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Bactericidal Action of Binary and Ternary
Mixtures of Carvacrol, Thymol, and Eugenol
against Listeria innocua
Rebeca Garca-Garca, Aurelio L opez-Malo, and Enrique Palou
Abstract: The bactericidal effect of 3 natural agents (carvacrol, thymol, and eugenol) was evaluated as well as their binary
and ternary mixtures on Listeria innocua inactivation in liquid model systems. Minimal bactericidal concentrations (MBC)
of these agents were determined, and then binary and ternary mixtures were evaluated. Culture media were inoculated
with L. innocua and incubated for 72 h at 35

C. Turbidity of studied systems were determined every 24 h. The most
effective individual antimicrobial agent was carvacrol, followed by thymol and then eugenol with MBCs of 150, 250, and
450 mg kg
1
, respectively. It was observed that the most effective binary mixture was 75 mg kg
1
carvacrol and 62.5 mg
kg
1
thymol. Furthermore, the ternary mixture carvacrolthymoleugenol in concentrations of 75, 31.25, and 56.25 mg
kg
1
, correspondingly, was the most effective for L. innocua inactivation. Several binary and ternary mixtures of these 3
natural antimicrobial agents worked adequately to inactivate L. innocua.
Keywords: bactericidal mixtures, carvacrol, eugenol, Listeria innocua, thymol
Introduction
Research on natural antimicrobial agents has increased in recent
years due to the incidence of pathogenic microorganisms and the
need to control the presence of food spoilage ora, altogether with
the consumer demand for natural foods (L opez-Malo and others
2005a). Antimicrobial agents are compounds that exist in foods,
or are added to them, to inactivate microorganisms or delay their
growth (L opez-Malo and others 2000; Zhou and others 2007;
Ayala-Zavala and others 2009; Pei and others 2009; Palianappan
and Holley 2010; Schirmer and Langsrud 2010).
The use of antimicrobials is one of the oldest food preservation
techniques (L opez-Malo and others 2005a). Only one antimi-
crobial agent was traditionally used to preserve foods (Busta and
Foegeding 1983); however, it has become a trend in recent years
to use mixtures of agents for such a purpose (Lambert and others
2001; 2003, Lambert and Lambert 2003; Santiesteban-L opez and
others 2006). In theory, using mixtures of antimicrobial agents
provides a wider range of activity, which results in the increase of
antimicrobial activity against pathogenic or deteriorative microor-
ganisms. It is believed that a mixture of antimicrobial agents can
act upon different microbial species, or act on several vital points
(or targets) inside the cells of similar microbial species, which can
enable a better control of the microorganisms in food, compared
to the use of an individual antimicrobial agent (Santiesteban-L opez
and others 2006).
It is necessary to evaluate the combination of antimicrobial
agents because one microorganism may prove resistant to the in-
hibition and/or inactivation performed by conventional doses of a
MS 20100449 Submitted 4/24/2010, Accepted 11/21/2010. Authors are with
Dept. de Ingeniera Qumica, Alimentos y Ambiental, Univ. de las Am ericas
Puebla, Cholula, Puebla 72820, Mexico. Direct inquiries to author Palou (E-mail:
enrique.palou@udlap.mx).
single antimicrobial agent. However, when the microorganism is
exposed to a combination of agents, it could become less resistant
and may consequently die or be inhibited (Le on-Cruz and others
2003). Common methods to test the mixtures of antimicrobial
agents usually include: agar diffusion, agar or broth dilution, and
death curves. Dilution methods render quantitative data, which is
usually obtained from the combination of different dose concen-
trations following a checkerboard array. This experimental design
is the most common technique to test antimicrobial agents mix-
tures in vitro because: (1) it is easy to understand; (2) the math
calculations to understand and interpret results are simple; (3) it
can be relatively easily performed in the laboratory; and (4) it has
been the technique most widely used in studies on synergistic
interactions of antibiotics in clinical treatments (Eliopoulos and
Moellering 1991).
Studies of mixtures of antimicrobial agents also have the pur-
pose to determine the specic type of interaction among the
agents that are combined. The terms additive, antagonistic, and
synergistic are traditionally used to describe the possible inter-
actions of such agents (Barry 1976). The Fractional Inhibitory
Concentration (FIC) is commonly used to interpret these types
of interaction. To calculate FICs, minimum inhibitory concen-
trations (MICs) of the antimicrobials are determined. FIC is de-
ned as the concentration of an antimicrobial agent (expressed as
a fraction of its MIC) required to inhibit the growth of a mi-
croorganism when it is combined with a known concentration of
other agent(s). However, it is also of interest to determine those
concentrations that are bactericidal (MBC). Correspondingly, a
fractional bactericidal concentration (FBC) is dened as the con-
centration of an antimicrobial agent (expressed as a fraction of its
MBC) required to inactivate (reduce 99.9%) the growth of a
microorganism when it is combined with a known concentration
of other agent(s) (L opez-Malo and others 2005b).
Santiesteban-L opez and others (2006), and Garca-Garca
and others (2007) tested the inhibitory effect of mixtures of
C
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Bactericidal mixtures. . .
natural (carvacrol, thymol, eugenol) and synthetic (potassium sor-
bate) antimicrobial agents against food-relevant bacteria such as
Staphylococcus aureus, Salmonella typhimurium, L. innocua, and E. coli.
Nevertheless, since it is not easy to anticipate the effects or the
interaction of mixtures of antimicrobial agents on microorganisms,
the main object of this study was to test binary and ternary mix-
tures of natural antimicrobial agents such as carvacrol, thymol, and
eugenol in liquid model systems to inactivate L. innocua.
Materials and Methods
L. innocua
The strain of L. innocua (ATCC 51742) was obtained from
the Food Microbiology Lab. at the Univ. Aut onoma de San Luis
Potos (Mexico). The strain was grown in tripticase soy agar (TSA;
Merck, Mexico City, Mexico) at 35

