An original method for producing acetaldehyde and diacetyl by yeast fermentation

h tt p : / / w w w . b j m i c r o b i o l . c o m . b r /

Industrial

Microbiology

An

original

method

for

producing

acetaldehyde

and

diacetyl

by

yeast

fermentation

Irina

Rosca

_,

_Anca

_Roxana

_Petrovici

_,

_Mihai

_Brebu

_,

_Irina

_Stoica

_,

_Bogdan

_Minea

_,

Narcisa

Marangoci

a_{“PetruPoni”InstituteofMacromolecularChemistry,AdvancedResearchCenterforBionanoconjugatesandBiopolymers,} AleeaGrigoreGhicaVoda,Iasi,Romania

b_{SCZeelandiaSRL,R&DDepartment,ValeaLupului,Iasi,Romania} c_{“GrigoreT.Popa”UniversityofMedicineandPharmacy,Iasi,Romania}

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Articlehistory:

Received2June2015 Accepted29February2016 Availableonline25July2016 AssociateEditor:JorgeGonzalo FariasAvendano

Keywords:

Acetaldehyde Diacetyl

YADculturemedium Yeastfermentation

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Inthisstudyanaturalculturemediumthatmimicsthesyntheticyeastpeptoneglucose medium usedforyeastfermentationswasdesignedtoscreenandselectyeastscapable ofproducinghighlevelsofdiacetylandacetaldehyde.Thepresenceofwheypowderand sodiumcitrateinthemediumalongwithmanganeseandmagnesiumsulfateenhanced bothbiomassandaromadevelopment.Atotalof52yeastsstrainswerecultivatedintwo differentculturemedia,namely,yeastpeptoneglucosemediumandyeast acetaldehyde-diacetylmedium.Theinitialscreeningofthestrainswasbasedonthequalitativereaction oftheacetaldehydewithSchiff’sreagent(violetcolor)anddiacetylwithBrady’sreagent (yellowprecipitate).Thefermentedculturemediaof10yeaststrainsweresubsequently analyzedbygaschromatographytoquantifytheconcentrationofacetaldehydeanddiacetyl synthesized.Totaltitratableacidityvaluesindicatedthatatotaltitratableacidityof5.5◦SH, implyingculturemediumatbasicpH,wasmorefavorablefortheacetaldehydebiosynthesis usingstrainD15(Candidalipolytica;96.05mgL−1acetaldehyde)whileatotaltitratableacidity valueof7◦SHfacilitateddiacetylflavorsynthesisbystrainD38(Candidaglobosa;3.58mgL−1 diacetyl).Importantly,theresultspresentedheresuggestthatthiscanbepotentiallyused inthebakingindustry.

©2016SociedadeBrasileiradeMicrobiologia.PublishedbyElsevierEditoraLtda.Thisis anopenaccessarticleundertheCCBY-NC-NDlicense(http://creativecommons.org/ licenses/by-nc-nd/4.0/).

Introduction

Yeastsarecommonlyusedasstarterculturesforincreasing product-specificaromaproductionduringvarious fermenta-tionprocesses(cheese,kefir,sourdough,wine,beer,etc.),as

∗ _{Correspondingauthor.}

E-mail:petrovici.anca@icmpp.ro(A.R.Petrovici).

theyarecapableofsynthesizingnaturalflavorslike acetalde-hyde (ethanal) or diacetyl (2,3-butanedione) which inturn servetoenhancethequalityofthefood.1_{Acetaldehydeisthe}

mostimportantcarbonyl compoundproduced during alco-holicfermentationwithfinalconcentrationstypicallyvarying between10and200mgL−1dependingontechnologicalfactors

http://dx.doi.org/10.1016/j.bjm.2016.07.005

1517-8382/©2016SociedadeBrasileiradeMicrobiologia.PublishedbyElsevierEditoraLtda.ThisisanopenaccessarticleundertheCC BY-NC-NDlicense(http://creativecommons.org/licenses/by-nc-nd/4.0/).

