effectiveness of electrolyzed oxidizing water

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Enero-junio de 2005UNIVERSITAS SCIENTIARUMRevista de la Facultad de CienciasPONTIFICIA UNIVERSIDAD JAVERIANA Vol. 10, No. 1, 97-108

EFFECTIVENESS OF ELECTROLYZED OXIDIZING WATERFOR INACTIVATING

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 Microbiología2Departamento de Física, Facultad de Ciencias, Pontificia Universidad Javeriana,

Cra. 7 No. 40-62 Bogotá, Colombia

ABSTRACT

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.

RESUMEN

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

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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.

INTRODUCTION

Nowadays the consumption of vegetablesin Colombia has become a critical factor inthe development of a wide variety ofenteric, parasitic and viral diseases, ofdifferent levels of seriousness like typhoidfever, amoeba infections and listeriosis (thislast is statistically ascribed in 12% of thecases to the consumption of lettuce in theCundiboyacense highlands) among others.Listeria monocytogenes is a food-bornepathogenic microorganism that has a highrate of mortality (23%); it is resistant to stressconditions, which makes it difficult todestroy, and it is easy to swallow throughthe consumption of vegetables; thismicroorganism is the causal agent of ageneralized disease called Listeriosis(ICMSF, 1996). To solve this problem,washing processes and disinfection of foodincluding the use of chemical substanceslike chlorine, organic acids and ozone havebeen implemented; in some cases thesesubstances have reduced effects onpathogenic microorganisms, because thosethat have not been removed multiply; dueto this problem, there is a need for aneffective method to inactivate food-bornepathogens in food.

Electrolyzed Oxidizing Water (EO water)is the product of a new concept developedin Japan and it could be an effectivealternative for disinfectant treatment offresh products like vegetables(Venkitaranayanan et al., 1999a and Kosekiet al., 2003). Electrolysis of deionized water

containing a low concentration of sodiumchloride in an electrolysis chamber whereanode and cathode electrodes are separatedby a diaphragm imparted strongbactericidal and virucidal properties to thewater collected from the anode (EO water).Water from the anode normally has a pH of2.7 or lower, an oxidation-reductionpotential (ORP) greater than 1,100 mV, anda free chlorine concentration of 10 to 80ppm in the form of hypochlorous acid. Thisuse of electrolyzed oxidizing water in thedisinfection of vegetables substantiallyeliminates food-borne pathogens, avoidingtransmission of diseases (Cháves et al.,2004).

Previously, the effectiveness of EO waterhas been evaluated for inactivatingdifferent pathogens like Escherichia coli,Salmonella enterit idis, and Listeriamonocytogenes, obtaining a considerablereduction in logarithmic units of CFU incomparison with the initial population intomatoes, lettuce, kitchen cutting boardsand in vitro experiments, at differenttemperatures and storage conditions.Cháves et al in 2004 designed anelectrolytic chlorinator to produce EO waterthat was used efficiently for the eliminationof microorganisms present in lettuce. Theobjective of this study was to evaluate theeffectiveness of EO water produced in anelectrolytic cell to inactivate L.monocytogenes in suspension and in lettuceleaves, and in this way, to standardize theaction time required to inactivate the L.monocytogenes suspension.

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2. MATERIALS AND METHODS

2.1. Electrolytic cell and EO watercharacterization

Following the parameters of Cháves et al.,2004, with the modification that two late-ral holes were added for the exit of the twotypes of water, a 3.3 L glass cell was builtwith a PVC membrane (Darnel®) in orderto separate the acid and alkalineelectrolytes, allowing the ions to move tothe electrodes. The material used aselectrodes was graphite, the applied voltagewas 14 volts and the current was 0.4amperes. A 5% solution of sodium chloridewas prepared to feed the electrolytic cell.During the electrolysis of water, the pH inboth acid and alkaline water wasdetermined at 3, 5, 7 and 10 minutes; a pH-meter previously calibrated made by theCenter of Interfaculty Equipment (CEIF) ofthe Universidad Nacional de Colombia wasused for these evaluations. The freeavailable chlorine present in acidicelectrolyzing water was also determined atthe same times using, a photometric DPDkit (ref. 1.00598.0001 Merck, Darmstadt,Germany).

