低甲醇酿酒酵母的选育与酿造酒的发酵

ReductionofMethodAtmosphericofMethanolandRoom-TemperatureinBrewedWinebyPlasmatheUseMaltwithandDifferenttheCombinationAdjuncts

OptimizationofMing-HuaLiang,Ying-JieLiang,Jiang-YanChai,Shi-ShuiZhou,andJian-GuoJiang

Abstract:

Methanol,oftengeneratedinbrewedwine,ishighlytoxicforhumanhealth.Todecreasethemethanol

contentofthebrewedwine,atmosphericandroom-temperatureplasma(ARTP)wasusedasanewmutagenesistooltogenerateamutantofSaccharomycescerevisiaewithlowermethanolcontent.Headspacegaschromatographywasusedtodeterminetheidentityandconcentrationofmethanolwithbutylacetateasinternalstandardinbrewedwine.With47.4%higherand26.3%positivemutationrateswereobtained,theARTPjetexhibitedastrongeffectonmutationbreedingofS.cerevisiae.ThemutantS.cerevisiaeS12exhibitedthelowestmethanolcontent,whichwasdecreasedby72.54%comparedwiththatofthewild-typestrain.Subsequently,themutantS.cerevisiaeS12wasusedtofermentdifferentcombinationsofmaltandadjunctsforlowermethanolcontentandhigheralcoholiccontent.Itwasshownthattheculture6#,whichwas60%malt,20%wheat,and20%corn,wasthebestcombinationsofmaltandadjuncts,withthelowestmethanolcontent(104.8mg/L),andarelativelyhigheralcoholiccontent(15.3%,v/v).Theoptimalmalt–adjunctculture6#,treatedwiththeglucoamylasedoseof0.04U/mgofgrainreleasedthehighestreducingsugars(201.6mg/mL).ItwasindicatedthatM:thevariationinreducingsugarsamongthecombinationsofmaltanddifferentadjunctscouldbeduetothedoseof&Foodexogenousenzymes.

Safety

MicrobiologyKeywords:adjunct,atmosphericandroom-temperatureplasma,brewedwine,headspacegaschromatography,methanol

Introduction

combinationofmaltwithdifferentadjuncts.Also,manymethodsMethanol(CH3OH,methylalcohol)isahighlytoxicandin-havebeendevelopedtodetectmethanolinbeverageproducts, ammablematerialwhoseingestionorinhalationcancauseblind-suchasbiosensordetection(LiuandKirchhoff2007),highper-nessordeath(Singkongandothers2012).Methanolcontentisformanceliquidchromatography(HPLC;Kuoandothers2002),oneofthecriticalparametersthatshouldbecontrolledduringtheandgaschromatography(GC;Wangandothers2004).Thecom-alcoholicfermentationfortheproductionofsafewine.

plexbrewedwinesamplematricescanbelargelyeliminatedbyIthasbeenpointedoutthatmanyfactors,suchaspectinasetheproposedheadspace(HS)samplingtechnique,coupledwithtreatment,oenologicalpractices,fermentationconditions,andtheanalysisbyGC.Inaddition,sampleclean-upandpreconcentra-yeaststrainused,canin uencemethanolproductioninbrewedtionarenotnecessary,andtediousanderror-producingstepscanwine(VanRensburgandPretorius2000;Cabaroglu2005).Sev-beeliminated(Chaiandothers1998).Thus,methanolinbrewederalmethodsforreducingthemethanolcontentofbrewedwineswinescanbeanalyzedeasilyandquicklybyHSGC.

havebeentried:byoptimizingthetreatmentconditionsoftheARTPisanewkindofatmosphericpressurenonequilibriumpectinaseenzyme(ReddyandReddy2005),byaddinggallicdischargeplasmasource(Parkandothers2000;Liandothersacidorcoumaricacid(Houandothers2008),byselectingthe2007).TheARTPmutationsystemhasbeenobservedtoproducetypeofyeastwhichdoesnotformpectinesterasis(Niki´cevi´candpersebreakagesinplasmidDNAandoligonucleotideswiththeTeˇsevi´c2005),byapplyingdemethanolizationcolumn(Niki´cevi´cvariationintheplasmadosage(Liandothers2008).TheARTPandTeˇsevi´c2005),andbypackedbedcolumnsforbatchpro-equipmentiscompactandtheoperationcanbeeasilyperformedductionsofwinedistillations(Carvalloandothers2011),andsoundermildconditions(Luandothers2011),andhasbeenusedon.However,thesewaysarecostlyordif culttooperate,ornotinmanyaspectsrecently(Wangandothers2010;Fangandotherseffectiveenoughtoreducethemethanolcontentatthesource.2013;Liuandothers2013).

