Lebensm.-Wiss. u.-Technol., 35, 720–729 (2002)

Efficacy of Chlorine Dioxide, Ozone, and ThymeEssential Oil or a Sequential Washing in Killing

Escherichia coli O157:H7 on Lettuce and Baby Carrots

N. Singh, R. K. Singh*, A. K. Bhunia and R. L. Stroshine

N. Singh, R. K. Singh: University, of Georgia, Department of Food Science & Technology, Food Science Building,Athens, GA 30602-7610 (U.S.A.)

A. K. Bhunia: Purdue University, Department of Food Science, West Lafayette, IN 47907-1160 (U.S.A.)R. L. Stroshine: Purdue University, Department of Agricultural and Biological Engineering,

West Lafayette, IN 47907-1160 (U.S.A.)(Received July 23, 2002; accepted August 6, 2002)

Chlorine dioxide (ClO2), ozone, and thyme essential oil has been found to be effective in reducing pathogens, including Escherichiacoli O157:H7, on selected produce. The efficacy of these sanitizers was evaluated, alone or through their sequential washing toachieve a 3 or more log reduction of mixed strains of E. coli O157:H7 on shredded lettuce and baby carrots. Samples sprinkleinoculated with mixed strains of E. coli O157:H7 were air-dried for 1 h at 2272 1C in a biosafety cabinet, stored at 4 1C for 24 h, andthen treated with different concentrations of disinfectants and exposure time. Sterile deionized water washing resulted inapproximately 1log reduction of E. coli O157:H7 after 10min washing of lettuce and baby carrots. Gaseous treatments resulted inhigher log reductions in comparison to aqueous washing. However, decolorization of lettuce leaves was observed during long exposuretime. A logarithmic reduction of 1.48–1.97log10 cfu/g was obtained using aqueous ClO2 (10.0mg/L for 10min) ozonated water(9.7mg/L for 10min) or thyme oil suspension (1.0mL/L for 5min) on lettuce and baby carrots. Of the three sequential washingtreatments used in this study, thyme oil followed by aqueous ClO2/ozonated water, or ozonated water/aqueous ClO2 weresignificantly (Po0.05) more effective in reducing E. coli O157:H7 (3.75 and 3.99log, and 3.83 and 4.34 log reduction) on lettuceand baby carrots, respectively. The results obtained from this study indicate that sequential washing treatments could achieve 3–4logreduction of E. coli O157:H7 on shredded lettuce and baby carrots.

r 2002 Elsevier Science Ltd. All rights reserved.

Keywords: chlorine dioxide; ozone; thyme oil; E. coli O157:H7; lettuce; baby carrots

Introduction

Pathogenic microorganisms of most concern in mini-mally processed fresh produce include Escherichia coliO157:H7, Listeria monocytogenes, Shigella, Salmonella,and hepatitis A virus. These organisms have all beenimplicated in outbreaks of food-borne illnesses linked tothe consumption of contaminated fresh vegetables[Davis et al., 1988; Farber et al., 1990; Centers forDisease Control and Prevention (CDC), 1997a,b]. E. coliO157:H7, which is known to cause hemorrhagic colitisand hemolytic uremic syndrome, has emerged as a food-borne pathogen of major public health concern (Padhyeand Doyle, 1991). A wide variety of foods includingmeat, milk, fruit juices, and vegetables are possible

*To whom correspondence should be addressed.E-mail: rsingh@arches.uga.edu

0023-6438/02/$35.00r 2002 Elsevier Science Ltd. All rights reserved. All art

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vehicles of E. coli O157:H7. The E. coli O157:H7outbreaks occurring from the consumption of fresh-cutvegetables (FCVs) have led investigators to search fornovel methods of controlling E. coli O157:H7 contam-ination of FCVs.Water containing 50–200mg/L of chlorine is widelyused in food processing plants to sanitize whole fruitsand vegetables as well as fresh-cut produce, but thistreatment only results in a reduction of bacterialpopulations of less than 2log cfu/g of fruits andvegetables (Beuchat, 1992, 1999; Brackett, 1992; Cherry,1999; Taormina and Beuchat, 1999). Zhang and Farber(1996) reported that maximum log reduction ofL. monocytogenes on fresh-cut lettuce and cabbagetreated with 200mg/L of chlorine was 1.3–1.7 and0.9–1.2log cfu/g, respectively. In addition to suchlimited efficacy, the possible formation of carcinogenicchlorinated compounds in water (chloramines and

doi:10.1006/fstl.2002.0933icles available online at http://www.idealibrary.com on

