ThePrimaryAminoNitrogen(PANOPA)AssayKit issuitableforthemeasurementandanalysisofprimaryaminonitrogeningrapejuice/mustandwine.
Grapeandwineanalysis:Oenologiststoexploitadvancedtestkits.
Charnock,S.C.&McCleary,B.V.(2005).RevuedesEnology,117,1-5.
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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.
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ManyoftheenzymatictestkitsareofficialmethodsofprestigiousorganisationssuchastheAssociationofOfficialAnalyticalChemicals(AOAC)andtheAmericanAssociationofCerealChemists(AACC)inresponsetotheinterestfromoenologists.Megazymedecidedtouseitslonghistoryofenzymaticbio-analysistomakeasignificantcontributiontothewineindustry,bythedevelopmentofarangeofadvancedenzymatictestkits.Thistaskhasnowbeensuccessfullycompletedthroughthestrategicandcomprehensiveprocessofidentifyinglimitationsofexistingenzymaticbio-analysistestkitswheretheyoccurred,andthenusingadvancedtechniques,suchasmolecular
BIOLOGy(
photo1),torapidlyovercomethem.Noveltestkitshavealsobeendevelopedforanalytesofemerginginteresttotheoenologist,suchasyeastavailablenitrogen(
YAN;seepages2-3ofissue117article),orwherepreviouslyenzymesweresimplyeithernotavailable,orweretooexpensivetoemploy,suchasforD-mannitolanalysis.
FermentationofstalkjuicesfromdifferentNigeriansorghumcultivarstoethanol.
Nasidi,M.,Agu,R.,YusufDeeni,Y.&Walker,G.(2013).Bioethanol,1(1),20-27.
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Forimprovedproductionofethanolfromsorghumstalkjuicefermentation,cultivationlocationandcultivartypeareimportantfactorstoconsider.Inthepresentstudy,SSV2andKSV8sorghumcultivarswerecultivatedinKanoandKadunastatesinNigeriathatexhibitnotablydifferentrainprecipitationanddiurnaltemperatures.Thecrudestalkjuices(withoutpre-treatmentornutrientsupplementation)wereextractedfromthesesorghumsamplesandfermentedwithadistiller’sstrainoftheyeast,Saccharomycescerevisiae.SugarconsumptionandalcoholproductionweredeterminedbyHPLCandGC-MS,respectively.WhenitwasgrownintheKadunasite,SSV2wasidentifiedasthehighestyieldingsorghumcultivarfromwhichweextractedthemaximumlevelsofextractablesugars(161.50gl-1)thatyieldedfavourableethanollevelsof80.56gl-1followingfermentation.Ourfindingsshowthatrelativelycolderandwettercultivationsitesarepreferredforsorghumstalkjuicedestinedforbioethanolproduction.
SensorcombinationandchemometricvariableselectionforonlinemonitoringofStreptomycescoelicolorfed-batchcultivations.
Ödman,P.,Johansen,C.L.,Olsson,L.,Gernaey,K.V.&Lantz,A.E.(2010).AppliedMicrobiologyandBiotechnology,86(6),1745-1759.
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Fed-batchcultivationsofStreptomycescoelicolor,producingtheantibioticactinorhodin,weremonitoredonlinebymultiwavelengthfluorescencespectroscopyandoff-gasanalysis.Partialleastsquares(PLS),locallyweightedregression,andmultilinearPLS(N-PLS)modelswerebuiltforpredictionofbiomassandsubstrate(casaminoacids)concentrations,respectively.Theeffectofcombinationoffluorescenceandgasanalyzerdataaswellasofdifferentvariableselectionmethodswasinvestigated.Improvedpredictionmodelswereobtainedbycombinationofdatafromthetwosensorsandbyvariableselectionusingageneticalgorithm,intervalPLS,andtheprincipalvariablesmethod,respectively.Astepwisevariableeliminationmethodwasappliedtothethree-wayfluorescencedata,resultinginsimplerandmoreaccurateN-PLSmodels.Thepredictionmodelswerevalidatedusingleave-one-batch-outcross-validation,andthebestmodelshadrootmeansquareerrorofcross-validationvaluesof1.02gl-1biomassand0.8gl-1totalaminoacids,respectively.Thefluorescencedatawerealsoexploredbyparallelfactoranalysis.Theanalysisrevealedfourspectralprofilespresentinthefluorescencedata,threeofwhichwereidentifiedaspyridoxine,NAD(P)H,andflavinnucleotides,respectively.
Analysisofproteinandtotalusablenitrogeninbeerandwineusingamicrowellninhydrinassay.
Abernathy,D.G.,Spedding,G.&Starcher,B.(2009).JournaloftheInstituteofBrewing,115(2),122-127.
