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A New Frontier in Soy Bioactive Peptides that May Prevent Age-related Chronic Diseases

Wenyi Wang and Elvira Gonzalez de Mejia ABSTRA CT :D ur ing gastr ointestinal digestion or food pr ocessing of pr oteins , small peptides can be r eleased and may act as ABSTRACT CT: Dur uring gastrointestinal processing proteins oteins, released regulatory compounds with hormone-like activities. Numerous biologically active peptides (bioactive peptides) have been identified. Most bioactive peptides are derived from milk and dairy products, with the most common being angiotensin converting enzyme inhibitory peptides. Soybean protein and soybean derived peptides also play an important role in ev ention of chr so ybean physiological activities , par ticularly those r elated to the pr onic diseases .H owev er , the bioactiv e evention chronic soybean activities, particularly related prev diseases. Ho ever er, bioactive potential of soybean derived bioactive peptides is yet to be fully appreciated. After a general introduction of approaches and advances in bioactive peptides from food sources, this review focuses on bioactive peptides derived from soybean pr oteins and their physiological pr oper ties . Technological appr oaches to gener ate bioactiv e peptides , their isolation, proteins proper operties ties. approaches generate bioactive peptides, purification, characterization, and quantification, and further application in food and drug design are also presented. Safety concer ns , such as potential to xicity , aller genicity , and sensor y aspect of these peptides ar e likewise discussed. concerns ns, toxicity xicity, allergenicity genicity, sensory are Keywor ds: bioactiv e peptides , so ybean, antiobesity , hypocholester olemic, antihyper e eywords: bioactive peptides, soybean, antiobesity, hypocholesterolemic, antihypertensiv tensive tensiv

Introduction
In living organisms, endogenous peptides often function as hormones and neurotransmitters and play important physiological roles. Through hormone-receptor interactions and signaling cascades, they exert their actions on regulating metabolism (water, mineral, and other nutrients), controlling gland excretion, adjusting blood pressure, and impacting body growth. They may also exert effects on sleep, learning, memory, pain, sexual behavior, appetite, and stress via effects on the central nervous system (CNS). In humans, many peptide hormones are involved in the hypothalamus hormones cascade. For example, corticoliberin (CRH, 41-peptide amide) and thyroliberin (TRH, pGlu-His-ProNH2) function as hypothalamic hormones. They can stimulate the secretion of 2 pituitary hormones, corticotropin (acth, 39aa), and tryrotropin (glycoprotein, chain 96aa, chain 112 aa), respectively. Furthermore, various gastro-enteropancreatic peptides, such as insulin and glucagons, play very important roles in the regulation of the metabolism (Sewald and Jakubke 2002). In the past several decades, researchers have found that bioactive peptides can also be derived from dietary proteins. They may be present as independent entities or encrypted in the parent protein. It is known that during gastrointestinal (GI) digestion or food processing, these peptides are released from the parent protein
CRFSFS 20050128 Submitted 2/28/05, Revised 4/27/05, Accepted 8/19/ 05. Authors Wang and Mejia are with Dept. of Food Science and Human Nutrition, Univ. of Illinois at Urbana-Champaign, 228 ERML, MC-051, 1202 West Gregory Drive, Urbana-Champaign, IL 61801. Direct inquiries to author Gonzalez de Mejia (E-mail: edemejia@uiuc.edu).

and act as regulatory compounds with hormone-like activities (Korhonen and Pihlanto 2003). In 1950, Mellander suggested that casein-derived phosphorylated peptides (CPP) enhanced vitamin D-independent bone calcification in rachitic infants (Mellander 1950). This early observation is considered as the first indication of food derived bioactive peptides (Korhonen and Pihlanto 2003). Since then, numerous peptides with various bioactive functions have been identified. In a database named Biopep, more than 1500 different bioactive peptides have been presented (Dziuba and others 2003). Among them, angiotensin-converting enzyme (ACE) inhibitors and dipeptidyl peptidase IV inhibitors, which show antihypertensive activity, are the most common (Ahn and others 2000; Gobbetti and others 2002; Rhyu and others 2002; Clare and others 2003). Peptides with other biological activities, such as opioid agonistic and antagonistic, antioxidative, anticancer, and immunomodulatory actions have also been identified. Food-derived bioactive peptides commonly contain 2 to 9 amino acids (Kitts and Weiler 2003). However, this range may be extended to 20 or more amino acid units (Korhonen and Pihlanto 2003). For example lunasin, a food-derived peptide with proved anticancer bioactivity, contains 43 amino acids with a molecular weight (MW) of 5400 Da (Jeong and others 2002). Milk and other dairy products are among the best precursors of bioactive peptides and have been extensively studied (Floris and others 2003). However, bioactive peptides have also been isolated and characterized from other food protein sources, including egg, fish, oyster, cereal (rice, wheat, buckwheat, barley, corn), soybean, and radish seeds (Matsui and others 1993; Li and others 2002; Yoshikawa and others 2003).
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Soybean is an important protein source and a potential source of bioactive peptides. On average, soybean contains about 40% protein (Nielsen 1996) conformed by a complex mixture of different protein types. In ExPASy databases, up to date there are a total of 1411 protein entries (266 Swiss-Prot entries and 1145 TrEMBL entries) listed for soybean (Glycine max). The major components of soy proteins are storage proteins known as -conglycinin and glycinin, which account for 65% to 80% of total seed proteins. In addition, there are many enzymes (such as lipoxygenase, chalcone synthase, catalase, urease) in soybean, but only a relatively small number of them exceed 1% of total seed protein (Nielsen 1996). According to their rate of sedimentation during centrifugation, soy proteins can be classified as 2S, 7S, 11S, and 15S (Table 1). S means Svedberg unit, which is a unit of sedimentation rate computed as the rate of sedimentation per unit field of centrifugation strength. Among them, 11S (glycinin) and 15S (a polymer of glycinin) are pure proteins, whereas 2S and 7S are composed of several proteins. Figure 1a presents the crystal structure of soybean conglycinin homotrimer and Figure 1b the crystal structure of glycinin A3B4 subunits of hexamer. -Conglycinin is a trimer with a MW of about 180 kDa. It is composed of 3 subunits, (63 kDa), (67kDa) and (48kDa) (Nielsen 1996; Liu 1997; Apweiler and others 2004). These subunits share a large degree of amino acid homologies. Figure 2 presents the multiple sequence alignments of 3 subunits (, , ) of soybean beta-conglycinin. Furthermore, -conglycinins with different subunit composition have also been identified. It is likely that the trimers are composed of randomly assembled mixture of subunits (Nielsen 1996). Glycinin, on the other hand, is a hexamer with MW of about 320 to 375 kDa and with 5 major subunits, G1, G2, G3, G4, and G5. Each subunit consists of 2 polypeptide chains, an acidic chain (about 40 kDa) and a basic chain (about 20 kDa), joined by a single intra-chain disulphide bond. G1, G2, and G3 can be grouped as they share about 90% sequence homologies. Similarity, G4 and G5 share about 90% sequence homologies. However, sequence homologies between these 2 groups (G1, G2, G3 and G4, G5) are only about 50% (Nielsen 1996). From the epidemiological point of view, studies suggest that populations consuming high levels of soybean products have both lower cancer incidence and lower mortality rates for the major cancer types commonly found in the Western hemisphere (Nagata and others 2002; Wu and others 2002; Spector and others 2003). Thus, as the main components of soybean, soy proteins are receiving more and more attention with respect to their health effects. Bowman Birk inhibitor (BBI), a 2S soy protein component, has been shown to suppress carcinogenesis in animals (Kennedy 1995; Kennedy and others 2002) and in human prostate cancer cells (Kennedy and Wan 2002). BBI has also been the subject of promising clinical trials in cancer patients (Armstrong and others 2000, 2003; Meyskens 2001). A Phase IIb randomized, placebo-controlled clinical trial to determine the clinical effectiveness of Bowman-Birk inhibitor concentrate is currently under way. Similarly, lunasin has been found to suppress chemically induced carcinogenic transformation in mammalian cells using mice as a model (de Lumen 2005). This novel peptide can be found in amounts ranging from 0.10 to 1.33 g/100 g flour in different soybean varieties and in commonly available soy proteins (Jeong and others 2003). As described, soybean proteins and soybean-derived peptides may play an important role in disease prevention and treatment. Food processing and in vivo enzyme di-

(a)

(b) Table 1Soybean protein classificationa Ultrafiltration protein fractionb 2S Percent in extractable protein 20%

Proteins in the fraction Kunitz typsin inhibitors Bowman-Birk typsin inhibitors Cytochrome C AL1 and AL3 -Conglycinin -Conglycinin -Conglycinin -Amylase Lipoxygenase Hemagglutinins (or lectins) Soybean vacuolar protein P34 Pure protein: glycinin Pure protein: polymer of glycinin Figure 1 (a) Crystal structure of soybean -conglycinin homotrimer. Printed with permission (Maruyama and others 2004). (b) Crystal structure of glycinin A3B4 subunits of hexamer. Printed with permission (Adachi and others 2003).

7S

33%

11S 15S

33% 10%

aAdapted from: Catsimpoolas and Ekenstam 1969; Wolf 1970; Nielsen 1985;

Odani and others 1987; Kalinski and others 1990; Burks and others 1991; Samoto and others 1994; Liu 1997; Lin and others 2004. bS = Svedberg unit. A unit of sedimentation rate computed as the rate of sedimentation per unit field of centrifugation strength.