suchasculturemediumcomposition,pH,fermentation tem-perature,aerationandSO2 concentration,andonthe yeast

strainused.2,3_{Acetaldehydeisfoundinavarietyoffoodsand}

beveragessuchascheese,yogurt,beer,and wine.4,5

Gener-ally,acetaldehydelevelsreachtheirpeakvaluesintheearly fermentationphase;itisthenpartlyreutilizedbytheyeasts duringtherest ofthefermentationprocess.6 _Acetaldehyde

isresponsibleforthegreenapplearomainalimentary prod-ucts and, usually, the added acetaldehyde is produced by chemicalsynthesis.Contrarily,byaddingusingacetaldehyde producedbybiosynthesisrenderstheproductsafeforhuman consumption, and the inadvertent addition of the byprod-ucts of chemical acetaldehyde synthesis can be avoided. Diacetylisalmostexclusivelysynthesizedbylacticacid bacte-riaand isthe key flavor compound naturally produced by theLeuconostocsp.7,8 _{Diacetylisanimportantflavoring}

com-pound that determines specific characteristics of products suchasfermentedmilksand,atverylowconcentrations(up to5mgL−1), isalsoresponsible for the characteristic “but-tery”aromaofmilkproducts.9_{Furthermore,itisknownthat}

whileSaccharomycescerevisiaeproduces132.4mgL−1 acetalde-hyde,lessthan1mgL−1diacetylisproducedduringredwine fermentation.10–12 _{Thus, by using a potential fermentation}

mixthatcanproducebothacetaldehydeanddiacetylduring the fermentationprocess,inplace ofachemically synthe-sizedproduct,itispossibletoconfirmtothecurrentglobal trendofreplacingsyntheticproductswithnaturallyobtained products,whileconcurrentlysatisfyingconsumerdemandfor naturalcompoundsinalimentaryproducts.

Therefore,themainobjectiveofthisstudywastoidentify yeastscapableofbiosynthesizinghighamountsof acetalde-hydeanddiacetylandtoassesstheirproposeduseasnatural productsinfermentationproductsduringindustrial produc-tion.Thisisnecessarybecause anyproducton themarket thatalreadycontains thisaromacannotbefurtherusedin patisserieproducts.

Materials

and

methods

Yeaststrains

Theyeastsstrainsutilizedinthisstudywereisolatedfrom var-ioussourcesinthelaboratoriesofBIOALIMENT(biotechnology appliedinfoodindustry–integratedcenterforresearchand education),“DunareadeJos”,UniversityofGalati,Facultyof FoodScienceand Engineering. Everystrainwas assigned a uniquecodeintheMIUGcollectionofthe“DunareadeJos” UniversityofGalati,and simultaneously,auniquecode for themicroorganismcollectionofthe“PetruPoni”Instituteof MacromolecularChemistry(ICMPP).Purestrainswerestored at−80◦_{C in YPG culturemedium supplemented with20%}

glycerol.13_{Further,thetaxonomicclassificationoftheisolated}

yeastswasdeterminedandflavorproductionwasfollowedby gaschromatography(GS).

Cultureconditions

Inthisstudy,wedesignedandusedanaturalculturemedium, yeastacetaldehyde-diacetylmedium(YAD),thatcouldmimic the synthetic YPG culture medium, to select yeast strains

