ISO TS 04985-2023.docx

ISO/TSTECHNICA1.4985SPECIFICATIONIDF/RM255Firstedition2023-07Mi1.kandmi1.kproductsDeterminationofa1.ka1.inephosphataseactivityF1.uorimetricmicrop1.atemethodIS(Jff6icncc49H511htt(E)IDF/RM255：2023(E)COPYRIGHTPROTECTEDDOCUMENT©ISOandIDF2023Mrtf1.hreduscftvdi1.HikwjkNhCrWiminapviiiim1.>rbyqmFitttacmh8>>ni<w4cUMnbEdtne崛qniN911hi6cnpyip<bMMUntionmaytheinternetoranint11net1.withoutpriorwrittenpermission.PermissioncanberequestedInxncitherISOattheaddressbe1.oworISO'smemberbodyintheCoUntryOfthCrequester.fib包酬丽ndonnet8CH1214Vcmicr,GenevaPhone：*41227490111Emai1.：CoPyrh田EErgWebsitc八,.7,so.o11gsff1.Ht1.d三EaWM够UonReyerS70/BB1030Brusse1.sPhone：*3223256740Fax：*3223256741Emai1.：MEHWebsite：www.ft1.dfx>rgPub1.ishedinSwitzer1.and©ISOandIDF2023-A1.1.nghtsreservedContentsForewords1010.2.2Cheescsamp1.es.12AnnexB(informative)Methodcomparison8.1.3A1.ka1.inephosphatase-freecheese9.1.3Qua1.ity1.inearity-69.2.3Preparationoftheca1.ibrationstandard._.一.69.3.3Cheesesamp1.es8iii8.1.1Genera1.8.1.2PageScopeNormativereferences一.11Termsanddcnitions.1Princip1.e1Reagents.2Apparatusandmateria1.s3Samp1.ingPreparation一448.1PreparationOfa1.ka1.incphosphatase-freesamp1.e.448.2Preparationoftestsamp1.e»«««t«Mt(M(WtM(M(«(*Mt(««««Iteett55ProcedureS9.1Instrument9.1.1Instrumentsettings59.29.1.2Testthecontro1.Ca1.ibrationrangeoftheinstrument69.2.1Genera1.i*M(i21M*MM>4i*M*MMaMM2*9.3Determinationworkingcurveso1.utions79.3.1Mi1.ksamp1.es79.3.2P1.ate1.ayoutandf1.uorescencemeasurement9.4Testsamp1.e-re1.atedcontro1.s99.4.1Recommendednegativeandpositivecontro1.s9Ca1.cu1.ationandexpressionofresu1.ts1010.1 Ca1.ibrationcurve1010.2 Ca1.cu1.ationofA1.PactivityOfunknownsamp1.es11Mi1.k10.2.3Ca1.cu1.ationexamp1.e1310.3 Expressionofresu1.ts14Precision1411.1 Inter1.aboratorytest1411.2 2Repeatabi1.ity-14TestreportISAnnexA(informative)Precisiondata16Bib1.iography.21©ISOandIDF2023-A1.1.rightsreservedForewordsstandardsthrough(ISOISOmittees.workofPreparingbody1.ntemationa1.Standardsisnorma1.1.ynatterswork.e1.ectrotechnica1.StandardizationAheInternationa1.E1.ectrotechnica1.Commission(IEC)ona1.1.describedtypesofISO/IECDirectivesrShouIdbeParHCU1.ar,documentwasapprova1.accordancewiththeISO（八）patent(三).attcntion1.akcs1.hcnopossibiHtyconcerningi11p1.enentationvahdityapp1.icabi1.ityinvo1.vcc1.aimedcautioncdavai1.ab1.cWWW0iwg/PaUHKdatcstinformation,Whichresponsib1.eidentifyinganypatcntconstitutenameendorsement,documentisinformationgivenfortheconvenienceofusersanddoesnotexpressionsre1.ated。伸nization(WTO)assessment,asinwe1.1.asinfomationBaierstoTradeadherenceseeThisdocumen1.protucf5preparcdIntcrnationa1.ComrnitteeFcdcra1.ion(IDF).