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

7/27/2019 A New Frontier in Soy Bioactive Peptides That Prevent Age-related Chronic Diseases

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Vol. 4, 2005COMPREHENSIVE REVIEWS IN FOOD SCIENCE AND FOOD SAFETY 63 2005 Institute of Food Technologists

A New Frontierin Soy Bioactive

Peptides thatMay PreventAge-related

Chronic DiseasesWenyi Wang and Elvira Gonzalez de Mejia

ABSTRAABSTRAABSTRAABSTRAABSTRACTCTCTCTCT: D: D: D: D: Durururururing gastring gastring gastring gastring gastrointestinal digestion or food prointestinal digestion or food prointestinal digestion or food prointestinal digestion or food prointestinal digestion or food processing of processing of processing of processing of processing of proteinsoteinsoteinsoteinsoteins, small peptides can be r, small peptides can be r, small peptides can be r, small peptides can be r, small peptides can be released and may act aseleased and may act aseleased and may act aseleased and may act aseleased and may act asregulatory compounds with hormone-like activities. Numerous biologically active peptides (bioactive peptides) have beenregulatory compounds with hormone-like activities. Numerous biologically active peptides (bioactive peptides) have beenregulatory compounds with hormone-like activities. Numerous biologically active peptides (bioactive peptides) have beenregulatory compounds with hormone-like activities. Numerous biologically active peptides (bioactive peptides) have beenregulatory compounds with hormone-like activities. Numerous biologically active peptides (bioactive peptides) have beenidentified. Most bioactive peptides are derived from milk and dairy products, with the most common being angiotensinidentified. Most bioactive peptides are derived from milk and dairy products, with the most common being angiotensinidentified. Most bioactive peptides are derived from milk and dairy products, with the most common being angiotensinidentified. Most bioactive peptides are derived from milk and dairy products, with the most common being angiotensinidentified. 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 inconverting enzyme inhibitory peptides. Soybean protein and soybean derived peptides also play an important role inconverting enzyme inhibitory peptides. Soybean protein and soybean derived peptides also play an important role inconverting enzyme inhibitory peptides. Soybean protein and soybean derived peptides also play an important role inconverting enzyme inhibitory peptides. Soybean protein and soybean derived peptides also play an important role insososososoybean physiological activitiesybean physiological activitiesybean physiological activitiesybean physiological activitiesybean physiological activities, par, par, par, par, particularly those rticularly those rticularly those rticularly those rticularly those related to the prelated to the prelated to the prelated to the prelated to the prevevevevevention of chrention of chrention of chrention of chrention of chronic diseasesonic diseasesonic diseasesonic diseasesonic diseases. H. H. H. H. Hooooowwwwwevevevevevererererer, the bioactiv, the bioactiv, the bioactiv, the bioactiv, the bioactiveeeeepotential of soybean derived bioactive peptides is yet to be fully appreciated. After a general introduction of approachespotential of soybean derived bioactive peptides is yet to be fully appreciated. After a general introduction of approachespotential of soybean derived bioactive peptides is yet to be fully appreciated. After a general introduction of approachespotential of soybean derived bioactive peptides is yet to be fully appreciated. After a general introduction of approachespotential of soybean derived bioactive peptides is yet to be fully appreciated. After a general introduction of approachesand advances in bioactive peptides from food sources, this review focuses on bioactive peptides derived from soybeanand advances in bioactive peptides from food sources, this review focuses on bioactive peptides derived from soybeanand advances in bioactive peptides from food sources, this review focuses on bioactive peptides derived from soybeanand advances in bioactive peptides from food sources, this review focuses on bioactive peptides derived from soybeanand advances in bioactive peptides from food sources, this review focuses on bioactive peptides derived from soybeanprprprprproteins and their physiological proteins and their physiological proteins and their physiological proteins and their physiological proteins and their physiological properoperoperoperopertiestiestiestiesties..... TTTTTechnological apprechnological apprechnological apprechnological apprechnological approaches to generoaches to generoaches to generoaches to generoaches to generate bioactivate bioactivate bioactivate bioactivate bioactive peptidese peptidese peptidese peptidese peptides, their isolation,, their isolation,, their isolation,, their isolation,, their isolation,purification, characterization, and quantification, and further application in food and drug design are also presented.purification, characterization, and quantification, and further application in food and drug design are also presented.purification, characterization, and quantification, and further application in food and drug design are also presented.purification, characterization, and quantification, and further application in food and drug design are also presented.purification, characterization, and quantification, and further application in food and drug design are also presented.SSSSSafety concerafety concerafety concerafety concerafety concernsnsnsnsns, such as potential to, such as potential to, such as potential to, such as potential to, such as potential toxicityxicityxicityxicityxicity, aller, aller, aller, aller, allergenicitygenicitygenicitygenicitygenicity, and sensor, and sensor, and sensor, and sensor, and sensory aspect of these peptides ary aspect of these peptides ary aspect of these peptides ary aspect of these peptides ary aspect of these peptides are likewise discussed.e likewise discussed.e likewise discussed.e likewise discussed.e likewise discussed.

KKKKKeyworeyworeyworeyworeywords: bioactivds: bioactivds: bioactivds: bioactivds: bioactive peptidese peptidese peptidese peptidese peptides, so, so, so, so, soybean, antiobesityybean, antiobesityybean, antiobesityybean, antiobesityybean, antiobesity, hypocholester, hypocholester, hypocholester, hypocholester, hypocholesterolemic, antihyperolemic, antihyperolemic, antihyperolemic, antihyperolemic, antihypertensivtensivtensivtensivtensiveeeee

Introduction

In living organisms, endogenous peptides often function as hor-mones and neurotransmitters and play important physiologicalroles. Through hormone-receptor interactions and signaling cas-cades, they exert their actions on regulating metabolism (water,mineral, and other nutrients), controlling gland excretion, adjust-ing blood pressure, and impacting body growth. They may alsoexert 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 thehypothalamus hormones cascade. For example, corticoliberin(CRH, 41-peptide amide) and thyroliberin (TRH, pGlu-His-Pro-NH2) function as hypothalamic hormones. They can stimulate thesecretion of 2 pituitary hormones, corticotropin (acth, 39aa), andtryrotropin (glycoprotein, chain 96aa, chain 112 aa), respec-tively. Furthermore, various gastro-enteropancreatic peptides,

such as insulin and glucagons, play very important roles in theregulation of the metabolism (Sewald and Jakubke 2002).

In the past several decades, researchers have found that bioac-tive peptides can also be derived from dietary proteins. They maybe present as independent entities or encrypted in the parent pro-tein. It is known that during gastrointestinal (GI) digestion or foodprocessing, these peptides are released from the parent protein

and act as regulatory compounds with hormone-like activities

(Korhonen and Pihlanto 2003). In 1950, Mellander suggested thatcasein-derived phosphorylated peptides (CPP) enhanced vitaminD-independent bone calcification in rachitic infants (Mellander1950). This early observation is considered as the first indicationof food derived bioactive peptides (Korhonen and Pihlanto 2003).Since then, numerous peptides with various bioactive functionshave been identified. In a database named Biopep, more than1500 different bioactive peptides have been presented (Dziubaand others 2003). Among them, angiotensin-converting enzyme(ACE) inhibitors and dipeptidyl peptidase IV inhibitors, whichshow antihypertensive activity, are the most common (Ahn andothers 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, antican-cer, and immunomodulatory actions have also been identified.

Food-derived bioactive peptides commonly contain 2 to 9 ami-no acids (Kitts and Weiler 2003). However, this range may be ex-tended to 20 or more amino acid units (Korhonen and Pihlanto2003). For example lunasin, a food-derived peptide with provedanticancer bioactivity, contains 43 amino acids with a molecularweight (MW) of 5400 Da (Jeong and others 2002).

Milk and other dairy products are among the best precursors ofbioactive peptides and have been extensively studied (Floris andothers 2003). However, bioactive peptides have also been isolat-ed and characterized from other food protein sources, includingegg, fish, oyster, cereal (rice, wheat, buckwheat, barley, corn), soy-bean, and radish seeds (Matsui and others 1993; Li and others2002; Yoshikawa and others 2003).

