Food Chemistry 116 (2009) 982–989

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Food Chemistry
journal homepage: www.elsevier.com/locate/foodchem

Antimicrobial activity in the vapour phase of a combination of cinnamon

and clove essential oils
P. Goñi a, P. López b,*, C. Sánchez c, R. Gómez-Lus a, R. Becerril a, C. Nerín b
a
Department of Microbiology, Preventive Medicine and Public Health, Faculty of Medicine, University of Zaragoza, C/Domingo Miral s/n, E-50009 Zaragoza, Spain
b
Department of Analytical Chemistry, Aragón Institute of Engineering Research, i3A, CPS-University of Zaragoza, María de Luna St. 3, E-50018 Zaragoza, Spain
c
ARTIBAL S.A., Department of I + D + i, Cañada Real St. 12, E-22600 Sabiñánigo, Spain

a r t i c l e i n f o a b s t r a c t

Article history: The antimicrobial activity of the vapour generated by a combination of cinnamon and clove essential oils
Received 7 December 2008 against the growth of four Gram-negative (Escherichia coli, Yersinia enterocolitica, Pseudomonas aeruogin-
Received in revised form 8 February 2009 osa and Salmonella choleraesuis) and four Gram-positive bacteria (Staphylococcus aureus, Listeria monocyt-
Accepted 16 March 2009
ogenes, Bacillus cereus and Enterococcus faecalis) was assessed by means of the fractional inhibitory
concentration index (FIC) of the mixture. The presence of synergism or antagonism effects depended
on the reference parameter used to estimate such an index. If the minimal inhibitory concentrations were
Keywords:
applied, the vapours of the combination of essential oils exerted an antagonistic effect on the growth of
Essential oils
Cinnamon
E. coli, while they wielded a synergistic effect for the inhibition of L. monocytogenes, B. cereus and Y.
Clove enterocolitica when the concentrations of maximal inhibition were used. This fact revealed a clear concen-
Fractional inhibitory concentration index tration-dependent interaction.
Synergy The headspace of the cinnamon and clove essential oils and their combination was sampled by solid-
Vapour phase phase microextraction (SPME) and the constituents identified and quantified by gas chromatography–ion
Antimicrobial activity trap mass spectrometry (GC/ITMS). Eugenol was the most abundant compound for the three antibacterial
atmospheres. The differences in behaviour could be attributed to minor compounds. The combined head-
space contained slightly larger amounts of 1,8-cineole and camphor, which are believed to enhance the
eugenol activity. The mechanisms responsible for the antagonism are, however, less known and much
further investigation is required.
To the best of our knowledge this is the first time a combination of essential oils in the vapour phase
has been tested as a preservative method to prevent microorganism proliferation.
Ó 2009 Elsevier Ltd. All rights reserved.

1. Introduction the use of EOs for prevention of the transmission of resistant and
harmful pathogens strains, such as methicillin-resistant S. aureus
Numerous food products require protection against microbial (MRSA) (Penalver et al., 2005).
spoilage during their shelf life. The growing demand of consumers Despite the high efficiency of the EOs and their constituents
for safe and natural products, without chemical preservatives, has against food-borne pathogens and spoilage microorganisms when
resulted in thorough investigations from food authorities and in vitro tests are conducted (Chorianopoulos et al., 2004; Fisher &
researchers to assess the feasibility of mild preservation techniques Phillips, 2006), the same effect in food is only achieved with higher
and to improve the microbial quality and safety of products, while concentration of EOs (Burt, 2004; Hulin, Mathot, Mafart, & Dufosse,
maintaining their good nutritional and organoleptic properties. 1998). This fact may imply an organoleptic impact, caused by alter-
Essential oils (EOs) are volatile oily liquids obtained from differ- ing the natural taste of the food by exceeding the acceptable fla-
ent plant parts and widely used as food flavours (Burt, 2004). In vour thresholds (Hsieh, Mau, & Huang, 2001; Nazer, Kobilinsky,
spite of having been long recognised for their antibacterial, anti- Tholozan, & Dubois-Brissonnet, 2005). Few approaches have been
fungal, antiviral, insecticidal and antioxidant properties (Kordali proposed to minimise EO concentrations and reduce the sensory
et al., 2005; Pezo, Salafranca, & Nerin, 2006), the recent interest effect.
in alternative natural substances has lead to a new scientific One solution would consist of combining plant extracts.
awareness of these substances. Some authors have even suggested Although EOs were concluded to have greater activity than mix-
tures of their major components (Gill, Delaquis, Russo, & Holley,
* Corresponding author. Tel.: +34 976761873x5296; fax: +34 9762388. 2002; Mourey & Canillac, 2002), the combination of these major
E-mail address: [email protected] (P. López). components with other constituents with a weaker activity might

0308-8146/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.foodchem.2009.03.058
 P. Goñi et al. / Food Chemistry 116 (2009) 982–989 983