HereweusedanewwaystodecreasethemethanolcontentattheDuetoeconomicreasons,cheaprawmaterialsareusedasad-source,byselectingthetypeofyeastwithlowmethanolbyatmo-junctforpartialreplacementofmaltinwortproduction.Starchysphericandroomtemperatureplasma(ARTP),andoptimizingthe

grainssuchascorn,wheat,rice,andbarleycanbeusedasadjuncts(PriestandStewart2006).Table1showsthecharacteristicsandcomponentsofmaltandadjuncts.Malt,whosediastaticpowerMS20140833Submitted5/17/2014,Accepted8/21/2014.AuthorsLiang,ishigherthanothergrains,hasahighlevelofmaltoseproduc-Liang,Chai,andZhouarewithSchoolofBiologicalScience&Engineering,Southingbyβ-amylase,thekeyenzymeinbreakingdownmaltstarchChinaUniv.ofTechnology,Guangzhou,510006,China.AuthorsLiangandJiangarewithCollegeofFoodScienceandEngineering,SouthChinaUniv.ofTech-(Malomoandothers2011).Betteravailabilityonthelocalmarket,nology,Guangzhou,510640,China.DirectinquiriestoauthorsZhou(E-mail: avorimprovement,andsensorymodi cationofthewinearealsohgzhouss@http://www.77cn.com.cn),Jiang(E-mail:jgjiang@http://www.77cn.com.cn).

thereasonsfortheapplicationofadjunctsinadditiontoorinsteadofmalt(Poredaandothers2014).Theproportionofadjunctsused

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doi:10.1111/1750-3841.12660

Furtherreproductionwithoutpermissionisprohibited

Reductionofmethanolinbrewedwine...

Table1–Thecharacteristicsandcomponentsofmaltandadjuncts.Rawmaterial

Gelatinizationtemperature(°C)Starch(%)Protein(%)Lipids(%)

α-Amylase(U/g)β-Amylase(U/g)

Barleymalt61–65–––280920

Barley60–6569–739–132.50.62350

Wheat55–6572–7711–1520.42454

Corn64–8273–778–115–6NotdetectedNotdetected

Rice68–8484–885–90.50.1257.4

Sorghum68–7578–8011–123.70.36252

OperatingmethodfortheARTPbreedingsystem

MediumforS.cerevisiaecultivationisYPD:2%glucose,1%yeastextract,2%peptone;fermentationmedium:1%yeastextract,andthenwhitegranulatedsugarwasaddedtomakeupto18Brix.TheARTPoperatingparameterswereasfollows:(1)theradio-MaterialsandMethods

frequencypowerinputwas115W,(2)thedistancebetweenthe

Materialsplasmatorchnozzleexitandthesampleplatewas2.5mm,(3)theMalt,rice,wheat,corn,sorghum our,andwhitegranulatedtemperatureoftheplasmajetwas25to35°C,and(4)theheliumsugarwerecommerciallyavailable.Saccharomycescerevisiaewaskeptgas owratewas10SLPM.inthelabsofSouthChinaUniv.ofTechnology.MethanolandethanolareHPLCgrade.Butylacetateisanalyticalgrade.

