1、A Referencenumbe r ISO13965:1998 (E) INTERNATIONAL STANDARD ISO 13965 Firstedition 19980915 MeatandmeatproductsDetermination ofstarchandglucosecontents Enzymaticmethod ViandeetproduitsbasedeviandeDterminationdesteneursen amidonetenglucoseMthodeenzymatiqueISO13965:1998(E) ISO1998 Allrightsreserved.Un
2、lessotherwisespecified,nopartofthispublicationmaybereproduced orutilizedinanyformorbyanymeans,electronicormechanical,includingphotocopyingand microfilm,withoutpermissioninwritingfromthepublisher. InternationalOrganizationforStandardization Casepostale56 CH1211Genve20 Switzerland Internet isoiso.ch P
3、rintedinSwitzerland ii Foreword ISO(theInternationalOrganizationforStandardization)isaworldwide federationofnationalstandardsbodies(ISOmemberbodies).Theworkof preparingInternationalStandardsisnormallycarriedoutthroughISO technicalcommittees.Eachmemberbodyinterestedinasubjectforwhich atechnicalcommit
4、teehasbeenestablishedhastherighttoberepresented onthatcommittee.Internationalorganizations,governmentalandnon governmental,inliaisonwithISO,alsotakepartinthework.ISO collaboratescloselywiththeInternationalElectrotechnicalCommission (IEC)onallmattersofelectrotechnicalstandardization. DraftInternation
5、alStandardsadoptedbythetechnicalcommitteesare circulatedtothememberbodiesforvoting.PublicationasanInternational Standardrequiresapprovalbyatleast75%ofthememberbodiescasting avote. InternationalStandardISO13965waspreparedbyTechnicalCommittee ISO/TC34, Agriculturalfoodproducts ,SubcommitteeSC6, Meatan
6、dmeat products. AnnexesAandBofthisInternationalStandardareforinformationonly.INTERNATIONALSTANDARD ISO ISO13965:1998(E) 1 MeatandmeatproductsDeterminationofstarchand glucosecontentsEnzymaticmethod 1 Scope ThisInternationalStandardspecifiesanenzymaticmethodforthedeterminationofwaterfreestarchcontenta
7、nd glucosecontentofallkindsofmeatandmeatproducts,includingpoultry. Themethodissuitableforthequantitativedeterminationofstarchandglucosecontentsdowntolevelsof 0,30%( m/m). Themethodisnotapplicableforchemicallymodifiedstarchesortheirderivatives. 2 Normativereference Thefollowingstandardcontainsprovisi
8、onswhich,throughreferenceinthistext,constituteprovisionsofthis InternationalStandard.Atthetimeofpublication,theeditionindicatedwasvalid.Allstandardsaresubjectto revision,andpartiestoagreementsbasedonthisInternationalStandardareencouragedtoinvestigatethe possibilityofapplyingthemostrecenteditionofthe
9、standardindicatedbelow.MembersofIECandISOmaintain registersofcurrentlyvalidInternationalStandards. ISO3696:1987, WaterforanalyticallaboratoryuseSpecificationandtestmethods. 3 Definitions ForthepurposesofthisInternationalStandard,thefollowingdefinitionsapply. 3.1 starchcontentofmeatandmeatproducts st
10、archcontentdeterminedinaccordancewiththeproceduredescribedinthisInternationalStandard,and expressedasapercentagebymass 3.2 glucosecontentofmeatandmeatproducts glucosecontentdeterminedinaccordancewiththeproceduredescribedinthisInternationalStandard,and expressedasapercentagebymass 4 Principle 4.1 Hyd
11、rolysisofthestarchpresentinatestportionwiththeenzyme aamylase at pH=5,0 for 15min. Determinationofthestarchcontentusingthefollowingenzymaticreactions.ISO13965:1998(E) ISO 2 4.2 Hydrolysisofthesolubilizedstarchtoyieldglucoseusingamyloglucosidase(AGS): starch+( n21)H 2 O glucose Forthedeterminationoft
12、heglucosecontent,thisstepisomitted. 4.3 Phosphorylationoftheglucosegeneratedbymeansofadenosine5triphosphate(ATP)toyieldglucose 6phosphate(G6P)usinghexokinase(HK): glucose+ATP glucose6phosphate+ADP 4.4 Oxidationofglucose6phosphate(G6P)bymeansofnicotinamideadeninedinucleotidephosphate (NADP)togluconat
13、e6phosphateusingglucose6phosphatedehydrogenase(G6PDH): glucose6phosphate+NADP + gluconate6phosphate+NADPH+H + 4.5 Spectrometricmeasurementoftheamountofreducednicotinamidedinucleotidephosphate(NADPH)ata wavelengthof340nm. 5 Reagents Useonlyreagentsofrecognizedanalyticalgrade,unlessotherwisespecified.