result in a synergistic, additive or antagonist effect (Ultee, Kets, mixtures: trans-cinnamaldehyde (99%, CAS 14371-10-9), b-caryo-
Alberda, Hoekstra, & Smid, 2000). The combination of clove and phyllene (99.5%, CAS 87-44-5), bornyl acetate (95%, CAS 5655-61-
rosemary exerted all three effects, depending on the corresponding 8), estragol (98%, CAS 140-67-0), borneol (98%, CAS 464-43-7), R-
microorganism (Fu et al., 2007). Oregano EO combined with thyme (b)-pinene (98%, CAS 80-56-8), thymol (99.5%, CAS 89-83-8), 1,8-
EO at low doses has been reported as a potential means of control- cineole (99%, CAS 470-82-6), D-limonene (97%, CAS 5989-27-5),
ling the growth of pathogens and spoilage microorganisms, camphor (96%, CAS 76-22-2), benzyl benzoate (99%, CAS 126-51-
whereas the combinations of oregano with marjoram EO or thyme 4), linalool (97%, CAS 78-70-6), eugenol (99%, CAS 97-53-0), a-
with sage EO might be useful for targeted control of key Gram-neg- pinene (99%, CAS 8172-673), camphene (95%, CAS 79-92-5), a-
ative or Gram-positive bacteria, respectively (Gutierrez, Barry- humulene (99.5%, CAS 6753-98-6) supplied by Sigma–Aldrich (St.
Ryan, & Bourke, 2008). Since EOs are generally recognised as safe Louis, MO); a-cubenene (97%, CAS 17699-14-8), a-copaene (90%,
(GRAS) (Kabara, 1991), the possibility of reinforcing their natural CAS 3856-25-5), ()-verbenone (97%, CAS 1196-01-6), c-terpinene
antimicrobial effects by the addition of small amounts of other nat- (97%, CAS 99-85-4), a-terpinolene (97%, 582-67-9), a-phellandrene
ural preservatives may be a way of attaining a balance between (95%, CAS 4221-98-1), a-terpinene (95%, CAS 99-86-5) supplied by
sensory acceptability and antimicrobial efficiency. Fluka (Bellefonte, PA), and a-terpineol (98%, CAS 562-74-3) sup-
Lopez et al. have assessed the antimicrobial activity in the va- plied by Chem Service (West Chester, PA).
pour phase of a wide number of EOs and their main constituents
(Lopez, Sanchez, Batlle, & Nerin, 2005, 2007b) with promising re- 2.4. Antimicrobial activity test
sults, which concluded with the development of an antimicrobial
packaging (Lopez, Sanchez, Batlle, & Nerin, 2007a; Rodriguez, Bat- Solid diffusion tests: The susceptibility of the bacteria to the EOs
lle, & Nerin, 2007), which achieved similar inhibition for the in vitro was determined by an agar diffusion disc method (Lopez et al.,
tests and the assays conducted with food (Rodriguez, Nerin, & Bat- 2005). The appropriate solidified medium was inoculated with
lle, 2008). The innovative film creates a protective atmosphere 100 ll of bacterial suspension containing 105 cfu/ml of the micro-
with a negligible organoleptic alteration. Nevertheless, and to the organism under study. Afterwards, 10 ll of each dilution of either
best of our knowledge, there is no published report regarding the the pure essential oil or their combinations (1:1) were added to
antimicrobial effectiveness of combinations of EOs in the vapour 10 mm sterile blank filter discs and placed in direct contact with
phase. the agar medium. Previously, it was estimated that 10 ll of pure
The aim of the present study was the assessment of the suscep- essential oil corresponded to 10 mg weight on the disc.
tibility of various strains of microorganisms to cinnamon, clove Vapour diffusion tests: Solidified medium was inoculated with
and a mixture of cinnamon and clove EOs, all in the vapour phase, 100 ll of bacterial suspension containing 105 cfu/ml of the micro-
to detect synergistic, additive or antagonistic effects. The atmo- organism under study. Each pure essential oils or their combina-
sphere generated by the different antimicrobial natural agents tions (1:1) were diluted in ethyl ether (GC quality, Merck,
has been sampled by solid-phase microextraction (SPME) and ana- Darmstadt, Germany) to obtain serial dilutions. Then, 10 ll of each
lysed by gas chromatography–ion trap mass spectrometry (GC– dilution were added to 10-mm diameter sterile blank filter discs
ITMS). Finally, a correlation between the chemical composition and placed in the centre of the lid of the Petri dish (Lopez et al.,
and the antimicrobial activity has been proposed. 2005). The Petri dishes were then sealed using sterile adhesive tape
(Deltalab, Rubi, Spain). No hermetic sealing was needed because
experiments were designed to simulate a worst-case situation,
2. Experimental section when leaking of the active components to the atmosphere can oc-
cur, thus increasing the probability for microorganism contamina-
2.1. Bacterial strains tion. Blanks were prepared by adding 10 ll of ethyl ether to the
filter discs, which was demonstrated to have no effect on the via-
The following food-borne bacterial strains were selected, due to bility of any of the tested bacteria. Analyses were carried out in
their relevance in the food industry: the Gram-negative bacteria triplicate. The concentration of essential oil was expressed as
Escherichia coli (American Culture Collection, ATCC 29252), Yersinia weight per unit volume (mg/l air). The tested concentrations varied
enterocolitica (Colección Española de Cultivos Tipo, CECT 4315), from 180 to 1.8 mg/l in the headspace of the Petri dish.
Salmonella choleraesuis (CECT 4000), and Pseudomonas aeruginosa The effectiveness of the essential oil was calculated by measur-
(ATCC 27853); the Gram-positive bacteria Bacillus cereus (CECT ing the diameter (in mm) of the zone of microorganism growth
495), Listeria monocytogenes (ATCC 7644), Enterococcus faecalis inhibition above the disc. The size of the zone with visible growth
(ATCC 29212) and Staphylococcus aureus (ATCC 29213). The strains reduction around the inhibition zone was also measured.
were cultured on Mueller-Hinton agar (MHA, Bio-Rad, La Coquette, The minimum inhibitory concentration (MIC) was defined
France) at 30 °C for 48 and 24 h for Gram-positive and Gram-neg- as the lowest essential oil concentration resulting in the lack of
ative microorganisms, respectively, and stored at 80 °C in sterile visible microorganism growth. The reduction concentration (RC)
skimmed milk. was defined as the lowest essential oil concentration resulting in
a visible microorganism growth reduction. The essential oil con-
2.2. Essential oils centration which yielded the biggest inhibition zone was named
as Cmax.
The essential oils (EOs) were supplied by ARTIBAL (Sabiñanigo, A fractional inhibition concentration index (FIC) was estimated
Spain). Oils from the following plant species were tested in this for all tested microorganisms to determine the antimicrobial effect
work: Cinnamon zeylanicum [cinnamon, Chemical Abstract Service of the mixture of cinnamon and clove EOs, within the limits of the
(CAS), registry number 8015-91-6], and Syzgium aromaticum method used (White, Burgess, Manduru, & Bosso, 1996). The FIC
(clove, CAS No. 8000-34-6). was calculated by the following equation:

2.3. Chemicals FIC ¼ ½concentration of essential oil combination

which inhibits bacteria=½concentration of pure
The following chemicals were used as standards to identify the
composition of the atmospheres generated by the EOs and their essential oil which inhibits bacteria:
984 P. Goñi et al. / Food Chemistry 116 (2009) 982–989

Table 1
Inhibition and growth reduction zones, in millimetres, provided by cinnamon EO (CI), clove EO (CL) and their mixture (CI–CL). Results are expressed as mean ± standard deviation.

