TheUrea/Ammonia(Rapid)testkitissuitablefor thespecificandrapidmeasurementandanalysisofureaandammoniainwater,beverages,milkandfoodproducts.
Grapeandwineanalysis:Oenologiststoexploitadvancedtestkits.
Charnock,S.C.&McCleary,B.V.(2005).RevuedesEnology,117,1-5.
LinktoArticle
ReadAbstract
Itiswithoutdoubtthattestingplaysapivotalrolethroughoutthewholeofthevinificationprocess.Toproducethebestposs
IBLequalitywineandtominimiseprocessproblemssuchas“stuck”fermentationortroublesomeinfections,itisnowrecognisedthatifpossibletestingshouldbeginpriortoharvestingofthegrapesandcontinuethroughtobottling.Tr
ADItionalmethodsofwineanalysisareoftenexpensive,timeconsuming,requireeitherelaborateequipmentorspecialistexpertiseandfrequentlylackaccuracy.However,enzymaticbio-analysisenablestheaccuratemeasurementofthevastmajorityofanalytesofinteresttothewinemaker,usingjustonepieceofapparatus,thespectrophotometer(
seepreviousissueNo.116foradetailedtechnicalreview).Grapejuiceandwineareamenabletoenzymatictestingasbeingliquidstheyarehomogenous,easytomanipulate,andcangenerallybeanalysedwithoutanysamplepreparation.
Megazyme“advanced”winetestkitsgeneralcharacteristicsandvalidation.
Charnock,S.J.,McCleary,B.V.,Daverede,C.&Gallant,P.(2006).ReveuedesOenologues,120,1-5.
LinktoArticle
ReadAbstract
ManyoftheenzymatictestkitsareofficialmethodsofprestigiousorganisationssuchastheAssociationofOfficialAnalyticalChemicals(AOAC)andtheAmericanAssociationofCerealChemists(AACC)inresponsetotheinterestfromoenologists.Megazymedecidedtouseitslonghistoryofenzymaticbio-analysistomakeasignificantcontributiontothewineindustry,bythedevelopmentofarangeofadvancedenzymatictestkits.Thistaskhasnowbeensuccessfullycompletedthroughthestrategicandcomprehensiveprocessofidentifyinglimitationsofexistingenzymaticbio-analysistestkitswheretheyoccurred,andthenusingadvancedtechniques,suchasmolecular
BIOLOGy(
photo1),torapidlyovercomethem.Noveltestkitshavealsobeendevelopedforanalytesofemerginginteresttotheoenologist,suchasyeastavailablenitrogen(
YAN;seepages2-3ofissue117article),orwherepreviouslyenzymesweresimplyeithernotavailable,orweretooexpensivetoemploy,suchasforD-mannitolanalysis.
Determinationofureausinghigh-performanceliquidchromatographywithfluorescencedetectionafterautomatedderivatisationwithxanthydrol.
Clark,S.,Francis,P.S.,Conlan,X.A.&Barnett,N.W.(2007).JournalofChromatographyA,1161(1-2),207-213.
LinktoArticle
ReadAbstract
Ahigh-performanceliquidchromatography(HPLC)methodforthedeterminationofureathatincorporatesautomatedderivatisationwithxanthydrol(9H-xanthen-9-ol)isdescribed.Unliketheclassicxanthydrolapproachforthedeterminationofurea,whichinvolvestheprecipitationofdixanthylurea(N,N′-di-9H-xanthen-9-ylurea),thederivatisationprocedureemployedinthismethodproducesN-9H-xanthen-9-ylurea,whichremainsinsolutionandcanbequantifiedusingfluorescencedetection(λex=213nm;λem=308nm)afterchromatographicseparationfrominterferences.Thelimitofdetectionforureawas5×10-8M(0.003mgL-1).Thismethodwasappliedtothedeterminationofureainhumanandanimalurineandinwine.
Ureadegradationinsomewhitewinesbyimmobilizedacidureaseinastirredbioreactor.
Andrich,L.,Esti,M.&Moresi,M.(2010).JournalofAgriculturalandFoodChemistry,58(11),6747-6753.