2.2. Selection and growth curve of L.monocytogenes

Ten strains of L. monocytogenes suppliedby the Laboratorio de Microbiología deAlimentos de la Pontificia UniversidadJaveriana, which were previously isolatedby Correa and Fonseca in 2004 fromprocessed chicken, were used. To build thegrowth curve, 3 ml of suspension weretaken every 2 hours during the fermentationprocess of L. monocytogenes in Colum-bia broth supplemented with 50 mg/mlof nalidixic acid in order to determinethe biomass by optical density at 540 nm,to establish the beginning of the logphase.

2.3. Effect of EO water for inactivatingL. monocytogenes in culture

One ml aliquots from L. monocytogenesculture in the log phase were taken and se-rial dilutions in 9 ml of EO water from 10-1

to 10-6 were performed. The bactericidalactivity of the EO water was evaluated at 5,10, 15 and 20 min of exposure time at roomtemperature. After incubation time, thesurviving cells of L. monocytogenes in eachtreatment were determined; 1 ml of eachdilution was taken in order to do a recounton Columbia agar plates. L. monocytogenescolonies were counted after 48 h ofincubation at 37°C. The recount was donein duplicate (Venkitanarayanan et al.,1999).

2.4. Effect of EO water for inactivatingL. monocytogenes inoculated onlettuce leaves

The five strains most resistant to sodiumhypochlorite were chosen from among theinitial ten strains by a diffusion test usingWaltman No. 3 paper disks saturated withhypochlorite concentrations (5250, 52.5and 0.525 ppm), and these were selectedand used in a strain mixture for theinoculation of 35 lettuce samples by thedip inoculation method as described byPark et al., 2001, Bari et al., 2003 andKoseki et al., 2003. Sterile distilled waterwas used as a control; each strain of L.monocytogenes was cultured in 10 ml ofColumbia broth supplemented with 50 µg/ml of nalidixic acid at 37 °C during 5 hoursat 120 rpm. Then, the suspension wastransferred to 100 ml of Columbia broth ina 250 ml Erlenmeyer flask, and it wasincubated under the same conditionsduring 12 hours. The cells were collectedby centrifugation (3.500 rpm, 15 min atroom temperature) and the resulting pelletwas resuspended in 14 ml of sterile peptonewater (0.1%, pH 7.5) distributed in seventubes of 15 ml each. From each suspension,

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equal volumes were taken and thencombined to form an inoculum mix fromthe five strains with a final volume of 125ml. The inoculum was maintained at roomtemperature, and it was applied the lettucewithin one hour of preparation. Thepopulation of the L. monocytogenesinoculum was determined from a 10-8

dilution of the dip by recounting on Co-lumbia agar plates in duplicate.

Thirty five units of lettuce were purchasedfrom a local supermarket and were stored at4 °C before being inoculated, within amaximum time of two days (Österblad etal., 1999). Following the inoculationmethod described by Koseki et al in 2003,treatment of the inoculated lettuce leaveswas performed by immersing 6.25 g oflettuce in 375 ml of EO water or distilledwater (control) in plastic bags during 5minutes.

The previously treated lettuce samples werewashed with sterile peptone water andmacerated for one minute. 10-2 and 10-3

dilutions were made from the resultantsolution from the EO water treatment and10-4 and 10-6 dilutions were made from thedistilled water treatment. 0.1% sterilepeptone water was used for the dilutions.One ml of each dilution was taken for arecounting of the surviving population ofL. monocytogenes. A dip recount on Co-lumbia agar plates was carried out induplicate, and the plates were incubated at37 °C for 48 hours.

2.5. EO water and 0.6% acetic acidcombined effect on L. monocytogenesinoculated on lettuce

Three treated lettuce samples were thenexposed to a vinegar solution after EO watertreatment or distilled water treatment.The microbiologic analysis was done asdescribed earlier.

2.6. Minimal Inhibitory Concentration(MIC) Test

Ten 1:2 serial dilutions were made from a5.6 % sodium hypochlorite solution usingsterile distilled water; dilutions wereinoculated with 10 µl containing L.monocytogenes suspension in lag phase;after a 5 minute exposure time, 10 µl fromeach dilution was transferred to tubescontaining 2 ml of Columbia brothsupplemented with 50 µg/ml of nalidixicacid. These tubes were incubated at 37 °Cfor 48 hours. After this, MIC was determinedby turbidity at each sodium hypochloritedilution (Lúnden, 2004). The MIC test wasdone in triplicate. This procedure was alsodone on the most sensitive strain in orderto compare the results with the five strainsused in this study.

