Study On The Preparation Of Antioxidant Peptide From Walnut Protein And Its Antioxidant Activity

Walnut meal,as a high-quality protein resource,is only used as feed or fertilizer,and its utilization rate is very low.In order to make full use of walnut protein resources,it is necessary to make intensive processing of walnut meal,and to prepare functional peptides of walnut protein by enzymatic hydrolysis,which is beneficial to improve its commercial added value and prolong walnut industry chain,and its will play a certain role in promoting the walnut industry in china.In this paper,walnut was used as raw material,and the walnut meal was degreased by soxhlet extraction method.The crude protein was further purified by alkali-soluble acid precipitation method.The obtained walnut protein was lyophilized for enzymatic hydrolysis.The hydrolysis conditions of walnut protein were optimized by alkaline protease and trypsin,and the optimum conditions for preparation of antioxidant peptide by synergistic hydrolysis of walnut protein were studied.The results showed that the optimum hydrolysis conditions of alcalase were as follows:hydrolysis temperature 50?,pH 8.5,enzyme dosage 3%(E/S)and reaction time 3 h.Under this condition,the degree of hydrolysis of walnut protein was 8.83%.The optimal reaction conditions of Trypsin were as follows:enzymolysis temperature 40?,pH 8.0,enzyme dosage 3%(E/S)and reaction time 3 h;under this condition,the degree of hydrolysis of walnut protein could reach 8.93%.Finally,the antioxidant activity of Alcalase+Trypsin was higher than that of Trypsin+Alcalase,and the hydrolysis degree and DPPH radical scavenging rate of walnut protein hydrolyzed by Alcalase+Trypsin at 6 h were reaching the highest value of the whole hydrolysis process,respectively 14.11%and 76.88%.Antioxidant experiments on walnut protein hydrolyzate was established in vivo by D-Galactose aging mice.The walnut protein hydrolyzate was given to mice for 167 mg/kg,333 mg/kg,1000 mg/kg daily as low,middle and high-dose groups,and the control group was treated with Vitamin C.The results showed that walnut protein hydrolysates of high dose group is closed to Vitamin C group of experimental results,which can significantly improve D-galactose induced aging mice liver and serum superoxide dismutase and glutathione peroxidase activity(p<0.05),decreased the content of malondialdehyde(p<0.05)in vivo.The results showed that walnut protein hydrolysates had antioxidant and anti-aging effects on mice.The walnut protein hydrolysates were purified by chromatography.Three components were separated by Sephadex G-25 gel chromatography.The scavenging rate of DPPH radical of F3 was the highest among the components,which could reach 71%.Then,F3 was separated by agarose gel Sepharose XL,and five components were obtained.Among them,F3-3 had the highest scavenging rate of DPPH radical,which was 82.7%.Finally,F3-3 was purified by Sephadex G-15 gel column,and three components were obtained,among the three components,F3-3-2 had the highest scavenging rate of DPPH radical,reaching 87%.Component F3-3-2 was identified by LC-MS-MS,and 14 potential antioxidant peptides were identified,respectively:TNPSDSAPGTIR,EDFGGGHPDPN,MLPHHKDAESVAVVTRGRAT,HFREGDVIAFPAGVAH,DIVAIPAGVAH,VREIREGDVVAIPAGVAH,LVYIEQGEGLLGL,ATGEGFEWVSFK,LPHHKDAES VAVVTRGRAT,RGDIVAIPAGVAH,REGDVIAFPAGVAH,GLRGEEMEEMVQSA,EGDIIAFPAGVAH,FMLPHHKDAESVAVVTRGRAT.