C during 48 h, and was then
stored at 4

C.
Inoculum
A loop of the strain from TSA was taken to prepare the in-
oculum; it was grown in tripticase soy broth (Merck, Mexico
City, Mexico) at 35

C. Fresh inocula were prepared every 24
h to maintain the exponential growth phase (10
9
CFU mL
1
,
approximately).
Model systems
Liquid model systems were prepared (by triplicate) in 10 mL
glass tubes, using tripticase soy broth. These systems were sterilized
at 121

C for 15 min. Ethanol solutions (0.5% w/v) of carvacrol,
thymol, and eugenol were prepared. These solutions were lter-
sterilized with a cellulose membrane, 0.45 m pore size (Micro
Filtration Systems, Dublin, Calif., U.S.A.), and stored at 4

C in
jars covered with aluminum foil.
Minimal bactericidal concentration (MBC) of the individual
antimicrobial agents
A death kinetics study was performed to determine the MBC of
individual antimicrobial agents (thymol, carvacrol, and eugenol).
For this purpose, the microorganism was grown at 35

C for
24 h in 100 mL of tripticase soy broth. Once the stationary phase
was reached, 1 mL of the inoculum was transferred to 9 mL
of tripticase soy broth, where the antimicrobial agent was added
in the following concentrations: 100 and 150 mg kg
1
for car-
vacrol; 150, 200, and 350 mg kg
1
for thymol; and 250, 350, and
450 mg kg
1
for eugenol. Controls with ethanol were also evalu-
ated. Inoculated broths were incubated at 35

C for 72 h. Samples
(100 L) were taken from this medium every hour during the
rst 6 h, then at 24, 48, and 72 h. Every sample was diluted and
plated on TSA, incubated for 48 h at 35

