1215

Journalof Food Protection, Vol. 65, No. 8, 2002, Pages 1215– 1220 Copyright Q,InternationalAssociation for FoodProtection

Inactivationof Escherichia coli O157:H7, Salmonellaenterica Serotype Enteritidis,and Listeria monocytogenes onLettuce by Hydrogen Peroxide andLactic Acid andby Hydrogen Peroxide withMild Heat

CHIA-MIN LIN, 1 SARAH S. MOON, 1† MICHAEL P.DOYLE, 1,2* AND KAY H. MCWATTERS2

1Center forFood Safety and 2Departmentof Food Science andTechnology, University ofGeorgia, GrifŽ n, Georgia 30223-1797, USA Downloaded from http://meridian.allenpress.com/jfp/article-pdf/65/8/1215/2004957/0362-028x-65_8_1215.pdf by guest on 23 September 2021

MS01-477:Received 13December 2001/Accepted 16March 2002

ABSTRACT

Iceberglettuce is a majorcomponent in vegetable salad and has been associated with many outbreaks of foodborne illnesses.In this study, several combinations of lactic acid and hydrogen peroxide were tested to obtaineffective antibacterial activitywithout adverse effects on sensor ycharacteristics.A Žve-strainmixture of Escherichiacoli O157:H7, Salmonella enterica serotypeEnteritidis, and Listeriamonocytogenes wasinoculated separately onto fresh-cut lettuce leaves, which were latertreated with 1.5% lactic acid plus 1.5% hydrogen peroxide (H 2O2) at 408Cfor15 min, 1.5% lactic acid plus 2% H 2O2 at 228Cfor5 min,and 2% H 2O2 at 508Cfor60 or 90 s. Control lettuce leaves were treated with deionized water under the sameconditions. A 4-logreduction was obtained for lettuce treated with the combinations of lactic acid and H 2O2 for E. coli O157:H7 and Salmonella Enteritidis,and a 3-logreduction was obtained for L.monocytogenes. However,the sensory char- acteristicsof lettuce were compromised by these treatments. The treatment of lettuce leaves with 2% H 2O2 at 508C was effectivenot only in reducing pathogenic bacteria but also in maintaining good sensory quality for up to 15 days. A #4-log reductionof E. coli O157:H7 and Salmonella Enteritidiswas achieved with the 2% H 2O2 treatment,whereas a 3-logreduction of L.monocytogenes wasobtained. There was no signiŽ cant difference ( P . 0.05)between pathogen population reductions obtainedwith 2% H 2O2 with60- and 90-s exposure times. Hydrogen peroxide residue was undetectable (the minimum level ofsensitivity was 2 ppm)on lettuce surfaces after the treated lettuce was rinsed with cold water and centrifuged with a salad spinner.Hence,the treatment of lettucewith 2% H 2O2 at 508Cfor60 siseffectivein initially reducing substantial populations offoodborne pathogens and maintaining high product quality.

