Food Control 22 (2011) 1707e1714

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Food Control
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Antimicrobial potential and chemical composition of Mentha piperita oil in liquid

and vapour phase against food spoiling microorganisms
Amit Kumar Tyagi a, b, *, Anushree Malik a
a
Applied Microbiology laboratory, Centre for Rural Development and Technology, Indian Institute of Technology Delhi, New Delhi 110016, India
b
Dipartimento di Scienze degli Alimenti, Università degli Studi di Bologna, Sede di Cesena, Piazza G. Goidanich, 60, 47023 Cesena, Italy

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

Article history: Antimicrobial potential of Mentha piperita oil in liquid and vapour phase against different bacterial
Received 18 December 2010 strains (Escherichia coli aDH5, Escherichia coli ATCC 25922, Pseudomonas aeruginosa, Pseudomonas fluo-
Received in revised form rescens, Bacillus subtilis and Staphylococcus aureus), fungal strains (Penicillium digitatum, Aspergillus flavus,
27 March 2011
Aspergillus niger, Mucor spp, and Fusarium oxysporum) and yeasts (Candida albicans and Sacchromyces
Accepted 5 April 2011
cerevisiae) was determined by agar dilution method, well diffusion method and disc volatilization
method, respectively. Minimum inhibitory concentration (MIC) and Minimum bactericidal and fungicidal
Keywords:
concentration (MBC/MFC) of M. piperita oil varied from 1.13 to 2.25 mg/ml and 2.25 to 9 mg/ml for
Antimicrobial potential
Chemical composition
bacterial strains, 1.13 to 2.25 mg/ml and 2.25 to 4.5 mg/ml for fungal strains and 1.13 mg/ml and 2.25 mg/
Mentha piperita ml for yeasts, respectively. Bacterial inhibition zone due to M. piperita oil (40 ml/well) varied from 13 mm
GC (P. aeruginosa) to 22 mm (B. subtilis). Bacterial inhibition zone due to M. piperita oil (40 ml) vapour varied
GCeMS from 22 mm (P. fluorescens) to 35 mm (B. subtilis) while almost complete growth inhibition occurred in
SPME GCeMS case of fungal strains. In the kill time assays, 100% reduction in viability of C. albicans and B. subtilis was
Kill time assay found within 8 h exposure to M. piperita oil vapour. Significant morphological alterations due to the
effect of M. piperita oil and oil vapour on B. Subtilis have also been observed by scanning electron
microscope.
Chemical constituents of the M. piperita essential oil and oil vapour have been identified by gas
chromatography (GC), gas chromatography/mass spectrometry (GCeMS) and Solid phase micro
extraction-gas chromatography mass spectrometry (SPME GCeMS), respectively.
Ó 2011 Elsevier Ltd. All rights reserved.

1. Introduction spoilage microorganisms, including Listeria monocytogenes, Clos-

Foodborne diseases mediated by food spoilage microorganisms
are a major challenge in developing as well as developed countries
(Mead et al., 1999). In this regard, certain plant extracts or essential
oils with notable antimicrobial activity can be used to delay or
inhibit the growth of pathogenic and/or toxin producing microor-
ganisms in food (Marino, Bersani, & Comi, 2001). Increasing
concern about potentially harmful synthetic additives coupled with
low mammalian toxicity and less environmental effects of the
natural ingredients has lead to their wide public acceptance
(Paranagama, Abeysekera, Abeywickrama, & Nugaliyadd, 2003;
Reische, Lillard, & Eitenmiller, 1998). Many essential oils have
been demonstrated to have antimicrobial activity against food
* Corresponding author. Current address. Dipartimento di Scienze degli Alimenti,
Università degli Studi di Bologna, Sede di Cesena, Piazza G. Goidanich, 60, 47023
Cesena, Italy. Tel.: þ39 0547 338145; fax: þ39 9547 382348.
E-mail addresses: amit.kumar@unibo.it, amittyagiiitd@gmail.com (A.K. Tyagi).
tridium botulinum, Enterococcus faecalis, Staphylococcus spp.,
Bacillus spp., enterobacteria, Vibrio parahaemolyticus, Pseudomonas
fluorescens, Sacchromyces cerevisiae and Aspergillus spp. (Beuchat,
1976; Cerruti & Alzamora, 1996; Pandit & Shelef, 1994; Paster,
Menasherov, Ravid, & Juven, 1995; Shelef, 1984; Ultee, Kets, &
Smid, 1999).
Mentha (commonly known as mint or pudina) is a well-known
genus (family Lamiaceae) for medicinal and aromatic value. The
genus Mentha includes 25e30 species that are under cultivation
from tropical to temperate climate of America, Europe, China,
Brazil, India, etc. India fulfils 80% of total global demand with
production of 16,000 tons of mint oil (Dorman, Kosar, Kahlos, Holm,
& Hiltunen, 2003; Khanuja, 2007). Mentha spp. from different parts
of the world has been previously investigated for their essential oil
compositions and biochemical activities (Iscan, Kirimer,
Kurkcuoglu, Baser, & Demirci, 2002; Mahboubi & Haghi, 2008).
The antimicrobial efficacy of Mentha essential oils has been found
to vary from moderate to significant often correlating with the
composition of the oil (Iscan et al., 2002; Mahboubi & Haghi, 2008;