Direct contact Vapour phase

CI CL CI–CL CI CL CI–CL
Gram-negative
E. coli 18 ± 1 18 ± 1 18 ± 1 22 ± 3 (27)a 30 ± 1b 25 ± 3 (29)
Y. enterocolitica 21 ± 2 23 ± 3 22 ± 2 32 ± 3 (35) 35 ± 2 (38) 50 ± 2 (54)
S. choleraesuis 21 ± 1 23 ± 2 22 ± 1 13 ± 3 13 ± 4 (18) 16 ± 4 (28)
P. aeruginosa 12 ± 1 0±1 12 ± 1 0 0 0
Gram-positive
B. cereus 30 ± 2 32 ± 1 30 ± 1 26 ± 4 (32) 21 ± 3 (24) 32 ± 2 (42)
L. monocytogenes 19 ± 1 18 ± 2 20 ± 1 26 ± 2 (32) 13 ± 1 (22) 25 ± 3 (29)
E. faecalis 14 ± 1 15 ± 0 17 ± 1 15 ± 5 (18) 12 ± 3 (17) 12 ± 3 (14)
S. aureus 19 ± 2 19 ± 1 20 ± 1 24 ± 3 (28) 23 ± 3 (24) 25 ± 1 (29)
a
The growth reduction zone in brackets. 90 mm means total inhibition.
b
Significant different results in bold.

Table 2
Minimum inhibitory concentration (MIC), reduction concentration (RC) and concentration of maximal inhibition (Cmax), expressed as mg EO/l headspace in vapour phase of
cinnamon EO (CI), clove EO (CL) and their combination (CI–CL).

CI CL CI–CL (1:1)
MIC RC Cmax MIC RC Cmax MIC RC Cmax
Gram-negative
E. coli 18 ± 0 18 ± 0 54 ± 0 27 ± 0 27 ± 0 180 ± 0 90 ± 0 90 ± 0 90 ± 0
Y. enterocolitica 18 ± 0 13 ± 2 90 ± 0 9±5 9±2 180 ± 25 18 ± 0 18 ± 0 90 ± 10
S. choleraesuis 136 ± 25 54 ± 10 181 ± 0 54 ± 20 35 ± 10 180 ± 25 135 ± 0 36 ± 0 180 ± 0
P. aeruginosa 0 0 0 0 0 0 0 0 0
Gram-positive
B. cereus 18 ± 5 13 ± 2 181 ± 25 18 ± 5 18 ± 5 135 ± 25 36 ± 0 36 ± 0 135 ± 0
L. monocytogenes 54 ± 0 54 ± 2 181 ± 25 18 ± 5 18 ± 0 135 ± 25 90 ± 0 90 ± 0 180 ± 0
E. faecalis 54 ± 0 54 ± 0 181 ± 0 90 ± 0 35 ± 0 180 ± 25 90 ± 5 90 ± 0 135 ± 25
S. aureus 36 ± 5 27 ± 5 136 ± 25 27 ± 0 27 ± 0 90 ± 0 54 ± 0 36 ± 0 135 ± 25

2.5. Solid-phase microextraction (SPME) min to 90 °C; 5 °C/min to 170 °C; then 5 °C/min to 200 °C, and held
for 15 min. For cinnamon, and the mixture of clove–cinnamon EOs,
Fully-retracted SPME fibres (Supelco, Bellefonte, PA), coated the injector temperature was held at 250 °C, whereas the oven
with an 85-lm layer of polyacrylate (PA) for clove EO or a 100- temperature was initially held at 45 °C for 1 min, raised at 2 °C/
lm layer of polydimethylsiloxane (PMDS) for cinnamon EO and min to 85 °C, by 5 °C/min to 170 °C, then finally at 15 °C/min to
the clove–cinnamon mixture were used. The optimum conditions 200 °C, and held for 2 min.
as well as the type of fibre used were based on the results of pre- The MS was operated in electron impact (EI) ionisation mode,
vious studies (Lopez, Huerga, Batlle, & Nerin, 2006). The atmo- and complete scans from 40 to 350 amu were recorded. Com-
sphere generated in the vapour diffusion tests was measured by pounds were identified by matching their mass spectra with those
placing a fully-retracted SPME fibre into the headspace of the Petri
dish (Lopez et al., 2005). Sampling was performed over 24 h. The
volume of the atmosphere sampled on every occasion was Table 3
57.3 cm3. The atmosphere composition inside the Petri dishes Fractional inhibitory concentration (FIC) index for each individual EO and its
was estimated for the pure EOs and/or mixture under the same combination (CI, cinnamon EO; CL, clove EO; A, antagonism; S, synergism; I,
indifferent effect; ad, additive effect).
conditions of temperature and culture medium as the microbiolog-
ical tests, but without inoculating any strain. FICCI FICCL FICCI + FICCL
(a) Considering MIC values as reference
2.6. Gas chromatography–ion trap mass spectrometric (GC–ITMS) E. coli 2.5 (A) 1.7 (I) 4.2 (A)a
analysis Y. enterocolitica 0.5 (S) 1.0 (I) 1.5 (ad)
S. choleraesuis 0.5 (S) 1.3 (I) 1.8 (ad)
B. cereus 1.0 (I) 1.0 (I) 2.0 (ad)
GC–ITMS analyses were carried out using a Varian CP 3800 gas L. monocytogenes 0.8 (I) 2.5 (A) 3.3 (ad)
chromatograph (Varian Inc., Palo Alto, CA) equipped with a VF-5 E. faecalis 0.8 (I) 0.5 (S) 1.3 (ad)
MS (Varian) column (60 m  0.25 mm, 0.25 lm film thickness) S. aureus 0.7 (I) 1.0 (I) 1.8 (ad)
coupled to a Saturn 2000 ITMS detector; a split–splitless injector (b) Considering concentrations of maximal inhibition (Cmax) as reference
operated in splitless mode, (splitless time 2 min) with a 0.8 mm E. coli 0.8 (I) 0.3 (S) 1.1 (ad)
Y. enterocolitica 0.1 (S) 0.1 (S) 0.2 (S)b
id SPME-specific liner (Varian), and an MS version 6.03 Chemsta-
S. choleraesuis 0.5 (S) 0.5 (S) 1.0 (ad)
tion. The carrier gas was helium (C-50, Carburos Metálicos, Zara- B. cereus 0.2 (S) 0.2 (S) 0.4 (S)
goza, Spain) at a constant flow rate of 1.0 ml/min. L. monocytogenes 0.2 (S) 0.3 (S) 0.5 (S)
Two different sets of chromatographic conditions were used, E. faecalis 0.5 (S) 0.5 (S) 1.0 (ad)
according to a previous study (Lopez et al., 2006). For clove analy- S. aureus 0.4 (S) 0.8 (I) 1.1 (ad)