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Food bioactive peptides . . .

Figure 2Multiple sequence alignments of 3 subunits: alpha (), beta (), alpha ( ) of soybean -conglycinin. The amino acid sequences were obtained from Swiss-Prot protein knowledgebase (accession number P13916, P25974, P11827) (Apweiler and others 2004). The alignments were carried out by T-Coffee (Notredame and others 2000). Sequence regions from high to low consensus (cons) were colored from blue to red ( ). * = the residues in that column are identical in all sequences in the alignment. : = conserved substitutions have been observed. . = semi-conserved substitutions are observed. Amino acid nomenclature: C = Cys, Cystein; H = His, Histidine; I = Ile, Isoleucine; M = Met, Methionine: S = Ser, Serine; V = Val, Valine; A = Ala, Alanine; G = Gly, Glycine; L = Leu, Leucine; P = Pro, Proline; T = Thr, Threonine; F = Phe, Phenylalanine; R = Arg, Arginine; Y = Tyr, Tyrosine; W = Trp, Tryptophan; D = Asp, Aspartic acid; N = Asn, Asparagine; B = Asx, Either of D or N; E = Glu, Glutamic acid; Q = Gln, Glutamine; Z = Glx, either of E or Q; K = Lys, Lysine; X = Undetermined amino acid. Vol. 4, 2005COMPREHENSIVE REVIEWS IN FOOD SCIENCE AND FOOD SAFETY 65

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gestion of soy proteins may release peptides, which exert diverse biological functions by interacting with cell receptors, functioning as hormones, regulating enzymes or interfering cell cycles. Therefore, an overview on the scientific advancement and technical aspects of bioactive peptides derived from soybean products would be helpful for a better understanding of their bioactive potential and for stimulating further research in this area. After a general introduction of bioactive peptides from several food sources, this review will provide an overview of the role of bioactive peptides derived specifically from soybean proteins and their physiological properties. Technological approaches to generate bioactive peptides, their isolation, purification, characterization, and quantification, as well as their application in food and drug design will also be presented. Meat. Chicken meat was found to be a source of antihypertensive peptides. For example, with the action of the enzyme thermolysin, 2 antihypertensive, Ile-Lys-Trp and Leu-Lys-Pro have been detected (Korhonen and Pihlanto 2003). Fish and seafood. Bioactive peptides can also be found in fish products, such as sardine muscle, tuna muscle, bonito (Yamamoto and others 2003), and Alaska Pollack skin (Korhonen and Pihlanto 2003). Different enzymes have been used to generate bioactive peptides (Fujita and Yoshikawa 1999). For example, LKPNM, isolated from the thermolysin digest of dried bonito, was activated 8-fold by ACE itself and showed a prolonged effect after its oral administration to animal models (Yoshikawa and others 2000). After 17 h of hydrolysis of sardine protein by Bacillus licheniformis alkaline protease, the ethanol fraction obtained with a chromatographic resin showed strong ACE-inhibitory activity (IC50 = 0.015 mg protein/mL). This fraction was confirmed to have a significant in vivo depressor effect in mild hypertensive volunteers after 4 wk of oral administration (Matsui and others 1993). In genetically obese Zucker rats, fish protein hydrolysate (FPH) reduced the plasma cholesterol level. Furthermore, the HDL cholesterol:total cholesterol ratio was greater in these rats and in Wistar rats fed FPH compared with those fed casein (Wergedahl and others 2004). These observations suggest that FPH may have a role as a cardioprotective nutrient. Insects. Royal jelly (RJ), a bee product rich in proteins, has also been found as a good source of ACE-inhibitory peptides. Matsui and others (2002) found that intact RJ and its protein fraction did not show ACE-inhibitory activity. However, after pepsin and the subsequent trypsin and chymotrypsin hydrolysis, ACE inhibitory capacity was developed (IC50 = 0.099 mg protein/mL). Furthermore, single oral administration of this GI RJ hydrolysate significantly lowered systolic blood pressure in spontaneous hypertensive rats (SHR). They further fractionated the hydrolysate and identified 8 additional peptides with IC50 value of < 10 mM.
Plant origin

Food Sources of Bioactive Peptides


Animal origin

Dairy products. Milk is a particularly rich source of bioactive peptides. These peptides are encrypted in both casein (s, , , casein) and whey proteins (-lactalbumin, -lactoglobulin, lactoferrin, and immunoglobulins) (Belem and others 1999; Gobbetti and others 2002) and can be released by enzyme hydrolysis or microbial fermentation. For enzyme hydrolysis, single enzymes or a combination of proteinases have been used, such as trypsin, alcalase, chymotrypsin, carboxypeptidase, pancreatin, pepsin, and enzymes from bacterial or fungal origin (for example, proteinase K from Tritirachium album). Bioactive peptides have been identified from milk fermented by lactic acid bacteria, such as Lactococcus lactis subsp. cremoris, Lactobacillus helveticus, Lactobacillus GG strain, Lactobacillus delbruskii subsp. Bulgaricus (Gobbetti and others 2002; LeBlanc and others 2002; Korhonen and Pihlanto 2003). Milk-derived bioactive peptides have shown various physiological activities. For example, -casokinins (derived from , casein) showed antihypertensive and immunomodulatory activities; lactoferricin (f 17-41 of lactoferrin) antimicrobial activity; casomorphins (derived from , -casein) and -casein exorphin (f 90-96 of s1-casein) opioid activity; caseinophoshopeptides (derived from post-translational phosphorylated s, -casein) mineral binding activity and peptide KRDS (f 17-41 of lactoferrin) antithrombotic activity (Korhonen and others 1998; Gobbetti and others 2002; Meisel and FitzGerald 2003). Milk processing conditions affect the formation of milk-derived bioactive peptides. In fermented milk products, peptide activities depend on type of bacterial starter cultures and degree of proteolysis. Adequate proteolysis can facilitate the release of bioactive peptides, but once it exceeds certain level, it will decrease the bioactivity. For example, in products with low degree of proteolysis, such as yogurt and fresh cheese, ACE-inhibitory activity is low, while cheese with longer ripening time such as middle aged gouda has a higher ACE-inhibitory action (Korhonen and Pihlanto 2003). Hypocholesterolemic peptides have also been identified; for example, Ile-Ile-Ala-Glu-Lys, a peptide from a -lactoglobulin tryptic hydrolysate (LTH), was found to lower serum and liver cholesterol level, at least partly, by inhibiting micellar solubility of cholesterol (Nagaoka and others 2001). Egg. Several bioactive peptides with vasodilatation, ACE-inhibitory activities have been found in egg ovalbumin treated with chymotrypsin and pepsin (Korhonen and Pihlanto 2003). For example, 2 vasorelaxing peptides, RADHPF (f 359 to 364 of ovalbumin) and ovokinin (FRADHPFL, f 358 to 365 of ovalbumin), were isolated from chymotryptic and peptic digest of ovalbumin, respectively (Fujita and others 1995; Matoba 1999). Although they are released from the same region of ovalbumin, it was believed that they showed different modes of vasorelaxing activities.
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As indicated in Table 2, soybean, wheat, corn, rice, barley, buckwheat, and sunflower are all plant sources of bioactive peptides. It can be observed that the main approach used to produce peptides is by enzymatic hydrolysis. These peptides are then further isolated by either ultrafiltration or by cationic exchange resins. Thus, peptides with different amino acid sequences can be obtained that possess various biological activities. It is interesting to observe that depending on the initial protein source, enzyme used, and processing conditions, the biological activities of the peptides are different. As shown in the table, when different enzymes hydrolyze soy protein, it yields either antioxidant peptides (Pena-Ramos and Xiong 2002), peptides with anticancer properties (Kim and others 2000), or peptides with hypotensive activity (Wu and Ding 2001). In addition to studies listed in Table 2, Matsui and others (1999) isolated 16 ACE inhibitory peptides with IC50 value of less than 20 mM, composed of 2 to 7 amino acid residues from wheat germ hydrolysate. Ile-Val-Tyr was identified as a main contributor to the ACE inhibition of the hydrolysate. An opioid peptide with similar structure to endogenous opioid peptides gluten exorphin A5 (Gly-Tyr-Pro-Thr) has also been isolated from the enzymatic digests of wheat gluten (Yoshikawa and others 2003). Also, peptides derived from a spinach constituent, Rubisco (D-ribulose-1,5-bisphosphate carboxylase/oxygenase; EC 4.1.1.39), were found to have opioid activity (Teschemacher 2003). Based on a bioactive peptide sequence database analysis, rice prolamin is believed to be one of the best precursors of antihypertensive peptides (Dziuba and others 2003). Similarly, ACE-inhibitory peptides, such as LQP, LLP, LSP, LAA, FY, have been reported in -zein thermolysin hydrolysates (Yamamoto and others 2003).

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Food bioactive peptides . . .


