The Screening Of Protein Indicators For Nile Tilapia Freshness,Properties Study And Application In The Quality Assessment Research

With the improvement of human living standards, people have more requirements for nutrition and diversification of daily diet. Nile tilapia(Oreochromis niloticus) is one of the main species in cultured freshwater fishes, and is regarded as one of the main animal protein sources in the future. Freshness is an important indicator of the quality and safety of fish products, which also is a main reference for consumers. How to evaluate fish freshness quickly and accurately during storage, processing and circulation is essential. Sensory analysis, physical, chemical and microbial assessment are the common methods used to evaluate the freshness of fish. Due to the high content of protein in fish, the degradation and degeneration of protein cause the decline of fish freshness. However, there was little research about the relationship between protein and freshness.Proteomics research regards two-dimensional electrophoresis(2-DE), mass spectrometry(MS) and bioinformatics as main technologies, which is able to analysis the whole protein of the research object, and has been gradually applied in the studies of aquatic products. Researchers applied proteomics technology to screen the differently expressed protein during storage and regarded them as indicators of freshness, which provide new thoughts and methods for aquatic products freshness assessmen. Little information is available for the proteomics research of Nile tilapia.In view of above reasons, iced stored Nile tilapia fillets were chosen as research object to investigated the freshness variation by evaluating the indexes: sensory evaluation, color, p H, total volatile basic nitrogen(TVB-N), thiobarbituric acid(TBA) and total viable counts(TVC), which would be provided as a reference for the follow-up study of muscle protein changes. Changes in the content and composition of Nile tilapia muscle protein during iced storage were studied. Two-dimensional electrophoresis(2-DE) system was established and optimized, which was suitable for the following Nile tilapia muscle protein proteomics research. The 2-DE and MS combined with database retrieval were applied to study the changes of muscle protein during iced storage in order to find indicators of freshness. The main results are showed as follows:1. Sensory evaluation score, TVB-N value, TVC value can be considered as suitable indicators to evaluate the freshness of Nile tilapia fillet, p H and TBA value can be taken as reference index to freshness evaluation. The data of color analyses have no significant relationship with freshness. The shelf life of iced stored Nile tilapia fillet was suggested as 12 d.2. The muscle protein of Nile tilapia fillets have obviously changed during iced storage. The content of 3 different soluble proteins declined with the extension of storage time. In the 3 different structures of proteins, the contents of myofibrillar protein and myosinogen declined significantly(P<0.05), while myostromin had no obvious change(P>0.05).The molecular weight of Nile tilapia muscle protein distribution between 10 to 200 KDa, myosin heavy chain(200 KDa), actin(45 KDa), tropomyosin(35 KDa) and some other protein bands in SDS-PAGE patterns indicate the degradation of these proteins. There were also some new bands appearing in the last phase of storage.3. The 2-DE system of Nile tilapia muscle protein was optimized and established. The results showed that by combining liquid nitrogen grinding with acetone precipitation to prepare protein sample, using 24 cm p H 4~7 IPG gel strips for isoelectric focusing(IEF) and 12.5% gel for the second SDS-PAGE, dying the gels by silver staining can get optimized 2-DE map which presented clear protein points, high resolution and little cross band. The 2-DE technical system was successfully established, which could be used for the separation of proteins in Nile tilapia muscle proteomics research.4. Ten proteins were found differently expressed among Nile tilapia fillets with 3 different freshness conditions by 2-DE and MALDI-TOF-MS technique. Three of the 10 proteins were identified by comparing the message in databases. The 3 proteins were predicted as phosphodiesterase 4C isoform, myosin heavy chain and beta-actin respectively, which were participated in energy metabolism, movement and structure maintenance of cells, respectively.