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 HumanNutrition, Univ. of Illinois at Urbana-Champaign, 228 ERML, MC-051, 1202West Gregory Drive, Urbana-Champaign, IL 61801. Direct inquiries to au-thor Gonzalez de Mejia (E-mail: [email protected]).
mailto:[email protected]:[email protected]:[email protected]:[email protected]


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64 COMPREHENSIVE REVIEWS IN FOOD SCIENCE AND FOOD SAFETYVol. 4, 2005

CRFSFS:Comprehensive Reviews in Food Science and Food Safety

Soybean is an important protein source and a potential source ofbioactive peptides. On average, soybean contains about 40% pro-tein (Nielsen 1996) conformed by a complex mixture of differentprotein types. In ExPASy databases, up to date there are a total of1411 protein entries (266 Swiss-Prot entries and 1145 TrEMBL en-tries) listed for soybean (Glycine max). The major components ofsoy proteins are storage proteins known as -conglycinin and gly-cinin, which account for 65% to 80% of total seed proteins. In ad-dition, there are many enzymes (such as lipoxygenase, chalcone

synthase, catalase, urease) in soybean, but only a relatively smallnumber 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). Smeans Svedberg unit, which is a unit of sedimentation rate com-puted as the rate of sedimentation per unit field of centrifugationstrength. Among them, 11S (glycinin) and 15S (a polymer of glyci-nin) are pure proteins, whereas 2S and 7S are composed of sever-al proteins. Figure 1a presents the crystal structure of soybean -conglycinin homotrimer and Figure 1b the crystal structure ofglycinin 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 sub-units share a large degree of amino acid homologies. Figure 2presents the multiple sequence alignments of 3 subunits (, , )of soybean beta-conglycinin. Furthermore, -conglycinins withdifferent subunit composition have also been identified. It is likelythat the trimers are composed of randomly assembled mixture ofsubunits (Nielsen 1996).

Glycinin, on the other hand, is a hexamer with MW of about320 to 375 kDa and with 5 major subunits, G1, G2, G3, G4, andG5. Each subunit consists of 2 polypeptide chains, an acidicchain (about 40 kDa) and a basic chain (about 20 kDa), joined bya single intra-chain disulphide bond. G1, G2, and G3 can begrouped as they share about 90% sequence homologies. Similari-ty, G4 and G5 share about 90% sequence homologies. However,sequence homologies between these 2 groups (G1, G2, G3 andG4, G5) are only about 50% (Nielsen 1996).

From the epidemiological point of view, studies suggest that popu-lations consuming high levels of soybean products have both lowercancer incidence and lower mortality rates for the major cancer typescommonly found in the Western hemisphere (Nagata and others

2002; Wu and others 2002; Spector and others 2003). Thus, as themain components of soybean, soy proteins are receiving more andmore attention with respect to their health effects. Bowman Birk in-hibitor (BBI), a 2S soy protein component, has been shown to sup-press carcinogenesis in animals (Kennedy 1995; Kennedy and oth-ers 2002) and in human prostate cancer cells (Kennedy and Wan2002). BBI has also been the subject of promising clinical trials incancer patients (Armstrong and others 2000, 2003; Meyskens2001). A Phase IIb randomized, placebo-controlled clinical trial to

determine the clinical effectiveness of Bowman-Birk inhibitor con-centrate is currently under way. Similarly, lunasin has been found tosuppress chemically induced carcinogenic transformation in mam-malian cells using mice as a model (de Lumen 2005). This novelpeptide can be found in amounts ranging from 0.10 to 1.33 g/100 gflour in different soybean varieties and in commonly available soyproteins (Jeong and others 2003). As described, soybean proteinsand soybean-derived peptides may play an important role in diseaseprevention and treatment. Food processing and in vivo enzyme di-

Table 1Soybean protein classificationa

Percent inUltrafiltration extractable

protein fractionb protein Proteins in the fraction

2S 20% Kunitz typsin inhibitorsBowman-Birk typsin inhibitorsCytochrome CAL1 and AL3

-Conglycinin7S 33% -Conglycinin

-Conglycinin-AmylaseLipoxygenaseHemagglutinins (or lectins)Soybean vacuolar protein P34

11S 33% Pure protein: glycinin

15S 10% Pure protein: polymer ofglycinin

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 ofsedimentation per unit field of centrifugation strength.

Figure 1 (a) Crystal structure of soybean -conglycinin -homotrimer. Printed with permission (Maruyama and others2004). (b) Crystal structure of glycinin A3B4 subunits of hex-amer. Printed with permission (Adachi and others 2003).

(a)

(b)


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Vol. 4, 2005COMPREHENSIVE REVIEWS IN FOOD SCIENCE AND FOOD SAFETY 65

Food bioactive peptides . . .

Figure 2Multiple sequence alignments of 3 subunits: alpha (), beta (), alpha () of soybean -conglycinin. The aminoacid 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 regionsfrom 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 Dor N; E = Glu, Glutamic acid; Q = Gln, Glutamine; Z = Glx, either of E or Q; K = Lys, Lysine; X = Undetermined amino acid.


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gestion of soy proteins may release peptides, which exert diverse bi-ological functions by interacting with cell receptors, functioning ashormones, regulating enzymes or interfering cell cycles. Therefore,an overview on the scientific advancement and technical aspects ofbioactive peptides derived from soybean products would be helpfulfor a better understanding of their bioactive potential and for stimu-lating further research in this area.

After a general introduction of bioactive peptides from severalfood sources, this review will provide an overview of the role of

bioactive peptides derived specifically from soybean proteins andtheir physiological properties. Technological approaches to gener-ate bioactive peptides, their isolation, purification, characteriza-tion, and quantification, as well as their application in food anddrug design will also be presented.

Food Sources of Bioactive Peptides

Animal origin

Dairy products. Milk is a particularly rich source of bioactivepeptides. These peptides are encrypted in both casein (s, , , -casein) and whey proteins (-lactalbumin, -lactoglobulin, lactofer-rin, and immunoglobulins) (Belem and others 1999; Gobbetti andothers 2002) and can be released by enzyme hydrolysis or micro-bial fermentation. For enzyme hydrolysis, single enzymes or a com-bination of proteinases have been used, such as trypsin, alcalase,chymotrypsin, carboxypeptidase, pancreatin, pepsin, and enzymesfrom bacterial or fungal origin (for example, proteinase K from Triti-rachium album). Bioactive peptides have been identified from milkfermented by lactic acid bacteria, such as Lactococcus lactissubsp.cremoris, Lactobacillus helveticus, LactobacillusGG strain, Lacto-bacillus delbruskii subsp. Bulgaricus (Gobbetti and others 2002;LeBlanc and others 2002; Korhonen and Pihlanto 2003).

Milk-derived bioactive peptides have shown various physiolog-ical activities. For example, -casokinins (derived from , -casein) showed antihypertensive and immunomodulatory activi-ties; lactoferricin (f 17-41 of lactoferrin) antimicrobial activity; ca-somorphins (derived from , -casein) and -casein exorphin (f

90-96 ofs1-casein) opioid activity; caseinophoshopeptides (de-rived from post-translational phosphorylated s, -casein) mineralbinding activity and peptide KRDS (f 17-41 of lactoferrin) anti-thrombotic activity (Korhonen and others 1998; Gobbetti andothers 2002; Meisel and FitzGerald 2003).

Milk processing conditions affect the formation of milk-derivedbioactive peptides. In fermented milk products, peptide activitiesdepend on type of bacterial starter cultures and degree of proteoly-sis. Adequate proteolysis can facilitate the release of bioactive pep-tides, but once it exceeds certain level, it will decrease the bioactivi-ty. For example, in products with low degree of proteolysis, such asyogurt and fresh cheese, ACE-inhibitory activity is low, whilecheese with longer ripening time such as middle aged gouda has ahigher ACE-inhibitory action (Korhonen and Pihlanto 2003).

Hypocholesterolemic peptides have also been identified; for

example, Ile-Ile-Ala-Glu-Lys, a peptide from a -lactoglobulintryptic hydrolysate (LTH), was found to lower serum and liver cho-lesterol level, at least partly, by inhibiting micellar solubility ofcholesterol (Nagaoka and others 2001).

Egg. Several bioactive peptides with vasodilatation, ACE-inhibi-tory activities have been found in egg ovalbumin treated with chy-motrypsin and pepsin (Korhonen and Pihlanto 2003). For exam-ple, 2 vasorelaxing peptides, RADHPF (f 359 to 364 of ovalbu-min) and ovokinin (FRADHPFL, f 358 to 365 of ovalbumin), wereisolated from chymotryptic and peptic digest of ovalbumin, re-spectively (Fujitaand others 1995; Matoba 1999). Although theyare released from the same region of ovalbumin, it was believedthat they showed different modes of vasorelaxing activities.

Meat. Chicken meat was found to be a source of antihyperten-sive peptides. For example, with the action of the enzyme ther-molysin, 2 antihypertensive, Ile-Lys-Trp and Leu-Lys-Pro havebeen detected (Korhonen and Pihlanto 2003).

Fish and seafood. Bioactive peptides can also be found in fishproducts, such as sardine muscle, tuna muscle, bonito (Yamamo-to and others 2003), and Alaska Pollack skin (Korhonen andPihlanto 2003). Different enzymes have been used to generatebioactive peptides (Fujita and Yoshikawa 1999). For example, LKP-

NM, isolated from the thermolysin digest of dried bonito, was acti-vated 8-fold by ACE itself and showed a prolonged effect after itsoral administration to animal models (Yoshikawa and others2000). After 17 h of hydrolysis of sardine protein by Bacillus li-cheniformisalkaline protease, the ethanol fraction obtained with achromatographic resin showed strong ACE-inhibitory activity(IC50 = 0.015 mg protein/mL). This fraction was confirmed to havea significant in vivo depressor effect in mild hypertensive volun-teers 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 HDLcholesterol:total cholesterol ratio was greater in these rats and inWistar rats fed FPH compared with those fed casein (Wergedahland others 2004). These observations suggest that FPH may havea role as a cardioprotective nutrient.

