The effects of different metal ions at various concentrations (0, 5, 25 ppm) on lipid oxidation, discolouration and physicochemical properties of muscle protein in cuttlefish (Sepia pharaonis) subjected to multiple freeze–thaw cycles, were investigated. Lipid oxidation of all treatments increased as the freeze–thaw cycle increased. However, the rate of the TBARS increases varied, depending on concentration, type and valency of the metal ion. Fe(II) induced lipid oxidation most effectively and its prooxidative effect was in a concentration-dependent manner. Cu(I), Cu(II) and Cd(II) showed negligible effects on lipid oxidation. The increased lipid oxidation of cuttlefish with added iron was coincidental with the increase in b* values (yellowness), especially with increasing freeze–thaw cycles. Cu(I) and Cu(II) altered cuttlefish protein sulfhydryl content and the protein solubility decreased with a concomitant increase in the disulfide bond content. The oxidative changes of proteins were observed only when a concentration of metal ions of 25 ppm was used. Those changes were more intense with increasing freeze–thaw cycles. The Ca2+, Mg2+, and Mg2+–Ca2+–ATPase activities of cuttlefish natural actomyosin decreased markedly in the presence of copper, whereas the Mg2+–EGTA–ATPase was increased. SDS-PAGE revealed that Cu(I) and Cu(II) induced the polymerization of muscle proteins stabilised by disulfide bond formation. However, Fe(II), Fe(III) and Cd(II) exhibited no pronounced effect on the oxidation of cuttlefish muscle proteins. Therefore, copper mainly caused the oxidation of protein, while iron induced lipid oxidation and the formation of a yellow colour in cuttlefish muscle, particularly with multiple freeze–thaw cycles.
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