Intact buckwheat was found to exhibit ACE inhibitory activity having an IC50 value of 3.0 mg/mL. After pepsin, chymotrypsin, and trypsin hydrolysis, ACE inhibitory activity was significantly increased (IC50 0.14mg protein/mL) compared with IC50 before hydrolysis (0.36 mg protein/mL). Several di-/tri-peptide fractions (TyrGln-Tyr and Pro-Ser-Tyr) of the buckwheat digest were identified considering the magnitude of their ACE inhibitory action (Li and others 2002). Immunomodulatory peptides derived from tryptic hydrolysates of rice and soybean proteins act to stimulate superoxide anions (reactive oxygen species-ROS), which triggers nonspecific immune defense systems (Kitts and Weiler 2003). A 9 amino acid

Table 2Examples of biologically functional peptides derived from plant proteins Source Native and heated soy protein isolate Preparation Purified proteases: pepsin, papain, and chymotrypsin and crude proteases: alcalase, Protamex, and Flavourzyme Peptides Degree of hydrolysis of SPI hydrolysates ranged from 1.7% to 20.6% Activity Antioxidant activities. Both hydrolyzed and nonhydrolyzed SPI decreased TBARS (by 28% to 65%), except for papain-hydrolyzed samples. Samples of chymotrypsin- and Flavourzymehydrolyzed (0.5 h) preheated SPI had the greatest inhibitory effect on lipid oxidation Up-regulate the uptake and degradation of LDL by the HepG2 cell receptors Reference Pena-Ramos and Xiong 2002

Soybean protein concentrate, Crocksoy 70, extracted by 80% ethanol Soy flour or wheat flour

Porcine, pepsin and bovine pancreatic trypsin or only trypsin

Peptides with different molecular weights were separated from the digested material by ultrafiltration Soluble peptides were separated from the hydrolysate by ultrafiltration X-Met-Leu-Pro-Ser-TyrSer-Pro-Tyr Soluble hydrolyzed sample was further fractionated on cationic exchange resin (1) Tyr-Val-Val-Phe-Lys (2) Pro-Asn-Asn-Lys-ProPhe-Gln (3) Asn-Trp-GlyPro-Leu-Val (4) Ile-Pro-ProGly-Val-Pro-Tyr-Trp-Thr (5) Thr-Pro-Arg-Val-Phe. PGTAVFK LPYPR Soymetide-13: MITLAIPVNKPGR Soymetide-9: MITLAIPVN; Soymetide-4: MITL LLPHH; RPLKPW

Arnoldi and others 2001

Enzymes of non-animal origin, papain or pronase

Defatted soy protein Thermolase Defatted soy meal Alcalase enzyme

Growth-promoting and production enhancing activities when tested on a mouse hybridoma culture in protein-free medium Anticancer Hypotensive

Franek and others 2000

Kim and others 2000 Wu and Ding 2001 Kodera and Nio 2002

Soy protein

Protease D3

Hypotensive

Soybean Soybean glycinin -Conglycinin

Protease from Bacillus subtilis Peptide derived from soybean glycinin Derived from subunit of -conglycinin

Antihypertensive IC50 = 26.5 M Hypocholesterolemic peptide Immunostimulating peptide by FPR receptor; Sometide-9 is the most active in stimulating phagocytosis in vitro Antioxidative; Antihypertensive

Kitts and Weiler 2003 Yoshikawa and others 2000 Yoshikawa and others 2000

Genetically modified soybean protein Chymotrypsin Korean fermented soybean paste Rubisco from spinach

Proteinase S; alcalase; trypsin Fermentation

Korhonen and Pihlanto 2003 Shin and others 2001 Yang 2001

HHL

Antihypertensive peptide: IC50 = 2.2 M IC 50 = 51.0 M and 24.4 M in mouse assay; 2.09 M and 0.93 M in receptor binding assay using [3H] Deltorphin II as radioligand, respectively GEA5 showed more potent -opioid activity than GEA4. GEB5 was more potent than GEB4. GEC was more potent than GEA but weaker than GEB.

Synthesis; Pepsin and leucine aminopeptidase (LAP) Derived from gluten

Rubiscolin-5 YPLDL, Rubiscolin-6 YPLDLF

Gluten

Buck wheat -Zein Sunflower Protein isolates

Pepsin, chymotrypsin and trypsin Thermolysin Sequential treatment with pepsin and pancreatin

Gluten exorphin: GEA5:GYYPT GEA4:GYYP GEB5:YGGWL GEB4:YGGW GEC: YPISL VK, FY, YQY, PSY LQP, LLP, LSP, LAA, FY FVNPQAGS

Yoshikawa and others 2003

ACE inhibitory peptides. IC50 = 13, 25, 4, 16 M, respectively. ACE inhibitory peptides: IC50 = 1.9, 57, 1.7, 13, 25 M, respectively. ACE inhibitory peptides 5.7 g/mL

Liam and others 2002 Yamamoto and others 2003 Megias and others 2004 67

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Table 3Activity and structural homology of selected bioactive peptides Activity Opioid Structural homology YPX Remarks X- Aromatic amino acid (Phe, Trp) Or aliphatic amino acid (As in GYYPT, YPISL, YPVEPF, YPLDL, YPLDLF Typical opioid peptides. Originate from: proopiomelanocortin, proenkephalin, and prodynorphin. Atypical opioid peptides. Tyrosine residue located at the amino terminal or bioactive site. x-F,Y,I,V Can lower blood pressure, stimulate isolated smooth muscle and cause salivary secretion (tachykinins) High content of P residues make the peptides resistant to proteolytic attack Usually short and resistant to the action of digestive-tract endopeptidases. Reference Yang 2001

Opioid

N-terminal YGGF

Opioid Opioid Antihypertensive

N-terminal YXF, YXXF Y at N-terminal C-terminal FXGLM-NH 2 N-terminal Y in b-casomorphins Rich in hydrophobic amino acid and have a Pro, Lys, or Arg as a C-terminal Peptides with PHH

Pihlanto-Leppl 2001 Pihlanto-Leppl 2001 Kitts and Weiler 2003 Lin and Lin 2001

ACE-inhibitory ACE-inhibitory

Haileselassie and others 1999 Kitts and Weiler 2003

Antioxidant

Peptides with PHH have greatest antioxidant activity among all tested Kitts and Weiler tested peptides and had synergistic effects with nonpeptidic antioxidants. 2003

peptide (Gly-Tyr-Pro-Met-Tyr-Pro-Leu-Pro-Arg) with ileum contracting and immunostimulatory activities has also been found in rice albumin trypsin hydrolysates. Table 3 presents examples of activity and structural homology of selected bioactive peptides such as opioid, antihypertensive, ACE inhibitor, and antioxidant capacity. It is interesting to notice that high content of proline residues makes peptides resistant to proteolytic attack (Haileselassie and others 1999).

Biological Activities of Soybean Peptides


Antihypertensive peptides

Antihypertensive peptides are the most extensively studied bioactive peptides in foods. They show their activity by inhibiting angiotensin-converting enzyme. ACE is a nonspecific dipeptidyl carboxypeptidase associated with the regulation of blood pressure by modulating the rennin-angiotensin system. This enzyme converts the decapeptide angiotensin I into the potent vasoconstricting octapeptide angiotensin II, which leads to an increase in blood pressure. Therefore, inhibition of the ACE will result in an antihypertensive effect (Natesh and others 2003). Using spontaneously hypertensive rat (SHR) model, it has been found that ACE inhibitory bioactive peptides lower systolic blood pressure and ACE activity in the aorta (Li and others 2002). Several ACE inhibitory bioactive peptides have been found in enzyme hydrolysates of soy proteins. Chen and others (2003, 2004) identified the angiotensin I-converting enzyme inhibitory peptides in the peptic digest of soybean protein. Peptide fractions, which inhibited ACE activity, were separated from peptidic digests of soybean proteins by ion exchange chromatography and gel filtration. Further separation by reversed-phase ODS high-performance liquid chromatography (HPLC) led to 4 active ACE inhibitory peptides, the amino acid sequences of which were identified by the Edman procedure as: Ile-Ala (IC50 153 mM), Tyr-Leu-AlaGly-Asn-Gln (IC5014 mM), Phe-Phe-Leu (IC50 37 mM), and Ile-TyrLeu-Leu (IC50 42 mM). The peptide fractions given orally to SHR at a level of 2.0 g/kg body weight markedly lowered their blood pressure. Antihypertensive peptides were also found in soybean alcalase digest (Wu and Ding 2001). Oral doses of these peptides significantly (P < 0.05) decreased systolic blood pressure of SHR
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in a dose-dependent manner. However, the peptides had little effects on blood pressure of normotensive rats even at highest dose tested (1000 mg/kg of body weight/d). Kodera and Nio (2002) have produced peptides from soybeans with ACE inhibitory activity and favorable taste. Namely, the following 5 peptides were generated by soybean protein digestion with protease D3: (1) Tyr-Val-Val-Phe-Lys, (2) Pro-Asn-Asn-LysPro-Phe-Gln, (3) Asn-Trp-Gly-Pro-Leu-Val, (4) Ile-Pro-Pro-Gly-ValPro-Tyr-Trp-Thr, and (5) Thr-Pro-Arg-Val-Phe. Yoshikawa and others (2002) introduced a new physiological function into soy protein by genetic engineering. RPLKPW, a highly potent anti-hypertensive peptide, was introduced into 3 homologous sites in soybean -conglycinin -subunit by site-directed mutagenesis. In this experiment, the mutated -subunit expressed in Escherichia coli exerted an anti-hypertensive effect in SHR at a dose of 10 mg/kg. When the 4th RPLKPW sequence was introduced to the subunit, the antihypertensive activity of subunit was further improved. Fermented soybean products are a good source of ACE inhibitory bioactive peptides. From fermentation of soybean containing medium with Bacillus natto or B. subtilis, several ACE inhibitory peptides, such as Val-Ala-His-Ile-Asn-Val-Gly-Lys or Tyr-Val-TrpLys, were isolated (Kimura and others 2000). This experiment showed the potential of fermented soybean meal as a source of antihypertensive peptides. ACE inhibitory peptides have also been found in many traditional Asian fermented soy foods, such as soybean paste (His-His-Leu) (Shin and others 2001), soy sauce (Okamoto and others 1995), natto, and tempeh (Gibbs and others 2004). Korhonen and Pihlanto (2003) discuss in their review of an antihypertensive peptide from chunggugjang, a traditional Korean soybean product fermented with Bacillus subtilis CH-1023. The optimum incubation condition for the generation of antihypertensive peptide from chunggugjang was 60 h at 40 C. The crude extract was partially purified by Amicon YM-3 membrane filtration and Sephadex G-10, G-25 gel filtration. The purified peptide (0.5 mg) showed an inhibitory rate of 94.3%. The most prominent amino acid composition of the peptide from chunggugjang was Ala, followed by Phe, and His (Cho and others 2000).
Hypocholesterolemic

