1、BRITISH STANDARD BS EN 14131:2003 Foodstuffs Determination of folate by microbiological assay The European Standard EN 14131:2003 has the status of a British Standard ICS 07.100.30 BS EN 14131:2003 This British Standard, was published under the authority of the Standards Policy and Strategy Committe
2、e on 12 June 2003 BSI 12 June 2003 ISBN 0 580 42040 X National foreword This British Standard is the official English language version of EN 14131:2003. The UK participation in its preparation was entrusted to Technical Committee AW/-/3, Horizontal analysis, which has the responsibility to: A list o
3、f organizations represented on this committee can be obtained on request to its secretary. Cross-references The British Standards which implement international or European publications referred to in this document may be found in the BSI Standards Catalogue under the section entitled “International
4、Standards Correspondence Index”, or by using the “Find” facility of the BSI Standards Electronic Catalogue. A British Standard does not purport to include all the necessary provisions of a contract. Users of British Standards are responsible for their correct application. Compliance with a British S
5、tandard does not of itself confer immunity from legal obligations. aid enquirers to understand the text; present to the responsible European committee any enquiries on the interpretation, or proposals for change, and keep the UK interests informed; monitor related international and European developm
6、ents 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 19 and a back cover. The BSI copyright date displayed in this document indicates when the document was last issued. Amendments issued since publication Amd
7、. No. Date CommentsEUROPEANSTANDARD NORMEEUROPENNE EUROPISCHENORM EN14131 June2003 ICS07.100.30 Englishversion FoodstuffsDeterminationoffolatebymicrobiologicalassay ProduitsalimentairesDterminationdesfolatesparessai microbiologique LebensmittelMikrobiologischeBestimmungvonFolat ThisEuropeanStandardw
8、asapprovedbyCENon21April2003. CENmembersareboundtocomplywiththeCEN/CENELECInternalRegulationswhichstipulatetheconditionsforgivingthisEurope an Standardthestatusofanationalstandardwithoutanyalteration.Uptodatelistsandbibliographicalreferencesconcernings uchnational standardsmaybeobtainedonapplication
9、totheManagementCentreortoanyCENmember. ThisEuropeanStandardexistsinthreeofficialversions(English,French,German).Aversioninanyotherlanguagemadebytra nslation undertheresponsibilityofaCENmemberintoitsownlanguageandnotifiedtotheManagementCentrehasthesamestatusasthe official versions. CENmembersarethena
10、tionalstandardsbodiesofAustria,Belgium,CzechRepublic,Denmark,Finland,France,Germany,Greece, Hungary,Iceland,Ireland,Italy,Luxembourg,Malta,Netherlands,Norway,Portugal,Slovakia,Spain,Sweden,SwitzerlandandUn ited Kingdom. EUROPEANCOMMITTEEFORSTANDARDIZATION COMITEUROPENDENORMALISATION EUROPISCHESKOMIT
11、EEFRNORMUNG ManagementCentre:ruedeStassart,36B1050Brussels 2003CEN Allrightsofexploitationinanyformandbyanymeansreserved worldwideforCENnationalMembers. Ref.No.EN14131:2003EEN14131:2003(E) 2 Contents page Foreword. 3 1 Scope . 3 2 Normativereferences . 3 3 Principle. 3 4 Reagents 4 5 Apparatus 8 6 P
12、rocedure 9 7 Calculation. 13 8 Precision 14 9 Testreport . 14 AnnexA (informative) Optionalenzymetreatment. 16 AnnexB (informative) Resultsofinterlaboratorytests 19 Bibliography . 20EN14131:2003(E) 3 Foreword Thisdocument(EN14131:2003)hasbeenpreparedbyTechnicalCommitteeCEN/TC275“Foodanalysis Horizon
