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
College of Life Sciences, Shanxi Normal University, 1 Gongyuan Street, Linfen City 041004, PR China
b
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: Fennel (Foeniculum vulgare Mill.) is widely cultivated and used as a culinary spice. In this work, the
Received 1 April 2013 chemical composition of the essential oil obtained by hydrodistillation of fennel seeds was analyzed by
Received in revised form gas chromatography-mass spectrometry (GCeMS), and 28 components were identified. Trans-anethole
25 June 2013
(68.53%) and estragole (10.42%) were found to be the major components. The antibacterial activity,
Accepted 26 June 2013
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
Keywords:
gram negative strains of bacteria had different sensitivities to essential oil of fennel seeds, the essential
Foeniculum vulgare Mill.
Antibacterial activity
oil exhibited antibacterial activity against Staphylococcus albus, Bacillus subtilis, Salmonella typhimurium,
Essential oil Shigella dysenteriae and Escherichia coli according to the results of MIC and MBC. Among these bacteria,
Mechanism of action 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 sig-
nificant 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.
Ó 2013 Elsevier Ltd. All rights reserved.

1. Introduction agents in food systems may be considered as additional intrinsic

Food poisoning and food spoilage caused by microorganisms are
still the most important issues facing the food industry and con-
sumers (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 con-
sumers’ 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.: þ86 357 2051247; fax: þ86
357 2051000.
E-mail address: xjg71@163.com (J.-G. Xu).
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 (Díaz-Maroto, Pérez-Coello,
Esteban, & Sanz, 2006; Rather, Dar, Sofi, Bhat, & Qurishi, 2012).
Mature fennel fruit and its essential oil are used as flavoring agents
in food products such as liqueurs, bread, pickles, pastries, and
cheese. They are also used as a constituent in cosmetic and phar-
maceutical 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 (Díaz-Maroto
et al., 2006), extraction methods (Díaz-Maroto, Díaz-Maroto,
Sánchez-Palomo, & Pérez-Coello, 2005), maturation stages (Telci
et al., 2009), and parts of the fennel (Díaz-Maroto et al., 2006;
Guillén & Manzanos, 1996), and reports on various biological ac-
tivities of the essential oil, such as hepatoprotective effect (Özbek

0956-7135/$ e see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.foodcont.2013.06.056
110 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-inflammatory
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
significant 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 compo-
sition 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 anal-
ysis. 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 modifications. Briefly, 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 analyt-
ical 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 Sal-
monella 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 paraffin 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 modifications. Briefly, 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 so-
dium 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 specific 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 flame ionization detector (FID) and DB-5 capil-
lary column (30 m  0.25 mm; film 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 flow 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; film thickness, 0.25 mm) and a HP
5972 mass selective detector for the separation. The mass se-
lective 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 ho-
mologous series of n-alkanes C8eC22. The essential oil constit-
uents were identified by comparing their GC retention indices,
mass spectra with publish data (Adams, 2001, pp. 1e40) and
National Institute of Standards and Technology mass spectra li-
brary 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
filtration through 0.22 mm Millipore filters. 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 in-
cubation 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 modifications. Briefly, stock solution of essential oil was
prepared in DMSO. Two fold serial dilutions of essential oil were
filtered through 0.22 mm Millipore filters 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 microorgan-
isms 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 111

