Megazyme/βGlucan (Barley; Medium Viscosity)/PBGBM/5 grams

Asimpleandquantitativemethodforthedeterminationof(1→3)(1→4)-β-D-glucaninbarleyflourandmaltisdescribed.Themethodallowsdirectanalysisofβ-glucaninflourandmaltslurries.Mixed-linkageβ-glucanisspecificallydepolymerizedwithahighlypurified(1→3)(1→4)-β-D-glucanase(lichenase),fromBacillussubtilis,totri-,tetra-andhigherdegreeofpolymerization(d.p.)oligosaccharides.Theseoligosaccharidesarethenspecificallyandquantitativelyhydrolysedtoglucoseusingpurifiedβ-D-glucosidase.Theglucoseisthenspecificallydeterminedusingglucoseoxidase/peroxidasereagent.Sincebarleyflourscontainonlylowlevelsofglucose,andmaltosaccharidesdonotinterferewiththeassay,removaloflowd.p.sugarsisnotnecessary.Blankvaluesaredeterminedforeachsampleallowingthedirectmeasurementofβ-glucaninmaltsamples.α-Amylasedoesnotinterferewiththeassay.Themethodissuitablefortheroutineanalysisofβ-glucaninbarleysamplesderivedfrombreedingprograms;50samplescanbeanalysedbyasingleoperatorinaday.Evaluationofthetechniqueondifferentdayshasindicatedameanstandarderrorof0–1forbarleyfloursamplescontaining3–8and4–6%(w/w)β-glucancontent.

Amethoddevelopedforthequantificationof(1→3)(1→4)-β-D-glucaninbarleyflourhasbeenmodifiedtoallowitsuseinthemeasurementofthiscomponentinmalt,wort,beerandspentgrain.Formaltsamples,freeD-glucosewasfirstremovedwithaqueousethanol.Quantificationofthepolymerinwortandbeersamplesinvolvedprecipitationoftheβ-glucanwithammoniumsulphatefollowedbywashingwithaqueousethanoltoremovefreeD-glucose.Spentgrainwaslyophilisedandmilledandthenanalysedbythemethoddevelopedformalt.Inallcases,theβ-glucanwasdepolymerisedwithlichenaseandtheresultantβ-gluco-oligosaccharideshydrolysedtoD-glucosewithβ-D-glucosidase.ThereleasedD-glucosewasthenspecificallydeterminedusingglucoseoxidase-peroxidasereagent.

Mixedlinkageβ-glucaneandpentosanes(mainlyarabinoxylanes)arethemajorendospermcell-wallpolysaccharidesofbarleyandwheatrespectively.Thesepolysaccharides,althoughminorcomponentsofthewholegrain,significantlyaffecttheindustrialutilizationofthesecereals.Themodificationofbarleycornsduringmaltingrequiresthedissolutionoftheβ-glucaneinthecell-wallofthestarchendosperm.Highβ-glucaneconcentrationinwortandbeereffecttherateoffiltrationandcanalsoleadtoprecipitateorgelformationinthefinalproduct.Inasimilarmanner,pentosaneisthoughttocausefiltrationproblemswithwheatstarchhydrolysatesbyincreasingviscosityandbyproducinggelatinousprecipitatewhichblocksfilters.Ironically,itisthissameviscositybuildingandwaterbindingcapacitywhichisconsideredtorenderpentosanesofconsiderablevalueindoughdevelopmentandbreadstorage(anti-stalingfunctions).Inthecurrentpaper,someaspectsofthebeneficialanddetrimentaleffectsofpentosanesandβ-glucaneintheindustrialutilizationofwheatandbarleyarediscussed.Morespecifically,enzymicmethodsforthepreparation,analysisandidentificationofthesepolysaccharidesandfortheremovaloftheirfunctionalproperties,aredescribedindetail.

Acommerciallyavailableenzymicmethodforthequantitativemeasurementof(1→3),(1→4)-β-glucanhasbeensimplifiedtoallowanalysisofupto10grainsamplesin70minorof100–200samplesbyasingleoperatorinaday.Theseimprovementshavebeenachievedwithnolossinaccuracyorprecisionandwithanincreaseinreliability.Theglucoseoxidase/peroxidasereagenthasbeensignificantlyimprovedtoensurecolourstabilityforperiodsofupto1hafterdevelopment.Someproblemsexperiencedwiththeoriginalmethodhavebeenaddressedandresolved,andfurtherexperimentstodemonstratethequantitativenatureoftheassayhavebeendesignedandperformed.