¾ocproductsSubcommitteebySC1.SOcomp1.eteIistingquestionshodiesthiscandocumentWwwTki<herg三effiberstH)natiQna1.standardsbody.AOandIDF2023-A1.1.11ghtsreservedISO(theInternationa1.OrganizationforStandardization)isawor1.dwidefederationofnationa1.carriedoutbodiesmemberbodies).TheEmitteehasbeenestab1.ishedhastherighttoberepresentedonthatcommittee.Internationa1.organizations,governmenta1.andnon-governmenta1.,in1.iaisonwithISO,a1.sotakepartintheofISOco1.1.aboratesc1.ose1.yvrithTheproceduresusedtodeve1.opthisdocumentandthoseintendedforitsfurthermaintenancearedifferentinthedocumentPart1.Innoted.ThiSthediffereddraftedincriteriaforeditoria1.ru1.esoftheISO/IECDirectives,Part2(seewww.iso.org/directives).vofdrawsISOtopositionthatthetheevidence,ofthisordocumentmayofanytheusepatentrightsinrespectthereof.Asofthedateofpub1.icationofthisdocument,ISOhadnotreceivednoticeof（八）PatCnt(三)whichmayberequiredtoimp1.ementthisdocument.However,imp1.ementorsaredatabasethatthisatmaythe.ISOsha1.1.behe1.dmayforobtainedfromtheora1.suchpatentrights.AnytradeusedinthisForanexp1.anationofthevo1.untarynatureOfstandards1.themeaningofISOspecifictermsandtheWor1.dTradeconformityprincip1.estheTechnica1.about1.SO*s(TBT)1towww.iso.org/iso/foreword.htm1.Mi1.kandmi1.kwas,andthebyTechnica1.DairyISO/TC34rItisbeing,pub1.ishedjoint1.y5,andIDF.Anyfeedbackoroftheseonbefoundatshou1.ddirecteduser's.1.fiF<(thHiMn1.MHCnaIStfihiiy)IderSFdd呷Ift1.Pn)IhItgiOb中，。位IpXU油ectnembe*sn曲agi1.&u&pmiahtiMtheCommittees,whicharenationa1.associationscomposedofrepresentativesofdairy-re1.atednationa1.interestgroupsinc1.udingdairyfanners,dairyprocessingindustry,dairysupp1.iers,academicsandgovernments/foodcontro1.authorities.ISOandIDFco1.1.aboratec1.ose1.yona1.1.mattersofstandardizationre1.atingtomethodsofana1.ysisandsamp)ingformi1.kandmi1.kproducts.Since2001,ISOandIDFjoint1.ypub1.ishtheirInternationa1.Standardsusingthe1.ogosandreferencenumbersofbothorganizations.Attentionisdrawntothepossibi1.itythatsomeofthee1.ementsofthisdocumentmaybethesubjectofpatentrights.IDFsha1.1.notbehe1.dresponsib1.eforidentifyinganyora1.1.suchpatentrights.Detai1.sof9时摘能41.M3西阳fiedd8曲式ec用迪甘制辑Hts).beintheIntroductionand/orAnytradenameusedinthisdocumentisinformationgivenfortheconvenienceofusersanddoesnotconstituteanendorsement.ThisdocumentwaspreparedbytheIDFStandingCommiUeeonAna1.ytica1.MethodsforProcessingAidsandIndicatorsandISOTechnica1.CommitteeISO/TC34,Foodproducts,SubcommitteeSC5,Mi1.kandmi1.kproducts.Itisbeingpub1.ishedjoint1.ybyISOandIDF.Theworkwascarriedoutbythe1DF1SOActionTeamonP20oftheStandingCommitteeonAna1.ytica1.MethodsforProcessingAidsandIndicatorsundertheaegisofitsproject1.eaderDrC.Egger(CH).TECHNICA1.SPECIFICATIONMi1.kandmi1.kproductsDeterminationofa1.ka1.inephosphataseactivityF1.uorimetricmicrop1.atemethod1 ScopeThisdocumentspecifiesaf1.uorimetricmicrop1.atemethodforthedeterminationofa1.ka1.inephosphatase(Addh,EC114ds11i1.1.0atyiciRriS.andheat-treatedwho1.emi1.k,semi-skimmedmi1.kskimmedmi1.k.Thismethodisapp1.icab1.etomi1.kandmi1.k-baseddrinksfromcows,sheepandgoats.A1.thoughthe用0蹴ii1.ar”阀曲tion11除价际Sh脚就躲&es需根%ucha1.sH1i1.