Food Research International 44 (2011) 30573064

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Food Research International

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

In vitro antimicrobial effects and mechanism of action of selected plant essential oil
combinations against four food-related microorganisms
Fei Lv a, Hao Liang a,, Qipeng Yuan a, Chunfang Li b
a
State Key Laboratory of Chemical Resource Engineering, Beijing University of Chemical Technology, Beijing 100029, People's Republic of China
b
Beijing Industrial Technician College, Beijing 100023, People's Republic of China

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

Article history: The aim of this study was to evaluate the antimicrobial efcacy of selected plant essential oil (EO)
Received 22 December 2010 combinations against four food-related microorganisms. Ten EOs were initially screened against Escherichia
Accepted 20 July 2011 coli, Staphylococcus aureus, Bacillus subtilis and Saccharomyces cerevisiae using agar disk diffusion and broth
dilution methods. The highest efcacy against all the tested strains was shown when testing the oregano EO.
Keywords:
EOs of basil and bergamot were active against the Gram-positive bacteria (S. aureus and B. subtilis), while
Essential oils
Food-related microorganisms
perilla EO strongly inhibited the growth of yeast (S. cerevisiae). The chemical components of selected EOs
Antimicrobial activity were also analyzed by GC/MS. Phenols and terpenes were the major antimicrobial compounds in oregano and
GC/MS basil EOs. The dominant active components of bergamot EO were alcohols, esters and terpenes. For perilla EO,
Synergism the major active constituents were mainly ketones. The checkerboard method was then used to investigate
Action mechanisms the antimicrobial efcacy of EO combinations by means of the fractional inhibitory concentration index (FICI).
Based on an overall consideration of antimicrobial activity, organoleptic impact and cost, four EO
combinations were selected and their MIC values were listed as follows: oreganobasil (0.3130.313 l/ml)
for E. coli, basilbergamot (0.3130.156 l/ml) for S. aureus, oreganobergamot (0.3130.313 l/ml) for B.
subtilis and oreganoperilla (0.3130.156 l/ml) for S. cerevisiae. Furthermore, the mechanisms of the
antimicrobial action of EO combinations to the tested organisms were studied by the electronic microscopy
observations of the cells and the measurement of the release of cell constituents. The electron micrographs of
damaged cells and the signicant increase of the cell constituents' release demonstrated that all EO
combinations affected the cell membrane integrity.
2011 Elsevier Ltd. All rights reserved.

1. Introduction
In spite of modern improvements in food production and preserva-
tion techniques, such as genetic engineering, irradiation of food, and
modied-atmosphere packaging (WHO, 2002), food safety is a growing
public health concern. The survival of microorganisms in food is an
important problem, which can lead to spoilage and deteriorate the
quality of food products (Celiktas et al., 2007) or cause infection and
illness (Jacob, Mathiasen, & Powell, 2010). It is estimated that food-
borne diarrheal diseases have already caused about 4 to 6 million deaths
per year, with most of these occurring in young children (WHO, 2003).
Although chemical preservative has been used for years, there is much
controversial because they have been shown to cause respiratory or
other health problems (Fleming-Jones & Smith, 2003). Therefore, it is
necessary to nd a novel way to reduce or eliminate food-related
microorganisms during the shelf life of food products. In the meantime,
Corresponding author at: P.O. Box 75, Beijing University of Chemical Technology,
No. 15, Bei San Huan Dong Rd., Beijing 100029, People's Republic of China. Tel.: + 86 10
64431557; fax: + 86 10 64437610.
E-mail address: starslh@163.com (H. Liang).
the increasing demand of consumers for natural products has led to
research on new antimicrobial agents from plants to improve the safety
of products (Goni et al., 2009).
Essential oils (EOs) are the volatile oily liquids of the secondary
metabolism of scented plants, which are obtained from different plant
parts, such as owers, leaves, seeds, bark, fruits and roots (Burt, 2004).
Although some EOs have long been researched for their antibacterial,
antifungal, antiviral, and antioxidant properties (Gao et al., 2011; Gilles,
Zhao, An, & Agboola, 2010; Kordali et al., 2005; Mourey & Canillac, 2002;
Prakash et al., 2011; Sylvestre, Pichette, Longtin, Nagau, & Legault,
2006), the recent enhancement of interest in green consumerism has
given rise to a renewal scientic awareness of them. It has been
demonstrated that various medicine plants, spices and herbs containing
EOs signicantly inhibit a wide range of microorganisms. Moreover,
different results in antimicrobial test were observed depending on
measurement conditions, tested strains and the source of the
antimicrobial compound (Turgis, Han, Caillet, & Lacroix, 2009).
Considering their excellent antimicrobial function, EOs possess great
potential as natural additives for food preservation.
Although desired antimicrobial activity of several EOs against
pathogenic and spoilage microorganisms is performed in vitro test, it

