Study On The Preparation,Purification And Identification Of Antioxidant Peptides From Grass Carp Skin

Grass carp is one of the most popular freshwater fish in China,and its consumption is the highest in all aquatic products.In the process of production,a large number of byproducts can be formed.However,fish skin,with large amounts of collagen,is a good raw material for preparation of bioactive peptides.Protease hydrolysate from grass carp contains some peptides with physiological activities,such as decreased blood pressure and had antioxidant activity.Only through this method can we solve the problem of high quality protein resource waste,and also provide reliability for the development of functional food and medicine industry.In this study,grass carp skin was used as material which was hydrolyzed to prepare antioxidant peptides.The content includes the effect of protease types on the hydrolysate,optimization of enzymatic conditions,separation and purification of antioxidant peptides from skin protein hydrolysate.Then primary structure of peptides were identified using sequencing technology.Meanwhile,in vitro antioxidant experiments were used to evaluate their antioxidant abilities to explore the relationship between antioxidant abilities and the amino acid sequence of peptides.The main research conclusions are as follows:(1)The protein content of grass carp skin is 31.31%(wet basis).We analysed the composition of amino acid and found that glycine(Gly),alanine(Ala)and proline(Pro)are rich in skin protein,as well as seven kinds of human body essential amino acids are in it,which can be considered as a kind of high quality protein.Acetic acid and pepsin were used to extract the acid soluble collagen(ASC)and pepsin soluble collagen(PSC)from grass carp skin.Higher yield(17.8%)was obtained from PSC than that of ASC(8.9%)based on wet weight.The ultraviolet absorption spectrum showed that ASC and PSC might be pure collagens.The infrared spectra for the ASC and PSC were similar and some bands vibration indicated their structural integrity in the secondary structure.SDS-polyacrylamide gel electrophoresis showed that ASC and PSC should be most likely classified as type I collagen,but there were some differences in the proportion of a and ? components.The results of scanning electron microscopy indicated that ASC looks like disorder fiber porous beam and PSC had fiber filaments with orderly arrangement.In different NaCl concentrations and pH two systems,PSC had a better solubility than ASC.(2)Six proteases,such as alcalase,papain protease,complex protease,neutral protease,flavor protease and pepsin,were chose to hydrolyze the protein of grass carp skin.The degree of hydrolysis(DH),peptide content and antioxidant activity were determined.Finally,based on the DPPH radical scavenging ability,alcalase was selected as the best hydrolytic enzyme.On the basis of single factor tests,response surface design experiment was cond ucted using four factors three levels with DPPH radical scavenging activity as the main index.The optimal conditions of alcalase were as follows:enzyme addition amount of 6.3%,material-liquid ratio of 1:40.6,hydrolysis temperature of 51.3?,and hydrolysis time of 115.1 min,and the scavenging activity on DPPH radical under above conditions reached 54.09%.While under the actual conditions scavenging activity on DPPH radical was 54.98%,and DH was 13.55%.(3)The antioxidant peptides were separated and purified from alcalase protein hydrolysate(GPH)by scavenging activity on DPPH radical.The GPH was fractionated using a Sephadex G-15 column.Fractions were labeled as A,B,C,D and E,pooled separately.The antio xidant activity of fraction E was significantly higher than other fractions.Five peaks(El,E2,E3,E4 and E5)were eluted from fraction E by reverse phase high-performance liquid chromatography(RP-HPLC).E2 was further purified by RP-HPLC once again and separated into two peaks(E21 and E22).However,E21,E4 and E5 displayed significantly higher antioxidant activity.The peptide sequences and molecular masses of purified GPH-E21,GPH-E4 and GPH-E5 were measured using a protein/peptide sequencer,respectively.The sequences of GPH-E21,GPH-E4 and GPH-E5 were determined to be Pro-Tyr-Ser-Phe-Lys(PYSFK,640.74 u),Gly-Phe-Gly-Pro-Glu-Leu(GFGPEL,618.89 u)and Val-Gly-Gly-Arg-Pro(VGGRP,484.56 u).(4)In this study,some in vitro assays were conducted to evaluate their antioxidant abilities.All samples scavenged radicals in a dose-dependent manner.GPH-E21,GPH-E4 and GPH-E5 were able to scavenge DPPH radical and showed concentration-dependent anti-DPPH radical activity with IC50 of 2.459,3.634 and 6.063 mM,respectively.The IC50 of GPH-E21,GPH-E4 and GPH-E5 scavenging activity on hydroxyl radical were 3.563,2.606 and 4.241 mM,respectively.The IC50 of GPH-E21,GPH-E4 and GPH-E5 scavenging ABTS radicals were 0.281,0.530 and 0.960 mM,respectively.Meanwhile,three antioxidant peptides showed excellent inhibition on linoleic acid peroxidation,whereas GPH-E5 could effectively inhibite lipid peroxidation in linoleic acid emulsion system up to 7 days,and the activity was similar to that of glutathione,followed by GPH-E4 and GPH-E21.