Isolation, identification and characterization of regional indigenous Saccharomyces cerevisiae strains

ht t p : / / w w w . b j m i c r o b i o l . c o m . b r /

Food

Microbiology

Isolation,

identification

and

characterization

of

regional

indigenous

Saccharomyces

cerevisiae

strains

Hana ˇSuranská

,

Dana

Vránová,

Jiˇrina

Omelková

BrnoUniversityofTechnology,FacultyofChemistry,DepartmentofFoodScienceandBiotechnology,Purky ˇnova464/118,61200Brno, CzechRepublic

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t

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f

o

Received29July2014 Accepted19May2015

AssociateEditor:MaricêNogueira deOliveira Keywords: Saccharomycesgenus Oenologicalproperties ITS-PCR-RFLP InterdeltaPCRtyping Species-specificprimers

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InthepresentworkweisolatedandidentifiedvariousindigenousSaccharomycescerevisiae strainsandscreenedthemfortheselectedoenologicalproperties.TheseS.cerevisiaestrains wereisolatedfromberriesandspontaneouslyfermentedmusts.Thegrapeberries (Sauvi-gnonblancandPinotnoir)weregrownundertheintegratedandorganicmodeoffarming intheSouthMoravia(CzechRepublic)wineregion.Moderngenotypingtechniquessuchas PCR-fingerprintingandinterdeltaPCRtypingwereemployedtodifferentiateamong indige-nousS.cerevisiaestrains.Thiscombinationofthemethodsprovidesarapidandrelatively simpleapproachforidentificationofyeastofS.cerevisiaeatstrainlevel.Intotal,120isolates wereidentifiedandgroupedbymolecularapproachesand45oftherepresentativestrains weretestedforselectedimportantoenologicalpropertiesincludingethanol,sulfurdioxide andosmoticstresstolerance,intensityofflocculationanddesirableenzymaticactivities. Theirabilitytoproduceandutilizeacetic/malicacidwasexaminedaswell;inaddition, H2Sproductionasanundesirablepropertywasscreened.Theoenologicalcharacteristics

ofindigenousisolateswerecomparedtoacommerciallyavailableS.cerevisiaeBS6strain, whichiscommonlyusedasthestarterculture.Finally,someindigenousstrainscoming fromorganicallytreatedgrapeberrieswerechosenfortheirpromisingoenological proper-tiesandthesestrainswillbeusedasthestarterculture,becauseapplicationofaselected indigenousS.cerevisiaestraincanenhancetheregionalcharacterofthewines.

©2015SociedadeBrasileiradeMicrobiologia.PublishedbyElsevierEditoraLtda.Thisis anopenaccessarticleundertheCCBY-NC-NDlicense (http://creativecommons.org/licenses/by-nc-nd/4.0/).

Introduction

The quality of fermented foods and beverages is partially determinedbythemicroorganismsusedfortheirproduction.

∗ _{Correspondingauthor.}

E-mail:[email protected](H. ˇSuranská).

Forinstance,thesecondarycharacterofwineisdetermined bysensorycharacteristicsthatarisefromthedirectactionof microorganismsonthesubstrate.Thefermentationofgrape mustintowineisanecologicallycomplexprocess,inwhich bacteriaandothermicroorganisms,especiallyyeasts,playa

http://dx.doi.org/10.1016/j.bjm.2015.11.010

1517-8382/©2015SociedadeBrasileiradeMicrobiologia.PublishedbyElsevierEditoraLtda.ThisisanopenaccessarticleundertheCC BY-NC-NDlicense(http://creativecommons.org/licenses/by-nc-nd/4.0/).

crucialrole.ThestrainsofSaccharomycescerevisiae involved infermentationplayanimportantpartinthecharacteristics ofthe finalproductandthediversityofS.cerevisiaestrains presentinspontaneousfermentationcontributetothe chem-icalcompositionandsensoryqualitiesoftheresultingwine.1

Therefore, one of the most important technological advancesinwine-makingwastheinoculationofgrapejuice withselectedculturesofS.cerevisiae.2_{Thisapproachisbased}

ontheevidencethatmicrobiologicalcontrolofthe fermen-tation process allows better management ofthis alcoholic fermentation.ItisknownthatselectedstrainsofS.cerevisiae suppressindigenousnon-Saccharomycesspeciesanddominate thefermentationprocess.3–5

Nowadays, novel biotechnological approaches in wine-making are used in several aspects of the fermentation industry.Apartfrom themonitoringofthemicrobial popu-lations and the controlof the spoilage yeasts, attention is focusedalsoontheselectionandutilizationofthestarter cul-turescomingfrom one’sownvineyard,whichcanenhance theregionalcharacterofthewine.6,7_Themetabolic

peculiari-tiesandthephysiologicalpropertiesofS.cerevisiaeyeastmay leadtotheformationofmetabolitesandthetransformation ofgrapesubstancesthatmayenrichthewineflavor.7_Certain

criterianeedtobemetinordertoguaranteethedesirable fea-turesoftheyeaststrainsselected.Themostimportantones are:tolerancetoethanol;growthathighsugarconcentrations; resistancetosulfurdioxide;lowproductionofhydrogen sul-fide;productionofkillertoxinsorsomeenzymaticactivities.8