Per capitaconsumption of fresh vegetablesand fruits tion,the sensory quality of lettuce can be improvedby mild intheUnited States almost doubled over the past 2 decades. heattreatments (10,11, 17). Hence,the treatment of lettuce However,reportedfoodborne outbreaks associated with withhydrogen peroxide with or without lactic acid at ele- fresh producehave also increased (15).Escherichia coli vatedtemperatures may be a practicalapproach to inacti- O157:H7, Listeriamonocytogenes, Salmonella spp., and vatingpathogenic bacteria without compromising sensory Shigellasonnei were responsiblefor recentoutbreaks as- quality.The objective of thisstudy was todetermine (i) the sociatedwith the consumption of fresh produce (1, 2, 8). bactericidalefŽ cacy of hydrogenperoxide with and without Hence,suitable prevention or decontaminationmethods that lacticacid at elevated temperatures on lettuce and (ii) the minimizethe risk of microbialinfection are needed. Several effectsof theseantibacterial treatments on thesensory char- differentwashing procedures for fresh produceare being acteristicsof lettuce. usedto decontaminate fruits and vegetables. W ashingwith coldchlorinated water is themost widely used method, but MATERIALS AND METHODS itsefŽ cacy in inactivating pathogenic bacteria on the sur- Bacterialcultures. Fivestrains each of E. coli O157:H7 facesof fresh produceis minimal (6,16, 19, 21). (E06,E08, E10, E16, and E22), Salmonellaenterica serotypeEn- Lacticacid and hydrogen peroxide are generally rec- teritidis(E-565-88, E-180-88, E-457-88, SE61696, and E-1294), ognizedas safe chemicalsfor speciŽc usesin foods. The and L.monocytogenes (LM109,LM116, LM201, LM ATCC antibacterialactivity of hydrogenperoxide alone or incom- 19117,and LM H7550serotype 4b) from the culture collection binationwith lactic acid on fresh producehas been reported ofthe UGA Centerfor Food Safety were used. The bacterial (18–22). TheefŽ cacy of a combinationof hydrogen per- strainswere cultured individually in 10 ml of tryptic soy broth oxideand lactic acid in killing microorganisms is greatly (DifcoLaboratories, Detroit, Mich.) for three successive 24-h in- enhancedby an increase in temperature (22, 23). In addi- tervalsat 37 8C.Afterincubation, each culture was sedimented twiceby centrifugationat 4,000 3 g for10 min and then washed *Authorfor correspond ence. Tel:770-228- 7284;Fax: 770-229- 3216; andresuspended in 10 ml of sterile0.1% peptone water .Foreach E-mail: [email protected]Žn.peachnet .edu. pathogen,equal portions of each bacterial strain were combined †Presentaddress: Plant Genetic Resources ConservationUnit, U.S. De- andused as the inoculum (ca. 10 8 CFU/ml).The bacterial count partmentof Agriculture,GrifŽ n, GA 30223,USA. ofthe Ž ve-strainmixture of each pathogen was determined by 1216 LIN ET AL. J.FoodProt., Vol. 65, No. 8 plating0.1-ml portions of appropriate dilutions onto duplicate species.Colonies randomly selected from the colonies with typical platesof trypticsoy agar (TSA; Difco),which were then incubated characteristicsof theinoculated bacteria on TSA andon selective at 378C for 24 h. mediawere conŽ rmed serologically with Oxoid (Ogdensburg, N.Y.)latexagglutination assays for E. coli O157:H7 and Salmo- Inoculationand antimicrobialtreatments. Iceberglettuce nella Enteritidisor biochemically with API strips(bioMe ´rieux, waspurchased from a localgrocery store and stored at 4 8C on Hazelwood,Mo.) for L.monocytogenes. Thecolonies were se- theday before the experiment. On the day of theexperiment, two lectedfrom plates with the highest dilution on which there was tothree layers of outside leaves were removed and discarded. growth. Intactinside leaves were removed and cut into pieces of 11.5 by 8cm.The lettuce leaves were placed in a laminarhood, and 100 Appearanceof lettuce leaves and occurrenceof residual mlofbacterial suspension was delivered in 10 drops onto the hydrogenperoxide. Uninoculatedleaves were used to determine surfaceof each lettuce leaf. Bacterial suspensions on the lettuce thesensory quality of lettuce after the antibacterial treatments. leaveswere dried in a laminarhood for 45 to 55 min at room Afterbeing cut and treated as describedpreviously, lettuce leaves temperature(22 8C).After drying, each inoculated leaf was placed werewashed with cold tap water at 16 to 20 8Cfor10 min. The Downloaded from http://meridian.allenpress.com/jfp/article-pdf/65/8/1215/2004957/0362-028x-65_8_1215.pdf by guest on 23 September 2021 intoa quart-sizeZiploc bag (Johnson & Son,Racine, Wis.). T o washedlettuce leaves were dried on layers of paper towels and obtainbaseline data, 200 ml of 0.1% peptone water was added to thencentrifuged with a handsalad spinner for 2 min.Three hun- thelettuce in theZiploc bag, and then the bag was agitated on an dredgrams of cutlettuce leaves was packed into a Ziplocbag and orbitalshaker at 300 rpm for 2 min.Three different treatments stored at 48C.Theappearance of the lettuce leaves was observed wereevaluated. For the Ž rsttreatment, lettuce leaves were im- 