R: Concise Reviews in Food Science
Bioactive Peptides and Hydrolysates from Pulses and Their Potential Use as Functional Ingredients
Bioactive peptides (BPs) are amino acid sequences derived from food proteins. Their relevance lies in the biological activities they have once they are released from the parent protein. BPs or protein hydrolysates can be commercialized as nutraceutical products or functional ingredients according to their activities. Different food protein sources have been researched for their potential to generate BPs. However, with the exception of lunasin (derived from soy), animal protein sources have been predominantly exploited as commercial BPs sources. On the other hand, pulses have shown diverse BP contents without further impact on their commercialization. Pulses are a rich source of protein in the human diet and their consumption has been associated with the prevention of chronic diseases. The beneficial effect in human health has been related to their micronutrients, phytochemical bioactive compounds, and recently BPs. This article reviews the current literature about pulse protein hydrolysates and BPs with proved angiotensin converting enzyme inhibitory, antioxidant, cancer preventing, and other health promoting activities. Proteolysis process is commonly achieved by digestive and microorganism enzymes. BP purification and identification has consisted mainly on size segregation procedures followed by mass spectrometry techniques. Hydrolysis time, peptide size, and hydrophobicity are employed as process variants and structural features relevant for the BP activities. Finally, some considerations about industrial processing and BPs used as functional food ingredients were reviewed.
According to the Food and Agricultural Organization (FAO) pulses are dry seeds of annual legume plants belonging to the Fabaceae family (or Leguminosae family). This definition excludes vegetable cultivars (harvested green for food), the crops used for oil extraction like soybean (Glycine max) and peanuts (Arachis hypogaea), and the ones used for sowing, such as clover (Trifolium sp) and alfalfa (Medicago sativa). FAO covers 11 primary pulses: dry beans, dry broad beans, dry peas, chickpeas, dry cowpeas, pigeon peas, lentils, bambara beans, vetches, lupins, and/or minor pulses (FAO 2007).
Nutritionally, pulses are an important source of proteins, which, in spite of being deficient in sulfur-containing amino acids and tryptophan, possess greater amounts of lysine, arginine, glutamic, and aspartic acidamides in comparison with cereals grains. Beyond their known nutritional benefits, pulse consumption has recently been associated with protective or therapeutic effects on chronic health conditions, such as cardiovascular diseases, diabetes, cancer, overweight, and obesity (Bazzano and others 2001; Lanza and others 2006; Bean and others 2008; Patterson and Maskus 2009; Marinangeli and Jones 2011; Zhu and others 2012).
Owing to their low cost and easy adaptation to grow in poor conditions, in several low-income countries pulses are used as staple foods and serve as a principal source of both protein and calories (Duranti 2006; FAO 2012). Otherwise, in America and Europe, high-income countries, pulses consumption is low and efforts are being made to promote their intake. Health organizations recommend pulse consumption as part of a healthy diet and initiatives have been developed to improve their production, intake, and uses (Leterme 2002; American Pulse Association 2010).
The protein content of pulses ranges from 20% to 40% dry weight, within these percentages, the most abundant are seed storage proteins (SSPs). Depending on the source, seeds usually present 1 or 2 predominant type of SSP (Table 1). The rest are minor or housekeeping proteins, which include enzymes, protease, and amylase inhibitors, lectins, lipooxygenase, defense proteins, and others (Duranti 2006; Roy and others 2010).
Table 1. Predominant globulins on different pulses
|Phaseolus spp. (Dry beans)||Phaseolin||50||(Emani and Hall 2008)|
|Vicia faba (Dry broad beans)||Vicilin||30||(Bassuner and others 1987)|
|Pisum spp. (Dry peas)||Vicilin and legumin||26.3-52 and 5.9-24.5||(Tzitzikas and others 2006)|
|Cicer arietinum (Chickpea)||Legumin and vicilin||66.5 and 12.2||(Mittal and others 2012)|
|Vigna unguiculata spp. Dekindtiana (Dry cowpea, blackeye pea, blackeye bean)||α-vignin, β-vignin, γ-vignin||51||(Freitas and others 2004)|
|Cajanus cajan (Pigeon pea, cajan pea, congo bean)||Globulin||68||(Krishna and others 1977)|
|Lens culinaris (Lentil)||Vicilin and legumin||72 and 11||(Scippa and others 2010)|
|Vigna subterranea (Bambara groundnut, earth pea)||SSP B and vicilin||17.5-21.1||(Okpuzor and others 2009)|
|Vicia sativa (Vetch, common vetch)||α-vicilin and β-vicilin||73 and 50.8||(Ribeiro and others 2004)|
|Lupinus spp. (Lupins)||α –Conglutin and β –Conglutin||36 and 44.5||(Duranti and others 2008)|
Based on their solubility properties, SSPs are classified as globulins, albumins, glutelins, and prolamins. Globulins are soluble in salt–water solutions, albumins in water, glutelins in diluted acids or bases, and prolamins in ethanol–water solutions. Globulins and albumins comprise the storage proteins of dicots-like pulses, where they represent approximately 70% of the total protein (Table 1). Legume globulins are generally categorized as 7S vicilin-type and 11S legumin-type according to their sedimentation coefficients, S. Globulins commonly found in legumes are legumin, vicilin, and convicilin (Duranti 2006; Roy and others 2010; Marambe and Wanasundara 2012).
Legume seeds also contain proteins considered as antinutritional compounds (ANCs) due to their effect on diet quality. In pulses, the most abundant ANCs are lectins and protease inhibitors, which have shown antinutritional and even toxic effects on animal models and humans (Lajolo and Genovese 2002; Campos-Vega and others 2010). Nevertheless, the harmful effects of these compounds can be easily inactivated after cooking or processes like fermentation, germination, or dehulling (Champ 2002).