2.7. Statistical Analysis

The results obtained were analyzed througha t student hypothesis test.

3. RESULTS AND DISCUSSION

3.1. Electrolytic cell and EO water

The electrolyzed water was obtained in anelectrolytic cell that was built according tosection 2.1. EO water was produced in arelatively short time with low powerconsumption, around 6 watts, (14 volts and0.4 amperes). The required EO water shouldhave had the same characteristics as thatused in previous studies to inactivate L.monocytogenes, where EO water was usedwith a pH of 2.5 and 72 ppm residualchlorine, and with a pH of 2.63 and 43ppm or 48.5 ppm residual chlorine(Venkitanarayanan et al.,1999,Venkitanarayanan et al., 1999a), with a pHof 2.5 and 45 ppm residual chlorine (Parket al., 2001) and with a pH of 2.6 and 30ppm residual chlorine (Bari et al., 2003).Additionally, a disinfectant solution thatwould not change the lettuce flavor wassought.

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First, 2.5%, 3.0%, 3.5%, 4.0%, 4.5%, 5.0%and 5.5% NaCl concentrations wereevaluated. After 5 minutes of electrolysis,the pH was lower than 3.0 (see figure 1);therefore, at this time the first water samplewas taken to determine the residualchlorine concentration present, and thesecond was taken after 10 minutes ofelectrolysis. The highest residual chlorineconcentration (47.68 ppm) was obtained ina 5% NaCl solution, after 10 minutes ofelectrolysis, and the lowest (0.38 ppm) wasobtained in a 2.5% NaCl solution after 5minutes of electrolysis (see figure 2). Thehighest residual chlorine concentrationobtained between 2.5% and 3.5% NaClsolutions was 5.83 ppm, obtained in a 3.5%NaCl solution; these solutions werediscarded. A 5% NaCl solution was used inthis study, because it had the highestchlorine concentration.

Once electrolysis time and NaClconcentration were determined, pH and re-sidual chlorine stability were evaluatedbetween 24 and 96 hours. A new electrolysiswas done using a 5% NaCl solution, and aresidual chlorine concentration of 47.33ppm and a pH of 2.31 were obtained. ThepH value remained on the same scale witha mean of 2.3. In contrast, the chlorinemeasurements were unstable, because thelevels decreased drastically after 24hours of s torage, and addi t ional ly,values presented variability; therefore,measurements were stopped at 48 hours ofstorage because at this time the residualchlorine concentration was 5.48 ppm.These results were consistent with thosedescribed by Kirk et al. in 1962 who statedthat in acid or alkaline solutions, thehypochlorous acid dissociates, forminghydrochloric and chloric acids, and instrongly acid solutions, chlorine, or chlorineand oxygen are released. Additionally, Ro-jas and Guevara, 1998, concluded thathypochlorous acid ionization has anequilibrium constant that depends on the

pH solutions, which confirms the resultsobtained by Len et al. in 2002, who foundthat upon adjusting the pH of water from2.5 to 4.0 using acetic acid solution, thestability of chlorine was greater by a nine-fold factor.

3.2. Effect of EO water for inactivatingL. monocytogenes in culture

The bacterial population was reduced toundetectable levels in a 10-2 dilution (asdetermined by dip recount) after 5 minutesof EO water treatment; whereas in the con-trol treatment, the logarithmic units of CFU/ml (log CFU/ml) were constant (see figure3). The highest reduction was obtainedwith strain 227, which decreased toundetectable levels in a 10-1 dilution (asdetermined by dip recount), and the lowestreduction was obtained with strain 244,where after 5 minutes of exposure time, thepopulation decreased by 5.0 log CFU/ml,and only after 10 minutes of exposure time,growth in 10-2 dilutions was not detected.An exposure time of 5 min reduced thepopulations by approximately 6.6 logCFU/ml. According to these results, it waspossible to conclude that the most resistantstrain to EO water effect was strain 244 andthe most sensitive one was strain 227. Theseresults were later confirmed by the MinimalInhibitory Concentration test, using disks.

Between 5 and 20 minutes of exposure,there were no changes in L. monocytogenespopulations. The microorganism recount ina 10-1 dilution was inconsistent, so only the10-2 dilution values were taken.