C, and then counts were
performed. MBC was dened (following Skandamis and Nychas
2001) as the lowest concentration tested that reduced L. innocua at
least 6 log cycles, and no growth (recovery) after 24, 48, and 72 h
was detected.
Carvacrol, thymol, and eugenol binary and ternary mixtures
Once the MBC of the antimicrobial agents was individually
determined, the effect of the carvacrolthymol, thymoleugenol,
and carvacroleugenol binary mixtures was tested by triplicate.
The effect of the carvacrolthymoleugenol ternary mixture was
also evaluated (by triplicate) using selected concentrations of each
of the agents according to a checkerboard design (Davidson and
Parish 1989; L opez-Malo and others 2005b). Combinations of
the tested concentrations of antimicrobial agents for binary and
ternary mixtures included combinations of MBC, 1/2 MBC, 1/4
MBC, 1/8 MBC, and 0 MBC of the tested antimicrobials.
Inoculation of the model systems to test binary and ternary
mixtures
A total of 1 mL of the inoculum (10
9
CFU mL
1
) was trans-
ferred to 9 mL of tripticase soy broth (the liquid model system)
containing the antimicrobial amounts previously specied. Sys-
tems were incubated for 72 h at 35

C, and the turbidity of every
system was determined every 24 h by comparison against a con-
trol using a turbidimeter, to identify whether there was growth
(G) or no growth (NG) of the microorganism. Microbial growth
was registered for the model systems that exhibited a change in
turbidity. Microbial counts were determined by taking a 100 L
sample and appropriate dilutions were plated and counted.
Bactericidal effect
A sample (100 L) was taken from the liquid model systems that
exhibited no turbidity change and 10
1
, 10
2
, and 10
3
dilutions
were made. A 1-mL sample was taken form each dilution and
plated on TSA. Determination was performed (by duplicate) after
24, 48, and 72 h of incubation at 35

C. Inoculated dishes were
incubated for 48 h at 35

C and then counts were performed. If
no turbidity change and no growth after plating were observed
the effect was registered as bactericidal.
Fractional bactericidal concentration (FBC)
MBC of each tested agent in the presence of another agent were
determined in the liquid systems where no turbidity change (and
no growth) was detected after 72 h. Then FBC values were calcu-
lated with equations 1, 2, and 3. The FBC index was determined
with Eq. 4, to test whether the studied mixture exhibited an addi-
tive, antagonistic, or synergistic effect. The formulas reported by
L opez-Malo and others (2005b) are commonly utilized to obtain
FIC or FBC values for a combination of 3 agents:
FBC
A
= (MBCof Ain presence of B&C)/
(MBCof individual A)
(1)
FBC
B
= (MBCof Bin presence of A&C)/
(MBCof individual B)
(2)
FBC
C
= (MBCof Cin presence of A&B)/
(MBCof individual C)
(3)
Once FBC values are obtained, the FBC index can be deter-
mined as follows:
FBCindex = FBC
A
+FBC
B
+FBC
C
(4)
Results and Discussion
Determination of the individual MBCs of carvacrol, thymol,
and eugenol against L. innocua
For individual MBC determination, in the case of carvacrol, 2
concentrations were studied (100 and 150 mg kg
1
), and it was
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determined that the 100 mg kg
1
concentration did not inacti-
vate L. innocua after 72 h at 35

