FOOD

MICROBIOLOGY
www.elsevier.nl/locate/jnlabr/yfmic
Food Microbiology 21 (2004) 475479
Short communication
Efcacy of ozone to reduce bacterial populations in the presence of
food components
Zeynep G. uzel-Seydim
a,
*, Paul I. Bever Jr.
b
, Annel K. Greene
b
a
Department of Food Engineering, Faculty of Agriculture, S. uleyman Demirel University, Cunur, 32260 Isparta, Turkey
b
Department of Animal and Veterinary Sciences, Clemson University, 121 Poole Agricultural Center, Box 340361, Clemson, SC 29634-0361, USA
Received 7 July 2003; accepted 12 October 2003
Abstract
Ozone is a strong oxidant and potent disinfecting agent. Even though it is new for the US, it has been utilized in European
countries for a long time. Ozone use may have many advantages in the food industry. There are numerous application areas of
ozone in food industry such as food surface hygiene, sanitation of food plant equipment and reuse of waste water. While the
destruction of bacteria by ozone has been studied extensively, relatively little information is available on the effect of various food
components on the bactericidal activity of ozone. In this study, it was aimed to compare the ozone-induced destruction of a
sporeformer, a Gram-positive bacterium, and a Gram-negative bacterium in the presence of fat, protein, and carbohydrate sources.
The efcacy of ozone to reduce bacterial populations in food components was evaluated using sterile Class C buffer, whipping
cream, and 1% solutions of locust bean gum, soluble starch, and sodium caseinate. These substrates were inoculated with spores of
Bacillus stearothermophilus or vegetative cells of Escherichia coli or Staphylococcus aureus and ozonated at 0.4 ppm for 10 min. In
general, it has been recorded that the starch provided little or no protective effects compared to the buffer control. The locust bean
gum provided an intermediate level of protection, while the caseinate and whipping cream provided the greatest levels of protection
to the bacterial populations.
r 2003 Elsevier Ltd. All rights reserved.
Keywords: Ozone; Bacillus stearothermophilus; Escherichia coli; Staphylococcus aureus; Food components; Starch; Locust bean gum
1. Introduction
Ozone (O
3
) is the highly unstable triatomic oxygen
molecule that is formed by addition of an oxygen atom
to molecular diatomic oxygen (O
2
). Ozone is generated
commercially by passing oxygen molecules (O
2
) through
an electrical charge. Thus, molecular oxygen is split into
two atoms of oxygen which are highly reactive moieties.
When a free oxygen atom (O

) encounters molecular
oxygen (O
2
), it combines to form the highly unstable
ozone molecule (O
3
). Because ozone is unstable, it
rapidly degrades back to molecular oxygen (O
2
) with the
released free oxygen atom (O

