Study On Antioxidive Peptides From Fermented Soybean Meal

Soybean antioxidative peptides have been reported to have antioxidant functions of scavenging hydroxyl free radical, terminating single oxygen formation, trapping free radical by chelating heavy metal ion etc. By now soybean antioxidative peptides have been prepared by methods of enzymatic hydrolysis of soybean, and most research on preparation and purification of soybean antioxidative peptides have been carried out at laboratory scale with the disadvantages of low efficiency, high cost, no applicability to industrial production.Hydrolyzation,purification and antioxidant activities determination of soybean antioxidative peptides were studied in detail in this paper. In order to achieve the highest hydrolysis efficiency, the best strain called Jb009 with enzymatic activity of 2.28 u/mL was screened out from 12 proteinase-producing strains in our laboratory. The Jb009 strain was used to carry on the liquid and solid fermentation. The antioxidant activity of the fermentation products in the liquid fermentation was 621.68 U/g bean cake and hydrolysis degree was 14.85%, while in the solid fermentation product they were respectively 462.81U/g bean cake and 15.39%.The solid state fermention conditions of soybean antioxidative peptides was optimized. Using the single factor experiments, the effects of medium composition, fermentation temperature, inoculative rate, primary pH value and water addition on total antioxidant activity were investigated. The reactive temperature and pH were optimized by secondary regression common rotary combination design. Results showed that suitable fermentation conditions of solid state fermentation for soybean antioxidative peptides were: the proportion of bean residue and bran 4:1,water content 66.7%,inoculative rate 25.0%, age of inoculum 48h, fermentation temperature 33.7°C, primary pH value 8.49, and fermentation time 36h. Under the optimal conditions, the total antioxidant activity of fermentation products reached to 568.9U/g bean residue and increased nearly 30.4%. Experimental value was basically agreed with predicted value.In order to optimize the liquid state fermentation conditions of soybean antioxidative peptides, the single factor experiments and factorial experiment design for medium rate, fermentation temperature, inoculative rate, primary pH value and water addition were carried out using total antioxidant activity as index. Then the reactive temperature and pH were optimized by steepest ascent design and secondary regression common rotary combination design. Results showed that suitable fermentation conditions of liquid state fermentation for soybean antioxidative peptides were: the medium rate 4%, inoculative rate 5.19%, age of inoculum 36h, fermentation temperature 37.0°C, primary pH value 7.39, and fermentation time 36h. Under the optimal conditions, the total antioxidant activity of fermentation products reached to 795.75U/g bean residue and increased nearly 52.9%。Experimental value was basically in agreement with predicted value.Compared with the liquid fermentation and solid fermentation, the liquid fermentation was chosen to produce soybean antioxidative peptides.The antioxidant activities of soybean antioxidative peptides from liquid state fermentationc were systematically analyzed. The total antioxidant activity was measured by the T-AOC method, the scavenging ability of·OH and O2- was determined by the salicylic acid method and the pyrogallic acid method respectively. The effect of soybean antioxidative peptides on the liposome oxidation and lipid oxidation processes were investigated by the lecithin method and the POV method, respectively. The scavenging ability of alkyl peroxid, DPPH and hydroxyl free radials was analyzed by linoleic acid autooxidation method, DPPH method and deoxyribose method, respectively. The results were as follow:the total antioxidative activity was 50.07U,the ability of scaveng- ine·OH and O2- was respectively 85.68% and 23.61 U/mL, the ability of restraining liposome oxidation and lipid oxidation was respectively 34.58% and 28.52%, the ability of scavenging alkyl peroxide, DPPH and hydroxyl free radials was respectively 72.27%, 35% and 38.77%.The soybean antioxidative peptides were filtered by 10kDa ultrafiltration membrane and freezing-dried. Then, they were purified by DEAE-Sepharose ion-exchange chromatography, and Sephadex G-25 Medium Gel permeation chromatography. The purity of soy antioxidative peptides was determined by SDS-Page. Two compositions named PtIV-1 and PtVII-2 with high purity were obtained respectively with molecular weight of 6kDa and 8kDa. The amino acid content of PtIV-1 and PtVII-2 was measured. The aspartic acid content in PtIV-1 was 27.85%, increased by 207.5% and 89.46% respectively compared with that in bean cake and in hydrolysis solution before purification, while the glutamic acid content was 30.41% increased by 101.79 and 28.80%. The threonine acid content in PtVII-2 was 9.41%, increased by 131.77% and 141.28% respectively compared with that in bean cake and in hydrolysis solution before purification, while the serine acid content was 10.41%, increased by 100.19% and 103.32%.