 from *Cerastes cerastes* viper are non-toxic and acidic proteins. They have high inhibitory effects on platelet aggregation and coagulation. In addition, CC-PLA2-1 and CC-PLA2-2 inhibit the adhesion of the human fibrosarcoma (HT1080) and melanoma (IGR39) cells to fibrinogen and fibronectin. In the same direction, CC-PLA2-1 and CC-PLA2-2 potently reduces HT1080 cell migration to fibrinogen and fibronectin with nearly similar IC50 values [93]. This anti-adhesive effect was due to the inhibition of α5β1 and αv-containing integrins [94]. A recent report demonstrated that Bth-A-I, a non-toxic PLA2 isolated from *Bothrops jararacussu* venom display an anti-tumoral effect upon breast adenocarcinoma as well as upon human leukaemia T and Erlich ascetic

**Proteins Snake Integrins Effects References** 

*jararacussu* - Anti-tumor activity on

α5β1,αv Inhibits migration and

α5β1,αv Inhibits adhesion and

and *in vitro*.

adhesion of fibrosarcoma and melanoma cells

adenocarcinoma and leukaemia cells

migration of human microvascular cells and inhibits angiogenesis *in vivo* 


[93,94]

[95]

[96]

[97]

when an Aspartic acid of native PLA2 is replaced by another amino acid [80,84].

activities [63,85-92].

tumor [95].

CCPLA2-1; CCPLA2-2

> Bth-A-I-PLA2

> > MVL-PLA2

*Cerastes cerastes* 

*Bothrops* 

*Macrovipera lebetina* 

flavoviridis

BP II Prothobotrops

**Table 3.** PLA2s targeting tumor cells

Lebestatin is an example of a non toxic KTS-disintegrin isolated from *Macrovipera lebetina*  that inhibits migration and VEGF-induced *in vivo* angiogenesis [56]. The presence of a WGD motif in CC8, a heterodimeric disintegrin from *Echis carinatus*, increases its inhibitory effect on αvβ3 and α5β1 integrins [57].

There are few reports regarding the effects of ECD-disintegrins on endothelial cell migration. Acurhagin-C, dose-dependently blocked HUVEC migration toward a vitronectin-coated membrane. Furthermore, acurhagin-C elicited endothelial anoïkis *via* disruption of the αvβ3/FAK/PI3K survival cascade and subsequent initiation of the procaspase-3 apoptotic signaling pathway [58].

Eristostatin, an RGD-disintegrin from *Eristocophis macmahoni* was tested on individual metastasis steps such as cell arrest, extravasation and migration [59]. Eristostatin treatment did not prevent tumor cell extravasation or migration [60]. However, it was shown later that eristostatin inhibited melanoma cell motility, an effect mediated by fibronectin-binding integrins [61]. Interestingly, this disintegrin, contrary to other RGD-disintegrins, did not inhibit angiogenesis, as stated before [61]. DisBa-01, a αvβ3 integrin-blocking RGDdisintegrin, inhibits not only migration of endothelial cells *in vivo* [62] but also *in vitro*  migratory ability of fibroblasts and two tumor cell lines.

Since integrin receptors are also quite indiscriminate as they support cell adhesion to several substrates, it seems highly reasonable that the general RGD-disintegrin scaffold of the integrin-binding motif could be employed as a prototype for drug design for new antimetastatic therapies *via* blocking both tumor cell adhesion and tumor angiogenesis.

### **2.3. The snake venom phospholipases**

Snake venom is one of the most abundant sources of secretory phospholipases A2 (PLA2), which are one of the potent molecules in snake venoms [63-65].

PLA2 (EC 3.1.1.4)—are enzymes that catalyze the hydrolysis of sn-2-acyl bond of sn-3 phospholipids, generating free fatty acids and lysophospholipids as products [66]. They are currently classified in 15 groups and many subgroups that include five distinct types of enzymes, namely secreted PLA2 (sPLA2), cytosolic PLA2 (cPLA2), Ca2+ independent PLA2s (iPLA2), platelet-activating factor acetyl-hydrolases (PAF-AH), lysosomal PLA2, and a recently identified adipose-specific PLA2 [65,67]. PLA2 are low molecular weight proteins with molecular masses ranging from 13-19 kDa that generally require Ca2+ for their activities [69,70]. Snake venom sPLA2 are secreted enzymes belonging to only two groups that are based on their primary structure and disulfide bridge pattern [68,71,72]. Those of group I are similar to pancreatic sPLA2 present in mammals, were found in venom of *Elapidae* snakes, while group II PLA2s belong to the *Viperidae* and are similar to mammals nonpancreatic, inflammatory sPLA2s [73,74]. The group II can be subdivided mainly in two subgroups, depending on the residue at position 49 in the primary structure: Aspartic acid-49 PLA2s are enzymatically active, while Lysine 49 present low or no enzymatic activity [75]. There are other subgroups, such as Asparagine-49, Serine-49, Glutamine-49 and Arginine -49 [76-83]. Studies have found that catalytic activity is reduced or even abolished when an Aspartic acid of native PLA2 is replaced by another amino acid [80,84].

Despite a high identity of their amino acid sequences, sPLA2 exhibit a wide variety of pharmacological properties such as anticoagulant, haemolytic, neurotoxic, myotoxic, oedema-inducing, hemorrhagic, cytolytic, cardiotoxic, muscarinic inhibitor and antiplatelet activities [63,85-92].

Recently, PLA2s have been shown to possess anti-tumor and anti-angiogenic properties (Table 3). CC-PLA2-1 and CC-PLA2-2 from *Cerastes cerastes* viper are non-toxic and acidic proteins. They have high inhibitory effects on platelet aggregation and coagulation. In addition, CC-PLA2-1 and CC-PLA2-2 inhibit the adhesion of the human fibrosarcoma (HT1080) and melanoma (IGR39) cells to fibrinogen and fibronectin. In the same direction, CC-PLA2-1 and CC-PLA2-2 potently reduces HT1080 cell migration to fibrinogen and fibronectin with nearly similar IC50 values [93]. This anti-adhesive effect was due to the inhibition of α5β1 and αv-containing integrins [94]. A recent report demonstrated that Bth-A-I, a non-toxic PLA2 isolated from *Bothrops jararacussu* venom display an anti-tumoral effect upon breast adenocarcinoma as well as upon human leukaemia T and Erlich ascetic tumor [95].


**Table 3.** PLA2s targeting tumor cells

224 Bioactive Food Peptides in Health and Disease

prostate cancer cells [55].

on αvβ3 and α5β1 integrins [57].

procaspase-3 apoptotic signaling pathway [58].

migratory ability of fibroblasts and two tumor cell lines.

which are one of the potent molecules in snake venoms [63-65].

**2.3. The snake venom phospholipases** 

Contortrostatin, a disintegrin isolated from the venom of the southern copperhead snake, exhibits anti-cancer activity in a variety of tumor cells [48-50]. It does not display cytotoxic activity *in vitro* nor animals upon injection. Contortrostatin inhibits adhesion, migration, invasion, metastatic and angiogenesis of tumor and endothelial cells mediated by αvβ3,α5β1 and αvβ5 [48,50-54]. Recently, contortrostatin showed an additive inhibitory effect in combination with docetaxel on the growth of xenograft tumors derived from

Lebestatin is an example of a non toxic KTS-disintegrin isolated from *Macrovipera lebetina*  that inhibits migration and VEGF-induced *in vivo* angiogenesis [56]. The presence of a WGD motif in CC8, a heterodimeric disintegrin from *Echis carinatus*, increases its inhibitory effect

There are few reports regarding the effects of ECD-disintegrins on endothelial cell migration. Acurhagin-C, dose-dependently blocked HUVEC migration toward a vitronectin-coated membrane. Furthermore, acurhagin-C elicited endothelial anoïkis *via* disruption of the αvβ3/FAK/PI3K survival cascade and subsequent initiation of the

Eristostatin, an RGD-disintegrin from *Eristocophis macmahoni* was tested on individual metastasis steps such as cell arrest, extravasation and migration [59]. Eristostatin treatment did not prevent tumor cell extravasation or migration [60]. However, it was shown later that eristostatin inhibited melanoma cell motility, an effect mediated by fibronectin-binding integrins [61]. Interestingly, this disintegrin, contrary to other RGD-disintegrins, did not inhibit angiogenesis, as stated before [61]. DisBa-01, a αvβ3 integrin-blocking RGDdisintegrin, inhibits not only migration of endothelial cells *in vivo* [62] but also *in vitro* 

Since integrin receptors are also quite indiscriminate as they support cell adhesion to several substrates, it seems highly reasonable that the general RGD-disintegrin scaffold of the integrin-binding motif could be employed as a prototype for drug design for new anti-

Snake venom is one of the most abundant sources of secretory phospholipases A2 (PLA2),

PLA2 (EC 3.1.1.4)—are enzymes that catalyze the hydrolysis of sn-2-acyl bond of sn-3 phospholipids, generating free fatty acids and lysophospholipids as products [66]. They are currently classified in 15 groups and many subgroups that include five distinct types of enzymes, namely secreted PLA2 (sPLA2), cytosolic PLA2 (cPLA2), Ca2+ independent PLA2s (iPLA2), platelet-activating factor acetyl-hydrolases (PAF-AH), lysosomal PLA2, and a recently identified adipose-specific PLA2 [65,67]. PLA2 are low molecular weight proteins with molecular masses ranging from 13-19 kDa that generally require Ca2+ for their activities

metastatic therapies *via* blocking both tumor cell adhesion and tumor angiogenesis.

MVL-PLA2 is a snake venom phospholipase purified from Macrovipera lebetina venom that inhibited adhesion and migration of human microvascular endothelial cells (HMEC-1) without being cytotoxic. Using MatrigelTM and chick chorioallantoic membrane assays, MVL-PLA2, as well as its catalytically inactivated form, significantly inhibited angiogenesis both *in vitr*o and *in vivo*. Also, the actin cytoskeleton and the distribution of αvβ3 integrin, a critical regulator of angiogenesis and a major component of focal adhesions, were disturbed after MVL-PLA2 treatment. The enhancement of microtubule dynamics of HMEC-1 cells, in consequence of treatments by MVL-PLA2, may explain the alterations in the formation of focal adhesions, leading to inhibition of cell adhesion and migration [96].

Snake Venom Peptides: Promising Molecules with Anti-Tumor Effects 227

angiogenic efficacy yet described for snake venom-derived peptides [108,109]. These

Extensive researches have been shown that cell adhesion activities in cancer disease are deregulated. According to this idea, it was also reported that lebectin inhibits these alterations by promoting N-cadherin/catenin complex reorganisation at cell-cell contacts,

Another snaclec, BJcuL isolated from *Bothrops jararacussa* venom, was also described for its anti-tumor, but the receptor or integrin implicated has not been determined yet. This homodimeric protein inhibits proliferation of several cell lines of renal, pancreatic, prostate and melanoma origin, but no effect was observed on colon or breast cancer cells [111]. BJcuL also affects the viability of some tumor cell lines of different origins, but has no effect on the growth of K562 and T24 cells, suggesting that these cells do not express the receptor recognized by the lectin. BJcuL induces apoptosis in human gastric carcinoma cells

accompanied by inhibition of cell adhesion and actin cytoskeleton disassembly [112].

**Proteins Snake Integrins Effects References** 

α5β1,αv Inhibits adhesion, migration and

inhibits angiogenesis

invasion of human tumor cells;

 Inhibits tumor cell and endothelial cell growth; induces apoptosis of human gastric carcinoma cells; inhibits cell adhesion and actin cytoskeleton disassembly

α2β1 Inhibits adhesion and migration of

HUVEC cells

Venoms are a rich source of molecules endowed with diverse pharmacological effects. Most part of these molecules act *via* the adhesion molecules. The intervention of the scientists and the clinicians in the pharmaceutical development field would employ these molecules as

Until now, no medicine was produced from a native molecule purified from venom. However, several peptidomimetics were designed by basing on the structure of these molecules. The benefits of these peptidomimetics compared to antibodies that can be used for the treatment of certain diseases are: a shorter half-life, reversible inhibition, easier to control a problem and very low immunogenicity. For example, the antihypertensive drug captopril, modelled from the venom of the Brazilian arrowhead viper (*Bothrops jaracusa*); the anticoagulant Integrilin (eptifibatide), a heptapeptide derived from a protein found in the

therapeutic agents for several pathologies such as cancer, thrombosis, diabetes....

[106]

[111,112]

[104]

observed effects are mediated by α5β1 and αv integrins [107].

inducing a strengthening of intercellular adhesion [110].

Lebecetin, lebectin

*Macrovipera lebetina* 

*jararacussu* 

*multisquamatus*

**Table 4.** Snaclecs and their effects on tumor cells

**3. Potential application of snake venom compounds** 

BJcuL *Bothrops* 

EM16 *Echis* 

A cell death activity was discovered in Lysine 49-PLA2 called BPII. It induces caspaseindependent cell death in human leukaemia cells regardless of its depressed enzymatic activity [97].

## **2.4. The C-type lectins**

The C-type lectins are abundant components of snake venom with various function. Typically, these proteins bind calcium and sugar residues. However, the C-type lectin like proteins from snake venom (termed actually snaclec) does not contain the classic calcium/sugar binding loop and have evolved to bind a wide range of physiologically important proteins and receptors [98].

