guidelines for using enterococcus faecium nrrl b-2354 as a … · 2018-02-28 · b-2354 as a...
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A L M O N D B OA R D O F C A L I F O R N I A G U I D E L I N E 7/ 1 4 | 1
Guidelines for Using Enterococcus faecium NRRL B-2354 as a Surrogate Microorganism in Almond Process Validation
JULY 2014
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CONTENTS
1 SURROGATE CULTURE – BACKGROUND AND BIOSAFETY CONSIDERATIONS
2 INOCULUM AND INOCULATED ALMONDS – PREPARATION, HANDLING AND STORAGE
2.1 Materials
2.2 Preparationtimeline
2.3 Inoculumpreparation
2.4 Inoculationprocedureanddrying
2.5 Heatresistance,storageandtransportofinoculatedalmonds
3 USE OF SURROGATE IN VALIDATION
3.1 Challengetestingwithinoculatedalmonds
3.2 Recoveryandenumerationofinoculatedmicroorganisms
3.3 Dilutionandplatingschemes
3.4 Datareporting
3.5 Datacalculationexamples
4 REFERENCES
CONTRIBUTORS:Almond Board of California Technical Expert Review Panel (TERP)
Ken Stevenson, Ph.D., Chairman ABC TERP
Keith Ito, University of California, Davis (Retired) and Consultant
Larry Beuchat, Ph.D., Distinguished Research Professor, Center for Food Safety, University of Georgia
Bradley P. Marks, Ph.D., P.E., Professor, Biosystems Engineering, Michigan State University
Dave Ashton, Ph.D., Food Safety Consultant
Others
Linda J. Harris, Ph.D., Specialist in Cooperative Extension, University of California, Davis
Guangwei Huang, Associate Director, Food Science and Technology, Almond Board of California
Tim Birmingham, Director Quality Assurance and Industry Services, Almond Board of California
EDITORIAL ASSISTANCESylvia Yada, Consultant
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S E C T I O N 1 :
SURROGATE CULTURE – BACKGROUND AND BIOSAFETY CONSIDERATIONS
Enterococcus faeciumNRRLB-2354hasbeenidentifiedasasuitablesurrogateorganismforSalmonellaEnteritidisPhageType30andotherSalmonellaeinvalidationstudiesofthermaltreatmentprocessesforalmonds.Thegoalofalmondprocessvalidationstudiesistodetermineifthetreatmenttechnologyandequipmentcanachievethemandatedminimum4-logreductionofSalmonellainCalifornia-grownalmonds(Federal Register,2007;AlmondsgrowninCalifornia,7CFRpart981).
ThroughstudiesfundedbytheAlmondBoardofCalifornia(ABC),theuseofE. faeciumNRRLB-2354asasurrogatehasbeendeemedappropriateforuseinalmondprocessvalidationstudiesforthefollowingtypesofprocesses:•Dry-heat processes,suchasdryroast,brineandpre-wetdryroast,dryroastflavoring,dryplasticizing,etc.
•Moist air or steam processes(ambientorvacuum)(Jeongetal.,2011)
Enterococcus faeciumNRRLB-2354mayalsobeanappropriatesurrogateforalternativeprocesses,suchasinfraredheating,microwave,radiofrequencyheatingandothers.However,beforethesurrogateisusedinvalidationofothertypesofprocesses,studiesmustbeconductedanddatagatheredtodemonstrateappropriateresistanceofE. faeciumNRRLB-2354comparedwithSalmonellaEnteritidisPhageType30onalmondsforthespecificprocess.Inaddition,studiescomparingtheresistanceofE. faeciumNRRLB-2354andSalmonellaEnteritidisPhageType30(orotherpathogensofconcern)shouldbeconductedbeforeusingE. faeciumNRRLB-2354asasurrogateforproductsotherthanalmonds.Furthermore,protocolsandguidelinesestablishedforuseofthissurrogateonalmondsshouldnotbeconsideredappropriateforotherproductswithoutadditionalscientificdatatosupportsuchapplication.
ThestrainEnterococcusfaeciumNRRLB-2354isavailablefromtheculturecollectionoftheUSDANationalCenterforAgriculturalUtilizationResearch(NCAUR).TheABCTechnicalExpertReviewPanel(TERP)recommendstheuseofthisstrainforalmondprocessvalidation.
NRRLB-2354culturescanbeobtainedthroughNCAURfornochargeviatheonlineorderingsystemforstrainsinthepublicaccesscatalog:http://nrrl.ncaur.usda.gov/.