13、talmethods”,thesecretariatofwhichisheldbyDIN. ThisEuropeanStandardshallbegiventhestatusofanationalstandard,eitherbypublicationofanidentical textorbyendorsement,atthelatestbyDecember2003,andconflictingnationalstandardsshallbewithdrawn atthelatestbyDecember2003. AnnexesAandBareinformative. Accordingto
14、theCEN/CENELECInternalRegulations,thenationalstandardsorganizationsofthefollowing countriesareboundtoimplementthisEuropeanStandard:Austria,Belgium,CzechRepublic,Denmark, Finland,France,Germany,Greece,Hungary,Iceland,Ireland,Italy,Luxembourg,Malta,Netherlands, Norway,Portugal,Slovakia,Spain,Sweden,Sw
15、itzerlandandtheUnitedKingdom. 1Scope ThisEuropeanStandardspecifiesamicrobiologicalmethodforthedeterminationofthetotalfolatecontentof foodstuffsbyturbidimetricdetectionofthegrowthofthemicroorganism Lactobacilluscasei, subsp. rhamnosus(ATCC7469). Themethodallowsforthedeterminationoffolatesinfoodstuffs
16、,includingnaturallyoccurringfolatesand addedfolicacid(pteroylglutamicacid). 2 Normativereferences ThisEuropeanStandardincorporatesbydatedorundatedreference,provisionsfromotherpublications. Thesenormativereferencesarecitedattheappropriateplacesinthetextandthepublicationsarelisted hereafter.Fordatedre
17、ferences,subsequentamendmentstoorrevisionsofanyofthesepublicationsapplyto thisEuropeanStandardonlywhenincorporatedinitbyamendmentorrevision.Forundatedreferencesthe latesteditionofthepublicationreferredtoapplies(includingamendments). ENISO3696, WaterforanalyticallaboratoryuseSpecificationsandtestmeth
18、ods(ISO3696:1987). 3Principle treatmentmaybeusedtofurtherdigestthefoodmatrix.Naturallyoccurringfolylpolyglutamatesare 1tofolylmonoorfolyldiglutamates. Extractedfolatesaredilutedwithbasalmediumcontainingallrequiredgrowthnutrientsexceptfolate.The growthresponseof Lactobacilluscasei ,subsp. rhamnosus(A
19、TCC7469)toextractedfolatesisfollowed turbidimetricallyandiscomparedtothegrowthresponsetocalibrantsolutionswithknownconcentration. Themethodallowsfortheoptionaluseofasemiautomatedliquidhandlingsystemandofamicroplateortest tubesforincubationofthemicroorganism.EN14131:2003(E) 4 4Reagents 4.1General Dur
20、inganalysis,unlessotherwisestated,useonlyreagentsofrecognisedanalyticalgradeandwaterofat leastgrade1asdefinedinENISO3696.Thewaterusedforreagentpreparationshallbeglassdistilled. 4.2Solventsandchemicals 4.2.1Glycerol, w(C 3 H 8 O 3 )=80% Mix120mlofglycerolwith30mlofglassdistilledwater. 4.2.21Octanol,C
21、 8 H 18 O 4.2.3Toluene, C 7 H 8 4.2.42Mercaptoethanol,c(C 2 H 6 OS)=0,1mol/l Add70lof2mercaptoethanolto10mlofwater. 4.2.5 Sodiumascorbate, C 6 H 7 O 6 Na Sodiumascorbateisusedasareagentinseveralsolutionsspecifiedinthisdraftstandard.Ascorbicacidmay equallywellbeused,butproceduresforpHadjustmentmaynee
22、dtobemodified. 4.2.6 Hydrochloricacid, c(HCl)=1mol/l 4.2.7 Sodiumhydroxide, w(NaOH)=40% Dissolve400gofsodiumhydroxideinwateranddiluteto1l. 4.2.8 Ammoniumsulfate, H 8 N 2 O 4 S 4.2.9Sodium phosphate, monobasic,anhydrous,NaH 2 PO 4 Theamountsofmonobasicsodiumphosphateusedforbufferpreparation(4.3)haveb
23、eencalculatedforthe anhydroussubstance.Themonoordihydratedsubstancemayalsobeused,withtheproceduresadjusted accordingly. 4.2.10 2(NCyclohexylamino)ethanesulfonicacid(CHES), C 8 H 17 NO 3 S 4.2.11 N(2Hydroxyethyl)piperazineN(2ethanesulfonicacid )(HEPES), C 8 H 18 N 2 O 4 S 4.2.12Carbonpowder, acidwash
