Bioluminescence ATP Assay for Estimating Total Plate Counts Of
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813 Journalof Food Protection, Vol. 64, No. 6, 2001, Pages 813– 819 Bioluminescence ATPAssay for EstimatingT otalPlate Counts ofSurface Microora ofWhole Cantaloupe and Determining Efcacy ofW ashingTreatments † DIKE O.UKUKU, * VLASTA PILIZOTA, AND GERALD M.SAPERS U.S.Department of Agriculture,Agricultural Research Service, EasternRegional Research Center, 600East Mermaid Lane, Wyndmoor, Pennsylvania 19038, USA MS00-226:Received 11July 2000/ Accepted 19December 2000 ABSTRACT Thesurface micro ora of cantaloupes were estimated using a bioluminescenceA TPassay,and results were compared to platecount data. Cantaloupes were treated as follows: (i) water washed, or (ii) washed in solutions of sodium hypochlorite (1,000mg/ liter)or hydrogen peroxide (5%) for 5 min.Bioluminescence A TPassayresults showed differences in ATP level/ cm2 ofcantaloupes dipped in chlorine or hydrogen peroxide solution; A TPlevelsin these washed samples were lower than incontrols due to antimicrobial action of the treatments on the cantaloupe surface. Linear correlations were found between thebioluminescence A TPassayand aerobic plate counts of unwashed cantaloupe ( r 2 5 0.995)and those washed with water (r 2 5 0.990)determined before storage. Lower correlations between the bioluminescence ATP assayand the aerobic plate countswere obser vedon cantaloupes stored for 120 h at20 8C (r 2 5 0.751)than at 4 8C (r 2 5 0.980)without washing treatment.Lower correlation at 20 8Cmaybe the result of clusters or growth that occurred in chains. A TPlevelsof washed cantaloupescorrelated well with bacterial plate counts ( r 2 5 0.999).A reliableminimum detectable threshold using the 2 2 bioluminescenceA TPassaywas established at 3log 10 fg/cm correspondingto 4log 10 CFU/cm .BioluminescenceA TPassay isnot recommended for washed samples where the microbial load is near or below the threshold. Therefore, the biolumines- cenceATP assaywill be recommendedfor quick estimation of totalmicrobial load on cantaloupesurfaces where the population isexpected to exceed this threshold. The assay can save the industry time by eliminating the required incubation required by theconventional methods. Themicro ora of all food items are ofpractical sig- ducemicrobial populations if done properly (7). Washing nicance to producers (1, 4, 25), processors (3,12, 15, 31), usuallyconsists of spraying with potable water or may in- andconsumers (16,20, 22). Fruitsand vegetables are fre- volveuse of disinfection by application of chlorine solu- quentlyin contact with soil, insects, animals, and humans tions.Chlorination of washwater hasbeen reported to pre- duringgrowing, harvesting (32), andin theprocessing plant ventmicrobial contamination in produce processing lines (21). Thus,their surfaces are notfree from naturalcontam- (36). However,widevarieties of organicmaterials are sub- inants,and by the time they reach the packing house, most jectto oxidationand chlorination reactions in aqueouschlo- fresh produceretain populations of 10 4 to 106 microorgan- rinesolutions. Chlorination reactions are of particular con- isms/g (6, 7). Thelevel of sanitation and the microbiolog- cernin foods because of formation of potentially toxic icalload are of primary importance to the quality, shelf chlororganiccompounds (37). Inthis study, the ef cacy of stability,and safety of fresh produce (7, 8). Therefore, hydrogenperoxide as analternative to chlorinein reducing knowledgeof the level of micro ora on fruit or vegetable surfacemicro ora of cantaloupe was investigated.While surfacesshould help processors in implementing hazard thereare numerous reports in the literature on the use of analysisand critical control point (HACCP) plansand good chlorineas a sanitizer,informationon the use of hydrogen manufacturingpractices. However ,estimationof themicro- peroxideon produce is limited. bialload of a foodstuffis problematic, particularly where Recoveryof bacteria or other microorganisms from thesurface of interest is uneven, as in the case of canta- cantaloupesurfaces may not represent a truevalue due to loupe.Accordingly, the problem of obtaining a represen- thesurface roughness of these fruits. The surface roughness tativesample for examinationis often dif cult (24). favorsmicrobial attachment and complicates detachment. Washingis one of the very rst processingoperations Faveroet al. (11) reviewedmicrobiological sampling meth- towhich a fruitor vegetable is subjected. W ashingcan re- odsfor surfacesand summarized them into four basic meth- ods:(i) therinse, (ii) the swab rinse, (iii) agar contact, and *Authorfor correspondence. Tel: 215-233-6427; Fax: 215-233-6406; (iv)direct surface agar plating. Methods 2 through4 are E-mail: [email protected]. notsuitable