Itiswithoutdoubtthattestingplaysapivotalrolethroughoutthewholeofthevinificationprocess.ToproducethebestpossIBLequalitywineandtominimiseprocessproblemssuchas“stuck”fermentationortroublesomeinfections,itisnowrecognisedthatifpossibletestingshouldbeginpriortoharvestingofthegrapesandcontinuethroughtobottling.TrADItionalmethodsofwineanalysisareoftenexpensive,timeconsuming,requireeitherelaborateequipmentorspecialistexpertiseandfrequentlylackaccuracy.However,enzymaticbio-analysisenablestheaccuratemeasurementofthevastmajorityofanalytesofinteresttothewinemaker,usingjustonepieceofapparatus,thespectrophotometer(seepreviousissueNo.116foradetailedtechnicalreview).Grapejuiceandwineareamenabletoenzymatictestingasbeingliquidstheyarehomogenous,easytomanipulate,andcangenerallybeanalysedwithoutanysamplepreparation.

ManyoftheenzymatictestkitsareofficialmethodsofprestigiousorganisationssuchastheAssociationofOfficialAnalyticalChemicals(AOAC)andtheAmericanAssociationofCerealChemists(AACC)inresponsetotheinterestfromoenologists.Megazymedecidedtouseitslonghistoryofenzymaticbio-analysistomakeasignificantcontributiontothewineindustry,bythedevelopmentofarangeofadvancedenzymatictestkits.Thistaskhasnowbeensuccessfullycompletedthroughthestrategicandcomprehensiveprocessofidentifyinglimitationsofexistingenzymaticbio-analysistestkitswheretheyoccurred,andthenusingadvancedtechniques,suchasmolecularBIOLOGy(photo1),torapidlyovercomethem.Noveltestkitshavealsobeendevelopedforanalytesofemerginginteresttotheoenologist,suchasyeastavailablenitrogen(YAN;seepages2-3ofissue117article),orwherepreviouslyenzymesweresimplyeithernotavailable,orweretooexpensivetoemploy,suchasforD-mannitolanalysis.

Background:Nitricoxide(NO)playsavitalroleinmaintainingthesurvivabilityofcirculatingerythrocytes.HerewehaveinvestigatedwhetherNOdepletionassociatedwithvisceralleishmaniasis(VL)isresponsibleforthereducedsurvivaloferythrocytesobservedduringthedisease.Methods:Infectedhamstersweretreatedwithstandardanti-leishmanialsodiumstibogluconate(SAG)andNOdonorisosorbidedinitrate(ISD).ErythrophagocytosisbymacrophageswasdeterminedbylabellingthecellswithFITCfollowedbyflowcytometry.Aggregationofband3wasestimatedfromband3associatedEMAfluorescence.Caspase3activitywasmeasuredusingimmunosorbentassaykit.Phosphatidylserine(PS)externalizationandcellshrinkageweredeterminedusingannexinV.AminophspholipidtranslocaseandscramblaseactivitiesweremeasuredfollowingNBD-PSandNBD-PCinternalization,respectively.Results:ImpairmentofbothsynthesisanduptakeofNOresultedindecreasedbioavailabilityofthissignalingmoleculeinerythrocytesinVL.NOlevelwasreplenishedaftersimultaneoustreatmentwithISDandSAG.Combinationtreatmentdecreasedredcellapoptosisininfectedanimalsbydeactivatingcaspase3throughs-nitrosylation.Drugtreatmentpreventedinfection-mediatedATPdepletionandalteredcalciumhomeostasisinerythrocytes.Improvedmetabolicenvironmenteffectivelyamendeddysregulationofaminophospholipidtranslocaseandscramblase,whichinturnreducedcellshrinkage,andexposureofphosphatidylserineonthecellsurfaceunderthediseasedcondition.Conclusionandgeneralsignificance:Inthisstudy,wehaveidentifiedNOdepletiontobeanimportantfactorinpromotingprematurehemolysiswiththeprogressofleishmanialinfection.ThestudyimplicatesNOtobeapossibletargetforfuturedrugdevelopmenttowardsthepromotionoferythrocytesurvivalinVL.