An exposure time of 5 min reduced thepopulations by approximately 6.6 logCFU/ml, a high bactericidal effect,according to Delgado et al in 2003, whosuggested that a disinfectant solution hasto reduce the population by 5.0 log CFU.In this study, EO water with 28. 72 ppm ofresidual chlorine and pH 2.83 was used. Our

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results are supported by Venkitanarayananet al in 1999; in their study, the effect ofEO water to inactivate L. monocytogeneswas evaluated, and they determined thatafter 5 minutes of exposure time to EO water(48.5 ppm residual chlorine and pH of 2.63),the population decreased by 6.64 log CFU/ml.

From the results obtained in this phase, itwas possible to determine that the treatmenttime for the evaluation of the EO water effecton L. moncytogenes inoculated on lettuceshould be 5 minutes. Moreover, the mostresistant strains to chlorine were selectedby the MIC test using disks; with thesestrains, a mixed inoculum to inoculatelettuce samples was made. The mostresistant strains were 155, 244, 91, 131 and163.

3.3. Effect of EO water for inactivatingL. monocytogenes inoculated onlettuce leaves

The five strain mixtures had a populationmean of 9.56 log CFU/ml (109 CFU). Thereduction mean of L. monocytogenesinoculated on lettuce was 3.92 log CFU/g(see figure 5). In sample number 31, thehighest reduction was obtained at 5.82 logCFU/g, and in sample number 2 the lowestreduction was obtained at 2.48 log CFU/g.These results show that the EO water’s effecton the L. monocytogenes culture is higherthan the effect on L. monocytogenesinoculated on lettuce because thehypochlorous acid reacts with organicmatter present in the lettuce, losing itsdisinfectant power (Snoeyink and Jenkins,2002). However, the population reductionwas statistically significant (p=0.00001).

Results of the present work are consistentwith Oomori et al. in 2000, whodemonstrated that available chlorine wastransformed to N- Chlorates compounds byaminoacids and proteins present. A removal

of available chlorine was observed byoxidation-reduction reactions with somevitamins, lipids and minerals. They alsodetermined that the combined chlorinebactericidal effect on E. coli was lower thanthe free chlorine bactericidal effect. Theseauthors suggest that for practical use of EOwater in the food industry, the residualchlorine concentration must be increasedto avoid the organic matter effect.Furthermore, the results obtained in lettucetreatments suggest that the action time ofEO water must be increased to obtain agreater microbial reduction. The results ofthis study show the need of increasing theavailable chlorine concentration in EOwater produced in an electrolytic cell forinactivation of L. monocytogenes toreduction levels lower than those observedby Park et al. in 2001, where after 3 minutesof exposure to EO water (45 ppm residualchlorine and pH 2.5) the L. monocytogenespopulation inoculated on lettuce decreasedby 5.5 log CFU/g; in the present study theresidual chlorine concentration was higherby 16 ppm. In the Bari et al., 2003, study,chlorinated water (200 ppm) and EO water(30.3 ppm residual chlorine and pH of 2.6)effects were evaluated on L. monocytogenesinoculated on tomatoes. They obtained areduction in population by 4.76 logCFU/g and 7.54 log CFU/g per tomatorespectively. However, Beuchat and Brakettin 1990 proved the low effectiveness ofchlorine solutions prepared with from200 to 250m ppm of free chlorine forinactivation of L. monocytogenesinoculated on lettuce, reducing thepopulation only by 1.36 log CFU/g. Theseresults were lower than the ones obtainedwith EO water, due to its higher effect onmicroorganisms than a conventionalchlorine solution, because of its propertieslike low pH and high ORP (Kim et al., 2000and Koseki et al., 2002).

A population mean reduction of 2.46 logCFU/ml was observed in the control

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treatment, obtained through the washingprocess of lettuce, in which manymicroorganisms were removed by water.Another reason could be osmotic stress dueto hypotonic characteristics of distilledwater; namely, a low solute concentrationthat produces cellular lysis (Madigan 2001).Additionally, the pH of distilled water wasbetween 5 and 6, which contributed to thepopulation decrease.

