936 WANG ET AL.: JOURNAl. OF AOAC INTERNATIONAL VOL.

91, No. 4, 2008

SPECIAL GUEST EDITOR SECTION

Analysis of Soybean Protein-Derived Peptides and the Effect of Cultivar, Environmental Conditions, and Processing on Lunasin Concentration in Soybean and Soy Products
WENYI WANG, VERMONT P. DIA, MIGUEL VASCONF:Z, and ELVIRA GONZALEZ 1W MEJIA1

University of Illinois, Department of Food Science and Human Nutrition, 228 ERML, 1201 W. Gregory Dr, Urbana, IL 61801
RANDALL L NELSON

U.S. Department of Agriculture, Agricultural Research Service, Soybean/Maize Gerrnplasm, Pathology, and Genetics Research Unit, Department of Crop Sciences, 1101 W. Peabody Dr. University of Illinois, Urbana, IL 61801

Soybean, an important source of food proteins, has received increasing interest from the public because of its reported health benefits. These health benefits are attributed to its components, including isoflavones, saponins, proteins, and peptides. Lunasin, Bowman-Birk inhibitor, lectin, and 3-conglycinin are some of the biologically active peptides and proteins found in soybean. This article provides a comprehensive review on the recently used techniques in the analysis and characterization of food bioactive peptides, with emphasis on soybean peptides. The methods used to isolate and purify lunasin from defatted soybean flour were ion-exchange chromatography, ultrafiltration, and gel filtration chromatography. The identity of lunasin was established by sodium dodecyl sulfate-polyacrylam ide gel electrophoresis, Western blot, matrix-assisted laser desorption ionizationtime of flight, and liquid chromatography. The results on the effect of soybean cultivar and environmental factors on lunasin concentration are also reported. The highest lunasin concentration, 11.7 0.3 mg/g flour, was found in Loda soybean cultivar grown at 23C; the lowest concentration, 5.4 0.4 mg/g flour, was found in Imari soybean cultivar grown at 28C. Lunasin concentration was affected by cultivartemperature, cultivarsoil moisture, and cultivartemperaturesoil moisture interactions. The variation on lunasin concentration suggests that its content can be improved by breeding, and by optimization of growing conditions. In summary, bioactive peptides can be accurately identified and
Guest edited as a special report on Accurate Methodology for Amino Acids and Bioactive Peptides in Functional Foods and Dietary Supplements for Assessing Protein Adequacy and health Effects" by (i Sarwar Gilani and Paul J. Moughan. Mention of a trademark, proprietary product, or vendor does not constitute a guarantee or warranty of the product by the USDA or the University of Illinois and does not imply its approval to the exclusion of other products or vendors that may also be suitable. Author to whom correspondence should be addressed; e-mail: edemejiaiuiuc.edu

quantified by using different techniques and conditions. In addition, lunasin concentration in soybean depends mainly on cultivar and to some extent on environmental factors, particularly temperature. Lunasin concentration in soy products was also affected by processing conditions.

ood proteins have long been recognized as a source of essential amino acids required for the maintenance of F life. They also serve certain functions as ingredients in foods and food preparations. For the past 2 to 3 decades, attention has been given to other aspects of food proteins, such as their biologically active peptides derived either from enzymatically digested proteins or by protein fermentation. In vitro and in vivo studies have shown that these bioactive peptides can perform certain beneficial biological functions in the body, such as antihypertensive and antioxidant activities, cancer prevention, hypocholesterolemic, antiobesity, and immunomodulatory actions (I). It is therefore important to have precise and accurate methods for quantification and characterization of these bioactive peptides present in foods. The most studied food sources of bioactive peptides are milk and soybean. Lunasin is a novel bioactive peptide found in soybean. It is composed of 43 amino acids with 9 aspartic acid residues on its carboxyl end. Its bioactive properties are attributed to its capability to arrest cell division in cancer cells and to inhibit core histone acetylation in mammalian cells (2). This paper reviews the methods used in the analysis of bioactive peptides in soybean and the effect of processing. cultivar, and environmental factors on lunasin concentration in soybean and soy products.
Techniques Used in the Analysis of Soybean Peptides

Protein is the most abundant component in soybean. On average, soybean contains 40% protein conformed by a complex mixture of different protein types (3). The major components of so y proteins are seed storage proteins known as

WANG FT AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 91, No. 4, 2008 937

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938 WANG ETAL.: JOIJRNALOF AOAC INTERNATIONAL VOL. 91, No. 4,2008

