Growth dynamics of Clostridium perfringens superdormant spores in cooked ground chicken by ultrasound-assisted mild heat treatment with nutrient

The present study aimed to refine 'Germination-inactivation' strategy for Clostridium perfringens by integrating mild heat, ultrasound, and nutrient stimulation: Samples were first subjected 75 °C for 20 min (heat shock) and a refined thermosonication condition (55 °C; 60 min; 20.0 W/mL) and nutrient concertation of 50 mM, then inactivation of spores at an elevated temperature (80 °C for 20 min). Under optimal thermosonication condition and nutrient concertation, the majority of spores (≈ 3.8 log₁₀ CFU/mL, with germination rate of 99.98 %) were germinated and inactivated, yet some superdormant (SD) spores (≈ 3.2 log₁₀ CFU/mL) remaining. Flow-cytometry and phase-contrast imaging revealed population heterogeneity, a phase-bright to phase-dark change was found during germination. Four distinct subpopulations identified, and SD fraction (mHT-US-SD, R1) was isolated. mHT-US-SD spores were inoculated into cooked chicken, incubated at 20, 25, 30, 36, or 45 °C and exhibited markedly lower maximum specific growth rates, prolonged lag phases, and an extended estimated shelf-life (time to ≤ 1 log₁₀ CFU/g increase) compared with untreated dormant (D) spores. The temperature dependence of growth was accurately captured by both square-root and exponential secondary models and showed lower minimum growth temperature. Collectively, this study demonstrates that ultrasound-assisted mild heat treatment with nutrient as a novel strategy for controlling C. perfringens by both facilitating germination and characterizing the growth behavior of SD spores.