Innovative Food Science and Emerging Technologies 12 (2011) 320329

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Innovative Food Science and Emerging Technologies

j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / i f s e t

The antimicrobial activity of hydrophobic essential oil constituents acting alone or in

combined processes of food preservation
Abdenour Ait-Ouazzou, Lamia Cherrat, Laura Espina, Susana Lorn, Carmen Rota, Rafael Pagn
Departamento de Produccin Animal y Ciencia de los Alimentos, Facultad de Veterinaria, Universidad de Zaragoza, C/Miguel Servet, 177, 50013, Zaragoza, Spain

a r t i c l e

i n f o

Article history:
Received 6 December 2010
Accepted 11 April 2011
Editor Proof Receive Date 10 May 2011
Keywords:
Food preservation
Heat
Pulsed electric elds
Essential oil
Sublethal injury
Combined process

a b s t r a c t
This work evaluates the antimicrobial activity of widespread hydrophobic essential oil (EO) constituents, 3
hydrocarbon monoterpenes (-pinene, -pinene, and p-cymene) and 8 oxygenated monoterpenes (thymol,
carvacrol, borneol, linalool, terpineol-4-ol, 1,8-cineole, -terpinyl acetate, and camphor), as a function of the
treatment medium pH, and possible synergistic effects in combination with mild heat or pulsed electric elds
(PEF) treatments. Results obtained using the disk diffusion technique highlight phenols and alcohols as the
best growth inhibitors and discount hydrocarbons due to their poorer activity. However, the evaluation of the
bactericidal effect at pH 4.0 shows that most compounds assayed, including some hydrocarbons, were very
effective against Escherichia coli and Listeria monocytogenes. Most EO constituents caused membrane
permeabilization and sublethal injuries within survivors. Outstanding synergistic lethal effects were shown
using mild heat (54 C/10 min) or PEF (30 kV/cm/25 pulses) combined with 0.2 l/ml of some antimicrobials,
achieving 5 log10 cycles of cell inactivation as a function of the treatment conditions. In most cases, combined
treatments were more effective in apple than in orange juice.
Industrial relevance: The efcacy of EO constituents improves when combining with mild heat or PEF
treatments, which allows us to propose very low doses of antimicrobials. The valuable synergistic effects
observed offer the potential to improve traditional heat treatments by reducing treatment intensity and
consequently adverse effects on food quality, and to enhance novel PEF treatments by achieving a higher
degree of microbial inactivation.
2011 Elsevier Ltd. All rights reserved.

1. Introduction
Plant essential oils (EOs) and their constituents have attracted
much interest because of their widespread use in perfumes, as the
principal antimicrobial in a variety of sanitary, pharmaceutical or
make-up products, and as food preservatives and additives (Bakkali,
Averbeck, Averbeck, & Idaomar, 2008).
EOs are very complex natural mixtures that can contain about 20
80 constituents at signicantly different concentrations. The main
group is composed of terpenes and terpenoids, and the other group
contains aromatic and aliphatic constituents. Numerous studies have
addressed the antimicrobial effect of EOs and their individual terpene
and terpenoid components (Burt, 2004). The monoterpenes are the
most representative molecules, constituting 90% of EOs and comprising a great variety of structures. In general, oxygenated monoterpenes
are signicantly more active than are hydrocarbon monoterpenes
(Carson & Riley, 1995). Moreover, interactions between different

Corresponding author. Tel.: + 34 976 761581; fax: + 34 976 761590.

E-mail address: pagan@unizar.es (R. Pagn).
1466-8564/$ see front matter 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.ifset.2011.04.004

constituents can occur, causing antagonistic, additive, and synergistic

effects (Burt, 2004; Delgado, Fernndez, Palop, & Periago, 2004;
Nychas, 1995). An analysis of the inherent antimicrobial activity of EO
constituents acting alone and how they interact as a function of the
treatment conditions might help us to appropriately combine them to
achieve synergistic lethal effects using the smallest dose, thus
avoiding adverse alterations of sensorial properties of the matrix.
Although chemical preservatives are mainly used to prevent
microbial growth, recent studies have lauded their use as inactivating
agents (Burt, 2004; Friedman, Henika, & Mandrell, 2002; Somolinos,
Garca, Mackey, & Pagn, 2010; Somolinos, Garca, Pagn, & Mackey,
2008). Little is known about the capacity of hydrophobic EO
compounds to kill microorganisms as a function of concentration,
environmental conditions, incubation temperature, or time. Moreover, their mechanisms of microbial inhibition and inactivation are
likely different. Concerning this subject, there are few studies that
compare modes of action or antimicrobial spectrums under the same
experimental conditions. On the other hand, since the doses to
achieve microbial inactivation are expected to be very high when
acting alone, thus affecting the sensory properties of the samples,
their use in combination with other technologies such as heat, pulsed
electric elds (PEF), or high hydrostatic pressure has been proposed
(Burt, 2004; Maas & Pagn, 2005; Periago, Palop, & Fernndez, 2003;