Insects. Royal jelly (RJ), a bee product rich in proteins, has alsobeen found as a good source of ACE-inhibitory peptides. Matsuiand others (2002) found that intact RJ and its protein fraction didnot show ACE-inhibitory activity. However, after pepsin and thesubsequent trypsin and chymotrypsin hydrolysis, ACE inhibitorycapacity was developed (IC50 = 0.099 mg protein/mL). Further-more, single oral administration of this GI RJ hydrolysate signifi-cantly lowered systolic blood pressure in spontaneous hyperten-sive rats (SHR). They further fractionated the hydrolysate and iden-tified 8 additional peptides with IC50 value of < 10 mM.

Plant origin

As indicated in Table 2, soybean, wheat, corn, rice, barley,buckwheat, and sunflower are all plant sources of bioactive pep-

tides. It can be observed that the main approach used to producepeptides is by enzymatic hydrolysis. These peptides are then fur-ther isolated by either ultrafiltration or by cationic exchange res-ins. Thus, peptides with different amino acid sequences can beobtained that possess various biological activities. It is interestingto observe that depending on the initial protein source, enzymeused, and processing conditions, the biological activities of thepeptides are different. As shown in the table, when different en-zymes hydrolyze soy protein, it yields either antioxidant peptides(Pena-Ramos and Xiong 2002), peptides with anticancer proper-ties (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 20mM, composed of 2 to 7 amino acid residues from wheat germ

hydrolysate. Ile-Val-Tyr was identified as a main contributor to theACE inhibition of the hydrolysate. An opioid peptide with similarstructure to endogenous opioid peptides gluten exorphin A5(Gly-Tyr-Pro-Thr) has also been isolated from the enzymatic di-gests of wheat gluten (Yoshikawa and others 2003). Also, peptidesderived from a spinach constituent, Rubisco (D-ribulose-1,5-bis-phosphate carboxylase/oxygenase; EC 4.1.1.39), were found tohave opioid activity (Teschemacher 2003).

Based on a bioactive peptide sequence database analysis, riceprolamin is believed to be one of the best precursors of antihyper-tensive peptides (Dziuba and others 2003). Similarly, ACE-inhibi-tory peptides, such as LQP, LLP, LSP, LAA, FY, have been reportedin -zein thermolysin hydrolysates (Yamamoto and others 2003).


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Intact buckwheat was found to exhibit ACE inhibitory activityhaving an IC50 value of 3.0 mg/mL. After pepsin, chymotrypsin,and trypsin hydrolysis, ACE inhibitory activity was significantly in-creased (IC50 0.14mg protein/mL) compared with IC50 before hy-drolysis (0.36 mg protein/mL). Several di-/tri-peptide fractions (Tyr-Gln-Tyr and Pro-Ser-Tyr) of the buckwheat digest were identified

considering the magnitude of their ACE inhibitory action (Li andothers 2002).

Immunomodulatory peptides derived from tryptic hydrolysatesof rice and soybean proteins act to stimulate superoxide anions(reactive oxygen species-ROS), which triggers nonspecific im-mune defense systems (Kitts and Weiler 2003). A 9 amino acid

Table 2Examples of biologically functional peptides derived from plant proteinsSource Preparation Peptides Activity Reference

Native and heated Purified proteases: pepsin, Degree of hydrolysis of SPI Antioxidant activities. Both hydrolyzed Pena-Ramossoy protein isolate papain, and chymotrypsin hydrolysates ranged from and nonhydrolyzed SPI decreased and Xiong 2002

and crude proteases: 1.7% to 20.6% TBARS (by 28% to 65%), except foralcalase, Protamex, and papain-hydrolyzed samples. SamplesFlavourzyme of chymotrypsin- and Flavourzyme-

hydrolyzed (0.5 h) preheated SPI hadthe greatest inhibitory effect onlipid oxidation

Soybean protein Porcine, pepsin and bovine Peptides with different Up-regulate the uptake and degradation Arnoldi andconcentrate, pancreatic trypsin or only molecular weights were of LDL by the HepG2 cell receptors others 2001Crocksoy 70, trypsin separated from the digestedextracted by 80% material by ultrafiltrationethanol

Soy flour or Enzymes of non-animal Soluble peptides were Growth-promoting and production Franek andwheat flour origin, papain or pronase separated from the hydro- enhancing activities when tested others 2000lysate by ul trafi ltration on a mouse hybridoma cul ture in

protein-free medium

Defatted soy protein Thermolase X-Met-Leu-Pro-Ser-Tyr- Anticancer Kim andSer-Pro-Tyr others 2000

Defatted soy meal Alcalase enzyme Soluble hydrolyzed sample Hypotensive Wu and Dingwas further fractionated on 2001cationic exchange resin

Soy protein Protease D3 (1) Tyr-Val-Val-Phe-Lys Hypotensive Kodera and(2) Pro-Asn-Asn-Lys-Pro- Nio 2002Phe-Gln (3) Asn-Trp-Gly-Pro-Leu-Val (4) Ile-Pro-Pro-Gly-Val-Pro-Tyr-Trp-Thr(5) Thr-Pro-Arg-Val-Phe.

Soybean Protease from PGTAVFK Antihypertensive IC50

= 26.5 M Kitts and

Bacillus subtilis Weiler 2003Soybean glycinin Peptide derived from LPYPR Hypocholesterolemic peptide Yoshikawa and

soybean glycinin others 2000

-Conglycinin Derived from subunit Soymetide-13: Immunostimulating peptide by FPR Yoshikawa andof -conglycinin MITLAIPVNKPGR receptor; Sometide-9 is the most others 2000

Soymetide-9: MITLAIPVN; active in stimulating phagocytosisSoymetide-4: MITL in vitro

Genetically Proteinase S; alcalase; LLPHH; RPLKPW Antioxidative; Antihypertensive Korhonen andmodified soybean trypsin Pihlanto 2003protein

Chymotrypsin Fermentation HHL Antihypertensive peptide: IC50

= 2.2 M Shin and othersKorean fermented 2001soybean paste

Rubisco from Synthesis; Pepsin and Rubiscolin-5 YPLDL, IC50

= 51.0 Mand 24.4 Min mouse Yang 2001spinach leucine aminopeptidase Rubiscolin-6 YPLDLF assay; 2.09 Mand 0.93 Min -

(LAP) receptor binding assay using [3

H]Deltorphin II as radioligand, respectively

Gluten Derived from gluten Gluten exorphin: GEA5 showed more potent -opioid Yoshikawa andGEA5:GYYPT activity than GEA4. GEB5 was more others 2003GEA4:GYYP potent than GEB4. GEC was moreGEB5:YGGWL potent than GEA but weaker than GEB.GEB4:YGGWGEC: YPISL

Buck wheat Pepsin, chymotrypsin VK, FY, YQY, PSY ACE inhibitory peptides. IC50

= 13, Liam andand trypsin 25, 4, 16 M, respectively. others 2002

-Zein Thermolysin LQP, LLP, LSP, LAA, FY ACE inhibitory peptides: IC50

= 1.9, 57, Yamamoto and1.7, 13, 25 M, respectively. others 2003

Sunflower Protein Sequential treatment with FVNPQAGS ACE inhibitory peptides Megias andisolates pepsin and pancreatin 5.7 g/mL others 2004


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peptide (Gly-Tyr-Pro-Met-Tyr-Pro-Leu-Pro-Arg) with ileum con-tracting and immunostimulatory activities has also been found inrice albumin trypsin hydrolysates.

Table 3 presents examples of activity and structural homologyof selected bioactive peptides such as opioid, antihypertensive,ACE inhibitor, and antioxidant capacity. It is interesting to noticethat high content of proline residues makes peptides resistant toproteolytic attack (Haileselassie and others 1999).

Biological Activities of Soybean Peptides

Antihypertensive peptides

Antihypertensive peptides are the most extensively studied bio-active peptides in foods. They show their activity by inhibiting an-giotensin-converting enzyme. ACE is a nonspecific dipeptidyl car-boxypeptidase associated with the regulation of blood pressureby modulating the rennin-angiotensin system. This enzyme con-verts the decapeptide angiotensin I into the potent vasoconstrict-ing octapeptide angiotensin II, which leads to an increase inblood pressure. Therefore, inhibition of the ACE will result in anantihypertensive effect (Natesh and others 2003). Using spontane-ously hypertensive rat (SHR) model, it has been found that ACE in-hibitory bioactive peptides lower systolic blood pressure and ACEactivity in the aorta (Li and others 2002).

