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MicrobiologicalAnalysisof FoodsandFoodProcessing Environments Thispageintentionallyleftblank
MicrobiologicalAnalysisof FoodsandFoodProcessing Environments OsmanErkmen
DepartmentofFoodEngineering,FacultyofEngineering, UniversityofGaziantep,Gaziantep,Turkey
AcademicPressisanimprintofElsevier 125LondonWall,LondonEC2Y5AS,UnitedKingdom 525BStreet,Suite1650,SanDiego,CA92101,UnitedStates 50HampshireStreet,5thFloor,Cambridge,MA02139,UnitedStates TheBoulevard,LangfordLane,Kidlington,OxfordOX51GB,UnitedKingdom
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ThisbookandtheindividualcontributionscontainedinitareprotectedundercopyrightbythePublisher(otherthan asmaybenotedherein).
Notices
Knowledgeandbestpracticeinthisfieldareconstantlychanging.Asnewresearchandexperiencebroadenour understanding,changesinresearchmethods,professionalpractices,ormedicaltreatmentmaybecomenecessary.
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SectionIICountingofimportantmicrobialgroups fromfoodproducts SectionIIIIsolationandcountingofindicator 17.4
SectionIVDetectionoftoxigenicfungi,viruses,andparasites SectionVIdentificationoffoodssafetyandquality Thispageintentionallyleftblank
Abouttheauthor OsmanErkmen Bornin1955inKonya,Turkey,OsmanErkmenhasbeenaprofessorof FoodMicrobiologyintheDepartmentofFoodEngineeringatGaziantep University(Gaziantep,Turkey)since2004.HereceivedhisBScdegreein Biology(1985)andMScdegreeinFoodMicrobiology(1987)fromthe MiddleEastTechnicalUniversity(Ankara,Turkey).HegainedhisPhD fromtheDepartmentofMicrobiologyandClinicalMicrobiology,Faculty ofMedicine,GaziantepUniversityin1994.Hestartedhiscareerasa researchassistantattheDepartmentofFoodEngineeringin1985andlater becameassistantProfessorin1994andassociateProfessorofFood Microbiologyin1999.Since2004,hehasbeenworkingasaprofessorin thisdepartment.AttheDepartmentofFoodEngineering,heexpandedhis researchontheusesofnonthermalprocessesandnaturalantimicrobialsinfoodpreservation;inthe productionoffermentedfoods;inthemicrobialproductionoflycopene,thiamin,alcohol,andcitric acidfromindustrialwastes;andinmicrobialinactivationandmodeling.Hehasreceivedfunding forresearchfromtheUniversityofGaziantepFoundation,theScientificandTechnological ResearchCouncil,andtheRepublicofTurkeyStatePlanningOrganization.Hehasstudiedthe combinedeffectofnonthermalprocesses,naturalantimicrobialsinthedestructionofmicrobial cellsandspores,theirapplicationinfoodpreservation,andthecharacteristicsofwhiteandred winesproductionfromGaziantepGrapes.HeteachescoursesintheFoodEngineeringDepartment andtheNutritionandDieteticsDepartment.HeteachescoursesinGeneralMicrobiology,Food Microbiology,FoodSanitation,andFoodToxicology.ProfessorErkmenhaspublishedover150 researcharticles,reviews,bookchapters,proceedingspapers,andpopulararticlesinthefieldsof FoodMicrobiology,GeneralMicrobiology,FoodToxicology,andFoodSanitationwithmorethan 3500citations.Heistheeditoroftwobooks:“GıdaMikrobiyolojisi”(FoodMicrobiology;5thedition)and“FermenteU ¨ runlerTeknolojisiveMikrobiyolojisi”(FermentedProductsTechnologyand Microbiology)intheTurkishlanguage;andistheauthorofthreebooks:“FoodMicrobiology: PrinciplesintoPractice(twovolumes)”(JohnWileyandSons,Ltd),“LaboratoryPracticein Microbiology”(AcademicPress),and“FoodSafety(NobelAcademicPublishing).Heisanadvisor toPhDandMScgraduates.Hehasmorethan10patents,hasorganizedmorethan20international scientificsymposiums,andhasparticipatedmorethan65internationalsymposiums.Hehasheld administrativepositionsasDirectoroftheInstituteofNaturalandAppliedSciences,Directorof theInstituteofSocialSciences,DirectoroftheTechnicalSciencesVocationalSchool,andasHead oftheDepartmentofFoodSciences.
