Megazyme/Amylazyme Tablets/TAMZ1000T/1,000 Tablets

Newchromogenicsubstrateshavebeendevelopedforthequantitativeassayofalpha-amylaseand(1→4)-β-D-glucanase.Thesewerepreparedbychemicallymodifyingamyloseorcellulosebeforedyeing,toincreasesolubility.Afterdyeing,thesubstrateswereeithersolubleorcouldbereadilydispersedtoformfine,gelatinoussuspensions.Assaysbasedontheuseofthesesubstratesaresensitiveandhighlyspecificforeitheralpha-amylaseor(1→4)-β-D-glucanase.Themethodofpreparationcanalsobeappliedtoobtainsubstratesforotherendo-hydrolases.

Hydrolysisofmannan-typepolysaccharidesbyβ-mannanaseisdependentonsubstitutiononandwithinthemain-chainaswellasthesourceoftheβ-mannanaseemployed.Characterisationofreactionproductscanbeusedtodefinethesub-sitebindingrequirementsoftheenzymesaswellasthefine-structuresofthepolysaccharides.Actionofendo-arabinanaseandendo-galactanaseonarabinansandarabinogalactansisdescribed.Specificassaysforendo-arabinanaseandarabinan(infruit-juiceconcentrates)arereported.

Enzymicdegradationofcarbohydratesisofmajorsignificanceintheindustrialprocessingofcerealsandfruits.Intheproductionofbeer,barleyisgerminatedunderwelldefinedconditions(malting)toinducemaximumenzymesynthesiswithminimumrespirationofreservecarbohydrates.Thegrainsaredriedandthenextractedwithwaterundercontrolledconditions.Theamylolyticenzymessynthesizedduringmalting,aswellasthosepresentintheoriginalbarley,convertthestarchreservestofermentablesugars.Otherenzymesactonthecellwallpolysaccharides,mixed-linkageβ-glucanandarabinoxylan,reducingtheviscosityandthusaidingfiltration,andreducingthepossibilityofsubsequentprecipitationofpolymericmaterial.Inbaking,β-amylaseandα-amylasegivecontrolleddegradationofstarchtofermentablesugarssoastosustainyeastgrowthandgasproduction.Excessquantitiesofα-amylaseintheflourresultinexcessivedegradationofstarchduringbakingwhichinturngivesastickycrumbtextureandsubsequentproblemswithbreadslicing.Juiceyieldfromfruitpulpissignificantlyimprovedifcell-walldegradingenzymesareusedtodestroythethree-dimensionalstructureandwaterbindingcapacityofthepecticpolysaccharidecomponentsofthecellwalls.Problemsofroutineandreliableassayofcarbohydratedegradingenzymesinthepresenceofhighlevelsofsugarcompoundsareexperiencedwithsuchindustrialprocess.

Afinebalanceexistsbetweenenzymeactivityandtheadverseeffectsassociatedwithfeedprocessing.Accurateestimationofenzymeactivityinthefeedisapre-requisitetooptimisingtheresponse.

InGeneral,thedevelopmentofmethodsformeasuringα-amylaseispioneeredintheclinicalchemistryfieldandthentranslatedtootherindustries,suchasthecerealsandfermentationindustries.Inmanyinstances,thistransferoftechnologyhasbeendifficultorimpossibletoachieveduetothepresenceofinterferingenzymesorsugarsandtodifferencesinthepropertiesoftheenzymesbeinganalysed.Thisarticledescribesmanyofthecommonlyusedmethodsformeasuringα-amylaseinthecereals,food,andfermentationindustriesanddiscussessomeoftheadvantagesandlimitationsofeach.

Deoxynivalenol(DON)isamycotoxinwhichcanbeproducedincerealgrainsinfectedbyFusariumHeadBlight(FHB).AlterationofDONbytheplantofteninvolvesconjugationoftherespectivemycotoxintocertainfunctionalgroupsormolecules.ConjugationofDONwithglucoseresultsinDeoxynivalenol-3-β-D-glucopyranoside(D3G),whichhasbeenfoundtobethemainDONmetaboliteinwheat.TheobjectiveofthisresearchwastoidentifythefateofD3GandDONduringwheatprocessingusingLC–MS–MSandGC,respectively.Therewasanapproximatereductionof61.8%inthedetectedDONleveloftheflourcomparedtothewholewheat.DONlevelsdetectedduringthefermentationstageweresignificantlyhigher(P<0.05)=""than=""the=""mixed=""dough.=""d3g=""detected=""in=""the=""flour=""was=""23.7%=""lower=""than=""detected=""in=""the=""whole=""wheat.=""there=""were=""no=""significant=""differences="">P<0.05)=""in=""the=""d3g=""detected=""in=""the=""dough=""samples.=""however,=""the=""baked=""bread=""had=""significantly="">P<0.05)=""less=""d3g=""detected=""than=""the=""dough.=""experiments=""were=""conducted=""to=""determine=""the=""effect=""of=""enzyme=""hydrolysis=""on=""don=""detection=""in=""whole=""wheat.=""there=""were=""significant=""differences="">P<0.05)=""between=""the=""wheat=""treated=""with=""α-amylase,=""cellulase,=""protease,=""and=""xylanase.=""don=""detection=""levels=""were=""significantly="">P<0.05)=""higher=""after=""treatment=""with=""protease=""(16%)=""and=""xylanase=""(39%)=""compared=""to=""the=""wheat=""composite.=""these=""results=""suggest=""that=""don=""may=""be=""bound=""or=""embedded=""to=""the=""cell=""wall=""matrix=""or=""protein=""component=""of=""the=""wheat=""kernel=""due=""to=""the=""rise=""in=""detection=""of=""don=""after=""these=""enzyme=""treatments.="">

