1595

Journal of Food Protection Vol 66 No 9 2003 Pages 1595ndash1598Copyright q International Association for Food Protection

Survival of Escherichia coli O157H7 Listeria monocytogenesand Salmonella in Juice Concentrates

OMAR A OYARZABAL1dagger MARA C L NOGUEIRA23 AND DAVID E GOMBAS2

1Neogen Corporation 620 Lesher Place Lansing Michigan USA 2National Food Processors Association 1350 I Street NW Suite 300Washington DC 20005 USA and 3Department of Microbiology Biologic Sciences Institute University of Minas Gerais Belo Horizonte Brazil

MS 02-461 Received 13 December 2002Accepted 4 April 2003

ABSTRACT

The survival of Escherichia coli O157H7 Listeria monocytogenes and Salmonella was studied in apple orange pine-apple and white grape juice concentrates and banana puree Pouches of juice concentrate or puree were inoculated withpathogens at a level $103 CFUg and stored at 2238C (2108F) Pathogen survival was monitored at 6 and 24 h once a weekfor four consecutive weeks and biweekly thereafter until 12 weeks When pathogens were not detectable by direct platingsamples were enriched in universal preenrichment broth for 72 h and plated on selective media Results showed that E coliO157H7 L monocytogenes and Salmonella were recoverable from all ve concentrates through 12 weeks of storage at2238C

TABLE 1 Characteristics of juice concentrates used in the ex-periments

Juiceconcentrate 8Brixa pH

titratableacidityb

AppleBananaOrangePineappleWhite grape

549389634598662

3755373636

1004383209

a Corrected at 238Cb Calculated as g citric acidg sample

Several incidents of foodborne disease have been as-sociated with juices Escherichia coli O157H7 (4 7 8) andSalmonella enterica serotype Typhimurium (5) have beeninvolved in foodborne outbreaks transmitted by unpasteur-ized apple cider S enterica serotypes Anatum EnteritidisGaminara Hartford Muenchen and Rubislaw have beenlinked to outbreaks transmitted by the consumption of un-pasteurized orange juice (6 9 10 17) It is known that Ecoli O157H7 can survive in single-strength apple juice (47 8) and fruit pulps (21) and Salmonella serovars havecaused several outbreaks associated with unpasteurized or-ange juice (9 22) but little is known about the survival ofthese pathogens in juice concentrates To date no outbreakshave been linked to juice concentrates

The Food and Drug Administration has mandated theapplication of Hazard Analysis and Critical Control Pointprinciples for the safe processing of fruit and vegetablejuices This regulation (10) became effective January 2002for large juice processors and requires processors to includea 5-log pathogen reduction step prior to packaging for retaildistribution This pathogen reduction often in the form ofthermal processing applies to juice concentrates under cer-tain circumstances Questions have been raised as to howlong pathogens would survive in juice concentrates if acontamination occurred eg during transportation of con-centrates prior to nal packaging The goal of this studywas to determine the survival of E coli O157H7 Listeriamonocytogenes and Salmonella in apple orange pineap-ple and white grape juice concentrates and banana pureeFor simplicity banana puree is referred to as a juice con-centrate in this paper

Author for correspondence Tel 202-639-5978 Fax 202-639-5991E-mail dgombasnfpa-foodorg

dagger Present address Department of Poultry Science Auburn University Au-burn AL 36849 USA

MATERIALS AND METHODS

Juice concentrates Concentrates at 8Brix levels commonlyproduced by industry were obtained from Member companies ofthe National Food Processors Association The 8Brix levels of theconcentrates were determined with a refractometer (Bausch andLomb Rochester NY) the pH with an Orion 620 pH meter (Ori-on Research Inc Boston Mass) and the titratable acidity ( wtwt as citric acid) by NaOH titration to a pH 81 endpoint Table1 shows the characteristics of the juice concentrates used in thestudy

Test strains and inoculum preparation Five strains eachof E coli O157H7 L monocytogenes and Salmonella were acidadapted and handled as described previously (22) Each pathogencomposite was prepared by combining 1 ml from each of the vestrains in a sterile tube just prior to inoculation Each strain andcomposite was enumerated by serial dilution in 01 peptone wa-ter and spread plating on tryptic soy agar and incubating at 358Cfor 24 h

Inoculation protocol Each pathogen-concentrate combina-tion was prepared and tested twice Juice concentrates were dis-pensed into sterile plastic bags (Whirl-Pak Nasco Fort AtkinsonWisc) in 45- or 95-g aliquots and held at 2238C until inocula-

J Food Prot Vol 66 No 91596 OYARZABAL ET AL

FIGURE 1 Survival of E coli O157H7 (closed circles) Salmonella (open circles) and L monocytogenes (triangles) in (A) apple (B)banana (C) orange (D) pineapple and (E) white grape concentrates at 2238C Symbols represent average of samples from twoexperiments bars represent range y axis P positive on enrichment N negative on enrichment x axis Inocula calculated fromenumeration of individual strains inoculated AI within 15 min after inoculation Note that the time scale is not linear

tion Each bag was inoculated at 10 (volvol) with a pathogencomposite to achieve 103 to 104 CFUg (ie $105 CFU100 gconcentrate) Negative controls were prepared with sterile citratebuffer Inoculated samples were stomached brie y At 2238Cconcentrates were solid which might have hindered homogeneousdistribution of the cells After inoculation samples were quicklyreturned to 2238C and kept at this temperature for the durationof the experiment

Monitoring of survival Each pathogen-concentrate combi-nation was monitored for surviving cells by randomly drawingtwo 5-g samples for direct enumeration within 15 min after in-

oculation at 6 and 24 h once a week for four consecutive weeksand biweekly thereafter until 12 weeks At 12 weeks one 100-gsample was also tested At each sampling point the entire samplewas tested to account for potential heterogeneous distribution ofthe inoculum