DeterminationofthemutagenesisdosagefortheARTP

DeterminationofthemethanolcontentwithbutylacetateUndertheconditionsabove,themutagenesisdosagebyARTPasinternalstandardbyHSGCwasdependentonthetreatmentperiod.TodeterminetheoptimalThemethanolconcentrationofeachwinesamplewascalculatedtreatmentperiod,S.cerevisiaegrownatOD600=1.5,whichwasbyinternalstandardmethodwithrespecttotheinternalstandardwithinthelog-phase,wasselectedtoperformARTPtreatment.fromresponsefactor,sothatHSGCisqualitative(bytheuseofForthemutation,10μLoftheculturewasdippedintothestainlessretentiontime)andquantitative(bytheuseofsignalstrength).ThesteelplateandthenexposedtoARTPjetfor0,30,45,60,90andproceduretoobtainacalibrationcurvewasasfollowed:20%(v/v)120s,respectively.ethanolsolutionwasaddedto10mL,containinginternalstandardAftertreatmentofthebacterialsamples,theplatewasputintobutylacetate(300mg/L)anddifferentmethanolconcentrationsatube ttedwith1mLofclearmedium,diluted104timeswith(0,100,200,400and800mg/L),respectively.Theresponsefromsterilewater.Subsequently,100μLofthediluentwerespreadonthemethanolpeakwascomparedtotheinternalstandardpeak.asolidagarplate.Afterincubationat28°Cfor48h,thecolonyHereradiofrequency(RF)wasde nedtotheratioofthepeaknumberswerecalculated.areaofthemethanoltothepeakareaofinternalstandard.ThenInordertoascertainanoptimummutationdosage,alethalityplottheresultswithRFontheY-axisandthemethanolcon-rateofS.cerevisiaeusinganARTPbiologicalbreedingsystemwascentrationontheX-axis,and tthisdatatoobtainacalibrationdrawn.Ahighrateofcelllethalitymakesforeffectivemutation.curve.ThenthesamplepreparationandmeasurementproceduresStrainsfromsolidagarplateswithhighlethalityratewereselectedwereasfollows:10mLofbrewedwinesamplewith300mg/Lasaninoculantforlowermethanolcontentandhigheralcoholicbutylacetatewereaddedinto20mLheadspacevials,thenclosecontents.Thelethalityrateofthesporesunderdifferentmutationthevials.Thesamplethenunderwentanequilibrationperiodtodosageswasevaluatedbasedonthefollowingequation:achievevapor–liquidphaseequilibrium.Onehundredmicrolitersoftheheadspacevaporwasputintotheheadspacesamplerfor

U T

measurement.LR(%)=×100%

UHSGCmeasurementswerecarriedoutwithanautomatic

headspacesampler(DANIHS86.50,Italy)andaGCsystem(Ag-ilentGC7890A,UnitedStates)equippedwitha ameionizationwhereLRisthelethalityrate(%),Uisthetotalcolonycountofdetector(FID;H2:30mL/minandair:350mL/min),andaDB-thesamplewithouttreatment,andTisthetotalcolonycountafterFFAPcapillarycolumn(30m×0.53mm×1.00μm),operatingtreatmentwithARTPonthesolidmedium.Allthecolonynum-withnitrogencarriergas( owrate=1.0mL/min).Oventemper-berswereobtainedbythecolonyformingunits(CFUs)methodaturewascontrolledwithatemperatureelevationprogramduringonthesolidmedium.

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variesfrom10%to25%inEurope,35%to45%inUnitedStates,andashighas50%to70%incertainAfricancountries(Ogbeide2011).

Inthisstudy,ARTPwasusedasamutagenesistooltogenerateamutantofSaccharomycescerevisiaewithlowmethanol.HSGCwasusedtodeterminetheconcentrationofmethanolwithbutylac-etateasinternalstandard.ThemutantofS.cerevisiaewasselectedasaninoculanttofermentdifferentcombinationsofmaltandad-junctsinordertoestimatetheethanol,methanol,andtotalesterscontents.Theoptimizedcombinationofmaltwithdifferentad-junctswithlowmethanolandhighalcoholiccontentwas nallydeterminated.Theeffectofglucoamylaseadditionwastestedonthereducingsugarscontentofthewortproductionfromtheop-timalmalt–adjunctcombination.

analysis,whichwasinitiallysetat35°Cfor4min,elevatedto180°Cattherateof20°C/minandmaintainedfor5min.Thetemperaturesatthedetectorandvaporizerweresetat250and150°C,respectively,and50:1splitratioinsplitmodewasused.Headspaceoperatingconditionswereasfollows:25minofstrongshakingforthesampleequilibrationat80°C;samplelooptemperature=85°C;transferlinetemperature=90°C;vialpres-surizationtime=12s;sampleloop lltime=30s;andtransfertime=6s.