LinktoArticle
ReadAbstract
ApurifiedacidureasepreparationwascovalentlyimmobilizedontoeitherEupergitC250Lorglutaraldehyde-cross-linkedchitosan-derivativebeads(i.e.,ChitopearlsBCW-3003andBCW-3010).ThekineticsofureadegradationintwotargetItalianwhite(i.e.,GrechettoandSauvignonBlanc)wines,aswellasinamodelwinesolution,byusingtheaboveEupergitC250L-,BCW-3003-,orBCW-3010-basedbiocatalysts,wasconfirmedtobeofthepseudofirstorderwithrespecttotheureaconcentrationintheliquidbulkandnotlimitedbyureamasstransfer.InGrechettoandSauvignonBlancwines,thecorrespondingkineticrateconstantswerequitesimilar,beingabout7,18,or17%ofthatobservedforfreeenzymeinthemodelwinesolution,respectively.Owingtotheirminorsensitivitytothephenoliccontentofthewinestested,thechitosan-basedbiocatalystsmightbepotentiallyemployableinthemakeupofpacked-bedcartridgestocontinuouslyremoveureafromcommercialwines.
Thedeterminationofureainwine–areview.
Francis,P.S.(2006).Australianjournalofgrapeandwineresearch,12(2),97-106.
LinktoArticle
ReadAbstract
TheconcentrationofureainwineisnotroutinelymeasuredinAustralianlaboratories,buthasbeenexaminedinstudiesofyeastmetabolismandtheformationofethylcarbamate,aknowncarcinogen.Foralcoholicbeveragesthatmaycontainhighlevelsofurea,stepshavebeentakentoreducetheconcentrationofureaandthereforepreventethylcarbamateproduction.Methodsforthedeterminationofureainwinecanbegroupedintothreecategoriesthatindicatehowselectivityforureaisachieved;thosebasedoncolour-formingreactions,enzymatichydrolysisandchromatographicseparation.Thetwodominantmethodsusedbyresearchgroupsoverthepastfifteenyearsforthedeterminationofureainwinearebasedontheurea/ammoniatestkitavailablefromBoeringerMannheim/R-Biopharmandthereactionofureawith1-phenyl-1,2-propanedione-2-oxime;botharetime-consumingandlabour-intensive,butinvolverelativelystraightforwardandwell-establishedprocedures.However,otheroptionsareavailablethatmaybebettersuitedtothedesiredapplicationandtheinstrumentationavailableinanyparticularlaboratory.
Energymetabolismofleukemiacells:glycolysisversusoxidativephosphorylation.
Suganuma,K.,Miwa,H.,Imai,N.,Shikami,M.,Gotou,M.,Goto,M.,Mizuno,S.,Takahashi,M.,Yamamoto,H.,Hiramatsu,A.,Wakabayashi,M.,Watarai,M.,Hanamura,I.,Imamura,A.,Mihara,H.&Nitta,M.(2010).Leukemia&Lymphoma,51(11),2112-2119.
LinktoArticle
ReadAbstract
Forgenerationofenergy,cancercellsutilizeglycolysismorevigorouslythanoxidativephosphorylationinmitochondria(Warburgeffect).Weexaminedtheenergymetabolismoffourleukemiacelllinesbyusingglycolysisinhibitor,2-deoxy-D-glucose(2-DG)andinhibitorofoxidativephosphorylation,oligomycin.NB4wasrelativelysensitiveto2-DG(IC50:5.75 mM),consumedmoreglucoseandproducedmorelactate(wasteproductofglycolysis)thanthethreeothercelllines.Consequently,NB4wasconsideredasa“glycolytic”leukemiacellline.DependencyonglycolysisinNB4wasconfirmedbythefactthatglucose(+)FCS(−)mediumshowedmoregrowthandsurvivalthanglucose(−)FCS(+)medium.Alternatively,THP-1,mostresistantto2-DG(IC50:16.14 mM),wasmostsensitivetooligomycin.Thus,THP-1wasrecognizedtobedependentonoxidativephosphorylation.InTHP-1,glucose(−)FCS(+)mediumshowedmoregrowthandsurvivalthanglucose(+)FCS(−)medium.ThedependencyofTHP-1onFCSwasexplained,atleastpartly,byfattyacidoxidationbecauseinhibitoroffattyacidβ-oxidation,etomoxir,augmentedthegrowthsuppressionofTHP-1by2-DG.WealsoexaminedthemechanismsbywhichTHP-1wasresistantto,andNB4wassensitiveto2-DGtreatment.InTHP-1,AMPkinase(AMPK),whichisactivatedwhenATPbecomeslimiting,wasrapidlyphosphorylatedby2-DG,andexpressionofBcl-2wasaugmented,whichmightresultinresistanceto2-DG.Ontheotherhand,AMPKphosphorylationandaugmentationofBcl-2expressionby2-DGwerenotobservedinNB4,whichis2-DGsensitive.Theseresultswillfacilitatethefutureleukemiatherapytargetingmetabolicpathways.
