BS EN 13783-2002 Foodstuffs - Detection of irradiated food using Direct Epifluorescent Filter Technique Aerobic Plate Count (DEFT APC) - Screening method《食品 用直接外荧光过滤器技术 供氧盘计数法检测经辐照.pdf

1、BRITISH STANDARD BS EN 13783:2002 The European Standard EN 13783:2001 has the status of a British Standard ICS 67.050 NO COPYING WITHOUT BSI PERMISSION EXCEPT AS PERMITTED BY COPYRIGHT LAWBS EN 13783:2002 This British Standard, having been prepared under the direction of the Consumer Products and Se

2、rvices Sector Policy and Strategy Committee, was published under the authority of the Standards Policy and Strategy Committee on 24 January 2002 BSI 24 January 2002 ISBN 0 580 38936 7 National foreword This British Standard is the official English language version of EN 13783:2001. The UK participat

3、ion in its preparation was entrusted to Technical Committee AW/-/3, Food analysis Horizontal methods, which has the responsibility to: A list of organizations represented on this committee can be obtained on request to its secretary. Cross-references The British Standards which implement internation

4、al or European publications referred to in this document may be found in the BSI Standards Catalogue under the section entitled “International Standards Correspondence Index”, or by using the “Find” facility of the BSI Standards Electronic Catalogue. A British Standard does not purport to include al

5、l the necessary provisions of a contract. Users of British Standards are responsible for their correct application. Compliance with a British Standard does not of itself confer immunity from legal obligations. aid enquirers to understand the text; present to the responsible European committee any en

6、quiries on the interpretation, or proposals for change, and keep the UK interests informed; monitor related international and European developments and promulgate them in the UK. Summary of pages This document comprises a front cover, an inside front cover, the EN title page, pages 2 to 15 and a bac

7、k cover. The BSI copyright date displayed in this document indicates when the document was last issued. Amendments issued since publication Amd. No. Date CommentsEUROPEANSTANDARD NORMEEUROPENNE EUROPISCHENORM EN13783 November2001 ICS07.100.30;67.050 Englishversion FoodstuffsDetectionofirradiatedfood

8、usingDirect EpifluorescentFilterTechnique/AerobicPlateCount (DEFT/APC)Screeningmethod ProduitsalimentairesDtectiondalimentsionissen utilisantlatechniquedpifluorescenceaprsfiltrationet dnombrementdelaflorearobiesurmilieuglos (DEFT/APC)Mthodeparcriblage LebensmittelNachweisderBestrahlungvon Lebensmitt

9、elnmitEpifluoreszenzFiltertechnik/aerober mesophilerKeimzahl(DEFT/APC)Screeningverfahren ThisEuropeanStandardwasapprovedbyCENon29September2001. CENmembersareboundtocomplywiththeCEN/CENELECInternalRegulationswhichstipulatetheconditionsforgivingthisEurope an Standardthestatusofanationalstandardwithout

10、anyalteration.Uptodatelistsandbibliographicalreferencesconcernings uchnational standardsmaybeobtainedonapplicationtotheManagementCentreortoanyCENmember. ThisEuropeanStandardexistsinthreeofficialversions(English,French,German).Aversioninanyotherlanguagemadebytra nslation undertheresponsibilityofaCENm

11、emberintoitsownlanguageandnotifiedtotheManagementCentrehasthesamestatusasthe official versions. CENmembersarethenationalstandardsbodiesofAustria,Belgium,CzechRepublic,Denmark,Finland,France,Germany,Greece, Iceland,Ireland,Italy,Luxembourg,Netherlands,Norway,Portugal,Spain,Sweden,SwitzerlandandUnited

12、Kingdom. EUROPEANCOMMITTEEFORSTANDARDIZATION COMITEUROPENDENORMALISATION EUROPISCHESKOMITEEFRNORMUNG ManagementCentre:ruedeStassart,36B1050Brussels 2001CEN Allrightsofexploitationinanyformandbyanymeansreserved worldwideforCENnationalMembers. Ref.No.EN13783:2001EEN13783:2001(E) 2 Contents page Forewo

