Letters in Applied Microbiology ISSN 0266-8254

ORIGINAL ARTICLE

Inhibitory activity of natural antimicrobial compounds

alone or in combination with nisin against Enterobacter
sakazakii
S.-Y. Lee and H.-H. Jin
Department of Food and Nutrition, Chung-Ang University, Anseong-si, Gyeonggi-do, South Korea

Keywords
combined effect, Enterobacter sakazakii,
growth inhibition, natural antimicrobials,
nisin.
Correspondence
Sun-Young Lee, 72-1 Nae-ri, Daedeok-myeon,
Anseong-si, Gyeonggi-do, 456-756, South
Korea. E-mail: nina6026@gmail.com
2007 ⁄ 1172: received 23 July 2007, revised
11 February 2008 and accepted 5 June 2008
doi:10.1111/j.1472-765X.2008.02432.x
Abstract
Aim: To determine the antimicrobial activity of natural organic compounds
alone and in combination with nisin on the growth of Enterobacter sakazakii in
laboratory media.
Methods and Results: The minimum inhibitory concentrations (MIC) of five
natural organic compounds were determined, and their effects in combination
with nisin were evaluated by comparing treatment with each natural organic
compound alone and in combination with 25 mg ml)1 nisin in tryptic soy
broth. Among the tested natural organic compounds, the MIC of carvacrol and
thymol was 1Æ25 mmol l)1 and showed the strongest inhibitory activity against
E. sakazakii, whereas the MIC of cinnamic acid was higher than 5 mmol l)1,
and therefore showed the weakest inhibitory activity. However, the combina-
tion of each compound with nisin did not result in the enhancement of their
antimicrobial activities except when nisin was combined with diacetyl.
Conclusions: The order of inhibition attributed to natural organic compounds
was carvacrol = thymol > eugenol > diacetyl > cinnamic acid, and only the
combination of diacetyl and nisin showed a synergistic effect of inhibiting the
growth of E. sakazakii.
Significance and Impact of the Study: This study shows the potential of natural
organic compounds for controlling E. sakazakii.

E. sakazakii world-wide (Nair et al. 2004). Moreover, E. sak-

Introduction
Enterobacter sakazakii is a member of the family Enterobac-
teriaceae, genus Enterobacter, and is a motile peritrichous
Gram-negative bacillus and an emerging pathogen of neo-
nates with mortality rates of 40–80% (Nazarowec-White
et al. 1997). Enterobacter sakazakii has been implicated in
severe forms of neonatal infections, such as meningitis,
bacteremia, sepsis, and necrotizing enterocolitis (Kleiman
et al. 1981). Although the natural habitat of E. sakazakii is
unknown, dried infant formula has been epidemiologically
identified as the source of transmission (Nazarowec-White
and Farber 1997). More than 70 cases of E. sakazakii infec-
tion have been reported and more than 1Æ5 million cans of
dry infant formula recalled because of contamination with
azakii has been found in several types of foods, food-pro-
cessing plants, and the environment (Iversen and Forsythe
2003). While E. sakazakii has not been reported to cause
illnesses linked to the consumption of fresh produce, it has
been isolated from lettuce, fruits, and other vegetables,
thereby representing a potential risk of causing human
infections (Kim and Beucaht 2005). Enterobacter sakazakii
possess the characteristic of resistance or tolerance against
several stresses that enable it to survive in foods and the
environment and cause foodborne illness. Several studies
have shown the stress response and resistance of E. sak-
azakii against several challenges, such as chilling, heating,
osmotic stress and drying, and natural antibiotics (Breeu-
wer et al. 2003).