sis, the injector temperature was 265 °C, and the oven programme a
Antagonism effect in bold.
b
was as follows: initial temperature, 45 °C, held for 1 min; 15 °C/ Synergism effect in bold.
 P. Goñi et al. / Food Chemistry 116 (2009) 982–989 985

in the NIST commercial library (purity criterion, >85%). When against the eight microbial species described in Section 2.1, was
available, the retention times and fragmentation spectra of pure qualitatively and quantitatively assessed by the presence or ab-
standards (>95%) were obtained for confirmation. All analyses sence of inhibition zone. The diameter of the inhibition zone is gi-
were carried out in triplicate. ven in Table 1.
The mass of tested EO was 10.4 mg, which corresponded to a
2.7. Statistical analysis concentration of 181 mg/l in the vapour phase. No significant dif-
ferences were observed among the size of the inhibition zone for
The triplicate data obtained are presented in Tables 1 and 2 as the mixture and the individual EOs when direct contact, while
means ± standard deviation (SD). Significant differences were the combination provided a significant increase of the activity for
determined by ANOVA at the 95% significance level using STAT- Y. enterocolitica, B. cereus and L. monocytogenes.
GRAPHICS Plus 5.1 (Statistical Graphic Corporation, Warrenton, Essential oils showed higher activity in the vapour phase. P.
VA). aeruginosa was the only microorganism that was better inhibited
when in direct contact. The size of the inhibition zone in the va-
3. Results pour phase generally increased in the following order:
Cinnamon EO: S. choleraesuis < E. faecalis < L. monocytoge-
3.1. Antimicrobial activity nes  E. coli  S. aureus  B. cereus < Y. enterocolitica; clove EO: S.
choleraesuis  L. monocytogenes  E. faecalis < S. aureus  B.
3.1.1. Direct contact versus vapour phase cereus < E. coli < Y. enterocolitica; and cinnamon–clove combina-
The antimicrobial activity of cinnamon and clove EOs and their tion: E. faecalis < S. choleraesuis < S. aureus  L. monocytoge-
combination (1:1), both by direct contact or through vapour phase, nes  E. coli  B. cereus < Y. enterocolitica.

Y. enterocolitica E. coli
60 35
50 30
Inhibition (mm)

Inhibition (mm)

40 25
30 20
15
20
10
10 5
0 0
0 50 100 150 200 0 50 100 150 200
mg EO / L headspace mg EO / L headspace

S. choleraesuis S. aureus
20 30
25
Inhibition (mm)

15
Inhibition (mm)

20

10 15
10
5 5

0 0
0 50 100 150 200 0 50 100 150 200
mg EO / L headspace mg EO / L headspace

B. cereus L. monocytogenes
20 30

15
Inhibition (mm)

20
Inhibition (mm)

10
10
5

0 0
0 50 100 150 200 0 50 100 150 200
mg EO / L headspace mg EO / L headspace

E. faecalis
40

30 Cinnamon EO
Inhibition (mm)

20 Clove EO
10
Cinnamon-clove (1:1)
0
0 50 100 150 200
mg EO / L headspace

Fig. 1. Antimicrobial activity of the single EOs and their combination in the vapour phase.
986 P. Goñi et al. / Food Chemistry 116 (2009) 982–989

However, no relationship between the Gram-positive or Gram- Table 4

negative structure and the activity of the essential oils was ob- Composition of the atmosphere generated by cinnamon EO, clove EO and their
combination (1:1), expressed as percentages of total ion counts identified (n = 3).
served, with the exception of P. aeruginosa, which provided a slight
activity in direct contact with cinnamon. Peak number Compound CIa CLa CI–CL
1 a-Pinene 1.1 ± 0.4 – 0.3 ± 0.0
3.1.2. Minimum inhibitory (MIC), reduction concentration (RC) and 2 Camphene 0.4 ± 0.2 – 0.1 ± 0.0
concentration of maximal inhibition (Cmax) in vapour phase 3 b-Pinene 0.8 ± 0.2 – 0.2 ± 0.0
4 a-Phellandrene 1.2 ± 0.6 – 0.3 ± 0.1
Table 2 shows the minimum inhibitory concentration (MIC), the 5 p-Cymene 1.2 ± 0.1 – 0.6 ± 0.2
reduction concentration (RC) and the concentration of maximal 6 Limonene 0.9 ± 0.5 – 0.3 ± 0.1
inhibition for the atmosphere generated by cinnamon and clove 7 1,8-Cineole 1.2 ± 0.1 – 6.2 ± 1.7
EO and their mixture (1:1). 8 Linalool 3.0 ± 0.4 – 2.5 ± 0.3
9 Camphor 0.4 ± 0.3 – 1.3 ± 0.5
Regarding the MIC values, Y. enterocolitica was the most sensi-
10 Citronellal 0.2 ± 0.1 – 0.4 ± 0.1
tive microorganism, providing the lowest microorganism growth, 11 Borneol 0.2 ± 0.1 – 0.2 ± 0.0
followed by B. cereus and S. aureus whatever the tested EO. 12 1-Terpinen-4-ol 0.3 ± 0.2 – –
Although cinnamon EO and its combination with clove EO pro- 13 a-Terpineol 0.5 ± 0.1 – 0.2 ± 0.1
vided the same MIC values against Y. enterocolitica, an increase in 14 Estragol 1.7 ± 0.5 0.2 ± 0.1 2.6 ± 0.5
15 (E)-Cinnamaldehyde 0.4 ± 0.0 – 0.2 ± 0.0
the inhibition zone, without bacterial growth, was observed for
16 Safrol 2.8 ± 0.9 – 1.8 ± 0.1
the combination of EOs. As expected, P. aeruginosa was the least 17 Thymol 0.1 ± 0.0 0.1 ± 0.0 –
susceptible strain. 18 (E)-Cinnamyl alcohol 0.1 ± 0.0 – –
As far as the Cmax-values were concerned, the EO mixture was 19 a-Cubebene 0.1 ± 0.0 – –
20 Eugenol 67 ± 12 82 ± 2 61 ± 4
more effective than the single EOs against E. faecalis, Y. enterocoli-
21 Benzenepropy lacetate 0.1 ± 0.0 – 0.1 ± 0.0
tica or B. cereus growth. In the cases of Y. enterocolita and B. cereus, 22 a-Copaene 2.1 ± 0.7 – 1.2 ± 0.2
the Cmax-value of the combination was equal to that of one of the 23 b-Caryophyllene 8.6 ± 1.7 10 ± 2 14 ± 2
single oils; however, the inhibition zone was larger. 25 a-Aromadendrene 0.1 ± 0.0 – –
26 (E)-Cinnamyl acetate 0.7 ± 0.1 – 0.6 ± 0.1
27 a-Humelene 1.7 ± 0.6 2.9 ± 0.5 2.6 ± 1.2
3.1.3. Synergistic, antagonistic or additive effect
28 c-Muurolene 0.2 ± 0.0 – –
An additive effect is observed when the combined effect is equal 29 Ledene 0.2 ± 0.0 – –
to the sum of the individual effects. Synergism is observed when 30 a-Muurolene 0.1 ± 0.0 – –
the effect of the combined substances is greater than the sum of 31 Eugenol acetate 0.6 ± 0.5 0.5 ± 0.3 0.5 ± 0.2
32 d-Cadinene 0.3 ± 0.1 0.4 ± 0.1 0.5 ± 0.2
the individual effects. Antagonism is observed when the effect of
33 Calamenene 0.1 ± 0.0 0.3 ± 0.0 0.1 ± 0.0
one or both compounds is less when they are applied together than 34 Benzyl benzoate 1.1 ± 0.4 – 1.0 ± 0.1
when individually applied (Davidson & Parish, 1989). Not identified 0.5 ± 0.2 0.3 ± 0.0 0.9 ± 0.4
In order to numerically define the influence of the concentra- Total 100 ± 1 100 ± 3 100 ± 0
tion in the antagonistic/synergistic effects, the fraction inhibitory
a
concentration index (FIC index) was calculated for each EO in the CI, cinnamon EO; CL, clove EO.