0956-7135/$ e see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.foodcont.2011.04.002
1708 A.K. Tyagi, A. Malik / Food Control 22 (2011) 1707e1714
Mimica-Dukic, Bozin, Sokovic, Mihajlovic, & Matavulj, 2003; Schelz,
Molnar, & Hohmann, 2006; Yadegarinia et al., 2006). Besides, the
antimicrobial and biofilm formation preventive properties of
Mentha piperita essential oil against Streptococcus mutans and
Streptococcus pyogenes in vitro and in vivo have also been assessed
(Rasooli, Shayegh, Taghizadeh, & Astaneh, 2008). Due to the rela-
tively low antimicrobial activity of essential oils, the recent reports
have followed a more rational approach of integrating the essential
oils/plant extracts with conventional antibiotics. For example,
Coutinho, Costa, Lima, Falcao-Silva, and Siqueira-Junior (2008)
reported the antibiotic resistance-modifying activity of the etha-
nolic extract of Mentha arvensis. On the other hand, Van Vuuren,
Suliman, and Viljoen (2009) while investigating the antimicrobial
activity of essential oils in combination with conventional antimi-
crobials reported that M. piperita essential oils with amphotericin B
mainly depicted antagonistic profiles against C. albicans. Hence,
alternative means should be explored to enhance the efficacy of
essential oils.
Although the essential oils have high efficiency against the
foodborne pathogen and spoilage microorganisms in liquid phase
but this effect in food is only achieved with higher concentration of
essential oils as compared to the MIC in nutrient media (Burt, 2004;
Hulin, Mathot, Mafart, & Dufosse, 1998). This fact may imply an
organoleptic impact, caused by altering the natural taste of the food
by exceeding the acceptable flavour thresholds (Nazer, Kobilinsky,
Tholozana, & Dubois-Brissonneta, 2005). Hence, for reducing the
sensory effect, one of the alternative approaches may be the use of
essential oil in the vapour phase. Essential oil in vapour phase could
be highly effective against foodborne pathogens and spoilage
bacteria at relatively lower concentrations than the liquid phase,
thereby causing minimum effect on organoleptic properties. Lopez,
Saanchez, Battle, & Nerian, 2005, have assessed the antimicrobial
activity in the vapour phase of a wide number of essential oils
(cinnamon, clove, basil, rosemary, dill and ginger) and their main
constituents with promising results, which concluded with the
development of an antimicrobial packaging (Lopez, Sanchez, Batlle,
& Nerin, 2007; Rodriguez, Batlle, & Nerin, 2007). Fisher and Phillips
(2009) also found the effect of citrus essential oils and its vapours
against Enterococcus faecium and E. faecalis and try to investigate
the mechanism of action by which a blend of citrus essential oil and
essential oil vapours inhibits the growth of Enterococcus sp. But to
the best of our knowledge, there is no report demonstrating anti-
microbial activity of M. piperita oil vapour in vivo or in vitro.
Therefore, in the present study, the antimicrobial effect of
M. piperita oil against bacteria (gram negative, gram positive),
fungus and yeasts in liquid as well as in vapour phase has been
studied by employing various antimicrobial assays. Morphological
alteration in B. subtilis due to treatment with M. piperita oil in
different phase has been studied by scanning electron microscope.
To further validate the efficacy of the essential oil vapours, kill time
assay has been conducted. Chemical constituents of M. piperita
essential oil have been analysed by GC, GCeMS and M. piperita oil
vapours by SPME GCeMS.
2. Materials and methods
2.1. Chemicals and strains
The essential oils were procured from Natural Aromatics Private
Limited, New Delhi (India) and stored in airtight sealed glass bottles
at 4  C till further use. Growth media, DMSO and Tween-80 were
purchased from Himedia and Qualigens (India), respectively, while
ethanol and diethyl ether were purchased from Merck, India.
Different bacterial strains (E. coli aDH5, E. coli ATCC 25922,
P. aeruginosa, P. fluorescens, B. subtilis and S. aureus), fungal strains
(P. digitatum, A. flavus, A. niger, Mucor spp. and F. oxysporum) and
yeasts (C. albicans and S. cerevisiae) were collected from the Central
Microbial Culture Facility, Department of Biotechnology &
Biochemical Engineering, Indian Institute of Technology Delhi, New
Delhi (India) and used to evaluate the effect of essential oils.
2.2. Inoculum preparation
The bacterial and fungal strains used in this study were grown in
Mueller Hinton broth (MHB) and Potato Dextrose broth (PDB)
medium, respectively at 30  C for 24 h in an orbital shaking incu-
bator (Scigenics India Pvt. Ltd., India) at 180 rpm. Cells were har-
vested by centrifugation, suspended in sterile distilled water and
used immediately for the antimicrobial assays.
2.3. Antimicrobial assays