Snaclecs have a basic heterodimeric structure with two subunits, nearly always linked covalently, *via* a disulphide bond. The heterodimers are often further multimerized either non-covalently or covalently *via* additional disulphide bonds, to form larger structures [99]. The two subunits form a concave surface between them [100] thus constituting the main site of ligand binding [101,102]. The subunits have a high structural degree of homology between them and with other snaclecs [103]. Despite their highly conserved primary structure, the snaclecs are characterized by various biological activities. They were and are still considered as modulators of platelet aggregation by targeting vWF, GPIb-IX-V, GPVI and possibly other platelet receptors.

Recently, novel activities of snaclecs were highlighted. They were described for their potential anti-tumor effect by blocking adhesion, migration, proliferation and invasion of different cancer cell lines (Table 4). Among these proteins, EMS16, a heterodimer isolated from the venom of *Echis multisquamatus*, inhibits the adhesion of HUVECs cells on ECM proteins and their migration by inhibiting the binding of integrin α2β1 to collagen [104].

Lebecetin and lebectin, purified from *Macrovipera lebetina* venom, are the only snaclecs, until today, with an evident anti-tumor effect in addition to their anti-aggregation activity on platelets. Indeed, these two non cytotoxic proteins inhibit the adhesion of various cancer cell lines: melanoma (IGR39), adenocarcinoma (HT29-D4), fibrosarcoma (HT1080) and leukemia cells (K562) on different ECM proteins. They also inhibit the proliferation, migration and invasion of HT1080 cells [105,106]. Lebectin also displays anti-angiogenic activity at very low concentrations both *in vitro* and *in vivo* [107]. Thus, lebectin presents the best antiangiogenic efficacy yet described for snake venom-derived peptides [108,109]. These observed effects are mediated by α5β1 and αv integrins [107].

Extensive researches have been shown that cell adhesion activities in cancer disease are deregulated. According to this idea, it was also reported that lebectin inhibits these alterations by promoting N-cadherin/catenin complex reorganisation at cell-cell contacts, inducing a strengthening of intercellular adhesion [110].

Another snaclec, BJcuL isolated from *Bothrops jararacussa* venom, was also described for its anti-tumor, but the receptor or integrin implicated has not been determined yet. This homodimeric protein inhibits proliferation of several cell lines of renal, pancreatic, prostate and melanoma origin, but no effect was observed on colon or breast cancer cells [111]. BJcuL also affects the viability of some tumor cell lines of different origins, but has no effect on the growth of K562 and T24 cells, suggesting that these cells do not express the receptor recognized by the lectin. BJcuL induces apoptosis in human gastric carcinoma cells accompanied by inhibition of cell adhesion and actin cytoskeleton disassembly [112].


**Table 4.** Snaclecs and their effects on tumor cells

226 Bioactive Food Peptides in Health and Disease

activity [97].

**2.4. The C-type lectins** 

important proteins and receptors [98].

and possibly other platelet receptors.

MVL-PLA2 is a snake venom phospholipase purified from Macrovipera lebetina venom that inhibited adhesion and migration of human microvascular endothelial cells (HMEC-1) without being cytotoxic. Using MatrigelTM and chick chorioallantoic membrane assays, MVL-PLA2, as well as its catalytically inactivated form, significantly inhibited angiogenesis both *in vitr*o and *in vivo*. Also, the actin cytoskeleton and the distribution of αvβ3 integrin, a critical regulator of angiogenesis and a major component of focal adhesions, were disturbed after MVL-PLA2 treatment. The enhancement of microtubule dynamics of HMEC-1 cells, in consequence of treatments by MVL-PLA2, may explain the alterations in the formation of

A cell death activity was discovered in Lysine 49-PLA2 called BPII. It induces caspaseindependent cell death in human leukaemia cells regardless of its depressed enzymatic

The C-type lectins are abundant components of snake venom with various function. Typically, these proteins bind calcium and sugar residues. However, the C-type lectin like proteins from snake venom (termed actually snaclec) does not contain the classic calcium/sugar binding loop and have evolved to bind a wide range of physiologically

Snaclecs have a basic heterodimeric structure with two subunits, nearly always linked covalently, *via* a disulphide bond. The heterodimers are often further multimerized either non-covalently or covalently *via* additional disulphide bonds, to form larger structures [99]. The two subunits form a concave surface between them [100] thus constituting the main site of ligand binding [101,102]. The subunits have a high structural degree of homology between them and with other snaclecs [103]. Despite their highly conserved primary structure, the snaclecs are characterized by various biological activities. They were and are still considered as modulators of platelet aggregation by targeting vWF, GPIb-IX-V, GPVI

Recently, novel activities of snaclecs were highlighted. They were described for their potential anti-tumor effect by blocking adhesion, migration, proliferation and invasion of different cancer cell lines (Table 4). Among these proteins, EMS16, a heterodimer isolated from the venom of *Echis multisquamatus*, inhibits the adhesion of HUVECs cells on ECM proteins and their migration by inhibiting the binding of integrin α2β1 to collagen [104].

Lebecetin and lebectin, purified from *Macrovipera lebetina* venom, are the only snaclecs, until today, with an evident anti-tumor effect in addition to their anti-aggregation activity on platelets. Indeed, these two non cytotoxic proteins inhibit the adhesion of various cancer cell lines: melanoma (IGR39), adenocarcinoma (HT29-D4), fibrosarcoma (HT1080) and leukemia cells (K562) on different ECM proteins. They also inhibit the proliferation, migration and invasion of HT1080 cells [105,106]. Lebectin also displays anti-angiogenic activity at very low concentrations both *in vitro* and *in vivo* [107]. Thus, lebectin presents the best anti-

focal adhesions, leading to inhibition of cell adhesion and migration [96].

## **3. Potential application of snake venom compounds**

Venoms are a rich source of molecules endowed with diverse pharmacological effects. Most part of these molecules act *via* the adhesion molecules. The intervention of the scientists and the clinicians in the pharmaceutical development field would employ these molecules as therapeutic agents for several pathologies such as cancer, thrombosis, diabetes....

Until now, no medicine was produced from a native molecule purified from venom. However, several peptidomimetics were designed by basing on the structure of these molecules. The benefits of these peptidomimetics compared to antibodies that can be used for the treatment of certain diseases are: a shorter half-life, reversible inhibition, easier to control a problem and very low immunogenicity. For example, the antihypertensive drug captopril, modelled from the venom of the Brazilian arrowhead viper (*Bothrops jaracusa*); the anticoagulant Integrilin (eptifibatide), a heptapeptide derived from a protein found in the venom of the American southeastern pygmy rattlesnake (*Sistrurus miliarius barbouri*); Ancrod, a compound isolated from the venom of the Malaysian pit viper (*Agkistrodon rhodostoma*) for use in the treatment of heparin-induced thrombocytopenia and stroke and alfimeprase, a novel fibrinolytic metalloproteinase for thrombolysis derived from southern copperhead snake (*Agkistrodon contortrix contortrix*) venom (Table 5). Two venom proteins from the Australian brown snake, *Pseudonaja textilis*, are currently in development as human therapeutics (QRxPharma). The first is a single agent procoagulant that is a homolog of mammalian Factor Xa prothrombin activator, whereas the other is a plasmin inhibitor, named Textilinin-1, with antihemorrhagic properties.

Snake Venom Peptides: Promising Molecules with Anti-Tumor Effects 229

Actually, most of the current anticancer therapies (radiotherapy, chemotherapy) are not specific and are targeting at both tumor cells and healthy cells. However, in recent years, new treatments tend to focus on the tumor microenvironment and particularly on the inhibition of tumor angiogenesis. These treatments are based on several active and non toxic proteins from snake venom, as for example contortrostatin from *Agkistrodon contortrix contortrix* and eristostatin from *Eristocophis macmahoo.* Although all these molecules are still currently in clinical trials, they could in the future open new ways of healing and could be

From the initial discovery of captopril, the first oral ACE inhibitor, to the recent application of disintegrins for the potential treatment of cancer, the various components of snake venoms have never failed to reveal amazing new properties. While the original native snake venom compounds are usually unsuitable as therapeutics, interventions by medicinal chemists as well as scientists and clinicians in pharmaceutical R&D have made it possible to use the snake venom proteins as potential drugs for multiple disorders or scaffolds for drug

*Research Center of Biologic Oncology and Oncopharmacology (CRO2), INSERM UMR 911,* 

[1] Felding-Habermann, B. Integrin adhesion receptors in tumor metastasis. Clinical and

[2] Fidler IJ. Biological behavior of malignant melanoma cells correlated to their survival in

[3] Barczyk M, Carracedo S, Gullberg D. Integrins. Cell Tissue Research 2010;339(1) 269-280. [4] Berrier AL and Yamada KM. Cell Matrix. Journal of cellular physiology 2007;213(3) 565-

used as drugs.

design.

**4. Conclusions** 

**Author details** 

*Pasteur Institute of Tunis, Tunis Belvedere, Tunisie Faculty of Science, University of Tunis El Manar, Tunisie* 

*University of Aix-Marseille, Marseille, France*  Mohamed El Ayeb and Naziha Marrakchi *Pasteur Institute of Tunis, Tunis Belvedere, Tunisie* 

Experimental Metastasis 2003;20(3) 203–213.

vivo. Cancer Research 1975; 35(1) 218–224.

Sameh Sarray\*

*Marseille, France* 

**5. References** 

573

Corresponding Author

 \*

Jose Luis


**Table 5.** Drugs and clinical diagnostic kits from snake venom

Actually, most of the current anticancer therapies (radiotherapy, chemotherapy) are not specific and are targeting at both tumor cells and healthy cells. However, in recent years, new treatments tend to focus on the tumor microenvironment and particularly on the inhibition of tumor angiogenesis. These treatments are based on several active and non toxic proteins from snake venom, as for example contortrostatin from *Agkistrodon contortrix contortrix* and eristostatin from *Eristocophis macmahoo.* Although all these molecules are still currently in clinical trials, they could in the future open new ways of healing and could be used as drugs.

## **4. Conclusions**

228 Bioactive Food Peptides in Health and Disease

Capoten ®

Integrilin ®

Aggrastat ®

Exanta

Ancrod ® (viprinex)

Protac/ Protein C activator

named Textilinin-1, with antihemorrhagic properties.

**Name Snake Target and** 

*barbouri*

*Cobra* 

contortrix contortrix)

*Agkistrodon rhodostoma*

*Agkistrodon contortrix contortrix* 

**Table 5.** Drugs and clinical diagnostic kits from snake venom

Reptilase *Bothrops jaraca* Diagnosis of blood

Ecarin *Echis carinatus* Prothrombin activator/

(Captropil) *Bothrops jaracusa*

(Eptifibatide) *Sisturus miliarus* 

(tirofiban) *Echis carinatus* 

Alfimeprase (Agkistrodon

hemocoagulase *Bothrops atrox* 

venom of the American southeastern pygmy rattlesnake (*Sistrurus miliarius barbouri*); Ancrod, a compound isolated from the venom of the Malaysian pit viper (*Agkistrodon rhodostoma*) for use in the treatment of heparin-induced thrombocytopenia and stroke and alfimeprase, a novel fibrinolytic metalloproteinase for thrombolysis derived from southern copperhead snake (*Agkistrodon contortrix contortrix*) venom (Table 5). Two venom proteins from the Australian brown snake, *Pseudonaja textilis*, are currently in development as human therapeutics (QRxPharma). The first is a single agent procoagulant that is a homolog of mammalian Factor Xa prothrombin activator, whereas the other is a plasmin inhibitor,

**function/treatment** 

Platelet aggregation inhibitor/acute coronary

GPIIb-IIIa inhibitor/ myocardial infarct, refractory ischemia

Thrombin inhibitor/ arterial fibrillation and

Thrombolytic/ Acute ischemic stroke, acute peripheral arterial

Thrombin-like effect and thromboplastin activity/ prevention and treatment

Protein C activator/clinical diagnosis of haemostatic

of haemorrhage

disorder

Fibrinogen inhibitor/ stroke Phase III

coagulation disorder Granted FDA approval

diagnostic Granted FDA approval

syndrome

blood

occlusion

Angiotensin converted enzyme (ACE) inhibitor/ high blood pressure

**Clinical stage** 

Granted FDA approval

Granted FDA approval

Approved for use with heparin and aspirin for the treatment of acute coronary syndrome

Seeking FDA approval

Phase III

Phase III

Granted FDA approval

From the initial discovery of captopril, the first oral ACE inhibitor, to the recent application of disintegrins for the potential treatment of cancer, the various components of snake venoms have never failed to reveal amazing new properties. While the original native snake venom compounds are usually unsuitable as therapeutics, interventions by medicinal chemists as well as scientists and clinicians in pharmaceutical R&D have made it possible to use the snake venom proteins as potential drugs for multiple disorders or scaffolds for drug design.

## **Author details**

Sameh Sarray\* *Pasteur Institute of Tunis, Tunis Belvedere, Tunisie Faculty of Science, University of Tunis El Manar, Tunisie* 

Jose Luis *Research Center of Biologic Oncology and Oncopharmacology (CRO2), INSERM UMR 911, Marseille, France University of Aix-Marseille, Marseille, France* 

Mohamed El Ayeb and Naziha Marrakchi *Pasteur Institute of Tunis, Tunis Belvedere, Tunisie* 

## **5. References**


<sup>\*</sup> Corresponding Author

[5] Kren A, Baeriswyl V, Lehembre F, Wunderlin C, Strittmatter K, Antoniadis H, Fässler R, Cavallaro U, Christofori G. Increased tumor cell dissemination and cellular senescence in the absence of β1-integrin function. The EMBO Journal 2007;26(12) 2832–2842.

