Professional Documents
Culture Documents
Tyagi a.K. Và Cs (2011) Kháng Khuẩn
Tyagi a.K. Và Cs (2011) Kháng Khuẩn
Food Control
journal homepage: www.elsevier.com/locate/foodcont
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.
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, (P. digitatum, A. flavus, A. niger, Mucor spp. and F. oxysporum) and
Molnar, & Hohmann, 2006; Yadegarinia et al., 2006). Besides, the yeasts (C. albicans and S. cerevisiae) were collected from the Central
antimicrobial and biofilm formation preventive properties of Microbial Culture Facility, Department of Biotechnology &
Mentha piperita essential oil against Streptococcus mutans and Biochemical Engineering, Indian Institute of Technology Delhi, New
Streptococcus pyogenes in vitro and in vivo have also been assessed Delhi (India) and used to evaluate the effect of essential oils.
(Rasooli, Shayegh, Taghizadeh, & Astaneh, 2008). Due to the rela-
tively low antimicrobial activity of essential oils, the recent reports
2.2. Inoculum preparation
have followed a more rational approach of integrating the essential
oils/plant extracts with conventional antibiotics. For example,
The bacterial and fungal strains used in this study were grown in
Coutinho, Costa, Lima, Falcao-Silva, and Siqueira-Junior (2008)
Mueller Hinton broth (MHB) and Potato Dextrose broth (PDB)
reported the antibiotic resistance-modifying activity of the etha-
medium, respectively at 30 C for 24 h in an orbital shaking incu-
nolic extract of Mentha arvensis. On the other hand, Van Vuuren,
bator (Scigenics India Pvt. Ltd., India) at 180 rpm. Cells were har-
Suliman, and Viljoen (2009) while investigating the antimicrobial
vested by centrifugation, suspended in sterile distilled water and
activity of essential oils in combination with conventional antimi-
used immediately for the antimicrobial assays.
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 2.3. Antimicrobial assays
essential oils.
Although the essential oils have high efficiency against the 2.3.1. Determination of MIC by agar dilution method
foodborne pathogen and spoilage microorganisms in liquid phase Minimum Inhibitory Concentration (MIC) of essential oil was
but this effect in food is only achieved with higher concentration of determined by agar dilution assay (Griffin, Markham, & Leach,
essential oils as compared to the MIC in nutrient media (Burt, 2004; 2000). The agar plates prepared using yeast Potato Dextrose agar
Hulin, Mathot, Mafart, & Dufosse, 1998). This fact may imply an (PDA) for fungi and Mueller Hinton agar (MHA) for bacteria were
organoleptic impact, caused by altering the natural taste of the food amended with various concentrations of M. piperita essential oil
by exceeding the acceptable flavour thresholds (Nazer, Kobilinsky, (i.e. 0.14e18.0 mg/ml). For enhancing the oil solubility, Tween-80,
Tholozana, & Dubois-Brissonneta, 2005). Hence, for reducing the 0.5% (v/v) was added. A particular concentration of the M. piperita
sensory effect, one of the alternative approaches may be the use of oil was incorporated in the large amount (45 ml) of melted culture
essential oil in the vapour phase. Essential oil in vapour phase could media (PDA/MHA þ Tween-80) and homogenized for making an
be highly effective against foodborne pathogens and spoilage emulsion prior to distribution in petri dish (15 ml each). These
bacteria at relatively lower concentrations than the liquid phase, plates were inoculated with 100 ml cell suspension (106 cfu/ml) of
thereby causing minimum effect on organoleptic properties. Lopez, test strains. The plates (triplicate for each run) were incubated at
Saanchez, Battle, & Nerian, 2005, have assessed the antimicrobial 30 C. Plates with Tween-80 but without essential oil were used as
activity in the vapour phase of a wide number of essential oils control. Observation of the plates for microbial growth was done at
(cinnamon, clove, basil, rosemary, dill and ginger) and their main a time interval of 12 h up to 24 h (bacterial strains) and 48 h (fungal
constituents with promising results, which concluded with the strains). The MIC values were determined as the lowest concen-
development of an antimicrobial packaging (Lopez, Sanchez, Batlle, tration of oil preventing visible growth of microorganisms.