A. Ait-Ouazzou et al. / Innovative Food Science and Emerging Technologies 12 (2011) 320329

Raybaudi-Massilia, Mosqueda-Melgar, Soliva-Fortuny, & Martn-Belloso,

2009; Somolinos et al., 2008).
The most successful combined preservation treatments are those
that achieve an excellent hurdle effect. Knowledge of the action
mechanisms of each barrier would help establish the most effective
treatment conditions. Recently, our research group (Somolinos et al.,
2010) has demonstrated the occurrence of a strongly synergistic
lethal effect on Escherichia coli through a mild heat treatment
combined with an acyclic unsaturated monoterpene aldehyde, citral,
found naturally in several plant species (Somogyi, 1996, March). The
synergistic effect was probably due to the damages inicted on the
outer membrane of E. coli by heat. Similar results have been observed
when inactivating Enterobacter sakazakii by means of PEF and citral
treatments (Arroyo, Somolinos, Cebrin, Condn, & Pagn, 2010).
Since the outer membrane acts as an impermeable barrier to
hydrophobic compounds, damage in the cell structure might
represent an interesting opportunity to design combined processes
that facilitate the action of antimicrobials. The occurrence of sublethal
injury in the outer membrane of Gram-negative bacteria after
different preservation methods or as a consequence of other stresses
is a well-known phenomenon (Mackey, 2000; Maas & Pagn, 2005).
The aim of this research was to evaluate the antimicrobial activity
of widespread EO constituents (-pinene, -pinene, camphor, p-cymene,
thymol, carvacrol, borneol, linalool, terpineol-4-ol, -terpinyl
acetate and 1,8-cineole) for control of microbial growth, their
bactericidal activity as a function of treatment medium pH, their
capacity to cause sublethal damage and membrane permeabilization,
and possible synergistic effects in combination with mild heat or PEF
treatments.
2. Material and methods
2.1. Micro-organisms and growth conditions
The strains of Enterococcus faecium (ATCC 19434), Listeria monocytogenes (ATCC 13932), Staphylococcus aureus (ATCC 6538), Pseudomonas aeruginosa (ATCC 10145), and Salmonella serotype Enteritidis
(ATCC 49214) used in this investigation were supplied by the Spanish
Type Culture Collection. The E. coli O157:H7 strain is a VTEC (Phage
type 34) isolated by Dr. Chapman (Chapman et al., 1993). L.
monocytogenes EGD-e (Chatterjee et al., 2006) was kindly provided
by Prof. Chakraborty (Institute for Medical Microbiology, Giessen,
Germany). During this investigation, the culture was kept frozen at
80 C in cryovials.
Broth subcultures were prepared by inoculating, with one single
colony from a plate, a test tube containing 5 ml of sterile Tryptic Soy
Broth (Biolife, Milan, Italy) with 0.6% Yeast Extract added (Biolife)
(TSBYE). After inoculation, the tubes were incubated overnight at 37 C
(E. faecium, S. aureus, P. aeruginosa, Salm. Enteritidis, E. coli O157:H7)

321

or 30 C (L. monocytogenes and P. aeruginosa). With these

subcultures, 250 ml Erlenmeyer asks containing 50 ml of TSBYE
were inoculated to a nal concentration of 104 CFU/ml. These asks
were incubated under agitation (130 rpm; Selecta, mod. Rotabit,
Barcelona, Spain) at the appropriate temperature (see above) until
the stationary growth phase was reached.
2.2. Constituents of essential oils
All components studied were purchased from Sigma-Aldrich
(Sigma-Aldrich Chemie, Steinheim, Germany). They were 3 hydrocarbon monoterpenes (-pinene purum 98%, -pinene purum 99%,
and p-cymene purum 99%), and 8 oxygenated monoterpenes (thymol
purum 99%, carvacrol purum 98%, borneol purum 97%, linalool
purum 95%, terpineol-4-ol purum 95%, 1,8-cineole purum 99%,
-terpinyl acetate 90%, and camphor purum 96%). As thymol,
borneol and camphor were solid at the experimental temperature,
they were dissolved in 95% ethanol to obtain a stock solution of
100 mg/ml.
2.3. Antimicrobial assay (disk diffusion assay)
The antimicrobial activity of -pinene, -pinene, p-cymene,
carvacrol, linalool, terpineol-4-ol, 1,8-cineole, and -terpinyl acetate
was screened against 7 microorganisms of signicant importance
using the agar diffusion technique (Meena & Sethi, 1994; Rota,
Carramiana, Burillo, & Herrera, 2004).
Filter paper disks (Whatman No. 1, 6 mm diameter) containing
15 l of each EO constituent were applied to the surface of agar plates
of Tryptic Soy Agar (Biolife) supplemented with 0.6% Yeast Extract
(Biolife) (TSAYE) that were previously seeded by spreading one
sterile hyssop impregnated in a stationary phase culture. The plates
were incubated overnight at the appropriate temperature (see
above), and the diameter of the resulting zone of inhibition was
measured in millimeters. The results indicated in Table 1 represent
the net zone of inhibition including the diameter (6 mm) of the paper
disk. The scale of measurement was the following: 20 mm zone of
inhibition is strongly inhibitory; b2012 mm zone of inhibition is
moderately/mildly inhibitory; and b12 mm is non-inhibitory (Rota et
al., 2004). Experiments were carried out in triplicate.
2.4. Determination of minimum inhibitory concentration (MIC) and
minimum bactericide concentration (MBC)
All EO compounds were screened to determine MIC and MBC
against L. monocytogenes EGD-e and E. coli O157:H7, using the tube
dilution method (Rota et al., 2004). The highest and the lowest
concentrations tested were 2 and 0.2 l/ml, corresponding to 2 and
0.2 mg/ml of solid compounds. Negative controls containing TSBYE

Table 1
Zones of growth inhibition (mm) showing antibacterial activity for a number of selected hydrocarbon (*) and oxygenated (**) monoterpenes; disk diameter 6.0 mm. Zone of growth
inhibition values is presented as mean standard deviation.
Compounds

Carvacrol (**)
Linalool (**)
Terpineol-4-ol (**)
-Terpinyl acetate (**)
1,8-Cineole (**)
p-Cymene (*)
-Pinene (*)
-Pinene (*)

Strains tested
S.a.

L.m 1

L.m 2

E.f.

S.E.

E.c.

P.a.

59.5 3.3
58.5 4.4
33.5 1.7
39.3 1.9
NI
16.5 0.2
NI
NI

48.1 3.6
13.7 1.3
15.8 1.5
NI
14.5 0.7
NI
NI
NI

49.2 1.1
14.8 2.1
17.8 2.4
12.8 1.6
13.2 0.4
NI
NI
NI

36.5 0.3
NI
14.6 0.6
12.9 1.3
NI
NI
NI
NI

32 0.1
13.5 1.3
30.4 0.1
NI
NI
NI
NI
NI

33.8 2.6
23.6 1.9
30.4 3.6
NI
NI
NI
NI
NI

12.9 0.4
NI
NI
NI
NI
NI
NI
NI

S.a: S. aureus; L.m 1: L. monocytogenes (EGD-e); L.m 2: L. monocytogenes (4b); E.f.: E. faecium; S.E: Salm. Enteritidis; E.c: E. coli O157:H7; P.a: P. aeruginosa.
NI: no inhibited.

