Inhibition of Quorum Sensing-Mediated Biofilm Formation and Spoilage Factors in <em>P</em><em>seudomonas fluorescens</em> by Plasma-Activated Water

Plasma-activated water (PAW) is an emerging disinfectant, but few studies have explored its role as a quorum sensing inhibitor (QSI) for biofilm suppression, and the mechanisms of action remain unclear. Herein, we investigated the effects of PAW on biofilm formation and spoilage factors secretion in Pseudomonas fluorescens under sub-inhibitory conditions. The results showed that PAW-60 treatment achieved a maximum reduction of 1.29 log CFU/mL in P. fluorescens biofilm after 12 h incubation. Furthermore, PAW-60 completely inhibited protease production (100%) and decreased siderophore production by 31.87%. C4-HSL was identified as the dominant signaling molecule, and its production was reduced by 34.34% to 84.07% after selected PAW treatments. Meanwhile, C4-HSL activity was significantly decreased by 42.58% to 65.38% (P &lt; 0.05). FTIR analysis detected the formation of a new C=O group after PAW treatment, indicating that PAW degraded AHLs by oxidative modification. Exogenous C4-HSL gradually restored biofilm biomass, spoilage factors production, and QS-related gene expression levels, implying that the inhibitory effects of PAW primarily resulted from the disruption of AHLs transduction. Molecular docking showed that long-lived reactive species in PAW could bind to AHLs synthetic protein (FadD1) and receptor protein (LuxR) via hydrogen bonding. PAW-60 treatment effectively reduced spoilage activity of P. fluorescens inoculated to fish muscle juice and extended its shelf life from 8 to 10 days during 4 °C storage. These findings revealed that PAW mitigated biofilm formation and the spoilage process of food by blocking signaling transduction, which involved AHLs production inhibition, degradation, and recognition disruption.