Once inactivated, lectins or protease inhibitors may present potential health benefits. Protease inhibitors are prospective anti-inflammatory and anticancer agents. Whereas, lectins have demonstrated to play a key role preventing certain cancers and the activation of certain innate defense mechanisms. Besides, lectins have also been proposed as therapeutic agents for preventing or controlling obesity (Roy and others 2010).
Bioactive Peptides (BPs)
BPs are defined as small amino acid sequences derived from food proteins that possess potential physiological properties beyond normal and adequate nutrition. Within the precursor proteins, the amino acid sequence conforming the BP is inactive, once the peptide gets released it can display diverse biological activities (Udenigwe and Aluko 2012).
Proteolysis is necessary to generate BPs, which may occur through enzymatic digestion of the precursor protein, either in vitro or in the digestive tract (in vivo). Also, food processing and enzymes from microorganism or plants can cause proteolysis and release of potential BPs. The biological activities of the released peptides depend on the initial protein source, the enzyme employed, and the processing conditions used (Mine and others 2010). Many BPs have common structural properties, including a relatively short residue length, hydrophobic amino acid residues, and the presence of Arg, Lys, and Pro (Marambe and Wanasundara 2012).
Nowadays, at least 18 companies worldwide produce and sell food or nutraceutical products based on hydrolyzed proteins or BPs (Table 2). Functional foods and nutraceuticals contain BPs, either added or enriched by modification of the usual manufacturing process. Furthermore, BPs, such as casein phosphopeptides, can be included in nonfood matrices to provide certain health-enhancing effects. These phosphopeptides have proved anticariogenic effect and used in combination with amorphous calcium phosphate to mouth rinse solution, toothpaste, or chewing gum (Llena and others 2009).
Table 2. Commercial products with bioactive peptides or hydrolysates as their fundamental constituents
|Lunasin||Food ingredient/ dietary supplement||Soy (Glycine max)||43 amino acid peptide (SKWQHQQDSCRKQLQGVNLTPCEKHIMEKIQGRGDDDDDDDDD)||Naturally occurring||Cholesterol lowering and cancer preventive properties||Reliv, USA|
|Vasotensin®/ PeptACE®/ LevenormTM||Dietary supplement||Bonito fish (Sarda orientalis)||LKPNM peptide named “Katshuobushi Oligopeptide.” The peptide is converted into its active form LKP in the digestive system||Thermolysin treatment and converted to “LKP” in the digestive system||Blood pressure reduction||Metagenics, USA/Swanson, USA/Ocean Nutrition, Canada|
|Lapis support/Valtyron®||Dietary supplement||Sardine (Sardina pilchardus)||LKP tripeptides||Bacillus licheniformis alkaline proteolysis||Blood pressure reduction||Tokiwa Yakuhin, Japan/Senmi Ekisu, Japan|
|Calpis||Sour milk||β-casein and к-casein||Ile-Pro-Pro and Val-Pro-Pro||Fermentation with acid lactic bacterias||Blood pressure reduction||Calpis Co., Japan|
|Ameal||Dietary supplement|| || || || ||Swanson, USA|
|Evolus||Calcium enriched fermented milk drink|| || || || ||Valio Oy, Finland|
|Biopure GMP||Food and supplement ingredient||Fresh cheese whey||Glycomacropeptide (GMP)(MAIPPKKNQDKTEIPTI-NTIASGEPTSTPTIEAVESTVATL-EASPEVIESPPEINTVQVTSTAV)||Hydrolysis with rennet||Prevention of dental caries, influence the clotting of blood, protection against viruses and bacteria||Davisco, USA.|
|Lactium®||Food ingredient||αs1-casein||Decapeptide (YLGYLEQLLR)||Tryptic hydrolysate||Stress symptoms moderation||Ingredia, France|
|Seacure®||Dietary supplement||Pacific whiting (Merluccios productus)||Fish hydrolysates derived from the controlled proteolytic yeast fermentation||Yeast fermentation||Gastrointestinal health improvement||Proper nutrition, USA|
|Protizen®||Dietary supplement||Whitefish (Coregonus lavaretus)||Hydrolysates||NA||Stress relief action||Copalis, France|
|Stabilium®||Dietary supplement||Atlantic fish||Autolysate|| ||Stress relief action||Yalacta, France|
|Biozate||Food ingredient||Whey protein isolate||β -lactoglobulin fragments from hydrolysate||NA||Blood pressure reduction||Davisco, USA.|
|Verisol®/Fortibone®/Fortigel®||Food and cosmetic ingredients||Collagen||Hydrolysates||NA||Improvement functionality of joints and bones, aids in weight reduction or rejuvenates the appearance of aging skin.||GELITA, USA|
|Fortidium®||Dietary supplement||Maruca fish (Molva molva)||Autolysate|| ||Prevents oxidative stress||Biothalassol, France|
|Nutripeptin®||Food ingredient||Cod fish||Hydrolysates||NA||Reduces glycemic index||Copalis, France/Nutrimarine, Norway|
|Peptibal®||Food ingredient||Shark protein||Hydrolysates||Trypsin-chemotrypsin hydrolysis||Stimulates the immune system||InnoVactiv, Canada|
All food proteins are capable of releasing BPs, but animal protein sources, primarily dairy products, have been extensively researched for their BPs. Milk-derived BPs have demonstrated in vivo and in vitro activities over the digestive, endocrine, cardiovascular, immune, and nervous systems (Haque and others 2008; Choi and others 2012). Meat and fish BPs also have antioxidant (AOX), antimicrobial, and antiproliferative effects (Ryan and others 2011).