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trihalomethanes) (Page et al., 1976; Dychdala, 1991; Weiet al., 1999) has also raised questions on the use ofchlorine in food-processing plants. Chlorine, even whenused at low concentration, may cause taste and odordefect in treated products. Due to these problems, thereis a great interest in developing alternative sanitizers forwashing.Research and commercial applications have revealedthat chlorine dioxide, ozone, and natural antimicrobialsolutions can replace traditional sanitizing agents andprovide other benefits (Graham, 1997; Cherry, 1999).Chlorine dioxide (ClO2) is a strong oxidizing agent andhas a broad biocidal effectiveness. It has about 2.5 timesthe oxidation capacity of chlorine (Benarde et al., 1965).In addition, it does not react with nitrogen-containingcompounds or ammonia to form dangerous chloraminecompounds (White, 1972). Furthermore, ClO2 does notreact with phenolic compounds to produce foul smellingand tasting chlorophenols as does chlorine (Ward,1979). Food and Drug Administration (FDA) (1998)has allowed the use of aqueous chlorine dioxide inwashing fruits and vegetables. Chlorine dioxide has beenused as a drinking water treatment agent since 1944 andis a widely used bactericidal (Aieta and Berg, 1986).Lillard (1979) compared the effectiveness of chlorine andchlorine dioxide in reducing the number of bacteriapresent in poultry-processing water. It was found that5mg/L chlorine dioxide was as effective as 34mg/Lchlorine. Han et al. (1999) found that all the spoilagemicroorganisms inoculated onto the surface of a modelstorage tank with a relative humidity (RH) above 90%were completely killed after a treatment by 10mg/Lchlorine dioxide gas for 30min at 9–28 1C. Zhang andFarber (1996) reported that chlorine dioxide solutiontreatment (5mg/L, 10min) at 4 and 22 1C resulted in 1.1and 0.8log reductions of L. monocytogenes, respectively,on cut lettuce. Han et al. (2000) also studied the effectsof washing and chlorine dioxide gas treatment on thesurvivability and attachment of E. coli O157:H7 onuninjured and injured green pepper surfaces. Theyobtained 3.03 and 6.45log reductions of E. coliO157:H7 after treatments by 0.62 and 1.24mg/Lchlorine dioxide, respectively, for 30min at 22 1C and90–95% RH on surface-injured green peppers. Ozonehas recently been declared as a generally recognized assafe (GRAS) substance by an expert panel for use infood processing (Graham, 1997). This affirmationencouraged broader use of this gas in food industry.The biocidal effect of ozone is caused by a combinationof its high oxidation potential, reacting with organicmaterial up to 3,000 times faster than chlorine (EPRI,1997), and its ability to diffuse through biological cellmembranes. Kim et al. (1999) treated shredded lettucewith ozone and reported that bubbling ozone gas(49mg/L, 0.5 L/min) in a lettuce–water mixturedecreased the natural microbial load by 1.5–1.9log in5min. Essential oils and extracts from some herbs andspices have been shown to affect the growth andmultiplication of bacteria (Conner and Beuchat, 1984;Naitoh and Shiga, 1989; Paster et al., 1990; Aureli et al.,1992; Kandil et al., 1994; Lis-Balchin et al., 1996;

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Quattara et al., 1997; Hao et al., 1998; Reddy et al.,1998; Smith-Palmer et al., 1998). Spices and herbsare widely used in food industry and are consideredGRAS substances. Among them, thyme essential oilhas been reported to have antibacterial, antimycotic,antioxidative and food preservative properties (Deansand Ritchie, 1987). The means by which microorganismsare inhibited by essential oils seems to involve differentmodes of action. The most frequent inhibitions involvephenolic components of oils that sensitize the phospho-lipid bilayer of the cell membrane, causing an increaseof permeability and leakage of vital intracellularconstitutes (Juven et al., 1994; Kim et al., 1995)or impairment of bacterial enzyme systems (Faraget al., 1989; Wendakoon and Sakaguchi, 1995). Wanet al. (1998) reported promising results when naturallycontaminated lettuce was washed with solutions con-taining oil of basil, with comparable results to thoseachieved with 125mg/L of chlorine.Food preservation can be assisted not only by naturallyoccurring chemical agents with individual antimicrobialeffects but also by interactions among multiple anti-microbial factors resulting in additive or synergisticeffects. Venkitanarayanan et al. (1999) studied theefficacy of a combination of various GRAS chemicalsand moderate temperature to kill E. coli O157:H7 in1.0 g/L peptone solutions. They reported that at 22 1C,complete inactivation of E. coli O157:H7 was observedafter 20min of exposure to 15mL/L lactic acid plus1.0mL/L hydrogen peroxide, whereas a reduction of5log10 cfu/ml was observed with a treatment of 15mL/Llactic acid plus glycerol monolaurate. Liyanage et al.(1997) evaluated a two-step disinfection process forcontrol of Cryptosporidium parvum in 0.05mol/Lphosphate buffer at pH 8 and temperature 22 1C. Theyreported that the sequential treatment of oocytes byozone followed by chlorine dioxide resulted in addi-tional inactivation of C. parvum due to the synergism ofthe two disinfectants.The majority of this work has been conducted in culturemedia, consequently little is understood about theireffectiveness in the food substrates. The present studywas done to evaluate the effectiveness of aqueous andgaseous forms of chlorine dioxide and ozone, and thymeessential oil as potential antibacterial surface treatmentsto eliminate E. coli O157:H7 inoculated on shreddedlettuce and baby carrots. The efficacy of these treat-ments being applied individually and sequentially wasalso investigated.

Materials and Methods

Lettuce/baby carrotRomaine lettuce and baby carrots were purchased froma local supermarket (West Lafayette, IN, U.S.A.) andstored at 7 1C. The outer three or four leaves of lettuceand core were removed and discarded. The remainingleaves and baby carrots were then rinsed with cold tapwater for 1min at 22 1C. Intact and unwilted portions ofleaves were cut into pieces (3� 3 cm2, approximately 1 g

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each) using sterile knife and used in experiments. Thelettuce pieces and baby carrots were treated by UV-light(30W, 50 cm irradiation distance) in a class II biosafetycabinet (Labcono Corporation, Kansas city, MO,U.S.A.) for 30min (15min for each side) to reduce thenaturally existing microflora.