13、rd2 1 Scope 3 2 Normativereferences 3 3 Principle3 4 Reagents.3 5 Apparatus .6 6 Samplingtechnique .7 7 Procedure .8 8 Evaluation.9 9 Limitations10 10 Validation10 11 Testreport 11 AnnexA (informative) Furtherinformationontheapplicability.12 AnnexB (informative) Practicalexampleforcalculationofthemi

14、croscopefactor.13 AnnexC (informative) Flowdiagramoftheprocedure14 Bibliography 15 Foreword ThisEuropeanStandardhasbeenpreparedbyTechnicalCommitteeCEN/TC275“FoodAnalysisHorizontal Methods“,thesecretariatofwhichisheldbyDIN. ThisEuropeanStandardshallbegiventhestatusofanationalstandard,eitherbypublicat

15、ionofanidenticaltextor byendorsement,atthelatestbyMay2002,andconflictingnationalstandardsshallbewithdrawnatthelatestby May2002. ThisEuropeanStandardwaselaboratedonthebasisofaprotocoldevelopedfollowingaconcertedaction supportedbytheCommissionofEuropeanUnion(XIIC.)(BCR),andascreeninginvestigationcarri

16、edoutbythe DanishNationalFoodAgencyandthelocalEnvironmentandfoodAgency,MLKFYN,DKOdense.Expertsand laboratoriesfromE.U.andEFTAcountries,contributedjointlytothedevelopmentoftheconcertedactionprotocol. WARNING:Theuseofthisstandardmayinvolvehazardousmaterials,operationsandequipment.Thisstandard doesnotp

17、urporttoaddressallthesafetyproblemsassociatedwithitsuse.Itistheresponsibilityoftheuserofthis standardtoestablishappropriatesafetyandhealthpracticesanddeterminetheapplicabilityofregulatory limitationspriortouse. TheannexesA,BandCareinformative. ThisstandardincludesaBibliography. AccordingtotheCEN/CEN

18、ELECInternalRegulations,thenationalstandardsorganizationsofthefollowing countriesareboundtoimplementthisEuropeanStandard:Austria,Belgium,CzechRepublic,Denmark,Finland, France,Germany,Greece,Iceland,Ireland,Italy,Luxembourg,Netherlands,Norway,Portugal,Spain,Sweden, SwitzerlandandtheUnitedKingdom.EN13

19、783:2001(E) 3 1Scope ThisEuropeanStandardspecifiesamicrobiologicalscreeningmethodforthedetectionofirradiationtreatmentof herbsandspices,usingthecombineddirectepifluorescentfiltertechnique(DEFT)andaerobicplatecount(APC). TheDEFT/APCtechniqueisnotradiationspecific,therefore,itisrecommendedtoconfirmpos

20、itiveresultsusinga standardisedmethod(e.g.EN1788,prEN13751)tospecificallyproveanirradiationtreatmentofthesuspected food. Themethodhasbeensuccessfullytestedininterlaboratorytestswithherbsandspices1to5. 2 Normativereferences ThisEuropeanStandardincorporatesbydatedorundatedreference,provisionsfromother

21、publications.These normativereferencesarecitedattheappropriateplacesinthetext,andthepublicationsarelistedhereafter.For datedreferences,subsequentamendmentstoorrevisionsofanyofthesepublicationsapplytothisEuropean Standardonlywhenincorporatedinitbyamendmentorrevision.Forundatedreferencesthelatestediti

22、onofthe publicationreferredtoapplies(includingamendments). ISO4833,MicrobiologyGeneralguidancefortheenumerationofmicroorganismsColonycounttechniqueat 30C. ISO7218, MicrobiologyoffoodandanimalfeedingstuffsGeneralrulesformicrobiologicalexaminations. 3Principle ThemethodisbasedonthecomparisonoftheAPCwi

23、ththecountobtainedusingDEFT.TheAPCgivesthe numberofviablemicroorganismsinthesampleafterapossibleirradiationandtheDEFTcountindicatesthetotal numberofmicroorganisms,includingnonviablecells,presentinthesample.ThedifferencebetweentheDEFT countandtheAPCcountinspicestreatedwithdosesof5kGyto10kGyisgenerall