During the manufacture of milk products, processes like fermentation or cheese ripening causes the release of BPs. As a result, great variety of naturally formed BPs have been found in end –products, such as fermented dairy products, yogurt, sour milk, and cheese. Moreover, industrial-scale technologies suitable for the production of bioactive milk peptides have been developed recently (Haque and others 2008; Korhonen 2009).
To our knowledge, only 1 BP from nonanimal food sources has reached the market. Lunasin, derived from soybean (G. max), is commercialized as ingredient of soy drinks and as dietary supplement in capsules or powder (Table 2). Lunasin is a 43-amino acid peptide with anticancer, anti-inflammatory, AOX, and cholesterol lowering activities (Hernández-Ledesma and others 2013).
Plant foods studied for their BPs include soy, oat, wheat, hemp seed, canola, flaxseed, and pulses. Particularly, pulse seeds hydrolysates and BPs have been described with in vitro activities toward cancer, cardiovascular conditions, or their physiological elements like oxidative damage, inflammation, hypertension, and high cholesterol (Oseguera-Toledo and others 2011; Yust and others 2012; Ajibola and others 2013; Kou and others 2013).
Production and identification of BPs
To assess if BPs are produced naturally during gastrointestinal (GI) digestion, in vitro digestions are commonly carried out employing the proteinases pepsin, trypsin, chymotrypsin, and pancreatin. As well, enzymatic hydrolysis performed with microbial or plant enzymes, such as alcalase, flavourzyme, papain, and bromelain, have been used to obtain BPs. Less common but equally effective, for seed sources, some researchers have used germination as a natural hydrolytic process that generates intrinsic enzymes (Bamdad and others 2009).
After proteolysis process, hydrolysates must be purified, based on their activities, in order to identify specific bioactive sequences. Purification steps include ultrafiltration, various types of chromatography, activated carbon columns, and electrodialysis ultrafiltration (Udenigwe and Aluko 2012). Industrially, nanofiltration and ultrafiltration techniques are now employed to yield ingredients, which contain specific BPs based on casein or whey protein hydrolysates (Korhonen 2009).
Finally, to characterize the structure and quantify the BPs, mass spectrometry (MS)-based techniques are the most effective and frequently used. The recent development of soft ionization techniques, electrospray, and matrix-assisted laser desorption/ionization, have revolutionized MS analysis of biomolecules like BPs (Contreras and others 2008; Mamone and others 2009).
In silico approaches
Correctly documented BPs can be forecasted from a known protein amino acid sequence. Computer-assisted databases are available for predicting BPs located within a parent protein. Other databases predict precursor protein of a BP from a known amino acid sequence (Marambe and Wanasundara 2012).
BIOPEP is a peptide sequences database integrated with a program that allows classifying food proteins as potential sources of BPs. Using this database and common bean (Phaseolus vulgaris) proteins sequences from UniPort database, Carrasco-Castilla and others (2012) identified sequences with 12 different biological activities corresponding to 15 seed proteins.
The common bean proteins identified with the highest abundance of BPs were phytohemagglutinin, phaseolin, and Bowman–Birk type protease inhibitor. So it was expected that the purified phaseolin hydrolysates were more active than other hydrolysates. Nonetheless, the highest AOX activity was generated by the complete protein isolate hydrolysate instead of the specific phaseolin or lectin-derived hydrolysates. As explained by the authors, the AOX potential can be affected by factors, such as synergistic AOX effects, the limited number of peptides liberated by the enzymes, cell permeability, and absorption. So even though in silico approaches enable the identification of BPs from the protein sequences (Majumder and Wu 2010; Cavazos and others 2013), it may not be the best method to search for the optimal hydrolysates activity and its use is obviously restricted to the search of BPs previously described.
Angiotensin-Converting Enzyme Inhibitory Pulse Peptides
Inhibition of angiotensin converting enzyme (ACE) results in an overall antihypertensive effect that has been exploited by commercial drugs like captopril, enalapril, and lisinopril (Hernández-Ledesma and others 2011). This fact has triggered the research for natural sources of ACE inhibitory agents, which in turn has led to the description of a large amount of BPs with presumably antihypertensive activity.
Renin inhibition is another target in the search for antihypertensive compounds and it is thought that inhibition of renin could provide a more effective treatment for hypertension than ACE inhibition (Norris and FitzGerald 2013). However, most of the research has focused on ACE inhibition.
In general, the majority of ACE inhibitory peptides described from all kinds of food sources are relatively short sequences containing from 2 to 12 amino acids (Hernández-Ledesma and others 2011). Also several publications, compiled by Norris and FitzGerald (2013), have identified structural features from the C-terminal tripeptide residue that play a predominant role in competitive binding to the active site of ACE. Such features are the presence of bulky hydrophobic residues, aromatic or branched side chains, proline at 1 or more positions, positively charged Arg and Lys residues in position 2, Tyr, Phe, and Trp residues, and L-configured residues in position 3.
The hydrolysis time is a very important factor related to the peptide size, in most cases, the hydrolysates ACE inhibitory potency increases with the hydrolysis time, implying thus that the smaller peptides are more active (Hernández-Álvarez and others 2012; Medina-Godoy and others 2012; Wan and others 2013). Correspondingly, in publications where hydrolysates were fractioned by ultrafiltration, the highest activities were found in the smaller molecular weight fractions, in all cases <1 kDa fractions (Segura-Campos and others 2010, 2011, 2013; Wan and others 2013; Ruiz-Ruiz and others 2013). This is related to the active sites containing the sequence His-Glu-XX-His, which are located within the cleft of the 2 ACE domains (C- and N-). The active sites are protected by an N-terminal “lid” that blocks the access of large polypeptides (Norris and FitzGerald 2013). Thus, small peptides are more effective inhibiting ACE activity.