0963-9969/$ see front matter 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.foodres.2011.07.030
3058 F. Lv et al. / Food Research International 44 (2011) 30573064
has generally been found that a higher concentration is needed to
achieve the same effect in foods (Shelef, Jyothi, & Bulgarelli, 1984).
This fact may lead to an organoleptic impact as the use of natural
preservatives can alter the taste of food and exceed the avor
threshold acceptable to consumers (Hsieh, Mau, & Huang, 2001;
Nazer, Kobilinsky, Tholozan, & Dubois-Brissonnet, 2005). Gutierrez,
Rodriguez, Barry-Ryan, and Bourke (2008) found that lettuce samples
treated with 500 or 1000 ppm concentration of thyme and lemon
balm EOs were rejected because their bad effects on the organoleptic
acceptability of foods.
A number of studies have focused on the synergistic activity of the
EO in combination with antibiotic to minimize the side effects of the
antibiotic (Mahboubi & Bidgoli, 2010; Rosato et al., 2009; Tohidpour,
Sattari, Omidbaigi, Yadegar, & Nazemi, 2010), while the combinations
of EO with other natural antibacterial compounds (e.g., nisin) was
used in food to reduce the minimum effective dose of EO (Solomakos,
Govaris, Koidis, & Botsoglou, 2008). However, a few studies regarding
the synergistic effects of EO combinations to obtain effective
antimicrobial activity at sufciently low concentrations and conse-
quently reduce negative sensory impact were performed (Gutierrez,
Barry-Ryan, & Bourke, 2009). EOs in plants generally are mixtures of
abundant components (Burt, 2004), therefore, the synergistic effects
are perhaps more likely to achieve in EO combinations.
The present study aimed to: (1) screen the antimicrobial
properties of ten plant EOs against four common food-related bacteria
and yeasts, including Escherichia coli, Staphylococcus aureus, Bacillus
subtilis and Saccharomyces cerevisiae; (2) determine the chemical
composition of the selected EOs by GC/MS; and (3) access the efcacy
of selected EO combinations against tested microorganisms to
determine potential for their synergistic at low doses. The effects of
selected EO combinations on the integrity of cell membranes were
also studied in order to elucidate the antimicrobial mechanisms of
action.
2. Material and methods
2.1. Antimicrobial agents
2.1.1. Essential oils
The commercial EOs used in this study were purchased from
Guanxiang Chemicals Trading Co. Ltd. (Changsha, China), including
patchouli EO (Pogostemon cablin), clary sage EO (Salvia sclarea),
rosemary EO (Rosmarinus ofcinalis), basil EO (Ocimum basilicum),
spearmint EO (Mentha spicata), oregano EO (Origanum vulgare), perilla
EO (Perilla arguta), absinthe EO (Artemisia absinthium), bergamot EO
(Citrus bergamia) and lavender EO (Lavandula angustifolia). The details
of ten commercial plants EOs were listed in Table 1 and all EOs were
stored at the temperature of 46 C before analysis.
2.1.2. Antibiotic and chemical preservatives
The antibiotic selected in the study was kanamycin sulfate, which
was purchased from Conba Pharmaceutical Co. Ltd. (Jinhua, Zhejiang,
Table 1
The details of ten commercial plant essential oils.
Plant species Common name Distilled part
Pogostemon cablin Patchouli Leaf
Salvia sclarea Clary sage Leafower
Rosmarinus ofcinalis Rosemary Branch
Ocimum basilicum Basil Leafower
Mentha spicata Spearmint Stemleaf
Origanum vulgare Oregano Whole plant
Perilla arguta Perilla Leaf
Artemisia absinthium Absinthe Leafbranch
Citrus bergamia Bergamot Peel
Lavandula angustifolia Lavender Flower
China). Three common chemical preservatives (sodium benzoate,
potassium sorbate and methylparaben) were obtained from Sinopharm
Chemical Reagent Beijing Co. Ltd. (Beijing, China).
2.2. Antimicrobial activity
2.2.1. Microbial strains and growth conditions
Four food-related microorganisms were used to assess the
antimicrobial properties, including the Gram-positive S. aureus ATCC
6538, B. subtilis ATCC 6633, the Gram-negative E. coli ATCC 8739, and
the yeast S. cerevisiae ATCC 9763. All strains were obtained from China
General Microbiological Culture Collection Center and maintained on
slants of Nutrient Agar (NA, Abxing, Beijing, China) for bacteria and
Yeast Peptone Dextrose Agar (YPDA, Abxing, Beijing, China) for the
yeast at 4 C.
Active cultures were prepared by transferring a loop of cells from
the agar slant to a test tube containing 5 ml of Nutrient Broth for
bacteria and YPD Broth for the yeast. They were then incubated
overnight to the logarithmic phase of growth at 37 C for bacteria (6
10 h) and 30 C for the yeast (1216 h). Culture purity was examined
by streaking each culture on plates of Nutrient Agar for bacteria and
YPD Agar for yeast (Gilles et al., 2010). The turbidity of the cell
suspensions was measured at 600 nm and adjusted to the required
concentration of 10 510 6 CFU/ml using the McFarland standard
(Firuzi, Asadollahi, Gholami, & Javidnia, 2010).
2.2.2. Agar disk diffusion assay
