| Freshness is the most important criterion for judging the quality of aquatic products, and the development of reliable methods for freshness assessment is of outstanding interest in the field of food science. Loss of freshness is the result of a combination of complex chemical, physiological, biochemical and microbiological processes. Protein is a crucial factor causing aquatic quality deterioration, but the evaluation method of freshness based on muscle protein has received relatively little attention. In recent years, proteomics has become a popular way to study protein, especially differential proteomics. By seeking, screening and identifying the potential biochemical markers enabling monitoring and evaluating of fish freshness, differential proteomics comprehensive and in-depth insights into the formation mechanism of the decline of freshness. The study provides a new and scientific basis for further explanation of fish freshness differences. Therefore, there is a very important significance with proteomics method to assess fish freshness.So, in order to est ablish the quality indices and indicators model of freshness base on muscle protein of tuna(Thunnus obesus), tuna muscle during storage at 4℃ and 15℃ was assessed by physicochemical, sensory and microbiological methods. Moreover, changes in biochemical characteristics of muscle were studied based on the denaturation of protein. Combined the analysis in protein composition based on SDS-PAGE, and myofibrils ultrastructure observation through transmission electron microscope (TEM), the degradation of muscle protein was studied during refrigerated storage. Finally, the system of two-dimensional electrophoresis gels (2-DE) was applied to study the changes in muscle in muscle protein and research the indicators of freshness based on muscle protein. The transcriptional levels of genes of the identified proteins were also measured by real-time PCR. The main results were as followed:1. Traditional assessment methods were used to evaluate the tuna freshness. Sensory score, K value, hardness, springiness and a*/b* displayed strong correlations with storage time and were chosen as the indicators for evaluating freshness, while pH value could be used as a reference index to reflect the freshness of the fish. Sensory score and microorganisms (Mesophilic and Psychrophilic counts) could also be indices for spoilage evaluation. The shelf-lives of tuna muscle stored at 4℃ and 15℃ were 4 and 1.5 d, respectively. The comprehensive models for assessing tuna freshness and quality were constructed by Factor Analysis, which provided a reference for quality assessment.2. The biochemical characteristics of tuna muscle underwent obvious changes during storage. The salt-solubilities, T-SH contents, A-SH contents and Ca2+-ATPase activities of actomyosin significantly decreased with storage time, while the surface hydrophobicity increased obviously in the earlier stage of storage. T-SH and A-SH contents displayed strong correlations with storage time, and they may be considered potential quality indices of freshness. Refrigerated storage of 4℃ could inhibit the denaturation of tuna muscle protein.3. The contents of myofibrils protein significantly decreased with storage time (P<0.05), while the contents of alkai-soluble protein and TCA-soluble peptide increased during the storage. The muscle protein electrophoresis showed that postmortem storage can lead to protein degradation of the different degree, the most obvious was MHC. Observed by transmission electron microscope, we found that after 6 d storage the muscle fibrils structure was completely disrupted, Z line ruptured, M line and H zone disappeared completely, sarcomere as well as the gaping area increased. According to the correlation analysis results, we detected that the relative content of myofibrils protein, alkali-soluble protein, TCA-soluble peptide, MHC, actin, hardness and springiness had very significant correlated (P＜0.01). The contents of myofibrils protein, TCA-soluble peptide and MHC could be considered as the potential indicator of freshness.4. A proteomic reference map with wide pH/Mr range, high resolution and good reproduction for tuna muscle protein was obtained using 2-DE by using 24 cm (pH 4-7) gel strips. By MALDI-TOF/TOF and NCBI database search method,15 spots (13 proteins) were successfully identified. Among these proteins,12 protein spots were down-regulated, including myosin heavy chain 1(MHC-1), 6-phosphofructokinase (6-PFK), phosphoglycerate kinase (PGK), pyruvate kinase muscle isozyme-like, Enolase, β-Enolase (2 spots), triose phosphate isomerase B(TIM), glycogen phosphorylase (2 spots), myosin light chain 1(MLC-1), myosin light chain 3(MLC-3), a-Actin and β-Actin, while Type I Keratin S8 was up-regulated.5. The transcription levels of 13 spots in postmortem changes during storage period were confirmed by RT-PCR, and 8 spots were successfully identified. Meanwhile, the expression of 5 proteins (such as Enolase, TIM, MHC-1, a-Actin and P-Actin) abundance changes and expression quantity of genes had good correlation.6. Based on GO terms and annotations, the analysis showed that 10 potential protein indicators of freshness affected quality deterioration of tuna muscle through two pathways.(1) Affecting quality characteristics of tuna muscle through the glycolytic metabolic pathwayDown-regulation of the five glycolysis related metabolic enzymes (6-PFK, PGK, pyruvate kinase muscle isozyme-like, Enolase, TIM, glycogen phosphorylase) indicated that the glycolytic metabolic reaction was accelerated while the metabolic enzymes were consumed rapidly during refrigerated storage. In this case, these five metabolic enzymes could be considered as the protein indicators of freshness.(2) Affecting quality characteristics of tuna muscle through the Cathepsins pathwayCytoskeletal proteins (MLC-1, MLC-3, MHC-1, α-Actin, β-Actin) were down-regulated during storage. This indicated that the effect of Cathepsins on myofibril protein was significant, and these cytoskeletal proteins were degraded rapidly. As a result, hardness declined rapidly and finally sacrificed the fish quality. Accordingly, these cytoskeletal proteins could be considered as the protein indicators of freshness. |
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