A novel non-thermal hurdle strategy: Cold stress potentiated cold plasma to control Pseudomonas aeruginosa via integrated oxidative and metabolic disruption.

This study developed a synergistic sterilization strategy combining cold plasma (CP) and cold stress (CS) to control Pseudomonas aeruginosa (P. aeruginosa), a major spoilage bacterium in refrigerated foods. The optimized sequence of CS pretreatment (4 °C, 24 h) followed by CP treatment (50 kV, 180 s) achieved 88.90 ± 3.85% sterilization efficiency and induced irreversible cellular damage. The synergistic effect (CSCP24) initiated with CS-induced physiological sensitization, leading to severe membrane disruption (97.92% PI-positive cells), biomolecule leakage, and ultrastructural collapse. This was accompanied by a cascade of intracellular damage: CSCP24 triggered a redox homeostasis collapse through ROS burst and inactivation of key antioxidant enzymes (SOD, CAT, GSH-PX), along with energy metabolism failure evidenced by ATP depletion and critical enzyme activity loss (Na+K+-ATPase, MDH). Molecular docking revealed that CP-generated reactive species (H₂O₂, O₃, ·OH, NO·) specifically inhibited essential bacterial targets, including DNA gyrase, dihydrofolate reductase, catalase, and cold shock proteins, thereby blocking key metabolic and stress-response pathways. The treatment also attenuated virulence by inhibiting motility, auto-aggregation, and pyocyanin production. Validation in a pasteurized milk model confirmed that CSCP24 effectively suppressed microbial recovery throughout refrigerated storage. Collectively, this work establishes the sequential CSCP synergy as a potent multi-target intervention that systematically disrupts membrane integrity, oxidative defense, and energy metabolism, demonstrating its potential as a novel non-thermal processing step to enhance microbial control in the food industry.