Aims:The minimum inhibitory concentration (MIC) of oregano essential oil (OEO) and two of its principle components, i.e. thymol and carvacrol, against Pseudomonas aeruginosa and Staphylococcus aureus was assessed by using an innovative technique. The mechanism of action of the above substances was also investigated. Methods and Results: The applied technique uses 100-well microtitre plate and collects turbidimetric growth data. To produce the inhibition pro®les, a wide range of concentrations were tested for each of the three compounds, as well as for carvacrol±thymol mixtures. Following a speci®c mathematical analysis of the observed inhibition pro®les from all compounds, it was suggested that mixtures of carvacrol and thymol gave an additive effect and that the overall inhibition by OEO can be attributed mainly to the additive antimicrobial action of these two compounds. Addition of low amounts of each additive: (a) increased permeability of cells to the nuclear stain EB, (b) dissipated pH gradients as indicated by the CFDA-SĒ uorescent probe irrespective of glucose availability and (c) caused leakage of inorganic ions. Conclusions: Mixing carvacrol and thymol at proper amounts may exert the total inhibition that is evident by oregano essential oil. Such inhibition is due to damage in membrane integrity, which further affects pH homeostasis and equilibrium of inorganic ions. Signi®cance and Impact of the Study: The knowledge of extent and mode of inhibition of speci®c compounds, which are present in plant extracts, may contribute to the successful application of such natural preservatives in foods, since certain combinations of carvacrol± thymol provide as high inhibition as oregano essential oil with a smaller¯avour impact.

Measuring the minimum inhibitory concentration (MIC) of a substance by current methods is straightforward, whereas obtaining useful comparative information from the tests can be more difficult. A simple technique and a method of data analysis are reported which give the experimentalist more useful information from susceptibility testing. This method makes use of a 100‐well microtitre plate and the analysis uses all the growth information, obtained by turbidometry, from each and every well of the microtitre plate. A modified Gompertz function is used to fit the data, from which a more exact value can be obtained for the MIC. The technique also showed that at certain concentrations of inhibitor, there was no effect on growth relative to a control well (zero inhibitor). Above a threshold value, which has been termed the non‐inhibitory concentration or NIC, growth becomes limiting until it reaches the MIC, where no growth relative to the control is observed.

R .J . L A MB ER T AN D M . ST RA T FO RD . 1999. Weak-acid preservatives are widely used to prevent microbial spoilage of acidic foods and beverages. Characteristically, weak-acid preservatives do not kill micro-organisms but inhibit growth, causing very extended lag phases. Preservatives are more effective at low pH values where solutions contain increased concentrations of undissociated acids. Inhibition by weak-acids involves rapid diffusion of undissociated molecules through the plasma membrane; dissociation of these molecules within cells liberates protons, thus acidifying the cytoplasm and preventing growth. By modelling preservative action in yeast, using a thermodynamic and kinetic approach, it was possible to demonstrate that: (i) inhibition depends more on the degree to which individual preservatives are concentrated within cells, rather than on undissociated acid concentration per se; (ii) it is entirely feasible for microbes to pump protons out of the cell during extended lag phase and raise internal pH (pH i ), despite further influx of preservatives; (iii) the duration of the lag phase can be predicted from the model, using a Gaussian fit of proton-pumping H ¦ -ATPase activity against pH i ; (iv) theoretical ATP consumption for proton pumping can be directly correlated with the reduction in cell yield observed in glucose-limited cultures.

No abstract