Samples were serially diluted in universal preenrichmentbroth (UPB Difco Laboratories Detroit Mich) and spread platedin duplicate on tryptic soy agar and selective media SorbitolMacConkey (Difco) agar plates and EMB (Difco) were used forE coli O157H7 Palcam (Difco) plates were used for L mono-cytogenes and xylose-lysine-desoxycholate agar (Difco) plateswere used for Salmonella All plates were incubated at 358C for

J Food Prot Vol 66 No 9 SURVIVAL OF PATHOGENS IN JUICE CONCENTRATES 1597

FIGURE 2 Survival of E coli O157H7 inoculated at a lower level in juice concentrates and held at 2238C

24 h The initial UPB dilution was also incubated (358C for 72h) to detect pathogen survival if negative results were obtainedfrom direct plating After incubation UPB enrichments werestreaked on the selective agar appropriate to the pathogen inocu-lated in the sample

Identi cation of isolates Isolates were identi ed by growthon selective plating media If the target organism was not recov-ered directly on selective media presumptive isolates on trypticsoy agar were transferred to selective plating media and incubatedat 358C for 24 h Gram staining and Vitek 32 (bioMerieux Ha-zelwood Mo) were used for further con rmation of isolates ifgrowth on selective plates was atypical

RESULTS AND DISCUSSION

Our research was aimed at determining the potentialfor survival of E coli O157H7 L monocytogenes andSalmonella in different juice concentrates Juices have notbeen implicated in any listeriosis outbreak However weincluded L monocytogenes in our studies because it hasbeen isolated from unpasteurized apple juice and becauseit can survive in acidic foods (13 25)

Pathogens in citrate buffer were adapted to trigger theacid tolerance response that is known to confer enhancedcell survival to E coli O157H7 Listeria and Salmonella(11 13 14 16 18 20) This preconditioning was done tomimic a worst-case scenario in which acid-adapted cellscontaminate the concentrates postprocessing or duringtransportation The temperature at which samples werestored (2238C) was determined by surveying the NationalFood Processors Associationrsquos Member companies and rep-resents the lowest temperature typically used during trans-portation of juice concentrates

Our choice of UPB as the enrichment broth was based

on the ability of this medium to recover sublethally injuredbacteria (3) A recent study reported that orange juice sam-ples enriched with UPB yielded better recovery of inocu-lated Salmonella serovars when compared to samples en-riched with lactose broth (15)

Enrichment of samples was needed for the recovery ofL monocytogenes in banana as soon as 6 h after inoculationand for the duration of the study Enrichment was alsoneeded for the recovery of Salmonella in orange and pine-apple 12 weeks after inoculation and in white grape 10weeks after inoculation and for the recovery of E coliO157H7 in pineapple and white grape concentrates 12weeks after inoculation

E coli O157H7 survived through 12 weeks at detect-able levels in apple banana orange pineapple and whitegrape concentrates stored at 2238C (Fig 1) Previous re-search with orange juice concentrate stored at 2108F hasshown that E coli can be detected at levels of 103 CFU100 ml 147 d after inoculating with 107 CFU100 ml ofjuice (19) These results are not unexpected consideringother bacteria such as Aerobacter spp Klebsiella pneu-moniae and Streptococcus spp have been isolated fromfrozen orange juice concentrate (12 19 24 26)

In general Salmonella did not survive in concentratesas well as E coli O157H7 However Salmonella still sur-vived for at least 12 weeks at detectable levels (Fig 1)Reports suggest that S enterica serovar Enteritidis can sur-vive for 90 d in passion fruit nectar (pH 28 to 32) storedat 2208C (1 2) However other research has reported thatin orange juice stored at pH 38 and 08C S enterica ser-ovars Gaminara Hartford Rubislaw and Typhimurium de-clined from 106 CFUml to below detection level (02

J Food Prot Vol 66 No 91598 OYARZABAL ET AL

most probable numberml) at 24 45 39 and 29 d respec-tively (23)

In all concentrates but banana L monocytogenes gen-erally survived at higher levels than Salmonella or E coliO157H7 (Fig 1) In banana concentrate L monocytogenesdropped quickly to levels that required enrichment of thesamples for recovery and remained essentially at the samelow level throughout the 12 weeks (Fig 1b) The bananaconcentrate used in this study was not acidi ed nor did itcontain added preservatives

Pathogen recoveries were highly variable between runsand between samples of the same run as indicated by thebars in Figure 1 Some samples required enrichment fordetection whereas others remained at countable levels induplicate samples However none of the pathogens wereeliminated during storage in these juice concentrates underthe conditions of the study

To determine whether these juice concentrates mighthave a more lethal effect on lower levels of bacteria anexperiment was performed in which E coli O157H7 wasinoculated at 195 log CFUg of juice concentrate AlthoughE coli O157H7 showed a fast decline in some juice con-centrates survivors were still recoverable from all ve con-centrates 4 weeks after inoculation (Fig 2)

Storage temperature could be critical for the survivalof bacteria in juices and concentrates Aea and Bushnell (2)found that passion fruit nectar at room temperature has alethal effect for E coli and Salmonella inoculated at levelsof 104 to 105 CFUml of nectar Two hours after inocula-tion bacteria could not be detected in plating media or sam-ples enriched in tetrathionate broth Although bacteria werequickly inactivated at room temperature passion fruit nec-tar held at 2208C allowed S enterica serovar Enteritidis tosurvive at detectable levels for 90 d Furthermore bacteriacould persist for 1 year or longer in nectar stored at 2208C(1) Although higher temperatures might enhance the le-thality of juice concentrates to pathogens the practicalityof storage at such higher temperatures would need to beconsidered

