Abstract
Alterations in the protein and lipid components of lean fish species were studied to elucidate the nature of protein denaturation in Gadus morhua and Melanogrammus aeglefinus during frozen storage. Frozen storage of lean fish, Gadus morhua and Melanogrammus aeglefinus led to the formation of ice crystals, which contributed to protein denaturation. Ice crystals were larger in fish fillets stored at -10°C compared to matching fillets stored at -30°C as studied by light microscopy at -20°C, which indicated damaged muscle fibre by compression. Protein denaturation was also attributed to the effect of lipid oxidation products. The presence of oxygen in the muscle system and high, hydrolytic enzyme and lipoxygenase activity led to increased free fatty acids and lipid oxidation respectively. The peroxide value (PV), conjugated dienes, thiobarbituric acid reactive substances (TEARS) and olefinic to aliphatic protons ratio by 1H NMR spectroscopy was indicative of oxidative deterioration of the lipid components of Gadus morhua and Melanogarmmus aeglefinus during frozen storage especially at -10°C. The ratio of the C=C to the aliphatic groups as assessed by FT-Raman spectroscopy also decreased progressively over the frozen storage period. The 1H NMR, conjugated diene and FT-Raman spectroscopic measurements were found to be effective and less labour intensive techniques for finger printing lipid oxidation than traditional methods. Intact muscle analysis using differential scanning calorimetry (DSC) showed that protein components of the muscle were denatured resulting in altered Tm and DeltaH values. FT-Raman spectroscopy of fish tissue confirmed changes in the proteins and showed decreased levels of alpha-helix and increased ?beta-sheet content (%) as well as changes in hydrophobic groups after frozen storage. These changes were pronounced in samples stored at -10°C compared to samples stored at -30°C. Model systems of protein-lipid complexes were studied using DSC, FT-Raman spectroscopy and ELISA. The effect of lipids and the primary and secondary oxidation products altered the conformation of myosin, collagen and water soluble proteins during freezing and frozen storage. DSC parameters namely Tm and DeltaH values indicated the degree of denaturation of fish proteins, when frozen in the presence and absence of lipids. It is proposed that ice crystal formation resulted in the removal of the hydration shell of the proteins and the overall rearrangement of the stabilising forces; this allowed protein-lipid interaction to take place and induced further protein denaturation. Reduced immune affinity of the myosin-lipid systems towards the myosin antibody compared to the control native myosin indicated conformational changes of the myosin molecule. The addition of lipids (DHA, EPA, extracted fish oil and hexanal) induced secondary structure changes in myosin over and above those caused by freezing. This was evidenced by decreased alpha-helix content with a concomitant increase of beta-sheet structure, indicating myosin polymerisation. A decrease in the tryptophan band, and increase in the ratio of the dityrosine bands indicated changes in hydrophobic groups. The model studies and the analysis of intact fish muscle of Gadus morhua and Melanogrammus aeglefinus suggest that protein denaturation occurred due to the concerted action of ice crystals, supercooled water molecules (unfrozen), high solute concentration, free fatty acids, and primary and secondary lipid oxidation products on the fish muscle proteins. Possible intervention schemes include, the addition of appropriate antifreeze glycoproteins, cryoprotectants and antioxidants.