The beneficial effects of soybean on cardiovascular diseases were

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Food bioactive peptides . . .


first considered through its impact on blood cholesterol. Among all soybean components, soy proteins and isoflavones were believed to be main factors. A large body of literature indicates that soy proteins can reduce blood cholesterol concentrations in experimental animals and humans (Potter 1995). For example, Sagara and others (2004) found that dietary intakes of soy protein (at least 20 g) and isoflavones (at least 80 mg) for 5 wk would be effective in reducing CHD risk among high-risk, middle-aged men. More recently, studies in rats have demonstrated that SPI intake could modulate lipid and energy metabolism, including the synthesis and degradation of cholesterol (Tachibana and others 2005). Soy protein is known to exert hypocholesterolemic effects when ingested, prompting the Food and Drug Administration to approve a health claim linking foods that are naturally rich in soy protein to a reduction in coronary heart disease (Anderson and others 1995). Within this context, Wang and others (2004) showed that soy protein reduces circulating triglycerides and cholesterol in hypercholesterolemic individuals. However, the mechanisms underlying the hypocholesterolemic properties of soy protein remain unclear (Desroches and others 2004). Adams and others (2002) compared the effect of SPI in 2 genetically engineered mouse models of atherosclerosis, quantifying the aortic content of esterified cholesterol. It was found that both preparations, alcohol-washed soy protein isolate (total isoflavones = 0.04 mg/g) and intact soy protein isolate (total isoflavones = 1.72 mg/g) inhibited atherosclerosis in comparison to casein control. However, the effect was enhanced in mice fed intact SPI relative to those fed alcohol-washed soy protein. The effect was independent of plasma lipoprotein concentrations and the presence or absence of LDL receptors. More recently, it has been shown that dietary soy -conglycinin inhibits atherosclerosis in mice (Adams and others 2004; Moriyama and others 2004). Soy protein can also shift LDL particle distribution to a less atherogenic pattern in an isoflavone independent manner (Desroches and others 2004). Compared with animal protein control, soy protein (with or without isoflavone) significantly decreased the cholesterol levels in LDL < 25.5 nm by 12.3% (P < 0.001) and increased cholesterol levels in LDL > 26.0 nm by 14.3% (P < 0.05) and therefore shifting LDL particle distribution to a less atherogenic pattern. Another hypothesis is that soy proteins might bind with bile acids inhibiting their reabsorption and therefore lowering blood cholesterol level. In most animal studies and clinical trials, soy proteins have been given to animals or human subjects by oral administration. Thus, these proteins have been subjected to protease digestion in the GI tract releasing the bioactive peptides, which then may lower cholesterol levels. Based on these observations, it is likely that soy peptides may be responsible, at least in part, for the hypocholesterolemic benefits of soy protein. It has been reported that a soy protein peptic hydrolysate (SPH) has a stronger serum cholesterol lowering effect than intact soy protein in rats (Sugano and others 1990). Compared with casein, soy protein hydrolysate significantly decreased the serum cholesterol level as well as promoted fecal excretion of steroids. These data indicate that soy protein hydrolysate may indeed inhibit cholesterol absorption. In the GI system, cholesterol is rendered soluble in bile salt-mixed micelles and then absorbed. In an in vitro study, it was found that the micellar cholesterol solubility was significantly lower in the presence of SPH compared with the cholesterol micelles containing soy protein (Nagaoka and others 1999). In vitro, cholesterol absorption in Caco-2 cells showed a cholesterol uptake from micelles containing SPH, which was significantly lower than that containing soy protein. The incorporation of [3H]-cholesterol into the serum, liver, and intestine of rats was also significantly lower in SPH groups than in soy protein groups (Nagaoka and others 1999). These results indicated soybean peptides have stronger hypocholesterolemic effects than soy protein by inhibiting cholesterol absorption due to the suppression of micellar solubility of cholesterol. To narrow down the active moiety of soy protein, the LDL receptor up-regulation effects of -conglycinin and glycinin were studied in human hepatoma cells (HepG2). The results showed that -conglycinin was markedly more effective than glycinin (Lovati and others 1992). Follow-up research found that + subunits from -conglycinin had higher LDL receptor up-regulation activity than subunit. Incubation of HepG2 cells with purified + subunits sharply increased uptake and degradation of 125ILDL added to the culture medium, whereas the subunit was ineffective (Lovati and others 1998); the subunit was believed to contribute more than subunits (Manzoni and others 1998). These observations led to the development of an enzymatic modification process for the hydrolisis of soy -conglycinin subunit, for use as a hypocholesterolemic agent (Duranti and Morazzoni 2003). In rats, the administration by gavage of 20 mg/kg body weight/d of this hydrolysate for 28 d resulted in a 36% decrease in plasma cholesterol; a greater effect than when using 100 mg/kg body weight/d of whole -conglycinin (Duranti and others 2004). Comparing among amino acid sequences, 2 regions present in + but absent in , were further examined. A synthetic peptide (104 mol/L, MW 2271 Da), corresponding to positions 127 to 150 of -conglycinin was found to markedly (P 0.05) increase 125I-LDL uptake and degradation in HepG2 cells (Lovati and others 2000). To determine active soy protein components in the regulation of cholesterol homeostasis in HepG2 cells, a soybean protein concentrate (CrocksoyR 70), was subjected to pepsin-trypsin digestion and fractionation. It was found that, at 0.125 g/L, the MW > 3000 Da fraction significantly up-regulated the uptake and degradation of LDL by receptor pathways while the MW < 1000 fraction and MW between 1000 and 3000 fraction had no effect on LDL catabolism (Lovati and others 2000). Although some researchers suggest that small peptides can cross the intestinal wall intact, the in vivo effect of larger molecule peptide fractions, particularly those from soy proteins, still needs to be investigated (Scanff and other 1992; Chabance and others 1998; Haupt and others 2002). On the other hand, hypocholesterolemic effect has been also found in glycinin. Leu-Pro-Tyr-Pro-Arg, a fragment peptide derived from soybean glycinin was found to reduce serum cholesterol in mice after oral administration at a dose of 50 mg/kg, without isoflavones, for 2 d (25.4% in total cholesterol and 30.6% in LDLcholesterol) (Yoshikawa and others 2000). This peptide has structural homology to enterostatin (Val-Pro-Asp-Pro-Arg). Although both have hypocholesterolemic activities, enterostatin did not increased excretion of bile acids in feces, suggesting that they may act by different mechanisms (Takenaka and others 2000, 2004). Soy peptides may also bind to phospholipids and exert serum cholesterol lowering activity in humans (Hori and others 2001). Because ethanol washed soy protein, containing no isoflavones, show less cholesterol-lowering capacity, some researchers believe that this effect is due to the presence of these flavonoids (Ali and others 2004; Zhan and Ho 2005). Paradoxically, isoflavones alone do not have a cholesterol lowering effect in 5-wk-old male Sprague-Dawley rats (Fukui and others 2002). Therefore, it has been postulated that the effect of soy protein in lowering cholesterol levels may be due to an isoflavone-protein interaction (Peluso and others 2000; Simons and others 2000; Hsu and others 2001). Although all these observations in experimental animals have not been found in human feeding studies, some researchers have postulated that in human, soy protein may in some way up-regulate LDL receptors depressed by hypercholesterolemia or by dietary cholesterol administration (Sirtori and others 1995).
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Antiobesity