ACKNOWLEDGMENT

The authors thank Sandra Arze for her technical assistance

REFERENCES

1 Aea R T E and O A Bushnell 1962 The micro ora of frozenpassionfruit nectar base Appl Microbiol 10272ndash276

2 Aea R T E and O A Bushnell 1962 Survival times of selectedenteropathogenic bacteria in frozen passionfruit nectar base ApplMicrobiol 10277ndash279

3 Bailey J S and N A Cox 1992 Universal preenrichment brothfor the simultaneous detection of Salmonella and Listeria in foodsJ Food Prot 55256ndash259

4 Besser R E S M Lett J T Weber M P Doyle T J Barrett JG Wells and P M Grif n 1993 An outbreak of diarrhea and he-molytic uremic syndrome from Escherichia coli O157H7 in fresh-pressed apple cider JAMA 2692217ndash2220

5 Centers for Disease Control and Prevention 1975 Salmonella Ty-

phimurium outbreak traced to a commercial apple cidermdashNew Jer-sey Morb Mortal Wkly Rep 2487ndash92

6 Centers for Disease Control and Prevention 1995 Outbreak of Sal-monella Hartford infections among travelers to Orlando FloridaEPI-AID Trip Rep 95ndash62

7 Centers for Disease Control and Prevention 1996 Outbreak of Esch-erichia coli O157H7 infections associated with drinking unpasteur-ized commercial apple juicemdashBritish Columbia California Colo-rado and Washington Morb Mortal Wkly Rep 45975

8 Centers for Disease Control and Prevention 1997 Outbreak of Esch-erichia coli O157H7 infections and cryptosporidiosis associatedwith drinking unpasteurized apple juicemdashConnecticut and NewYork October 1996 Morb Mortal Wkly Rep 464ndash8

9 Centers for Disease Control and Prevention 1999 Outbreak of Sal-monella serotype Muenchen infections associated with unpasteurizedorange juice United States and Canada June 1999 Morb MortalWkly Rep 48582ndash585

10 Food and Drug Administration 2001 Hazard analysis and criticalcontrol point (HAACP) procedures for the safe and sanitary pro-cessing and importing of juice nal rule Fed Regist 666137ndash6202

11 Foster J W and H K Hall 1990 Adaptive acidi cation toleranceresponse of Salmonella Typhimurium J Bacteriol 172771ndash778

12 Fuentes F A T C Hazen A J Lopez-Torres and P Rechani 1985Klebsiella pneumoniae in orange juice concentrate Appl EnvironMicrobiol 491527ndash1529

13 Gaham C G M B OrsquoDriscoll and C Hill 1996 Acid adaptationof Listeria monocytogenes can enhance survival in acidic foods andduring milk fermentation Appl Environ Microbiol 623128ndash3132

14 Garren D M M A Harrison and S M Russell 1997 Retentionof acid tolerance and acid shock responses of Escherichia coli O157H7 and non-O157H7 isolates J Food Prot 601478ndash1482

15 Hammack T S R M Amaguana and W H Andrews 2001 Animproved method for the recovery of Salmonella serovars from or-ange juice using universal preenrichment broth J Food Prot 64659ndash663

16 Hill C B OrsquoDriscoll and I Booth 1995 Acid adaptation and foodpoisoning microorganisms Int J Food Microbiol 28245ndash254

17 Krause G R Terzagian and R Hammond 2002 Outbreak of Sal-monella serotype Anatum infection associated with unpasteurizedorange juice South Med J 941168ndash1172

18 Kroll R G and R A Patchett 1992 Induced acid tolerance inListeria monocytogenes Lett Appl Microbiol 14224ndash227

19 Larkin E P W Litsky and J E Fuller 1955 Fecal streptococci infrozen foods III Effect of freezing on Escherichia coli Streptococ-cus faecalis and Streptococcus liquefaciens inoculated into orangeconcentrate Appl Microbiol 3104ndash106

20 Leyer G J L L Wang and E A Johnson 1995 Acid adaptationof Escherichia coli O157H7 increases survival in acidic foods ApplEnviron Microbiol 613752ndash3755

21 Marques P A H F D Worcman-Barninka S C Lannes and MLandgraf 2001 Acid tolerance and survival of Escherichia coliO157H7 inoculated in fruit pulps stored under refrigeration J FoodProt 641674ndash1678

22 Nogueira M C L O A Oyarzabal and D E Gombas 2003Inactivation of Eschericha coli O157H7 Listeria monocytogenesand Salmonella in cranberry lemon and lime juice concentrates JFood Prot 661637ndash1641

23 Parish M E J A Narciso and L M Friedrich 1997 Survival ofsalmonellae in orange juice J Food Saf 17273ndash281

24 Patrick R 1953 Coliform bacteria from orange concentrate anddamaged oranges Food Tech 7157ndash159

25 Sado P N K C Jinneman G J Husby S M Sorg and C JOmiecinski 1998 Identi cation of Listeria monocytogenes from un-pasteurized apple juice using rapid test kits J Food Prot 611199ndash1202

26 Wolford E R 1956 Certain aspects of the microbiology of frozenconcentrated orange juice Am J Public Health 46708ndash715

J Food Prot Vol 66 No 91596 OYARZABAL ET AL

FIGURE 1 Survival of E coli O157H7 (closed circles) Salmonella (open circles) and L monocytogenes (triangles) in (A) apple (B)banana (C) orange (D) pineapple and (E) white grape concentrates at 2238C Symbols represent average of samples from twoexperiments bars represent range y axis P positive on enrichment N negative on enrichment x axis Inocula calculated fromenumeration of individual strains inoculated AI within 15 min after inoculation Note that the time scale is not linear

tion Each bag was inoculated at 10 (volvol) with a pathogencomposite to achieve 103 to 104 CFUg (ie $105 CFU100 gconcentrate) Negative controls were prepared with sterile citratebuffer Inoculated samples were stomached brie y At 2238Cconcentrates were solid which might have hindered homogeneousdistribution of the cells After inoculation samples were quicklyreturned to 2238C and kept at this temperature for the durationof the experiment

