Proteomics Studies On The Protein Indicators Of Freshness In Turbot(Scophthalmus Maximus)Muscle

Aquatic products such as fish are rich in nutrients and water,and owing to its endogenous proteases,aquatic would lost freshness easily,eventually spoilage.Freshness is one of the important quality attributes of aquatic products that will determine the final value of the product.The processes above are very complex physiological and biochemical processes,changes in protein have the most direct influence on aquatic products freshness.With proteomics widely used,researches about the qualities of aquatic products during storage increased.However,researches about changes on quality or freshness for turbot are not reported.In this paper,a differential proteomics platform in turbot muscle was developed,based on the changes of freshness and biochemical properities of muscle proteins.The changes of proteins in refrigerated process were analyzed by proteomics techniques to filter protein indicators of freshness.The aim of this work is to provide a theoretical basis for the evaluation of freshness for turbot.And the changes in quality from the perspective of the protein were explains.The results are as follows:1.In this article,the freshness of turbot stored at 4? were determined by the quality index method(QIM)combined with TVB-N,K value,TBA,TPA,total viable counts(TVC).The results showed that QI value had a highly linear relationship with the storage time and could be represented by the equation QI=1.175×days-0.395(R2=0.988).It could be assumed that the QI could predict the remaining shelf life with an accuracy of±1 day(0.816)under the 95%confidence interval.At the 15th day,the sensory quality was considered unacceptable.TVB-N value,K value and the TVC reached 31.34 mg N/100g,72.24%and 6.45 log cfu/g at 15th day,respectively,which had reached the reference limit standard of fish shelf life.The QIM result had a good coincidence with freshness indices,indicating that QIM was a promising rapid detection method of freshness and was applied to study on shelf life.Meanwhile,a shelf life of 15 days was obtained for turbot stored at 4?.2.In this paper,The changes on the refrigerated turbot muscle proteins were studied The results showed that the salt solubility of myofibrillar protein Ca2+-ATPase decreased significantly(P<0.05),while surface hydrophobicity and TCA soluble peptides content increased significantly(P<0.05).The total sulphur content increased at the beginning of storage then decreased with time extended.SDS-PAGE electrophoresis showed that 25-26 Ku band in total protein,38-39 Ku band and 41 Ku band in low salt soluble protein and MHC,tropomyosin,37 Ku band in high salt soluble protein,etc,had regularly changes regularly.TVB-N and TVC increased gradually during storage,which indicating a shelf life 15 days for turbot stored at 4?.Correlation analysis showed that the salt solubility of myofibrillar protein,Ca2+-ATPase,surface hydrophobicity and TCA-soluble peptides were markedly correlated with TVB-N and TVC,and can be used as auxiliary freshness indexes of refrigerated turbot.Meanwhile,band III and band IV in low salt soluble protein,band I in total protein and MHC,tropomyosin,band II in high salt soluble protein are also obviously correlated with TVB-N and TVC,thus they could be used as freshness indicators of turbot.3.Objective of the research is aimed to construct treatment method of muscle proteins of Scophthalmus maximus for two-dimensional electrophoresis(2-DE).Under the process of IEF III(active hydration at 50 V for 16 h,at 500 V for 1.5 h,1,000 V for 2 h,4,000 V for 2 h,8,000 V for 1.5 h,and 10,000 V for 1h),protein samples processed by lysis buffer III(containing 8 mol/L urea,2 mol/L thiourea,4%(w/v)chaps,0.5%(v/v)ampholyte,65 mmol/L dithiothreitol,40 mmol/L Tris-base)got satisfactory 2-DE patterns with high quality and repetition.And the IPG strip with pH 4-7 was chosen in the experiment.Then,Proteins were separated on 12%SDS-PAGE in the second dimension.4.The post mortem changes of proteins in turbot muscle were detected by two-dimensional electrophoresis(2-DE)and MALDI-TOF/TOF MS after storage for 0,3,6,9,12,15 days.The results of 2-DE revealed that 7 differentially expressed protein spots were identified successfully and the abundance of these spots have significant correlation with storage time,QI,K value,TVB-N and TVC,which indicated that these spots could be regarded as potential freshness indicators of turbot.Six kinds of proteins were obtained using MALDI-TOF/TOF MS,including 2 metabolic proteins namely phosphoglucomutase-1,pyruvate kinase,and 5 cytoskeletal proteins namely kinesin-1 heavy chain,Troponin T(two spots),desmin,actin.Muscle cell metabolism has undergone significant changes,mainly in the myofibrillar proteins,cytoskeleton structure and metabolic enzymes in turbot muscle stored at 4?.Under hypoxic conditions,glycolysis process occurred primarily,as the energy metabolism gradually weakening,phosphoglucomutase-1 and pyruvate kinase nvolved in glycolysis were consumed.Owing to endogenous enzymes,cytoskeleton of muscle was broke,the content of kinesin-1 heavy chain and actin increased,the content of troponin T and desmin decreased.Therefore,the freshness indicator proteins which were screened were closely related to muscle glycolysis and protein degradation pathway,which would provide evidence for explaining mechanism of turbot quality change during storage.