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-conglycinin and glycinin, which account for 50-70% of total seed proteins (4, 5). 3-Conglycinin is a trimer with a molecular weight (MW) of! 50-200 kilodaltons (kDa). It is composed of 3 subunits: a, a', and 3(6). The a and a' subunits consist of core regions with high degree of homology (86.8%) and extension regions (a, 125 residues; a', 141 residues) exhibiting lower homologies (57.3%), whereas the f3 subunit consists only of a core region that has homology with the a and a' core regions (75.5 and 71.4%, respectively; 7). Glycinin is a hexamer with MW of 320-375 kDa and with 5 major subunits (G I, G2, G3, G4, and G5). Each subunit consists of an acidic chain (about 40 kDa) and a basic chain (about 20 kDa), joined by disulfide bonds. G 1, G2, and G3 can be grouped as they share 90% sequence homologies. Similarily, G4 and G5 share 90% sequence homologies. However, sequence homologies between these 2 groups (G I, G2, G3, and G4, G5) are only 50%. In addition, there are many enzymes (such as lipoxygenase, chalcone synthase, catalase, urease) in soybean, but only a relatively small number of them exceed 1% of total seed protein. Upon ingestion, soy proteins are digested to peptides by gastrointestinal enzymes and have been found to be bioactive (I). Soybean has been extensively studied for the presence of biologically active peptides. Table I summarizes some of the analytical techniques used recently in the analysis of bioactive peptides from soybean and soy products. Table 2 provides examples of the biological activities of some of the peptides recently isolated from soybean and soy products. Combined with mass spectrometry (MS) techniques, gel-based separations provide valuable information on protein/peptide composition. Natarajan et al. (8) compared the presence of Kunitz tlypsin inhibitor (KTI) from wild and cultivated soybean using 2-dimensional polyacrylamide gel electrophoresis (2D/PAGE) and matrix-assisted laser desorption ionization/time of flight/mass spectrometry (MALDI/TOF/MS). The results showed that the number and intensity of the protein spots between wild and cultivated genotypes varied even though overall distribution patterns of KTI protein spots appeared similar. Due to the complexity of peptides present in protein digests, or fermented foods, various chromatographic techniques are often coupled together to achieve desired separations. Lo and Li-Chan (9) used anion-exchange chromatography, ultrafiltration, reversed-phase high-performance liquid chromatography (RP-HPLC), and immobilized metal affinity chromatography (IMAC) to study angiotensin converting enzyme (ACE) inhibitors from in vitro pepsin-pancreatin digest of soy proteins. They showed that many different peptides with ACE inhibitory activity were produced by in vitro enzyme treatment and speculated that physiological gastrointestinal digestion could also yield ACE inhibitory peptides from soy protein. Zhang et al. (10) used gel filtration chromatography (GFC) and HPLC to purify and separate peptides with ACE inhibitory function from douchi, a Chinese fermented soybean product, and showed that a peptide composed of phenylalanine/isoleucine/glycine in a 1:2:5 ratio exhibited the highest ACE inhibitory activity. Multiple HPLC, MS, and amino acid analyses were used by Gibbs et al. (11) to

'I

WANG ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 91, No. 4,2008 939

B
10 1 Ser-Lys-Try-GIn-His-GIn-GIn-Asp-Ser-Cys20 Arg-Lys-GIn-Leu-GIn-GIy-VaI-Asn-Leu-Thr30 Pro-Cys-GIu-Lys-His-I le-Met-GIu-Lys-I le39 GIn-GIy-Arg-GIy-Asp-Asp- Asp- Asp- Asp43 Asp- Asp- Asp- AspFigure 1. Lunasin predicted structure. (A) Helix with structural homology to a conserved region of chromatin-binding proteins; (B) 43 amino acid peptide that contains an arginine-glycine-aspartate motif (ref. 2).

analyze ACE inhibitory peptides from soy hydrolysate and fermented soy foods. The results showed that endoproteases with lower specificity produced more oligopeptides with higher biological activities. GFC and RP-HPLC were also used by Kuba et al. (12) to isolate 2 ACE inhibitors from the tofuyo extract. RP-HPLC and ion-pair chromatography coupled with MALDI/TOF were used by Mal likarjun Gouda et al. (13) to study ACE inhibitory peptides in different enzymatic hydrolysates of glycinin. A potent ACE inhibitory peptide, Val-Leu-Ile-Val-Pro, was identified from the protease P hydrolysate of glycinin. GFC and RP-HPLC with electrospray ionization with tandem MS (ESJ/MS/MS) were used by Kodera and Nio (14) to study soybean protein hydrolysates produced by protease D3. Kuba et al. (15) hydrolyzed glycinin and 3-conglycinin from soybean using an acid proteinase from Monascus pupureus to produce ACE inhibitory peptides. They used DIAION HP2ISS resin, ultrafiltration, and RP-HPLC to purify active fractions from glycinin and 3-conglycinin. They were able to isolate and analyze 4 ACE inhibitors, 2 from each soybean seed storage protein. Due to the relatively small size of bioactive peptides, protein desalting procedures, such as dialysis, may not be applicable for peptide concentration. Zhong et al. (16) studied