24、ed 4.2.13Saline, sterile Dissolve9gofsodiumchloridein1000mlofwater.Dispense10mlportionsinto20mmx150mmtesttubes. Captubesandheatat+121Cfor15min.Coolandstorerefrigerated.EN14131:2003(E) 5 4.2.14 Folicacidfreebasalmediumsolution, doublestrength Foreach100mlneeded,suspendtherecommendedamountofbasalmediu
25、m(BactoFolicAcidCasei Mediumorequivalent 1) )in100mlofglassdistilledwater.Add0,050gofsodiumascorbate(4.2.5)andheatto boilingfor1minto2min.AllowcoolingtoroomtemperatureandadjustpHto6,10,1. 4.2.15 Folicacidstandardsubstance Folicacidcanbeobtainedfromvarioussuppliersandmaycontainupto8%water.Thepurityof
26、thefolicacid standardmayvaryanditisthereforenecessarytodeterminetheconcentrationofthecalibrationsolutionby UVabsorptionmeasurement(seeprocedureforstandardisationin6.4.2). 4.3Buffers 4.3.1 Phosphatebuffer, pH5,0( c=0,002mol/l) Dissolve0,24gofsodiumphosphate,monobasic(4.2.9)in900mlofwater.AdjustpHto5,
27、00,1with sodiumhydroxide(4.2.7)anddiluteto1000mlwithwater. 4.3.2 Phosphatebuffer, pH7,0( c=0,1mol/l) Dissolve12,0gofsodiumphosphate,monobasic(4.2.9)in900mlofwater.AdjustpHto7,00,1with sodiumhydroxide(4.2.7)anddiluteto1000mlwithwater. 4.3.3 Phosphatebuffer, pH5,0( c=0,1mol/l)with2mercaptoethanol( c=1
28、0mmol/l) Dissolve12,0gofsodiumphosphate,monobasic(4.2.9)in900mlofwater.AdjustpHto5,00,1,add 0,70mlof2mercaptoethanol(4.2.4)anddiluteto1000mlwithwater. 4.3.4 Phosphatebuffer, pH4,5( c=0,1mol/l)withascorbate(1%) Dissolve12,0gofsodiumphosphate,monobasic(4.2.9)and10gofsodiumascorbate(4.2.5)in900mlof wat
29、er.AdjustpHto4,50,1withsodiumhydroxide(4.2.7)anddiluteto1000mlwithwater.Preparefreshon dayofuse. 4.3.5 Phosphatebuffer, pH6,1( c=0,1mol/l)withascorbate(1%) Dissolve12,0gofsodiumphosphate,monobasic(4.2.9)and10gofsodiumascorbate(4.2.5)in900mlof water.AdjustpHto6,10,1withsodiumhydroxide(4.2.7)anddilute
30、to1000mlwithwater.Preparefreshon dayofuse. 4.3.6 Phosphatebuffer, pH7,8( c=0,1mol/l)withascorbate(1%) Dissolve12,0gofsodiumphosphate,monobasic(4.2.9)and10gofsodiumascorbate(4.2.5)in900mlof water.AdjustpHto7,80,1withsodiumhydroxide(4.2.7)anddiluteto1000mlwithwater.Preparefreshon dayofuse. 1) BactoFol
31、icAcidCaseiMediumisthetradenameofaproductsuppliedbyDifco.Thisinformationisgivenforthe convenienceofusersofthisEuropeanStandardanddoesnotconstituteanendorsementbyCENoftheproductnamed. Equivalentproductsmaybeusediftheycanbeshowntoleadtothesameresults.EN14131:2003(E) 6 4.3.7CHES/HEPES buffer, pH7,85( c
32、=0,05mol/l)withascorbateand2mercaptoethanol(4.2.4)forthe dialysisofratbloodplasma. Dissolve23,8gofHEPES(4.2.11),20,7gofCHES(4.2.10),40gofsodiumascorbate(4.2.5)and1,4mlof 2mercaptoethanol(4.2.4)in1900mlofwater.AdjustpHto7,850,1withsodiumhydroxide(4.2.7)and diluteto2000mlwithwater.Add4gofacidwashedcar
33、bonpowder(4.2.12).Preparefreshondayofuse. 4.4Enzymes 4.4.1 Additionalenzymetreatment(optional) ProceduresforadditionalenzymetreatmentarefurtherdiscussedinAnnexA. 4.4.2 4.4.2.1General 0fromoneofseveralsources.An 4.4.2.2). A. The appropriatenessofthechosenenzymepreparationshallbecheckedwithasuitablete
34、chnique. NOTE Ayeastpowder,lyophilisedpigsliver(e.g.BCRCRM4872),ortoalimitedextentpteroyltriglutamicacidcan beappropriatesamplestouseforthecheckingoftheenzymepreparation. 4.4.2.2 3 Homogenise250goffreshhogkidneyat+2Cin750mlofphosphatebufferwith2mercaptoethanol(4.3.3). Centrifugeat+2C(18000 g,20min).
