Food Control 35 (2014) 109e116

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Food Control
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Chemical composition, antibacterial activity and mechanism of action

of essential oil from seeds of fennel (Foeniculum vulgare Mill.)
Wen-Rui Diao a, Qing-Ping Hu a, Hong Zhang a, Jian-Guo Xu a, b, *
a
b

College of Life Sciences, Shanxi Normal University, 1 Gongyuan Street, Linfen City 041004, PR China
College of Engineering, Shanxi Normal University, 1 Gongyuan Street, Linfen City 041004, PR China

a r t i c l e i n f o

a b s t r a c t

Article history:
Received 1 April 2013
Received in revised form
25 June 2013
Accepted 26 June 2013

Fennel (Foeniculum vulgare Mill.) is widely cultivated and used as a culinary spice. In this work, the
chemical composition of the essential oil obtained by hydrodistillation of fennel seeds was analyzed by
gas chromatography-mass spectrometry (GCeMS), and 28 components were identied. Trans-anethole
(68.53%) and estragole (10.42%) were found to be the major components. The antibacterial activity,
minimum inhibitory concentration (MIC), and minimum bactericide concentration (MBC) of essential oil
against several food-borne pathogens were evaluated. The results showed that the gram positive and
gram negative strains of bacteria had different sensitivities to essential oil of fennel seeds, the essential
oil exhibited antibacterial activity against Staphylococcus albus, Bacillus subtilis, Salmonella typhimurium,
Shigella dysenteriae and Escherichia coli according to the results of MIC and MBC. Among these bacteria,
S. dysenteriae was the most sensitive to essential oil, showing the lowest MIC and MBC values of 0.125
and 0.25 mg/mL respectively. In addition, kill-time assay also showed that the essential oil had a signicant effect on the growth rate of surviving S. dysenteriae. We concluded that the mechanism of action
of the essential oil against S. dysenteriae might be described as essential oil acting on membrane integrity
according to the results of the leakage of electrolytes, the losses of contents (proteins, reducing sugars
and 260 nm absorbing materials) assays and electron microscopy observation.
2013 Elsevier Ltd. All rights reserved.

Keywords:
Foeniculum vulgare Mill.
Antibacterial activity
Essential oil
Mechanism of action

1. Introduction
Food poisoning and food spoilage caused by microorganisms are
still the most important issues facing the food industry and consumers (Sokmen et al., 2004; Walker, 1988), even in developed
countries (Sokmen et al., 2004). To avoid food contamination the
food industry has used synthetic additives to diminish microbial
growth or inhibit microorganisms (Al-Reza, Rahman, Lee, & Kang,
2010; Bajpai, Baek, & Kang, 2012). However, because of consumers growing concerns over the safety of foods containing
synthetic chemicals, much attention has been paid to use natural
antibacterial products for food preservation (Alzoreky & Nakahara,
2003). Recently, spices have also received attention in their useful
physiological functions and antimicrobial activity. There are a lot of
reports about antimicrobial activity of spice extracts and its
essential oils, and use of natural essential oils as antimicrobial

* Corresponding author. College of Life Sciences, Shanxi Normal University,

1 Gongyuan Street, Linfen City 041004, PR China. Tel.: 86 357 2051247; fax: 86
357 2051000.
E-mail address: xjg71@163.com (J.-G. Xu).
0956-7135/$ e see front matter 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.foodcont.2013.06.056

agents in food systems may be considered as additional intrinsic

determinant to increase the safety and shelf life of foods (Sagdic,
Karahan, Ozcan, & Ozkan, 2003; Salgueiro, Martins, & Correia,
2010).
Fennel (Foeniculum vulgare Mill.) is a small genus of annual,
biennial or perennial herbs distributed in central Europe and
Mediterranean region. It is widely cultivated throughout the
temperate and tropical regions of the world for its aromatic fruits,
which are used as a culinary spice (Daz-Maroto, Prez-Coello,
Esteban, & Sanz, 2006; Rather, Dar, So, Bhat, & Qurishi, 2012).
Mature fennel fruit and its essential oil are used as avoring agents
in food products such as liqueurs, bread, pickles, pastries, and
cheese. They are also used as a constituent in cosmetic and pharmaceutical products (Rather et al., 2012; Telci, Demirtas, & Sahin,
2009). There have been several reports on fennel oils, including
reports on the relative concentration of fennel oil compounds
considerably depending on the geographical origins (Daz-Maroto
et al., 2006), extraction methods (Daz-Maroto, Daz-Maroto,
Snchez-Palomo, & Prez-Coello, 2005), maturation stages (Telci
et al., 2009), and parts of the fennel (Daz-Maroto et al., 2006;
Guilln & Manzanos, 1996), and reports on various biological activities of the essential oil, such as hepatoprotective effect (zbek

