doi: 10.1111/1471-0307.

12015

ORIGINAL
RESEARCH Antibacterial activity of zinc oxide nanoparticle
suspensions on food-borne pathogens
M A H B O U B E H M I R H O S S E I N I * and F A T E M E H B F I R O U Z A B A D I
Department of Biology, Payam Noor University, Iran

Nanoparticles (NPs) are increasingly recognised for their utility in biological applications including
nanomedicine and food safety. The present study investigated the antibacterial activity of zinc oxide
(ZnO) when tested against the Gram-negative bacteria Escherichia coli as well as the Gram-positive
bacterium Staphylococcus aureus, and the effect was more pronounced with the Gram-positive than
with the Gram-negative bacteria. ZnO NPs also exhibited a preferential ability to suppress growth
of E. coli and S. aureus in milk. This study suggested that the application of ZnO NPs as antibacte-
rial agents in food systems and medicine may be effective at inhibiting certain pathogens.
Keywords Foods, Nanoparticles, Pathogens, Antibacterial agents, Zinc oxide.

of the important links of nanotechnology to the

INTRODUCTION
The increased resistance of micro-organisms to
antibiotics and the adaptation of the pathogenic
germs necessitate the preparation of composi-
tions to destroy the organism without the possi-
bility of adaptations appearing. Even though the
mechanism of action is not clearly elucidated,
the preparation of new components with antibac-
terial properties is increasingly necessary (Raz-
van et al. 2010).
Recently, nontraditional antibiotic agents have
been of tremendous interest in overcoming resis-
tance which is developed by several pathogenic
micro-organisms against most of the commonly
used antibiotics. In particular, several classes of
antimicrobial nanoparticles (NPs) and nanosized
carriers for antibiotics delivery have proved their
effectiveness for treating infectious diseases,
including antibiotics-resistant ones, in vitro as
well as in animal models (Huh and Kwon
2011). Some NPs made of metal oxides are sta-
ble under harsh processing conditions and have
selective toxicity to bacteria but also exhibit a
minimal effect on human and animal cells (Fu
et al. 2005; Brayner et al. 2006). Nanotechnol-
*Author for ogy has also the potential to impact many
correspondence. E-mail: aspects of food and agricultural systems, includ-
m.mirhossaini@gmail. ing food security, disease treatment delivery
com
methods, new tools for molecular and cellular
© 2012 Society of biology, new materials for pathogen detection
Dairy Technology and protection of the environment, are examples
science and engineering of agriculture and food
systems (Weiss et al. 2006). Nanoparticles have
been reported for the application in nanosensor
and nanotracer (Moraru et al. 2003; Jin et al.
2009).
Currently, there are very few reports related to
the application of NPs in food safety, for exam-
ple, Zinc oxide (ZnO) quantum dots were used
as the antimicrobial treatments in liquid egg
white samples. Depending on the concentration,
ZnO quantum dots could significantly inhibit or
reduce Listeria monocytogenes and Salmonella
enteritidis in liquid egg white (Jin et al. 2009).
ZnO is one of the five zinc compounds that
are currently listed as generally recognised as
safe (GRAS) by the U.S. Food and Drug
Administration (21CFR182.8991). Zinc salt has
been used for the treatment of zinc deficiency
(Saldamli et al. 1996; Lopez et al. 2002; Jin
et al. 2009). ZnO powder has been used for a
long time as an active ingredient for dermato-
logical applications in creams, lotions and
ointments on account of its antibacterial proper-
ties (Sawai 2003). Also, ZnO NPs, which are
nontoxic and biocompatible, have been utilised
as drug carriers and medical filling materials
(Zhou et al. 2006; Jiang et al. 2007). ZnO NPs
are believed to destruct lipids and proteins of
the bacterial cell membrane, resulting in a
leakage of intracellular contents and eventually
the death of bacterial cells (Huang et al. 2008;