Fermentationofβ-glucanfractionsfrombarley[averagemolecularmass(MM),of243,172,and137kDa]andoats(averageMMof230and150kDa)bythehumanfaecalmicrobiotawasinvestigated.FractionsweresupplementedtopH-controlledanaerobicbatchculturefermentersinoculatedwithhumanfaecalsamplesfromthreedonors,intriplicate,foreachsubstrate.Microbiotachangesweremonitoredbyfluorescentinsituhybridization;groupsenumeratedwere:Bifidobacteriumgenus,BacteroidesandPrevotellagroup,Clostridiumhistolyticumsubgroup,Ruminococcus-Eubacterium-Clostridium(REC)cluster,Lactobacillus-Enterococcusgroup,Atopobiumcluster,andclostridialclusterIX.Short-chainfattyacidsandlacticacidweremeasuredbyHPLC.TheC.histolyticumsubgroupincreasedsignificantlyinallvesselsandclostridialclusterIXmaintainedhighpopulationswithallfractions.TheBacteroides-Prevotellagroupincreasedwithallbutthe243-kDabarleyand230-kDaoatsubstrates.Ingeneralβ-glucansdisplayednoapparentprebioticpotential.TheSCFAprofile(51:32:17;acetate:propionate:butyrate)wasconsideredpropionate-rich.Inafurtherstudyaβ-glucanoligosaccharidefractionwasproducedwithadegreeofpolymerizationof3-4.Thisfractionwassupplementedtosmall-scalefaecalbatchculturesandgavesignificantincreasesintheLactobacillus-Enterococcusgroup;however,theprebioticpotentialofthisfractionwasmarginalcomparedwiththatofinulin.

CommercialxylanasepreparationShearzyme®,whichcontainstheglycosidehydrolasefamily10endo-1,4-β-D-xylanasefromAspergillusaculeatus,wasusedtoprepareshort-chainarabinoxylo-oligosaccharides(AXOS)fromryearabinoxylan(AX).AmajorAXOSwasformedasahydrolysisproduct.LongerAXOSwerealsoproducedasminorproducts.ThepureGH10xylanasefromA.aculeatuswasusedasacomparisontoensurethattheformedAXOSwereconsequenceoftheendoxylanase‘sfunctioninsteadofsomesideenzymespresentinShearzyme.ThemajorAXOSwaspurifiedandthestructureconfirmedwithvariousanalysismethods(TLC,HPAEC-PAD,MALDI-TOF-MS,andone-andtwo-dimensionalNMRspectroscopywithnano-probe)asα-L-Araf-(1→3)-β-D-Xylp-(1→4)-D-Xylp(arabinoxylobiose).Thisisthefirstreporton¹³CNMRdataofpurearabinoxylobiose.Theyieldofarabinoxylobiosewas12%fromthequantifiedhydrolysisproducts.Inconclusion,GH10endoxylanasefromA.aculeatusisthusabletocutefficientlythexylosidiclinkagenexttothearabinofuranosyl-substitutedxyloseunitwhichisnottypicalforalltheGH10endoxylanases.Interestingly,pureA.aculeatusxylanaseshowednotablyactivitytowardsp-nitrophenyl-β-Dxylopyranose.InpreviouslystudieslongerAXOShavebeenproducedwithShearzymebuttheformationofshort-chainAXOSbyA.aculeatusGH10xylanasehasnotbeenstudiedbefore.

Arabinoxylans(AX)arethepredominantcell-wallpolysaccharidesinwheatflour.Water-extractableAXareessentialfordoughandbreadpropertiesandperformance.However,thereisnospecificandaccuratewayofdeterminingthecontentandstructureofAX.Anenzyme-assistedmethodemployinganefficientenzymemixtureforthetotalhydrolysisofAXwasdevelopedinthepresentwork.Enzymatichydrolysis(EH)isagentlemethodduringwhichnounwantedsugardestructionoccurs.FollowingEH,liberatedmonosaccharideswereanalysedbygaschromatography(GC)andliquidchromatographyusingHPAEC–PAD.Theresultswerecomparedwithacidmethanolysis(AM)andacidhydrolysis(AH).EHperformedbetteroncommerciallyisolatedAXsamplesthanthereferencemethodAM.ItsactioninthewaterextractfromwheatflourwasalsomoreefficientthanthatofAMandcomparabletotheefficiencyofAH.HPAEC–PADrevealedasignificantamountoffructoseinthewaterextractfollowingEH,originatingfromfructansinwheatflournotdetectedintheGCanalysis.Thewheatflourexaminedcontained0.29%water-extractableAX.Thearabinose/xyloseratiowas0.32.Theenzyme-basedmethoddevelopedisapplicableforcomparisonofdifferentwheatfloursandcanbeusedinevaluatingtheeffectofprocessingonthecontentandstructureofwater-extractableAX.