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W.-R. Diao et al. / Food Control 35 (2014) 109e116

et al., 2003), antioxidant activity (Ruberto, Baratta, Deans, &

Dorman, 2000; Singh, Maurya, de Lampasona, & Catalan, 2006),
antithrombotic activity (Tognolini et al., 2007), anti-inammatory
activity (Choi & Hwang, 2004), antidiabetic activity (El-Soud
et al., 2011), antitumour activity (Pradhan et al., 2008), acaricidal
activity (Lee, 2004). In addition, the essential oil of fennel seeds had
signicant antifungal activity (Singh et al., 2006; Soylu, Yigitbas,
Soylu, & Kurt, 2007) as well as antibacterial activity (Dadalioglu &
Evrendilek, 2004; Lo Cantore, Iacobellis, De Marco, Capasso, &
Senatore, 2004; Ruberto et al., 2000). Although the chemical
compositions and antimicrobial properties of essential oil from
fennel have been studied, these informations are still limited. Most
importantly, to the best of our knowledge, little work has been
reported on the mechanism of action of essential oil from fennel
seeds on the growth of microorganisms. Therefore, the aim of the
present study was conducted to investigate the chemical composition and antibacterial activity of essential oil from fennel seeds on
several food-borne pathogens, and to further evaluate the possible
mechanism of action responsible for the antibacterial activity
against sensitive strains by kill-time analysis, the permeability and
integrity of cell membrane assays as well as scanning electron
microscopy observation.
2. Material and methods
2.1. Plant materials
The Foeniculum vulgare grows in Yaodu County of Sichuan
Province on 2012. Fennel seeds were harvested, and then obtained
as a commercial product from the local market in mid-October,
2012. Dried whole fennel seeds were stored at 20  C until analysis. The moisture content was 7.6% for fennel seeds, which was
determined according to the method described by Xu, Hu, Duan,
and Tian (2010) with some modications. Briey, fennel seeds
about 10 g were dried using a laboratory oven at 110  C for 4 h.
2.2. Chemicals
Dimethyl sulfoxide (DMSO), n-alkanes C8eC22 was purchased
from Sigma (USA). Nutrient agar (NA), nutrient broth (NB) and
tryptone soy agar mediums were from Beijing Aoboxing Bio-tech
Co. Ltd. (Beijing, China). Other chemicals used were all of analytical grade.
2.3. Microbial strains and culture
The antimicrobial activity of the essential oil was tested against
seven different microorganisms. Three Gram-positive strains were
Staphylococcus aureus ATCC 25923, Staphylococcus albus ATCC 8799,
Bacillus subtilis ATCC 6051. Four Gram-negative bacteria were Salmonella typhimurium ATCC 19430, Pseudomonas aeruginosa ATCC
9027, Shigella dysenteriae CMCC (B) 51252, and Escherichia coli ATCC
25922. These strains were provided by the College of Life Science,
Shanxi Normal University, and stored with liquid parafn wax at
4  C. All strains were cultured at 37  C on Nutrient agar (NA) or
nutrient broth (NB) mediums.
2.4. Extraction of essential oil
The essential oil was extracted according to the method described
by Viuda-Martos et al. (2011) with minor modications. Briey, the
dried fennel seeds (500 g) were ground and hydrodistilled for 4 h
using a Clevenger-type apparatus. The oily layer obtained on top of
the aqueous distillate was separated and dried with anhydrous sodium sulfate. The oil obtained was stored in tightly closed dark vials