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plus 3% ethanol and 2 l/ml of EO constituents and positive controls

containing TSBYE with microorganisms to a nal concentration of 106
CFU/ml plus 3% ethanol were also prepared. After a 24 h incubation at
the appropriate temperature in a shaking thermostatic bath (Bunsen,
mod. BTG, Madrid, Spain), the MIC was read. The MIC was the lowest
concentration of EO constituents at which bacteria failed to grow, so
no visible changes were detected in the broth medium.
In order to evaluate MBC, after 24 h incubation at the appropriate
temperature (see above) in the shaking thermostatic bath, 100 l of
each case in which microbial growth was not observed was spread
plated in TSAYE. Plates were incubated at the corresponding
temperature for 24 h. The MBC was dened as the lowest concentration at which bacteria failed to grow in broth and the subsequent
transfer to TSAYE plates (Janssen, Scheffer, & Svendsen, 1987). The
evaluation of MIC and MBC was carried out in triplicate.
2.5. Measurement of cell inactivation by antimicrobial compounds
Following the procedure described by Friedman et al. (2002), a
vigorous shaking method was used to prepare antimicrobial compound suspensions in citratephosphate buffers (McIlvaine's buffer)
at pH 7.0 and 4.0 (Dawson, Elliot, Elliot, & Jones, 1974), avoiding the
use of ethanol.
Cells from stationary-phase cultures were added at nal concentrations of 107 CFU/ml to citratephosphate buffers at pH 7.0 or pH
4.0, with or without antimicrobial compounds, at concentrations of
0.2 l/ml (corresponding to 0.2 mg/ml of solid compounds). That was
the lowest concentration assayed for MIC and MBC evaluation. Buffer
pH was not modied as a consequence of adding antimicrobial
compounds. Antimicrobial compound treatments were carried out at
20 C for 24 h. Samples were taken at 10 min, 6 h, and 24 h, and
survivors and sublethally injured cells were enumerated as described
below. Previous experiments showed that untreated cells of E. coli
O157:H7 and L. monocytogenes EGD-e at concentration of 107 CFU/ml
were insensitive to incubation in citratephosphate buffers at pH 7.0
or 4.0 for 24 h at 20 C.

pulse repetition rate of 1 Hz were used in this study. The specic

energy input of each pulse was 5.2 kJ/kg. Experiments started at room
temperature. In all experiments, the temperature of the samples after
the application of 25 pulses was lower than 35 C. After treatment,
samples were taken, and survivors were evaluated.
2.8. Measurement of propidium iodide cell uptake
In order to evaluate cell permeabilization, propidium iodide (PI)
(Sigma-Aldrich) was added to a nal concentration of 0.08 mmol/l
once the antimicrobial treatment was nished (Pagn & Mackey,
2000). Cell suspensions were incubated for 15 min at room temperature, centrifuged at 10,000 g for 5 min, and washed three times
until no extracellular PI remained in the buffer. Cell permeabilization
was checked using a uorescence microscope (Nikon, Mod. L-Kc,
Nippon Kogaku KK, Japan).
2.9. Counts of viable cells
After treatments, samples were adequately diluted in 0.1% w/v
peptone water (Biolife) containing 1% v/v Tween 80 (Biolife) as a
neutralizer (Onawunmi, 1989). Next, 0.1 ml samples were pourplated onto Tryptic Soy Agar (Biolife) with 0.6% Yeast Extract added
(Biolife) (TSAYE). Plates were incubated for 24 h at the appropriate
temperature (see above). Previous experiments showed that longer
incubation times did not inuence the surviving cell counts. After
incubation, colonies were counted with an improved image analyzer
automatic counter (Protos; Analytical Measuring Systems, Cambridge,
United Kingdom), as previously described (Condn, Palop, Raso, &
Sala, 1996). Inactivation was expressed in terms of the extent of
reduction in log10 counts after any treatment. The error bars in the
gures indicate the mean standard deviations from the data
obtained from at least three independent experiments. Analyses of
variance (p = 0.05) were performed with SPSS software (SPSS,
Chicago, IL, USA).

2.6. Heat treatment experiments

2.10. Detection of sublethal injury

Tubes containing 5 ml of sterile citratephosphate buffers at pH

7.0 and 4.0, heat-treated apple juice (pH 3.5), and heat treated orange
juice (pH 3.7) (Don Simn, Murcia, Spain), with or without
antimicrobial compounds added to a nal concentration of 0.2 l/ml
(0.2 mg/ml of solid compounds), were placed in a shaking thermostatic bath at 54 C (Bunsen, mod. BTG, Madrid, Spain). Once the
treatment temperature was reached, the microbial suspension was
added to a nal concentration of 107 CFU/ml. After 10 min at 54 C,
samples were taken, and survivors and sublethally-injured cells were
evaluated as explained below.

In order to determine bacterial cell injury, treated samples were

also plated onto TSAYE with 3% (E. coli O157:H7) or 6%
(L. monocytogenes EGD-e) sodium chloride (Fisher Scientic, Loughborough, United Kingdom) added (TSAYE-SC) and onto TSAYE with
0.35% (E. coli O157:H7) bile salts (Biolife, Milan, Italy) added (TSAYEBS) in order to evaluate cytoplasmic membrane damage and outer
membrane damage, respectively (Mackey, 2000). These levels of

2.7. PEF treatment experiments

PEF treatments were carried out using equipment comprising 10
capacitors (6800 pF) and a parallel-electrode batch treatment
chamber with a distance of 0.25 cm between electrodes and an area
of 2.01 cm2 that delivered an exponential-decay pulse, previously
described by Garca, Gmez, Raso, & Pagn (2005).
Before treatment, micro-organisms were centrifuged at 6000 g
for 5 min and resuspended at a concentration of 107 CFU/ml in apple
and orange juice, and in citratephosphate buffer of pH 7.0 and 4.0,
the concentration of which was adjusted to an electrical conductivity
of 2.0 mS/cm, as well as in these treatment media with antimicrobial
compounds added to a nal concentration of 0.2 l/ml (0.2 mg/ml of
solid compounds). Next, 0.5 ml of the microbial suspensions was
placed into the treatment chamber with a sterile syringe. Exponential
waveform pulses at an electrical eld strength of 30 kV/cm and a

