9-effectiveness.p65
UNIVERSITAS SCIENTIARUM
Enero-junio de 2005
Revista de la Facultad de CienciasPONTIFICIA UNIVERSIDAD JAVERIANA
Vol. 10, No. 1, 97-108
EFFECTIVENESS OF ELECTROLYZED OXIDIZING WATER
Listeria monocytogenes IN LETTUCE
Casadiego Laíd Paola1, Cuartas Vivian Rocío1, Mercado Marcela 1,
Díaz Milciades2 y Carrascal Ana Karina1
1Laboratorio de Microbiología de Alimentos, Departamento de Microbiología
2Departamento de Física, Facultad de Ciencias, Pontificia Universidad Javeriana,
Cra. 7 No. 40-62 Bogotá, Colombia
The effectiveness of electrolyzed oxidizing (EO) water for the inactivation of
L. monocytogenes insuspension and when inoculated on lettuce leaves was evaluated. An electrolytic cell for the production ofEO water was built and a solution of 5% NaCl was used. The EO water produced had a residual chlorineconcentration of 29 parts per million (ppm) and pH 2.83. Ten strains of
L. monocytogenes isolated fromprocessed chicken (109 CFU/ml) were inoculated into 9 ml of EO water or 9 ml of deionized water(control) and incubated at 15°C for 5, 10, 15 and 20 min. The surviving population of each strain wasdetermined on Columbia agar. An exposure time of 5 min reduced the populations by approximately 6.6log CFU/ml. The most resistant strains to sodium hypochlorite (NaOCl) were selected and used in a strainmixture (9.56 log CFU/ml, 109UFC/ml approximately) for the inoculation of 35 lettuce samples, by thedip inoculation method using distilled water as control. The population mean of
L. monocytogenes aftertreatment with EO water and distilled water was reduced by 3.92 and 2.46 log CFU/ml respectively (p=0.00001). EO water and 6% acetic acid (vinegar) were combined to improve the EO water effect on
L.
monocytogenes inoculated in lettuce; the effectiveness of this combination was examined. The resultsshowed that there was a synergistic effect of both antimicrobial agents (population reduction by 5.49 logCFU/ml approximately) on the viability of
L. monocytogenes cells.
Keywords: Disinfectant, EO water, Lettuce,
L. monocytogenes.
En este estudio se evaluó la efectividad del agua electrolizada oxidadora (EO) en la inactivación de
Listeria monocytogenes en suspensión e inoculada en lechuga, para lo cual se construyó una celdaelectrolítica que permitiera la producción de agua EO a partir de una solución de NaCl al 5%, con unaconcentración de cloro residual de 29 partes por millón (ppm) y pH 2.83. Inicialmente se tomaron 10cepas de
L. monocytogenes aisladas de pollo procesado, las cuales fueron inoculadas en 9 ml de agua EOo 9 ml de agua desionizada estéril (control) e incubadas a 15°C durante 5, 10, 15 y 20 minutos. Lapoblación sobreviviente de cada cepa se determinó por recuento en placa en agar Columbia, obteniéndoseuna reducción de 6.6 UL en promedio a los 5 minutos de exposición. A partir de estas cepas se seleccio-naron las cinco más resistentes a la acción del hipoclorito de sodio, las cuales fueron utilizadas comosuspensión mixta (9.56 UL, 109UFC/ml aproximadamente), para inocular 35 lechugas por el método deinmersión. Después de la inoculación se sumergieron 6.25 g de cada lechuga en 375 ml de agua EO oagua destilada (control) a 15 °C durante 5 minutos. La población promedio de
L. monocytogenes despuésdel tratamiento con agua EO y con agua destilada, se redujo en 3.92 y 2. 46 UL respectivamente. Se
Universitas Scientiarum Vol 10, No. 1, 97-108
demostró que el agua EO tiene un efecto bactericida estadísticamente significativo (p=0.00001) Paramejorar el efecto del agua EO sobre
L. monocytogenes inoculada en lechuga, se evaluó su efectividad encombinación con ácido acético al 6% (vinagre). Los resultados obtenidos (reducción de la población en5.49 UL aproximadamente) muestran que hay un efecto sinergista de ambos agentes antimicrobianossobre la viabilidad de las células de
L. monocytogenes.
Palabras clave: Agua EO, Desinfectante, Lechuga,
L. monocytogenes.
containing a low concentration of sodiumchloride in an electrolysis chamber where
Nowadays the consumption of vegetables
anode and cathode electrodes are separated
in Colombia has become a critical factor in
by a diaphragm imparted strong
the development of a wide variety of
bactericidal and virucidal properties to the
enteric, parasitic and viral diseases, of
water collected from the anode (EO water).