Snake Venom Peptides: Promising Molecules with Anti-Tumor Effects 231

activator (ecarin) from Kenyan Echis carinatus venom. Biochemistry 1995;34 (5) 1771–

[20] Yamada D, Sekiya F, Morita T. Isolation and characterization of carinactivase, a novel prothrombin activator in Echis carinatus venom with a unique catalytic mechanism.

[21] Kress LF and Paroski EA. Enzymatic inactivation of human serum proteinase inhibitors by snake venom proteinases. Biochemical and Biophysics Research Communications

[22] Hamako J, Matsui T, Nishida S, Nomura S, Fujimura Y, Ito M, Ozeki Y, Titani K. Purification and characterization of kaouthiagin, a von Willebrand factor-binding and – cleaving metalloproteinase from Naja kaouthia cobra venom, Thrombosis and

[23] Laing GD, Moura-da-Silva AM. Jararhagin and its multiple effects on hemostasis.

[24] Kamiguti AS. Platelets as targets of snake venom metalloproteinases. Toxicon 2005;45(8)

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jararacussu venom. Protein Journal 2004; 23(4) 273-285.

and disorganization of focal adhesions. PLoS One 2010; 5(4):e10124.


[112] Nolte S, de Castro Damasio D, Baréa AC, Gomes J, Magalhães A, Mello Zischler LF, Stuelp-Campelo PM, Elífio-Esposito SL, Roque-Barreira MC, Reis CA, Moreno-Amaral AN. BJcuL, a lectin purified from Bothrops jararacussu venom, induces apoptosis in human gastric carcinoma cells accompanied by inhibition of cell adhesion and actin cytoskeleton disassembly. Toxicon 2012; 59(1) 81-85.

**Section 3** 

**Production of Bioactive Food Peptides** 


**Production of Bioactive Food Peptides** 

238 Bioactive Food Peptides in Health and Disease

480-489.

29(2)117-126.

cytoskeleton disassembly. Toxicon 2012; 59(1) 81-85.

Biophysica Acta 1998; 1425(3) 493-504.

[112] Nolte S, de Castro Damasio D, Baréa AC, Gomes J, Magalhães A, Mello Zischler LF, Stuelp-Campelo PM, Elífio-Esposito SL, Roque-Barreira MC, Reis CA, Moreno-Amaral AN. BJcuL, a lectin purified from Bothrops jararacussu venom, induces apoptosis in human gastric carcinoma cells accompanied by inhibition of cell adhesion and actin

[113] Wan SG, Jin Y, Lee WH, Zhang Y. A snake venom metalloproteinase that inhibited cell proliferation and induced morphological changes of ECV304 cells. Toxicon 2006; 47(4)

[114] Limam I, Bazaa A, Srairi-Abid N, Taboubi S, Jebali J, Zouari-Kessentini R, Kallech-Ziri O, Mejdoub H, Hammami A, El Ayeb M, Luis J, Marrakchi N. Leberagin-C, A disintegrin-like/cysteine-rich protein from Macrovipera lebetina transmediterranea venom, inhibits alphavbeta3 integrin-mediated cell adhesion. Matrix Biology 2010;

[115] Yeh CH, Peng HC, Yih JB, Huang TF A new short chain RGD-containing disintegrin, accutin, inhibits the common pathway of human platelet aggregation. Biochemica

**Chapter 10** 

© 2013 Muro 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 Muro 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.

**Advancements in the Fractionation of Milk** 

Claudia Muro, Francisco Riera and Ayoa Fernández

reasonable price on an industrial scale (e.g. β-lactoglobulin, β-Lg).

**2. Overview of techniques used for peptide fractionation** 

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/53674

**1. Introduction** 

**Biopeptides by Means of Membrane Processes** 

Nowadays the most common way to obtain bioactive peptides is by enzymatic hydrolysis of protein solutions. The most studied substrates used to produce bioactive peptides are milk proteins in the form of co-products from dairy industries: caseins, cheese whey, buttermilk, whey protein concentrates and isolates or even pure single proteins that can be obtained at a

Different specific and non-specific enzymes are used to obtain hydrolysates (trypsin, pepsin, pancreatin and alcalase). The catalytic activity of some of them is quite specific and the composition of the hydrolysate is predictable when substrates are quite pure [1]. In other cases, the activity of the enzyme is non-specific and produces a complex mixture of peptides and amino acids in which individual effect of each molecule in the subsequent fractionation process is difficult to demonstrate and quantify. The design of an efficient fractionation methodology is then of paramount importance for peptides separation and even more, when the process must applied on an industrial scale. Separation technologies, which discriminate small differences in charge, size and hydrophobicity, can be employed to fractionate protein hydrolysates and obtain peptide fractions with higher functionality or higher nutritional value in a more purified form. Membrane separation techniques seem to be well suited for this purpose. These processes are based upon selective permeability of one or more of the liquid constituents through the membrane according to the driving forces.

Due to the demonstration of their impact on human health, the market for functional food and nutraceuticals containing bioactive peptides is increasing very rapidly and, consequently, the food and bio-pharmaceutic industries are looking for processes allowing

## **Advancements in the Fractionation of Milk Biopeptides by Means of Membrane Processes**

Claudia Muro, Francisco Riera and Ayoa Fernández

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/53674

## **1. Introduction**

Nowadays the most common way to obtain bioactive peptides is by enzymatic hydrolysis of protein solutions. The most studied substrates used to produce bioactive peptides are milk proteins in the form of co-products from dairy industries: caseins, cheese whey, buttermilk, whey protein concentrates and isolates or even pure single proteins that can be obtained at a reasonable price on an industrial scale (e.g. β-lactoglobulin, β-Lg).

Different specific and non-specific enzymes are used to obtain hydrolysates (trypsin, pepsin, pancreatin and alcalase). The catalytic activity of some of them is quite specific and the composition of the hydrolysate is predictable when substrates are quite pure [1]. In other cases, the activity of the enzyme is non-specific and produces a complex mixture of peptides and amino acids in which individual effect of each molecule in the subsequent fractionation process is difficult to demonstrate and quantify. The design of an efficient fractionation methodology is then of paramount importance for peptides separation and even more, when the process must applied on an industrial scale. Separation technologies, which discriminate small differences in charge, size and hydrophobicity, can be employed to fractionate protein hydrolysates and obtain peptide fractions with higher functionality or higher nutritional value in a more purified form. Membrane separation techniques seem to be well suited for this purpose. These processes are based upon selective permeability of one or more of the liquid constituents through the membrane according to the driving forces.

## **2. Overview of techniques used for peptide fractionation**

Due to the demonstration of their impact on human health, the market for functional food and nutraceuticals containing bioactive peptides is increasing very rapidly and, consequently, the food and bio-pharmaceutic industries are looking for processes allowing

the production of this kind of products from natural sources. Considering that most functional peptides are present in complex mixtures containing a large number of hydrolysed protein fractions, their separation and purification are required.

Advancements in the Fractionation of Milk Biopeptides by Means of Membrane Processes 243

scale. In addition, membrane processes are especially suitable for the food industry, because of the mild working conditions, relatively easy scale up and low processing costs in

The separation of peptides by UF mainly depends on the molecular weight (MW) cut-off (MWCO) of the membrane. However, when the MW of the peptides involved in the process is quite similar, their isolation is a hard subject; in these cases, NF is the best membrane separation technique [18]. The fact that NF membranes are usually charged offers the possibility of separating solutes through a combination of size and charge mechanisms.

Membrane processes are now viewed as efficient tools for the development of new valueadded products by separating minor compounds such as bioactive peptides [19]. These separation processes are based upon selective permeability of one or more of the liquid constituents through the membrane according to the pressure difference. Amongst the pressure-driven membrane techniques, which main features are summarized in Figure 1, UF and NF have been tested for the fractionation of protein hydrolysates due to the fact that the molecular weight of most bioactive peptides is within the normal pore size range of these

UF is commonly applied to prepare enriched bioactive solutions from protein hydrolysates and improve the bioactivity of peptides. This process is also used to separate peptides with a size lower than 7 kDa [20]. The fractions are collected by subsequently filtration in two or three streams to obtain peptides with different size [21]. For example, amino acids and small peptides can be separated at pH 4.6 into four ranges of molecular mass (I<30 kDa, II>30 kDa (protein fraction), III>10 kDa (protein fraction), IV>0.3 kDa) [22]. Recent results on fractionation peptides by UF-membranes show that crude yoghurt fractions obtained after ion exchange can be separated into four fractions by successive UF using membranes with

**3. Membrane technology applied to peptide fractionation** 

comparison to chromatographic techniques.

**Figure 1.** Pressure-driven membrane processes

membranes.

The methodologies commonly used for peptide fractionation and enrichment include: selective precipitation, membrane filtration, ion exchange, gel filtration technologies and liquid chromatography [1]. However, significant differences concerning the number and type of extracted peptides occur among extraction procedures. Additionally, undesired peptides, such as allergenic or bitter-tasting peptides, could be enriched in the process when using some of those techniques [2].

Fractionation methods involving precipitation steps are carried out by means of the addition of organic solvents like ethanol, methanol or acetone; adding acids like trichloroacetic acid (TCA), sulphosalicylic acid or phosphotungstic acid (PPTA); by means of the addition of salts (ammonium sulphate) or just by adjusting the pH to the isoelectric point. Precipitation often results in a selective fractionation of peptides depending on their solubility in the precipitating agent [3]; however the addition of chemical compounds causes in some cases peptide degradation and changes in the biological and physical properties.

Chromatographic methods for peptide separation are currently used at lab-scale: high performance liquid chromatography (HPLC), fast protein liquid chromatography (FPLC), isoelectric focusing (IEF) and ion exchange chromatography (IEC) are some of them. In most cases, one or two cycles of successive HPLC separation had been adequate to isolate peptides one by one. In the same way, IEC has been used for the enrichment of casein phosphopeptides from casein hydrolysates or for the isolation of cationic antibacterial peptides from lactoferrin. However, although chromatographic processes can provide good separation selectivity, the low productivity and high production costs involved in these processes make impossible its use at industrial scale.

Size exclusion chromatography (SEC) and more frequently Ultrafiltration-Nanofiltration (UF-NF) are the main techniques used to isolate peptides according to their molecular size [4-10]. In addition it is possible to obtain more purified hydrolysate samples by removing salts and other interfering components by means of UF membranes [11]. In fact, investigations into these methodologies under optimized conditions to reduce time and cost are ongoing [12].

Pressure-driven membrane-based processes, such as UF and NF, are used to fractionate peptide mixtures and amino acids [13]. These types of membrane have been widely used to fractionate milk protein hydrolysates with the aim of enhancing their biological or functional properties [14-15]. It has been shown that variations in operating conditions may favor the permeation of bioactive peptides [16-17].

Membrane technology has become an important separation technology in recent decades probably because their main advantages (it works without the addition of chemicals, with a relatively low use of energy, it has low processing costs, the scale-up is an easy subject and the process lines are well arranged) make it the ideal technology for use on an industrial scale. In addition, membrane processes are especially suitable for the food industry, because of the mild working conditions, relatively easy scale up and low processing costs in comparison to chromatographic techniques.

The separation of peptides by UF mainly depends on the molecular weight (MW) cut-off (MWCO) of the membrane. However, when the MW of the peptides involved in the process is quite similar, their isolation is a hard subject; in these cases, NF is the best membrane separation technique [18]. The fact that NF membranes are usually charged offers the possibility of separating solutes through a combination of size and charge mechanisms.

## **3. Membrane technology applied to peptide fractionation**

Membrane processes are now viewed as efficient tools for the development of new valueadded products by separating minor compounds such as bioactive peptides [19]. These separation processes are based upon selective permeability of one or more of the liquid constituents through the membrane according to the pressure difference. Amongst the pressure-driven membrane techniques, which main features are summarized in Figure 1, UF and NF have been tested for the fractionation of protein hydrolysates due to the fact that the molecular weight of most bioactive peptides is within the normal pore size range of these membranes.