14、 5.1 Water,complyingwithatleastgrade3inaccordancewithISO3696. 5.2 aAmylase(EC3.2.1.1), enzymesuspension. Aliquidenzymepreparationofaheatstable aamylaseproducedfrom Bacilluslicheniformis (Termamyl120L) 1) . 5.3 Sodiumhydroxidesolution, c(NaOH)=5mol/l. Dissolve200gofsodiumhydroxideinwater.Cooltoroomte
15、mperature,diluteto1000mlandmix. 5.4 Sodiumhydroxidesolution, c(NaOH)=0,5mol/l. Dissolve20gofsodiumhydroxideinwater.Cooltoroomtemperature,diluteto1000mlandmix. 5.5 Ammoniumsulfatesolution, c(NH 4 ) 2 SO 4 =3,2mol/l. Dissolve422gofammoniumsulfateinwater.Diluteto1000mlandmix. 5.6 Acetatebuffer, c(CH 3
16、CO 2 Na)=0,1mol/l,pH=5,0. Dissolve6,80gofsodiumacetatetrihydrate(CH 3 CO 2 Na3H 2 O)in400mlofwater.AdjustthepHto5,0with hydrochloricacidorsodiumhydroxidesolutionwithapHmeter(6.2).Dilutewithwaterto500mlandmix. Thesolutionisstableforatleast3monthsat+4Cinthedark. 5.7 Citratebuffer, c(citrate)=0,05mol/l
17、,pH=4,6. Dissolve440 mgofcitricacidmonohydrate(C 6 H 8 O 7 H 2 O)and850 mgoftrisodiumcitratedihydrate (C 6 H 5 Na 3 O 7 2H 2 O)inwater.Dilutewithwaterto100mlandmix.CheckthepHwithapHmeter(6.2)andadjustif necessarywithhydrochloricacidorsodiumhydroxidesolution. Thesolutionisstableforatleast3monthsat+4C
18、inthedark. 1)Termamyl120LisanexampleofasuitableproductavailablecommerciallyfromNovo,Denmark,andTecator,Sweden. ThisinformationisgivenfortheconvenienceofusersofthisInternationalStandardanddoesnotconstituteanendorsementby ISOofthisproduct.ISO ISO13965:1998(E) 3 5.8 Triethanolaminebuffer, c(triethanola
19、mine)=0,75mol/l,pH=7,6. Dissolve14,0goftriethanolaminehydrochloride(C 6 H 15 NO 3 HCl)and0,25gofmagnesiumsulfateheptahydrate (MgSO 4 7H 2 O)in80mlofwater.SetthepHat7,6withapHmeter,usingthesodiumhydroxidesolutions(5.3and 5.4).Dilutewithwaterto100mlandmix. Thesolutionisstablefor4weeksat+4Cinthedark. 5
20、.9 Nicotinamideadeninedinucleotidephosphatesolution, c(bNADPNa 2 )=12,7x10 - 3 mol/l. Dissolve100mgofNADPdisodiumsaltin10,0mlofwaterandmix. Thesolutionisstablefor4weeksat+4Cinthedark. 5.10 Adenosine5triphosphatesolution, c(5ATPNa 2 H 2 3H 2 O) 81x10 - 3 mol/l. Dissolve500mgof5ATPNa 2 H 2 3H 2 Oand50
21、0mgofanhydrousmonosodiumhydrogencarbonate(NaHCO 3 )in 10,0mlofwaterandmix. Thesolutionisstablefor4weeksat+4Cinthedark. 5.11 Amyloglucosidase(AGS;EC 3.2.1.3), enzymesuspensioninammoniumsulfatesolution(5.5), r(AGS)=10mg/ml. Thespecificactivityoftheenzymeshallbe14unitspermilligram. Thesuspensionisstabl
22、efor1yearat+4Cinthedark. 5.12 Hexokinase(HK;EC2.7.1.1)/glucose6phosphatedehydrogenase(G6PDH;EC1.1.1.49), enzyme suspensioninammoniumsulfatesolution(5.5), r(HK)=2mg/mland r(G6PDH)=1mg/ml. Thespecificactivityofbothenzymesshallbe140unitspermilligram. Thesuspensionisstablefor1yearat+4Cinthedark. 6 Appar
23、atus Usuallaboratoryapparatusand,inparticular,thefollowing. 6.1 Mechanicalorelectricalequipment, capableofhomogenizingthelaboratorysample. Thisincludesahighspeedrotationalcutter,oramincerfittedwithaplatewithaperturesnotexceeding4,0mmin diameter. 6.2 pHmeter. 6.3 Flutedfilterpapers, glucosefree,ofdia