3,5, 7, 10, and 15 days after treatment. Residual hydrogen per- mersedin acombinationof 1.5%lactic acid (30%; Sigma Chem- oxidewas determined by pressinga teststrip of theRe ectoquant icalCo., St. Louis, Mo.) and 1.5% hydrogen peroxide (30%; Sig- system(EM ScienceInc., Gibbstown, N.J.) against a moistspot ma)solution (prepared with sterilized deionized water on a vol/ ona lettuceleaf. At leastthree areas on a lettuceleaf were as- volbasis) at 40 8Cfor15 min.The second treatment solution con- sayed,with the detection limit of theRe ectoquant system being tained1.5% lactic acid and 2% hydrogen peroxide, and lettuce 2ppm.The presence of residual hydrogen peroxide was deter- leaveswere treated at 22 8Cfor5 min.For the third treatment, minedimmediately after centrifugation and on eachday on which lettuceleaves were treated only with 2% hydrogen peroxide at lettucewas evaluated for appearance. 508Cfor60 or 90 s. Lettuce leaves treated with sterile deionized waterunder the same temperature-time conditions were used for Statisticalanalysis. Datafrom independent replicate trials controlsamples. Two hundredmilliliters of fresh-madetreatment werepooled, and the mean value and standard deviation were solutionor sterile deionized water was added to the Ziploc bag, determinedwith Microsoft Excel 97 for each pathogen. The sig- whichwas then immersed in a waterbath at the designated tem- niŽcance of differences between treatments was determined by a perature.Control samples were tested side-by-side with treated t testwith the Statistical Analysis System (SAS) program. samples.The treatment solution and sterile deionized water were RESULTS AND DISCUSSION warmedto the designated temperature in the water bath prior to theexperiment. Three leaves were used for each treatment, and Weusedspot inoculation instead of dip inoculation to trialswere duplicated for each treatment condition and bacterial applypathogens to the lettuce because spot inoculation species. morerealistically represents the mode by which lettuce Enumerationof bacterial populations. Aftertreatment, couldbe contaminated in the Ž eld.In addition, the spot eachlettuce leaf was transferred into a secondZiploc bag con- inoculationmethod offered more consistent and reproduc- taining200 ml of Dey-Engley (DE) neutralizingbroth. The bags ibleresults for theinoculation of a knownnumber of bac- containinglettuce leaves and DE brothwere agitated on anorbital terialcells on lettuce surfaces in our laboratory (4). shakerat 300rpm for 2min.The DE brothin thebags was serially Alargerreduction of bacterial populations was ob- (1:10)diluted in 0.1%peptone water. For undiluted samples, qua- servedfor lettucetreated with a combinationof lactic acid druplicate0.25-ml and duplicate 0.1-ml samples of DEbrothwere andhydrogen peroxide than for lettucetreated with hydro- plated.Duplicate 0.1-ml portions of diluted samples were also genperoxide alone. More than 4 logCFU of E. coli O157: plated.All plates were incubated at 37 8Cfor24 h. A volumeof H7 and Salmonella Enteritidisper lettuce leaf and about 3 2mlof undiluted DE brothwas transferred to a 250-mlErlen- log CFU of L.monocytogenes perlettuce leaf were inac- meyer askcontaining 48 ml of tryptic soy broth. Following en- tivated(T ables1 and2) by the combination of lactic acid richmentfor 24 h at37 8C,theculture in each  askwas streaked ontoduplicate selective agar plates. Bacterial populations in treat- andhydrogen peroxide for bothtime-temperatures condi- mentand control solutions were enumerated by plating and were tions.The treatment of lettucewith 2% hydrogen peroxide detectedby enrichmentaccording to the same procedures used for at 508Cfor 90s reduced Salmonella Enteritidis, E. coli thelettuce samples. The pH values of the treatment solution, the O157:H7,and L.monocytogenes populationsby 4.5, 4.7, deionizedwater ,andthe DE brothwere measured with a pHmeter and 2.7 log10 CFU perleaf, respectively. Slightly less ex- afterthe lettuce was rinsed (Accumet Basic, Fisher ScientiŽ c, tensivebut not statistically signiŽ cantly different ( P . Pittsburgh,Pa.). 0.05)pathogen reduction was obtainedwith a 60-sexposure time(T ables3 through5). A reductionof approximately 1 Bacterialmedia and conŽrmation. Allmedia were pur- logCFU ofpathogen per leaf was observedfor lettuce chasedfrom Difco. Xylose lysine desoxycholate (XLD), sorbitol MacConkeyagar (SMA), and modiŽ ed Oxford (MOX) agarwere treatedwith sterile deionized water (control) under all tem- usedas selective media for Salmonella Enteritidis, E. coli O157: perature-timeconditions. Previous studies on the use of H7, and L.monocytogenes, respectively.TSA wasused as anon- chlorineon lettuceand other types of producealso revealed selectivemedium to recover injured bacterial cells. T rypticsoy thatincreasing the exposure time from 1to10 min did not brothwas used as a generalenrichment broth for allthree bacterial signiŽcantly further reduce the populations of inoculated E. J.FoodProt., Vol. 65, No. 8 INACTIVATION OFPATHOGENSON LETTUCE 1217