**Figure 1.** Pressure-driven membrane processes

242 Bioactive Food Peptides in Health and Disease

using some of those techniques [2].

the production of this kind of products from natural sources. Considering that most functional peptides are present in complex mixtures containing a large number of

The methodologies commonly used for peptide fractionation and enrichment include: selective precipitation, membrane filtration, ion exchange, gel filtration technologies and liquid chromatography [1]. However, significant differences concerning the number and type of extracted peptides occur among extraction procedures. Additionally, undesired peptides, such as allergenic or bitter-tasting peptides, could be enriched in the process when

Fractionation methods involving precipitation steps are carried out by means of the addition of organic solvents like ethanol, methanol or acetone; adding acids like trichloroacetic acid (TCA), sulphosalicylic acid or phosphotungstic acid (PPTA); by means of the addition of salts (ammonium sulphate) or just by adjusting the pH to the isoelectric point. Precipitation often results in a selective fractionation of peptides depending on their solubility in the precipitating agent [3]; however the addition of chemical compounds causes in some cases

Chromatographic methods for peptide separation are currently used at lab-scale: high performance liquid chromatography (HPLC), fast protein liquid chromatography (FPLC), isoelectric focusing (IEF) and ion exchange chromatography (IEC) are some of them. In most cases, one or two cycles of successive HPLC separation had been adequate to isolate peptides one by one. In the same way, IEC has been used for the enrichment of casein phosphopeptides from casein hydrolysates or for the isolation of cationic antibacterial peptides from lactoferrin. However, although chromatographic processes can provide good separation selectivity, the low productivity and high production costs involved in these

Size exclusion chromatography (SEC) and more frequently Ultrafiltration-Nanofiltration (UF-NF) are the main techniques used to isolate peptides according to their molecular size [4-10]. In addition it is possible to obtain more purified hydrolysate samples by removing salts and other interfering components by means of UF membranes [11]. In fact, investigations into these methodologies under optimized conditions to reduce time and cost

Pressure-driven membrane-based processes, such as UF and NF, are used to fractionate peptide mixtures and amino acids [13]. These types of membrane have been widely used to fractionate milk protein hydrolysates with the aim of enhancing their biological or functional properties [14-15]. It has been shown that variations in operating conditions may

Membrane technology has become an important separation technology in recent decades probably because their main advantages (it works without the addition of chemicals, with a relatively low use of energy, it has low processing costs, the scale-up is an easy subject and the process lines are well arranged) make it the ideal technology for use on an industrial

hydrolysed protein fractions, their separation and purification are required.

peptide degradation and changes in the biological and physical properties.

processes make impossible its use at industrial scale.

favor the permeation of bioactive peptides [16-17].

are ongoing [12].

UF is commonly applied to prepare enriched bioactive solutions from protein hydrolysates and improve the bioactivity of peptides. This process is also used to separate peptides with a size lower than 7 kDa [20]. The fractions are collected by subsequently filtration in two or three streams to obtain peptides with different size [21]. For example, amino acids and small peptides can be separated at pH 4.6 into four ranges of molecular mass (I<30 kDa, II>30 kDa (protein fraction), III>10 kDa (protein fraction), IV>0.3 kDa) [22]. Recent results on fractionation peptides by UF-membranes show that crude yoghurt fractions obtained after ion exchange can be separated into four fractions by successive UF using membranes with

molecular cut off sizes of 30, 10 and 3 kDa [23]; whereas UF membranes < 1 kDa are efficient for peptides fractionation from milk hydrolysates if the last permeate contains free amino acids [21].

Advancements in the Fractionation of Milk Biopeptides by Means of Membrane Processes 245

of specific peptide sequences by means a selective membrane, which is used to separate the biocatalyst from the reaction products and the peptides fractionation [29]. At present, EMR is used when working on an industrial scale. This technology for peptides separation is gaining interest, because it is a specific mode for running batch or continuous processes in which enzymes are separated from end products with the help of a selective membrane. By that way, it is possible to obtain complete retention of the enzyme without deactivation problems typical of enzyme immobilization. Furthermore, EMR have been shown to improve the efficiency of

EMR technology has been investigated for the production and separation of peptides since the 90´s. Antithrombotic peptides derived from hydrolysed CMP can be recovered by UF membranes [32-33] and Lactorphin have been successfully produced through continuous hydrolysis of whey in an UF-reactor [34-35]. Multicompartment EMR has also been designed for the continuous hydrolysis of milk proteins. Nowadays, this technique is operated under an electric field for continuous harvesting of some biologically active peptides, such as phosphopeptides and precursors of casomorphins from the tryptic digest of β-casein [36]. Special attention had also had the study of the hydrolysis of whey protein isolates (WPI) using a tangential flow filter membrane (TFF) of 10 kDa in EMR [37]. The factors influencing on the operation of the EMR (substrate concentration, ionic strength, and transmembrane pressure) have been studied and discussed in other research works [30, 38]. In recent years, the use of EMR has emerged as an exciting area of research due to their low production cost, product safety and easy scaled up [39]. Table 1 summarizes some examples of processes for the separation or concentration of bioactive peptides by means of UF membranes. UF offers possibilities for a large-scale production of bioactive peptides but seems limited because of fouling and poor selectivity. Another drawback of UF membranes is their pore size, because the large pores are not selective enough to fractionate small peptides MW of bioactive peptides is usually smaller than 1 kDa). To sum up, with the use of an EMR equipped with UF membranes, the first peptide fractionation is achieved but if a more purified permeate is required; NF membranes should be used as an additional step

NF is a pressure-driven membrane technique in which the pore size of the membrane is in the nanometers range. As can be observed in Figure 1, this technique is an intermediate step between reverse osmosis (RO) and UF and it is useful to separate/fractionate solutes with MW lower than 5 kDa. Transmembrane pressure in NF is lower than in RO and the permeate flux is usually higher, which represents an important energetic advantage in industrial applications. NF membranes of cut-off < 1 kDa are particularly useful for the

The selectivity of NF membranes is based on both size and charge characteristics of the solutes and on the interaction between charged solutes and membrane surface. Hydrodynamic parameters (mainly transmembrane pressure and linear velocities) and

enzyme-catalyzed bioconversion and to increase product yields [13, 30-31].

instead of UF membranes.

**3.2. NF membranes and peptide fractionation** 

filtration of the smaller peptides from hydrolysates solutions.

The combination of membrane processes (UF and NF) is also often used to separation of peptides. The first step of these processes consists in the UF of the hydrolysate in order to obtain complete rejection of intact proteins and intermediate peptides. The resulting permeate fractions is then subjected to a fractionation by NF and a peptide fraction having a molar mass < 1 kDa is isolated of the mixture by means of these membranes.

In this case, permeates obtained after UF could be adjusted at two pH values (9.5 and 3.0) that corresponded to the different charged states of the membrane and of the peptides to improve of separation of polypeptides of molar mass < 1 kDa [23-24].

Recently a method that couple UF and HPLC has also been applied on milk hydrolysate samples for enhance the peptides separation. A current study showed that an UF-membrane was enough to concentrate peptides and subsequently, both permeate and retentate were fractioned by SE-HPLC to obtain small peptides with biological activity [25].

There are also other important UF-processes to separate specific compounds of whey as caseinomacropeptide (CMP). A first method was designed to obtain CMP fractions trough UF membranes with MWCO 20-50 kDa by two diafiltration steps [26]. The method is based on the ability of CMP to form non-covalent linked polymers with a molecular weight up to 50 kDa at neutral pH, which dissociate at acid conditions. The dissociated form of CMP permeates through the UF-membrane at pH 3.5, whereas the majority of whey proteins such as β-Lg, α-lactalbumin (α-La), immunoglobulins (IGs) and bovine serum albumin (BSA) are held back. At pH 7.0, permeate containing CMP can be concentrated by means of the same membrane; however a low permeation rate is obtained with this technique. A second method for separation of CMP can be seen in [27]. Thermal stability of CMP is used in comparison to that of the rest of whey proteins. Complete denaturation and aggregation of proteins is obtained by treating whey at 90°C for 1h; with this method, the denatured proteins can be removed by centrifugation at 5200 g and 4°C for 15 min and the supernatant containing CMP can be concentrated by UF with MWCO 10 kDa after pH adjustment to 7.0; however whey proteins lose part of their functionality due to the denaturation.

Another method for separation of CMP consists in the pretreatment of whey protein concentrate with the enzyme transglutaminase (Tgase) followed by microfiltration [28]. The amino acid sequence of CMP includes two glutamine and three lysine residues, whereby this peptide can be cross-linked by tranglutaminase. The covalent linked CMP aggregates can be removed be means of microfiltration or diafiltration to obtain CMP-free whey protein.

## **3.1. Enzymatic membrane reactor equipped with membranes: first step to peptide fractionation**

Enzymatic membrane reactor (EMR) consists on a coupling of a membrane separation process with an enzymatic reaction. EMR allows the continuous production and separation of specific peptide sequences by means a selective membrane, which is used to separate the biocatalyst from the reaction products and the peptides fractionation [29]. At present, EMR is used when working on an industrial scale. This technology for peptides separation is gaining interest, because it is a specific mode for running batch or continuous processes in which enzymes are separated from end products with the help of a selective membrane. By that way, it is possible to obtain complete retention of the enzyme without deactivation problems typical of enzyme immobilization. Furthermore, EMR have been shown to improve the efficiency of enzyme-catalyzed bioconversion and to increase product yields [13, 30-31].

EMR technology has been investigated for the production and separation of peptides since the 90´s. Antithrombotic peptides derived from hydrolysed CMP can be recovered by UF membranes [32-33] and Lactorphin have been successfully produced through continuous hydrolysis of whey in an UF-reactor [34-35]. Multicompartment EMR has also been designed for the continuous hydrolysis of milk proteins. Nowadays, this technique is operated under an electric field for continuous harvesting of some biologically active peptides, such as phosphopeptides and precursors of casomorphins from the tryptic digest of β-casein [36]. Special attention had also had the study of the hydrolysis of whey protein isolates (WPI) using a tangential flow filter membrane (TFF) of 10 kDa in EMR [37]. The factors influencing on the operation of the EMR (substrate concentration, ionic strength, and transmembrane pressure) have been studied and discussed in other research works [30, 38]. In recent years, the use of EMR has emerged as an exciting area of research due to their low production cost, product safety and easy scaled up [39]. Table 1 summarizes some examples of processes for the separation or concentration of bioactive peptides by means of UF membranes. UF offers possibilities for a large-scale production of bioactive peptides but seems limited because of fouling and poor selectivity. Another drawback of UF membranes is their pore size, because the large pores are not selective enough to fractionate small peptides MW of bioactive peptides is usually smaller than 1 kDa). To sum up, with the use of an EMR equipped with UF membranes, the first peptide fractionation is achieved but if a more purified permeate is required; NF membranes should be used as an additional step instead of UF membranes.

#### **3.2. NF membranes and peptide fractionation**

244 Bioactive Food Peptides in Health and Disease

acids [21].

molecular cut off sizes of 30, 10 and 3 kDa [23]; whereas UF membranes < 1 kDa are efficient for peptides fractionation from milk hydrolysates if the last permeate contains free amino

The combination of membrane processes (UF and NF) is also often used to separation of peptides. The first step of these processes consists in the UF of the hydrolysate in order to obtain complete rejection of intact proteins and intermediate peptides. The resulting permeate fractions is then subjected to a fractionation by NF and a peptide fraction having a

In this case, permeates obtained after UF could be adjusted at two pH values (9.5 and 3.0) that corresponded to the different charged states of the membrane and of the peptides to

Recently a method that couple UF and HPLC has also been applied on milk hydrolysate samples for enhance the peptides separation. A current study showed that an UF-membrane was enough to concentrate peptides and subsequently, both permeate and retentate were

There are also other important UF-processes to separate specific compounds of whey as caseinomacropeptide (CMP). A first method was designed to obtain CMP fractions trough UF membranes with MWCO 20-50 kDa by two diafiltration steps [26]. The method is based on the ability of CMP to form non-covalent linked polymers with a molecular weight up to 50 kDa at neutral pH, which dissociate at acid conditions. The dissociated form of CMP permeates through the UF-membrane at pH 3.5, whereas the majority of whey proteins such as β-Lg, α-lactalbumin (α-La), immunoglobulins (IGs) and bovine serum albumin (BSA) are held back. At pH 7.0, permeate containing CMP can be concentrated by means of the same membrane; however a low permeation rate is obtained with this technique. A second method for separation of CMP can be seen in [27]. Thermal stability of CMP is used in comparison to that of the rest of whey proteins. Complete denaturation and aggregation of proteins is obtained by treating whey at 90°C for 1h; with this method, the denatured proteins can be removed by centrifugation at 5200 g and 4°C for 15 min and the supernatant containing CMP can be concentrated by UF with MWCO 10 kDa after pH adjustment to 7.0;

molar mass < 1 kDa is isolated of the mixture by means of these membranes.

fractioned by SE-HPLC to obtain small peptides with biological activity [25].

however whey proteins lose part of their functionality due to the denaturation.

removed be means of microfiltration or diafiltration to obtain CMP-free whey protein.

**3.1. Enzymatic membrane reactor equipped with membranes: first step to** 

**peptide fractionation** 

Another method for separation of CMP consists in the pretreatment of whey protein concentrate with the enzyme transglutaminase (Tgase) followed by microfiltration [28]. The amino acid sequence of CMP includes two glutamine and three lysine residues, whereby this peptide can be cross-linked by tranglutaminase. The covalent linked CMP aggregates can be

Enzymatic membrane reactor (EMR) consists on a coupling of a membrane separation process with an enzymatic reaction. EMR allows the continuous production and separation

improve of separation of polypeptides of molar mass < 1 kDa [23-24].

NF is a pressure-driven membrane technique in which the pore size of the membrane is in the nanometers range. As can be observed in Figure 1, this technique is an intermediate step between reverse osmosis (RO) and UF and it is useful to separate/fractionate solutes with MW lower than 5 kDa. Transmembrane pressure in NF is lower than in RO and the permeate flux is usually higher, which represents an important energetic advantage in industrial applications. NF membranes of cut-off < 1 kDa are particularly useful for the filtration of the smaller peptides from hydrolysates solutions.

The selectivity of NF membranes is based on both size and charge characteristics of the solutes and on the interaction between charged solutes and membrane surface. Hydrodynamic parameters (mainly transmembrane pressure and linear velocities) and membrane material exert influence on membrane selectivity too. NF membranes have a slightly charged surface; because the dimensions of the pores are less than one order of magnitude larger than the size of ions [54].