Asurrogateselectedforprocessvalidationstudiesinfoodprocessingandpilotplantfacilitiesmustbenonpathogenictohumans.E. faeciumNRRLB-2354hasbeenusedinthefoodindustryasanonpathogenictestorganismformanydecades,alsopreviouslyundervariousothernamesandstraindesignationsincludingMicrococcus freudenreichiiATCC8459,Pediococcussp.NRRLB-2354andE. faeciumATCC8459.Arecentreview(Kornacki,2012)indicatestheusefulnessofE. faeciumNRRLB-2354asasurrogate.Furthermore,astudyfundedbytheAlmondBoardofCaliforniaexaminingthegenomicandfunctionalcharacteristicsofE. faeciumNRRLB-2354hasshownthatthisstrainisasafesurrogate,appropriateforuseinprocessvalidation(Kopitetal.,2014).
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S E C T I O N 2 :
INOCULUM AND INOCULATED ALMONDS – PREPARATION, HANDLING AND STORAGE
Thefollowingguidelinesdescribethematerialsandstep-wisepreparationandhandlingproceduresforusingE. faeciumNRRLB-2354foranalmondprocessvalidationstudy,includinginoculumpreparationandthestorageandtransportofinoculatedalmondsamples.
2.1 MATERIALS
ALMOND KERNELS
•Nonpareilvariety,gradeU.S.No.1,size27/30*(*Ifthissizeisnotavailable,contactABC.)
Toensurealowbackgroundmicrobialloadonsamples,usealmondspasteurizedbytreatmentwithpropyleneoxide(PPO)andwith<300ppmPPOresidue
Moisturecontentofthekernelsmustbe4.0–5.5%priortoinoculation
Temperatureofthekernelsshouldbe21–24°C(70–75°F)priortoinoculation
CULTURE
•E. faecium NRRLB-2354
EQUIPMENT
•Plasticpetridishes(standardand150-mmdiameter)•Pipettes•Testtubes•Glassspreaders•Magneticstirplateandbars•Incubatorat35°C(95°F)•Refrigeratorat4°C(40°F)•Polyethylene(PE)samplebags,mediumsize(710mL/24oz.)•PEsamplebagswithzipperclosure,largesize(30×30cm/16×16in.)•Filterpapersheets(46×57cm;P8grade)•Metaldryingrack•Plasticstoragebinwithlid,sterile•Metalmeshtray•Laboratoryoven,convection/forcedair•Laboratorypaddleblender(e.g.,Stomacherlabblenderorequivalent)•Containerstoholdinoculatednutsfortreatment(e.g.,thermostablebagsorbaskets)
MEDIA
•Trypticsoyagar(TSA)•Trypticsoybroth(TSB)•0.1%peptonewater•Butterfield’sphosphatebuffer(BPB)
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2.2 PREPARATION TIMELINE
Days1–5: Prepareinoculum
Days5–6: Inoculatealmonds,assessinitialinoculationlevels,anddetermineheatresistance
Days7–14: Beginvalidationtrialswithinoculatedalmonds.Note:Useinoculatedalmondswithin14daysafterinoculationunless>7.0logCFU/gisconfirmed(seeSection3.2)andadditionalheatresistancetestingconfirmsadequateresistance(seeSection2.5)
2.3 INOCULUM PREPARATION
Thefollowingprocedurewillyielda25-mLsuspensionofcells,whichisasufficientvolumetoinoculateone400-gportionofalmonds.
Theamountofalmondstoinoculateisdeterminedbytheexperimentaldesign:50gpersample×numberofsamplingpoints×numberofreplicatesateachsamplingpoint.Typicalvalidationstudieswillutilize>2,400gofinoculatedalmondsincludingtravelingcontrolsandheatresistancetestsamples.Thetotalinoculumvolumeneededdependsontheamountofalmondstobeinoculated.Makeanappropriatenumberof25-mLinoculumpreparationsandpoolasdescribedbelow.
Streak culture (active or frozen) onto TSA plates
Incubate at 35° C for 24 ± 2 hours
Transfer cells from isolated typical colonies into TSB (10 mL)
Incubate at 35° C for 24 ± 2 hours
Transfer loop of broth culture into TSB (10 mL)
Incubate at 35° C overnight (18 ± 2 hours)
Spread overnight culture (1 mL/plate) over large TSA plates (150 × 15 mm); prepare 5 plates
Incubate at 35° C for 24 ± 2 hours
Add 6 mL of 0.1% peptone to each plate, loosen bacterial lawn with a sterile spreader, and use a sterile pipette to collect cells into a sterile container. (Add additional peptone as needed for total volume of 25 mL per 5 petri dishes.)