TABLE 1. Populationsof E. coli O157:H7, Salmonella Enteritidis,and L. monocytogenes onlettuce leaves treated with 1.5% lactic acidand 1.5% H 2O2 at 408Cfor15 min and in treatment and control solutions Count fora:

Treatment Salmonella Enteritidis E. coli O157:H7 L.monocytogenes

Nonselectiveagar b Untreatedlettuce 7.19 6 0.31 7.12 6 0.27 6.95 6 0.10 Treatedlettuce 2.84 6 0.44 2.36 6 0.18 3.77 6 0.37 Treatmentsolution NDc ND ND Controllettuce 6.08 6 0.61 5.81 6 0.71 6.26 6 0.23 Controlsolution d 6.92 6 0.22 5.09 6 0.36 6.87 6 0.31 e

Selectiveagar Downloaded from http://meridian.allenpress.com/jfp/article-pdf/65/8/1215/2004957/0362-028x-65_8_1215.pdf by guest on 23 September 2021 Untreatedlettuce 7.09 6 0.41 6.92 6 0.28 6.45 6 0.50 Treatedlettuce 2.54 6 0.34 2.26 6 0.33 3.47 6 0.36 Treatmentsolution ND ND ND Controllettuce 6.18 6 0.75 5.44 6 0.51 6.02 6 0.45 Controlsolution 6.77 6 0.46 5.11 6 0.26 6.22 6 0.51 a Log10 CFU perleaf for lettuce; log 10 CFU/mlfor solution; average 6 standarddeviation. Data were obtained from duplicate trials; eachtrial involved three samples. b TSA wasthe nonselective agar used to enumerate pathogens. c ND, notdetectable. The minimum level of detection was 50 CFU perleaf. d Steriledeionized water was the control solution used to treat lettuce leaves. e Selectiveagars used to enumerate pathogens were XLD, SMA, andMOX for Salmonella Enteritidis, E. coli O157:H7,and L. mono- cytogenes, respectively. coli O157:H7, L.monocytogenes, and Salmonella spp. (3, largerthan those recovered from theselective agars, with 5). Beuchatet al. (5) concludedthat inactivation of most bacterialcounts for TSA averaging0.39 log 10 CFU per leaf microorganismson produceoccurred within 1 minafter the higherthan those for selectivemedia. However ,therewas applicationof chlorine. Similar mechanisms may apply to nostatistically signiŽ cant difference ( P $ 0.05)between hydrogenperoxide, which is also a strongoxidizing agent. pathogenpopulations recovered from TSAandthose re- As abeneŽt, it ismoreconvenient to applyan antibacterial coveredfrom selectivemedia. None of the three bacterial treatmentfor ashorterperiod. specieswas detectedin treatment solutions, but large cell Thepathogen populations recovered from TSAwere populations( .5 log10 CFU/ml) were recoveredfrom con-

