Abstract: With the aim of understanding the mechanism of action of cystatin C of silver carp (HmCystatin C) as a potential natural biological preservative, we investigated the effect of HmCystatin C on maintaining the texture quality of refrigerated fish fillets and the regulatory effect on myocyte apoptosis mediated by mitochondrial pathway. Western blot was used to verify the cellular permeability of HmCystatin C in grass carp fillets. Quality changes during refrigeration for 72 h at 4 ℃ were monitored with respect to texture, histological characteristics, cathepsin B/L activity and reactive oxygen species (ROS) levels. Myocyte apoptosis, the opening degree of mitochondrial permeability transition pore (MPTP), mitochondrial membrane potential and mitochondrial morphological damage were detected by immunohistochemistry, TUNEL staining, spectrophotometry, and transmission electron microscopy (TEM). Meanwhile, the activities and protein expression of caspase-3 and caspase-9 were analyzed by fluorescence assay and Western blot, respectively. The results demonstrated that HmCystatin C effectively penetrated into and persistently existed in cells. Compared with the control group, shear force and hardness obviously increased in the Cys treated group, gaps between myocytes, cathepsin B/L activity and ROS levels decreased, and texture deterioration was postponed by at least 24 h. Additionally, the damage degree of mitochondrial function and morphology was suppressed significantly in the Cys group. Meanwhile, the activities of caspase-3 and caspase-9 were inhibited to different extents and the protein expression levels were lower than that in the control group. In conclusion, the inhibition of mitochondrial pathway-mediated apoptosis and relevant caspases activities by HmCystatin C might contribute to its effect on delaying the quality deterioration of refrigerated grass carp fillets.

Key words: cystatin C of silver carp; refrigerated grass carp fillets; texture characteristics; mitochondrial pathway-mediated apoptosis; caspases