Study On Quality Evaluation,Protein Changes And Mechanism Of Endogenous Protease Of Grouper(Epinephelus Coioides) During Refrigerated Storage

Grouper?Epinephelus spp?,which belongs to the order Perciformes and the family Serranidae.As the change of biochemical characteristics of proteins in muscleis is an important reason for the deterioration of grouper'quality during refrigerated storage,in response to this problem,this study investigated the quality evaluation,protein changes and the action mechanism of endogenous protease in grouper?Epinephelus coioides?during refrigeration.First of all,the quality changes of grouper were analyzed,and the physicochemical and microbial changes of grouper fillet were studied.Through the establishment of QIM evaluation scheme and the establishment of regression model using NIR and LF-NMR technology,the fast and nondestructive evaluation of frozen grouper fillet was realized.That provide technical support for quality control of frozen grouper fillet.Secondly,the mechanism of grouper quality change was revealed from the perspective of protein biochemical changes by studying the oxidation and degradation of grouper muscle protein in different packaging methods during refrigeration.Thirdly,the proteome changes of frozen grouper fillets were obtained by proteomics and the potential freshness indicator proteins were determined.The potential mechanism of quality degradation was revealed by correlation analysis between differential proteins and quality indicators.Fourthly,the contribution of endogenous proteases to texture deterioration and flavor change of grouper muscle during cold storage was studied.The role of endogenous proteases in muscle quality degradation of grouper and the key proteases causing texture deterioration were determined.Finally,the recombinant cathepsin B,L,D and calcium activated protease were used to establish a simulation system with the extracted grouper myofibril and the role of protease in myofibril degradation was investigated by liquid chromatography-tandem mass spectrometry?LC MS/MS?.The research contents and results are described as follows:1.The rapid detection on the freshness of grouper fillets was obtained by establishing quality index method?QIM?scheme,near-infrared analysis and LF-NMR model.Between the QI score and storage time showed a linear relationship?QI=1.179×t–1.157,R2=0.98?which indicated that shelf life of grouper fillets was 12 days under 4?.Partial least squares?PLS?analysis showed the mean squared error between predict days and measure days was almost 1 day?MSE=0.984?.Correlation analysis between QI value and freshness indices found that QI score had a high correlation with total volatile base nitrogen?TVB-N?.Partial least squares?PLS?,principal component analysis?PCA?and multiple linear regression?MLR?methods were used to establish near-infrared spectroscopy?NIRs?prediction models for TVB-N,various spectral pretreatment methods such as the first derivative?1st?,vector normalization?SNV?,and multi-scatter correction?MSC?were adopted.The results showed that SNV combined with PLS had the best acceptable fitting accuracy and predictive ability,the coefficients of prediction?Rp?was 0.968 and RMSEP was 1.381 for TVB-N.The total results revealed the feasibility of using NIRS and QIM scheme to detect freshness in grouper fillets.Multiple regression for TVB-N measured values versus predicted values for LF-NMR relaxation time of grouper fillets had a root mean square error of 1.883 and R²=0.96,which can well predict the TVB-N content of grouper muscles.Moisture status enabled non-destructive rapid detection of freshness of refrigerated grouper fillets.2.The effect of different packaging methods,namely,air packaging?AP?,vacuum packaging?VP?,and modified atmosphere packaging?MAP?were investigated,on the protein oxidation and degradation of grouper?Epinephelus coioides?fillets during refrigerated storage.The carbonyl group,myofibril fragmentation index,free amino acids,FTIR of myofibrillar proteins,and total protein SDS-PAGE were determined.The results showed that the protein oxidation degree of the fillets gradually increasing as the storage time increased.The FTIR results indicated that the secondary structure transformed from an?-helix to an irregular curl.SDS-PAGE confirmed the degradation of the myosin heavy chain and that myosin gradually occurred during refrigerated storage.Meanwhile,protein oxidation and degradation were highly correlated.Protein degradation was accelerated by protein oxidation in myofibrils,which included the increase of protein surface hydrophobicity and changes of the secondary structure.In fact,the protein oxidation and degradation of the grouper fillets were effectively inhibited by MAP and VP during refrigerated storage,and MAP?30%N₂and 70%CO₂?had the best results.3.A TMT?Tandem Mass Tag?-based strategy was applied to elucidate proteins that change in proteomes of grouper fillets during refrigerated storage.In addition,quality analyses on p H,centrifugal loss,color?L*,a*,b*?and texture?hardness,chewiness,and gumminess?for grouper fillets were carried out.A total of 64differentially significant expressed proteins?DSEP?were found in the results in the Day0 vs.Day 6 group comparison and the Day 0 vs.Day 12 group comparison.It was worth mentioning that more proteome changes were found in the Day 0 vs.Day 12comparisons.Bioinformatics was utilized to analyze the DSEP.Uni Prot KB,Gene Ontology?GO?enrichment,Kyoto Encyclopedia of Genes and Genomes?KEGG?and protein interaction network analysis were adopted.All DSEPs were classified into seven areas by function:binding proteins,calcium handling,enzymes,heat shock protein,protein turnover,structural proteins and miscellaneous.The numbers of proteins that correlated closely with p H,centrifugal loss,color?L*,a*,b*?and texture?hardness,chewiness,and gumminess?were 4,3,6 and 8,respectively.4.The purpose of this study was to inquire into the effects of endogenous cathepsin and microorganisms on texture emollescence and flavor changes in refrigerated grouper fillets.Iodoacetic acid and procin300 were used to inhibit endogenous protease activity and microbial growth separately.Iodoacetic acid can well inhibit the activity of cathepsin B,L,and calpain.Moreover,iodoacetic acid did not significantly affect the growth of microorganisms.The total number of bacteria and pseudomonas spp in the samples treated with proclin300 were less than 2 log CFU g^-1 and 1 log CFU g^-1 on the18th day,which did not significantly affect the activity of the protease.On the 6th day,the hardness of the iodoacetic acid treatment group decreased by 8%,while the proclin300 treatment group decreased by 28%,and the changes of free amino acids and volatile substances significantly exceeded that of the iodoacetic acid treatment group,indicating that endogenous protease was the main factor of texture deterioration.The first-order exponential decay model indicated that cathepsin L was the most important protease for reducing the hardness of grouper fillets,while changes in the content of free amino acids and volatile substances indicated that microorganisms played a major role in the deterioration of flavor substances.The effect of microorganisms on the texture of grouper is almost negligible,but its effect on flavor changes is stronger than that of endogenous protease.5.The degradation mechanism of endogenous protease to muscle of grouper was studied.In this chapter,the recombinant cathepsin B,L,D and calpain were combined with the extracted grouper myofibrin to establish a simulation system to explore the role model of protease in myofibril degradation.Meanwhile,liquid chromatography-tandem mass spectrometry?LC/MS/MS?technology was used to identify all the identifiable proteins and degradation products produced by each enzyme in the degradation of myofibril,so as to determine the role of each enzyme in the degradation of myofibrin.The protein identification results of LC/MS/MS showed that the hydrolysis ability of myofibrin myosin heavy chain,myosin light chain and actin in grouper was cathepsin L>cathepsin B>cathepsin D>calpain.Cathepsin L had the strongest ability to degrade grouper myofibril alone,and could significantly degrade myosin heavy chain,myosin light chain,heat shock protein?HSP70?and LIM domain binding protein.Calpain had the ability to degrade troponin T and desmin,while cathepsin L degraded heat shock protein HSP 70,which was a competitive substrate of calpain,so there was a synergistic effect between cathepsin L and calpain.