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Applied and Environmental Microbiology, September 1999, p. 4276-4279, Vol. 65, No. 9
0099-2240/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Efficacy of Electrolyzed Oxidizing Water for
Inactivating Escherichia coli O157:H7, Salmonella
enteritidis, and Listeria monocytogenes
Kumar S.
Venkitanarayanan,1
Gabriel O.
Ezeike,2
Yen-Con
Hung,2 and
Michael P.
Doyle2,*
Department of Animal Science, University of
Connecticut, Storrs, Connecticut 06269,1 and
Center for Food Safety and Quality Enhancement, College of
Agricultural and Environmental Sciences, University of Georgia,
Griffin, Georgia 30223-17972
Received 14 December 1998/Accepted 18 June 1999
 |
ABSTRACT |
The efficacy of electrolyzed oxidizing water for inactivating
Escherichia coli O157:H7, Salmonella
enteritidis, and Listeria monocytogenes was
evaluated. A five-strain mixture of E. coli O157:H7,
S. enteritidis, or L. monocytogenes of
approximately 108 CFU/ml was inoculated in 9 ml of
electrolyzed oxidizing water (treatment) or 9 ml of sterile, deionized
water (control) and incubated at 4 or 23°C for 0, 5, 10, and 15 min;
at 35°C for 0, 2, 4, and 6 min; or at 45°C for 0, 1, 3, and 5 min.
The surviving population of each pathogen at each sampling time was
determined on tryptic soy agar. At 4 or 23°C, an exposure time of 5 min reduced the populations of all three pathogens in the treatment
samples by approximately 7 log CFU/ml, with complete inactivation by 10 min of exposure. A reduction of 
7 log CFU/ml in the levels of the
three pathogens occurred in the treatment samples incubated for 1 min
at 45°C or for 2 min at 35°C. The bacterial counts of all three
pathogens in control samples remained the same throughout the
incubation at all four temperatures. Results indicate that electrolyzed
oxidizing water may be a useful disinfectant, but appropriate
applications need to be validated.
| |
TEXT |
Enterohemorrhagic Escherichia
coli O157:H7, Salmonella enteritidis, and
Listeria monocytogenes are food-borne pathogens of major
public health concern in the United States. A variety of foods,
including poultry, eggs, meat, milk, fruits, and vegetables, have been
implicated as vehicles of one or more of these pathogens in outbreaks
of food-borne illness (2, 4, 5). The Pathogen Reduction
program of the U.S. Department of Agriculture Food Safety and
Inspection Service recommends antimicrobial treatments as a method for
reducing or inactivating pathogenic bacteria in foods (13).
Effective methods of reducing or eliminating pathogens in foods are
important to the successful implementation of Hazard Analysis and
Critical Control Point (HACCP) programs by the food industry and for
the establishment of critical control points in restaurants,
homes, and other food service units. Washing of raw agricultural
produce with water is practiced in the industry; however, washing alone
does not render the product completely free from pathogens. Although
many chemicals generally recognized as safe (GRAS), including organic
acids, possess antimicrobial activity against food-borne pathogens,
none can eliminate high populations of pathogens when they are used
individually at concentrations acceptable in foods. Treatments of
fruits and vegetables with water containing sanitizers, including
chlorine, may reduce but not eliminate pathogens on the surface of
produce (2, 14). Hence, there is a need for, and interest
in, developing practical and effective antimicrobial treatments for the
inactivation of pathogenic microorganisms on foods.
Electrolyzed oxidizing water (EO water) is the product of a new concept
developed in Japan. Research carried out in Japan revealed that
electrolysis of deionized water containing a low concentration of
sodium chloride (0.1%) in an electrolysis chamber where anode and
cathode electrodes were separated by a diaphragm imparted strong
bactericidal and virucidal properties to the water collected from the
anode (EO water). Water from the anode normally has a pH of 2.7 or
lower, an oxidation-reduction potential (ORP) greater than 1,100 mV,
and a free-chlorine concentration of 10 to 80 ppm (10). EO
water has been experimentally used in Japan by medical and dental
professionals for treating wounds or disinfecting medical equipment.