and covered with aluminum foil at 4  C until further analysis. The

essential oil was obtained as a light yellow transparent liquid and
had specic fennel aroma with a 1.74% (v/w) yield.
2.5. GC-FID analysis
The essential oil was analyzed using HewlettePackard 5890 II
GC equipped with ame ionization detector (FID) and DB-5 capillary column (30 m  0.25 mm; lm thickness, 0.25 mm), whose
injector and detector temperatures were maintained at 250  C. The
oven temperature was programmed from 40  C for 2 min, raised to
250  C at a rate of 3  C/min, and isotherm at 250  C for 5 min.
Helium was the carrier gas, at a ow rate of 1 mL/min. A sample of
0.1 mL of essential oil was injected manually (in split mode 50:1).
2.6. GCeMS analysis
The analysis of the essential oil was performed using a
HewlettePackard 5890 II GC, equipped with a DB-5 MS capillary
column (30 m  0.25 mm; lm thickness, 0.25 mm) and a HP
5972 mass selective detector for the separation. The mass selective detector was operated in electron-impact ionization (EI)
mode with a mass scan range from m/z 50 to 350 at 70 eV. GC
conditions were the same as described above. The retention
indices were calculated, for all volatile constituents using a homologous series of n-alkanes C8eC22. The essential oil constituents were identied by comparing their GC retention indices,
mass spectra with publish data (Adams, 2001, pp. 1e40) and
National Institute of Standards and Technology mass spectra library data provided by the software of GCeMS system. Essential
oil components are reported as a relative percent of the total oil
by peak area.
2.7. Antimicrobial activity
The essential oil was dissolved in DMSO and sterilized by
ltration through 0.22 mm Millipore lters. Antimicrobial tests
were then carried out by the Oxford cup method (Wang, Tang,
Jiang, Fang, & Liu, 2006) using 100 mL of suspension containing
1  107 colony forming units (CFU)/mL of bacteria determined by
blood count assay spread on nutrient agar (NA) medium. Oxford
cups (6 mm in diameter) were placed on the inoculated agar, and
then 100 mL of essential oil was added with a micropipette. The
diameter of inhibition zone (DIZ) was measured after 24 h of incubation at 37  C, and DMSO was used as a negative control. Tests
were performed in triplicate.
2.8. Minimum inhibitory concentration (MIC) and minimum
bactericide concentration (MBC)
MIC and MBC were determined according to the method
described by Kubo, Fujita, Kubo, Nihei, and Ogura (2004) with
minor modications. Briey, stock solution of essential oil was
prepared in DMSO. Two fold serial dilutions of essential oil were
ltered through 0.22 mm Millipore lters and prepared in sterile NB
medium ranging from 0.125 to 10 mg/mL. To each tube, 50 mL of the
inoculum containing approximately 1  107 CFU/mL microorganisms determined by blood count assay were added. A control test
containing inoculated broth supplemented with only DMSO was
also performed. The tubes were then incubated at 37  C and
examined for evidence of the growth. The MIC was determined as
the lowest concentration of the essential oil that demonstrated no
visible growth for incubating for 24 h, while the MBC was the
lowest concentration of the test essential oil that showed no visible
growth in the culture incubating at 37  C for 48 h.

W.-R. Diao et al. / Food Control 35 (2014) 109e116

2.9. Kill-time analysis

The kill-time curve assay method was used to investigate the
bactericidal effects of the essential oil according to the technique
described by Joray, del Rolln, Ruiz, Palacios, and Carpinella (2011).
The cultivation with the essential oil was done the same as the
above MIC assay and controls containing only DMSO were simultaneously run. At selected time intervals, samples from the test
culture were taken, serially diluted in sterile water, and plated in
Plate Count Agar (PCA) medium. All plates were then incubated for
24 h at 37  C, and CFU were counted.
2.10. Cell membrane permeability
The permeability of bacteria membrane is expressed in the
relative electric conductivity and determined according to the
method described by Kong et al. (2008). After incubated at 37  C for
10 h, S. dysenteriae strains were separated by centrifugation at
5000 rpm for 10min. Then the bacteria were washed with 5% of
glucose until their electric conductivities were near to that of 5%
glucose, and they were the case for isotonic bacteria. The essential
oils at two different concentrations (MIC, and 2  MIC) were added
to 5% glucose and the electric conductivities of the mixtures were
marked as L1. Then different concentrations of essential oils were
added into the isotonic bacteria solution. After completely mixed,
the samples were incubated at 37  C for 8 h, and then the conductivities were measured and marked as L2. The conductivity of
bacteria in 5% glucose treated in boiling water for 5 min was served
as the control and marked as L0. The permeability of bacteria
membrane is calculated according to the formula, the relative
electric conductivity (%) 100  (L2 L1)/L0.

