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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 deListeria 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 onL. 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.
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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.

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