) combining with another

free oxygen atom (O

) to form molecular oxygen (O

2
) or
combining with other chemical moieties to cause
oxidation. Upon release of the third oxygen atom,
ozone acts as a strong oxidizing agent.
It is this potent oxidation capacity that makes ozone
very effective in destroying micro-organisms. Ozone has
been demonstrated to destroy a wide range of viruses
including Venezuelan equine encephalomyelitis virus,
hepatitis A, inuenza A, vesicular stomatitis virus, and
infectious bovine rhinotracheitis virus as well as several
bacteriophage strains (Akey and Walton, 1985; Bolton
et al., 1982; Botzenhart et al., 1993; Hall and Sobsey,
1993; Kim et al., 1980). Oocysts of the protozoan
Cryptosporidium parvum are also susceptible to the
microbiocidal effects of ozone (Peeters et al., 1989). The
bacteriocidal effects of ozone have been documented on
a wide variety of organisms, including Gram-positive
and Gram-negative bacteria as well as spores and
vegetative cells (Foegeding, 1985; Restaino et al.,
1995). Ozone treatments have been evaluated under a
wide variety of conditions. Possible applications of
ozone in the food industry include reducing microbial
ARTICLE IN PRESS
*Corresponding author. Tel.: +90-246-211-1681; fax: +90-246-237-
0437.
E-mail address: zeyneps@ziraat.sdu.edu.tr (Z. G. uzel-Seydim).
0740-0020/$ - see front matter r 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/j.fm.2003.10.001
populations in biolms found on stainless-steel surfaces,
treating and possibly recycling poultry chill waters,
and depuration of shellsh (Chang, 1989; Greene
et al., 1993; Schneider et al., 1991; Sheldon and Brown,
1986).
Ozone toxicity is the most important criterion for
approval of ozone in food and dairy processing plants.
It is important to monitor people who might have
contact with ozone in industry. In humans, ozone
primarily affects the respiratory tract. Symptoms of
ozone toxicity include headache, dizziness, a burning
sensation in the eyes and throat, a sharp taste and smell,
and cough. Chronic toxicity symptoms might cause
headache, weakness, decreased memory, increased pre-
valence of bronchitis and increased muscular excitability
(Hoof, 1982). Although in low concentrations ozone is
not an extremely toxic gas, at high concentration ozone
may be fatal to humans. After 12 h exposure to ozone
(0.65 ppm) dogs exhibited rapid breathing whereas long
term (46 weeks) ozone exposure (0.2 ppm) to young
rats exhibited lung distension (Barlett et al., 1974). It
was found that 0.2 ppm and higher concentrations of
ozone can cause varying degrees of damage to the
respiratory tract, depending on exposure length
(Schwartz et al., 1976). Damage of pulmonary system
involves the trachea (Schwartz et al., 1976), bronchi
(Castleman et al., 1973), and alveoli (Schwartz et al.,
1976). In the US ozone application in the food industry
has not been widely used; however, the United States
Food and Drug Administration granted generally
recognized as safe (GRAS) status for use of ozone in
bottled water in 1982. Ozone use was approved by the
US Department of Agriculture for reconditioning
recycled poultry chilling water in 1997 (USDA, 1997).
After a year of reviewing the worldwide database on
ozone, an expert panel in 1997 decreed that ozone was a
GRAS substance for use as a disinfectant or sanitizer for
foods when used in accordance with good manufactur-
ing practices. Since the US Food and Drug Adminis-
tration did not object to the expert panels ndings,
ozone has now been approved for use as a disinfectant
or sanitizer in foods and food processing in the United
States.
While the destruction of bacteria by ozone has
been studied extensively, relatively little information
is available on the effect of various food components
on the bactericidal activity of ozone. A decrease in
the germicidal activity of ozone was observed with
some bacteria in the presence of 20 ppm of bovine
serum albumen but not in the presence of 20 ppm
of soluble starch (Restaino et al., 1995). The purpose
of this research, therefore, was to compare the oz-
one-induced destruction of a sporeformer, a Gram-
positive bacterium, and a Gram-negative bacterium
in the presence of fat, protein, and carbohydrate
sources.
2. Material and methods
2.1. Preparation of food components
The efcacy of ozone to reduce bacterial populations
in food components was evaluated using sterile Class C
buffer (APHA, 1993), locust bean gum (Sigma Chemical
Company, St. Louis, MO), soluble starch (J.T. Baker
Chemical Company, Phillipsburg, NJ), sodium caseinate
(Sigma Chemical Company, St. Louis, MO), and
whipping cream (obtained from a local grocery store).
Solutions (1%) of locust bean gum, starch, and case-
inate were prepared by adding 1 g of each substrate to a
total volume of 100 ml sterile HPLC grade deionized
water (dH
2
O). The sodium caseinate required gentle
heating with stirring to enter solution. The whipping
cream was not diluted. All ve components were stored
overnight at 4

C until use.
2.2. Preparation of inoculum
Three bacteria were utilized in this experiment: (1)
Bacillus stearothermophilus ATCC 10149 thermospore
suspension (Difco, Detroit, MI), (2) Escherchia coli
ATCC 4350, and (3) Staphylococcus aureus ATCC
27661. The thermospore suspension was stored under
refrigeration until immediately before use to prevent
spore germination. A 1 ml sample of the suspension was
transferred into 9 ml of sterile Class C buffer (APHA,
1993). The diluted spore suspension was used to
inoculate the samples in the ozonation experiment.
The E. coli culture was grown in nutrient broth
supplemented with 0.2% yeast extract (NBY). The S.
aureus culture was grown in brainheart infusion
supplemented with 0.2% yeast extract (BHIY). Both
cultures were grown at 32

