Overthepast8years,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.

Anewprocedurefortheassayofcerealα-amylasehasbeendeveloped.Thesubstrateisadefinedmaltosaccharidewithanα-linkednitrophenylgroupatthereducingendofthechain,andachemicalblockinggroupatthenon-reducingend.Thesubstrateiscompletelyresistanttoattackbyβ-amylase,glucoamylaseandα-glucosidaseandthusformsthebasisofahighlyspecificassayforα-amylase.Thereactionmixtureiscomposedofthesubstrateplusexcessquantitiesofα-glucosidaseandglucoamylase.Nitrophenyl-maltosaccharidesreleasedonactionofα-amylaseareinstantaneouslycleavedtoglucoseplusfreep-nitrophenolbytheglucoamylaseandα-glucosidase,suchthattherateofreleaseofp-nitrophenoldirectlycorrelateswithα-amylaseactivity.TheassayprocedureshowsanexcellentcorrelationwiththeFarrand,theFallingNumberandthePhadebasα-amylaseassayprocedures.

Aprocedureforthemeasurementoffungalandbacterialα-amylaseincrudeculturefiltratesandcommercialenzymepreparationsisdescribed.Theprocedureemploysend-blocked(non-reducingend)p-nitrophenylmaltoheptaosideinthepresenceofamyloglucosidaseandα-glucosidase,andisabsolutelyspecificforα-amylase.Theassayprocedureissimple,reliableandaccurate.

Animprovedenzymicmethodforthedeterminationofstarchdamageinwheatflourhasbeendevelopedandcharacterized.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.

Thisstudywasconductedtoevaluatethemethodperformanceofarapidprocedureforthemeasurementofα-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%.

Digestibility,gelatinization,retrogradationandpastingpropertiesofstarchinvariouscereal,tuberandlegumefloursweredetermined.RapidlyandslowlydigestIBLestarchandresistantstarchwerepresentin11selectedflours.Ingeneral,cerealstarchesweremoredigestiblethanlegumestarchesandtuberstarchescontainedahighamountofresistantstarch.Thermalandrheologicalpropertiesoffloursweredifferentdependingonthecropsource.

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%.

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.

ApreliminaryinterlaboratorystudywasconductedtoevaluatethevalidityofthemodifiedAOACmethodfordeterminationoftotaldietaryfiberbyTadaandInnami,inwhichthe3-stepenzymaticdigestionprocessinAOACMethod991.43ismodifiedtoa2-stepprocesswithoutpHadjustment.Totaldietaryfibercontentsin8representativefoodstuffsweremeasuredusingboththeoriginalAOACMethod991.43andthemodifiedmethodin6researchfacilitiesinJapan.Repeatabilityrelativestandarddeviations,reproducibilityrelativestandarddeviations,andHorwitzratiovaluesfromthemodifiedmethodwereequivalenttothosefromAOACMethod991.43,exceptinthericesample.However,thisexceptionalcaseshowninthemodifiedmethodwasentirelydissolvedbytheadditionof-amylasestabilizingagents.Themodifiedmethod,whichshortenstheprocessofenzymaticdigestionfrom3to2stepsandinwhichonlyreactiontemperatureisadjustedunderthesamepH,wasfoundnotonlytogiveaccuratevaluescomparabletotheoriginalmethod,butalsotosubstantiallyreducethelaborrequiredbythelaboratorystaffintheprocessofroutineanalysis.Thisstudyrevealedthatthevalidityofthemodifiedmethodwasfurtherensuredbyadding-amylasestabilizingagentstothereactionsystem.

NutritioNISTsrecommendincreasingtheintakeofsolubledietaryfiber(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.

Animprovedmethodfordirectdeterminationofavailablecarbohydratesinlow-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.

Folatecontentinsomegluten-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.

Wheatflourwasextrudedatdifferentconditionsofbarreltemperature(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.

Rapidlydigestible(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.

Cerealstarchgranuleswithhigh(>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.