Preparation of inoculaA three-strain cocktail of E. coli O157:H7 (C7927,EDL933, and 204P) was used in this study. E. coliO157:H7 C7927 was provided by Dr M. P. Doyle at theUniversity of Georgia, Athens, GA, U.S.A. and strainsEDL 933 and 204P were obtained from Dr A. K. Bhunia(Purdue University, IN, U.S.A). Bacterial cultures weremaintained at 5 1C on slants of tryptic soy agar (TSA)(Difco Laboratories, Detroit, MI, U.S.A.) and activatedby culturing in tryptic soy broth (TSB, pH 7.3) (DifcoLaboratories) at 37 1C at least twice at 24 h intervalsprior to being used in experiments. The 24 h cultures ofeach bacterium were washed three times by centrifuging(1,800� g, 10min, 21 1C) with 0.1mol/L sterile phos-phate buffer, pH 7.0 (PBS) and the cell pellets wereresuspended in the same buffer. A final inoculum(1� 109 cfu/mL) cocktail was prepared by mixing thethree bacterial suspensions in the same proportion, andwas used to inoculate the lettuce leaves and babycarrots. Bacterial populations in the inoculum weredetermined by surface plating duplicate samples on TSAafter serial dilution in 1.0 g/L peptone water. The plateswere incubated for 24 h at 37 1C before colony countswere obtained.

Inoculation of lettuce/baby carrotsShredded lettuce leaves, and baby carrots (100 g each)were placed into sterile bags and then sprinkle inocu-lated with the mixed inocula of E. coli O157:H7 (1mL)to obtain an initial level of 108 cfu/g. The samples wereshaken vigorously for 3min to evenly distribute theinoculum. To allow attachment of bacteria, inoculatedlettuce leaves and baby carrots were air-dried under aclass II biosafety cabinet for 1 h at 2272 1C and thenstored in a refrigerator at 4 1C for 24 h before exposingto various sanitizing treatments.

Chlorine dioxide (gas/aqueous) productionChlorine dioxide gas was generated from a CDGlaboratory generator (CDG Technology, Inc., NewYork, NY, U.S.A.) using 4% chlorine in nitrogengas (Matheson Gas Products Co., Joliet II). Thegenerated chlorine dioxide gas (115mg/L chlorinedioxide in nitrogen) was collected in 4.7 L Teflon PEPgas-sampling bag (Cole-Parmer Instrument Co., VernonHills, IL, U.S.A.). The sampling bag was placed ina light protected outer bag to prevent light decomposi-tion of chlorine dioxide. The concentration of chlorinedioxide gas was measured by a modified amperometricmethod (Greenberg et al., 1992). A certain volumeof freshly generated chlorine dioxide gas (5mL)

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was immediately dissolved in 1L deionized sterilewater in a brown bottle. Two hundred milliliters ofchlorine dioxide solution was used for measurementof ClO2 concentration following the procedures of theamperometric method. An aqueous solution of chlorinedioxide was prepared by dissolving, a certain volumeof chlorine dioxide gas into deionized sterile water usinga gas-sampling syringe. Before injecting the gas intothe water, the gas was first dissolved in water inthe syringe by drawing some water in and outrepeatedly. The chlorine dioxide concentration wasmeasured in triplicate and the data were recordedasmg/L ClO2.

Ozone (gas/aqueous) productionOzone gas was generated using a laboratory coronadischarge ozone generator (Clear Water Tech, Inc.,San Luis Obispo, CA, U.S.A.) from purified, extra-dry oxygen feed gas. The oxygen carrier gas containingozone was humidified by flowing through 75mL steriledeionized water in a 125mL gas-washing bottle (Chem-glass Inc., Vineland, NJ, U.S.A.). The humidifiedand ozonated oxygen was passed through the cylinderat a 1 L/min flow rate using a diaphragm vacuumpump (KNF Neuberger, Inc., Trenton, NJ, U.S.A.).The concentration of ozone gas in the treatment cylinderwas measured using a high-concentration ozone monitor(Model 450H, Advanced Pollution Instrumenta-tion, Inc., San Diego, CA, U.S.A.). For the productionof ozonated water, the ozone-containing oxygen carriergas was bubbled for a minimum of 15min at 22 1Cthrough 300mL sterile deionized water in a 500mL flaskat 1, 3, and 5 ozone output levels. The flask was thenconnected to a reservoir containing 20 g/L potassiumiodide (KI) solution capable of neutralizing excessozone. The ozone solution was used within 2min ofremoving it from the gas stream. To determine thedissolved ozone concentration in water, a modifiediodometric method was used. Ten millilitres of ozone-treated water was added to 90mL of a 20 g/L KIsolution, acidified to pH of just below 2 with 1N H2SO4,and 1mL of starch indicator was added. The solutionwas titrated with 0.0005mol/L thiosulfate solutionand the concentration of ozone was calculated accord-ing to Greenberg et al. (1985). All experimental workwith ozone was done in a chemical fume hood. Theconcentration of ozone in the air and water wasrecorded as mg/L.

Thyme oil suspensionThyme essential oil was obtained from Lebermuth Co.,Inc. (Mishwaka, IN, U.S.A.) and stored at refrigerationtemperature (571 1C). A suspension of thyme essentialoil (0.1, 1.0, and 10mL/L) was prepared by dispersingappropriate amount of thyme essential oil in 1L ofsterile deionized water. The suspension was thoroughlymixed by shaking vigorously for 5min at roomtemperature (2271 1C) before use.