Quantitative structure-activity relationship (QSAR) modeling and substrate docking can be used to asses in silico numerous peptide structures for their bioactivity potential. Wu and others (2006) reported a QSAR of ACE inhibitory di- and tripeptides. Based on a 168 dipeptide and 140 tripeptide database constructed from published literature, 2 models were computed using partial least-squares regression. The dipeptide model indicated that amino acids with bulky and hydrophobic side chains were preferred by ACE. The tripeptide model suggested that C-terminal aromatic residues, positively charged residues in position 2, and hydrophobic residues at the amino terminus were preferred. Excepting the positively charged residues on position 2 (or the middle of the chain), these results are on concordance with the structural characteristics compiled by Norris and FitzGerald (2013).
The potency of ACE inhibitory activity is normally measured as an IC50 value, which indicates the concentration of inhibitory peptide or hydrolysates needed for 50% inhibition of ACE activity (Roy and others 2010). The next phase to establish if a BP is in fact hypotensive is through trials with small animals such as spontaneously hypertensive rats (SHRs). Finally, to reach the market bearing a health claim, BPs must be subjected to human clinical trials.
Milk-derived tripeptides Ile-Pro-Pro and Val-Pro-Pro (IPP and VPP) are the best known antihypertensive peptides. They are found in several dairy functional products, such as the fermented drink Calpis® from Japan (Table 2). Numerous human clinical trials have been done with IPP and VPP to demonstrate significant decreases in systolic and diastolic blood pressure (Xu and others 2008). Nonetheless, no health claim has been granted to IPP and VPP peptides due to methodological weakness of the human clinical trials and no convincing evidence (Tetens 2012).
The oligopeptide from fish bonito (Sarda orientalis) Leu-Lys-Pro-Asn-Met possess the FOSHU (Food for Specified Health Uses) approval from Japan as blood pressure reducing agent, and is currently commercialized as a dietary supplement under the commercial names of Vasotensin®, PeptACE®, and LevenormTM (Table 2). Bonito oligopeptide is converted into Leu-Lys-Pro, ACE inhibitory molecule, in the digestive system.
Chickpea (Cicer arietinum), field pea (Pisum sativum), mung bean (Vigna radiata), and kidney bean (P. vulgaris) among others, have yielded ACE-I inhibitory hydrolysates (Table 3) or BPs (Table 4).
Table 3. ACE inhibitory hydrolysates from pulses
|Cicer arietinum||Chickpea (kabuli)||-Alcalase/Flavourzyme||316||(Barbana and Boye 2010)|
| || ||-Gastrointestinal simulation: Pepsin/trypsin-α-chymotrypsin||229|| |
| || ||-Papain||282|| |
| ||Chickpea (desi)||-Alcalase/Flavourzyme||228|| |
| || ||-Gastrointestinal simulation: Pepsin/trypsin-α-chymotrypsin||140|| |
| || ||-Papain||180|| |
| ||Fresh chickpea grains (globulin fraction)||-Alcalase||0.307||(Medina-Godoy and others 2012)|
| || ||-Papain||0.016|| |
| || ||-Pancreatin||0.585|| |
| ||Hard to cook chickpea grains (globulin fraction)||-Alcalase||0.039|| |
| || ||-Papain||0.049|| |
| || ||-Pancreatin||0.116|| |
|Phaseolus lunatus||Lima bean||-Alcalase||56||(Torruco-Uco and others 2009)|
| || ||-Flavourzyme||6.9|| |
| ||Raw lima bean||-Gastrointestinal simulation: Pepsin/Pancreatin||250||(Chel-Guerrero and others 2012)|
| ||Germinated lima bean||Gastrointestinal simulation: Pepsin/Pancreatin||280|| |
|Phaseolus vulgaris||Jamapa bean||-Alcalase||61||(Torruco-Uco and others 2009)|
| || ||-Flavourzyme||127|| |
| ||Common bean pre heated at 121 °C for 50 min||-Gastrointestinal simulation: Pepsin/pancreatin/bile||770||(Akıllıoğlu and Karakaya 2009)|
| ||Pinto bean preheated at 121 °C for 30 min|| ||150|| |
| ||Navy bean||-Alcalase/Papain||68||(Rui and others 2012)|
| ||Black bean|| ||83|| |
| ||Red bean|| ||78|| |
| ||Anthracnose-damaged Jamapa beans||-Alcalase||19.1||(Hernández-Álvarez and others 2012)|
| ||Hard-to-cook bean||-Alcalase/Flavourzyme||2.7||(Ruiz-Ruiz and others 2013)|
| ||Navy bean||-Trypsin||∼200||(Rui and Barbana 2012)|
| ||Pink bean|| ||∼275|| |
| ||Pinto bean|| ||∼225|| |
| ||Cranberry bean|| ||∼275|| |
| ||Black bean|| ||∼406|| |
| ||Great northern bean|| ||∼225|| |
| ||Light red kidney|| ||∼275|| |
| ||Dark red kidney|| ||∼375|| |
| ||Small red|| ||∼175|| |
|Phaseolus vulgaris||Navy bean||-Gastrointestinal simulation: α-amylase/pepsin/trypsin/α-chymotrypsin||137||(Rui and Barbana 2012)|
| ||Pink bean|| ||∼175|| |
| ||Pinto bean|| ||198|| |
| ||Cranberry bean|| ||∼175|| |
| ||Black bean|| ||∼150|| |
| ||Great northern bean|| ||∼150|| |
| ||Light red kidney|| ||∼125|| |
| ||Dark red kidney|| ||199|| |
| ||Small red|| ||∼125|| |
|Lens culinaris||Green lentils with heat treatment at 121 °C for 50 min||-Gastrointestinal simulation: Pepsin/pancreatin/bile||0.8||(Akıllıoğlu and Karakaya 2009)|