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brewedwinessampleswasdeterminedwiththehelpofERMAEvaluationofmutagenesisofS.cerevisiaebyARTPjet

Themutationrateandthepositivemutationratewerecalculatedhandrefractometerhavingrangeof0to32Brixat20°C.The

ethanolwasestimatedbyportablealcoholmeter.Theproceduresusingthefollowingequations:

oftheestimationoftotalesterswereasfollowed:1stfreeacidsin

PMthewinesamplewereneutralizedbyNaOHsolution,andthen

×100%,PMR=×100%MR=

acertainamountofNaOHsolutionwasaddedforsaponi ca-TM

tionoftheesters.Finally,excessNaOHsolutionwasaddedfor

whereMRisthemutationrate,PMRisthepositivemutationantititrationwithacid.rate,MisthetotalCFUofthemutantstrains,andPistheCFUof

themutantswiththemethanolcontentwhichis20%lowerthanStatisticalanalysisthatofthewild-typestrain(WT),andTisthetotalcolonycount

Valueswereexpressedasmean±standarddeviationof3parallel

aftertreatmentwithARTPonthesolidmedium.

measurements.Thesigni canceofdifferencesbetweengroupswasassessedbyone-wayanalysesofvariance.P<0.05indicatedthe

Detectionofthemutant’sgeneticstability

presenceofastatisticallysigni cantdifference,andP<0.01was

Thegeneticstabilityofthemutants,whichisessentialforthe

consideredhighlysigni cant.

fermentationindustryandalsore ectstheoccurrenceofmuta-tionsatthegenelevel,wasexaminedbysubculturing.First,the

Results

mutantwasstreakedandcultivatedonthesolidmediumfor48

h(the1stsubculture).Next,severalsinglecolonieswereselectedDeterminationofmethanolcontentinbrewedwinebyandstreakedontonewplateswiththesolidmediumforthe2ndHSGC48-hcultivation(the2ndsubculture).Thesameprocedurewasre-WithHSGC,butylacetateasinternalstandardand20%(v/v)

peatedforatotalof5subcultures.Aftereachsubculture,methanolethanolsolutionasthesolvent,weestablishedanequation:Y=contentwasevaluatedbyfermentation.0.0019X+0.0056,R2=0.9998.YrepresentsRF,andXrep-resentsthecontentofmethanol.TherepeatabilityoftheHSGCPreparationofwortextractionmethodwastestedusingthestandardsample(methanolconcen-Inordertocomparetheresultswithastandard100%malttration=200mg/L).Arelativestandarddeviationofmeasuredforbrewing,a100%maltstandardmashwasperformedusingmethanolcontentislessthan2.0%forthe5timestested,indicat-thefollowingmashingprogramme:malt our(50g)wasmixedingthatthetechniqueofHSGCtodetectthemethanolcontentwith200mLofdistilledwater,55°Cfor90min,65°Cforhasexcellentrepeatabilityandhighprecisionandaccuracy.60min,and72°Cfor30min.Nocommercialenzymeswereadded.LethalityrateofS.cerevisiaebyARTPjetMalt ourandmilledadjunctweremixedinaproportionsuchWhenthebacterialsolutionwastreatedwithARTPjetfor30,thatXgmalt+Ygofadjunctgavea50gsample.Milledadjuncts45,60,and90s,thelethalityratesofthetreatedyeastsincreasedto(rice,wheat,corn,andsorghum),Yg(20%to60%or10to3044.4%,59.3%,70.4%,and92.6%,respectively(Figure1).Wheng),containedinastainlessmashingbeaker,wasseparatelymixedtheexposuretimewasextendedto120s,thelethalityrateraisedwith80mLofdistilledwater,precookedatthegelatinizationslightly(96.3%).Sotheexposuretimeemployedinthisstudywastemperaturefor30mininawaterbath(asforwheat,precooking120stoobtainadesirableandsuitablelethalityrateforscreeningwasskipped),thencookedat105°Cfor15mininawaterbathS.cerevisiaemutant.togelatinizethestarchandthencooledto85°C.Thermostable