Advancements in the Fractionation of Milk Biopeptides by Means of Membrane Processes 247

process (transmembrane pressure, lineal velocities and solute concentration). d) Membrane characteristics (manufacture process, surface roughness, porosity, film layer material and hydrophilic/hydrophobic surface). All these aspects must be considered in order to estimate

Especially in NF membranes involving peptide fractionation from mixtures, charge exclusion mechanisms are predominant in the separation. The charge effects affect membrane-peptide and peptide-peptide interactions in the mixture or at the membrane surface. The transport mechanism through the pores is governed by convective and diffusive fluxes as well as by electromigrative flux. These phenomena make the prediction

The current state of science the knowledge of the NF process is not sufficient to make a model fulfilling the requirements. The difficulties in modeling permeate flow rates and solute rejection come from the scale at which the different phenomena takes place at the membrane surface and through the membrane pores, where most of the hydrodynamic and macroscopic interactions begin to break down. However, simplified approaches could be

The solute transfer through the membrane follow two main steps: distribution of ionic species at the selective interface according to their charge (both solutes and membrane) and transfer by a complex combination among diffusion, convection and electrophoretic mobility through the membrane, at least at low feed concentrations [13]. According to Donnan theory, the passage of charged solutes through a charged NF membrane is likely to be different whether they are considered to be co-ions, i.e. with the same charge of the membrane, or counter-ions, i.e. with a charge of opposite sign. In fact, due to electrostatic repulsive/attractive forces between the membrane and the solutes the concentration of coions will be lower in the membrane than in the solutions. On the contrary, the counter-ions have a higher concentration in the membrane than in the solution. This concentration difference of the ions generates a potential difference at the interface between the membrane and the solution, which is called Donnan potential. Under equilibrium conditions, electroneutrality and equality of electrochemical potentials are maintained through the system. The Donnan equilibrium depends on the ion concentration, the fixed charge concentration in the membrane and the valences of the co-ions and counter-ions. Figure 2 shows an adapted schematic representation [57] of the influence of the electrostatic interactions in the

Because of the electro-neutrality principle, and on the assumption that the charge density of the membrane is quite higher than the net charge of the co-ions, is possible to calculate the distribution of the co-ion resulting from a binary electrolyte *A B z z ABA B* between the membrane surface and the solution as a function of the charge density of the membrane.

<sup>=</sup> ��� ���)

������

�����

(2)

� � �� ��� �)

used to explain qualitatively the experimental results obtained, as can be seen below.

transmission of charged peptides through a charged NF membrane.

K = �� � ��

the viability of a peptide fractionation process.

of the separation selectivity a difficult objective.


**Table 1.** Bioactive peptides obtained by means of UF membranes

#### *3.2.1. NF transport mechanism*

The mechanism behind the selectivity of membrane processes is generally the size of the component. This mainly applies in the case of UF membranes and in the case of NF membranes with uncharged solutes. Charge effects are minimized in this case and the transmission of the solutes depends largely on the size exclusion effects of the membrane. This sieving effect is usually modeled and corrected [55] using continues hydrodynamic models such as originally proposed by Ferry. In this model, the membrane is assumed to be a network of perfectly cylindrical and parallel pores in which solvent velocity follows Poiseuille's law with a parabolic profile and solutes are assimilated to hard spheres. The transmission coefficient (*Tr*) of a given solute can be calculated according to equation (1) However, the selectivity of NF membranes is based on both size and charge characteristics of the solutes and on the interaction between charged solutes and membrane surface [56].

$$\text{Tr} = (1 \text{-} (\lambda (\lambda \text{-} 2))^2 \exp \text{ (-0.7146 } \lambda 2) \tag{1}$$

Where λ is the relation between the radius of the solute and the radius of the pore.

The selectivity of the separation when using NF membranes is based on the following factors: a) Solute (peptide) size, shape and charge. b) Membrane pore size and surface charge (sign and surface charge density). c) Hydrodynamic conditions of the fractionation process (transmembrane pressure, lineal velocities and solute concentration). d) Membrane characteristics (manufacture process, surface roughness, porosity, film layer material and hydrophilic/hydrophobic surface). All these aspects must be considered in order to estimate the viability of a peptide fractionation process.

246 Bioactive Food Peptides in Health and Disease

magnitude larger than the size of ions [54].

membrane material exert influence on membrane selectivity too. NF membranes have a slightly charged surface; because the dimensions of the pores are less than one order of

Bone and teeth mineralization

The mechanism behind the selectivity of membrane processes is generally the size of the component. This mainly applies in the case of UF membranes and in the case of NF membranes with uncharged solutes. Charge effects are minimized in this case and the transmission of the solutes depends largely on the size exclusion effects of the membrane. This sieving effect is usually modeled and corrected [55] using continues hydrodynamic models such as originally proposed by Ferry. In this model, the membrane is assumed to be a network of perfectly cylindrical and parallel pores in which solvent velocity follows Poiseuille's law with a parabolic profile and solutes are assimilated to hard spheres. The transmission coefficient (*Tr*) of a given solute can be calculated according to equation (1) However, the selectivity of NF membranes is based on both size and charge characteristics of the solutes and on the interaction between charged solutes and membrane surface [56].

Where λ is the relation between the radius of the solute and the radius of the pore.

The selectivity of the separation when using NF membranes is based on the following factors: a) Solute (peptide) size, shape and charge. b) Membrane pore size and surface charge (sign and surface charge density). c) Hydrodynamic conditions of the fractionation

Tr = (1-( λ(λ-2))2 exp (-0.7146 λ2) (1)

Calcium bioavailability improvement [40]

Opioid [42] Anti microbial [43] Muscular contraction [44]

**Protein Hydrolysate Source Biological Activity References**  Bovine caseinomacropeptide Antithrombotic [32-33]

Bovine whey β-lactoglobulin ACE inhibitor [41]

Bovine whey α-lactalbumin ACE inhibitor [4] Fish protein ACE inhibitor [45] Alfalta white protein ACE inhibitor [46] Alfalta leaf protein Antioxidant [47] Wheat gluten ACE inhibitor [48] Soybean protein ACE inhibitor [49] Soybean β-conglycinin ACE inhibitor [50] Sea cucumber gelatin ACE inhibitor [51] Potato Antimicrobial [52] Potato Antimicrobial [53]

**Table 1.** Bioactive peptides obtained by means of UF membranes

*3.2.1. NF transport mechanism* 

Especially in NF membranes involving peptide fractionation from mixtures, charge exclusion mechanisms are predominant in the separation. The charge effects affect membrane-peptide and peptide-peptide interactions in the mixture or at the membrane surface. The transport mechanism through the pores is governed by convective and diffusive fluxes as well as by electromigrative flux. These phenomena make the prediction of the separation selectivity a difficult objective.

The current state of science the knowledge of the NF process is not sufficient to make a model fulfilling the requirements. The difficulties in modeling permeate flow rates and solute rejection come from the scale at which the different phenomena takes place at the membrane surface and through the membrane pores, where most of the hydrodynamic and macroscopic interactions begin to break down. However, simplified approaches could be used to explain qualitatively the experimental results obtained, as can be seen below.

The solute transfer through the membrane follow two main steps: distribution of ionic species at the selective interface according to their charge (both solutes and membrane) and transfer by a complex combination among diffusion, convection and electrophoretic mobility through the membrane, at least at low feed concentrations [13]. According to Donnan theory, the passage of charged solutes through a charged NF membrane is likely to be different whether they are considered to be co-ions, i.e. with the same charge of the membrane, or counter-ions, i.e. with a charge of opposite sign. In fact, due to electrostatic repulsive/attractive forces between the membrane and the solutes the concentration of coions will be lower in the membrane than in the solutions. On the contrary, the counter-ions have a higher concentration in the membrane than in the solution. This concentration difference of the ions generates a potential difference at the interface between the membrane and the solution, which is called Donnan potential. Under equilibrium conditions, electroneutrality and equality of electrochemical potentials are maintained through the system. The Donnan equilibrium depends on the ion concentration, the fixed charge concentration in the membrane and the valences of the co-ions and counter-ions. Figure 2 shows an adapted schematic representation [57] of the influence of the electrostatic interactions in the transmission of charged peptides through a charged NF membrane.

Because of the electro-neutrality principle, and on the assumption that the charge density of the membrane is quite higher than the net charge of the co-ions, is possible to calculate the distribution of the co-ion resulting from a binary electrolyte *A B z z ABA B* between the membrane surface and the solution as a function of the charge density of the membrane.

$$\mathbf{K} = \frac{\mathbf{c\_B^m}}{\mathbf{c\_B}} = \frac{\left(\mathbf{z\_B \cdot \mathbf{c\_B}}\right)^{\mathbf{z\_B}} \mathbf{z\_A}}{\left(\mathbf{z\_B \cdot \mathbf{c\_B^m} + \mathbf{z\_x} \cdot \mathbf{c\_x^m}}\right)}\tag{2}$$

Advancements in the Fractionation of Milk Biopeptides by Means of Membrane Processes 249

acid is electrically rejected by the charged active layer of the membrane. Simultaneously, the counter-ion is retained to ensure the balance of charges as the consequence of the electromigrative flow that opposes the convective one. However unfortunately, the extrapolation of Donnan theory to predict the behavior of individual solutes in mixed solutions containing several negative, neutral and positive solutes is very limited, mainly because of coupling and competitive effects. For this reason, NF process of complex mixtures of amino acids and peptides is a difficult object for mathematical modeling [64].

To clarify the mechanisms involved in the separation of biomolecules by NF membranes several fundamental researches have been published. Table 2 shows relevant NF studies involving amino acids and peptides. The data obtained are relative at different factors affecting the separation of single amino acid (AA) solutions, peptides mixtures and protein hydrolysates. For example, the influence of pH in the retention of amino acids through NF membranes was studied to analyse the separation of small peptides (only two amino acids). In this case, different isoelectric points (pI) by adjusting the pH of the mixture were considered in peptides rejection [65]. Another report showed the separation of a mixture of nine amino acids on the basis of electrostatic interactions of solutes-membrane [66]. According to results, pH has the greater influence on membrane selectivity. In addition the content of inorganic ions compared to the content of ionized amino acids affects also the separation. Therefore these variables are crucial for optimization of membrane selectivity.

*3.2.2. NF Applied to amino acid and peptide fractionation: Review* 

Reference **Solution Experiments Membrane** 

[66] Mixtures of AA Separation of a mixture of 9

pH variation experiments Separation experiments of mixed dipeptides

> AAs on the basis of differential electrostatic interactions with the

Membrane selectivity as a function of pH, AA concentration and Ionic

membrane

Strength

Flat-sheet membranes **Materials:** Phosphatidic Acid (PA), Thin Film Composite (TFC), Sulfonated

Polyethersulfone SPES)

and Sulfonated Polystyrene (SPE) **MWCO**: 0.2-3 kDa **Charge at pH 7**: negative (SPES, SPE and TFC) or amphoteric (PA)

**Material:** Inorganic membrane, chemical modification of the ZrO2 layer of a UF membrane with cross linked Polyetherimide (PEI) **Charge:** positive

[65] Single AA solutions

Mixtures of dipeptides

**Figure 2.** Schematic representation of solute flows across a negatively charged NF membranes. Je: electromigrative flow as a consequence of the transitory electric field. Tr: transmission of the solute. Attractive (>> <<) and repulsive (<< >>) electrostatic interactions between charged solutes and the membrane are also represented.

CBm and CB represent the concentration of co-ions B in the membrane and in the solution respectively. The coefficient of distribution, K, can be used to predict the rejection value of a binary electrolyte if the ionic transport is mainly due to convection and size exclusion effects are negligible. Under these conditions, K will mainly depend on: the co-ion valence (zB), the counter-ion valence (zA), the membrane charge (Cxm), its valence (zx) and the concentration of the co-ion in the solution (CB).

According to equation 2, Donnan equilibrium predicts that an increase in the concentration of co-ions in the global solution and/or a decrease in the membrane charge density lead to a decrease in the exclusion of co-ions from the membrane surface (K is increased) and to a decrease in the retention of the binary salt (co-ion and cointer-ion) in order to maintain electroneutrality in both sides of the membrane [58-59]. The concentration of co-ions in the membrane will change according to the valence of the co-ion and counter-ions present in the solution. Thus, if the valence of the co-ion (zB) has a lower value and the valence of the counter-ion (zA) is increased, the concentration of co-ions in the membrane will be favored. For example, the retention of some common salts by descending order (Na2SO4 > NaCl > CaCl2) through a negatively charged NF membrane can be predicted according to these principles [60-63].

Donnan theory is generally used to describe the permeability and selectivity of NF membranes using solutions containing only one amino acid. For example, for an amino acid co-ion and its associated counter-ion, in accordance with the Donnan equilibrium, the amino acid is electrically rejected by the charged active layer of the membrane. Simultaneously, the counter-ion is retained to ensure the balance of charges as the consequence of the electromigrative flow that opposes the convective one. However unfortunately, the extrapolation of Donnan theory to predict the behavior of individual solutes in mixed solutions containing several negative, neutral and positive solutes is very limited, mainly because of coupling and competitive effects. For this reason, NF process of complex mixtures of amino acids and peptides is a difficult object for mathematical modeling [64].

## *3.2.2. NF Applied to amino acid and peptide fractionation: Review*

248 Bioactive Food Peptides in Health and Disease

membrane are also represented.

of the co-ion in the solution (CB).

principles [60-63].

