Solvent engineering as a tool in enzymatic indicator development for mild high pressure pasteurization processing

When developing an indicator to detect temperature heterogeneities in a high pressure (HP) vessel, the temperature sensitivity and the broadness of the application window have to be weighed against each other. In this study, the potential of a Bacillus amyloliquefaciens α-amylase (BAA)-based indicator for mild pasteurization conditions was evaluated. Solvent engineering was successfully used to reach two objectives: (i) to shift the pressure,temperature-stability of BAA in the HP pasteurization range; (ii) to differentiate the pressure,temperature-range of BAA inactivation from the pressure,temperature-range of inactivation of a previously developed Bacillus subtilis α-amylase (BSA)-based indicator system (1 g/L – 0.05 M MES pH 5.0), towards milder processing conditions. The BAA-system (0.1 g/L – 0.1 M MES pH 5.0) was kinetically calibrated and modeled under static conditions. As these model parameters were inadequate to predict the residual α-amylase activity of BAA under dynamic conditions correctly, parameters were determined under dynamic conditions and subsequently successfully validated on an independent dynamic data set. Based on the kinetic model and parameters valid under dynamic conditions, isorate contour plots for different inactivation levels were constructed, demonstrating clearly the different application windows of the BAA- and BSA-systems.