Biochemical and rheological characterization of a protease from fruits of Withania coagulans with a milk-clotting activity

A protease from Withania coagulans fruits was evaluated for milk-clotting properties. The optimum temperature and pH for enzyme activity were 70°C and 4, respectively. The protease also exhibited an excellent thermal stability at 60°C for 30 min. Fractional precipitation using ammonium sulfate showed that the 40–50% fraction (F5) possessed the highest milk-clotting activity. The F5 fraction showed a Mw band of 66 kDa by SDS-PAGE. Gel formation was monitored using low amplitude oscillatory rheology at different temperatures. An Arrhenius plot was used to describe the temperature dependence of the gelation rate parameter. Time sweep testing showed that an increase in temperature was accompanied by a decrease in the gelation onset time, a higher gel formation rate, and higher values for final gel strength. W.coagulans fruits have potential for use as a rennet substitute.