national standard of the people’s republic of...

GB4789.26-2013FoodMicrobiologicalExaminationCommercialSterilizationExamination

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NationalStandardofthePeople’sRepublicofChina

GB4789.26-2013

NationalFoodSafetyStandard

FoodMicrobiologicalExamination CommercialSterilization

Examination

食品安全国家标准

食品微生物学检验商业无菌检验

• Releasedon2013-11-29

• Implementedon2014-06-01

• IssuedbyNHFPC

DISCLAIMER: The English version is an unofficial translation of the original in Chinese forinformationandreferencepurposesonly. Incaseofadiscrepancy theChineseoriginal standardwillprevail.


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ForewordThestandardreplacesGB/T4789.26---2003FoodHygieneMicrobiologicalExaminationCommercialSterilizationExaminationofCannedFood.ComparedwithGB/T4789.26--2003,themainchangesareasfollows:— TheChinesenameoftheStandardarerevised;— Thescopeisrevised;— Normativereferencesaredeleted;— Termsanddefinitionsarerevised;— Equipmentandmaterialsarerevised;— Theculturemediumandreagentsarerevised;— Inspectionprocedurechartsareadded;— Teststepsarerevised;— Resultdeterminationisrevised;— AppendixAandAppendixBarerevised.


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NationalFoodSafetyStandard

FoodMicrobiologicalExaminationCommercialSterilizationExamination1. ScopeThis standard stipulates the basic requirements, operation procedures and result determination of thecommercialsterilizationexaminationoffood.Thisstandardisapplicabletothecommercialsterilizationexaminationoffood.2. TermsanddefinitionsThefollowingtermsandtheirdefinitionsareapplicabletotheStandard.2.1 LowacidcannedfoodsInadditiontoalcoholicbeverages,theacidulatedlow-acidcannedfoodsaremadefromlow-acidfruit,vegetablesorvegetableproducts,pHvalueofwhicharereducedbyaddingacidforthepurposeofheatsterilization,whoseequilibriumpHvalueshouldbegreaterthan4.6afterbeingsterilizedandthewateractivityshouldbemorethan0.85belongtolowacidcannedfoods.2.2 AcidcannedfoodsThecannedfoodsofwhichtheequilibriumpHvaluesareequal toorbelow4.6afterbeingsterilizedbelongtoacidcannedfoods.Tomatoes,pears,pineapplesandjuicesprocessedfromthemofwhichpHvaluesarebelow4.7aswellasfigswiththepHvalueofbelow4.9areacidcannedfoods.3. EquipmentandmaterialsBesides the regularequipment for sterilizationand culture inmicrobiology lab,otherequipmentandmaterialsrequiredareasfollows:

a) Refrigerator:2℃~5℃;

b) Constanttemperatureincubator:30℃±1℃;36℃±1℃;55℃±1℃;

c) Constanttemperaturewaterbathcase:55℃±1℃;

d) Homogenizer,homogeneousbag,homogeneouscupandpestle;e) PotentialpHmeter(accuracy:pH0.05unit);f) Microscope(10~100-fold);g) Canopenerandpuncherforcan;h) Electronicscaleorbenchbalance;i) SupercleanbenchorClass100cleanlab.4. Culturemediaandreagents

4.1 Sterilesalinesolution:seeA.1inappendixA.4.2 Crystalvioletstainingsolution:seeA.24.3 Dimethylbenzene4.4 Ethanolsolutioncontaining4%iodine:dissolve4giodinein100mLof70%ethanolsolution.5. Inspectionprocedures


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CommercialsterilizationinspectionproceduresareshowninFigure1.