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Vol 66, No 2 May 2013
Liu et al. 2009). In addition, generation of hydrogen perox-
ide and Zn2+ ions were suggested to be key antibacterial
mechanisms of ZnO NPs (Sawai 2003). Increased mem-
brane permeability, cellular internalisation and intracellular
structural changes of polyvinyl alcohol (PVA)-coated ZnO
NPs were also reported (Huang et al. 2008).
The inclusion of the metallic oxides in vitreous oxidic
matrices is a structural goal that leads to the obtaining of
compounds with new physical properties that can be used in
the medical fields. In the case of ZnO, it was proved that
granulation under which is used may have different effects.
The use of inorganic oxides in small quantities as an antimi-
crobial agent has also the advantage that these microele-
ments are essential for people. (Padmavathy and
Vijayaraghavan 2008). However, there are few data that
demonstrate antimicrobial efficacy of ZnO in foods. In the
present study, ZnO in the form of powder was prepared and
its antibacterial activities in culture media and milk were
investigated. The objectives of this study were to evaluate
different approaches, that is, direct additions of ZnO and
potential applications of ZnO at inhibiting two food-borne
pathogens.
MATERIALS AND METHODS
Bacterial strains, media and materials
The following bacterial strains were used in this study:
Escherichia coli PTCC1394 and Staphylococcus aureus
PTCC1431 were obtained from the culture collection of the
I.R. Department. Stock cultures were maintained at 80 °C.
The stains were propagated on Tryptic Soy Agar (TSA;
Merck, Darmstadt, Germany) at 37 °C and maintained at 0–
2 °C until use. Zinc oxide nanoparticle was purchased from
TECONAN, Spain (20–25 nm particle diameter). ZnO NPs
were more than 99.98% pure. ZnO nanoparticle was resus-
pended in sterile double-distilled water and briefly sonicated
so that they dispersed and formed a uniform colloidal sus-
pension. All the experiments were performed from a freshly
prepared colloidal suspension.
Determination of the antibacterial activity of the ZnO
nanoparticle
Spot on the lawn method
For testing antibacterial activity of ZnO NPs, spot on the
lawn method was employed. Antibacterial activity of ZnO
nanoparticle was tested by spotting 20 lL of the ZnO nano-
particle solution (0, 0.5, 2, 5, 10 mM) onto soft agar lawn
(0.6%) seeded with 107 cell/mL E. coli PTCC1394 and
S. aureus PTCC1431, respectively. Each concentration of
ZnO was placed on surface-inoculated TSA agars and
incubated at 37 °C for 24 h. Inhibition zone around speci-
mens was used to indicate antibacterial activity of each ZnO
concentration (Mirhosseini and Emtiazi 2011).
292
Detection of inhibitory activity
The Tryptic Soy Broth (TSB; Merck) culture (TSB culture;
Merck) contained (0, 0.5, 2, 5, 10 mM) of ZnO nanoparticle
inoculated with 107 cell/mL of E. coli PTCC1394 and
S. aureus PTCC1431, respectively. The bottles were shaken
at 50 rpm at 37 °C. Following inoculation, the OD
(600 nm) of the cultures was serially monitored every hour
or every 2 h up to 10–12 h, with a final reading at 24 h
(Nicole et al. 2008). Cultures of nanoparticle-free medium
under the same growth conditions were used as a control.
To avoid potential optical interference during optical
measurements of the growing cultures caused by the light-
scattering properties of the NPs, the same liquid medium
without micro-organisms but containing the same concentra-
tion of NPs cultured under the same conditions as blank
controls.
Application of ZnO nanoparticles in food safety
Two concentrations of ZnO were used as the antimicrobial
treatments in milk samples: 5 and 10 mM. The milk
contained (5, 10 mM) ZnO nanoparticle inoculated with
107 cell/mL of E. coli PTCC1394 and S. aureus
PTCC1431, respectively. The bottles were shaken at 50 rpm
at 25 °C. Following inoculation, the number of bacteria
determined every 4 h up to 8 h by plate count agar method
(Jin et al. 2009).
Statistical analysis
Antimicrobial experiments were conducted in triplicate.
Data points were expressed as the mean  SD. Data were
analysed using analysis of variance (ANOVA) from SAS
software. Duncan’s multiple range tests were used to deter-
mine the significant difference between mean values. Unless
stated otherwise, significance was expressed at 5% level.
RESULTS
Antibacterial tests
Antibacterial properties of (0, 0.5, 2, 5, 10 mM) ZnO are
measured according to the inhibition zone method against
E. coli PTCC1394 and S. aureus PTCC1431. Table 1
shows the results of inhibition zone of different concentra-
tions of ZnO. Results showed that (0, 0.5 and 2 mM) ZnO
had no inhibition zone on S. aureus and E. coli. Also,
results showed that (5 and 10 mM) ZnO had 10 and 12 mm
inhibition zone against S. aureus, respectively, but caused
growth reduction in E. coli. As it can be seen, the inhibition
area increased once with the molar content of ZnO. Also,
results showed that the larger sensitivity of the S. aureus
compared with E. coli (Table 1).
Figure 1 shows the effect of ZnO treatment on the growth
of S. aureus and E. coli in TSB broth at 37 °C. Treatments
(5, 10 mM) concentration of ZnO powder significant inhibi-
tory effect on the growth of S. aureus and E. coli during
© 2012 Society of Dairy Technology
Vol 66, No 2 May 2013