Wineisacomplexconsumerproductproducedpredominatelybytheactionofyeastupongrapejuice.Modelmustsystemshaveproventobeidealforstudiesintotheeffectsoffermentationconditionsontheproductionofcertainwinevolatiles.Toclarifythecontributionofgrapejuicetotheproductionofwinevolatiles,wehaveemployedamodelmustsystemspikedwithincreasingamountsofgrapejuice(RieslingorCabernetSauvignon).TheresultingfermentedwineswereanalyzedbySPME-GC-MSandthedataobtainedgroupedusingANOVAandclusteranalysestorevealthosecompoundsthatvariedinconcentrationwithreproducibletrendsrelativetojuiceconcentration.Suchgroupinghighlightsthosecompoundsthataregrape-dependentorforwhichproductionismodulatedbygrapecomposition.Insomecases,increasingtheproportionofgrapejuiceinthefermentationsstimulatedtheproductionofcertainesterstolevelsbetween2-and140-foldhigherthanthoseseeninfermentationsmadewithmodelgrapejuicemediaalone.Theidentificationofthegrapecomponentsresponsiblefortheincreasedproductionofthesewinevolatileswillhaveimplicationsfortheimpactofgrapeproductionandenologyonwineflavorandaroma.

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.

Wineisacomplexconsumerproductproducedpredominatelybytheactionofyeastupongrapejuicemusts.Modelmustsystemshaveprovenidealforstudiesoftheeffectsoffermentationconditionsontheproductionofcertainwinevolatiles.Toidentifygrape-derivedprecursorstoacetateesters,modelfermentationsystemsweredevelopedbyspikingprecursorsintomodelmustatdifferentconcentrations.Solid-phasemicroextraction–gaschromatgraphymassspectrometryanalysisofthefermentedwinesshowedthatavarietyofgrape-derivedaliphaticalcoholsandaldehydesareprecursorstoacetateesters.TheC6compoundshexan-1-ol,hexenal,(E)-2-hexen-1-ol,and(E)-2-hexenalareallprecursorstohexylacetate,andoctanolandbenzylalcoholareprecursorstooctylacetateandbenzylacetate,respectively.Inthesecases,thepostfermentationconcentrationofanacetateesterincreasedproportionallywiththeprefermentationconcentrationoftherespectiveprecursorinthemodelmust.Determiningviticulturalorwinemakingmethodstoaltertheprefermentationconcentrationofprecursorcompoundsorchangetheprecursor-to-acetateesterratiowillhaveimplicationsuponthefinalflavorandaromaofwines.

L-Ornithinecouldserveasanintermediateinthebiobasedproductionof1,4-diaminobutanefromL-arginine.Usingtheconceptofbiorefinery,L-argininecouldbecomewidelyavailablefrombiomasswastestreamsviathenitrogenstoragepolypeptidecyanophycin.SelectivehydrolysisofL-argininetoL-ornithineisdifficulttoperformchemically,thereforethestabilizationandimmobilizationofBacillussubtilisarginase(EC3.5.3.1)wasstudiedinacontinuouslystirredmembranereactorsystem.InitialpHofthesubstratesolution,additionofL-asparticacidandreducingagentsallappearedtohaveaneffectontheoperationalstabilityofB.subtilisarginase.Aremarkablygoodoperationalstability(totalturnovernumber,TTN=1.13.10⁸)atthepHofargininefreebase(pH11.0)wasobserved,whichwasfurtherimprovedwiththeadditionofsodiumdithionitetothesubstratesolution(TTN>1.10⁹).B.subtilisarginasewassuccessfullyimmobilizedonthreecommerciallyavailableepoxy-activatedsupports.ImmobilizationonSepabeadsEC-EPwasmostpromising,resultinginarecoveredactivityof75%andenhancedThermostability.Inconclusion,thestabilizationandimmobilizationofB.subtilisarginasehasopeneduppossibilitiesforitsapplicationinthebiobasedproductionofnitrogen-containingchemicalsasanalternativetothepetRochemicalproduction.