C. In contrast, no growth was
observed in the 150 mg kg
1
concentration (Figure 1) after 4 h,
and this condition prevailed even after 72 h. In the case of thymol,
3 concentrations (150, 200, and 250 mg kg
1
) were studied. It
was observed that in a concentration of 200 mg kg
1
, thymol did
not inactivate L. innocua because after the rst 6 h, the microor-
ganism recovered and exhibited microbial growth at 24, 48, and
72 h (data not shown). However, L. innocua was inactivated and
no later growth was observed when a 250-mg kg
1
concentration
of thymol was tested (Figure 1). In the case of eugenol, 3 con-
centrations (250, 350, and 450 mg kg
1
) were studied. A total of
350 mg kg
1
of eugenol did not inactivate L. innocua. However, a
450-mg kg
1
concentration of this antimicrobial agent (Figure 1)
inactivated the microorganism in the rst few hours, and this con-
dition prevailed after 72 h. This result indicates that the 450-mg
kg
1
eugenol concentration was the MBC for L. innocua. Control
broths formulated with ethanol exhibited growth.
Summing up, minimal bactericidal concentrations for L. in-
nocua in a liquid culture media (pH 7.0 and a
w
0.99) were 150
mg kg
1
for carvacrol, 250 mg kg
1
for thymol, and 450 mg
kg
1
for eugenol. These results are similar to the ones reported by
Santiesteban-L opez and others (2006) in their study on S. aureus,
L. innocua, E. coli, and S. typhimurium, where they observed that
carvacrol was the most effective antimicrobial agent for the inhi-
bition of the growth of those bacteria, followed by thymol and
eugenol. Santiesteban-L opez and others (2006) observed that the
MIC of carvacrol against L. innocua at pH 4.5 and a
w
0.97 was 100
mg kg
1
in solid culture media. Garca-Garca and others (2007)
reported that the MIC of carvacrol for L. innocua in liquid culture
media was 150 mg kg
1
. It is important to mention that in liquid
media, bacteria have a better ability to move and thus be more in
contact with the antimicrobial agents (Pelczar and others 1995).
According to Quinn (1986) and Suutari and Laakso (1994),
in general terms, 3 physical layers can be distinguished in cel-
lular membranes: a gel phase bilayer (ordered lipid chain), a
-8
-6
-4
-2
0
0 10 20 30 40 50 60 70
Carvacrol
Thymol
Eugenol
Log (N/No)
Time (h)
Figure 1L. innocua death kinetics at 35