111

4  C. After this, the cells were dehydrated using sequential exposure per ethanol concentrations ranging from 30 to 100% and the
ethanol was replaced by tertiary butyl alcohol at last. Then, cells
after centrifugation were dried at critical point in liquid CO2 under 95 bar pressure, and samples were gold-covered by cathodic
spraying. Finally, morphology of the bacterial cells was observed on
a scanning electronic microscope (JSM-7500F, JEOL Ltd., Japan).
2.13. Statistical analysis
One-way analysis of variance (ANOVA) and Duncans multiple
range tests were carried out to determine signicant differences
(p < 0.05) between the means by Data Processing System (DPS,
version 7.05) and EXCEL program.
3. Results
3.1. Chemical compositions of the essential oil
The essential oil was obtained by hydrodistillation of air-dried
sample with a yield of 1.74% (v/w). The chemical compositions of
essential oil were analyzed by GC and GCeMS and the result was
presented in Table 1. In total, 28 components were identied, representing 95.8% of the total amount. Trans-anethole (68.53%), a
phenylpropanoid, was found as the main component. Estragole
(10.42%) was the second major phenylpropanoid detected in fennel
oil, followed by limonene (6.24%), fenchone (5.45%), and others were
found to be the minor components in the essential oil of fennel seeds.
The prole obtained in the present study was very similar to the
previous results reported by Viuda-Martos et al. (2011) who identied 20 components and also found that trans-anethole (65.59%),
estragole (13.11%), limonene (8.54%) and fenchone (7.76%) were the

2.11. Integrity of cell membrane

The cell integrity of S. dysenteriae strains is examined by
determining the release of cell constituents into supernatant according to the method described by Du, Sun, Liang, Han, and Yu
(2012), with slight modications. Cells from the 100 mL working
culture of tested microorganisms were collected by centrifuged for
15 min at 5000 rpm, washed three times, and resuspended in 0.1 M
phosphate buffer solution (PBS, pH 7.4). One hundred milliliters of
cell suspension were incubated at 37  C under agitation for 4 h in
the presence of essential oil at three different concentrations
(control, MIC and 2  MIC). Then, 25 mL of samples were collected
and centrifuged at 11,000 g for 5 min. And then the concentrations
of proteins and reducing sugars in supernatant were determined
according to the method described by Xu, Hu, Wang, et al. (2010). In
addition, to determine the concentration of the released constituents consisting largely of nucleic acids, 3 mL supernatant was used
to measure UV absorption at 260 nm. Correction was made for the
absorption of the suspension with the same PBS containing the
same concentration of essential oil after 2 min of contact with
tested strains. The untreated cells were corrected with pH 7.4 PBS.
2.12. Scanning electron microscope (SEM)
To determine the efcacy of the essential oil and the morphological changes of S. dysenteriae strains, SEM observation was performed on the tested bacteria. The bacteria cells were incubated in
nutrient broth at 37  C for 10 h. The suspensions were added 0  ,
1  and 2  MIC of essential oil, respectively; control culture was
left untreated. Next the suspensions were incubated at 37  C for 4
and 8 h respectively, and then the suspensions were centrifuged.
The precipitated cells were washed twice with 0.1 M PBS (pH 7.4)
and xed with 2.5% (v/v) glutaraldehyde in 0.1 M PBS overnight at

Table 1
Chemical composition of essential oil from fennel seeds.
Compounds

RIa

RIb

Peak
area (%)c

Idd

Styrene
a-Thujene
a-Pinene
Camphene
Sabinene
b-Pinene
Myrcene
a-Phellandrene
o-Cymene
Limonene
d-3-Carene
Fenchone
2, 4, 6-Octatriene, 3, 4-dimethyl
Camphor
p-Menth-1-en-4-ol
Estragole
Fenchyl acetate
p-Anisaldehyde
Cis-anethole
Trans-anethole
1-(p-Methoxyphenyl)-2-propanone
Isocaryophillene
(E)-b-Farnesene
1-(3-methoxyphenyl)-1-propanone
A germacrene
Cadina-1(2), 4-diene
(E)-Nerolidol
Palustrol

893
928
939
952
975
979
991
1003
1026
1029
1031
1087

893
927
938
950
972
976
990
1006
1026
1031
1032
1090
1130
1148
1177
1202
1230
1252
1258
1288
1390
1437
1458
1462
1502
1537
1562
1733

0.05
Trace
0.42
Trace
Trace
0.35
0.21
0.12
0.63
6.24
1.21
5.45
0.14
0.16
0.27
10.42
0.08
0.31
0.47
68.53
0.18
0.12
0.09
Trace
0.21
0.04
Trace
0.10

GCeMS,
GCeMS,
GCeMS,
GCeMS,
GCeMS,
GCeMS,
GCeMS,
GCeMS,
GCeMS,
GCeMS,
GCeMS,
GCeMS,
GCeMS
GCeMS,
GCeMS,
GCeMS,
GCeMS,
GCeMS,
GCeMS,
GCeMS,
GCeMS
GCeMS,
GCeMS,
GCeMS
GCeMS,
GCeMS,
GCeMS,
GCeMS,

1146
1177
1200
1233
1250
1253
1285
1438
1457
1509
1435
1563
1731

KI
KI
KI
KI
KI
KI
KI
KI
KI
KI
KI
KI
KI
KI
KI
KI
KI
KI
KI
KI
KI
KI
KI
KI
KI

RI, the retention index published by Adams.

