Preparation,Isolation And Purification Of Peanut Antioxidant Peptide And Their Antifatigue Activity In Mice

Peanut meal,as a by-product of peanut oil,has been used in feed and other low-value products.Peanut meal is high in protein content and rich in amino acids,which is a high quality plant protein raw material.An increasing number of antioxidant peptides have been identified from food plant sources as well as from plant-based agricultural products and food processing by-products.In this study,the enzymatic hydrolysis process,separation and purification of arachidin and the function activity of alleviating fatigue in mice were studied,so as to provide a basis for the maximum utilization of peanut meal and other by-products.(1)after extrusion and ultrasonic pretreatment of peanut meal as raw material,using the alkaline protease enzymolysis,hydrolysis degree as the index,by single factor and response surface optimization,for best enzyme hydrolysis of peanut protein peptide:p H 9.5,plus the amount of enzyme 7200 U/g,120 g/L,initial concentration of enzyme solution temperature of55?,120 min,enzymolysis time under the condition of the hydrolysis degree of 39.15%.Comprehensive consideration,in conjunction with antioxidant indicators selected with high activity and high degree of hydrolysis enzyme solution,thus determine the peanut antioxidant peptide best digestion process:p H 9.5,plus enzyme quantity is 8000 U/g,90 g/L substrate concentration,enzymolysis temperature 55?,enzymolysis time 120 min,under the condition of the hydrolysis degree was 38.10%,hydroxyl radical scavenging capacity of 79.56%.(2)The enzymatic hydrolysates of peanut meal were separated by ultrafiltration to obtain three components with molecular weight<3 k Da,3-10 k Da and>10 k Da,and their antioxidant activity was determined in vitro.In ABTS and DPPH radical scavenging experiment,the antioxidant activity of 3-10 k Da component was the best.In the hydroxyl radical scavenging experiment,when the mass concentration of>10 k Da reached 9 mg/L,the scavenging ability of>10 k Da was similar to that of glutathione.In the experiments of Fe²⁺chelating capacity,oxygen free radical absorption capacity(ORAC)and reducing power,the antioxidant capacity of<3 k Da component is the best.The results showed that<3 k Da had better antioxidant activity.The<3 k Da fractions were separated and purified by Sephadex G-25,F1,F2,F3 and F4 were collected,and the antioxidant activity was determined.In the DPPH free radical scavenging experiment,the scavenging ability of F2 was better than that of the other three components.In the experiments of Fe²⁺chelating ability,F3 and F4 fractions chelate metal ions better than the other two fractions.In ABTS cation,hydroxyl radical scavenging,ORAC and reducing power experiments,F4 polypeptide had the strongest free radical scavenging activity.Overall,the antioxidant activity of F4 component was better.C1and C2 were obtained from the separation of F4 components by Sephadex G-15.In DPPH and hydroxyl radical scavenging experiments,the antioxidant activity of component C1 was higher than that of component C2.The antioxidant activity of C2 component was better than C1 in ABTS cation,Fe²⁺chelating ability,ORAC and reducing power tests.(3)The relieving effect of peanut antioxidant peptide(<3 k Da)on fatigue model mice was evaluated by swimming experiment in mice.The results showed that using exhaustive swimming time as fatigue index,endogenous antioxidant enzymes included superoxide dismutase(SOD),malondialdehyde(MDA),catalase(CAT),total antioxidant enzyme(T-AOC),and anti-fatigue indexes including lactic acid(LA),lactate dehydrogenase(LDH),urea nitrogen(BUN),liver glycogen,interferon-?(IFN-?),interleukin-4(IL-4).The results of this experiment showed that the peanut antioxidant peptide<3 k Da could better increase the swimming time of mice.The levels of SOD,CAT,T-AOC and LDH in low and high dose groups were up-regulated,liver glycogen storage was increased,MDA level was down-regulated,the accumulation of La and BUN in mice was decreased,and the contents of anti-inflammatory factors IFN-?and IL-4 were increased.These results indicated that<3 k Da peanut antioxidant peptide could improve antioxidant capacity and relieve fatigue in mice.