Adsorption isotherms at 25 °C and 38 °C for microencapsulated microbial transglutaminase (mTG) powder produced by ultrasonic spray–freeze–drying (USFD), conventional spray drying (CSD), and conventional freeze–drying (CFD) methods and USFD–crude (without coating materials) were obtained using the standard static gravimetric method, and experimental data were fitted to the mathematical models. The sorption isotherms exhibited type III behavior, and the Ferro–Fontán model was selected as the best fit, having an R 2 of 0.920–0.995. The sorption isotherms of mTG powder depended on temperature and drying methods. The equilibrium moisture content decreased with increasing temperature, and the monolayer moisture content of the USFD sample was lower than other microencapsulated samples (M 0 = 0.133 kg/kg dry solids for 25 °C and M 0 = 0.080 kg/kg dry solids for 38 °C from Guggenheim–Anderson–deBoer model), followed by CFD and CSD. Furthermore, the storage stability of mTG powders produced by different methods in terms of quality parameters including enzyme activity, moisture content, water activity, color, density, porosity, flowability, and caking degree value was studied in aluminum-laminated polyethylene (ALPE) pouches under storage conditions of 25 °C, 50% relative humidity (RH) for 180 days and 38 °C, 90% RH for 90 days. Inactivation kinetics, the effect of drying methods, and application of the microencapsulation process were also examined on mTG powder during the storage period. The first-order kinetic reaction was successfully applied to the relative activity values of all samples for both storage conditions. Both the drying method and application of microencapsulation showed a significant effect on enzyme activity. When comparing the drying techniques, the USFD samples were the most stable, having 816 and 465 days of half-life times for both storage conditions, respectively. Storage at 25 °C, 50% RH increased the half-life twofold compared with that of the USFD samples kept at 38 °C, 90% RH. The physical properties of mTG powder obtained either USFD, USFD–crude, CFD, or CSD showed that moisture content and wettability values increased, while caking degree and enzyme activity values decreased during storage. USFD samples had the lowest moisture content (5.48%) and Hausner ratio (1.12) and the highest relative activity (86.2%) compared with other samples after 180 days storage at 25 °C, 50% RH. When all quality characteristics and enzyme stability were considered, the USFD method was superior to others, and it was determined to be the best method for the production of microencapsulated mTG powders. mTG powders stored at 25 °C, 50% RH exhibited a longer shelf life.
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