The objective of this study was to evaluate the efficacy of EO water
for killing E. coli O157:H7, S. enteritidis, and
L. monocytogenes with a view to its potential application to
foods and food contact surfaces as an antimicrobial treatment.
Bacterial culture and media.
Five strains each of E. coli O157:H7, S. enteritidis, and L. monocytogenes were used for the study. The five strains of
E. coli O157:H7 (with origins in parentheses following
strain designations) were E06 (milk), E08 (meat), E10 (meat), E16
(meat), and E22 (calf feces). The S. enteritidis
isolates included SE180 (human), SE457 (egg), SE565 (salad),
SE294 (egg), and SE1697 (human). The five strains of L. monocytogenes were LM ATCC 19117 (sheep), LM101 (salami), LM109 (pepperoni), LM116 (cheese), and LM201
(milk). The E. coli O157:H7 and L. monocytogenes strains, but not ATCC 19117, were isolated by one of
the authors, whereas the S. enteritidis isolates were
obtained from the Centers for Disease Control and Prevention, Atlanta,
Ga. The strains of each pathogen were cultured separately in 100 ml of
sterile tryptic soy broth (TSB) (Difco Laboratories, Detroit, Mich.) in
250-ml Erlenmeyer flasks at 37°C for 24 h with agitation (150 rpm). Following incubation, 10 ml of each culture was sedimented by
centrifugation (4,000 × g for 20 min), washed, and
resuspended in 10 ml of 0.1% peptone water (pH 7.1). The optical
density of the suspension was determined and adjusted with 0.1%
peptone water to 0.5 at 640 nm (representing approximately
109 CFU/ml). The bacterial population in each culture was
confirmed by plating 0.1-ml portions of appropriately diluted culture
on tryptic soy agar (TSA) (Difco Laboratories) plates and incubating the plates at 37°C for 48 h. For each pathogen, equal portions from each of the five strains were combined, and 1 ml of the suspension was used as the inoculum (109 CFU).
EO water.
EO water was generated with a model ROX-20TA EO
water generator (Hoshizaki Electric Company Ltd., Toyoake, Aichi,
Japan). The current passing through the EO water generator and the
voltage between the electrodes were set at 19.8 A and 10 V,
respectively. A 12% solution of sodium chloride (Sigma Chemical Co.,
St. Louis, Mo.) and deionized water from the laboratory supply line
were simultaneously pumped into the equipment. The display indicator was activated and observed until the machine stabilized at a reading of
19.8 A. The EO water was collected from the appropriate outlet in
sterile containers and was used within 5 min for the microbial study.
Samples for determination of the pH, ORP, and free-chlorine concentration also were collected simultaneously.
Sample inoculation and treatments.
A volume of 9 ml of EO
water (treatment) or sterile deionized water (control) was transferred
to separate, sterile screw-cap tubes, and the caps were tightly closed.
The tubes were placed in a water bath in order to prewarm the water
samples to the desired temperature. To each tube containing 9 ml of EO
water or deionized water, 1 ml (equivalent to 109 CFU) of
the five-strain mixture of E. coli O157:H7, S. enteritidis, or L. monocytogenes was added, and the
samples were incubated in a water bath (Pharmacia LKB, Piscataway,
N.J.) at 4°C for 0, 5, 10, and 15 min; at 23°C for 0, 5, 10, and 15 min; at 35°C for 0, 2, 4, and 6 min; and at 45°C for 0, 1, 3, and 5 min. Following each incubation, the number of viable cells in each
sample was determined by plating 0.1-ml portions directly or after
serial (1:10) dilutions in 0.1% peptone water on duplicate TSA plates. Colonies of the inoculated pathogen were enumerated on TSA plates after
incubation at 37°C for 48 h. A volume of 1 ml of the inoculated solution (treatment or control) after exposure to each temperature-time combination was also transferred to separate 250-ml Erlenmeyer flasks containing 100 ml of sterile TSB and incubated at 37°C for
24 h. Following enrichment in TSB, the culture was streaked on
either sorbitol MacConkey agar no. 3 (Oxoid Division, Unipath Co.,
Ogdensburg, N.Y.) (for E. coli O157:H7), xylose lysine
deoxycholate agar (Gene-Trak, Framingham, Mass.) (for S. enteritidis), or Oxford agar (Gene-Trak) (for L. monocytogenes), and the plates were incubated at 37°C for
24 h. Representative colonies of E. coli O157:H7 and S. enteritidis from the respective plates were confirmed by
the E. coli O157:H7 latex agglutination assay (Remel
Microbiology Products, Lenexa, Kans.) and the
Salmonella latex test (Oxoid), respectively. The
colonies of L. monocytogenes on Oxford agar were confirmed
by the API-20E diagnostic test kit (Biomerieux, Hazelwood, Mo.). At
least duplicate samples of treatments and controls were assayed at each
sampling time, and the entire study with each pathogen was replicated
three times.