C for 18 h with 100 RPM

agitation provided by a rotary shaker (New Brunswick
Scientic, New Brunswick, NJ). Fresh cultures were
prepared for each replication of the experiment. A 1 ml
sample of each culture was diluted in 99 ml of Class C
buffer (APHA, 1993). The diluted cultures were used to
inoculate samples in the ozonation experiment.
2.3. Ozonation of inoculated components
For each of the ve components used in this
experiment (buffer, 1% locust bean gum solution, 1%
soluble starch, 1% sodium caseinate, and whipping
cream), a 14 ml sample was placed in an impinger
(Wheaton #753332, Wheaton Scientic, Millville, NJ).
The samples were inoculated with 1 ml of diluted
thermospore suspension, diluted E. coli culture, or
diluted S. aureus culture and mixed by drawing the
solution in and out of a pipette ten times. Ozone was
bubbled into the impinger using a Pure Power O
3
ozonator (Longmark Ozone Industries, Yreka, CA)
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Z. G. uzel-Seydim et al. / Food Microbiology 21 (2004) 475479 476
with 2 Tetra Luft Pumps (Tetra/Second Nature,
Blacksburg, VA) connected in tandem. Samples were
collected from the impinger at 0, 2, and 10 min after the
commencement of ozonation. This procedure was
repeated 5 times for each bacterium.
The amount of ozone generated in dH
2
O was
determined using an Ozone Test Kit (Model OZ-2,
Hach Company, Loveland, CO) with DPD Reagent
Powder Pillows (Hach Company, Loveland, CO). These
tests indicated that at 2 and 10 min after beginning the
ozonation process, the amount of ozone present in the
dH
2
O was 0.4 ppm.
2.4. Microbiological analyses
The samples inoculated with B. stearothermophilus
were enumerated by plating in NBY agar and incubated
at 55

C for 30 h. The samples inoculated with E. coli

were plated in NBY agar and incubated at 32

C for
48 h. The samples inoculated with S. aureus were plated
using BHIY agar and incubated at 32

