Highpurityα-Amylase(Bacilluslicheniformis)foruseinresearch,biochemicalenzymeassaysandinvitrodiagnosticanalysis.EC3.2.1.1CAZyFamily:GH13CAS:9000-90-2/ 9000-85-5alpha-amylase;4-alpha-D-glucanglucanohydrolaseHighlypurified.FromBacilluslicheniformis.StABIlisedsolution.Forusein MegazymeTotalStarchandDietaryFiber methods,suitableforuseatpH6.5andabove.E-BLAAM-A-100mLspecificallytobeusedwithANKOMTDFDietaryFiberAnalyzer.Specificactivity:~55U/mg(40oC,pH6.5onCeralphareagent).Stability:>4yearsat4oC.DatabookletsforeachpacksizearelocatedintheDocumentationtab.ViewMegazyme’slatestGuideforDietaryFiberAnalysis.Newdevelopmentsinthemeasurementofα-amylase,endo-protease,β-glucanaseandβ-xylanase.McCleary,B.V.&Monaghan,D.(2000).“ProceedingsoftheSecondEuropeanSymposiumonEnzymesinGrainProcessing”,(M.Tenkanen,Ed.),VTTInformationService,pp.31-38.LinktoArticleReadAbstractOverthepast8years,wehavebeenactivelyinvolvedinthedevelopmentofsimpleandreliableassayprocedures,forthemeasurementofenzymesofinteresttothecerealsandrelatedindustries.Insomeinstances,differentprocedureshavebeendevelopedforthemeasurementofthesameenzymeactivity(e.g.α-amylase)inarangeofdifferentmaterials(e.g.malt,cerealgrainsandfungalpreparations).Thereasonsfordifferentproceduresmaydependonseveralfactors,suchastheneedforsensitivity,easeofuse,robustnessofthesubstratemixture,orthepossibilityforautomation.Inthispresentation,wewillpresentinformationonourmostup-to-dateproceduresforthemeasurementofα-amylase,endo-protease,β-glucanaseandβ-xylanase,withspecialreferencetotheuseofparticularassayformatsinparticularapplications.Measurementofcerealα-Amylase:Anewassayprocedure.McCleary,B.V.&Sheehan,H.(1987).JournalofCerealScience,6(3),237-251.LinktoArticleReadAbstractAnewprocedurefortheassayofcerealα-amylasehasbeendeveloped.Thesubstrateisadefinedmaltosaccharidewithanα-linkednitrophenylgroupatthereducingendofthechain,andachemicalblockinggroupatthenon-reducingend.Thesubstrateiscompletelyresistanttoattackbyβ-amylase,glucoamylaseandα-glucosidaseandthusformsthebasisofahighlyspecificassayforα-amylase.Thereactionmixtureiscomposedofthesubstrateplusexcessquantitiesofα-glucosidaseandglucoamylase.Nitrophenyl-maltosaccharidesreleasedonactionofα-amylaseareinstantaneouslycleavedtoglucoseplusfreep-nitrophenolbytheglucoamylaseandα-glucosidase,suchthattherateofreleaseofp-nitrophenoldirectlycorrelateswithα-amylaseactivity.TheassayprocedureshowsanexcellentcorrelationwiththeFarrand,theFallingNumberandthePhadebasα-amylaseassayprocedures.Anewprocedureforthemeasurementoffungalandbacterialα-amylase.Sheehan,H.&McCleary,B.V.(1988).BiotechnologyTechniques,2(4),289-292.LinktoArticleReadAbstractAprocedureforthemeasurementoffungalandbacterialα-amylaseincrudeculturefiltratesandcommercialenzymepreparationsisdescribed.Theprocedureemploysend-blocked(non-reducingend)p-nitrophenylmaltoheptaosideinthepresenceofamyloglucosidaseandα-glucosidase,andisabsolutelyspecificforα-amylase.Theassayprocedureissimple,reliableandaccurate.Animprovedenzymicmethodforthemeasurementofstarchdamageinwheatflour.Gibson,T.S.,AlQalla,H.&McCleary,B.V.