Casein zymography‐based detection and one‐step purification for simultaneous quantification of calcium‐induced endogenous proteases in breast and thigh muscles from different chicken breeds

Abstract

Identification and separation of endogenous proteases are important to find out their role in changes of biochemical process in post-mortem muscle. Diverse studies were conducted to find out the activity of these enzymes but only a very few studies were carried out on avian species. In this study, a simple casein zymography method was explained for detection of calpain-1 and calpain-2 with the standardization of extraction buffer and that were quantified biochemically after one-step separation and purification on anion exchange chromatography. DEAE Sephacel matrix was used to purify calpain-1, calpain-2, and calpastatin. SDS-PAGE analysis showed native and autolyzed forms of calpains. Biochemical test indicated concentrations of calpain-1, calpain-2, and calpastatin being higher in breast than thigh muscles across all four breeds of chicken. A simple, effective, and low-cost extraction, purification, and separation technique was developed for accurate identification of calpain-1and calpain-2 on casein zymography method and quantification by biochemical test. Novelty Impact Statement Identification and isolation of endogenous proteases are necessary to determine their significance in the PM aging of chicken meat from various breeds. In this study, a straightforward casein zymography approach for in-vitro detection of calpain-1 and calpain-2 activity is devised. This will aid in the optimization of PM aging duration for meat tenderization.