different levels of seriousness like typhoid
Water from the anode normally has a pH of
fever, amoeba infections and listeriosis (this
2.7 or lower, an oxidation-reduction
last is statistically ascribed in 12% of the
potential (ORP) greater than 1,100 mV, and
cases to the consumption of lettuce in the
a free chlorine concentration of 10 to 80
Cundiboyacense highlands) among others.
ppm in the form of hypochlorous acid. This
Listeria monocytogenes is a food-borne
use of electrolyzed oxidizing water in the
pathogenic microorganism that has a high
disinfection of vegetables substantially
rate of mortality (23%); it is resistant to stress
eliminates food-borne pathogens, avoiding
conditions, which makes it difficult to
transmission of diseases (Cháves
et al.,
destroy, and it is easy to swallow through
the consumption of vegetables; thismicroorganism is the causal agent of a
Previously, the effectiveness of EO water
generalized disease called Listeriosis
has been evaluated for inactivating
(ICMSF, 1996). To solve this problem,
different pathogens like
Escherichia coli,
washing processes and disinfection of food
Salmonella enteritidis, and Listeria
including the use of chemical substances
monocytogenes, obtaining a considerable
like chlorine, organic acids and ozone have
reduction in logarithmic units of CFU in
been implemented; in some cases these
comparison with the initial population in
substances have reduced effects on
tomatoes, lettuce, kitchen cutting boards
pathogenic microorganisms, because those
and
in vitro experiments, at different
that have not been removed multiply; due
temperatures and storage conditions.
to this problem, there is a need for an
Cháves
et al in 2004 designed an
effective method to inactivate food-borne
electrolytic chlorinator to produce EO water
pathogens in food.
that was used efficiently for the eliminationof microorganisms present in lettuce. The
Electrolyzed Oxidizing Water (EO water)
objective of this study was to evaluate the
is the product of a new concept developed
effectiveness of EO water produced in an
in Japan and it could be an effective
electrolytic cell to inactivate
L.
alternative for disinfectant treatment of
monocytogenes
in suspension and in lettuce
fresh products like vegetables
leaves, and in this way, to standardize the
(Venkitaranayanan
et al., 1999a and Koseki
action time required to inactivate the
L.
et al., 2003). Electrolysis of deionized water
Enero-junio de 2005
MATERIALS AND METHODS
2.3. Effect of EO water for inactivating
L. monocytogenes in culture
2.1. Electrolytic cell and EO water
characterization
One ml aliquots from
L. monocytogenesculture in the log phase were taken and se-
Following the parameters of Cháves
et al.,
rial dilutions in 9 ml of EO water from 10-1
2004, with the modification that two late-
to 10-6 were performed. The bactericidal
ral holes were added for the exit of the two
activity of the EO water was evaluated at 5,
types of water, a 3.3 L glass cell was built
10, 15 and 20 min of exposure time at room
with a PVC membrane (Darnel®) in order
temperature. After incubation time, the
to separate the acid and alkaline
surviving cells of
L. monocytogenes in each
electrolytes, allowing the ions to move to
treatment were determined; 1 ml of each
the electrodes. The material used as
dilution was taken in order to do a recount
electrodes was graphite, the applied voltage
on Columbia agar plates.