(1992).JournalofCerealScience,15(1),15-27.LinktoArticleReadAbstractAnimprovedenzymicmethodforthedeterminationofstarchdamageinwheatflourhasbeendevelopedandcharacterized.Theproposedmethodissimpleandreliable,andenablesupto20samplestobemeasuredinduplicatein2h.Asingleassaytakesapproximately40min.Theassayprotocolisintwophases.Inthefirst,thefloursampleisincubatedwithpurifiedfungalalpha-amylasetoliberatedamagedstarchgranulesassolubleoligosaccharides.Aftercentrifugation,theoligosaccharidesinthesupernatantarehydrolysedbyamyloglucosidasetoglucoseinphase2.Theglucoseisthenquantifiedwithaglucoseoxidase/peroxidasereagent.Theproposedmethodthereforeavoidspotentialerrorsassociatedwithexistingstandardassays,whichemployunpurifiedamylasepreparationsandnon-specificreducinggroupmethodstoquantifythehydrolyticproducts.Despitetheuseofpurifiedassaycomponents,theproposedstarchdamagemethoddidnotexhibitanabsoluteend-pointtotheactionofalpha-amylaseinphase1.Thiswasduetoalowrateofhydrolysisofundamagedgranules,andisafeatureofenzymicmethodsforstarchdamagedetermination.Otheramylolyticenzymes,includingbeta-amylase,isoamylaseandpullulanase,andcombinationsoftheseenzymes,wereevaluatedasalternativestoalpha-amylaseintheproposedmethod.Theseenzymes,whenusedalone,gavenobenefitsovertheuseofalpha-amylase.Whenusedincombinationwithalpha-amylase,therewasasynergisticactiononundamagedgranules.Atestkitbasedontheassayformatdescribedinthispaperisthesubjectofaninternationalinterlaboratoryevaluation.Measurementofα-amylaseactivityinwhitewheatflour,milledmalt,andmicrobialenzymepreparations,usingtheceralphaassay:Collaborativestudy.McCleary,B.V.,McNally,M.,Monaghan,D.&Mugford,D.C.(2002).JournalofAOACInternational,85(5),1096-1102.LinktoArticleReadAbstractThisstudywasconductedtoevaluatethemethodperformanceofarapidprocedureforthemeasurementofα-amylaseactivityinfloursandmicrobialenzymepreparations.Samplesweremilled(ifnecessary)topassa0.5mmsieveandthenextractedwithabuffer/saltsolution,andtheextractswereclarifiedanddiluted.Aliquotsofdilutedextract(containingα-amylase)wereincubatedwithsubstratemixtureunderdefinedconditionsofpH,temperature,andtime.Thesubstrateusedwasnonreducingend-blockedp-nitrophenylmaltoheptaoside(BPNPG7)inthepresenceofexcessquantitiesofThermostableα-glucosidase.TheblockinggroupinBPNPG7preventshydrolysisofthissubstratebyexo-actingenzymessuchasamyloglucosidase,α-glucosidase,andβ-amylase.Whenthesubstrateiscleavedbyendo-actingα-amylase,thenitrophenyloligosaccharideisimmediatelyandcompletelyhydrolyzedtop-nitrophenolandfreeglucosebytheexcessquantitiesofα-glucosidasepresentinthesubstratemixture.Thereactionisterminated,andthephenolatecolordevelopedbytheadditionofanalkalinesolutionismeasuredat400nm.AmylaseactivityisexpressedintermsofCeralphaunits;1unitisdefinedastheamountofenzymerequiredtorelease1µmolp-nitrophenyl(inthepresenceofexcessquantitiesofα-glucosidase)in1minat40°C.Inthepresentstudy,15laboratoriesanalyzed16samplesasblindduplicates.Theanalyzedsampleswerewhitewheatflour,whitewheatflourtowhichfungalα-amylasehadbeenadded,milledmalt,andfungalandbacterialenzymepreparations.Repeatabilityrelativestandarddeviationsrangedfrom1.4to14.4%,andreproducibilityrelativestandarddeviationsrangedfrom5.0to16.7%.Thephysicochemicalpropertiesandinvitrodigestibilityofselectedcereals,tubersandlegumesgrowninChina.Liu,Q.,Donner,E.,Yin,Y.,Huang,R.L.&Fan,M.Z.