α-amylaseenzyme(1.0U/mgofgrain)wasaddedforliquefactionThemutationandscreeningofARTPforS.cerevisiaefor30min.Duringthecookingprocessoftheadjunct,nomaltAftertheyeastssolutionwastreatedbyARTPjetfor120sandwasadded.Aftercoolingat60°C,glucoamylasewasaddedcultivatedonthesolidmediumfor48h,therewereabout30tothecookedadjunctinthemashingvesselfor30minforsinglecoloniesgrowingon4plates,andthenthecoloniesweresacchari cation,thenXgmalt120mLofdistilledwaterweretransferredtosolidmediumrespectively.Afterincubationon

theaddedat65°Cfor60minand72°Cfor30min.0,4,8,16,and32μL/gofgrainofliquidglucoamylase(5000U/mL)wasadded.Aftercompletionofthemashingprocedures,theseparationofwortandspentgrainsinasmallscalelautertunwascarriedout.Thewortwasfurthercooledto23°C,pitchedwithS.cerevisiae(5×107cells/mL),andfermentedat23°Cfor3d.

Sixgristcombinationswithmalttoadjunct(rice,wheat,cornandsorghum)proportionsof100:0,80:20,70:30,60:40,50:50,and40:60wereassessedasfollowed:A,100%malt;B,80%malt+20%adjunct(rice,wheat,corn,andsorghum);C,70%malt+30%adjunct(rice,wheat,corn,andsorghum);D,60%malt+40%adjunct(rice,wheat,corn,andsorghum);E,50%malt+50%adjunct(rice,wheat,corn,andsorghum);andF,40%malt+60%adjunct(rice,wheat,corn,andsorghum).

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Analyticalproceduresforthebrewedwine

Thereducingsugarswereestimatedby3,5-dinitrosalicylicacidFigure1–LethalityrateofS.cerevisiaebyARTPfordifferentexposuretime.(DNS)method(Miller1959).Totalsolublesolids(TSS)oftheTheexposuretimeemployedinthisstudywas120s(lethalityrate96.3%).

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platesfor48h,19singlecolonies(S1toS19strains)grewwellinsubcultureandweredeterminedbyfermentationexperiments(glucoseassubstrate).Finally,themethanolandalcoholcontentsinthebrewedwinebyfermentationofS1toS19strainswereshowninFigure2.Inordertoavoidtheeffectofrandomer-ror,onlythestrainswithmorethan20%decreaseinmethanolyieldcomparedwiththeWTstrainwereconsideredaspositivemutants.FromFigure2,itwasdisplayedthat9strains(S2,S5,S9,S10,S12,S13,S15,S17,andS19)producedlowermethanolyield,comparedwiththeWTstrain.Amongthe9strains,5strains(S2,S9,S10,S12,andS19)satis edtobepositivemutants,andS12strainsproducedthelowestmethanolcontent(30.7mg/L),witha72.54%decreaseofthatoftheWTstrain(111.8mg/L).Bycalculationaccordingtheequationsintheevaluationofmuta-genesisofS.cerevisiaebyARTPjetoftheMaterialsandmethodssection,themutationandpositivemutationratewere47.4%and26.3%,respectively.Asforalcoholcontents,itwasfoundthattherewerenotobviouschangesbetweenthemutantsandtheWTstrain.S.cerevisiaeS12couldproducejust8.89%morealcoholcontentthantheWTstrain.Inaddition,themutantS.cerevisiaeS12wasisolatedtostudyitsgeneticstability.Aftera5-generationculture,S.cerevisiaeS12stillmaintainedlowmethanolproductiv-ityinfermentation.S.cerevisiaeS12wasusedforthefollowingfermentation.

sorghumandcornasadjuncts,thebrewedwinescontainedhighertotalesters.