Soy has long been used in the management of obesity (Anderson and Moore 2004) and it is believed that soy protein may contribute to lower the human obesity epidemics by decreasing hunger, increasing metabolic rate and promoting weight loss (Allison and others 2003; Fontaine and others 2003; Ohr 2005). The consumption of soy protein may also lead to low hepatic deposits of triglycerides (Ascencio and others 2004). Soy protein isolate was found to lower plasma triglycerides, increase adiponectin (Nagasawa and others 2002, 2003), accelerate lipid metabolism, and decrease body fat in obese rats and mice (Aoyama 2000a, 2000b). On the other hand, in male Zucker diabetic fatty rats, the low-isoflavone soy diet decreased plasma lipids and increased body weight, but did not change liver weight or carcass adiposity. High-isoflavone soy decreased plasma lipids, liver weight, and body weight (Banz and others 2004). After hydrolysis of soybean flakes by an alkaline protease, the obtained hydrolysate was fed to Wistar male rats and found to decrease blood lipids and body weight. Although the caloric content of the experimental diets was not indicated, this hydrolysate lowered the concentration of rats serum triglycerides by 11% at a low dosage (10 mL/kg/d). A high dosage (20 mL/kg/d), lowered serum triglycerides by 30% and decreased body weight of the animals (Zhang and others 1998). Several anorectic peptides have been already identified to exert antiobesity activity through decreasing food intake, fat and lean body mass, and body weight (Challis and others 2004). For example, Leu-Pro-Tyr-Pro-Arg, a peptide from soybean glycinin A5A4B3 subunit (Takenaka and others 2000) and Pro-Gly-Pro have been found to have anorectic activities. Based on the antiobesity activity of soy and soy peptides, various foods and beverages have been developed. For example, a soy protein meal replacement formula (Scan Diet) has been found to be effective for weight loss and fat mass reduction in obese subjects (Allison and others 2003). Other products include antiobesity formulas containing soy proteins, water-soluble fibers and gelatins, basic amino acids, and/or basic peptides (Fujita 2000). A sugar-free coffee containing soybean protein hydrolysate was also developed (Miura 2002). This beverage contained oligopeptides with 3 to 6 amino acid residues prepared by enzymatic hydrolysis of soybean protein. Compared with baseline values, ingestion of this kind of sugar-free coffee for 8 wk led to a 4% to 7% body weight reduction in human volunteers. Soybean peptides have also been used as body fat-decreasing agents in foods. It has been observed in humans that body fat, serum glycerides, and cholesterol can be decreased by peptides without decreasing body proteins (Inaba and others 2002). The satiety inducing effect of soy protein has been linked to independent activation of both opioid and cholecystokinin (CCK)-A receptors in rats. It has been found that protein digestion into peptides stimulates satiety (PupoVac and Anderson 2002). The soybean -conglycinin pepsin hydrolysates were also found to suppress food intake and gastric emptying by direct action on rat small intestinal mucosal cells. The digestion of proteins gives rise to peptides that may initiate several satiety signals from the gut and these signals may be dependent on dietary protein sources. Intraduodenal infusion of -conglycinin hydrolysates inhibited food intake of rats in a dose-dependent manner and this suppression can be abolished by intravenous injection of devazepide, a selective peripheral CCK receptor antagonist (Nishi and others 2003a). The arginine residue in protein structures was shown to be responsible for CCK release through direct action on the intestinal cells. Regarding the relationship between arginine and binding activity to brush border membrane, synthetic model peptides with 1 arginine (GGGRGGG and GGGGGGR) showed no activity. The binding activity of synthetic peptides containing 2 arginine
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residues, depended on the position of arginine residues, GGRGRGG, GRGGRGG, and GRGGGRG can bind to the brush border membrane while GGGRRGG cannot bind. GRGRGRG, a synthetic peptide containing 3 arginine residues had stronger binding ability (Nishi and others 2003b). Comparing between several arginineconcentrated fragments of -conglycinin on their abilities to bind to the intestinal cell component, the fragment from 51 to 63 of the subunit was found to have the highest binding affinity affecting also food intake in rats (Nishi and others 2003b). All these observations in experimental animals are encouraging. However, the effect of antiobesity peptides in humans remains to be determined.
Antioxidant

Several amino acids, such as Tyr, Met, His, Lys, and Trp, are generally accepted to be antioxidants. Saito and others (2003) constructed series of tripeptide libraries to explore antioxidative properties of peptides; one was composed of 108 peptides containing either 2 His or Tyr residues and the other 114 peptides structurally related to Pro-His-His. The antioxidative activities of the tripeptide libraries were examined by several methods, including the antioxidative activity against the peroxidation of linoleic acid, the reducing activity, the radical scavenging activity, and the peroxynitrite scavenging activity. Tripeptides containing Trp or Tyr residues at the C-terminus had strong radical scavenging activities, but very weak peroxynitrite scavenging activity. The results explained why protein digests have such a variety of antioxidative properties. They also found that the antioxidative peptides may exert a strong synergistic effects with some other antioxidants, such as phenolic compounds (Saito and others 2003). During hydrolysis, the soy protein structure will be altered and more active amino acid R group will be exposed. Therefore, soybean peptides can have higher antioxidant activity than intact protein (Chen and others 1998). After enzyme digestion of -conglycinin and glycinin the radical-scavenging activities were increased 3 to 5 times. Heating did not change the activity of the proteins, indicating that forming peptides was more critical than maintaining protein structure (Matoba 2002). In a study using male Wistar rats as model, it was found that the intake of either soy protein isolate (SPI) or soy peptide, but not of an amino acid mixture resembling soy protein (SPAA), had the effect of reducing paraquat (PQ)-induced oxidative stress. In this experiment, both SPI and soy peptide prevented the elevation of the serum thiobarbituric acid-reactive substances (TBARS) concentration and tended to prevent the elevation of lung weight induced by PQ, while the SPAA intake had no effect (Takenaka and others 2003). Whether these test materials were isoflavone-free was not indicated. The antioxidant capacity of soy peptides is dependent on its structure and therefore affected by the hydrolysis procedures (Yang and others 2000). When comparing the antioxidant capacity of 28 structurally related peptides to Leu-Leu-Pro-His-His, isolated from soybean protein digests, Pro-His-His was identified as an active center. It was believed that His-containing peptides can act as metal-ion chelators, active-oxygen quenches, and hydroxy-radical scavengers and contribute to the antioxidative activity of peptides (Saito and others 2003). Different hydrolysis conditions (enzyme, temperature, sample preparation) resulted in peptide mixtures with different antioxidant properties. Native and heated soy protein isolate hydrolyzed with different enzymes, such as pepsin, papain, chymotrypsin, alcalase, Protamex, and flavourzyme, resulted in different degree of hydroylsis ranging from 1.7% to 20.6%. The antioxidant activity ranged from 28% to 65%, determined by the decrease in TBARS concentration after incubating with a liposome-oxidizing system (50 mM FeCl3/0.1 mM ascorbate, pH 7.0). Samples of chymotrypsin- and flavourzyme-hydrolyzed (0.5 h) preheated SPI had the greatest inhibitory effect (Pena-Ramos and Xiong 2002).

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Food bioactive peptides . . .


Liu and others (2005) demonstrated that soymilk-kefir possess significant antimutagenic and antioxidant activity and suggest that fermented soymilk may be considered among the more promising food components in terms of preventing mutagenic and oxidative damage. More research is needed to demonstrate if peptides produced during fermentation may play an important role in this biological activity. It has also been observed that radical scavenging ability of soy peptides plays an important role in the suppression of lipid oxidation in a preparation of encapsulated lipids (Park and others 2005). The data available strongly suggest that bioactive peptides from soy protein have a clear antioxidant capacity.
Anticancer

The evidence of soy proteins/peptides and cancer relationship is not scientifically strong yet. Birt and others (2001) concluded that flavones and isoflavones in soy may contribute to cancer prevention; however, further investigations are required to clarify the nature of the interaction between these constituents and peptides/ proteins. Anticancer activity of soy proteins has been suggested (St. Clair and others 1990; Messina and Barnes 1991). For example, soybean Kunitz trypsin inhibitor was reported to suppress ovarian cancer cell invasion by blocking urokinase up-regulation (Kobayashi and others 2004). Another study showed that SPI diet altered colonic global gene expression profile and enhanced somatostatin, a known antiproliferative agent for colon cancer cells, and therefore would inhibit tumorigenesis (Xiao and others 2005). Lifetime consumption of soy proteins reduced the incidence of azoxymethaneinduced colon tumors in rats (Hakkak and others 2001). It is possible that part of these anticancer activities may be attributed to bioactive peptides derived from soy proteins. In support of this hypothesis, it has been found, both in vitro and in experimental animals, that hydrophobic peptides from soy proteins may have anticancer activity. For example, peptides obtained by thermolase hydrolysis of defatted soy protein, further purified with ethanol and fractionated by gel filtration chromatography, showed an IC50 value of 0.16 mg/mL in vitro cytotoxicity on mouse monocyte macrophage cell line. At 1 mg/mL, this fraction significantly affected cell cycle progression by arresting the cells in G2/M phases. Further purification with C18 HPLC resulted in 1157 Da nonapeptide (X-MetLeu-Pro-Ser-Tyr-Ser-Pro-Tyr) (Kim and others 2000). Kim and others (1999a) have obtained a glycopeptide from ethanol fractions of bromelain-defatted soybeans hydrolysate composed mainly of Asp, Glu, Pro, Gly and Leu with strong cytotoxic activity against P388D1 mouse lymphoma cells. In addition, lunasin is a well-studied chemopreventive peptide found in 2S soybean albumin. It contains 43-amino acid, with a carboxyl end of 9 aspartic acids residues and a cell adhesion motif (RGD) (Galvez and de Lumen 1999). Lunasin has a motif that binds specifically to non-acetylated H3 and H4 histones and prevents their acetylation. This mechanism was believed to be responsible for the anti-carcinogenic property of this chromatinbinding peptide isolated from soybean seeds (de Lumen 2005). Lunasin was found to suppress chemically induced carcinogenic transformation in mammalian cells in mice. The chemopreventive properties of lunasin have also been confirmed by in vitro studies (Lam and others 2003). The lunasin gene was cloned from soybean and the chemically synthesized form of the lunasin peptide has been used experimentally (Jeong and others 2002).
Immunomodulatory

bean protein (Yoshikawa and others 2000; Tsuruki and others 2003, 2005; Tsuruki and Yoshikawa 2004). Regarding the phagocytosis-stimulating activity, an active peptide sequence (MITLAIPVNKPGR) has been isolated from trypsin digests of soybean proteins. It was found to be derived from the subunit of -conglycinin. Met at the N terminus of the peptide was found to be essential for its activity while the 3rd residue from the N terminus may affect the activity (Thr < Phe < Trp). For the subunit, the 1st residue is Ile, and the 3rd residue is Lys, and its activity was not observed. Through mutation methods, the 1st residue of the corresponding sequence of subunit was replaced to Met and the 3rd residue was replaced to Thr, Phe, or Trp. Without changing the protein confirmation, phagocytosis-stimulating activity was observed. The phagocytosis activities of the 3 mutants followed the expected order: wild type < I122M/K124T < I122M/K124F < I122M/K124W (Maruyama and others 2003).