The EOs were screened for antimicrobial activity using the agar disk
diffusion method (Rota, Carraminana, Burillo, & Herrera, 2004) against 4
microorganisms. Nutrient Agar and YPD Agar were sterilized in an
autoclave and cooled to 4550 C before being poured into 90 mm Petri
dishes. After solidifying, sterile blank lter disks (6 mm diameter)
containing 5 l of each EO were applied to the surface of agar plates
that were previously seeded by spreading of 200 l overnight fresh
inoculums suspension (logarithmic growth phase cells). One standard
antibiotic (kanamycin sulfate) and three chemical preservatives (sodium
benzoate, potassium sorbate and methylparaben) were used as positive
control. The inoculated plates were incubated for 24 h at 37 C for
bacterial and 48 h at 30 C for yeast. Microbial inhibition was visually
appraised as the diameter of the inhibition zones surrounding the disks
(disk diameter included) and recorded in millimeter. The diameters of the
inhibition zones were measured with a digital caliper. The agar disk
diffusion tests were performed in triplicate.
2.2.3. Determination of minimal inhibitory concentration
Minimal inhibitory concentration (MIC) is cited by the most
researchers as a measure of the antimicrobial performance of EOs.
Bacteria and yeast sensitive to the EOs in disk diffusion assay were
studied for their MIC values with some modications of the method
described by previous study (Weerakkody, Cafn, Turner, & Dykes,
2010).
The inoculums were prepared from overnight broth cultures
(logarithmic growth phase cells) and suspensions were adjusted to
the required microbial density (1 10 8 CFU/ml) using an Ultraspec
2000 spectrophotometer at 600 nm. After adding 20 l of EOs to the
rst tube containing 4 ml of broth, serial two-fold dilutions were
made in a concentration range of 0.0395 l/ml in 10 ml sterile test
tubes containing Nutrient broth for bacteria and YPD broth for the
yeast. A 400 l suspension (1 10 8 CFU/ml) of tested microorganisms
was added to each tube. A negative control tube contained broth and
microorganism. Meanwhile, a positive control tube contained
50 g/ml of kanamycin sulfate in broth and microorganism. MIC was
dened as the concentration in the lowest serial dilution of the EOs
which resulted in the lack of visible microorganism growth in tubes
after 24 h (bacteria) and 48 h (yeast) (Reza, Rahman, Lee, & Kang,
2010).
 F. Lv et al. / Food Research International 44 (2011) 30573064
2.3. Chemical compositions analysis of selected EOs
The selected EOs were analyzed by Agilent 7890A/5975C GC-MS
system, equipped with a DB-624 capillary column (30 m 0.25 mm;
lm thickness, 0.25 m). The oven temperature program was initiated
at 60 C, held for 3 min then raised up to 220 C at a rate of 5 C/min
held for 1 min. The injector temperature was 250 C. The amount of
injection was 0.2 l. Helium was used as carrier gas at ow rate of
1 ml/min, with a split ratio equal to 1:4. The MS was operated in EI
ionization mode at 70 ev, and complete scans from 40 to 350 amu
(atomic mass units) were recorded. The compounds of EOs were
identied by the comparison of their retention indices (RI) relative to
n-alkanes (C8C24) and mass spectra with those of National Institute
of Standards and Technology (NIST 08) commercial library as well as
with literature data (Adams, 2007). All analyses were carried out
triplicate.
2.4. Synergism testing: checkerboard method
The broth dilution checkerboard method, which was frequently
used to assess interactive inhibition in vitro, was used to determine
the antimicrobial effects of selected EO combinations obtained in
antimicrobial activity testing. The assay was arranged as follows: EOA
was diluted two-fold in vertical orientation, while EOB was diluted
two-fold in horizontal orientation. The concentrations of EOA and EOB
were prepared corresponding to 1/2, 1/4 and 1/8 of the MIC values,
respectively. Subsequently, 400 l suspension (logarithmic growth
phase cells) containing 1 10 8 CFU/ml of the indicator strain was
added to each tube. The inoculated tubes were incubated overnight at
37 C for bacteria and 30 C for the yeast, and then evaluated for their
microbial growth.
The checkerboard method is often combined with calculation of
fractional inhibitory concentration indices (FICI) to assess the
antimicrobial effects of EO combinations. The FICI was calculated as
FICA + FICB (Hemaiswarya, Kruthiventi, & Doble, 2008), where FICA =
(MICA of the combination / MICA alone) and FICB = (MICB of the
combination / MICB alone). The results were interpreted as synergy
(FICI b 0.5), addition (0.5 FICI 1), indifference (1 b FICI 4) or
antagonism (FICI N 4).
2.5. Mechanisms of action of the EO combinations against cell
membranes
2.5.1. Scanning electron microscopy (SEM) analysis
To examine the mechanisms of action of EO combinations against
cell membranes, SEM studies were carried out as previously reported
with some modications (Moosavy et al., 2008). Logarithmic growth
phase cells of 4 tested microorganisms (each approximately
