Preparation Of Antifreeze Peptides From Tilapia Skin Collagen By Enzymatic Hydrolysis And Preliminary Study On Their Acitivity Improvement

Protein freezing and denaturation occurs in frozen products of aquatic products,which causes loss of juice of aquatic products,deterioration of nutrients and flavor.Polyphosphates and sugars are the main antifreeze used in aquatic processing.However,accumulation of polyphosphate in the human body is likely to cause imbalance of calcium and phosphorus,and the addition of sucrose will affect the flavor of the product.The development of new antifreeze agents has important application prospects.In the current research,it is found that the antifreeze protein and the antifreeze polypeptides have the problems of low preparation rate and high use cost.In this study,tilapia skin collagen was used as raw material,and hydrolysis degree?DH?was used as an index to optimize the enzymatic hydrolysis process of collagen,evaluation of the antifreeze activity of the collagen polypeptides,and isolation and purification to obtain a polypeptides having high antifreeze activity,to explore whether supramolecular assembly can optimize collagen polypeptides antifreeze activity.Provide a theoretical basis for the development of a new antifreeze for peptides with high antifreeze activity.The main research contents and results are as follows:?1?The collagen in tilapia skin was extracted by hot water,and the preparation process of collagen peptides was optimized by using hydrolysis degree as index.The extracted high-purity collagen conforms to the characteristics of type I collagen.Hydrophilic amino acids accounted for 33.28%.On the basis of single factor experiment,orthogonal experimental analysis was performed to obtain the optimal conditions for enzymatic hydrolysis for flavourzyme shown as following:p H 7,temperature 50?,time 6 h,enzyme addition 5000 U/g,substrate concentration 40mg/m L and the degree of hydrolysis reached to 22.15±0.35%.The optimal conditions for enzymatic hydrolysis for alcalaseas following:p H 7,temperature 60?,time 3 h,enzyme addition 5000 U/g,substrate concentration 20 mg/m L and the degree of hydrolysis reached to 12.03±0.12%.?2?The antifreeze activity of collagen peptides was evaluated by thermal hysteresis activity,residual activity of catalase after freezing and biochemical and tissue structure changes in frozen muscles of scallop scallops.The collagen polypeptidea and the polyphosphate antifreeze have a freezing protection effect on the catalase,and the polypeptidea concentration is directly proportional to the antifreeze effect,the THA of the collagen alcalaseas hydrolysate is higher than that of the flavourzyme hydrolysate.The frozen squash of the scallop body was frozen and denatured.Compared with the blank control group,Collagen peptidea can inhibit the increase of thawing rate of scallop muscles and the decrease of salt-soluble protein content,total sulfhydryl content and Ca²⁺-ATPase activity after freezing,the effect is positively correlated with the added amount,When the amount of collagen polypeptides added is 3%,the effect is comparable to that of the polyphosphate antifreeze.Add polyphosphate antifreeze and collagen peptides can protect the structural integrity of the scallops after the frozen muscles.?3?The collagen polypeptides was isolated and purified by Sephadex G-25 gel column chromatography and C18 reversed-phase high performance liquid chromatography,and the antifreeze activity of the fraction was evaluated.The GF₂₄component has high antifreeze activity,its relative molecular weight is 731.34 Da,and its amino acid sequence is PGDGDSA.The molecular weight of the GA₃₃component is 454.26 Da,and the amino acid sequence of the GA₃₃component is NHGK.?4?The collagen polypeptides with exogenous proline and glycine under different proteases for supramolecular assembly.After GF assembly,,the content of free amino acid increased,the molecular weight of trypsin and papain treatment group increased in the range of 0.1-5 k Da,the residual activity of catalase decreased,and the THA of trypsin and papain treatment group increased slightly.The free amino acid content of GA added papain and trypsin decreased,the residual activity of catalase increased,and THA increased.The collagen polypeptides and glycine can undergo supramolecular assembly under added trypsin and papain.The increase of the specific gravity of the small molecule polypeptide can increase the antifreeze activity of the collagen polypeptides.