C in tripticase soy broth contain-

ing the minimal bactericidal concentrations of carvacrol (150 mg kg
1
),
thymol (250 mg kg
1
), and eugenol (450 mg kg
1
).
liquidcrystal phase bilayer (disordered lipid chain), and a hexago-
nal structure. For effective biological functioning, the membrane
must remain in liquid crystal uid state (Ingram 1976), and the
transition temperature (T
m
) from gel phase to liquid crystal phase
is the main inuence upon the exibility and stability of the mem-
brane (Suutari and Laakso 1994). The hydrophobic property of
carvacrol interacts with the cellular membrane of the microorgan-
ism, thus allowing the exit of lipopolysaccharides and increasing
the permeability of the plasma membrane.
At low carvacrol concentrations (100 mg kg
1
in this study),
microorganisms possess some adaptation mechanisms against the
damaging effects of cytotoxic components (Bygraves and Russell
1988; Russell and Fukunaga 1990; Weber and de Bont 1996), such
as: the ability to keep an adequate proportion of liquidcrystal
phase lipids in the membrane (membrane uidity restoration);
keep an appropriate balance between the phospholipid bilayer
and the phospholipid nonbilayer (preserving the membranes in-
tegrity); and the systems ability to expel strange matter (Bolhuis
and others 1994). When the concentration of carvacrol increases,
a greater amount of this component is accumulated in the mem-
brane, and consequently, the damage is more severe (Ultee and
others 2002). It is important to mention that the antimicrobial
action and sensitivity to thymol (as would happen with any other
antimicrobial agent) depends on certain factors such as the type of
microorganism, the mediums pH, and the incubation temperature
(Falcone and others 2005). This is the reason why there are studies
(Garca-Garca and others 2007), where the reported MIC value
for L. innocua in liquid culture media was 162.5 mg kg
1
, which
is lower than the one obtained in this study. This difference in the
MIC value can also be a consequence of the mediums pH, since,
as reported by Juven and others (1994), the inhibitory effect of
thymol was higher at a pH of 5.5 than at 6.5; this is due to the fact
than when pH values are low, the molecule of the antimicrobial
agent is not dissociated, and, therefore, it binds better to the hy-
drophobic parts of proteins and the dissolution of the lipid phase
of the membrane takes place more easily. The pH in the culture
media of our study was 7. Thymol mechanism of action resembles
the one of carvacrol because their chemical structures are similar,
except for the position of the hydroxyl group in the ring, so both
substances make the cellular membrane permeable (Lambert and
others 2001). Helander and others (1998) pointed out that thymol
releases lipopolysaccharides (LPS), thus increasing the permeabil-
ity of the membrane. Juven and others (1994) studied the effect
of thymol on S. typhimurium and S. aureus, and concluded that
this antimicrobial agent binds the hydrophobic proteins of the
membrane through hydrogen bridges, and consequently changes
its permeability characteristics. When Helander and others (1998)
tested the effect of carvacrol and thymol on E. coli O157:H7
and S. typhimurium, they noticed that these antimicrobial agents
acted by diminishing the adenosine triphosphate (ATP) intracel-
lular content of the microorganism and simultaneously increasing
the extracellular ATP, which resulted in the rupture of the cell
membrane. There are few studies about eugenol mechanism of
action. Yet, it is known that it inhibits the production of certain
enzymes, such as amylases and proteases (Farag and others 1989).
It has been observed on Enterobacter aerogenes that the hydroxyl
group of the eugenol molecule binds to proteins to prevent the
action of enzymes (Wendakoon and Sakaguchi 1995). Gill and
Holley (2004) reported that eugenol had a bactericidal effect on
L. monocytogenes, and they observed that this agent acted as an ion
transport, thus avoiding the increase of cellular ATP levels. Walsh
and others (2003) reported that E. coli and S. aureus cells that were
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treated with eugenol exhibited changes in their membrane perme-
ability as a consequence of the exit of potassium ions. Wendakoon
and Sakaguchi (1995) observed that eugenol concentrations higher
than 985 mg kg
1
inhibited the histidine decarboxylase enzyme in
E. aerogenes, so they concluded that eugenol inhibited the synthesis
of this enzyme, which has a direct effect upon metabolic energy.
Effect of binary and ternary mixtures of antimicrobial
agents against L. innocua
Growth (G) and no-growth (NG) responses of L. innocua in the