The pH and ORP of the EO water were measured in duplicate samples
immediately after sampling by using pH and ORP electrodes (model 50, ACCUMET meter; Denver Instrument Company, Denver, Colo.). The
free-chlorine concentration was determined by an iodometric method
using a digital titrator (model 16900; Hach Company, Loveland, Colo.).
The assay was verified periodically by using a 100 ± 0.05 ppm
chlorine standard solution (Orion Research Inc., Beverly, Mass.).
Statistical analysis.
For each treatment, the data from the
independent replicate trials were pooled and the mean value and
standard deviation were determined (11).
The mean pH, ORP, and free-chlorine concentration of EO water at the
different temperatures used for treatment are presented
in Tables
1
through
3. The mean pH and ORP of sterile deionized
water were 7.1 ± 0.15 and 355 ± 7.0 mV, respectively. No free
chlorine was
detected in deionized
water.
EO water had major antibacterial activity at 4 and 23°C on the
five-strain mixtures of
E. coli O157:H7,
S. enteritidis, and
L. monocytogenes (Table
1). At time zero, both treatment and
control samples for all three pathogens had approximate mean bacterial
counts of 8.0 log CFU/ml. At 5 min of exposure at 4°C, the
E. coli O157:H7 count in the treatment samples was reduced to less
than 1.0 log CFU/ml (detected only by enrichment in TSB for 24
h),
whereas the populations of
S. enteritidis and
L. monocytogenes were slightly greater than 1.0 log CFU/ml. All three
pathogens
decreased to undetectable levels (as determined by both
plating
and enrichment procedures) after 10 min of exposure to EO water
at 4°C. However, no differences in bacterial counts were observed
in
the control samples throughout the study. At 5 min of exposure
at
23°C, the populations of
E. coli O157:H7 and
S. enteritidis in the treatment samples decreased to less than 1.0 log CFU/ml,
whereas the
L. monocytogenes count was
reduced to 1.25 log CFU/ml.
In agreement with the results obtained at
4°C, all three pathogens
were undetectable after 10 min of contact
with EO water at 23°C.
E. coli O157:H7,
S. enteritidis, and
L. monocytogenes were more rapidly inactivated by EO water at 35 or
45°C (Tables
2 and
3) than at
4 or 23°C. At 35°C, the populations of
E. coli O157:H7
and
L. monocytogenes in the treated samples decreased to
undetectable
levels within 2 min of exposure to EO water, whereas
S. enteritidis was detected only by enrichment of the
treated sample in TSB.
After 1 min of exposure to EO water at 45°C,
E. coli O157:H7 was
killed completely (a reduction of
approximately 8.0 log CFU/ml),
whereas the populations of
S. enteritidis and
L. monocytogenes were reduced by
approximately 7.0 log CFU/ml. The bacterial counts
of all three
pathogens in control samples remained the same throughout
the
study at both 35 and 45°C.
The theoretical sequence of chemical reactions involved in the
production of EO water can be summarized as follows (
1).
During electrolysis, sodium chloride dissolved in deionized water
in
the electrolysis chamber dissociates into negatively charged
chloride
(Cl
) and hydroxy (OH
) ions and positively
charged sodium (Na
+) and hydrogen (H
+) ions.
The chloride and hydroxy ions are adsorbed to the anode,
with each ion
releasing an electron (e
) to become a radical. The
chloric and hydroxy radicals combine,
forming hypochlorous acid (HOCl),
which separates from the anode.