Ureadegradationkineticsinmodelwinesolutionsbyacidureaseimmobilisedontochitosan-derivativebeadsofdifferentsizes.
Andrich,L.,Esti,M.&Moresi,M.(2010).EnzymeandMicrobialTechnology,46(5),397-405.
LinktoArticle
ReadAbstract
Inthiswork,apurifiedacidureasepreparationwascovalentlyimmobilisedontoporouschitosanbeadsofdifferentsize.Thecovalentbindingmethodwasfoundtobemoreefficientthantheadsorptioncross-linkageonewhatevertheglutaraldehyde-to-chitosanbeadratio(YGA/CHI)used.AttheoptimalYGA/CHIratioof0.625gg-1,thespecificactivity(ABi)ofthebiocatalystsdecreasedfromcirca300to70IUg-1wetsupport,asthebeadaveragediameter(dP)increasedfrom0.14to2.2mm.Generally,ABireducedlessthan5%afterpreservationinthewetformat4°Cfor150–170days.OnlythebiocatalystpreparedusingtheChitopearlBCW-3001lostabout40%ofitsinitialactivity.Thekineticsofureadegradationinamodelwinesolutionusingthesebiocatalystswasofthepseudo-firstorderwithrespecttotheureaconcentrationintheliquidbulk,theapparentpseudo-firstorderkineticrateconstant(kIi)rangingfromabouttwothirdstoonefifthofthat(kIF)pertainingtofreeacidurease.Intheoperatingconditionstested,thereactionkineticswasestimatedasunaffectedbythecontributionoftheexternalfilmandintraparticlediffusionmass-transferresistances.Whenthemodelwinesolutionwasenrichedwiththehigh-inhibitorytanninsextractedfromgrapeseeds,atthemaximumleveltested(374±2gGAEm-3)kIireducedtonomorethan(58±9)%ofkIF),thisprovingquiteahigherprotectiveactionagainstsuchcompoundsforthechitosan-basedbiocatalyststowardsfreeorEupergit®C250L-immobilisedacidurease.
Comparativestudyofcolorectalhealthrelatedcompoundsindifferenttypesofbread:Analysisofbreadsamplespreandpostdigestioninabatchfermentationmodelofthehumanintestine.
Hiller,B.,Schlörmann,W.,Glei,M.&Lindhauer,M.G.(2011).FoodChemistry,125(4),1202-1212.
LinktoArticle
ReadAbstract
Sevendifferenttypesofwheatandryebreadwereanalysedforcolorectalhealthrelatedcompounds,preandpostdigestion,inbatchfermentationmodelofthehumanintestine.Predigestion,higheramountsofcolorectalhealth-relateddietaryfibrecompounds(soluble/insoluble/totaldietaryfibre,arabinoxylans,β-glucans)andphytochemicals(mono-/di-phenolicacids,phyticacid,hydroxymethylfurfural)weredetectedinwholemealthaninrefinedflourtypesofbread,aswellasinryeflourtypesthaninwheatflourtypesofbread.Postdigestion,faecalbacterialmetabolitesofcolorectalhealthpromoting(acetate/propionate/butyrate,lactate,freemono-/di-phenolicacids)andimpairing(aminometabolites,bileacidmetabolites)activitieswerefoundinfermentationsupernatantsofbreadsamples.Alltypesofbreadpositivelyaffectedfaecalbacterialmetabolism;amongthedifferenttypesofbread,thehigheststimulationoforganicacidproduction(acetate/propionate/butyrate,lactate)andthelowestdetrimentalbacterialenzymeactivities(β-glucuronidase,urease)weredetectedforwheatflourbread,whereasthestrongestretardationofbacterialbileaciddegradationandthestrongeststimulationofphenolicacidmetaboliterelease(phenylpropionic/phenylpropenoicacidderivatives)wereinducedbywholemealryebread.Thisstudyforthefirsttimepresentsaqualitativeandquantitativeoverviewoverthebroadspectrumofcolorectalhealthrelatedcompoundsinhigh-andlow-fibretypesofbread,preandpostinvitrodigestion,andhighlightsthesignificanceofbreadforthepreventivenutritionalinterventionofcolorectalcancer.
Assessingheterogeneityofthecompositionofmare"smilkinFlanders.
Naert,L.,VandeVyvere,B.,Verhoeven,G.,Duchateau,L.,DeSmet,S.&Coopman,F.(2013).VlaamsDiergeneeskundigTijdschrift,82(1),23-30.
