Evaluation of antibacterial and anti-biofilm properties of kojic acid against five food-related bacteria and related subcellular mechanisms of bacterial inactivation

Abstract

Although the antimicrobial properties of kojic acid have been recognized, the subcellular mechanism of bacterial inactivation caused by it has never been clearly elucidated. In the present study, the antibacterial and anti-biofilm activity of kojic acid was evaluated against five foodborne pathogens including Listeria monocytogenes, Bacillus subtilis, Staphylococcus aureus, Escherichia coli, and Salmonella typhimurium. The antibacterial activity was determined by minimum inhibitory concentration, minimum bactericidal concentration, and the time-kill assay. Among them, the susceptibility of Escherichia coli was significant with the lowest minimum inhibitory concentration and minimum bactericidal concentration values of 10 and 20 mM, respectively. Subcellular mechanism of bacterial inactivation related to kojic acid was revealed through comprehensive factors including cell morphology, membrane permeability, K⁺ leakage, zeta potential, intracellular enzyme, and DNA assay. Results demonstrated that bacterial inactivation caused by kojic acid, especially for Gram-negative bacteria, was primarily induced by the pronounced damage to the cell membrane integrity. Leakage of intracellular enzyme to the supernatants implied that the cell membrane permeability was compromised. Consequently, the release of K⁺ from the cytosol leads to the alterations of the zeta potential of cells, which would disturb the subcellular localization of some proteins and thereby cause the bacterial inactivation. The free −CH₂OH group at the C-2 of kojic acid could play more significant role in the antimicrobial performance of kojic acid against Gram-negative bacteria. Moreover, remarkable interaction with DNA was also observed. Kojic acid at sub-minimum inhibitory concentration inhibited biofilm formation by these bacteria.