Study On Enzymatic Hydrolysis Technology And Detected Method Of Soybean Minor Peptides

Objective:In the past few years,a large number of biologically active peptids have been isolated from bacterial,fungal,plant,and animal sources.some researehers found that small peptides derived from the hydrolysate of food protein play an important role in various physiological activities and regulating autonomic nervous system ,activating the cellular immunity function,ameliorating the cardiovascular function,antioxidizng, antihypertensive and antaging.The concept that peptides with potential biological activity might be present in food is not new.Partial proteolysis is effective for improving the functional properties of protein. Small peptides have some advantages compared to protein,such as low osmotic pressure,fast absorption,good taste and low antigenicity.Several growth stimulating substances have been investigated,most of which derived from human and bovine milk, however,the effect of peptides isolated from soybean has not been understood well.In this work.The new extraction technique of soybean bioactive minor peptides from defatted soy flakes hydrolysized by enzymatic method.In this study,soybean minor peptides were obtained from the pretreatment and hydrolyzing by crude complex trypsinase and neutral proteinase hydrolysis and purification from defatted soybean flakes.Optimal condition of pretreatment and hydrolyzing by crude complex trypsinase is given.And the quality standards of soybean bioactive minor peptides were also established.Method:The soybean minor peptides was prepared from defatted soy flakes(substrate ratio 5% of protein)by the procedure of as reported briefly below.Defatted soy flakes were finely ground at room temperature.Ground meals pretreated and hydrolyzed by crude complex trypsinase.In order to prepare small soybean peptides, The pH of the mixture was adjusted to 8.5and.The crude complex trypsinase 9000U.g-1 wasadded to start the hydrolysis at 55℃in a water bath shaker. During the reaction,the pH was kept constantly(pH8.5)by adding 1.0 mol.L-1 NaOH solution.The degree of the hydrolysis(DH)of the protein was monitored according to the amount of NaoH depleted during the hydrolysis. The time for the hydrolysis was controlled for 6hours.The hydrolysis was stoppedby keeping the hydrolysate solution in a water bath at 950C for 1 5 min.Then the hydrolysate solution was cooled down to room temperature and adjustedto pH 7.0 by adding a 1.0 mol.L-1 HC1 solution.The peptidesconcentrations were evaluated by the method ofFolin Phenol.Thenthe solution was adjusted to pH 7.5with1.0 mol.L-1 NaOH.Enzymatic hydrolysis was performed with neutral proteinase8500 U.g-1, was incubated for 2 h at 45℃.The reaction was terminated by heating at 900C for 30 min.and the solution was adjusted to pH 7 with 1 mol.L-1 NaOH.After centrifugation(20 min,3 000 r.min-1,4℃),the supernatant was collected and lyophilized.The peptidesconcentrations were evaluated by the method ofFollin Phenol.We also evaluated the standards of soybean minor peptides: The Molecule weight distribution of The soybean minor peptides was estimated by chromatography on a TSK-Gel G2000SWXL column(2 cm×66 cm ). Having been loaded on the column.the lyophilized powder dissolved in the acetic acid was then equilibrated and eluted with the same solvent at a flow rate of 0.9 mL.min-1. The elution was m onitored at 210 nm .Three main fractions(Peak I.Peak I and Peak 111)were collected respectively. Their average chain length were700. 500,300, respectively measured.The amino acid composition of soybean minor peptides. was estimated by amino acid analyzer.We used formular: amino acid of peptides = total amino acid– free amino acid.The soybean minor peptides are effective antioxide factors,based on experiments in old rate.Old Wistarrats(381.75±39.23g) used in all of the experiments were provided by The experimental animal center,Shenyang Medical College. They were housed in a room,and constant temperature.During the first week,all rats had a commercially available basal diet and then randomly assigned into two groups of 10 rats each.Each group was administered for 60d intragastrically with physiological saline(contro1) and soybean minor peptides(6.0g/kg/d) respectively.At the end of the experimental period,rats were killed.Blood samples were collected and liver brain were gotten. The level of SOD activitywas assayed by biochemical methods.Result:1. Optimal condition of pretreatment and hydrolyzing by crude complex trypsinase is given by enzyme quantity of 9000 U.g-1 ,temperature 55℃, pH 8.5 ,time 6h, under the condition of 5.0% substrate concentration.2. The result showed that the best conditions of Optimum condition of neutral proteinase hydrolysis of is given by enzyme quantity 8500 U.g-1, pH 7.5;temperature 45℃; treatment time 2h. Under this condition,the ratio of soybean minor peptides achieves 73.98%.3. The Molecule weight distribution of The soybean minor peptides was estimated by chromatography on a TSK-Gel G2000SWXL .Three main fractions(Peak I.Peak I and Peak 111)were collected respectively. Their average chain length were about 700. 500,300, respectively measured.4. The amino acid composition of soybean minor peptides was estimated by amino acid analyzer.We used formular: amino acid of peptides = total amino acid– free amino acid. The result was 91.2%.5. Thelevel of SOD activitywas assayed:Compared with control,the SODlevel of the blood samples of testing group were significantly increased (P<0.05).But no significant difference was found in the liver and brain.Conclusion:In this work.The new extraction technique of soybean bioactive minor peptides from defatted soy flakes hydrolysized by enzymatic method.In this study,soybean minor peptides were obtained from the pretreatment and hydrolyzing by crude complex trypsinase and neutral proteinase hydrolysis and purification from defatted soybean flakes.Optimal condition of pretreatment and hydrolyzing by crude complex trypsinase is given by enzyme quantity of 9000 U.g-1 ,temperature 55℃, pH 8.5 ,time 6h, under the condition of 5.0% substrate concentration. The result showed that the best conditions of Optimum condition of neutral proteinase hydrolysis of is given by enzyme quantity 8500 U.g-1, pH 7.5;temperature 45℃; treatment time 2h.Under this condition,the ratio of soybean minor peptides achieves 73.98%. And the quality standards of soybean bioactive minor peptides were also established.