| ||Lentils of small seeded Persian type.||-Gastrointestinal simulation: Pepsin/trypsin-α-chymotrypsin||87||(Bamdad and others 2009)|
| || ||-Germination for 5 d||78|| |
| ||Red lentil protein||-Trypsin||440||(Boye and others 2010)|
| ||Red lentil legumin|| ||476|| |
| ||Red lentil albumin|| ||509|| |
| ||Red lentil vicilin|| ||539|| |
| ||Red lentils||- Gastrointestinal simulation: Pepsin/trypsin- α-chymotrypsin||90||(Barbana and Boye 2011)|
| || ||-Papain||86|| |
| || ||-Alcalase/Flavourzyme||154|| |
| || ||-Bromelain||190|| |
| ||Green lentils||- Gastrointestinal simulation: Pepsin/trypsin-α-chymotrypsin||53|| |
| || ||-Papain||80|| |
| || ||-Alcalase/Flavourzyme||152|| |
| || ||-Bromelain||174|| |
|Pisum sativum||Yellow pea||-Alcalase/Flavourzyme||412||(Barbana and Boye 2010)|
| || ||-Gastrointestinal simulation: Pepsin/ trypsin- α-chymotrypsin||159|| |
| || ||-Papain||128|| |
| ||Cowpea||-Flavourzyme||0.04||(Segura-Campos and others 2011)|
|Vigna unguiculata||Yam bean||-Alcalase||∼100||(Ajibola and others 2013)|
|Psophocarpus tetragonolobus||Undefatted winged bean seeds||-Papain||64||(Wan and others 2013)|
| || ||-Bromelain||73|| |
| || ||-Flavourzyme||161|| |
| || ||-Alcalase||91|| |
| ||Defatted winged bean seeds||-Papain||249|| |
| || ||-Bromelain||293|| |
| || ||-Flavourzyme||377|| |
| || ||-Alcalase||374|| |
Table 4. Identified bioactive peptides with ACE inhibitory activity
|Mung bean (Vigna radiata)||-Alcalase||Lys-Asp-Tyr-Arg-Leu||17.9||(Li and others 2006)|
| || ||Val-Thr-Pro-Ala-Leu-Arg||52.55|| |
| || ||Lys-Leu-Pro-Ala-Gly-Thr-Leu-Phe||11.1|| |
|Red bean (Phaseolus vulgaris)||-Alcalase/Papain/ Pepsin/Trypsin-α-chymotrypsin||Pro-Val-Asn-Asn-Pro-Gln-Ile-His||186.03||(Rui and others 2013)|
|Chickpea (Cicer arietinum)||-Alcalase/Flavourzyme||Amino acid composition||Molar relative composition|| || |
| || ||Met-Asp||2.2:1.0||21||(Yust and others 2003)|
| || ||Met-Asp-Phe-Leu-Ile||2.9:0.9:1.2:1.0:1.3||11|| |
| || ||Met||1.0||20|| |
| || ||Met-Phe-Asp-Leu||3.1:2.0:0.9:0.9||13|| |
| || ||Met-Asp-Leu||3.0:1.0:1.0||21|| |
| || ||Met-Asp-Leu-Ala||2.7:1.0:1.4:0.8||13|| |
Hydrolysates from the same source can present different ACE inhibitory potencies depending on the enzymes used, the enzyme:substrate ratio, and the hydrolysis time. For example, yellow pea papain hydrolysates are 3 times more potent than the alcalase and flavourzyme hydrolysates (Table 3).
Hydrolysis can be performed using a particular extracted storage protein, instead of using all the protein content of the seed. As reported by Medina-Godoy and others (2012) the globulin hydrolysates of 2 varieties of chickpeas presented significantly higher ACE inhibitory activity than the albumin or total protein hydrolysates generated with alcalase or papain. However, in the hydrolysates generated with pepsin, such difference was not observed.
Hydrolysates themselves could be used as commercial sources of ACE inhibitory compounds, but it has been noted that inhibitory activity is higher in the purified peptides (Li and others 2006; Barbana and Boye 2011; Rui and others 2013). So, further research is necessary to identify specific active sequences from pulses hydrolysates. There are only 3 reports detailing the specific sequences or the molar composition of BPs inhibitors of ACE, derived from pulses (Table 4). The mentioned peptides from pulses and marine sources account for some of the characteristics described as relevant for the ACE inhibitory activity, such as peptide length (2 to 12 amino acid residues) and the bulky hydrophobic residues in the C-terminal position (Norris and FitzGerald 2013).
Chickpea BPs reported by Yust and others (2003) were generated from its legumin extract with a 30 min alcalase hydrolysis. Further purification with reverse phase (RP) high-performance liquid chromatography evidenced that hydrophobic peptides and peptides with higher number of different amino acids residues were stronger ACE inhibitors.
Li and others (2006) reported 3 mung bean peptides hydrolyzed also with alcalase. The 3 peptides described by Li and others (2006) are believed to be responsible for the antihypertensive effect showed by mung bean alcalase hydrolysates over SHRs on a previous study (Li and others 2005).
Most recently, Rui and others (2013) identified an ACE inhibitory octapeptide from small red bean. For the generation of this peptide, the bean protein extract was first submitted to a heat treatment (15 min in boiling water) and then hydrolyzed by sequential digestion with alcalase, papain, and an in vitro GI digestion with pepsin/trypsin-α-chymotrypsin. Enzymatic studies using the most active fraction demonstrated competitive inhibition with a low ki value, indicating high affinity between the inhibitors and the enzyme. Finally, the characterization of the fraction using tandem MS allowed the identification of an octapeptide originated from phaseolin, the major storage protein.