2.3.1. Determination of MIC by agar dilution method
Minimum Inhibitory Concentration (MIC) of essential oil was
determined by agar dilution assay (Griffin, Markham, & Leach,
2000). The agar plates prepared using yeast Potato Dextrose agar
(PDA) for fungi and Mueller Hinton agar (MHA) for bacteria were
amended with various concentrations of M. piperita essential oil
(i.e. 0.14e18.0 mg/ml). For enhancing the oil solubility, Tween-80,
0.5% (v/v) was added. A particular concentration of the M. piperita
oil was incorporated in the large amount (45 ml) of melted culture
media (PDA/MHA þ Tween-80) and homogenized for making an
emulsion prior to distribution in petri dish (15 ml each). These
plates were inoculated with 100 ml cell suspension (106 cfu/ml) of
test strains. The plates (triplicate for each run) were incubated at
30  C. Plates with Tween-80 but without essential oil were used as
control. Observation of the plates for microbial growth was done at
a time interval of 12 h up to 24 h (bacterial strains) and 48 h (fungal
strains). The MIC values were determined as the lowest concen-
tration of oil preventing visible growth of microorganisms.
2.3.2. Determination of MBC and MFC using broth dilution method
MBC and MFC of essential oils were determined by broth macro
dilution assay (Devkatte, Zore, & Karuppayil, 2005). A range of
essential oil concentrations (0.14e18 mg/ml) were prepared in
MHB and PDB medium for bacteria and fungi, respectively. To
enhance oil solubility, Tween-80 was included at a final concen-
tration of 0.5% (v/v). Each flask containing 100 ml media was
inoculated with 100 ml of cell suspension (106 cfu/ml) of the test
strain. Flasks containing only Tween-80 (but without essential oil)
were used as control. All flasks were incubated at 30  C, in an
orbital shaking incubator at 180 rpm for 48 h. One ml of culture was
taken and suitably diluted before streaking on MHA/PDA plates to
judge the viability. The plates were incubated at 30  C for 24 h and
48 h, respectively and were observed for MBC/MFCs.
2.3.3. Well diffusion method
Well diffusion method was employed for the determination of
antimicrobial activities of the essential oil (Baratta et al., 1998;
NCCLS, 1999). M. piperita were dissolved in 0.5% dimethylsulph-
oxide (DMSO) and filter-sterilised using a 0.45 mm membrane filter.
Each test strain was suspended in sterile double distilled water and
serially diluted to 106 cfu/ml concentration. A 100 ml portion of each
suspension was spread over the surface of MHA/PDA plate and
allowed to dry. The wells (8 mm in diameter) were cut from the
agar and different doses (10, 20, 30 and 40 ml) of essential oil
solutions mixed with requisite amount of diethyl ether (to make
the volume 50 ml) were delivered into them. After incubation for
24 h at 30  C, all plates were examined for any zones of growth
 A.K. Tyagi, A. Malik / Food Control 22 (2011) 1707e1714
inhibition, and the diameters of these zones were measured in
millimetres. All tests were performed in triplicate.
2.3.4. Disc volatilization method
Standard experimental set-up as described by Lopez et al.
(2005) was used. Briefly, a 100 ml portion of each suspension con-
taining approximately 106 cfu/ml was spread over the surface of
MHA/PDA plate and allowed to dry. A paper disc (diameter 6 mm,
Sigma Aldrich, India) was laid on the inside surface of the upper lid
and 10 ml essential oil was placed on each disc. The plate inoculated
with microorganisms were immediately inverted on top of the lid
and sealed with parafilm to prevent leakage of essential oil vapour.
Plates were incubated at 30  C for 24 h and the diameter of the
resulting inhibition zone in the bacterial/fungal lawn was
measured. Volume of essential oils tested was varied (20, 40 or
60 ml) by using appropriate number of sterile discs.
2.4. Determination of the kill time
These experiments were conducted for selected microorgan-
isms (E. coli, P. fluorescens, P. aeruginosa, B. subtilis and C. albicans) in
a compact chamber made up of acrylic material (size
50 cm  50 cm; W  L). The height of the chamber was 50 cm on
the back side and 25 cm at the front side. The total volume of the
chamber was 0.09375 m3 (93.75 l). The front side of the chamber
had gloves through which the objects inside the chamber could be
handled without opening the chamber. Prior to exposure the
chamber was cleaned with ethanol and UV sterilised. Two essential
oil evaporating machine (Khera instruments Pvt. Ltd, New Delhi,
India; evaporation rate ¼ 0.50 ml/h) were fixed in this chamber.
Appropriate serial dilution of the culture (to obtain 100e300 cfu)
was plated on PDA/MHA plates. Inoculated plates were opened
inside the chamber and fixed with double sided tape at the top
surface of chamber for exposure to essential oil vapours. After
a particular time period (0.5, 1, 2, 4, 8 and 12 h) the plates were