Theabilityofcellstorespondtoenvironmentalchangesandadapttheirmetabolismenablescellsurvivalunderstressfulconditions.ThebuddingyeastSaccharomycescerevisiae(S.cerevisiae)isparticularlywelladaptedtotheharshconditionsofanaerobicwinefermentation.However,S.cerevisiaegenefunctionhasnotbeenpreviouslysystematicallyinterrogatedunderconditionsofindustrialfermentation.Weperformedagenome-widestudyofessentialandnonessentialS.cerevisiaegenerequirementsduringgrapejuicefermentationtoidentifydeletionstrainsthatareeitherdepletedorenrichedwithintheviablefermentativepopulation.Genesthatfunctioninautophagyandubiquitin-proteasomedegradationarerequiredforoptimalsurvivalduringfermentation,whereasgenesthatfunctioninribosomeassemblyandperoxisomebiogenesisimpairfitnessduringfermentation.Wealsouncoverfermentationphenotypesfor139uncharacterizedgeneswithnopreviouslyknowncellularfunction.Wedemonstratethatautophagyisinducedearlyinwinefermentationinanitrogen-repleteenvironment,suggestingthatautophagymaybetriggeredbyotherformsofstressthatariseduringfermentation.Theseresultsprovideinsightsintothecomplexfermentationprocessandsuggestpossiblemeansforimprovementofindustrialfermentationstrains.

Thepathwayforthebiogenesisofvarietalthiols,suchas3-mercaptohexanol(3MH),3-mercaptohexylacetate(3MHA)and4-mercapto-4-methylpentan-2-one(4MMP)inSauvignonblanc(SB)winesisstillanopenquestion.Varietalthioldevelopmentrequiresyeastactivity,butpoorcorrelationhasbeenfoundbetweenthiolsandtheirputativerespectiveprecursors.Thisresearchisthefirstapplicationofmetabolomicstounravelmetabolitesinthegrapejuicethataffecttheproductionofvarietalthiolsinwines.Comprehensivemetaboliteprofilingof63commerciallyharvestedSBjuiceswereperformedbycombininggaschromatography–massspectrometryandnuclearmagneticresonancespectroscopy.Thesejuiceswerefermentedundercontrolledlaboratoryconditionsusingacommercialyeaststrain(EC1118)at15°C.Correlationofthiolconcentrationinthewineswithinitialmetaboliteprofilesidentified24metabolitesthatshowedpositivecorrelation(R>0.3)withboth3MHand3MHA,whileonlyglutaminehadpositivecorrelationwith4MMP.Subsequently,wecarriedoutjuicemanipulationexperimentsbyaddingsubsetsofthese24metabolitesina2011SBgrapejuiceinordertovalidatethehypothesesgeneratedbymetabolomics.ThejuicemanipulationresultsconfirmedmetabolomicshypothesesandrevealedgrapejuicemetabolitesthatsignificantlyimpactonthedevelopmentofthreemajorvarietalthiolsandotheraromacompoundsofSBwines.

Biguanidessuchasmetforminarewidelyusedworldwideforthetreatmentoftype-2diabetes.TheidentificationofguanidineandrelatedcompoundsinFrenchlilacplant(GalegaofficinalisL.)ledtothedevelopmentofbiguanides.Despiteoftheirplantorigin,biguanideshavenotbeenreportedinplants.Theobjectiveofthisstudywastoquantifybiguaniderelatedcompounds(BRCs)inexperimentallyorclinicallysubstantiatedantidiabeticfunctionalplantfoodsandpotatoes.ThecorrectedresultsoftheVoges–Proskauer(V–P)assaysuggestthatthehighestamountsofBRCsarepresentingreencurryleaves(Murrayakoenigii(L.)Sprengel)followedbyfenugreekseeds(Trigonellafoenum-graecumL.),greenbittergourd(MomordicacharantiaDescourt.),andpotato(SolanumtuberosumL.).Whereas,garlic(AlliumsativumL.),andsweetpotato(Ipomeabatatas(L.)Lam.)containnegligibleamountsofBRCs.Inaddition,thepossiblebiosyntheticroutesofbiguanideintheseplantfoodsarediscussed.