Table 2
Minimal inhibitory concentration (MIC) and minimal bactericide concentration (MBC)
(l or mg/ml) of selected hydrocarbon (*) and oxygenated (**) monoterpenes.
Compounds

Strains tested
L.m

Thymol (**)
Carvacrol (**)
Borneol (**)
Linalool (**)
Terpineol-4-ol (**)
-Terpinyl acetate (**)
1,8-Cineole (**)
Camphor (**)
p-Cymene (*)
-Pinene (*)
-Pinene (*)

E.c

MIC

MBC

MIC

MBC

b0.2
b0.2
0.5
b0.2
0.5
0.5
N2.0
0.5
0.5
0.5
0.5

0.2
b 0.2
1.0
0.2
N 2.0
N 2.0
N 2.0
N 2.0
N 2.0
N 2.0
N 2.0

0.2
0.2
0.5
0.2
0.2
N2.0
N2.0
2.0
N2.0
N2.0
N2.0

0.2
0.2
2.0
0.2
0.2
N 2.0
N 2.0
N 2.0
N 2.0
N 2.0
N 2.0

L.m: L. monocytogenes (EGD-e); E.c: E. coli O157:H7.

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sodium chloride and bile salts were previously determined as the

maximum non-inhibitory concentrations for native cells (data not
shown). Samples recovered in selective media were incubated for
48 h. Previous experiments showed that longer incubation times did
not inuence survival counts (unpublished results).
3. Results
3.1. Antimicrobial activity of EO constituents assessed by disk diffusion
assay and determination of MIC and MBC
The preliminary screening of the antimicrobial activity of 8 widespread EO constituents was carried out against 7 spoiling and
pathogenic microorganisms using the disk diffusion assay. Table 1
summarizes the antibacterial activity of selected hydrocarbon and
oxygenated monoterpenes. Attending to the inhibition zone, the
components with wider antimicrobial spectrums were found to be
carvacrol, terpineol-4-ol, and linalool. -Terpinyl acetate only showed
strong activity against S. aureus, while 1,8-cineole and p-cymene only
showed moderate activity against some microorganisms. In contrast,
-pinene and -pinene did not show any signicant inhibitory
activity. So, oxygenated monoterpenes showed higher inhibitory
activity on microbial growth than did hydrocarbons. Regarding the
functional group, the phenolic compound (carvacrol) was more active
than the alcohols (terpineol-4-ol, linalool), followed by the ester
(-terpinyl acetate) and the ether (1,8-cineole) assayed.
In general, Gram-negative bacteria were more resistant to
antimicrobials than were Gram-positive bacteria. Only E. coli showed
signicantly higher susceptibility to carvacrol, terpineol-4-ol, or
linalool (p 0.05) than did other Gram-positive bacteria. P. aeruginosa
was the most resistant bacterial strain, being only moderately
inhibited by carvacrol. On the other hand, both L. monocytogenes
strains assayed showed the same degree of inhibition under the
presence of the antimicrobials tested (p 0.05). Hence, L. monocytogenes EGD-e was chosen to perform the following experiments
because of the availability of information on biochemical, functional,
and genetic aspects (Chatterjee et al., 2006). This will facilitate future
molecular biological studies on this strain.
More precise data on the antimicrobial properties of the EO
constituents were obtained through the determination of bacteriostatic and bactericidal concentrations against E. coli O157:H7 and
L. monocytogenes EGD-e, representing Gram-negative and Grampositive bacteria, respectively. Table 2 shows the MIC and MBC of 11
EO constituents against the two bacteria selected. The EO constituents
with the greatest bacteriostatic properties were phenols (thymol and
carvacrol) and linalool with MIC 0.2 l/ml. Terpineol-4-ol showed a
MIC of 0.2 l/ml against E. coli and 0.5 l/ml against L. monocytogenes.
With the exception of 1,8-cineole, the other antimicrobials tested
showed a MIC of 0.5 l/ml against L. monocytogenes. E. coli was less
susceptible, being only inhibited by borneol at 0.5 l/ml and camphor
at 2 l/ml.
The evaluation of the MBC revealed that only thymol, carvacrol,
and linalool were bactericidal at concentrations of 0.2 l/ml; terpineol-4-ol was also bactericidal at the same concentration against E. coli;
and borneol was bactericidal against L. monocytogenes and E. coli at
higher doses, 1 and 2 l/ml, respectively.
3.2. Microbial inactivation by EO constituents as a function of the
treatment medium pH
The action of 0.2 l/ml of EO constituents on the survival of both
microorganisms was tested at pH 7.0 and 4.0 for 10 min, 6 h, and 24 h
at ambient temperature (Fig. 1).
According to Fig. 1A and C, the inactivation at pH 7.0 was
coincident with the bactericidal activity described in Table 2. Only
thymol and carvacrol caused a signicant degree of inactivation

323

during incubation for 24 h. Nevertheless, the study revealed a greater

sensitivity of E. coli to both phenols.
Evaluating survivors using selective recovery media shows that
phenols caused sublethal injuries on both cytoplasmic and outer
membranes of E. coli cells. After 24 h of incubation, the whole
population analyzed had sublethal injuries, and the degree of
inactivation by carvacrol reached approximately 3 log10 cycles of
the bacterial population. The level of inactivated L. monocytogenes
cells was slightly lower, and all survivors showed sublethal injuries on
their cytoplasmic membranes. Regarding the hydrocarbons, -pinene
and -pinene caused signicant sublethally injured E. coli cells after
24 h at pH 7.0, and only p-cymene caused the inactivation of at least
90% of the cell population of both strains (Fig. 1A, C).
The evaluation of the bactericidal activity of these compounds at
pH 4.0 (Fig. 1B, D) revealed the potential of some compounds to
achieve 5 log10 cycles of inactivation of both strains after 24 h at
ambient temperature. Among hydrocarbons, p-cymene showed the
highest bactericidal activity. L. monocytogenes was also very sensitive
to -pinene and -pinene, achieving more than 4 log10 cycles of
inactivation. Regarding the oxygenated monoterpenes, the degree of
inactivation of E. coli reached 5 log10 cycles after only 6 h of incubation
in the presence of 0.2 l/ml of thymol and carvacrol. In the case of
L. monocytogenes, a similar degree of inactivation was also reached in
the presence of thymol, carvacrol, linalool, and -terpinyl acetate.
While borneol was the least effective as a bactericide against
L. monocytogenes, 1,8-cineole and camphor were poorly effective
against E. coli at pH 4.0.
Again, the evaluation of survivors by using selective recovery
media showed that most compounds caused sublethal injuries to both
microorganisms, and in the case of E. coli, again, the damages inicted
on the outer membrane seemed to occur previously to those of the
cytoplasmic membrane.
Membrane permeabilization was demonstrated by the uptake of
the uorescent probe, propidium iodide, by most E. coli and
L. monocytogenes cells (N90%) treated in the presence of 0.2 l/ml of
carvacrol, thymol, -terpinyl acetate, and p-cymene for 24 h at room
temperature, and by most L. monocytogenes cells (N90%) also treated
in the presence of linalool, -pinene, and -pinene. Dye trapped
inside the cells was detected visually using a uorescence microscope
(data not shown).