3.4. EO water and 0.6% acetic acidcombined effect on L.monocytogenes inoculated on lettuce

Based on the previous results, the combinedeffect of EO water and 0.6% acetic acid onL. monocytogenes inoculated on lettucewas evaluated, and it was concluded that ahigh reduction in log CFU/g was obtainedwhen lettuce was treated with acetic acidafter EO water treatment. The L.monocytogenes population decreased by5.74, 5.14 and 5.6 log CFU/g for the samples36, 37 and 38 respectively; the mean valuereduction was 5.49 log CFU/g. Theseresults show that EO water can be employedas a disinfectant, but a combined treatmentwith another disinfectant like 0.6% aceticacid for improved bactericidal effect issuggested; other antimicrobial agentcombinations like nisin and essential oils(carvacrol or tymol) have shown asynergistic effect for the inactivation of L.monocytogenes (Pol and Smid, 1999 citedby Delgado et al., 2003)

3.5. CMI Test

Turbidity was observed from an 875 ppmNaClO dilution in strain 163, from 439.5ppm in strain 121, from 218.75 ppm instrains 91 and 244, and finally from 109.37ppm in strain 155. Strain 227 did not growin any NaClO dilution, which confirms itssensitivity.

Selected strains for inoculation can presentan acquired resistance to this disinfectant

because they were isolated from processedchicken, and these were exposed to ahigh chlorine concentration during theprechiller and chiller processes, indicatingthat probably the cells had been exposedto a sublethal disinfectant concentration.Resistance could be due to a flux bomb ormodifications in cell walls (Mc Donell andRussell, 1999).

3.6. Statistical Analysis

The reduction in L monocytogenespopulation was statistically significant after5 minutes of immersing lettuce leaves inEO water (p= 0.00001 and CI=95%).

4. CONCLUSIONS

A 5 % NaCl solution had the highest resi-dual chlorine concentration (mean of 29ppm) and a pH value of 2.83 during 10minutes of electrolysis.

The L. monocytogenes population had amean decrease of 6.6 log CFU/ml after 5minutes of exposure to EO water; howeverthis reduction can probably be obtained inless time.

When lettuce was treated with 0.6% aceticacid after EO water treatment, there was ahigher reduction in cell population (5.49log CFU/g), which indicates that thecombined treatment with anothermicrobicidal agent improves the EO watereffect.

According to MIC test results, trains usedin this study show an acquired resistanceto chlorine due to their place of origin.

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VENKITANARAYANAN K.S, EZEIKE G.O, HUNG Y.Cand DOYLE M. 1999. Efficacy ofElectrolyzed Oxidizing Water forInactiving Escherichia coli O157:H7,Salmonella enteritidis, and Listeriamonocytogenes. Applied andEnvironmental Microbiology. Vol. 65:.( 9), Pp: 4276-4279.

VENKITANARAYANAN K.S, EZEIKE G.O, HUNG Y.Cand DOYLE M. 1999a. Inactivation ofEscherichia coli O157: H7 andListeria monocytogenes on PlasticKitchen Cutting Boards byElectrolyzed Oxidizing Water. Journalof Food Protection. Vol. 62: (8), Pp:857-860.

Recibido: 6-07-2004Aceptado: 2-02-2005

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0

1 0

2 0

3 0

4 0

5 0

6 0

4 4 ,5 5 5 ,5

NaC l C oncentration (% )l

Chl

orin

eC

once

nrat

ion

(ppm

)

C h lo rin e (pp m ) at 5 m in . C h lo rin e (pp m ) at 1 0 m in.

0

2

4

6

8

10

4 4.5 5 5.5

NaCl Concentration (%)

pH

Initial pH Final pH at 5 min. Final pH at 10 min.

FIGURE 1. pH change of EO water during electrolysis process at different NaCl concentrations

FIGURE 2. Residual chlorine concentration after electrolysis at different NaCl concentrations

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01

2345

678

910

0 5 10 15 20 25

Time of exposure (min)

Log

CF

U/m

l

Control (Mean values) EO w ater 163 EO w ater 131EO w ater 227 EO w ater 91 EO w ater 96EO w ater 155 EO w ater 244 EO w ater 122EO w ater 121 EO w ater 127

FIGURE 3. EO water effect upon the inactivation of L. monocytogenes suspension. (logCFU/ml) Strains 163, 131, 227, 91, 155, 244, 121, 127 and 132.

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36 37 38

Lettuce No.

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CF

U/m

l

Initial recount Control EO + A.A

Figure 4. Combined effect of EO water and 0.6% acetic acid upon the inactivation of L.monocytogenes inoculated on lettuce.

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Lettuce sample

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CF

U/g

In itia l R ecount C ontro l EO w ater

FIGURE 5. EO water effect on L. monocytogenes population inoculated on lettuce.

effectiveness of electrolyzed oxidizing water

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