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 quantified by using different techniques and received increasing interest from the public conditions. In addition, lunasin concentration in because of its reported health benefits. These health soybean depends mainly on cultivar and to some benefits are attributed to its components, including extent on environmental factors, particularly isoflavones, saponins, proteins, and peptides. temperature. Lunasin concentration in soy products Lunasin, Bowman-Birk inhibitor, lectin, and was also affected by processing conditions. 3-conglycinin are some of the biologically active peptides and proteins found in soybean. This article provides a comprehensive review on the recently ood proteins have long been recognized as a source of used techniques in the analysis and essential amino acids required for the maintenance of characterization of food bioactive peptides, with F life. They also serve certain functions as ingredients in emphasis on soybean peptides. The methods used foods and food preparations. For the past 2 to 3 decades, to isolate and purify lunasin from defatted soybean attention has been given to other aspects of food proteins, flour were ion-exchange chromatography, such as their biologically active peptides derived either from ultrafiltration, and gel filtration chromatography. The enzymatically digested proteins or by protein fermentation. identity of lunasin was established by sodium In vitro and in vivo studies have shown that these bioactive dodecyl sulfate-polyacrylam ide gel electrophoresis, peptides can perform certain beneficial biological functions Western blot, matrix-assisted laser desorption in the body, such as antihypertensive and antioxidant ionization—time of flight, and liquid chromatography. activities, cancer prevention, hypocholesterolemic, The results on the effect of soybean cultivar and antiobesity, and immunomodulatory actions (I). It is environmental factors on lunasin concentration are therefore important to have precise and accurate methods for also reported. The highest lunasin concentration, quantification and characterization of these bioactive 11.7 ± 0.3 mg/g flour, was found in Loda soybean peptides present in foods. The most studied food sources of cultivar grown at 23°C; the lowest concentration, 5.4 bioactive peptides are milk and soybean. Lunasin is a novel ± 0.4 mg/g flour, was found in Imari soybean cultivar bioactive peptide found in soybean. It is composed of 43 grown at 28°C. Lunasin concentration was affected amino acids with 9 aspartic acid residues on its carboxyl end. by cultivar—temperature, cultivar—soil moisture, and Its bioactive properties are attributed to its capability to cultivar—temperature—soil moisture interactions. The arrest cell division in cancer cells and to inhibit core histone variation on lunasin concentration suggests that its acetylation in mammalian cells (2). content can be improved by breeding, and by This paper reviews the methods used in the analysis of optimization of growing conditions. In summary, bioactive peptides in soybean and the effect of processing. bioactive peptides can be accurately identified and cultivar, and environmental factors on lunasin concentration in soybean and soy products. 