Appl Environ Microbiol 65(10): 4606-4610

Mechanisms of Action of Carvacrol on the Food-Borne Pathogen <em>Bacillus cereus</em>

Agrotechnological Research Institute (ATO-DLO), 6700 AA Wageningen,^{and Wageningen Centre for Food Sciences (WCFS), 6700 AN Wageningen,}^{The Netherlands}

^{Corresponding author. Mailing address: Agrotechnological Research Institute (ATO-DLO), P.O. Box 17, 6700 AA Wageningen, The Netherlands. Phone: 31-317-475171. Fax: 31-317-475347. E-mail:}ln.old.ota@eetlU.A.

Received 1999 Mar 11; Accepted 1999 Jul 12.

Abstract

Carvacrol, a naturally occurring compound mainly present in the essential oil fraction of oregano and thyme, was studied for its effect on bioenergetic parameters of vegetative cells of the food-borne pathogen Bacillus cereus. Incubation for 30 min in the presence of 1 to 3 mM carvacrol reduced the viable cell numbers exponentially. Carvacrol (2 mM) significantly depleted the intracellular ATP pool to values close to 0 within 7 min. No proportional increase of the extracellular ATP pool was observed. Depletion of the internal ATP pool was associated with a change of the membrane potential (Δψ). At concentrations of 0.01 mM carvacrol and above, a significant reduction of Δψ was observed, leading to full dissipation of Δψ at concentrations of 0.15 mM and higher. Finally, an increase of the permeability of the cytoplasmic membrane for protons and potassium ions was observed (at 0.25 and 1 mM carvacrol, respectively). From this study, it could be concluded that carvacrol interacts with the membranes of B. cereus by changing its permeability for cations like H^{and K}^{. The dissipation of ion gradients leads to impairment of essential processes in the cell and finally to cell death.}

Abstract

Bacillus cereus is a spore-forming food-borne pathogen often associated with food products such as meat, vegetables, soup, rice, and milk and other dairy products. Between 1 and 20% of the total number of outbreaks of food infection in the world is caused by B. cereus (19). Growth of vegetative cells usually occurs within the temperature range of 10 to 50°C, with an optimum between 28 and 35°C. However, psychrotrophic variants of B. cereus, capable of growing at temperatures below 5°C, have been identified (6, 22). Although vegetative cells of B. cereus can easily be inactivated by heating, spores are considerably more resistant and can cause food spoilage after subsequent germination (6).

Mild preservation technologies are becoming more important in modern food industries. As a consequence, spore-forming microorganisms are likely to proliferate and hence become a serious food safety risk. Mild processes are often combined to obtain safe products with improved organoleptic quality. A novel way to reduce the proliferation of microorganisms is the use of essential oils. The antifungal and antibacterial effects of these components on different microorganisms have been described in several studies (5, 14, 16–18, 26–29). Among the diverse group of chemical components in essential oils, carvacrol exerts a distinct antimicrobial action. Carvacrol is the major component of the essential oil fraction of oregano (60 to 74% carvacrol) and thyme (45% carvacrol) (1, 20). In practice, carvacrol is added to different products, e.g., baked goods (15.75 ppm), nonalcoholic beverages (28.54 ppm/0.18 mM), chewing gum (8.42 ppm), etc. (8). However, not much is known about the mechanisms of action of this compound. A better knowledge of the mode of action is important regarding application in food systems. Recently, Ultee et al. (29) showed the antimicrobial effect of carvacrol on B. cereus. Hydrophobic compounds like carvacrol are likely to have an influence on biological membranes. The cytoplasmic membrane of bacteria has two principal functions: (i) barrier function and energy transduction, which allow the membrane to form ion gradients that can be used to drive various processes, and (ii) formation of a matrix for membrane-embedded proteins (such as the membrane-integrated F₀ complex of ATP-synthase) (12, 24). In the present study, changes in the energy-transducing processes of B. cereus caused by carvacrol were studied in more detail. The effect of carvacrol on the intracellular ATP pool, the membrane potential, the pH gradient across the cytoplasmic membrane, and the potassium gradient was evaluated.

ACKNOWLEDGMENTS

We thank R. A. Slump for his assistance with the viability tests and P. Breeuwer for his advice considering pH_in measurements.

This work was financially supported by the Commission of the European Union through contract FAIR CT 96-1066.

ACKNOWLEDGMENTS

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