capableofproducinghighlevelsofdiacetylandacetaldehyde. Importantly, the sodium citrate, manganese and magne-sium sulfate, which are part of the YAD culture medium, are consumed by the microorganisms during growth and biosynthesis, thereby rendering the YAD culture medium ‘natural’. For selecting yeast strains capable of producing diacetyl and acetaldehyde, wedesigned a culturemedium with the following composition: 10gL−1 dextrose; 50gL−1 yeast extract; 5gL−1 sodium citrate;10gL−1 whey powder; 0.05gL−1 manganese sulfate; 0.2gL−1 magnesium sulfate. ThepHwasadjustedbetween6and7using0.1NHCl.The culturemediumwassterilizedat120◦Cfor20min,andthis culturemediumwasdesignatedYAD(yeastdiacetyl acetalde-hyde). The dormant yeasts were activated by inoculating theminstandardYPGmediumandtransferredtoYAD cul-turemediumduringtheloggrowthphasebyinoculating5mL ofYADculturemediumwitha20%yeastsuspensionthatwas activatedinYPGmediumfor24handhadanabsorbanceof 0.5at600nm.Thecultureswereincubatedat30◦Cfor48h under static conditions inorder to reducethe evaporation ofvolatilesubstances. Afterfermentation,themediumwas filteredtoremovecellsandsubjectedtoGaschromatography for identification and quantification of acetaldehyde and diacetylproduction.

IdentificationoftheyeaststrainsusingID32CTM

ID32CTM_{isastandardizedsystemofyeastidentificationthat}

uses32miniaturizedassimilationtestsandadatabase.The ID32CTM_{stripconsistsof32cupules,eachcontaininga}

dehy-dratedcarbohydratesubstrate.Asemi-solid,minimalmedium isaddedalongwithasuspensionoftheyeasttobetested. After24–48hofincubation,growthineachcupulewasread usingtheATBExpression,theminiAPIinstrument,orvisually. IdentificationwasperformedusingtheApiwebTM

identifica-tionsoftware.

Detectionofdiacetylandacetaldehydeinculturemedium

DetectionofaldehydeswithSchiff’sreagent

Schiff’stestisaqualitativetestforthepresenceofaldehyde functional groups wherein,acolorlessSchiff reagent turns into acharacteristicvioletcolorwhen aldehydegroups are presentinthesampleadded.14_{Thereactionwasperformed}

byadding500␮LofSchiff’sreagenttothe5mLofmedium fer-mentedfor48hat30◦C.Themediasampleswereassigned scoresbetween0and5basedonreactionintensityat5safter reagent addition, with0denoting noacetaldehyde produc-tion;1–3denotinglowproductionofacetaldehyde,4denoting good productionofacetaldehyde,and5denotingverygood productionofacetaldehyde.Onlysampleswithhighaldehyde content(score5)werefurthertestedtodetermineif acetalde-hydewasindeedpresentinhighamounts.

DetectionofdiacetylwithBrady’sreagent

2,4-Dinitrophenylhydrazine (DNPH, Brady’s reagent) is a chemicalcompoundwhichcanbeusedtoqualitativelydetect the presenceofketoneor aldehyde functional groups.The test isconsidered positiveif ayellow(aliphatic), orange or red (aromatic) precipitate ispresent. DNPH does not react

withothercarbonyl-containingfunctionalgroupssuchas car-boxylic acids, amidesor esters.15 _{The test was performed}

by adding 250␮L of Brady’s reagent to 2.5mL of medium fermentedfor48hat30◦C.Asdiacetylisanaliphatic com-pound,onlythosestrainsthatgaveayellowprecipitatewere selectedforfurtherinvestigation.SimilartoSchiff’sreaction, thesampleswereevaluatedbasedonintensityoftheyellow precipitateandthis wasassignedascorebetween0and5, where0denotesnodiacetylproduction,1–3lowproduction ofdiacetyl,4goodproductionofdiacetyland5verygood pro-ductionofdiacetyl.Further,resultsfromboththeSchifftest andtheDNPHtestwereconsideredtogetherinordertoselect strainscapableofproducingbothacetaldehydeanddiacetyl. Foridentificationofdiacetylandacetaldehydeintheculture medium,thesamestrainwassimultaneouslyinoculatedinto bothYADandYPGmedia.