LinktoArticle
ReadAbstract
Inthisstudy,theeffectoffarm,time,seasonandhealthwasevaluatedonthecompositionofmare"smilksoldinFlanders.Thecontentoftheanalyzedcomponents(i.e.fat,fattyacids,protein,lactoferrin,lysozyme,lactoseandurea)differedsignificantly(p<0.0001)=""between=""farms,=""at=""a=""given=""moment=""in=""time.=""within=""each=""farm,=""large=""month-to-month=""variations=""for=""most=""milk=""components="">p<0.01 to="" 0.0001)="" were="" observed.="" the="" variation="" over="" time="" between="" different="" farms="" was="" smaller.="" these="" findings="" indicate="" that="" the="" composition="" of="" the="" mare"s="" milk="" consumer="" portions="" varies="" substantially="" between="" the="" different="" farms="" and="" also="" over="" time="" within="" each="" farm.="" season,="" nutrition,="" udder="" health="" and="" worm="" burden="" are="" believed="" to="" contribute="" significantly="" to="" this="" variation.="">0.01>
Comparativelipidproductionbyoleaginousyeastsinhydrolyzatesoflignocellulosicbiomassandprocessstrategyforhightiters.
Slininger,P.J.,Dien,B.S.,Kurtzman,C.P.,Moser,B.R.,Bakota,E.L.,Thompson,S.R.,,O"Bryan,P.J.,Cotta,M.A.,Balan,V.,Jin,M.,Sousa,L.D.C.&Dale,B.E.&Sousa,L.D.C.(2016).BiotechnologyandBioengineering,113(8),1676-1690.
LinktoArticle
ReadAbstract
Oleaginousyeastscanconvertsugarstolipidswithfattyacidprofilessimilartothoseofvegetableoils,makingthemattractiveforproductionofbiodiesel.Lignocellulosicbiomassisanattractivesourceofsugarsforyeastlipidproductionbecauseitisabundant,potentiallylowcost,andrenewable.However,lignocellulosichydrolyzatesareladenwithbyproductswhichinhibitmicrobialgrowthandmetabolism.Withthegoalofidentifyingoleaginousyeaststrainsabletoconvertplantbiomasstolipids,wescreened32strainsfromtheARSCultureCollection,Peoria,ILtoidentifyfourrobuststrainsabletoproducehighlipidconcentrationsfrombothacidandbase-pretreatedbiomass.Thescreeningwasarrangedintwotiersusingundetoxifiedenzymehydrolyzatesofammoniafiberexpansion(AFEX)-pretreatedcornstoverastheprimaryscreeningmediumandacid-pretreatedswitchgrassasthesecondaryscreeningmediumappliedtostrainspassingtheprimaryscreen.Hydrolyzateswerepreparedat~18–20%solidsloadingtoprovide~110 g/Lsugarsat~56:39:5massratioglucose:xylose:arabinose.AtwostageprocessboostingthemolarC:Nratiofrom60towellabove400inundetoxifiedswitchgrasshydrolyzatewasoptimizedwithrespecttonitrogensource,C:N,andcarbonloading.Usingthisprocessthreestrainswereabletoconsumeaceticacidandnearlyallavailablesugarstoaccumulate50–65%ofcellbiomassaslipid(w/w),toproduce25–30 g/Llipidat0.12–0.22 g/L/hand0.13–0.15 g/gor39–45%ofthetheoreticalyieldatpH6and7,aperformanceunprecedentedinlignocellulosichydrolyzates.Threeofthetopstrainshavenotpreviouslybeenreportedforthebioconversionoflignocellulosetolipids.Thesuccessfulidentificationanddevelopmentoftop-performinglipid-producingyeastinlignocellulosehydrolyzatesisexpectedtoadvancetheeconomicfeasibilityofhighqualitybiodieselandjetfuelsfromrenewablebiomass,expandingthemarketpotentialforlignocellulose-derivedfuelsbeyondethanolforautomobilestotheentireU.S.transportationmarket.
Functionalexpressionofaheterologousnickel-dependent,ATP-independentureaseinSaccharomycescerevisiae.
Milne,N.,Luttik,M.A.H.,Rojas,H.C.,Wahl,A.,VanMaris,A.J.A.,Pronk,J.T.&Daran,J.M.(2015).MetabolicEngineering,30,130-140.