& Nerin, 2007; Rodriguez, Batlle, & Nerin, 2007). Fisher and Phillips
(2009) also found the effect of citrus essential oils and its vapours 2.3.2. Determination of MBC and MFC using broth dilution method
against Enterococcus faecium and E. faecalis and try to investigate MBC and MFC of essential oils were determined by broth macro
the mechanism of action by which a blend of citrus essential oil and dilution assay (Devkatte, Zore, & Karuppayil, 2005). A range of
essential oil vapours inhibits the growth of Enterococcus sp. But to essential oil concentrations (0.14e18 mg/ml) were prepared in
the best of our knowledge, there is no report demonstrating anti- MHB and PDB medium for bacteria and fungi, respectively. To
microbial activity of M. piperita oil vapour in vivo or in vitro. enhance oil solubility, Tween-80 was included at a final concen-
Therefore, in the present study, the antimicrobial effect of tration of 0.5% (v/v). Each flask containing 100 ml media was
M. piperita oil against bacteria (gram negative, gram positive), inoculated with 100 ml of cell suspension (106 cfu/ml) of the test
fungus and yeasts in liquid as well as in vapour phase has been strain. Flasks containing only Tween-80 (but without essential oil)
studied by employing various antimicrobial assays. Morphological were used as control. All flasks were incubated at 30 C, in an
alteration in B. subtilis due to treatment with M. piperita oil in orbital shaking incubator at 180 rpm for 48 h. One ml of culture was
different phase has been studied by scanning electron microscope. taken and suitably diluted before streaking on MHA/PDA plates to
To further validate the efficacy of the essential oil vapours, kill time judge the viability. The plates were incubated at 30 C for 24 h and
assay has been conducted. Chemical constituents of M. piperita 48 h, respectively and were observed for MBC/MFCs.
essential oil have been analysed by GC, GCeMS and M. piperita oil
vapours by SPME GCeMS. 2.3.3. Well diffusion method
Well diffusion method was employed for the determination of
2. Materials and methods antimicrobial activities of the essential oil (Baratta et al., 1998;
NCCLS, 1999). M. piperita were dissolved in 0.5% dimethylsulph-
2.1. Chemicals and strains oxide (DMSO) and filter-sterilised using a 0.45 mm membrane filter.
Each test strain was suspended in sterile double distilled water and
The essential oils were procured from Natural Aromatics Private serially diluted to 106 cfu/ml concentration. A 100 ml portion of each
Limited, New Delhi (India) and stored in airtight sealed glass bottles suspension was spread over the surface of MHA/PDA plate and
at 4 C till further use. Growth media, DMSO and Tween-80 were allowed to dry. The wells (8 mm in diameter) were cut from the
purchased from Himedia and Qualigens (India), respectively, while agar and different doses (10, 20, 30 and 40 ml) of essential oil
ethanol and diethyl ether were purchased from Merck, India. solutions mixed with requisite amount of diethyl ether (to make
Different bacterial strains (E. coli aDH5, E. coli ATCC 25922, the volume 50 ml) were delivered into them. After incubation for
P. aeruginosa, P. fluorescens, B. subtilis and S. aureus), fungal strains 24 h at 30 C, all plates were examined for any zones of growth
A.K. Tyagi, A. Malik / Food Control 22 (2011) 1707e1714 1709
inhibition, and the diameters of these zones were measured in 2.6. Chemical composition of M. piperita oil and M. piperita oil
millimetres. All tests were performed in triplicate. vapour
2.3.4. Disc volatilization method 2.6.1. Gas chromatographic (GC) and gas chromatographic mass
Standard experimental set-up as described by Lopez et al. spectrometry (GCeMS) analysis
(2005) was used. Briefly, a 100 ml portion of each suspension con- The percentage composition of essential oil was determined by
taining approximately 106 cfu/ml was spread over the surface of GC-FID and the compounds were identified by GCeMS. GC analysis
MHA/PDA plate and allowed to dry. A paper disc (diameter 6 mm, was carried out on a Shimadzu 2010 Gas Chromatograph equipped
Sigma Aldrich, India) was laid on the inside surface of the upper lid with an FID and 25 m 0.25 mm 0.25 mm WCOT column coated
and 10 ml essential oil was placed on each disc. The plate inoculated with diethylene glycol (AB-Innowax, 7031428, Japan). Both injector
with microorganisms were immediately inverted on top of the lid and detector (FID) temperatures were maintained at 260 C.
