Elucidation of protein aggregation in frozen cod and haddock by transmission electron microscopy/immunocytochemistry,light microscopy and atomic force microscopy

Polyclonal antibodies raised to both native cod myosin and actin as well as to aggregated proteins obtained from frozen cod stored for 11 months at ?10 °C were used to investigate disposition of muscle proteins in frozen cod and haddock fillets by transmission electron microscopy. Specimens from cod and haddock fillets, stored at ?10 °C, treated with anti‐aggregate antibody as the primary antibody, showed significantly more gold particles, especially around the protein aggregates and muscle fibres compared with fish stored at ?30 °C. Samples that were treated with anti‐myosin or anti‐actin antibody showed opposite results. Similar binding properties were observed in ELISA experiments involving the reaction of actin and myosin to both native and aggregate antibodies; thus immunological tests can be used for monitoring aggregate and texture changes in frozen stored fish. In addition, atomic force microscopy images obtained from cod muscle also indicated structural changes in frozen cod muscle proteins. The mica surface was covered with a continuous layer of muscle proteins comprising mainly small globular particles and a few large particles for the control cod sample stored at ?30 °C for 11 months. In contrast, cod fillets stored at ?10 °C showed a thin layer of proteins with small holes and an increased number of large particles denoting aggregates. Formation of ice crystals between the muscle fibres of frozen cod and haddock muscle was monitored without thawing by light microscopy at ?20 °C. The micrographs showed a greater proportion of large ice crystals and extensive protein fibre changes in fillets stored at ?10 °C compared with the control at ?30 °C. Copyright © 2004 Society of Chemical Industry