Studies On Indicator Proteins Of Freshness For Pacific White Shrimp(Litopenaeus Vannamei)Contribute To Postmortem Changes Stored Under Vacuum Packing At4℃

Freshness has significantly contributed to the edible quality and safety of shrimp and other aquatic products, and its level also determines the edible value of aquatic products. With the enhance of our living standard, the require for the level of freshness for shrimp and other aquatic products seems to be more higher, which makes the assessment of freshness curcially meanful for the edible safety, transportation, storage and processing. How to evaluate the freshness of aqutic products objectively and accurately has become an hot issue to be studied in processing industry of aquatic products. Precisely, the decline of freshness and decomposition turn out to be a complex physical, chemical, biochemical and microorganism growth and metabolism process. Therefore, in the present work, the quality of shrimp (L. vannamei) stored under vacuum-packing at4℃in different time points was analyzed by physicochemical, sensory, microbiological methods. Moreover, differentially expressed proteins of the shrimp muscle involved in freshness and muscle quality during0,2,4,6,8and10days of postmortem storage at 4℃were searched and investigated by constructed and optimized proteomic approach for shrimp (L. vcmnamei). Statistical analysis were used to reveal the correlation between differently expressed protein and frenshness characters and quality. 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. Finally, the transcriptional levels of genes of the identified proteins were also measured by real-time PCR. The target of this study is to chose and identify the potential protein markers that are associated to quality characteristics changes, which may have important implications for understanding the molecular mechanism of postmortem changes stored under vacuum packaging at4℃and bring us a step closer to define predictive models suitable for determining the freshness of shrimp. The results are concluded as follows:1. Traditional assessment methods were used to evaluate the shrimp freshness. Texture, colour,TVB-N value were chosen to represent the quality changes. The major quality indices that reflecting freshness of Shrimp(L. vannamei) during the cool storage at4℃were texture、a*、c*ab、TVB-N、 TBA、TVC、pH、K、sensory characteristics, and the shelf life was5.5days. The comprehensive models for assessing shrimp freshness and quality were constructed by Principal Component Analysis, which provided a reference for quality assessment.2. The methods of2-DE were optimized and completed, differently expressed protein were selected by mass spectrum and searching the database. Samples for2-DE analysis were purified, and2-DE profiles were advanced by adding TBP in lysis buffer. Shrimp (L. vannamei) muscle protein isoelectric points are between4and7, so the2-DE system is established chosing17cm (pH4-7) IPG gel strips, loading the sample120μg, increasing the voltage and power of isoelectric focusing, preparing12.5%SDS-PAFE gel, and staining the gels by the silver. The proteomics technologies were used to study the changes in muscle protein of shrimp (Lvannamei) during vacuum package storage at4℃and25differently expressed protein spots were found in2-DE maps of samples stored at4℃. After the peptide mass fingerprinting were gained by mass spectrum, three proteins were indentified as arginine kinase, phosphopyruvate hydratase, actin T2by searching database, which could be potential protein indicators of shrimp freshness.3. To validate the proteins identified by2-DE and MALDI-TOF/TOF mass spectrometry, the expression of genes such as arginine kinase, phosphopyruvate hydratase and actin T2in postmortem changes during storage period were confirmed by the quantitative RT-PCR. And the expression of protein abundance changes and expression quantity of genes have good correlation, The expresson of genes in postmortem changes with three different storage approaches were statistically analysed, which further validate that potential protein indicator of freshness.