and sealed with parafilm to prevent leakage of essential oil vapour. Helium was used as carrier gas at a flow rate of 3.0 ml/min at
Plates were incubated at 30 C for 24 h and the diameter of the a column pressure of 152 kPa. Samples (0.2 ml) were injected into
resulting inhibition zone in the bacterial/fungal lawn was the column with a split ratio of 100:1. Component separation was
measured. Volume of essential oils tested was varied (20, 40 or achieved following a linear temperature program of 60e260 C at
60 ml) by using appropriate number of sterile discs. 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
2.4. Determination of the kill time samples were then analysed on same Shimadzu instrument fitted
with the same column and following the same temperature
These experiments were conducted for selected microorgan- program as above and the MS parameters used were; Ionisation
isms (E. coli, P. fluorescens, P. aeruginosa, B. subtilis and C. albicans) in Voltage (EI) 70 eV, peak width 2 s, mass range 40e700 m/z and
a compact chamber made up of acrylic material (size detector voltage 1.5 V.
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 2.6.2. SPME GCeMS analysis of M. piperita oil vapours
chamber was 0.09375 m3 (93.75 l). The front side of the chamber SPME optimisation analyses were carried out using a Shimadzu
had gloves through which the objects inside the chamber could be 2010 GC instrument equipped with a data processor and Shimadzu
handled without opening the chamber. Prior to exposure the GCeMS QP2010 Plus, Japan. An AB-Innowax 7031428 capillary
chamber was cleaned with ethanol and UV sterilised. Two essential column (60.0 m 0.25 mm i.d., 0.25 mm film thicknesses) was used
oil evaporating machine (Khera instruments Pvt. Ltd, New Delhi, for the separation of the sample components. M. piperita oil vola-
India; evaporation rate ¼ 0.50 ml/h) were fixed in this chamber. tiles from petri plates were extracted using Divinyl benzene/Car-
Appropriate serial dilution of the culture (to obtain 100e300 cfu) boxen/Poly dimethyl siloxane (DVB/CAR/PDMS) fibre. The fibre was
was plated on PDA/MHA plates. Inoculated plates were opened conditioned in a GC injector port as indicated by manufacturer
inside the chamber and fixed with double sided tape at the top (Supelco, France).
surface of chamber for exposure to essential oil vapours. After The injector was maintained at 260 C and operated in split less
a particular time period (0.5, 1, 2, 4, 8 and 12 h) the plates were injection mode with the split valve closed for 1 min. Helium gas
detached, closed and incubated at 30 C for 18e20 h. All the plates was used as the carrier gas at a constant pressure of 136.3 kPa. The
were used in triplicate. 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
2.5. Preparation of samples for scanning electron microscopy impact (70 eV). The mass selective detector was operated in the
scan mode between 20 and 700 m/z. Data acquisition started
B. subtilis cells were incubated for 14 h in MHB at 30 C and 4.0 min after injection.
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 2.6.3. Identification of chemical compounds
another portion was left untreated as a control. These suspensions Peak identification was carried out by comparison of the mass
were incubated at 30 C for 4 h. spectra with mass spectra available on database of NIST05, WILEY8
For investigating the effect of M. piperita oil vapour, 1 ml of libraries and those of pure standards.
