Abstract: Targeting the quorum sensing (QS) system of Hafnia alvei, this study aimed to screen for quorum sensing inhibitors (QSIs) that exhibit synergistic effects with penicillin G acylase (PGA). The QS quenching activity of PGA individually and in combination with chlorogenic acid (CGA), phenyl lactic acid (PLA), or epigallocatechin gallate (EGCG) were assessed using the agar plate diffusion method. By analyzing bacterial spoilage phenotypes and calculating the Jin Zhengjun Q value, QSIs exhibiting a synergistic effect with PGA were selected. The results demonstrated that PGA (2.4 mg/mL), CGA (2.0 mg/mL), PLA (0.6 mg/mL), and EGCG (1.5 mg/mL) degraded C8-HSL by (50.05 ± 0.12)%, (15.08 ± 0.10)%, (10.01 ± 0.11)%, and (8.02 ± 0.14)%, respectively. PGA combined with CGA, compared with PLA and EGCG, showed a higher degradation rate of 60.00% for C8-HSL. Additionally, the combined use of QSIs proved to be more effective in inhibiting bacterial spoilage phenotypes than individual treatments. The combination of PGA with CGA, relative to its combination with PLA or EGCG, more effectively inhibited violacein production, H. alvei biofilm, and swarming, with inhibition rates of (74.19 ± 0.15)%, (62.28 ± 2.21)%, and (54.10 ± 1.72)%, respectively. PGA combined with PLA showed the strongest inhibitory effect on exopolysaccharide production, with an inhibition rate of (37.87 ± 1.39)%. PGA combined with CGA had a synergistic inhibitory effect (Q > 1.15) on violacein production and swarming motility and an additive inhibitory effect (0.85 < Q < 1.15) on biofilm formation, exopolysaccharide production, and swimming motility. The combination of PGA with CGA, compared with its combination with PLA or EGCG, showed a stronger synergistic inhibitory effect on the QS of H. alvei. This study provides a theoretical foundation for the development of novel, green, and efficient QSIs.

Key words: penicillin G acylase; chlorogenic acid; Hafnia alvei; quorum sensing; spoilage phenotypes