Several ACE inhibitory bioactive peptides have been found inenzyme 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 digestsof soybean proteins by ion exchange chromatography and gel fil-tration. Further separation by reversed-phase ODS high-perfor-mance liquid chromatography (HPLC) led to 4 active ACE inhibi-tory peptides, the amino acid sequences of which were identifiedby the Edman procedure as: Ile-Ala (IC50 153 mM), Tyr-Leu-Ala-Gly-Asn-Gln (IC5014 mM), Phe-Phe-Leu (IC50 37 mM), and Ile-Tyr-Leu-Leu (IC50 42 mM). The peptide fractions given orally to SHR ata level of 2.0 g/kg body weight markedly lowered their bloodpressure. Antihypertensive peptides were also found in soybeanalcalase digest (Wu and Ding 2001). Oral doses of these peptidessignificantly (P< 0.05) decreased systolic blood pressure of SHR

in a dose-dependent manner. However, the peptides had little ef-fects on blood pressure of normotensive rats even at highest dosetested (1000 mg/kg of body weight/d).

Kodera and Nio (2002) have produced peptides from soybeanswith ACE inhibitory activity and favorable taste. Namely, the fol-lowing 5 peptides were generated by soybean protein digestionwith protease D3: (1) Tyr-Val-Val-Phe-Lys, (2) Pro-Asn-Asn-Lys-Pro-Phe-Gln, (3) Asn-Trp-Gly-Pro-Leu-Val, (4) Ile-Pro-Pro-Gly-Val-Pro-Tyr-Trp-Thr, and (5) Thr-Pro-Arg-Val-Phe.

Yoshikawaand others (2002) introduced a new physiologicalfunction into soy protein by genetic engineering. RPLKPW, a high-ly potent anti-hypertensive peptide, was introduced into 3 homol-ogous sites in soybean -conglycinin -subunit by site-directed

mutagenesis. In this experiment, the mutated -subunit ex-pressed in Escherichia coliexerted an anti-hypertensive effect inSHR at a dose of 10 mg/kg. When the 4th RPLKPW sequence wasintroduced to the subunit, the antihypertensive activity ofsubunit was further improved.

Fermented soybean products are a good source of ACE inhibi-tory bioactive peptides. From fermentation of soybean containingmedium with Bacillus nattoor B. subtilis, several ACE inhibitorypeptides, such as Val-Ala-His-Ile-Asn-Val-Gly-Lys or Tyr-Val-Trp-Lys, were isolated (Kimura and others 2000). This experimentshowed the potential of fermented soybean meal as a source ofantihypertensive peptides. ACE inhibitory peptides have alsobeen found in many traditional Asian fermented soy foods, suchas 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 ofan antihypertensive peptide from chunggugjang, a traditional Ko-rean soybean product fermented with Bacillus subtilisCH-1023.The optimum incubation condition for the generation of antihy-pertensive peptide from chunggugjang was 60 h at 40 C. Thecrude extract was partially purified by Amicon YM-3 membranefiltration and Sephadex G-10, G-25 gel filtration. The purified pep-tide (0.5 mg) showed an inhibitory rate of 94.3%. The most promi-nent amino acid composition of the peptide from chunggugjangwas Ala, followed by Phe, and His (Cho and others 2000).

Hypocholesterolemic

The beneficial effects of soybean on cardiovascular diseases were

Table 3Activity and structural homology of selected bioactive peptides

Activity Structural homology Remarks Reference

Opioid YPX X- Aromatic amino acid (Phe, Trp) Yang 2001Or aliphatic amino acid (As in GYYPT,YPISL, YPVEPF, YPLDL, YPLDLF

Opioid N-terminal YGGF Typical opioid peptides. Originate from:proopiomelanocortin, proenkephalin, and prodynorphin. Pihlanto-Leppl

2001

Opioid N-terminal Atypical opioid peptides. Pihlanto-Leppl

YXF, YXXF 2001Opioid Y at N-terminal Tyrosine residue located at the amino terminal or bioactive site. Kitts and Weiler

2003

Antihypertensive C-terminal x-F,Y,I,V Lin and Lin 2001FXGLM-NH

2Can lower blood pressure, stimulate isolated smooth muscleand cause salivary secretion (tachykinins)

ACE-inhibitory N-terminal Y High content of P residues make the peptides resistant to Haileselassiein b-casomorphins proteolytic attack and others 1999

ACE-inhibitory Rich in hydrophobic amino Usually short and resistant to the action of Kitts and Weileracid and have a Pro, Lys, digestive-tract endopeptidases. 2003or Arg as a C-terminal

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


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first considered through its impact on blood cholesterol. Among allsoybean components, soy proteins and isoflavones were believed tobe main factors. A large body of literature indicates that soy proteinscan reduce blood cholesterol concentrations in experimental ani-mals and humans (Potter 1995). For example, Sagara and others(2004) found that dietary intakes of soy protein (at least 20 g) andisoflavones (at least 80 mg) for 5 wk would be effective in reducingCHD risk among high-risk, middle-aged men. More recently, studiesin rats have demonstrated that SPI intake could modulate lipid and

energy metabolism, including the synthesis and degradation of cho-lesterol (Tachibana and others 2005).

Soy protein is known to exert hypocholesterolemic effectswhen ingested, prompting the Food and Drug Administration toapprove a health claim linking foods that are naturally rich in soyprotein to a reduction in coronary heart disease (Anderson andothers 1995). Within this context, Wang and others (2004)showed that soy protein reduces circulating triglycerides and cho-lesterol in hypercholesterolemic individuals. However, the mech-anisms underlying the hypocholesterolemic properties of soy pro-tein remain unclear (Desroches and others 2004).

Adams and others (2002) compared the effect of SPI in 2 genet-ically engineered mouse models of atherosclerosis, quantifyingthe aortic content of esterified cholesterol. It was found that bothpreparations, 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 con-trol. However, the effect was enhanced in mice fed intact SPI rela-tive to those fed alcohol-washed soy protein. The effect was inde-pendent of plasma lipoprotein concentrations and the presenceor absence of LDL receptors. More recently, it has been shownthat dietary soy -conglycinin inhibits atherosclerosis in mice (Ad-ams and others 2004; Moriyama and others 2004).

Soy protein can also shift LDL particle distribution to a lessatherogenic pattern in an isoflavone independent manner (Desro-ches and others 2004). Compared with animal protein control,soy protein (with or without isoflavone) significantly decreased thecholesterol levels in LDL < 25.5 nm by 12.3% (P< 0.001) and in-creased cholesterol levels in LDL > 26.0 nm by 14.3% (P< 0.05)

and therefore shifting LDL particle distribution to a less atherogen-ic pattern. Another hypothesis is that soy proteins might bind withbile acids inhibiting their reabsorption and therefore loweringblood cholesterol level.

In most animal studies and clinical trials, soy proteins havebeen given to animals or human subjects by oral administration.Thus, these proteins have been subjected to protease digestion inthe GI tract releasing the bioactive peptides, which then may low-er cholesterol levels. Based on these observations, it is likely thatsoy peptides may be responsible, at least in part, for the hypocho-lesterolemic benefits of soy protein.

It has been reported that a soy protein peptic hydrolysate (SPH)has a stronger serum cholesterol lowering effect than intact soyprotein in rats (Sugano and others 1990). Compared with casein,soy protein hydrolysate significantly decreased the serum choles-

terol level as well as promoted fecal excretion of steroids. Thesedata indicate that soy protein hydrolysate may indeed inhibit cho-lesterol absorption. In the GI system, cholesterol is rendered solu-ble in bile salt-mixed micelles and then absorbed. In an in vitrostudy, it was found that the micellar cholesterol solubility was sig-nificantly lower in the presence of SPH compared with the choles-terol micelles containing soy protein (Nagaoka and others 1999).In vitro, cholesterol absorption in Caco-2 cells showed a choles-terol uptake from micelles containing SPH, which was significant-ly lower than that containing soy protein. The incorporation of[3H]-cholesterol into the serum, liver, and intestine of rats was alsosignificantly lower in SPH groups than in soy protein groups (Na-gaoka and others 1999). These results indicated soybean peptides

have stronger hypocholesterolemic effects than soy protein by in-hibiting cholesterol absorption due to the suppression of micellarsolubility of cholesterol.

To narrow down the active moiety of soy protein, the LDL re-ceptor up-regulation effects of-conglycinin and glycinin werestudied in human hepatoma cells (HepG2). The results showedthat -conglycinin was markedly more effective than glycinin (Lo-vati and others 1992). Follow-up research found that + sub-units from -conglycinin had higher LDL receptor up-regulation

activity than subunit. Incubation of HepG2 cells with purified + subunits sharply increased uptake and degradation of125I-LDL added to the culture medium, whereas the subunit was in-effective (Lovati and others 1998); the subunit was believed tocontribute more than subunits (Manzoni and others 1998).These observations led to the development of an enzymatic modi-fication process for the hydrolisis of soy -conglycinin subunit,for use as a hypocholesterolemic agent (Duranti and Morazzoni2003). In rats, the administration by gavage of 20 mg/kg bodyweight/d of this hydrolysate for 28 d resulted in a 36% decreasein plasma cholesterol; a greater effect than when using 100 mg/kgbody 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 to150 of-conglycinin was found to markedly (P 0.05) increase125I-LDL uptake and degradation in HepG2 cells (Lovati and oth-ers 2000).

