Megazyme/Amyloglucosidase (Aspergillus niger) /E-AMGDF-100ML/100ML

CulturefiltratesofCladosporiumresinaeATCC20495containamixtureofenzymesabletoconvertstarchandpullulanefficientlyintoD-glucose.Cultureconditionsforoptimalproductionofthepullulan-degrADIngactivityhavebeenestablished.Theamylolyticenzymepreparationwasfractionatedbyion-exchangeandmolecular-sievechromatography,andshowntocontainα-D-glucosidase,α-amylase,andtwoglucoamylases.Theglucoamylaseshavebeenpurifiedtohomogeneityandtheirsubstratespecificitiesinvestigated.Oneoftheglucoamylases(termedP)readilyhydrolysesthe(1→6)-α-Dlinkagesinpullulan,amylopectin,isomaltose,panose,and6³-α-D-glucosylmaltotriose.Eachoftheglucoamylasescleavesthe(1→6)-α-Dlinkageinpanosemuchmorereadilythanthatinisomaltose.

Interestindietaryfibreisundergoingadramaticrevival,thanksinparttotheintroductionofnewcarbohydratesasdietaryfibrecomponents.Muchemphasisisbeingplacedondetermininghowmuchfibreispresentinafood.Linkingaparticularamountoffibretoaspecifichealthbenefitisnowanimportantareaofresearch.Theterm"dietaryfibre"firstappearedin1953,andreferredtohemicelluloses,cellulosesandlignin(Theandere/tf/.1995).Trowell(1974)recommendedthistermasareplacementforthenolongeracceptableterm"crudefibre".Burkitt(1995)haslikenedtheinterestindietaryfibretothegrowthofariverfromitsfirsttrickletoamightytorrentHeobservesthatdietaryfibre"wasfirstviewedasmerelythelessdigestIBLeconstituentoffoodwhichexertsalaxativeactionbyirritatingthegut",thusacquiringthedesignation"roughage"-atermlaterreplacedby"crudefibre"andultimatelyby"dietaryfibre".Variousdefinitionsofdietaryfibrehaveappearedovertheyears,partlyduetothevariousconceptsusedinderivingtheterm(i.e.originofmaterial,resistancetodigestion,fermentationinthecolon,etc.),andpartlytothedifficultiesassociatedwithitsmeasurementandlabelling(Mongeauetal.1999).Theprincipalcomponentsofdietaryfibre,astraditionallyunderstood,arenon-starchpolysaccharides(whichinplantfibreareprincipallyhemicellulosesandcelluloses),andthenon-carbohydratephenoliccomponents,cutin,suberinandwaxes,withwhichtheyareassociatedinnature.In1976,thedefinitionofdietaryfibrewasmodifiedtoincludegumsandsomepecticsubstances,basedontheresistancetodigestionofthesecomponentsintheupperintestinaltract.Forthepurposesoflabelling,Englystetal.(1987)proposedthatdietaryfibrebedefinedas"non-starchpolysaccharides(NSP)inthedietthatarenotdigestedbytheendogenoussecretionsofthehumandigestivetract".MethodswereconcurrentlydevelopedtospecificallymeasureNSP(Englystetal.1994).

Debatecontinuesonthedefinitionofdietaryfiber(DF),methodsformeasurementofDF,andmethodsformeasurementofthecarbohydratesthatarereadilyhydrolyzedandabsorbedinthehumansmallintestine.HenNEBergandStahmanndevelopedthe"Wende"proximatesystemforanalysisoffoodsin1860,andasetofvaluesobtainedusingthismethodwerepublishedbyAtwaterandBryantin1900.ThismethodisstillinuseintheUSAforthemeasurementoftotalcarbohydrate.Inthisprocedure,totalcarbohydrateismeasuredbydifferenceafterdeductingthemoisture,protein,fatandashfromthetotalweight.Carbohydratecalculatedinthiswaycontainsnotonlysugarandstarch,butalsothe"unavailablecarbohydrate"ofDF.However,thereareanumberofproblemswiththisapproach,asthe"bydifference"figureincludesanumberofnon-carbohydratecomponentssuchaslignin,organicacids,tannins,waxesandsomeMaillardproducts.Inadditiontothiserror,itcombinesalloftheanalyticalerrorsfromtheotheranalyses(FAO1997).AneedforinformationonthecarbohydratecompositionoffoodsfordiabeticspromptedMcCanceandLawrence(1929)toattempttomeasurecarbohydratecompositiontogainresultsthatwouldbeofBIOLOGicalsignificance.Theydividedthecarbohydratesinfoodsintotwobroadgroups,"available"and"unavailable".Theavailablecarbohydrates,thatis,sugarplusstarch,weredefinedasthosethataredigestedandabsorbedbymanandareglucogenic.Theunavailablecarbohydratesweredefinedasthosethatarenotdigestedbytheendogenoussecretionsofthehumandigestivetract.Inthemid1920s,McCanceobtainedagrantof£30peryearfromtheMedicalResearchCounciltoanalyserawandcookedfruitsandvegetablesfortotal"availablecarbohydrate";valuesneededforcalculatingdiabeticdiets.