RI the retention index was calculated for all volatile constituents using a homologous series of n-alkanes C8eC22 on DB-5 column.
c
Peak area obtained by GC-FID.
d
Identication methods. Trace (0.01%).
b

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W.-R. Diao et al. / Food Control 35 (2014) 109e116

major compounds in the essential oil from Egyptian fennel. Telci et al.
(2009) reported a higher value for trans-anethole (84.12%) and lower
for estragole, limonene and fenchone (4.19e5.53%; 2.96e4.69%;
1.17e2.65%, respectively) of sweet fennel cultivated in Turkey. DazMaroto et al. (2005) reported that fennel oil extracted by simultaneous distillation-extraction (SDE) and supercritical uid extraction
(SFE) showed similar compositions, with trans-anethole, estragole,
and fenchone as the main components; but there were signicant
differences in the contents between SDE and SFE. However, Napoli,
Curcuruto, and Ruberto (2010) reported that the main constituents
from wild Sicilian fennel were estragole, followed by oxygenated
monoterpene, fenchone, which was very different with the chemical
compositions obtained in this study. These differences in components and its content of essential oil from fennel may be concerned in
the geographical origins (Daz-Maroto et al., 2006), cultivated varieties, and maturity of fennel fruits, as well as extraction methods
(Daz-Maroto et al., 2005) and analysis conditions of the essential oil.

antibacterial mode of action of essential oil from fennel seeds. The

effect of the essential oil on the viable counts of tested bacterial
pathogen is shown in Fig. 1. As observed in Fig. 1, compared to the
control, susceptible S. dysenteriae treated with the essential oil at
the MIC value showed a slow decrease in the number of viable cells
over the rst 12 h period of the test, and the number of viable cells
decreased by 11.67% from 6.0 to 5.3 log10 CFU/mL at the cultivation
time of 12 h. Subsequently, an increase in cell numbers was
detected and increased to 6.3 log10 CFU/mL, which showed that
concentrations of the essential oil lower than MIC did not
completely inhibit growth of S. dysenteriae but did increase the lag
time in the growth curve (Fig. 1). Unlike the changing trend of the
number of viable cells at 1  MIC, in treatments at 2  MIC, the
number of viable cells decreased obviously from the rst hour after
cultivation and decreased by 97.5% to 0.15 log10 CFU/mL over 24 h of
incubation. These results showed that the treatment time and
concentration of essential oil had great inuences on antibacterial
effects.

3.2. DIZ, MIC and MBC of the essential oil

3.4. Cell membrane permeability
The DIZ, MIC, and MBC values of the essential oil from fennel
seeds are presented in Table 2. The results showed that the essential
oil had certain antibacterial activity on all of the tested food-borne
pathogens, including both Gram-positive and Gram-negative bacteria. The DIZ values for all tested bacterial strains were in the range
of 11.5e20.2 mm. The DIZ was the maximum value for
S. typhimurium, followed by E. coli, S. dysenteriae and S. albus, however, no difference in DIZ values was found among these bacterial
strains. In contrast with above strains, although the DIZ values were
lower for B. subtilis, P. aeruginosa and S. aureus, we could not deny
that the essential oil from fennel seeds had a better antibacterial
activity on these food-borne pathogens because the DIZ values were
much higher than that of negative control. The MIC and MBC values
for tested bacterial strains were in the range of 0.125e0.25 mg/mL
and 0.25e0.50 mg/mL, respectively. Unfortunately, the MIC and MBC
values of the essential oil for P. aeruginosa and S. aureus strains have
not been gained when the concentration of essential oil reached the
maximum in method system tested. Of these bacteria, the essential
oil from fennel seeds performed both a minimum MIC of 0.125 mg/
mL and a minimum MBC of 0.25 mg/mL against S. dysenteriae, which
indicated it was the most effective bacterial inhibitor and bactericide
against S. dysenteriae. Therefore, the antibacterial properties and
mechanism of action of essential oil from fennel seeds against S.
dysenteriae will be further investigated in this study.

