Food Microbiology 21 (2004) 327–334
Effect of thymol and cymene on Bacillus cereus vegetative cells
evaluated through the use of frequency distributions
a Delgado, Pablo S. Fern
andez, Alfredo Palop, Paula M. Periago*
Dpto. Ing. Alimentos y del Equipamiento Agr
ıcola, Escuela T
ecnica Superior de Ingenier
omica (ETSIA), Universidad Polit
ecnica de Cartagena,
Paseo Alfonso XIII 48, 30203 Cartagena (Murcia), Spain
Received 29 April 2003; accepted 18 July 2003
The bactericidal action of thymol and cymene on two Bacillus cereus strains (INRA-AVTZ415 and INRA-AVZ421) was studied.
Increasing concentrations of thymol (0.2–1.0 mmol l
) or cymene (0.2–2.0 mmol l
) showed higher bactericidal effect on
exponential growth phase B. cereus cells suspended in HEPES buffer (pH 7), at 30
C. The two strains tested presented different
sensitivity to these natural antimicrobials. When thymol and cymene were combined, it resulted in a greater bactericidal effect on
B. cereus cells than when these compounds in the essential oil fraction of aromatic plants were applied separately. There was a
synergistic effect of both natural antimicrobials on the viability of exponential growth phase B. cereus cells in pH 7 HEPES buffer, at
C. Survivor curves did not follow a ﬁrst-order kinetics, which makes it difﬁcult to establish processing conditions. Experimental
data were modeled using a frequency distribution function (Weibull). This model guaranteed a good description of the experimental
results and allowed predictions of time to a speciﬁc decrease in bacterial populations. This study indicates the potential use of
thymol and cymene applied separately or simultaneously for preservation of minimally processed foods, as well as the validity of the
Weibull distribution to describe the resulting nonlinear data and establish effective treatment conditions with these compounds.
r 2003 Elsevier Ltd. All rights reserved.
Keywords: Thymol; Cymene; Bacillus cereus; Food safety; Frequency distribution
Bacillus cereus is a spore-forming, facultative anae-
robe and Gram-positive rod. Many authors have widely
documented in the literature the presence of B. cereus in
raw and processed meat, vegetables, rice and dairy
products (Carlin et al., 2000; Dufrenne et al., 1994;
Hassan and Nabbut, 1996; Valero et al., 2002). B. cereus
is associated with two kinds of foodborne illnesses: a
diarrheal and an emetic type, caused by two distinct
toxins (Granum and Lund, 1997). Due to its capability
to produce these toxins, B. cereus is becoming one of the
most important causes of food-poisoning in the
industrialized world (Granum, 1997).
Food manufacturers and consumers demand additive
free, fresher and more natural tasting food products
(Dillon and Board, 1994; Gould, 1996) while maintain-
ing microbiological safety. The use of natural antimi-
crobial systems for preservation of foods could
accomplish this increasing demand. Herbs and spices
have been known for their antimicrobial activity since
antiquity. The safe use of herbs and spices and their
components (such as thymol and cymene) has led to
their current status of Generally Recognized as Safe
(GRAS) food ingredients. Although essential oil com-
ponents, including thymol and cymene, extracted from
several types of plants, have been used as ﬂavorings in
the food industry, they represent a highly interesting
source of natural antimicrobials for food preservation
due to their antimicrobial and antioxidant activity
(Beuchat, 1994; Lagouri et al., 1993).
The components thymol (a cyclohexane with a
bounded hydroxyl group) and cymene (a biosynthetic
precursor of thymol which lacks of the bounded
hydroxyl group) are compounds present in the essential
oil fraction of Origanum and Thymus genus (Lagouri
et al., 1993; Sivropoulou et al., 1996). These compounds
in the essential oil fraction are hydrophobic and they are
ARTICLE IN PRESS
*Corresponding author. Present address: Dpto. Ciencia y Tecnolog-
ıa de Alimentos, Centro de Edafolog
ıa y Biolog
ıa Aplicada del Segura
(CEBAS-CSIC), Apdo. Correos 4195, 30.080 Murcia, Spain.
Tel.: +34-968396248; fax: +34-968396213.
E-mail address: firstname.lastname@example.org (P.M. Periago).
0740-0020/$ - see front matter r 2003 Elsevier Ltd. All rights reserved.