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Preface Foodmicrobiologyisadynamicdisciplinecoveringmanyareasincludingmicrobiologicalanalysisoffoods,identificationoffoodbornepathogens,developmentoftechniquesforisolatingandenumeratingmicroorganisms,newfoodformulations,developmentofnewfoodprocessing, distributiontechniques,andcarryingoutimportantactivitiestoensurethequalityandsafetyof food.
Conductingmicrobiologicalresearchtopreservethequalityoffoodshasresultedinthedevelopmentofmanymicrobiologicaltechniques.Itisessentialthatthetechniquesusedinmicrobiologicalanalysisoffoodsshouldbethesameorequivalentprocedures.Otherwise,healthfood productioninaccordancewiththestandardsisnotpossibleandthusevaluationofthedataobtained fromtheanalysisoffoodsfromdifferentlaboratoriesorresearchcannotbeassured.Essentially, thereisaneedforaresourceinthefoodindustrywherestandardtechniquesformicroorganisms andfoodsareofferedfortheisolation,enumeration,andidentificationofmicroorganisms.This bookpresentsthemostaccurateandbasicmicrobiologicaltechniquesfordeterminingthemicrobiologicalqualityoffoodsinaccordancewiththestandards.Microbiologicalanalysesinfoodand foodproductionareasaredividedintofivepartsinthisbook.Inthefirstpart,generalfoodmicrobiologyanalysistechniquesaregiven.Techniquesforcountingimportantmicrobialgroupsinfoods areexplainedinthesecondchapter.Partthreeisconcernedwithtechniquesfortheisolation,counting,andidentificationofindicatorandpathogenicmicroorganisms.Partfourconsistsoftechniques forthedetectionoftoxigenicfungi,viruses,andparasites.Inthelastpart,microbiologicalanalyses offoodsareincludedinordertodeterminethemicrobiologicalqualitiesoffoods.Flowchartsare providedfortheisolation,counting,andidentificationofmicroorganisms,enablingtheprocessesto beunderstoodfasterandeasier.Visualsarealsoprovidedforthemorphologicalappearancesand testresultsofmicroorganismsthatfacilitatefasteridentification.Inaddition,thephenotypiccharacteristicsofmicroorganismsaregivenintables,providingeasydeterminationoftheircharacteristics andeasyunderstandingoftheirdistinctions.Thebookpresentsstandardandapplicabletechniques thatstudents,researchers,analysts,andteacherscanapplyinfoodmicrobiologycourses,thefood industry,thehealthfield,andinanalysislaboratories.
Corrections,technicalquestions,andotherinterestscouldbesentto: osmerkmen@gmail.com
OsmanErkmen
DepartmentofFoodEngineering,FacultyofEngineering, UniversityofGaziantep,Gaziantep,Turkey
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Acknowledgments Ialwaysthankmystudentswholikemicrobiologylessons.Thedesireofmystudentstolearnand accessinformationinspiredmetowritethisbook.IwouldliketothankmywonderfulwifeAssist.
Prof.Dr.Ay¸seErkmen;mydaughter,DisasterManagementspecialist,UmranErkmen;andmy sonsDr.Barı¸sErkmenandElectricElectronicEngineerHuseyinErkmenfortheirsupport,sincere encouragement,andendurance.
Finally,Iwouldliketothanktheubiquitousandinvisiblemicroorganisms,Iwroteandpublishedabookduetotheirexistence,andalsohadtojointhe“realworld.”
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Laboratoryrules Thelaboratoryisanimportantpartofmicrobiology.LaboratoriesareNOToptional.Themicrobiologylaboratoryinvolvesworkingwithlivingmicroorganisms.Propertechniqueswillbedemonstratedandshouldalwaysbeusedbeforemicrobiologicalanalysis.Personswillberequiredtoread therelatedpartofthisbookandtheyshouldunderstandtheinherentrisksinvolvedinworkingwith livingmicroorganisms.