24、meterabout15cm. 6.4 Pipettes,calibrated,forenzymaticanalysis,orautomaticmicropipettesofequivalentqualitywiththe followingvolumes:20 ml,50 ml,100 mland200 ml. 6.5 Smallplasticsspatula, formixingthecontentsofacuvette(6.9). 6.6 Waterbath, capableofbeingmaintainedat(602)C. 6.7 Hotplate.ISO13965:1998(E)
25、ISO 4 6.8 Spectrometer,capableofmeasuringatawavelengthof340nm. NOTE Whenaspectrometerfittedwithamercuryvapourlampisavailable,thereadingscanbecarriedoutat365nmand 334nm.Themolecularabsorptioncoefficient k forNADPHis3,51l mmol - 1 cm - 1 at365nmand6,18l mmol - 1 cm - 1 at334nm. 6.9 Cuvettes,madeofquar
26、tzorglass,withlid,ordisposablecuvettesforsingleuse,madeofpolymethacrylate, of10mmopticalpathlength. 6.10 Analyticalbalance, capableofweighingtothenearest0,1mg. 7 Sampling SamplingisnotpartofthemethodspecifiedinthisInternationalStandard.Arecommendedsamplingmethodis giveninISO310011. Itisimportantthat
27、thelaboratoryreceiveasamplewhichistrulyrepresentativeandhasnotbeendamagedor changedduringtransportorstorage. Startfromarepresentativesampleofatleast200g.Storethesampleinsuchawaythatdeteriorationandchange incompositionareprevented. 8 Preparationoftestsample Homogenizethelaboratorysamplewiththeappropr
28、iateequipment(6.1).Takecarethatthetemperatureofthe samplematerialdoesnotriseabove25C.Ifamincerisused,passthesampleatleasttwicethroughthe equipment. Fillasuitableairtightcontainerwiththepreparedsample.Closethecontainerandstoreinsuchawaythat deteriorationandchangeincompositionareprevented.Analysethesa
29、mpleassoonaspracticable,butalways within24hafterhomogenization. 9 Procedure NOTE Muscleglycogen,whichoccursnormallyin,forexample,sausages,doesnotinterferewiththedetermination.Maltose interferesbecausethisdisaccharideishydrolysedbyamyloglucosidaseintoglucose.Maltose(andglucose)can,however,be extracte
30、dfromthesamplewithalcohol. 9.1 Testportion Ifthetestsamplecontainsmaltose,makesurethatthewatercontentdoesnotexceed20%( m/m).Ifnecessary,dry thetestsample. Weighabout400mgofthepreparedtestsample(seeclause8)tothenearest0,1mgintoacentrifugetube. Proceedinaccordancewith9.2. Ifthetestsampledoesnotcontain
31、maltose,weighbetween100mgand1,0g( m)ofthepreparedtestsampletothe nearest0,1mgintoa100mlconicalflask. Add30mlofacetatebuffer(5.6)andproceedinaccordancewith9.3. 9.2 Extractionofmaltose(andglucose) Washthesamplethreetimeswith10mlof40%( V/V)ethanolandcentrifugeaftereachwashing.Filterthe supernatant.Comb
32、inetheprecipitateinthecentrifugetubewiththatonthefilterandtransfertoa100mlconical flaskusing4x5mlofacetatebuffer(5.6).Add10mlofacetatebuffer(5.6).ISO ISO13965:1998(E) 5 9.3 Preparationofextract Pipette(6.4)50 mlofaamylasesuspension(5.2)intotheconicalflaskcontainingthesample.Covertheconical flaskwith
33、aluminiumfoilandboilonahotplate(6.7)for15min.Shaketheflaskatintervals. Thenkeeptheconicalflaskat60Cinthewaterbath(6.6)for15min.Quantitativelytransferthecontentsofthe conicalflasktoa100mlvolumetricflask.Rinsetheconicalflaskwithwarmwaterandaddthewashingstothe volumetricflask.Allowtocooltoroomtemperatu