Anenzyme-linkedassayforthemeasurementofamyloglucosidaseincommercialenzymemixturesandcrudeculturefiltratesisdescribed.Amethodforthesynthesisofthesubstrateemployed,p-nitrophenylβ-D-maltoside,isalsodescribed.Thesubstrateisusedinthepresenceofsaturatinglevelsofβ-glucosidase.WitharangeofAspergillussp.culturefiltrates,anexcellentcorrelationwasfoundforvaluesobtainedwiththisassayandaconventionalassayemployingmaltoseassubstratewithmeasurementofreleasedglucose.

Interestindietaryfibreisundergoingadramaticrevivalthanksinparttotheintroductionofnewcarbohydratesasdietaryfibrecomponents.Muchemphasisisbeingplacedondetermininghowmuchfibreispresentinafood.Linkingaparticularamountoffibretoaspecifichealthbenefitisnowanimportantareaofresearch.TotalDietaryFibre.Theterm“dietaryfibre”firstappearedin1953andreferredtohemicelluloses,cellulosesandlignin(1).In1974,Trowell(2)recommendedthistermasareplacementforthenolongeracceptableterm“crudefibre”Burkitt(3)haslikenedtheinterestindietaryfibretothegrowthofariverfromitsfirsttrickletoamightytorrent.Heobservesthatdietaryfibre“wasviewedasmerelythelessdigestibleconstituentoffoodwhichexertsalaxativeactionbyirritatingthegut“thusacquiringthedesignation“roughage”atermwhichwaslaterreplacedby“crudefibre”andultimatelyby“dietaryfibre”Variousdefinitionsofdietaryfibrehaveappearedovertheyears,partlyduethevariousconceptsusedinderivingtheterm(i.e.originofmaterial,resistancetodigestion,fermentationinthecolonetc.),andpartlytothedifficultiesassociatedwithitsmeasurementandlabelling(4).Theprinciplecomponentsofdietaryfibre,astraditionallyunderstood,arenon-starchpolysaccharides,whichinplantfibreareprincipallyhemicellulosesandcelluloses,andthenon-carbohydratephenoliccomponents,cutin,suberinandwaxeswithwhichtheyareassociatedinNature.

Dietaryfiberismainlycomposedofplantcellwallpolysaccharidessuchascellulose,hemicellulose,andpecticsubstances,butitalsoincludesligninandotherminorcomponents(1).Basically,itcoversthepolysaccharidesthatarenothydrolyzedbytheendogenoussecretionsofthehumandigestivetract(2,3).Thisdefinitionhasservedasthetargetforthosedevelopinganalyticalproceduresforthemeasurementofdietaryfiberforqualitycontrolandregulatoryconsiderations(4).Mostproceduresforthemeasurementoftotaldietaryfiber(TDF),orspecificpolysaccharidecomponents,eitherinvolvesomeenzymetreatmentstepsoraremainlyenzyme-based.InthedevelopmentofTDFproceduressuchastheProskymethod(AOACInternational985.29,AACC32—05)(5),theUppsalamethod(AACC32-25)(6),andtheEnglystmethod(7),theaimwastoremovestarchandproteinthroughenzymetreatment,andtomeasuretheresidueasdietaryfiber(afterallowingforresidual,undigestedproteinandash).Dietaryfiberwasmeasuredeithergravimetricallyorbychemicalorinstrumentalprocedures.Manyoftheenzymetreatmentstepsineachofthemethods,particularlytheprosky(5)andtheUppsala(6)methodsareverysimilar.Asanewrangeofcarbohydratesisbeingintroducedaspotentialdietaryfibercomponents,theoriginalassayprocedureswillneedtobereexamined,andinsomecasesslightlymodified,toensureaccurateandquantitativemeasurementofthesecomponentsandofTDF.These“new”dietaryfibercomponentsincluderesistantnondigestibleoligosaccharides;nativeandchemicallymodifiedpolysaccharidesofplantandalgalorigin(galactomannan,chemicallymodifiedcelluloses,andagarsandcarrageenans);andresistantstarch.Tomeasurethesecomponentsaccurately,thepurity,activity,andspecificityoftheenzymesemployedwillbecomemuchmoreimportant.Aparticularexampleofthisisthemesurementoffructan.Thiscarbohydrateconsistsofafractionwithahighdegreeofpolymerization(DP)thatisprecipitatedinthestandardProskymethod(5,8)andalowDPfractionconsequentlyisnotmeasured(9).Resistantstarchposesaparticularproblem.Thiscomponentisonlypartiallyresistanttodegradationbyα-amylase,sothelevelofenzymeusedandtheincubationconditions(timeandtemperature)arecritical.