Figure1Inspectionproceduresofcommercialsterilization

6. Operationsteps6.1 SamplespreparationRemovetheexternaltags,anduseawaterproofpermanentmarkertomarkonthesurfaceofthecontainer.Recordthecontainer,serialnumber,productcharactersandleakageaswellasabnormalconditions,suchasholesonthecontainerorrust,indentationandexpansionandsoon.6.2 WeightPackedobjectsof1 kgor less shouldbeweighedwithanaccuracy to1g, thoseofmore than 1 kg should beweighedwithanaccuracyto2gandthoseover10kgshouldbeweighedwithanaccuracyto10g.Atthesametime,makerecordsofthem.6.3 HeatPreservation

6.3.1 Takeonesamplefromeachbatch,storethemintherefrigeratorat2℃-5℃asreferences,andpreservethe

othersamplesfor10daysunderthetemperatureof36℃±1℃.Duringheatpreservation,dailychecksshouldbe

conductedandthedilatantorleakedonesshouldbetakenoutimmediatelyforinspectiononcedetected.6.3.2 Weigh again andmake recordswhen heat preservation is finished, and compare theweights before and


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aftertheheatpreservationtocheckforanychanges.Thelighterweightsofsamplesindicatethatleakageoccursinsamples. On such conditions, keep all packed objects under room temperature until they are unpacked forinspection.6.4 Unpack

6.4.1Keepthedilatantsamplesintherefrigeratorat2℃-5℃forseveralhoursbeforeopening.

6.4.2Ifexpansionsoccur,cleanthesmoothsurfaceofsamplestobetestedwithcoldwateranddetergent.Afterbeing washed by water, dry the smooth surface with sterile towels. Soak the smooth surface in the ethanolsolution containing4% iodine for15minutes todisinfect, thendry itwith sterile towels, and light it inairtightcoveruntiltheethanolsolutioncontainingiodineremainedonthesurface isburnedoff.Don’tburnthedilatantsamplesandthosepackedwithcombustiblepackagingmaterials,andjustsoakthesmoothsurfaceintheethanolsolutioncontaining4%iodinefor30minutes,anddryitwithsteriletowels.6.4.3Unpack thesampleson thecleanbenchor in theClass100clean lab.Before that, thesampleswithsoupshouldbeproperly shaken.Openaholeof appropriate sizeon thesmoothsurfaceof the sterilizedcanwithasterileopener,andbreakingthecrimpingstructureisnotallowedduringthisprocess.Oneopenershallbeusedbyonlyonecan,andcross-use shallbebanned. If samplesarepackedwith softmaterials, theycouldbeopenwithsterilescissorsandtheseamsshouldnotbebroken.Sniffthesmell immediatelyafteropeningandmakearecord.Note:Theseriouslydilatantsamplesmayexplodeandtoxicsubstancesmayspray.Such hazardous events couldbe prevented bycovering thedilatantsampleswithsteriletowelsorsterilefunnel.6.5 SamplereservingAftertheunpacking,takeoutatleast30ml(g)ofthecontentswithsterilestrawsorotherpropertoolsandputin

sterilecontainersthroughsterileoperations.Storethecontents in therefrigeratorat2℃-5℃ for further test if

necessary.Aftertestingconclusionofthebatchofsamplesisachieved,thecontentscouldbethrownaway.Theunpackedsamplesshouldbeproperlypreservedforfutureexaminationsonthecontainers.6.6 SensorytestInthetestroomwithsufficientlight,cleanairandnobadsmell,pourthecontentsofsamplesintowhiteenameldish,thenobservethestructure,shapeandcolor,snifftheodor,andpressthefoodtoinspectitsproperties,andso that judgments could be made if the food has been spoiled. At the same time, observe the packagingcontainersfromoutsideandinsideandmakerecordsatthesametime.6.7 DeterminationofpHvalues6.7.1 Sampletreatment6.7.1.1 Mixtheliquidproductsforfurtheruse.Asfortheproductswithbothsolidandliquidphase,choosethewellmixedliquid-phasepartforuse.6.7.1.2 For the thickorhalf-thickproductsor those fromwhich it’sdifficult toextractjuice(suchassyrup, jam,jelly,greaseandsoon),takepartofthemandgrindinahomogenizerormortar.Ifthesamplesarestilltoothickafterbeinggrinded,addsameamountofsteriledistilledwaterintothemandfullymixforuse.6.7.2 Determination6.7.2.1 Asthesolutionstobetested, inserttheelectrodesintothesamplesolutionstobetested,andadjustthetemperaturecorrectorofpHmetertothesametemperature.Adjustthetemperatureofsolutionstobetestedto