Monitoring of survival Each pathogen-concentrate combi-nation was monitored for surviving cells by randomly drawingtwo 5-g samples for direct enumeration within 15 min after in-

oculation at 6 and 24 h once a week for four consecutive weeksand biweekly thereafter until 12 weeks At 12 weeks one 100-gsample was also tested At each sampling point the entire samplewas tested to account for potential heterogeneous distribution ofthe inoculum

Samples were serially diluted in universal preenrichmentbroth (UPB Difco Laboratories Detroit Mich) and spread platedin duplicate on tryptic soy agar and selective media SorbitolMacConkey (Difco) agar plates and EMB (Difco) were used forE coli O157H7 Palcam (Difco) plates were used for L mono-cytogenes and xylose-lysine-desoxycholate agar (Difco) plateswere used for Salmonella All plates were incubated at 358C for

J Food Prot Vol 66 No 9 SURVIVAL OF PATHOGENS IN JUICE CONCENTRATES 1597

FIGURE 2 Survival of E coli O157H7 inoculated at a lower level in juice concentrates and held at 2238C

24 h The initial UPB dilution was also incubated (358C for 72h) to detect pathogen survival if negative results were obtainedfrom direct plating After incubation UPB enrichments werestreaked on the selective agar appropriate to the pathogen inocu-lated in the sample

Identi cation of isolates Isolates were identi ed by growthon selective plating media If the target organism was not recov-ered directly on selective media presumptive isolates on trypticsoy agar were transferred to selective plating media and incubatedat 358C for 24 h Gram staining and Vitek 32 (bioMerieux Ha-zelwood Mo) were used for further con rmation of isolates ifgrowth on selective plates was atypical

RESULTS AND DISCUSSION

Our research was aimed at determining the potentialfor survival of E coli O157H7 L monocytogenes andSalmonella in different juice concentrates Juices have notbeen implicated in any listeriosis outbreak However weincluded L monocytogenes in our studies because it hasbeen isolated from unpasteurized apple juice and becauseit can survive in acidic foods (13 25)

Pathogens in citrate buffer were adapted to trigger theacid tolerance response that is known to confer enhancedcell survival to E coli O157H7 Listeria and Salmonella(11 13 14 16 18 20) This preconditioning was done tomimic a worst-case scenario in which acid-adapted cellscontaminate the concentrates postprocessing or duringtransportation The temperature at which samples werestored (2238C) was determined by surveying the NationalFood Processors Associationrsquos Member companies and rep-resents the lowest temperature typically used during trans-portation of juice concentrates

Our choice of UPB as the enrichment broth was based

on the ability of this medium to recover sublethally injuredbacteria (3) A recent study reported that orange juice sam-ples enriched with UPB yielded better recovery of inocu-lated Salmonella serovars when compared to samples en-riched with lactose broth (15)

Enrichment of samples was needed for the recovery ofL monocytogenes in banana as soon as 6 h after inoculationand for the duration of the study Enrichment was alsoneeded for the recovery of Salmonella in orange and pine-apple 12 weeks after inoculation and in white grape 10weeks after inoculation and for the recovery of E coliO157H7 in pineapple and white grape concentrates 12weeks after inoculation

E coli O157H7 survived through 12 weeks at detect-able levels in apple banana orange pineapple and whitegrape concentrates stored at 2238C (Fig 1) Previous re-search with orange juice concentrate stored at 2108F hasshown that E coli can be detected at levels of 103 CFU100 ml 147 d after inoculating with 107 CFU100 ml ofjuice (19) These results are not unexpected consideringother bacteria such as Aerobacter spp Klebsiella pneu-moniae and Streptococcus spp have been isolated fromfrozen orange juice concentrate (12 19 24 26)

In general Salmonella did not survive in concentratesas well as E coli O157H7 However Salmonella still sur-vived for at least 12 weeks at detectable levels (Fig 1)Reports suggest that S enterica serovar Enteritidis can sur-vive for 90 d in passion fruit nectar (pH 28 to 32) storedat 2208C (1 2) However other research has reported thatin orange juice stored at pH 38 and 08C S enterica ser-ovars Gaminara Hartford Rubislaw and Typhimurium de-clined from 106 CFUml to below detection level (02

J Food Prot Vol 66 No 91598 OYARZABAL ET AL

most probable numberml) at 24 45 39 and 29 d respec-tively (23)

In all concentrates but banana L monocytogenes gen-erally survived at higher levels than Salmonella or E coliO157H7 (Fig 1) In banana concentrate L monocytogenesdropped quickly to levels that required enrichment of thesamples for recovery and remained essentially at the samelow level throughout the 12 weeks (Fig 1b) The bananaconcentrate used in this study was not acidi ed nor did itcontain added preservatives

Pathogen recoveries were highly variable between runsand between samples of the same run as indicated by thebars in Figure 1 Some samples required enrichment fordetection whereas others remained at countable levels induplicate samples However none of the pathogens wereeliminated during storage in these juice concentrates underthe conditions of the study

To determine whether these juice concentrates mighthave a more lethal effect on lower levels of bacteria anexperiment was performed in which E coli O157H7 wasinoculated at 195 log CFUg of juice concentrate AlthoughE coli O157H7 showed a fast decline in some juice con-centrates survivors were still recoverable from all ve con-centrates 4 weeks after inoculation (Fig 2)