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Gaseous treatment of lettuce and baby carrotsChlorine dioxide gas treatments were carried outin a 10L Irvine Plexiglass cylinder with a stainless-steel shelf, on which shredded lettuce leaves, and babycarrots were placed. The following concentrationsof chlorine dioxide gas were used: 0.5, 0.75, and1.00mg/L for 5, 10, and 15min, under 80% RHand 22 1C, respectively. A 60mL plastic gas-samplingsyringe was used to deliver specific volumes of chlorinedioxide gas into the cylinder containing the shreddedlettuce leaves, or baby carrots. During treatment, thechlorine dioxide gas inside the cylinder was circulated bya diaphragm vacuum pump (KNF Neuberger, Inc.,Trenton, NJ, U.S.A.), to ensure that chlorine dioxidegas was evenly distributed throughout the sample.During the treatment, the cylinder was covered withaluminum foil to prevent light decomposition ofchlorine dioxide. Ozone gas treatment was also carriedout in the same treatment cylinder as used for chlorinedioxide gas treatment. Inoculated lettuce, and babycarrots were treated with 2.1, 5.2, and 7.6mg/L ozonegas for 5, 10, and 15min, under 80% RH and 22 1C,respectively.

Washing of lettuce and baby carrotsWashing treatment of inoculated lettuce and babycarrots was performed by immersing shredded lettuceleaves/baby carrots (10 g) in 200mL of each treatmentsolution (aqueous chlorine dioxide 5.0, 10.0, and20.0mg/L; ozonated water 5.2, 9.7, and 16.5mg/L;and thyme oil suspension 0.1, 1.0, and 10.0mL/L,for 1, 5, 10, and 15min) in a sterile bag with gentle,continuous agitation using a shaker (New BrunswickScientific Co., Edison, NJ, U.S.A.) at 120 rpm(2272 1C). At the end of contact time, the respectivetreatment solution was drained off, and the treatedsamples were rinsed with 200mL of neutralizingbuffer solution (Neutralizing buffer, Difco, Detroit,MI, U.S.A.) by shaking the bag for 30 s. For eachaqueous treatment, two controls were prepared. Thenegative control consisted of shredded lettuce leaves/baby carrots without inoculation and aqueous treat-ment. The positive control consisted of the inoculatedsamples without aqueous treatment. Water washing wasalso done to compare removal effect of water washingwith different treatments. The inoculated leaves (10 g)were blended in a Stomacher bag containing 200mLsterile deionized water, and microbial counts weredetermined.

Sequential washing of lettuce and baby carrotsBased on the preliminary results of the current study, atwo-, and three-step sequential washing system wasadopted for the inactivation of inoculated E. coliO157:H7 from lettuce leaves and baby carrots. Themethod used for multi-step washing was similar to theone described for single-step washing. The multi-stepsequential process consisted of washing the samples withaqueous chlorine dioxide (10mg/L for 10min), ozonated

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water (9.7mg/L for 10min), and thyme oil suspension(1.0mL/L for 5min) in the following order:(i) Two-step sequential washing: Aqueous ClO2/ozonatedwater; aqueous ClO2/thyme oil; ozonated water/aqu-eous ClO2; ozonated water/thyme oil; thyme oil/aqueous ClO2; thyme oil/ozonated water.(ii) Three-step sequential washing: Aqueous ClO2/ozo-nated water/thyme oil; aqueous ClO2/thyme oil/ozo-nated water; ozonated water/aqueous ClO2/thyme oil;ozonated water/thyme oil/aqueous ClO2; thyme oil/aqueous ClO2/ozonated water; thyme oil/ozonatedwater/aqueous ClO2.

Procedure for enumeration of microorganismsFor enumeration of E. coli O157:H7 from shreddedlettuce leaves/baby carrots, 10 g of the same wastransferred into sterile stomaching bags (Fisher Scien-tific Inc., Pittsburgh, PA, U.S.A.), with the aid of asterile stainless-steel spatula, combined with 90mL ofsterile peptone water (1.0 g/L) and then pummeled in aSeward 400 Stomacher (Seward Medical Co., London,England) at medium speed for 2min. One milliliter ofStomached sample was serially diluted in 9mL of sterilepeptone water (1.0 g/L). Serially diluted samples werespread-plated (0.1mL) in duplicates over a sterilepolycarbonate filter membrane (Osmonics Co., West-boro, MA, U.S.A.), which was previously placed on thesurface of a TSA plate. The coarse side of the membranefaced upwards. Plates were incubated at 37 1C for 4 h torepair injured cells. Then the membranes were gentlyand aseptically transferred onto Sorbitol Mac-Conkeyagar plates (SMAC) (Oxide Ltd., Basingstoke, Hamp-shire, England) supplemented with cefixime-tullerite(CT) (Dynal, Inc., Lake Success, NY, U.S.A.) usingsterile tweezers. The membrane-SMAC-CT plates werefurther incubated at 37 1C for 24 h. Presumptive E. coliO157:H7 colonies were counted. For confirmation, twocolonies per plate were picked and confirmed by an E.coli O157:H7 Latex Test (Oxoid Inc., Ogdensburg, NY,U.S.A.).

Statistical analysisAll the samples used for the enumeration of E. coliO157:H7 by colony enumeration methods were pre-pared in triplicate, and each test was replicated threetimes, making a total of nine samples analysed for eachtest parameter. Data were analysed using general linearmodel (GLM) and Duncan’s multiple range test (SASInstitute, Cary, NC, U.S.A.) to determine if significantdifferences (Po0.05) in populations of microorganismsexisted between mean values of treatments.