20℃±2℃andconductdeterminationbythestepsapplicableforallpHmetersiftemperaturecorrectionsystemis

notavailable,.Afterthenumericalreadingsbecomesteady,directlyreadthepHvaluesfromthescales,withtheaccuracyto0.05pHunit.6.7.2.2 Thesamepreparedsampleshouldbedeterminedatleasttwice,andthedifferencesbetweentworesultsshouldnotexceed0.1pHunit.Takethearithmeticmeanvalueofthetwotestsastheresult,andreportresultshallbewiththeaccuracyto0.05pHunit.6.7.3 Resultanalysis


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Compare the resultswith the refrigerated control samples of the samebatch to check for obvious differencesbetweenthem.ThedifferencesbetweenpHvaluesreaching0.5orabovecanbedeterminedasobviousones.6.8 Smearstainingmicroscopy6.8.1 SmearTake the contents of samples tomake smears. For sampleswith soup, take out some soup fromwhich by aninoculating loop, and smear it on glass slides. Solid products can be used tomake into smears directly or bedilutedbysmallamountofstroke-physiologicalsalinesolution(SPSS)andthenmadeintosmears.Thenitshouldbe fastenedwith fire afterdrying. For greasy food its smears shouldbedriednaturally and fastenedwith fire,thenwashedwithflowingdimethylbenzeneanddriednaturally.6.8.2 StainingmicroscopicexaminationThe smears in 6.8.1 should be conducted simple staining with crystal violet staining solution and carry outmicroscopic examination for them after drying. Observe at least five fields ofmicroscope and recordmycelialmorphologyandthebacterialcountwithineachfield.Comparethemwithrefrigeratedsamplesofthesamebatchtosee ifobviousmicrobialproliferationoccurs.Obviousproliferation isindicated if thebacterial countgrowsahundredfoldorabove.7. InterpretationofResultsThe product can be reported as commercial sterilization if there is no leakage within the samples after heatpreservationtest,andnomicrobialproliferation isobserved insensorytest,pHvaluedeterminationandsmearmicroscopicexamination.The product can be reported as non-commercial sterilization, if there are leakageswithin samples after heatpreservationtest,andmicrobialproliferationsareobserved in sensory test, pHvaluedeterminationand smearmicroscopicexamination.If it’s necessary to findout the reasons forexpansion,unusualpHvaluesor sensoryexperienceandmicrobialproliferation,takethereservedsamplestoinoculateandcultureandmakeareportasperAppendixB.Whenitisnecessarytodeterminewhetherthereisaleakageoccursinthecontainersofsamples,conductasealingtestfortheunpackedsamplesandmakeareportofit.


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AppendixA

CultureMediumandReagentA.1 Stroke-physiologicalsalinesolutionA.1.1 CompositionsSodiumchloride 8.5gDistilledwater 1000.0mLA.1.2 Preparationmethod

Weigh8.5gofsodiumchlorideanddissolvein1000mlofdistilledwater.Autoclaveat121℃for15minutes.

A.2 CrystalvioletstainingsolutionA.2.1 CompositionsCrystalviolet 1.0g95％Ethanol 20.0mL1％Ammoniumoxalatesolution 80.0mLA.2.2 PreparationmethodDissolve1.0gofCrystalVioletin95%ethanol,andthenmixwith1%ammoniumoxalatesolution.A.2.3 StainingmethodFixthesmearwiththeflamefromanalcoholburner.Addcrystalviolettothesmeardropbydropandstainfor1min,thenrinsewithwater.