The recovery of BPs from SSP reported for legumin and phaseolin, could implicate better yields on recovery of such BP, and the feasibility of using seeds as commercial sources of BPs, since SSP are highly abundant in them. It remains to be studied if the described peptides exert antihypertensive activity in vivo as well as optimization and scale up of the purification steps.
Antioxidants can avoid or inhibit oxidation by preventing generation of reactive oxygen species (ROS) in the metabolism or by inactivating them. Therefore, AOXs are of great importance in the human diet, as they can help the body to diminish oxidative damages (Kohen and Nyska 2002). There is abundant literature information on AOX hydrolysates or peptides, derived from several plant and animal food proteins. The interest on such AOX food-derived peptides lies on its 2 possible roles as dietary AOX supplements and/or as food preservatives (Ajibola and others 2011).
Similarly to the recovery of ACE inhibitory peptides, AOX hydrolysates have been generated on a different number of ways, and their activity is affected by the processing factors and structural properties of the resulting peptides. Structure activity studies have proposed peptide features relevant for their AOX activity. Regarding to the amino acid composition, it is known that basic amino acids possess chelating capacity for metallic ions, while aromatic and sulfur counterparts have the capacity to donate protons to free radicals. About the position of the amino acids in the peptide chain, branched amino acids valine and leucine have more AOX activity when they are found in the N-terminal position, as well as tryptophan and tyrosine in the C-terminal position (Medina-Godoy and others 2012). However, the specific contribution of individual amino acid residues to the AOX activity of a peptide depends largely on the nature of the ROS/free radical and the reaction medium (Udenigwe and Aluko 2012).
Several techniques have been employed on pulse protein hydrolysates to determinate their AOX capacities (Table 5). It can be noted that in some investigations, the same hydrolysates exhibit different AOX activities depending on the method for the AOX assay employed (Wongekalak and others 2011; Carrasco-Castilla and others 2012; Medina-Godoy and others 2012). Nonetheless, it may be possible to identify a particular hydrolysate that presents an overall AOX activity in all the methods analyzed, as in the case of the kidney bean hydrolysate fraction Fra-C reported by Ren and others (2010). Fra-C showed the highest 2,2-diphenyl-1-picrylhydrazyl (DPPH)/superoxide/hydroxyl radical scavenging activities from all the other fractions obtained by gel filtration.
Table 5. Antioxidant hydrolysates from pulse crops
|ABTS assay||P. lunatus||-Flavourzyme||11.55 mM TEAC/mg protein||(Torruco-Uco and others 2009)|
| ||P. vulgaris||-Alcalase||10.09 mM TEAC/mg protein|| |
| || ||-Pepsin/Pancreatin||6922 mM TEAC/mg protein||(Ruiz-Ruiz and others 2013)|
| ||V. unguiculata||-AlcalaseFlavourzymePepsin/ ancreatin||1457 mM TEAC/mg protein 200mM TEAC/mg protein 830 mMTEAC/mg protein||(Segura-Campos and others 2013)|
| ||C. arietinum||-AlcalasePapainPancreatin||27.73–95.61 mM TEAC/mg protein||(Medina-Godoy and others 2012)|
| ||V. radiate||-Trypsin||4.6e−4 mmol TEAC/mg protein||(Wongekalak and others 2011)|
|DPPH radical-scavenging activity||P. vulgaris||-Alkali/neutral protease||IC50avalue of 2.301 mg/mL||(Ren and others 2010)|
| ||V. unguiculata||-Alkaline proteinase M0126||IC50avalue of 6.38 mg/mL||(Xiong and others 2012)|
| ||S. stenocarpa||-Alcalase||∼40% 1 mg/mL||(Ajibola and others 2011)|
| ||C. arietinum||-AlcalasePapainPancreatin||18–17–46.76 mM TEAC/mg protein||(Medina-Godoy and others 2012)|
| || ||-Alcalase/Flavourzyme||∼50% to 80% at a 10 mg/mL assay||(Yust and others 2012)|
| ||P. sativum||-Thermolysin||∼20% 1 mg/mL||(Pownall and others 2010)|
|ORACFL||V. radiate||-Trypsin||6.7e−4 mmol TEAC/mg protein||(Wongekalak and others 2011)|
| ||P. vulgaris||-Alcalase||∼30 mmol TEAC/mg protein||(Oseguera-Toledo and others 2011)|
| || ||-Pepsin/pancreatin||∼42 mmol TEAC/mg protein|| |
|Reducing power||P. vulgaris||-Alkali/neutral protease||RP50 of 2.263 mg/mL||(Ren and others 2010)|
|Hydroxyl radical-scavenging activity||P. vulgaris||-Alkali/neutral protease||IC50avalue of 1.386 mg/mL|| |
| ||V. unguiculata||-Alkaline proteinase M0126||50% at a 2.03 mg/mL assay||(Xiong and others 2012)|
| ||S. stenocarpa||-Alcalase||∼30% at a 1 mg/mL assay||(Ajibola, and others Okpuzor and others 2011)|
| ||P. sativum||-Thermolysin||∼17.5% at a 1 mg/mL assay||(Pownall and others 2010)|
|O2− radical-scavenging activity||P. vulgaris||-Alkali/neutral protease||IC50avalue of 2.553 mg/mL||(Ren and others 2010)|
| ||P. sativum||-Thermolysin||∼30% at a 1 mg/mL assay||(Pownall and others 2010)|
|O2H2 radical-scavenging activity||P. sativum||-Thermolysin||∼70% at a 1 mg/mL assay||(Pownall and others 2010)|
|bAntioxidant activity of hydrolysates in Caco-2 cells||P. vulgaris||-Pepsin/ancreatin||71% inhibition at 0.250 mg/cells||(Carrasco-Castilla and others 2012)|
|Copper chelating activity||P. vulgaris||-Pepsin/ancreatin||53%|| |
|Iron chelating activity||P. vulgaris||-Pepsin/ ancreatin||81%|| |
|Superoxide radical-scavenging activity||S. stenocarpa||-Alcalase||∼40% 1 mg/mL||(Ajibola and others 2011)|
|Inhibition of linoleic acid oxidation||S. stenocarpa||-Alcalase||Dose-dependent inhibition of lipid at 0.25–1.00 mg/mL||(Ajibola and others 2011)|
| ||P. sativum||-Thermolysin||Inhibition of linoleic acid oxidation over 7 d||(Pownall and others 2010)|
|β-carotene bleaching method||C. arietinum||-Alcalase/Flavourzyme||26% protection index||(Yust and others 2012)|
Fractions <1 kDa from black bean, cowpea, and African yam bean, had the highest trolox equivalent antioxidant capacity (TEAC) values, in the DPPH, 2,2-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS) and hydroxyl radical scavenging activities as well in the Fe3+ reduction methods (Segura-Campos and others 2010; Ajibola and others 2011; Ruiz-Ruiz and others 2013; Segura-Campos and others 2013). This may reflect the enhanced accessibility of small peptides to the redox reaction system.