Results and Discussion

Effect of aqueous chlorine dioxidePopulations of E. coli O157:H7 on shredded lettuce andbaby carrots surviving after treatments with aqueouschlorine dioxide are shown in Table 1. No detectable

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Table 1 Effect of aqueous chlorine dioxide on thesurvival of Escherichia coli O157:H7 on shredded lettuceand baby carrots

Population (log10 cfu/g)Concentration(mg/L)

Exposuretime (min) Lettuce Baby carrots

0 1 7.7170.16a 7.1670.10a5 7.5470.16a 7.0370.11a10 7.1970.14b 6.7070.09b15 7.1870.20b 6.7070.06b

5 1 7.1570.16b 6.7070.14b5 7.1470.17b 6.6070.23b10 6.9270.23bc 6.3970.13bc15 6.9270.20bc 6.1670.18cd

10 1 6.9370.20bc 6.4270.21bc5 6.8170.12c 6.1670.22cd10 6.4570.18de 5.6470.21e15 6.4570.18de 5.6470.06e

20 1 6.8170.12c 6.1370.24cd5 6.7270.12cd 5.9170.24de10 6.4270.17de 5.3470.21f15 6.4070.17e 5.3170.19f

Initial inoculation levels for lettuce and baby carrots are 8.12and 7.85log10 cfu/g.Values are mean7SD population recovered (log cfu/g) (n=3).Values in the same row sharing a common letter are notsignificantly different (Po0.05).

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E. coli O157:H7 was found on uninoculated lettuceleaves or baby carrots. The initial population of mixedstrains of E. coli O157:H7 was 8.12 and 7.85log10 cfu/gon lettuce and baby carrots, respectively. Washinglettuce or baby carrots with sterile deionized water(control) for 10min resulted in a significant (Po0.05)reduction (0.93 and 1.15log10 cfu/g, respectively) of E.coli O157:H7 as compared to 1 or 5min washing.However, washing for 15min did not result in furthersignificant (Po0.05) reductions in E. coli O157:H7. Thissuggests that washing had a limited effectiveness inreducing bacterial population regardless of the washingtime. Standard washing of lettuce in tap water has beenshown to result in the removal of an average of 92% ofthe naturally occurring microflora (Adams et al., 1989).Han et al. (2000) also found that water washingachieved only 1.5log reduction on green peppers. Ourobservations on the effectiveness of deionized sterilewater (control) in removing pathogens from lettuce andbaby carrots concur with these reports. Increasing theconcentration of ClO2 in deionized water (5.0mg/L for 1and 5min) resulted in a significant (Po0.05) decrease inE. coli O157:H7 population on lettuce and baby carrotsin comparison to washing with deionized water (control)for the same period. However, Reina et al. (1995) foundthat bacterial populations present on cucumbers werenot greatly influenced by ClO2 treatment, even atconcentration of 5.1mg/L. The effect of aqueous ClO2

was in agreement with the results reported by Zang andFarber (1996) who found that the initial microbial loaddecreased by approximately 1log cycle for shreddedlettuce inoculated with L. monocytogenes at levels of

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5mg/L ClO2 in aqueous solution. Increasing thewashing period from 1 to 15min with aqueous ClO2

(5.0mg/L) showed no significant (Po0.05) reduction inthe population of E. coli O157:H7 on lettuce. However,a significant (Po0.05) reduction (1.69log10 cfu/g) in thepopulation of E. coli O157:H7 was observed afterwashing for 15min compared to 1 or 5min washing ofbaby carrots. This may be caused by the penetration ofmicroorganisms through cut edges into inaccessible sitesof shredded lettuce leaves. Both the studies of Adamset al. (1989) and Babic et al. (1996) also indicated thatdisinfectants in the washing solutions could not pene-trate into the protective hydrophobic pockets, folds orminute cracks on the surface of leafy vegetables.Washing of lettuce for 1 and 5min with aqueous ClO2

(10mg/L) had no significant (Po0.05) effect on thepopulation recovered, but increasing the washing time(10min) resulted in a significant (Po0.05) decrease(1.67log10 cfu/g reduction) in the E. coli O157:H7population. Similar results were also obtained withbaby carrots (Table 1). The results obtained in this studyshowed that 15min exposure of shredded lettuce orbaby carrots to 20mg/L of aqueous chlorine dioxidecaused a maximum reduction of 1.72 and 2.54log10 cfu/gof E. coli O157:H7, respectively. In general, greaterreductions of 0.54, 1.06, and 1.39log of E. coli O157:H7were observed with baby carrots in comparison toshredded lettuce (0.26, 0.73, and 0.76log10 cfu/g reduc-tion) when using 5, 10, or 20mg/L chlorine dioxide for15min, respectively, as compared to the correspondingsterile deionized water, indicating that cells adhered lesstenaciously to baby carrots surface as compared to theshredded lettuce. This might be due to concentration ofbacterial cells in the cut surfaces of the lettuce, whichmay have serious implications in relation to the efficacyof sanitization treatments. Once the pathogens areattached to the cut edges or entrapped in sides, itbecomes difficult to reduce the number of microorgan-isms by treatments. This was consistent with the findingsof Seo and Frank (1999) who found that some E. coliO157:H7 cells that attached to stomata and cut edgessurvived after chlorine treatment.