Astudywasmadeoftheeffectoftheactivityandpurityofenzymesintheassayoftotaldietaryfiber(AOACMethod985.29)andspecificdietaryfibercomponents:resistantstarch,fructan,andβ-glucan.Inthemeasurementoftotaldietaryfibercontentofresistantstarchsamples,theconcentrationofα-amylaseiscritical;however,variationsinthelevelofamyloglucosidasehavelittleeffect.Contaminationofamyloglucosidasepreparationswithcellulasecanresultinsignificantunderestimationofdietaryfibervaluesforsamplescontainingβ-glucan.Pureβ-glucanandcellulasepurifiedfromAspergillusnigeramyloglucosidasepreparationswereusedtodetermineacceptablecriticallevelsofcontamination.Sucrose,whichinterfereswiththemeasurementofinulinandfructooligosaccharidesinplantmaterialsandfoodproducts,mustberemovedbyhydrolysisofthesucrosetoglucoseandfructosewithaspecificenzyme(sucrase)followedbyborohydridereductionofthefreesugars.Unlikeinvertase,sucrasehasnoactiononlowdegreeofpolymerization(DP)fructooligosaccharides,suchaskestoseorkestotetraose.Fructanishydrolyzedtofructoseandglucosebythecombinedactionofhighlypurifiedexo-andendo-inulinases,andthesesugarsaremeasuredbythep-hydroxybenzoicacidhydrazidereducingsugarmethod.Specificmeasurementofβ-glucanincerealflourandfoodextractsrequirestheuseofhighlypurifiedendo-1,3:1,4β-glucanaseandA.nigerβ-glucosidase.β-glucosidasefromalmondsdoesnotcompletelyhydrolyzemixedlinkageβ-glucooligosaccharidesfrombarleyoroatβ-glucan.Contaminationoftheseenzymeswithstarch,maltosaccharide,orsucrose-hydrolyzingenzymesresultsinproductionoffreeglucosefromasourceotherthanβ-glucan,andthusanoverestimationofβ-glucancontent.Theglucoseoxidaseandperoxidaseusedintheglucosedeterminationreagentmustbeessentiallydevoidofcatalaseandα-andβ-glucosidase.

Enzymeactivityandpurityofthesetopics,theeasiesttodealwithistheimportanceofenzymepurityandactivity.Asascientistactivelyinvolvedinpolysaccharideresearchoverthepast25years,Ihavecometoappreciatetheimportanceofenzymepurityandspecificityinpolysaccharidemodificationandmeasurement(7).Thesefactorstranslatedirectlytodietaryfiber(DF)methodology,becausethemajorcomponentsofDFarecarbohydratepolymersandoligomers.ThecommitteereportpublishedintheMarchissueofCerealFOODSWORLDrefersonlytothemethodologyformeasuringenzymepurityandactivity(8)thatleduptheAOACmethod985.29(2).Inthisworkenzymepuritywasgaugedbythelackofhydrolysis(i.e.,completerecovery)ofaparticularDFcomponent(e.g.β-glucan,larchgalactanorcitruspectin).Enzymeactivitywasmeasuredbytheabilitytocompletelyhydrolyzerepresentativestarchandprotein(namelywheatstarchandcasein).Theserequirementsandrestrictionsonenzymepurityandactivitywereadequateatthetimethemethodwasinitiallydevelopedandservedasausefulworkingguide.However,itwasrecognizedthattherewasaneedformorestringentqualitydefinitionsandassayproceduresforenzymesusedinDFmeasurements.

The"goldstandard"methodforthemeasurementoftotaldietaryfibreisthatoftheAssociationofOfficialAnalyticalChemists(2000;method985.29).Thisprocedurehasbeenmodifiedtoallowmeasurementofsolubleandinsolubledietaryfibre,andbuffersemployedhavebeenimproved.However,therecognitionofthefactthatnon-digestibleoligosaccharidesandresistantstarchalsobehavephysiologicallyasdietaryfibrehasnecessitatedare-examinationofthedefinitionofdietaryfibre,andinturn,are-evaluationofthedietaryfibremethodsoftheAssociationofOfficialAnalyticalChemists.Withthisrealisation,theAmericanAssociationofCerealChemistsappointedascientificreviewcommitteeandchargeditwiththetaskofreviewingand,ifnecessary,updatingthedefinitionofdietaryfibre.Itorganisedvariousworkshopsandacceptedcommentsfrominterestedpartiesworldwidethroughaninteractivewebsite.Morerecently,the(US)FoodandNutritionBoardoftheInstituteofHealth,NationalAcademyofSciences,undertheoversightoftheStandingCommitteeontheScientificEvaluationofDietaryReferenceIntakes,assembledapaneltodevelopaproposeddefinition(s)ofdietaryfibre.Variouselementsofthesedefinitionswereinagreement,butnotall.Whatwasclearfrombothreviewsisthatthereisanimmediateneedtore-evaluatethemethodsthatareusedfordietaryfibremeasurementandtomakeappropriatechangeswhererequired,andtofindnewmethodstofillgaps.Inthispresentation,the"stateoftheart"inmeasurementoftotaldietaryfibreanddietaryfibrecomponentswillbedescribedanddiscussed,togetherwithsuggestionsforfutureresearch.