10 6 CFU/ml) were treated with selected EO combinations at MIC
value which obtained in checkerboard testing. The control without
EOs and samples containing EOs were incubated at room temperature
for 3 h. After incubation, cells were harvested by centrifugation for
10 min at 5000 g and washed twice with 0.1 M phosphate buffer
solution (PBS, pH 7.0), and then were resuspended in PBS containing
2.5% glutaraldeyde and kept for 2 h at 4 C to x the cells. After
centrifuging, the cells were further dehydrated in wateralcohol
solutions at various alcohol concentrations (30%, 50%, 70%, 80%, 90%
and 100%) for 10 min each. Finally, the samples were xed on SEM
support, and then sputter-coated with gold under vacuum, followed
by microscopic examinations using a scanning electron microscope
(Zeiss Supra 7M 55).
2.5.2. Cell constituents' release
The release of cell constituents into supernatant was examined
according to the method described by Rhayour, Bouchikhi, Elaraki,
Sendide, and Remmal (2003). Cells from the 100 ml working culture
3059
of 4 tested microorganisms were collected by centrifuged for 10 min
at 5000 g, washed three times, and resuspended in 0.1 M PBS (pH 7.0).
One hundred milliliters of cell suspension was incubated at 37 C
(yeast 30 C) under agitation in an environmental incubator shaker
for 1 h in the presence of EO combinations at three different
concentrations (control, MIC and 2 MIC). Then, 2 ml of samples
was collected and centrifuged at 12,000 g for 2 min. To determine the
concentration of the constituents' released, 1 ml supernatant was
used to measure UV absorption at 260 nm. Correction was made for
the absorption of the suspension with PBS (pH 7.0) containing the
same concentration of selected EO combinations after 2 min of contact
with 4 strains. The untreated cells (control) were corrected with PBS
(pH 7.0).
2.6. Statistical analysis
All experiments were performed in triplicate. The data were
recorded as mean standard deviation (SD) for the measurements of
inhibition zones in the antimicrobial activity tests and cell constituents'
release. Mean composition of essential oil was analyzed by standard
deviation (SD) and relative standard deviation (RSD). A statistical
package (SPSS, version 12.0 for Windows, SPSS Inc.) was used for the
data processing. Differences were considered signicant at p b 0.05.
3. Results and discussion
3.1. Antimicrobial activity
It is usually acknowledged that the logarithmic phase cells are
more sensitive to external stress factors, while stationary phase cells
are more resistant (Kheadr, Bernoussi, Lacroix, & Fliss, 2004; Phillips &
Duggan, 2002; Song, Hu, & Zhou, 2010). This phenomenon may be due
to the structural or conformational changes in cell wall, which is a
dynamic structure that can adapt to physiological changes (Aguilar-
Uscanga & Francois, 2003; Geciova, Bury, & Paul, 2002). For instance,
previous studies have shown that the peptidoglycan of E. coli cells
made obvious changes during the transition from logarithmic to
stationary phase, including the thickening of peptidoglycan layer and
the increase of cross-linkage degree (Leduc, Frehel, Siegel, & Van
Heijenoort, 1989; Pisabarro, Pedro, & Vazquez, 1985). This shows that
the increase of chemical bonds in the peptidoglycan makes the long-
cultured cell difcult to be damaged. In order to effectively evaluate
the antimicrobial effects of EOs, all cells used in this study were in the
logarithmic phase for its sharp metabolism and sensitivity to
environment properties.
Initial screening of the antimicrobial activity of the investigated
EOs was studied against four tested microorganisms using the agar
disk diffusion assay, which was assessed by the presence and absence
of inhibition zones. The antimicrobial activity of EOs can be classied
into three levels (Rota, Herrera, Martinez, Sotomayor, & Jordan,
2008): weak activity (inhibition zone 12 mm), moderate activity
(12 mm b inhibition zone b 20 mm) and strong activity (inhibition
zone 20 mm). Results from the agar disk diffusion tests for
antimicrobial activity of the EOs are shown in Table 2.
The EOs displayed a variable degree of antimicrobial activity
against different tested strains. The oregano EO showed the highest
activity and signicant broad spectrum against all the tested
microorganisms, especially for Gram-positive bacteria (S. aureus and
B. subtilis) and yeast (S. cerevisiae), whose zones of inhibition were
greater than 27 mm. It was also worth noting that the EOs of basil and
bergamot were active against the Gram-positive bacteria (S. aureus
and B. subtilis) with zones of inhibition larger than 22 mm, which
were even more effective than 30 g of kanamycin sulfate (less than
20 mm). Meanwhile, the EO of perilla strongly inhibited the growth of
the yeast (S. cerevisiae) with inhibition zones larger than 25 mm.
Comparatively, EOs from patchouli, clary sage, rosemary, spearmint,
3060 F. Lv et al. / Food Research International 44 (2011) 30573064