Generallaboratoryrules:
1. Wearappropriateclothes.Ingeneral,donotcometolabwithalotofskinexposed. Disposablepaperisrequired.DONOTtransportlabcoatsbackandforth/toandfromlab.
2. NEVEReat,drink,orapplycosmeticsinthelaboratory.
3. Avoidtouchingyourfacewhileworkingwithcultures.
4. AvoidcontactofyourhairwithBunsenburnersandcultures.
5. Reportallspills,accidents,orinjuriesimmediatelytotheinstructor.
6. Beforeyouleavethelabfortheday,disinfectantyourworkingarea.Showyourworkingarea toyourinstructorwithregardtocleanlinessbeforeleaving.
7. Scrubyourhandsbeforeyoubeginlabwork(tohelpminimizecontaminationofyour cultures)andscrubthembeforeyouleavetoremoveanythingyoumayhavepickedupinthe lab.
8. Oldculturetubesaretobeplacedintheracksprovidedsotheycanbeautoclaved.
9. OldPetriplatesaretobeplacedintheprovidedautoclavebags.
10. Microscopeslideswhicharenolongerneededaretobeplacedintheprovidedglassdisposal containers.
11. Donotremoveculturesorothermaterialfromthelab.
12. ALWAYSusethepropertechniquesforhandlingcultures.Thesetechniqueswillbe demonstratedduringthelaboratoryclasses.
13. Readlaboratoryexercisesbeforecomingtothelab.Itisagoodideatobeginyourwrite-ups beforecomingtoclass.
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SECTION I Generalfood microbiologyanalyzing practices Rapidandconventionaltechniquesareusedinthemicrobiologicalanalysisoffoods.These techniquesarewidelyusedinthecountingofmicroorganismsanddetectionofthemicrobiological qualityoffoods.Theyweredevelopedovermanyyearsandareusedastheofficialtechniquesin mostfoodmicrobiologylaboratories.Thesetechniquesareacceptedinternationallyand recommendedforthemicrobiologicalanalysisoffoods.Conventionalapproachesfortheisolation ofmicroorganismsinvolvevarioussolidandliquidmedia.Solidandliquidculturingtechniques areusedeitherforthetargetmicroorganismdetection(presence/absencetest)orquantitation.The rapiddirectmicroscopiccounttechniquesuseasmallvolume(e.g.,0.01mL)ofsamplebut membranefiltertechniqueinvolvesusingsamplesofupto100mLormore.Theplatecount techniqueshaveamaximumsampleuseofavolumeofabout1mL.Liquidculturingiscommonly usesthemostprobablenumbertechnique.Thistechniqueallowsflexiblesamplevolumeuse.The conventionaltechniquesusedtoisolate,count,andidentifyfoodbornemicroorganismsarebased ontheirculturalcharacteristics.Generally,platecountagarorothernonselectiveagarmediacan
beusedfornonspecifictotalviablecount.Selectiveand/ordifferentialmediaareusedforcounting andisolationofspecificgroupsorspeciesofmicroorganisms.Mostofthestandardsforthe indicationofsafetyandhygieneconditionsinfoodproductioninvolvecountingindicator microorganisms(e.g.,aerobicbacteria,coliforms,fecalcoliforms,yeasts,andmolds).Thissection describesthemostcommonlyusedfoodmicrobiologytechniquesintheisolationandcountingof microorganisms.
Practice1.SamplingandSamplePreparationTechniques
Practice2.PlateCountTechniques
Practice3.DirectMicroscopicCountTechniques
Practice4.MostProbableNumberTechnique
Practice5.MembraneFilterTechniques
Practice6.YeastsandMoldsCountingTechniques
Practice7.SanitationDetectionTechniquesinFoodProcessingPlant
1 Samplingandsamplepreparation techniques 1.1 Introduction Theobjectiveofthispracticeistoenabletheanalyzertohaverepresentativesamplesinthelaboratory thataremicrobiologicallyunchangedfromthetimeofsamplingandtopreparethesamplesforanalysis. Ifsamplesarecollectedincorrectly ordonotrepresenttheentireproduct,themicrobiologicalresults willbemeaningless.Thepersonwhocollectsthesamplesshouldabletoapplyanappropriatesampling plan,preventcontamination,andminimizemicrobialchangesduringtransport,storage,andhandling. Thestatisticalsamplingprocedureshouldbeperformedduringsamplingaccordingtothephysicalconditionofthefood(e.g.,solid,semisolid,viscous,orliquid).Aspecificsamplingproceduremayalsobe useddependingonthespecificmicroorganisms,typesoffoods,andotherfactors.Forsuchsampling, refertotherelatedtechniqueinthisbook.Sampling,samplepreparation,andallsamplingtechniques shouldbeperformedunderasepticconditionsinduplicateunlessotherwisespecified.Allsolutionsand equipmentthatmaycausecontaminationofmicroorganismsmustbesterilizedordisinfected.Someof theequipmentcanbedangerousandinadequatelysterilizedbydippingintoalcoholandflamingwith flame.Suchheat-labeledequipmentcanbedisinfectedusingdisinfectants.