Withtherecognitionthatresistantstarch(RS)andnondigestibleoligosaccharides(NDO)actphysiologicallyasdietaryfiber(DF),aneedhasdevelopedforspecificandreliableassayproceduresforthesecomponents.TheabilityofAOACDFmethodstoaccuratelymeasureRSisdependentonthenatureoftheRSbeinganalyzed.Ingeneral,NDOarenotmeasuredatallbyAOACDFMethods985.29or991.43,theoneexceptionbeingthehighmolecularweightfractionoffructo-oligosaccharides.ValuesobtainedforRS,ingeneral,arenotingoodagreementwithvaluesobtainedbyinvitroproceduresthatmorecloselyimitatetheinvivosituationinthehumandigestivetract.Consequently,specificmethodsfortheaccuratemeasurementofRSandNDOhavebeendevelopedandvalidatedthroughinterlaboratorystudies.Inthispaper,modificationstoAOACfructanMethod999.03toallowaccuratemeasurementofenzymicallyproducedfructo-oligosaccharidesaredescribed.SuggestedmodificationstoAOACDFmethodstoensurecompleteremovaloffructanandRS,andtosimplifypHadjustmentbeforeamyloglucosidaseaddition,arealsodescribed.

Amethodisdescribedforthemeasurementofdietaryfibre,includingresistantstarch(RS),non-digestibleoligosaccharides(NDO)andavailablecarbohydrates.Basically,thesampleisincubatedwithpancreaticα-amylaseandamyloglucosidaseunderconditionsverysimilartothosedescribedinAOACOfficialMethod2002.02(RS).Reactionisterminatedandhighmolecularweightresistantpolysaccharidesareprecipitatedfromsolutionwithalcoholandrecoveredbyfiltration.RecoveryofRS(formostRSsources)isinlinewithpublisheddatafromileostomystudies.Theaqueousethanolextractisconcentrated,desaltedandanalysedforNDObyhigh-performanceliquidchromatographybyamethodsimilartothatdescribedbyOkuma(AOACMethod2001.03),exceptthatforlogisticalreasons,D-sorbitolisusedastheinternalstandardinplaceofglycerol.Availablecarbohydrates,definedasD-glucose,D-fructose,sucrose,theD-glucosecomponentoflactose,maltodextrinsandnon-resistantstarch,aremeasuredasD-glucoseplusD-fructoseinthesampleafterhydrolysisofoligosaccharideswithamixtureofsucrase/maltaseplusβ-galactosidase.

Anintegratedtotaldietaryfibre(TDF)method,consistentwiththerecentlyacceptedCODEXdefinitionofdietaryfibre,hasbeendeveloped.TheCODEXCommitteeonNutritionandFoodsforSpecialDietaryUses(CCNFSDU)hasbeendeliberatingforthepast8yearsonadefinitionfordietaryfibrethatcorrectlyreflectsthecurrentconsensusthinkingonwhatshouldbeincludedinthisdefinition.Asthisdefinitionwasevolving,itbecameevidenttousthatneitherofthecurrentlyavailablemethodsforTDF(AOACOfficialMethods985.29and991.43),noracombinationoftheseandothermethods,couldmeettheserequirements.Consequently,wedevelopedanintegratedTDFprocedure,basedontheprincipalsofAOACOfficialMethods2002.02,991.43and2001.03,thatiscompliantwiththenewCODEXdefinition.Thisprocedurequantitateshigh-andlow-molecularweightdietaryfibresasdefined,givinganaccurateestimateofresistantstarchandnon-digestibleoligosaccharidesalsoreferredtoaslow-molecularweightsolubledietaryfibre.Inthispaper,themethodisdiscussed,modificationstothemethodtoimprovesimplicityandreproducibilityaredescribed,andtheresultsofthefirstroundsofinterlaboratoryevaluationarereported.

Amethodforthedeterminationoftotaldietaryfiber(TDF),asdefinedbytheCODEXAlimentarius,wasvalidatedinfoods.BasedupontheprinciplesofAOACOfficialMethods^SM985.29,991.43,2001.03,and2002.02,themethodquantitateshigh-andlow-molecular-weightdietaryfiber(HMWDFandLMWDF,respectively).In2007,McClearydescribedamethodofextendedenzymaticdigestionat37°CtosimulatehumanintestinaldigestionfollowedbygravimetricisolationandquantitationofHMWDFandtheuseofLCtoquantitatelow-molecular-weightsolubledietaryfiber(LMWSDF).Themethodthusquantitatesthecompleterangeofdietaryfibercomponentsfromresistantstarch(byutilizingthedigestionconditionsofAOACMethod2002.02)todigestionresistantoligosaccharides(byincorporatingthedeionizationandLCproceduresofAOACMethod2001.03).ThemethodwasevaluatedthroughanAOACcollaborativestudy.Eighteenlaboratoriesparticipatedwith16laboratoriesreturningvalidassaydatafor16testportions(eightblindduplicates)consistingofsampleswitharangeoftraditionaldietaryfiber,resistantstarch,andnondigestibleoligosaccharides.Thedietaryfibercontentoftheeighttestpairsrangedfrom11.57to47.83.DigestionofsamplesundertheconditionsofAOACMethod2002.02followedbytheisolationandgravimetricproceduresofAOACMethods985.29and991.43resultsinquantitationofHMWDF.ThefiltratefromthequantitationofHMWDFisconcentrated,deionized,concentratedagain,andanalyzedbyLCtodeterminetheLMWSDF,i.e.,allnondigestibleoligosaccharidesofdegreeofpolymerization3.TDFiscalculatedasthesumofHMWDFandLMWSDF.Repeatabilitystandarddeviations(S_r)rangedfrom0.41to1.43,andreproducibilitystandarddeviations(S_R)rangedfrom1.18to5.44.Theseresultsarecomparabletootherofficialdietaryfibermethods,andthemethodisrecommendedforadoptionasOfficialFirstAction.