Optimizationofadjunctscombinationsforlowermethanolcontent

Accordingtotheconclusionsabove,forbrewingwinesoflowmethanolcontent,wheatwasthe1stchoicetobeadjunct;forbrewingwinesofhighethanolcontent,wechosewheatorcornasadjunct;forbrewingwinesofhightotalester,wechosesorghumorcornasadjunct.Next,thecombinationsofdiffer-entkindsofadjuncts(wheat,corn,andsorghum)anddifferentproportions(Table2)weretestforlowermethanolandhighethanol.FromTable2,itwasfoundthattheculture6#,whichwas60%malt,20%wheatand20%corn(wheat:corn=1:1),wasthebestcombinationsofmaltandadjuncts,withthelowestmethanolcontent(104.8mg/L),andarelativelyhigheralcoholiccontent.

Effectofglucoamylasedoseontheconcentrationofreducingsugars

Withthemethodoftheestimationofreducingsugarsconcen-trationintheAnalyticalproceduresforthebrewedwineoftheMaterialsandMethodssection,weestablishedanequation:y=7.4265x+0.0507,R2=0.9979,inwhichyrepresentstheopticaldensityofthereactionsolutionscontainingthebrownreductionproduct,andxrepresentsthereducingsugarscontent.

Aftertheoptimalculture6#,whichwas60%malt,20%wheat,and20%corn(wheat:corn=1:1),wasdetermined,theeffectofglucoamylasedoseonreducingsugarsconcentrationofthewortwastested.Itwasfoundthatwhenglucoamylasewasaddedas0,4,8,16,and32μL/gofgrain,whichmeantglucoamylasedoseswere0,0.02,0.04,0.08,and0.16U/mgofgrain,as5000U/mLofliquidglucoamylasewasadded,theconcentrationsofreducingsugarsinthewortvaried(Table3),andamaximumreducingsugarsconcentrationof201.6mg/mLwasobservedwhentheglucoamylasedosewas8μL/gofgrain.

Fermentationfromonekindofstarchadjunct

Fivegristcombinations(B,C,D,E,andFinthepreparationofwortextractionoftheMaterialsandMethodssection)withmalttoadjunct(rice,wheat,corn,andsorghum)proportionsof80:20,70:30,60:40,50:50,and40:60wereassessed.Theeffectsofdifferentadjunctsanddifferentmalttoadjunctproportionsontheyieldsofmethanol,alcohol,andtotalesterwasshowninFigure3.Theresult(Figure3A)showedthatthemethanolcontentinthebrewedwineswiththeadditionofwheatasadjunctwaslowerasawhole,whilecomparedwithotheradjuncts.Especially,whenthemalttowheatproportionswas60:40,themethanolcontentwasthelowest.Asforalcoholyields,theresult(Figure3B)showedthatwiththeadditionofwheatandcornasadjuncts,thealcoholcontentswerehigherthanotheradjuncts.Especially,whenthemalttocornproportionswas80:20,thealcoholcontentwasthehighest.Sobrewingforwineswiththeadditionofwheatandcornasadjunctscouldcontributehigheralcoholcontents,suggestingthatwheatandcorncanbeef cientlyhydrolyzedintoreducingsugars.Asfortotalesters(Figure3C),itwasdisplayedthatusing

Discussion

Yeastspeciesareusedinmanyindustrialfermentationprocessesincludingalcoholicbeveragesproduction.Thequalityofwineproducedgreatlydependsontheyeaststrain(Okunowoandoth-ers2005).Itwasreportedthatthemethanolconcentrationvariedbetween9.51%withS.cerevisiaevar.ellipsoideusand14.93%withS.carlsbergensis(Okunowoandothers2007).Inthisstudy,withhigher(47.4%,over30%)andpositive(26.3%,over20%)mutation

rates,

Figure2–ThemethanolandalcoholcontentsinthebrewedwinebyfermentationofthescreeningstrainsandtheWTstrain.Thedashedlinewascorrespondingwiththe

methanolcontent=111.8mg/L,whichwasfermentedbytheWTstrain(controlgroup).Thesolidlinewascorrespondingwiththemethanolcontent=89.44mg/L,whichwas20%lowerthanthatoftheWTstrain.P<0.01.

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Table2–Thecombinationsofdifferentkindsanddifferentproportionsofadjunctsandthecorrespondingmethanolcontentsandalcoholiccontents.Theculture6#,whichwas60%malt,20%wheat,and20%corn,wasthebestcombinationsofmaltandadjuncts,withthelowestmethanolcontent(104.8mg/L).