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Journal compilation ª 2008 The Society for Applied Microbiology, Letters in Applied Microbiology 47 (2008) 315–321 315
E. sakazakii inhibition by compounds
There has been an increasing interest in the develop-
ment of effective natural antimicrobials as food preser-
vatives because of concerns regarding the safety of
synthetic compounds (Nair et al. 2004). Safety issues
are especially important when selecting antimicrobials
for use in infant foods. One useful approach is the
application of naturally occurring antimicrobials either
alone or in combination with other preservative strate-
gies to control the incidence of pathogens in foods
(Olasupo et al. 2004). There are several studies that
investigated the inhibitory effects of the naturally
occurring antimicrobials, such as animal derived (e.g.
lactoferrin, lysozyme), plant-derived (e.g. essential oils),
and micro-organism-derived (e.g. bacteriocin) antimi-
crobials against foodborne pathogens. Among these nat-
ural compounds, the antimicrobial properties of
essential oils derived from many plants have been
empirically recognized for centuries, but only scientifi-
cally confirmed in the last 30 years (Olasupo et al.
2004). Essential oils or their individual constituents, e.g.
carvacrol, eugenol, thymol, and cinnamic acid, are
shown to exhibit antimicrobial activity against food-
borne pathogens, such as Escherichia coli O157:H7, Sal-
monella Typhimurium, Listeria monocytogenes, and
Bacillus cereus, in addition to food-spoilage bacteria and
yeasts, and thus have potential for application as novel
food preservatives (Dorman and Deans 2000). Diacetyl
is also an antimicrobial metabolite produced by citrate-
fermenting lactic acid bacteria (Hugenholtz 1993). It is
a flavor compound found in several dairy products that
elicits a potent antimicrobial activity against various
foodborne pathogens and spoilage organisms (Kang and
Fung 1999).
Nisin is a lantibiotic class of bacteriocin produced by
strains of Lactococcus lactis, and it has found practical
application as a natural food preservative in the food
industry (Delves-Broughton et al. 1996). However, the
practical application of nisin is limited because of its
low stability, reduced activity at high pH, and poor
efficacy in certain food matrices (Pol and Smid 1999).
Combined treatment of nisin with other natural preser-
vatives, such as essential oils, might be helpful for over-
coming these limitations of nisin. Especially, an additive
or synergistic effect of combined treatment of nisin and
essential oils could result, because both nisin and essen-
tial oils act on the cytoplasmic membrane.
To date, there are no published reports of studies
investigating the effect of natural organic compounds,
such as essential oils and nisin, in inhibiting the levels of
E. sakazakii. Therefore, this study was conducted to inves-
tigate the effect of some natural organic compounds on
inhibiting the growth of E. sakazakii when applied alone
or in the combination with nisin in laboratory media.
316 Journal compilation ª 2008 The Society for Applied Microbiology, Letters in Applied Microbiology 47 (2008) 315–321
S.-Y. Lee and H.-H. Jin
Materials and methods
Bacterial strains and culture conditions
Enterobacter sakazakii ATCC 12868 and ATCC 29004
were obtained from the bacterial culture collection at
Washington State University (Pullman, WA, USA) and
used in this study. Each strain of E. sakazakii was grown
in tryptic soy broth (TSB; Difco laboratories, Detroit, MI,
USA) at 37C for 24 h.
Chemical preparation
The inhibitory compounds used in the assay included
nisin (HPLC-purified; Sigma, Korea) and five natural
organic compounds, namely diacetyl (Acros Organics,
Belgium) and the essential oils carvacrol (Sigma, Korea),
thymol (Daejung Chemicals, Korea), eugenol (Wako,
Japan) and cinnamic acid (Junsei, Japan). In all cases,
chemicals were of the highest grade available (98–100%
pure). Stock solutions (1 mol l)1) of the natural organic
compounds were prepared in 95% (v ⁄ v) ethanol, with the
exception of diacetyl that was prepared in sterile distilled
water. Further dilutions were made in the same solvents.
In the case of nisin, stock solutions (250 mg ml)1) and
dilutions were prepared in 0Æ02 N HCl. Solutions were
sterilized by filtration through a 0Æ22-lm membrane filter
(Millipore, Billerica, MA, USA).
Treatment with natural organic compounds and MIC
determination
Each strain of E. sakazakii (ATCC 12868 and ATCC
29004) was separately cultured in TSB for 18 h at 37C
and used to inoculate test tubes. Five millilitres of TSB
was placed in 8-mL tubes and autoclaved. After autoclav-
ing, natural organic compounds were added into test
tubes, corresponding to target levels of concentration (0–
5 mmol l)1) using stock solutions described earlier. For
controls, ethanol or sterile distilled water used for prepar-
ing stock solutions of natural organic compounds was
added using the same volume. Test tubes were inoculated
with 0Æ1-ml cultured E. sakazakii (c. 106 CFU ml)1) and
incubated at 37C. The optical densities of cultures at
600 nm (OD600) were measured at 0-, 3-, 6-, 12-, 24- and
48-h incubation following adjustment to 0 using the same
diluted media but without inoculation.
Combined treatment of natural organic compounds and
nisin
The two strains of E. sakazakii were cultured as described
