Study On Role And Mechanism Of Collagen Degradation During Textural Deterioration Of Refrigerated Grass Carp (Ctenopharyngodon Idellus) Fillets

Freshwater fish is regarded as a delicacy by consumers due to its advantage of high proteins and nutritional value.Along with changes in consumption habits and advances in coldchain logistics,there is an increasing demand for fish fillets.Nevertheless,the problem of muscle degradation always occurs during textural deterioration of refrigerated fillets because of its abundant water content and high activity of endogenous proteinases.At present,the role of myofibrillar protein in refrigerated freshwater fish has been reported,while the mechanism of textural deterioration in fillets is still not clear.Intramuscular connective tissues(IMCTs)are consisted of endomysium,perimysium and epimysium,surrounding muscle fibers,bundles of myofibers and the muscle,respectively.IMCTs have a less amount than myofibrils,while they play important roles in maintaining the integrity and mechanical properties of the muscle.Collagen,as the main component of IMCTs,its role and mechanism in textural deterioration of refrigerated freshwater fish is unknown.Therefore,this study was aimed to investigate the relationship between collagen degradation and textural deterioration of refrigerated grass carp(Ctenopharyngodon idellus),and clarify the contribution of collagen-degrading proteinases in collagen degradation.On this basis,the primary collagen-degrading proteinases were prepared and identified,and their roles in collagen degradation were also studies.The main contents and results were as follows:The relationship between collagen degradation and textural deterioration of refrigerated grass carp was studied,by analyzing the changes in shear force,water-holding ability,drip loss,p H value,microstructure,collagen solubility and correlation analysis.Meanwhile,the changes in shear force,cooking loss and heat soluble proteins of refrigerated grass carp after cooking were studies.At the first 5 days,a striking decrease of shear force to be 43.29% occurred in refrigerated fillets(P < 0.05),accompanied with a decrease of water-holding capacity and an increase of drip loss.Connective tissues were separated from muscle fibers at the first day,with a decline of total collagen and acid-insoluble collagen.In addition,the increase in heat-soluble collagen after cooking process exacerbated the decrease of shear force in refrigerated fillets.Peel correlation analysis showed that the contents of total collagen and acid-insoluble collagen were significantly correlated with shear force of refrigerated grass carp fillets(P < 0.05).In order to investigate the endogenous proteinases involved in collagen degradation,the types,molecular weight and optimum p H values of the collagen-degrading proteinases were measured by gelatin zymography.On this basis,to investigate the effects of collagen-degrading proteinases on collagen degradation,the models of different proteinase inhibitors(Phen,PMSF and E-64)on refrigerated fillets were established,by measuring collagen-degrading proteinase activity,total collagen content,the structure of connective tissue and shear force.Results showed that collagen-degrading proteinases in grass carp were identified as metalloproteinases(66 k Da and 68 k Da),serine proteinases(250 k Da)and cysteine proteinases(29 k Da).After Phen,PMSF and E-64 treatment,the activities of collagen-degrading proteinases in refrigerated fillets were significantly suppressed(P< 0.05).Collagen solubility indicated metalloproteinase and serine proteinase played critical roles in collagen breakdown during the first 3 days,and cysteine proteinase revealed its effect after 3 days.Meanwhile,during refrigerated storage for11 days,the final value of shear force increased 19.68% and 24.33% in PMSF and E-64 treatments when compared to control fillets respectively,whereas the increase after Phen treatment was 49.89%.The disintegration of collagen in post-mortem grass carp fillets was mainly mediated by metalloproteinase and a lesser extent by serine proteinase and cysteine proteinase.In order to elucidate the mechanism of collagen degradation by collagen-degrading proteinases in refrigerated fillets,the crucial enzymes were isolated and identified by mass spectrometry(LC-MS/MS).Results revealed that a 66 k Da metalloproteinase was isolated from by 30%～70% ammonium sulfate fractionation and sequential column chromatographies(DEAE-Sepharose,Phenyl Sepharose and Gelatin Sepharose 4B).After LC-MS/MS analysis,16 fragments(97 amino acids)were completely consistent with MMP-2 from grass carp,suggesting that the purified metalloproteinase was MMP-2.A serine proteinase(SP)with the molecular weight of 250 k Da was also isolated by DEAE-Sepharose and Gelatin Sepharose 4B.Furthermore,a N-glycosylated cysteine proteinase(29 k Da)was isolated by 70%～90%ammonium sulfate fractionation and column chromatographies of SP Sephadex C-50,SPSepharose and Con-A Sepharose.6 fragments(55 amino acids)were consistent with cathepsin L of grass carp,suggesting that the purified proteinase was cathepsin L(CL).The action mode of collagen-degrading proteinases in proteolysis of collagen fibers,collagen monomer and gelatin were investigated,using model system consisting of individual,multiple or sequential combination of CL,MMP-2 and SP.The changes of collagen before and after digestion were measured by scanning electron microscopy,Fourier transform infrared spectroscopy,SDS-PAGE and chemical analysis.Results revealed that CL,MMP-2 and SP could release GAGs by degrading the core protein in proteoglycan,while MMP-2 and SP could release PYD by removing the terminal peptides in collagen fibrils and degrade collagen monomer.Proteoglycan degradation could facilitate the degradation of collagen fibers by MMP-2 and SP.MMP-2 and SP had more collagenolytic activity than CL.For breakdown of collagen fibers,collagen monomer and gelatin,MMP-2 had the strongest degrading ability,and the weakest was CL.The degradation pattern of collagen in model system was basically the same as that of refrigerated flesh,confirming that MMP-2,SP and CL were the main collagendegrading proteinases involving in collagen degradation during textural deterioration of grass carp fillets.This study confirmed that collagen breakdown might be involved in textural deterioration of refrigerated grass carp,and collagen-degrading proteinases(metalloprotease,serine proteinase and cysteine proteinase)was identified as crucial importance.MMP-2,SP and CL were isolated and identified,and their roles in collagen breakdown were confirmed.This study could provide an important information for understanding and solving the mechanism during textural deterioration of refrigerated freshwater fish,and was of great significance for promoting the technological progress of fishery processing industry.