Lipid oxidation mechanisms and lipid-protein interactions in frozen mackerel (Scomber scombrus).

Atlantic mackerel (Scomber scombrus) is a pelagic fish widely distributed along the Northern coast of Great Britain. The lipid content of mackerel was found to be about 13% of the total body weight and 50% of total fatty acids were eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) (fatty acids which are reported to reduce the concentration of plasma triglycerides, LDL (low density lipoproteins) and cholesterol in humans and animals). The proximate analysis also showed that mackerel is a good source of protein (20% w/w). The poly unsaturated fatty acids (PUFA) are prone to oxidation during frozen storage leading to rancidity and protein damage. Thus the objective of this project was to prolong the shelf-life of mackerel by controlling and understanding lipid oxidation mechanisms. HPLC, GCMS and 13C NMR spectroscopy were used for the first time to monitor the production of hydroperoxides and their secondary products in fish matched pairs of mackerel fillets were stored at either -20&deg;C or -30&deg;C. In addition fillets were also stored with or without different antioxidants at -20&deg;C. The development of lipid oxidation products were recorded for up to 24 months. The oxidation products identified were mixtures of alcohol derivatives of hydroperoxides, namely: 13-hydroxy-9-trans, 11-cis-octadecadienoic, 13-hydroxy-9-trans, 11-trans-octadecadienoic, 9-hydroxy-10-cis, 12-transoctadecadienoic and 9-hydroxy-10-trans, 12-transoctadecadienoic acids. The amount of hydroxides produced were higher in fillets stored at -20&deg;C compared with fillets stored at -30&deg;C. Similarly, the hydroperoxides produced were considerably higher in samples stored without antioxidant than in fillets stored with vitamin E. In this study the transfer of radicals from lipid oxidation to proteins and subsequent formation of protein-cross-links has been reported for the first time. The interaction between lipids and proteins were examined by both ESR and fluoroscence spectroscopy. A central esr free radical (g )signal was observed in both simple systems (methyl linoleate and pure amino acids) and complex systems (fish lipid and pure proteins (lysozyme, ovalbumin) or fish protein (myosin)). The esr signal reached a maximum within a week and then started to decline and with a concomitant increase in a pinkish yellow chromogen. This chromogen which was soluble in organic solvent and fluoresced at an excitation wavelength 360 nm and emission wavelength 420 nm and indicated the formation of protein cross-links. Synthetic (BHT, BHA) and natural (vitamins E, C) antioxidants were capable of preventing both the radical transfer and protein cross-linking. In this study lipoxygenase was isolated from mackerel flesh and its involvement in lipid oxidation mechanism was established. The molecular weight of partially purified lipoxygenase was 119,000 Daltons. This enzyme was capable of oxidising arachidonic acid to 12-hydroeicosatetraenoic acid (12-HETE), which was identified by HPLC. This 12-HETE was absent in pure arachidonic acid and in samples to which boiled enzyme was added. Conventional inhibitors, synthetic and natural antioxidants also inhibited the formation of 12-HETE, indicating the importance of lipoxygenase in fish lipid oxidation. During frozen storage, protein solubility decreased and the texture deteriorated in Atlantic mackerel stored for 3, 6, 12 and 24 months at -20&deg;C and -30&deg;C. There was an increase in peroxide value and TBARS; decrease in myosin ATPase activity a decrease in myofibrillar protein solubility in high salt concentration as well as formation of high molecular weight aggregates which showed low thermal stability and high G' and G" modulus values. There were significant differences (P < 0.01) between samples stored at -20&deg;C and -30&deg;C, with greater deterioration evident in samples stored at -20&deg;C. Similarly, there were significant differences (P < 0.01) between samples stored with and without antioxidants; the samples stored without antioxidants deteriorated faster than samples stored with antioxidants. This suggests the involvement of lipid oxidation products in protein deterioration during frozen storage