Technological Approach to Generate Bioactive Peptides


Enzymatic and chemical hydrolysis

Soybean peptides with immunomodulatory activities have been identified from soybean protein hydrolysates. For example, immunostimulating peptide preventing the alopecia induced by cancer chemotherapy has been isolated from an enzymatic digest of soy-

Acidic hydrolysis and enzymatic hydrolysis are 2 main methods to generate soybean peptides. The acid hydrolysis method is relatively simple and less expensive, but it is more difficult to control and amino acid damage may occur. Enzymatic methods are easier to control, use mild conditions, and do not cause amino acid damage. Therefore, enzymatic hydrolysis is a commonly used method to produce food-grade protein hydrolysate and to release bioactive peptides from their protein precursors. The type of enzyme is also very important for the preparation of bioactive peptides. Proteinases (endopeptidases) such as trypsin, subtilisin, chymotrypsin, thermolysin, pepsin, proteinase K, papain, and plasmin are used commonly for the proteolysis of food proteins (Yamamoto and others 2003). Animal, plant, and microorganism are main sources of enzymes used for the production of bioactive peptides. Non-animal origin enzymes, papain or pronase, have been used to hydrolyze soy protein. Hydrolysates have shown growth-promoting and production-enhancing activities when tested on a mouse hybridoma culture in protein-free medium (Franek and others 2000). Papain has also been used to prepare healthy food containing nutritious protein hydrolysates (Cai and Cai 2001). Proteinases from microorganisms such as Mucor sp, Aspergillus oryzae, Bacillus subtilis 1389, Aterricola 3942 are also used to hydrolyze proteins and generate peptides. Aspergillus oryzae peptidase was used to hydrolyze soybean protein slurry to shortchain peptide material (peptide chains mostly with 7 peptides) (Korhonen and Pihlanto 2003). A Mucor piriformis enzyme was also used for the production of soybean peptides from isolated soybean protein (Li and others 2001). After 5 h of hydrolysis with 750 U enzyme at 45 C, pH 6.0, a yellowish product without any bitter or astringent odor was produced. The molecular weight of the enzymatic decomposition product was 1000 Da. To obtain desirable results, acid hydrolysis can be combined with enzymatic hydrolysis. To produce soybean protein hydrolysates in high concentration, defatted soybean flour was treated using a low hydrochloric acid solution before degradation by a protein catalyst, such as protease, to inhibit gelation of soybean protein during heat sterilization (Lee and Lee 2000). In the presence of carboxylic acids, peptides having average amino acid residues of 10 to 100 were produced by enzyme hydrolysis (Hirano and Koide 2000). Heat treatment of proteins can also affect the efficiency of enzyme hydrolysis. Fischer and others (2002) found that soybean meals heat-treated at high humidity had higher levels of aggregated peptides.
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Enzymes are often combined to produce bioactive peptides. Different enzyme combinations of proteases including alcalase, chymotrypsin, pancreatin, and pepsin as well as enzymes from bacterial and fungal sources have been used (Meisel and FitzGerald 2003). Trypsin/AS.1398 protease (1:1) was used for soybean protein hydrolysis at pH 7.0, 35 C, 5.0% substrate, 3000 U enzyme/ mL. The degree of hydrolysis of soybean protein after 8 h was 35% under optimum conditions (Li and others 2000). Sometimes combination of enzymes may demonstrate synergistic actions. Byun and others (2001) found that nonspecific monoaminopeptidase (AP; E.C. 3.4.11) and X-prolyl dipeptidyl aminopeptidase (X-PDAP; E.C. 3.4.14.5), both from Aspergillus oryzae, had strong synergism in hydrolyzing proline-containing peptides. Adding X-PDAP to AP can highly improve the hydrolysis of peptide Ala-Pro-Gly-Asp-ArgIle-Tyr-Val-His-Pro-Phe, whereas adding X-PDAP to the enzyme mixture of subtilisin (E.C. 3.4.21.62) and AP can markedly increase the degree of hydrolysis of soybean (from 54% to 72%).
Fermentation

agents has largely restricted its use. On the other hand, liquidphase synthesis is the preferred method for large-scale synthesis of relatively short peptides and for carrying out the condensation of peptide fragments (Gill 1996). Recombinant DNA technology is the preferred choice for relatively large peptides with up to several hundred amino acids (Gill 1996). Due to the low expression efficiencies obtained and difficulties encountered in product extraction and recovery, attempts to extend this approach to the preparation of short peptides have not yet been truly successful (Korhonen and Pihlanto 2003). Using genetic engineering techniques, Yoshikawa and others (2002) introduced RPLKPW, a highly potent antihypertensive peptide, into 3 homologous sites in soybean -conglycinin subunit by site-directed mutagenesis. In practice, enzymatic synthesis is currently limited to relatively short sequences.

Fermentation is considered to be an efficient way to produce bioactive peptides. Bioactive peptides can be released by the microbial activity of fermented food or through enzymes derived from microorganism (Korhonen and Pihlanto 2003). Fermented milk and cheese have been extensively studied to investigate their potential to form bioactive peptides. Interest in fermented soybean products, such as natto, tempeh, soy sauce, soy paste, has grown in recent years. Many bioactive peptides have been identified. For example, ACE-inhibitory peptides containing Ala, Phe, and His have been isolated from soybeans fermented by Bacillus natto and chunggugjang fermented by Bacillus subtilis (Korhonen and Pihlanto 2003). ACE-inhibitory peptides were also found in soybean paste (His-His-Leu) (Shin and others 2001), soy sauce (Okamoto and others 1995), natto, and tempeh (Gibbs and others 2004). Three potent ACE-inhibitors, 3 thrombin inhibitors, 5 peptides with surface-active properties, and 1 peptide with antibacterial activity were also found in enzymatic hydrolysates of soy fermented foods. They were all derived from glycinin, while -conglycinin was found more stable to proteolytic attack even by multi-enzyme preparations (Gibbs and others 2004). Fermentation is not enough to fully hydrolyze soybean proteins. Glycoproteins, phosphoproteins, and other post-translationally modified species or domains that contain a higher number of disulfide bridges are more difficult to hydrolyze. The proteases in Bacillus and Rhizopus strains can only cleave soybean proteins into large peptides. Further enzymatic degradations, such as pronase, trypsin, Glu C protease, plasma proteases, and kidney membrane proteases hydrolysis, are needed to produce peptides with high activities (Gibbs and others 2004). Fermentation may also synthesize new peptide sequences. In enzymatic hydrolysates of soy fermented foods, the precursor of a peptide sequence ELLVYLL with good surface active properties could not be identified and it was believed to be synthesized during fermentation (Gibbs and others 2004).
Synthesis

Isolation, Purification, Characterization, and Quantification of Bioactive Peptides


Isolation and purification

Peptides synthesis is a useful method to prepare bioactive peptides in large scale and also to study their mechanism of action. At present, 3 main approaches are available: (1) chemical synthesis, (2) recombinant DNA technology, and (3) enzymatic synthesis (Gill 1996). Chemical synthesis is the most widely used approach at laboratory scale, existing 2 variants, liquid-phase and solid phase. The solid phase approach is the most powerful method for synthesis of peptides composed of about 10 to over 100 residues on a small scale (most practical for sequences of intermediate lengths). However, the high cost of the instrumentation and re72

The isolation and purification techniques are very important in bioactive peptide research. For example, the importance of glutathione (Glu-Cys-Gly) has been noticed as early as 1888, but the bioactivities of peptides became apparent only after 1950s with the development of a new purification technology (Sewald and Jakubke 2002). Most protein isolation and purification techniques can be applied for bioactive peptide separation. However, because of relative small size and molecular weight, special consideration should be given to bioactive peptides purification. For example, the resolving power of size-exclusion chromatography (SEC) is somewhat limited (Sewald and Jakubke 2002). Salting out and solvent extraction are often used before further purification steps. For example, in our laboratory a mixture of water, acetonitrile and trifluoroacetic acid (TFA) is used to extract peptides from enzymatic hydrolysis of fermented soybean products. After centrifugation, the supernatant is filtered and lyophilized for liquid chromatography analysis. Chromatography is the most powerful technique to isolate and purify bioactive peptides. Based on different properties of peptides, different chromatography techniques have been developed. Among them, HPLC is the most commonly used separation method. Commercially available reversed-phase columns allow for rapid separation and detection of the peptides from a mixture, whereas normal phase liquid chromatography is used preferentially for the separation of hydrophilic peptides. Ion-exchange chromatography (IEC), capillary electrophoresis (CE), and capillary isoelectric focusing (CIEF) separate peptides based on their charge properties. Size-exclusion chromatography (SEC), which is also named gel filtration chromatography (GFC) in aqueous separation systems and gel-permeation chromatography in nonaqueous separation systems, is a separation method solely based on molecular size. Gel-permeation chromatography with a Superdex Peptide HR 10/30 column was used to obtain the di- and tri-peptide fraction from buckwheat digestion using 30% acetonitrile containing 0.1% TFA for elution (Li and others 2002). Ultrafiltration (UF), crystallization, counter-current distribution, partition chromatography, and low-pressure hydrophobic interaction chromatography (HIC) have also been used for protein fractionation and purification (Sewald and Jakubke 2002).
Characterization