the preparation, yield, and in vitro hypocholesterolemic activities of low MW soy protein hydrolysates. They found that the optimal parameters for desalting soy protein alcalase hydrolysates (SPAH) were the use of macroporous adsorption resin (DA210-C) at pH 4.5, with SPAH dispersion to resin ratio of 75:100, and a loading rate of I bed volume/h with 89.7% adsorption rate of peptide. This nonpolar resin adsorbed hydrophobic peptides and separated them from the salt in solution; because of its large capacity, it is very suitable in desalting peptides (16). Zhong et al. (17) also analyzed a novel hypocholesterolemic peptide from soy protein hydrolysate using fractionation by gradient ethanol elution from DA20 1-C resin. They further characterized the peptide by GFC using Sephadex G-15 and RP-HPLC. The hypocholesterolemic peptide structure was identified by HPLC/MS. Gianazza et al. (18) compared soy protein diets used for hypercholesterolemia studies in Europe and the United States by using 2D electrophoresis, and the identities were established by MALDI/MS. They showed that there were differences in the composition of the soy products used in these clinical studies; thus a comparison was difficult to establish. Lee et al. (19) developed a calcium-binding mediator using peptides derived from isolated soybean protein. The MW of the peptides was determined by GFC, and the results showed the possibility that soybean phosphopeptides can be potent calcium carriers; thus, they can be used to prevent poor absorption of dietary calcium in animals. Rho et al. (20) used size-exclusion chromatography-HPLC to confirm and quantify amino acid composition of peptides from black soybean. These peptides were analyzed by GFC to determine the MW distribution, and results showed that 80% of black soybean peptides had an MW <10 kDa. This study also showed the effect of these peptides on significant attenuation of body, liver, and adipose tissue weight (P < 0.05) as compared to casein groups in Sprague-Dawley male rats. A detailed review on the use of HPLC and capillary electrophoresis on the analysis of soybean proteins and peptides has been provided by Saz and Marina (2 1). These published articles show that a wide variety of techniques can be used in the isolation, purification, characterization, and identification of biologically active peptides in soybean. Choosing suitable methods depends on the objectives of the study and the chemical nature and stability of the target compound.
Lunasin: A Novel Bioactive Peptide in Soybean

Lunasin is a soybean peptide composed of 43 amino acid residues with an MW of 5.5 kDa. It contains 9 aspartic acid residues on its carboxyl end, a cell adhesion motif composed of arginine-glycine-aspartic acid residues, and a predicted helix with structural homology to a conserved region of chromatin-binding proteins (22). Earlier studies on animals showed that lunasin is not fully digested in the gastrointestinal system but is absorbed intact, reaching target tissues (23). The biological activity of lunasin depends on its concentration in

940 WANG ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 91, No. 4, 2008

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Figure 2. Chromatographic profiles for the isolation, identification, and purification of lunasin from defatted soy flour. (A) Lunasin profile of ion-exchange fractions of soy extract. The extract was eluted using TrisHCI as solvent Aand TrisHCI plus 2 M NaCl with step gradient from 0, 5, 10, 20, and 100% solvent B. (B) HPLC profiles of synthetic lunasin (B, dash line) and lunasin purified by IEC (A and C, solid line). Profile A corresponds to fraction 104 and profile C to fraction 88. The buffers used were 5% acetonitrile + 0.08% trifluoroacetic acid (buffer A) and 95% acetonitrile + 0.1% trifluoroacetic acid (buffer B) using linear gradient elution, and were detected at 215 nm. (C) Lunasin profile of gel filtration fractions after one void volume using active pooled fraction from IEC. The pooled fraction was eluted using TrisHCI plus 0.15 M NaCl.

the product, which in turn is affected by cultivar, environmental factors, and processing conditions. Figure 1 presents the predicted secondary structure of lunasin, its 43 amino acids, and the motif (2). Studying the structure of lunasin using native PAGE, nonreducing and reducing denaturing PAGE revealed that lunasin is a single polypeptide chain. Under those 3 different electrophoretic conditions, the same gel pattern was observed, implying that the 3 active bands corresponding to 5, 7, and

14 kDa are not in association with each other. More research is needed regarding the chemical structure of this peptide.

Identification and Quantification of Lunasin by Chromatography, Western Blot, and Enzyme-Linked Immunosorbent Assay
In our laboratory, a defatted soybean protein extract was prepared by extraction at pH 8.2, loaded in an ion-exchange chromatographic (IEC) column and run with TrisHCI and TrisHCI plus 2 M NaCI buffer. Fractions were collected

WANG El AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 91, No. 4, 2008 941

78kDa 14 0. 5D

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Figure 3. SOS-PAGE (A) and Western blot (B) of gel filtration fractions for identification of lunasin. Lanes 1-5 are examples from different GF fractions. Lane M is molecular weight standard.