Someamylasescandelaybreadstalingand/orstarch(amylopectin)retrogradation,butthemolecularbasisofthiseffectremainslittleunderstood.Inordertoincreaseourinsightintheseaspectsofamylasefunctionality,severalamylaseswereaddedinapurewheat-starch-containingmodelsystemandsubjectedtoaheatingstepcorrespondingtothatinthebakingphaseinbreadmaking.Next,theeffectsofthelimitedamylolyticdegradationontherapidviscoanalyser(RVA)rheologicalpropertiesofstarchwerestudiedandtheaccompanyingchangesintheamylopectinmolecularproperties(suchaschainlengthdistribution)investigated.Thedifferentamylasesclearlyaffectedthemolecularstructureofamylopectintoadifferentextent,whichcouldberelatedtotheirmodeofactionandtheenzymeactivitylevelsadded.BacillussubtilisandAspergillusoryzaeα-amylaseshadonlyalimitedimpactonthesidechaindistributionoftheamylopectinmolecules,presumablyduetotheirpreferentialhydrolysisofinternalchainsegmentsandthelowenzymeactivityaddedintheRVA.Incontrast,porcinepancreaticα-amylaseandBacillusstearothermophilusmaltogenicα-amylase,bothwithhigherdegreeofmultipleattackandusedathigherenzymeactivitylevels,hadamarkedinfluenceontheamylopectinmolecularstructure.Moreinparticular,underthetestconditions,themaltogenicα-amylasereducedtheaveragechainlengthoftheouterchainsby50%.Presumably,thiswillaffectamylopectinretrogradationtoalargeextent.Theresultscontributetoabetterunderstandingofamylasefunctionalityinstarchyfoods.

Unlikewheatbread,thedoughofwhichhasavisco-elasticnetworkandhighgas-holdingcapacity,oatbreadgenerallyhasalowvolumeandadensestructure.Weshowedearlierthatincludingryewater-extractablecomponentsinanoatbreadbatterrecipeincreasesloafvolumebyca.30%(PaulyandDelcour,submittedasback-to-backpublication).Weherereportoneffortstoidentifytheactivefactor(s).Anionexchangechromatographyallowedenrichingtheactivefactor(s).Thisandthefactthatonlyalimitedvolumeincreasewasobservedwhenoatbatterwassupplementedwithboiledryeextractindicatethatproteinsarelikelythemostimportantcomponentsresponsibleforthevolumeincrease.Whilethemostactivefactor(s)hadapIbelow4.5,componentswithpIvaluesbetween4.5and8.5alsocontributedtooatloafvolume.Alkalineryecomponents(pI>8.5)orryearabinoxylanhadnoimpact.Ryewater-extractablecomponentssmallerthan6–8kDaalsohadapositiveimpactonloafvolume.

Loafvolumeandcrumbstructureofoatbreadarenotcomparabletothoseofbreadfromwheatflour.Hydrocolloids,surfactantsand/orenzymesareoftenincludedinoatbatterrecipesforqualityenhancementreasons.Inthisstudy,weexaminedtheimpactofwater-extractablecomponentsfrombarley,oat,ryeandwheatflouronoatbreadquality.Wespeculatedthatsuchwaterextractscontaincomponentswhichalsowouldenhancethequalityofoatbread.Asexpected,extractprotein,non-starchpolysaccharide,lipidandenzymelevelsvariedwidelyamongstthedifferentcerealfloursused.Theextractsalsovariedinfoamingpropertiesandextractviscosities.Ryeflourcontainedthehighestlevelofwater-extractablecomponents.Inclusionofryeaqueousextractresultedinthelargestloafvolumeincreaseandinsoftercrumbthannotedforcontroloatbread.Rheofermentometeranalysesshowedthatthemomentofgascellopeningwasdelayedwhenryeextractwasadded,indicatingimprovedbattergascellstabilization,whilecollapseduringbakingwasnotaffected.Theoatbreadimprovingeffectoftheryeextractislikelyduetoacombinationoftheimpactofdifferentofitsconstituentssuchasenzymesandsurfaceactivecomponents.