Food safety and quality assurance face persistent challenges due to microbial contamination, especially in aquatic products characterized by high water activity and abundant nutrients such as free amino acids and nucleotides. These properties make them highly susceptible to spoilage by psychrotrophic Gram-negative bacteria, even under refrigerated conditions (Rathod et al., 2022). Hafnia alvei, a notable spoilage organism, has been isolated from diverse sources including plants, refrigerated meats, and aquatic products (Awolope et al., 2021; Litrenta and Oetgen, 2017). This bacterium is of particular concern due to its capacity to produce undesirable spoilage metabolites—including offensive odors and biogenic amines like putrescine—through quorum sensing (QS) communication primarily mediated by N-acyl homoserine lactones (AHLs), with C4-HSL and C6-HSL being the predominant signaling molecules in the LuxI/LuxR-type QS system of H. alvei H4 (Li et al., 2019). Additionally, its ability to form biofilms on food contact surfaces significantly enhances its persistence and spoilage potential (Li et al., 2025; Pang et al., 2022; Wang et al., 2024).

While natural antimicrobials such as ε-polylysine (PL) represent safer alternatives to synthetic preservatives (Li et al., 2022a), their efficacy against Gram-negative bacteria like H. alvei remains limited by dose-dependent effects. The cationic PL molecules must first traverse the formidable outer membrane barrier—rich in lipopolysaccharides (LPS)—to reach the cytoplasmic membrane. This often necessitates high concentrations (≥1.0 mg/mL) to achieve effective bactericidal action, raising concerns related to cost, potential sensory impacts, and environmental load (Jia et al., 2019; Chen et al., 2023; Zhang et al., 2024b). Consequently, developing innovative strategies to sensitize Gram-negative bacteria to lower, more practical doses of natural preservatives is urgently needed.

Quorum sensing inhibitors (QSIs), such as theaflavin-3,3′-digallate (TF3), offer a promising “anti-virulence” approach by disrupting bacterial communication pathways without inducing immediate lethal pressure (Chu et al., 2023; Zhang et al., 2024a; Zhang et al., 2025), thereby potentially mitigating resistance development. Previous studies have confirmed that TF3 effectively suppresses AHL production, biofilm formation, and motility in H. alvei H4 by targeting the QS circuitry (Li et al., 2023b). We therefore hypothesized that predisrupting the QS system using sub-inhibitory TF3 would compromise the coordinated defense and stress response mechanisms of H. alvei, effectively sensitizing the bacterial population and enhancing its susceptibility to the membrane-targeting action of PL. .

While existing research on synergistic antimicrobial combinations has often focused on direct antimicrobial interactions or pharmaceutical applications (Wang et al., 2021; Li et al., 2022b), the strategy proposed here is fundamentally different. Instead of directly enhancing killing, we introduce the concept of “pre-weakening” bacterial defenses by targeting the QS system. This approach aims to disarm the bacterium's coordinated virulence and stress response mechanisms before the application of a membrane-targeting preservative like PL. The theoretical breakthrough lies in shifting the paradigm from a direct “attack” to a “disarm-and-attack” strategy. Practically, this synergy allows for a substantial reduction in the required dosage of preservative, mitigating potential sensory impacts and cost, which represents a significant practical advancement for natural preservation in high-value seafood.

Thus, the objectives of this study were to: (1) evaluate the synergistic antibacterial effect of TF3 and PL against H. alvei H4; (2) elucidate the underlying mechanisms with emphasis on QS-associated phenotypes, physiological functions (membrane integrity, oxidative stress, and energy metabolism), and gene expression changes; and (3) validate the synergistic efficacy in refrigerated turbot fillets by monitoring microbial growth, spoilage metabolites, and sensory quality. This work provides mechanistic insights into a novel “pre-weaken and attack” strategy for controlling spoilage bacteria in seafood, offering a sustainable and resistance-mitigating paradigm for next-generation food preservation.