Wageningen University Dissertation No. 2799

Summary

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

Author: Annemieke Ultee
Date: 2 June 2000

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 B. cereus. Carvacrol shows a dose-related inhibition
of growth of B. cereus. 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 B. cereus 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 B. cereus 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, B. cereus 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 B. cereus 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+ and H+. Consequently, the
dissipation of the membrane potential ( ) and pH 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, is completely
dissipated, however the viable count of B. cereus is not affected.

Vegetative cells of B. cereus 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 (Tm) from 20.5°C to 28.3°C. Addition of carvacrol to cell
suspensions of adapted B. cereus cells decreases Tm 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 B. cereus. 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 B.
cereus 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 B. cereus 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 B. cereus 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 B. cereus, reduces the risk of food intoxication by
this pathogen.

In conclusion, carvacrol may play an important role in future as a
natural antimicrobial compound.