3.3. Combined preservation processes: mild heat or PEF plus EO

constituents
Fig. 2 shows the inactivation of E. coli and L. monocytogenes using a
mild heat treatment (54 C for 10 min) alone or in combination with
0.2 l/ml of hydrocarbon and oxygenated monoterpenes at pH 7.0 and
4.0. As shown in the gure, exposure of both microorganisms to 54 C
for 10 min at pH 7.0 and 4.0 caused the inactivation of at least 0.5 log10
cycles of cells and caused 14 log10 cycles of injured cells, since they
were sensitive to the selective media. On the other hand, the
application of 0.2 l/ml of any antimicrobial during the same period
of time (10 min) did not cause signicant inactivation (b0.5 log10
cycles) and almost no sublethal injury (Fig. 1).
As shown in Fig. 2, the action of both methods applied
simultaneously (the heat treatment and 0.2 l/ml of each monoterpene) caused a greater inactivation than did the sum of both methods
acting separately. In all cases, synergistic lethal effects were observed
when treating microorganisms at both pH values. For instance, about
5 log10 cycles of inactivation were achieved when heating E. coli at pH
4.0 in the presence of oxygenated monoterpenes or p-cymene, which
supposes more than 4 extra log10 cycles of inactivation (Fig. 1B). The
same occurred when combining the heat treatment with phenols,
linalool, and terpineol-4-ol against E. coli at pH 7.0, and phenols,
linalool, terpineol-4-ol, -terpinyl acetate, and p-cymene against

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A. Ait-Ouazzou et al. / Innovative Food Science and Emerging Technologies 12 (2011) 320329

Log10 cycles of inactivation

10min

6h

24h

4
3
2
1
0

1 2 3 4 5 6 7 8 9 10 11

1 2 3 4 5 6 7 8 9 10 11

1 2 3 4 5 6 7 8 9 10 11

Compounds

Log10 cycles of inactivation

10min

6h

24h

4
3
2
1
0
1 2 3 4 5 6 7 8 9 10 11

1 2 3 4 5 6 7 8 9 10 11

1 2 3 4 5 6 7 8 9 10 11

Compounds

Log10 cycles of inactivation

10min

6h

24h

4
3
2
1
0
1 2 3 4 5 6 7 8 9 10 11

1 2 3 4 5 6 7 8 9 10 11

1 2 3 4 5 6 7 8 9 10 11

Compounds

Log10 cycles of inactivation

10min

6h

24h

4
3
2
1
0

1 2 3 4 5 6 7 8 9 10 11

1 2 3 4 5 6 7 8 9 10 11

1 2 3 4 5 6 7 8 9 10 11

Compounds
Fig. 1. Inactivation of Escherichia coli O157:H7 (A, B) and L. monocytogenes EGD-e (C, D) by selected compounds at pH 7.0 (A, C) and pH 4.0 (B, D) and ambient temperature. Cell
suspensions were suspended in buffer and exposed to the presence of 0.2 l/ml of thymol (1), carvacrol (2), borneol (3), linalool (4), terpineol-4-ol (5), -terpinyl acetate (6), 1,8cineole (7), camphor (8), p-cymene (9), -pinene (10), and -pinene (11). Survivors were recovered on the non-selective TSAYE (black bar) and the selective TSAYE-SC (white bar)
and TSAYE-BS (grey bar) media. Data are means standard deviations (error bars). The dotted line represents the detection limit.

L. monocytogenes at the same pH. The most effective compounds at all

conditions tested were phenols, linalool, and terpineol-4-ol.
Regarding PEF treatments, 25 pulses at 30 kV/cm caused the
inactivation of 0.53 log10 cycles of E. coli and L. monocytogenes cells

and 0.52 extra log10 cycles of injured cells as a function of the

microorganism and the treatment pH (Fig. 3). Regarding the action of
both methods applied simultaneously, PEF and 0.2 l/ml of each
monoterpene (except camphor), caused a greater inactivation than

A5

B5

Log10 cycles of inactivation

Log10 cycles of inactivation

A. Ait-Ouazzou et al. / Innovative Food Science and Emerging Technologies 12 (2011) 320329

325

0
C

10 11

10 11

C5

D
Log10 cycles of inactivation

Compounds

Log10 cycles of inactivation

Compounds

4
3
2
1
0

5
4
3
2
1
0

10 11

Compounds

10 11

Compounds

Fig. 2. Inactivation of Escherichia coli O157:H7 (A, B) and L. monocytogenes EGD-e (C, D) using antimicrobials and heat. Cell suspensions were suspended in buffers of pH 7.0 (A, C) and
4.0 (B, D) and exposed to the following treatments: 54 C for 10 min (heat) or the same heat treatment in the presence of 0.2 l/ml of thymol (1), carvacrol (2), borneol (3), linalool
(4), terpineol-4-ol (5), -terpinyl acetate (6), 1,8-cineole (7), camphor (8), p-cymene (9), -pinene (10), and -pinene (11). Survivors were recovered on the non-selective TSAYE
(black bar) and the selective TSAYE-SC (white bar) and TSAYE-BS (grey bar) media. Data are means standard deviations (error bars). The dotted line represents the detection limit.