L. monocytogenes
was 14 volts and the current was 0.4
colonies were counted after 48 h of
amperes. A 5% solution of sodium chloride
incubation at 37°C. The recount was done
was prepared to feed the electrolytic cell.
in duplicate (Venkitanarayanan
et al.,
During the electrolysis of water, the pH in
both acid and alkaline water was
2.4. Effect of EO water for inactivating
determined at 3, 5, 7 and 10 minutes; a pH-
L. monocytogenes inoculated on
meter previously calibrated made by the
Center of Interfaculty Equipment (CEIF) ofthe Universidad Nacional de Colombia was
The five strains most resistant to sodium
used for these evaluations. The free
hypochlorite were chosen from among the
available chlorine present in acidic
initial ten strains by a diffusion test using
electrolyzing water was also determined at
Waltman No. 3 paper disks saturated with
the same times using, a photometric DPD
hypochlorite concentrations (5250, 52.5
kit (ref. 1.00598.0001 Merck, Darmstadt,
and 0.525 ppm), and these were selected
and used in a strain mixture for theinoculation of 35 lettuce samples by the
2.2. Selection and growth curve of L.
dip inoculation method as described by
Park
et al., 2001, Bari
et al., 2003 andKoseki
et al., 2003. Sterile distilled water
Ten strains of
L. monocytogenes supplied
was used as a control; each strain of
L.
by the Laboratorio de Microbiología de
monocytogenes was cultured in 10 ml of
Alimentos de la Pontificia Universidad
Columbia broth supplemented with 50 µg/
Javeriana, which were previously isolated
ml of nalidixic acid at 37 °C during 5 hours
by Correa and Fonseca in 2004 from
at 120 rpm. Then, the suspension was
processed chicken, were used. To build the
transferred to 100 ml of Columbia broth in
growth curve, 3 ml of suspension were
a 250 ml Erlenmeyer flask, and it was
taken every 2 hours during the fermentation
incubated under the same conditions
process of
L. monocytogenes in Colum-
during 12 hours. The cells were collected
bia broth supplemented with 50 mg/ml
by centrifugation (3.500 rpm, 15 min at
of nalidixic acid in order to determine
room temperature) and the resulting pellet
the biomass by optical density at 540 nm,
was resuspended in 14 ml of sterile peptone
to establish the beginning of the log
water (0.1%, pH 7.5) distributed in seven
tubes of 15 ml each. From each suspension,
Universitas Scientiarum Vol 10, No. 1, 97-108
equal volumes were taken and then
2.6. Minimal Inhibitory Concentration
combined to form an inoculum mix from
(MIC) Test
the five strains with a final volume of 125
Ten 1:2 serial dilutions were made from a
ml. The inoculum was maintained at room
5.6 % sodium hypochlorite solution using
temperature, and it was applied the lettuce
sterile distilled water; dilutions were
within one hour of preparation. The
inoculated with 10 µl containing
L.
population of the
L. monocytogenes
monocytogenes suspension in lag phase;
inoculum was determined from a 10-8
after a 5 minute exposure time, 10 µl from
dilution of the dip by recounting on Co-
each dilution was transferred to tubes
lumbia agar plates in duplicate.
containing 2 ml of Columbia brothsupplemented with 50 µg/ml of nalidixic
Thirty five units of lettuce were purchased
acid. These tubes were incubated at 37 °C
from a local supermarket and were stored at
for 48 hours. After this, MIC was determined
4 °C before being inoculated, within a
by turbidity at each sodium hypochlorite
maximum time of two days (Österblad
et
dilution (Lúnden, 2004). The MIC test was
al., 1999). Following the inoculation
done in triplicate. This procedure was also
method described by Koseki
et al in 2003,
done on the most sensitive strain in order
treatment of the inoculated lettuce leaves
to compare the results with the five strains
was performed by immersing 6.25 g of
used in this study.
lettuce in 375 ml of EO water or distilledwater (control) in plastic bags during 5
2.7. Statistical Analysis
The results obtained were analyzed througha t student hypothesis test.