**Figure 2.** Schematic representation of solute flows across a negatively charged NF membranes. Je: electromigrative flow as a consequence of the transitory electric field. Tr: transmission of the solute. Attractive (>> <<) and repulsive (<< >>) electrostatic interactions between charged solutes and the

CBm and CB represent the concentration of co-ions B in the membrane and in the solution respectively. The coefficient of distribution, K, can be used to predict the rejection value of a binary electrolyte if the ionic transport is mainly due to convection and size exclusion effects are negligible. Under these conditions, K will mainly depend on: the co-ion valence (zB), the counter-ion valence (zA), the membrane charge (Cxm), its valence (zx) and the concentration

According to equation 2, Donnan equilibrium predicts that an increase in the concentration of co-ions in the global solution and/or a decrease in the membrane charge density lead to a decrease in the exclusion of co-ions from the membrane surface (K is increased) and to a decrease in the retention of the binary salt (co-ion and cointer-ion) in order to maintain electroneutrality in both sides of the membrane [58-59]. The concentration of co-ions in the membrane will change according to the valence of the co-ion and counter-ions present in the solution. Thus, if the valence of the co-ion (zB) has a lower value and the valence of the counter-ion (zA) is increased, the concentration of co-ions in the membrane will be favored. For example, the retention of some common salts by descending order (Na2SO4 > NaCl > CaCl2) through a negatively charged NF membrane can be predicted according to these

Donnan theory is generally used to describe the permeability and selectivity of NF membranes using solutions containing only one amino acid. For example, for an amino acid co-ion and its associated counter-ion, in accordance with the Donnan equilibrium, the amino To clarify the mechanisms involved in the separation of biomolecules by NF membranes several fundamental researches have been published. Table 2 shows relevant NF studies involving amino acids and peptides. The data obtained are relative at different factors affecting the separation of single amino acid (AA) solutions, peptides mixtures and protein hydrolysates. For example, the influence of pH in the retention of amino acids through NF membranes was studied to analyse the separation of small peptides (only two amino acids). In this case, different isoelectric points (pI) by adjusting the pH of the mixture were considered in peptides rejection [65]. Another report showed the separation of a mixture of nine amino acids on the basis of electrostatic interactions of solutes-membrane [66]. According to results, pH has the greater influence on membrane selectivity. In addition the content of inorganic ions compared to the content of ionized amino acids affects also the separation. Therefore these variables are crucial for optimization of membrane selectivity.



Advancements in the Fractionation of Milk Biopeptides by Means of Membrane Processes 251

Flat sheet membrane **Material:** cellulose acetate **MWCO:** 2.500 kDa **Charge:** anionic charge characteristics at basic pH

**Material:** SPES **Charge:** high negative charge at neutral pH

Flat sheet membrane **Material:** PA (proprietary)

Flat sheet membrane **Material:** PA (proprietary)

**MWCO:** 2.5 kDa **Charge:** negatively charged at alkaline pH

**Material:** SPES **MWCO:** 1 kDa **Charge:** negatively charged at neutral pH

**Material:** Bilayers of Phosphatidylserine synthase (PSS) on porous

**MWCO:** 2.5 kDa **Charge:** negatively charged at alkaline pH

L-Asp

rejection

rejection)

phenomena: effect of hydrodynamic conditions on the *Tr* of selected peptides from the hydrolysate

Effect of pH, concentration and physicoquemical environment (ionic strength and kind of salt added) on single AA

Effect of operating pressure and concentration of fermentation broth on NF (selectivity and AA

pH, transmembrane pressure and feed velocity in the ability of a "loose" composite NF membrane to fractionate acid, neutral and basic peptides. Evaluation of the effect of peptides fouling on sieving

and electrostatic characteristics of the membrane: PEG and Effect of aggregating peptides on the fractionation of a protein hydrolysate

measurements.

fouling

using multilayer polyelectrolyte NF

separation peptides from lactose and effect of pH in

[71] Protein hydrolysate Concentration polarization

[14] Protein hydrolysate Effect of feed concentration,

[57] Protein hydrolysate NaCl retention

[23] Peptide mixture Selectivity estimation in the

[73] AA mixtures Separation of neutral AA

[72] Single AA solutions

Fermentation broth

[13] Single AA solutions

[67] Single AA solutions

[69] Single AA solutions

AA mixtures

AA mixtures Peptides (from protein hydrolysate)

AA mixtures

[68] Protein hydrolysate Separation of a mixture of 10

[16] Protein hydrolysate Effect of adjusting pH and

[70] AA mixtures Separation performance of

NF of charged AA (single solutions and mixtures) and peptides(similar MW

Influence of concentration and ionic composition (salt concentration and kind of salt added) on single AA

Separation of AA mixtures

Influence of physicochemical conditions (ionic strength

fractionation (permeate

ionic strength in the fractionation of the hydrolysate.

Influence of experimental conditions on the steady-

state regime pH effect on retention coefficients of single AA solutions and AA mixtures Influence of ionic strength and transmembrane pressure on retention coefficients of an AA

mixture

two different NF membranes.

of the separation. Simulation NF process system for separation and

Influence of pH and operation pressure on the selectivity

concentration of L-Phe and

**Material:** ZrO2 filtering layer on a mineral support **Charge:** weakly negative

**Material:** cellulose acetate,

**M5+PEI:** ZrO2 modified

microporous active layer

**Material and MWCO:** PA (2.5 kDa), cellulose acetate (0.5, 0.8, 1-5 and 8-10 kDa).

Cross-flow NF membrane **Material:** ceramic alumina γ with an average pore radius of 2.5 nm.

**Charge:** zero point charge in the range of pH 8-9. Positively charged in the

charge at pH 8.0

SPES, SPS and Polysulfunate (PS) **MWCO:** 35-45% (NaCl retention), 1 kDa, 3, 6 kDa

respectively

with PEI **Kerasep Solgel:**

of ZrO2

Flat sheet TFC membranes.

**Charge:** anionic characteristics

pH range tested.

SPS

CTF membranes with asymmetric structure **Material:** aromatic PA and

but different pI)

retention.

small peptides

and pH) on the

flux and *Tr*)



Advancements in the Fractionation of Milk Biopeptides by Means of Membrane Processes 253

reported as a function of concentration in a concentration range from 0.3 to 3% (w/v) while the

Permeation experiments of aqueous solutions of diprotic amino acids (L-glutamine and glycine) showed different data [75]. Amino acid rejection became more concentration dependant at higher pH values due to the increased net charge of the solutes. In this high concentration regime (up to 2 M of glycine) and under alkaline conditions, an important

Recent results were also found in the experiments of rejection of five amino acids by NF membranes, where experimental data were compared against a combined steric and charge rejection model [76]. Only positive charged amino acids showed good agreement with the model in all the concentration range studied while the behavior of negatively charged peptides only agree with the model at the highest concentration values and rejection of neutral amino acids was decreased due to its smaller net charge. Despite these data, the separation of bioactive peptides from natural sources and the prediction of their individual

At the other hand, the study of separation of tryptic β-casein peptides trough UF membranes showed that the separation of peptides is also affected by ionic strength by means a controlled dual mechanism: size exclusion and electrostatic repulsion [77]. Electrostatic interactions affect

Another subsequent work reported the interesting potential of NF membranes for separating peptides in the range of 0.3-1 kDa [68]. Specific conditions of ionic strength and especially pH promoted the separation of peptides because the membrane and peptides showed amphoteric properties. Three categories of peptides (acid, basic, neutral) were separated according to their pI. At optimum pH 8 this led to high transmissions of basic peptides (even over 100%), intermediate transmissions for neutral peptides, and low transmissions for acid peptides. The addition of multicharged cationic and anionic species in the hydrolysate induced a markedly enhanced selectivity when the polyelectrolyte was a

membrane co-ion and a complete reversion of selectivity when it was a membrane.

considered in order to explain the transmission of a given peptide.

An additional research was later performed in order to understand the separation of peptide mixtures through NF membranes [13]. In this case, the solution tested was a mixture of 4 small peptides (4-7 residues) obtained by trypsin hydrolysis of caseinomacropeptide. From above results, it was proposed the first comprehensive approach concerning at filtration of mixtures of peptides, under two principles: (i) electro-neutrality of the solutions is always recovered, which means that all charged solute transmission are interdependent, and (ii) the number of charges along the peptide sequence, rather than the global net charge, has to be

Afterwards, it was investigated the potential of organic NF membranes with a MWCO between 1 and 5 kDa for the fractionation of whey protein hydrolysates. The effect of adjusting pH and ionic strength on the separation properties of the membranes was also characterized in these tests [16]. Highest selectivity between basic and acidic peptides was

rejection of I-Glu decreased in the range from 0.1 to 0.85%.

decrease in amino acid rejection was observed in all tests.

behavior require previous NF studies of complex mixed solutions.

the peptides transport, especially if the ionic strength of the solution is low.

**Table 2.** NF studies involving amino acids and peptides

Influence of concentration and ionic composition (salt concentration and type of salt added) on single amino acid retention and on the separation of amino acid mixtures was also studied to explain peptides rejection [67]. The different results show that both parameters have a negative impact on the selectivity of the membrane when size effects are not dominant. Under these conditions, the membrane seems to be more permeable to charged components due to saturation of its charged sites which makes that repulsive/attractive force between the membrane and the charged peptides become weaker.

Other studies have showed that the mixture of amino acids and their concentration affect also the behavior of NF membranes. However very few works have focused on concentrated amino acid or peptide mixtures. The most NF studies involve highly diluted amino acid solutions, which are the most likely to be found in industrial processes, and the results obtained to date are not completely understood due to at the difference in the data. For example, the results of the separation of l-glutamine (l-Gln) from Gln fermentation broth by NF, showed the effects of various experimental parameters such as transmembrane pressure, pH and concentration of broth on the rejection of l-Gln and l-glutamate (l-Glu). However, the rejection of fermentation broth from a single l-Gln or l-Glu solution was mainly caused by the complex ionic composition of the real fermentation broth [72]. Increase of I-GIn rejection was reported as a function of concentration in a concentration range from 0.3 to 3% (w/v) while the rejection of I-Glu decreased in the range from 0.1 to 0.85%.

252 Bioactive Food Peptides in Health and Disease

[74] Protein hydrolysate Fractionation of small

[75] Single AA solutions Permeation of single AA

[76] Single AA solutions Study solute rejection versus

force between the membrane and the charged peptides become weaker.

**Table 2.** NF studies involving amino acids and peptides

membranes alumina support

Cross-flow filtration **Material:** cellulose acetate

**MWCO:** 1 kDa

Membrane discs **MWCO and material:**

0.15-0.30 kDa (proprietary), 1 kDa (proprietary), 2.5 kDa (proprietary), 0.15-0.30 kDa (permanently hydrophilic PES) and 1kDa (permanently hydrophilic PES)

**Material:** SPES **MWCO:** 1kDa

peptides using a 1 kDa NF

Influence of pH and ionic strength on *Tr*

> solutions in the whole range of their solubility with a stepwise pH scan ranging from 0 to -1 total

concentration of 5 different

Comparison of experimental data against a combined steric and charge rejection

membrane.

net charge

AA.

model.

Influence of concentration and ionic composition (salt concentration and type of salt added) on single amino acid retention and on the separation of amino acid mixtures was also studied to explain peptides rejection [67]. The different results show that both parameters have a negative impact on the selectivity of the membrane when size effects are not dominant. Under these conditions, the membrane seems to be more permeable to charged components due to saturation of its charged sites which makes that repulsive/attractive

Other studies have showed that the mixture of amino acids and their concentration affect also the behavior of NF membranes. However very few works have focused on concentrated amino acid or peptide mixtures. The most NF studies involve highly diluted amino acid solutions, which are the most likely to be found in industrial processes, and the results obtained to date are not completely understood due to at the difference in the data. For example, the results of the separation of l-glutamine (l-Gln) from Gln fermentation broth by NF, showed the effects of various experimental parameters such as transmembrane pressure, pH and concentration of broth on the rejection of l-Gln and l-glutamate (l-Glu). However, the rejection of fermentation broth from a single l-Gln or l-Glu solution was mainly caused by the complex ionic composition of the real fermentation broth [72]. Increase of I-GIn rejection was Permeation experiments of aqueous solutions of diprotic amino acids (L-glutamine and glycine) showed different data [75]. Amino acid rejection became more concentration dependant at higher pH values due to the increased net charge of the solutes. In this high concentration regime (up to 2 M of glycine) and under alkaline conditions, an important decrease in amino acid rejection was observed in all tests.

Recent results were also found in the experiments of rejection of five amino acids by NF membranes, where experimental data were compared against a combined steric and charge rejection model [76]. Only positive charged amino acids showed good agreement with the model in all the concentration range studied while the behavior of negatively charged peptides only agree with the model at the highest concentration values and rejection of neutral amino acids was decreased due to its smaller net charge. Despite these data, the separation of bioactive peptides from natural sources and the prediction of their individual behavior require previous NF studies of complex mixed solutions.

At the other hand, the study of separation of tryptic β-casein peptides trough UF membranes showed that the separation of peptides is also affected by ionic strength by means a controlled dual mechanism: size exclusion and electrostatic repulsion [77]. Electrostatic interactions affect the peptides transport, especially if the ionic strength of the solution is low.