(2006).FoodChemistry,99(3),470-477.LinktoArticleReadAbstractDigestibility,gelatinization,retrogradationandpastingpropertiesofstarchinvariouscereal,tuberandlegumefloursweredetermined.RapidlyandslowlydigestIBLestarchandresistantstarchwerepresentin11selectedflours.Ingeneral,cerealstarchesweremoredigestiblethanlegumestarchesandtuberstarchescontainedahighamountofresistantstarch.Thermalandrheologicalpropertiesoffloursweredifferentdependingonthecropsource.Determinationof“NetCarbohydrates”usinghigh-performanceanionexchangechromatography.Lilla,Z.,Sullivan,D.,Ellefson,W.,Welton,K.&Crowley,R.(2005).JournalofAOACInternational,88(3),714-719.LinktoArticleReadAbstractForlabelingpurposes,thecarbohydratecontentoffoodshastrADItionallybeendeterminedbydifference.Thisvalueincludessugars,starches,fiber,dextrins,sugaralcohols,polydextrose,andvariousotherorganiccompounds.Insomecases,thecurrentmethodmaylacksufficientspecificity,precision,andaccuracy.Thesearesubsequentlyquantitatedbyhighperformanceanionexchangechromatographywithpulsedamperometricdetectionandexpressedastotalnonfibersaccharidesorpercent“netcarbohydrates.”Inthisresearch,anewmethodwasdevelopedtoaddressthisneed.Themethodconsistsofenzymedigestionstoconvertstarches,dextrins,sugars,andpolysaccharidestotheirrespectivemonosaccharidecomponents.Thesearesubsequentlyquantifiedbyhigh-performanceanionexchangechromatographywithpulsedamperometricdetectorandexpressedastotalnonfibersaccharidesorpercent“netcarbohydrates.”Hydrolyzedendproductsofvariousnovelfibersandsimilarcarbohydrateshavebeenevaluatedtoensurethattheydonotregisterasfalsepositivesinthenewtestmethod.Thedatageneratedusingthe“netcarbohydrate”methodwere,inmanycases,significantlydifferentthanthevaluesproducedusingthetraditionalmethodology.Therecoveriesobtainedinafortifieddrinkmatrixrangedfrom94.9to105%.Thecoefficientofvariationwas3.3%.Physical,microscopicandchemicalcharacterisationofindustrialryeandwheatbransfromtheNordiccountries.Kamal-Eldin,A.,Lærke,H.N.,Knudsen,K.E.B.,Lampi,A.M.,Piironen,V.,Adlercreutz,H.,Katina,K.,Poutanen,K.&Ɨman,P.A.(2009).Food&nutritionresearch,53.LinktoArticleReadAbstractBackground:Epidemiologicalstudiesshowinverserelationshipbetweenintakeofwholegraincerealsandseveralchronicdiseases.Componentsandmechanismsbehindpossibleprotectiveeffectsofwholegraincerealsarepoorlyunderstood.Objective:Tocharacterisecommercialryebranpreparations,comparedtowheatbran,regardingstructureandcontentofnutrientsaswellasanumberofpresumablybioactivecompounds.Design:SixdifferentryebransfromSweden,DenmarkandFinlandwereanalysedandcomparedwithtwowheatbransregardingcolour,particlesizedistribution,microscopicstructuresandchemicalcompositionincludingproximalcomponents,vitamins,mineralsandbioactivecompounds.Results:Ryebransweregenerallygreenerincolourandsmallerinparticlesizethanwheatbrans.Theryebransvariedconsiderablyintheirstarchcontent(13.2–;28.3%),whichreflectedvariableinclusionofthestarchyendosperm.Althoughryeandwheatbranscontainedcomparablelevelsoftotaldietaryfibre,theydifferedintherelativeproportionsoffibrecomponents(i.e.arabinoxylan,β-glucan,cellulose,fructanandKlasonlignin).Generally,ryebranscontainedlesscelluloseandmoreβ-glucanandfructanthanwheatbrans.Withinsmallvariations,theryeandwheatbranswerecomparableregardingthecontentsoftocopherols/tocotrienols,totalfolate,sterols/stanols,phenolicacidsandlignans.Ryebranhadlessglycinebetaineandmorealkylresorcinolsthanwheatbrans.Conclusions:Theobservedvariationinthechemicalcompositionofindustriallyproducedryebranscallsfortheneedofstandardisationofthiscommodity,especiallywhenusedasafunctionalingredientinfoods.AsimplifiedmodificationoftheAOACofficialmethodfordeterminationoftotaldietaryfiberusingnewlydevelopedenzymes:preliminaryinterlaboratorystudy.Kanaya,K.,Tada,S.,Mori,B.,Takahashi,R.,Ikegami,S.,Kurasawa,S.,Okuzaki,M.,Mori,Y.,Innami,S.