382 Journalof Food Protection, Vol. 66, No. 3, 2003, Pages 382– 389 Viabilityof Clostridiumperfringens, Escherichia coli, and Listeria monocytogenes SurvivingMild Heat or AqueousOzone Treatment onBeef Followedby Heat, Alkali,or Salt Stress † JOHN S.NOVAK 1* AND JAMES T.C.YUAN 2 1U.S.Department of Agriculture, Agricultural Research Service, EasternRegional Research Center,Microbial Food Safety Research Unit,600 East MermaidLane, Wyndmoor, Pennsylvania 19038; and 2AmericanAir Liquide, Chicago Research Center,5230 South East Avenue, Countryside,Illinois 60525, USA Downloaded from http://meridian.allenpress.com/jfp/article-pdf/66/3/382/1674080/0362-028x-66_3_382.pdf by guest on 01 October 2021 MS02-200:Received 20June 2002/ Accepted 18October 2002 ABSTRACT Thethreat of pathogen survival following ozone treatment of meatnecessitates careful evaluation of themicroorganisms survivingunder such circumstances. The objective of this study was to determine whether sublethal aqueous ozone treatment (3 ppm of O3 for5 min)of microorganisms on beef surfaces would result in increased or decreased sur vivalwith respect to subsequentheat, alkali, or NaCl stress. A mildheat treatment (55 8Cfor30 min) was used for comparison. Reductions in three-straincocktails of Clostridiumperfringens, Escherichia coli O157:H7,and Listeriamonocytogenes onbeef following theheat treatment were 0.14, 0.77, and 1.47 log 10 CFU/g,respectively, whereas reductions following ozone treatment were 1.28,0.85, and 1.09 log 10 CFU/g,respectively. C.perfringens cellsexhibited elevated heat resistance at 60 8C (D60 [time at 608Crequiredto reduce the viable cell population by 1 log 10 unitsor 90%] 5 17.76min) following heat treatment of beef (558Cfor30 min) but exhibited reduced viability at 60 8Cfollowingozone treatment ( D60 5 7.64min) compared with the viabilityof untreated control cells ( D60 5 13.84min). The D60-values for L.monocytogenes and E. coli O157:H7following heatand ozone exposures were not signi cantly different ( P . 0.05). C.perfringens cellsthat sur vivedozone treatment did notexhibit increased resistance to pH (pH 6 to12) relative to non-ozone-treated cells when grown at 37 8Cfor24 h.Theheat treatmentalso resulted in decreased numbers of sur vivingcells above and below neutral pH valuesfor both E. coli O157:H7 and L.monocytogenes relativeto those of non-heat-treated cells grown at 37 8Cfor24 h. There were signi cant differences (P , 0.05) in C.perfringens reductionswith increasing NaCl concentrations. The effects of NaCl were less apparent for E. coli and L. monocytogenes survivors.It is concluded that pathogens surviving ozone treatment of beef are less likely to endangerfood safety than are those surviving sublethal heat treatments. Therecent U.S. Foodand Drug Administration ap- stituents,including proteins, unsaturated lipids, peptidogly- provalof ozone (O 3)for useas an antimicrobial agent in cans,enzymes, and nucleic acids (17). Ozone,a verypotent thestorage and processing of meats and produce under- oxidizingagent, readily inactivates microorganisms in scoresthe need for moreextensive research to evaluatethis aqueoussolutions; however ,infoods, ozone indiscrimi- technology (6). Over theyears, different studies have dem- natelyreacts with many organic components, resulting in onstratedthe success of ozonetreatment in the disinfection self-depletionand reduced biocidal effectiveness (5). The of shell sh (34), theinhibition of the overripening and de- sameresults can be expected of most other chemical oxi- cayof fruits (8, 19), andthe inhibition of microbialgrowth dants,such as hypochlorite and hydrogen peroxide. It has duringthe storage of meats (29). Thereis no information, beensuggested that ozonated water mightbe more appli- however,onthe effect of ozone treatment on the cross- cableto the killing of microorganisms on fruits or vegeta- protectionor cross-sensitizationof survivingbacterial cells bles,where interference by organic components might be withregard to subsequent stresses. The food industry must minimal (27). Theuse of increased ozone concentrations beprepared to incorporate this technology safely without (.3 ppm O3 for 5min)to inactivate pathogens attached to therisk of increasedhazards arising from survivingorgan- foodscan become problematic, because ozone may cause isms. theoxidation of fats and discoloration (18). Asalientfea- Bacterialcell death following oxidation brought about tureof ozone reactions is that one atom from ozoneis in- byozone treatment has been attributed to changes in cel- corporatedin theoxidized product, while the unincorporat- lularpermeability followed by lysis (10). However,ozone edreactant (O 2)isreleased as innocuous, nontoxic oxygen hasalso been reported to damage numerous cellular con- (16). Thisis