carvacrolthymol binary combination are presented in Table 1.
No microbial growth was observed in 12 combinations out of the
25 that were tested, these exhibited a bactericidal effect. Similarly,
the results obtained from the carvacroleugenol binary mixture
are also presented in Table 1. Only 9 combinations out of the 25
that were tested exhibited a bactericidal effect. The results of the
tests with the thymoleugenol binary mixture are also presented
in Table 1, where 12 combinations out of the 25 combinations
that were tested exhibited a bactericidal effect.
When analyzing the 3 binary mixtures that were tested, it
can be concluded that the most effective mixtures that inacti-
vated the growth of L. innocua in liquid model systems were:
(1) 75 mg kg
1
carvacrol and 62.5 mg kg
1
thymol; (2) 37.5 mg
kg
1
carvacrol and 125 mg kg
1
thymol; and (3) 125 mg kg
1
thymol and 56.25 mg kg
1
eugenol. Results obtained for ternary
mixtures are presented in Table 2. It can be observed that 14 com-
binations out of the 36 that were tested exhibited a bactericidal
effect. MBCs of the ternary mixtures established for L. innocua in
this study were: (1) 75 mg kg
1
carvacrol, 31.25 mg kg
1
thymol,
and 56.25 mg kg
1
eugenol; (2) 37.5 mg kg
1
carvacrol, 62.5 mg
kg
1
thymol, and 225 mg kg
1
eugenol; and (3) 18.75 mg kg
1
carvacrol, 125 mg kg
1
thymol, and 112.5 mg kg
1
eugenol.
FBCs and the FBC index were determined for the binary
and ternary combinations that effectively inactivated the growth
of the microorganism (bactericidal effect). Table 3 presents the
FBCs, FBC indices and the type of effect produced by the
different binary combinations that effectively inactivated L. in-
nocua. It can be observed that in terms of the bactericidal ef-
fect, the carvacrolthymol combination has the highest number of
synergistic combinations, followed by a single thymoleugenol
combination. Table 4 presents the results obtained for ternary
combinations. From 3 effective bactericidal mixtures, 2 have syn-
ergistic effect and only 1 exhibited an additive effect.
There are some critics towards this approach of FBC index
calculation to characterize the effects (synergy, addition, or antag-
onism) of mixtures of antimicrobial agents (Lambert and Lambert
2003; Lambert and others 2001, 2003). The basic assumption
of the method of FBC index calculation is that the concentra-
tion of an antimicrobial is proportional to its effect, which means
Table 2Bactericidal combinations of carvacrolthymoleugenol
on L. innocua after 72 h of incubation in TSB and after plating
in TSA without antimicrobials.
Eugenol 225 (mg kg
1
)
Thymol (mg kg
1
)
Carvacrol (mg kg
1
) 125 62.5 31.25
75 NG NG NG
37.5 NG NG G
18.75 NG G G
Eugenol 112.5 (mg kg
1
)
Thymol (mg kg
1
)
Carvacrol (mg kg
1
) 125 62.5 31.25
75 NG NG NG
37.5 NG G G
18.75 NG G G
Eugenol 56.25 (mg kg
1
)
Thymol (mg kg
1
)
Carvacrol (mg kg
1
) 125 62.5 31.25
75 NG NG NG
37.5 G G G
18.75 G G G
Eugenol 28.125 (mg kg
1
)
Thymol (mg kg
1
)
Carvacrol (mg kg
1
) 125 62.5 31.25
75 G G G
37.5 G G G
18.75 G G G
NG = no growth; G = growth.
Table 1Bactericidal combinations of carvacrolthymol, carvacroleugenol, and thymoleugenol on L. innocua after 72 h of incu-
bation in TSB and after plating in TSA without antimicrobials.
Thymol (mg kg
1
)
Carvacrol (mg kg
1
) 250 125 62.5 31.25 0
150 NG NG NG NG NG
75 NG NG NG G G
37.5 NG NG G G G
18.75 NG G G G G
0 NG G G G G
Eugenol (mg kg
1
)
Carvacrol (mg kg
1
) 450 225 112.5 56.25 0
150 NG NG NG NG NG
75 NG G G G G
37.5 NG G G G G
18.75 NG G G G G
0 NG G G G G
Eugenol (mg kg
1
)
Thymol (mg kg
1
) 450 225 112.5 56.25 0
250 NG NG NG NG NG
125 NG NG NG NG G
62.5 NG G G G G
31.25 NG G G G G
0 NG G G G G
NG = no growth; G = growth.
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Table 3Fractional bactericidal concentrations (FBC) for L. innocua using effective binary combinations of carvacrol, thymol, and
eugenol.
Carvacrol (mg kg
1
) Thymol (mg kg
1
) FBC carvacrol FBC thymol FBC Index Effect
75.00 62.50 0.50 0.25 0.75 Synergistic
37.50 125.00 0.25 0.50 0.75 Synergistic
Carvacrol (mg kg
1
) Thymol (mg kg
1
) FBC carvacrol FBC thymol FBC Index Effect
125.00 56.25 0.50 0.13 0.63 Synergistic
Table 4Fractional bactericidal concentrations (FBC) for L. innocua using effective ternary combinations of carvacrol, thymol, and
eugenol.
Carvacrol (mg kg
1
) Thymol (mg kg
1
) Eugenol (mg kg
1
) FBC carvacrol FBC thymol FBC eugenol FBC Index Effect
37.50 62.50 225.00 0.25 0.25 0.50 1.00 Additive
18.75 125.00 112.50 0.13 0.50 0.25 0.88 Synergistic
75.00 31.25 56.25 0.50 0.13 0.13 0.76 Synergistic
that there is a linear response in every inhibitor; the FBC index
method does not provide information on antimicrobial response
to different doses and therefore assumes that response proles of