24、yaboutorabove3to4log units.SimilardifferencesbetweenDEFTandAPCcountscanbeinducedbyothertreatmentsofthefoodsleading todeathofmicroorganisms,e.g.heat,thuspositiveresultsshallbeconfirmed. Aknownvolumeofsampleisfilteredthroughamembranefilteratreducedpressureinordertoconcentratethe microorganismsonthefil

25、ter.Themicroorganismsarestainedwithafluorochrome,acridineorange(AO),resulting inanorangeandorangeyellowfluorescenceunderilluminationwithbluelightat450nmto490nm.These microorganismsarecountedusinganepifluorescencemicroscopetogivetheDEFTcount.However, microorganismswhichwerenonviablebeforeirradiations

26、howgreenfluorescenceandarenotcounted.Inparallel, theAPCisdeterminedfromasecondportionofthesametestsample6to10. 4Reagents 4.1General Duringtheanalysisuseonlyreagentsofrecognizedanalyticalgrade.AllthereagentsusedintheDEFTandAPC determinationsshouldbesterilizedbymembranefiltrationthrough0,2mporesizemem

27、branefiltersorby autoclaving. 4.2Peptonesalinediluent 4.2.1Composition Sodiumchloride 8,5g Peptone 1,0g Distilledordemineralizedwater 1000mlEN13783:2001(E) 4 4.2.2Preparation Dissolvethecomponentsinthewater.AdjustthepH,ifnecessary,sothataftersterilizationthefinalpHis7,20,2 at20Cto25C.Sterilizeinthea

28、utoclave(5.13)at121C1Cfor15min.Thediluentmaybestoredinaglass bottleat4Cto6Cfornotmorethantwoweeks. 4.3Buffer, pH3,0 4.3.1 Citricacidsolution, substanceconcentration c(C 6 H 8 O 7 H 2 O)=0,1mol/l 4.3.1.1Composition Citricacidmonohydrate 21g Distilledordemineralizedwater 1000ml 4.3.1.2Preparation Diss

29、olvethecitricacidmonohydrateinthewater.Thesolutionmaybestoredinasterilizedglassbottleat4Cto6 Cfornotmorethanthreemonths. 4.3.2 Sodiumhydroxidesolution, c(NaOH)=0,1mol/l 4.3.2.1Composition Sodiumhydroxide 4,0gor Sodiumhydroxidesolution,1mol/l 100ml Distilledordemineralizedwater 900ml 4.3.2.2Preparati

30、on Dissolvethesodiumhydroxideordilutethesodiumhydroxidesolutioninthewater.Thesolutionmaybestoredina glassbottleat4Cto6Cfornotmorethanthreemonths. 4.3.3 Completebuffer, pH3,0 4.3.3.1 Composition Citricacidsolution(4.3.1) 100ml Sodiumhydroxidesolution(4.3.2) 54ml 4.3.3.2Preparation Mixthecitricacidsol

31、utionandthesodiumhydroxidesolution.AdjustpHto3,00,2withcitricacidsolutionor sodiumhydroxidesolution.Sterilizethebufferthroughamembranefilterofporesize0,2m(5.3)beforeuse.The solutionmaybestoredinaglassbottleat4Cto6Cfornotmorethanthreeweeks.EN13783:2001(E) 5 4.4Acridineorangesolution 4.4.1 Buffersolut

32、ion,pH6,6 4.4.1.1Composition Citricacidsolution(4.3.1) 35,5ml Sodiumhydroxidesolution(4.3.2) 100ml 4.4.1.2Preparation Mixthecitricacidsolutionandthesodiumhydroxidesolution.AdjustpHto6,60,2withcitricacidsolutionor sodiumhydroxidesolution.Sterilizethebufferthroughamembranefilterofporesize0,2m(5.3)befo

33、reuse.The buffersolutionmaybestoredinaglassbottleat4Cto6Cfornotmorethanthreeweeks. 4.4.2 Completeacridineorangesolution 4.4.2.1Composition Acridineorange 0,025g Buffer,pH6,6(4.4.1) 100ml 4.4.2.2Preparation Dissolvetheacridineorangeinthebuffersolution(4.4.1).Sterilizetheacridineorangesolutionthrougha