Table 2
Antimicrobial activity of investigated essential oils against the tested microorganisms.

Samples Inhibition zone diametera (mm)

Gram+ Gram Yeast

Staphylococcus aureus Bacillus subtilis Escherichia coli Saccharomyces cerevisiae

EOs Patchouli 10.1 0.4 10.4 0.3 ndb 8.1 0.3

Clary sage 15.4 0.4 15.0 0.6 nd 10.0 0.4
Rosemary 10.2 0.2 nd 8.6 0.2 12.3 0.5
Basil 23.3 0.4 22.2 0.3 15.2 0.5 18.3 0.6
Spearmint nd 16.1 0.6 nd 12.6 0.8
Oregano 27.4 0.5 27.4 0.7 18.2 0.8 27.2 0.6
Perilla nd 15.0 1.7 nd 25.9 0.2
Absinthe nd 15.2 0.6 nd 11.5 0.6
Bergamot 26.2 0.6 24.3 0.6 nd 15.2 0.4
Lavender nd 13.5 1.0 nd 11.0 0.4
Antibiotic KSc 19.5 0.4 18.4 0.6 24.3 0.6 21.0 0.3
Chemical SBd 10.3 0.4 7.8 0.2 10.7 0.4 9.8 0.3
preservatives PSe 10.6 0.3 8.7 0.2 11.9 0.4 8.5 0.3
MEf 12.6 0.3 11.4 0.4 11.8 0.3 8.8 0.4
a
Results are presented as mean standard deviation from the experiments in triplicate. The diameter of the disks ( = 6 mm) was included.
b
nd: not detected.
c
KS: kanamycin sulfate (30 g/disk).
d
SB: sodium benzoate (200 g/disk).
e
PS: potassium sorbate (200 g/disk).
f
ME: methylparaben (200 g/disk).

absinthe and lavender showed weak or moderate activity for tested MICs (0.625 l/ml) for all the tested microorganisms, while perilla EO
strains, with the inhibition zones less than 16 mm. Generally, Gram- exhibited the same strong effectiveness (MIC = 0.625 l/ml) against the
positive bacteria are more sensitive to the presence of EOs from spices yeast, S. cerevisiae. Basil and bergamot EOs displayed high effect against
and herbs than Gram-negative bacteria (Oussalah, Caillet, Saucier, & the Gram-positive bacteria, with MICs of 1.25 l/ml against S. aureus,
Lacroix, 2006). This is possibly because Gram-negative bacteria while the MICs of 0.625 l/ml and 1.25 l/ml against B. subtilis,
possess an outer membrane surrounding the cell wall, which restricts respectively. Oregano EO (O. vulgare) has been reported previously to
diffusion of hydrophobic compounds through its lipopolysaccharide have strong antimicrobial activity on a number of bacteria and yeasts
covering (Vaara, 1992). In this study, the Gram-negative bacteria E. (Burt & Reinders, 2003; Elgayyar, Draughon, Golden, & Mount, 2001;
coli was found to be the most resistant to the examined EOs, only the Gutierrez et al., 2008). In our study, the oregano EO showed its best
moderate inhibition (1519 mm) was observed for oregano and basil inhibitory activity in agar disk diffusion assay, and with lowest MIC
EOs. values of 0.625 l/ml for all strains. Similar results were reported by
It has been reported that the antimicrobial activity of EOs can be Hammer and coworkers (Hammer, Carson, & Riley, 1999) that MIC
attributed to the presence of a number of low molecular weight phenols, values of oregano EO against E. coli and S. aureus were from 0.5 to
terpenes and aldoketones, which also have been shown to exhibit 1.2 l/ml. Basil EO (O. basilicum) showed high activity on Gram-positive
antimicrobial activity in pure form (Ceylan & Fung, 2004; Panizzi, bacteria (MIC value: 0.6251.25 l/ml), which was several times
Flamini, Cioni, & Morelli, 1993). Therefore, a higher antimicrobial more active when compared to the previously study (MIC value:
activity of oregano, basil, bergamot and perilla EOs in this work could be 3.1412.5 l/ml) by Runyoro et al. (2010). O. basilicum existed in a
explained by the presence of a signicant amount of carvacrol, eugenol, number of chemotypes (Grayer et al., 1996), and this may be the reason
D-limonene and 2-pentanoylfuran, respectively (The particulars were for the various observed activity. To the best of our knowledge, only a
detailed in the results of GC/MS, Section 3.2). A number of compounds in few studies on the antimicrobial activity of bergamot (C. bergamia) and
relatively low concentrations, such as -pinene, -pinene, thymol, - perilla (P. arguta) EOs have been carried out (Friedman, Henika, Levin, &
terpinen, terpinolene, piperitone and perillene, could also be expected Mandrell, 2004; Yamamoto & Ogawa, 2002). Therefore, the notable
to make a signicant contribution to the antimicrobial activity of the antimicrobial effects of bergamot EO for Gram-positive bacteria and
tested EOs (Burt, 2004).Based on inhibition zone test results, oregano, perilla EO for yeast which were found in this work should be further
basil, bergamot and perilla EOs were selected for further study on MICs investigated.
as described previously. The MICs of the selected EOs against the tested
strains are presented in Table 3. As shown in the table, the EOs have
variable levels of inhibition. The MICs values conrmed the results 3.2. Chemical compositions analysis of selected EOs
obtained by the agar disk diffusion method. Oregano EO had the lowest
Chemical compositions of EOs of oregano (O. vulgare), basil (O.
Table 3 basilicum), bergamot (C. bergamia) and perilla (P. arguta) were then
Minimal inhibitory concentration (MIC) (l/ml) of selected essential oils against the analyzed by GC/MS technique. Table 4 shows the main chemical
tested microorganismsa. components of tested EOs, together with the retention indices (RI) of the
Microorganism MICb compounds. Other components not listed are present in amounts less
than 3%. It was found that the phenols and terpenes were the main two
Basil Oregano Bergamot Perilla KSc
groups of constituents in oregano and basil EOs. The major constituents
Gram+ Staphylococcus aureus 1.25 0.625 1.25 5.0 0.3125
of oregano EO were carvacrol (30.17%), p-cymene (15.20%), -terpinen
Bacillus subtilis 0.625 0.625 1.25 2.5 0.3125
Gram Escherichia coli 1.25 0.625 5.0 5.0 0.156 (12.44%) and thymol (8.62%), while the basil EO contained eugenol
Yeast Saccharomyces cerevisiae 2.5 0.625 2.5 0.625 0.3125 (62.60%) followed by caryophyllene (21.51%) and -caryophyllene
a
Results are means of three different experiments.
(4.85%). In the case of bergamot EO, bergamol (16.10%), linalool
b
MIC: minimal inhibitory concentration (l/ml). (14.02%) and D-limonene (13.76%) were found as the main volatile
c
KS: kanamycin sulfate (50 g/ml). constituents. In comparison, the dominant components of perilla EO
 F. Lv et al. / Food Research International 44 (2011) 30573064 3061