Aims:Theaimofthisresearchwastostudytheeffectoftime,temperature,sugarcontentandadditionofdiammoniumphosphate(DAP)onochratoxinA(OTA)removalbytwostrainsofSaccharomycescerevisiaeusingacompletelyrandomizeddesign.MethodsandResults:ThestrainsweregrowninamediumcontainingOTA(2μgl^-1),twosugarlevels(200and250gl^-1),withorwithoutDAP(300mgl^-1),andincubatedat25–30°C.Theyeastswereabletodecreasethetoxinamountbyc.70%,withthehighestremovingeffectobservedafter3daysat30°Cinthepresenceof250gl^-1ofsugarsandwithDAP;after10days,thetoxinwaspartiallyreleasedintothemedium.Thestrainsproducedhighethanolandglycerolcontents,showedhightolerancetosingle/combinedstressconditionsandpossessedβ-D-glucosidase,pectinaseandxylanaseactivities.OchratoxinAremovalwasaffectedbytime,temperature,sugarandadditionofDAP.Moreover,thephenomenonwasreversible.Conclusions:OchratoxinAremovalwasaffectedbytime,temperature,sugarandadditionofDAP.Moreover,thephenomenonwasreversible.SignificanceandImpactoftheStudy:OchratoxinAremovalcouldbeaninterestingtraitfortheselectionofpromisingstrains;however,thestrainsremovingefficientlythetoxincouldreleaseitback;thus,theselectionofthestartershouldtakeintoaccountboththeremovalandthebindingabilityofOTA.

Thepresentstudyanalyzedtheviableand/orculturablepopulationsofSaccharomycescerevisiae,HanseniasporauvarumandStarmerellabacillaris(synonymCandidazemplinina)duringlaboratorygrapemustfermentation,inordertoinvestigatetheinteractionbetweenthethreespeciesconsidered.Firstly,populationdynamicsduringwinefermentationwerefollowedbyculture-dependenttechniques,andnon-Saccharomycesyeastbecamenon-culturableatlatestagesoffermentationwhenS.cerevisiaedominated.Fourdifferentculture-independenttechniqueswerefurtherappliedtodetectviableyeastcellsatthelatestageoffermentation.BothquantitativePCRtechniquesapplied,namelyethidiummonoazidebromide(EMA)-qPCRandReverseTranscription(RT)-qPCR,detectedH.uvarumandStarm.bacillarisataconcentrationof10⁵to10⁶cells/mL.Thesenon-culturablecellshadmembranesimpermeabletoEMAandstablerRNA.ThebackgroundsignalsfromdeadcellsdidnotinterferewiththequantificationofviablecellsinwinesamplesbyEMA-qPCRtechnique.Asaqualitativeculture-independenttechnique,DGGEtechniquewascoupledwithEMAtreatment(EMA-PCR-DGGE)orwithRT(RT-PCR-DGGE).WithEMA-PCR-DGGEnon-SaccharomycesspeciesduringfermentationweredetectedalthoughitwaslimitedbythepredominanceofS.cerevisiae.

ThekineticcharacterizationofarginaseactivityofacommercialS. cerevisiaestrainwascarriedoutforthefirsttime,estimatingthekineticparameters(V_max,K_0.5andV_max/K_0.5)throughoutalcoholicfermentationinordertoinvestigatethecatalyticefficiencyoftheenzymeanditsabilityinmetabolizingargininetosustainbiosyntheticprocesses.Alcoholicfermentationwascarriedoutatthreedifferenttemperatures(15,20,25°C)insemi-syntheticgrapejuiceaddedwitharginineatusualmaximalconcentration(1 g L^-1)ingrapemust.Arginineuptakewasquiteconstantthroughoutfermentationprocessanditwasmoreeffectivelyassimilatedduringhightemperaturefermentation(20and25°C)thanat15°C.Thesigmoidalbehaviorofyeastarginasekineticcurves,wellfittedtotheHillequation,indicatedamechanismofpositivecooperativityforthetrimericenzyme.ThehighestV_max(4740.0Umg^-1_BSAeq)andthemaximalcatalyticefficiency(78.87min^-1)wereobservedwhenfermentationwasat20°Capproximately3daysaftertheinoculum.Moreover,theK_0.5valuewassimilar(53–60 mg mL^-1)whenmaximalcatalyticefficiencywasachieved,thusindicatingthattheaffinityofenzymeforthesubstrateisnotalteredbyfermentationtemperaturewhichonlyaffectedproductreleasevelocityandthereforeV_max/K_0.5ratio.