Totaltitratableacidity(TTA)

TTA was quantified according to the method of Soxhlet-Henkelusingasodiumhydroxidestandardsolutionto neu-tralizetheacidcontentofthesample(herein,suspensionof theculturemedium).16_{TheprocedureusedforestimatingTTA}

isasfollows.Asolutioncontaining5gofthesampleand45mL ofdistilledwaterintitrationflaskwasstirredonamagnetic stirrerfor2minand2mLofphenolphthaleinadded. Subse-quently,0.25MNaOHsolutionwasaddedtothissuspension untilthesolutionturnedalightpinkcolor.TTAwascalculated usingtheformulaTTA(◦SH)=2×2.5×1,where1isthe

vol-umeof0.25MNaOHconsumedforneutralizationoftheacids inthesample.Alltitrationswereperformedintriplicate.This methodwasalsousedtomonitorhowculturemediumacidity affectedaromabiosynthesisduringfermentation.

Quantificationofdiacetylandacetaldehydeintheculture

medium

Diacetylandacetaldehydeintheculturemediumwere quan-tifiedusinggaschromatography(GS)spectraoftheanalyzed samplesand from equations obtainedfrom thecalibration curvesforacetaldehydeanddiacetyl.Theconcentrationsused forobtainingthecalibrationcurveforacetaldehydewere:5,10, 20,40,80,and200␮LL−1,whilethosefordiacetylwere2.5,5, 10,25,and50␮LL−1.

GSspectrawereobtainedusinganAgilent6890NGas Chro-matographwithanFIDDetectorandaSuprawax-280(100% PEGbonded,crosslinked)60.0m×250␮m×0.25␮mcapillary columnwith the following parameters: He gas flow set at 2mLmin−1,inlettemperature150◦C,splitratio10:1;sample volume1␮L.Thetemperatureprogramstartedat40◦C (main-tainedfor1min),andwasfollowedbyheatingto110◦Cata rateof10◦Cmin−1,andthentill270◦Cat40◦Cmin−1.

Results

and

discussion

Taxonomicclassificationoftheisolatedyeasts

Atotalof52yeastisolatesweretaxonomicallyclassifiedusing theApiwebTM_{identificationsoftware(Table1).Thirtyisolates}

were identifiedasbelongingtothegenusCandidaspp., ten isolatesbelongedtogenusSaccharomycesspp.(S.cerevisiae),five isolateswereCryptococcusspp.,oneisolatebelongedtogenus

Geotrichum spp.(Geotrichumcapitatum),andsixisolateswere

Rhodotorulaspp.

Yeastselectionbydetectionofacetaldehydeanddiacetylin

culturemedium

Inordertoidentifyyeastisolatescapableofbothacetaldehyde and diacetyl biosynthesisduringfermentation, theisolates were inoculated into both YAD and YPG media and fla-vor biosynthesis was compared between these two media (Tables1 and2)using Schiff’s testand Brady’stest forthe determinationofacetaldehydeanddiacetyl,respectively.

SelectionofyeastsusingSchiff’sreagent

Acetaldehyde biosynthesis depends on the type of carbon and nitrogensourcesinthe medium,incubationtime, agi-tationrate,andincubationtemperature.Therefore,dextrose wasaddedtotheYADmediumasitisaveryaccessible car-bonsourceforyeasts.Wheypowderwasaddedasanatural andveryeconomicalsourceofnitrogen,whilemanganeseand magnesiumsulfatearenecessarygrowthfactorsforyeast cul-ture.Sodiumcitrateintheculturemediumactsasaprecursor forandstimulatesdiacetylbiosynthesis(Table1).17

Giventheaboveandascanobservedfromtheresults pre-sentedinTable1,yeastsinoculatedinYPGdidnotproduce acetaldehyde,whilefermentationinYADculturemediumled tothe biosynthesisofacetaldehydeduetothe presenceof thedextroseandwheypowderinthisculturemedium.Nine yeastsstrainsproduced highlevelsofacetaldehydeinYAD (Schiff’sreactionscore5),andwereselectedforfurther analy-sesbygaschromatography.Thenineyeaststrainsfoundtobe goodproducersofacetaldehydeareasfollows:threestrainsof

S.cerevisiae:D5,D37andD49,twostrainsofCandidalipolytica:

D15andD21,twostrainsofCandidacolliculosa:D17andD30, onestrainofCandidaglobosa:D38,andonestrainofCandida krusei:D50).

