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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


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


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).


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)


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)


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


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


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


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


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


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


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


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.


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.

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