SDS-PAGE can be used to determine the molecule weight and the purity of bioactive peptides. For example, after HPLC separation and further concentration, an antihypertensive peptide fraction with ACE

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Food bioactive peptides . . .


inhibitory activity (IC50 0.42 mg) was isolated from casein and resolved into a single band (6 kDa) on 15% SDS-PAGE gel (Devi and others 2002). SDS-PAGE is helpful for relatively large peptides. For small peptides, the resolution of SDS gel is usually low. Size-exclusion HPLC may provide more accurate estimation of the peptide size. With appropriate columns and conditions, HPLC may provide useful information for peptide characterization. For example, reverse-phase HPLC will indicate the hydrophilicity of peptides. Because several peptides may have similar retention times, it is not fully reliable to use the retention time to identify peptides even if the target peptide sequence is known and standards has been run. In this case, UV spectra (usually from 200 to 300 nm) may provide extra information to facilitate peptide identification. For example, UV-spectral comparison was used to identify an expected peptide from a complicated peptide mixture. This method was demonstrated to aid in the identification of haemorphins (Zhao and others 1995) and (1-23) peptide from hemoglobin hydrolysate (Choisnard and others 2002). HPLC and size-based analysis cannot give direct amino acid sequence information. For unknown peptides, amino acid analyzer and protein sequencer are commonly used to determine amino acid composition and sequence. In a study of ACE-inhibitory peptides from wheat germ, the amino acid composition was analyzed with a Shimadzu LC-6A amino acid analyzer after hydrolysis of 6 N HCl for 24 h at 110 C. The amino acid sequence was determined by automated Edman degradation using a Shimadzu PPSQ-21 protein sequencer (Matsui and others 1999). Automatic protein sequencer base on Erdman degradation method is still widely used in peptide sequencing (Li and others 2002; Chen and others 2003; Kuba and others 2003; Motoi and Kodama 2003; Megias and others 2004). Mass spectrometry methods, such as triple stage Model API-III (Haileselassie 1999; Gibbs and others 2004), ESI-MS/MS (Stapels and Barofsky 2004), and MALDITOF-MS (Kim and others 2000. Rejtar and others 2004) together with database search are becoming more and more popular.
Quantification

Blotto B. The membrane was washed again and prepared for detection using a chemiluminescence kit. Lunasin quantities in sample can be determined by comparison to standard curve (Jeong and others 2002). ELISA has also been used for the quantification of lunasin (Gonzalez de Mejia and others 2004).

Application of Bioactive Peptides in Food and Drug Design


Applications

With the increasing knowledge in physiological activities of bioactive peptides, the commercial interest to use them as active ingredients in food and drug keep growing. The development of technology for industrial-scale production of such peptides is in progress (Korhonen and others 1998). Some bioactive peptides are already used in food and drug design. Dental-care/hygiene products containing caseinophosphopeptides, targeted to have an anticariogenic effect, are currently available (Meisel and FritzGerald 2003). Soybean is regarded as one of the most important source for the preparation of bioactive peptides and amino acids for food applications such as drinks, yogurts, bars, and many others.

Sensory aspects
Generally, bitterness is not a concern for intact globular soy proteins. However, during the hydrolysis of soybean proteins to bioactive peptides, some hydrophobic groups in the protein molecule may be exposed developing a bitter taste. The bitterness of peptides has been related to processing procedures such as hydrolysis conditions, enzyme used and degree of hydrolysis. Also, properties of the final hydrolysate such as size distribution, peptide composition and hydrophobicity, position of hydrophobic residues in peptide play an important role in taste development (Cho and others 2004). Middle-size peptides tend to be bitterer than larger or very small peptides. In a study on the relation of peptide-size distribution and bitterness, commercial soy protein hydrolysates were fractionated into 4 fractions, based on size, by ultrafiltration (Cho and others 2004). The results showed that fractions with average MW at about 1900 to 3300 Da had the highest bitterness, whereas fractions with larger or smaller average MW showed lower bitterness. In another study on the bitterness of gel filtration fractions of soybean proglycinin A1aB1b subunit hydrolysate, it was found that fractions with a MW of 1700 Da had higher bitter intensity than fractions with larger (4300 to 9500 Da) or smaller (210 to 850 Da) MW (Kim and others 1999b). In contrast, fractions with very small average MW (<75 Da), believed to be composed of amino acids and ammonia, was as bitter as the 1700-Da fraction. Many small bitter peptides (<1000 Da) were found to have a charged residue present at either end, indicating certain structural requirement for taste development and perception. The understanding of the relationship between bitterness of soy peptides, hydrophobicity or spatial structure of the amino acids, and their mechanism of action, has allowed the development of different bitterness eliminating methods. The optimization of hydrolytic conditions was found to be an efficient way to reduce the production of bitter peptides. Also, debittering processes (enzyme, adsorbent) have been used. For instance, sugars and carboxylates have been used for controlling bitter taste of soybean peptides in food and beverages. The carboxylates are sodium salts of citric, malic, tartaric, gluconic, fumaric, and ascorbic acids. The sugars use are monosaccharides such as fructose and glucose, disaccharides such as sucrose and trehalose, and sugar alcohol like xylitol, lactitol, mannitol, sorbitol, maltitol, and erythritol (Araki 2000). Enzymatic debittering methods using different enzymes,
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Enzyme-linked immunosorbent assay for the quantification of bioactive peptides. Based on specific combination of antigens and antibodies, enzyme-linked immunosorbent assay (ELISA) has been used for a specific detection of very small quantities of peptides. High levels of specificity are achieved with such immunoassays due to the specific and high affinity reversible binding of antigens to antibodies (Cohen and others 2002). Substances <1000 Da are usually not immunogenic. In general, immunogenicity increases with structural complexity. Aromatic amino acids (tyrosine or phenylalanine) contribute more to immunogenicity than nonaromatic. A length of 6 to 25 amino acids is usually suggested to produce an antibody. An ELISA system has been developed for the quantification of bioactive peptides using an antirabbit secondary antibody conjugated to peroxidase for sandwich antibody assay. This method is more sensitive than HPLC and is as specific as radioimmune assay (RIA), without the inconvenience of use of radioactive materials (Cohen and others 2002). Electrophoresis and Western blots. Gel electrophoresis and Western blots are used for quantification of relatively large peptides such as lunasin. This technique is not easily applied to smaller peptides. For the analysis of lunasin, SDS-PAGE of seed extracts was performed using 15% tris-HCl ready gel. A goat antirabbit Western-compatible molecular weight standard was used. Gels were stained and transblotted to PVDF membranes. The PVDF membrane with the transferred protein was blocked for nonspecific binding in Blotto A, washed and incubated with the primary antibody Zymed R1 in Blotto B solution. After washing, the membrane was incubated with an antirabbit secondary antibody in