Storage temperature could be critical for the survivalof bacteria in juices and concentrates Aea and Bushnell (2)found that passion fruit nectar at room temperature has alethal effect for E coli and Salmonella inoculated at levelsof 104 to 105 CFUml of nectar Two hours after inocula-tion bacteria could not be detected in plating media or sam-ples enriched in tetrathionate broth Although bacteria werequickly inactivated at room temperature passion fruit nec-tar held at 2208C allowed S enterica serovar Enteritidis tosurvive at detectable levels for 90 d Furthermore bacteriacould persist for 1 year or longer in nectar stored at 2208C(1) Although higher temperatures might enhance the le-thality of juice concentrates to pathogens the practicalityof storage at such higher temperatures would need to beconsidered

ACKNOWLEDGMENT

The authors thank Sandra Arze for her technical assistance

REFERENCES

1 Aea R T E and O A Bushnell 1962 The micro ora of frozenpassionfruit nectar base Appl Microbiol 10272ndash276

2 Aea R T E and O A Bushnell 1962 Survival times of selectedenteropathogenic bacteria in frozen passionfruit nectar base ApplMicrobiol 10277ndash279

3 Bailey J S and N A Cox 1992 Universal preenrichment brothfor the simultaneous detection of Salmonella and Listeria in foodsJ Food Prot 55256ndash259

4 Besser R E S M Lett J T Weber M P Doyle T J Barrett JG Wells and P M Grif n 1993 An outbreak of diarrhea and he-molytic uremic syndrome from Escherichia coli O157H7 in fresh-pressed apple cider JAMA 2692217ndash2220

5 Centers for Disease Control and Prevention 1975 Salmonella Ty-

phimurium outbreak traced to a commercial apple cidermdashNew Jer-sey Morb Mortal Wkly Rep 2487ndash92

6 Centers for Disease Control and Prevention 1995 Outbreak of Sal-monella Hartford infections among travelers to Orlando FloridaEPI-AID Trip Rep 95ndash62

7 Centers for Disease Control and Prevention 1996 Outbreak of Esch-erichia coli O157H7 infections associated with drinking unpasteur-ized commercial apple juicemdashBritish Columbia California Colo-rado and Washington Morb Mortal Wkly Rep 45975

8 Centers for Disease Control and Prevention 1997 Outbreak of Esch-erichia coli O157H7 infections and cryptosporidiosis associatedwith drinking unpasteurized apple juicemdashConnecticut and NewYork October 1996 Morb Mortal Wkly Rep 464ndash8

9 Centers for Disease Control and Prevention 1999 Outbreak of Sal-monella serotype Muenchen infections associated with unpasteurizedorange juice United States and Canada June 1999 Morb MortalWkly Rep 48582ndash585

10 Food and Drug Administration 2001 Hazard analysis and criticalcontrol point (HAACP) procedures for the safe and sanitary pro-cessing and importing of juice nal rule Fed Regist 666137ndash6202

11 Foster J W and H K Hall 1990 Adaptive acidi cation toleranceresponse of Salmonella Typhimurium J Bacteriol 172771ndash778

12 Fuentes F A T C Hazen A J Lopez-Torres and P Rechani 1985Klebsiella pneumoniae in orange juice concentrate Appl EnvironMicrobiol 491527ndash1529

13 Gaham C G M B OrsquoDriscoll and C Hill 1996 Acid adaptationof Listeria monocytogenes can enhance survival in acidic foods andduring milk fermentation Appl Environ Microbiol 623128ndash3132

14 Garren D M M A Harrison and S M Russell 1997 Retentionof acid tolerance and acid shock responses of Escherichia coli O157H7 and non-O157H7 isolates J Food Prot 601478ndash1482

15 Hammack T S R M Amaguana and W H Andrews 2001 Animproved method for the recovery of Salmonella serovars from or-ange juice using universal preenrichment broth J Food Prot 64659ndash663

16 Hill C B OrsquoDriscoll and I Booth 1995 Acid adaptation and foodpoisoning microorganisms Int J Food Microbiol 28245ndash254

17 Krause G R Terzagian and R Hammond 2002 Outbreak of Sal-monella serotype Anatum infection associated with unpasteurizedorange juice South Med J 941168ndash1172

18 Kroll R G and R A Patchett 1992 Induced acid tolerance inListeria monocytogenes Lett Appl Microbiol 14224ndash227

19 Larkin E P W Litsky and J E Fuller 1955 Fecal streptococci infrozen foods III Effect of freezing on Escherichia coli Streptococ-cus faecalis and Streptococcus liquefaciens inoculated into orangeconcentrate Appl Microbiol 3104ndash106

20 Leyer G J L L Wang and E A Johnson 1995 Acid adaptationof Escherichia coli O157H7 increases survival in acidic foods ApplEnviron Microbiol 613752ndash3755

21 Marques P A H F D Worcman-Barninka S C Lannes and MLandgraf 2001 Acid tolerance and survival of Escherichia coliO157H7 inoculated in fruit pulps stored under refrigeration J FoodProt 641674ndash1678

22 Nogueira M C L O A Oyarzabal and D E Gombas 2003Inactivation of Eschericha coli O157H7 Listeria monocytogenesand Salmonella in cranberry lemon and lime juice concentrates JFood Prot 661637ndash1641

23 Parish M E J A Narciso and L M Friedrich 1997 Survival ofsalmonellae in orange juice J Food Saf 17273ndash281

24 Patrick R 1953 Coliform bacteria from orange concentrate anddamaged oranges Food Tech 7157ndash159

25 Sado P N K C Jinneman G J Husby S M Sorg and C JOmiecinski 1998 Identi cation of Listeria monocytogenes from un-pasteurized apple juice using rapid test kits J Food Prot 611199ndash1202

26 Wolford E R 1956 Certain aspects of the microbiology of frozenconcentrated orange juice Am J Public Health 46708ndash715