Before inoculating almonds, pool all 25-mL inoculum preparations and mix thoroughly for at least 1 minute using a magnetic stir bar and stir plate
Hold pooled inoculum on stir plate (for up to 0.5 hour) until all almond samples have been inoculated
DAY 1
DAY 2
DAY 3
DAY 4
DAY 5
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2.4 INOCULATION PROCEDURE AND DRYING
Thefollowinginoculationprocedureisforone400-gportionofalmondkernels.Topreparemoreinoculatedalmonds,separatelyinoculate400-gbatchesofalmondsandpoolafterdryingasdescribedbelow.
Mix contents of sealed bag by inverting the bag repeatedly by hand for 1 minute
Pour almonds out of bag and spread onto filter paper (4 layers) placed on a metal drying rack inside a lidded bin (sterile)
Allow inoculated almonds to dry in bin (with lid ajar) at ambient temperature (24 ± 2° C) for at least 24 hours
Weigh Nonpareil almonds (400 ± 1 g) into large PE bag (sterile), add 25 mL of pooled inoculum, and seal bag
Pool batches of dried inoculated almonds into sterile PE storage bag, and mix contents by shaking bag by hand for 2 minutes
Determine inoculation level
Determine heat resistance
Before inoculating: Hand-sort almonds to remove defects including broken and chipped/scratched nuts
Make sure that almond moisture content is 4.0–5.5% and almonds are tempered to room temperature (21–24° C)
To make four layers of filter paper, fold two large sheets in half
Almonds should be air dried until the moisture content is less than 5.5%. This may take up to 72 hours, depending on the ambient relative humidity
Level must be >7 log CFU/g
See Section 3.2 for enumeration methods
See Section 2.5 for heat resistance test
DAYS 6–7
DAY 5
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2.5 HEAT RESISTANCE, STORAGE AND TRANSPORT OF INOCULATED ALMONDS
Followthehandlingproceduresforinoculatedalmonds(anduninoculatedcontrols)asindicatedbelowbeforestartingchallengetesting.
Note: Test inoculated almonds immediately after drying and again before validation trials if dried samples are stored for more than 14 days
Step 1: Spread 25 g dried inoculated almonds on metal mesh tray and space kernels
Step 2: Heat in convection/forced air oven at 280° F (138° C) for 15 minutes
Step 3: Enumerate heated sample and control (unheated inoculated almonds): Section 3.2
Acceptable heat resistance = <2.5 log reduction
Store dried inoculated almonds at 4° C ± 1° C
Loosely pack almonds (50-g portions) into mesh bags, baskets, or other suitable container that can be incorporated into processing line
• Prepare enough sample packs for each validation run to cover all sampling points on line
• Also prepare triplicate inoculated sample packs to serve as “traveling” controls
Transport and handle dried inoculated samples and inoculated traveling controls in identical manner on day(s) of validation trial
>14 days post inoculation Use almonds in validation trials if testing confirms >7 log CFU/g and 4.0–5.5% moisture
0–14 days post-inoculation
Determine heat resistance
Heat resistance is determined by subtracting the lowest log value of survivors (heated samples) from highest log values of non-heated, inoculated controls
Or prepare sample packs after transporting stored almonds to processing site
If 50-g packs are not feasible with processing system, contact ABC
HEAT RESISTANCE TEST
STORAGE
INOCULATED SAMPLE PACKS
TRANSPORT
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Hold treated samples at 0–4°C.
Enumerate E. faecium in samples within 24 hours of treatment
S E C T I O N 3 :
USE OF SURROGATE IN VALIDATION
ThefollowingguidelinesdescribestepsinchallengetestingwithsurrogateorganismEnterococcus faeciumNRRLB-2354inanalmondprocessvalidationstudyaswellastheproceduresforrecoveryandenumeration.
3.1 CHALLENGE TESTING WITH INOCULATED ALMONDS
Torunmeaningfulprocessvalidationtrials,itisimportanttomapthetemperatureoftheprocessinglineorproductcontainerstoidentifypotentialcoldspotsbeforerunningvalidationtrials.Conductmicrobialchallengetestingatidentifiedcoldspotsandunderconditionsthatwillalwaysbeexceededduringnormaloperation.Forexample:Fordry-roastervalidation,conducttestingwithtemperaturesetpointslowered.Fornormalproduction,increasesetpointsandestablishtemperaturecriticalfactorsthatexceedthemaximumvaluesreachedduringvalidationtesting.
Condition inoculated almonds to teperature of carrier product in processling line.