a majorfactor for considerationwith regard tothe use of ozonerather than commonly used chlorinated antimicrobialagents that may result in ef uents containing *Authorfor correspondence. Tel: 215-836-3762; Fax: 215-233-6581; carcinogenicchlorinated by-products. E-mail: [email protected]. †Mentionof brandor rm names doesnot constitute an endorsement by Severalstudies have highlighted the limited microbial theU.S. Department ofAgriculture over others of a similar naturenot reductionsresulting from ozonetreatment of meatsurfaces mentioned. (7,11, 14, 29). Typicalaerobic plate count reductions ob- J.FoodProt., Vol. 66, No. 3 SURVIVAL AFTEROZONE TREATMENT 383 tainedby rinsing beef carcasses with ozonated water (2 sterileloops to culture tubes containing 10 ml of BHI (for E. coli 2 O157:H7 and L. monocytogenes cultures)or 10 ml of uid thio- ppm)at 28to 42 8Cwere 1to2log 10 CFU/cm ,reductions thatwere notany larger than those obtained with conven- glycolatemedium (Difco) (for C.perfringens cultures).Following tionalwashing procedures (26). Combinationtreatments in- 4to6 hofincubation at 37 8C,1-mlaliquots of eachof thethree E. cludingheat, irradiation, acidic pH, and competitive micro- strainsof each organism were transferred to 1 literof BHI (for coli O157:H7 and L.monocytogenes strainmixes) or 1 literof oraenhance microbial inactivation in foods (32). There- uidthioglycolate (for C.perfringens strainmixes). The cultures fore,a potentialuse for ozonemay be as an antimicrobial werethen incubated at 37 8Cfor16 h withoutaeration. treatmentin synergy with other treatments, such as heator irradiation.Others have reported that food preservation Beefinoculation and treatments. Londonbroil beef (top practicesinvolving heat, cold, irradiation, high pressure, os- roundcut from the steer ’slongissimusdorsi muscle) was pur- moticstress, and pH modi cationshave the potential to chasedfrom a localsupermarket, cut into sections (7.5 by 10.0 increasethe resistance and virulence of microorganisms (1, by1.0 cm), and weighed, and these sections were separately vac- uumsealed to 2kPain avacuumpackager (Multi-V acInc.,Model 2,4). Escherichia coli O157:H7that had been prestressed Downloaded from http://meridian.allenpress.com/jfp/article-pdf/66/3/382/1674080/0362-028x-66_3_382.pdf by guest on 01 October 2021 A300,Kansas City, Mo.). The meat was sterilized by irradiation ata lowpH for 60min at 37 8Cwas foundto be more witha self-containedgamma-radiation source of 137Cs (42 kGy, resistantto salt (28). Stressingof Listeriamonocytogenes 2308C)atthe ERRC facilityto eliminate contaminating micro- withthe addition of ethanol, hydrogen peroxide, hydro- organisms.Cocktails for a speci cbacterialspecies contained chloricacid, or sodium chloride to the growth medium for threestrains per species as described above and were grown sep- 60 min at 358Cresultedin increasedtolerance to heat (21). aratelyto 10 9 CFU/ml.Meat samples were immersed in the bac- Resultsof variousstudies have shown that microorganisms terialsuspensions for 30 sandthen drip dried for 15 s.Treatments willsurvive ozone treatment of meats; thus, it is possible usedfor the samples are shown in Figure 1. thatsurviving organisms may be cross-protectedfrom other Beefinoculation and treatments:control samples. Con- antimicrobialor food preservation treatments. trolswere inoculated and placed in separate sterile lter-lined Thepresent study is the rst toevaluate the ability of Stomacherbags (Nasco, Fort Atkinson, Wis.), to which 10 ml of differentfoodborne pathogens surviving ozone treatment 0.1%(wt/ vol)peptone water was added. The bags were hand mas- onbeef surfaces to resist subsequent applied stresses such sagedfor 30 s andthen pummeled for 2 minwith a Stomacher asheat, pH, and salt. Heat (55 8Cfor 30min) and ozone (3 lab-blender(Model 400, Spiral Systems, Inc., Cincinnati, Ohio). ppmfor 5min)treatments that did not visually damage Thenumber of bacteria in the inoculated meat ltratewas deter- meatwere usedto evaluate reductions in total viable path- minedby the spread plating of dilutions onto tryptic soy agar ogenson beef as well as thesusceptibility of thesurviving (TSA; Difco)(for E. coli O157:H7 and L.monocytogenes ) or bacteriato subsequent stresses. ShahidiFerguson perfringens (SFP; Difco)agar base medium withoverlay(for C.perfringens ) (Fig. 1). MATERIALS AND METHODS Beefinoculation and treatments:heat-treated samples. Microorganisms. Threedifferent strains were combined to Beefcuts were inoculated, placed individually into Stomacher l- forma cocktailfor each bacterial species, and this species-speci c terbags, vacuum sealed to 2 kPa,submerged in a waterbath at culturecocktail was then separately examined for its survival on 558Cfor30 min, and chilled on ice for 2 min.T