TABLE 2. Populationsof E. coli O157:H7, Salmonella Enteritidis,and L. monocytogenes onlettuce leaves treated with 1.5% lactic acidand 2.0% H 2O2 at 228Cfor5 minand in treatment and control solutions Count fora:

Treatment Salmonella Enteritidis E. coli O157:H7 L.monocytogenes

Nonselectiveagar b Untreatedlettuce 6.87 6 0.12 6.95 6 0.24 7.20 6 0.25 Treatedlettuce 2.04 6 0.34 2.16 6 0.46 3.73 6 1.48 Treatedsolution NDc ND ND Controllettuce 6.19 6 0.27 6.89 6 0.52 6.76 6 0.03 Controlsolution d 7.27 6 0.04 5.89 6 0.52 7.54 6 0.03 Selectiveagar e Untreatedlettuce 6.52 6 0.02 6.82 6 0.02 6.98 6 0.02 Treatedlettuce 1.97 6 0.14 2.01 6 0.23 3.52 6 0.42 Treatmentsolution ND ND ND Controllettuce 4.97 6 0.39 5.61 6 0.55 6.67 6 0.10 Controlsolution 3.85 6 0.39 5.80 6 0.28 7.45 6 0.05 a Log10 CFU perleaf for lettuce; log 10 CFU/mlfor solution; average 6 standarddeviation. Data were obtained from duplicate trials; eachtrial involved three samples. b TSA wasthe nonselective agar used to enumerate pathogens. c ND, notdetectable. The minimum level of detection was 50 CFU perleaf. d Steriledeionized water was the control solution used to treat lettuce leaves. e Selectiveagars used to enumerate pathogens were XLD, SMA, andMOX for Salmonella Enteritidis, E. coli O157:H7,and L. mono- cytogenes, respectively. 1218 LIN ET AL. J.FoodProt., Vol. 65, No. 8

TABLE 3. Populationsof Salmonella Enteritidison lettucetreat- TABLE 4. Populationsof E. coli O157:H7on lettucetreated with ed with 2% H2O2 (treated)and deionized water (control) at 50 8C 2% H2O2 (treated)and deionized water (control) at 50 8C for 60 for 60 or 90 sa or 90 sa