24-h incubation, as compared to the control. Among the
four concentrations of ZnO, the treatment of 10 mM of
ZnO was the most effective in S. aureus inhibition and
almost completely inhibited growth of S. aureus at 24 h.
Also, results showed that (0.5, 2 mM) ZnO had no inhibi-
tion effect against S. aureus at 24 h in TSB.
Results showed that 5 mM of ZnO had inhibitory effect
against S. aureus in TSB broth. The OD of S. aureus in
TSB broth with 5 mM ZnO was 0.705 at 8 h, 0.801 at 12 h
and 0.936 at 24 h, while the control values were 1.142 at
8 h, 1.277 at 12 h and 1.405 at 24 h, respectively. Results
showed that 5 mM ZnO caused 33.9% growth reduction in
Table 1 Inhibition zone diameters for different concentration of
ZnO.
Concentration of ZnO Staphylococcus aureus
mM mm
0
0.5
2 Gr
5 10  0.3
10 12  0.3
, no inhibition zone; Gr, Growth reduction.
1.6 (a)
1.4
1.2
1 S. aureus + 0.5 mM
OD (600 nm)
S. aureus after 24 h. Also, results showed that (0.5, 2 mM)
ZnO had no inhibition effect against E. coli. Results showed
that the OD of E. coli in TSB broth with 5 mM ZnO was
0.797 at 8 h, 0.782 at 12 h and 0.966 at 24 h, while the
control values were 1.154 at 8 h, 1.163 at 12 h and 1.304
at 24 h. These data showed that 5 mM ZnO caused 25.92%
growth reduction in E. coli at 24 h. Also, results showed
that the OD of E. coli in TSB broth with 10 mM ZnO was
0.458 at 8 h, 0.316 at 12 h and 0.269 at 24 h, while the
control values were 1.154 at 8 h, 1.163 at 12 h and 1.304
at 24 h. These data showed that 10 mM ZnO caused
79.38% growth reduction in E. coli at 24 h.
Results showed that ZnO had more inhibitory effect in
liquid medium than the solid culture against E. coli. Also,
results showed that ZnO showed strongest antibacterial
activity against S. aureus at 24 h. These data suggested that
the antibacterial activity of ZnO was concentration depen-
Escherichia coli dent which was further confirmed by the agar diffusion test;
mm 5 mM and 10 mM of ZnO have thus been selected for fur-
ther studies in milk, as they showed a signification growth
inhibition in TSB.
Gr Application of ZnO nanoparticles in food safety
Gr Two concentrations of ZnO were used as the antimicrobial
treatments in milk samples: (5, 10 mM). Figure 2 illustrates
the effect of ZnO against growth of S. aureus in milk dur-
ing 8-h storage at 25 °C. After an initial drop from 7
log CFU/mL at 8 h, S. aureus cells in the control reduced
S. aureus + 0 mM
ZnO
8 (a)

ZnO
7
0.8
S. aureus + 2 mM 6
0.6
Log CFU/mL

ZnO 5
0.4 S. aureus + 5 mM
4
ZnO
0.2
S. aureus + 10 mM 3
0 ZnO 2 S. aureus + 0 mM ZnO
–0.2 0 2 4 6 8 12 24
1 S. aureus + 5 mM ZnO
Time (h)
S. aureus + 10 mM ZnO
0
1.6
0 4 8
(b)
E. coli + 0 mM Time (h)
1.4
1.2 E. coli + 0.5 mM 12 (b)
OD (600 nm)

ZnO
1
10
E. coli + 2 mM
0.8
Log CFU/mL

ZnO 8
0.6 E. coli + 5 mM
6
0.4 ZnO
4 E. coli + 0 mM ZnO
E. coli + 10 mM
0.2 E. coli + 5 mM ZnO
ZnO
2
0 E. coli + 10 mM ZnO
0 2 4 6 8 12 24 0
Time (h) 0 4 8
Time (h)
Figure 1 Effect (0, 0.5, 2, 5, 10 mM) ZnO on growth of (a) Staphylo-
coccus aureus and (b) Escherichia coli in Tryptic Soy Broth broth at Figure 2 Effect (5, 10 mM) ZnO on growth of (a) Staphylococcus aureus
37 °C. and (b) Escherichia coli in milk at 25 °C.

