Study On Separation And Purification Of Antioxidant Peptides And Structure-Activity Relationship From Abalone Visceral

In recent years,China's abalone consumption market and its farming continues to expand,but the viscera is often used as waste.A large number of studies have shown that abalone offal also have a high nutritional value and utilization value.This subject is based on the abalone offal of Fujian Province,preparation of antioxidant peptides by enzymatic hydrolysis of protease,and through separation and purification,structural identification,peptides were used synthesis and its antioxidant activity was determined.To study its structure-activity relationship,for the study and development of high activity antioxidant peptide to provide a reference.The main results are as follows:The proteolytic conditions of trypsin,alkaline protease,neutral protease and papain hydrolyzate were optimized by response surface experiment with the degree of hydrolysis as the evaluation index.The optimum enzymolysis conditions of trypsin were as follows:the ratio of material to liquid was 1:20 g/mL,the amount of enzyme was 2500 U/g,the enzymolysis temperature was 45?and pH 8.0.The optimum conditions of alkaline protease were 1:30 g/mL,the amount of enzyme was 3000 U/g,the enzymolysis temperature was 55?,and the pH was 9.5.The optimal enzymolysis condition of neutral protease was 1:30 g/m L,the amount of enzyme was 3000 U/g The optimum conditions for the hydrolysis of papain were 1:30 g/m L,the amount of enzyme was 3000 U/g,the enzymolysis temperature was 60?and the pH was 6.5.Under the above conditions,The hydrolyzability of the hydrolyzate was 15.98±0.21,25.14±0.42,18.67±0.32 and 18.21±0.71%,respectively.The molecular weight of the hydrolyzate was mainly distributed in the range of 5000 to 400 Da,the four hydrolyzate DPPH scavenging rates were 38.58±0.56,29.65±1.21,28.39±1.88 and 44.81±1.78%.The enzymatic hydrolyzate of trypsin,alkaline protease,neutral protease and papain was isolated and purified by Sephadex G-15 gel chromatography and HW-40f medium pressure column.The macromolecular peptide components A,the medium molecular weight peptide component D and the small molecular weight peptide component E?AVH-TE,AVH-AE,AVH-NE and AVH-PE?were determined by in vitro antioxidant activity,and the small molecular weight peptide component E The antioxidant capacity was significantly higher than that of peptide component A and peptide component D.The IC₅₀ of remove DPPHfree radicals were0.832,1.935,1.793 and 1.138 mg/m L;The EC_A700nm=0.5 value of the reduction force was2.176,3.464,2.107 and 1.987 mg/mL,and the inhibition rate of linoleic acid was90.4±4.88,89.8±1.20,90.2±6.77 and 89.9±1.4%on the 7th day.Eight major target components were obtained by RP-HPLC separation.Sixteen target peptides were identified by structure identification:METY,YHGF,QCVR,QSCARF,AAPAVSGR,NRFGVSR,PVPPYKA,AAQYSRN,VHAEPTK,GCYVPKC,NSHVVR,AANNSTR,TIDCDR,CIGYDR,DDITRD,DVAFMR.In vitro antioxidant activity evaluation,the results showed that only the fragments containing Cys were obviously active in the removal of DPPH·free radical and reducing force.In the removal of ABTS·⁺free radicals,the peptide containing Cys and Tyr had obvious activity;In the inhibition of linoleic acid autoxidation,containing Tyr,Cys,His,acidic amino acids and basic amino acids showed inhibition of linoleic acid autoxidation.Through the correlation analysis also found that hydrophobic amino acids in the inhibition of linoleic acid oxidation also has a great contribution?correlation coefficient of-0.532?.QSAR analysis showed that the lateral volume of the N-terminal amino acid adjacent to the Cys residue,acidic amino acids and basic amino acids had a large effect on the DPPH·radical scavenging rate.The position of Tyr in the peptide chain,tyr ortho-amino acid species and basic amino acidshas a significant effect on the removal of ABTS·⁺free radicals.In cell antioxidant assays.The results showed that the four peptides had no toxic and side effects on the cells,and the activities of GSH-Px,CAT and SOD in the cells were increased to decrease the content of MDA and the supernatant of LDH in the supernatant.The results showed that the three synthetic peptides containing Tyr YHGF,GCYVPKC and CIGYDR could decrease the content of MDA and the supernatant of LDH in the cells were significantly stronger than those containing only Cys.The GCYVPCC,TIDCDR and CIGYDR containing Cys were significantly higher the activities of CAT,SOD,GSH-Px were significantly stronger than those without Cys.