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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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(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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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
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).
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
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
Growth-promoting and production enhancing activities when tested on a mouse hybridoma culture in protein-free medium Anticancer Hypotensive
Kim and others 2000 Wu and Ding 2001 Kodera and Nio 2002
Soy protein
Protease D3
Hypotensive
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
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.
Gluten
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
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
Opioid
N-terminal YGGF
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
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).
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
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).
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).
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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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
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
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).
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
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
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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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.
References
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