Amethodforthedeterminationofinsoluble(IDF),soluble(SDF),andtotaldietaryfiber(TDF),asdefinedbytheCODEXAlimentarius,wasvalidatedinfoods.BasedupontheprinciplesofAOACOfficialMethods^SM985.29,991.43,2001.03,and2002.02,themethodquantitateswater-insolubleandwater-solubledietaryfiber.ThismethodextendsthecapabilitiesofthepreviouslyadoptedAOACOfficialMethod2009.01,TotalDietaryFiberinFoods,Enzymatic-Gravimetric-LiquidChromatographicMethod,applicabletoplantmaterial,foods,andfoodingredientsconsistentwithCODEXDefinition2009,includingnaturallyoccurring,isolated,modified,andsyntheticpolymersmeetingthatdefinition.ThemethodwasevaluatedthroughanAOAC/AACCcollaborativestudy.Twenty-twolaboratoriesparticipated,with19laboratoriesreturningvalidassaydatafor16testportions(eightblindduplicates)consistingofsampleswitharangeoftraditionaldietaryfiber,resistantstarch,andnondigestibleoligosaccharides.Thedietaryfibercontentoftheeighttestpairsrangedfrom10.45to29.90%.DigestionofsamplesundertheconditionsofAOAC2002.02followedbytheisolation,fractionation,andgravimetricproceduresofAOAC985.29(anditsextensions991.42and993.19)and991.43resultsinquantitationofIDFandsolubledietaryfiberthatprecipitates(SDFP).Thefiltratefromthequantitationofwater-alcohol-insolubledietaryfiberisconcentrated,deionized,concentratedagain,andanalyzedbyLCtodeterminetheSDFthatremainssoluble(SDFS),i.e.,alldietaryfiberpolymersofdegreeofpolymerization=3andhigher,consistingprimarily,butnotexclusively,ofoligosaccharides.SDFiscalculatedasthesumofSDFPandSDFS.TDFiscalculatedasthesumofIDFandSDF.Thewithin-laboratoryvariability,repeatabilitySD(S_r),forIDFrangedfrom0.13to0.71,andthebetween-laboratoryvariability,reproducibilitySD(s_R),forIDFrangedfrom0.42to2.24.Thewithin-laboratoryvariabilitys_rforSDFrangedfrom0.28to1.03,andthebetween-laboratoryvariabilitysRforSDFrangedfrom0.85to1.66.Thewithin-laboratoryvariabilitysrforTDFrangedfrom0.47to1.41,andthebetween-laboratoryvariabilitys_RforTDFrangedfrom0.95to3.14.Thisiscomparabletootherofficialandapproveddietaryfibermethods,andthemethodisrecommendedforadoptionasOfficialFirstAction.

TheCodexCommitteeonMethodsofAnalysisandSamplingrecentlyrecommended14methodsformeasurementofdietaryfiber,eightofthesebeingtypeImethods.OfthesetypeImethods,AACCInternationalApprovedMethod32-45.01(AOACmethod2009.01)istheonlyprocedurethatmeasuresallofthedietaryfibercomponentsasdefinedbyCodexAlimentarius.OthermethodssuchastheProskymethod(AACCIApprovedMethod32-05.01)givesimilaranalyticaldataforthehigh-molecular-weightdietaryfibercontentsoffoodandvegetableproductslowinresistantstarch.Inthecurrentwork,AACCIApprovedMethod32-45.01hasbeenmodifiedtoallowaccuratemeasurementofsampleshighinparticularfructooligosaccharides:forexample,fructotriose,which,intheHPLCsystemused,chromatographsatthesamepointasdisaccharides,meaningthatitiscurrentlynotincludedinthemeasurement.Incubationoftheresistantoligosaccharidesfractionwithsucrase/β-galactosidaseremovesdisaccharidesthatinterferewiththequantitationofthisfraction.Thedietaryfibervalueforresistantstarchtype4(RS₄),variessignificantlywithdifferentanalyticalmethods,withmuchlowervaluesbeingobtainedwithAACCIApprovedMethod32-45.01thanwith32-05.01.ThisdifferenceresultsfromthegreatersusceptibilityofRS₄tohydrolysisbypancreaticα-amylasethanbybacterialα-amylase,andalsoagreatersusceptibilitytohydrolysisatlowertemperatures.OnhydrolysisofsampleshighinstarchintheassayformatofAACCIApprovedMethod32-45.01(AOACmethod2009.01),resistantmaltodextrinsareproduced.Themajorcomponentisaheptasaccharidethatishighlyresistanttohydrolysisbymostofthestarch-degradingenzymesstudied.However,itishydrolyzedbythemaltase/amyloglucosidase/isomaltaseenzymecomplexpresentinthebrushborderliningofthesmallintestine.Asaconsequence,AOACmethods2009.01and2011.25(AACCIApprovedMethods32-45.01and32-50.01,respectively)mustbeupdatedtoincludeanadditionalincubationwithamyloglucosidasetoremovetheseoligosaccharides.