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 Techniques Used in the Analysis of Soybean and Paul J. Moughan. Peptides 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 Protein is the most abundant component in soybean. On other products or vendors that may also be suitable. average, soybean contains 40% protein conformed by a Author to whom correspondence should be addressed; e-mail: complex mixture of different protein types (3). The major edemejiaiuiuc.edu components of so y proteins are seed storage proteins known as WANG FT AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 91, No. 4, 2008 937 C-)a,. a a ci) N- - C C,) (N (0)N 0) 0) (. ci) (U a, — a. II 0 cc ci) (U (U ci)—..----------- ci) (U (U (U CU (U ci) LU ow (0 -a CU . -ccS 0) a, CU ) CU (U CU CU CU CU CU — • ci) CU a © cC) (U00 CU a. a, a, a, Cl) a, a, a, CU< a CU C.) 0) E > a- a. a. a. a. a. a. E - cca, Da aJ a, a, 0 .2 .2.2 .2 .2 .2.? CU 0 : °a ci, (U V o - t t t3t (U 0 CO CU (U CU CU CU CU CU >.9 ci a c) a CU .Lc) — D 0) 0) 0) 0) 0) 0) 0) C 3 a . 6 E ci) - 0 +1 a, ci) a a CU o . :aa.5- .0 a - ci) > — - .2 : = 0 -C 0OD oci) 0 0 0 -D 70 D D - A .2 a, a, a, (D a, a, a, a, LX) Q C)) a, Cl) (U ci) o ci) CO (U Cl) ._(U :1 < 0 D ci) -D(D 0ci) -cc a_ a. 0 a) H 0_ a. a- a. .9 .9 .9 .9 a, 0 E .0 .0 CC) a. • 2 ci) C ci a, UJ - - LU w LU LU W LU 2.5. 0 LU LU LU LU m 0 0 0 0 0 0 ci, 0 0 a, 0 0 0 0 < _5 < < - <i < < < E, .0 .a r 0 C.) 0 2° 0 0 o CL a. >CL > Cl. II I I 90 a2 cr a a — Ca C aa. ci) ci) C 2 2 .2 O a, (1) 0 — CU a, CU— ..-. CUCU .0 >S a. 0) ci) 0 0 C) 0) 0) 0 ci) 0) > 0 a a, (U a. a. -.,- -- .0 .0_ 0 :6 :6 2 a^- a a a-i, >1 - 0 CD0 >O (U Oa, 2 am) M CL 0 U) U)Cl) C/) C/) a, 0) E Oo E E -J a a E< 0 a u E LL LUC E OH I— c0(N C 64 0 E E U - LO LO E C Ea N- - E C..) E 0 > a. a E DE > H E 0 0 d o a CU o + 04 1 E 0 0) + E(NE c CU cD E EZELU Z O- cc z a 2 0 CD LO 0 a 0 E 3E a 0 0 <--a ... LU EEE (Na CU 0)Q LU - C (00 LU U.) E E ,CciC) ci) ©© ,_ H Z H —2 , - OdZH + 0a ci a, (N (U LO 0 CU Z 0) Cc 0 CU N- C© -< 0 .0 0 —20 Z .2-2 .5. 3 a. 0 ci) + Z- 0- 0 Cl a LO (_) O -aCL - .© ' ' I -0 C) Z N. .— 00 —C C qzi ci) U 03 + < CD (N I LU 0 110 (0:i: (N Z 0 ) 0 H ci, LL .2) CU 0 0< QZ .6 -a CU CY5 (:)CD ci) CD 8-1 0 0 z..— 0 .011 CU C C) a 0 5. 03 ci) D a 0 Oa, . E ci) (U)< I a,a (U (N a co ? c-, 75 < - LO E 2 L 0 _ -o (N <0 c -E -2 CL 0 8. << E.ci (0.9 a. a,.a-Cl) 8 o o 2E5. C a, x-i &)0 0 -ö CL X0 CU lo Ln u 2© aD° LU = 0t = - 9. o - a 2 a a. a,© .0a, (N 0 E 0 aEE w5 a, a,o 0 <cc ci - E E 2 N 5. Lu 0cn En w .2 V) oo o 0 RH00 -35 C/) 00 0 pcc, aoa 1 0 0 -J 0 11 0> 03 0 I 0(Ua .0 0 LL 0 0 wO CU a, 0 u LL < 0 0 —CC)0 I— w o o 938 WANG ETAL.: JOIJRNALOF AOAC INTERNATIONAL VOL. 91, No. 4,2008 a) -conglycinin and glycinin, which account for 50-70% of total C.) C 5). 3-Conglycinin is a trimer with a molecular a) Q N (N (0 10 U) Lt) It) N. (N C)) C) N- seed proteins (4, a) 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 a) regions (a, 125 residues; a, 141 residues) exhibiting lower CO C homologies (57.3%), whereas the f3 subunit consists only of a 0 core region that has homology with the a and a core regions 0a) (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, :S! E .0 -J 0 0) C G4, and G5). Each subunit consists of an acidic chain (about E E C > 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% 0 > CO .2 C C a) H sequence homologies. Similarily, G4 and G5 share 90% sequence 0 (0E C Q Lt Ii co CC) 0 LO - homologies.