Bacillus cereus is a spore-forming foodborne pathogen known for its high resistance to conventional sterilization methods, posing a persistent threat to food safety. In this study, a novel inactivation system was developed by combining superheated steam (SHS) with cold plasma, aiming to achieve enhanced inactivation of B. cereus spores on stainless steel surfaces. The system was designed to inject cold plasma gas directly into the SHS flow, ensuring simultaneous exposure to thermal and oxidative stress. Combined SHS-plasma (SHS-P) treatment at 150°C for 18.88 min achieved an experimental reduction of 4.88 log CFU/cm2, that is sufficient to satisfy microbiological control requirements for spore-forming pathogens such as B. cereus and Clostridium perfringens in food processing applications. To clarify the inactivation mechanism, dipicolinic acid release, lipid peroxidation, intracellular reactive oxygen species generation, and DNA integrity were assessed. SHS-P induced early iROS accumulation and DNA damage, followed by increased DPA release and membrane lipid peroxidation over time. Transmission electron microscopy further confirmed structural disruption in endospores following SHS-P treatment. Notably, the synergistic effects of SHS-P were more pronounced at 150°C than at 200°C, possibly due to differences in relative humidity and reactive species stability. These findings suggest that SHS-P is a promising, efficient strategy for inactivating B. cereus spores under milder conditions, with potential application in improving the microbiological safety of food contact surfaces without excessive thermal or chemical inputs.