using step gradient elution at 10 mL/min. The lunasin profile of the ion-exchange fractions is shown in Figure 2A. From this collection, fractions 88 and 104 were taken and analyzed by HPLC due to the higher concentration of lunasin per total protein, as determined by enzyme-linked immunosorbent assay (ELISA) and Bradford protein assays. A 100 1.iL volume of filtered ion-exchange fraction was injected into an HPLC system equipped with a Vydac C4 column (25 cm x 4.6 mm, 10 I.tm) after system equilibration and stabilization, using diode array detection (215 nm). A mobile phase [5% acetonitrile and 0.08% trifluoroacetic acid (TFA). buffer A; and 95% acetonitrile and 0.1% TFA, buffer B] in linear gradient for 30 min at 1 mL/rnin was used. Lunasin was identified by retention time using synthetic lunasin (profile B) as shown in Figure 2B. The chromatogram shows that fraction 104 (profile A) contains fewer contaminating proteins than fraction 88 (profile Q. Active fractions from ion exchange were run through GFC to further purify lunasin. Gel filtration was performed by introducing 15 mL pooled active ion-exchange fractions in Superdex Prep Grade 75 column using 20 mM TrisHCI plus 0A5 M NaCl buffer as mobile phase at a flow rate of 4 mL/min. Fractions were collected every minute after the void volume. Lunasin concentration of the gel filtration fractions is shown in Figure 2C. From this figure, one active peak corresponding to fraction 14 was detected. Lunasin active fractions from IEC and GFC were analyzed by sodium dodecyl sulfate (SDS)-PAGE with high-density gels and trans-blotted to polyvinylidene difluoride (PVDF) membranes for Western blot analysis. The PVDF membrane, with the transferred proteins, was blocked and incubated for 16 h at 4C with lunasin monoclonal antibody (1:1000 dilution) provided by Ben 0. de Lumen, University of California, Berkeley. After washing, the membrane was incubated with an antimouse secondary antibody (1:10000 dilution) for 3 h and was examined using chemiluminescent reagent. Figure 3A and B show the

SDS-PAGE and Western blot results for the identification of lunasin in ion-exchange and gel filtration fractions. Three active bands can be seen from ion-exchange fractions 88 and 104, and gel filtration fractions 11, 14, and 16 corresponding to 5, 7, and 14 kDa peptides. Figure 4 shows the MALDI-TOF profile of synthetic lunasin (A), lunasin purified by IEC (B), and lunasin purified by GFC (C). Figure 4A and B show that 3 major peaks were found which corresponded to 5.1. 7.9, and 14.1 kDa peptides. MALDI-TOF profile of active fraction from GFC showed very few peaks as compared to ion-exchange fractions, suggesting further purification of the sample. The detailed procedure to quantify lunasin by ELISA in our laboratory has been described previously (24). Briefly, IOU p1 soy flour extract was plated on a 96-well plate and stored overnight at 4C; the plate was then washed and blocked with 5% bovine serum albumin for I h. After blocking and washing, 50 p1 lunasin monoclonal antibody (1:4000) was plated and incubated for 1 h. After washing, 50 .jL antimouse immunoglobulin G (1:7000) was plated and incubated for I h. The color was developed by adding 100 p1 p-nitrophenyl phosphate (pNPP) to each well. The color produced after 20 min was read using an ELISA plate reader at 405 rim. The reaction was stopped by adding 100 .tL 3 N NaOH after 25 min and read again at 35 mm. Lunasin concentration was quantified using a standard curve from different concentrations of synthetic lunasin ranging from 8 to 36 ng/mL (y = 0.0291x + 0. 1533, R 2 = 0.95). Effect of Soybean Cu/tivar and Environment on Lunasin Concentration In order to understand the effect of cultivar, temperature, and soil moisture on lunasin concentration of soybean, 5 cultivars (2 French, Imari and Queen, and 3 U.S., Dwight, Jack, and Loda) were studied (25). They were selected based on similar maturity from a greenhouse experiment by Lozovaya et al. (26) and Vasconez et al. (27). All entries were grown in the Plant Sciences Laboratory greenhouse on the campus of the University of Illinois; one plant per 30 cm diameter plastic pot under intermediate night/daytime temperatures of 18/28C (23C mean) with high soil moisture and under a 14.5 h photoperiod. When the plants reached the R6 growth stage, they were moved into one of 3 different temperature regimens until maturity: low. 13/23C (18'C mean); intermediate, 18/28C (23C mean); and high, 23/33C (28C mean). In each temperature treatment, one-half of the plants were grown in high soil moisture (approximately 70% of soil-holding capacity) and one-half in low soil moisture (approximately 30% of the high treatment). Out of a total of 5 replicates of cultivar/teniperature/soil moisture, a pooled sample was obtained; thus, a total of 30 samples was analyzed for lunasin concentration. The results showed that cultivar and temperature, but not soil moisture, significantly affected lunasin concentration in soy. Significant interactions of cultivar-temperature, cultivar-soil moisture, and cultivar-temperature-soil moisture were also detected (Table 3).