嘟IiCab隔U利蝙IR8½ids.1.ciesapp1.icab1.etomi1.kpowderafterreconstitutionandsoft,semi-hardandhardcheesesprovidedthatthemou1.dison1.yonthesurfaceofthecheeseandnota1.sointhei11nrpart(e.g.b1.ueveinedcheeses).For1.argehardcheeses,specificconditionsofsamp1.ingapp1.y(seeC1.ause7).NOTEThismethodwasadaptedfromReference6.2 NormativereferencesTherearenonormativereferencesinthisdocument.3 TermsanddefinitionsForthepurposesofthisdocument,thefo1.1.owingtermsanddefinitionsapp1.y.ISOandIECmaintaintermino1.ogydatabasesforuseinstandardizationatthefo1.1.owingaddresses:ISOOn1.inebrowsingp1.atform:avai1.ab1.eathttps:/www.iso.org/obpX1.IECE1.ectropedia:avai1.ab1.eathttps:WWWe1.eCtTopediaQr"a1.ka1.inephosphataseactivityA1.Pactivityamountofenzymethatiscapab1.eofcata1.ysingthetransformationof1rno1.ofsubstrateperminuteundertheconditionsofthespecifiedprocedurehkd出*owhyutrfFttdnzymeactiwynHkratiwppte<<dUgi1.iuiMt6eo<i冲QKSeJctiVityper1.itreofsamp1.e(mUi)4 Princip1.eThe1.Pactivityofthesamp1.eismeasuredbyacontinuousf1.uorimetricdirectkineticassay.non-ne8centa11wnftikJhrtH而Pid>bphoItheSmHtPhUvndEghyiihxtiZ好eR>neitsphosphate,ProdutiNH1.ftPhigh1.yf1.uorescentproduct4-methy1.umbe1.1.iferone(4-MU),F1.uorimetricmeasurementofA1.Pactivityismeasuredat37overa15minperiod.ffihemeaturedca1.cu1.ate1.tboEuuyai)a?aGtpndfu)rtiona1.totheconcentrationoftheemittedf1.uorescentproductand©ISOandIDF2023-A1.1.11ghtsreserved5 ReagentsUseon1.yreagentsofrecognizedana1.ytica1.grade.5.1Magnesiumch1.orideso1.ution,substanceconcentration,c(MgC1.z)=1mo1.1.Weigh50,8gofmagnewuwch1.oridehexahydrate(MgC1.z6HzO1.mo1.ecu1.armassis203,3gmo1.)inW0AQbEbeake(<d1.ia)nevAidis>totfeh11iiedwaterandtransferinavo1.umetricf1.askof250m1.Thisso1.utioncanbestoredina1.iquots(e.g.10m1.)atbe1.ow-20foroneyear.5.2Tergito1.iM15S9(CASRegistryNunberi)84133-50-6)2)so1.ution,c=100g1.Weigh25r0gOfTergito1.TM15-S-9ina150m1.g1.assbeaker(6.17)rdisso1.veby-airringgent1.ywithdjatdhMtQtent2S.0C±2c,Candtransferinavo1.umetricf1.askof250m1.(6.18).Adjustthevo1.umewithThisso1.utioncanbestoredina1.iquots(e.g.15m1.)atatemperaturebetween2and8foroneyear.5.32-amino-2-methy1.-1.-propano1.(MP)bufferso1.ution,c=0,11mo1.1.,pH=10,1.Weigh9,8gofAMP,purity>95%fmo1.ecu1.armassis89,14gmo1.rina600m1.g1.assbeaker(6.17),cHd1.)5Q05mVHKtHhfrfdrVHert00ektirwo1umrriia曲MK1.tejnkDytitHjastwithy<irqrh1.orixHnhk51.utionThisso1.utioncanbestoredina1.iquots(e.g,30m1.)atbe1.ow-20foroneyear.5.4Diethano1.amine(DEA)bufferso1.ution,<?