Searching for molecular markers of quality in fish reared in aquaculture.

One of the most unique characteristics of fish as food is that it is highly perishable. Consequently, freshness is fundamental to the quality of fish, and the time that has passed after catching it and the temperature ‘‘history” of fish are very often the key factors in determining the ultimate quality characteristics of such products. Therefore, the development of reliable methods to assess the freshness of fish, as well as the evaluation of quality, has been the goal of fish research for many years. During post mortem storage, fish muscle degrades and the flesh quality decreases rapidly, depending on the fish species. Loss of freshness, followed by spoilage, is the result of complex biochemical and microbiological processes that begin with a metabolic shift from an aerobic to an anaerobic state. This is followed by transformation of glycogen into lactic acid and the consequent reduction in pH and activation of different proteolytic reactions catalyzed by endogenous enzymes, which produce nutrients that promote subsequent bacterial proliferation. Post mortem tenderization is one of the most unfavourable quality changes in fish muscle and contrasts with mammalian and avian meats in which post mortem degradation of myofibrillar and cytoskeletal proteins is often desired to obtain a tender product. Previous studies on fish have investigated post mortem phenomena using chemical, physical, histological, and microbiological methods by focusing on indicators such as pH, lactic acid, adenine nucleotides and their degradation products, texture, firmness, and elasticity. Only few studies in the current literature have included a combination of molecular and proteomic investigation. Therefore the objective of the present Thesis was to investigate by molecular biology and proteomic techniques on how different methods of slaughtering and post mortem storage conditions influence the freshness quality of fish filet. The target species was cultured European sea bass (Dicentrarchus labrax) which is a marine teleost of great interest for Mediterranean aquaculture, representing an excellent fishfood product of high commercial value. Firstly, microfluidic capillary electrophoresis and real-time PCR were successfully applied to investigate the post mortem alterations in RNA extracted from sea bass muscular tissue in relation to three parameters: slaughtering method, and post mortem time and storage temperature (Chapter 3). In the course of this study, we first identified the sea bass cDNA sequences coding for endogenous proteases such as calpains and cathepsins which are assumed to play a major role in post mortem degradation of fish muscle. Then we determined the total RNA and ì-calpain- and cathepsin-L-specific mRNAs integrity over 5 days of storage at two different temperatures (1°C and 18°C) in sea bass slaughtered by three different methods (asphyxia in air, hypothermia/asphyxia in ice, and spinal cord transection). The results of this study showed that, RNA degradation is a slow process under the conditions investigated and, although post mortem storage temperature negatively affects the integrity of total RNA (higher degradation at 18°C), the transcripts of ì-calpain and cathepsin L are present for up to 5 days post mortem in the muscle of sea bass stored at either 1°C or 18°C without showing any significant slaughtering method-related degradation rates. In the light of these results, and in order to shed light on the influence of temperature on the status of sea bass muscle proteins during post mortem storage, a 2-D DIGE (2 Dimensional Difference Gel Electoforesis) and mass spectrometry study was performed on fish (Chapter 4). As expected, the greatest alterations in sea bass filet protein composition were observed upon post mortem storage at 18°C, with distinct changes appearing in the 2-D protein profile after 5 days of storage at this temperature. In particular, degradation of the myofibrillar protein myosin heavy chain and of the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase, among the most abundant muscle proteins, could be clearly observed upon storage at higher temperatures. Although to a lesser extent, however, several proteins were observed to vary in abundance also upon storage for 5 days at 1°C. In particular, one of the most interesting observations was the rapid and significant decrease in the abundance of nucleoside diphosphate kinase B and phosphoglycerate mutase 2, which was observed also at low storage temperatures and appeared to be temperature-independent. The results of this study offer new knowledge on changes occurring in sea bass muscle proteins during post mortem storage at different temperatures and provide indications on protein degradation trends that might be useful for monitoring freshness of fish and quality of storage conditions. Thirdly, 2D DIGE and mass spectrometry were applied to investigate the impact of slaughtering on the post mortem integrity of muscle tissue proteins in European sea bass (Chapter 5). Three different slaughtering techniques were evaluated: asphyxia in air (AA), asphyxia in ice (AI), and spinal cord severance (SCS). Principal components analysis (PCA) revealed a significant divergence of SCS samples, whereas AA and AI samples, although grouped separately, were less divergent and could be included in a single asphyxia cluster. In terms of single proteins, the most significant impact was seen on nucleoside diphosphate kinase B, which was consistently less affected when fish were slaughtered by SCS as compared to asphyxia. Integrity of the sarcomeric proteins myosin heavy chain and myosin binding protein C and of the cytosolic proteins fructose biphosphate aldolase, glyceraldehyde 3-phosphate dehydrogenase, and enolase 1 was also better preserved upon SCS slaughtering. Most interestingly, the influence on muscle protein integrity could be detected since the early post mortem phase. The results of this study demonstrated that slaughtering by SCS preserves protein integrity better than death by asphyxia, either in ice or in air. Both asphyxia conditions are comparably more adverse than SCS to muscle protein integrity, although a general trend favoring AI over AA is observed. The last objective of this Thesis was to realize a proteome map of the sea bass muscle (Chapter 6) for use in traceability and species authenticity studies, and as a reference for identification of possible freshness and quality markers. In the last study of this Thesis, the proteome profile of European sea bass (D.labrax) muscle was analyzed using 2-DE and tandem mass spectrometry (MS/MS) with the aim of providing a more detailed characterization of its specific protein expression profile. A highly populated and well resolved 2-DE map of the sea bass muscle tissue was generated, and the corresponding protein identity was provided for a total of 54 abundant protein spots. Upon Ingenuity Pathway Analysis, the proteins mapped in the sea bass muscle profile were mostly related to glycolysis and to the muscle myofibril structure, together with other biological activities crucial to fish muscle metabolism and contraction, and therefore to fish locomotor performance. The data obtained in this study provide important and novel information on the sea bass muscle tissue-specific protein expression, which can be useful for future studies aimed to improve seafood traceability, food safety/risk management and authentication analysis. In conclusion, the results of this Thesis indicate that, storage conditions are important for post mortem deterioration of fish muscle, and temperature is one of the factors with the strongest impact on this process. Pre-slaughter and slaughter stressful practices can have an important effect on the fish flesh quality. Therefore, the reduction of stress at slaughter might be a satisfactory strategy for both animal welfare and product quality. The molecular and proteomic methods used in this Thesis helped to identify nucleoside diphosphate kinase B and phosphoglycerate mutase 2 as candidate biomarkers for the evaluation of sea bass fillet freshness quality.