The Effects Of Three Endogenous Proteases On Storage Quality Of Tilapia And Its Mechanism

The quality of fish is the major factor that affects consumers' behavior.Many studies concluded that endogenous proteases are the most important factors affecting the fish quality in the early stage after slaughter.Calpains,cathepsins and proteasomes have been extensively studied.In recent years,caspases have drawed many attentions.The limited degradation of myofibrillar proteins by endogenous proteases causes softness,loss of juice,and changes in color,which affect the consumer's comsuption.Therefore,in the current study,tilapia,as the research object,was used to compare the changes in quality and shelf life during stored in atmosphere packaging(AP)and modified-atmosphere packing(MAP),as well as its changes in proteome when stored in AP.The effects of ?-calpain,cathepsin B,and caspase-3 on degradation of myofirillar proteins,and the activations of ?-calpain,cathepsin B,and caspase-3,as well as the interactions between ?-calpain and cathepsin B,and caspase-3 in tilapia meat after slaughter were studied.We aim to provide a theoretical basis for further improving the fish meat quality after slaughter.The major results are as follows:1.Tilapia fillets were packaged in AP and MAP(CO2/N2,30%/70%)and stored on ice with the temperature from-0.7? to 0?.The freshness and shelf life of fillets were investigated and valuated by sensory,textural,physicochemical parameters,microorganism,and endogenous enzyme activities.The results showed that values of the total volatile base nitrogen(TVB-N),myofbril fragmentation index(MFI),sensory scores,total viable count(TVC),and pH of tilapia fillets in MAP were lower than those in AP,while the salt-soluble protein(SSP)level and water holding capacity(WHC)of tilapia fillets in MAP were higher than those in AP fillets.The texture results showed that,fillets stored in MAP were harder and more elastic than those in AP,and thus had a greater chewiness.In addition,the three endogenous enzyme activities of tilapia fillets in MAP were always slower than those in AP at each measured time.The results of protein oxidation index showed that,the protein carbonyl groups of tilapia fillets in MAP were significantly lower than those in AP(P<0.05),while the total sulfhydryl content and protein solubility were significantly higher than those in AP(P<0.05).The Results showed that tilapia fillets stored on ice with MAP exhibited a significant fresh effect on tilapia fillets,the shelf lives of tilapia fillets stored in AP and MAP were 11 d and 14 d,respectively.It concluses that MAP can effectively reduce or inhibit the activity and reaction rate of endogenous proteases,and reduce the degree of protein oxidation and inhibit the proliferation of microbe.2.Here,we aimed to study the changes in proteome of tilapia during postmortem storage and it's relationship with fish tenderness.The effects of post-mortem storage time(0 and 7 d in ice storage)on the proteome of tilapia skeletal muscle was characterized by label-free proteomics.In total,902 proteins were identified,of which 34 proteins underwent alterations that were statistically significant(P<0.05).The 15 up-regulated and 19 down-regulated proteins were mainly structural proteins,followed by transcription and translation regulation,enzymes and stress proteins.The top three enriched Kyoto encyclopedia of genes and genomes(KEGG)pathway are metabolic pathways,tight junction,and ribosome.The STRING network interaction analysis was carried out on 34 differential abundance proteins(DAPs),among which 27 DAPs with interaction relationship formed 70 interaction relationships.A total of 295 absent or newly generated proteins during storage,mainly involved in metabolism,oxidation-reduction,and transcription and translation regulation.Two DAPs,pgml and ldha were selected for further verification using 0 d and 7 d iced storage fish muscle.Western blotting analysis showed significantly lower levels of two proteins,ldha and pgml,in tilapia fillets of 7 d ice-stored than in fresh.Our results speculate that fish tenderness is related to structural protein degradation and increased levels of lactic acid caused by enzymes and anaerobic conditions.and provide an important basis toward enabling further understanding of the molecular mechanisms responsible for differences in fish texture and meat tenderness between fresh and ice-stored fish.3.This study investigated the effect of three proteases,?-calpain,cathepsin B and caspase-3 on the degradation of tilapia muscle myofibrillar proteins.Tilapia muscle was soaked in MDL-28170,E-64,and AC-DEVD-CHO,specific inhibitors of calpain,cathepsin and caspase-3,respectively,for 14 d to determine the in vivo degradation of myofibrillar proteins.Actin was reduced after 14d,and the degradation rate in MDL-28170 and E-64 treated-groups were lower than that in the AC-DEVD-CHO treated-group and control group(P<0.05);Desmin and troponin T were significantly reduced after treated with inhibitors,and degradation degree of troponin T was control>MDL-28170>AC-DEVD-CHO>E-64,and that of desmin was control>AC-DEVD-CHO>E-64>MDL-28170.For in vitro analyses,myofibril was incubated with recombinant active?