=2rno1.1.,pH=9,8,c(Mg2)=0,5mmo1.1.Weigh210,3gofDEA,mo1.ecu1.armassis105r14gmo1.,ina1000m1.g1.assbeaker(6.17),add700m1.of口adiD四mteMgb1.teskihrtoB0而d街1皿四疝im(f1.WRb皿tajrfrirfUWjd±嫌hKaj6wh(mo1.1.uptothemark.Thisso1.utioncanbestoredina1.iquots(e.g.50m1.)atbe1.ow-20foroneyear.DEAextractionbuffer,c=1,511o1.1.,p1.i=9.8,c(Mg2)=1,5mmo1.1.C(TCrgitOITM)=Or1%.Weigh157,7gofDEAina600m1.g1.assbeaker(6.17),add500m1.ofdisti1.1.edwateronastirrerandadjust(HUuoHVdrtb1.owotBiS-9so1.utionc(而以=T1.m<nHKhShfi0iyum*1.u8netricof1.tU0ndadjustwithwateruptothemark.TNsso1.utioncanbestoredina1.iquots(e.g.200m1.)atbe1.ow-20foroneyear.4-nethy1.unbc11iferonesodiumsa1.t(4-MU)standardstockso1.ution,c=2,5mmo1.1.Weigh9,9mgof4MU.purity>98%,mo1.ecu1.armassnU8,16gmo1.,ina25m1.g1.assbeaker(6.1SaiifinIbpMdhinsemtfriHwiththetheAWftbuffer.KdeponscOftf1.dnX1.teahHfhrtoavo1.umetricf1.askof20m1.¼48)ThiS铀超嘛轴OnAWi僧氏&鹤也战朝的HfWSfttrthWW三-knftf服§。WMff1.%teChdHiiC翁日旅业膜鼠byISOoftheproductnamed.Equiva1.entproductsmaybeusediftheycanbeshownto1.eadtothesameresu1.ts.2,1.iJ涮豳咻Bfand蝴膻Pr蜩ituteaM触1.三瞅幌欹Thi端rn&游嗣8硒糊他姓enienceproductsmaybeusedIftheycanbeshownto1.eadcothesameresu1.ts.QISOandIDF2023-A1.1.11ghtsreservedThisso1.utioncanbestoredina1.iquots(c.g.2001.)atbe1.ow-20foroneyear.5.74nethy1.unbc1.1.iferonephosphate(4MUP)substrateso1.ution,c=2,5nmo1.1.Weigh12,8mgof4MUP,mo1.ecu1.arrnass-te.256,15gmo1.rina25m1.g1.assbeaker(6.1.2)ro1.ubiIizcandthtsemxkwiththesa11OBffer.so1.ution(5.4)rtransferinavo1.umetricf1.askof20m1.(6.18)andfi1.1.uptoPreparetheso1.utionfresh1.yandkeepinthedark.6Apparatusandmateria1.sUsua1.1.aboratoryequipmentand,inparticu1.ar,thefo1.1.owingsha1.1.beused.6.1F1.uorescencemicrop1.ateofreadingexcitatiowax,denphemis:37±1anda1.)ovringkineticmeasurements(e.g.onereadingperminutefor15min).6.2Microp1.atesrb1.ack,f1.atbottom,96we1.1.6.3PIpettefofcapacity201.to1001.6.4Pipette,ofcapacity1001.to10001.6.5Mu1.tichanne1.pipettor,capab1.eOfdispensing201.to1001.6.6Airdisp1.acementpipetterofcapacity5m1.Ag1.asspipettecana1.sobeused.6.7Microtubes,ofcapacity2m1.6.8Tubes,ofcapacity15m1.and50m1.6.9G1.asstesttube,ofapproximate1.ydiameter12mmand1.ength10cm.6.10 Waterbath,heatingb1.ockorincubatorsuitab1.eofmaintainingatemperatureof37±1,631XVon忌JC±1.613P¼AteMuM*f<)i1.othersuitab1.e1.aboratory-gradefi1.m.6.14 Grindingdevice.6.15 U1.TRATURRAX®4),OrOthCrhomogenizerprovidedwithasternofdiameterofapproximate1.y6mmto8mm.-多处11nttimMedo<?Othen凶n<tab1.edoH<udiRtQtta业IHR1.i1.f1.IXUnn)nnhs)1.1.yThis1.30fformatojiMproduct.forthe4) U1.TRA-TURRAXnisanexamp1.eofasuitab1.eproductavai1.ab1.ecommercia1.1.y.ThisinformationisgivenfortheconvenienceofusersofthisdocmentanddoesnotconstituteanendorsementbyISOorIDFofthisproduct.©ISOandIDF2023-A1.1.rightsreserved1.16 Centrifuge,capab1.eofcentrifugingat1OOOgat4for10m1.or15m1.and50m1.tubes.1.17 G1.assbeaker,ofcapacity5m1.(approximate1.ydiameter20mmand1.ength30mm),10m1.