J Food Prot Vol 66 No 9 SURVIVAL OF PATHOGENS IN JUICE CONCENTRATES 1597

FIGURE 2 Survival of E coli O157H7 inoculated at a lower level in juice concentrates and held at 2238C

24 h The initial UPB dilution was also incubated (358C for 72h) to detect pathogen survival if negative results were obtainedfrom direct plating After incubation UPB enrichments werestreaked on the selective agar appropriate to the pathogen inocu-lated in the sample

Identi cation of isolates Isolates were identi ed by growthon selective plating media If the target organism was not recov-ered directly on selective media presumptive isolates on trypticsoy agar were transferred to selective plating media and incubatedat 358C for 24 h Gram staining and Vitek 32 (bioMerieux Ha-zelwood Mo) were used for further con rmation of isolates ifgrowth on selective plates was atypical

RESULTS AND DISCUSSION

Our research was aimed at determining the potentialfor survival of E coli O157H7 L monocytogenes andSalmonella in different juice concentrates Juices have notbeen implicated in any listeriosis outbreak However weincluded L monocytogenes in our studies because it hasbeen isolated from unpasteurized apple juice and becauseit can survive in acidic foods (13 25)

Pathogens in citrate buffer were adapted to trigger theacid tolerance response that is known to confer enhancedcell survival to E coli O157H7 Listeria and Salmonella(11 13 14 16 18 20) This preconditioning was done tomimic a worst-case scenario in which acid-adapted cellscontaminate the concentrates postprocessing or duringtransportation The temperature at which samples werestored (2238C) was determined by surveying the NationalFood Processors Associationrsquos Member companies and rep-resents the lowest temperature typically used during trans-portation of juice concentrates

Our choice of UPB as the enrichment broth was based

on the ability of this medium to recover sublethally injuredbacteria (3) A recent study reported that orange juice sam-ples enriched with UPB yielded better recovery of inocu-lated Salmonella serovars when compared to samples en-riched with lactose broth (15)

Enrichment of samples was needed for the recovery ofL monocytogenes in banana as soon as 6 h after inoculationand for the duration of the study Enrichment was alsoneeded for the recovery of Salmonella in orange and pine-apple 12 weeks after inoculation and in white grape 10weeks after inoculation and for the recovery of E coliO157H7 in pineapple and white grape concentrates 12weeks after inoculation

E coli O157H7 survived through 12 weeks at detect-able levels in apple banana orange pineapple and whitegrape concentrates stored at 2238C (Fig 1) Previous re-search with orange juice concentrate stored at 2108F hasshown that E coli can be detected at levels of 103 CFU100 ml 147 d after inoculating with 107 CFU100 ml ofjuice (19) These results are not unexpected consideringother bacteria such as Aerobacter spp Klebsiella pneu-moniae and Streptococcus spp have been isolated fromfrozen orange juice concentrate (12 19 24 26)

In general Salmonella did not survive in concentratesas well as E coli O157H7 However Salmonella still sur-vived for at least 12 weeks at detectable levels (Fig 1)Reports suggest that S enterica serovar Enteritidis can sur-vive for 90 d in passion fruit nectar (pH 28 to 32) storedat 2208C (1 2) However other research has reported thatin orange juice stored at pH 38 and 08C S enterica ser-ovars Gaminara Hartford Rubislaw and Typhimurium de-clined from 106 CFUml to below detection level (02

J Food Prot Vol 66 No 91598 OYARZABAL ET AL

most probable numberml) at 24 45 39 and 29 d respec-tively (23)

In all concentrates but banana L monocytogenes gen-erally survived at higher levels than Salmonella or E coliO157H7 (Fig 1) In banana concentrate L monocytogenesdropped quickly to levels that required enrichment of thesamples for recovery and remained essentially at the samelow level throughout the 12 weeks (Fig 1b) The bananaconcentrate used in this study was not acidi ed nor did itcontain added preservatives

Pathogen recoveries were highly variable between runsand between samples of the same run as indicated by thebars in Figure 1 Some samples required enrichment fordetection whereas others remained at countable levels induplicate samples However none of the pathogens wereeliminated during storage in these juice concentrates underthe conditions of the study

To determine whether these juice concentrates mighthave a more lethal effect on lower levels of bacteria anexperiment was performed in which E coli O157H7 wasinoculated at 195 log CFUg of juice concentrate AlthoughE coli O157H7 showed a fast decline in some juice con-centrates survivors were still recoverable from all ve con-centrates 4 weeks after inoculation (Fig 2)

Storage temperature could be critical for the survivalof bacteria in juices and concentrates Aea and Bushnell (2)found that passion fruit nectar at room temperature has alethal effect for E coli and Salmonella inoculated at levelsof 104 to 105 CFUml of nectar Two hours after inocula-tion bacteria could not be detected in plating media or sam-ples enriched in tetrathionate broth Although bacteria werequickly inactivated at room temperature passion fruit nec-tar held at 2208C allowed S enterica serovar Enteritidis tosurvive at detectable levels for 90 d Furthermore bacteriacould persist for 1 year or longer in nectar stored at 2208C(1) Although higher temperatures might enhance the le-thality of juice concentrates to pathogens the practicalityof storage at such higher temperatures would need to beconsidered

ACKNOWLEDGMENT

The authors thank Sandra Arze for her technical assistance

REFERENCES

1 Aea R T E and O A Bushnell 1962 The micro ora of frozenpassionfruit nectar base Appl Microbiol 10272ndash276

2 Aea R T E and O A Bushnell 1962 Survival times of selectedenteropathogenic bacteria in frozen passionfruit nectar base ApplMicrobiol 10277ndash279

3 Bailey J S and N A Cox 1992 Universal preenrichment brothfor the simultaneous detection of Salmonella and Listeria in foodsJ Food Prot 55256ndash259

