Thiol-reactive natural antimicrobials and high pressure treatment synergistically enhance bacterial inactivation

Abstract Bacterial inactivation by high pressure (HP) treatment can be strongly enhanced in the presence of antimicrobial compounds, but the mechanism of this synergy is poorly understood. In the present study, screening of thirteen natural antimicrobial compounds (NACs) led to the identification of six NACs that exhibited synergy with HP treatment against eight Gram-negative and two Gram-positive bacteria. The strongest synergy was found with α,β-unsaturated aldehydes (t-cinnamaldehyde, t-2-hexenal, dimethylfumarate), isothiocyanates (allyl isothiocyanate, sulforaphane) and other electrophilic compounds (reuterin). The antibacterial activity of these compounds and their synergistic interaction with HP was linked to thiol reactivity based on (1) their reaction with cysteine in vitro, (2) their reduced minimal inhibitory concentration in an Escherichia coli gshA mutant which is defective in glutathione synthesis, and (3) the loss of synergy with HP in the presence of excess cysteine. These novel insights in the mechanisms leading to synergy will support the development of more effective hurdle technology applications of HP treatment and natural antimicrobials. Industrial relevance HP treatment is an emerging mild processing technology that offers a better balance between microbiological safety and stability and quality retention compared to traditional heat-based pasteurization methods. The efficacy of microbial inactivation by HP treatment is improved in the presence of natural antimicrobials, but the basis of this synergistic interaction is poorly understood. In the present work, a specific antibacterial mode of action is shown to result in synergy, and this insight will facilitate the development of more effective combined treatments.