Studies On Quality Assessment And Protein Indicators Of Freshness In Chinese Shrimp(Fenneropenaeus Chinensis) During Refrigerated Storage

Freshness makes a major contribution to the quality and processing suitability of aquatic products. Freshness assessment is essential for the safety, transportation, storage, and processing of aquatic products. Thus, how to quickly and objectively evaluate fish freshness has been a hot issue to be studied in aquatic products processing industiy and researchers. Protein is one of the most important substrates of spoilage for fish products. However, the evaluation method of freshness based on muscle protein has received little attention, and little information is available on the quality indices and protein indicators of freshness.In this paper, to search the quality indices and indicators of freshness based on muscle protein of Chinese shrimp (Fenneropenaeus chinensis), freshness of the shrimp stored under vacuum-packing at4℃,0℃and -2℃was assessed by physicochemical, sensory and microbiological methods. Changes in biochemical characteristics and tissue microstructure of Chinese shrimp muscle were studied based on the denaturation of protein. Changes in protein composition and patterns were studied based on the degradation of muscle protein. Two-dimensional gel electrophoresis (2-DE) related techniques of muscle proteome of Chinese shrimp were constructed and optimized. The proteomics technologies (2-DE and MS) were applied to study the changes in muscle protein and research the indicators of freshness based on muscle protein. The work aims to provide a new thought and reference for the assessment of seafood freshness, to discuss the post-mortem change mechanisms of the quality, and to give a further explanation of the basic reason for the spoilage of the muscle. The results are showed as follows:1. The major quality indices that reflecting freshness of Chinese Shrimp during the refrigerated storage at4℃,0℃and-2℃were sensory score, chemical indices, texture and color attributes and microorganisms. Sensory score, TVB-N, HxR, Hx, K value, hardness, springiness, cohesiveness, Gumminess, and chewiness displayed strong correlations with storage time, and they may be considered suitable indicators for evaluating freshness. Sensory score, TVB-N, Hx could also be indices for evaluating spoilage. The comprehensive models for assessing shrimp freshness and quality were constructed by Principal Component Analysis, which provided a reference for quality assessment. The shelf-lives of vacuum package Chinese shrimp stored at4℃,0℃and-2℃were5.5-5.7,7.9-8.0and16.0-16.8days respectively. Superchilled storage could significantly extend the shelf-life of the shrimp.2. The biochemical characteristics and tissue microstructure of Chinese shrimp muscle suffered obvious changes during refrigerated storage. The salt-solubilities, SH contents and Ca2+-ATPase activities of actomyosin significantly decreased with storage time, while the surface hydrophobicity increased. The microstructure of the muscle was disrupted, including the fracture and fragmentation of the muscle fiber, the significant decrease of the fiber area, and the increase of gaping and the gaping area. SH contents and Ca2+-ATPase activities displayed strong correlations with storage time, and they may be considered potential quality indices of freshness. Superchilled storage could inhibit the denaturation of protein and disruption of the muscle fiber.3. Changes in protein composition and patterns indicated the degradation of the shrimp muscle protein during refrigerated storage. The contents of myofibrillar protein and stroma significant decreased with storage, and the contents of sarcoplasmic protein slight decreased, while the contents of alkali-soluble protein increased during the storage. MHC actin, tropomysion, troponin I, troponin C and the proteins of30-31KDa,6.5KDa showed a different degradation during the storage. The density of23-25KDa protein band in total soluble proteins,176-178KDa protein band in water soluble proteins,67-68KDa protein band in low-salt soluble proteins, and the relative contents of fraction I and fraction V in acid soluble nitrogen displayed strong correlations with storage time, so they may be the potential protein indicators of freshness. The contents of TCA soluble peptide and tryptophan could be considered as quality indices of Chinese shrimp.4. Most of shrimp muscle protein isoelectric points were between4and7. The2-DE related techniques was constructed and optimized in muscle proteome of Chinese shrimp by comparative tests on different extraction methods, IPG gel strips, isoelectric focusing programs, salt bridge, and sample volume, etc. The results showed that the resolution and reproducibility of2-DE profiles was significantly improved by adding Tris and TBP in lysis buffer, active rehydrating of17cm (pH4-7) IPG gel strips, loading the sample120μ,g, prolonging the time of desalting, increasing the voltage and power of isoelectric focusing, employed the salt bridge, preparing12.5%SDS-PAFE gel, and were dying the gels by the silver stained. This work provided a technical basis for the further study on mechanism of muscle quality changes and differential proteomics on muscle tissue of Chinese shrimp. 5. The proteomics technologies were used to study the changes in muscle protein of Chinese shrimp during refrigerated storage at4℃and-2℃. Six differently expressed protein spots were found in2-DE maps of samples stored at4℃, while11different spots were found in the samples stored at-2℃. The density of spot1501,1304and1405correlated positively with storage time (correlation coefficients were0.949,0.947and0.940, respectively), while that of7202and7205correlated negatively with storage time (correlation coefficients were0.901and, respectively). Thus those five proteins could consider as the indicators of freshness. The peptide mass fingerprinting were obtained by MALDI-TOF-MS. Spot6001and7205was identified to be SH2domain-containing protein6and adenosylcobyric acid synthase, while other9spots were uncharacterized. SH2domain-containing protein6might be a key protein related to tyrosinase activity, which make great contribution to shrimp melanosis. Adenosylcobyric acid synthase (Spot7205) could be a potential protein indicator of freshness, because of a strong correlation between the density and storage time.