Althoughvasculardementiaisthesecondleadingcauseofdementiaandoftenunderdiagnosed,therearenodrugsyetapprovedforthetreatmentofvasculardementia.Inthisstudy,itisdemonstratedthatwaterextractofTriticumaestivumL.(TALE)andsomeofitscomponentshaveprotectiveeffectsagainstvasculardementia-induceddamagebypreservingthemyelinsheathandinhibitingastrocyticactivation.Thememorytestusedavasculardementiamodelutilizingbilateralligationofthecarotidarteriesofrats.TALE,someofitscomponents,suchasstarch,totaldietaryfiber(TDF),arabinoxylan,β-glucan,anddegradedproductsofarabinoxylan,suchasarabinoseandxylose,wereadministeredtotheanimalsfromday8today14,followingthesurgery.Twenty-onedaysafterthesurgery,thewatermazetestwasperformedfor5days,andthetimetakentofindtheplatformduringtrainingtrials(meanescapelatency)wasmeasured.ThemeanescapelatencywasdecreasedconsistentlyintheTALE-,starch-,TDF-,arabinoxylan-,andarabinose-treatedgroups,comparedwiththatinthevasculardementiagroup.Tomeasurebraindamage,Luxolfastbluestainingandimmunohistochemistryofmyelinbasicprotein(MBP)wereperformedtoobservemyelinsheathinthewhitematter,andimmunohistochemistryofglialfibrillaryacidicprotein(GFAP)wasperformedtoobservetheastrocyticreaction.VasculardementiareducedtheMBPlevelandincreasedtheGFAPlevel.ArabinoseeffectivelyinhibitedtheMBPandGFAPchange,whereasarabinoxylaninhibitedtheGFAPchangeonly.TheseresultssuggestthatTALEandsomeofitscomponentscanbeusedasamedicinalmaterialforthedevelopmentofneuroprotectiveagentsagainstvasculardementia.

Themaltingecosystemconsistsoftwocomponents:thegerminatingcerealgrainsandthecomplexmicrobialcommunity.Yeastsandyeast-likefungiareanimportantpartofthisecosystem,butthecompositionandtheeffectsofthismicrobialgrouphavebeenlargelyunknown.Inthisstudywesurveyedthedevelopmentofyeastsandyeast-likefungiinfourindustrialscalemaltingprocesses.Atotalof136maltingprocesssampleswerecollectedandexaminedforthepresenceofyeastsgrowingat15,25and37°C.Morethan700colonieswereisolatedandcharacterized.TheisolateswerediscriminatedbyPCR-fingerprintingwithmicrosatelliteprimer(M13).YeastsrepresentingdifferentfingerprinttypeswereidentifiedbysequenceanalysisoftheD1/D2domainofthe26SrRNAgene.Furthermore,identifiedyeastswerescreenedfortheproductionofα-amylase,β-glucanase,cellulaseandxylanase.Anumerousanddiverseyeastcommunityconsistingofbothascomycetous(25)andbasidiomycetous(18)specieswasdetectedinthevariousstagesofthemaltingprocess.ThemostfrequentlyisolatedascomycetousyeastsbelongedtothegeneraCandida,Clavispora,Galactomyces,Hanseniaspora,Issatchenkia,Pichia,SaccharomycesandWilliopsisandthebasidiomycetousyeaststoBulleromyces,Filobasidium,Cryptococcus,Rhodotorula,SporobolomycesandTrichosporon.Inaddition,twoascomycetousyeast-likefungi(blackyeasts)belongingtothegeneraAureobasidiumandExophialawerecommonlydetected.Yeastsandyeast-likefungiproducedextracellularhydrolyticenzymeswithapotentiallypositivecontributiontothemaltenzymespectrum.Knowledgeofthemicrobialdiversityprovidesabasisformicrofloramanagementandunderstandingoftheroleofmicrobesinthecerealgerminationprocess.