detached, closed and incubated at 30  C for 18e20 h. All the plates
were used in triplicate.
2.5. Preparation of samples for scanning electron microscopy
B. subtilis cells were incubated for 14 h in MHB at 30  C and
180 rpm. The suspension was divided into two portions. In one
portion, M. piperita oil at MIC level (1.13 mg/ml) was added and
another portion was left untreated as a control. These suspensions
were incubated at 30  C for 4 h.
For investigating the effect of M. piperita oil vapour, 1 ml of
B. subtilis cell suspension (1  106 cells/ml) was inoculated on an
MHA plate and incubated at 30  C for 12 h. These Pre-grown cells
were treated with M. piperita oil vapours (98.28 mg/ml) for 4 h. The
treated cells were then collected gently with the help of brush from
the petri plate and collected in separate test tube. All the treated
cells were harvested by centrifugation and were prefixed with
a 2.5% glutaraldehyde solution overnight at 4  C. After this, the cells
were again harvested by centrifugation and washed three times
with 0.1 M sodium phosphate buffer solution (pH 7.2). Now each
resuspension was serially dehydrated with 25, 50, 75, 90, and 100%
ethanol, respectively. Then, cells were dried at “critical point”. A
thin film of cells was smeared on a silver stub for SEM observation.
The samples were gold-covered by cathodic spraying (Polaron
gold). Finally, morphology of the B. subtilis cells was observed on
a scanning electronic microscope (ZEISS EVO 50). The SEM obser-
vation was done under the following analytical condition:
EHT ¼ 20.00 kV, WD ¼ 9.5 mm, Signal A ¼ SE1.
1709
2.6. Chemical composition of M. piperita oil and M. piperita oil
vapour
2.6.1. Gas chromatographic (GC) and gas chromatographic mass
spectrometry (GCeMS) analysis
The percentage composition of essential oil was determined by
GC-FID and the compounds were identified by GCeMS. GC analysis
was carried out on a Shimadzu 2010 Gas Chromatograph equipped
with an FID and 25 m  0.25 mm  0.25 mm WCOT column coated
with diethylene glycol (AB-Innowax, 7031428, Japan). Both injector
and detector (FID) temperatures were maintained at 260  C.
Helium was used as carrier gas at a flow rate of 3.0 ml/min at
a column pressure of 152 kPa. Samples (0.2 ml) were injected into
the column with a split ratio of 100:1. Component separation was
achieved following a linear temperature program of 60e260  C at
3  C/min and then held at 260  C for 10 min, with a total run time of
76 min. The percentage composition was calculated using peak
normalization method assuming equal detector response. The
samples were then analysed on same Shimadzu instrument fitted
with the same column and following the same temperature
program as above and the MS parameters used were; Ionisation
Voltage (EI) 70 eV, peak width 2 s, mass range 40e700 m/z and
detector voltage 1.5 V.
2.6.2. SPME GCeMS analysis of M. piperita oil vapours
SPME optimisation analyses were carried out using a Shimadzu
2010 GC instrument equipped with a data processor and Shimadzu
GCeMS QP2010 Plus, Japan. An AB-Innowax 7031428 capillary
column (60.0 m  0.25 mm i.d., 0.25 mm film thicknesses) was used
for the separation of the sample components. M. piperita oil vola-
tiles from petri plates were extracted using Divinyl benzene/Car-
boxen/Poly dimethyl siloxane (DVB/CAR/PDMS) fibre. The fibre was
conditioned in a GC injector port as indicated by manufacturer
(Supelco, France).
The injector was maintained at 260  C and operated in split less
injection mode with the split valve closed for 1 min. Helium gas
was used as the carrier gas at a constant pressure of 136.3 kPa. The
column oven was initially maintained at 40  C for 2 min, raised to
180  C at 8  C/min, then to 230  C at 4  C/min. The interface
temperature was 260  C and the ionisation mode was electron
impact (70 eV). The mass selective detector was operated in the
scan mode between 20 and 700 m/z. Data acquisition started
4.0 min after injection.
2.6.3. Identification of chemical compounds
Peak identification was carried out by comparison of the mass
spectra with mass spectra available on database of NIST05, WILEY8
libraries and those of pure standards.
2.7. Statistical analyses
All the experiments were done in triplicate and the data pre-
sented here represents the mean of these replicates. Data related to
the zone of inhibition due to the M. piperita oil were subjected to
analysis of variance (one way ANOVA) in Duncan multiple range
test using SPSS (version 10) statistical software. The differences
with p < 0.05 were considered significant.
3. Results
3.1. Antimicrobial activity
3.1.1. MIC and MBC/MFC of M. piperita oil
MIC of the M. piperita essential oil was determined against
various gram negative (E. coli aDH5, E. coli ATCC 25922, P. aeruginosa,
1710 A.K. Tyagi, A. Malik / Food Control 22 (2011) 1707e1714