Table 4 chemotypes of O. basilicum species, such as methyl cinnamate, linalool,

The main components of the essential oils of oregano (O. vulgare), basil (O. basilicum), methyl chavicol and methyl eugenol chemotypes (Vina & Murillo, 2003).
bergamot (C. bergamia) and perilla (P. arguta).
There were quite a few reports on the chemical components of bergamot
NO. RIa Compounds GC area RSD and perilla EOs. In our study, the major composition of bergamol (16.10%)
(%)b (%)c for bergamot EO and 2-pentanoylfuran (23.42%) for perilla EO signi-
Oregano EO cantly differed from previously published data (Fang, Goto, Sasaki, &
1 936 -Pinene 4.09 0.03 0.61 Hirose, 2004; Figoli et al., 2006; Nitta, Kobayashi, Ohnishi-Kameyama,
2 1026 p-cymene 15.20 0.25 1.64
Nagamine, & Yoshida, 2006). This observed differences in the chemical
3 1060 -Terpinen 12.44 0.19 1.49
4 1098 Linalool 5.27 0.06 1.14 compositions may be attributed to occurrence of chemotypes, geograph-
5 1291 Thymol 8.62 0.17 1.96 ical locations, season at the time of collection, stage of development,
6 1299 Carvacrol 30.17 0.14 0.46 culture climate and other culture conditions, which may affect biological
7 1431 Caryophyllene 4.33 0.06 1.35 activities (Calvey, White, Matusil, Sha, & Block, 1998; Runyoro et al., 2010).
Basil EO
1 1360 Eugenol 62.60 0.62 1.00 3.3. Synergism testing: checkerboard method
2 1431 Caryophyllene 21.51 0.43 1.98
3 1450 -Caryophyllene 4.85 0.09 1.85 To explore the possibility of reducing undesirable impacts of EOs
on organoleptic properties of food, we extended our investigation to
Bergamot EO
1 1035 D-limonene 13.76 0.23 1.69 study the synergism among the selected EOs. According to the
2 1058 -Terpinene 4.49 0.02 0.45 checkerboard test, the FIC indices for the EO combinations are shown
3 1091 Terpinolene 3.20 0.09 2.82 in Table 5. With reference to the FICI scale, all of the tested
4 1098 Linalool 14.02 0.16 1.12 combinations displayed a synergism (FICI = 0.375) for S. aureus in
5 1187 -Terpineol 4.76 0.14 2.98
this study. EO combinations of oregano with basil or bergamot both
6 1197 Ethanone,1-(1,3-dimethyl 5.69 0.03 0.44
3-cyclohexen-1-yl) exhibited a useful addictive effect with the FICI of 0.75 against B.
7 1260 Bergamol 16.10 0.29 1.77 subtilis. EO combinations of oregano with basil or oregano with parilla
also had additive effects (FICI = 0.75) against E. coli and S. cerevisiae,
Perilla EO
respectively. No indifference and antagonism was observed for any of the
1 1098 Linalool 3.88 0.10 2.46
2 1181 2-Pentanoylfuran 23.42 0.15 0.64
combinations evaluated. Moreover, sensory analysis was performed by
3 1243 Perillaldehyde 3.17 0.04 1.38 our team member according to the method described by previous study
4 1255 Benzene,1-methoxy-4 - 13.31 0.11 0.80 (Gutierrez et al., 2008), and the sensory acceptability of EOs followed the
(1-methylpropyl)- order: bergamotN oreganoN basilN perilla. Based on an overall consider-
5 1399 -Bergamotene 4.81 0.08 0.61
ation of antimicrobial activity, organoleptic impact and cost (the cost of
a
RI, retention indices relative to C8-C24 n-alkanes on the DB-624 column. EOs followed the order: perillaN oreganoN basilN bergamot), four EO
b
Relative proportions as percent of the total peak area, and results are presented as
combinations were selected and their MIC values were listed as follows:
mean standard deviation from the experiments in triplicate.
c
Relative standard deviation (standard deviation divided by the mean). oreganobasil (0.3130.313 l/ml) for E. coli, basilbergamot (0.313
0.156 l/ml) for S. aureus, oreganobergamot (0.3130.313 l/ml) for B.
subtilis and oreganoperilla (0.3130.156 l/ml) for S. cerevisiae.
were mainly ketones, such as 2-Pentanoylfuran, reaching percentages of Some studies have concluded that whole EOs have a greater
23.42%. antibacterial activity than the major components mixed (Gill,
It was worth noting that the composition contents of oregano EO in Delaquis, Russo, & Holley, 2002). Similarly, Burt (2004) and Ultee,
this work differed greatly from previous reports. Bozen, Dukic, Simin, and Kets, Alberda, Hoekstra, and Smid (2000) also suggested that the
Anackov (2006) reported that the major content of carvacrol and thymol minor components present in the EOs were more critical to the
representing 61.30% and 13.90% of the total EO, respectively, whereas the activity than EOs main components mixed, and the combination of
studied EO of oregano was shown to contain carvacrol (30.17%) and major components with other minor components that have a weaker
thymol (8.62%), probably depending on the different plant material activity may achieve a synergistic effect. For instance, previous studies
investigated. In our study, we used fresh whole plant material of oregano, reported that the acetate compounds may positively inuence the
whereas Bozen et al. used aerial parts of cultivated owering plants. A activity of marjoram EO (Dorman & Deans, 2000). Moreover, the
similar situation also occurred in basil EO. 1,8-cineole (54.30%), -pinene general components of many plant EOs, which are camphor and
(8.15%) and -terpineol (6.60%) (Runyoro et al., 2010), which were eucalyptol, possess oxygen functions in their structure and these
reported as major compounds in the EO of basil, were in minor quantities functions are indentied to increase the antimicrobial activities of
in our sample. The reasons of these differences may be the various terpenoids (Naigre, Kalck, Roques, Roux, & Michel, 1996). Considering