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AppendixB

AbnormalCauseAnalysis(OptionalItem)B.1 CulturemediumandreagentB.1.1 BromcresolpurpledextrosebrothB.1.1.1 CompositionsPeptone 10.0gBeefextract 3.0gGlucose 10.0gSodiumchloride 5.0gBromocresolPurple 0.04g(or1.6％ethanolsolution,2.0mL)Distilledwater1000.0mLB.1.1.2 PreparationmethodHeat,mix and dissolve all the compositions excluding Bromocresol Purple. Adjust pH to 7.0±0.2 and then addBromocresolPurpleintothemixedsolution.Dispenseitintotesttubeswithsmallinversedtesttubes.Andeachtube

shouldbefiledwithl10mLofthemixedsolutionandautoclavedat121℃for10minutes.

B.1.2 CookedmeatmediumB.1.2.1 CompositionsBeefinfusionbroth 1000.0mLPeptone 30.0gYeastextract 5.0gGlucose 3.0gSodiumdihydrogenphosphate 5.0gSolublestarch 2.0gMeatresidue appropriateamountB.1.2.2 PreparationmethodB.1.2.2.1 Weigh500goffreshmincedbeefwithoutfatandfascia,thenmixedwith1000mLofdistilledwaterand25.0mL of 1moL/L sodium hydroxide solution. Stir the solution and boil it for 15 minutes, and cool it down.Removetheexternalfat,clarifyandfilterthesolution.Add1000mLofwatertogetthebeefinfusionbroth.AddinallthecompositionsexceptmeatresiduereferredtoinB.1.2.1,andthenadjustthepHvalueto7.8±0.2.B.1.2.2.2 Wash the meat residue with water and make it half-dry. Respectively fill it into test tubes(15mm×150mm)whichisabout2cmto3cmhigh.Add0.1gto0.2greducedironpowderorafewironfilingsintoeachtesttube.FilleverytesttubewiththepreparedliquidculturemediumofB.1.2.2.1,makingtheliquidabout1cmhigherthanthesurfaceofmeatresidue.Itisthentoppedwithdissolvedpetrolatumorliquidparaffin(0.3cm

to0.4cmhigh).Autoclaveat121℃for15minutes.

B.1.3 Nutrientagar


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B.1.3.1 CompositionsPeptone 10.0gBeefextract 3.0gSodiumchloride 5.0gAgar 15.0g-20.0gDistilledwater 1000.0mLB.1.3.2 PreparationmethodDissolveallthecompositionsexceptagarindistilledwater,andthenaddinabout2mLof15%sodiumhydroxidesolution.AdjustpH to7.2 to7.4. Thenadd inagarandheat toboiling todissolveagar.Dispense theobtainedsolutionintoflasksortesttubes(13mm×130mm)respectively.Autoclaveat121℃for15minutes.B.1.4 AcidbrothB.1.4.1 CompositionsPolypetone 5.0gYeastextract 5.0gGlucose 5.0gPotassiumdihydrogenphosphate 5.0gDistilledwater 1000.0mLB.1.4.2 Preparationmethod

Heat,stiranddissolveallthecompositionsreferredtoinB.1.4.1.AdjustpHto5.0±0.2.Autoclaveat121℃for15

minutes.B.1.5 MaltextractbrothB.1.5.1 CompositionsMaltextract 15.0gDistilledwater 1000.0mLB.1.5.2 PreparationmethodDissolvemalt extract fully indistilledwater, then filterby filterpaper.AdjustpH to4.7±0.2 and dispense it to

severalcontainers.Autoclaveat121℃for15minutes.

B.1.6 Sabouraud'sdextroseagarB.1.6.1 CompositionsPeptone 10.0gAgar 15.0gGlucose 40.0gDistilledwater 1000.0mL


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B.1.6.2 Preparationmethod

Dissolveallthecompositionsindistilledwaterandheattoboiling.AdjustpHto5.6±0.2.Autoclaveat121℃for15

minutes.B.1.7 LivervealagarB.1.7.1 CompositionsLiverextract 50.0gVealextract 500.0gProteosepeptone 20.0gNeopeptone 1.3gTryptone 1.3gGlucose 5.0gSolublestarch 10.0gPlasmacasein 2.0gSodiumchloride 5.0gSodiumnitrate 2.0gGelatin 20.0gAgar 15.0gDistilledwater 1000.0mLB.1.7.2 Preparationmethod

Dissolveallthecompositionsindistilledwater.AdjustpHto7.3±0.2.Autoclaveat121℃for15minutes.