P. fluorescens), gram positive (B. subtilis and S. aureus) bacterial F. oxysporum and C. albicans were completely inhibited in presence
strains, fungal strains (P. digitatum, A. flavus, A. niger, Mucor spp. and of the vapours generated by 40 ml M. piperita oil (Fig. 2b).
F. oxysporum) and yeasts (C. albicans and S. cerevisiae). These MIC
values are shown in Table 1. The oil exhibited concentration- 3.2. Kill time assay
dependent inhibition of growth.
For the bacterial strains MIC varied from 1.13 mg/ml to 2.25 mg/ Based on the MIC/MBC/MFC and Zone of inhibition data, kill
ml (Table 1). MIC of gram positive bacteria (B. subtilis and S. aureus) time assay was conducted for E. coli, P. fluorescens, P. aeruginosa,
was less than gram negative bacteria (E. coli aDH5, E. coli ATCC B. subtilis and C. albicans. The selection included both gram positive/
25922, P. aeruginosa, P. fluorescens). Highest MIC (2.25 mg/ml) was negative bacteria as well as yeast with differential sensitivity
shown by Pseudomonads. MBC of bacterial strains varied from towards oils/vapours. These studies were conducted to investigate
2.25 mg/ml to 9 mg/ml and showed the similar pattern i.e. the appropriate pre-exposure time for achieving 100% reduction in
P. aeruginosa, P. fluorescens (9 mg/ml) > E. coli aDH5, E. coli ATCC viability (no viable cells detected) of the selected representative
25922 (4.5 mg/ml) > B. subtilis and S. aureus (2.25 mg/ml). strains through vapour phase treatment. The inoculated plates
MIC for fungal strains varied from 1.13 mg/ml to 2.25 mg/ml were exposed to M. piperita oil vapour in the closed airtight
(Table 1). MIC for P. digitatum was higher (i.e. 2.25 mg/ml) than chamber for different time periods till 12 h prior to incubation.
other fungus (i.e. 1.13 mg/ml). MFC of fungal strain varied from Reduction in viability (%) was calculated based on the reduction in
2.25 mg/ml to 4.5 mg/ml and showed the similar pattern i.e. colony count in M. piperita vapour exposed plates with respected to
P. digitatum, (4.5 mg/ml) > A. niger, A. flavus, F. oxysporum and Mucor the control (unexposed) plates. Results are shown in Fig. 3.
spp (2.25 mg/ml). MIC and MFC of yeasts (C. albicans and Within 8 h of M. piperita oil vapour exposure, 100% reduction in
S. cerevisiae) was 1.13 mg/ml and 2.25 mg/ml, respectively. viability was observed in B. subtilis and C. albicans while only 76.9,
74.2 and 85% reduction in viability was observed in P. aeruginosa,
3.1.2. Well diffusion method P. fluorescens and E. coli, respectively after 12 h exposure (Fig. 3). In the
Antimicrobial potential of the M. piperita essential oil was also initial 4 h the percentage reduction rate in viable count was higher
observed in terms of zone of inhibition generated by the diffusion of than the percentage reduction rate after 4 h. In initial 4 h more than
the essential oil components into the microorganisms inoculated agar 50% reduction was observed even in the most resistant bacterial
plate. The zone of inhibition increased with the increasing concen- strain (Pseudomonads) while 92, 88 and 66.8% reduction in viability
tration (i.e. 10, 20, 30 and 40 ml) of M. piperita oil in each well (Fig. 1). was observed in B. subtilis, C. albicans and E. coli, respectively.
The trend observed here with respect to the antibacterial activity was
similar to that noticed in MIC determinations i.e. the zone of inhibi- 3.3. Scanning electron microscope (SEM) observation
tion in case of gram negative bacteria was less than that for the gram
positive bacteria. The inhibition zone due to 40 ml, Mentha oil was Bacterial cells treated with M. piperita oil at MIC level displayed
P. aeruginosa (13 mm) < P. fluorescens (14 mm) < E. coli aDH5 considerable morphological alterations in comparison to the
(15 mm) < E. coli ATCC 25922 (16 mm) < B. subtilis (22 mm). However, control when observed by a scanning electron microscope (Fig. 4).
no clear zone of inhibition could be observed at all the tested Control B. subtilis cells appeared intact, rod shaped, separated from
concentrations in case of fungal and yeast strains. each other, turgid and whole with smooth surface (Fig. 4a) while
the M. piperita oil (1.13 mg/ml) treated cells appeared to be partially
3.1.3. Disc volatilization method deformed with frequent depressions on the cell surface (Fig. 4b).
The zone of inhibition resulting from the exposure to M. piperita The cells were completely and uniformly destroyed when exposed
essential oil vapours is shown in Fig. 2. As observed in the earlier to M. piperita oil vapour (Fig. 4c). All the cells were shrunken
assays using M. piperita essential oil in liquid phase, the zone of completely and appeared like cellular debris (Fig. 4c). Hence change
inhibition due to M. piperita oil vapours also increased with in morphology and destruction of the B. subtilis cells appeared more
increasing concentration of the oil and followed the same trend in vapour phase exposure than that in the broth phase treatment.
with respect to the different bacterial strains (Fig. 2). However, as
compared to the oil, the vapours resulted in significantly (1.5e1.7 3.4. Chemical composition of M. piperita oil and M. piperita oil
times) larger zone of inhibition in all the bacterial strains tested. vapour
Hence, the antimicrobial activity of 20 ml M. piperita oil vapour was
higher than 40 ml liquid oil. Remarkable efficacy of vapours was Qualitative and quantitative analysis of the M. piperita oil is lis-
seen in case of fungal strains (Fig. 2b). A. niger, Mucor spp., ted in Table 2. In M. piperita oil, 47 components were identified,
which represented 27% monoterpene hydrocarbons, 63.3%
Table 1 oxygenated monoterpenes, 1.8% susquiterpine hydrocarbons and
Antimicrobial activity of M. piperita essential oil.
1.4% oxygenated sesquiterpenes, about 97.85% of the total detected
Microorganism Microbial strains MIC (mg/ml) MBC/MFC (mg/ml) constituents. A portion (2.15%) of total composition was not iden-
Bacteria Escherichia coli aDH5 1.13 4.5 tified. The major constituents of the oil were menthol (19.1%), iso-
Escherichia coli ATCC 25922 1.13 4.5 menthone (14.8%), limonene (10.6%), iso-menthanol (8.8%),
Pseudomonas aeruginosa 2.25 9 menthyl acetate (6.6%), b-pinene (5.6%), a-pinene (4.8%), 1,8-cineole
Pseudomonas fluorescens 2.25 9
Bacillus subtilis 1.13 2.25
(3.5%), isopulegol (3%), pulegone (2.3%), piperitone (2.1%), and
Staphylococcus aureus 1.13 2.25 b-phellandrene (2.8%). Other components were present in amounts
Fungi Penicillium digitatum 2.25 4.5 less than 2%.
Aspergillus flavus 1.13 2.25 In the M. piperita oil vapour, 18 compounds constituting 62.8%
Aspergillus niger 1.13 2.25
monoterpene hydrocarbon and 29.3% oxygenated monoterpenes
Mucor spp. 1.13 2.25
Fusarium oxysporum 1.13 2.25 (about 92.1% of the vapour), were identified. These compounds
Yeasts Candida albicans 1.13 2.25 were a-pinene (17.3%), limonene (18.4%), b-pinene (13.9%), iso-
Sacchromyces cerevisiae 1.13 2.25 menthone (9%), menthyl acetate (6.6%), b-phellandrene (5.8%),
MIC: Minimum inhibitory concentration; MBC: Minimum bactericidal concentra- menthol (4.8%), myrcene (3.8%), iso-menthanol (1.8%), isopulegol
tion; MFC: Minimum fungicidal concentration. (1.8%), and piperitone (2.1%).
 A.K. Tyagi, A. Malik / Food Control 22 (2011) 1707e1714 1711