Effect of gaseous chlorine dioxideSummarized in Table 2 are results from experimentsdone to determine the efficacy of gaseous ClO2 in killingE. coli O157:H7 on lettuce and baby carrots. GaseousClO2 treatments of lettuce leaves with 0.5, 0.75, or1.00mg/L for 5min did not have any significant(Po0.05) effect on reducing populations of E. coliO157:H7. Increasing the exposure time (from 5 to10min) of lettuce leaves with gaseous ClO2 (0.5mg/L)did not result in any significant (Po0.05) decrease inpopulation of E. coli O157:H7. However, treatment oflettuce leaves with gaseous ClO2 (0.75 or 1.00mg/L) for10min resulted in a significant (Po0.05) reduction (1.67and 1.91log10cfu/g reduction, respectively) of microbialpopulation compared to 5min exposure time, butincreasing the exposure time (from 10 to 15min) didnot have any further significant (Po0.05) effect on the

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Table 2 Effect of gaseous chlorine dioxide on thesurvival of E. coli O157:H7 on shredded lettuce andbaby carrots

Population (log10 cfu/g)Concentration(mg/L)

Exposuretime (min) Lettuce Baby carrots

0 5 8.1070.36a 7.7270.20a10 8.1770.28a 7.8070.18a15 8.2070.15a 7.8070.19a

0.5 5 7.3770.32b 6.9270.24b10 6.9370.23bc 6.2770.24cd15 6.8570.28cd 6.0070.16de

0.75 5 7.2970.32bc 6.5770.27bc10 6.4370.26ed 5.8370.24e15 6.20*70.23ef 5.4170.22f

1.00 5 7.0670.20bc 6.3370.20cd10 6.19*70.27ef 5.1870.17f15 5.89*70.20f 4.7270.19g

*Decolorization of lettuce leaves.Initial inoculation levels for lettuce and baby carrots are 8.10and 7.72log10cfu/g.Values are mean7SD population recovered (log cfu/g) (n=3).Values in the same row sharing a common letter are notsignificantly different (Po0.05).

Table 3 Effect of ozonated water on the survival of E.coli O157:H7 on shredded lettuce and baby carrots

Population (log10 cfu/g)Concentration(mg/L)

Exposuretime (min) Lettuce Baby carrots

0 1 7.6070.17a 7.1470.10a5 7.5370.13a 6.9270.14ab10 7.1470.10bc 6.5770.15cd15 7.0870.16bc 6.5470.12cd

5.2 1 7.3270.15ab 6.9270.13ab5 7.2070.22bc 6.8170.12bc10 6.9970.25bc 6.4870.15d15 7.0070.23bc 6.4870.19d

9.7 1 7.3070.17a–c 6.9170.19ab5 7.1870.16bc 6.7170.14b–d10 6.6970.14d 6.1470.08e15 6.6770.19d 6.1170.18e

16.5 1 7.1870.14bc 6.8170.12bc5 7.1270.18bc 6.7070.11b–d10 6.6870.12d 6.0270.27e15 6.5970.11d 5.9770.16e

Initial inoculation levels for lettuce and baby carrots are 8.10and 7.82log10 cfu/g.Values are mean7SD population recovered (log cfu/g) (n=3).Values in the same row sharing a common letter are notsignificantly different (Po0.05).

lwt/vol. 35 (2002) No. 8

efficacy of gaseous ClO2 to reduce the microbialpopulation. However, significant (Po0.05) differencein numbers of E. coli O157:H7 population on babycarrots was observed during 10 and 15min exposure tobetween 0.75 and 1.00mg/L gaseous ClO2 concentra-tion, respectively. In general, somewhat greater reduc-tions in populations of E. coli O157:H7 on baby carrotsoccurred as compared to reductions on lettuce treatedwith gaseous ClO2, indicating that microorganisms oncut lettuce leaves are somehow protected from ClO2.The logarithmic reduction of E. coli O157:H7 on lettuceand baby carrots using gaseous ClO2 contrasts with theresults of Han et al. (2000) who reported a greaterreduction (3.03 and 6.45log) of E. coli O157:H7 oninjured green pepper surface after treatments by 0.62and 1.24mg/L ClO2, respectively, for 30min at 22 1Cand at 90–95% RH. These differences could be due todifferences in method of inoculation, initial microbialpopulation, exposure time, and type of produce. Thecolor of lettuce leaves also changed (decolorize) during15min exposure at 0.75mg/L gaseous ClO2. This maybe due to the oxidation of chlorophyll content duringlonger exposure times at higher concentrations ofgaseous ClO2. Similar results were observed with1.0mg/L gaseous ClO2 treatment on lettuce leaves for10 or 15min exposure time.

Effect of ozonated waterThe antimicrobial effects of ozonated deionized wateron E. coli O157:H7 inoculated onto lettuce and babycarrots are presented in Table 3. The results obtained inthis study have shown that treatment with ozonatedwater (5.2mg/L) did not result in any significant(Po0.05) reduction in E. coli O157:H7 populations

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during 1, 5, 10, or 15min of washing. However, asignificant (Po0.05) reduction in microbial populationson baby carrots was observed after 10min exposure to5.2mg/L ozonated water compared to 1 or 5min. Thereduced efficacy of ozonated water during lettucewashing might be due to more ozone demand of organicmaterial in the medium. The study of Restaino et al.(1995) also indicated that the type of organic materialpresent during ozonation is more important than theamount present. They reported that residual ozonelevels in deionized water were significantly reduced inthe presence of BSA. In the washing treatmentscontaining 9.7 or 16.5mg/L ozonated water, E. coliO157:H7 populations were reduced significantly(Po0.05) (1.41, 1.42, and 1.68 and 1.8log10 reductionscfu/g, respectively) on lettuce and baby carrots onlyafter 10min washing. Kim et al. (1999) treated shreddedlettuce with ozone and reported that bubbling ozone gas(49mg/L, 0.5 L/min) in a lettuce (1.0 g)–water (20.0 g)mixture decreased the natural microbial load by1.5–1.9log in 3min.

