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International Journal of Food Microbiology 123 (2008) 288 – 292

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Short communication
Development of TiO2 powder-coated food packaging film and its ability to
inactivate Escherichia coli in vitro and in actual tests
Chamorn Chawengkijwanich a,⁎, Yasuyoshi Hayata b
a
National Nanotechnology Center, National Science and Technology Development Agency, Thailand Science Park, Klong luang, Pathumthani 12120, Thailand
b
School of Agriculture, Meiji University, 1-1-1 Higashi-mita, Tama-ku, Kawasaki-shi, Kanagawa 214-8571, Japan
Received 8 August 2007; received in revised form 11 December 2007; accepted 18 December 2007

Abstract

Titanium dioxide (TiO2) has attracted a great deal of attention as a photocatalytic disinfecting material in the food and environmental industry.
TiO2 has been used to inactivate a wide variety of microorganisms in many applications. In the present study, we aimed to develop a TiO2 powder-
coated packaging film and clarify its ability to inactivate Escherichia coli both in vitro and in actual tests, using two different particle sizes and
two types of illumination at different intensities. No inhibition effect of the testing method itself on the growth of E. coli was observed. The cells
of E. coli were found to have decreased 3 log CFU/ml after 180 min of illumination by two 20 W black-light bulbs (wavelength of 300–400 nm)
on TiO2-coated oriented-polypropylene (OPP) film, while E. coli decreased 1 log CFU/m with black-light illumination of uncoated OPP film. The
results showed that both ultraviolet A (UVA; wavelength of 315–400 nm) alone and TiO2-coated OPP film combined with UVA reduced the
number of E. coli cell in vitro, but that the reduction of E. coli cell numbers was greater by TiO2-coated OPP film combined with UVA. The
antimicrobial effect of TiO2-coated film is dependent on the UVA light intensity (0, b0.05 and 1 mW/cm2) and the kind of artificial light (black-
light and daylight fluorescent bulbs), but it is independent of the particle size of TiO2 coating on the surface of OPP film. The surviving cell
numbers of E. coli on TiO2-coated film decreased 3 log and 0.35 log CFU/ml after 180 min of illumination by two 20 W black bulbs and two
20 W daylight fluorescent bulbs, respectively. Despite the lesser efficacy of the photocatalytic method with fluorescent lights, the survival of E.
coli cells using this method was 50% of that using fluorescent lights alone. In the actual test, the number of E. coli cells from cut lettuce stored in a
TiO2-coated film bag irradiated with UVA light decreased from 6.4 on Day 0 to 4.9 log CFU/g on Day 1, while that of an uncoated film bag
irradiated with UVA light decreased from 6.4 to 6.1 log CFU/g after 1 day of storage. The result shows that the TiO2-coated film could reduce the
microbial contamination on the surface of solid food products and thus reduce the risks of microbial growth on fresh-cut produce.
© 2007 Elsevier B.V. All rights reserved.

Keywords: Photocatalytic disinfection; Titanium dioxide; Escherichia coli; Antimicrobial packaging film

1. Introduction
Titanium dioxide (TiO2) is a photocatalyst and widely uti-
lized as a self-cleaning and self-disinfecting material for surface
coatings in many applications (Fujishima et al., 1999, 2000).
The photocatalytic reaction of TiO2 has been used to inactivate a
wide spectrum of microorganisms (Matsunaga et al., 1988;
Fujishima et al., 1999; Kim et al., 2003; Duffy et al., 2004;
⁎ Corresponding author. Tel.: +66 02 564 7100; fax: +66 02 564 6981.
E-mail address: chamorn@nanotec.or.th (C. Chawengkijwanich).
0168-1605/$ - see front matter © 2007 Elsevier B.V. All rights reserved.
doi:10.1016/j.ijfoodmicro.2007.12.017
Maneerat and Hayata, 2006). The first work on the micro-
biocidal effect of TiO2 photocatalyst was carried out with
Escherichia coli in water (Matsunaga et al., 1985). These authors
reported that E. coli was killed by contact with a TiO2 photo-
catalyst upon illumination with light. Hydroxyl radicals (•OH)
and reactive oxygen species (ROS) generated on the illuminated
TiO2 surface play a role in inactivating microorganisms by oxi-
dizing the polyunsaturated phospholipid component of the cell
membrane of microbes (Saito et al., 1992; Fujishima et al., 1999;
Manees et al., 1999; Huang et al., 2000; Kuhn et al., 2003; Cho
et al., 2004). OH radicals are approximately one thousand or
possibly ten thousand times more effective for E. coli inactivation
 C. Chawengkijwanich, Y. Hayata / International Journal of Food Microbiology 123 (2008) 288–292
than common disinfectants such as chlorine, ozone and chlorine
dioxide (Cho et al., 2004).
