Study On Correlation Of Endogenous Proteases With Texture Deterioration Of Grass Carp (Ctenopharyngodon Idella) During Chilled Storage And Quality Control

Due to its succulent tenderness and rich nutrients as a source of high-quality protein,freshwater fish is very popular among consumers throughout China.Quality deterioration often occurs as a result of bacteriological,endogenous enzymatic activities and chemical changes of freshwater fish.Therefore,efficient preservation technology is needed for extending shelf-life of freshwater fish commodities.Ice-storage and superchilling are widely used for freshness preservation of aquatic products,which could inhibit microbial activities and chemical changes.However,both marine fish and freshwater fish inevitably experience texture deterioration to different content during chilled storage.Furthermore,the mechanism underlying the deterioration process is still unclear.Although the role of endogenous proteases in texture deterioration of marine fish and the enzymatic characteristics of endogenous proteases of freshwater fish are well understood by researchers,the contribution of endogenous proteases to texture deterioration of freshwater fish during chilled storage is still unknown.In the absence of theoretical basis,the quality deterioration problem of freshwater fish demands prompt solution.Therefore,grass carp which is one of the main freshwater fish species in China was selected as subject.Changes in endogenous protease activities and proteolysis,as well as its correlation with texture of grass carp fillets were investigated in order to elucidate major endogenous protease responsible for the texture deterioration and illuminate the proteolysis process of major structural protein induced by endogenous protease of grass carp fillets during chilled storage,as well as develop freshness preservation technology based on endogenous protease inhibition.The present study is of great importance for understanding the mechanism underlying the quality deterioration of freshwater fish during chilled storage.And it also provides a solid theoretical basis for development of high quality freshwater fish products.The changes in protein and its correlation with texture of grass carp fillet during chilled storage were first investigated by analyzing the changes in shear force,proteolytic products,protein composition,myofibrillar structure regulator as well as EDC-crosslinking coupling SDS-PAGE and correlation analysis.Results showed that texture deterioration of grass carp fillets during ice-storage and superchilling was characterized by the decrease of shear force by 45%-51% within 6 days of storage.During storage of grass carp fillet,myofibrillar fragmented.Insoluble protein and salt soluble protein decreased with the corresponding increase of water soluble protein and the accumulation of ?-amino nitrogen,TCA-soluble peptides and non-protein nitrogen.Moreover,the dissociation of actomyosin was attributed to loss of heat shock protein(HSP)including HSP27 and ?B-crystallin and dephosphorylation of myosin light chain.The dissociation of myosin,which released myosin heavy chain(MHC)and light chain(MLC),was mainly ascribed to the loss of UNC45 and HSP90 in myofibrillar proteins.The major changes that induced texture deterioration of grass carp fillets were the dissociation and degradation of MHC and MLC.Also,the dissociation of actomyosin contributed to the decrease in shear force of grass carp fillet within storage of 1 day.The role of endogenous proteases in texture deterioration of grass carp fillet during ice-storage was investigated.From an overall perspective,comparison between the contribution of microorganism and endogenous proteases to texture deterioration was conducted using microorganism-dominated and protease-dominated model systems utilizing sodium azide and iodoacetate acid followed by analyzing microbial changes,endogenous protease activities,proteolysis and shear force.Further investigation into the major protease involved in the texture deterioration of ice-stored grass carp fillets was performed by analyzing the changes in activities and distribution of endogenous proteases and their specific endogenous inhibitors in myocyte,as well as correlation analysis.Results showed that endogenous protease was mainly responsible for texture deterioration of ice-stored grass carp fillets.Remarkably,cathepsin B,L and calpain were the proteases which played major roles in the texture deterioration.Due to the release from lysosome into sarcoplasma and the loss of inhibitor(cystatin)activity,cathepsin B and L activities in sarcplasma and myofibrillar increased by 1.3 and 1.9 folds(cathepsin B),6.0 and 2.4 folds(cathepsin L)respectively,followed by slight decrease and stabilized afterwards.Therefore,cathepsin B and L mainly contributed to softening of grass carp fillet throughout the storage.Additionally,calpain activity increased by 1.0 fold after 12 h and then diminished after 2 days owing to suppression by its active endogenous inhibitor(calpastatin).Therefore,calpain only contributed to texture deterioration of grass carp fillets within 2 days of storage.The action mode of endogenous proteases in proteolysis and dissociation process of myofibrillar proteins of grass carp was investigated using model system consisting of individual,multiple or sequential combination of recombinant proteases and myofibrillar protein of grass carp followed by analysis of EDC-crosslinking coupling SDS-PAGE and HSP,Results indicated that the synergistic mode of cathepsin B,L and calpain in proteolysis and dissociation of myofibrillar proteins was characterized by sequential action.By accelerating the loss of UNC45 and HSP90,cathepsin promoted the dissociation of myosin heavy chain and light chain from thick filament of myofibrillar protein,thus facilitating the further proteolysis action of calpain.Additionally,through boosting the loss of HSP27 and ?B-crystallin in myofibrillar protein which resulting in dissociation of thin filament,cathepsin L facilitated the further proteolysis action of calpain.Moreover,through degrading 140 kDa and 150 kDa fragments of myosin heavy chain which were produced by cathepsin B and L,calpain facilitated the continuous proteolytic action of cathepsin B and L.Furthermore,the degradation mode of myofibrillar protein in model system was in line with that of ice-stored grass carp fillets,convincing the texture deterioration of ice-stored grass carp fillets was mainly caused by proteolysis and dissociation of myofibrillar protein induced by endogenous proteases.Freshness preservation technology for freshwater fish was developed on the basis of protease inhibition.Preliminarily,the effectiveness of allicin and aqueous extracts of Alliums for inhibiting endogenous protease activities and their proteolysis of myofibrillar protein of grass carp was investigated by analyzing residual protease activities and SDS-PAGE.Further detailed investigation into the effective concentration of allicin and type of aqueous extracts of Alliums for alleviating the texture deterioration was conducted by injection of allicin into fillets or immersion of fillets in aqueous extracts of Alliums before ice-storage,followed by analyzing the endogenous protease activities,proteolysis and texture of fillets during storage.Results showed that the effective concentration of allicin for inhibiting the texture deterioration of ice-stored grass carp fillets was 10-100 mmol/L,with shear force that was 39%-51% higher than that of control after 21 days.Additionally,cathepsin B,L and calpain activities of grass carp fillets treated with 10-100 mmol/L allicin decreased to below 11%,40% and 48% of the initial level after 6 days,leading to less extent of proteolysis and dissociation of myofibrillar protein.Among aqueous extracts of Alliums,scallion and garlic with thiosulfinate content of more than 0.9 mmol/g inhibited the cathepsin B,L and calpain activities to below 50%,43% and 36% of the initial level,resulting in significant alleviation of myofibrillar proteolysis and texture deterioration of ice-stored grass carp fillets,as indicated by shear force that were 36% and 22% higher than that of control after 21 days.In the present study,it was convinced that the major protein changes involved in texture deterioration of ice-stored grass carp fillet was the dissociation of myosin and degradation of myosin heavy chain and light chain.It was convinced that the major endogenous proteases responsible for texture deterioration of ice-stored grass carp fillet were cathepsin B,L and calpain.The synergistic mode of cathepsin B,L and calpain in the degradation and dissociation of myofibrillar protein was also illuminated at molecular levels,elucidating the mechanism underlying the texture deterioration of ice-stored grass carp fillet.In addition,freshness preservation technology for freshwater fish was developed based on protease inhibition.