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such as exopeptidases (Lovsin-Kukmanand and others 1996; Maehashi and Arai 2002; Raksakulthai and Haard 2003), aminopeptidase from the edible basidiomycete Grifola frondosa (Nishiwaki and others 2002), have also been developed. These studies indicate that as the incubation time elapses, the amount of free amino acids released increases and the bitterness of the reaction mixtures decreases. ease, cancer, obesity, and decreased immune function. Bioactive peptides are released from proteins by either food processing or by GI digestion. Indirect evidence also suggests that these peptides can be absorbed by the GI system thus exerting their action on specific target organs. Other peptides do not need to be absorbed and act at the intestinal level. However, understanding whether digestion of food proteins in vivo releases the same peptide fragments as the ones in vitro experiments is an important question. Also, the effective plasma levels of bioactive peptides are unknown and need to be determined. In comparison with milk, research on bioactive peptides from soybean is far from complete. Crude enzyme hydrolysates have been used in many studies. Functional peptides have not always been identified; there is still a great potential for discovery. A bioactive peptide database has been developed to predict the biological activity of protein fragments using sequence alignments between proteins and biologically active peptides (Dziuba and others 1999). This database may also be helpful to reveal the amino acid sequence-activity relationship. Of course, besides primary structure, the secondary or tertiary structure of bioactive peptides may also be important for their activity. Figure 3 presents an example of the potential biological peptides that can be produced from -conglycinin -subunit, as determined by our group, after searching the Biopep database (Dziuba and others 1999). It can be observed that this subunit can be the source of peptides with various biological activities as those indicated in this figure. As shown in Figure 3, many potential bioactive peptide sequences are embedded in conglycinin -subunit. Of course protease specificity plays an important role in determining the bioactivities of protein hydrolysates. For example, antioxidant peptide VIPAGYP may be released from conglycinin hydrolysate. However, if the protease can effectively cut the peptide bond between V and I, antihypertensive peptide IPAGYP will be released. Similarly, further digestion may release the dipeptidyl-aminopeptidase IV inhibitory peptide, PA. Using a similar approach, we have identified many potential bioactive peptides in the major soy proteins which includes, subunits of glycinin and -conglycinin, as well as in Kunitz inhibitor, Bowman-Birk inhibitor (Figure 4, Gonzalez de Mejia and others 2004). We found that the profile of peptides in soy protein demonstrates amino acid sequences with antihypertensive, dipeptidyl peptidase IV (DPPIV) inhibition, antithrombotic, immunostimulatory, antiamnestic, opioid, and antioxidant activities among others. Antihypertensive activity and DPPIV inhibition were the most common. As it can be seen in Figure 4, soy protein components are good potential sources of bioactive peptides. For example glycinin G1 precursor (soybean source nr 1) intercepts with antihypertensive activity (activity A) showing the highest frequency of corresponding amino acid sequence (20). Considering the diversity and complexity of protein sequences, there are still many possibilities to generate new bioactive peptides with higher activity or unrevealed activities. By this means, an efficient hydrolysis-separation-screening protocol will be very important. Structure-function relationship is always important for a better understanding and utilization of bioactive peptides. For example, Cheung and others (1980) reported that the hydrophobicity of the carboxyl terminal amino acid was the most important factor affecting the overall binding of the peptides to the active site of ACE. The sequence is protected from proteolysis because of its high hydrophobicity and the presence of proline residues (Meisel and FritzGerald 2003). Research and medical trials have demonstrated the biological activities of bioactive peptides, but the corresponding molecular mechanism of action is still not completely clear. A better understanding of how these bioactive peptides work and how they are regulated will be helpful. Understanding whether natural in vivo

Bioavailability of peptides
Depending on the exerted function, bioactive peptides may not need to be absorbed by the intestine or pass into systemic circulation. In the case of anorectic peptides, their action is at the intestinal level where they stimulate opioid and hormonal receptors, which induce satiety (Pupovac and Anderson 2002; Nishi and others 2003a, 2003b). However, other functions such as hypotensive or anticancer activities would require passage of the bioactive peptides through the intestinal barrier and their transport to target organs. Studies of the kinetics of digestion of milk peptides in experimental animals have shown that active peptides can still be present in the intestine even after the action of pancreatic enzymes (Scanff and others 1992). These observations suggest the availability of these substances for intestinal absorption (Shimizu 2004). Chabance and others (1998) demonstrated that peptides are released and passed to the blood with human digestion of milk or yogurt. In this study, 2 long peptides, the -caseinglycopeptide and the N-terminal peptide from -S1-casein were detected in plasma. It is also known that due to a more efficient and rapid absorption of peptides in comparison to free amino acids, peptide mixtures and protein hydrolysates are recommended to deliver nitrogen to patients suffering from malnourishment or problems of protein digestion (Gill and others 1996). Although more mechanistic studies are needed, these results support the concept that food-born peptides can be absorbed and have physiological activities in various human organs.

Safety Concerns
Potential toxicity

Peptides are normally generated during protein digestion in the GI tract. Because soybean and fermented soybean have been safely used as food for thousands of years without apparent harmful effects, the risk of toxicity caused by peptides formed during this process is practically nil. Although peptides can be absorbed into the blood, there have been no reports about toxic soybean peptides to date. However, considering the complexity of bioactive peptides preparation procedures, it becomes necessary to keep this safety concern in mind.

Allergenicity
Soybean proteins can be allergenic. However, most allergenic proteins have relatively high molecular weight. Goodman and others (2005) present a comprehensive review on allergens in genetically modified soybean with conventional soybean. Bioactive peptides contain 2 to 40 amino acids and their MW ranges from 200 to 5000 Da. Thus, the possibility of allergenicity is very low. No soybean allergic peptides have been reported in this molecular weight range.

Conclusions
Soybean proteins can be a source of bioactive peptides with diverse and unique health benefits that can be used in the prevention of age-related chronic disorders such as cardiovascular dis74

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digestion of food proteins releases the same peptide fragments as the ones in vitro experiments is important (Pellegrini 2003). How we can manage to generate desired bioactive peptides in the GI tract while preventing digestion damaging the desired peptides constitutes an important question. Chemical modification of peptides to make them more resistant to degradation is 1 of the approaches. N-methylation of the peptide bond, C-terminal esterification, the use of infusion pumps for peptide delivery, and encap-

Figure 3Potential biological peptides in -conglycinin, -subunit. The amino acid sequences were obtained from SwissProt protein knowledgebase (accession number P13916) (Apweiler and others 2004). The potential bioactive peptides and their possible function were determined by searching the Biopep database (Dziuba and others 2003). Amino acid nomenclature: C = Cys, Cystein; H = His, Histidine; I = Ile, Isoleucine; M = Met, Methionine: S = Ser, Serine; V = Val, Valine; A = Ala, Alanine; G = Gly, Glycine; L = Leu, Leucine; P = Pro, Proline; T = Thr, Threonine; F = Phe, Phenylalanine; R = Arg, Arginine; Y = Tyr, Tyrosine; W = Trp, Tryptophan; D = Asp, Aspartic acid; N = Asn, Asparagine; B = Asx, Either of D or N; E = Glu, Glutamic acid; Q = Gln, Glutamine; Z = Glx, either of E or Q; K = Lys, Lysine; X = Undetermined amino acid.

Figure 4Predicted profiles of peptides in soy protein with potential biological activities. The amino acid sequences were obtained from Swiss-Prot protein knowledgebase (accession number P13916) (Apweiler and others 2004). The potential bioactive peptides and their possible function were determined by searching the Biopep database (Dziuba and others 2003). Soybean sources: 1 = Glycinin G1precursor; 2 = Glycinin G2 precursor; 3 = Glycinin G3 precursor; 4 = Glycinin G4 precursor; 5 = Glycinin G5 precursor; 6 = Conglycinin chain; 7 = -Conglycinin chain; 8 = Conglycinin chain; 9 =Trypsin inhibitor A/C precursor; 10 = Trypsin inhibitor B; 11 = Trypsin inhibitor KTI1 precursor; 12 = Trypsin inhibitor KTI2 precursor; 13 = Bowman-Birk inhibitor precursor; 14 = Bowman-Birk inhibitor C-II precursor; 15 = Bowman-Birk inhibitor DII precursor. Predicted activities: A = Antihypertensive; B = Dipeptidyl peptidase IV inhibitor; C = Antithrombotic; D = Opioid; E = Immunostimulating; F = Regulating; G = Ligand; H = Antiamnestic; I = Activating ubiquitin-mediated proteolysis; J = Antioxidative; K = Opioid agonist.

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sulation of peptides in various carriers such as liposomes are also useful methods (Lee and Kim 2000). Understanding this question is also helpful for delivery of bioactive peptides used as drugs by oral administration. Improvements in peptides purification, qualification, and synthesis techniques are always important. Largescale bioactive peptides production is still a challenge. Enzymes from microorganisms produced during fermentation are very helpful to release bioactive peptides. New peptide sequences may also be generated during fermentation. A better understanding of this phenomenon is important for discovering of new active peptides as well as for determining their safety. The mechanism of the physiological activities of the small peptides from soybean needs to be further investigated. Studies on the impact of soy processing on the generation of bioactive peptides are lacking. It is also important to discover new peptides with health benefits in soy-hydrolysates and fermented foods. The identification of these compounds will contribute toward the development of new functional foods and the prevention of disease. In summary, there are opportunities in this field for the industrial exploitation of soy value-added bioactive peptides that can be used to enhance health and prevent disease.
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Acknowledgments
The authors express their gratefulness to the USDA-Future Foods Initiative and Hatch funds for their support.

Abbreviations
aa = amino acid; ACE = angiotensin-converting enzyme; APP = acid-precipitated soy protein; BBI = Bowman-Birk inhibitor; BP = bioactive peptides; CCK = cholecystokinin; CE = capillary electrophoresis; CHD = coronary heart disease; CIEF = capillary isoelectric focusing; CNS = central nervous system; CPP = casein-derived phosphorylated peptides; DH = degree of hydrolysis; ED = effective dose; ELISA = enzyme-linked immunosorbent assay; FPH = fish protein hydrolysate; GFC = gel filtration chromatography; GI = gastrointestinal tract; GM = genetic modification; HIC = hydrophobic interaction chromatography; HPLC = high-pressure liquid chromatography; IC50 = 50% inhibitory concentration; IEC = ion exchange chromatography; IG = immunoglobulin; kDa = thousand daltons; KTI = Kunitz trypsin inhibitor; LDL = low-density lipoproteins; MAC = metal affinity chromatography; MW = molecular weight; PQ = paraquat; PEP = soy peptide; PVDF = polyvinylidene fluoride; S = Svedberg unit; SDS-PAGE = sodium dodecyl sulfate-polyacrylamide gel electrophoresis; SEC = size exclusion cromatography; SHR = spontaneously hypertensive rats; SPI = soy protein isolate; SPA = soy protein allergy; SPHF = soy protein hydrolysate formulas; TBARS = thiobarbituric acid-reactive substances; UF = ultrafiltration; WG = wheat germ.

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