1.2 Samplingplan Asamplingplanisasystematicwaytoassessthemicrobiologicalqualityoffoods.Asamplingplan includesboththesamplingprocedureandthedecisioncriteria.Toexamineafoodforthepresenceof microorganisms,arepresentativesamplemustbeexaminedbydefinedprocedures.Aquantity(“lot”) ofproductisproduced,handled,andstoredwithinalimitedtimeperiodunderuniformconditions.It isimpracticaltoexamineallproducts.Insteadofthis,acertainnumberandsizeofsampleunitsfrom thelotareanalyzed.Thesamplesshouldbetakenfromthelotindependentlyandrandomly.Indevelopingasamplingplan,anumberoffactorsshouldbeconsidered:propertiesoffood,productionprocesses,storageconditions,associatedrisks,targetedconsumers,andlimitations.Eachfoodproduct shouldbeconsideredindividually.Asamplingplanincludesthefollowingelements:
1. Microorganismorgroupofmicroorganismsofconcern;
2. Numberofsamplestobetested(n);
3. Testingtechnique(s);
4. Microbiologicallimit(s); m and M:
a. Acceptable( , m);
b. Marginallyacceptable( . m and , M);
c. Unacceptable( . M);
5. Samplingprocedure.
MicrobiologicalAnalysisofFoodsandFoodProcessingEnvironments.DOI: https://doi.org/10.1016/B978-0-323-91651-6.00037-9 © 2022ElsevierInc.Allrightsreserved.
Twotypesofsamplingplanscanbeusedtoindicatelimit(s)forproduct:two-classsampling planandthree-classsamplingplan.
Atwo-classsamplingplanconsistsofthefollowingspecifications: n,c,m
Athree-classsamplingplanconsistsofthefollowingspecifications: n,c,m,M. where
n 5 numberofsampleunits(packages,beefpatties,andsoforth)fromalot.
c 5 themaximumacceptablenumberormaximumallowablenumberofsampleunits.When thisnumberofsampleunitsexceedsthemicrobiologicalcriterion,thelotisrejected.
m 5 maximumnumberofmicroorganisms(criteria)perg;valuesabovethislevelareeither marginallyacceptableorunacceptable.Itisusedtoseparateacceptablefromunacceptablefoodsin atwo-classsamplingplanorseparategoodqualityfrommarginallyacceptablequalityfoodsina three-classsampleplan.
M 5 aquantitythatisusedtoseparatemarginallyacceptablequalityfromunacceptablefood.It isusedonlyinthree-classsamplingplans.Valuesabove M foranysampleareunacceptable,indicatinghealthhazards,sanitaryindicators,andspoilagepotential.
Inatwo-classsamplingplan,onlyonemicrobiologicallimit“m”isinvolved,thereforethetwoclasssamplingplanattributes , m and . m bymaximumallowablenumberof c sample(s).Thelotwill beacceptedorrejectedaccordingto thetwo-classsamplingplanasillustratedinthefollowingdiagram.
Atwo-classplanisusedtoacceptorrejectabatch(lot)offoodinapresence/absencedecision byaplan.Asamplingplanfor Salmonella is n 5 5, c 5 0,where n 5 5meansthatfiveindividual samplesofthelotareexaminedmicrobiologicallyforthepresenceof Salmonella,and c 5 0means thatallfiveunitsmustbefreeofthebacteriaforthelottobeacceptable.Ifanyunitispositivefor Salmonella,theentirelotisrejected.