-calpain,cathepsin B,and caspase-3,significant degradation of actin was observed when myofibril was incubated with cathepsin B and caspase-3(P<0.05).Desmin were significantly reduced when incubated with recombinant proteases(P<0.05),and the degradation was ?-calpain>caspase-3>cathepsin B;however,troponin T showed significant degradation only in the ?-calpain and cathepsin B treatment groups(P<0.05).Our results reveal that ?-calpain,cathespin B have similar degradation patterns,while caspase-3 have partly similar degradation for myofibril between in vivo and in vitro,suggesting that ?-calpain,cathespin B are the mainly proteases responsible for post-mortem myofibril degradation,while caspase-3 is minor.4.The chapter mainly focus on the activation of three proteases after fish slaughter by investigating the changes in activities of three proteases and their related influencing factors during postmoterm storage.(1)Calpain is known to be the most important endogenous protease responsible for degrading myofibril protein,thus affecting meat quality.This study was aimed to investigate the activation of the calpain and its potential influencing factor in fish after slaughter.Calpain activity,expressions of the calpain inhibitor and activator(calpastatin and UK114,respectively),and effects of influencing factors on calpain activity and on the necessary Ca2+ activation concentration was investigated.Calpain activity increased significantly during the first 5 h storage followed by rapid decline(P<0.05).Three calpastatin expression bands were detected,and the 120 kDa calpastatin significantly was down-regulated with increasing of storage time(P<0.05),while UK114 level of tilapia muscle was significantly up-regulated during postmortem storage(P<0.05).Calpastatin and UK114 showed opposite effects on calpain activity;a significant decrease and increase in calpain activity were observed when crude calpain was incubated with calpastatin and UK114,respectively(P<0.05).Furthermore,a significant increase was observed in calpain activity following co-treatment with calpastatin and UK114(P<0.05),but lower than that of treatment with UK114.The calpain activity in the UK114-treated group was higher than that of the control and calpastatin with the same Ca2+ concentration.Calpastatin inhibited calpain activation,whereas UK114 promoted the activation of calpain by reducing the concentration of Ca2+ required.In addition,we speculated that UK114 may increase calpain activity by inhibiting that of calpastatin.(2)The activation of the apoptosis pathway in tilapia muscles during postmortem storage was studied.Changes in caspase-3 activity,ATP content,mitochondria and cytosol cytochrome c levels,and ratio of Bcl-2/Bax level of tilapia muscle were observed during postmortem storage at 20?.Caspase-3 activity was found to be significantly increased at first,followed by a decrease(P<0.05);caspase-3 activity was highest at lh.The ATP content decreased significantly during postmortem storage(P<0.05).The cytochrome c level in the cytosol showed a significant increase after 5 h of storage(P<0.05),while the mitochondrial cytochrome c levels showed a decrease.The Bcl-2/Bax ratio was stable from 0-5 h,followed by a rapid decrease at 10-20 h and a significant increase after 20 h(P<0.05).The apoptosis process occurred until 20 h of postmortem storage.Thus,we speculated that the availability of ATP and the increase in cytosolic cytochrome c levels are essential for the activation of caspase-3,and that the former partly limits caspase-3 activity.(3)The results of activation of cathepsin B showed that,the changes in expressions of cathepsin B,membrane-associated membrane protein(LAMP-1)and Hsp70 had the similar trends,which were significantly up-regulated with increasing of storage time(P<0.05),and then significantly down-regulated later(P<0.05);the highest expression level of cathepsin B was observed at 10 h,while the highest expression level LAMP-1 was observed at 5 h and 10 h,and Hsp70 reached the highest expression level at 5 h.We speculated that the lysosomal membrane began to permeabilize at 5 h and gradually released and activated cathepsin B,and completely permeabilize at 10 h,when cathepsin B had the highest expression.5.In this section,we investigated the interactions between ?-calpain and cathepsin B,and between ?-calpain and caspase-3 by co-immunoprecipitation(CO-IP).The expression levels of the three proteases were detected by western blot and the apoptosis was observed by TUNEL staining.Based on the above results,the CO-IP was performed using the tilapia muscle at the time point of 1 h after slaughter.At the first experiment,?-calpain was as the bait protein and cathepsin B as the target protein.The results showed that bait protein was detected in Input group,but no found in IP group,indicating that there was no direct interaction between ?-calpain and cathepsin B at 1 h.And then,caspase-3 was used to be the bait protein,and ?-calpain was as the target protein.The same results were observed as above,indicating that there is no direct interaction between ?-calpain and caspase-3 at 1 h.The results indicate that there is no direct interaction between ?-calpain and cathepsin B,and bwtween ?-calpain and caspase-3 in physiological cells.