&Negishi,Y.(2007).JournalofAOACInternational,90(1),225-237.LinktoArticleReadAbstractApreliminaryinterlaboratorystudywasconductedtoevaluatethevalidityofthemodifiedAOACmethodfordeterminationoftotaldietaryfiberbyTadaandInnami,inwhichthe3-stepenzymaticdigestionprocessinAOACMethod991.43ismodifiedtoa2-stepprocesswithoutpHadjustment.Totaldietaryfibercontentsin8representativefoodstuffsweremeasuredusingboththeoriginalAOACMethod991.43andthemodifiedmethodin6researchfacilitiesinJapan.Repeatabilityrelativestandarddeviations,reproducibilityrelativestandarddeviations,andHorwitzratiovaluesfromthemodifiedmethodwereequivalenttothosefromAOACMethod991.43,exceptinthericesample.However,thisexceptionalcaseshowninthemodifiedmethodwasentirelydissolvedbytheadditionof-amylasestabilizingagents.Themodifiedmethod,whichshortenstheprocessofenzymaticdigestionfrom3to2stepsandinwhichonlyreactiontemperatureisadjustedunderthesamepH,wasfoundnotonlytogiveaccuratevaluescomparabletotheoriginalmethod,butalsotosubstantiallyreducethelaborrequiredbythelaboratorystaffintheprocessofroutineanalysis.Thisstudyrevealedthatthevalidityofthemodifiedmethodwasfurtherensuredbyadding-amylasestabilizingagentstothereactionsystem.TreatmentofcerealproductswithatailoredpreparationofTrichodermaenzymesincreasestheamountofsolubledietaryfiber.Napolitano,A.,Lanzuise,S.,Ruocco,M.,Arlotti,G.,Ranieri,R.,Knutsen,S.H.,Lorito,M.&Fogliano,V.(2006).JournalofAgriculturalandFoodChemistry,54(20),7863-7869.LinktoArticleReadAbstractNutritioNISTsrecommendincreasingtheintakeofsolubledietaryfiber(SDF),whichisverylowinmostcereal-basedproducts.ConversionofinsolubleDF(IDF)intoSDFcanbeachievedbychemicaltreatments,butthisaffectsthesensorialpropertiesoftheproducts.Inthisstudy,thepossibilityofgettingasubstantialincreaseofSDFfromcerealproductsusingatailoredpreparationofTrichodermaenzymesisreported.EnzymeswereproducedcultivatingTrichodermausingdurumwheatfiber(DWF)andbarleyspentgrain(BSG)asuniquecarbonsources.ManyTrichodermastrainswerescreened,andthehydrolysisconditionsabletoincreasebyenzymatictreatmenttheamountofSDFinDWFandBSGweredetermined.ResultsdemonstrateinbothproductsthatitispossibletotripletheamountofSDFwithoutamarkeddecreaseoftotalDF.Theenzymatictreatmentalsocausesthereleaseofhydroxycinnamicacids,mainlyferulicacid,thatarelinkedtothepolysaccharideschains.Thisincreasesthefreephenolicconcentration,thewater-solubleantioxidantactivity,and,inturn,thephenolcompoundsbioavailability.Methodforthedirectdeterminationofavailablecarbohydratesinlow-carbohydrateproductsusinghigh-performanceanionexchangechromatography.Ellingson,D.,Potts,B.,Anderson,P.,Burkhardt,G.,Ellefson,W.,Sullivan,D.,Jacobs,W.&Ragan,R.