earlier, combined, and used for inoculation for this study.
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S.-Y. Lee and H.-H. Jin
All experiments were performed using the same method
described earlier, except for the addition of 25 mg ml)1
nisin along with the natural organic compounds, and the
combined culture was used for inoculation. After inocula-
tion with E. sakazakii, optical densities at 600 nm
(OD600) were measured at 0-, 3-, 6-, 12-, 18- and 24-h
incubation. Ethanol, sterile distilled water, and 0Æ02 N
HCl were used as controls.
Statistical analysis
All experiments were repeated three to five times. Data
were analysed by analysis of variance using the anova
procedure of SAS (SAS Institute, Cary, NC, USA) for a
completely randomized design (treatment, storage time,
and treatment · storage). When the effect was significant
(P < 0Æ05), means were separated using Duncan’s multi-
ple range test.
Results and Discussion
The growth of E. sakazakii ATCC 12868 (Fig. 1) and
ATCC 29004 (data not shown) was evaluated when trea-
ted with five natural organic compounds in laboratory
media (TSB) and incubated at 37C. Carvacrol and thy-
mol were the most effective compounds for inhibiting
growth of both strains of E. sakazakii. Growth of E. sak-
azakii was observed only in the sample treated with
0Æ0625 mmol l)1 carvacrol or thymol, whereas E. sak-
azakii did not grow in samples containing higher con-
centrations of carvacrol or thymol within 48 h.
Therefore, the minimum inhibitory concentration (MIC)
of both carvacrol and thymol was 1Æ25 mmol l)1 based
on the concentrations evaluated in this study (Table 1,
Fig. 1). Although growth of E. sakazakii occurred, the
growth rate in samples treated with 0Æ625 mmol l)1 car-
vacrol or thymol was significantly lower during 48-h
incubation compared with the control (P < 0Æ05). In
contrast, cinnamic acid was the least effective at inhibit-
ing the growth of E. sakazakii among tested compounds.
Growth of E. sakazakii was observed at all tested concen-
trations of cinnamic acid. However, growth levels were
significantly lower than controls when samples were trea-
ted with higher than 1Æ25 mmol l)1 cinnamic acid
(P < 0Æ05). When samples were amended with diacetyl,
E. sakazakii failed to grow only in samples containing
5 mmol l)1 of the compound. Therefore, the MIC of di-
acetyl was 5 mmol l)1. The MIC of eugenol was also
5 mmol l)1 (Table 1). However, the inhibitory effect of
eugenol seemed to be greater than that of diacetyl
because the growth levels of E. sakazakii were lower in
samples treated with eugenol than with diacetyl at con-
centrations of 1Æ25 and 2Æ5 mmol l)1. Although the
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Journal compilation ª 2008 The Society for Applied Microbiology, Letters in Applied Microbiology 47 (2008) 315–321
E. sakazakii inhibition by compounds
results for one strain (ATCC 29004) were not shown,
the growth curves of the two E. sakazakii strains were
very similar, showing no significant differences
(P > 0Æ05). The order of inhibition resulting from the
addition of natural organic compounds was carva-
crol = thymol > eugenol > diacetyl > cinnamic acid
(Table 1).
To date, the inhibitory effects of the natural organic
compounds, such as the ones used in this study, against
E. sakazakii have not been investigated. However, several
studies reported the inhibitory effect of the same com-
pounds used in this study against other pathogens, such
as E. coli and S. Typhimurium. Olasupo et al. (2003)
investigated the inhibitory effects of the same natural
antimicrobial compounds used in our study against E. coli
and S. Typhimurium in L broth for 16 h at 37C. The
MIC values of thymol and carvacrol were 1Æ0 and
1Æ5 mmol l)1, or 1Æ0 and 1Æ2 mmol l)1 against E. coli and
S. Typhimurium, respectively. From our results, the MIC
values of both thymol and carvacrol were 1Æ25 mmol l)1;
therefore, similar MIC values were observed. In their
study, the order of inhibition of the natural organic com-
pounds against E. coli and S. Typhimurium was thy-
mol > carvacrol > eugenol > cinnamic acid > diacetyl.
These results were also similar to our results except that
diacetyl was more effective at inhibiting the growth of E.
sakazakii than was cinnamic acid in our study. In another
study, higher MIC values of 3Æ0 mmol l)1 of carvacrol
and thymol against E. coli were observed (Helander et al.
1998). Kang and Fung (1999) reported that diacetyl con-
centrations of 0Æ57 and 3Æ41 mmol l)1 were sufficient to
inhibit the growth of E. coli O157:H7 and S. Typhimuri-
um, respectively, in laboratory media and during meat
fermentation. Olasupo et al. (2003) observed MIC values
of 5Æ0 and 12Æ5 mmol l)1 for diacetyl against E. coli and
S. Typhimurium, respectively. Friedman et al. (2002)
observed MIC values of 2Æ5 and 3Æ0 mmol l)1 for eugenol
against E. coli and S. Typhimurium, respectively. Their
Table 1 Minimum inhibitory concentrations (MIC)* of five natural
organic compounds against two strains of Enterobacter sakazakii
MIC (mmol l)1)
E. sakazakii E. sakazakii
Bacterial strains ATCC 12868 ATCC 29004
Carvacrol 1Æ25 1Æ25
Thymol 1Æ25 1Æ25
Diacetyl 5Æ00 5Æ00
Eugenol 5Æ00 5Æ00
Cinnamic acid >5Æ00 >5Æ00
*The minimum concentration at which microbial growth was not
observed within 48 h at 37C in laboratory media.
317
E. sakazakii inhibition by compounds S.-Y. Lee and H.-H. Jin