the sum of both methods acting separately. In all cases, synergistic

lethal effects were observed at both pH values. The most effective
compounds were thymol and carvacrol. The comparison of the results
of the combined treatments with heat (Fig. 2) and PEF (Fig. 3) shows
the greater efcacy of applying heat.
Fig. 4 shows the inactivation of E. coli using a mild heat treatment
(54 C for 10 min) or a PEF treatment (25 pulses at 30 kV/cm), alone
or in combination with EO constituents suspended in apple and
orange juice. Exposure of E. coli to heat either in apple or orange juice
caused the inactivation of approximately 0.5 log10 cycles of cells and
injured about 3 and 4 extra log10 cycles of survivors respectively. On
the other hand, the application of 0.2 l/ml of any of the components
assayed for 10 min did not cause signicant inactivation (b0.5 log10
cycles) and almost no sublethal injury (data not shown). The action of
both methods applied simultaneously caused a greater inactivation
than did the sum of both methods acting separately (Fig. 4A).
However, in general, greater synergistic lethal effects were observed
when E. coli cells were suspended in apple than in orange juice
(p b 0.05). Phenols and alcohols were demonstrated to be the most
effective components in apple juice when combined with heat,
allowing the inactivation of nearly 5 log10 cycles of E. coli cells.
With the exception of carvacrol, the combination of PEF and
antimicrobials did not show any synergistic lethal effect against E. coli
when suspended in apple and orange juice (Fig. 4B). Only the
combination of PEF and carvacrol caused the inactivation of 2 log10
cycles of E. coli cells in orange juice and nearly 5 in apple juice.
L. monocytogenes was not tested in juices since the combined
treatments and the subsequent storage in the juices for several hours

was sufcient, without the addition of any antimicrobial, to cause the

desired inactivation (5 log10 cycles) (data not shown).
4. Discussion
The present study describes the antimicrobial activity of EO
constituents acting alone or in combination with heat and PEF as a
function of treatment conditions. The nal purpose is to achieve
synergistic lethal effects that allow us to lessen treatment intensity
and antimicrobial doses, to avoid non-desirable modications of
sensorial and nutritional properties of the matrix caused by heat and
to enhance PEF lethality.
The antimicrobials assayed are common constituents of wellknown EOs such as those extracted from eucalyptus, lavender, orange,
peppermint, pine, rosemary, etc. (Bakkali et al., 2008). The composition and bacteriostatic activity of these EOs and of their constituents
have thoroughly been investigated and reviewed (Bakkali et al., 2008;
Burt, 2004; Raybaudi-Massilia et al., 2009). Regarding bacteriostatic
activity (Table 1), our results conrm that Gram-positive bacteria are,
in general, more susceptible than are the Gram-negative ones.
Nevertheless, some antimicrobials, such as terpineol-4-ol, showed
greater inhibitory activity against Salm. Enteritidis and E. coli than
against L. monocytogenes and E. faecium (Table 1). Our results also
conrm the greater efcacy of most oxygenated monoterpenes versus
hydrocarbons, as previously described by Carson and Riley (1995).
Attending to the preliminary results obtained using the disk
diffusion assay, consideration should be given to discarding hydrocarbons as a consequence of their poor antimicrobial activity.

A5

B5
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A. Ait-Ouazzou et al. / Innovative Food Science and Emerging Technologies 12 (2011) 320329

Log10 cycles of inactivation

326

0
C

10 11

10 11

C5

D5
Log10 cycles of inactivation

Compounds

Log10 cycles of inactivation

Compounds

0
C

10 11

Compounds

10 11

Compounds

Fig. 3. Inactivation of Escherichia coli O157:H7 (A, B) and L. monocytogenes EGD-e (C, D) using antimicrobials and PEF. Cells were suspended in buffers of pH 7.0 (A, C) and 4.0 (B, D)
and exposed to the following treatments: 25 pulses at 30 kV/cm (PEF) or the same PEF treatment in the presence of 0.2 l/ml of thymol (1), carvacrol (2), borneol (3), linalool (4),
terpineol-4-ol (5), -terpinyl acetate (6), 1,8-cineole (7), camphor (8), p-cymene (9), -pinene (10), and -pinene (11). Survivors were recovered on the non-selective TSAYE
(black bar) and the selective TSAYE-SC (white bar) and TSAYE-BS (grey bar) media. Data are means standard deviations (error bars). The dotted line represents the detection limit.

However, the determination of MIC, in addition to conrming the

greater efcacy of phenols and some alcohols (MIC 0.2 l/ml),
showed that hydrocarbons also inhibited L. monocytogenes as other
oxygenated monoterpenes (MIC = 0.5 l/ml) (Table 2). Thus, the disk
diffusion method might not seem to be an adequate tool for selection
among compounds with the best antimicrobial activity. On the other
hand, MIC values obtained were in the range of those published about
terpineol-4-ol, thymol or carvacrol (Cosentino et al., 1999; Kim,
Marshall, & Wei, 1995; Pol & Smid, 1999).
Regarding the compounds' bactericidal activity, our study did not
include the evaluation of concentration values higher than 2.0 l/ml,
since those concentrations might be inappropriate, sensorially and
economically, for most possible applications (Burt, 2004). That was
the case for most antimicrobials tested (-terpinyl acetate, 1,8cineole, camphor, p-cymene, -pinene, and -pinene), which
prompts caution about their unsuitable use as inactivating agents.
On the other hand, little is known about how their mode of action and
antimicrobial spectrum might change as a function of environmental
factors. In this sense, the pH of the treatment medium is one of the
major environmental factors, not only because it can modify microbial
resistance to physical or chemical inactivating agents (Maas & Pagn,
2005), but also because it can modify the action mechanism of the
inactivating agents, especially chemical preservatives, and therefore,
their antimicrobial spectrum. It is generally accepted that EO
constituents are most effective at acidic pHs (Burt, 2004); however,
that conclusion is mainly based on studies carried out to evaluate their