Count (log10 CFU/leaf) for Count (log10 CFU/leaf) for exposuretime exposuretime

Treatment 60 s 90 s Treatment 60 s 90 s

Nonselectiveagar Nonselectiveagar Untreatedlettuce 7.66 6 0.33 7.67 6 0.32 Untreatedlettuce 7.75 6 0.40 7.68 6 0.54 Treatedlettuce 3.95 6 0.41 3.18 6 0.45 Treatedlettuce 3.38 6 0.74 3.11 6 0.38 Controllettuce 6.67 6 0.37 7.11 6 0.53 Controllettuce 6.26 6 0.53 6.85 6 0.13 Selectiveagar Selectiveagar Downloaded from http://meridian.allenpress.com/jfp/article-pdf/65/8/1215/2004957/0362-028x-65_8_1215.pdf by guest on 23 September 2021 Untreatedlettuce 7.59 6 0.32 7.25 6 0.38 Untreatedlettuce 7.54 6 0.32 7.11 6 0.18 Treatedlettuce 3.68 6 0.57 2.98 6 0.24 Treatedlettuce 2.94 6 1.06 2.82 6 0.23 Controllettuce 6.64 6 0.40 6.70 6 0.25 Controllettuce 6.15 6 0.46 6.05 6 0.68 a Valuesshown are averages 6 standarddeviations. Data were a Valuesshown are averages 6 standarddeviations. Data were obtainedfrom duplicate trials; each trial involved three samples. obtainedfrom duplicate trials; each trial involved three samples. Therewere no statistically signiŽ cant differences ( P . 0.05) in Therewere no statistically signiŽ cant differences ( P . 0.05) in Salmonella Enteritidiscounts for the 60- and 90-s treatments. E. coli O157:H7counts for the 60- and 90-s treatments. The Thenonselective agar was TSA, andthe selective agar was nonselectiveagar was TSA, andthe selective agar was SMA. XLD. treatmentsolutions of 1.5% lactic acid plus 1.5 or 2% hy- trolsolutions. Similar results were obtainedfrom ourpre- drogenperoxide, 2% hydrogenperoxide, and deionized wa- viousstudy on fruits (23). Hence,produce wash water may terboth before and after treatment and of DE brothafter containpathogens from contaminatedproduce and should theneutralization of treated lettuce are shown in T able7. notbe reused without proper sanitation. ThepH values obtained for DEbrothwere similarfor all Althoughmore extensive bactericidal activity was ob- treatments.Hence, the buffering capacity of DE brothwas servedwhen lactic acid was usedin combination with hy- sufŽcient to neutralizethe acidity of thetreatment solutions. drogenperoxide, the sensory quality of lettuce was com- Hydrogenperoxide was removedfrom thelettuce surface promisedby the inclusion of lactic acid in the treatment. afterrinsing and centrifugation, with only trace amounts (5 Severebrowning was observedon lettuce 3 to5 daysafter to10 ppm) of hydrogenperoxide being detected after rins- treatmentwith the combination of lacticacid and hydrogen ingwith cold water and no residue ( ,2ppm)being de- peroxidefor bothtemperature-time treatments evaluated tectedimmediately after centrifugation with the salad spin- (14). Consumersin an extensive sensory panel study de- ner. terminedthat lettuce leaves treated with a combinationof Thetreatment involving a combinationof hydrogen lacticacid and hydrogen peroxide were notacceptable (14). peroxideand mild heat also improved the visual quality of Inour previous studies, apples, oranges, and tomatoes treat- edwith a combinationof lacticacid and hydrogen peroxide retainedgood sensory qualities (13, 22). Thus,different an- TABLE 5. Populationsof L.monocytogenes onlettuce treated with 2% H O (treated)and deionized water (control) at 50 C for timicrobialtreatments may be neededfor differenttypes of 2 2 8 60 or 90 sa produce. Lettucetreated with 2% hydrogenperoxide at 50 8C for Count (log10 CFU/leaf) for 90shada betterappearance than the control lettuce, which exposuretime was treatedat 50 8Cindeionizedwater (Table6). Both treat- Treatment 60 s 90 s edand control lettuce leaves were slightlybrown after 10 daysof storage at 4 8C.However,browningof control let- Nonselectiveagar tucewas moreextensive than that of treated samples. All Untreatedlettuce 7.27 6 0.26 7.26 6 0.44 lettuceleaves retained their Ž rmnesseven after 15 days of Treatedlettuce 4.76 6 0.01 4.53 6 0.48 storage.Similar results were obtainedfor lettucetreated Controllettuce 6.48 6 0.40 6.96 6 0.53 with2% hydrogen peroxide at 50 8Cfor 60s onthe basis Selectiveagar oftheobservations of laboratorypersonnel and a consumer Untreatedlettuce 6.96 6 0.18 7.06 6 0.64 sensorypanel (12). After 10days, treated lettuce was rated Treatedlettuce 4.39 6 0.06 4.10 6 0.21 asgood as or better than untreated lettuce, depending on Controllettuce 6.22 6 0.40 6.23 6 0.64 thecolor of the leaves. Leaves initially having a morein- a tensegreen color were ratedbetter than leaves of a less Valuesshown are averages 6 standarddeviations. Data were obtainedfrom duplicate trials; each trial involved three samples. intensegreen. After 15days, treated lettuce was superior Therewere no statistically signiŽ cant differences ( P . 0.05) in tountreatedlettuce in sensory quality provided that the let- L.monocytogenes countsfor the 60- and 90-s treatments. The tuceinitially was intenselygreen. The pH values of the nonselectiveagar was TSA, andthe selective agar was MOX. J.FoodProt., Vol. 65, No. 8 INACTIVATION OFPATHOGENSON LETTUCE 1219