tested combination (White et al., 1996) using two approaches.

The MIC values were considered as the reference in the first ap-
proach, while the concentrations of maximal inhibition (Cmax) were 3.2. Chemical composition of the antibacterial atmosphere
the key parameters in the second approach. For instance, the FIC of
cinnamon EO (FICCI) was equal to the MIC of cinnamon EO in com- The identified components of the atmosphere generated by cin-
bination divided by the MIC of cinnamon EO alone, following the namon EO, clove EO and their combination are listed in Table 4. As
first approach (Table 3a), and equal to Cmax of cinnamon EO in com- can be seen in Fig. 2, the antibacterial headspaces were character-
bination divided by Cmax of cinnamon EO alone, following the sec- ised with a prominent (>60%) concentration of eugenol. In addition
ond approach (Table 3b). Synergistic effect was defined as an FIC to eugenol, the headspace created by clove EO also contained b-
index less than or equal to 0.5; additive effect when FIC = 0.5– caryophyllene (10%) and a-humulene (3%) as main constituents
0.75, indifferent effect when FIC = 0.76–2.0; and antagonistic effect and traces of estragol, eugenol acetate, d-cadinene and calamen-
when FIC was greater than or equal to two. The FIC mixture index ene. Cinnamon EO headspace was characterised by the presence
was estimated as the sum of the individual FIC values for each EO. of a larger number of volatile compounds, most of them terpenes
An FIC mixture index between 0.5 and 4 provided additive (non- at trace level. The main constituents were eugenol (67%), b-caryo-
antagonistic) interactions. Antagonistic interactions were defined phyllene (8.6%), linalool (3%) and safrol (2.8%). Finally, the com-
as those with FIC index greater than four and synergistic effect as bined mixture provided an antibacterial atmosphere more similar
FIC < 0.5. to the one generated by single cinnamon EO.
Following the MIC approach, no synergistic effect was obtained
for the combination of EOs. On the other hand, such a mixture pro- 4. Discussion
vided an antagonism on the inhibition of E. coli. Nevertheless, at
higher concentrations (see Fig. 1) the individual EOs and the mix- The present study has demonstrated the potential of the combi-
ture possessed the same activity against the growth of E. coli. nation of cinnamon EO and clove EO (1:1, v/v) as an antibacterial
Fig. 1 also shows a clear concentration-dependent interaction agent in the vapour phase. The effectiveness of these two EOs
between the individual EOs. Like E. coli, L. monocytogenes and B. against microbial growth when in direct contact with the inocu-
cereus required higher concentrations of the mixture to be inhib- lated culture had already been widely reported (Ouattara, Simard,
ited; however, they were more sensitive to the mixture than to Holley, Piette, & Begin, 1997; Smith-Palmer, Stewart, & Fyfe, 1998;
the single EO, even achieving a synergism effect at Cmax. Consider- Valero & Salmeron, 2003), although their application in the vapour
ing the Cmax approach, a synergistic effect was also observed for the phase entails a relative innovative approach (Lopez et al., 2005). In
inhibition of Y. enterocolitica. accordance with other authors, when bacteria were exposed to the
As for the rest of the tested bacteria, the combination of the sin- vapours of essential oils, the inhibitory effects were noticeably dif-
gle EOs did not contribute to improving the inhibition. ferent from those found by direct contact (Edwards-Jones, Buck,
 P. Goñi et al. / Food Chemistry 116 (2009) 982–989 987

Fig. 2. GC–ITMS chromatograms of the two essential oils (cinnamon (CI) and clove (CL)) and their combination (CI–CL) evaluated in this study as antimicrobial agents in the
vapour phase. For peak identification, see Table 4.