4 Besser R E S M Lett J T Weber M P Doyle T J Barrett JG Wells and P M Grif n 1993 An outbreak of diarrhea and he-molytic uremic syndrome from Escherichia coli O157H7 in fresh-pressed apple cider JAMA 2692217ndash2220

5 Centers for Disease Control and Prevention 1975 Salmonella Ty-

phimurium outbreak traced to a commercial apple cidermdashNew Jer-sey Morb Mortal Wkly Rep 2487ndash92

6 Centers for Disease Control and Prevention 1995 Outbreak of Sal-monella Hartford infections among travelers to Orlando FloridaEPI-AID Trip Rep 95ndash62

7 Centers for Disease Control and Prevention 1996 Outbreak of Esch-erichia coli O157H7 infections associated with drinking unpasteur-ized commercial apple juicemdashBritish Columbia California Colo-rado and Washington Morb Mortal Wkly Rep 45975

8 Centers for Disease Control and Prevention 1997 Outbreak of Esch-erichia coli O157H7 infections and cryptosporidiosis associatedwith drinking unpasteurized apple juicemdashConnecticut and NewYork October 1996 Morb Mortal Wkly Rep 464ndash8

9 Centers for Disease Control and Prevention 1999 Outbreak of Sal-monella serotype Muenchen infections associated with unpasteurizedorange juice United States and Canada June 1999 Morb MortalWkly Rep 48582ndash585

10 Food and Drug Administration 2001 Hazard analysis and criticalcontrol point (HAACP) procedures for the safe and sanitary pro-cessing and importing of juice nal rule Fed Regist 666137ndash6202

11 Foster J W and H K Hall 1990 Adaptive acidi cation toleranceresponse of Salmonella Typhimurium J Bacteriol 172771ndash778

12 Fuentes F A T C Hazen A J Lopez-Torres and P Rechani 1985Klebsiella pneumoniae in orange juice concentrate Appl EnvironMicrobiol 491527ndash1529

13 Gaham C G M B OrsquoDriscoll and C Hill 1996 Acid adaptationof Listeria monocytogenes can enhance survival in acidic foods andduring milk fermentation Appl Environ Microbiol 623128ndash3132

14 Garren D M M A Harrison and S M Russell 1997 Retentionof acid tolerance and acid shock responses of Escherichia coli O157H7 and non-O157H7 isolates J Food Prot 601478ndash1482

15 Hammack T S R M Amaguana and W H Andrews 2001 Animproved method for the recovery of Salmonella serovars from or-ange juice using universal preenrichment broth J Food Prot 64659ndash663

16 Hill C B OrsquoDriscoll and I Booth 1995 Acid adaptation and foodpoisoning microorganisms Int J Food Microbiol 28245ndash254

17 Krause G R Terzagian and R Hammond 2002 Outbreak of Sal-monella serotype Anatum infection associated with unpasteurizedorange juice South Med J 941168ndash1172

18 Kroll R G and R A Patchett 1992 Induced acid tolerance inListeria monocytogenes Lett Appl Microbiol 14224ndash227

19 Larkin E P W Litsky and J E Fuller 1955 Fecal streptococci infrozen foods III Effect of freezing on Escherichia coli Streptococ-cus faecalis and Streptococcus liquefaciens inoculated into orangeconcentrate Appl Microbiol 3104ndash106

20 Leyer G J L L Wang and E A Johnson 1995 Acid adaptationof Escherichia coli O157H7 increases survival in acidic foods ApplEnviron Microbiol 613752ndash3755

21 Marques P A H F D Worcman-Barninka S C Lannes and MLandgraf 2001 Acid tolerance and survival of Escherichia coliO157H7 inoculated in fruit pulps stored under refrigeration J FoodProt 641674ndash1678

22 Nogueira M C L O A Oyarzabal and D E Gombas 2003Inactivation of Eschericha coli O157H7 Listeria monocytogenesand Salmonella in cranberry lemon and lime juice concentrates JFood Prot 661637ndash1641

23 Parish M E J A Narciso and L M Friedrich 1997 Survival ofsalmonellae in orange juice J Food Saf 17273ndash281

24 Patrick R 1953 Coliform bacteria from orange concentrate anddamaged oranges Food Tech 7157ndash159

25 Sado P N K C Jinneman G J Husby S M Sorg and C JOmiecinski 1998 Identi cation of Listeria monocytogenes from un-pasteurized apple juice using rapid test kits J Food Prot 611199ndash1202

26 Wolford E R 1956 Certain aspects of the microbiology of frozenconcentrated orange juice Am J Public Health 46708ndash715

J Food Prot Vol 66 No 91598 OYARZABAL ET AL

most probable numberml) at 24 45 39 and 29 d respec-tively (23)

In all concentrates but banana L monocytogenes gen-erally survived at higher levels than Salmonella or E coliO157H7 (Fig 1) In banana concentrate L monocytogenesdropped quickly to levels that required enrichment of thesamples for recovery and remained essentially at the samelow level throughout the 12 weeks (Fig 1b) The bananaconcentrate used in this study was not acidi ed nor did itcontain added preservatives

Pathogen recoveries were highly variable between runsand between samples of the same run as indicated by thebars in Figure 1 Some samples required enrichment fordetection whereas others remained at countable levels induplicate samples However none of the pathogens wereeliminated during storage in these juice concentrates underthe conditions of the study

To determine whether these juice concentrates mighthave a more lethal effect on lower levels of bacteria anexperiment was performed in which E coli O157H7 wasinoculated at 195 log CFUg of juice concentrate AlthoughE coli O157H7 showed a fast decline in some juice con-centrates survivors were still recoverable from all ve con-centrates 4 weeks after inoculation (Fig 2)