SelectionoftheyeastsusingBrady’sreagent

Similartoaldehydeproduction,yeastsinoculatedinYPGdid notsynthesizediacetyl,andonlyonestrainproduceddiacetyl when inoculated in to YAD medium, even though sodium citratewasadded tothemedium.Theyeastisolatewitha scoreof5fordiacetylbiosynthesis(C.lipolytica;D13)wasthen selectedforfurtheranalysisbyGC.

Gaschromatographicanalysis

The acetaldehyde and diacetyl concentrations obtainedby GC,andTTAvaluesoftheselectedisolatesarepresentedin

Table2.

The data presented in Table 2 show that consider-able amounts of acetaldehyde (75.07mgL−1) and diacetyl (3.58mgL−1)wereobtainedwhentheD38isolate(identified asC.globosabytheAPItest)wasusedforfermentationinthe YADmedium.

GC spectrumpeak area of acetaldehyde(retention time approx.4.3min)anddiacetyl(retentiontimeapprox.7.0min)

Table1–TheyeastsisolatesidentifiedwithID32CTM_{andtheselectionoftheyeaststrainsforacetaldehydeanddiacetyl}

biosynthesisinliquidculturemedium.

No. ICMPP ID code Species ID32CTM (%) YPGculture medium Schiff’sreaction foracetaldehydea YADculture medium Schiff’sreaction foracetaldehydea YPGculture medium Brady’sreaction fordiacetyla YADculture medium Brady’sreaction fordiacetyla 1 D1 Candidafamata 98.7 0 2 1 1 2 D2 Candidarugosa 82.9 0 1 1 1 3 D3 Rhodotorulaminuta 99.9 0 2 1 1 4 D4 Rhodotorulamucilaginosa 91.9 0 1 1 1 5 D5 Saccharomycescerevisiae 98.5 0 5 1 1 6 D6 Candidapelliculosa 99.9 0 4 1 1 7 D7 Cryptococcusuniguttulatus 99.9 0 2 1 1 8 D8 Cryptococcusterreus 99.9 0 1 1 1 9 D9 Candidafamata 99.2 0 2 1 1 10 D10 Candidafamata 98.7 0 2 1 1 11 D11 Candidalipolytica 99.9 0 2 1 1 12 D12 Candidakrusei 85.7 0 1 1 1 13 D13 Candidalipolytica 99.9 0 4 1 1 14 D14 Candidalipolytica 99.9 0 2 1 1 15 D15 Candidalipolytica 99.9 0 5 1 5 16 D16 Candidalipolytica 99.9 0 1 1 1 17 D17 Candidacolliculosa 99.9 0 5 1 1 18 D19 Candidaholmii 87.9 0 2 1 1 19 D20 Candidapelliculosa 87.0 0 1 1 1 20 D21 Candidalipolytica 99.9 0 5 1 1 21 D22 Candidapelliculosa 99.9 0 3 1 1 22 D23 Candidacolliculosa 99.9 0 2 1 1 23 D24 Candidafamata 96.9 0 2 1 1 24 D25 Cryptococcusalbidus 99.5 0 4 1 1 25 D26 Candidalipolytica 99.9 0 3 1 1 26 D27 Candidasphaerica 97.2 0 4 1 1 27 D28 Geotrichumcapitatum 98.1 0 2 1 1 28 D29 Candidalambica 92.7 0 4 1 1 29 D30 Candidacolliculosa 99.9 0 5 1 1 30 D31 Cryptococcusterreus 99.9 0 2 1 1 31 D32 Candidalambica 99.9 0 2 1 1 32 D33 Rhodotorulaminuta 99.9 0 4 1 1 33 D34 Candidalambica 96.4 0 3 1 1 34 D35 Saccharomycescerevisiae 99.6 0 3 1 1 35 D36 Saccharomycescerevisiae 99.7 0 1 1 1 36 D37 Saccharomycescerevisiae 99.7 0 5 1 1 37 D38 Candidaglobosa 99.9% 0 5 1 1 38 D39 Saccharomycescerevisiae 99.6% 0 3 1 1 39 D47 Saccharomycescerevisiae 99.9 0 2 1 1 40 D49 Candidalipolytica 99.9 0 5 1 1 41 D50 Candidakrusei 85.7 0 5 1 1 42 D51 Candidasilvicola 99.9 0 2 1 1 43 D52 Rhodotorulaglutinis 83.1 0 2 1 1 44 D53 Candidafamata 93.2 0 4 1 1 45 D54 Saccharomycescerevisiae 99.9 0 4 1 1 46 D55 Saccharomycescerevisiae 99.4 0 2 1 1 47 D56 Saccharomycescerevisiae 99.9 0 2 1 1 48 D57 Cryptococcuscurvatus 99.9 0 2 1 1 49 D58 Candidapelliculosa 99.9 0 2 1 1 50 D59 Rhodotorulamucilaginosa 88.2 0 3 1 1 51 D60 Rhodotorulamucilaginosa 86.6 0 1 1 1 52 D61 Saccharomycescerevisiae 99.9 0 3 1 1

