Bactericidal action of carvacrol towards the food pathogen Bacillus cereus : a case study of a novel approach to mild food preservation

A new trend in food preservation is the use of mild preservation systems, instead of more severe techniques such as heating, freezing or addition of chemical preservatives. Carvacrol, a phenolic compound present in the essential oil fraction of oreganum and thyme, is known for its antimicrobial activity since ancient times. This thesis describes a study of the antimicrobial activity of carvacrol towards the foodborne pathogen <em>B. cereus</em> . Carvacrol shows a dose-related inhibition of growth of <em>B. cereus</em> . Concentrations of 0.75 mM and higher inhibit growth completely at 8°C. Below 0.75 mM, carvacrol extends the lag-phase and reduces the specific growth rate as well as the final population density. Exposure to 0.75-3 mM carvacrol decreases the number of viable cells of <em>B. cereus</em> exponentially. Spores are approximately two fold more resistant towards carvacrol than vegetative cells.The incubation and exposure temperature have a significant influence on the sensitivity of <em>B. cereus</em> to carvacrol. An increase of the growth temperature from 8°C to 30°C decreases the fluidity of the membrane of vegetative cells and as a consequence, <em>B. cereus</em> becomes less sensitive to carvacrol. The change in membrane fluidity is probably the result of a higher percentage of lower melting lipids in the membranes at 8°C (chemical process) as an adaptation to lower temperature. Cells need to maintain an adequate proportion of the liquid-crystalline lipid in the membrane, as this is the ideal physical state of the membrane. On the other hand, an increase of the exposure temperature from 8 to 30°C, reduces the viability again. This can be explained by an increase of the membrane fluidity at a higher temperature as a result of melting of the lipids (physical process). At a higher membrane fluidity, relatively more carvacrol can dissolve in the membrane and the cells will be exposed to relatively higher concentrations than at a lower membrane fluidity.Not only the temperature plays a role in the activity of carvacrol, also pH is an important factor. The sensitivity of <em>B. cereus</em> to carvacrol is reduced at pH 7, compared to other pH-values between pH 4.5 and 8.5.Carvacrol interacts with the cytoplasmic membrane by changing its permeability for cations such as K <sup>+</SUP>and H <sup>+</SUP>. Consequently, the dissipation of the membrane potential (Δψ) andΔpH leads to inhibition of essential processes in the cell, such as ATP synthesis, and finally to cell death. At carvacrol concentrations as low as 0.15 mM,Δψis completely dissipated, however the viable count of <em>B. cereus</em> is not affected.Vegetative cells of <em>B. cereus</em> can adapt to carvacrol when the compound is present at concentrations below the MIC-value. Compared to non-adapted cells, lower concentrations of carvacrol are needed to obtain the same reduction in viable count of adapted cells. Adapted cells were found to have a lower membrane fluidity, caused by a change in the fatty acid composition and head group composition of the phospholipids in the cytoplasmic membrane. Adaptation to 0.4 mM carvacrol increases the phase transition temperature of the lipid bilayer (T _m ) from 20.5°C to 28.3°C. Addition of carvacrol to cell suspensions of adapted <em>B. cereus</em> cells decreases T _m again to 19.5°C, approximately the same value as was found for non-adapted cells in the absence of carvacrol.Incubation of cooked rice in the presence of different carvacrol concentrations results in a dose-related reduction of the viable count of <em>B. cereus</em> . Concentrations of 0.15 mg/g and above, reduce the viable count, leading to full suppression of growth at 0.38 mg/g. The influence of carvacrol on the viable count is dependent on the initial inoculum size. Although carvacrol is an effective inhibitor of growth of <em>B. cereus</em> in rice, it could affect the flavour and taste of the product at concentrations where full suppression of growth is observed. However, strong synergistic activity is observed when carvacrol is combined with the biosynthetic precursor cymene or the flavour enhancer soya sauce. This makes it possible to use lower carvacrol concentrations and consequently a smaller influence on the sensoric properties of the rice is expected.Besides its influence on the viability of vegetative cells, carvacrol also shows inhibition of diarrhoeal toxin production by <em>B. cereus</em> at concentrations below the MIC-value. Addition of 0.06 mg/ml carvacrol to the growth medium, inhibits the toxin to 21% of the control (no carvacrol added). The inhibition correlates with the reduction of the viable count of <em>B. cereus</em> in the presence of carvacrol. At the same time, the total amount of cells did not change. In mushroom soup, also an inhibition of the toxin production was observed, however, the viable count did not change. This effect on the toxin production is most probably caused by a lack of sufficient metabolic energy, since carvacrol affects ATP synthesis. The cell will use its low levels of ATP to maintain its viability, rather than using it for toxin production or excretion. It could also be possible that the decreased toxin synthesis in BHI was the result of the lower amount of viable cells. The inhibition of toxin production at carvacrol concentrations which do permit growth of <em>B. cereus</em> , reduces the risk of food intoxication by this pathogen.In conclusion, carvacrol may play an important role in future as a natural antimicrobial compound. However, its application will most probably be in combination with other natural antimicrobial systems.