Shawcross, Dawson, & Dunn, 2004). Whereas Y. enterocolitica and phase experiments are more reliable in determining the antibacte-
S. choleraesuis were the most sensitive strains and showed similar rial properties of EOs.
behaviour when in direct contact, S. choleraesuis was more resis- The mode of action of antimicrobial agents depends on the type
tant to the vapours. These differences may be explained by consid- of microorganism and evidence indicates that in the case of EOs it
ering the physicochemical properties of the antimicrobial agents is mainly associated with cell membrane damage. Their chemical
and the culture media and how the contact between the microor- constituents are characteristically hydrophobic and will accumu-
ganism and the agent occurs. The antimicrobial effect in direct- late in the lipid-rich environments of cell membrane structures
contact experiments is mostly due to the activity of the more and cause structural and functional damage (Cox et al., 2000; Lam-
hydrophilic (water-soluble) and less volatile substances, whereas bert, Skandamis, Coote, & Nychas, 2001; Sikkema, Debont, & Pool-
an equilibrium is attained during the vapour-phase experiments man, 1995). However, hydrophobicity and ability to damage cell
among the volatile compounds released in the headspace (both membrane structures are not the only factors involved (Becerril,
hydrophilic and hydrophobic) and part of the more hydrophilic Gomez-Lus, Goni, Lopez, & Nerin, 2007) and it is clear that toxicity
ones, absorbed in the agar surface. The agar diffusion method is is linked to an optimum range of hydrophobicity. It has been sug-
considered unsuitable in estimating the antimicrobial activity of gested that aqueous solubility is a factor that limits the extent to
EOs since the active volatile components are likely to be evapo- which hydrophobic compounds can accumulate to lethal levels in
rated, together with the dispersing solvent, and their apolar nature cell membranes (Cox, Mann, & Markham, 2001). Antimicrobial
prevents them from diffusion through the agar media (Kalemba & monoterpenes typically have aqueous solubilities ranging from
Kunicka, 2003; Kubo, Muroi, & Kubo, 1995). Therefore, the vapour around 800–2000 ppm (Griffin, Wyllie, Markham, & Leach, 1999).
988 P. Goñi et al. / Food Chemistry 116 (2009) 982–989

However, it is also clear that other factors should be considered for of microorganisms still need to be clearly defined, even when the
inhibiting the growth of P. aeruginosa, which displays an intrinsic antimicrobials are used individually (Alzamora, López-Malo, Guer-
resistance to a wide variety of EOs and their constituents (Cox & rero, & Palou, 2003).
Markham, 2007). All the tested combinations were prepared at 50% in volume,
The antimicrobial activity of the EOs and their combination in but the study of the effect with different proportions is now in pro-
vapour phase is closely associated with the composition of the gress. It is remarkable the low probability of appearance of resis-
headspace. The phenolic compounds are widely reported to pos- tances, as was demonstrated by the fact that some bacteria, like
sess high levels of antimicrobial activity (Baydar, Sagdic, Ozkan, Y. enterocolitica or S. Choleraesuis, were not able to develop resis-
& Karadogan, 2004; Dorman & Deans, 2000; Lambert et al., tance after more than 20 passes at subinhibitory concentrations
2001). Eugenol, which is the main component of the three charac- of cinnamon, either by direct contact or by vapour phase (data
terised atmospheres, exhibited both antimicrobial and antifungal not shown).
activity in the vapour phase (Lopez et al., 2007b; Matan et al.,
2006; Valverde et al., 2005). Bactericidal properties of clove oil 5. Conclusion
are comparable to those of disinfectants applied in hospitals and
eugenol has been proved to kill even L. monocytogenes, E. coli and This work enables us to evaluate and compare the antimicrobial
some antibiotic-resistant bacteria (Gill & Holley, 2006; Nostro activity of the combination of cinnamon and clove essential oils at
et al., 2004). The atmosphere of cinnamon EO and hence the atmo- 50% in volume against a wide range of bacteria in vapour phase.
sphere of the cinnamon–clove combination, comprised other com- The results showed that a synergistic effect could be achieved for
pounds in trace levels, such as cinnamaldehyde, a known powerful some of the tested microorganisms, this effect being concentra-
antimicrobial agent (Lopez et al., 2007b; Valero & Giner, 2006), and tion-dependent.
linalool, 1,8-cineole, p-cymene and a-pinene (Bagamboula, Uytten- The experimental results also provide the first approach to
daele, & Debevere, 2004; Belaiche, TantaouiElaraki, & Ibrahimy, developing an antimicrobial packaging with less active concentra-
1995; Santoyo et al., 2005), which are less effective in inhibiting tions of the active essential oils. This fact is of paramount impor-
the growth of microorganisms. tance from the point of view of food safety and food organoleptic
The minimum inhibitory concentration (MIC) is cited by most properties.
researchers as a measure of the antimicrobial performance of
EOs. Among all the different definitions found in literature (Burt, Acknowledgements
2004), the concepts applied in this work were the lowest concen-
tration inhibiting visible growth of the test organism (here defined This study has been financed by the Project Naturalpack (EU