(2010).JournalofAOACInternational,93(6),1897-1904.LinktoArticleReadAbstractAnimprovedmethodfordirectdeterminationofavailablecarbohydratesinlow-levelproductshasbeendevelopedandvalidatedforalow-carbohydratesoyinfantformula.Themethodinvolvesmodificationofanexistingdirectdeterminationmethodtoimprovespecificity,accuracy,detectionlevels,andruntimesthroughamoreextensiveenzymaticdigestiontocaptureallavailable(orpotentiallyavailable)carbohydrates.Thedigestionhydrolyzesallcommonsugars,starch,andstarchderivativesdowntotheirmonosaccharidecomponents,glucose,fructose,andgalactose,whicharethenquantitatedbyhigh-performanceanion-exchangechromatographywithphotodiodearraydetection.Methodvalidationconsistedofspecificitytestingand10daysofanalyzingvariousspikelevelsofmixedsugars,maltodextrin,andcornstarch.TheoverallRSDwas4.0acrossallsampletypes,whichcontainedwithin-dayandday-to-daycomponentsof3.6and3.4,respectively.Overallaveragerecoverywas99.4(n=10).Averagerecoveryforindividualspikedsamplesrangedfrom94.1to106(n=10).Itisexpectedthatthemethodcouldbeappliedtoavarietyoflow-carbohydratefoodsandbeverages.Lowfolatecontentingluten-freecerealproductsandtheirmainingredients.Yazynina,E.,Johansson,M.,Jägerstad,M.&Jastrebova,J.(2008).FoodChemistry,111(1),236-242.LinktoArticleReadAbstractFolatecontentinsomegluten-freecerealproductsandtheirmainingredientswasdeterminedusingavalidatedmethodbasedonreversed-phasehighperformanceliquidchromatography(HPLC)withfluorescenceanddiodearraydetection.Themainfolateformsfoundingluten-freeproductswere5-methyl-tetrahydrofolateandtetrahydrofolate.Starchesandlowproteinflourscommonlyusedasmaincomponentsingluten-freeproductsappearedtobepoorfolatesourceswithfolatecontent≤6µg/100gfreshweight.Folatecontentingluten-freebreadswashigher(15.1–35.9µgfolate/100gfreshweight)duetouseofbakeryyeastwhichisarichfolatesource.Overall,folatecontentingluten-freeproductswaslowerthanintheirgluten-containingcounterparts.Therefore,fortificationofgluten-freeproductswithfolicacidorenrichmentoftheseproductswithnutrient-densefractionsofcerealsnaturallyfreefromgluten(suchasbuckwheat,quinoa,amaranthormillet)canbeofinterest.Starchtransformationinbran-enrichedextrudedwheatflour.Robin,F.,Théoduloz,C.,Gianfrancesco,A.,Pineau,N.,Schuchmann,H.P.&Palzer,S.(2011).CarbohydratePolymers,85(1),65-74.LinktoArticleReadAbstractWheatflourwasextrudedatdifferentconditionsofbarreltemperature(120°Cand180°C),watercontent(18%and22%)andscrewspeed(400rpmand800rpm)withanincreasingconcentrationofwheatbranfibers(2.8%,12.6%and24.4%).Inthetestedextrusionconditions,starchcrystalliteswerefullydissociated.Theestimatedstarchsolubilitywasinfluencedbytheprocessconditionsandrangedfrom24.1%to63.1%.Atsameprocessconditions,thestarchsolubilitywasincreasedonlyatthehighestbranlevel.Thebranconcentrationinfluencedtheglasstransitiontemperature,meltingtemperatureandsorptionisothermoftheunprocessedwheatflour.Attheextrusionconditions,itshowedthathigherbranlevelsledtoahigheramountoffreewaterandadecreaseinstarchglasstransitiontemperatureofupto13K.Thedifferencesinstarchtransformation,inducedbytheconcentrationofbran,mightcontributetothemodulationoftheexpansionpropertiesofbran-containingstarchyfoams.Potatogenotypedifferencesinnutritionallydistinctstarchfractionsaftercooking,andcookingplusstoringcool.Monro,J.,Mishra,S.,Blandford,E.,Anderson,J.&Genet,R.