for wholecantaloupe or other fruits with large †Mentionof brand or rm name doesnot constitute an endorsement by theU.S. Department ofAgriculture over others of similar naturenot surfacearea for thefollowing reasons: (i) largeamounts of mentioned. mediawould be required;(ii) sampling would be time-con- 814 UKUKU ET AL. J.FoodProt., Vol. 64, No. 6 TABLE 1. Comparisonof extracting media for the biolumines- sitive.The purposes of thisstudy were toexaminea method cenceATP assayof cantaloupe surfaces a thatis suitable for extractingsurface microbial A TP of Log10 Log10 cantaloupesand then use the method to monitor changes in Extractingmedia CFU/cm2b ATP (fg/cm2) thesurface A TPlevelof cantaloupesstored at differenttem- peratures.The correlation between log femtogram (fg) ATP Extralight 7.10 6 0.12 5.14 6 0.05 andlog CFU was investigatedto determine the reliability Chloroform-water(30% vol/ vol) 7.00 6 0.03 5.43 6 0.47 ofthe bioluminescent A TP assay.Finally, the assay was Tris-EDTAc 7.18 6 0.10 5.40 6 0.04 usedto compare the ef cacy of hydrogen peroxide and a Valuesare means of vetrials with duplicate determinations. All chlorinesolutions in reducing the surface micro ora of ATPextractionswith the exception of Extralight involved boil- cantaloupe. ing step. b Determinationswere performed immediately before A TPextrac- MATERIALS AND METHODS tionand determination. c 0.1M Trisin 2 mM EDTA. Preparationof sanitizers. Clorox,a commercialbleach con- taining5.25% sodium hypochlorite (NaOCl; Clorox Company, Oakland,Calif.), was diluted in sterile water to provide the desired suming;and (iii) these methods may not give an accurate concentration(1,000 mg/ liter)of chlorine in the wash solution. indicationof total microbial counts on the surface. The ThepH was adjusted downward to 6.4 6 0.1by adding citric rinsemethod involving the use of water would only be acid.Free chlorine in the solution was determined with a chlorine suitablefor cantaloupeif adheringbacteria were allde- testkit (Hach Co., Ames, Iowa) that has been approved by the tachedby the rinse and then enumerated in the rinse water . U.S. EnvironmentalProtection Agency. Hydrogen peroxide was Sincethis is unlikely, new methods that can take into ac- preparedfrom a 30%stock solution (Fisher Scienti c, Suwannee, countsize, shape, and surface properties of cantaloupe are Ga.),which was diluted in sterilewater to providea concentration of5% in the wash solution. needed. Estimationof microbial numbers in foods by conven- Cantaloupes. Cantaloupespurchased from a localsupermar- tionalmicrobiological techniques takes at least2 to3days, ketand stored at roomtemperature ( ;208C) or 48Cfor0, 24,72, andthere is a needfor fastermethods that can give results or120 h weregrouped (six per treatment) into the following cat- inminutes. The bioluminescent A TP determinationwith egories:(i) unwashed; (ii) washed with tap water; and (iii) washed rey luciferaseprovides a sensitiveindicator for thepres- insodium hypochlorite (1,000 mg/ liter)or in hydrogen peroxide enceand growth of bacteria in biological material. The (5%)for 5 min.The concentrations of sodium hypochlorite and presenceof bacteria in various media can be estimated us- hydrogenperoxide were chosen based on the results of our pre- ingthe bioluminescent A TP technique (2,5, 13, 14, 18, viouslaboratory work (27). Chlorine-or hydrogen peroxide-treat- 34). Its usefor rapidmeasurement of microbial cell num- edsamples were rinsed by dipping in sterile water and agitating bersis based on the premise that all living things contain witha glove-coveredhand for 1 min,then air dried for 1 hat roomtemperature before sampling. ATPandthat the intracellular A TP levelshave to be con- stantfor cellsto maintain normal physiological activities. Samplepreparation for microbiology. Asterilizedstainless Therefore,the level of ATPdeterminedin a sampleis pro- steelcork borer was used to cut randomly through the cantaloupe portionalto theactual cell number (30, 33). Theassay does surfaceto produce rind plugs of 22 mm diameterwith a surface notrequire incubation and is therefore very rapid and sen- area (pr2) of 3.80 cm2.Atotalof 152 rind plugs per cantaloupe FIGURE 1. Relationshipbetween the plate counttechnique and the bioluminescence ATP assayof surface micro ora of canta- loupesstored at 20 8Cfor24 h. Determi- nationsof ATP wereperformed using 0.1 MTrisin 2 mM EDTA, 30%chloroform- water(vol/ vol),and Extralight, a commer- cialdetergent from Turner Design. Values aremeans of threedeterminations 6 stan- darddeviation. A correlation( r2 5 0.990) betweenplate count numbers and ATP val- ueswas noted. J.FoodProt., Vol. 64, No. 6 CANTALOUPE SURFACEMICROFLORA ESTIMATION USING ATPBIOLUMINESCENCE 815 TABLE 2. BioluminescenceATP estimationof the surface micro ora of cantaloupe enumerated from different media