Protein Oxidation,Cathepsins Activity And The Quality Changes Of Bighead Carp(Aristichthysnobilis)during Low-temperature Storage

The protein oxidation and endogenous protease are main reasons for quality changes of freshwater fish during storage.There is no clear conclusion about the effects of protein oxidation on water-holding capacity(WHC)and texture of fish.At the same time,the impact of cathepsins on microstructure of muscle myofibrils is not comprehensively studied.We selected the bighead carp(Aristichthys nobilis),as the research object in the present study,explored the effects of protein oxidation and cathepsins on the protein degradation of fish under low-temperature storage,established the linear correlation between protein oxidation,WHC and texture,investigated the cathepsin activities in lysosomes,heavy mitochondria,myofibrils and sarcoplasma and their damaging effects on microstructure of myofibrils,analyzed the effects of the protein oxidation and cathepsin on the quality of fish muscle and deterioration of protein structure.Based on that,we investigated the influence of protein oxidation on the gel properties.(1)The protein oxidation parameters(salt-soluble and water-soluble protein contents,total sulfhydryl and disulfide content,Ca2+-ATPase activity,surface hydrophobicity and carbonyl contents),WHC(drip loss)and texture of bighead carp under 4℃ and-3℃ were measured and the linear correlation between these indicators were analyzed.The result indicated that for the fish under same storage temperature,less protein oxidation indicated the better texture and WHC for fish.There existed significant linear relationship between protein oxidation parameters with WHC and texture characteristics for fillets under 4℃ and-3℃.It showed less protein oxidation but better WHC and texture in fish at-3℃ than at 4℃,which was probably caused by ice crystals formed in fish at-3℃.(2)The changes of cathepsin B,cathepsin B+L activities,SDS-PAGE of sarcoplasmic and myofibrillar proteins,texture,microstructure of myofibrils in bighead carp fillets at 4℃ and-3℃ were measured during 0-72 h after slaughter.The results indicated that fillets at-3℃ showed higher cathepsin activity in sarcoplasm and lower cathepsin activity in myofibrils compared with fillets at 4℃.Correspondingly,we observed degradation of a-actinin by cathepsin L in fillets at 4℃ and degradation of creatine kinase by cathepsin B in fillets at-3℃ during the storage.The lower cathepsin activity in myofibrils for fillets at-3℃ might induce a more intact microstructure of myofibrils than fillets at 4℃.(3)The protein oxidation(salt-soluble and water-soluble protein contents,total sulfhydryl and disulfide content,Ca2+-ATPase activity,surface hydrophobicity and carbonyl contents),WHC(drip loss,NMR-T2 and NMR-P2)and texture of fillets at-12,-20 and-28℃ were evaluated during 0-16 weeks and the linear correlation between these indicators were analyzed.The result indicated that the texture and WHC of fillets were better when frozen temperature was lower,which followed by less protein oxidation.Namely the fillets at-28℃ showed better WHC and texture than that of fillets at-12 and-20℃,correspondingly the fillets at-28℃ showed less protein oxidation than that of fillets at-12 and-20℃.There existed significant linear relationship between protein oxidation parameters with WHC and texture characteristics for fillets under frozen storage.(4)The changes of cathepsin B,cathepsin B+L activities,SDS-PAGE of sarcoplasmic and myofibrillar proteins,microstructure of myofibrils in bighead carp fillets at-12、-20 and-28℃ were measured during 0-16w after slaughter.And we also hydrolyzed the isolated myofibrillar protein from fillets at-20℃ using exogenous cathepsin B and L.The results indicated that frozen storage would inhibit the cathepsins activity during 0-8w,while the inhibition would be diminished during 8-16w.At 16w,the cathepsin activity in myofibrils and sarcoplasm of fillets at-12℃ were higher than that of fillets at-20 and-28℃.Correspondingly,we observed more serious degradation of myofibrillar and sarcoplasmic protein occurred to fillets at-12℃ than that of fillets at-20 and-28℃.At the same time,the microstructure of fish at-12℃ was more damaged than that of-20 and-28℃.The degradation of MHC from frozen fillets by exogenous cathepsins was less than the raw fresh MHC(Ow),which indicated that the sensitivity of myosin to the cathepsins was decreased when protein oxidation existed.(5)The changes in protein oxidation and gel texture and water-holding capacity(WHC)of isolated myofibrillar protein from bighead carp fillets were estimated during frozen storage and under different H2O2 concentrations,which were used to represent in vivo and in vitro conditions,respectively.The results indicated that there existed similar protein oxidation condition for myofibrillar protein under frozen storage and hydroxyl radicals.We found a certain range in content of disulfide crosslinks(0.91 mol/105 g protein)would promote gel hardness.Mild protein oxidation caused by a certain degree of frozen storage and hydroxyl radicals can promote gel texture and WHC.