To determine active soy protein components in the regulation ofcholesterol homeostasis in HepG2 cells, a soybean protein con-centrate (CrocksoyR 70), was subjected to pepsin-trypsin digestionand fractionation. It was found that, at 0.125 g/L, the MW > 3000Da fraction significantly up-regulated the uptake and degradation ofLDL by receptor pathways while the MW < 1000 fraction and MWbetween 1000 and 3000 fraction had no effect on LDL catabolism(Lovati and others 2000). Although some researchers suggest thatsmall peptides can cross the intestinal wall intact, the in vivo effectof larger molecule peptide fractions, particularly those from soyproteins, still needs to be investigated (Scanff and other 1992; Cha-

bance and others 1998; Haupt and others 2002).On the other hand, hypocholesterolemic effect has been alsofound in glycinin. Leu-Pro-Tyr-Pro-Arg, a fragment peptide derivedfrom soybean glycinin was found to reduce serum cholesterol inmice after oral administration at a dose of 50 mg/kg, without isofla-vones, for 2 d (25.4% in total cholesterol and 30.6% in LDL-cholesterol) (Yoshikawa and others 2000). This peptide has structur-al homology to enterostatin (Val-Pro-Asp-Pro-Arg). Although bothhave hypocholesterolemic activities, enterostatin did not increasedexcretion of bile acids in feces, suggesting that they may act by dif-ferent mechanisms (Takenaka and others 2000, 2004).

Soy peptides may also bind to phospholipids and exert serumcholesterol lowering activity in humans (Hori and others 2001).

Because ethanol washed soy protein, containing no isofla-vones, 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 low-ering effect in 5-wk-old male Sprague-Dawley rats (Fukui and oth-ers 2002). Therefore, it has been postulated that the effect of soyprotein in lowering cholesterol levels may be due to an isofla-vone-protein interaction (Peluso and others 2000; Simons andothers 2000; Hsu and others 2001). Although all these observa-tions in experimental animals have not been found in humanfeeding studies, some researchers have postulated that in human,soy protein may in some way up-regulate LDL receptors de-pressed by hypercholesterolemia or by dietary cholesterol admin-istration (Sirtori and others 1995).


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Antiobesity

Soy has long been used in the management of obesity (Ander-son and Moore 2004) and it is believed that soy protein may con-tribute to lower the human obesity epidemics by decreasing hun-ger, increasing metabolic rate and promoting weight loss (Allisonand others 2003; Fontaine and others 2003; Ohr 2005). The con-sumption of soy protein may also lead to low hepatic deposits oftriglycerides (Ascencio and others 2004). Soy protein isolate wasfound to lower plasma triglycerides, increase adiponectin (Na-

gasawa and others 2002, 2003), accelerate lipid metabolism, anddecrease body fat in obese rats and mice (Aoyama 2000a,2000b). On the other hand, in male Zucker diabetic fatty rats, thelow-isoflavone soy diet decreased plasma lipids and increasedbody weight, but did not change liver weight or carcass adiposity.High-isoflavone soy decreased plasma lipids, liver weight, andbody weight (Banz and others 2004).

After hydrolysis of soybean flakes by an alkaline protease, theobtained hydrolysate was fed to Wistar male rats and found to de-crease blood lipids and body weight. Although the caloric con-tent of the experimental diets was not indicated, this hydrolysatelowered the concentration of rats serum triglycerides by 11% at alow dosage (10 mL/kg/d). A high dosage (20 mL/kg/d), lowered se-rum triglycerides by 30% and decreased body weight of the ani-mals (Zhang and others 1998).

Several anorectic peptides have been already identified to exertantiobesity activity through decreasing food intake, fat and leanbody mass, and body weight (Challis and others 2004). For exam-ple, Leu-Pro-Tyr-Pro-Arg, a peptide from soybean glycininA5A4B3 subunit (Takenaka and others 2000) and Pro-Gly-Prohave been found to have anorectic activities.

Based on the antiobesity activity of soy and soy peptides, vari-ous foods and beverages have been developed. For example, asoy protein meal replacement formula (Scan Diet) has been foundto be effective for weight loss and fat mass reduction in obesesubjects (Allison and others 2003). Other products include anti-obesity formulas containing soy proteins, water-soluble fibers andgelatins, basic amino acids, and/or basic peptides (Fujita 2000). Asugar-free coffee containing soybean protein hydrolysate was also

developed (Miura 2002). This beverage contained oligopeptideswith 3 to 6 amino acid residues prepared by enzymatic hydrolysisof soybean protein. Compared with baseline values, ingestion ofthis kind of sugar-free coffee for 8 wk led to a 4% to 7% bodyweight reduction in human volunteers. Soybean peptides havealso been used as body fat-decreasing agents in foods. It has beenobserved in humans that body fat, serum glycerides, and choles-terol can be decreased by peptides without decreasing body pro-teins (Inaba and others 2002).

The satiety inducing effect of soy protein has been linked to in-dependent activation of both opioid and cholecystokinin (CCK)-Areceptors in rats. It has been found that protein digestion into pep-tides stimulates satiety (PupoVac and Anderson 2002). The soy-bean -conglycinin pepsin hydrolysates were also found to sup-press food intake and gastric emptying by direct action on rat

small intestinal mucosal cells. The digestion of proteins gives riseto peptides that may initiate several satiety signals from the gutand these signals may be dependent on dietary protein sources.

Intraduodenal infusion of-conglycinin hydrolysates inhibitedfood intake of rats in a dose-dependent manner and this suppres-sion can be abolished by intravenous injection of devazepide, aselective peripheral CCK receptor antagonist (Nishi and others2003a). The arginine residue in protein structures was shown tobe responsible for CCK release through direct action on the intes-tinal cells. Regarding the relationship between arginine and bind-ing activity to brush border membrane, synthetic model peptideswith 1 arginine (GGGRGGG and GGGGGGR) showed no activity.The binding activity of synthetic peptides containing 2 arginine

residues, depended on the position of arginine residues, GGR-GRGG, GRGGRGG, and GRGGGRG can bind to the brush bordermembrane while GGGRRGG cannot bind. GRGRGRG, a syntheticpeptide containing 3 arginine residues had stronger binding abili-ty (Nishi and others 2003b). Comparing between several arginine-concentrated fragments of-conglycinin on their abilities to bindto the intestinal cell component, the fragment from 51 to 63 of the subunit was found to have the highest binding affinity affectingalso food intake in rats (Nishi and others 2003b).

All these observations in experimental animals are encourag-ing. However, the effect of antiobesity peptides in humans re-mains to be determined.

Antioxidant

Several amino acids, such as Tyr, Met, His, Lys, and Trp, aregenerally accepted to be antioxidants. Saito and others (2003)constructed series of tripeptide libraries to explore antioxidativeproperties of peptides; one was composed of 108 peptides con-taining either 2 His or Tyr residues and the other 114 peptidesstructurally related to Pro-His-His. The antioxidative activities ofthe tripeptide libraries were examined by several methods, in-cluding the antioxidative activity against the peroxidation of li-noleic acid, the reducing activity, the radical scavenging activity,and the peroxynitrite scavenging activity. Tripeptides containingTrp or Tyr residues at the C-terminus had strong radical scaveng-ing activities, but very weak peroxynitrite scavenging activity. Theresults explained why protein digests have such a variety of anti-oxidative properties. They also found that the antioxidative pep-tides may exert a strong synergistic effects with some other antiox-idants, such as phenolic compounds (Saito and others 2003).

During hydrolysis, the soy protein structure will be altered andmore active amino acid R group will be exposed. Therefore, soy-bean peptides can have higher antioxidant activity than intactprotein (Chen and others 1998). After enzyme digestion of-con-glycinin and glycinin the radical-scavenging activities were in-creased 3 to 5 times. Heating did not change the activity of theproteins, indicating that forming peptides was more critical thanmaintaining protein structure (Matoba 2002).

In a study using male Wistar rats as model, it was found that theintake of either soy protein isolate (SPI) or soy peptide, but not of anamino acid mixture resembling soy protein (SPAA), had the effect ofreducing paraquat (PQ)-induced oxidative stress. In this experi-ment, both SPI and soy peptide prevented the elevation of the se-rum thiobarbituric acid-reactive substances (TBARS) concentrationand 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 itsstructure and therefore affected by the hydrolysis procedures (Yangand others 2000). When comparing the antioxidant capacity of 28structurally related peptides to Leu-Leu-Pro-His-His, isolated fromsoybean protein digests, Pro-His-His was identified as an activecenter. It was believed that His-containing peptides can act as met-

al-ion chelators, active-oxygen quenches, and hydroxy-radicalscavengers and contribute to the antioxidative activity of peptides(Saito and others 2003). Different hydrolysis conditions (enzyme,temperature, sample preparation) resulted in peptide mixtures withdifferent antioxidant properties. Native and heated soy protein iso-late hydrolyzed with different enzymes, such as pepsin, papain,chymotrypsin, alcalase, Protamex, and flavourzyme, resulted in dif-ferent degree of hydroylsis ranging from 1.7% to 20.6%. The anti-oxidant activity ranged from 28% to 65%, determined by the de-crease in TBARS concentration after incubating with a liposome-ox-idizing system (50 mMFeCl3/0.1 mMascorbate, pH 7.0). Samplesof chymotrypsin- and flavourzyme-hydrolyzed (0.5 h) preheatedSPI had the greatest inhibitory effect (Pena-Ramos and Xiong 2002).