34、reanddilutetothemarkwithwater.Filter(6.3)atleast10mlofthe sampleextract,discardingthefirstfewmillilitres,andimmediatelyproceedinaccordancewith9.4. Keepsamplescontainingfatfor1hintherefrigeratorbeforefiltration. 9.4 Determination 9.4.1 Prepareareagentblanksolutionasfollows.Pipette(6.4)200 mlofcitrate
35、buffer(5.7)and100 mlofwater intoacuvette(6.9)containingaspatula(6.5). 9.4.2 Prepareatestsolutionfortheglucosedeterminationasfollows.Pipette(6.4)200 mlofcitratebuffer(5.7) and100 ml( V 2 )ofthesampleextract(9.3)intoacuvette(6.9)containingaspatula(6.5). 9.4.3 Prepareatestsolutionforthestarchdeterminat
36、ionasfollows.Pipette(6.4)200 mlofcitratebuffer(5.7)and 100 ml( V 2 )ofthesampleextract(9.3)intoacuvette(6.9)containingaspatula(6.5). Iftheconcentrationofstarchinthesamplesolutionexceeds0,4g/l,diluteitbeforeanalysis. 9.4.4 Pipette(6.4)20 mlofAGSsuspension(5.11)intothecuvettecontainingthereagentblanks
37、olution(9.4.1) andintothecuvettecontainingthetestsolutionforthestarchdetermination(9.4.3).Pipette20 mlofwaterintothe cuvettecontainingthetestsolutionfortheglucosedetermination(9.4.2). Mixthecontentsofthecuvettesbyswirlingorbymovingthespatulaupanddown. Closethecuvetteswiththelidorotherwise(e.g.paraff
38、insheet).Keepthecuvettesinthewaterbath(6.6)for15min at(602)C. Theabovepipetteprocedureisschematicallypresentedbelow: Reagent Reagentblank solution Testsolutionfor glucose determination Testsolutionfor starch determination Citratebuffer(5.7) 200l 200l 200l Sampleextract(9.3) 100l 100l Water(5.1) 100l
39、 20l AGSsuspension(5.11) 20l 20l Thevolumeofsampleextractpipettedintothecuvettemaybeincreasedupto1,0mlofaqueoussolution.Inthis case,adjustthepHofthefiltratetopH=4topH=5.Accordinglyreducethevolumeof1,50mlofwatertobe addedtothereactionmixturein9.4.5,inordertoobtainthesamefinalvolume V 1 in9.4.6. 9.4.5
40、 Coolthecuvettesto(22,52,5)Candcleantheiroutsidesurfaces.Pipette(6.4)successivelytoall cuvettes1,00mlofthetriethanolaminebuffer(5.8),100 mloftheNADPsolution(5.9),100 mloftheATPsolution (5.10)and1,50mlofwater.Mixcarefullybyswirlingorwiththespatula(6.5). Readtheabsorbance A 1 (6.8)ofeachcuvetteatawave
41、lengthof340nmagainstwaterafter3min. 9.4.6 Pipette(6.4)intoeachofthecuvettes20 mlofHK/G6PDHsuspension(5.12).Mixthecontentsofthe cuvettesbymovingthespatulaupanddown.Thefinalcuvettevolumeis3,04ml( V 1 ). Readtheabsorbance A 2 ofeachcuvetteatawavelengthof340nmagainstwaterafter15min.ISO13965:1998(E) ISO
42、6 NOTE Thereactionisnormallycompletedwithin5minto10min.Ifthereactiondoesnotstopwithinthistime,repeatthis readingevery2minuntilaconstantincreaseofabsorbanceevery2minisdetected.Extrapolatetheabsorbancetothetimeof additionoftheenzymeHK/G6PDH(foranexample,seefigureA.1). 10 Calculation 10.1 Waterfreestarchcontent 10.1.1 Absorbancedifference Calculatetheabsorbancedifferenceusingtheequation () () () D AAAAAAA s 2s 1s 2g 1g 2b 1b =-