For conveyor lines—embed sample packs among product on bed
For non-conveyor lines—add sample packs to mix with product flow
Conduct thermal treatment trial with inoculated sample packs in processing line
Retrieve treated inoculated sample packs from line—chill hot sample packs immediately in an ice bath or chilled water
Inoculated sample packs should be placed uniformly in the process and in all identified coldspots/zones
Perform a minimum the three validation runs to address each process variation
Results may dictate need for additional testing
To chill, double bag sample pack in sterile sample bags. Seal and place in ice bath or chilled water
See Section 3.2 for enumeration methods
CONDITIONING
INOCULATED SAMPLE PACK POSITIONING
THERMAL TREATMENT
SAMPLE RETRIEVAL
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Add almonds to TSB (at ambient temperature) in a 1:2 w/v ratio in a sterile medium-size bag—i.e., 25 g of almonds to 50 mL TSB for heat test samples, and 50 g almonds to 100 mL TSB for validation study
Mix sample in bag with paddle blender for 2 minutes, let stand 3–5 minutes
Shake bag vigorously 5 times in a 30 cm (1 ft) arc just before making serial dilutions in BPB
Perform serial dilutions in BPB
Option 1: Spread 0.1 mL aliquots from each dilution on TSA plates in duplicate
Option 2: To improve detection spread 1.0 mL from each dilution over four TSA plates (0.25 mL each)
Incubate plates at 35° C for 48 hours
Count colonies on plates by hand
Add almonds to BPB (at ambient temperature) in a 1:1 w/v ratio in a sterile medium-size bag — i.e., 25 g of almonds to 25 mL BPB for heat test samples, and 50 g almonds to 50 mL BPB for validation study
Shake container vigorously 50 times in a 30 cm (1 ft) arc, let stand 3–5 minutes
Shake container vigorously 5 times in a 30 cm arc just before making serial dilutions in BPB
3.2 RECOVERY AND ENUMERATION OF INOCULATED MICROORGANISMS
RecoverandenumerateinoculatedE. faeciumonallsamples(inoculatedanduninoculated[control]almonds)byfollowingtheABCprotocolortheproceduredescribedintheFDA Bacteriological Analytical Manual(BAM)(AndrewsandHammack,2003).
• Controls (uninoculated)
• Inoculated Traveling Controls (untreated)
• Inoculated Samples (treated)
Before plating, temper TSA plates for >4 hours to ambient temp, or make plates the previous day and hold at ambient temp overnight.
Follow guidelines in the Compendium of Methods for the Micobiological Examinationof Foods (Swanson et al., 2001)
See Section 3.3 for dilution scheme
ABC PROTOCOL FDA BAM PROCEDURE
ALMOND SAMPLES
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3.3 DILUTION AND PLATING SCHEMES
3.4 DATA REPORTING
IncludethefollowingitemsintheprocessvalidationreporttobeevaluatedbyTERP:•AllrawdataofmicrobiologicalcountsandrespectivelogCFU/gvalues(seeexamplesonpage11)•Averageandminimumlogreductionvalues(seeexamplesonpage11):
Logreduction=initialcounts–survivors=logCFU/ginuntreatedinoculatedsamples(travelingcontrols)−logCFU/gintreatedinoculatedsamples
Tocalculateminimumlogreduction,subtractthehighestlogofthenumberofsurvivorsintheinoculatedtreatedsamplesforeachprocessparameterfromthelowestlogofinitialcountsinthecorrespondinguntreatedinoculatedsamples(travelingcontrols).
Pleasenote:Datamustbeconvertedtobase10BEFOREanycalculationsaredone.Althoughaveragevaluesareusefulininterpretingresults,foralmondvalidationpurposes,theleastlogreductionvaluesachievedmustmeettheminimum4-logdestructionrequirement.
•Date(s)ofalmondinoculation,heatresistancetestresultsandpre-/post-inoculationalmondmoisture•Validationtestdate(s)andenumerationdate(s)
DILUTIONS50 g almonds +
100 mL TSB (ABC) or 50 mL BPB (BAM)
(100 dilution)*
9.0 mL BPB per tube
OPTION 1: 0.1 mL per plate
OPTION 2: 0.25 mL per plate
10-1
1.0 mL 1.0 mL 1.0 mL
10-2 10-3
*ABC Procedure–A correction factor of x2 is needed in calculations for 1:2 w/v dilution
100–10-3 dilutions
DILUTION SCHEME
SPREAD PLATING FOR SERIAL DILUTIONS
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3.5 DATA CALCULATION EXAMPLES
BelowaretwoexamplesofcalculationstodeterminelogCFU/gandlogreductionofE. faecium.RefertoSection3.3forthedilutionscheme.Fordilutioncalculations,assumethatalmondsarenothomogenizedandTSBorBPBarenotabsorbedbythealmonds.Intheexamplesgiven,assumethatthelowestcountforuntreatedinoculatedsamplesis7.4logCFU/g.