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 2.7. Statistical analyses
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 All the experiments were done in triplicate and the data pre-
the petri plate and collected in separate test tube. All the treated sented here represents the mean of these replicates. Data related to
cells were harvested by centrifugation and were prefixed with the zone of inhibition due to the M. piperita oil were subjected to
a 2.5% glutaraldehyde solution overnight at 4 C. After this, the cells analysis of variance (one way ANOVA) in Duncan multiple range
were again harvested by centrifugation and washed three times test using SPSS (version 10) statistical software. The differences
with 0.1 M sodium phosphate buffer solution (pH 7.2). Now each with p < 0.05 were considered significant.
resuspension was serially dehydrated with 25, 50, 75, 90, and 100%
ethanol, respectively. Then, cells were dried at “critical point”. A 3. Results
thin film of cells was smeared on a silver stub for SEM observation.
The samples were gold-covered by cathodic spraying (Polaron 3.1. Antimicrobial activity
gold). Finally, morphology of the B. subtilis cells was observed on
a scanning electronic microscope (ZEISS EVO 50). The SEM obser- 3.1.1. MIC and MBC/MFC of M. piperita oil
vation was done under the following analytical condition: MIC of the M. piperita essential oil was determined against
EHT ¼ 20.00 kV, WD ¼ 9.5 mm, Signal A ¼ SE1. 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
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)
4. Discussion
application and no alteration in organoleptic properties of the edible borne bacterial and fungal strains. Journal of Agricultural and Food Chemistry,
53, 6939e6946.
material/food. With respect to above-mentioned data, our findings
Lopez, P., Sanchez, C., Batlle, R., & Nerin, C. (2007). Development of flexible anti-
demonstrate that M. piperita oil vapour may be considered as microbial films using essential oils as active agents. Journal of Agricultural and
a potential agent for preventing microbial mediated food spoilage. A Food Chemistry, 55, 8814e8824.
further study in vivo condition is warranted to confirm the antimi- Mahboubi, M., & Haghi, G. (2008). Antimicrobial activity and chemical compo-
sition of Mentha pulegium L. essential oil. Journal of Ethnopharmacology, 119,
crobial activity of M. piperita, which may be used for preservation 325e327.
and/or extension the shelf life of raw and processed food. Marino, M., Bersani, C., & Comi, G. (2001). Impedance measurements to study the
antimicrobial activity of essential oils from Lamiaceae and Compositae. Inter-
national Journal of Food Microbiology, 67, 187e195.
Acknowledgement Mead, P. S., Slutsker, L., Detz, V., McCaig, L. F., Breese, J. S., Shapiro, C., et al. (1999).
Food related illness and dead in the United States. Emerging Infectious Diseases,
5, 607e625.
The present work was financially supported by CSIR SRF to
Meincken, M., Holroyd, D. L., & Rautenbach, M. (2005). Atomic force microscopy
AKT and compilation of this work was done during India4EU study of the effect of antimicrobial peptides on the cell envelope of Escherichia
(EMECW13c) research exchange fellowship to AKT. Mr. D.C. Sharma coli. Antimicrobial Agents and Chemotherapy, 49, 4085e4092.
(IIT Delhi, India), Mr. Ajai Kumar (AIRF JNU, India) and Sabal Singh Mimica-Dukic, N., Bozin, B., Sokovic, M., Mihajlovic, B., & Matavulj, M. (2003).
Antimicrobial and antioxidant activities of three Mentha species essential oils.
(IIT Delhi, India) are also acknowledged for their technical support Planta Medicine, 69, 413e419.
in SEM, GC/GCeMS and experimental work, respectively. Nazer, A. I., Kobilinsky, A., Tholozana, J. L., & Dubois-Brissonneta, F. (2005).
Combinations of food antimicrobials at low levels to inhibit the growth of
Salmonella sv. Typhimurium: a synergistic effect. Food Microbiology, 22,
References 391e398.