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Liu and others (2005) demonstrated that soymilk-kefir possesssignificant antimutagenic and antioxidant activity and suggest thatfermented soymilk may be considered among the more promisingfood components in terms of preventing mutagenic and oxidativedamage. More research is needed to demonstrate if peptides pro-duced during fermentation may play an important role in this bio-logical activity. It has also been observed that radical scavengingability of soy peptides plays an important role in the suppressionof 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 relationshipis not scientifically strong yet. Birt and others (2001) concludedthat flavones and isoflavones in soy may contribute to cancer pre-vention; however, further investigations are required to clarify thenature of the interaction between these constituents and peptides/proteins.

Anticancer activity of soy proteins has been suggested (St. Clairand others 1990; Messina and Barnes 1991). For example, soy-bean Kunitz trypsin inhibitor was reported to suppress ovarian can-cer cell invasion by blocking urokinase up-regulation (Kobayashiand others 2004). Another study showed that SPI diet altered co-lonic global gene expression profile and enhanced somatostatin, aknown antiproliferative agent for colon cancer cells, and thereforewould inhibit tumorigenesis (Xiao and others 2005). Lifetime con-sumption of soy proteins reduced the incidence of azoxymethane-induced colon tumors in rats (Hakkak and others 2001). It is possi-ble that part of these anticancer activities may be attributed to bio-active peptides derived from soy proteins. In support of this hy-pothesis, it has been found, both in vitro and in experimental ani-mals, that hydrophobic peptides from soy proteins may have anti-cancer activity. For example, peptides obtained by thermolase hy-drolysis of defatted soy protein, further purified with ethanol andfractionated by gel filtration chromatography, showed an IC50 valueof 0.16 mg/mL in vitro cytotoxicity on mouse monocyte macroph-

age cell line. At 1 mg/mL, this fraction significantly affected cell cy-cle progression by arresting the cells in G2/M phases. Further puri-fication with C18 HPLC resulted in 1157 Da nonapeptide (X-Met-Leu-Pro-Ser-Tyr-Ser-Pro-Tyr) (Kim and others 2000).

Kim and others (1999a) have obtained a glycopeptide from eth-anol fractions of bromelain-defatted soybeans hydrolysate com-posed mainly of Asp, Glu, Pro, Gly and Leu with strong cytotoxicactivity against P388D1 mouse lymphoma cells.

In addition, lunasin is a well-studied chemopreventive peptidefound in 2S soybean albumin. It contains 43-amino acid, with acarboxyl end of 9 aspartic acids residues and a cell adhesion mo-tif (RGD) (Galvez and de Lumen 1999). Lunasin has a motif thatbinds specifically to non-acetylated H3 and H4 histones and pre-vents their acetylation. This mechanism was believed to be re-sponsible for the anti-carcinogenic property of this chromatin-

binding peptide isolated from soybean seeds (de Lumen 2005).Lunasin was found to suppress chemically induced carcinogenictransformation in mammalian cells in mice. The chemopreventiveproperties of lunasin have also been confirmed by in vitro studies(Lam and others 2003). The lunasin gene was cloned from soy-bean and the chemically synthesized form of the lunasin peptidehas been used experimentally (Jeong and others 2002).

Immunomodulatory

Soybean peptides with immunomodulatory activities have beenidentified from soybean protein hydrolysates. For example, immu-nostimulating peptide preventing the alopecia induced by cancerchemotherapy has been isolated from an enzymatic digest of soy-

bean protein (Yoshikawa and others 2000; Tsuruki and others2003, 2005; Tsuruki and Yoshikawa 2004).

Regarding the phagocytosis-stimulating activity, an active peptidesequence (MITLAIPVNKPGR) has been isolated from trypsin digestsof soybean proteins. It was found to be derived from the subunitof-conglycinin. Met at the N terminus of the peptide was found tobe essential for its activity while the 3rd residue from the N terminusmay affect the activity (Thr < Phe < Trp). For the subunit, the 1stresidue is Ile, and the 3rd residue is Lys, and its activity was not ob-

served. Through mutation methods, the 1st residue of the corre-sponding sequence of subunit was replaced to Met and the 3rdresidue was replaced to Thr, Phe, or Trp. Without changing the pro-tein confirmation, phagocytosis-stimulating activity was observed.The phagocytosis activities of the 3 mutants followed the expectedorder: wild type < I122M/K124T < I122M/K124F < I122M/K124W(Maruyama and others 2003).

Technological Approach to Generate Bioactive Peptides

Enzymatic and chemical hydrolysis

Acidic hydrolysis and enzymatic hydrolysis are 2 main meth-ods to generate soybean peptides. The acid hydrolysis method isrelatively simple and less expensive, but it is more difficult to con-trol and amino acid damage may occur. Enzymatic methods areeasier to control, use mild conditions, and do not cause aminoacid damage. Therefore, enzymatic hydrolysis is a commonlyused method to produce food-grade protein hydrolysate and torelease bioactive peptides from their protein precursors.

The type of enzyme is also very important for the preparation ofbioactive peptides. Proteinases (endopeptidases) such as trypsin,subtilisin, chymotrypsin, thermolysin, pepsin, proteinase K, pa-pain, and plasmin are used commonly for the proteolysis of foodproteins (Yamamoto and others 2003).

Animal, plant, and microorganism are main sources of en-zymes used for the production of bioactive peptides. Non-animalorigin enzymes, papain or pronase, have been used to hydrolyzesoy protein. Hydrolysates have shown growth-promoting and

production-enhancing activities when tested on a mouse hybri-doma culture in protein-free medium (Franek and others 2000).Papain has also been used to prepare healthy food containing nu-tritious protein hydrolysates (Cai and Cai 2001).

Proteinases from microorganisms such as Mucorsp, Aspergil-lus oryzae, Bacillus subtilis1389, Aterricola 3942 are also usedto hydrolyze proteins and generate peptides. Aspergillus oryzaepeptidase was used to hydrolyze soybean protein slurry to short-chain peptide material (peptide chains mostly with 7 peptides)(Korhonen and Pihlanto 2003). A Mucor piriformisenzyme wasalso used for the production of soybean peptides from isolatedsoybean protein (Li and others 2001). After 5 h of hydrolysis with750 U enzyme at 45 C, pH 6.0, a yellowish product without anybitter or astringent odor was produced. The molecular weight ofthe enzymatic decomposition product was 1000 Da.

To obtain desirable results, acid hydrolysis can be combinedwith enzymatic hydrolysis. To produce soybean protein hydroly-sates in high concentration, defatted soybean flour was treated us-ing a low hydrochloric acid solution before degradation by a pro-tein catalyst, such as protease, to inhibit gelation of soybean pro-tein during heat sterilization (Lee and Lee 2000). In the presenceof carboxylic acids, peptides having average amino acid residuesof 10 to 100 were produced by enzyme hydrolysis (Hirano andKoide 2000).

Heat treatment of proteins can also affect the efficiency of en-zyme hydrolysis. Fischer and others (2002) found that soybeanmeals heat-treated at high humidity had higher levels of aggregat-ed peptides.


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Enzymes are often combined to produce bioactive peptides. Dif-ferent enzyme combinations of proteases including alcalase, chy-motrypsin, pancreatin, and pepsin as well as enzymes from bacteri-al and fungal sources have been used (Meisel and FitzGerald2003). Trypsin/AS.1398 protease (1:1) was used for soybean pro-tein 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 com-bination 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 synergismin hydrolyzing proline-containing peptides. Adding X-PDAP to APcan highly improve the hydrolysis of peptide Ala-Pro-Gly-Asp-Arg-Ile-Tyr-Val-His-Pro-Phe, whereas adding X-PDAP to the enzymemixture of subtilisin (E.C. 3.4.21.62) and AP can markedly increasethe degree of hydrolysis of soybean (from 54% to 72%).

Fermentation

Fermentation is considered to be an efficient way to producebioactive peptides. Bioactive peptides can be released by the mi-crobial activity of fermented food or through enzymes derivedfrom microorganism (Korhonen and Pihlanto 2003). Fermentedmilk and cheese have been extensively studied to investigate theirpotential to form bioactive peptides. Interest in fermented soybeanproducts, such as natto, tempeh, soy sauce, soy paste, has grownin recent years. Many bioactive peptides have been identified. Forexample, ACE-inhibitory peptides containing Ala, Phe, and Hishave been isolated from soybeans fermented by Bacillus nattoand chunggugjang fermented by Bacillus subtilis (Korhonen andPihlanto 2003). ACE-inhibitory peptides were also found in soy-bean paste (His-His-Leu) (Shin and others 2001), soy sauce (Oka-moto and others 1995), natto, and tempeh (Gibbs and others2004).