50
10 μl
45
20μl
30 μl
40
40 μl

Zone of inhibition (mm)

35

30

25

20

15

10

0
E. Coli αDH5 E. Coli ATCC P. aeruginosa P. flourescens B. subtilis
Bacterial strains

Fig. 1. Zone of inhibition (mm) due to different concentration (10, 20, 30 and 40 ml/well) of M. piperita oil against bacterial strains (E. coli aDH5, E. coli ATCC 25922, P. aeruginosa, P.
fluorescens and B. subtilis). (Standard error of mean indicated, n ¼ 3).

100
a
90

80
Zone of inhibition (mm)

70

60

50

40

30

20
20 μl
10 40 μl
60 μl
0
E. Coli αDH5 E. Coli ATCC P. aeruginosa P. flourescens B. subtilis

Bacterial strains

b 100
90
Zone of inhibition (mm)

80
70
60
50
40
30
20
20 μl
10 40 μl
60 μl
0
A. flavus A. niger Mucor Fusarium C. albicans
Fungal strains

Fig. 2. Antimicrobial potential of Mentha oil vapour; Disc volatilization method (a) Zone of inhibition (mm) due to different concentration (20, 40 and 60 ml) of Mentha oil vapour
against bacterial strains (E. coli aDH5, E. coli ATCC 25922, P. aeruginosa, P. fluorescens and B. subtilis) (b) Zone of inhibition (mm) due to different concentration (20, 40 and 60 ml) of
Mentha oil vapour against different fungal strains (A. flavus, A. niger, Mucor spp., Rhizopus, Fusarium and C. albicans). (Standard error of mean indicated, n ¼ 3).
1712 A.K. Tyagi, A. Malik / Food Control 22 (2011) 1707e1714

120
110
100
Reduction in viability (%)

90
80
70
60
50
40
E. coli
30 P. fluorescens
P. aeruginosa
20 B. subtilis
10 C. albicans

0
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14
Time (h)

Fig. 3. Kill Time Assay; percentage reduction in viability of E. coli, P. aeruginosa, P.

fluorescens, B. subtilis and C. albicans due to pre-incubation exposure to M. piperita oil
vapour for different time durations. (Standard error of mean indicated, n ¼ 3).