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InthisstudywepresentaninhydrinbasedmicrowellassaythatcanbeutilizedinplaceofthetraditionalKjeldahlmethodforthedeterminationoftheproteincontentofbeerorwine.Inaddition,theassayisidealforthedeterminationoffreeaminoacidsinbeer(FAN),atermunderstoodandusedbybrewers,andyeastassimilablenitrogen(YAN)usedbyenologists.Theassayonlymeasuresalphaaminoacidsandammoniasoothernitrogensourcesarenotdetected,resultingina30%reductionintotalproteinofavarietyofbeerscomparedtotheKjeldahlmethod,whichmeasuresnitrogenfromallsources.Theresultsalsoshowedthatonly25%ofthetotal“protein”inbeerisactuallyderivedfrompeptideslargerthan3,500Kd.AnalysisofbeerorwinewiththemicrowellassayfortotalusablenitrogenwascomparedtothestandardFANandYANmethodsandconditionsweredeterminedformaximalefficiencyandprecision.SuperiorresultswereobtainedwithlowreactionvolumesandastablesodiumacetatebufferedninhydrinreagentatpH5.5.Asanalternative,forusewithcuvettes,areducedvolumeFANassayusingthesamepH5.5sodiumacetatebufferedninhydrinreagentgavecomparableresults.Theassayiseconomical,rapid,accurateandapplicabletolargenumbersofsamples.
Genedeliveryusingdendrimer-entrappedgoldnanoparticlesasnonviralvectors.
Shan,Y.,Luo,T.,Peng,C.,Sheng,R.,Cao,A.,Cao,X.,Shen,M.,Guo,R.,Tomas,H.&Shi,X.(2012).
Biomaterials,33(10),3025-3035.
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Developmentofhighlyefficientnonviralgenedeliveryvectorsstillremainsagreatchallenge.Inthisstudy,wereportanewgenedeliveryvectorbasedondendrimer-entrappedgoldnanoparticles(AuDENPs)withsignificantlyhighergenetransfectionefficiencythanthatofdendrimerswithoutAuNPsentrapped.Amine-terminatedgeneration5poly(amidoamine)(PAMAM)dendrimers(G5.NH<>)wereutilizedastemplatestosynthesizeAuNPswithdifferentAuatom/dendrimermolarratios(25:1,50:1,75:1,and100:1,respectively).TheformedAuDENPswereusedtocomplextwodifferentpDNAsencodingluciferase(Luc)andenhancedgreenfluorescentprotein(EGFP),respectivelyforgenetransfectionstudies.TheAuDENPs/pDNApolyplexeswithdifferentN/PratiosandcompositionsofAuDENPswerecharacterizedbygelretardationassay,lightscattering,zetapotentialmeasurements,andatomicforcemicroscopicimaging.WeshowthattheAuDENPscaneffectivelycompactthepDNA,allowingforhighlyefficientgenetransfectionintotheselectedcelllinesasdemonstratedbybothLucassayandfluorescencemicroscopicimagingoftheEGFPexpression.ThetransfectionefficiencyofAuDENPswithAuatom/dendrimermolarratioof25:1wasatleast100timeshigherthanthatofG5.NH2dendrimerswithoutAuNPsentrappedattheN/Pratioof2.5:1.ThehighergenetransfectionefficiencyofAuDENPsisprimarilyduetothefactthattheentrapmentofAuNPshelpspreservethe3-dimensionalsphericalmorphologyofdendrimers,allowingformoreefficientinteractionbetweendendrimersandDNA.WiththelesscytotoxicitythanthatofG5.NH2dendrimersdemonstratedbythiazoylbluetetrazoliumbromideassayandhighergenetransfectionefficiency,itisexpectedthatAuDENPsmaybeusedasanewgenedeliveryvectorforhighlyefficienttransfectionofdifferentgenesforvariousbiomedicalapplications.
Grapecontributiontowinearoma:productionofhexylacetate,octylacetate,andbenzylacetateduringyeastfermentationisdependentuponprecursorsinthemust.
Dennis,E.G.,Keyzers,R.A.,Kalua,C.M.,Maffei,S.M.,Nicholson,E.L.&Boss,P.K.(2012).JournalofAgriculturalandFoodChemistry,60(10),2638-2646.
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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.
Dendrimer-entrappedgoldnanoparticlesmodifiedwithfolicacidfortargetedgenedeliveryapplications.
Xiao,T.,Hou,W.,Cao,X.,Wen,S.,Shen,M.&Shi,X.(2013).BiomaterialsScience,1(11),1172-1180.