Theboldvaluesareindicatingtheverygoodproducersofacetaldehyde/diacetyl.

a _{Theyeaststrainswerenotedas:0,theyeaststrainthatdoesnotproduceacetaldehyde/diacetyl;1to3,lowproducersofacetaldehyde/diacetyl;} 4,goodproducersofacetaldehyde/diacetyland5,verygoodproducersofacetaldehyde/diacetyl.

Table2–Gaschromatographyandtotaltitratableacidity(TTA)valuesforselectedfermentedmediums. No ICMPPID codea ALDb_retention timefromGC chromatogram (min) ALDc (␮LL−1) ALDd (mgL−1) DAe_retention timefromGC chromatogram (min) DAf (␮LL−1) DAg (mgL−1) TTA (◦SH) 1 D5 4.394 109.85 86.23 6.914 1.72 1.69 23.5 2 D13 4.409 98.41 77.25 6.931 0.82 0.8 30 3 D15 4.388 122.36 96.05 6.964 0.11 0.107 5.5 4 D17 4.386 66.86 52.49 6.923 0.97 0.951 10.5 5 D21 4.387 66.78 52.42 6.999 0.127 0.124 6.5 6 D30 4.402 69.17 54.3 6.938 0.22 0.215 10 7 D37 4.402 60.66 47.62 6.991 0.26 0.255 8.5 8 D38 4.391 95.63 75.07 6.896 3.65 3.58 7 9 D49 4.391 98.47 77.3 6.987 2.07 2.03 6.5 10 D50 4.391 79.56 62.45 6.929 0.072 0.07 7

a _{Thecodegiventotheyeastsstrainsinmicroorganismscollectionof“PetruPoni”InstituteofMacromolecularChemistry(ICMPP).} b _{Acetaldehyde.}

c _{Theconcentrationofacetaldehydewascalculateusingcalibrationequation.} d _{TheconversionoftheacetaldehydeconcentrationinmgL}−1_.

e _Diacetyl.

f _{Theconcentrationofdiacetylcalculatedusingcalibrationequation.} g _{TheconversionofthediacetylconcentrationinmgL}−1_.

30 000 25 000 20 000 15 000 10 000 4.00 4.50 5.00 5.50 6.00 6.50 7.00 Time Standards Response D38 Diacetyl Acetal deh yde

Fig.1–GSspectrumofmediumresultedafterthe fermentationofD38yeaststraininYADmedium.