Regarding hydrophobicity, Ajibola and others (2013) found that the ability to scavenge hydroxyl radicals was independent of the hydrophobic character of African yam bean hydrolysates. As well, the reducing power (ability to donate electrons) from pea hydrolysates was not related to the hydrophobic character (Pownall and others 2010). Nonetheless, most of the literature refers that a high level of hydrophobicity is important for peptides AOX activity (Torruco-Uco and others 2009; Pownall and others 2010; Ren and others 2010; Ajibola and others 2011). It is also believed that an increase in hydrophobicity of peptides increases their solubility in lipid and therefore enhances their AOX activity (Ren and others 2010). Kou and others (2013) also suggest that hydrophobic amino acids with bulky and aromatic side chains may act as hydrogen donors and AOXs. The author and his colleagues identified the peptide sequence Arg-Gln-Ser-His-Phe-Ala-Asn-Ala-Gln-Pro, responsible of the radical scavenging activity and reducing power of chickpea albumin hydrolysates.
In an effort to understand the structure–function relationship of AOX BPs, Pownall and others (2011) evaluated the in vitro AOX activity of pea hydrolysates. Generally, they concluded that the cationic property of amino acids contributed negatively to the AOX activity of the peptide fractions.
Most of the research related to BPs and cancer has been focused in lunasin and peptides derived from marine sources (Udenigwe and Aluko 2012). Publications evidencing pulse hydrolysates or BPs active toward cancer or related syndromes have recently appeared. Girón-Calle and others (2010) evaluated cancer cell proliferation after treatment with hydrolysates from chickpea digested with pepsin/pancreatin. Hydrolysates <3 kDa inhibited human epithelial colorectal adenocarcinoma cells (Caco-2) and monocytics leukemia cells (THP-1) up to 48% and 78%, respectively. Interestingly, the authors determined the in vitro bioavailability and found a dramatic change in the inhibitory activity of the hydrolysates. A model of the intestinal absorption was made by cocultivating THP-1 cells with Caco-1 monolayers grown on inserts (representing the intestinal lumen). After the addition of hydrolysate to the inserts, the growth of THP-1 cells was promoted up to 66%. This effect could be attributed to various factors, including the hydrolysis of the BPs by peptidases expressed in the intestine. So, further research is required to assess and improve the in vivo activity of chickpea pepsin/pancreatin hydrolysates.
Inflammation and cancer are linked, chronic inflammation predisposes individuals to various types of cancer and inflammatory mediators and cells are involved in the migration, invasion, and metastasis of malignant cells (Mantovani and others 2008). It has been suggested that suppression of the pro-inflammatory pathways may provide opportunities for both prevention and treatment of cancer (Aggarwal and others 2009). Hydrolysates of the common bean (P. vulgaris) varieties Negro 8025 and Pinto Durango inhibited inflammation in Lipopolysaccharide-induced macrophages through suppression of NF-κB pathways. Protein isolates from both varieties hydrolyzed with alcalase for 120 min, in a concentration range of 3.7 to 61.3 μM, inhibited the markers of inflammation: cyclooxygenase-2 expression, prostaglandin E2 production, inducible nitric oxide synthase expression, and nitric oxide production. Also, hydrolysates significantly inhibited the transactivation of NF-κB and the nuclear translocation of the NF-κB p65 subunit (Oseguera-Toledo and others 2011).
Infections with Helicobacter pylori cause inflammation and gastric ulcers, which can precede gastric cancer, hence H. pylori is considered a group I carcinogen. The risk of infection and inflammation can be prevented by inhibiting bacterial adhesion. Niehues and others (2010) identified anti-adhesive peptides against H. pylori from peas hydrolyzed with trypsin, and fractioned by size exclusion and RP chromatography. An undecapeptide was particularly effective inhibiting bacterial adhesion in 2 different assays: a quantitative in vitro assay evaluating bacterial binding by flow cytometry on human AGS cells (adherent gastric adenocarcinoma epithelial), and a semiquantitative in situ adhesion assay using fluorescein isothiocyanate(FITC)-labeled H. pylori on human stomach tissue sections. The authors also identified that the undecapeptide interacted specifically with H. pylori outer membrane proteins involved in H. pylori adhesion to gastric epithelial cells named adhesins, particularly Baba, SabA, HpaA, and a fibronectin-binding adhesin.