35、Incubatesupernatantat+50Cfor2hwithgentleagitationand repeatcentrifugation.Fractionatethesupernatantbyprecipitationwithsaturatedammoniumsulfate(4.2.8). Collectthefractionprecipitatedbetween50%and75%saturationwithammoniumsulfate.Suspend precipitateinaminimalvolumeofphosphatebuffer(4.3.1).Dialyseagains
36、tthesamebuffer2x24hand centrifuge(18000 g,20min).Transfer1mlaliquotstovialsandlyophilise. 4.5Inoculum 4.5.1 Testorganism Lyophilisedcultureof Lactobacilluscasei subsp. rhamnosus(ATCC7469) 2) . 4.5.2 Culturemedium Dilute50mlofdoublestrengthfolicacidfreebasalmediumsolution(4.2.14)to100mlwithglassdisti
37、lled water.Add0,5mlofdilutedfolicacidstocksolution(6.4.4),mixandsterilefilterorheatat+121Cfor15min andrapidlycooltoroomtemperature. 4.5.3 Cryoprotectedinoculum Aseptically,add1mlofthepreparedculturemedium(4.5.2)tothelyophilisedculture(4.5.1)andtransfer 0,15mloftheresultingsuspensiontotheculturemediu
38、maccordingto4.5.2.Incubateat+37Cfor18h. 2) DistributorsincludeNationalCollectionofIndustrialandMarineBacteriaLtd(Aberdeen,UK)andCultureCollection, UniversityofGteborg(Gothenburg,Sweden).ThisinformationisgivenfortheconvenienceofusersofthisEuropean StandardanddoesnotconstituteanendorsementbyCENofthedi
39、stributorsnamed.EN14131:2003(E) 7 Heat150mlofglycerol(4.2.1)at+121Cfor15minandcoolinicebath.Coolincubatedbacterialculturein icebathandadd100mlsterilisedandcooledglycerol.Mixgently.Dispense2mlaliquotsintosterilevials. Storeat20Cforuptothreemonthsorat70Cforuptosixmonths. NOTE Itisessentialtomaintainas
40、epticconditionsthroughoutthewholeprocess. 4.5.4 Workinginoculum 4.5.4.1 Workinginoculumfortubecultures Dilute2mlcryoprotectedinoculum(4.5.3)to50mlwithsterilesaline(4.2.13).Vortexmix. 4.5.4.2 Workinginoculumformicroplatecultures(optional) Add5mlofsterilesaline (4.2.13)to2mlcryoprotectedinoculum(4.5.3
41、).Vortexmix. 4.5.5 Inoculatedfolicacidfreebasalmediumsolution, formicroplateassay(optional). Add1lworkinginoculum(4.5.4.2)per1mlfolicacidfreebasalmediumsolution(4.2.14).Mixthoroughly. 5Apparatus Usuallaboratoryapparatus,glasswareand,inparticular,thefollowing: 5.1Centrifuge,cooled, forpreparationofhogkidneyconjugase(4.4.2.2),suitabl