942 WANG ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 91, No. 4, 2008

Figure 4. MALDI-TOF profile of synthetic lunasin (A), lunasin purified by IEC (B), and lunasin purified by GFC (C) using the following parameters: linear mode of operation, positive polarity, and 3000-20 000 Da scanning range. Defatted soybean extract was loaded in an ion-exchange column and run with TrisHCI and TrisHCI plus 2 M NaCl buffer. Fractions were collected corresponding to 5-20% TrisHCI plus 2 M NaCl using step gradient elution. Active fractions from IEC were pooled and passed through a gel filtration chromatographic system using TrisHCI plus 0.15 M NaCl as mobile phase.

Soybean genotype had a significant effect on lunasin concentration. Across all temperatures and soil moisture traits, the concentration of lunasin in the 5 cultivars ranged from 7.5 to 10.4 nig/g flour. The 3 U.S. cultivars. Loda, Jack, and Dwight, had a higher concentration of lunasin than did the 2 French cultivars, Queen and Imari (Figure 5A). Loda had a 38% higher ILmasin concentration than Imari, which demonstrated genetic differences in lunasin concentration. Analysis of lunasin concentration of 144 selected, diverse soybean accessions from the U.S. Department of Agriculture (USDA) Soybean Germplasm Collection showed that the

concentration of lunasin in soybean accessions ranged from 1.0 to 13.3 mg/g flour (24). These genetic differences of lunasin should make it possible to modify lunasin concentration in high-yielding cultivars. Temperature also had a significant effect on lunasin concentration (Figure 513). High temperature led to significantly lower concentration of lunasin (8.08 1.62 mg!g flour) than did intermediate (9.15 1.64 mg/g flour) and low temperatures (8.83 1.38 rng/g flour). This trend was not predictable by either the agronomic data (maturity date, plant height, seed yield. 100-seed weight), or the isoflavone concentration (26). Lunasin concentration of

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Table 3. Analysis of variance of lunasin concentration of 5 soybean cultivars grown under controlled temperature and soil moisture conditions Source Cultivar Temperature Soil moisture Cultivar-temperature Cultivar-moisture Temperature-moisture Cultivar-temperature-moisture
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DFa Type I SS b Mean square F-value Probability> F

4 71.29 17.82 6.11 1.25 2.63 5.12 1.18 1.67 29.48 10.11 2.07 4.36 8.47 1.95 2.76 <.0001 0.0004c 0.1603 0.0014C 0.0001 0.1594 0.0206

12.22 1.25

8 2

21.06

20.48 2.36 13.35

DF = Degrees of freedom. Type I SS = Sum of squares. Significant at the 0.01 probability level.

values are similar to our findings, which ranged from 10.72 to soybean grown under interniediate temperature had the 23.36 mg lunasinig protein (27). highest value, although it was not significantly different from In summary. we have demonstrated the effect of genetics that grown at low temperature. A 14 1N) increase in lunasin and environmental conditions on lunasin concentration. The concentration was observed when soybean was grown at variation in lunasin concentration of 5 soybean cultivars intermediate temperature in comparison to high temperature. grown in different environmental conditions indicates that the Soil moisture did not show a significant effect on lunasin concentration of this important bioactive component in concentration; however, high soil moisture delayed maturity soybean can be manipulated by using genetics and different and increased isoflavone concentration, while the plant height growing conditions. and 100-seed weight remained the same (26). Considering the relatively large moisture difference between the 2 groups, the Effect of Processing on Lunasin Concentration results indicated that lunasin concentration is not very The effect of processing on lunasin concentration in sensitive to soil moisture conditions. soybean products has been demonstrated by de Melia et A significant interaction between cultivar and temperature al. (24) and Jeong Ct al. (29). de Mejia et al. (24) quantified was observed for lunasin concentration (Table 4). For Jack, Loda, and Queen. lunasin concentration did not change with temperature (P > 0.05); however, high temperature led to significantly lower (P< 0.05) lunasin concentration for Dwight 14 and Imari. o12Although soil moisture did not show a significant effect on A Es lunasin concentration, significant cultivar-soil moisture o 5interactions were observed (Table 5). For Imari, high soil =4 moisture led to significant higher concentration of lunasin '2 5 0 (P < 0.05) than did dry conditions, while the trend was Loda Jack Dwght Queen inri reversed for Jack. The temperature-soil moisture interaction Cultivar was not significant. Temperature and soil moisture combinations did not significantly affect lunasin 12 a concentration for all cultivars. However, for individual b 110 cultivars, significant difference was observed. This agreed B with the significant interaction observed for cultivar x 6- I I S I r temperature x soil moisture (Table 6). For each cultivar, -J I I I lunasin concentration can vary from 35 to 75% when the most E2 0favorable temperature and moisture conditions are compared Intermediate Low High with the least favorable. Highest percentage difference was Temperature observed in Imari (75%), and decreased in the order of Queen (52%). Jack (45%), Dwight (38%), and Loda (35%). Figure 5. Lunasin concentration (mg/g flour) of soy Recently, Jeong et al. (28) found that lunasin concentration flour as affected by (A) cultivar, and (B) temperature. of 8 Korean soybean cultivars ranged from 4.40 0.32 to Average temperatures: intermediate (23 CC), low (18C), 70.49 1.38 mg lunasinlg protein, and the amounts correlated and high (28CC). with the extent of inhibition of core historic acetylation. These
'0 ' S -J C,

