Enzymatic Preparation Of Antioxidant Oligopeptides From Paphia Undulata And Their Exogenous Amino Acids Stress Modification

P.undulata is a marine shellfish resource with high-protein and low-fat.With the increased in production,the problem we face its' low utilization.The predominantly way was shelled fresh sales,it makes P.undulata development slowly.With the improvement of people's living standards,bioactive peptides become hot spot.Used the low price protein resources as raw materials to prepared bioactive peptides solves the contradiction problem we faced.In this paper,P.undulata as raw materials prepared antioxidant peptides,figure out the whole process of antioxidant oligopeptides release regularity and used coercion by enzymatic modification techniques to further improve the activity of antioxidant peptides and explore the reactions mechanism.Lastly,the modified products were isolated and purified,and evaluated the isolated components antioxidant activity.The main findings are as follows:?1?The degree of DH,DPPH radical and hydroxyl radical scavenging rate evaluation were used as index to screen the optimal protease.Results indicated that the DH,DPPH radical and hydroxyl radical scavenging activity prepared by Alkaline protease hydrolysates was significantly higher than the other three proteases?P<0.05?,so choose Alkaline protease as experimental enzyme.Through single factor experiment and response surface optimization of enzyme hydrolysis conditions to determine the optimal process conditions:temperature,55?,the amount of enzyme,3300U/g,time,220 min.?2?Analyzed the change of DH and radical scavenging rate in the whole enzymatic hydrolysis process,established first-order hydrolytic kinetic model:DH=12.20ln?t+31.48?-42.59,?R²=0.9874?.Also first-order oligopeptides release kinetic model established by molecular weight distribution?HPSEC?statistics,Q=59.655[1-exp?-0.0098?t+96.42??],?R²=0.9673?,and the oligopeptides release constant?0.0098?indicating that the oligopeptide release is an relatively slow process.Combine RP-HPLC and TOF-MS showing:with reaction time the extension of the low molecular weight oligopeptide content increases,the kinds of hydrophobic peptides increased and antioxidant activity increased.After 220 min,because of less than 3000 Da molecular weight peptides continue be digested,some active fragment or sequence was cut off,resulting in the antioxidant activity decreased.?3?The degree of reduce the amount of free amino acids,DPPH radical and hydroxyl radical scavenging rate evaluation were used as index to screen the optimal protease and amino acids,chosen Papain as experimental enzyme and selected of Cys?Lys=3:7 as experiments amino acids.Through single factor experiment and response surface optimization of enzymatic modification conditions to determine the optimal process conditions:enzyme dosage,3800U/g,the amount of amino acids added 0.56g/g,reaction temperature,34?.?4?Compared analysis the substrates and product's antioxidant activity of exogenous amino acids stress modification reaction,it found the DPPH radical and hydroxyl radical scavenging rates were enhance from 38.67%and 38.60%to 90.24%and 50.44%?P<0.05?.RP-HPLC and TOF-MS's result showing,in the reaction products,the molecular weight is reduced in the whole.The 3kDa⁵kDa molecular weight peptides were't appear significantly decreased,while the contents of 1kDa³kDa molecular weight peptides have increased significantly.And exogenous amino acids stress modification reaction makes peptide species increased,new antioxidant peptides was appeared.?5?The Sephacryl S-100 have better separating effect and isolated four peaks P₁-P₄,where the highest antioxidant activity peak P₂ molecular weight range of 9225-521 Da,DPPH radical and hydroxyl radical scavenging rates were 82.16%and 41.63%respectively.