2.9. Kill-time analysis 4  C. After this, the cells were dehydrated using sequential expo-
sure per ethanol concentrations ranging from 30 to 100% and the
The kill-time curve assay method was used to investigate the ethanol was replaced by tertiary butyl alcohol at last. Then, cells
bactericidal effects of the essential oil according to the technique after centrifugation were dried at “critical point” in liquid CO2 un-
described by Joray, del Rollán, Ruiz, Palacios, and Carpinella (2011). der 95 bar pressure, and samples were gold-covered by cathodic
The cultivation with the essential oil was done the same as the spraying. Finally, morphology of the bacterial cells was observed on
above MIC assay and controls containing only DMSO were simul- a scanning electronic microscope (JSM-7500F, JEOL Ltd., Japan).
taneously run. At selected time intervals, samples from the test
culture were taken, serially diluted in sterile water, and plated in 2.13. Statistical analysis
Plate Count Agar (PCA) medium. All plates were then incubated for
24 h at 37  C, and CFU were counted. One-way analysis of variance (ANOVA) and Duncan’s multiple
range tests were carried out to determine significant differences
2.10. Cell membrane permeability (p < 0.05) between the means by Data Processing System (DPS,
version 7.05) and EXCEL program.
The permeability of bacteria membrane is expressed in the
relative electric conductivity and determined according to the 3. Results
method described by Kong et al. (2008). After incubated at 37  C for
10 h, S. dysenteriae strains were separated by centrifugation at 3.1. Chemical compositions of the essential oil
5000 rpm for 10min. Then the bacteria were washed with 5% of
glucose until their electric conductivities were near to that of 5% The essential oil was obtained by hydrodistillation of air-dried
glucose, and they were the case for isotonic bacteria. The essential sample with a yield of 1.74% (v/w). The chemical compositions of
oils at two different concentrations (MIC, and 2  MIC) were added essential oil were analyzed by GC and GCeMS and the result was
to 5% glucose and the electric conductivities of the mixtures were presented in Table 1. In total, 28 components were identified, rep-
marked as L1. Then different concentrations of essential oils were resenting 95.8% of the total amount. Trans-anethole (68.53%), a
added into the isotonic bacteria solution. After completely mixed, phenylpropanoid, was found as the main component. Estragole
the samples were incubated at 37  C for 8 h, and then the con- (10.42%) was the second major phenylpropanoid detected in fennel
ductivities were measured and marked as L2. The conductivity of oil, followed by limonene (6.24%), fenchone (5.45%), and others were
bacteria in 5% glucose treated in boiling water for 5 min was served found to be the minor components in the essential oil of fennel seeds.
as the control and marked as L0. The permeability of bacteria The profile obtained in the present study was very similar to the
membrane is calculated according to the formula, the relative previous results reported by Viuda-Martos et al. (2011) who identi-
electric conductivity (%) ¼ 100  (L2 L1)/L0. fied 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
Table 1
The cell integrity of S. dysenteriae strains is examined by Chemical composition of essential oil from fennel seeds.
determining the release of cell constituents into supernatant ac- Compounds RIa RIb Peak Idd
cording to the method described by Du, Sun, Liang, Han, and Yu area (%)c
(2012), with slight modifications. Cells from the 100 mL working Styrene 893 893 0.05 GCeMS, KI
culture of tested microorganisms were collected by centrifuged for a-Thujene 928 927 Trace GCeMS, KI
15 min at 5000 rpm, washed three times, and resuspended in 0.1 M a-Pinene 939 938 0.42 GCeMS, KI
phosphate buffer solution (PBS, pH 7.4). One hundred milliliters of Camphene 952 950 Trace GCeMS, KI
Sabinene 975 972 Trace GCeMS, KI
cell suspension were incubated at 37  C under agitation for 4 h in
b-Pinene 979 976 0.35 GCeMS, KI
the presence of essential oil at three different concentrations Myrcene 991 990 0.21 GCeMS, KI
(control, MIC and 2  MIC). Then, 25 mL of samples were collected a-Phellandrene 1003 1006 0.12 GCeMS, KI
and centrifuged at 11,000 g for 5 min. And then the concentrations o-Cymene 1026 1026 0.63 GCeMS, KI
of proteins and reducing sugars in supernatant were determined Limonene 1029 1031 6.24 GCeMS, KI
d-3-Carene 1031 1032 1.21 GCeMS, KI
according to the method described by Xu, Hu, Wang, et al. (2010). In Fenchone 1087 1090 5.45 GCeMS, KI
addition, to determine the concentration of the released constitu- 2, 4, 6-Octatriene, 3, 4-dimethyl 1130 0.14 GCeMS
ents consisting largely of nucleic acids, 3 mL supernatant was used Camphor 1146 1148 0.16 GCeMS, KI
to measure UV absorption at 260 nm. Correction was made for the p-Menth-1-en-4-ol 1177 1177 0.27 GCeMS, KI
Estragole 1200 1202 10.42 GCeMS, KI
absorption of the suspension with the same PBS containing the
Fenchyl acetate 1233 1230 0.08 GCeMS, KI
same concentration of essential oil after 2 min of contact with p-Anisaldehyde 1250 1252 0.31 GCeMS, KI
tested strains. The untreated cells were corrected with pH 7.4 PBS. Cis-anethole 1253 1258 0.47 GCeMS, KI
Trans-anethole 1285 1288 68.53 GCeMS, KI
2.12. Scanning electron microscope (SEM) 1-(p-Methoxyphenyl)-2-propanone 1390 0.18 GCeMS
Isocaryophillene 1438 1437 0.12 GCeMS, KI
(E)-b-Farnesene 1457 1458 0.09 GCeMS, KI
To determine the efficacy of the essential oil and the morpho- 1-(3-methoxyphenyl)-1-propanone 1462 Trace GCeMS
logical changes of S. dysenteriae strains, SEM observation was per- A germacrene 1509 1502 0.21 GCeMS, KI
formed on the tested bacteria. The bacteria cells were incubated in Cadina-1(2), 4-diene 1435 1537 0.04 GCeMS, KI
(E)-Nerolidol 1563 1562 Trace GCeMS, KI
nutrient broth at 37  C for 10 h. The suspensions were added 0  ,
Palustrol 1731 1733 0.10 GCeMS, KI
1  and 2  MIC of essential oil, respectively; control culture was
a
left untreated. Next the suspensions were incubated at 37  C for 4 b
RI, the retention index published by Adams.
RI the retention index was calculated for all volatile constituents using a ho-
and 8 h respectively, and then the suspensions were centrifuged. mologous series of n-alkanes C8eC22 on DB-5 column.
The precipitated cells were washed twice with 0.1 M PBS (pH 7.4) c
Peak area obtained by GC-FID.
and fixed with 2.5% (v/v) glutaraldehyde in 0.1 M PBS overnight at d
Identification methods. Trace (0.01%).
112 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. Díaz-
Maroto et al. (2005) reported that fennel oil extracted by simulta-
neous distillation-extraction (SDE) and supercritical fluid extraction
(SFE) showed similar compositions, with trans-anethole, estragole,
and fenchone as the main components; but there were significant
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 compo-
nents and its content of essential oil from fennel may be concerned in
the geographical origins (Díaz-Maroto et al., 2006), cultivated vari-
eties, and maturity of fennel fruits, as well as extraction methods
(Díaz-Maroto et al., 2005) and analysis conditions of the essential oil.
3.2. DIZ, MIC and MBC of the essential oil
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 bac-
teria. 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, how-
ever, 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.
3.3. Kill-time analysis
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 confirm the
Table 2
DIZ, MIC, and MBC of the essential oil from fennel seeds.
Microorganisms DIZa (mm) MIC (mg/mL)
Gram-positive
S. aureus 11.5  0.7 c >10.0
S. albus 17.4  1.2 ab 0.25
B. subtilis 15.8  1.4 bc 0.25
Gram-negative
S. typhimurium 20.2  2.4 a 0.25
P. aeruginosa 12.3  0.8 c >10.0
S. dysenteriae 17.8  1.5 ab 0.125
E. coli 19.1  2.2 ab 0.25
a
Values represent means of three independent replicates  SD.
b
NT, not tested. Different letters within a column indicate statistically significant
differences between the means (p < 0.05) for DIZ.
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 first 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 first 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 influences on antibacterial
effects.
3.4. Cell membrane permeability
Further antibacterial mode of action of essential oil against the
tested food-borne pathogens was confirmed 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 first 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 af-
ter 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 corre-
spondingly, which caused the leakage of intracellular ingredient,
especially losses of electrolytes including Kþ, Ca2þ, Naþ and so on.
3.5. Integrity of cell membrane
The integrity of cell membrane were determined by the mea-
surement 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
10
control
1×MIC
8 2×MIC
Log CFU/mL
6
MBC (mg/mL)
4
NTb
0.50
0.50 2
0.25
NT 0
0.25
0 1 3 6 12 24
0.50
Time (h)
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 113