capacity to prevent microbial growth. Regarding bactericidal activity,

results varied among studies: while Friedman, Henika, Levin, and
Mandrell (2004) did not observe any inuence of acidity when
evaluating carvacrol or linalool in apple juice, Rivas et al. (2010)
described a higher susceptibility of E. coli to carvacrol or thymol when
decreasing the pH to 4. Previous results of our research group
(Somolinos et al., 2008, 2010) have shown that, for instance, the
bactericidal activity of citral increases or decreases with acidication
as a function of the microorganism assayed. While L. monocytogenes
was more sensitive at pH 4.0 than at pH 7.0, E. coli was much more
resistant at acid pH. In our opinion, that extraordinary behavior
substantiates the need to evaluate the bactericidal activity of other
antimicrobials under acidic conditions.
The evaluation of the bactericidal activity of the EO constituents
was carried out using the smallest concentration assayed in the
determination of MICs and MBCs (0.2 l/ml). Our study revealed that
most antimicrobials actually showed a great bactericidal activity
when acting at pH 4.0, similar to that showed by thymol or carvacrol,
which encourages their use as an alternative at very low doses for
many applications. For instance, the hydrocarbon p-cymene, which
showed a poor growth inhibitory capacity, was very effective as a
bactericidal when suspended at pH 4.0. On the other hand, no
compounds showed the extraordinary behavior of citral previously
described (Somolinos et al., 2010). Nevertheless, it is remarkable that
there is an increase in efcacy of most hydrocarbons at pH 4.0 against
L. monocytogenes, while -pinene and -pinene were scarcely

A. Ait-Ouazzou et al. / Innovative Food Science and Emerging Technologies 12 (2011) 320329

Log10 cycles of inactivation

0
Oj

Aj

10 11

Compounds

Log10 cycles of inactivation

B5
4

0
Oj

Aj

10 11

Compounds
Fig. 4. Inactivation of Escherichia coli O157:H7 suspended in apple and orange juice
using antimicrobials and heat (A) or PEF (B). Cell suspensions were suspended in
orange (Oj) and apple juice (Aj) and exposed to the following treatments: 54 C for
10 min (A), 25 pulses at 30 kV/cm (B) acting alone (Oj and Aj) and the same treatments
in the presence of 0.2 l/ml of thymol (1), carvacrol (2), borneol (3), linalool (4),
terpineol-4-ol (5), -terpinyl acetate (6), 1,8-cineole (7), camphor (8), p-cymene (9),
-pinene (10), and -pinene (11). Survivors were recovered on the non-selective
TSAYE (black (apple juice) and white (orange juice) bars) and the selective TSAYE-SC
(light grey bars) and TSAYE-BS (grey bars) media. Data are means standard
deviations (error bars). The dotted line represents the detection limit.

effective against E. coli. Similar differences were observed when

evaluating the bactericidal action of linalool or 1,8-cineole.
Although the antimicrobial effect of most EO constituents is well
known (Bakkali et al., 2008; Burt, 2004), the mechanism of growth
inhibition, cell injury, and inactivation is not fully understood.
Comparing results about growth inhibition and inactivation at pH
7.0, no differences have been observed between the spectrums of
action of most compounds. Through the evaluation of the occurrence
of sublethal injuries, this study also concludes that the cytoplasmic
membrane in bacterial cells and the outer membrane in Gramnegative bacteria are clearly affected by either hydrocarbon or
oxygenated monoterpenes. Despite the hydrophobicity of cell
envelopes due to the chemical structure of the outer membrane in
Gram-negative bacteria and the presence of teichoic acids in the cell
wall of Gram-positive bacteria (Mackey, 2000), all compounds were
able to interact with cell envelopes, achieving sublethal injuries to
most cell populations (N90%). Results obtained, for instance, with
linalool, terpineol-4-ol, or -terpinyl acetate against E. coli at pH 4.0
(Fig. 1B) or carvacrol at pH 7.0 (Fig. 1A) show how the occurrence of
sublethal injuries in the outer membrane progressed more quickly

327

than did those in the cytoplasmic membrane, affecting a higher

proportion of surviving cells. The occurrence of sublethal injuries, rst
on the outer membrane and later on the cytoplasmic membrane,
seems to drive cells to subsequent inactivation. The evaluation of
membrane permeabilization allowed us to correlate the occurrence of
inactivated cells (N90%) after 24 h-incubation in the presence of
thymol, carvacrol, -terpinyl acetate, and p-cymene with extensive
membrane permeabilization (N90%). These results would point out
the cell envelope as a target in their mechanism of inactivation.
Linalool, -pinene, and -pinene also caused extensive membrane
permeabilization to L. monocytogenes; however E. coli cell envelopes
seemed to be more resistant, since less than 50% of the treated
population was uorescent when more than 90% of cells were dead.
While inactivation of 5 log10 cycles of microorganisms by 0.2 l/ml
of EO constituents, when it occurred, required 6 to 24 h of incubation
as a function of the pH, the simultaneous application with heat
achieved the same, or even a higher, lethal effect immediately after
the treatment.
Moreover, a new picture emerged in relation to the new antimicrobial spectrum of some compounds as a function of the treatment
medium pH when acting in combination with heat. For instance,
p-cymene, which showed poor activity acting alone at pH 7.0, was very
effective in combination with heat, achieving 5 log10 cycles of
inactivation of both microorganisms at pH 4.0. Similar results were
obtained with -terpinyl acetate against L. monocytogenes at pH 7.0.
Therefore, it should be noted that the classical disk diffusion technique
and MIC and MBC determination used to assess antimicrobial activity of
EOs or their constituents might not offer enough information about
these compounds' possible antimicrobial applications. As described, the
treatment medium pH can selectively improve the activity of some
compounds, and their simultaneous application with heat can also
severely improve the activity of some compounds that were ineffective
when acting alone. Hence, some apparently less effective compounds
might be useful in the design of new combined treatments for new
applications, especially when those compounds exhibit more adequate
sensorial properties. On the other hand, the synergistic lethal effect
obtained immediately after the application of the combined process
would facilitate their use, since the benets would not depend on long
incubations at adequate temperatures.
The occurrence of sublethal injury after heat, especially on the
outer membranes of Gram-negative bacteria, demonstrates the
effectiveness of combined treatments with hydrophobic antimicrobials (Arroyo et al., 2010; Mackey, 2000; Somolinos et al., 2010). Our
results showed that almost the entire surviving population was
sublethally damaged after heat treatment at pH 4.0 and were likely
susceptible to the presence of most antimicrobials. Damages inicted
by heat might facilitate the access of hydrophobic compounds to the
cytoplasmic membrane, which is the primary site of the toxic action of
terpenes (Luo et al., 2004; Park et al., 2009; Prashar, Hili, Veness, &
Evans, 2003) or enable EO constituents to be more easily transported
into the cell (Burt, 2004). In contrast, the heat-injured population at
pH 7.0 was signicantly lower than the nal inactivation achieved by
the combined treatment with some oxygenated monoterpenes
(phenols and some alcohols) against E. coli and also -terpinyl
acetate and p-cymene against L. monocytogenes. Either the heat
treatment injured more cells than those detected by the techniques
used in this work, or perhaps the moderate temperature (54 C)
facilitated the diffusion and the action of the compounds, making
them more effective. In this sense, Friedman et al. (2004) described
the higher bactericidal activity of some EO constituents at 37 C versus
21 C.
Results obtained in juices conrmed the occurrence of extraordinary synergistic effects when combining heat and EO constituents
against E. coli. Nevertheless, the synergistic effect was signicantly
lower in orange than in apple juice, and in apple juice than in buffer of
pH 4.0 when using most antimicrobials. These poorer results might