Three potent ACE-inhibitors, 3 thrombin inhibitors, 5 peptideswith surface-active properties, and 1 peptide with antibacterial ac-tivity were also found in enzymatic hydrolysates of soy fermentedfoods. They were all derived from glycinin, while -conglycinin

was found more stable to proteolytic attack even by multi-enzymepreparations (Gibbs and others 2004).Fermentation is not enough to fully hydrolyze soybean pro-

teins. Glycoproteins, phosphoproteins, and other post-transla-tionally modified species or domains that contain a higher num-ber of disulfide bridges are more difficult to hydrolyze. The pro-teases in Bacillusand Rhizopusstrains can only cleave soybeanproteins into large peptides. Further enzymatic degradations, suchas pronase, trypsin, Glu C protease, plasma proteases, and kidneymembrane proteases hydrolysis, are needed to produce peptideswith high activities (Gibbs and others 2004). Fermentation mayalso synthesize new peptide sequences. In enzymatic hydroly-sates of soy fermented foods, the precursor of a peptide sequenceELLVYLL with good surface active properties could not be identi-fied and it was believed to be synthesized during fermentation

(Gibbs and others 2004).

Synthesis

Peptides synthesis is a useful method to prepare bioactive pep-tides in large scale and also to study their mechanism of action. Atpresent, 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 approachat laboratory scale, existing 2 variants, liquid-phase and solidphase. The solid phase approach is the most powerful method forsynthesis of peptides composed of about 10 to over 100 residueson a small scale (most practical for sequences of intermediatelengths). However, the high cost of the instrumentation and re-

agents has largely restricted its use. On the other hand, liquid-phase synthesis is the preferred method for large-scale synthesisof relatively short peptides and for carrying out the condensationof peptide fragments (Gill 1996).

Recombinant DNA technology is the preferred choice for rela-tively large peptides with up to several hundred amino acids (Gill1996). Due to the low expression efficiencies obtained and diffi-culties encountered in product extraction and recovery, attemptsto extend this approach to the preparation of short peptides have

not yet been truly successful (Korhonen and Pihlanto 2003). Us-ing genetic engineering techniques, Yoshikawa and others (2002)introduced RPLKPW, a highly potent antihypertensive peptide,into 3 homologous sites in soybean -conglycinin subunit bysite-directed mutagenesis.

In practice, enzymatic synthesis is currently limited to relativelyshort sequences.

Isolation, Purification, Characterization,and Quantification of Bioactive Peptides

Isolation and purification

The isolation and purification techniques are very important inbioactive peptide research. For example, the importance of glu-

tathione (Glu-Cys-Gly) has been noticed as early as 1888, but thebioactivities of peptides became apparent only after 1950s withthe development of a new purification technology (Sewald and

Jakubke 2002). Most protein isolation and purification techniquescan be applied for bioactive peptide separation. However, be-cause of relative small size and molecular weight, special consid-eration should be given to bioactive peptides purification. For ex-ample, the resolving power of size-exclusion chromatography(SEC) is somewhat limited (Sewald and Jakubke 2002).

Salting out and solvent extraction are often used before furtherpurification steps. For example, in our laboratory a mixture of wa-ter, acetonitrile and trifluoroacetic acid (TFA) is used to extractpeptides from enzymatic hydrolysis of fermented soybean prod-ucts. After centrifugation, the supernatant is filtered and lyo-

philized for liquid chromatography analysis.Chromatography is the most powerful technique to isolate andpurify bioactive peptides. Based on different properties of pep-tides, different chromatography techniques have been developed.Among them, HPLC is the most commonly used separation meth-od. Commercially available reversed-phase columns allow forrapid separation and detection of the peptides from a mixture,whereas normal phase liquid chromatography is used preferen-tially for the separation of hydrophilic peptides. Ion-exchangechromatography (IEC), capillary electrophoresis (CE), and capillaryisoelectric focusing (CIEF) separate peptides based on their chargeproperties. Size-exclusion chromatography (SEC), which is alsonamed gel filtration chromatography (GFC) in aqueous separationsystems and gel-permeation chromatography in nonaqueous sep-aration systems, is a separation method solely based on molecu-

lar size. Gel-permeation chromatography with a Superdex PeptideHR 10/30 column was used to obtain the di- and tri-peptide frac-tion from buckwheat digestion using 30% acetonitrile containing0.1% TFA for elution (Li and others 2002). Ultrafiltration (UF),crystallization, counter-current distribution, partition chromatog-raphy, and low-pressure hydrophobic interaction chromatogra-phy (HIC) have also been used for protein fractionation and purifi-cation (Sewald and Jakubke 2002).

Characterization

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


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inhibitory activity (IC50 0.42 mg) was isolated from casein and re-solved into a single band (6 kDa) on 15% SDS-PAGE gel (Devi andothers 2002). SDS-PAGE is helpful for relatively large peptides. Forsmall peptides, the resolution of SDS gel is usually low. Size-exclu-sion HPLC may provide more accurate estimation of the peptide size.

With appropriate columns and conditions, HPLC may provideuseful information for peptide characterization. For example, re-verse-phase HPLC will indicate the hydrophilicity of peptides. Be-cause several peptides may have similar retention times, it is not

fully reliable to use the retention time to identify peptides even ifthe target peptide sequence is known and standards has beenrun. In this case, UV spectra (usually from 200 to 300 nm) mayprovide extra information to facilitate peptide identification. Forexample, UV-spectral comparison was used to identify an expect-ed peptide from a complicated peptide mixture. This method wasdemonstrated to aid in the identification of haemorphins (Zhaoand others 1995) and (1-23) peptide from hemoglobin hydroly-sate (Choisnardand others 2002).

HPLC and size-based analysis cannot give direct amino acidsequence information. For unknown peptides, amino acid analyz-er and protein sequencer are commonly used to determine aminoacid composition and sequence. In a study of ACE-inhibitorypeptides from wheat germ, the amino acid composition was ana-lyzed with a Shimadzu LC-6A amino acid analyzer after hydroly-sis of 6 NHCl for 24 h at 110 C. The amino acid sequence wasdetermined by automated Edman degradation using a ShimadzuPPSQ-21 protein sequencer (Matsui and others 1999). Automaticprotein sequencer base on Erdman degradation method is stillwidely used in peptide sequencing (Li and others 2002; Chenand others 2003; Kuba and others 2003; Motoi and Kodama2003; Megias and others 2004). Mass spectrometry methods,such as triple stage Model API-III (Haileselassie 1999; Gibbs andothers 2004), ESI-MS/MS (Stapels and Barofsky 2004), and MALDI-TOF-MS (Kim and others 2000. Rejtar and others 2004) togetherwith database search are becoming more and more popular.

Quantification

Enzyme-linked immunosorbent assay for the quantification of

bioactive peptides. Based on specific combination of antigensand antibodies, enzyme-linked immunosorbent assay (ELISA) hasbeen used for a specific detection of very small quantities of pep-tides. High levels of specificity are achieved with such immunoas-says due to the specific and high affinity reversible binding of anti-gens to antibodies (Cohen and others 2002). Substances


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such as exopeptidases (Lovsin-Kukmanand and others 1996;Maehashi and Arai 2002; Raksakulthai and Haard 2003), ami-nopeptidase from the edible basidiomycete Grifola frondosa(Nishiwaki and others 2002), have also been developed. Thesestudies indicate that as the incubation time elapses, the amount offree amino acids released increases and the bitterness of the reac-tion mixtures decreases.

Bioavailability of peptidesDepending on the exerted function, bioactive peptides may notneed to be absorbed by the intestine or pass into systemic circula-tion. In the case of anorectic peptides, their action is at the intesti-nal level where they stimulate opioid and hormonal receptors,which induce satiety (Pupovac and Anderson 2002; Nishi andothers 2003a, 2003b). However, other functions such as hy-potensive or anticancer activities would require passage of thebioactive peptides through the intestinal barrier and their trans-port to target organs. Studies of the kinetics of digestion of milkpeptides in experimental animals have shown that active peptidescan still be present in the intestine even after the action of pancre-atic enzymes (Scanff and others 1992). These observations sug-gest the availability of these substances for intestinal absorption(Shimizu 2004). Chabance and others (1998) demonstrated thatpeptides are released and passed to the blood with human diges-tion of milk or yogurt. In this study, 2 long peptides, the -casein-glycopeptide and the N-terminal peptide from -S1-casein weredetected in plasma. It is also known that due to a more efficientand rapid absorption of peptides in comparison to free amino ac-ids, peptide mixtures and protein hydrolysates are recommendedto deliver nitrogen to patients suffering from malnourishment orproblems of protein digestion (Gill and others 1996). Althoughmore mechanistic studies are needed, these results support theconcept that food-born peptides can be absorbed and have phys-iological activities in various human organs.

