Synergistic antibacterial effects and mechanisms of different phenolic acids against Shewanella putrefaciens from fish, with emphasis on the combination of gallic acid and caffeic acid

The strong spoilage capability and cold adaptability of Shewanella putrefaciens present a serious threat to the quality and safety of fish. This study aimed to investigate the synergic antibacterial effects of six phenolic acids (Gallic acid, GA; Caffeic acid, CA; Protocatechuic acid; Ferulic acid; Chlorogenic acid; p-Coumaric acid) in pairs against S . putrefaciens , and to illustrate the antibacterial mechanisms. The fractional inhibitory concentration index (FICI) of 15 combinations of phenolic acids against S. putrefaciens ranged from 0.5-0.75, suggesting synergistic or partial synergistic antibacterial effects of these phenolic acids. Time-kill curves and morphologic observation demonstrated that GA and CA (GC) exhibited the strongest synergistic effect against S. putrefaciens . GC synergistically damaged the structure integrity of S. putrefaciens , leading to the leakage of intracellular substances and inhibiting the activity of Na + K + -ATPase and respiratory chain dehydrogenase. Both GA and CA could bind to DNA and cause changes in DNA content. Proteomics analysis revealed that 399 proteins were affected in the GC groups, a number significantly higher than that in the single GA and CA groups. GC inhibited dissimilatory nitrate and sulfate reduction, and enhanced fatty acid degradation and assimilatory sulfate reduction, which might cause the imbalance of energy supply and ROS accumulation. Meanwhile, the antioxidant-related enzymes, efflux systems and some two-components systems were upregulated, while chemotaxis were inhibited. GC synergistically inhibited S. putrefaciens in turbot, and retarded the spoilage process. The results revealed multi-targeted synergistic antibacterial mechanisms of GC against S. putrefaciens , suggesting GC might be a good strategy for controlling S. putrefaciens and fish spoilage.