(ofapproximate1.ydiameter25mmand1.ength30min),25m1.,50m1.,150m1.,600m1.and1000m1.1.18 One-markvo1.umetricf1.asks,ofcapacity20m1.,25m1.,250m1.and1000m1.1.19 Ana1.ytica1.ba1.ance.1.20 pHmeter.7 Samp1.ingArepresentativesamp1.eshou1.dhavebeensenttothe1.aboratory.Itshou1.dnothavebeendamagedorchangedduringtransportorstorage.Samp1.ingisnotpartofthmethodspecifiedinthisdocument.Arecommendedsamp1.ingmethodisgiveninISO707IDF50.hwW(W<蚓Ch«t««hhrdCheeSMAhS6瞧,解bftrjthMxturehashomogenous1.ydistributedwithinthesecheeses.Theactivityishighintheouter1.ayerofthecheesewhee1.,between0cmto4Cmbe1.owtherindoftheroundside,butVery1.oworevenundetectab1.einthecore.Samp1.esof1.argehardcheeses,therefore,1.a1.1.besamp1.edbytakingaportionof1cm,takenat0,5cmbe1.owtherindoftheroundside(seeFigureC.1).11piftsdoubtdesrwrtii)fgaghdiweses.betweenahardandasemi-hardcheese,proceedtothe8 Preparation8.1 Preparationofa1.ka1.inephosphatase-freesamp1.e8.1.1 Genera1.The1.P-freesamp1.eisusedassamp1.eb1.ank,forca1.ibrationandforsamp1.edi1.utionifnecessary.8.1.2 A1.ka1.inephosphatase-freemi1.kPreparephosphatase-freemi1.kofthetypetobetestedbycarefu1.1.ydispensingthedesiredportiononiier.intofiovriesttubeOrOEIIttntdinCTIaHitttjrJinge她UringmiIkthatPOrtionndIB1.ftredhMtherimwatIhatHeSOftIrthetheheatingb1.ock(6.10)setat95.Preheatthemi1.kportionto95,beforestartingits5minheatingperiodatthattemperature.Checkthetemperaturebyusingathermometerorthermistorprobep1.acedheatin龈隔也隘HWWh腺朗棚怫册网住浏的陆帕电斶明明豌immediate1.ystartits5min8.1.3 A1.ka1.inephosphatase-freecheeseForeachtypU4eesetobetested,prepareaphosphatase-freecheesefromthesupernatantoftheCheeSCSHfr候疝A)byheatingaportionofthesupernatantasdescribedin&.1.2,rep1.acingthemi1.kby8.2 Preparationoftestsamp1.e8.2.1 Mi1.ksamp1.es8.2.1.1 Genera1.Carefu1.1.ymixa1.1.testsamp1.espriortouse.NOTEItisusua1.1.ynotnecessarytoprewa11ntestsamp1.es.8.2.1.2 Pasteurizedtestsamp1.esUsepasteurizedtestsamp1.esasde1.ivered,inamountsasrequired.8.2.1.3 Di1.utionoftestsamp1.eswithhighA1.Pva1.uesPreparedi1.utionsofthetestsamp1.esofmi1.kusingPhoSPhataSe-fr4WiIk(see8.1.2)inordertobring§?2!2A1.PCritiifesthe1.inearityrangeoftheinstrument(see9.1.2).Mixthedi1.utedso1.utionswe1.1.8.2.2.1 Genera1.Removetherindorthesurfacefromthetestsamp1.ewithadeanknife.Ensurethatthetestsamp1.eiR<k1.edc6utiM(Ua1.CdreViChai1.扯Ifamindn1.tdn>f1.to)shR9崎dRppPicj0Qn.ESqmvtHHy的。MiChhEfth*tfatwith1.ayerunderthemou1.dsurface(seeAnnexC).For1.argehardcheeses,proceedas-describedinC1.ause7.Grindthetestsamp1.ebymeansofagrindingmi1.1.orotherappropriatedevice(6.14)andmixthorough1.y.Keepthepreparedsamp1.einanairtightcontainer.8.2.2.2 Di1.utionoftestsamp1.eswithhighA1.Pva1.uesPrepaMM1.i1.utionsofthesupernatantofthecheesesamp1.es(see9.3.2.6)usingphosphatasc-frue-dwcse(bedSi1.ddsOhdtoiissbeihgtheir