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We aimed to investigate the antibacterial activities of carvacrol, thymol, eugenol, cinnamaldehyde, and lantibiotic nisin against standard bacterial strains of the milk pathogens Staphylococcus aureus ATCC 25923 and Listeria monocytogenes ATCC 15313 in cow milk. The minimum inhibitory concentrations (MIC) of these substances were recorded. The synergistic effects were also assessed in culture medium (time kill curve) and in a food model (cow milk) during the storage period (4 °C for 6 d) after inoculation with S. aureus and L. monocytogenes individually by combining nisin and the phenolic compounds at proportions of 1/4 + 1/4 the MIC (determined in a previous in vitro assay) in the culture medium and 1/4 + 1/4 of MIC in the food model. Inhibitory activities of nisin and the tested compounds, as well as synergism in the combinations, were found against both bacteria assayed. Bacteriostatic effects were found with all combinations and a significant difference in L. monocytogenes reduction was found compared with the control assays. Thus, the antibacterial activity of nisin combined with phenolic compounds was confirmed against these pathogenic bacteria that are important in the milk industry, or more broadly in food science, with potential applications for milk preservation.

Food safety has been an established research field for many decades. This report describes the antibacterial and anti-staphylococcal enterotoxin properties of major phenolic compounds found in essential oils (cinnamaldehyde, citronellol, eugenol, geraniol and terpineol). The determination of minimum inhibitory concentrations (MIC) against Gram-negative and Gram-positive strains, including methicillin-resistant Staphylococcus aureus (MRSA) and enterotoxin-producing S. aureus (enterotoxins A, B, C and D) were evaluated. Cinnamaldehyde displayed MIC values ranging from 100 to 400 μg/mL against Gram-positive and Gram-negative bacteria, while the other four compounds showed similar values only against Gram-negative bacteria. For Grampositive strains, MIC values ranged from approximately 800-1750 μg/mL. Interactions between the compounds and antibacterial drugs were evaluated by disc diffusion and time-kill curve assays against MRSA. Combinations of phenolic compounds that included gentamicin showed the greatest synergistic effect. In vitro treatments with subinhibitory concentrations of phenolic compounds resulted in a decreased production of enterotoxins B and C (SEB and SEC). Transmission electron microscopy was performed to evaluate mechanisms of action for cinnamaldehyde and geraniol against E. coli and MRSA. Cells treated with compounds showed complete loss of membrane integrity, separation of the cytoplasmic membrane from the cell wall, cytoplasmic content leakage and cytoplasmic polarization. Thereby, this work showed in vitro potential of using combinations of phenolic compounds and antimicrobial drugs against S. aureus and the virulence of S. aureus enterotoxin producers. Industrial relevance: Antimicrobial compounds derived from plants are a focus of renewed interest as potential substitutes for artificial food preservatives. In our study subinhibitory concentrations of phenolic compounds had a significant effect on the quantity of enterotoxins produced by Staphylococcus aureus and inhibited the growth of Escherichia coli, Salmonella Enteritidis and other bacteria in microbiological media.

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