B.1.8 GramstainsolutionB.1.8.1 CrystalvioletstainingsolutionB.1.8.1.1 CompositionsCrystalviolet 1.0g95％Ethanol 20.0mL1％Ammoniumoxalatesolution 80.0mLB.1.8.1.2 PreparationmethodDissolve1.0gofcrystalvioletfullyin95％ethanolandthenmixwith1％ammoniumoxalatesolution.B.1.8.2 Gram’siodinesolutionB.1.8.2.1 CompositionsIodine 1.0gPotassiumiodide 2.0gDistilledwater 300mLB.1.8.2.2 PreparationmethodFirst,mix1.0gofiodidewith2.0gofpotassiumiodide.Thenaddalittledistilledwaterintothemixtureandshake


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well.Afteritisfullydissolved,adddistilledwaterto300mL.B.1.8.3 Safranine(counterstain)B.1.8.3.1 CompositionsSafranine 0.25g95％Ethanol 10.0mLDistilledwater 90.0mLB.1.8.3.2 PreparationMethodDissolve0.25gofsafranineinethanolandthendilutewithdistilledwater.B.1.8.4 StainingMethodB.1.8.4.1 Fixasmearontheflamefromanalcoholburner.Addcrystalviolettothesmeardropbydropandstainfor1min,thenrinsewithwater.B.1.8.4.2 Addgram’siodinedropbydrop,retainfor1minuteandthenrinsewithwater.B.1.8.4.3 Add 95% ethanol drop by drop, decolor for about 15~30s until the staining solution is washed off.Decoloringtoomuchisnotallowed,andthenrinsewithwater.B.1.8.4.4 Addcounterstainliquiddropbydrop,re-dyefor1minuteandrinsewithwater.Conductmicroscopyafterdrying.B.2 InoculatedCultureofLow-acidCannedFood(pH>4.6)B.2.1 Forlow-acidcannedfood,inoculateeachofitssamplein4testtubesofcookedmeatmediumpreheatedto

100℃andrapidlycooleddowntoroomtemperature;Meanwhile,inoculatein4testtubesofbromcresolpurple

dextrosebroth. Inoculate1mL(g)~2mL(g)of samples (1mL~2mL for liquid samples, 1g~2g for solidsamples, ifbothexist,halfofeachforboth)ineachtesttube.TheconditionsofcultureareshowninTableB.1.

TableB.1CookedMeatMediumandBromcresolPurpleDextroseBrothforInoculationofLow-acidCanned

Food(pH>4.6)

CultureMedium NumberofTubes CultureTemperature/℃ CultureTime/h

CookedMeatMedium 2 36±1 96~120

CookedMeatMedium 2 55±1 24~72

BromcresolPurpleDextroseBroth

2 55±1 24~48

BromcresolPurpleDextroseBroth

2 36±1 96~120


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B.2.2 AfterculturinginaccordancewiththeconditionsshowninTableB.1,recordstatesofmicrobialgrowthineachtube.Ifnomicrobialgrowthisobservedinatube,discarditafterrecording.B.2.3 Ifthereismicrobialgrowthinatube,pickliquidwithaninoculatinglooptomakeasmearandconductGramstainingmicroscopyforit.Ifdifferentmicrobialmorphologiesorasinglecoccalorfungalmorphologyisobservedina tubeofbromcresolpurpledextrosebroth, thendiscard itafter recording. If there isnobacillus, andyeast,myceteandtheirmixturesareobserved inatubeofcookedmeatmedium,discard itafterrecording. Inoculatetheotherpositivetubesofbromcresolpurpledextrosebrothandcookedmeatmediuminwhichmicrobialgrowthisobservedontwolivervealagarornutrientagarstreakplates,oneforaerobiccultivation,anotherforanaerobiccultivation.ThecultivationshallrefertoFigureB.1.