AOACOfficialMethods2009.01and2011.25havebeenmodifiedtoallowremovalofresistantmaltodextrinsproducedonhydrolysisofvariousstarchesbythecombinationofpancreaticα-amylaseandamyloglucosidase(AMG)usedintheseassayprocedures.Themajorresistantmaltodextrin,6³,6⁵-di-α-D-glucosylmaltopentaose,ishighlyresistanttohydrolysisbymicrobialα-glucosidases,isoamylase,pullulanase,pancreatic,bacterialandfungalα-amylaseandAMG.However,thisoligosaccharideishydrolyzedbythemucosalα-glucosidasecomplexofthepigsmallintestine(whichissimilartothehumansmallintestine),andthusmustberemovedintheanalyticalprocedure.HydrolysisoftheseoligosaccharideshasbeenbyincubationwithahighconcentrationofapurifiedAMGat60°C.ThisincubationresultsinnohydrolysisorlossofotherresistantoligosaccharidessuchasFOS,GOS,XOS,resistantmaltodextrins(e.g.,Fibersol2)orpolydextrose.TheeffectofthisadditionalincubationwithAMGonthemeasuredleveloflowmolecularweightsolubledietaryfiber(SDFS)andoftotaldietaryfiberinabroadrangeofsamplesisreported.Resultsfromthisstudydemonstratethattheproposedmodificationcanbeusedwithconfidenceinthemeasurementofdietaryfiber.

Background: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.

Fructansareimportantinthesurvivalofplantsandalsovaluableforhumansaspotentiallyhealthpromotingfoodingredients.Inthisstudyfructancontentandcompositionweredeterminedingrainsof20barleybreedinglinesandcultivarswithawidevariationinchemicalcomposition,morphologyandcountryoforigin,grownatonesiteinChile.Therewassignificantgenotypicvariationingrainfructancontentrangingfrom0.9%to4.2%ofgraindryweight.Fructandegreeofpolymerisation(DP)wasanalysedusinghigh-performanceanion-exchangechromatographywithpulsedamperometricdetection(HPAEC-PAD).Changesinthedistributionofdifferentchainlengthsandthepatternofstructuresoffructanweredetectedwithincreasingamountoffructaninthedifferentbarleys.Apositivecorrelationwasfoundbetweenfructancontentandtherelativeamountoflongchainfructan(DP>9)(r=0.54,p=0.021).Ourresultsprovideabasisforselectingpromisingbarleylinesandcultivarsforfurtherresearchonfructaninbarleybreedingwiththeaimtoproducehealthyfoodproducts.

Extractabledietaryfiber(DF)playsanimportantroleinnutrition.ThisstudyonporridgemakingwithwholegrainryeinvestigatedtheeffectofresttimeofflourslurriesatroomtemperaturebeforecookingandamountofflourandsaltintherecipeonthecontentofDFcomponentsandmolecularweightdistributionofextractablefructan,mixedlinkage(1→3)(1→4)-β-D-glucan(β-glucan)andarabinoxylan(AX)intheporridge.ThecontentoftotalDFwasincreased(fromabout20%to23%ofdrymatter)duringporridgemakingduetoformationofinsolubleresistantstarch.Asmallbutsignificantincreaseintheextractabilityofβ-glucan(P=0.016)andAX(P=0.002)duetoresttimewasalsonoted.ThemolecularweightofextractablefructanandAXremainedstableduringporridgemaking.However,incubationoftheryeflourslurriesatincreasedtemperatureresultedinasignificantdecreaseinextractableAXmolecularweight.Themolecularweightofextractableβ-glucandecreasedgreatlyduringaresttimebeforecooking,mostlikelybytheactionofendogenousenzymes.Theamountofsaltandflourusedintherecipehadsmallbutsignificanteffectsonthemolecularweightofβ-glucan.TheseresultsshowthatwholegrainryeporridgemadewithoutaresttimebeforecookingcontainsextractableDFcomponentsmaintaininghighmolecularweights.Highmolecularweightismostlikelyofnutritionalimportance.

Glycaemicresponsestofoodsreflectthebalancebetweenglucoseloadinginto,anditsclearancefrom,theblood.Currentinvitromethodsforglycaemicanalysisdonottakeintoaccountthekeyroleofglucosedisposal.Thepresentstudyaimedtodevelopafoodintake-sensitivemethodformeasuringtheglycaemicimpactoffoodquantitiesusuallyconsumed,asthedifferencebetweenreleaseofglucoseequivalents(GGE)fromfoodduringinvitrodigestionandacorrespondingestimateofclearanceofthemfromtheblood.Fivefoods–whitebread,fruitbread,mueslibar,mashedpotatoandchickpeas–wereconsumedonthreeoccasionsbytwentyvolunteerstoprovidebloodglucoseresponse(BGR)curves.GGEreleaseduringinvitrodigestionofthefoodswasalsoplotted.GlucosedisposalratesestimatedfromdownwardslopesoftheBGRcurvesallowedGGEdose-dependentcumulativeglucosedisposaltobecalculated.BysubtractingcumulativeglucosedisposalfromcumulativeinvitroGGErelease,accuracyinpredictingtheinvitroglycaemiceffectfrominvitroGGEvalueswasgreatlyimproved.GGE_invivo=0·99GGE_invitro+0·75(R²0·88).FurThermore,thedifferencebetweenthecurvesofcumulativeGGEreleaseanddisposalcloselymimickedinvivoincrementalBGRcurves.Weconcludethatvalidmeasurementoftheglycaemicimpactoffoodsmaybeobtainedinvitro,andexpressedasgramsofglucoseequivalentsperfoodquantity,bytakingaccountnotonlyofGGEreleasefromfoodduringinvitrodigestion,butalsoofbloodglucoseclearanceinresponsetothefoodquantity.