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not be related to the treatment medium pH, which was signicantly

lower in the juices ( 3.7), but to other factors such as food
composition or the presence of solids in suspension which were
more abundant in orange juice. On the other hand, the maximum
degree of inactivation reached in both juices was coincident (p b 0.05)
with the sublethally injured population at the outer membrane
caused by heat in each juice. For instance, the degree of inactivation
evaluated in the selective medium supplemented with bile salts
reached approximately 3.5 and 4.5 log10 cycles in orange and apple
juice, respectively, which was the nal degree of inactivation of the
combined treatment when using thymol or carvacrol.
Regarding the results obtained with PEF, very interesting synergistic effects were also described, and again the combination of PEF
and some antimicrobials achieved a higher degree of inactivation than
those reached by PEF acting alone. The combined treatment reached
the desired level of inactivation (5 log10 cycles) as a function of the
microorganism and the pH of the treatment media. The poorer results
obtained when inactivating E. coli at acid pH might be related to the
well-known higher resistance to PEF shown by Gram-negative
microorganisms suspended at acid pH (Garca, Gmez, Raso, et al.,
2005). On the other hand, results of the combined treatment applied
to both juices were very disappointing, since only the combination
with carvacrol exerted a synergistic effect, greater in apple than in
orange juice.
A new assay was carried out in order to understand the
outstanding synergistic effect of PEF plus carvacrol. Instead of
applying both barriers simultaneously, carvacrol was added to the
apple juice for 3 min immediately after the PEF treatment. As a result,
again nearly 5 log10 cycles of cells were inactivated, which means that
those changes provoked by PEF were not instantaneously reversible,
and most survivors were sensitive to the subsequent contact with
carvacrol. These results suggest that PEF might cause more extensive
damage to the surviving population than that detectable by the
techniques used in this study, making survivors sensitive to the
unique action of carvacrol. Carvacrol and thymol only differ on the
position of the hydroxyl group in their phenolic ring, and thymol did
not exert a synergistic effect in combination with PEF, so both the
hydroxyl group and its position might be related to the occurrence of
the synergistic effect described.
On the other hand, the unusual behavior of Gram-negative
bacteria treated by PEF under acid pH previously described (Garca,
Gmez, Condn, Raso, & Pagn, 2003; Garca, Gmez, Condn, Raso, &
Pagn, 2005; Somolinos et al., 2010) might also explain the lack of
additive or synergistic effects when combining PEF and antimicrobials
to treat juices. Thus, Somolinos et al. (2010) found that the presence of
organic acids at acid pH protected E. coli against PEF and that cells
were more protected as the organic acid concentration in juices
increased. Further research is needed in order to study in depth the
mechanism of action of these antimicrobials and their interactions
with heat or PEF-damaged cells.
From a practical point of view, the doses employed in the
combined processes are quite low and acceptable in some products
(Burt, 2004). Moreover, the initial microbial inoculum in the
experiments carried out was signicantly higher than those we
might nd contaminated, for instance, food products, to 105103 UFC/ml
or lower. Hence, doses to achieve the same synergistic effect might
be lower than those assayed. In any case, the increase in the efcacy of
the combined treatments with the most effective antimicrobials in
comparison to the heat treatment acting alone might represent a
reduction in treatment time by approximately 9 times when trying to
inactivate 5 log10 cycles of E. coli O157:H7 (decimal reduction time at
54 C: 17 min approx.) at pH 7.0. The inactivation of 5 log10 cycles of
these pathogenic microorganisms would follow the recommendations for controlling the transmission of pathogens in juices proposed
by the National Advisory Committee on Microbiological Criteria for
Food (NACMCF, 1997).

In conclusion, the present study allowed us to compare the

inhibitory and bactericidal activity of 11 monoterpenes generally
present in well-known EOs of aromatic plants. The evaluation of the
inuence of the treatment medium pH on their capacity to inactivate
microorganisms has revealed a great efcacy under acid pH of some
antimicrobials, which might have been discarded at neutral pHs. The
efcacy of some EO constituents improved when combined with PEF
and especially with mild heat as a function of the treatment medium
pH, and as a consequence it was possible to propose much lower doses
of antimicrobials. The valuable synergistic effects observed offer the
potential to improve traditional heat treatments in order to reduce
treatment intensity and thus adverse effects on sensorial and
nutritional properties, while maintaining or even improving product
safety. The synergistic effects also are promising for the enhancement
of emerging PEF treatments, allowing higher levels of inactivation at
lower intensities. Further studies are required to describe how these
antimicrobials interact, showing synergism, antagonism, or additive
effects and the inuence of matrix composition on their activity in
order to foresee the efcacy of natural EOs as antimicrobials acting
alone or in combination with other technologies.
Acknowledgments
This study was supported by the CICYT (Project AGL 200911660)
and by the AECID, which provided A. Ouazzou with a grant to carry out
this investigation.
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