Safety Concerns

Potential toxicity

Peptides are normally generated during protein digestion in theGI tract. Because soybean and fermented soybean have beensafely used as food for thousands of years without apparent harm-ful effects, the risk of toxicity caused by peptides formed duringthis process is practically nil. Although peptides can be absorbedinto the blood, there have been no reports about toxic soybeanpeptides to date. However, considering the complexity of bioac-tive peptides preparation procedures, it becomes necessary tokeep this safety concern in mind.

AllergenicitySoybean proteins can be allergenic. However, most allergenic

proteins have relatively high molecular weight. Goodman andothers (2005) present a comprehensive review on allergens in ge-netically modified soybean with conventional soybean. Bioactivepeptides contain 2 to 40 amino acids and their MW ranges from200 to 5000 Da. Thus, the possibility of allergenicity is very low.No soybean allergic peptides have been reported in this molecu-lar weight range.

ConclusionsSoybean proteins can be a source of bioactive peptides with di-

verse and unique health benefits that can be used in the preven-tion of age-related chronic disorders such as cardiovascular dis-

ease, cancer, obesity, and decreased immune function. Bioactivepeptides are released from proteins by either food processing orby GI digestion. Indirect evidence also suggests that these pep-tides can be absorbed by the GI system thus exerting their actionon specific target organs. Other peptides do not need to be ab-sorbed and act at the intestinal level. However, understandingwhether digestion of food proteins in vivo releases the same pep-tide fragments as the ones in vitro experiments is an importantquestion. 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 havebeen used in many studies. Functional peptides have not alwaysbeen identified; there is still a great potential for discovery. A bioac-tive peptide database has been developed to predict the biologicalactivity of protein fragments using sequence alignments betweenproteins and biologically active peptides (Dziuba and others 1999).This database may also be helpful to reveal the amino acid se-quence-activity relationship. Of course, besides primary structure,the secondary or tertiary structure of bioactive peptides may also beimportant for their activity. Figure 3 presents an example of the po-tential biological peptides that can be produced from -conglyci-nin -subunit, as determined by our group, after searching the Bio-pep database (Dziuba and others 1999). It can be observed thatthis subunit can be the source of peptides with various biologicalactivities 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 im-portant role in determining the bioactivities of protein hydrolysates.For example, antioxidant peptide VIPAGYP may be released from -conglycinin hydrolysate. However, if the protease can effectivelycut the peptide bond between V and I, antihypertensive peptide IP-AGYP will be released. Similarly, further digestion may release thedipeptidyl-aminopeptidase IV inhibitory peptide, PA.

Using a similar approach, we have identified many potentialbioactive peptides in the major soy proteins which includes, sub-units 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 dem-onstrates amino acid sequences with antihypertensive, dipeptidylpeptidase IV (DPPIV) inhibition, antithrombotic, immunostimula-tory, antiamnestic, opioid, and antioxidant activities among oth-ers. Antihypertensive activity and DPPIV inhibition were the mostcommon. As it can be seen in Figure 4, soy protein componentsare good potential sources of bioactive peptides. For example gly-cinin G1 precursor (soybean source nr 1) intercepts with antihy-pertensive activity (activity A) showing the highest frequency ofcorresponding amino acid sequence (20).

Considering the diversity and complexity of protein sequences,there are still many possibilities to generate new bioactive pep-tides with higher activity or unrevealed activities. By this means,an efficient hydrolysis-separation-screening protocol will be veryimportant.

Structure-function relationship is always important for a betterunderstanding and utilization of bioactive peptides. For example,Cheung and others (1980) reported that the hydrophobicity of thecarboxyl terminal amino acid was the most important factor affect-ing the overall binding of the peptides to the active site of ACE.The sequence is protected from proteolysis because of its high hy-drophobicity and the presence of proline residues (Meisel andFritzGerald 2003).

Research and medical trials have demonstrated the biologicalactivities of bioactive peptides, but the corresponding molecularmechanism of action is still not completely clear. A better under-standing of how these bioactive peptides work and how they areregulated will be helpful. Understanding whether natural in vivo


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digestion of food proteins releases the same peptide fragments asthe ones in vitro experiments is important (Pellegrini 2003). Howwe can manage to generate desired bioactive peptides in the GItract while preventing digestion damaging the desired peptides

constitutes an important question. Chemical modification of pep-tides to make them more resistant to degradation is 1 of the ap-proaches. N-methylation of the peptide bond, C-terminal esterifi-cation, the use of infusion pumps for peptide delivery, and encap-

Figure 3Potential biologicalpeptides in -conglycinin, -sub-

unit. The amino acid sequenc-es were obtained from Swiss-Prot protein knowledgebase (ac-cession number P13916) (Ap-

wei ler and others 2004). Thepotential bioactive peptides andtheir possible function were de-termined by searching the Bio-pep database (Dziuba and oth-ers 2003).

Amino acid nomenclature: C =Cys, Cystein; H = His, Histidine;I = Ile, Isoleucine; M = Met, Me-thionine: 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, As-partic acid; N = Asn, Aspar-agine; B = Asx, Either of D or N;E = Glu, Glutamic acid; Q = Gln,Glutamine; Z = Glx, either of Eor Q; K = Lys, Lysine; X = Unde-termined amino acid.

Figure 4Predicted profiles of peptides in soy proteinwith potential biological activities. The amino acid se-quences were obtained from Swiss-Prot protein knowl-edgebase (accession number P13916) (Apweiler andothers 2004). The potential bioactive peptides and theirpossible function were determined by searching theBiopep database (Dziuba and others 2003).Soybean sources: 1 = Glycinin G1precursor; 2 = Glyci-nin G2 precursor; 3 = Glycinin G3 precursor; 4 = Glyci-

nin G4 precursor; 5 = Glycinin G5 precursor; 6 = -Conglycinin chain; 7 = -Conglycinin chain; 8 = Conglycinin chain; 9 =Trypsin inhibitor A/C precur-sor; 10 = Trypsin inhibitor B; 11 = Trypsin inhibitor KTI1precursor; 12 = Trypsin inhibitor KTI2 precursor; 13 =Bowman-Birk inhibitor precursor; 14 = Bowman-Birkinhibitor C-II precursor; 15 = Bowman-Birk inhibitor D-II precursor.Predicted activities: A = Antihypertensive; B = Dipepti-dyl peptidase IV inhibitor; C = Antithrombotic; D = Opi-oid; E = Immunostimulating; F = Regulating; G = Ligand;H = Antiamnestic; I = Activating ubiquitin-mediated pro-teolysis; J = Antioxidative; K = Opioid agonist.


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sulation of peptides in various carriers such as liposomes are alsouseful methods (Lee and Kim 2000). Understanding this questionis also helpful for delivery of bioactive peptides used as drugs byoral administration. Improvements in peptides purification, quali-fication, and synthesis techniques are always important. Large-scale bioactive peptides production is still a challenge. Enzymesfrom microorganisms produced during fermentation are veryhelpful to release bioactive peptides. New peptide sequences mayalso be generated during fermentation. A better understanding of

this phenomenon is important for discovering of new active pep-tides as well as for determining their safety.

The mechanism of the physiological activities of the small pep-tides from soybean needs to be further investigated. Studies onthe impact of soy processing on the generation of bioactive pep-tides are lacking. It is also important to discover new peptideswith health benefits in soy-hydrolysates and fermented foods. Theidentification of these compounds will contribute toward the de-velopment of new functional foods and the prevention of disease.

In summary, there are opportunities in this field for the industri-al exploitation of soy value-added bioactive peptides that can beused to enhance health and prevent disease.

AcknowledgmentsThe authors express their gratefulness to the USDA-Future

Foods Initiative and Hatch funds for their support.

Abbreviationsaa = amino acid; ACE = angiotensin-converting enzyme; APP =

acid-precipitated soy protein; BBI = Bowman-Birk inhibitor; BP =bioactive peptides; CCK = cholecystokinin; CE = capillary electro-phoresis; CHD = coronary heart disease; CIEF = capillary isoelec-tric focusing; CNS = central nervous system; CPP = casein-derivedphosphorylated peptides; DH = degree of hydrolysis; ED = effec-tive dose; ELISA = enzyme-linked immunosorbent assay; FPH =fish protein hydrolysate; GFC = gel filtration chromatography; GI =gastrointestinal tract; GM = genetic modification; HIC = hydro-

phobic interaction chromatography; HPLC = high-pressure liquidchromatography; IC50 = 50% inhibitory concentration; IEC = ionexchange chromatography; IG = immunoglobulin; kDa = thou-sand daltons; KTI = Kunitz trypsin inhibitor; LDL = low-density li-poproteins; MAC = metal affinity chromatography; MW = molec-ular weight; PQ = paraquat; PEP = soy peptide; PVDF = polyvi-nylidene fluoride; S = Svedberg unit; SDS-PAGE = sodium dodecylsulfate-polyacrylamide gel electrophoresis; SEC = size exclusioncromatography; SHR = spontaneously hypertensive rats; SPI = soyprotein isolate; SPA = soy protein allergy; SPHF = soy protein hy-drolysate formulas; TBARS = thiobarbituric acid-reactive substanc-es; UF = ultrafiltration; WG = wheat germ.

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