The Bioactivities And Structural Characteristions Of Black Soybean Protein Hydrolysates

Preparation of bioactive peptides from soybean protein by enzymatic hydrolysis plays animportant role in deep processing of soybean. Up to now, numerous researches about soybeanpeptides at home and abroad were reported, which were used ordinary soy protein as rawmaterials, but the papers about black soybean peptides (BSP) were rare and not systematicand thorough. Black soybean protein is a good plant protein resource, the subunit structureand amino acid compositions were basically similar to ordinary soy protein. In this paper,black soybean protein were hydrolyzed with different combinations and steps of three kindsof commercial proteases (the Alcalase protease, Neutrase and Flavourzyme protease) on thebasis of the single enzyme hydrolysis experiments, and the BSP fractions, which displayedbest antioxidant activity and hypocholestermic effect were separated and purified byultrafiltration (UF) and macroporous adsorption resin (MAR), and their antioxidant activity,relieviing physical fatigue and hypolipidemic effect in mice were investigated. In order topreparated highly purified targeted BSP fractons, high-speed countercurrent chromatography(HSCCC), gel filtration and reverse-phase HPLC were used, and the amino acid sequences ofBSPs were analyzed by N-terminal amino acid sequence analysis as well.The SDS-PAGE and photodnsitometry were used to analyzed black soybean proteinisolate and commercial soybean protein isolate, the results showed that all samples had thesame subunit electrophoresis band number, compared with the commercial soybean proteinisolate, the bands of-subunit and Basic-subunit of black soybean protein were wider and haddeeper color, the value of11S/7S of black soybean protein was higher. Moreover, the contentof11S globulin of late-maturing black soybean protein was higher than early-maturing cultivar.Amino acid composition analyed showed that the amino acid compositions of black soybeanprotein were basically similar to ordinary soy protein, but the contents of total essential aminoacids and hydrophobic amino acids were higher than ordinary soy protein.To clear the hydrolysis properties of different protease, black soybean protein werehydrolyzed by using single enzyme. The results showed that the Alcalase protease enzymatic hydrolysates possessed the highest DPPH·, O2－· scavenging capability and inhibitory rates ofcholesterol micelle solubility (CMSIR), that by Neutrase took the second place, and that ofFlavourzyme was the lowest. On the basis of single enzyme hydrolysis expriments, thedifferent combinations and steps of multienzyme hydrolysis processing were investigated, theresults showed that the BSPs hydrolyzed by multienzyme (the Alcalase protease, Neutraseand Flavourzyme protease) for2h had better DPPH·, O2－·scavenging capability and CMSIRthan BSPs hydrolyzed by single enzyme for3h, it indicated that the hydrolysates produced byreasonable combinations with different enzymes had better antioxidant activity andhypocholesterolemic effect, and it can also save hydrolysis time. Compared with theearly-maturing cultivar, the late-maturing BSPs had better antioxidant activity andhypocholesterolemic effect using the same hydrolysis processing, which may be that thelate-maturing black soybean protein had greater contents of hydrophobic amino acids, and theactive groups on side chains of hydrophobic amino acids would expose when hydrolysed byenzymes.The UF and MARs were used to separation and purification of targeted enzymatichydrolysates, the results showed that the OABSP-MR-Ⅲ and DCBSP-MR-Ⅲ fractions withMr＜3000eluted by75%ethanol displayed strongest antioxidant activity andhypocholesterolemic effect, respectively. The antioxidant activity and relieving physicalfatigue effect were carried out with OABSP-MR-Ⅲ fractions in mice, the results showed thatthe contents of MDA,GSH-Px in serum and hepar in mice were positive, and the content ofSOD in hepar in mice were positive as well, when oral administrated500mg·kg-1·d-1(middledosage) and1000mg·kg-1·d-1(high dosage) BSPs, this impled that the BSPs can inhibitlipid peroxidative injury and impove the activity of endogenous antioxidant enzymes in vivo.Compared with the control group, the middle and high dose group can significantly (P＜0.01)prolong loaded-swimming time, blood lactic acid (BLA) can be reduced notable (P＜0.01),and improving hepatic glycogen (HG) storage status greatly (P＜0.01), it indicated that oraladministration of the BSP can relieve physical fatigue in mice. Furthermore, Mice withhyperlipidemia were made by feeding high fat feed in order to evaluate the effect of theDCBSP-MR-Ⅲ fraction in regulating serum cholesterol. Results showed that compared withthe model control group, high dosage group of DCBSP-MR-Ⅲ could notable（P＜0.01）reduce the levels of TC,TG and LDL-C in serum, it hinted that the DCBSP-MR-Ⅲ hadhypolipidemic effect in mice.The antioxidant capacities of BSPs were evaluated by oxyen radical absorbance capacity(ORAC), the fraction, AOBSP-MR-Ⅲ, exhibited the highest antioxidative activity in vitio andin vivo was further purified using consecutive methods on HSCCC, Sephadex G-25column, reversed phase high-performance liquid chromatography (RH-HPLC) and size exclusionchromatography (SEC), then the highly purified antioxidant peptides named SEC-P3andSEC-P2were obtained, and the sequences were Trp－Asn－Pro and Tyr－Asn－Ile,respectively. In addition, the CMSIR was chosen as an index to isolate highly purifitedhypocholesterolemic peptides from the DCBSP-MR-III fraction, which demonstrated goodhyocholesterolemic effect in mice, the methods of HSCCC, Sephadex G-25column,RH-HPLC and SEC were applied, and then the highly purifited hypocholesterolemic peptidesnamed SEC-P2and SEC-P5were got, which isolated from RP-HPLC-P5and RP-HPLC-P7fractions, respectively. Moreover, the sequences of SEC-P2and SEC-P5were Ala－Phe－Pro－Lys－Asp and Ile－Leu－Ser－Tyr－Ala－Met－Asp－Gly, respectively.