Preparation,Isolation,Identification And Activity Of Antioxidant Peptides From Grass Carp(Ctenopharyngodon Idella) Muscle

In this paper,the meat of grass carp?Ctenopharyngodon Idella?was used as raw materials to prepare hydrolysates with high antioxidant activity by two-step enzymatic hydrolysis,and best controllable enzymolysis technics was obtained through step-by-step single-factor experiments.Then,the Grass carp fish hydrolysates?GCHs?was purified by Sephadex G-25 to obtain the target fraction which had the highest antioxidant activity.In addition,Nano–LC–ESI–Q–Orbitrap–MS/MS analysis was conduct to identify the amino sequence of target peptides,and the peptides matching the characteristics of antioxidant peptides was screened out to verify the antioxidant activity.Finally,synergistic and antagonistic effects among antioxidant peptides was studied in order to provide some theoretical basis for its application in health food.?1?Single-factor experiments were used to optimize the conditions of the two-step enzymatic hydrolysis to prepare hydrolysates originated from grass carp meat.The in vitro antioxidant activity(ABTS·⁺and O₂·^-scavenging ability,Fe²⁺chelating ability)and the amino acid composition of GCHs were determined.The results showed that the optimal conditions were fixed as follows:solid-liquid ratio,4%;pH,9.0;volume of enzyme,6000 U/g;temperature,50?;hydrolysis time,2 h.GCHs prepared under this best conditions was rich in hydrophobic and acidic amino acids,which exhibited strong ABTS·⁺and O₂·^-scavenging ability,Fe²⁺chelating ability,with the IC₅₀ value of0.21?5.60 and 2.47 mg/mL,respectively.?2?According to the molecular weight distribution of GCHs,Sephadex-G25 was used to purify GCHs oriented by antioxidant activity,and then Nano–LC–ESI–Q–Orbitrap–MS/MS analysis result to identification of the target peptides.The results showed that GCHs were mainly composed of small molecular peptides with molecular weight distribution below 5400 Da.Five fractions were obtained after purification by Sephadex-G25,in which fraction 3?F3?had the strongest ABTS·⁺and O₂·^-scavenging ability.The IC₅₀ values of F3 scavenging ABTS·⁺and O₂·^-were 0.11 and 0.47 mg/mL,which were 1.91 and 11.91 times higher than that before,respectively.From F3,26peptides with ALC>95%was identified through Nano–LC–ESI–Q–Orbitrap–MS/MS,containing 15 peptides matching the characteristics of antioxidant peptides.?3?Fifteen synthetic peptides were prepared to verify the antioxidant activity as well as synergistic and antagonistic effects by determining ABTS·⁺,DPPH·and O₂·^-scavenging ability.The results indicated that VAGW,WEPPR,and LLPDDGDH had the best ABTS·⁺,DPPH·and O₂·^-scavenging ability,with the GSH equivalent values of 324.83,139.77,529.06?mol GSH/mg,respectively.Trp?Tyr?Pro and Arg are possibly dominated amino acid for peptides to scavenge ABTS·⁺and DPPH·,while Asp seems to be highly correlated with O₂·^-scavenging ability.There were both synergistic and antagonistic effects between synthetic peptides.