Further antibacterial mode of action of essential oil against the

tested food-borne pathogens was conrmed using the assay of cell
membrane permeability. Fig. 2 showed the effect of essential oil
from fennel seeds on the membrane permeability of S. dysenteriae.
There was little change in the relative electric conductivity of the
control during the rst 12 h period of the test, and then an increase
in the relative electric conductivity was found, which may be due to
normal lysis and death of bacteria, resulting in the rise in the
relative electric conductivity. Compared to the control, the relative
electric conductivity of the suspensions increased immediately after the addition of essential oil at greater than or equal to MIC
concentration and it also increased rapidly with the increasing
treatment time and concentration of essential oil. It meant that the
permeability of bacteria membrane would be increased correspondingly, which caused the leakage of intracellular ingredient,
especially losses of electrolytes including K, Ca2, Na and so on.
3.5. Integrity of cell membrane
The integrity of cell membrane were determined by the measurement of the release of cell constituents including protein,
reducing sugar and the absorbance at 260 nm of the supernatant of
tested bacteria. Table 3 showed the results when S. dysenteriae were

3.3. Kill-time analysis

10
Based on the sensitivity of the tested food-borne pathogens, one
Gram-negative bacterium (S. dysenteriae CMCC (B) 51252) was
selected as the model organisms for further study to conrm the

Microorganisms
Gram-positive
S. aureus
S. albus
B. subtilis
Gram-negative
S. typhimurium
P. aeruginosa
S. dysenteriae
E. coli

DIZa (mm)

MIC (mg/mL)

MBC (mg/mL)

11.5  0.7 c
17.4  1.2 ab
15.8  1.4 bc

>10.0
0.25
0.25

NTb
0.50
0.50

0.25
>10.0
0.125
0.25

0.25
NT
0.25
0.50

20.2
12.3
17.8
19.1






2.4
0.8
1.5
2.2

a
c
ab
ab

Log CFU/mL

Table 2
DIZ, MIC, and MBC of the essential oil from fennel seeds.

control
1MIC
2MIC

0
0

12

24

Time (h)

Values represent means of three independent replicates  SD.

NT, not tested. Different letters within a column indicate statistically signicant
differences between the means (p < 0.05) for DIZ.
b

Fig. 1. Effect of the essential oil from fennel seeds on the viability of the tested
S. dysenteriae.

W.-R. Diao et al. / Food Control 35 (2014) 109e116

membrane assays, and indicated that the essential oil from fennel
seeds may have severe effects on the cell wall and cytoplasmic
membrane. However, these changes in more details still need to be
further observed by transmission electron microscopy (TEM).

Relative conductivity (%)

50
control
1MIC
2MIC

40

4. Discussion

30
20
10
0
0

Time (h)
Fig. 2. Effect of the essential oil from fennel seeds on the impermeability of cell
membrane of tested S. dysenteriae.

treated with different concentrations of essential oil from fennel

seeds for 4 h, respectively. The results indicated that after adding
the corresponding essential oil to strains, the cell constituents
release increased signicantly with the increased concentration of
the essential oil. Compared to control, the concentration of proteins, reducing sugars and cell constituents (OD260nm) in suspensions treated with 1  MIC essential oil increased by 8.11, 2.43, 10.63
times respectively, while they increased by 13.03, 4.07, 16.64 times
respectively when treatment at 2  MIC. These results indicated
that the irreversible damage to the cytoplasmic membranes might
occur, which led to the losses of cell constituents such as protein
and some essential molecules and to cell death.
3.6. Electron microscope observation
The S. dysenteriae bacteria were treated with the essential oil
from fennel seeds at 1  and 2  MIC for 4, 8 h respectively, and
then the morphological and physical changes of treated
S. dysenteriae bacteria were observed by SEM. Fig. 3 showed the
SEM images of the treated and untreated bacteria. These images
directly illustrated the destructive effects of the essential oil on the
tested bacteria. The surfaces of the treated strains underwent
obvious morphological changes compared with the untreated
controls. Untreated cells were rod shaped, regular, and intact (Fig. 3,
a, and b), while some bacterial cells treated with the essential oil
became deformed, pitted, shriveled, adhesive to each other and
parts of the cell were broken (Fig. 3, A4, A8, B4, and B8), which may
give rise to the leaching out of nutrient and genetic materials. And
the changes were more frequent, evident with the increase of
concentration and treatment time of essential oil. This supported
the results of the kill-time study, permeability and integrity of cell

Table 3
The effect of the essential oil on cell constituents release of tested S. dysenteriae.
Concentrations

Control
1  MIC
2  MIC

113

Cell constituents release

Protein (mg/mL)

Reducing
sugar (mg/mL)

Cell constituents
(OD260nm)

10.3  1.1 c
93.8  9.5 b
144.5  14.3 a

18.2  1.6 c
62.6  4.5 b
92.4  6.1 a

0.022  0.007 c
0.256  0.016 b
0.388  0.021 a

Values represent means of three independent replicates  SD. Different letters

within a column indicate statistically signicant differences between the means
(p < 0.05).