NCCLS e National Committee for Clinical Laboratory Standards. (1999). Methods for
Bakkali, F., Averbeck, S., Averbeck, D., & Idaomar, M. (2008). Biological effects of determining bacterial activity of antimicrobial agents. (Wayne, PA).
essential oils e a review. Food and Chemical Toxicology, 46, 446e475. Oussalah, M., Caillet, S., & Lacroix, M. (2006). Mechanism of action of Spanish
Baratta, M. T., Dorman, H. J. D., Deans, S. G., Figueiredo, A. C., Barroso, J. G., & oregano, Chinese cinnamon, and savory essential oils against cell membranes
Ruberto, G. (1998). Antimicrobial and antioxidant properties of some and walls of Escherichia coli O157:H7 and Listeria monocytogenes. Journal of Food
commercial essential oils. Flavour and Fragrance Journal, 13, 235e244. Protection, 69, 1046e1055.
Beuchat, L. R. (1976). Sensitivity of vibrio parahaemolyticus to spices and organic Pandit, V. A., & Shelef, L. A. (1994). Sensitivity of Listeria monocytogenes to rosemary
acids. Journal of Food Science, 41, 899e902. Rosmarinus officinalis L. Food Microbiology, 11, 57e63.
Burt, S. (2004). Essential oils: their antibacterial properties and potential applications Paranagama, P. A., Abeysekera, K. H. T., Abeywickrama, K., & Nugaliyadd, L. (2003).
in foods e a review. International Journal of Food Microbiology, 94, 223e253. Fungicidal and anti-aflatoxigenic effects of the essential oil of Cymbopogon
Cerruti, P., & Alzamora, S. M. (1996). Inhibitory effects of valinnin on some food citratus (DC.) Stapf. (lemongrass) against Aspergillus flavus link isolated from
spoilage yeasts in laboratory media and fruit purees. International Journal of stored rice. Letters in Applied Microbiology, 37, 86e90.
Food Microbiology, 29, 379e383. Paster, N., Menasherov, M., Ravid, U., & Juven, B. (1995). Antifungal activity of
Coutinho, H. D., Costa, J. G., Lima, E. O., Falcao-Silva, V. S., & Siqueira-Junior, J. P. (2008). oregano and thyme essential oils applied as fumigants against fungi attacking
Enhancement of the antibiotic activity against a multi-resistant Escherichia coli by stored grain. Journal of Food Protection, 58, 81e85.
Mentha arvensis L. and chlorpromazine. Chemotherapy, 54(4), 328e330. Paulo, L., Ferreira, S., Gallardo, E., Queiroz, J. A., & Domingues, F. (2010). Antimi-
Cox, S. D., Mann, C. M., Markham, J. L., Bell, H. C., Gustafson, J. E., & Warmington, J. R. crobial activity and effects of resveratrol on human pathogenic bacteria. World
(2000). The mode of antimicrobial action of the essential oil of Melaleuca Journal of Microbiology and Biotechnology, 26, 1533e1538.
alternifolia (tea tree oil). Journal of Applied Microbiology, 88, 170e175. Pibiri, M. C. (2006). Assainissement microbiologique de l’air et des systèmes de
Devkatte, A. N., Zore, G. B., & Karuppayil, S. M. (2005). Potential of plant oils as ventilation au moyen d’huiles essentielles. PhD thesis. Lausanne, France: Federal
inhibitors of Candida albicans growth. FEMS Yeast Research, 5, 867e873. Polytechnic Institute.
Dorman, H. J., Kosar, M., Kahlos, K., Holm, Y., & Hiltunen, R. (2003). Antioxidant Rasooli, I., Shayegh, S., Taghizadeh, M., & Astaneh, S. D. A. (2008). Phytother-
prosperities and composition of aqueous extracts from Mentha species, hybrids, apeutic prevention of dental biofilm formation. Phytotherapy Research, 22,
varieties and cultivars. Journal of Agricultural and Food Chemistry, 51, 4563e4569. 1162e1167.
Ebrahimabadi, A. H., Ebrahimabadi, E. H., Bidgoli, Z. D., Kashi, F. J., Mazoochi, A., & Reische, D. W., Lillard, D. A., & Eitenmiller, R. R. (1998). Antioxidants in food lipids.