TiO2 is non-toxic and has been approved by the American
Food and Drug Administration (FDA) for use in human food,
drugs, cosmetics and food contact materials. Currently there is
considerable interest in the self-disinfecting property of TiO2 for
meeting hygienic design requirements in food processing and
packaging surfaces. Bactericidal and fungicidal effects of TiO2
on E. coli, Salmonella choleraesuis, Vibrio parahaemolyticus,
Listeria monocytogenes, Pseudomonas aeruginosa, Stayphylo-
coccus aureus, Diaporthe actinidiae and Penicillium expansum
have been reported (Matsunaga et al., 1985, 1988; Wei et al.,
1994; Kikuchi et al., 1997; Horie et al., 1998; Sunada et al.,
1998; Maness et al., 1999; Choi and Kim, 2000; Wist et al.,
2002; Kim et al., 2003; Cho et al., 2004; Hur et al., 2005;
Maneerat and Hayata, 2006). Application of TiO2 photocata-
lytic disinfection for drinking water production was investi-
gated in Wist et al. (2002). The development of TiO2-coated or
-incorporated food packaging and food preparing equipment
has also received attention.
The aim of the present study was to develop TiO2-coated
oriented-polypropylene (OPP) film, clarify its antimicrobial ac-
tivity and causative factors and assess its potential use in food
packaging. The TiO2-coated OPP packaging films were devel-
oped with two different particle sizes of TiO2 powder. The an-
timicrobial capacity of TiO2-coated packaging film against E. coli
was examined both in vitro and in actual tests under two kinds of
artificial light.
2. Materials and methods
2.1. Preparation of TiO2-coated packaging film
Nanoparticle TiO2 powder (anatase-phase crystal structure
with a 7-nm particle size) from Ishihara Sangyo Company, Ltd.,
Tokyo, Japan, and microparticle TiO2 powder (anatase-phase
crystal structure with a 5-μm particle size) from Wako Pure
Chemical Industries, Ltd., Tokyo, Japan, were used. An oriented-
polypropyrene (OPP) plastic film with a thickness of 30 μm was
obtained from Utsunomiya Pack Co., Ltd, Hiroshima, Japan.
TiO2 powder was mixed with organic solvents (normally ethyl
methyl ketone, toluene, isopropyl alcohol), and this TiO2
suspension was manually coated onto one side of the OPP film
using a bar coater (R.D.S., Webster, N.Y.) at room temperature.
The TiO2-coated OPP films were dried in air for 5–10 min.
2.2. Preparation of E. coli cells
E. coli (ATCC 11775) was purchased from JCM (Japan
Collection of Microorganisms, Saitama, Japan). E. coli cells
were grown in a 300 ml conical glass flask containing 200 ml of
nutrient broth (Nihon Pharmaceutical Co., Ltd., Tokyo, Japan).
The culture flask was aerobically incubated on a rotary shaker at
37 °C for 16 h. E. coli cells were harvested by centrifugation at
10,000 ×g for 10 min and washed twice with sterile deionized
water. E. coli stock solution was prepared by resuspending the
final pellets in sterile deionized water. The initial population of
289
E. coli was approximately ~ 107 CFU/ml using a colony count
method.
2.3. In vitro test of film antimicrobial activity
To assess the antimicrobial activity of TiO2-coated OPP
packaging film, test pieces (a square of 5 cm) of uncoated and
TiO2-coated film were placed in sterilized Petri dishes under
aseptic conditions. One ml of the E. coli solution was pipetted
onto each test piece in its Petri dish; the inoculated area was
covered with overhead projector (OHP) film; and the Petri dish
was covered with a lid (Fig. 1). Petri dishes containing the test
piece inoculated with E. coli were placed under light illu-
mination from above with two 20 W black-light bulbs (FL-20S
BL-B, Toshiba Co., Tokyo) or two 20 W daylight fluorescent
bulbs (FL20S, Toshiba Co., Tokyo) at 20 °C. The ultraviolet A
(UVA light, wavelength 315–400 nm) intensities on the surface
of the film test piece, as measured by a UVR-400 radiometer
(S-365 UV-sensor, Iuchi, Osaka, Japan), were 1 and b 0.05 mW/
cm2 for the black-light and fluorescent-light bulbs, respectively.
Samples were taken in three replicates at 60 min intervals for 3 h.