From a different approach, Wongekalak and others (2011) explored the potential use of hydrolysates as cell-penetrating vehicles to bioactive compounds. The mung bean (V. radiata) tryptic hydrolysates, described previously with AOX activities (Table 5), were successfully used as a carrier for anticancer Asiatic acid (AA). The pentacyclic triterpene AA was freeze dried with mung bean protein hydrolysates and lactose before assaying on hepatoblastoma (HepG2) cells. The inhibition concentration (IC50) of AA in HepG2 was lowered from 58.5 to 38.5 μg/mL with the AA-hydrolysates complex.
Chickpea AOX hydrolysates described by Yust and others (2012) also showed hypocholesterolemic activity (Table 5). After 60 min of treatment with alcalase followed by 30 min of Flavourzyme, the resulting chickpea hydrolysates showed a cholesterol micellar solubility inhibition of 50% (Yust and others 2012).
Incorporation into Food Products
Pure peptides tend to present low bioavailability and are rapidly metabolized, so they are considered poor drug candidates. Still, peptides present key advantages that have resulted on a renewed interest as potential drug candidates for the pharmaceutical companies. Peptides present high bioactivity and specificity to targets, wide spectrum of therapeutic action, low levels of toxicity, structural diversity, and absence or low levels of accumulation in body tissues. Nowadays an unexpected and considerable number of peptides are available as drugs (Vlieghe and others 2010).
Therapeutic peptides from 5 to 50 amino acid residues are produced by large-scale chemical synthesis and recombinant transgenic methods can also be employed. However, these manufacturing methods are very expensive and represent a hindrance for the employment of therapeutic peptides (Agyei and others2011). The alternative option for taking advantage of the peptides benefits as health-enhancing elements is through the exploitation of BPs from foods proteins.
Besides BPs commercialization on pharmaceuticals presentations like capsules and powders, protein hydrolysates, or purified BPs can be incorporated as “health-enhancing” ingredients in functional foods. Pulses flours have already been studied as adjuvant ingredients into baked products, tortillas, and snacks to improve functionality and nutritional quality (Anton and others 2008, 2009; Betancur-Ancona and others 2009; Borsuk 2011). The main challenge on BP incorporation relies on their sustained activity and reaching the specific target in the organism.
To sustain the physiological effects, BPs must show stability against GI proteases and be absorbed through the enterocytes to the serum without being degraded by peptidases from the brush border and serum. There is an exception with peptides that act in the GI tract and do not have to be absorbed to exert their biological properties (for example, cholesterol-binding and anoretic peptides; Udenigwe and Aluko 2012).
Certain protein/peptide structures are resistant to GI digestion, which favors their bioavailability. Peptides containing Pro and hydroxyl Pro residues have been found to be resistant to hydrolysis. Also, glycosylated peptides and peptides which have undergone the formation of Maillard reaction products resist GI tract enzyme cleavage (Norris and FitzGerald 2013).
For the industrial application and incorporation into food, the main aspects to be considered are the organoleptic characteristics and the resistance to processing conditions. Common processes such as dehydration and thermal processing can negatively affect the food protein hydrolysates or active peptides. For example, bitterness is a negative sensory attribute associated with low molecular weight peptides composed mainly of hydrophobic amino acids (Saha and Hayashi 2001; Hernández-Ledesma and others 2011).
Various methods have been developed to remove or mask the bitterness of food containing protein hydrolysates. The methods designed to physically eliminate the hydrophobic peptides includes treatment with activated carbon, extraction with alcohol, isoelectric precipitation, and chromatographic separation. Enzymatically, the use of exopeptidases has also reduced significantly the bitterness in digestion of food proteins (Saha and Hayashi 2001; FitzGerald and O'Cuinn 2006). Nonetheless, these physical and enzymatic techniques are not suitable for the biologically active hydrolysates or peptides because they can affect their functionality.
Encapsulation has emerged as an effective strategy to mask bitterness and to improve stability of bioactive compounds in food matrices. Spray drying is the most common technology to produce microencapsulated food particles, since equipment is accessible and production costs are lower (Hernández-Ledesma and others 2011). Also, the use of glutamate and several glutamyl oligopeptides are methods proposed for masking the bitterness (Saha and Hayashi 2001). Encapsulation can also be used to generate a controlled release of bioactive compounds, ensuring efficacy during the shelf life of food products.
In addition to the bitterness, there could be undesirable high ash build up in the hydrolysates and the development of salty off-flavors as result of the neutralization required after the hydrolysis process (Hernández-Ledesma and others 2011).
Pulse crops have long been known to be an excellent source of protein. Based on the latest amount of research pointing out the activity of their hydrolysates or BPs, it is suggested that these legumes may be excellent sources for the development of new protein-derived products. As it has been evidenced, BPs or hydrolysates can be obtained from underutilized vegetable sources, like hard-to-cook beans, generating value-added food ingredients with health promoting characteristics.
Special attention has been given to the antihypertensive and AOX activities, but recent publications indicate that pulse-derived hydrolysates or BPs can be active toward several other conditions like cancer, inflammation, and hypercholesterolemia.
The bioactivities of pulses-derived peptides or hydrolysates have been investigated mainly on in vitro studies. In vivo studies in animal models and sequential human clinical trials to confirm such bioactivities are needed. Further research is also required to understand the absorption into the blood stream, migration to the target sites, and the mechanism of the activity exerted.
Finally, it is also required to improve the processes of production and incorporation into foods, since practically all of the reviewed research was made on a laboratory scale, using equipment and methodologies difficult to scale up to industrial scale.
This research was supported by the Research Chair Funds of Nutraceutical Foods (CAT-005) and Nutrigenomics-FEMSA. The scholarship of Lidia López-Barrios (CVU-295031) was provided by Consejo Nacional de Ciencia y Tecnología (CONACYT).
Lidia López-Barrios collected information and drafted the manuscript. Sergio Serna-Saldívar structured the first sections and Janet Gutiérrez-Uribe helped designed Table 1 to 4.