Two chloric radicals can also combine
to produce chlorine gas.
In the cathode section, each positively
charged sodium ion receives
an electron and becomes metallic sodium.
The metallic sodium combines
with water molecules, forming sodium
hydroxide and hydrogen gas.
A bipolar membrane separating the
electrodes enhances the electrolysis
of water to produce strong acidic
and alkali waters from the anode
and cathode,
respectively.
The antagonistic effects of chlorine and low pH on microorganisms are
well documented. Although organic acids (with low pH)
and hypochlorite
solution (with free chlorine) have been used
widely in treatments for
killing food-borne bacteria in the food
industry, systems involving
high ORP values, greater than 1,000
mV, have not normally been used.
The ORP of a solution is an indicator
of its ability to oxidize or
reduce, with positive and higher
ORP values correlated to greater
oxidizing strength (
6,
8,
9). An ORP of +200 to +800 mV is
optimal for growth of aerobic
microorganisms, whereas an optimum range
of
200 to
400 mV is
favored for growth of anaerobic microorganisms
(
6). Since the
ORP of EO water in this study was greater
than 1,100 mV, the ORP
likely played an influential role, in
combination with low pH
and free chlorine, in killing microorganisms. A
possible explanation
for the high ORP of EO water is the oxygen
released by the rupture
of the weak and unstable bond between hydroxy
and chloric radicals
(
1). It is hypothesized that the low pH
in EO water sensitizes
the outer membranes of bacterial cells, thereby
enabling hypochlorous
acid to enter the bacterial cells more
efficiently. Acid-adapted
cells of
Salmonella typhimurium
were reported to be more sensitive
to inactivation by hypochlorous acid
than nonadapted cells, due
to increased outer membrane sensitivity to
hypochlorous acid in
acid-adapted cells (
7). Experiments to
identify the contributions
of the different components of EO water to
its antimicrobial activity
are under way in our
laboratory.
The effects of EO water on the three pathogens were evaluated at low
and moderate temperatures in the interest of developing
potential
antibacterial dip treatments for unprocessed agricultural
foods. No
differences in the inactivation rates of the three pathogens
were
observed between treatment at 4°C and treatment at 23°C.
However at
35 and 45°C, much higher rates of inactivation were
observed for all
three
pathogens.
Since chlorine is one of the antimicrobial components of EO
water, we evaluated the survival of
E. coli O157:H7
and
L. monocytogenes in sterile deionized water
containing a free-chlorine concentration
of 70 to 80 ppm, which was
similar to that present in EO water.
Results revealed reductions in the
bacterial counts of both pathogens
similar to those observed with EO
water, indicating that the concentration
of free chlorine present in EO
water is sufficient to bring about
the reductions in bacterial counts
achieved by EO water. Although
chlorine is highly effective in killing
pathogenic microorganisms
in simple aqueous systems, its antibacterial
effect on microorganisms
on foods is minimal, especially in the
presence of organic materials
which convert chlorine into inactive
forms (
3). For example,
treatment of fresh produce with 200 ppm chlorine results in a
reduction in the
L. monocytogenes count of less than 2 log CFU/g
(
15).
Studies comparing the efficacies of chlorinated water
and EO water for
inactivating
E. coli O157:H7 on apples are in
progress
in our
laboratory.
Results revealed that EO water is highly effective in killing
E. coli O157:H7,
S. enteritidis, and
L. monocytogenes, indicating
its potential application for
decontamination of food and food
contact surfaces. An advantage of EO
water is that it can be produced
with tap water, with no added
chemicals other than sodium
chloride.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Center for Food
Safety and Quality Enhancement, College of Agricultural and
Environmental Sciences, University of Georgia, 1109 Experiment St.,
Griffin, GA 30223-1797. Phone: (770) 228-7284. Fax: (770)
229-3216. E-mail: mdoyle{at}cfsqe.griffin.peachnet.edu.
| |
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Applied and Environmental Microbiology, September 1999, p. 4276-4279, Vol. 65, No. 9
0099-2240/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
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