Storage temperature could be critical for the survivalof bacteria in juices and concentrates Aea and Bushnell (2)found that passion fruit nectar at room temperature has alethal effect for E coli and Salmonella inoculated at levelsof 104 to 105 CFUml of nectar Two hours after inocula-tion bacteria could not be detected in plating media or sam-ples enriched in tetrathionate broth Although bacteria werequickly inactivated at room temperature passion fruit nec-tar held at 2208C allowed S enterica serovar Enteritidis tosurvive at detectable levels for 90 d Furthermore bacteriacould persist for 1 year or longer in nectar stored at 2208C(1) Although higher temperatures might enhance the le-thality of juice concentrates to pathogens the practicalityof storage at such higher temperatures would need to beconsidered

ACKNOWLEDGMENT

The authors thank Sandra Arze for her technical assistance

REFERENCES

1 Aea R T E and O A Bushnell 1962 The micro ora of frozenpassionfruit nectar base Appl Microbiol 10272ndash276

2 Aea R T E and O A Bushnell 1962 Survival times of selectedenteropathogenic bacteria in frozen passionfruit nectar base ApplMicrobiol 10277ndash279

3 Bailey J S and N A Cox 1992 Universal preenrichment brothfor the simultaneous detection of Salmonella and Listeria in foodsJ Food Prot 55256ndash259

4 Besser R E S M Lett J T Weber M P Doyle T J Barrett JG Wells and P M Grif n 1993 An outbreak of diarrhea and he-molytic uremic syndrome from Escherichia coli O157H7 in fresh-pressed apple cider JAMA 2692217ndash2220

5 Centers for Disease Control and Prevention 1975 Salmonella Ty-

phimurium outbreak traced to a commercial apple cidermdashNew Jer-sey Morb Mortal Wkly Rep 2487ndash92

6 Centers for Disease Control and Prevention 1995 Outbreak of Sal-monella Hartford infections among travelers to Orlando FloridaEPI-AID Trip Rep 95ndash62

7 Centers for Disease Control and Prevention 1996 Outbreak of Esch-erichia coli O157H7 infections associated with drinking unpasteur-ized commercial apple juicemdashBritish Columbia California Colo-rado and Washington Morb Mortal Wkly Rep 45975

8 Centers for Disease Control and Prevention 1997 Outbreak of Esch-erichia coli O157H7 infections and cryptosporidiosis associatedwith drinking unpasteurized apple juicemdashConnecticut and NewYork October 1996 Morb Mortal Wkly Rep 464ndash8

9 Centers for Disease Control and Prevention 1999 Outbreak of Sal-monella serotype Muenchen infections associated with unpasteurizedorange juice United States and Canada June 1999 Morb MortalWkly Rep 48582ndash585

10 Food and Drug Administration 2001 Hazard analysis and criticalcontrol point (HAACP) procedures for the safe and sanitary pro-cessing and importing of juice nal rule Fed Regist 666137ndash6202

11 Foster J W and H K Hall 1990 Adaptive acidi cation toleranceresponse of Salmonella Typhimurium J Bacteriol 172771ndash778

12 Fuentes F A T C Hazen A J Lopez-Torres and P Rechani 1985Klebsiella pneumoniae in orange juice concentrate Appl EnvironMicrobiol 491527ndash1529

13 Gaham C G M B OrsquoDriscoll and C Hill 1996 Acid adaptationof Listeria monocytogenes can enhance survival in acidic foods andduring milk fermentation Appl Environ Microbiol 623128ndash3132

14 Garren D M M A Harrison and S M Russell 1997 Retentionof acid tolerance and acid shock responses of Escherichia coli O157H7 and non-O157H7 isolates J Food Prot 601478ndash1482

15 Hammack T S R M Amaguana and W H Andrews 2001 Animproved method for the recovery of Salmonella serovars from or-ange juice using universal preenrichment broth J Food Prot 64659ndash663

16 Hill C B OrsquoDriscoll and I Booth 1995 Acid adaptation and foodpoisoning microorganisms Int J Food Microbiol 28245ndash254

17 Krause G R Terzagian and R Hammond 2002 Outbreak of Sal-monella serotype Anatum infection associated with unpasteurizedorange juice South Med J 941168ndash1172

18 Kroll R G and R A Patchett 1992 Induced acid tolerance inListeria monocytogenes Lett Appl Microbiol 14224ndash227

19 Larkin E P W Litsky and J E Fuller 1955 Fecal streptococci infrozen foods III Effect of freezing on Escherichia coli Streptococ-cus faecalis and Streptococcus liquefaciens inoculated into orangeconcentrate Appl Microbiol 3104ndash106

20 Leyer G J L L Wang and E A Johnson 1995 Acid adaptationof Escherichia coli O157H7 increases survival in acidic foods ApplEnviron Microbiol 613752ndash3755

21 Marques P A H F D Worcman-Barninka S C Lannes and MLandgraf 2001 Acid tolerance and survival of Escherichia coliO157H7 inoculated in fruit pulps stored under refrigeration J FoodProt 641674ndash1678

22 Nogueira M C L O A Oyarzabal and D E Gombas 2003Inactivation of Eschericha coli O157H7 Listeria monocytogenesand Salmonella in cranberry lemon and lime juice concentrates JFood Prot 661637ndash1641

23 Parish M E J A Narciso and L M Friedrich 1997 Survival ofsalmonellae in orange juice J Food Saf 17273ndash281

24 Patrick R 1953 Coliform bacteria from orange concentrate anddamaged oranges Food Tech 7157ndash159

25 Sado P N K C Jinneman G J Husby S M Sorg and C JOmiecinski 1998 Identi cation of Listeria monocytogenes from un-pasteurized apple juice using rapid test kits J Food Prot 611199ndash1202

26 Wolford E R 1956 Certain aspects of the microbiology of frozenconcentrated orange juice Am J Public Health 46708ndash715