It is generally known that the growth of food spoilage and foodborne pathogens can decrease nutritional quality of the food by
consuming nutrients including fat, protein and carbohydrate that
are present in the food, subsequently causes food discoloration,
mustiness, biochemical changes, weight loss and toxicity, which
can adversely affect the health of humans. The method of microbial
growth inhibition most appropriate to food is the use of food preservatives. Essential oils are volatile and odorous principles of plant
secondary metabolism which have wide applications in food
avoring and preservation industries (Bajpai et al., 2012). Recently,
some researchers have reported that monoterpene or sesquiterpene hydrocarbons and their oxygenated derivatives, which are the
major components of essential oils, exhibit potential antimicrobial
activities (Cakir, Kordali, Zenghin, Izumi, & Hirata, 2004). These
ndings strongly supported the results of this study as the essential
oil from fennel seeds was also found to contain these components,
which conrmed its efcacy as natural antimicrobial agent.
The results from the Oxford cup method, followed by measurement of MIC and MBC indicated that the essential oil from fennel
seeds had strong and consistent inhibitory effects against all of the
tested food-borne pathogens including both Gram-positive and
Gram-negative bacteria as conrmed by the inhibitory effect of
essential oil showing different susceptibility rate against the tested
food-borne pathogens, and S. dysenteriae was found to be the most
sensitive microorganism tested, showing larger inhibition zone and
the lowest MIC and MBC values. Some studies reported that the
essential oil extracted from fennel seeds exhibited antibacterial effect against food-borne pathogens such as E. coli, Bacillus megaterium, S. aureus (Dadalioglu & Evrendilek, 2004; Mohsenzadeh,
2007); Ozcan, Chalchat, Arslan, Ates, and Unver (2006) also found
that fennel essential oils possessed an inhibitory effect against a
wide range of Bacillus species, which supported our results in the
present study, indicating that essential oil of fennel seeds was a
potent bacterial inhibitor with a broad antibacterial spectrum.
Furthermore, the effects of essential oil on the growth of S. dysenteriae were investigated by measuring the viable cell counts and the
results revealed that exposure of essential oil had a rigorous effect
on the cell viability of the tested bacterial pathogens. A minor
amount of essential oil could prolong the lag phase of S. dysenteriae,
while the essential oil at 2  MIC exerted its maximum bactericidal
activity as evident by the signicant reduction in microbial counts
and complete inhibition of S. dysenteriae cell viable counts at 24 h
exposure, which indicated that the essential oil have perfect antibacterial activity. Besides, the effects of essential oil on the growth of
other bacterial strains except for S. aureus and P. aeruginosa were
also investigated and the results were similar to that on
S. dysenteriae, where they differed was that the extent of changes in
the number of viable cells and the time of controlling the lag phase
were different for different bacteria treated by essential oil (not
shown). Similar to our ndings, other essential oils from plants also
exhibited inhibitory effects against various food-borne pathogens
(Al-Reza et al., 2010; Bajpai, Sharma, & Baek, 2013).
A number of authors have mentioned the antimicrobial activity
of essential oils, however, the mechanism of action has not been
studied in great detail (Tajkarimi, Ibrahima, & Cliver, 2010). The
previous ndings showed that terpenes, phenols, aldehydes and
ketones are the major components of essential oils (Ceylan & Fung,
2004), and it is generally believed that essential oils principally

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W.-R. Diao et al. / Food Control 35 (2014) 109e116

Fig. 3. The photography of SEM of S. dysenteriae for a and b, untreated bacteria cultured for 14, 18 h, respectively; A4 and A8, bacteria treated with the essential oil at 1  MIC for 4,
8 h, respectively; B4 and B8, bacteria treated with the essential oil at 2  MIC for 4, 8 h, respectively.