Another subsequent work reported the interesting potential of NF membranes for separating peptides in the range of 0.3-1 kDa [68]. Specific conditions of ionic strength and especially pH promoted the separation of peptides because the membrane and peptides showed amphoteric properties. Three categories of peptides (acid, basic, neutral) were separated according to their pI. At optimum pH 8 this led to high transmissions of basic peptides (even over 100%), intermediate transmissions for neutral peptides, and low transmissions for acid peptides. The addition of multicharged cationic and anionic species in the hydrolysate induced a markedly enhanced selectivity when the polyelectrolyte was a membrane co-ion and a complete reversion of selectivity when it was a membrane.

An additional research was later performed in order to understand the separation of peptide mixtures through NF membranes [13]. In this case, the solution tested was a mixture of 4 small peptides (4-7 residues) obtained by trypsin hydrolysis of caseinomacropeptide. From above results, it was proposed the first comprehensive approach concerning at filtration of mixtures of peptides, under two principles: (i) electro-neutrality of the solutions is always recovered, which means that all charged solute transmission are interdependent, and (ii) the number of charges along the peptide sequence, rather than the global net charge, has to be considered in order to explain the transmission of a given peptide.

Afterwards, it was investigated the potential of organic NF membranes with a MWCO between 1 and 5 kDa for the fractionation of whey protein hydrolysates. The effect of adjusting pH and ionic strength on the separation properties of the membranes was also characterized in these tests [16]. Highest selectivity between basic and acidic peptides was

found at alkaline conditions without the addition of NaCl. In addition the authors demonstrated that two peptides differing by only one amino acid are transmitted differently. Consequently a single change in the amino acid sequence can affect peptides transmission.

Advancements in the Fractionation of Milk Biopeptides by Means of Membrane Processes 255

characteristics are of paramount importance in order to predict and optimize the

The interactions of peptide-peptide and peptide-membrane affect the separation process performance and thus it is difficult to predict the selectivity of the membranes when the objective is the fractionation of complex peptide mixtures. According the literature, the most important parameters that cause effect on membrane selectivity are pH, ionic strength,

1. The pH of solution is an important control variable in NF processes for the fractionation of complex peptide mixtures, because peptides are molecules that have at least one carboxylic group ( *R COOH R COO* and one amine group 3 2 (*R NH R NH* ). The total number of acid and basic groups depends on its primary structure (amino acid sequence) and it determines the pH value at which the peptides have the same number of negative than positive charges, i.e., its pI. Peptides can be classified in three different groups according to their pI: acidic peptides (pI ≤ 5), neutral peptides (5 < pI ≤ 7) and basic peptides (pI > 7). Their net charge depends on the pH of the solution, as well as the charge density of the NF membrane. This last value will vary because of the ionization of its functional groups

In NF, the transmission of amino acids and peptides reaches its maximum value when the pH is equal to the pI. Under these conditions, repulsive electrostatic interactions are minimized. That way, the modification of NF membranes transmission is possible by

In the case of protein hydrolysates, which composition is more complex, there will be a pH value at which the fractionation of acid, neutral and basic peptides is maximized. For example, it has been shown that the separation factor between basic and acid peptides reaches its maximum value when the pH of the mixture is alkaline [16]. However, literature published on this topic only describes the behavior of "tracer" peptides in the hydrolysate

2. The ionic strength of peptides solution affects the selectivity of NF membranes. In an aqueous medium the increase of the ionic strength, for example by the addition of NaCl, results in a decrease of zeta potential of the NF membrane [80-83] as well as a decrease in the electrophoretic mobility of proteins and peptides [84]. According to these observations, electrostatic interactions between the membrane and the peptides become less intense,

Several authors have demonstrated the preponderance of a selectivity based on electrostatic interactions at low ionic strength values [16, 68, 85]. The fact that electrostatic interactions membrane-peptide lose significance at high ionic strength values results in a decrease of the double selectivity size/charge in processes involving NF membranes. In addition to the effect over the charge density of the membrane, ionic strength also influences the effective

performance of NF membranes for the fractionation of complex peptide mixtures.

*3.2.3. Main parameters influencing peptide fractionation using NF membranes* 

polarization layer and fouling.

(acidic and basic).

changing the pH of the mixture.

and this limits the scope of the separation factor calculated.

which usually leads to better transmission values of the peptides.

Influence of peptide interactions on peptide separation was also established in some studies of NF membranes. The data show that the same peptide could be transmitted differently when issued from different hydrolysates, reflecting the importance of surrounding peptides, and, hence, the possible occurrence of peptide-peptide interactions [78]. Therefore hydrophobic interactions between peptides when the pH of the solution is close to their pI can lead to their aggregation and subsequent fouling of the NF membrane.

By means of NF experiments on fractionation of β-Lg tryptic hydrolysate, it was shown that peptide-peptide interactions are mainly driven by hydrophobic interactions and that some peptides are aggregated at acidic pH [14]. The morphology of these aggregates avoids the neutralization of the negative charge of the membrane surface with the alkaline peptides in the bulk. Therefore, higher permeability and higher transmission of small positive peptides is obtained under these conditions.

Furthermore peptide aggregates contribute at the polarization concentration on the membrane surface. In this case, the peptides can interact in the polarized layer during the filtration process and their transmission decreases with the time under specific conditions [79].

Other successive tests demonstrated that although physico-chemical parameters such as pH and ionic strength are the dominant ones in the case of NF membranes, operational parameters which determine permeate flux through the membrane, and in particular transmembrane pressure, have also an important influence on the retention of peptides and therefore on the selectivity of the membrane [71]. Furthermore, it should be noted that the resulting sieving properties of some NF membranes could depend on the fouled peptide layer and the composition of this layer interacting with the membrane is pH dependant [28].

The combination of membrane processes (UF and NF) was also recently used in the fractionation of whey hydrolysates to study peptides transmission [29]. The first step of this process consisted in the UF of the hydrolysate in order to obtain complete rejection of intact proteins and intermediate peptides. The resulting permeate fractions were then subjected to a fractionation by NF and a peptide fraction having a molar mass range of 5-2 kDa was isolated in this step. Transmission of peptides, amino acids and lactose were found to be mainly affected by the permeability of the fouling layer showing the effect of peptide aggregates.

Comparison of results of NF peptides using a single amino acid solutions, amino acid mixtures and peptide mixtures, had enabled to conclude that whatever the complexity of the solution: the charge is the most important criterion for the separation of peptides having similar molecular weight. The pH value of the solution is the parameter, which has the greatest effect on the separation. Addition of salts (increase of ionic strength) could decrease the intensity of charge effects. The determination of both the membrane and the mixture characteristics are of paramount importance in order to predict and optimize the performance of NF membranes for the fractionation of complex peptide mixtures.

#### *3.2.3. Main parameters influencing peptide fractionation using NF membranes*

254 Bioactive Food Peptides in Health and Disease

is obtained under these conditions.

transmission.

found at alkaline conditions without the addition of NaCl. In addition the authors demonstrated that two peptides differing by only one amino acid are transmitted differently. Consequently a single change in the amino acid sequence can affect peptides

Influence of peptide interactions on peptide separation was also established in some studies of NF membranes. The data show that the same peptide could be transmitted differently when issued from different hydrolysates, reflecting the importance of surrounding peptides, and, hence, the possible occurrence of peptide-peptide interactions [78]. Therefore hydrophobic interactions between peptides when the pH of the solution is close to their pI

By means of NF experiments on fractionation of β-Lg tryptic hydrolysate, it was shown that peptide-peptide interactions are mainly driven by hydrophobic interactions and that some peptides are aggregated at acidic pH [14]. The morphology of these aggregates avoids the neutralization of the negative charge of the membrane surface with the alkaline peptides in the bulk. Therefore, higher permeability and higher transmission of small positive peptides

Furthermore peptide aggregates contribute at the polarization concentration on the membrane surface. In this case, the peptides can interact in the polarized layer during the filtration process and their transmission decreases with the time under specific conditions [79]. Other successive tests demonstrated that although physico-chemical parameters such as pH and ionic strength are the dominant ones in the case of NF membranes, operational parameters which determine permeate flux through the membrane, and in particular transmembrane pressure, have also an important influence on the retention of peptides and therefore on the selectivity of the membrane [71]. Furthermore, it should be noted that the resulting sieving properties of some NF membranes could depend on the fouled peptide layer and the composition of this layer interacting with the membrane is pH dependant [28].

The combination of membrane processes (UF and NF) was also recently used in the fractionation of whey hydrolysates to study peptides transmission [29]. The first step of this process consisted in the UF of the hydrolysate in order to obtain complete rejection of intact proteins and intermediate peptides. The resulting permeate fractions were then subjected to a fractionation by NF and a peptide fraction having a molar mass range of 5-2 kDa was isolated in this step. Transmission of peptides, amino acids and lactose were found to be mainly affected by the permeability of the fouling layer showing the effect of peptide aggregates.

Comparison of results of NF peptides using a single amino acid solutions, amino acid mixtures and peptide mixtures, had enabled to conclude that whatever the complexity of the solution: the charge is the most important criterion for the separation of peptides having similar molecular weight. The pH value of the solution is the parameter, which has the greatest effect on the separation. Addition of salts (increase of ionic strength) could decrease the intensity of charge effects. The determination of both the membrane and the mixture

can lead to their aggregation and subsequent fouling of the NF membrane.

The interactions of peptide-peptide and peptide-membrane affect the separation process performance and thus it is difficult to predict the selectivity of the membranes when the objective is the fractionation of complex peptide mixtures. According the literature, the most important parameters that cause effect on membrane selectivity are pH, ionic strength, polarization layer and fouling.

1. The pH of solution is an important control variable in NF processes for the fractionation of complex peptide mixtures, because peptides are molecules that have at least one carboxylic group ( *R COOH R COO* and one amine group 3 2 (*R NH R NH* ). The total number of acid and basic groups depends on its primary structure (amino acid sequence) and it determines the pH value at which the peptides have the same number of negative than positive charges, i.e., its pI. Peptides can be classified in three different groups according to their pI: acidic peptides (pI ≤ 5), neutral peptides (5 < pI ≤ 7) and basic peptides (pI > 7). Their net charge depends on the pH of the solution, as well as the charge density of the NF membrane. This last value will vary because of the ionization of its functional groups (acidic and basic).

In NF, the transmission of amino acids and peptides reaches its maximum value when the pH is equal to the pI. Under these conditions, repulsive electrostatic interactions are minimized. That way, the modification of NF membranes transmission is possible by changing the pH of the mixture.

In the case of protein hydrolysates, which composition is more complex, there will be a pH value at which the fractionation of acid, neutral and basic peptides is maximized. For example, it has been shown that the separation factor between basic and acid peptides reaches its maximum value when the pH of the mixture is alkaline [16]. However, literature published on this topic only describes the behavior of "tracer" peptides in the hydrolysate and this limits the scope of the separation factor calculated.

2. The ionic strength of peptides solution affects the selectivity of NF membranes. In an aqueous medium the increase of the ionic strength, for example by the addition of NaCl, results in a decrease of zeta potential of the NF membrane [80-83] as well as a decrease in the electrophoretic mobility of proteins and peptides [84]. According to these observations, electrostatic interactions between the membrane and the peptides become less intense, which usually leads to better transmission values of the peptides.

Several authors have demonstrated the preponderance of a selectivity based on electrostatic interactions at low ionic strength values [16, 68, 85]. The fact that electrostatic interactions membrane-peptide lose significance at high ionic strength values results in a decrease of the double selectivity size/charge in processes involving NF membranes. In addition to the effect over the charge density of the membrane, ionic strength also influences the effective hydrodynamic volume of charged proteins and peptides [86]. A charged protein is surrounded by a diffuse ion cloud, typically called the electrical double layer, and the thickness of this layer is characterized by the Debye length (LD):

$$L\_D = 0.304 \, I^{-1/2} \tag{3}$$

Advancements in the Fractionation of Milk Biopeptides by Means of Membrane Processes 257

(5)

(6)

Concentration polarization is one of the consequences of selective solute transport through membranes. The constituents of the solution that are retained by the membrane tend to accumulate over its surface and this creates a concentration gradient in the area called polarization boundary layer. This phenomenon is quickly established at the beginning of the process and leads to a modification in the efficiency of membrane processes as well as a change in the composition of the permeate stream. The management of hydrodynamic conditions could minimize its effects. In addition size exclusion properties related to the pore size of the membrane could be completely modified due to pore blocking by the peptides. Fouling is a general term for any accumulation of deposits and materials over the membrane surface or within the pores. Two kinds of fouling can be defined: reversible fouling, the one which can be reduced by adjusting hydrodynamic conditions (velocity or transmembrane

pressure), and irreversible fouling, which effect can´t be avoid by cleaning procedures.

clean water flux rate (JW) through a membrane is defined by equations (5).

Equation 6 allows the calculation of the resistance associated to fouling (Rf).

resistance of the membrane.

JW' = ��

be expressed as:

membranes [92-93].