FigureB.1TheProceduresoftheInoculationandCultureofLow-acidCannedFoodB.2.4 Pickasinglecolonyinaerobiccultureandinoculateitonthenutrientagarsmallslant,whichwillbeusedforthelaterGram’sstainingmicroscopicexamination.Pickasinglecolonyinanaerobicculturetomakeasmearandconduct Gram’s staining microscopy for it. Inoculate single colonies respectively picked from aerobic andanaerobiccultureintocookedmeatmediumtoconductpureculture.B.2.5 Pick cultures from nutrient agar small slant and anaerobically cultured cookedmeatmedium tomake asmearandconductmicroscopyforit.B.2.6 Pickaerobicculturesfrompurecultivationandinoculateinlivervealagarornutrientagarplatetoconductanaerobic cultivation for them; pick anaerobic culturesfrompurecultivationand inoculate in livervealagarornutrientagarplatetoconductaerobiccultivationforthem,toidentify whethertheculturesarefacultativeanaerobesornot.B.2.7 Iftestingthebotulinumtoxinofclostridiumisrequired,typicalcoloniesshouldbeselectedforpureculture


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incookedmeatmedium.Cultivateat36℃for5days.ThenconductbotulinumtoxintestinaccordancewithGB/T

4789.12.B.3 TheInoculationandCultureofAcidCannedFood(pH≤4.6)B.3.1 Inoculateeachsampleinfourtesttubesofacidbrothandtwomaltextractbroth.Inoculate1mL(g)~2mL(g)ofsamples(1mL~2mLforliquidsamples,1g~2gforsolidsamples,ifbothexist,halfofeachforboth)ineachtesttube.TheconditionsofcultureareshowninTableB.2.

TableB.2TheAcidBrothandMaltExtractBrothforInoculationofAcidCannedFood(pH≤4.6)

CultureMedium NumberofTubes CultureTemperature/℃ CultureTime/h

AcidBroth 2 55±1 48

AcidBroth 2 30±1 96

MaltExtractBroth 2 30±1 96

B.3.2 Afterculturing inaccordancewiththeconditionsshowninTableB.2, recordstatesofmicrobialgrowth ineachtube.Discarditafterrecordingifnomicrobialgrowthisobservedinatube.

B.3.3 If there ismicrobial growth in a culture tube, pick the cultured content tomake a smear and conductGramstainingmicroscopyforitandrecordthemicroorganismsobserved.

B.3.4 Ifmicrobialgrowthisfoundinacidbrothormaltextractbrothunderthecultureconditionof30℃, inoculate

the contents of each positive tube respectively in 2 nutrient agar or Sabouraud dextrose agar plates, one foraerobiccultivation,theotherforanaerobiccultivation.

B.3.5 Ifmicrobialgrowthisfoundinacidbrothunderthecultureconditionof55℃,inoculatethecontentsofeach

positivetubesrespectively in2nutrientagarorSabourauddextroseagarplates,oneforaerobiccultivation,theotherforanaerobiccultivation.Conductsmearstainingmicroscopyfortheplatewherethereismicrobialgrowthandreportthemicrobialtypesobservedinmicroscopy.ThecultivationproceduresareshowninFigureB.2.


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FigureB.2TheProceduresofInoculationandCultureofAcidCannedFood

B.3.6 Picksinglecolonies fromthenutrientagarorsabourauddextroseagarplatesaerobicallyculturedat30℃

andinoculatetheminthenutrientagarsmallslant,whichwillbeusedforthelaterGram’sstainingmicroscopy.Meanwhile,inoculateinacidbrothormaltextractbrothandperformpureculture.

Picksinglecoloniesfromthenutrientagarorsabourauddextroseagarplatesanaerobicallyculturedat30℃and

inoculatetheminacidbrothormaltextractbrothandperformpureculture.

