Studies On The Enzymatic Preparation Of Low Value Fish Oligopeptides And Its Antioxidant And Anti-fatigue Activities

At present,the annual output of aquatic products in china has reached 70million tons.About 80%of the sea fishing catch is low value marine fish,which is not suitable for hurnan food because of its backwardness of processing and recycling means.It’s usually used as to produce low value-added products such as fish meal,and even discarded directly as waste.Thus natural resources are wasted.The paper aimed at the development and utilization of ocean regenerative resource,to find small molecular peptides with biological activity,which will be beneficial to the development of marine drugs and functional marine health care products,turning waste into treasure.This study dealt with the preparation of oligopeptide from high protein low value marine fish meal by enzymatic hydrolysis.Chemical models in vitro were used to evaluate their antioxidant activities.Ultrafiltration,gel filtration chromagraphy and reversed-phase high performance liquid chromatography were applied to the separation of peptides and LC-MS/MS technology was used to identify the amino acid sequence of the peptide.Finally,animal models in vivo was used to evaluate the antifatigue effect of target peptides,and the relationship between antioxidant activity and antifatigue effect of target peptides were discussed.The research results of this study were as follows:In order to optimize the production process of functional polypeptides from low value marine fish meal protein,six different enzymes（including alcalase,protamex,flavourzyme,papain,trypsin,neutral protease）were used to hydrolyze the low value marine fish meal.The effects of temperature,pH,reaction time,enzyme amount,mass ratio of alkaline protease and neutral protease on the degree of hydrolysis（DH）were researched by using single factor experiment and response surface methodology.The results showed the optimal enzymatic hydrolysis conditions with alkaline protease and neutral protease enzyme activity ratio of 6:4 successively joined,E/S=2500 U/g.The alkaline protease kept hydrolyzing for 5 h when the pH was 8.Until the pH value dropped to 7.0,the neutral protease was added and continued stirring for another 6 h,enzymolysis temperature 50°C.The hydrolysis degree of low value marine fish meal protein under these conditions could be41.84%.The relative molecular weight distribution of oligopeptides from low value marine fish after enzymatic hydrolysis was concentrated between 180¹000 Da,which indicated that the low molecular peptide instead of free amino acids was the main ingredient,although the low value marine fish meal protein was enzymatic hydrolysis by depth.Four different chemical models in vitro were used to evaluate the antioxidant activities of low value marine fish meal oligopeptide（LVMFMO）.Results showed that its reductive potential was as high as that of glutathione（GSH）.The1,1-diphenyl-2-picrylhydrazyl（DPPH）and superoxide anion activities of LVMFMO were a little bit less than that of GSH,while the hydroxyl radical scavenging activity of LVMFMO was much higher than that of GSH.LVMFMO also displayed activities on inhibiting oxidative damage of DNA.Low value marine fish meal oligopeptide was isolated and purified by ultrafiltration,gel filtration and reverse phrase high pressure liquid chromatogram（RP-HPLC）.The purified peptide showed a 2.06-fold higher scavenging activity for hydroxyl radical compared with the crude LVMFMO(the IC₅₀ value of scavenging activity for hydroxyl radical is 2.47±0.09 mg/mL).Finally,the purified antioxidant peptide was identified as Thr-Pro-Ser-Ala-Gly-Lys（559.3 Da）with the help of mass spectrometry of flight time for matrix aided laser absorption ionization.The purified peptide is the first hexapeptide to be found which has significant antioxidant activities.The antifatigue effects of low value marine fish meal oligopeptide IV-4-1 were evaluated.The results showed that low value marine fish meal oligopeptide IV-4-1had no effect on body weight and growth of mice;it could significantly prolong the exhaustion swimming time,the LVMFMO IV-4-1-H,LVMFMO IV-4-1-M and LVMFMO IV-4-1-L groups were 1.53-fold,1.69-fold and 2.13-fold of the control group,and all groups showed a dose-dependent behavior;compared with the control group,the levels of lactic acid and blood urea nitrogen decreased by 22.99-34.77%and 19.63-35.94%,respectively.It also increased the liver glycogen contents of mice and the endogenous cellular antioxidant enzymes in mice by increasing the activities of superoxide dismutase（SOD）,catalase（CAT）and glutathione peroxidase（GSH-Px）.Therefore,low value marine fish meal oligopeptide IV-4-1 could improve the mice’s endurance to exercise and has antifatigue effects.Results also showed that the antioxidant activities of LVMFMO IV-4-1 had good relationship with its antifatigue effect.Results also showed that the antioxidant activities of low value marine fish meal oligopeptide IV-4-1 had good relationship with its antifatigue effect.