as MIC) and the lowest concentration required for complete inhibi- Project INTERREG IIA-5-326-C) and by the AGL2004-07545 from
tion (here defined as Cmax). Given that the effect of the mixture of the Spanish Ministry of Education and Universities as well as by
cinnamon and clove EO (synergistic, antagonistic or additive) de- FEDER funds. P. López and R. Becerril gratefully acknowledge the
pended on the concentrations of the single EOs (see Fig. 1), it is Regional Government of Aragón and the European Social Fund
important to specify which value (either MIC or Cmax) was used for their Grants (Ref. B006/2003 and B025/2006, respectively).
to calculate the FIC index. Using MIC values, the mixture of cinna-
mon and clove EO (1:1, v/v) exhibited a clear antagonistic effect
against E. coli, whereas it revealed a synergistic effect against Y. References
enterocolitica, L. monocytogenes and B. cereus when Cmax was used.
Alzamora, S. M., López-Malo, A., Guerrero, S., & Palou, E. (2003). Plant antimicrobials
Burt (2004) also suggested that the minor components of EOs are combined with conventional preservatives for fruit products. In S. Roller (Ed.),
more critical to the activity than mixtures of the main EO compo- Natural Antimicrobials for the Minimal Processing of Foods (pp. 235–249).
nents, and may have either an additive or synergistic effect. For in- Cambridge: Woodhead Publishing Ltd..
Bagamboula, C. F., Uyttendaele, M., & Debevere, J. (2004). Inhibitory effect of thyme
stance, terpinen-4-ol is thought to diffuse into and damage cell and basil essential oils, carvacrol, thymol, estragol, linalool and p-cymene
membrane structures, causing increased fluidity or disordering towards Shigella sonnei and S-flexneri. Food Microbiology, 21(1), 33–42.
membrane structure and inhibition of membrane-bound enzymes Baydar, H., Sagdic, O., Ozkan, G., & Karadogan, T. (2004). Antibacterial activity and
composition of essential oils from Origanum, Thymbra and Satureja species
(Sikkema et al., 1995). Camphor and 1,8-cineole, whose concentra-
with commercial importance in Turkey. Food Control, 15(3), 169–172.
tion in the mixture headspace was five times greater than for the Becerril, R., Gomez-Lus, R., Goni, P., Lopez, P., & Nerin, C. (2007). Combination of
single cinnamon EO, could contribute to the enhancement of euge- analytical and microbiological techniques to study the antimicrobial activity of
nol activity, since the oxygenated terpenes are believed to present a new active food packaging containing cinnamon or oregano against E-coli and
S-aureus. Analytical and Bioanalytical Chemistry, 388(5–6), 1003–1011.
a higher activity than the terpene hydrocarbons (Caccioni & Guiz- Belaiche, T., TantaouiElaraki, A., & Ibrahimy, A. (1995). Application of a two levels
zardi, 1994; Knobloch, Pauli, & Iberl, 1989). factorial design to the study of the antimicrobial activity of three terpenes.
There are some generally accepted mechanisms of antimicro- Sciences Des Aliments, 15(6), 571–578.
Burt, S. (2004). Essential oils: Their antibacterial properties and potential
bial interaction that produce synergism: sequential inhibition of applications in foods – A review. International Journal of Food Microbiology,
a common biochemical pathway, inhibition of protective enzymes, 94(3), 223–253.
combinations of cell wall active agents, and use of cell wall active Caccioni, D. R. L., & Guizzardi, M. (1994). Inhibition of germination and growth of
fruit and vegetable postharvest pathogenic fungi by essential oil components.
agents to enhance the uptake of other antimicrobials (Santieste- Journal of Essential Oil Research, 6(2), 173–179.
ban-Lopez, Palou, & López-Malo, 2007). Mechanisms of antimicro- Chorianopoulos, N., Kalpoutzakis, E., Aligiannis, N., Mitaku, S., Nychas, G. J., &
bial interaction that produce antagonism are less known, although Haroutounian, S. A. (2004). Essential oils of Satureja, Origanum, and
Thymus species: Chemical composition and antibacterial activities against
they include combinations of bactericidal and bacteriostatic
foodborne pathogens. Journal of Agricultural and Food Chemistry, 52(26),
agents, use of compounds that act on the same target of the micro- 8261–8267.
organism and chemical (direct or indirect) interactions among Cox, S. D., Mann, C. M., & Markham, J. L. (2001). Interactions between components of
the essential oil of Melaleuca alternifolia. Journal of Applied Microbiology, 91(3),
compounds. For instance, non-oxygenated monoterpene hydrocar-
492–497.
bons such as c-terpinene and p-cymene appear to produce antag- Cox, S. D., Mann, C. M., Markham, J. L., Bell, H. C., Gustafson, J. E., Warmington, J. R.,
onistic effects, since they reduce the aqueous terpene solubility et al. (2000). The mode of antimicrobial action of the essential oil of Melaleuca
and, therefore, the microbial availability of the active components alternifolia (tea tree oil). Journal of Applied Microbiology, 88(1), 170–175.
Cox, S. D., & Markham, J. L. (2007). Susceptibility and intrinsic tolerance of
(Cox et al., 2001). Therefore, specific modes of action of plant con- Pseudomonas aeruginosa to selected plant volatile compounds. Journal of Applied
stituents with antimicrobial properties on the metabolic activities Microbiology, 103, 930–936.
 P. Goñi et al. / Food Chemistry 116 (2009) 982–989 989