© 2012 Society of Dairy Technology 293

Vol 66, No 2 May 2013
and reached to 6 log CFU/mL, while the cells in milk sam-
ple treated with (5, 10 mM) ZnO reduced and reached to
4 log CFU/mL. Also, results showed that ZnO NPs could
inhibit or reduce E. coli in milk. After an initial drop from
7 log CFU/mL at 8 h, E. coli cells in the control increased
and reached to 10 log CFU/mL, while the cells in milk
sample treated with (5, 10 mM) ZnO increased and reached
to 9 log CFU/mL. Figure 2 shows the antibacterial effect of
ZnO on S. aureus stronger than on E. coli in milk. These
findings imply that the treatment efficacy was more like that
obtained with ZnO in the first set of experiments (Figure 1).
DISCUSSION
The present study investigated the antibacterial activity of
ZnO was tested against the Gram-negative bacteria E. coli
as well as the Gram-positive bacterium S. aureus. Among
the four concentrations of ZnO, the treatment of 10 mM of
ZnO was the most effective in S. aureus inhibition and
E. coli inhibition. These data showed that 10 mM ZnO
almost completely inhibited the growth of S. aureus at
24 h, but these data showed that 10 mM ZnO caused
79.38% growth reduction in E. coli at 24 h. ZnO showed
the strongest antibacterial activity against S. aureus at 24 h.
These data suggested that the antibacterial activity of ZnO
was concentration dependent which was further confirmed
by the agar diffusion test. Antibacterial effect was more
pronounced with the Gram-positive (S. aureus) than the
Gram-negative (E. coli) bacteria. In recent years, the use of
inorganic antimicrobial agents in nonfood applications has
attracted interest for the control of microbes (Okouchi et al.
1995; Wilczynski 2000). Sawai et al. (1995) have evaluated
the antibacterial activity of 26 ceramic powders, and 10
were found to inhibit bacterial growth. Among these active
powders, MgO, CaO and ZnO exhibited strong antibacterial
activity (Sawai et al. 1995, 1998, 1999, 2000). It was found
that the treatment with ZnO formulation caused a net reduc-
tion in bacterial cells of 78% and 62% in the case of treated
cotton and cotton/polyester fabrics, while the net reduction
in fungi was calculated to be 80.7% and 32%, respectively
(Zohdy et al. 2003). Antibacterial activities of metal oxide
(ZnO, MgO and CaO) powders against S. aureus, E. coli or
fungi were quantitatively evaluated in culture media (Sawai
2003). ZnO NPs also exhibited a preferential ability to
growth suppression E. coli and S. aureus in milk.
Recently, ZnO NPs were found to have antibacterial
activity against important food-borne pathogens, such as
E. coli O157:H7 and enterotoxigenic E. coli (Roselli et al.
2003; Brayner et al. 2006; Liu et al. 2009). These studies
suggested that the application of ZnO NPs may be effective
for preserving agricultural products and food. ZnO NPs
have advantages over Ag NPs, such as a low production
cost, a white appearance and UV-blocking properties (Das-
tjerdi and Montazer 2010).
294
The recommended maximum dietary allowance for zinc
was 40 mg per day for adult (Jin et al. 2009), which is
equivalent to 100 mL of milk daily intake if 0.4 mg of ZnO
per mL of food is used. Therefore, further research is neces-
sary to investigate the efficacy of ZnO at a lower concentra-
tion level to inactivate pathogens in foods. The combined
usage of ZnO with another antimicrobial agent, such as
nisin, may be an effective approach to reduce the amount of
ZnO used per volume of food while maintaining or increas-
ing its effectiveness in microbial inactivation.
When ZnO powders were used, ZnO particles were visu-
ally observed on the bottom of bottles during incubation
because of ZnO sedimentation, which probably is the reason
for their lower antibacterial activity. The above-mentioned
results suggested that it was essential for ZnO molecules to
contact or penetrate into microbial cells to express their anti-
bacterial activities. These data might also be interpreted as a
requirement for interaction of ZnO with the bacterial cell
wall or membrane for expression of antibacterial activity.
Further study is needed to confirm the above conjecture.
The inhibition action of ZnO in microbial culture media
may come from two parts: killing and growth suppression.
At higher ZnO concentration, ZnO treatments initially kill a
sensitive subpopulation of cells and reduce the total micro-
bial population and then retard the growth by a resistant
surviving subpopulation. At lower ZnO concentration, such
as Figure 1, ZnO treatments only suppress the growth of
bacteria and result in a 4- or 6-h lag time before growth.
Our results revealed that the uniform distribution or disper-
sion of ZnO in a liquid medium (culture medium or food)
was critical to increase the efficiency of microbial inhibition.
Availability of ZnO in a medium would be more important
than its total concentration used. ZnO sedimentation in a
liquid system should be reduced or avoided.
CONCLUSION
Results showed that ZnO NPs exhibited a preferential ability
to growth suppression E. coli and S. aureus in TSB and
milk. Therefore, in the future, ZnO nanoparticle containing
formulations may be utilised for food safety and external
uses as antibacterial agents in ointments, lotions, mouth-
washes and surface coatings on various substrates to prevent
micro-organisms from attaching, colonising, spreading and
forming biofilms in indwelling medical devices.
ACKNOWLEDGEMENTS
This work was supported by Payam Noor University.
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