Maltandadjunctsproportions

Test1#2#3#4#5#6#7#8#9#

Malt80%80%80%60%60%60%40%40%40%

Wheat2%16%10%4%32%20%6%48%30%

Corn16%2%10%32%4%20%48%6%30%

Sorghum2%2%–4%4%–6%6%–

Methanolcontent

(mg/L)178.6171.2144.3175.3111.2104.8177.4122.7108.4

Alcoholiccontent

(%v/v)16.517.515.513.216.015.314.213.313.0

Table3–Theeffectofglucoamylasedoseaddedontheconcentrationofreducingsugar.Theoptimalmalt–adjunctculture6#,treatedwiththeglucoamylasedoseof0.04U/mgofgrainreleasedthehighestreducingsugars(201.6mg/mL).Parameter

Glucoamylasedose(μL/gofgrain)Glucoamylasedose(U/mgofgrain)

Opticaldensityinthecolorimeterreading

Concentrationofreducingsugarsbycalculatedbytheequation(mg/mL)Concentrationofreducingsugarsinwort(mg/mL)

001.8050.250150.0

40.021.5710.218130.8

Themeasuredvalues

80.042.4460.336201.6

160.081.9080.264158.4

320.161.4410.201120.6

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http://www.77cn.com.cnparedwiththeWTstrain,themethanolcontentofS.cerevisiaeS12wasdecreasedby72.54%,andtheal-coholiccontentwasincreasedby8.89%.ItwasdisplayedthattheARTPjetyieldedhightotal(31.6%)andpositive(26.0%)muta-tionratesonStreptomycesalbulusandamutatedstrain,designatedasS.albulusA-29,showedamaximumε-poly-l-lysineproductivityof1.59±0.08mg/mL,whichwas4timesasmuchasthatofthewildstraininthesameculturecondition(Zongandothers2010).Wangandothers(2010)demonstratedthatARTPjethashigherpositivegenotoxicresponsethanconventionalmutationmethods,andtheARTPshowsdifferentgenotoxiccharacteristicsdependingonthetreatmenttime.BythetreatmentofS.avermi-tiliswithAPGD,thelethalityrateofthecellsincreasedwiththeexposuretime,andatthelethalityofaround98.2%,amutationrateof30%andpositivemutationrateof21%werereached.Itwascon rmedthatradiofrequencyAPGDjethasastrongmutageniceffectonS.avermitilisandamutatedstrainwithhighproductiv-ityofavermectinB1aandageneticstabilitywasobtained(Wangandothers2010).Inaddition,inordertoimprovethecarbo-hydrateproductivityofSpirulinaplatensisbygeneratingmutantswithincreasedcarbohydratecontentandgrowthrate,ARTPwasusedasanewmutagenesistoolandthetotalmutationfrequencyandpositivemutationfrequencyreached45%and25%,respec-tively(Fangandothers2013).AlltheseshowthattheARTPjetcanbedevelopedasapromisingandconvenientmutationtoolforthefermentationindustryandforuseinbiotechnologyresearch.

Inthisstudy,thecombinationsofdifferentproportionsofmaltandadjunctswereestimatedonethanol,methanol,andtotalesterscontents.FromFigure3,forbrewingwinesoflowmethanolcontent,wheatwasthe1stchoiceofthe4adjuncts;forbrewingwinesofhighethanolcontent,wheatorcornhadanadvantageovertheothers;forbrewingwinesofhightotalester,sorghum,orcornwassuperiortotheothers.Thenthecombinationsofdifferentkindsofadjuncts(wheat,cornandsorghum)anddifferentproportions(Table2)wereoptimizedforlowermethanolandhighethanol.Itwasfoundthattheculture6#,including60%malt,20%wheat,and20%corn(wheat:corn=1:1),wastheoptimalcultureforfermentationwiththelowestmethanolcontent(104.8mg/L),andarelativelyhigheralcoholiccontent.ItwasreportedthatfermentationoftheorangejuicebyS.cerevisiaefromyamandS.cerevisiaefromsugarcanemolassesresultedinproductswithdifferentconcentrationsofethanolandmethanol,despitethefactthatthefermentingorganismsareofthesamespecies.Thisindicatesthatthesourceoftheyeastmayin uencethealcoholpro leofthewineproduced(Okunowoandothers2007).