Effect of gaseous ozoneResults in Table 4 show that the gaseous ozone exerts alethal effect toward E. coli O157:H7 inoculated ontolettuce and baby carrots. Ozone treatments (2.1 to7.6mg/L) inactivated E. coli O157:H7 by 0.79–1.79, and1.11–2.64log cfu/g on lettuce and baby carrots, respec-tively. The bactericidal effect increases with concentra-tion, and length of exposure to gaseous ozone on lettuceand baby carrots. Ozone treatment of lettuce leaves at2.1, 5.2, or 7.6mg/L did not decrease the population ofE. coli O157:H7 significantly (Po0.05) during 5 or

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Table 4 Effect of gaseous ozone on the survival ofE. coli O157:H7 on shredded lettuce and baby carrots

Population (log10 cfu/g)Concentration(mg/L)

Exposuretime (min) Lettuce Baby carrots

0 5 8.0770.23a 7.8070.18a10 8.0770.22a 7.8070.18a15 8.1970.21a 7.8670.29a

2.1 5 7.2170.20b 6.6970.11b10 7.1170.22b 6.5270.20bc15 6.5870.20c 5.9670.18d

5.2 5 7.1170.22b 6.5170.20bc10 6.91*70.13bc 6.3170.20c15 6.54*70.19cd 5.3270.20e

7.6 5 7.0870.16b 6.4870.19bc10 6.90*70.20bc 6.3270.20c15 6.21*70.24d 5.1670.15e

*Decolorization of lettuce leaves.Initial inoculation levels for lettuce and baby carrots are 8.00and 7.80log10 cfu/g.Values are mean7SD population recovered (log cfu/g) (n=3).Values in the same row sharing a common letter are notsignificantly different (Po0.05).

Table 5 Effect of thyme essential oil on the survival ofE. coli O157:H7 on shredded lettuce and baby carrots

Population (log10 cfu/g)Concentration(mL/L)

Exposuretime (min) Lettuce Baby carrots

0 1 7.4370.14a 6.9470.12a5 7.4170.11a 6.9070.17a10 7.0070.16b 6.6170.09bc15 6.9070.17b 6.6270.14bc

0.1 1 7.4370.21a 6.9070.17a5 7.4070.19a 6.9070.17a10 6.9570.29b 6.4670.11c15 6.8770.15b 6.4170.19c

1.0 1 7.1270.16ab 6.7370.11ab5 6.2170.15c 5.6470.11d10 5.9570.15cd 5.4870.19d15 5.9570.17cd 5.4370.12d

10.0 1 7.0070.16b 6.5770.10bc5 5.7270.12de 5.0070.16e10 5.5270.17e 4.8970.11e15 5.4570.13e 4.7770.15e

Initial inoculation levels for lettuce and baby carrots are 7.86and 7.54log10 cfu/g.Values are mean7SD population recovered (log cfu/g) (n=3).Values in the same row sharing a common letter are notsignificantly different (Po0.05).

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10min of exposure; however, a significant reduction(1.42 to 1.79log cfu/g) in populations of E. coli O157:H7was observed after 15min of exposure. Decolorizationof lettuce leaves was also observed during 10 or 15minexposure at 5.2 and 7.6mg/L of ozone concentration.Similarly, ozone treatment of baby carrots did not resultin any significant (Po0.05) change in population of E.coli O157:H7 during 5 or 10min exposure time at 2.1,5.2, or 7.6mg/L concentration level. However, theincrease in exposure time (15min) resulted in asignificant decrease (1.84 to 2.64log cfu/g) in population.

Effect of thyme essential oilThe effects of thyme essential oil washing of lettuce andbaby carrots, inoculated with E. coli O157:H7 are shownin Table 5. Deionized water washing for 10min resultedin significant reductions (Po0.05) in population ofE. coli O157:H7 (7.00 and 6.61log cfu/g, respectively) onlettuce and baby carrots in comparison to 1 or 5minwashing. However, increasing the washing time (15min)did not result in any significant (Po0.05) change inmicrobial population. The results also revealed thatthyme oil washing (0.1mL/L) did not lead to anysignificant (Po0.05) difference in numbers of E. coliO157:H7 on lettuce and baby carrots as compared withdeionized water washing. A significant (Po0.05) reduc-tion (1.65 and 1.90log cfu/g, respectively) in populationof E. coli O157:H7 on lettuce and baby carrots wasobserved when using thyme oil (1.0mL/L for 5min)compared to deionized water or 0.1mL/L, thyme oil forsame period of time. Washing time from 5 to 10 or15min did not result in any significant (Po0.05)reduction (thyme oil 1.0mL/L) in microbial population.Wan et al. (1998) also reported that washing lettuce with

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1.0 and 10.0mL/L suspensions of basil essential oilresulted in 2.0 and 2.3log reductions of visible bacteriaon fresh-cut lettuce, respectively. Increasing the con-centration of thyme oil (10.0mL/L for 5, 10, or 15min)resulted in a further decrease in microbial population onlettuce and baby carrots, compared to deionized water(control), 0.1 or 1.0mL/L thyme oil concentration forthe same period of time. In general, numbers of E. coliO157:H7 decreased only marginally with increasingexposure time (from 5 to 15min), regardless of thymeoil concentration in the washing solution.

