The Effect Of Calpain 2,20S Proteasome And Cathepsin B+L On Postmortem Pork Water-holding Capacity

It was well known that postmortem proteolysis played a role in meat tenderization.Especially degradation of key myofibrillar proteins influenced tenderness and water holding capacity.However,the relationship of proteolysis and postmortem meat quality was not been studied well,mechanism was not clear.The main endogenous protease systems to be considered in postmortem proteolysis included calpains,proteasome and cathepsins.In the present thesis,gene expression of CAPN2,PSMB1,PSMB2,PSMB5,CTSB and CTSL were investigated by real time qualification PCR,the activity changes of calpain2,20 S proteasome,cathepsin B+L and the changes of desmin,?-actinin,water holding capacity during postmortem storage were investigated.The relationship between genes expression of the endogenous enzymes,activity of the three protease systems and water holding capacity was analyzed.In addition,methods(inculding homogenization,ion exchange chromatography,salting out,gel filtration chromatography and concentration)of purification the three proteases from pork and their characters were studied.Besides,myofibrils were incubated with endogenous enzymes as a model system,and the influence of proteolysis on myofibrillar water holding capacity was investigated.The aim of the thesis was to increase our knowledge about effect of endogenous proteolysis on postmortem meat quality and its mechanism.This may help us to improve meat quality and take advantage of meat resource.The main results included four parts:1.The order of gene expression in pork from more to less was PSMB2>CTSB>CTSL>CAPN2>PSMB1>PSMB5.The relationships between gene expression of PSMB1,PSMB5 and rate of tenderization were 0.563 and 0.513(P<0.05).The relationship between gene expression of PSMB1,PSMB5,CAPN2 and myofibrillar water controlling development were-0.597,-0.037 and 0.746(P<0.05).2.The activities of calpain2 and 20 S proteasome decreased,activity of cathepsin B+L increased during postmortem storage.The activity changes of calpain2 were positively correlated with intensity of intact desmin(P<0.01),with shear force(P<0.05)and negatively with purge loss(P<0.05).The activity changes of 20 S proteasome were negatively correlated with released ?-actinin(P<0.05),purge loss(P<0.01)and positively with shear force(P<0.01).The activity changes of cathepsin were positively with myofibrillar water controlling(P<0.05).In addition,there was positive correlation between intensity of intact desmin and shear force(P<0.01),as well as released ?-actinin and myofibrillar water controlling(P<0.05).3.The molecular mass of purified Calpain2 was 80 kDa,specific activity was 38.87U/mg protein,purified fold was 36.33,yield coefficient was 24.76%;the molecular mass of purified 20 S proteasome was 700 kDa,specific activity was 109.2U/mg protein,purified fold was 266.34,yield coefficient was 12.42%.The molecular mass of purified Cathepsin B+L was 32-35 kDa,specific activity was 206.67U/mg protein,purified fold was 120.86,and yield coefficient was 16.97%.The activity of calpain2 reached highest level when pH was 7.0,7.5 and temperature was 20,25?.The KCl decreased calpain activity.For 20 S proteasome,chymotrypsin like activity and PGPH reached highest level when pH was 7.0,7.5,trypsin like activity reached highest level when pH was 8.0,8.5.The optimum temperature was around 37?.The KCl decreased 20 S proteasome activity.The activity of cathepsin B+L reached highest level when pH was 5.5 and temperature was around 45?.The less than 300 mM KCl developed cathepsin activity.4.All three proteolytic systems improved the relative water-holding and generally there was a larger effect with increasing amount of enzymes in the incubation.The improved water-holding occurred in parallel to degradation of myofibrillar proteins.Desmin was degraded by calpain-2 as well as by cathepsin B+L and ?-actinin was released by the proteasome.In addition,20 S proteasome preferred to degrade low molecular weight proteins in myofibrils or acted after calpain degradation,and 20 S proteasome could take part in proteolysis in meat during pH decreased from 7.0 to 5.5 after slaughter and influenced water holdingAbove all,calpain,proteasome and cathepsin have important effects on pork quality during aging.The proteolysis of myofibrillar proteins changes structure in meat,influences meat quality and then contributes to tenderization.