The previously treated lettuce samples werewashed with sterile peptone water and
3. RESULTS AND DISCUSSION
macerated for one minute. 10-2 and 10-3dilutions were made from the resultant
3.1. Electrolytic cell and EO water
solution from the EO water treatment and
The electrolyzed water was obtained in an
10-4 and 10-6 dilutions were made from the
electrolytic cell that was built according to
distilled water treatment. 0.1% sterile
section 2.1. EO water was produced in a
peptone water was used for the dilutions.
relatively short time with low power
One ml of each dilution was taken for a
consumption, around 6 watts, (14 volts and
recounting of the surviving population of
0.4 amperes). The required EO water should
L. monocytogenes. A dip recount on Co-
have had the same characteristics as that
lumbia agar plates was carried out in
used in previous studies to inactivate
L.
duplicate, and the plates were incubated at
monocytogenes, where EO water was used
37 °C for 48 hours.
with a pH of 2.5 and 72 ppm residualchlorine, and with a pH of 2.63 and 43
2.5. EO water and 0.6% acetic acid
ppm or 48.5 ppm residual chlorine
combined effect on L. monocytogenes
(Venkitanarayanan
et al.,1999,
inoculated on lettuce
Venkitanarayanan
et al., 1999a), with a pH
of 2.5 and 45 ppm residual chlorine (Park
Three treated lettuce samples were then
et al., 2001) and with a pH of 2.6 and 30
exposed to a vinegar solution after EO water
ppm residual chlorine (Bari
et al., 2003).
treatment or distilled water treatment.
Additionally, a disinfectant solution that
The microbiologic analysis was done as
would not change the lettuce flavor was
described earlier.
Enero-junio de 2005
First, 2.5%, 3.0%, 3.5%, 4.0%, 4.5%, 5.0%
pH solutions, which confirms the results
and 5.5% NaCl concentrations were
obtained by Len
et al. in 2002, who found
evaluated. After 5 minutes of electrolysis,
that upon adjusting the pH of water from
the pH was lower than 3.0 (see figure 1);
2.5 to 4.0 using acetic acid solution, the
therefore, at this time the first water sample
stability of chlorine was greater by a nine-
was taken to determine the residual
fold factor.
chlorine concentration present, and thesecond was taken after 10 minutes of
3.2. Effect of EO water for inactivating
electrolysis. The highest residual chlorine
L. monocytogenes in culture
concentration (47.68 ppm) was obtained ina 5% NaCl solution, after 10 minutes of
The bacterial population was reduced to
electrolysis, and the lowest (0.38 ppm) was
undetectable levels in a 10-2 dilution (as
obtained in a 2.5% NaCl solution after 5
determined by dip recount) after 5 minutes
minutes of electrolysis (see figure 2). The
of EO water treatment; whereas in the con-
highest residual chlorine concentration
trol treatment, the logarithmic units of CFU/
obtained between 2.5% and 3.5% NaCl
ml (log CFU/ml) were constant (see figure
solutions was 5.83 ppm, obtained in a 3.5%
3). The highest reduction was obtained
NaCl solution; these solutions were
with strain 227, which decreased to
discarded. A 5% NaCl solution was used in
undetectable levels in a 10-1 dilution (as
this study, because it had the highest
determined by dip recount), and the lowest
reduction was obtained with strain 244,where after 5 minutes of exposure time, the
Once electrolysis time and NaCl
population decreased by 5.0 log CFU/ml,
concentration were determined, pH and re-
and only after 10 minutes of exposure time,
sidual chlorine stability were evaluated
growth in 10-2 dilutions was not detected.
between 24 and 96 hours. A new electrolysis
An exposure time of 5 min reduced the
was done using a 5% NaCl solution, and a
populations by approximately 6.6 log
residual chlorine concentration of 47.33
CFU/ml. According to these results, it was
ppm and a pH of 2.31 were obtained. The
possible to conclude that the most resistant
pH value remained on the same scale with
strain to EO water effect was strain 244 and
a mean of 2.3. In contrast, the chlorine
the most sensitive one was strain 227. These
measurements were unstable, because the
results were later confirmed by the Minimal
levels decreased drastically after 24
Inhibitory Concentration test, using disks.
