Extraction,Structural Characterization And Activity Evaluation Of The Antioxidant Peptide From Loach Mucus

In this paper,loach mucus freeze-dried powder was used to prepare a novel antioxidant peptide from enzymatic hydrolysis of loach mucus.Three separation methods were used to isolate and purify the peptide from loach mucus.The RP-HPLC and MALDITOF MS were used to identify the amino acid sequence of the peptide.And FTIR was used to analyze the secondary structure of the peptide.Finally,it was studied that the antioxidant activity and thermal stability of the peptide from loach mucus.The protein content of loach mucus freeze-dried powder is 71.48%.The content of amino acids in loach mucus is rich and the composition is reasonable.It is abundant in essential amino acids,flavor amino acids,antioxidant amino acids and branched chain amino acids.It can be used for protease hydrolysis to produce bioactive peptides.It was used that trypsin,papain,alcalase,neutral protease and flavor protease to hydrolyze loach mucus.It was taken the degree of hydrolysis and antioxidant capacity as indicators.In addition,alcalase was determined to be the most suitable protease for preparing the antioxidant peptide from loach mucus.On this basis,it was analyzed that the effects of temperature,pH,substrate concentration,enzymatic time and enzyme addition on alcalase hydrolysis of loach mucus freeze-dried powder.The results showed that the optimal enzymolysis conditions were temperature 50 ℃,pH 8.5,substrate concentration 8.77 mg/mL,enzymatic time 5.5 h,enzyme addition 8625 U/g protein,the degree of hydrolysis was 35.14%,the DPPH radical scavenging activity was 58.32%,there was no significant difference with the model prediction value(P>0.05).The peptide from loach mucus was isolated and purified by ultrafiltration,gel filtration chromatography and prep-HPLC.The antioxidant capacity of the peptide increased by more than 3 times than that before separation,indicating that isolation and purification can significantly enrich the antioxidant peptide from loach mucus.Finally,it was identified by MALDI-TOF MS that the amino acid sequence of the peptide from loach mucus.And it was analyzed that the amino acid composition of the peptide was analyzed.The amino acid sequence of the purified peptide,an antioxidant 12-mer peptide with molecular weight of 1352.10 Da,was identified as SLIGRTLVVHEK.Three chemical models in vitro were used to evaluate the antioxidant activity of the mucopolysaccharide peptide.The results showed that the peptide had strong antioxidant activity,and the DPPH radical scavenging ability was 44.65 ± 0.23%,the hydroxyl free radical scavenging ability was 34.12 ±0.09%,and the reducing power was 0.124±0.004,which were lower than that of ascorbic acid at a concentration of 1 mg/mL.The secondary structure of the peptide was characterized by FTIR.The results showed that the relative content of α-helix structure,β-fold structure,β-corner structure and irregular curl structure was 8.78%,22.32%,54.98%and 13.93%,separately.The microstructure of the peptide from loach mucus was observed by SEM and its thermal stability was analyzed by TGA.The SEM results showed that the peptide from loach mucus presented irregular lamellar and porous structure.The TGA results showed that the weight loss rate and degradation temperature of myxopeptide were 75.45%and 293.46℃,respectively.It indicates that the peptide from loach mucus had good thermal stability.