_ r1 Li

944 WANG FT AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 91, NC). 4, 2008

Table 4. Effect of cultivar and temperature on lunasin concentration (mglg) Cuttivar Temperaturea Mean SDb Dwight
I 7.98 0.64 Ell 9.54 1.10 BDCE 7.41 0761GH 8.38 1.4 CDEll 8.11 O.gE1 6.101.14H 962216BDC 978114ABC 8.98 079BDCEF

L
H

Imari

L
H

Jack

L
H

Loda

L
H

9.7Ol.27ABC 1029099AB 8.57 123CDEFG 7.01 097GH 7.61 0,26FGH

Queen

L
H

Average temperature of growth: I = intermediate temperature (23C); L = low temperature (18C); and H = high temperature
(28C).

Values, mean standard deviation (SD), followed by the same letter(s) are not significantly different from each other (P> 0.05); n = 4.

lunasin concentration on commercially available soy protein and isofiavone-cnriclied products. They showed that lunasin concentration of commercially available soy protein ranged from 13 2 mg lunasinlg flour (soy flour and soy flakes) to 44 6 nig lunasinlg flour. Lunasin concentration in lunasin-enriched flour was 27.3 rng/g flour. However, the isoflavone-enriched products contained from zero to a low lunasin concentration, possibly due to the poor solubility of lunasin in ethanol, which was used in the extraction of soybean isoflavones. Jcong et al. (29) found that defatted soy flour had the lowest lunasin concentration (5.48 0.17 mg/g protein) when compared to soy concentrate (8.72 0.19 to 16.52 0.23 mg lunasin/g protein) and soy isolate (6.92 0.14 mg lunasin/g protein). They also showed that water-washed soy concentrate had a higher concentration of lunasin (16.52 0.23 mg lunasin/g protein) than alcohol-washed concentrate (8.72 + 0.19 mg lunasin'g protein). It has been demonstrated that lunasin concentration in soy products is affected by processing conditions.

Bowman Birk inhibitor (BBI) was considerably higher than that of KTI. BBI values ranged from 0.6 to 6.3% of total extractable protein, whereas that of KTI ranged from 4.3 to 6.9% of total extractable protein. Also, the variety with the highest KTI (Krajina) also had the highest BBI, and the variety with the lowest Ku (Vojvodjanka) also had the lowest BRI. Genotypes with a high percentage of trypsin inhibitor; especially BBI, could have a significant role from the nutraceutica] point of view and might be used in cancer prevention and therapy. Deshimaru ci al. (31) were able to isolate 9 protcinasc inhibitors from wild soy seeds. Two inhibitors were classified as a soybean KI family and the others were stable to heat and extreme pH, suggesting that these belonged to the BBI family. Vollmann et al. (32) studied environmental influences and the effects of nitrogen and sulfur supply on TIA of soybean. TIA was affected significantly by environment (geographical location), fertilization type, and genotype. They used 3 macroenvironmcnts (of different elevations in 3 years), and the environmental means of TIA was 69.5-104.8 mg tiypsin inhibitcdlg defatted soy flour. Their results showed that significant genetic variation in TIA was found within the genotype and suggested that TIA of soybean may be modified considerably by genetic improvement and appropriate agronomic management. Friedman et al. (33) studied Williams 82 standard soybean cultivar, an isoline (L81-4590) lacking the KTI and 13 cultivars from the USDA soybean gerrnplasrn collection. L81-4590 had only 54% TIA and 79 1N, chymotmypsin inhibitory activity. All 13 cultivars exhibited values ranging from 4.7 to 13.2 mg chymotmypsin inhibited/- flour and 37.2 to 61.4 11mg TIAIg. Becker-Ritt et al. (34) found significant differences in the proteinase inhibitor content of 6 soybean cultivars. Differences in the inhibitory activity towards tlypsin might be related to different levels of the BBI since the levels of KTI appeared to be similar, as

Table 5. Effect of cultivar and soil moisture on lunasin concentration (mglg) Level of cultivar Moisturea Mean SDb Dwight man Jack Loda Queen L
H 8.01 1.25 BCD 8.61 6.86 820151BC

L
H H H

L 1044123A
8.48 1062104A 7.22 0.82 82411OBI

L 10. 18 1.29A L
H

Other Biologically Active Proteins and Peptides in Soybean

Pesic et al. (30) reported the influence of different genotypes on the level of trypsin inhibitory activity (TIA) in soybean. Significant differences were found among 12 soybean genotypes used in the study. Also, the extent of variation in

Soil moisture conditions: L = low soil moisture; H = high soil moisture. Values, mean SD, followed by the same letter(s) are not significantly different from each other (P> 0.05); n = 6.