Picksinglecoloniesfromthenutrientagarplatesaerobicallyculturedat55℃andinoculatetheminnutrientagar

smallslant,whichwillbeusedforthelaterGram’sstainingmicroscopy.Meanwhile, inoculate inacidbrothforpureculture.

Picksinglecoloniesfromthenutrientagarplatesanaerobicallyculturedat55℃andinoculatetheminacidbroth

forpureculture.B.3.7 Pick cultures from nutrient agar small slant to make a smear and take a look under a microscope. Pick

culturesfromacidbrothormaltextractanaerobicallyculturedat30℃andfromacidbrothanaerobicallycultured

at55℃,respectivelymakeasmearforthemandtakealookunderamicroscope.

B.3.8 Inoculatethepureculturesaerobicallyculturedat30℃ innutrientagarorsabourauddextroseagarplate

and conduct anaerobic culture. Inoculate the pure cultures anaerobically cultured at 30℃ in nutrient agar or

sabourauddextroseagarplateandconductaerobicculture.Inoculatethepureculturesaerobicallyculturedat55

℃innutrientagarandconductanaerobicculture.Inoculatethepureculturesanaerobicallyculturedat30℃in

nutrientagarandconductaerobicculture.Eachstepisforidentifyingwhetherthepureculturesbelongtofacultativeanaerobes.B.3.9 ResultsAnalysisB.3.9.1 Ifthegrowthofmicroorganismscannotbefoundininflatedsamples,theexpansionmaybecausedbythehydrogen which is generated by the reaction between the content and packaging. The amount of generatedhydrogenvarieswiththelengthofstoragetimeandconditionsaswell.Fillingexcessivecontentsinpackagesmayalsogiverisetoslightexpansion,whichcanbedeterminedbyweighing.If the mixture of a large number of microorganism is observed in direct smear examination and thesemicroorganismscannotgrowafterculture,itindicatesthatthereisputrefactionbeforesterilization.ThepH,smellandmorphologicalstructureofproductsarefoundaberrantduetothemicrobialgrowthbeforesealpacking.

B.3.9.2 Onlythegrowthofbacillifoundunderthecultureconditionof36℃withpackagingcontainersperfectly

sealedandtheheatresistantperformanceofsuchbacilliarenotbetterthanthatofclostridiumbotulinum,whichindicatesthatunder-sterilizationoccursduringtheproductionprocess.B.3.9.3 If the mixed colonies of bacilli, cocci and fungi appear after culture, it indicates there is a leakage inpackagingcontainers.Itmightresultfromunder-sterilizationaswell,butinthiscondition,theexpansionrateofthesamebatchofproductswouldberatherhigh.B.3.9.4 Observe the situations of acid production and gas production in bromcresol purple dextrose broth


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culturedat36℃or55℃.Ifacidisproduced,itindicatesthegrowthofmesophilicmicroorganisms(e.g.mesophilic

acid-resistingbacteria)orthermophilicmicroorganisms(e.g.Bacillusstearothermophilus).

Bacteria growth and gas with putrid odour production in cooked meat medium at 55℃ indicate that the

putrefactionofsamplesresultsfromthermophilicanaerobicclostridium.

Bacteriagrowthandgaswithputridodourproduction incookedmeatmediumat36℃andsporesobserved in

microscopy indicatethattheputrefactionmaybecausedbyclostridiumbotulinum,clostridiumsporogenesandclostridiumperfringens.Botulinumtoxincanbefurthertestedifnecessary.B.3.9.5 Thespoilageofacidcannedfoodisusuallycausedbylactobacilluswithnosporesandyeast.Generally,ifitspHislowerthan4.6,thedeteriorationcausedbybacillusdoesnothappen.Thespoiledketchuporcanned tomato saucewouldn’t be expanded, but putrid odorwith orwithout pH getting lowmay occur. It isusuallycausedbyaerobicbacillus.B.3.9.6 Thethermophilicbacteriaexistinmanycannedfoods.Theydon’tgrowinnormalstorageconditions,but

they will grow and result in putrefaction when the products are exposed to high temperature (50℃~55℃).