Batooli, H. (2010). Composition and antioxidant and antimicrobial activity of the In C. C. Ahoh, & D. B. Min (Eds.), Chemistry, nutrition and biotechnology (pp.
essential oil and extracts of Stachys inflata Benth from Iran. Food Chemistry, 119, 423e448). New York: Marcel Dekker.
452e458. Rodriguez, A., Batlle, R., & Nerin, C. (2007). The use of natural essential oils as
Fisher, K., & Phillips, C. (2009). The mechanism of action of a citrus oil blend against antimicrobial solutions in paper packaging. Part II. Progress in Organic Coatings,
Enterococcus faecium and Enterococcus faecalis. Journal of Applied Microbiology, 60, 33e38.
106, 1343e1349. Rohloff, J. (1999). Monoterpene composition of essential oil from peppermint
Gaunt, L., Higgins, S., & Hughes, J. (2005). Decontamination of surface borne (Mentha piperita L.) with regard to leaf position using solid-phase micro-
bacteria by ionized antimicrobial vapours. Journal of Electrostatics, 63, 809e814. extraction and gas chromatography/mass spectrometry analysis. Journal of
Gilles, M., Zhao, J., An, M., & Agboola, S. (2010). Chemical composition and anti- Agricultural and Food Chemistry, 47, 3782e3786.
microbial properties of essential oils of three Australian Eucalyptus species. Food Schelz, Z., Molnar, J., & Hohmann, J. (2006). Antimicrobial and antiplasmid activities
Chemistry, 119, 731e737. of essential oils. Fitoterapia, 77, 279e285.
Griffin, S. G., Markham, J. L., & Leach, D. N. (2000). An agar dilution method for the Shelef, L. A. (1984). Antimicrobial effects of spices. Journal of Food Safety, 6, 29e44.
determination of the minimum inhibitory concentration of essential oils. Sikkema, J., Debont, J. A. M., & Poolman, B. (1995). Mechanisms of membrane
Journal of Essential Oil Research, 12, 249e255. toxicity of hydrocarbons. Microbiololy Review, 59, 201e222.
Hammer, K. A., Carson, C. F., & Riley, T. V. (1999). Antimicrobial activity of essential Takahashi, T., Kokubo, R., & Sakaino, M. (2004). Antimicrobial activities of Euca-
oils and other plant extracts. Journal of Applied Microbiology, 86, 985e990. lyptus leaf extracts and flavonoids from Eucalyptus maculate. Letters in Applied
Hulin, V., Mathot, A. G., Mafart, P., & Dufosse, L. (1998). Antimicrobial properties of Microbiology, 39, 60e64.
essential oils and flavor compounds. Science Alimentory, 18, 563e582. Ultee, A., Kets, E. P. W., & Smid, E. J. (1999). Mechanisms of action of carvacrol on the
Iscan, G., Kirimer, N., Kurkcuoglu, M., Baser, K. H., & Demirci, F. (2002). Antimicrobial food-borne pathogen Bacillus cereus. Applied and Environmental Microbiology,
screening of Mentha piperita essential oils. Journal of Agricultural and Food 65, 4606e4610.
Chemistry, 50, 3943e3946. Van Vuuren, S. F., Suliman, S., & Viljoen, A. M. (2009). The antimicrobial activity of
Khanuja, S. P. S. (2007). Employ contract farming to boost area under cultivation for four commercial essential oils in combination with conventional antimicrobials.
essential oil bearing crops. In Business enabling of aromatic plants and products, 21e22 Letters in Applied Microbiology, 48, 440e446.
November 2007 at HRDI Dehradun. Chemical weekly, 25 December (pp. 180e184). Yadegarinia, D., Gachkar, L., Rezaei, M. B., Taghizadeh, M., Astaneh, S. A., & Rasooli, I.
Lopez, P., Saanchez, C., Battle, R., & Nerian, C. (2005). Solid- and vapor-phase (2006). Biochemical activities of Iranian Mentha piperita L. and Myrtus com-
antimicrobial activities of six essential oils: susceptibility of selected food- munis L. essential oils. Phytochemistry, 67, 1249e1255.