LIIWANG El AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 91, No. 4.2008 945

Table 6. Effect of cultivar, temperature of growth, and soil moisture on lunasin concentration (mglg) Cultivar Temperaturea Moisturea Mean 8Db Dwight I L
7.71 079JKL1 8.24 057FGHIJ}<

I H

BAPNA as substrate, Voliman et al. (32) used azocasein as one of the substrates and detennined TIA by measuring the residual activity of bovine trypsin used as standard. In summary, other biologically active peptides and proteins present in soybean are also affected by cultivar and growing conditions, the same observations found regarding lunasin concentration in soybean. Conclusions This paper discusses techniques recently used for the analysis of biologically active peptides in soybeans. It is apparent that these techniques are available for general use and require relatively expensive equipment, laboratory supplies, and highly trained personnel. Choosing a method should be based on chemical composition and stability of the material to be analyzed. As shown in this review, the best method for the analysis of different bioactive peptides in food includes a combination of different LC techniques because one method increases the efficacy and efficiency of other methods. Also, this review reports the effect of cultivar, environmental factors and processing on lunasin concentration of soybean and soybean products. Soybean cultivars and environmental conditions where soybean was grown also affected the concentration of lunasin in soy flour. The interactions of cultivars, soil moisture conditions, and growing temperature significantly affected lunasin concentration, suggesting that its concentration in soybean can be manipulated. Other bioactive peptides and proteins present in soybean were also affected by cultivar and environmental conditions. The significant effect of processing was shown by the differences in the concentration of lunasin in defatted soy flour, soy isolate, alcohol- and water-washed soy concentrates, and isoflavone products. Acknowledgments We acknowledge the financial support of the Illinois Soybean Association and the USDA Functional Foods Initiative. References (1) Wang. W, & (IC Mcjia, E.G. (2005) Comp. Rev Food Sd. Food Sate/v 4, 63-78 (2) de Lumen. B.O. (2005) Nun: Rev 63, 16-21 (3) Nielsen. N.C. (1996) So ybean: Genetics, MolecularBiolog' and Biotechnology. D.P.S. Verna & R.C. Shoemaker (Eds), CAB International, Wallingford, UK, pp 127-163 (4) Panthce. DR., Kwanyucn, P., Sams, CE., West, DR., Saxton, AM.. & Pantalone, V.R. (2004) .J. Am. Oil Chem. Soc. 81, 1005-1012 (5) Ji, M.P., Cai, T.D., & Chang, K.C. (1999) J. Food Sci. 64,
763-767 (6) Liu. K.S. (1997) Soybeans: Chemistry, Technology and UiiIi:atzon. Chapman and Hall, New York. NY. 532 pp

L L 9.03 1.52 11F1H1 10.05 051B0CE L H H L 7.281.27'7.53 023JKL1 H H L 7.40 083JKL marl I I H 9.361.16c0EF 7.82 001HIJKL

L L L H H L

8.41 145FGHIJK 5.36 040M

H H 6.851.25KML L 11.401.17AB Jack I 7.85 008GHIJKL H 1053127ABCD

L L L H H L
H H

9.03 9.40 072CDEFGH 8.57 081EFGHIJ 1Q77085ABC 1168034A 8.64 + 046EFGHIJ

Loda I

I H

ABC L H 10.76 1115013AB H L 9.43 000CF0 H H L 7.63 054JKL1 Queen I 6.28 053LM L L 7.75 061JKL1 L H IKLI H L 7.76 H H H

L L

7.45 006JKL1

L = Low; I = intermediate; H = high temperature. Values, mean SD, followed by the same letter(s) are not significantly different from each other (P> 0.05); n = 2.

detected by immun ob lotting. Vasconcelos etal. (35) studied the antinutritional and toxic proteins present in 2 Brazilian soybean cultivars. Rio Balsas and Bays. Their results showed that the Bays cultivar presented significantly higher, about 2-fold, TIA than the Rio Balsas cultivar. Giami (36) reported the chemical composition and antinutritional attributes of 3 promising advanced breeding lines of soybean in Nigeria. The raw seeds contained 8.6-18.2 rng trypsin inhibitor/g defatted flour. The assay used in the analysis of TIA used a-N-benzoyl-DL-argininc-p-nitroafllhde hydrochloride (BAPNA) as substrate, and the activity was determined by measuring the change in absorbance at 40 5 -420 nm. In addition to the use of

