Study On Purification And Structure-Activity Relationship Of Antioxidant Peptides From Perilla Seeds

Perilla and its seed are Chinese traditional herbal medicines which were homology of medicine and food,and possess multiple physiological functions.In this thesis,perilla seed protein?PSP?was extracted from perilla seed meal,which is a by-product from the process of perilla seed oil extraction,and then hydrolyzed and purified.Further,the antioxidant activity in vitro and structure-activity relationship of derived antioxidant peptides were explored.PSP was extracted by alkali extraction and acid precipitation from perilla seed meal.Based on single factor test,the technological conditions for protein extraction were optimized by response surface methodology?RSM?.The optimal result showed as follows:temperature was 58.00?,pH was 9.40,ratio of liquid to solid was 11.60,time was 60.00 min and extraction rate was 36.08%.The protein precipitated excellently at pH 4.0,approximately.PSP had higher emulsifyingability?EA?,emulsifying stability?ES?,water absorption capacity?WAC?and oil absorption capacity?OAC?,but lower foaming capacity?FC?,and foaming stability?FS?.The EA and ES were 122.93±2.34 m²/g and 99.60±2.27%at pH 10.0.The WAC and OAC were 5.02±0.26 g/g and 4.14±0.12 g/g at 40?,respectively.PSP was hydrolyzed by four proteases?acid protease,alkaline protease,neutral protease and papain?,and the optimum enzyme was alkaline protease.A Box-Behnken Design was used to optimize the hydrolysis technology and the optimal enzymolysis conditions were time of 5.00 h,protease dosage of 3000 U/g,pH of 9.80 and temperature of 50?.Under the optimal enzymolysis conditions,DPPH radical scavenging rate of the hydrolysates was 73.64%.Perilla seed protein hydrolysate?PSPH?was isolated by Sephadex G-25 molecular sieve chromatography and RP-HPLC orderly.The antioxidant activities were detected by DPPH radical scavenging activity during the following processes.Finally,four peptides were obtained and with strong antioxidant activity,and their amino acid sequence were measured as Ser-Gly-Pro-Val-Gly-Leu-Trp?SGPVGLW?,Tyr-Leu?YL?,Phe-Tyr?FY?and Phe-Asp-Ala-Asp-Ser?FDADS?by ESI-MS/MS analysis.Multiple antioxidant models in vitro were applied to assess the activities of perilla seed peptides.The results showed that all peptides had antioxidant activity inordinately.Not only could perilla seed peptides effectively quench all kinds of free radicals,and inhibit linoleic acid peroxidation and lipid peroxidation in rat liver.Especially,two dipeptides YL and FY exhibited the highest antioxidant activity.CHO cell and HepG-2 cell were incubated with certain concentration of the antioxidant peptides respectively.Results showed that all peptides did not have cytotoxicity,except high concentration of FY to HepG-2 cell.An oxidative damage model of HepG-2 cells produced by H₂O₂ was established,the antioxidant peptides possessed protective effects against H₂O₂-induced oxidative stress.Trp was replaced with Gly in the SGPVGLW sequence to get a new mutational peptide SGPVGLG.Tyr of YL and FY sequence was removed,and obtained another mutational peptide FL.Compared with maternal peptides,the antioxidant activity of mutational peptide SGPVGLG and FL decreased significantly,which indicated that Trp and Tyr made great contributions to antioxidant activity.