4. Discussion

The in vitro antimicrobial activities of M. piperita oil against the

studied microorganisms were assessed in liquid as well as in
vapour phase by the inhibition zone diameters, and MIC, MBC/MFC
values.
As can be seen in Table 1, M. piperita oil was found to have moderate
antimicrobial activities against all microorganisms tested. MIC/MBC of
M. piperita oil for bacterial strains (Pseudomonads > E. coli > B. subtilis/
S. aureus) was higher than the MIC/MFC of fungi (P. digitatum > A. niger/
Mucor spp./F. oxysporum) and yeasts (C. albicans/S. cerevisiae). MBC/
MFC is defined as the lowest concentration of oil resulting in the death
of 99.9% of the inoculum (Burt, 2004). In general, it has been observed
that the MBC/MFC was higher than MIC (Table 1). According to MICs
and MBCs, gram negative bacteria were more resistant than gram
positive bacteria. This may be due to the different nature of gram
negative cell envelope that makes access to membrane more restricted
in gram negative bacteria. The effect of components of essential oil on
cell membrane integrity of gram positive and gram negative bacteria
has been previously reported (Cox et al., 2000; Oussalah, Caillet, &
Lacroix, 2006). Mahboubi and Haghi (2008) screened the antimicro-
bial activity of essential oil from flowering aerial parts of Iranian
M. pulegium L. against different microorganisms. They reported
significant activity against gram positive bacteria with MIC values in
the range of 0.25e4 ml/ml whereas the least susceptible were gram
negative bacteria, especially E. coli. The essential oil of M. piperita was Fig. 4. Scanning electron micrographs of untreated and treated (24 h) B. subtilis cells:
(a) Untreated cells with normal smooth surfaces (25.00 K), (b) Shrinked and
earlier found to display good to excellent antimicrobial activities (MIC
deshaped M. piperita oil treated cells (25.00 K), (c) Non-uniform/deformed and
values 1e8 ml/ml) against E. coli, S. aureus and C. albicans (Yadegarinia ruptured M. piperita oil vapour treated cells (25.00 K).
et al., 2006). C. albicans was the more sensitive micro organism as
compared to E. coli. Mimica-Dukic et al. (2003) reported low MIC (4 ml/ the zone of inhibition resulting from the exposure to Mentha oil
ml) of M. piperita oil for E. coli and 8 ml/ml for C. albicans. Hammer, vapours in disc volatilization method was significantly larger than
Carson, and Riley (1999) reported cidal activity of M. piperita oil at that due to same concentration of essential oil in liquid phase
0.25% (v/v) equivalent to 25 ml/ml for E. coli and C. albicans and 12 ml/ml measured via the well diffusion method. This indicates that the
for S. aureus. Similar results were observed by many researchers earlier substances in the well diffusion method were less efficient than
(Bakkali, Averbeck, Averbeck, & Idaomar, 2008; Takahashi, Kokubo, & that in the disc volatilization method. Hence, smaller doses of
Sakaino, 2004). Gilles, Zhao, An, & Agboola, 2010 also reported that essential oil in vapour phase can be inhibitory to the spoilage
the gram negative spoilage bacterium P. aeruginosa was most resistant bacteria. Pibiri (2006) has also demonstrated that the essential oils
to the M. piperita oil tested. of Satureja Montana, Thymus, Origanum vulgaris and cinnamon bark
In the present study, inhibition zone due to M. piperita oil vapour in gaseous phase have a lethal effect on S. aureus and P. aeruginosa,
was more in case of fungi than the bacterial strain. In case of even in small doses. This demonstrates that the antimicrobial
bacteria, zone of inhibition due to 60 ml M. piperita oil vapour varied activity of the different essential oil vapours can be achieved at
from 28 (Pseudomonads) to 46 mm (gram positive bacteria) while lesser amount than essential oil in liquid phase.
presence of 40 ml M. piperita oil vapour almost inhibited the To understand the higher efficacy of vapours as compared to
complete growth of the fungi (Fig. 2). Also, in all the tested strains, essential oil, detailed chemical characterization of this oil and oil
 A.K. Tyagi, A. Malik / Food Control 22 (2011) 1707e1714 1713