Between 5 and 20 minutes of exposure,
measurements were stopped at 48 hours of
there were no changes in
L. monocytogenes
storage because at this time the residual
populations. The microorganism recount in
chlorine concentration was 5.48 ppm.
a 10-1 dilution was inconsistent, so only the
These results were consistent with those
10-2 dilution values were taken.
described by Kirk
et al. in 1962 who statedthat in acid or alkaline solutions, the
An exposure time of 5 min reduced the
hypochlorous acid dissociates, forming
populations by approximately 6.6 log
hydrochloric and chloric acids, and in
CFU/ml, a high bactericidal effect,
strongly acid solutions, chlorine, or chlorine
according to Delgado
et al in 2003, who
and oxygen are released. Additionally, Ro-
suggested that a disinfectant solution has
jas and Guevara, 1998, concluded that
to reduce the population by 5.0 log CFU.
hypochlorous acid ionization has an
In this study, EO water with 28. 72 ppm of
equilibrium constant that depends on the
residual chlorine and pH 2.83 was used. Our
Universitas Scientiarum Vol 10, No. 1, 97-108
results are supported by Venkitanarayanan
of available chlorine was observed by
et al in 1999; in their study, the effect of
oxidation-reduction reactions with some
EO water to inactivate
L. monocytogenes
vitamins, lipids and minerals. They also
was evaluated, and they determined that
determined that the combined chlorine
after 5 minutes of exposure time to EO water
bactericidal effect on
E. coli was lower than
(48.5 ppm residual chlorine and pH of 2.63),
the free chlorine bactericidal effect. These
the population decreased by 6.64 log CFU/
authors suggest that for practical use of EO
water in the food industry, the residualchlorine concentration must be increased
From the results obtained in this phase, it
to avoid the organic matter effect.
was possible to determine that the treatment
Furthermore, the results obtained in lettuce
time for the evaluation of the EO water effect
treatments suggest that the action time of
on
L. moncytogenes inoculated on lettuce
EO water must be increased to obtain a
should be 5 minutes. Moreover, the most
greater microbial reduction. The results of
resistant strains to chlorine were selected
this study show the need of increasing the
by the MIC test using disks; with these
available chlorine concentration in EO
strains, a mixed inoculum to inoculate
water produced in an electrolytic cell for
lettuce samples was made. The most
inactivation of
L. monocytogenes to
resistant strains were 155, 244, 91, 131 and
reduction levels lower than those observed
by Park
et al. in 2001, where after 3 minutesof exposure to EO water (45 ppm residual
3.3. Effect of EO water for inactivating
chlorine and pH 2.5) the
L. monocytogenes
L. monocytogenes inoculated on
population inoculated on lettuce decreased
by 5.5 log CFU/g; in the present study theresidual chlorine concentration was higher
The five strain mixtures had a population
by 16 ppm. In the Bari
et al., 2003
, study,
mean of 9.56 log CFU/ml (109 CFU). The
chlorinated water (200 ppm) and EO water
reduction mean of
L. monocytogenes
(30.3 ppm residual chlorine and pH of 2.6)
inoculated on lettuce was 3.92 log CFU/g
effects were evaluated on
L. monocytogenes
(see figure 5). In sample number 31, the
inoculated on tomatoes. They obtained a
highest reduction was obtained at 5.82 log
reduction in population by 4.76 log
CFU/g, and in sample number 2 the lowest
CFU/g and 7.54 log CFU/g per tomato
reduction was obtained at 2.48 log CFU/g.
respectively. However, Beuchat and Brakett
These results show that the EO water's effect
in 1990 proved the low effectiveness of
on the
L. monocytogenes culture is higher
chlorine solutions prepared with from
than the effect on
L. monocytogenes
200 to 250m ppm of free chlorine for
inoculated on lettuce because the
inactivation of
L. monocytogenes
hypochlorous acid reacts with organic
inoculated on lettuce, reducing the
matter present in the lettuce, losing its
population only by 1.36 log CFU/g. These
disinfectant power (Snoeyink and Jenkins,
results were lower than the ones obtained
2002). However, the population reduction
with EO water, due to its higher effect on
was statistically significant (p=0.00001).
microorganisms than a conventionalchlorine solution, because of its properties
Results of the present work are consistent
like low pH and high ORP (Kim
et al., 2000
with Oomori
et al. in 2000, who
and Koseki
et al., 2002).
demonstrated that available chlorine wastransformed to N- Chlorates compounds by
A population mean reduction of 2.46 log
aminoacids and proteins present. A removal
CFU/ml was observed in the control
Enero-junio de 2005
treatment, obtained through the washing
because they were isolated from processed
process of lettuce, in which many
chicken, and these were exposed to a
microorganisms were removed by water.
high chlorine concentration during the
Another reason could be osmotic stress due
prechiller and chiller processes, indicating
to hypotonic characteristics of distilled
that probably the cells had been exposed
water; namely, a low solute concentration
to a sublethal disinfectant concentration.
that produces cellular lysis (Madigan 2001).