34、0,2m poresizemembranefilter(5.3)beforeuse.Thesolutionmaybestoredinabrownglassbottleat4Cto6Cfor notmorethanoneweek. NOTE1Concentratedacridineorangesolutioniscommerciallyavailableandisrecommendedforsafetyreasons. NOTE2Asacridineorangeisregardedasamutagenicsubstance,disposableglovesandfacemasksshouldbe

35、usedwhen weighingthestain. 4.52Propanol 4.6TritonX100 1) ,1%cleaningsolution 4.6.1Composition TritonX100 10ml Distilledordemineralizedwater 1000ml 4.6.2Preparation MixTritonX100withwarm(80C)water.Sterilizethesolutionthrougha0,2mporesizemembranefilter(5.3). Thesolutionmaybestoredinaglassbottleat4Cto6

36、Cfornotmorethanthreeweeks. 1) TritonX100isanexampleofasuitableproductavailablecommercially.Thisinformationisonlygivenfortheconvenience ofusersofthisInternationalStandardanddoesnotconstituteanendorsementbyCENofthisproduct.EN13783:2001(E) 6 4.7TryptoneYeastExtractGlucoseAgar(Platecountagar) 4.7.1Compo

37、sition Tryptone 5,0g YeastExtract 2,5g Dextrose(Glucose) 1,0g Agar(accordingtothegelstrengthoftheagarused) 12gto18g Distilledordemineralizedwater 1000ml 4.7.2Preparation Dissolvethecomponentsordehydratedcompletemediuminthewaterwhileheatinguntilboiling.AdjustthepH,if necessary,sothataftersterilizatio

38、nthefinalpHis7,20,2at20Cto25C.Sterilizeintheautoclave(5.13)at 121C1Cfor15min. 5Apparatus UsuallaboratoryapparatusinaccordancewithISO7218and,inparticular,thefollowing: 5.1Apparatusformembranefiltrationofsamplesuspensions Filtrationequipmentmadeofstainlesssteelorglassshouldbeused.Thebottomfiltershould

39、beofsinteredglass orstainlesssteelintendedforfilterswithadiameterof25mm(5.5).Thefiltertowervolumeshouldbeatleast 10ml.Thefiltrationequipmentisplacedverticallyonasuctionflaskoramanifoldconnectedtoawaterpumpor vacuumpumpwithapressureregulator.Thevacuumduringfiltrationshouldusuallybeapproximately70kPa.

40、 NOTE Theuseofawaterpumpisnotverysuitableifthewaterpressurecannotberegulated. 5.2Filterfunnel andsuitablesuctionflasksmadeofglassforsterilefiltrationofreagentsanddiluent. 5.3Membranefilters, celluloseesterorsimilar,poresize0,2m,e.g.diameter30mmand/or47mm,forsterilefiltrationofreagents. 5.4Membranefi

41、lters, polypropylene,diameter25mm,poresize10m,forprefiltrationofsamples. 5.5Membranefilters, whitepolycarbonate,diameter25mm,poresize0,6mformembranefiltrationofsampletestsolution. 5.6Sterilefastfilterpaper, forfiltrationofspicesamples. 5.7Epifluorescencemicroscope, withsuitablelightandfiltercombinat

42、ion(450nmto490nm) 5.8Optics, 100ximmersionobjective,ocularwithmagnificationof10x.(Usingatubemagnificationof1,25atotal magnificationof1250xisachieved.) 5.9Microscopeslide, e.g.76mmx26mm.EN13783:2001(E) 7 5.10Coverslip, e.g.25mmx50mm,withathicknesscorrespondingtotherequirementsoftheobjective. 5.11 Imm

43、ersionoil, nonfluorescing.Refractiveindex1,515to1,518. 5.12Stagemicrometer, withgraduationsof0,01mm,formeasuringthediameterofthemicroscopefieldofview. 5.13Autoclave, forsterilizationofdiluentandculturemedium. 5.14Oven, fordryheatsterilizationofglassware. 5.15Whirlmixer, formixingsamplesuspensionanddiluent. 5.16Waterbath, formaintainingtheculturemediaattheappropriatetemperature. 5.17Incubator, capableofmaintainingatemperatureof30C1C. 5.18 Glassbo