Thevariationinreducingsugarsamongthecombinationsofmaltanddifferentadjunctscouldbeduetothefactthatthereactionofexogenousenzymesmaybebasedonthecompositionofthegrains.FromTable1,riceandsorghumhavemorestarchcontentthanotheradjuncts.Riceandcornadjunctscontributelittleornosolubleproteintothewort,whileotheradjuncts,suchaswheat,sorghum,andbarley,havehigherlevelsofsolubleprotein.Sotheeffectofdifferentglucoamylasedoseswastestedontheconcentra-tionofreducingsugarsofthewortproduction.Reducingsugarsareprimecomponentoffermentation,afterconsumptionofwhich,alcoholisproduced.Theprocessstepsinconvertingstarchtoglucosearegelatinization,liquefaction,andsacchari cation.Starchprocessinginvolvesthegelatinizationoftheslurrywhichisheatedtoatemperatureof105°C.Thisincreasestheviscosityoftheslurryandposesproblemswithmixingandpumping.Toover-comesuchviscosity-associatedproblems,themostablebacterialα-amylaseisaddedwhichcanliquefystarchathighertemperaturesgenerallyintherangeof70to90°Cormore.Itwouldbedif culttoprocessifanα-amylasewasnotaddedtopartiallyhydrolyzethestarchtodextrins,asα-amylaseservestoreducetheviscosityofthesolution.Forthoseadjunctswhosegelatinizationtemperatureishigherthanthatofmalt(thatiscorn,rice,andsorghum,seeninTable1),theyarepregelatinizedinadjunctcookingvesselsbeforetheyareintroducedintothemash(Lloyd1986).

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Lique edstarchisthensacchari edusingglucoamylaseat50to60°C(Soniandothers2003).Itwasreportedthatamaximumconversionef ciencyof94.01%forthehydrolysisofwheatstarchwasobservedafter72hwhentheα-amylasedosewas1.0U/mgsolidsduringprecooking(at75°C),2.0U/mgsolidsatpostcookingliquefaction(at65°C)andthelevelofglucoamylasewas0.02U/mgofsolids(Soniandothers2003).Inaddition,responsesurfacemethodologywasadoptedtodeterminetheop-timumvaluesforthetestedvariablesforthemaximumconversionef ciency96.25%ofmaizestarchtoglucosewere:precookingα-amylasedose2.243U/mgsolids,postcookingα-amylasedose3.383U/mgsolids,glucoamylasedose0.073U/mgsolids,andasacchari cationtemperatureof55.1°C(Kunamneni

and

Singh2005).Soitshouldbetakenintoconsiderationseriouslythatanα-amylaselevelthatistoolowwillgiveincompletestarchsolubilizationandconcomitantlossofethanolyield.Aglucoamylaselevelthatistoolowwillextendthefermentationtimefortheconcurrentsacchari cationfermentationprocessbecauseglucoseisnotproducedasrapidlyastheyeastcanconvertittoethanol,thenwillalsoreducetheethanolyield.Maltcouldalsobeamajorsourceofhydrolyticenzymesrequiredfortheconversionofstarchtosimplesugars.Buttheconversionofstarchyadjunctstoalcoholbyexogenousenzymes,couldaccomplishahighlyef cientdegradationofthestoredstarchintomonosac-charides,afterwhichitisfermentedwithyeasttoproduceethanol.

Conclusions

Takentogether,amutantS.cerevisiaeS12withlowermethanolcontentwasobtainedbyARTP.TheARTPjetexhibitsastrongeffectonmutationbreedingofS.cerevisiae.Inaddition,itwasshownthattheculture6#,whichwas60%malt,20%wheat,and20%corn,wasthebestcombinationsofmaltandadjuncts,withthelowestmethanolcontent,andarelativelyhigheralcoholiccontent.Theoptimalmalt–adjunctculture6#,treatedwiththeglucoamylasedoseof0.04U/mgofgrainre-leasedthehighestreducingsugars,whichwasindicatedthatthevariationinreducingsugarsamongthecombinationsofmaltanddifferentadjunctscouldbeduetothedoseofexogenousenzymes.

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