TABLE 6. Appearanceof lettuce leaves treated with 2% H 2O2 (treated)and deionized water (control) at 50 8Cfor90 s andstored for 3,5, 7, 10, and 15 days at 4 8Ca Appearance onday:

Treatment 3 5 7 10 15

Treated No browning No browning No browning Slightbrowning Slightbrowning on cut edge Control No browning No browning No browning Slightbrowning Slightbrowning a Datawere obtained from duplicate trials; each trial involved three samples for each treatment.

fresh-cutcantaloupe. The visual quality of cantaloupecubes warm water(47 8C)containing1,000 ppm chlorine reduced Downloaded from http://meridian.allenpress.com/jfp/article-pdf/65/8/1215/2004957/0362-028x-65_8_1215.pdf by guest on 23 September 2021 preparedfrom wholefruits treated with 5% hydrogen per- thepopulation of natural contaminating microbes by 3 log oxide at 508Cfor 1minwas betterthan that of cubes pre- CFU/gonlettuce, no data for pathogenicbacteria were re- paredfrom fruitstreated with 1,000 ppm chlorine at 20 8C ported,and the visual quality of thetreated fresh-cut lettuce for 1 min (20). Populationsof natural contaminating mi- was retainedfor only7 days (7). crobeson the surface of the cantaloupe treated with 5% Consumptionof fresh-cut produce likely will continue 2 hydrogenperoxide were 2log 10 CFU/cm smallerthan toincrease, and a widervariety of products will be intro- thoseon thechlorine-treated and control samples after stor- ducedinto the market. The treatment of lettuce with 2% age at 48Cfor 15days. A 3-to 4-log reduction of non- hydrogenperoxide and mild heat followed by a coldwater pathogenic E. coli was achievedby treating whole apples rinsecould confer a marginof microbiological safety on witha combinationof 5% hydrogen peroxide and acid sur- theproduct and could result in the retention of, and a pos- factantat 50 8C for 1 min (19). However,thesensory char- sibleimprovement in, the sensory quality of fresh-cut let- acteristicsof the treated apples and of pressed apple cider tuce.Commercial implementation of thistreatment will re- were notdescribed. quireregulatory approval. Browningreactions on lettuce leaves are enzymatic. ACKNOWLEDGMENTS Theenzymatic activities of browning enzymes released from injuredlettuce cells are greatly reduced by mild heat Thisstudy was sponsoredby a grant(FD-U-001631-01-1) from the treatment (10, 17). Phenylalanineammonia-lyase is theŽ rst U.S.Food and Drug Administration, Washington, D.C., and the State of enzymeinvolved in the browning reaction, and its activity Georgia’s TraditionalIndustries Program for Food Processing. isgreatly retarded by mild heat treatment applied either REFERENCES beforeor aftercutting (10). Thus,heat-treated lettuce leaves retainbetter visual quality than non– heat-treated leaves do. 1.Ackers, M.-L.,B. E.Mahon,E. Leahy,B. Goode,T .Damrow, P.S. However,treatinglettuce with 20 ppmchlorine or deionized Hayes, W.F.Bibb,D. H.Rice, T.J.Barrett, L.Hutwagner,P.M. GrifŽn, andL. Slutsker.1998. An outbreak of Escherichiacoli O157: water at 508Cfor 90s doesnot effectively inactivate in- H7infections associated withleaf lettuce consumption.J. Infect.Dis. oculated E. coli O157:H7 (9). Inaddition, larger popula- 177:1588–1593. tions of E. coli O157:H7were recoveredfrom heat-treated 2.Beuchat, L. R.1996. 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