Effect of sequential washingResults of the efficacy of washes containing aqueousClO2, ozonated water, and thyme oil alone or theirsequential treatments (two and three-step sequentialwashing), on the survival of E. coli O157:H7 on lettuceleaves and baby carrots can be seen in Table 6. Washingof lettuce leaves with aqueous ClO2, ozonated water orthyme oil led to a significant (Po0.05) reduction innumbers of E. coli O157:H7, as compared with thesterile deionized water (control). In general, reductionsranged from 1.48–1.61log depending upon the type ofsanitizers used. Whereas, water washing reduced thepopulation by 1.06log. Similar results were seen withinoculated baby carrots (1.61–1.97log reduction). Atwo-step sequential washing further reduced the popula-tions of E. coli O157:H7, as compared to single step-washing. The efficacy of thyme oil/aqueous ClO2 orthyme oil/ozonated water, however, proved to besignificantly more effective (3.15 and 3.00log reductionon lettuce and 3.47 and 3.25log reduction on baby

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Table 6 Efficacy of washes containing different sanitizers (aqueous ClO2, ozonated water, or thyme oilsuspension) alone or their sequential treatments on decontamination of shredded lettuce and baby carrotsinoculated with E. coli O157:H7

Population (log10 cfu/g)

Treatments* Lettuce Baby carrots

Unwashed 7.9370.17a 7.5470.13aDeionized water 6.8770.27b 6.3270.11bAqueous chlorine dioxide 6.3370.20c 5.6670.11cdOzonated water 6.4570.13c 5.9370.21cThyme oil 6.3270.20c 5.5770.21dAqueous ClO2/ozonated water 5.7270.15d 4.8570.23eAqueous ClO2/thyme oil 5.5570.17d 4.9670.18eOzonated water/aqueous ClO2 5.5470.18d 4.6270.11eOzonated water/thyme oil 5.5270.20d 4.8370.13eThyme oil/aqueous ClO2 4.7870.19e 4.0770.19fThyme oil/ozonated water 4.9370.23e 4.2970.20fAqueous ClO2/ozonated water/thyme oil 5.5570.24d 4.9170.26eAqueous ClO2/thyme oil/ozonated water 5.5670.16d 4.9170.08eOzonated water/aqueous ClO2/thyme oil 5.4270.07d 4.7770.17eOzonated water/thyme oil/aqueous ClO2 5.3870.10d 4.6870.22eThyme oil/aqueous ClO2/ozonated water 4.1870.12f 3.7170.10gThyme oil/ozonated water/aqueous ClO2 3.9470.13f 3.2070.20h

*Deionized water: 10min; aqueous chlorine dioxide: 10.0mg/L, 10min; ozonated water: 9.3mg/L, 10min; thyme oil suspension:1.0mL/L, 5min.Values are mean7SD population recovered (log cfu/g) (n=3).Values in the same row sharing a common letter are not significantly different (Po0.05).

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carrots, respectively) in killing microbial population. Itis, however, interesting to note that during the three-step sequential washing treatments used in this study,thyme oil followed by aqueous ClO2/ozonated water,or ozonated water/aqueous ClO2 were significantly(Po0.05) more effective in reducing E. coli O157:H7(3.75 and 3.99log, and 3.83 and 4.34log reduction) onlettuce and baby carrots, respectively. It could thereforebe inferred from the results of this study that thyme oilwashing followed by other sanitizers is of greatadvantage. The populations of E. coli O157:H7 onshredded lettuce were significantly decreased whenwashing regime was repeated (Singh et al., 2002). Themaximum reduction in E. coli O157:H7 populations onlettuce and baby carrots during two- or three-stepsequential washing by thyme oil followed by aqueousClO2 or ozonated water might be due to the phenoliccomponents of thyme oil, which may increase thepermeability and leakage of cell membrane, therebyenhancing the efficacy of other sanitizers.

Conclusions

The results obtained in this study show that waterwashing or using different sanitizers alone is not effectivein eliminating E. coli O157:H7 contamination ofshredded lettuce leaves and baby carrots’ surfaces.Therefore, a sequential washing may be an importantcomponent of overall contamination reduction processin fresh-cut vegetables. These findings conclude thatsequential washing is very effective in killing E. coliO157:H7, when thyme oil washing is followed byaqueous ClO2, and ozonated water, although a much-

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detailed study is anticipated. However, the use of plantextracts, aqueous ClO2, and ozonated water mayadversely affect the organoleptic properties of food.Further studies are needed to determine the accept-ability of fruits or vegetables treated with lowerconcentrations of sanitizers to reduce contaminationwith lower microbial population. In addition, differentfood constituents may influence the sequential washing(synergistic action) of thyme oil/ozonated water/aqu-eous chlorine dioxide, and as they appeared to have animportant role in the interaction with sanitizers, sucheffects on various fruits and vegetables need to beinvestigated.

Acknowledgements

This research was supported by USDA-ARS grant insupport of the Food Safety and Engineering Project atPurdue University.

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