After the sample test piece was removed from the light, it was
immediately washed, placed in a sterile cup with 9 ml of sterile
deionized water, and shaken for 10 min. One milliliter of so-
lution was withdrawn at each sampling and diluted to 1/10, 1/
100, 1/1000, and 1/10,000 with sterile distilled water. Finally,
0.1 ml of the undiluted and diluted solutions was plated onto
desoxycholate agar (Nissui Pharmaceutical Co., Ltd., Tokyo,
Japan) for recovery of undamaged cells of E. coli. Three rep-
licate plates were used for each solution. The plates were in-
cubated for 24 h at 37 °C, and the colony-forming units (CFU)
were then counted. The effect of light alone on the E. coli was
likewise determined on uncoated OPP film. The stability of the
E. coli during the experiment was measured in the dark.
2.4. Effect of TiO2-coated film on E. coli inoculated onto cut
lettuce in actual test
Fresh lettuce (Lactuca astiva) was purchased from a local
produce market and cooled overnight at 4°C. The older, dam-
aged, outer leaves were discarded, and the inner leaves were cut
and used for the experiment. The cut lettuce pieces were dipped
in 50 ppm sodium hypochlorite solution (Wako Pure Chemical
Fig. 1. Experiment on antimicrobial activity of TiO2-coated packaging film. The
test film placed into a sterilized Petri dish was inoculated with E. coli, and the
inoculated area was covered with overhead projector (OHP) film before being
exposed to light illumination.
290 C. Chawengkijwanich, Y. Hayata / International Journal of Food Microbiology 123 (2008) 288–292
Industries, Ltd., Tokyo, Japan) for 2 min, and excess solution
was removed. The cut lettuce pieces were then dip-inoculated
with E. coli at a concentration of ~ 107 CFU/ml for 2 min at
room temperature, and the excess solution was shaken off.
Approximately 25 g of cut lettuce pieces were randomly packed
into uncoated and TiO2-coated OPP film bags. Three bags were
used for each sampling per treatment. The disinfection
experiment was carried out under black-light illumination at
room temperature for 48 h.
In order to analyze the population of E. coli, 25 g of lettuce
was placed into 225 ml of sterile deionized water and mixed
using a stomacher bag (180 mm × 300 mm, Kanto Chemical
Co., Inc., Tokyo, Japan) for 120 s. Decimal dilutions were made
to create countable dilutions in sterile deionized water. Finally,
0.1 ml of the undiluted and diluted solutions was plated onto the
plate. Three replicate plates were used for each dilution. The
plates were incubated for 24 h at 37 °C. The number of viable E.
coli cells was presented as CFU/g cut lettuce.
3. Results and discussion
The experiment on the antimicrobial activity of the TiO2-
coated OPP film was carried out as shown in Fig. 1. The
surviving number of E. coli cells on the surface of uncoated and
TiO2-coated OPP film in the dark condition did not decrease
after 3 h of experimental period (data not shown). This suggests
that the antimicrobial testing method itself did not inactivate E.
coli during the experiment. When exposed to black-light
illumination, the TiO2-coated OPP film exhibited photocatalytic
inactivation of E. coli (Fig. 2). The cells of E. coli were found to
have decreased 3 log CFU/ml and 1 log CFU/ml after 180 min
of black-light illumination on TiO2-coated OPP film and
uncoated OPP film, respectively. This result showed that
inactivation of E. coli both by the photocatalytic disinfection
of TiO2-coated film and by photo-activation of UVA light
derived from black-light bulbs. The effect of UVA light
intensity on the inactivation of several bacteria including E.
coli has been previously reported in the literature (Fujita and
Fig. 2. Inactivation of Escherichia coli in an in vitro test with uncoated OPP film
and TiO2-coated OPP packaging film under black-light illumination (UVA light
intensity of 1 mW/cm2). Each value corresponds to the mean of three replicates,
three plates per replicate. The vertical bar indicates the standard error (SE).
Fig. 3. Inactivation of Escherichia coli with microparticle and nanoparticle
TiO2-coated OPP packaging film under black-light illumination. Each value
corresponds to the mean of three replicates, three plates per replicate. Vertical
bar indicates the standard error (SE).
Suzuki, 1978; Tuveson et al., 1988; Yoshimura et al., 1996;
Bintsis et al., 2000). With relatively low energy, UVA light
damages cells through the oxidative stress caused by oxygen
radicals within the cells (Bock et al., 1998). However, our data
show that the inactivation rate of E. coli with TiO2-coated OPP
film plus UVA light is higher than that with UVA photo-
inactivation alone. This suggests that the synergetic effect of
TiO2-coated packaging film with UVA light provides a greater
advantage. Using UVA irradiation of 0–2 mW/cm2 did not
induce abnormal ripening behavior, oxidative stress, fruit tem-
perature or physical damage in tomatoes (Maneerat et al., 2003).