Thermophilicacid-resistingbacillusandbacillusstearothermophilusmaycausetheputrefactionrespectivelyin

acidandlow-acidfood,buttherewouldn’tbeexpansion inthepackagingcontainers.Cultureat55℃willnot

giverisetothechangeoftheappearanceofpackagingcontainers,yetproducingtheputridodorwithandwithoutlowerpH.Thespoilageoftomatoes,pears,figsandpineapplesissometimescausedbyclostridiumpasteurianum.Asakindof thermophilicanaerobicbacteria,Clostridiumthermosaccharolyticumcancausetheexpansionandproducts’putridodor.Thermophilic anaerobicbacteria canproduce the gas aswell, because it proliferatesquickly afterbeginning togrow. Itmaybe confusingwhether the expansion is causedby hydrogenor the gas producedby thermophilicanaerobic bacteria. Chemical decomposition generates carbon dioxide, which commonly occurs especially insugary and sour food, for example, tomato sauce, molasses, mincemeat and sugary canned fruits. Thedecompositionspeedsupwiththetemperatureincreasing.B.3.9.7 Laboratory pollution should be considered if any microorganism is isolated from the direct smears ofsterile vacuum package and normal products.To confirmwhether it is lab pollution, inoculate the isolatedlivingmicroorganismunderasepticconditionstoanothernormalcontrolsample,thensealandcultureat36

℃ for14days. It is likelythatthesemicroorganismsarenot fromtheoriginalsamples ifexpansionorthe

deteriorationofproductsoccurs.Openthepackagesofsamplesunderasepticconditionsandre-cultureaspertheproceduresmentionedaboveifthesamplesarestillflat.Ifthesamekindofmicroorganismisfoundagainandtheproductsarenormal,theproductswouldbeconsideredcommerciallysterilebecausethiskindofmicroorganismswouldn’tgrowduringthenormalstorageandtransportation.B.3.9.8 Determinacyconclusionscannotbemadethroughbrothcultureifthereiscorruptioninthefooditself.Inthissituation,furthercultureisnecessarytoconfirmwhetherthereisgrowthofmicroorganism.B.4 LeakproofnessTestMethodforEmptyFoodCanMadeofTinnedSteelSheetB.4.1 Pressure-reduceLeakproofnessTest

Wash thepackaging cansusedas sampleswell, anddry it at the temperatureof36℃. Fill thedried canswith

cleanwateraccountingfor80%~90%ofeachcan.Placetheperspexsheetwitharubberringontotheopencurledendofcanstokeepitsealed.Startvacuumpumpandclosethegateforreleasinggas.Pressthecoverboardwith


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handsandcontroltheairbleed.Makesurethatthevacuumgaugerisingfrom0Pato6.8×104Pa(510mmHg)lasts

morethan1minuteandthevacuumdegreeof6.8×104Pa(510mmHg)lastsmorethan1minute.Tiltthecanandcarefullyobserveitinordertoseewhetherthereisbubblegenerated,especiallythehemmingandcommissure.Itcanbejudgedtobeleakyifthebubblesareconstantlyproducedatthesamesite.Recordthetimeofleakageandvacuumdegreeandmarkthesitewherethereisleakage.B.4.2 Pressure-increaseLeakproofnessTest

Cleanthecanusedassampleanddryitunderthetemperatureof36℃.Plugthemouthoftheemptycanwitha

rubberstoppers,soaktheemptycanintheglassjar fullofwater, start theaircompressorandslowlyopenthe

valves,tograduallyincreasethepressureinthecanto6.8x104Paandkeepthepressurefortwominutes.Carefulobservation of the can should be made, the hemming and commissure in particular, to see if bubbles areproduced.Itcanbejudgedtobeleakyifthebubblesareconstantlyproducedatthesamesite.Recordthetimeofleakageandpressure,andmarkthesitewherethereisleakage.

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