Reactive oxygen species-mediated inactivation of Listeria monocytogenes and Staphylococcus aureus induced by pulsed plasma treatment: influence of duty ratio and unit time

Reactive oxygen species (ROS) are key mediators of plasma-induced microbial inactivation. This study investigated the influence of pulsed plasma parameters, specifically duty ratio and unit time, on the inactivation of Listeria monocytogenes and Staphylococcus aureus . Pulsed plasma-activated water PAW was generated under controlled discharge conditions, where the unit time, defined as the duration of a single plasma discharge cycle, and the duty ratio, the fraction of the unit time during which the plasma is actively on, were systematically varied to optimize bactericidal efficacy. Optimization of the parameters significantly enhanced inactivation, achieving reductions of 5.88 ± 0.10 log CFU for L. monocytogenes and 5.89 ± 0.09 log CFU for S. aureus . The optimal treatment condition (duty ratio 0.5 and unit time 2 min) resulted in a 2.7-fold increase in intracellular ROS compared with continuous plasma exposure. Fluorescence- and spectrophotometry-based analyses confirmed substantial ROS accumulation, accompanied by a reduction in bacterial viability as determined by plate counts. In situ evaluation using fresh-cut lettuce demonstrated 5.25 ± 0.15 and 5.18 ± 0.19 log CFU reductions for L. monocytogenes and S. aureus , respectively, without adversely affecting physicochemical quality. These findings demonstrate that ROS-mediated oxidative stress is the primary mechanism of pulsed plasma–driven microbial inactivation and highlight that both duty ratio and unit time critically regulate ROS dynamics and bacterial susceptibility. This study provides mechanistic insight into plasma–microbe interactions and supports the practical implementation of pulsed plasma technology for microbial control in food systems.