Table 5
FIC indices (FICI) of essential oil combinations against the tested microorganismsa.

Combination Escherichia coli Bacillus subtilis Staphylococcus aureus Saccharomyces cerevisiae

FIC FICI FIC FICI FIC FICI FIC FICI

1. Oreganobasil 0.5 0.75 (A)b 0.5 0.75 (A) 0.125 0.375 (S)
0.25 0.25 0.25
2. Oreganobergamot 0.5 0.75 (A) 0.25 0.375 (S)
0.25 0.125
3. Basilbergamot 0.5 1 (A) 0.25 0.375 (S)
0.5 0.125
4. Oreganoperilla 0.5 0.75 (A)
0.25
a
Results are means of three different experiments.
b
The results were interpreted as synergy (S, FICI b 0.5), addition (A, 0.5 FICI 1), indifference (I, 1 b FICI 4) or antagonism (AN, FICI N 4).
3062 F. Lv et al. / Food Research International 44 (2011) 30573064

Fig. 1. Scanning electron micrographs of E. coli cells: (A) untreated (magnication 30,000); (B) treated with the combinations of oregano oil and basil oil at MIC value for 3 h
(magnication 20,000).

Fig. 2. Scanning electron micrographs of S. aureus cells: (A) untreated (magnication 100,000); (B) treated with the combinations of basil oil and bergamot oil at MIC value for 3 h
(magnication 50,000).

the above mentioned interaction of EO compositions, the EO
combinations which consist of different biochemical components
may increase the antimicrobial efcacy distinctly. It is reported that
the FIC indices for the EO combination of O. vulgare and R. ofcinalis
were 0.5 for L. monocytogenes, Y. enterocolitica and A. hydrophilla
suggesting a synergism of the essential oils against these bacteria
(Azeredo et al., 2011).
In the majority of cases, the antimicrobial efcacy of plant EOs
decreased in the food model media, by comparison with the in vitro
control media. For instance, Uhart and coworkers found that very low
antimicrobial activity or no effect against Salmonella when EOs were
applied to beef (Uhart, Maks, & Ravishankar, 2006). Therefore, higher
concentrations of plant EOs are generally required to achieve useful
antimicrobial activity when added to food. However, in that case,
unacceptable levels of inappropriate odors and changes in textural
quality of food may occur (Devlieghere, Vermeulen, & Debevere, 2004).
In our work, the results revealed that EO combinations selected can
efciently inhibit the growth of tested strains at lower concentrations
than required for the individual EO, and therefore there is a potential for
practical application in food industry to reduce undesirable impacts on
sensory properties and extend the shelf-life of food.
3.4. Mechanisms of action of the EO combinations against cell
membranes
A number of authors have mentioned the antimicrobial activity of
EOs, however, the mechanism of action has not been studied in great
detail (Tajkarimi, Ibrahima, & Cliver, 2010). Chemical analysis of EOs
showed that the major active EO components are phenols, terpenes,
aldehydes and ketones (Ceylan & Fung, 2004), and it is generally
believed that EOs principally performed against the cell cytoplasmic
membrane of microorganism. The hydrophobicity is an important
characteristic of EOs and their components (Sikkema, De Bont, &
Poolman, 1995), which enables them to accumulate in cell membranes,
disturbing the structures and causing an increase of permeability.
Leakage of intracellular constituents and impairment of microbial
enzyme systems can then occur (Carson, Mee, & Riley, 2002; Lambert,
Skandamis, Coote, & Nychas, 2001), and extensive loss of the cell

