Optimizing thermosonication treatment for Escherichia coli, Staphylococcus aureus, and endospores of Geobacillus stearothermophilus inactivation and fresh milk sterilization

Ultrasonication is a non-thermal technology capable of inactivating bacteria in food without compromising its quality. When combined with mild heat (thermosonication), the process becomes even more effective for bacterial inactivation. This study optimized the thermosonication process by determining the optimal combination of frequency, temperature, and exposure duration using an ultrasonicator prototype with adjustable frequency, power, and temperature. Response surface methodology with a three-factor Box–Behnken design was employed to inactivate Escherichia coli and Staphylococcus aureus cells. Based on the modeling results, the optimal thermosonication conditions for inactivating E. coli were 44.55 kHz, 50 °C, and 30 min, with a power density of 1.2 W/cm 3 , while for S. aureus , the optimal conditions were 38.79 kHz, 50 °C, and 30 min at the same power density. Under these conditions, the predicted log reductions were 3.3 and 2.8 CFU/mL for E. coli and S. aureus , respectively. Furthermore, the optimal thermosonication conditions also reduced the log number of Geobacillus stearothermophilus endospores by 1.4 CFU/mL at the same power density. These reductions were statistically significant (P ≤ 0.05) and demonstrated a strong fit between the predicted and experimental data, as indicated by high coefficients of determination (R² ≥ 0.97). When applied to milk, the optimized thermosonication treatment resulted in a greater reduction of total bacterial count compared to pasteurization. Additionally, compared with pasteurization, thermosonication extended the shelf life of local fresh milk up to 14 days while maintaining total bacterial counts and pH values below the Indonesian National Standard (SNI) thresholds. • A multi-frequency ultrasonication prototype with adjustable temperature and power was successfully developed and evaluated. • Thermosonication showed synergistic effects of ultrasound and heat in inactivating bacteria and endospores. • Thermosonication caused more severe cell damage compared with ultrasonication or thermal treatment alone. • Thermosonication extended milk shelf life while maintaining microbial and pH levels within Indonesian quality standards.