Study On The Preparation And Antioxidant, Calcium Chelating Activities Of Collagen Polypeptides From Silver Carp Bone

According to the China Fishery Statistical Yearbook,the output of freshwater aquaculture reached 29.5984 million tons in 2018,increasing 42.81%than 2008.With the continuous increase of freshwater aquaculture area and production,the freshwater product processing industry has also developed rapidly,such as surimi processing industry.However,at the same time,a large number of by-products produced by the freshwater fish processing industry have not been effectively used,and the resources have been waste aslo makes the processed product more expensive.Fishbone is one of the main by-products of the surimi processing industry.It is rich in nutrients such as protein,unsaturated fatty acids,calcium,and phosphorus.The ratio of calcium to phosphorus is close to 2:1,which easily absorbed by the body and is an excellent nutrition food resource.In order to realize the high-value application of resources,then taking the aim of transforming the waste into the treasure and promoting the sustainable development of freshwater fishery.This article uses silver carp bone as raw material,adopts enzymolysis technology to optimize the preparation of collagen polypeptide,and separation and purification them by Sephadex G-25 molecular chromatography,obtain polypeptide components of different molecular weights and analyze their amino acid composition.Study the antioxidant activity,calcium ion bingding activity of collagen polypeptide and their components,laying a certain foundation for their application.LC-MS/MS was used to identify the structure of the component with the strongest antioxidant activity and high calcium binding activity,and explore the relationship between structure and antioxidant activity,calcium ion chelation.The main research results are as follows:?1?The optimal enzyme for preparing collagen polypeptide by enzymatic hydrolysis of silver carp bones meal selected from neutral protease,alkaline protease,flavor protease,trypsin and composite protease.The yield of collagen polypeptide combined with degree of hydrolysis as evaluation index and examines the influence of factors such as solid-liquid ratio,enzyme addition amount,pH,enzymolysis temperature and time on the preparation of collagen polypeptide by the optimal enzyme.The results showed that the enzyme activity of the composite protease was the highest,reaching 16.80×10⁴ U/g,and the efficiency of preparing collagen polypeptide by the composite protease was the highest under the same enzyme activity and enzymolysis time.Therefore,the composite protease was determined as the optimal enzyme.Through the single factor experiment and response surface optimization were carried out to determine the optimal process for preparing collagen polypeptide by enzymatic hydrolysis of silver carp bone meal with composite protease:the solid-liquid ratio was 1:10?g:mL?,the enzyme addition amount was 8220 U/g,pH8,the enzymolysis temperature was 51?,the enzymolysis time was 4.5 h,at which time the yield of collagen polypeptide was 49.00%.?2?The enzymolysis liquid determined by HPSEC was composed of five kinds of collagen polypeptides with different molecular weights,and the molecular weight was mainly 246 to 4183 Da.After separation and purification by Sephadex G-25,the contents of Gly,Glu,Asp,Ala and Pro in collagen polypeptide and their first four components were relatively high,and the amino acid composition conformed to the characteristics of collagen.The results of antioxidant activity showed that collagen polypeptide and their components all had certain ABTS⁺·scavenging capacity and reducing power,and component F₄ had the strongest,with IC₅₀/OD_1.0 values of 0.28mg/mL and 10.47 mg/mL,respectively.The structural identification of component F₄showed that WGEP,WPDA,GPGPCVW and GGPW at component F₄ had high antioxidant activity and searched for new polypeptide sequences through the BIOPEP database.?3?The results of calcium binding activity of collagen polypeptide and their components showed that collagen polypeptide and their components all had certain calcium binding ability,and component F₃ reached the highest,with chelating rate and calcium mass fraction were 50.10%and 14.01%,respectively.The structure of peptide-calcium complex characterized,the results showed that component F₃ and peptide-calcium complex had certain differences in chemical structure and belonged to different substances,indicating the formation of new substances.The structure of component F₃ was identified,based on the analysis of the relationship between polypeptide and calcium chelating activity,ten peptides with high calcium binding activity were screened out,which were WGGPP,WPTP,YGLP,WLEE,YPMP,QPAGPR,WPGP,LPPP,GPPGYP and LPMAGP in sequence,and the new polypeptide sequence searched according to the BIOPEP database.