C for 24 h. All
samples were plated in duplicate.
2.5. Statistical analysis
Experimental results for each organism were analysed
using ANOVA (SAS, Cary, NC, 1996) in a completely
randomized block design.
3. Results and discussion
The objective of these experiments was to evaluate the
efcacy of ozone to reduce bacterial populations in the
food components using Class C buffer as a control. For
each replication of the experiment, the same amount of
inoculum was added to a constant volume of each
substrate. Because the buffer was sterilized before use,
the inoculated buffer at time 0 min represented the total
population of each micro-organism plus any additional
micro-organisms in the food components. All plate
count data obtained were transformed into log
10
values
before statistical analysis.
The mean bacterial counts for B. stearothermophilus
after 0, 2, and 10 min of ozonation are illustrated in
Fig. 1. The initial bacterial counts for all components
were not statistically different (P>0.05) and ranged
from 6.00 to 6.04 log CFU/ml. Statistically signicant
(Po0.05) reductions in bacterial counts were observed
in the buffer at 2 and 10 min (1.21 and 4.93 log cycles,
respectively). B. stearothermophilus population reduc-
tions in the starch were statistically signicant (Po0.05)
at time 2 min (0.49 log
10
cycles) and 10 (4.56 log cycles).
Reductions in the bacterial counts in the locust bean
gum, the sodium caseinate, and the whipping cream at
2 min were not statistically different (P>0.05) from the
corresponding initial counts. At 10 min, however, the
reductions for locust bean gum (0.95 log
10
cycles) and
the caseinate (0.24 log
10
cycles) were statistically signi-
cant (Po0.05) from the analogous values at 0 and 2 min.
The B. stearothermophilus bacterial count for whipping
cream at 10 min was not statistically different (P>0.05)
from the corresponding counts at 0 and 2 min. Based on
the differences among the nal bacterial populations at
10 min, the greatest reduction occurred in the buffer,
followed in decreasing order by the starch, the locust
bean gum, the sodium caseinate, and the whipping
cream.
The mean bacterial populations for E. coli in each
component after ozonation are shown in Fig. 2. The
initial values for the counts in the buffer and the starch
were not statistically different (P>0.05). The starting
counts in the locust bean gum and the whipping cream
were signicantly different from the initial value for the
buffer, but not statistically different from each other at a
signicance level of 0.05. The bacterial counts in the
caseinate and the whipping cream were not statistically
different from one another, but were signicantly
different from the bacterial population in the buffer at
0 min. Statistically signicant (Po0.05) reductions of
6.02 and 6.10 log
10
cycles were observed in the buffer at
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Fig. 1. Mean bacterial counts in log CFU/ml (7 mean standard error
= 0.087) of B. stearothermophilus ATCC 10149 after 0, 2, and 10 min
of ozonation.
a, b, c, d, e, f
Means followed by the same letter are not
statistically different (a 0:05).
Z. G. uzel-Seydim et al. / Food Microbiology 21 (2004) 475479 477
2 and 10 min, respectively. In the starch, similar
statistically signicant (Po0.05) population reductions
were observed at 2 (5.25 log cycles) and 10 (6.11 log -
cycles) min. E. coli population reductions at 2 min in the
locust bean gum, the caseinate, and the whipping cream
were not statistically different (P>0.05) from the
corresponding values at 0 min. The reductions in the
mean bacterial counts for the locust bean gum
(3.86 log cycles), the caseinate (3.76 log
10
cycles), and
the whipping cream (1.98 log
10
cycles) were statistically
different (Po0.05) from the respective values at
0 min. At 10 min, the mean bacterial populations
for E. coli in the buffer and the starch had been redu-
ced to 0 log CFU/ml. The mean bacterial counts in
the locust bean gum, the caseinate, and the whipping
cream were statistically different (Po0.05) from
one another; however, comparisons among the reduc-
tions of these means were biased by the different initial
values.
The mean bacterial counts for S. aureus for each
substrate after 0, 2, and 10 min of ozonation are
summarized in Fig. 3. The initial values of the bacterial
populations in the ve substrates were not statistically
different (P>0.05). The mean bacterial counts at 2 and
10 min for the buffer (0.12 and 0.00 log CFU/ml,
respectively) and the starch (0.97 and 0.00 log CFU/ml,
respectively) were not statistically different (P>0.05)
from one another, but were statistically different
(Po0.05) from the mean values at 0 min. The reduction
in mean bacterial counts for the locust bean gum at 0
(0.94 log
10
cycles) and 10 (4.94 log
10
cycles) min were
statistically signicant (Po0.05) from each other and
the initial value. The mean S. aureus bacterial popula-
tions for the caseinate were statistically different
(Po0.05) at 0, 2, and 10 min and resulted in total
population reductions of 0.49 and 1.47 log cycles at 2
and 10 min, respectively. The bacterial population
reductions in the whipping cream at 2 (0.47 log cycles)
and 10 (1.02 log
10
cycles) min were statistically signi-
cant (Po0.05). At 10 min, the mean bacterial counts of
S. aureus were not statistically different (P>0.05)
between the buffer and the starch and between the
caseinate and the whipping cream. Based on the
differences among the nal bacterial populations at
10 min, the greatest population reduction of S. aureus
occurred in the buffer and the starch, followed in
decreasing order by the locust bean gum, and the
sodium caseinate and the whipping cream grouping.
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Fig. 2. Mean bacterial counts in log CFU/ml (7 mean standard
error = 0.11) of E. coli ATCC 4350 after 0, 2, and 10 min of ozon-
ation.
a, b, c, d, e, f, g, h, i, j
Means followed by the same letter are not
statistically different (a 0:05).
Fig. 3. Mean bacterial counts in log CFU/ml (7 mean standard
error = 0.13) of S. aureus ATCC 27661 after 0, 2, and 10 min of ozo-
nation.
a, b, c, d, e, f, g
Means followed by the same letter are not
statistically different (a 0:05).
Z. G. uzel-Seydim et al. / Food Microbiology 21 (2004) 475479 478
4. Conclusions
The experimental results of this research indicated
that the food components had signicant effects on the
bacteriocidal power of ozone against sporeformer, a
Gram-negative rod, and a Gram-positive cocci. These
protective effects must be considered when designing a
process which will rely on ozone for bacterial destruc-
tion. For example, in the processing of food plant waste
waters, the composition of the waste water will likely
affect the amount of ozone and the duration of the
treatment required to achieve desired bacterial popula-
tion reductions. In general, the presence of starch did
not result in a signicant protective effect at the level
used in these experiments. The presence of locust bean
gum resulted in an intermediate level of protection,
while the presence of high levels of protein (caseinate)
and fat (whipping cream) resulted in the greatest
amount of protection to spores of B. stearothermophilus
and vegetative cells of E. coli and S. aureus. Further
research is required to ascertain the precise mechanism
of the protective effects of these substrates; however, it is
hypothesized that the ozone more readily oxidized the
substrate components than the bacterial cells. Because
B. stearothermophilus spores were more resistant to the
effects of ozone in this experiment than vegetative cells,
these spores serve as a better model for designing an
ozonation treatment designed to completely eliminate
bacterial populations.
Acknowledgements
The authors wish to thank Fletcher Biggs and Perry
Strait of Anbroco, Inc., Stanley, NC, for providing the
Pure Power O
3
Ozonator.
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