946 WANG ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 91, No. 4, 2008 (7) Maruyama, N., Katsube, T., Wada, Y., Oh, M.H., Barba De La Rosa, A.P., Okuda, E., Nakagawa, S., & Utsumi, S. (1998) Eu,: 1. Bioc/zern. 258,854-862 Natarajan. S., Xu. C., Bae, H., & Bailey, B. (2007)1. Plant Phvsio/. 164, 756--763 Lo, W.M.Y.. & Li-Chan, E.C. (2005) J. Agric. Food Ghe,n. 53,3369-3376 Zhang, J.H., Tatsumi, E., Deng. C.. & Li, L. (2006) Food Chern. 48, 551-557 Gibbs, B.F., Zougman. A., Masse, R., & Mulligan, C. (2004) Food Res. Int. 37. 123-131 Kuba, M., Tanaka, K., Tawata, S., Takeda, Y., & Yasuda. M. (2003) Biosci. Biotechnol. Biochern. 67, 1278-1283 Mallikaijun Gouda, KG., Gowda, L.R., Appu Rao, A.G., & Prakash, V. (2006)1 Agric. Food Chain. 54, 4568-4573 Kodera, T., & Nio, N. (2006)1 Food Sc!. 71, C164C173 Kuba, M., Tana, C., Tawata, S., & Yasuda, M. (2005) Process Biochern. 40,2191 21196 Zhong, F., Liu, J., Ma, J., & Shoemaker, C.F. (2007) Food Res. Int. 40, 661-667 Zhong, F., Zhang, X., Ma, J., & Shoemaker, C.F. (2007) Food Res. Int. 40, 756-762 Gianazza, E., Eberini, 1., Arnoldi, A., Wait, R., & Sirton, C. (2003)1. Note 133, 9-14 Lee, S.H., Yang, J.1., Hong, S.M., Hamh, D.H., Lee, S.Y., Kim, 1.1-I., & Choi, S.Y. (2005) Biofctocs 23, 121-128 Rho, S.J., Park, S., Ahn, C.W., Shin, J.K., & Lee, H.G. (2007) 1 Sci. Food Agric. 87, 908-913 Saz. J.M.. & Marina. M.L. (2007)1 Sep. Sci. 30, 431-451 Galvez, A., Chen, N., Macasieb, J.. & de Lumen, B.O. (2001) Cancer Res. 61, 7473-7478 (23) (24) (25) (26) (27) (28) (29) (30) (31) (32) (33) (34) (35) de Mejia. E.G. Bradford, T.. & Hasler. C. (2003) Nut,: Rei 61,239-246 de Mejia, E.G., Vasconez, M.. de Lumen. B.O.. & Nelson, R. (2004)1 Agiic. Food Chem. 52,5882-5887 Vasconcz-Costa, M. (2004) Wasters Thesis, University of Illinois. Urbana-Champaign. IL Lozovaya, V.V., Lygin, A.V., Ulanov, A.V., Nelson. R.L., Dayde. J., & Widholm. iD. (2005) Crop Sc!. 45. 1934-1940 Vasconcz, M., Nelson. R.. & dc Mcjia, E.G (2005) lust. Food Tee/no!. 85, 8 Jeong, H.J., Jeong, J.B., Kim. D.S., & de Lumen, B.O. (2007)1. Agric. Fooc/ C/tern. 55, 632--637 Jeong, H.J., Park, J.H., Lam, Y.. & de Lumen, B.O. (2003)1. Aguk. Food Chem. 51. 7901-7906 Pesic, MB., Vucelic-Radovic, B y., Barrac, M.B., Stanojevic. S.P.. & Ncdovic, V.A. (2007) Sensors 7. 67-74 Deshimaru, M.. 1-lanamoto. R., Kusano. C., Yoshimi. S.. & Terada, S. (2002) Biotech. Biochern. 66. 1897-1903 Vollmann, I., Grausguber. H., Wagentristl, H., Wohlescr. H., & Michele. P. (2003)1. Sci. Food Agric. 83, 1581-1586 Friedman, M., Brandon. DL., Bates, A.H., & Hymowitz, T. (1991)1 Agile. Food C/tern. 39. 327-335 Becker-Ritt, A.B., Mulinari, F., Vasconcelos. TM.. & Carlini. C . R. (2004) 1 Sc!. FoodAgric. 84, 263-270 Vasconcelos, I.M., Maia, A.A.B., Siebra, E.A., Oliveira, J.T.A., Carvalho, A.F.F.U., Melo, V.M.M., Carlini, CR.. & Castelar, L.I.M. (200 1).J. NuIr. Biochem. 12, 55-62 Giami, S.Y. (2002)1 Sd. FoodAgric. 82, 1735-1739 Tsuruki, T., Kishi, K., Takahashi, M., Tanaka, M., Matsukawa, T., & Yoshikawa, M. (2003) FEBS Leil. 540, 206-210

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