Davidson, P. M., & Parish, M. E. (1989). Methods for testing the efficacy of food foodborne microorganisms. Journal of Agricultural and Food Chemistry, 55(11),
antimicrobials. Food Technology, 43(1), 148–155. 4348–4356.
Dorman, H. J. D., & Deans, S. G. (2000). Antimicrobial agents from plants: Matan, N., Rimkeeree, H., Mawson, A. J., Chompreeda, P., Haruthaithanasan, V., &
Antibacterial activity of plant volatile oils. Journal of Applied Microbiology, Parker, M. (2006). Antimicrobial activity of cinnamon and clove oils under
88(2), 308–316. modified atmosphere conditions. International Journal of Food Microbiology,
Edwards-Jones, V., Buck, R., Shawcross, S. G., Dawson, M. M., & Dunn, K. (2004). The 107(2), 180–185.
effect of essential oils on methicillin-resistant Staphylococcus aureus using a Mourey, A., & Canillac, N. (2002). Anti-Listeria monocytogenes activity of essential
dressing model. Burns, 30(8), 772–777. oils components of conifers. Food Control, 13(4–5), 289–292.
Fisher, K., & Phillips, C. A. (2006). The effect of lemon, orange and bergamot essential Nazer, A. I., Kobilinsky, A., Tholozan, J. L., & Dubois-Brissonnet, F. (2005).
oils and their components on the survival of Campylobacter jejuni, Escherichia Combinations of food antimicrobials at low levels to inhibit the growth of
coli O157, Listeria monocytogenes, Bacillus cereus and Staphylococcus aureus Salmonella sv. Typhimurium: A synergistic effect? Food Microbiology, 22(5),
in vitro and in food systems. Journal of Applied Microbiology, 101(6), 1232–1240. 391–398.
Fu, Y. J., Zu, Y. G., Chen, L. Y., Shi, X. G., Wang, Z., Sun, S., et al. (2007). Antimicrobial Nostro, A., Blanco, A. R., Cannatelli, M. A., Enea, V., Flamini, G., Morelli, I., et al.
activity of clove and rosemary essential oils alone and in combination. (2004). Susceptibility of methicillin-resistant staphylococci to oregano essential
Phytotherapy Research, 21, 989–994. oil, carvacrol and thymol. Fems Microbiology Letters, 230(2), 191–195.
Gill, A. O., Delaquis, P., Russo, P., & Holley, R. A. (2002). Evaluation of antilisterial Ouattara, B., Simard, R. E., Holley, R. A., Piette, G. J. P., & Begin, A. (1997).
action of cilantro oil on vacuum packed ham. International Journal of Food Antibacterial activity of selected fatty acids and essential oils against six meat
Microbiology, 73(1), 83–92. spoilage organisms. International Journal of Food Microbiology, 37(2–3),
Gill, A. O., & Holley, R. A. (2006). Disruption of Escherichia coli, Listeria 155–162.
monocytogenes and Lactobacillus sakei cellular membranes by plant oil Penalver, P., Huerta, B., Borge, C., Astorga, R., Romero, R., & Perea, A. (2005).
aromatics. International Journal of Food Microbiology, 108(1), 1–9. Antimicrobial activity of five essential oils against origin strains of the
Griffin, S. G., Wyllie, S. G., Markham, J. L., & Leach, D. N. (1999). The role of structure Enterobacteriaceae family. APMIS, 113(1), 1–6.
and molecular properties of terpenoids in determining their antimicrobial Pezo, D., Salafranca, J., & Nerin, C. (2006). Design of a method for generation of gas-
activity. Flavour and Fragrance Journal, 14(5), 322–332. phase hydroxyl radicals, and use of HPLC with fluorescence detection to assess
Gutierrez, J., Barry-Ryan, C., & Bourke, R. (2008). The antimicrobial efficacy of plant the antioxidant capacity of natural essential oils. Analytical and Bioanalytical
essential oil combinations and interactions with food ingredients. International Chemistry, 385(7), 1241–1246.
Journal of Food Microbiology, 124(1), 91–97. Rodriguez, A., Batlle, R., & Nerin, C. (2007). The use of natural essential oils as
Hsieh, P. C., Mau, J. L., & Huang, S. H. (2001). Antimicrobial effect of various antimicrobial solutions in paper packaging. Part II. Progress in Organic Coatings,
combinations of plant extracts. Food Microbiology, 18(1), 35–43. 60, 33–38.
Hulin, V., Mathot, A. G., Mafart, P., & Dufosse, L. (1998). Antimicrobial properties of Rodriguez, A., Nerin, C., & Batlle, R. (2008). New cinnamon-based active paper
essential oils and flavour compounds. Sciences Des Aliments, 18(6), 563–582. packaging against Rhizopusstolonifer food spoilage. Journal of Agricultural and
Kabara, J. J. (1991). Phenols and chelators. In E. N. J. Russell & G. W. Gould (Eds.), Food Chemistry, 56(15), 6364–6369.
Food preservatives (pp. 200–214). London: Blackie. Santiesteban-Lopez, A., Palou, E., & López-Malo, A. (2007). Susceptibility of food-
Kalemba, D., & Kunicka, A. (2003). Antibacterial and antifungal properties of borne bacteria to binary combinations of antimicrobials at selected a(w) and
essential oils. Current Medicinal Chemistry, 10(10), 813–829. pH. Journal of Applied Microbiology, 102(2), 486–497.
Knobloch, K., Pauli, A., & Iberl, B. (1989). Antibacterial and antifungal properties of Santoyo, S., Cavero, S., Jaime, L., Ibanez, E., Senorans, F. J., & Reglero, G. (2005).
essential oil components. Journal of Essential Oil Research, 1, 119–128. Chemical composition and antimicrobial activity of Rosmarinus officinalis L.
Kordali, S., Kotan, R., Mavi, A., Cakir, A., Ala, A., & Yildirim, A. (2005). Determination essential oil obtained via supercritical fluid extraction. Journal of Food Protection,
of the chemical composition and antioxidant activity of the essential oil of 68(4), 790–795.
Artemisia dracunculus and of the antifungal and antibacterial activities of Sikkema, J., Debont, J. A. M., & Poolman, B. (1995). Mechanisms of membrane
Turkish Artemisia absinthium, A-dracunculus, Artemisia santonicum, and Artemisia toxicity of hydrocarbons. Microbiological Reviews, 59(2), 201–222.
spicigera essential oils. Journal of Agricultural and Food Chemistry, 53(24), Smith-Palmer, A., Stewart, J., & Fyfe, L. (1998). Antimicrobial properties of plant
9452–9458. essential oils and essences against five important food-borne pathogens. Letters
Kubo, I., Muroi, H., & Kubo, A. (1995). Structural functions of antimicrobial long- in Applied Microbiology, 26(2), 118–122.
chain alcohols and phenols. Bioorganic and Medicinal Chemistry, 3(7), 873–880. Ultee, A., Kets, E. P. W., Alberda, M., Hoekstra, F. A., & Smid, E. J. (2000). Adaptation of
Lambert, R. J. W., Skandamis, P. N., Coote, P. J., & Nychas, G. J. E. (2001). A study of the food-borne pathogen Bacillus cereus to carvacrol. Archives of Microbiology,
the minimum inhibitory concentration and mode of action of oregano essential 174(4), 233–238.
oil, thymol and carvacrol. Journal of Applied Microbiology, 91(3), 453–462. Valero, M., & Giner, M. J. (2006). Effects of antimicrobial components of essential
Lopez, P., Huerga, M. A., Batlle, R., & Nerin, C. (2006). Use of solid phase oils on growth of Bacillus cereus INRA L2104 in and the sensory qualities of
microextraction in diffusive sampling of the atmosphere generated by carrot broth. International Journal of Food Microbiology, 106(1), 90–94.
different essential oils. Analytica Chimica Acta, 559(1), 97–104. Valero, M., & Salmeron, M. C. (2003). Antibacterial activity of 11 essential oils
Lopez, P., Sanchez, C., Batlle, R., & Nerin, C. (2005). Solid- and vapor-phase against Bacillus cereus in tyndallized carrot broth. International Journal of Food
antimicrobial activities of six essential oils: Susceptibility of selected foodborne Microbiology, 85(1–2), 73–81.
bacterial and fungal strains. Journal of Agricultural and Food Chemistry, 53(17), Valverde, J. M., Guillen, F., Martinez-Romero, D., Castillo, S., Serrano, M., & Valero, D.
6939–6946. (2005). Improvement of table grapes quality and safety by the combination of
Lopez, P., Sanchez, C., Batlle, R., & Nerin, C. (2007a). Development of flexible modified atmosphere packaging (MAP) and eugenol, menthol, or thymol.
antimicrobial films using essential oils as active agents. Journal of Agricultural Journal of Agricultural and Food Chemistry, 53(19), 7458–7464.
and Food Chemistry, 55, 8814–8824. White, R. L., Burgess, D. S., Manduru, M., & Bosso, J. A. (1996). Comparison of three
Lopez, P., Sanchez, C., Batlle, R., & Nerin, C. (2007b). Vapor-phase activities of different in vitro methods of detecting synergy: Time-kill, checkerboard, and E
cinnamon, thyme, and oregano essential oils and key constituents against test. Antimicrobial Agents and Chemotherapy, 40(8), 1914–1918.