Graduate Thesis Or Dissertation

 

Endogenous proteinase and myosin gelation of arrowtooth flounder (Atheresthes stomias) Public Deposited

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  • Proteolytic degradation of fish flesh occurring at elevated temperatures is the primary limitation for the commercial utilization of arrowtooth flounder (ATF). Characterization of the autolytic activity of ATF muscle incubated at various pHs and temperatures indicated the involvement of heat-activated proteinases active at acidic and alkaline pHs. Further characterization of the proteinase extract from fish muscle indicated the proteinase was more active at acidic pH than at alkaline pH in hydrolysis of Z-Phe-Arg-NMec and all types of protein substrates tested. Based on molecular weight and hydrolytic properties, activity peak separated on size exclusion chromatography, or activity bands observed on activity-stained substrate gels were presumed to be cathepsin L or like. A muscle proteinase showing similar hydrolytic properties to a proteinase extract was purified to electrophoretic homogeneity and subsequently confirmed by kinetic studies to be cathepsin L. Therefore, the results clearly indicated that cathepsin L is primarily responsible for autolytic activity of ATF muscle and surimi at the elevated temperatures. Gelation of fish myofibrillar proteins, mainly myosin, is an important process for surimi production. Elucidation of the gelation mechanism and the effect of proteolysis on myosin provide information regarding protein interactions that improve ATF product quality. Heat-induced changes in physicochemical properties of myosin, free of endogenous proteinases, indicated myosin gelation consisted of two processes, denaturation and aggregation. ATF myosin was shown to be extremely sensitive to heat, resulting in denaturation at a lower temperature than other fish myosins. Denaturation began at 25°C and was initiated by the unfolding of the α-helical region. Following denaturation was the exposure of the hydrophobic and sulfhydryl residues, which were subsequently involved in aggregation and the gelation process. Changes in dynamic properties indicated ATF myosin formed a gel in three different stages, as shown by the first increase in gel rigidity at 28°C, followed by a decrease at 35°C and a second increase at 42°C. A model system using ATF myosin and papain was developed to investigate how proteolysis affects the heat-induced gelation of fish myosin. The addition of papain decreased the onset temperature and the rate at which G' developed during heating. DSC thermograms indicated papain significantly decreased the enthalpy required to induce myosin denaturation with no significant changes in the onset or the maximum temperature. Thermal denaturation kinetics indicated a decrease in both the activation energy of the denaturation process and the denaturation rate of myosin. Although myosin gels could be formed, structural disruption caused by proteolysis, i.e., reduction in molecular size and loss in structural domain, resulted in lowering of the gelling ability of myosin and rigidity of the formed gels.
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