(a) (b)
1·2 1·2

1·0 1·0

0·8 0·8
O.D. (610 nm)

O.D. (610 nm)

0·6 0·6

0·4 0·4

0·2 0·2

0·0 0·0

–0·2 –0·2
0 10 20 30 40 50 60 0 10 20 30 40 50 60
Treatment time (h) Treatment time (h
(c) (d)
1·2 1·2

1·0 1·0

0·8 O.D. (610 nm) 0·8

O.D. (610 nm)

0·6 0·6

0·4 0·4

0·2 0·2

0·0 0·0

–0·2 –0·2
0 10 20 30 40 50 60 0 10 20 30 40 50 60
Treatment time (h) Treatment time (h)
(e)
1·2

1·0

0·8
O.D. (610 nm)

0·6

0·4

0·2

0·0

–0·2
0 10 20 30 40 50 60
Treatment time (h)

Figure 1 Inhibition of various natural organic compounds against Enterobacter sakazakii ATCC 12868. (a) Carvacrol; (b) thymol; (c) diacetyl; (d)
eugenol, (e) cinnamic acid; and (•) 0 mmol l)1 (control); (s) 0Æ625 mmol l)1; (.) 1Æ25 mmol l)1; (,) 2Æ5 mmol l)1; ( ) 5 mmol l)1.

values were somewhat lower than our result of 5Æ0 mmol l)1 were not tested. For cinnamic acid, Olasupo
5Æ0 mmol l)1 for eugenol against E. sakazakii. However, et al. (2003) reported the MIC values to be 1Æ5
in our study, various concentrations between 0Æ5 and and 7Æ5 mmol l)1 against E. coli and S. Typhimurium,

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318 Journal compilation ª 2008 The Society for Applied Microbiology, Letters in Applied Microbiology 47 (2008) 315–321
S.-Y. Lee and H.-H. Jin E. sakazakii inhibition by compounds

(a) (b)
1·0 1·0

0·8 0·8

0·6 0·6
O.D. (610 nm)

O.D. (610 nm)

0·4 0·4

0·2 0·2

0·0 0·0

–0·2 –0·2
0 5 10 15 20 25 30 0 5 10 15 20 25 30
Treatment time (h) Treatment time (h)