Samplesmaycontaincoliformswithasamplingplan n 5 5, c 5 2.Bythisplan,ifthreeor moreofthefive-unitsamplescontainedcolif orms,theentirelotwouldberejected.Ifupto 100coliformspergintwoofthefiveunits isallowed,thesamplingplanwouldbe n 5 5, c 5 2, m 5 10 2 .Afterthefiveunitshavebeen examinedforcoliforms,thelotisacceptableifnomore thantwoofthefivecontainasmanyas10 2 coliformspergbutisrejectedifthreeormoreof thefiveunitscontain10 2 coliformsperg.
Athree-classsamplingplanisusedtoindicateacceptable/marginallyacceptable/ unacceptablefoods.Assumethatforagivenfoodproduct,thestandardplatecount(SPC)shallnot exceed106 cfuperg 1 (M)orbehigherthan105 colonyformingunit(cfu)pergfromthreeor moreoffiveunitsexamined.Thespecificationsbecome n 5 5, c 5 2, m 5 105, M 5 106.Ifanyof thefiveunitsexceeds106 cfuperg,thelotisrejected(unacceptable).Ifanysampleunitcannot giveresultsabove m,thelotisacceptable.Iftwoofthefiveunitsexceeds m anddonotexceed M, thelotismarginallyaccepted.
Inathree-classsamplingplan,twomicrobiologicallimits, m and M,areset.Themicrobiologicallimit m commonlyreflectstheupperlimitofagoodmanufacturingpractice.Thecriterion M marksthelimitbeyondwhichthelevelofcontaminationishazardousorunacceptable.Thelotwill beevaluatedaccordingtoathree-classsamplingplanasshowninthefollowingdiagram: Collect Sample
Ingeneral,atwo-classsamplingplanispreferredwhenthemicroorganismofconcernisnot permittedinfoodsamples.Ifanumberofmicroorganismsisallowable,athree-classsamplingplan isusuallyprepared.Toenhancefoodsafetyandimprovefoodquality,morestringentmicrobiologicallimits(m and/or M)shouldbeadopted.
1.3 Sampling Containersusedforplacingsamplesshouldbeleak-proof,wide-mouthed,clean,anddry,buttheir mouthmustbesuitabletoprotectagainstmicrobialcontamination.Appropriatesamplesshouldbetaken torepresentthefoodorothersamplingsource.Laboratoryresultsandinterpretationareonlyvalid whenappropriatesamplesareanalyzed.Asamplingunitshouldbeatleast100gormL.Whenliquid samplesareremoved,anadditionalliquidsamplemustbetakenforatemperaturecontrol.Thesamples arecollectedunderappropriate conditions.Samplesobtainedfrom foodsorothersourcesshouldbe deliveredtothelaboratoryassoonaspossible.Uponarrivalatthelaboratory,thetimeofarrivalshould berecorded.Thesamplesarepreparedatthestartofmicrobiologicalanalysis.Duringstorageandtransportofsamples,microbialcontaminationshouldbepreventedfromtheexternalenvironment,including theair,samplecontainer,samplingdevice,andthe shipmentvehicle.Thepackagedfoodshouldonly beopenedinthelaboratory.Aformshouldbelabeledwiththefollowinginformation:samplename, dataconditions,place,timeofcollection,name ofpeople,andtemperatureatthesamplingtime.
1.3.1 Liquidsamples Thermometersusedincontrollingthetemperatureofliquidfoodshouldbesanitizedbeforeuse.Metal thermometersarepreferredsincethebreakageofa mercurythermometerwouldcontaminatetheproduct.Beforeremovingasample,asepticallymixtheliquidfoods(e.g.,milk,icecream,fruitjuice,water, andsirups)tohomogenizethecontentsofthecontainerasmuchaspossible.Inthelaboratory,theliquidshouldbemixedthoroughlybeforeremovingtherequiredsamplesforanalysis.Samplingofliquid foodsfromin-processmayinvolvetheuseofasterilemetaltube.Aspeciallinesamplingtechnique involvestheuseofadisposablesterilehypodermicneedleandsyringe.Theneedleisinsertedintoa rubbercloserofastainless-steelnipple.Samplingcocksmaybeusedonholdingtanksandproduct