Fig. 3. Scanning electron micrographs of B. subtilis cells: (A) untreated (magnication 30,000); (B) treated with the combinations of oregano oil and bergamot oil at MIC value for
3 h (magnication 50,000).
 F. Lv et al. / Food Research International 44 (2011) 30573064
Fig. 4. Scanning electron micrographs of S. cerevisiae cells: (A) untreated (magnication 20,000); (B) treated with the combinations of oregano oil and perilla oil at MIC value for 3 h
(magnication 30,000).
contents will cause the death of cell (Moreira, Ponce, Del Valle, & Roura,
2005). Furthermore, there are some generally accepted mechanisms of
antimicrobial interaction for synergy: sequential inhibition of a common
biochemical pathway, inhibition of protective enzymes, combinations of
cell wall active agents, and use of cell wall active agents to enhance the
uptake of other antimicrobials (Santiesteban-Lopez, Palou, & Lopez-
Malo, 2007). Therefore, it is necessary to clarify the specic modes of
action of EOs constituents with antimicrobial properties on the
metabolic pathway of microorganisms (Goni et al., 2009).
The electron micrographs of both untreated (control) and EO
combinations treated microbial cells are presented in Figs. 14. In
control groups, the untreated cells had the typical structure of
themselves, showing a striated wall for E. coli (Fig. 1A) and B. subtilis
(Fig. 3A), and a smooth wall for S. aureus (Fig. 2A) and S. cerevisiae
(Fig. 4A). In contrast, obvious detrimental effects on the morphology
of cell membranes were shown when strains were treated with the EO
combinations at MIC value. Microstructural observations demonstrat-
ed that the EOs caused an increase in the permeabilization of the cells
and disrupted the membrane integrity. An incomplete and deformed
shape was observed in treated E. coli, S. aureus and S. cerevisiae,
accompanied by the lack of cell walls (Figs. 1B, 2B and 4B). More
deformation was observed in treated B. subtilis, showing rupture and
lysis of the membranes (Fig. 3B).
The cell constituents' release was determined by the measurement
of the absorbance at 260 nm of the supernatant of four tested strains.
Table 6 shows the results when E. coli, S. cerevisiae, S. aureus and B.
subtilis were treated with EO combinations for 1 h, respectively. The
results indicated that after adding the corresponding EO combinations
to strains, the cell constituents' release increased visibly with the
increased concentration of the EOs compared to the control group.
The maximum cell constituents' release was observed when B. subtilis
Table 6
The effect of essential oil combinations on cell constituents' release of the tested
microorganisms.
Microorganism EOs combinations Cell constituents' release
concentration (l/ml) (OD260nm)a
Staphylococcus aureus 0 0.018 0.002
MIC 0.153 0.007
MIC 2 0.260 0.014
Bacillus subtilis 0 0.025 0.002
MIC 0.276 0.012
MIC 2 0.438 0.017
Escherichia coli 0 0.008 0.001
MIC 0.177 0.007
MIC 2 0.302 0.008
Saccharomyces cerevisiae 0 0.056 0.004
MIC 0.142 0.008
MIC 2 0.247 0.012
Means within a same strain, which are not followed by a common letter, are
signicantly different (P b 0.05).
a
Results are presented as mean standard deviation from the experiments in
triplicate. OD260nm, optical density at 260 nm.
3063
cells were treated with EO combination at 2 MIC, showing an
absorbance of 0.438. An important loss of cell constituents means that
irreversible damage to the cytoplasmic membranes occurred, which is
in accordance with the results of SEM.
4. Conclusion
The antimicrobial activities of ten plant EOs against four food-
related microorganisms were evaluated and compared in this work.
Oregano, basil, bergamot and perilla EOs showed high effect against
the test microorganisms, and these four EOs have a large variation in
their chemical composition. Four kinds of EO combinations were
selected as oreganobasil for E. coli, basilbergamot for S. aureus,
oreganobergamot for B. subtilis and oreganoperilla for S. cerevisiae
by synergism testing. Scanning electron microscopy observation and
the determination of cell constituents' release demonstrated that EO
combinations destroyed the integrity of cell membrane, resulting in
the death of microorganisms. In conclusion, EO combinations can
effectively act against food-related microorganisms at low combined
concentrations. Further studies are needed to evaluate the efcacy of
these EO combinations in suitable food systems.
Acknowledgments
This study was supported by the National Natural Science
Foundation of China (Grant No. 20806005) and Chinese Universities
Scientic Fund (ZZ1025). The authors are grateful to Beijing Industrial
Technician College, for the technical collaboration
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