50 membrane assays, and indicated that the essential oil from fennel
control seeds may have severe effects on the cell wall and cytoplasmic
1×MIC membrane. However, these changes in more details still need to be
Relative conductivity (%)

40 2×MIC further observed by transmission electron microscopy (TEM).

30
20
10
0
0 1 2 4
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 significantly with the increased concentration of
the essential oil. Compared to control, the concentration of pro-
teins, reducing sugars and cell constituents (OD260nm) in suspen-
sions 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 Cell constituents’ release
Protein (mg/mL) Reducing
sugar (mg/mL)
Control 10.3  1.1 c 18.2  1.6 c
1  MIC 93.8  9.5 b 62.6  4.5 b
2  MIC 144.5  14.3 a 92.4  6.1 a
Values represent means of three independent replicates  SD. Different letters
within a column indicate statistically significant differences between the means
(p < 0.05).
4. Discussion
It is generally known that the growth of food spoilage and food-
borne 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 pre-
servatives. Essential oils are volatile and odorous principles of plant
6 8 secondary metabolism which have wide applications in food
flavoring and preservation industries (Bajpai et al., 2012). Recently,
some researchers have reported that monoterpene or sesquiter-
pene hydrocarbons and their oxygenated derivatives, which are the
major components of essential oils, exhibit potential antimicrobial
activities (Cakir, Kordali, Zenghin, Izumi, & Hirata, 2004). These
findings strongly supported the results of this study as the essential
oil from fennel seeds was also found to contain these components,
which confirmed its efficacy as natural antimicrobial agent.
The results from the Oxford cup method, followed by measure-
ment 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 confirmed 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 ef-
fect against food-borne pathogens such as E. coli, Bacillus mega-
terium, 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. dysen-
teriae 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 significant 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 anti-
bacterial 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 findings, other essential oils from plants also
exhibited inhibitory effects against various food-borne pathogens
Cell constituents
(OD260nm) (Al-Reza et al., 2010; Bajpai, Sharma, & Baek, 2013).
A number of authors have mentioned the antimicrobial activity
0.022  0.007 c
0.256  0.016 b of essential oils, however, the mechanism of action has not been
0.388  0.021 a studied in great detail (Tajkarimi, Ibrahima, & Cliver, 2010). The
previous findings 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
114 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 microorgan-
isms. 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). There-
fore, 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 deter-
mination 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
confirmed on the basis of leakage of the electrolytes from S. dys-
enteriae 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 Kþ, Naþ
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 perme-
ability 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 corre-
spondingly, causing the leakage of electrolytes and leading to cell
death.
Another apparent antimicrobial mode of action of essential oil
was visualized by the confirmation 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 inte-
rior 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
specific cell leakage markers including proteins and 260 nm
absorbing materials is an indicator of membrane integrity to spe-
cific 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 cyto-
plasmic 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 bac-
terial 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 al-
terations 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 organ-
isms 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 intracel-
lular 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 firstly 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 hetero-
geneous 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 financially 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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