Preparation, Isolation And Purification Of Peanut Peptides From Peanuts Protein By Enzymatic Hydrolysis And Antioxidant Activity

The peanut was very abundant in our country. Peanut protein with high nutritional value,containing eight essential amino acids in the human body.However, peanut production process backward, resulting in severe degeneration of peanut protein. At present,the exploiture of functional peptide has been a research focus.It was studied that the application of enzymatic hydrolysis to prepare peanut antioxidant peptides in this paper.In this paper,firstly,the five enzymes concluding trypsinase,flavourzyme, papain,pepsin and alcalase were individual applied to hydrolyze the peanut protein. The temperature,reaction time,protease amount,substrate concentration and pH effected the hydrolysis degree(DH) and trichloroacetic acid solbule nitrogen content(TCA-NSI) of peanut protein.Based on it,Alcalase and flavourzyme were chose to hydrolyze peanut protein.Then a predictive polynomial quadratic model was set up by means of central composite design to optimize the enzymolysis technology of peanut protein. The optimal condition of alcalase was concluded as follows:temperature 55℃, pH 8.4, substrate concentration 4.31%, protease amount 3.39%, ime 4h.Under this condition TCA-NSI reached to 74.88%,DH was 23.40%. The optimal condition of flavourzyme was:temperature 59.3℃, pH7.45, substrate concentration 5.60%, protease amount 5.03%,time 2h. Under this condition, TCA-NSI was 77.17%,DH was 22.84%.Under the optimal conditions, Chose 15,30,60,90,120,150,180 minutes respectively as hydrolysis time.Then the antioxidant activity under different time of hydrolysis was studied. The reasults showed that two protease exhibited the stronger power of deoxidization capacity, antioxidation capacity and free radical scavenging capacity.And hydrolysis degree, raduce capacity,antiodxidation activity, free radical scavenging rate increased with hydrolysis time. Antiodxidation activity of alcalase hydrolyzed had stranger than flavourzyme hydrolyzed.The two protease of antioxidant activity stronge with the concentration increased.and in the same concentration conditions, Vc had the strongest antioxidant activity,the antioxidant activity of alcalase hydrolyze had stronger than flavourzyme hydrolyzed.The dialysis bag of 8K Da,3.5K Da and 1K Da were used to classify the hydrolysate derived from alcalase and flavourzyme.Different enzymatic hydrolysate collcated same thickness.The content of<1K Da fraction were 76.21% and 83.42% of the total hydrolysate from alcalase and flavourzyme respectively.All hydrolysate with different molecular grades were both appeared free radical scavenging capacity and which was increased with the lowering of molecular mass. The<1KDa exhibited the higher radical scavenging capacity of 78.49%(alcalase) and 67.83%(flavourzyme),respectively.The scavenging capacity of fraction<1KDa from alcalase was higher than flavourzyme.The Tricine-SDS-PAGE showed that the calssification by dialysis bag was apparently and the molecular mass of the three grades was obviously less than that of the original peanut protein,what's more,the molecular mass distribution of them gave a descending tendency.The antioxidant peptides J22 and F22 isolated by continuous chromatographic technique of Sephadex G-25 column and Sephadex G-15 column from the<1K Da fraction of alcalase hydrolysate and flavourzyme hydrolysate exhibited the radical scavenging capacity of 85.16%(J22) and 76.83%(F22), respectively.The analysis of automatic amino acids showed that amino acid compositions of J22, F22 fraction from peanut isolate protein changed. Fifteen amino acids were detected in peanut isolate protein,nine amino acids were detected in J22 fraction and F22 fraction, respectively.Gly didn't measured in peanut isolate protein,but Gly,Glu,Ala,Val,Leu and Phe didn't measured in J22 fraction and F22 fraction, respectively.Meanwhile,Cys,Met,Ile,Tyr and His of percentage composition in the in J22 fraction and F22 fraction were higher than peanut isolate protein, respectively.The highly active antioxidant peptides J22 frction and F22 fraction were analyzed by HPLC-MS and the results indicated that the single component separated from gel column were the maxiure of peptides of different molecular weights,and their molecular weights were manily distributed in the range of 200-400Da, respectively.According to the average molecular weight of 128 amino acids,thus it can be inferried J22 fraction and F22 fraction were componented of two or three amino acids of small peptides, respectively.