Theeffectofanumberoflaboratory-scalepretreatmentsontheproportionsofrapidlydigested(RDS),slowlydigested(SDS)andresistantstarch(RS)inrawandcookedpotatohasbeenexaminedusinganinvitrodigestionprocedure.PotatoesofthevarietyFrisiawerepreparedinthreestates:raw,cooked,andcookedfollowedbyacoldtreatment(4°C,twodays).Eachpreparationwasthensubjectedintriplicatetofreeze-drying,coarselymincing,pasting,freezing,dry-millingafterfreeze-drying,in22differentcombinations,beforedigesting.Inrawpotato,verylittleRDSandSDS(<5% total="" starch="" (ts))="" were="" present,="" and="" the="" mechanical="" treatments="" of="" the="" potato="" did="" not="" affect="" the="" amounts="" of="" rds="" and="" sds.="" cooking="" resulted="" in="" an="" almost="" complete="" conversion="" to="" rds="" (="">95%TS)infreshly-cookedpotato,butafterpost-cookingcoldtreatmentmuchoftheRDStransformedtoSDS,whichreachedamaximumofabout45%TS.SDSformationwasindependentofthedegreeoftissuedisruptionaftercooking,andwasgenerallyassociatedwithformationofRS,however,freezingaftercookingallowedSDSformationwithoutprolongedcoldtreatmentandwithverylittleassociatedRS(SDS35%andRS4%ofTS).Freeze-dryingcausedanincreaseinRSinmosttreatmentsofthecookedpotatoes.Theobservedeffectsprovidedguidanceforsamplehandlinginpotatoresearch,butalsosuggestedseveralapproachestotheenrichmentofSDSand/orRS,withaconcurrentreductioninRDS,thatcouldbeusedtoimprovethenutritionalprofileofpotatoproductsbydecreasingRDS(loweredglycaemicimpact),andincreasingSDS(moresustainedenergyavailability)andRS(prebioticbenefits).

Inthisstudy,thefunctionalpropertiesofA-andB-typewheatstarchgranulesfromtwocommercialwheatflourswereinvestigatedfordigestibilityinvitro,chemicalcomposition(e.g.,amylose,protein,andashcontent),gelatinization,retrogradation,andpastingproperties.ThebranchchainlengthandchainlengthdistributionoftheseA-andB-typewheatstarchgranuleswerealsodeterminedusinghigh-performanceanionexchangechromatography(HPAEC).Wheatstarcheswithdifferentgranularsizesnotonlyhaddifferentdegreesofenzymatichydrolysisandthermalandpastingproperties,butalsodifferentmolecularcharacteristics.Differentamylosecontent,proteincontent,andbranchchainlengthofamylopectininA-andB-typewheatstarchgranulescouldalsobethemajorfactorsbesidesgranularsizefordifferentdigestibilityandotherfunctionalpropertiesofstarch.ThedataindicatethatdifferentwheatcultivarswithdifferentproportionofA-andB-typegranularstarchcouldresultindifferentdigestibilityinwheatproducts.

Wehaveproposedthetermshort-chaincarbohydrates(SCC)forthosespecies,otherthanthefreesugars,thataresolublein80%ethanolunderwell-definedconditions.WedescribeatechniqueforthemeasurementofresistantSCC(RSCC),whicharenotsusceptibletopancreaticamylaseorthebrushborderenzymesandthereforesometimestermednon-digestibleoligosaccharides.Intheprocedure,alpha-glucans(starchandmaltodextrins)arehydrolysedenzymaticallytoglucoseandthenon-starchpolysaccharides(NSP)areprecipitatedinethanol.Fructansarehydrolysedenzymaticallyandthemonosaccharideconstituentsarereducedtoacid-stablealditolderivativesbeforetheremainingRSCCarehydrolysedwithsulphuricacid.Alltheconstituentsugarsaremeasuredasalditolacetatederivativesbygas–liquidchromatography.TheprotocolallowsboththemeasurementoftotalRSCCandaseparate,specificmeasurementoffructans.

Digestibility,gelatinization,retrogradationandpastingpropertiesofstarchinvariouscereal,tuberandlegumefloursweredetermined.Rapidlyandslowlydigestiblestarchandresistantstarchwerepresentin11selectedflours.Ingeneral,cerealstarchesweremoredigestiblethanlegumestarchesandtuberstarchescontainedahighamountofresistantstarch.Thermalandrheologicalpropertiesoffloursweredifferentdependingonthecropsource.

