The Research On Biological Preparation Technology And Stability Of Antioxidant Peptide From Soybean

Supported by Project of National Key Technology Research and DevelopmentProgram for the12th Five-year Plan (NO.2012BAD33B03).Soybean concentrateprotein powder was acted as row material, microwave assisted digestion technology,ultrafiltration separation technology, dextran gel purification technology was used,based on in vitro chemical antioxidant activity with different mechanism and theprotection of HEK-293complete cell oxidative damage caused by H2O2preparingantioxidant peptide, obtaining the following conduction:Soybean concentrate protein poweder was processed by microwave assistedenzymatic digestion used of Alcalase protease, both one-factor-at-a-time (OFAT) andBox-Behnken design (BBD) of response surface methodology (RSM) were used tooptimize the experimental design. ABTS radical scavenging activity, hydroxyl freeradical scavenging activity and reducing power was acted as evaluation index, theoptimal process parameters for preparation of soybean antioxidant peptide wasobtained: microwave assisted enzymatic digestion time37min, temperature56℃,pH value8.17, the amount of enzyme5:100, substrate concentration50mg/mL,microwave power500W.The stability of antioxidant peptide from soybean protein obtained the followingconclusions: soybean antioxidant peptide in the lower temperature range of25-60℃,activity retention rate was relatively high, and when the temperature reached80℃,activity retention rate was significantly reduced, and the processing time was longer,the greater activity loss. The activity retention rate of soybean antioxidant peptide wasaffected by stronger acid and alkali environment, and the effects of alkali environmentare more obvious, at6,7,8of the pH value, activity retention rate was higher. Theeffects of NaCl solution with mass fraction0.5%-5.0%on soybean antioxidant peptide are not obvious, glucose solution with mass fraction1.0%-8.0%made theactivity dramaticly decline, citric acid solution with mass fraction0.04%-0.20%onABTS radical were not obvious, but lower mass fraction made the hydroxyl radicalscavenging activity becoming higher, higher mass fraction can restrain the hydroxylradical scavenging activity. The effects of metal ions on soybean antioxidant peptidewere obvious, Zn2+had the most significant influence on on ABTS radical scavenging,Ca2+had the most significant influence on hydroxy free radical scavenging, whereasthe impact of K+on the two’s are relatively litlle. Effects of gastrointestinal digestionliquid on the antioxidant activity of soybean were obvious.The enzymolysis liquid that obtained from soybean concentrate protein powederprocessed by microwave assisted enzymatic digestion intercepted by the ultrafiltrationmembrane of1kDa、3kDa、10kDa、30kDa, getting SAP-I (molecule weight>30kDa)，SAP-II (10kDa-30kDa)，SAP-III (3kDa-10kDa)，SAP-IV (1kDa-3kDa)，SAP-V(molecule weight <1kDa) with rate were19.53%,4.28%,17.12%,31.46%,17.12%.The DPPH radical scavenging, superoxide anion radical scavenging, ferrousion chelating rate, reducing power, inhibition of linoleic acid were acted as evaluationindex, synthetically evaluate the antioxidant activity of above separated and mixedcomponents. The SAP-IV (molecule weight1kDa-3kDa), SAP-V (molecule weight<1kDa) can be selected, in order to the futher research.SAP-IV and SAP-V were separated and purified by Sephadex G-25, obtainedF1、F2、F3、F4、F5、F6at the maximum absorption wavelength of280nm. The sixcomponents at concentration of less than1000μg/mL had no toxicity to HEK-293cells, F5showed the best protect on HEK-293cells injuried by H2O2, which could beselected as the soybean antioxidant peptide.