EXAMPLE 1:
•Countsontwoplatesonwhich0.1-mLsamplesofa10-1dilutionwereplatedare30and42colonies.Use36(theaveragenumberofsurvivorsinthissample)tocalculatetheminimumlogreduction.
Sincethiscountwasobtainedbyplatinga0.1-mLsampleofa10-1dilutionofTSBorBPBintheprimaryTSB/almondmixtureorBPB/almondmixture,respectively,thecountinTSBorBPBis100x36=3,600CFU/mL.(Ifthecountwasfroma10-2dilution,multiplyby1,000insteadof100.)
TocalculatelogCFU/gvalues:IftheABCprotocol(Section3.2)wasused,multiply3,600by2=7,200CFU/gofalmonds(3.9logCFU/g).IftheFDABAMprocedurewasused,thecountis3,600CFU/g(3.6logCFU/g);thereisnoconversionfactor.
Tocalculatelogreduction:FortheABCprotocolexampleabove,7.4logCFU/g(untreatedinoculatedalmonds[travelingcontrols])−3.9logCFU/g(treatedinoculatedalmonds)=3.5logCFU/g(logreduction).IftheFDABAMprocedurewasused,thelogreductionis7.4logCFU/g−3.6logCFU/g=3.8logCFU/g.
EXAMPLE 2:
•Countsfromquadruplicateplatesonwhich0.25-mLsamplesofa100dilutionwereplatedare5,7,13and0colonies.Addthecounts(total=25).
Thistotalcountisthecountin1mLofTSBorBPBfromtheTSB/almondorBPB/almondmixture(=25CFU/mL).
TocalculatelogCFU/gvalues:IftheABCprotocolwasused,multiply25by2=50CFU/gofalmonds(1.7logCFU/g).IftheFDABAMprocedurewasused,thecountis25CFU/g(1.4logCFU/g);thereisnoconversionfactor.
Tocalculatelogreduction:FortheABCprotocolexampleabove,7.4logCFU/g(untreatedinoculatedsamples[travelingcontrols])−1.7logCFU/g(treatedinoculatedalmonds)=5.7logCFU/g(logreduction).IftheFDABAMprocedurewasused,thelogreductionis7.4logCFU/g−1.4logCFU/g=6.0logCFU/g.
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S E C T I O N 4 :
REFERENCES
Andrews, W.H., and T.S. Hammack. 2003. Food sampling and preparation of sample homogenate, chap. 1. In U.S. Food and Drug Administration Bacteriological Analytical Manual [online]. Available at: http://www.fda.gov/Food/FoodScienceResearch/LaboratoryMethods/ucm063335.htm.
Federal Register. 2007. Almonds grown in California; outgoing quality control requirements. Fed. Reg. 72:15021–15036 (7 CFR part 981). Available at: http://www.gpoaccess.gov/fr/retrieve.html.
Jeong, S., B.P. Marks, and E.T. Ryser. 2011. Quantifying the performance of Pediococcus sp. (NRRL B-2354: Enterococcus faecium) as a nonpathogenic surrogate for Salmonella Enteritidis PT30 during moist-air convection heating of almonds. Journal of Food Protection 74:603–609.
Kopit, L.M., E.B. Kim, R.J. Siezen, L.J. Harris, and M.L. Marco. 2014. Safety of the surrogate microorganism Enterococcus faecium NRRL B-2354 for use in thermal process validation. Applied and Environmental Microbiology 80(6):1899–1909.
Kornacki, J.L. 2012. Enterococcus faecium NRRL B-2354: Tempest in a teapot or serious foodborne pathogen? Food Safety Magazine April/May 2012. Available at: http://www.foodsafetymagazine.com/magazine-archive1/april-may-2012/enterococcus-faecium-and-b-2354-tempest-in-a-teapot-or-serious-foodborne-pathogen/.
Swanson, K.M.J., R.L. Petran, and J.H. Hanlin. 2001. Culture methods for enumeration of microorganisms, pp. 53–62. In F.P. Downes and K. Ito (eds.), Compendium of Methods for the Microbiological Examination of Foods, 4th ed. American Public Health Association, Washington, D.C.