**techniques** 

In practice, the series resistance model is widely used for fouling quantification in membrane processes. This approach derives from Darcy's phenomenological equation. The

> �� <sup>=</sup> �� ����

Where PT is the transmembrane pressure, μW the water viscosity and RM the intrinsic

The measurement of water flux rate through the same membrane after being used (JW') can

Studies involving peptides transmission or retention don´t usually take into account the polarization and fouling phenomena but it has been demonstrated that these phenomena are crucial in the case of protein hydrolysates, especially at acid pH values [16, 68]. Complex peptide mixtures contain peptides, which with different physicochemical characteristics (pI, hydrophobicity, charge) promote the creation of strong interactions with filtration

**4. Future potential of peptides fractionation by means of membrane** 

Currently, conventional membrane separation techniques can be employed to obtain peptide fractions in purified form with higher functionality and higher nutritional value. Special properties of the NF membranes make possible novel peptide separations. However, the specific separation of one or more peptides from a raw hydrolysate is a difficult subject because ionic interactions between peptides and membranes can markedly influence on peptides fractionation. In addition these pressure-driven processes involve the accumulation of particles on membrane leading formation of a fouling and to the modification of the

��������)

Where *I* is the ionic strength (mol/L) and LD is in nm. According to equation 3 the higher the ionic strength the narrower the Debye length.

In addition, the effect of the electrical double layer could be described in terms of an increase in the effective protein radius Reff:

$$R\_{eff} = r\_\text{s} + 0.045 \, Z^2 \, \frac{L\_D}{r\_\text{s}} \tag{4}$$

Where rs is the hard-sphere radius of the uncharged protein or peptide (in nm) and z is the surface charge of the protein (in electronic charge units).

Equations (3) and (4) indicate that relatively low salt concentration is needed in order to enhance the magnitude of the electrostatic interactions. However, the increase in the ionic strength leads to an increase in the transmission of charged peptides though the membrane.

This last observation, which is well known and it has been applied to explain the selectivity of several protein separation processes using UF membranes, is not usually mentioned in works involving the separation of peptides by NF membranes. The effects of the ionic strength over the charge density of the membrane and over the effective hydrodynamic volume of charged peptides are complementary and both of them contribute in the explanation of experimental results.

Variation of these parameters has been applied by some authors [86-88] to obtain good selectivity values in the fractionation of different proteins with similar sizes. The wise combination between membranes, pH and ionic strength is called HPTFF (Highperformance-tangential flow filtration) and it is effective when proteins or peptides to be fractionated show different pI and when low or medium protein concentration is processed.

3. Concentration polarization and fouling is also a condition affecting the peptides separation. Physico-chemical parameters such as pH and ionic strength are of paramount importance in NF processes because they modulate the electrostatic interactions on which the selectivity of these membranes is supported. In addition electrostatic interactions may partly explain the distribution of a peptide between the whole solution and the membrane interface [89-90]. However, when using porous membranes, peptides are involved in a convective transport flux and its rejection is therefore the result of (i) electrostatic interactions between the membrane and the peptides plus (ii) a steric mechanism through the porous. In this sense, hydrodynamic parameters have influence in peptide rejection [91]. Thus, for example, when the MWCO of the membrane and the molecular weight of the peptide have similar values or in the presence of electrostatic interactions, an increase in transmembrane pressure will result in an increase of amino acid retention.

Concentration polarization is one of the consequences of selective solute transport through membranes. The constituents of the solution that are retained by the membrane tend to accumulate over its surface and this creates a concentration gradient in the area called polarization boundary layer. This phenomenon is quickly established at the beginning of the process and leads to a modification in the efficiency of membrane processes as well as a change in the composition of the permeate stream. The management of hydrodynamic conditions could minimize its effects. In addition size exclusion properties related to the pore size of the membrane could be completely modified due to pore blocking by the peptides. Fouling is a general term for any accumulation of deposits and materials over the membrane surface or within the pores. Two kinds of fouling can be defined: reversible fouling, the one which can be reduced by adjusting hydrodynamic conditions (velocity or transmembrane pressure), and irreversible fouling, which effect can´t be avoid by cleaning procedures.

256 Bioactive Food Peptides in Health and Disease

ionic strength the narrower the Debye length.

surface charge of the protein (in electronic charge units).

increase in the effective protein radius Reff:

explanation of experimental results.

hydrodynamic volume of charged proteins and peptides [86]. A charged protein is surrounded by a diffuse ion cloud, typically called the electrical double layer, and the

Where *I* is the ionic strength (mol/L) and LD is in nm. According to equation 3 the higher the

In addition, the effect of the electrical double layer could be described in terms of an

���� � �� � ����� �� ��

Where rs is the hard-sphere radius of the uncharged protein or peptide (in nm) and z is the

Equations (3) and (4) indicate that relatively low salt concentration is needed in order to enhance the magnitude of the electrostatic interactions. However, the increase in the ionic strength leads to an increase in the transmission of charged peptides though the membrane.

This last observation, which is well known and it has been applied to explain the selectivity of several protein separation processes using UF membranes, is not usually mentioned in works involving the separation of peptides by NF membranes. The effects of the ionic strength over the charge density of the membrane and over the effective hydrodynamic volume of charged peptides are complementary and both of them contribute in the

Variation of these parameters has been applied by some authors [86-88] to obtain good selectivity values in the fractionation of different proteins with similar sizes. The wise combination between membranes, pH and ionic strength is called HPTFF (Highperformance-tangential flow filtration) and it is effective when proteins or peptides to be fractionated show different pI and when low or medium protein concentration is processed.

3. Concentration polarization and fouling is also a condition affecting the peptides separation. Physico-chemical parameters such as pH and ionic strength are of paramount importance in NF processes because they modulate the electrostatic interactions on which the selectivity of these membranes is supported. In addition electrostatic interactions may partly explain the distribution of a peptide between the whole solution and the membrane interface [89-90]. However, when using porous membranes, peptides are involved in a convective transport flux and its rejection is therefore the result of (i) electrostatic interactions between the membrane and the peptides plus (ii) a steric mechanism through the porous. In this sense, hydrodynamic parameters have influence in peptide rejection [91]. Thus, for example, when the MWCO of the membrane and the molecular weight of the peptide have similar values or in the presence of electrostatic interactions, an increase in

transmembrane pressure will result in an increase of amino acid retention.

�� � ����� ����� (3)

(4)

��

thickness of this layer is characterized by the Debye length (LD):

In practice, the series resistance model is widely used for fouling quantification in membrane processes. This approach derives from Darcy's phenomenological equation. The clean water flux rate (JW) through a membrane is defined by equations (5).

$$J\_W = \frac{P\_T}{\mu\_W R\_M} \tag{5}$$

Where PT is the transmembrane pressure, μW the water viscosity and RM the intrinsic resistance of the membrane.

The measurement of water flux rate through the same membrane after being used (JW') can be expressed as:

$$\text{Jw}' = \frac{P\_T}{\mu\_W (R\_M + R\_f)} \tag{6}$$

Equation 6 allows the calculation of the resistance associated to fouling (Rf).

Studies involving peptides transmission or retention don´t usually take into account the polarization and fouling phenomena but it has been demonstrated that these phenomena are crucial in the case of protein hydrolysates, especially at acid pH values [16, 68]. Complex peptide mixtures contain peptides, which with different physicochemical characteristics (pI, hydrophobicity, charge) promote the creation of strong interactions with filtration membranes [92-93].

## **4. Future potential of peptides fractionation by means of membrane techniques**

Currently, conventional membrane separation techniques can be employed to obtain peptide fractions in purified form with higher functionality and higher nutritional value. Special properties of the NF membranes make possible novel peptide separations. However, the specific separation of one or more peptides from a raw hydrolysate is a difficult subject because ionic interactions between peptides and membranes can markedly influence on peptides fractionation. In addition these pressure-driven processes involve the accumulation of particles on membrane leading formation of a fouling and to the modification of the

membrane transport selectivity. Therefore, it is clear that NF still has to grow more in terms of understanding, materials, and process control. In addition modeling studies are necessary to predict of the process performance in all circumstances.

Advancements in the Fractionation of Milk Biopeptides by Means of Membrane Processes 259

[1] Muro-Urista C, Álvarez-Fernández R, Riera-Rodríguez F, Arana-Cuenca A, Téllez-Jurado. A. Review: Production and Functionality of Active Peptides from Milk. Food

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Alternatively the application of an external electrical field, which acts as an additional driving force to the pressure gradient, can be seen as a technique that could improve the efficiency of the conventional membrane processes for the separation of charged bioactive molecules. In this sense, two different configurations can be distinguished: electrically-enhanced filtration, which can be used with conventional pressure driven membrane filtration, and forced-flow membrane electrophoresis, which is conducted in an electrophoretic cell. Intensive researches on these membrane processes have been carried out including electromembrane filtration (EMF) [94-95], electrodialysis with UF membranes (EDUF) [96-99] and forced-flow electrophoresis (FFE) [100] for the separation of charged bioactive molecules.

EDUF couples size exclusion capabilities of UF membranes with the charge selectivity of electrodyalysis (ED) allowing separation of molecules according to their electric charges and to their molecular mass (membrane filtration cut-off). The feasibility of peptide fractionation by EDUF was demonstrated notably with β-Lg tryptic hydrolysate solutions and was suggested to improve the separation between basic and neutral peptides [97]. Actually, EDUF process also allowed a selective and a simultaneous separation of anionic and cationic peptides presents in an uncharacterized concentrated polypeptide mixture of snow crab byproducts hydrolysate [101].

Recently a comparative study on NF and EDUF was performed in terms of flux and mass balance [102]. The results showed that NF provides a greater mass flux while when using EDUF a wider range of peptides and more polar amino acids are recovered. EDUF can be seen to be a promising separation technology, but further scale-up developments will be necessary to confirm its feasibility at large scale.

EMF combines the separation mechanisms of membrane filtration and electrophoresis. Ion exchange membranes are replaced by UF in a conventional electrodialysis cell. In electrophoretic separators, a porous membrane is used to put into contact two flowing liquids between which an electrically driven mass transfer takes place. During this process the mass transport is affected by electrostatic interactions taking place at the membrane solution interface. The perspectives in the field of peptide fractionation will be the complete understanding of the interactions of peptides and membrane as well as the development of new membrane materials of gels limiting or increasing these interactions to improve the selectivity and the yield of production of specific peptides [100].

## **Author details**

Claudia Muro *Institute Technological of Toluca, México* 

Francisco Riera and Ayoa Fernández *University of Oviedo, Spain* 

#### **5. References**

258 Bioactive Food Peptides in Health and Disease

products hydrolysate [101].

**Author details** 

*University of Oviedo, Spain* 

*Institute Technological of Toluca, México*  Francisco Riera and Ayoa Fernández

Claudia Muro

necessary to confirm its feasibility at large scale.

selectivity and the yield of production of specific peptides [100].

to predict of the process performance in all circumstances.

membrane transport selectivity. Therefore, it is clear that NF still has to grow more in terms of understanding, materials, and process control. In addition modeling studies are necessary

Alternatively the application of an external electrical field, which acts as an additional driving force to the pressure gradient, can be seen as a technique that could improve the efficiency of the conventional membrane processes for the separation of charged bioactive molecules. In this sense, two different configurations can be distinguished: electrically-enhanced filtration, which can be used with conventional pressure driven membrane filtration, and forced-flow membrane electrophoresis, which is conducted in an electrophoretic cell. Intensive researches on these membrane processes have been carried out including electromembrane filtration (EMF) [94-95], electrodialysis with UF membranes (EDUF) [96-99] and forced-flow

EDUF couples size exclusion capabilities of UF membranes with the charge selectivity of electrodyalysis (ED) allowing separation of molecules according to their electric charges and to their molecular mass (membrane filtration cut-off). The feasibility of peptide fractionation by EDUF was demonstrated notably with β-Lg tryptic hydrolysate solutions and was suggested to improve the separation between basic and neutral peptides [97]. Actually, EDUF process also allowed a selective and a simultaneous separation of anionic and cationic peptides presents in an uncharacterized concentrated polypeptide mixture of snow crab by-

Recently a comparative study on NF and EDUF was performed in terms of flux and mass balance [102]. The results showed that NF provides a greater mass flux while when using EDUF a wider range of peptides and more polar amino acids are recovered. EDUF can be seen to be a promising separation technology, but further scale-up developments will be

EMF combines the separation mechanisms of membrane filtration and electrophoresis. Ion exchange membranes are replaced by UF in a conventional electrodialysis cell. In electrophoretic separators, a porous membrane is used to put into contact two flowing liquids between which an electrically driven mass transfer takes place. During this process the mass transport is affected by electrostatic interactions taking place at the membrane solution interface. The perspectives in the field of peptide fractionation will be the complete understanding of the interactions of peptides and membrane as well as the development of new membrane materials of gels limiting or increasing these interactions to improve the

electrophoresis (FFE) [100] for the separation of charged bioactive molecules.


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## *Edited by Blanca Hernandez-Ledesma and Chia-Chien Hsieh*

"Bioactive Food Peptides in Health and Disease" highlights recent developments on bioactive food peptides for the promotion of human health and the prevention/ management of chronic diseases. The book provides a comprehensive revision of bioactive peptides obtained from both animal and plant food sources. Aspects related to their bioactivity, mechanism of action, and bioavailability are extensively described along the different chapters. Also, the chapters describe the impact of bioactive peptides on the physiological absorption, regulation and disease prevention. The book also covers the recent technological advances for the production of food peptides. Bioactive Food Peptides in Health and Disease provides updated and interesting information, being a good reference book for nutritional and food scientists, biochemists, industry producers, and consumers.

Photo by Anna\_Gavrylova / iStock

Bioactive Food Peptides in Health and Disease

Bioactive Food Peptides in

Health and Disease

*Edited by Blanca Hernandez-Ledesma* 

*and Chia-Chien Hsieh*