Thepurposeofthisstudywastoinvestigateandcompareadjuvanteffectsofsolubleβ-glucansfrombarleyandSaccharomychescerevisiaininductionofantigenspecifichumoralimmuneresponses.Micewereimmunizedwithconalbuminatarelativelylowconcentrationinthepresenceofbetaglucans.Anti-conalbuminantibodiesintheseraofimmunizedandcontrolmicewerequantifiedbyELISA.Athighdoses,bothofglucansincreasedeffectivelylevelsofcirculatingIgMandIgGantibodieswhichwerespecificforconalbumin.However,coadministrationofglucanfrombarleyat1μgdoseresultedinloweryieldinIgG1,IgG2a,IgG2b,andIgAlevelsincomparisontothatofyeast-derivedglucanatthesamedose.Wealsofoundthatantigen(conalbumin)specificantibodylevelsenhancedbyβ-glucanfromSaccharomychescerevisiawerealwayshigherthanthoseoftheglucanfrombarley.Basedonthesefindings,weconcludedthat(1,3),(1,6)-β-D-glucanfromyeastcellwallmighthavesuperiorimmunostimulantactivityininductionofantigenspecifichumoralimmuneresponsesover(1,3),(1,4)-β-D-glucanfrombarley.

Purifiedandsemi-purifiedpolysaccharidescharacteristicofcerealswerefermentedinvitrowithapigfaecalinoculum,usingthecumulativegasproductiontechnique,toexaminethekineticsandend-productsoffermentationafter48h.Itwasshownthatarabinoxylanandmixedlinkage(1,3;1,4)β-glucanwererapidlyfermentedifsoluble,whilelesssolublesubstrates(insolublearabinoxylan,maizeandwheatstarchgranules,andbacterialcellulose)weremoreslowlyfermented.Relevantmonosaccharideswerefermentedatverysimilarratestosolublepolymericarabinoxylanandβ-glucan,showingthatdepolymerisationwasnotalimitingstep,incontrasttosomepreviousstudies.Bacterialcelluloseisshowntobeausefulmodelsubstrateforfermentationofplantcellulosewhichisdifficulttoobtainwithoutharshchemicaltreatments.Fermentationend-productswererelatedtokinetics,withslowcarbohydratefermentationresultinginincreasedproteinfermentation.Ratiosofshort-chainfattyacidproductsweresimilarforallarabinoxylanandβ-glucansubstrates.

(1,3)(1,4)-β-D-Glucan(mixed-linkageglucanorMLG),acharacteristichemicelluloseinprimarycellwallsofgrasses,wasinvestigatedtodeterminebothitsroleincellwallsanditsinteractionwithcelluloseandothercellwallpolysaccharidesinvitro.BindingisothermsshowedthatMLGadsorptionontomicrocrystallinecelluloseisslow,irreversible,andtemperature-dependent.MeasurementsusingquartzcrystalmicrobalancewithdissipationmonitoringshowedthatMLGadsorbedirreversiblyontoamorphousregeneratedcellulose,formingathickhydrogel.Oligosaccharideprofilingusingendo-(1,3)(1,4)-β-glucanaseindicatedthattherewasnodifferenceinthefrequencyanddistributionof(1,3)and(1,4)linksinboundandunboundMLG.ThebindingofMLGtocellulosewasreducedifthecellulosesampleswerefirsttreatedwithcertaincellwallpolysaccharides,suchasxyloglucanandglucuronoarabinoxylan.ThetetheringfunctionofMLGincellwallswastestedbyapplyingendo-(1,3)(1,4)-β-glucanasetowallsamplesinaconstantforceextensometer.Cellwallextensionwasnotinduced,whichindicatesthatenzyme-accessibleMLGdoesnottethercellulosefibrilsintoaload-bearingnetwork.