LinktoArticle
ReadAbstract
Inmicrobialprocessesforproductionofproteins,biomassandnitrogen-containingcommoditychemicals,ATPrequirementsfornitrogenassimilationaffectproductyieldsontheenergyproducingsubstrate.InSaccharomycescerevisiae,acurrenthostforheterologousproteinproductionandpotentialplatformforproductionofnitrogen-containingchemicals,uptakeandassimilationofammoniumrequires1ATPperincorporatedNH3.Ureaassimilationbythisyeastismoreenergyefficientbutstillrequires0.5ATPperNH3 produced.TodecreaseATPcostsfornitrogenassimilation,theS.cerevisiaegeneencodingATP-dependenturease(DUR1,2)wasreplacedbyaSchizosaccharomycespombevgeneencodingATP-independenturease(ure2),alongwithitsaccessorygenesureD,ureFandureG.SinceS.pombeure2isaNi2+-dependentenzymeandSaccharomycescerevisiaedoesnotexpressnativeNi2+-dependentenzymes,theS.pombehigh-affinitynickel-transportergene(nic1)wasalsoexpressed.ExpressionoftheS.pombegenesintodur1,2ΔS.cerevisiaeyieldedaninvitroATP-independentureaseactivityof0.44±0.01µmolmin-1 mgprotein-1andrestoredgrowthonureaassolenitrogensource.FunctionalexpressionoftheNic1transporterwasessentialforgrowthonureaatlowNi2+concentrations.ThemaximumspecificgrowthratesoftheengineeredstrainonureaandammoniumwerelowerthanthoseofaDUR1,2referencestrain.Inglucose-limitedchemostatcultureswithureaasnitrogensource,theengineeredstrainexhibitedanincreasedreleaseofammoniaandreducednitrogencontentofthebiomass.Ourresultsindicateanewstrategyforimprovingyeast-basedproductionofnitrogen-containingchemicalsanddemonstratethatNi2+-dependentenzymescanbefunctionallyexpressedinS.cerevisiae.
Ureaandlipidextractiontreatmenteffectsonδ15Nandδ13Cvaluesinpelagicsharks.
Li,Y.,Zhang,Y.,Hussey,N.E.&Dai,X.(2016).RapidCommunicationsinMassSpectrometry,30(1),1-8.
LinktoArticle
ReadAbstract
Rationale:Stableisotopeanalysis(SIA)providesapowerfultooltoinvestigatediverseecologicalquestionsformarinespecies,butstandardizedvaluesarerequiredforcomparativeassessments.Forelasmobranchs,theiruniqueosmoregulatorystrategyinvolvesretentionof15N-depletedureainbodytissuesandthismaybiasδ15Nvalues.Thismaybeaparticularproblemforlargepredatoryspecies,whereδ15Ndiscriminationbetweenpredatorandconsumedpreycanbesmall.Methods:Weevaluatedthreetreatments(deionizedwaterrinsing[DW],chloroform/methanol[LE]andcombinedchloroform/methanolanddeionizedwaterrinsing[LE+DW])appliedtowhitemuscletissueof125individualsfromsevenpelagicsharkspeciesto(i)assessureaandlipideffectsonstableisotopevaluesdeterminedbyIRMSand(ii)investigatemathematicalnormalizationofthesevalues.Results:Forallspeciesexamined,theδ15NvaluesandC:Nratiosincreasedsignificantlyfollowingallthreetreatments,identifyingthaturearemovalisrequiredpriortoSIAofpelagicsharks.Themoremarkedchangeinδ15NvaluesfollowingDW(1.3±0.4‰)andLE+DW(1.2±0.6‰)thanfollowingLEalone(0.7±0.4‰)indicatedthatwaterrinsingwasmoreeffectiveatremovingurea.TheDWandLE+DWtreatmentsloweredthe%Nvalues,resultinginanincreaseinC:Nratiosfromtheunexpectedlowvaluesof<2.6 in="" bulk="" samples="" to="" ~3.1="" ±="" 0.1,="" the="" expected="" value="" of="" protein.="" the="">2.6>13NvaluesofallspeciesalsoincreasedsignificantlyfollowingLEandLE+DWtreatments.Conclusions:Giventhemeanchangeinδ15N(1.2±0.6‰)andδ13Nvalues(0.7±0.4‰)acrosspelagicsharkspecies,itisrecommendedthatmuscletissuesamplesbetreatedwithLE+DWtoefficientlyextractbothureaandlipidstostandardizeisotopicvalues.Mathematicalnormalizationofureaandlipid-extractedδ15NLE+DWandδ13CLE+DWvaluesusingthelipid-extractedδ15NLEandδ13CLEdatawereestablishedforallpelagicsharkspecies.