The cell numbers of E. coli in the in vitro test using micro-
particle TiO2-coated film and nanoparticle TiO2-coated film were
similar (Fig. 3). The data showed that the antimicrobial effect of
TiO2-coated film is independent of the particle size of TiO2 on the
surface of plastic film. This result maybe because the TiO2
particles were partially embedded and agglomerated on the
surface of the plastic film, so that the higher surface area of TiO2
nanoparticles could not significantly enhance the E. coli
inactivation effect. Generally, it is accepted that the high surface
area of TiO2 nanoparticles is important in increasing the gas-phase
photocatalytic activity of volatile organic compounds (VOCs)
(Anpo et al., 1987; Maira et al., 2000). In our preliminary study,
the activity of nanoparticle TiO2-coated OPP film on gas-phase
photocatalytic oxidation of ethylene was higher than that of
microparticle TiO2-coated film (data not shown); however, no
such trend was observed in the photocatalytic disinfection of E.
coli in the present work. This difference may be due to the fact that
Table 1
Surviving cells of Escherichia coli in vitro test under black-light lamp and
fluorescent-light lamp illuminations for 3 h
Sample film Artificial lamp UVA light Starting cell Cell numbers
intensity numbers after 3 h
(mW/cm2) (log CFU/ml) (log CFU/ml)
TiO2-coated film Black light 1 7.8 ± 0.18a 4.8 ± 0.19b
TiO2-coated film Fluorescent light b0.05 7.8 ± 0.18a 7.45 ± 0.98a
Uncoated film Fluorescent light b0.05 7.8 ± 0.18a 7.7 ± 0.75a
Values represent mean (±S.D.) of measurement recorded on nine independent
sample pieces per treatment. Values followed by the same letter did not differ
significantly according to the LSD test (p b 0.05).
 C. Chawengkijwanich, Y. Hayata / International Journal of Food Microbiology 123 (2008) 288–292
gaseous ethylene could diffuse into the embedded and agglom-
erated TiO2 particles while the liquid suspension of E. coli cells
only contacted the surface of TiO2 agglomerates. Thus, the effect
of particle size of TiO2 powder-coated packaging film on
photocatalytic disinfection of E. coli was not significantly
observed.
The inactivation rate of E. coli with TiO2-coated film was
dependent on the UVA light intensity derived from two kinds of
artificial light sources (Table 1). When the UVA light intensity
was decreased from 1 to b 0.05 mW/cm2 the antimicrobial
efficacy of TiO2-coated OPP film was decreased. Similar
observations were made by Onoda et al. (1988) and Horie et al.
(1998) on Streptococcus mutans and E. coli with TiO2 powder
and TiO2 slurry. This can be explained by the fact that the intensity
of UVA light obtained from black-light bulbs illumination was
higher than that obtained from the fluorescent-light bulbs,
resulting in greater OH radical formation on the surface of the
TiO2-coated film. The linear correlation between inactivation of
E. coli and OH radical concentration in TiO2 photocatalytic
disinfection has been reported (Cho et al., 2004).
In the actual test, the cell number of E. coli obtained from the
cut lettuce packed in a TiO2-coated OPP film bag decreased from
6.4 to 4.9 log CFU/g after 1 day of storage while that obtained
from cut lettuce packed in an uncoated film bag only decreased
from 6.4 to 6.1 log CFU/g (data not shown). In addition, the
number of E. coli from cut lettuce packed in uncoated film after
2 days was higher than the initial concentration of E. coli while
that obtained from TiO2-coated OPP film remained lower than the
initial concentration (data not shown). The result suggests that the
TiO2-coated film can reduce the microbial contamination on the
surface area of solid food products and thus may reduce the risk of
microbial growth in food packaging.
The results of this study show that, in both in vitro and actual
tests, it was technically feasible for the TiO2-coated film to cause
E. coli inactivation. The TiO2-coated OPP film exhibited a
bactericidal effect when exposed to UVA light. The extent of E.
coli inactivation of TiO2-coated film was relevant to the UVA
intensity and kind of artificial light sources. The photocatalytic
disinfection activity of TiO2-coated film under black-light bulb
illumination was 8 times higher than under the fluorescent-light
bulb illumination. The number of E. coli on cut lettuce packed in a
TiO2-coated film plus UVA light was lower than that subjected to
UVA light alone.
Acknowledgments
This work was fully supported by the JSPS postdoctoral
fellowship program for foreign researchers of the Japan Society
for the Promotion of Science (JSPS). We are also grateful to the
Utsunomiya Pack Company, Hiroshima, Japan, for the
assistance in preparation of a TiO2-coated plastic film.
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