(Figs. 1 and 2)demonstrated that yeaststrain D15 (identi-fied as C. lipolytica by API test) was the best producer of acetaldehyde (96.05mgL−1) in YAD medium. However, the amount ofdiacetyl producedby thisyeast strain wasvery low(0.107mgL−1)eventhoughtheBradyreactiondata pre-sentedinTable1implyotherwise.Thiscanbeexplainedby thefactthattheBradyreagentreactswithbothketonesand aldehydes,andthepresenceofagreateramountofprecipitate wasduetothepresenceofhighamountsoftheacetaldehyde, ratherthandiacetyl.Therefore,thevolatilesproducedbythis

30 000 25 000 20 000 15 000 10 000 4.00 4.50 5.00 5.50 6.00 6.50 7.00 Time Standards Response D15 Diacetyl Acetal deh yde

Fig.2–GSspectrumofthemediumresultedfromthe fermentationofD15yeaststraininYADmedium.

strain werefurtheranalyzedbyGCinordertoidentifythe typeandconcentrationofthecompoundsproducedduring fermentation.IsolateD38(C.globosa)wasthebestproducerof diacetyl(3.58mgL−1)inthesameculturemedium(YAD), con-firmingthe resultsobtainedwithBrady’sandSchiff’s tests. Importantly,thisD38strainisalsocapableofbiosynthesizing asubstantialamountofacetaldehyde(75.07mgL−1)whichis, albeit,slightlylesserthanthatoftheD15strain.

Giventhattheliteratureonthebiosynthesisofaromafrom yeastsisscant,atbestonthebasisofourresults,wecould dis-cernonlyfewbasicinferences.ThedatapresentedinTable2

suggestthat,withrespecttotheyeaststrainD15,analkaline pHismorefavorablefortheacetaldehydebiosynthesisasthe culturemediumhadaTTAof5.5◦SH.Suchalkalinityis, how-ever,notoptimalforthesynthesisofdiacetyl,astheTTAof themediumfermentedwithD38was7◦SH,implyingthata neutralpHfavorsdiacetylbiosynthesis.

Conclusions

Weproposeanewandoriginalmethodtoproduce acetalde-hydeanddiacetylforuseinthebakeryindustryusingyeast fermentation.Anaturalculturemediumthatmimicsthe syn-theticyeastculture(YPG)wasdevelopedandusedsuccessfully toscreenpotentialisolatesandproducesubstantialamounts ofbothacetaldehydeand diacetyl.Theyeastsspecieswere identifiedusingID32CTM_{tests;andgaschromatographywas}

used toconfirm acetaldehydeand diacetyl production and accuratelyquantifythe amountproducedduring fermenta-tion.AlthoughC.lipolyticawascapableofsynthesizinghigh amountsofacetaldehyde,C.globosacouldbiosynthesize sub-stantial amounts of bothdiacetyl and acetaldehyde under neutralpHconditions.Inthelightoftheseresults,we pro-posethissystemtobetestedforbiotechnologicaltransferto industry-levelfermentersandtobeusedduringthe produc-tionoffermentedproducts.

Conflicts

of

interest

Theauthorsdeclarenoconflictsofinterest.

Acknowledgments

Thispublicationbenefitedfromthefinancialsupportofthe project“ProgramofExcellencyinthemultidisciplinary doc-toralandpost-doctoralresearchofchronicdiseases”,contract no.POSDRU/159/1.5/S/133377, beneficiary“Gr.T.Popa” Uni-versityofMedicineandPharmacyofIasi,projectco-financed from the EuropeanSocialFund throughthe Sectoral Oper-ationalProgramHumanResourcesDevelopment (SOPHRD) 2007–2013,andbyRomanianNationalAuthorityforScientific Research,CNCS–UEFISCDI, projectnumber PN-II-ID-PCCE-2011-2-0028.

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