(2009).JournalofFoodCompositionandAnalysis,22(6),539-545.LinktoArticleReadAbstractRapidlydigestible(RDS),slowlydigestible(SDS)andresistantstarch(RS)weremeasuredin9NewZealandsupermarketpotatoesandin37linesfromapotatobreedingprogrambyinvitrodigestionimmediatelyaftercooking,andafterstoringat4°Cfor44hpost-cooking.TheaimwastomeasuretherangeinthetendencytoformSDSandRSinthepotatogenepoolinNewZealand.Immediatelyaftercooking,thepotatoescontained(meanandacross-cultivarrange,drymatterbasis)68%RDS(range62–73%),3%SDS(range0–8.5%),and3.9%RS(range3–6.4%).Coolstorageaftercookingalteredthedistributionandrangesto44%RDS(range33–53%),23%SDS(range15–34%)and7%RS(range4.7–15.8%).TherewasnosignificantrelationshipbetweenRSandSDSinthecooked-cooledpotatoes.Inthe37potatolines,SDSrangedfrom7to37%oftotalstarch,RSfrom12to27%oftotalstarchafterthepost-cookingcooltreatment.Theresultssuggestthattheglycaemicimpactofsomepotatoesmaybesubstantiallyreducedbycool-storingaftercooking,andthatthedifferencesbetweencultivarsinthetendencytoformcold-inducedSDSandRSaresufficientforthesetraitstobeusedinconventionalplantbreeding.Molecular,mesoscopicandmicroscopicstructureevolutionduringamylasedigestionofmaizestarchgranules.Shrestha,A.K.,Blazek,J.,Flanagan,B.M.,Dhital,S.,Larroque,O.,Morell,M.K.,Gilbert,E.P.&Gilbert,M.J.(2012).CarbohydratePolymers,90(1),23-33.LinktoArticleReadAbstractCerealstarchgranuleswithhigh(>50%)amylosecontentareapromisingsourceofnutritionallydesirableresistantstarch,i.e.starchthatescapesdigestioninthesmallintestine,butthestructuralfeaturesresponsiblearenotfullyunderstood.Wereporttheeffectsofpartialenzymedigestionofmaizestarchgranulesonamylopectinbranchlengthprofiles,doubleandsinglehelixcontents,gelatinisationproperties,crystallinityandlamellarperiodicity.Comparingresultsforthreemaizestarches(27,57,and84%amylose)thatdifferinbothstructuralfeaturesandamylase-sensitivityallowsconclusionstobedrawnconcerningtherate-determiningfeaturesoperatingunderthedigestionconditionsused.Allstarchesarefoundtobedigestedbyaside-by-sidemechanisminwhichthereisnomajorpreferenceduringenzymeattackforamylopectinbranchlengths,helixform,crystallinityorlamellarorganisation.Weconcludethatthemajorfactorcontrollingenzymesusceptibilityisgranulearchitecture,withshorterlengthscalesnotplayingamajorroleasinferredfromthelargelyinvariantnatureofnumerousstructuralmeasuresduringthedigestionprocess(XRD,NMR,SAXS,DSC,FACE).Resultsareconsistentwithdigestionratesbeingcontrolledbyrestricteddiffusionofenzymeswithindenselypackedgranularstructures,withaneffectivesurfaceareaforenzymeattackdeterminedbyexternaldimensions(57or84%amylose–relativelyslow)orinternalchannelsandpores(27%amylose–relativelyfast).Althoughtheprocessofgranuledigestionistoafirstapproximationnon-discriminatorywithrespecttostructureatmolecularandmesoscopiclengthscales,secondaryeffectsnotedinclude(i)partialcrystallisationofV-typehelicesduringdigestionof27%amylosestarch,(ii)preferentialhydrolysisoflongamylopectinbranchesduringtheearlystagehydrolysisof27%and57%butnot84%amylosestarches,linkedwithdisruptionoflamellarrepeatingstructureand(iii)partialB-typerecrystallisationafterprolongedenzymeincubationfor57%and84%amylosestarchesbutnot27%amylosestarch.DESCRIPTIONα-Amylase(Bacilluslicheniformis)EC3.2.1.1CAZyFamily:GH13CAS: 9000-90-2/ 9000-85-5Synonyms:alpha-amylase;4-alpha-D-glucanglucanohydrolaseForm:Stabilisedsolution.Stability: >4yearsat4oC.Specificactivity:~55U/mg(40oC,pH6.5onCeralphareagent).Unitdefinition:OneUnitofα-amylaseistheamountofenzymerequiredtoreleaseoneμmoleof p-nitrophenolfromblockedp-nitrophenyl-maltoheptaosideperminute(inthepresenceofexcessα-glucosidase)atpH6.0and40oC.Specificity:endo-hydrolysisofα-1,4-D-glucosidiclinkagesinstarch.Applications:ForuseinMegazymeTotalStarchandDietaryFibermethods.

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