Study On The Changes In Key Proteins Of Grass Carp Muscle During Cold Storage Period

Grass carp, one of “the four major Chinese carps”, ranks first in the aquaculture production, consumption and output value in freshwater fish. Because of tender muscle tissue, abundant nutrients, 80% of moisture content and 18% of protein content, it is extremely susceptible to corruption during storage, transportation and processing. Nowdays, cold storage is a widely used preservation method, but it is still difficult to break the bottleneck that the shelf life of fish meat is too short. Protein is the main component of muscle tissue, its functional and structural integrity directly determines the quality of fish muscle. However, the change details and rules of those key proteins associated with corruption of grass carp was poorly understood and required an in-depth study.The changes in composition, structural and physicochemical properties of myofibrillar protein, which changed most significantly duing cold storage of grass carp, were investigated using electrophoresis, chromatography, electron microscope and infrared spectroscopy and other related technologies. And the key muscle proteins related with fish corruption was identified using proteomic technologies. This revealed the fish corruption pattern and mechanisms during cold storage from protein change of view, provided a theoretical basis for preservation researches of freshwater fish. The main results were as follows:?1? The changes in polyacrylamide gel electrophoresis?SDS-PAGE? map, microstructure, sulfhydryl?SH? and disulfide bond?S-S? content, secondary structure, surface hydrophobicity, thermodynamic properties, surface hydrophobicity and Ca²⁺-ATPase activity of myofibrillar proteins during grass carp cold storage were investigated. The results showed that with the extension of chilling storage time, the Ca2+-ATPase activity of myosin was gradually declined, but there was no apparent change in thermodynamic properties of the myofibrillar protein of different storage time; SDS-PAGE analysis revealed that there showed little change in subunits composition of myofibrillar protein, but its total content increased at first but later decreased, and at the same time, part of the protein band signal was occasionally detected, suggesting some degree of degradation with macromolecular subunits and polymerization with small molecular subunit might coexist during cold storage period. The surface hydrophobicity and content of ?-sheet increased, but content of disulfide bond and ?-helix decreased, and the microstructure became distorted but still relatively complete at the first 4 days, indicating the Main changes of myofibrillar protein was degeneration during this period. From the 6th day, the content of random coil increased but content of ?-sheet decreased, and the protein particles was gradually fractured or broken, suggesting the degeneration and disorder degree of myofibrillar protein was increased, and then gradually degraded.?2? Proteome differences between muscle protein samples of grass carp refrigerated for 0 days?fresh sample? and 8 days were analyzed by two-dimensional gel electrophoresis?2-DE? and the key proteins were identified using mass spectrometry?MS?. The results showed that there were 1494 protein spots with good repeatability in 2DE map of the fresh sample and 1790 in the 8 days sample. There were 110 main difference spots between 2-DE map of the two samples, and compared with fresh samples, 85 protein spots in 2-DE map of sample refrigerated for 8 days decreased in abundance or disappeared, of the sample, and 125 protein spots increased in abundance or newly added.?3? 79 typically different protein spots were selected to cut from 2-DE gel, digested and then anazyled with matrix assisted laser desorption ionization/time of flight mass spectrometry?MADL-TOF MS?. Finally, 16 kinds of proteins related to corruption of grass carp were identified as follows: myosin heavy chain, actin, tubulin beta 1, desmin, alpha-1,4 glucan phosphorylase, NADH dehydrogenase?Ubiquinone? Fe-S protein 3, NADH-coenzyme Q reductase, apolipoprotein A-IV, adenylosuccinate lyase, Si:dkey-180p18.9 protein, enolase 1?Alpha?, warm temperature acclimation-related 65 kDa protein?Fragment? and some uncharacterized proteins. Those proteins could be considered as the indicators of grass carp corruption during cold storage period.