performed against the cell cytoplasmic membrane of microorganisms. The hydrophobicity is an important characteristic of essential
oils 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 (Bajpai et al., 2013; Carson, Mee, & Riley,
2002; Lambert, Skandamis, Coote, & Nychas, 2001), and extensive
losses of the cell contents will cause the death of cell (Lv, Liang,
Yuan, & Li, 2011; Moreira, Ponce, Del Valle, & Roura, 2005). Therefore, it is necessary to clarify the mode of action of essential oil of
fennel seeds against the tested bacterial pathogens by investigating
changes in the permeability and integrity of cell membrane and
surface characteristic parameters which included determination of
relative electric conductivity, loss of 260 nm absorbing materials
and leakage of soluble protein, reducing sugars as well as determination of effect of essential oil on cell wall structure of the tested
food-borne pathogens using SEM analysis associated with
morphological changes and surface characteristics.
The antimicrobial mode of action of essential oil was also
conrmed on the basis of leakage of the electrolytes from S. dysenteriae cells when exposed to essential oil at 1  and 2  MIC
concentrations. The bacterial plasma membrane provides a
permeability barrier to the passage of small ions such as K, Na
which are necessary electrolytes, facilitate cell membrane functions
and maintain proper enzyme activity. This impermeability to small
ions is maintained and even regulated by the structural and
chemical compositions of the membrane itself. Increases in the
leakage of electrolytes will indicate a disruption of this permeability barrier. Maintaining ion homeostasis is integral to the
maintenance of the energy status of the cell as well as solute
transport, regulation of metabolism, control of turgor pressure and
motility (Cox et al., 2001). Therefore, even relatively slight changes
to the structural integrity of cell membranes can detrimentally
affect cell metabolism and lead to cell death (Cox et al., 2001). The
results in this study showed that the relative electric conductivity
of the suspension increased rapidly with the increasing treatment
time and concentration of essential oil, which meant that the
permeability of bacteria membrane would be increased correspondingly, causing the leakage of electrolytes and leading to cell
death.

Another apparent antimicrobial mode of action of essential oil

was visualized by the conrmation on the leakage of protein,
reducing sugar and 260 nm absorbing materials when the tested
food-borne pathogens exposed to the essential oils at 1  and
2  MIC concentrations. The macromolecules of a bacterial cell
including protein, nucleic acids which reside throughout the interior of the cell, in the cytoplasm, are the key structural components.
The transfer of cellular information through the processes of
translation, transcription and DNA replication occur within the
same compartment and can interact with other cytoplasmic
structures (Kohanski, Dwyer, & Collins, 2010). Measurement of
specic cell leakage markers including proteins and 260 nm
absorbing materials is an indicator of membrane integrity to specic antimicrobial agent in relationship to unexposed cells (Bajpai
et al., 2013). Our experimental results showed that the essential
oil of fennel seeds apparently caused rapid losses of proteins,
reducing sugars and 260 nm absorbing materials from the treated
bacteria, which indicated that irreversible damage to the cytoplasmic membranes occurred, which was in accordance with the
results of SEM. So it could be conferred that integrity of cell
membrane would also be an important factor to inhibit the bacterial growth. But it is still a mystery where the damage takes place,
on the lipopolysaccharide or membrane proteins in cell wall.
The SEM micrograph of S. dysenteriae cells treated with the
essential oil of fennel seeds showed that severe morphological alterations appeared in the cell wall and membrane, and the SEM
micrograph of treated cells showed many fragmentary bacteria,
which have also been observed for various kinds of tested organisms when treated with different essential oils (Bajpai et al., 2013;
Du et al., 2012). The changes in physical and morphological of
bacterial cells might have occurred due to the effect of essential oil
on the permeability and integrity of membrane, thereby resulting
in the lysis of bacterial cell wall followed by the losses of intracellular dense materials on the surface of treated cells. However, as
state before, a more detail cell wall changes still need to be further
observed by TEM.
5. Conclusion
Based on the present research, the essential oil from fennel
seeds, which was rich in trans-anethole, possessed good

W.-R. Diao et al. / Food Control 35 (2014) 109e116

antibacterial activity against selected food-borne pathogens in this

study. We concluded that the mechanism of action of essential oil
from fennel seeds against S. dysenteriae may be described as
essential oil making rstly a break through the permeability of cell
membrane associated with generalized the integrality of
membrane-disrupting effects, leading to the leakage of electrolytes
as well as losses of proteins, reducing sugars, and 260 nm absorbing
materials. These changes resulted in cell decomposition and death
eventually, and this corresponded to a simultaneous reduction in
the number of viable bacteria. Moreover, the SEM observation also
supported the above hypothesis. However, because of the heterogeneous compositions of essential oils, it seems unlikely that there
is only one mechanism of action or that only one component is
responsible for the antimicrobial action. Therefore, further research
is still necessary to fully understand the mechanisms involved
including against other food-borne pathogens, TEM observation as
well as the interactions with other food ingredients in order to
justify the real applications of essential oil from fennel seeds in food
practices as a natural antibacterial agent.
Acknowledgments
This work was nancially supported by a Project of the Natural
Science Foundation of Shanxi Province, China (Project No.
2012011031-3), and a Program for the Top Young Academic Leaders
of Higher Learning Institutions of Shanxi.
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