(c) 1·0 (d) 1·0

0·8 0·8

0·6 0·6
O.D. (610 nm)

O.D. (610 nm)

0·4 0·4

0·2 0·2

0·0 0·0

–0·2 –0·2
0 5 10 15 20 25 30 0 5 10 15 20 25 30
Treatment time (h) Treatment time (h)
(e)
1·0

0·8

0·6
O.D. (610 nm)

0·4

0·2

0·0

–0·2
0 5 10 15 20 25 30
Treatment time (h)

Figure 2 Inhibition of various natural organic compounds with or without nisin (25 mg ml)1) against Enterobacter sakazakii. (a) Carvacrol
(0Æ625 mmol l)1); (b) thymol (0Æ625 mmol l)1); (c) diacetyl (2Æ5 mmol l)1); (d) eugenol (2Æ5 mmol l)1); (e) cinnamic acid (2Æ5 mmol l)1); and (•)
control; (s) nisin alone; (.) natural organic compounds alone; (,) natural organic compounds with nisin.

respectively. From our results, treatment with 5 mmol l)1 The combined effects of nisin with natural organic
cinnamic acid did not prevent the growth of E. sakazakii compounds against a mixture of both strains of
within 48 h. E. sakazakii were investigated (Fig. 2). Treatment with

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Journal compilation ª 2008 The Society for Applied Microbiology, Letters in Applied Microbiology 47 (2008) 315–321 319
E. sakazakii inhibition by compounds
25 mg ml)1 nisin alone did not affect the growth of
E. sakazakii. However, the combination of diacetyl and
nisin showed the synergistic effect for inhibiting growth
of E. sakazakii. When 2Æ5 mmol l)1 diacetyl was com-
bined with 25 mg ml)1 nisin, growth of E. sakazakii did
not occur after 12-h incubation, whereas growth was
observed in samples containing diacetyl alone. In contrast,
combinations of nisin with other organic compounds
failed to enhance its antimicrobial activity.
Nisin is an antimicrobial compound known to be more
effective at inhibiting Gram-positive bacteria compared
with Gram-negative bacteria (Boziaris and Adams 1999).
The nisin resistance of Gram-negative bacteria is attributed
to the protective outer membrane that forms the outermost
layer of the cell envelope (Helander et al. 1998). Natural
organic compounds, such as carvacrol and thymol, are
known to have prominent outer membrane disintegrating
properties (Helander et al. 1998). Therefore, it could be
expected that combining potentially membrane-active
organic compounds with nisin may enhance the inhibitory
effect against Gram-negative bacteria by allowing nisin to
pass through the outer membrane (Olasupo et al. 2003).
However, the combination of nisin with several organic
compounds did not enhance its effectiveness at inhibiting
E. sakazakii in our study. Furthermore, other researchers
reported that there was no synergistic effect in the combi-
nation of nisin with organic compounds against E. coli and
S. Typhimurium (Olasupo et al. 2003). However, the syner-
gistic effects of nisin with organic compounds, such as car-
vacrol or thymol, against Bacillus cereus and L.
monocytogenes were reported by other researchers (Ettayebi
et al. 2002; Pol and Smid 1999).
The results of this study indicate that natural organic
compounds can substantially inhibit the growth of E. sak-
azakii in laboratory media. As the resistance or tolerance
responses of E. sakazakii against several stresses, such as
chilling, heating, osmotic stress and drying, have been
reported in numerous studies, and have been implicated
in illnesses traced to baby foods, the development of an
effective and safe approach for reducing E. sakazakii is
paramount. Hence, the use of natural organic compounds
as food additives could be one possible way to control
E. sakazakii in various types of foods. However, further
studies investigating their inhibitory activities in foods
and their effects on the organoleptic properties of foods
are required prior to commercial application.
Acknowledgment
This work was supported by the Korea Research Founda-
tion Grant funded by the Korean Government
(MOEHRD, Basic Research Promotion Fund) (KRF
2006-331-F00055).
320 Journal compilation ª 2008 The Society for Applied Microbiology, Letters in Applied Microbiology 47 (2008) 315–321
S.-Y. Lee and H.-H. Jin
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