Combined antimicrobial effect of plasma-activated water and mild heat against Penicillium expansum spores

This study aimed to examine the combined antimicrobial action of plasma-activated water (PAW) and mild heat (30, 40, 50, and 60 °C) against Penicillium expansum spores in phosphate-buffered saline (PBS). The combined treatment showed stronger antimicrobial activity against P. expansum spores. P. expansum spores decreased more than 6.76 log₁₀ CFU/mL after exposure to PAW at 60 °C for 10 min, which was higher than that of samples treated by PAW at 25 °C (0.80-log reduction) or mild heat treatment alone at 60 °C (2.27-log reduction). After exposure to PAW + mild heat, a high amount of intracellular nucleic acids and proteins were released from spores, suggesting the loss of cell membrane integrity. P. expansum spores also exhibited a large increase in intracellular reactive oxygen species (ROS) and a reduction in mitochondrial membrane potential after exposure to PAW + mild heat. Moreover, antioxidants glutathione and N-acetylcysteine effectively inhibited spores death induced by PAW + mild heat, suggesting that ROS was involved in the antifungal action of PAW and mild heat. In summary, PAW + mild heat exhibits excellent antimicrobial activity against P. expansum spores by destroying cellular membranes, reducing mitochondrial membrane potential, and causing oxidative damage.