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Wereportanewuseofdendrimer-entrappedgoldnanoparticles(AuDENPs)modifiedwithfolicacid(FA)asanon-viralvectorfortargetedgenedeliveryapplications.Inthisstudy,amine-terminatedgeneration5poly(amidoamine)dendrimersmodifiedwithFAviacovalentconjugationwereusedastemplatestosynthesizegoldnanoparticleswithanAusalt/dendrimermolarratioof25:1.ThesynthesizedFA-modifiedAuDENPs(AuDENPs-FA)wereusedasanon-viralvectorforthedeliveryofplasmidDNA(pDNA)intoamodelcancercellline(HeLacells)overexpressinghigh-affinityFAreceptors(FAR).TheDNAcompaction
ABIlityoftheformedAuDENPs-FAwassystematicallycharacterizedusingagelretardationassay,zetapotential,anddynamiclightscattering.WeshowthatsimilartotheAuDENPsvectorwithoutFA,theAuDENPs-FAvectorwasabletocompactthepDNAencodingenhancedgreenfluorescentprotein(EGFP)atanN/Pratioof0.5.TransfectionresultsshowthattheAuDENPs-FAvectorenablesmuchhigherluciferaseandEGFPgeneexpressioninHeLacellsoverexpressingFARthantheAuDENPswithoutFA,demonstratingtheroleplayedbyFA-mediatedtargetingforenhancedgenetransfectionintargetcells.WithalowercytotoxicitythanthatoftheAuDENPswithoutFAprovenbyacellviabilityassay,thedevelopedFA-modifiedAuDENPsmaybeusedasapromisingnon-viralvectorforsafeandtargetedgenetherapyapplications.
GrowthandlipidproductionofUmbelopsisisabellinaonasolidsubstrate—MechaNISTicmodelingandvalidation.
Meeuwse,P.,Klok,A.J.,Haemers,S.,Tramper,J.&Rinzema,A.(2012).ProcessBiochemistry,47(8),1228-1242.
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Microbiallipidsareaninterestingfeedstockforbiodiesel.Theirproductionfromagriculturalwastestreamsbyfungicultivatedinsolid-statefermentationmaybeattractive,buttheyieldofthisprocessisstillquitelow.Inthisarticle,amechanisticmodelispresentedthatdescribesgrowth,lipidproductionandlipidturnoverinacultureofUmbelopsisisabellinaonκ-carrageenanplatescontainingthemonomersglucoseandalanineasC-sourceandN-source,respectively,andimprovestheunderstandingofthecomplexsolid-statesystem.Themodelincludesreactionkineticsanddiffusionofglucose,alanineandoxygen.Itisvalidatedempiricallyanddescribesthedifferentphasesofthecultureverywell:exponentialgrowth,lineargrowthbecauseofoxygenlimitation,accumulationoflipidsandcarbohydratesafterlocalN-depletionandturnoveroflipidsafterlocalC-depletion.Extendingthemodelwithanunidentifiedextracellularproductimprovedthefitofthemodeltothedata.Themodelshowsthatoxygenlimitationisextremelyimportantinsolid-stateculturesusingmonomers,andexplainsthedifferenceinproductionratewithsubmergedcultures.However,theresultsalsoshowthatthespecificlipidproductionrateinsolid-stateculturesismuchlowerthaninsubmergedcultures,whichresultsinalowlipidyield.
Sauvignonblancmetabolomics:grapejuicemetabolitesaffectingthedevelopmentofvarietalthiolsandotheraromacompoundsinwines.
Pinu,F.R.,Edwards,P.J.B.,Jouanneau,S.,Kilmartin,P.A.,Gardner,R.C.&Villas-Boas,S.G.(2014).Metabolomics,10(4),556-573.
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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.
Changesinthevolatilecompoundproductionoffermentationsmadefrommustswithincreasinggrapecontent.
Keyzers,R.A.&Boss,P.K.(2009).JournalofAgriculturalandFoodChemistry,58(2),1153-1164.
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Wineisacomplexconsumerproductproducedpredominatelybytheactionofyeastupongrapejuice.Modelmustsystemshaveproventobeidealforstudiesintotheeffectsoffermentationconditionsontheproductionofcertainwinevolatiles.Toclarifythecontributionofgrapejuicetotheproductionofwinevolatiles,wehaveemployedamodelmustsystemspikedwithincreasingamountsofgrapejuice(RieslingorCabernetSauvignon).TheresultingfermentedwineswereanalyzedbySPME-GC-MSandthedataobtainedgroupedusingANOVAandclusteranalysestorevealthosecompoundsthatvariedinconcentrationwithreproducibletrendsrelativetojuiceconcentration.Suchgroupinghighlightsthosecompoundsthataregrape-dependentorforwhichproductionismodulatedbygrapecomposition.Insomecases,increasingtheproportionofgrapejuiceinthefermentationsstimulatedtheproductionofcertainesterstolevelsbetween2-and140-foldhigherthanthoseseeninfermentationsmadewithmodelgrapejuicemediaalone.Theidentificationofthegrapecomponentsresponsiblefortheincreasedproductionofthesewinevolatileswillhaveimplicationsfortheimpactofgrapeproductionandenologyonwineflavorandaroma.