antimicrobials are similar. In our case, we assume that this con-
dition was accomplished since the chemical characteristics of the
compounds tested are similar. Lambert and Lambert (2003) made
a comparison of the synergistic effect of thymol and carvacrol on
S. aureus and P. aeruginosa and they observed that their proposed
method (LambertPearson model; Lambert and Pearson 2000) is
entirely consistent with the FBC index methodology.
In general terms, among the combinations that were tested, the
least effective mixture in accomplishing a bactericidal effect was
carvacroleugenol. Garca-Garca and others (2007) reported that
only 40 out of 125 binary and ternary combinations of carvacrol,
thymol, and potassium sorbate that were tested by them, effectively
inhibited L. innocua, and among these, 29 presented a bacterici-
dal effect and 11 a bacteriostatic effect. Their results conrm the
ones obtained in our study, where we observed several binary and
ternary mixtures of the tested natural antimicrobial agents that
effectively inhibited this microorganism. Similarly, Santiesteban-
L opez and others (2006) carried out studies with L. innocua, S.
typhimurium, and E. coli, where binary combinations of carvacrol,
thymol, or eugenol with potassium sorbate were observed to ef-
fectively inhibit those microorganisms. They also pointed out that,
generally speaking, the natural antimicrobials (carvacrol, thymol,
and eugenol) were more effective than potassium sorbate within
the pH and a
w
conditions they tested in their study, and that these
natural antimicrobial agents were less dependent on pH and a
w
than potassium sorbate. Santiesteban-L opez and others (2006) ob-
served that there are synergistic mixtures of carvacrol or thymol
with potassium sorbate that effectively inhibited the growth of
L. innocua. In our case, in most cases, the synergistic binary com-
binations contributed to the synergistic effect in ternary mixtures
with the same antimicrobial agents. Santiesteban-L opez and oth-
ers (2006) reported that in a mixture, the inhibitory antimicrobial
agents and its concentrations depend on the a
w
, pH, and type of
bacteria that is expected to be inhibited. They also mentioned that
the antimicrobial agents have higher possibilities of eliminating a
wide variety of bacteria when they are combined rather than when
they are acting alone.
Several reports evaluated the effects on the sensory attributes of
the addition of essential oils (or their components) in various prod-
ucts such as sh (Goulas and Kotominas 2007), and ready-to-eat
(RTE) foods such as low fat products based RTE-meat or sausages
(Busatta and others 2008; Gutierrez and others 2008, 2009). These
researchers reported that for products where the avor of oils or
their compounds is compatible with the sensory attributes of the
products in which where added, the sensory evaluation of them
has been satisfactory. Another point that has been identied as
important for incorporation of essential oils or its components to
foods is to combine them with other conservation factors to use
lower concentrations (Goulas and Kotominas 2007; Gutierrez and
others 2008; 2009). Our results provide the possibility to decrease
concentrations of eugenol, thymol and/or carvacrol by using them
in binary or ternary mixtures.
Conclusions
Binary and ternary mixtures of carvacrol, thymol, and eugenol
that effectively inactivated L. innocua in liquid model systems were
determined. Despite the fact there is very little scientic knowl-
edge about the interaction mechanisms of antimicrobial agents,
many of their synergistic combinations can help inactivate mi-
crobial growth. Besides, the interest in nding effective synergistic
mixtures should rise, since the use of most of the essential oils or its
components (that is, carvacrol, thymol, or eugenol) with antimi-
crobial activity is limited because they may directly inuence the
avor of foods. The concentrations that effectively inactivate mi-
croorganisms may surpass an acceptable sensory level. This is why
it is so important to continue the research to determine the mini-
mal inhibitory and bactericidal concentrations of the antimicrobial
agents in synergistic mixtures, to establish a balance between their
antibacterial efciency and possible sensory acceptance.
Acknowledgments
Authors acknowledge nancial support from the Natl. Coun-
cil for Science and Technology of Mexico (CONACyT) Project
84859: Combinaci on de Factores Fsicos y Qumicos para la
Inactivaci on de Microorganismos Relacionados con Alimentos.
Author Garca-Garca gratefully acknowledges nancial support
for her PhD studies from CONACyT and Univ. de las Am ericas
Puebla.
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