Screening,Separation And Purification,Structure Identification Of Peptides With Antioxidant And Anti-aging Effects

The aggravating trend of aging population in China,a series of health,medical and social problems have gradually emerged because of aging.There is a close relationship between aging and oxidative stress damage caused by free radicals.Researching and discoverying functional substances with antioxidant and anti-aging effect is an important way to deal with aging and its related diseases.Most of the bioactive peptides have biological properties beneficial to the human body,such as the advantages of absorption easy,high bioavailability and biological functions.It is promising functional factors.Soy protein isolate and sea cucumber protein are typical representatives of plant and animal proteins.They both have a rich and reasonable amino acid composition.In this paper,soy protein isolate and two sea cucumbers were used as raw materials for enzymatic hydrolysis,in order to screening out the enzymatic hydrolysate with strong anti-aging activity.Then,using the method of separating,purifiing and dentifiing to analyzing the peptides structure.Finally,verifiing the antioxidant activity of the identified polypeptide by oxidative damage model.Alkaline protease was used to hydrolyze two species of sea cucumber protein,then two sea cucumber hydrolysates?HP-1 and HP-2?were obtained.The yields of the peptides were37.19±2.03%and 30.13±1.68%,respectively.Soy protein isolate was hydrolyzed by Alkaline protease and flavorzyme,then soybean isolated proteinase?SP?was obtained.Its average yield of the polypeptide was 42.31±2.04%.Then,a model of aging mice induced by D-galactose were used to evaluate the anti-aging effects of the three hydrolysates.The results showed that HP-1,HP-2 and SP had the ability of anti-aging and improving memory,and SP had the best effect.In the shuttle box experiment,the total shock time of mice which ate the SP?34.21±6.71s?were significantly reduction compared with the model group?53.98±7.97s?and close to the normal group?37.75±6.67s?.Other indicators also tend to normal transition.In the water maze experiment,the latency of the mice which ate the SP?14.26±3.35s?was significantly reduction compared with the model group?35.40±9.28s?and close to the normal group?37.75±6.67s?.Other indicators also tend to normal transition.In addition,the results of biochemical indicators test showed that these three hydrolysates could effectively stabilized the levels of SOD,MDA,GSH-Px,AGEs in vivo and tended to recur a normal level.Similarly,SP had the best effect.All results indicated that the anti-aging activity sequence of the three hydrolysates was SP>HP-2>HP-1,so SP was selected for further analysis.The experiment results of screening antioxidant models in vitro showed that only the sequence of DPPH·clearance activity was SP>HP-2>HP-1 which is the same with the result of animal experiment.So,it was selected as a guide for next experiments.The hydrolysates SP was separated by DEAE-52 and obtained three fragments?fa,fb,fc?.Molecular weight distribution measured by HPLC method showed that 80.37%and 84.26%of the peptides had a molecular weight of less than 1000Da in SP and fa,respectively.The fa with the best anti-oxidative capacity conducted by DPPH·clearance were selected for Sephadex G15 experiment.After fa was separated and purified by Sephadex G-15,fd were selected for UPLC-MS/MS experiments Two peptides from Soybean were identified and their sequences were WPK and AYLH.The two peptides were obtained by solid-phase synthesis under the standard Fomc strategy.And the purity was above 99%.Peptides had no significant effect on o PC12 cells within a certain concentration range.After pre experiment,the modeling concentration was selected H₂O₂ at 0.4mmol/L.WPK and AYLH all markedly increased the viabilities of the PC12cells.The two peptides also could inhibit H2O2-induce reactive oxygen species formation in PC12 cells.That indicated the protective effect of WPK and AYLH on H₂O₂-induced PC12injury.In conclusion,SP and soybean peptide?WPK,AYLH?were proved to be a potential functional food for anti-aging and anti-oxidation.