Table 2 (14.5%), pipertinone oxide (19.3%), and b-caryophyllene (7.6%) as

Chemical composition of M. piperita oil and oil vapour. the major compounds. Hence it can be deduced that chemical
S. N. Name of Percentage in Percentage in compounds other than menthol may have contributed to the
Compound liquid sample vapour sample antimicrobial property of M. piperita essential oil in this study.
1 a-pinene 4.8 17.3 While comparing the composition of M. piperita essential oil and
2 Camphene 0.3 1.1 its vapours, it is observed that both are rich in oxygenated mono-
3 b-pinene 5.7 13.9
terpenes and monoterpene hydrocarbon. However, in M. piperita
4 b-phellandrene 2.8 5.8
5 Sabinene 0.1 e oil, the content of oxygenated monoterpenes and monoterpenes
6 d-3-carene 0.4 1.3 hydrocarbon is 63.3 and 27.0% (Table 2), respectively while in
7 Myrcene 1.3 3.8 M. piperita oil vapour these are 29.3% and 62.8%, respectively (i.e.
8 Limonene 10.6 18.4 approximately reverse as in M. piperita oil). Similar results have
9 1,8-cineole 3.6 e
10 3-octanone 0.2 0.6
been found by Rohloff (1999). He analysed the chemical composi-
11 o-cymene 0.6 1.2 tion of M. piperita oil sample through SPME and solvent-based
12 a-terpinolene 0.3 0.6 sample. In SPME observation the percentage of monoterpene
13 3-octanol 3.5 2.0 hydrocarbon is around three times that of the solvent based sample
14 Perillene 0.1 e
analysis. The author explained that in SPME based analysis, high-
15 n-hexanyl iso-valerate 0.1 0.6
16 Limonene oxide 0.1 e volatile compounds are favoured and this results in higher fibre
17 iso-menthone 14.8 9.0 extraction and detection of monoterpenes such as a- and b-pinene,
18 a-bourbonene 0.5 e limonene, and 1,8-cineole. Probably due to this reason, reduction in
19 Linalool 0.1 e amount of menthol and enrichment of a-pinene, b-pinene and
20 Menthyl acetate 6.6 6.6
21 Isopulegol 3.0 1.8
limonene has been recorded in the SPME analysis of M. piperita oil
22 Germacrene-D 0.3 e vapours in the present study. Earlier studies report that the pres-
23 Iso-menthanol 6.4 1.8 ence of active monoterpene constituents, such as b-pinene has the
24 Caryophyllene 0.8 e membrane damaging effects on microbes (Sikkema, Debont, &
25 Neo-iso-menthol 1.5 e
Poolman, 1995). As they diffuse into and damage cell membrane
26 Menthol 19.1 4.8
27 Pulegone 2.3 e structures (Meincken, Holroyd, & Rautenbach, 2005). The antimi-
28 Pinocarveol 0.2 e crobial action of such molecules depends on their presence in
29 Thujol 0.4 e gaseous form facilitating their solubilization in cell membranes
30 Geranyl formate 0.6 e (Ebrahimabadi et al., 2010). Therefore, higher cell damage is
31 Neryl acetate 0.4 e
32 Copaene 0.1 e
expected to occur in M. piperita oil vapour treated cells as the
33 a-terpineol 0.8 e vapour contains 62.8% monoterpenes hydrocarbon (Table 2). Gaunt,
34 Sabinyl acetate 0.2 e Higgins, and Hughes (2005) also reported significant antibacterial
35 Piperitone 2.1 2.1 effects of b-pinene vapours (generated by burning of b-pinene
36 Carvone 0.3 e
containing candles) against E. coli and S. aureus. Hence, our results
37 Cubenol 0.2 e
38 Muurolene 0.1 e suggest that menthol does not seem to be the sole agent respon-
39 Myrtenol 0.2 e sible for antimicrobial property of M. piperita essential oil vapours,
40 Carveol 0.2 e rather the monoterpene hydrocarbon may play important role.
41 p-cymen-8-ol 0.1 e To confirm this effect and to clarify the mechanism of action of
42 Neryl acetate 0.1 e
43 Carveol 0.1 e
M. piperita oil in liquid and vapour phase, SEM was employed.
44 Verbenone 0.1 e Majority of the B. subtilis cells treated with M. piperita oil at MIC
45 Caryophyllene oxide 0.2 e level in broth show partial deformation and frequent depressions in
46 Epi-globulol 0.8 e the SEM. A more extensive damage is observed in B. subtilis cells
47 Spathulenol 0.2 e
treated with M. piperita oil vapour which appears ruptured and
Monoterpene hydrocabons 27.0 62.8
Oxygenated monoterpene 63.3 29.3 uniformly degraded (Fig. 4). These results are supported by the
Susquiterpine hydrocarbons 1.8 e observation that terpenes alter cell permeability causing changes in
Oxygenated sesquiterpenes 1.4 e membrane properties and functions by increasing membrane
Total 97.85 92.1 fluidity and altering membrane permeability. Also, the morpho-
Retention Indices on AB-Innowax column, Relative area percentage without using logical observations made here are quite different from those
the FID response correction factor, (Results are based on GC-FID; MS acquisition reported by Paulo, Ferreira, Gallardo, Queiroz, and Domingues
started after 4 min).
(2010) where the treatment of B. cereus with resveratrol (200 mg/
Bold values represent major compounds of Mentha essential oil which play an
important role in antimicrobial activity and significantly different in Liquid as well ml) leads to the change in the cells, from the typical long rod shape
as in vapour phase. to short rods or even to a coccus-like shape. This could be explained
on the basis of different mechanism of action of the two antimi-
vapour was done by GCeMS and SPME GCeMS, respectively. The crobial compounds as resveratrol tends to stop the cell division and
results confirmed that the antimicrobial activity of M. piperita affect the bacterial cell cycle while M. piperita oil and vapours
essential oil recorded here could be correlated to the presence of induce bactericidal effect through membrane damage.
menthol (19.1%) and menthone (14.8%). These constituents have
been reported to possess significant antimicrobial activity. Iscan 5. Conclusions
et al. (2002) found that the antimicrobial efficacy of four samples
of peppermint oils from various locations in the world could be The results of this work have shown that M. piperita oil vapour is
mainly correlated with the relative percentage of menthol more potent inhibitor of food spoiling microbial growth, leading to
(28e42%) and menthone (18e28%). However, another study deleterious morphological changes in cellular structures and cell
(Yadegarinia et al., 2006) revealed higher antimicrobial property of surface alterations as compared to M. piperita oil. The use of
M. piperita essential oil with menthol concentration as low as 3.6% M. piperita oil in vapour phase could have additional advantages
but with a-terpinene (19.7%), iso-menthone (10.3%), trans-carveol such as efficacy without requiring direct contact resulting in ease of
1714 A.K. Tyagi, A. Malik / Food Control 22 (2011) 1707e1714
application and no alteration in organoleptic properties of the edible
material/food. With respect to above-mentioned data, our findings
demonstrate that M. piperita oil vapour may be considered as
a potential agent for preventing microbial mediated food spoilage. A
further study in vivo condition is warranted to confirm the antimi-
crobial activity of M. piperita, which may be used for preservation
and/or extension the shelf life of raw and processed food.
Acknowledgement
The present work was financially supported by CSIR SRF to
AKT and compilation of this work was done during India4EU
(EMECW13c) research exchange fellowship to AKT. Mr. D.C. Sharma
(IIT Delhi, India), Mr. Ajai Kumar (AIRF JNU, India) and Sabal Singh
(IIT Delhi, India) are also acknowledged for their technical support
in SEM, GC/GCeMS and experimental work, respectively.
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