Thedigestionofdietaryfibersisamajorfunctionofthehumanintestinalmicrobiota.Sofarthisfunctionhasbeenattributedtothemicroorganismsinhabitingthecolon,andmanystudieshavefocusedonthisdistalpartofthegastrointestinaltractusingeasilyaccessiblefecalmaterial.However,microbialfermentations,supportedbythepresenceofshort-chainfattyacids,aresuspectedtooccurintheuppersmallintestine,particularlyintheileum.Usingafosmidlibraryfromthehumanilealmucosa,wescreened20,000clonesfortheiractivitiesagainstcarboxymethylcelluloseandxylanschosenasmodelsofthemajorplantcellwall(PCW)polysaccharidesfromdietaryfibres.ElevenpositiveclonesrevealedabroadrangeofCAZymeencodinggenesfromBacteroidesandClostridialesspecies,aswellasPolysaccharideUtilizationLoci(PULs).Thefunctionalglycosidehydrolasegeneswereidentified,andoligosaccharidebreak-downproductsexaminedfromdifferentpolysaccharidesincludingmixed-linkageβ-glucans.CAZymesandPULswerealsoexaminedfortheirprevalenceinhumangutmicrobiome.Severalclustersofgenesoflowprevalenceinfecalmicrobiomesuggestedtheybelongtounidentifiedstrainsratherspecificallyestablishedupstreamthecolon,intheileum.Thus,theilealmucosa-associatedmicrobiotaencompassestheenzymaticpotentialforPCWpolysaccharidedegradationinthesmallintestine.

Mixed-linkageglucan:xyloglucanendotransglucosylase(MXE)isoneofthethreeactivitiesoftherecentlycharacterisedhetero-trans-β-glucanase(HTG),whichamongland-plantsisknownonlyfromEquisetumspecies.ThebiochemicaldetailsoftheMXEreactionwereincompletelyunderstood-detailsthatwouldpromoteunderstandingofMXEsroleinvivoandenableitsfulltechnologicalexploitation.WeinvestigatedHTGssiteofattackononeofitsdonorsubstrates,mixed-linkage(1→3),(1→4)-β-D-glucan(MLG),withradioactiveoligosaccharidesofxyloglucanasacceptorsubstrate.ComparingthreedifferentMLGpreparations,weshowedthattheenzymefavoursthosewithahighcontentofcellotetraoseblocks.Thereactionproductswereanalysedbyenzymicdigestion,thin-layerchromatography,HPLCandgel-permeationchromatography.EquisetumHTGconsistentlycleavedtheMLGatthethirdconsecutiveβ-(1→4)-bondfollowing(towardsthereducingterminus)aβ-(1→3)-bond.Itthenformedaβ-(1→4)-bondbetweentheMLGandthenon-reducingterminalglucoseresidueofthexyloglucanoligosaccharide,consistentwithitsXTHsubfamilymembership.Usingsize-homogeneousbarleyMLGasdonorsubstrate,weshowedthatHTGdoesnotfavouranyparticularregionoftheMLGchainrelativethepolysaccharidesreducingandnon-reducingtermini;rather,itselectsitstargetcellotetraosylunitstochasticallyalongtheMLGmolecule.Thisworkimprovesourunderstandingofhowenzymescanexhibitpromiscuoussubstratespecificitiesandprovidesthefoundationstoexplorestrategiesforengineeringnovelsubstratespecificitiesintotransglycanases.

Theinteractionsofcellulosewithothermajorplantcellwallpolysaccharides-arabinoxylan(AX),xyloglucan(XG)andmixedlinkageglucans(MLG)-havebeeninvestigatedbycharacterisingthearchitectureofcompositedeuteratedcellulosehydrogelsbymeansofSAXSandSANS,combinedwithXRD,NMRandmicroscopy.Theresultsindicatethatcellulose-AXinteractions,limitedtotheribbonssurface,takeplaceviaanon-specificadsorptionmechanism.Incontrast,XGandMLGinteractspecificallywithcellulose,formingtwodifferentfractions:(i)interfibrillardomainsinteractingwiththecellulosemicrofibrilsand(ii)surfacedomains,responsibleforthecross-linkingofribbons.XGco-crystalliseswithcellulose,promotingtheformationofI_β-richermicrofibrilsandformingintercalatedamorphousregions.Ontheotherhand,MLGinteractswithcelluloseformingaparacrystallinecoatinglayer.ThisstructuralroleofXGandMLGinpreventingmicrofibrilaggregationmayhelpexplaintheirkeyfunctioninthecellexpansionprocessofgrowingplanttissues.