Resistance could be due to a flux bomb or
Additionally, the pH of distilled water was
modifications in cell walls (Mc Donell and
between 5 and 6, which contributed to the
Russell, 1999).
3.6. Statistical Analysis
3.4. EO water and 0.6% acetic acid
combined effect on L.
The reduction in
L monocytogenes
monocytogenes inoculated on lettuce
population was statistically significant after5 minutes of immersing lettuce leaves in
Based on the previous results, the combined
EO water (p= 0.00001 and CI=95%).
effect of EO water and 0.6% acetic acid on
L. monocytogenes inoculated on lettuce
was evaluated, and it was concluded that ahigh reduction in log CFU/g was obtained
A 5 % NaCl solution had the highest resi-
when lettuce was treated with acetic acid
dual chlorine concentration (mean of 29
after EO water treatment. The
L.
ppm) and a pH value of 2.83 during 10
monocytogenes population decreased by
minutes of electrolysis.
5.74, 5.14 and 5.6 log CFU/g for the samples36, 37 and 38 respectively; the mean value
The L. monocytogenes population had a
reduction was 5.49 log CFU/g. These
mean decrease of 6.6 log CFU/ml after 5
results show that EO water can be employed
minutes of exposure to EO water; however
as a disinfectant, but a combined treatment
this reduction can probably be obtained in
with another disinfectant like 0.6% acetic
acid for improved bactericidal effect issuggested; other antimicrobial agent
When lettuce was treated with 0.6% acetic
combinations like nisin and essential oils
acid after EO water treatment, there was a
(carvacrol or tymol) have shown a
higher reduction in cell population (5.49
synergistic effect for the inactivation of
L.
log CFU/g), which indicates that the
monocytogenes (Pol and Smid, 1999 cited
combined treatment with another
by Delgado
et al., 2003)
microbicidal agent improves the EO watereffect.
3.5. CMI Test
According to MIC test results, trains used
Turbidity was observed from an 875 ppm
in this study show an acquired resistance
NaClO dilution in strain 163, from 439.5
to chlorine due to their place of origin.
ppm in strain 121, from 218.75 ppm instrains 91 and 244, and finally from 109.37
ppm in strain 155. Strain 227 did not growin any NaClO dilution, which confirms its
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NaCl Concentration (%)
Final pH at 5 min.
Final pH at 10 min.
FIGURE 1. pH change of EO water during electrolysis process at different NaCl concentrations
p
(p
n 5 0
tio 40
NaC l C oncentration (% )l
FIGURE 2. Residual chlorine concentration after electrolysis at different NaCl concentrations
Enero-junio de 2005
Tim e of exposure (m in)
Control (Mean values)
FIGURE 3. EO water effect upon the inactivation of
L. monocytogenes suspension. (log
CFU/ml) Strains 163, 131, 227, 91, 155, 244, 121, 127 and 132.
Lettuce No.
Figure 4. Combined effect of EO water and 0.6% acetic acid upon the inactivation of
L.
monocytogenes inoculated on lettuce.
Universitas Scientiarum Vol 10, No. 1, 97-108
Lettuce sam ple
In itial R ecou nt
FIGURE 5. EO water effect on
L. monocytogenes population
inoculated on lettuce.
Source: http://www.watersource.com.sg/files/clinic_study_acidic/clinical-study-acidic-06.pdf
PURIM / Una historia de tragos amargos y final feliz Autor del Proyecto: Rabino Marcelo Polakoff Este proyecto fue producido por JCCenters.org Cuando Noé empezó a plantar, vino Satán y le Una historia de tragos A no confundirse. dijo"¿Qué plantas?" amargos y final feliz
3.02 Understand the functions and disorders of the nervous 3.02 Understand the functions and disorders of the nervous system 3.02 Essential Questions What are the functions of the nervous What are some disorders of the nervous How are nervous system disorders treated? How does the nervous system relate to the body's communication systems?