Resistantstarchhasdrawnbroadinterestforbothpotentialhealthbenefitsandfunctionalproperties.Inthisstudy,atechnologywasdevelopedtoincreaseresistantstarchcontentofcornstarchusingesterificationwithcitricacidatelevatedtemperature.Waxycorn,normalcornandhigh-amylosecornstarcheswereusedasmodelstarches.Citricacid(40%ofstarchdryweight)wasreactedwithcornstarchatdifferenttemperatures(120–150°C)fordifferentreactiontimes(3–9h).Theeffectofreactionconditionsonresistantstarchcontentinthecitratecornstarchwasinvestigated.Whenconductingthereactionat140°Cfor7h,thehighestresistantstarchcontentwasfoundinwaxycorncitratestarch(87.5%)withthehighestdegreeofsubstitution(DS,0.16)ofallstarches.High-amylosecornstarchhad86.4%resistantstarchcontentand0.14DS,andnormalcornstarchhad78.8%resistantstarchand0.12DS.Thephysicochemicalpropertiesofthesecitratestarcheswerecharacterizedusingvariousanalyticaltechniques.Inthepresenceofexcesswateruponheating,citratestarchmadefromwaxycornstarchhadnopeakintheDSCthermogram,andsmallpeakswerefoundfornormalcornstarch(0.4J/g)andHylonVIIstarch(3.0J/g)inthethermograms.Thisindicatesthatcitratesubstitutionchangesgranuleproperties.Therearenoretrogradationpeaksinthethermogramswhenstarchwasreheatedafter2weeksstorageat5°C.AllthecitratestarchesshowednopeaksinRVApastingcurves,indicatingcitratesubstitutionchangesthepastingpropertiesofcornstarchaswell.Moreover,citratestarchfromwaxycornismorethermallystablethantheothercitratestarches.

Forlabelingpurposes,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%.

Barleyhusks,ryebran,andafiberresiduefromoatmilkproductionwereprocessedbyheatpretreatment,variousseparationsteps,andtreatmentwithanendoxylanaseinordertoimprovetheprebioticpotentialofthesecerealbyproducts.Metabolicfunctionswereintendedtoimprovealongwithimprovedmicrobialactivity.Theproductsobtainedwereincludedinahigh-fatmousedietsothatalldietscontained5%dietaryfiber.Inaddition,high-fatandlow-fatcontrolsaswellaspartiallyhydrolyzedguargumwereincludedinthestudy.Thesolublefiberproductobtainedfromryebrancausedasignificantincreaseinthebifidobacteria(logcopiesof16SrRNAgenes;median(25–75percentile):6.38(6.04–6.66)and7.47(7.30–7.74),respectively;p<0.001)=""in=""parallel=""with=""a=""tendency=""of=""increased=""production=""of=""propionic=""acid=""and=""indications=""of=""improved=""metabolic=""function=""compared=""with=""high-fat=""fed=""control=""mice.=""the=""oat-derived=""product=""caused=""an=""increase=""in=""the=""pool=""of=""cecal=""propionic=""(from=""0.62=""±=""0.12=""to=""0.94=""±=""0.08)=""and=""butyric=""acid=""(from=""0.38=""±=""0.04=""to=""0.60=""±=""0.04)=""compared=""with=""the=""high-fat=""control,=""and=""it=""caused=""a=""significant=""increase=""in=""lactobacilli=""(log=""copies=""of=""16s=""rrna=""genes;=""median=""(25–75=""percentile):=""6.83=""(6.65–7.53)=""and=""8.04=""(7.86–8.33),=""respectively;="">p<0.01)=""in=""the=""cecal=""mucosa.=""however,=""no=""changes=""in=""measured=""metabolic=""parameters=""were=""observed=""by=""either=""oat=""or=""barley=""products.="">

Extractionprotocolsforβ-glucanfromoatflourweretestedtodetermineoptimalconditionsforβ-glucanqualitytesting,whichincludedextractabilityandmolecularweight.Wefoundmassyieldsofβ-glucanwereconstantatalltemperatures,pHvalues,andflour-to-waterratios,aslongassufficienttimeandenoughrepeatextractionswereperformedandnohydrolyticenzymeswerepresent.Extractscontainedabout30–60%β-glucan,withlowerproportionsassociatedwithhigherextractiontemperaturesinwhichmorestarchandproteinwereextracted.Allcommercialstarchhydrolyticenzymestested,eventhosethatareconsideredhomogenous,degradedβ-glucanapparentmolecularweightasevaluatedbysize-exclusionchromatography.Higherconcentrationβ-glucansolutionscouldbepreparedbycontrollingtheflour-to-waterratioinextractions.Eightgramsofflourper50mLofwatergeneratedthehighestnativeβ-glucanconcentrations.Routineextractionscontained2gofenzyme-inactivatedflourin50mLofwaterwith5mMsodiumazide(asanantimicrobial),whichwerestirredovernight,centrifuged,andthesupernatantboiledfor10min.Thepolymerextractedhadamolecularweightofabout2millionandwasstableatroomtemperatureforatleastamonth.