低甲醇酿酒酵母的选育与酿造酒的发酵
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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
C2014InstituteofFoodTechnologists
RM2308
JournalofFoodSciencerVol.79,Nr.11,2014
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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Reductionofmethanolinbrewedwine...
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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Reductionofmethanolinbrewedwine...
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.
References
CabarogluT.2005.MethanolcontentsofTurkishvarietalwinesandeffectofprocessing.FoodControl16:177–81.CarvalloJ,LabbeM,P´erez-CorreaJR,ZarorC,WisniakJ.2011.Modellingmethanolrecoveryinwinedistillationstillswithpackingcolumns.FoodControl22:1322–32.
ChaiX,DhasmanaB,ZhuJ.1998.DeterminationofvolatileorganiccompoundcontentsinKraftmillstreamsusingheadspacegaschromatography.JPulpPaperSci24:50–4.
FangM,JinL,ZhangC,TanY,JiangP,GeN,LiH,XingX.2013.RapidmutationofSpirulinaplatensisbyanewmutagenesissystemofatmosphericandroomtemperatureplasmas(ARTP)andgenerationofamutantlibrarywithpersephenotypes.PLoSOne8:e77046.
HouCY,LinYS,WangYT,JiangCM,LinKT,WuMC.2008.Additionofphenolicacidsonthereductionofmethanolcontentinwine.JFoodSci73:C432–7.
KunamneniA,SinghS.2005.Responsesurfaceoptimizationofenzymatichydrolysisofmaizestarchforhigherglucoseproduction.BiochemEngJ27:179–90.
KuoC,WenY,HuangCM,WuHL,WuSS.2002.AremovablederivatizationHPLCforanalysisofmethanolinChineseliquormedicine.JFoodDrugAnal10:101–6.
LiG,LiHP,WangLY,WangS,ZhaoHX,SunWT,XingXH,BaoCY.2008.Geneticeffectsofradio-frequency,atmospheric-pressureglowdischargeswithhelium.ApplPhysLett92:221504.
LiHP,SunWT,WangHB,LiG,BaoCY.2007.Electricalfeaturesofradio-frequency,atmospheric-pressure,bare-metallic-electrodeglowdischarges.PlasmaChemPlasmaProcess27:529–45.
LiuQ,KirchhoffJR.2007.Amperometricdetectionofmethanolwithamethanoldehydrogenasemodi edelectrodesensor.JElectroanalChem601:125–31.
LiuR,LiangL,MaJ,RenX,JiangM,ChenK,WeiP,OuyangP.2013.AnengineeringEscherichiacolimutantwithhighsuccinicacidproductioninthede nedmediumobtainedbytheatmosphericandroomtemperatureplasma.ProcessBiochem48:1603–9.LloydW.1986.Adjuncts.JInstBrew92:336–45.
LuY,WangL,MaK,LiG,ZhangC,ZhaoH,LaiQ,LiHP,XingXH.2011.CharacteristicsofhydrogenproductionofanEnterobacteraerogenesmutantgeneratedbyanewatmosphericandroomtemperatureplasma(ARTP).BiochemEngJ55:17–22.
MalomoO,OgunmoyelaOAB,OluwajobaSO,AdigunMO,DanielT.2011.Sensoryassess-mentofsorghumbrewadjunctandbarleybrewlagerbeer.JBrewingDistilling2:62–http://www.77cn.com.cneofdinitrosalicylicacidreagentfordeterminationofreducingsugar.AnalChem31:426–8.Niki´cevi´cN,Teˇsevi´cV.2005.Possibilitiesformethanolcontentreductioninplumbrandy.JAgricSci50:49–60.
OgbeideS.2011.InvestigatingtheuseofsorghumasmaltedBarleyadjunctinbrewingprocess.JEmergTrendsEngApplSci2:369–78.
OkunowoWO,OkotoreRO,OsuntokiAA.2005.Thealcoholicfermentativeef ciencyofindigenousyeaststrainsofdifferentoriginonorangejuice.AfrJBiotechnol4:1290–96.
OkunowoWO,OsuntokiAA.2007.Quantitationofalcoholsinorangewinefermentedbyfourstrainsofyeast.AfricanJBiochemRes1:95–100.
ParkJ,HeninsI,HerrmannH,SelwynG,JeongJ,HicksR,ShimD,ChangC.2000.Anatmosphericpressureplasmasource.ApplPhysLett76:288–90.
PoredaA,CzarnikA,ZdaniewiczM,JakubowskiM,AntkiewiczP.2014.Corngristadjunct—applicationandin uenceonthebrewingprocessandbeerquality.JInstitBrewing120:77–81.
PriestFG,StewartGG.2006.Handbookofbrewing.BocaRaton,Fla.:CRCPress.
Figure3–Theeffectsofsinglekindofadjunctontheyieldsofmethanol,alcohol,andtotalester.(A)Theeffectsofsinglekindofadjunctontheyieldsofmethanol.Wheatwastheadjunctforlowermethanolcontent;P<0.01.(B)Theeffectsofsinglekindofadjunctonalcoholiccontent.Wheatandcornweretheadjunctsforhigheralcoholiccontent;P<0.05.(C)Theeffectsofsinglekindofadjunctontheyieldsoftotalesters.Sorghumandcornweretheadjunctsforhighertotalesters;P<0.05.
Vol.79,Nr.11,2014rJournalofFoodScienceM2313
M:FoodMicrobiology&Safety
Reductionofmethanolinbrewedwine...
ReddyL,ReddyO.2005.Productionandcharacterizationofwinefrommangofruit(MangiferaindicaL).WorldJMicrobiolBiotechnol21:1345–50.
SingkongW,RattanapunB,http://www.77cn.com.cnnJFoodAgro-Ind5:61–70.
SoniSK,KaurA,GuptaJK.2003.Asolidstatefermentationbasedbacterialα-amylaseandfungalglucoamylasesystemanditssuitabilityforthehydrolysisofwheatstarch.ProcessBiochem39:185–92.
VanRP,PretoriusIS.2000.Enzymesinwinemaking:harnessingnaturalcatalystsforef cientbiotransformations:areview.SAfrJEnolVitic21:52–73.
WangLY,HuangZL,LiG,ZhaoHX,XingXH,SunWT,LiHP,GouZX,BaoCY.2010.NovelmutationbreedingmethodforStreptomycesavermitilisusinganatmosphericpressureglowdischargeplasma.JApplMicrobiol108:851–8.
WangML,WangJT,ChoongYM.2004.Arapidandaccuratemethodfordeterminationofmethanolinalcoholicbeveragebydirectinjectioncapillarygaschromatography.JFoodComposAnal17:187–96.
ZongH,ZhanY,LiX,PengL,FengF,LiD.2010.AnewmutationbreedingmethodforStreptomycesalbulusbyanatmosphericandroomtemperatureplasma.AfrJMicrobiolRes6:3154–8.
M:FoodMicrobiology&Safety
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