Furthermore,onlyreliableandrapididentificationofthe yeast species during the process and the quality control enables enologists to assess the role of yeasts as a main protagonistofalcoholic fermentationoras acontaminant. The utilization of molecular methods enabled rapid and preciseidentification ofthe yeasts atthe speciesor strain level.1 _{Mercado et al.}9 _{reported that Saccharomyces}

popula-tionsarerepresentedbymultiplestrains,evenininoculated fermentations.Therefore,itisimportanttohavesimpleand appropriatemethodsthatallowdiscriminationatthestrain level.

This study is focused on indigenous S. cerevisiae strain (i)selection,(ii)identificationand (iii)technological charac-terization.Yeastswereisolatedfromgrapesandmustsduring theproductionofwines.Weselectedtwotypesofwine vari-eties–SauvignonblancandPinotnoircomingfromanorganic and integratedtreated vineyardsituated inSouthMoravia, CzechRepublic.Ourobjectivewasalsofocusedonthe selec-tionoftheidentificationapproachesthatwillbesimpleand suitable for rapid and reliable strain identification. There-fore,isolatesofS.cerevisiaewere identifiedand groupedby several molecular approaches such as ITS-PCR-RFLP, PCR-fingerprinting, species-specific primers and interdelta PCR typing.Thecombination ofthesetechniquesenabledrapid detection, identification andtyping ofdifferentS. cerevisiae strains.Finally,theisolatedstrainswerescreenedforselected technological propertiesimportantinthe winemaking pro-cessand for furtherapplication asthe starter cultures. To sum up, this study has demonstrated the importance of selection of an appropriate and rapid identification tech-niqueandalsodeterminationofsomeimportantoenological properties.

Methods

Yeastspeciesisolationandcultivation

Autochthonous (indigenous) strains belonging to Saccha-romycesgenuswereisolatedfromgrapeberriesandalsofrom spontaneouslyfermentedmustsindifferentstagesofthe fer-mentationprocess.

GrapeswerecollectedattheIva ˇnvineyardsituatedinthe SouthMoraviaregionandbelongtotheMikulovwineregion, Czech Republic. Red wine Pinot Noir (Pn) and white wine Sauvignon blanc(Sg) cultivarsofVitis viniferawere chosen. Bothvarieties,typicalcultivarsforwhiteand,respectively,red Moravianwineproduction,werecultivatedusingtheorganic (O)andalsointegrated(I)farmingprocedure.Grapeberries (healthy and undamaged)were collected beforeharvest (in September2009,2010,2011)intosterileglasses.Immediately aftertransportationtothelaboratory,15–20grapeberrieswere placedinto150mLofMaltextractmediumMEM(Himedia;2% maltextract,0.1%peptone,2%dextrose,2%agar)and culti-vated for10daysatlaboratorytemperature(approx.23◦C). Afterthat,culturemedia(300␮L)inoculatedontoMEMagar plates supplemented with 250mgL−1 streptomycin sulfate (Himedia, India) were incubated at26◦C for3–5 days. The singlecolonieswereobtainedbyKoch’sdilutionmethod.

The fermentation process was performed in the cellar in Iva ˇn (during the vintage 2009, 2010, 2011), which was separatedfromcommonfermentationsofcommercialwine production.Thefermentationprocessfollowingthestandard procedurewasconductedina1000Lbarrel.Thecellar tem-perature was approx. 10◦C and the temperature of the fermentationwasapprox.18◦C.Themustwasspontaneously fermentedandthesampleswerecollected3timesperweek duringthewholefermentationprocess.

Yeastpopulationsfrom mustwere isolatedas described previously10 _{and cultivatedon maltextract medium(MEM)}

supplementedwith 250mgL−1 streptomycinsulfate (Hime-dia, India).Thesinglecolonies(pureculture)wereobtained byKoch’sdilutionmethod.

In total, 120Saccharomyces sp.strains were isolated and identified.PurecultureswerepreservedonMEMagarunder paraffinoil.Themostpromisingstrainswillbedepositedin theyeastculturecollectionCCYBratislava(strainS.cerevisiae 1-09hasalreadybeendepositedthere).

DNAisolation

Genomic DNA wasisolated from singlecolonies using the commercial kit UltraCleanTM _{Microbial DNA Isolation Kit}

(MoBio,USA)accordingtothemanufacturer’sprotocol.

ITS-PCR-RFLP

TodistinguishSaccharomycessp.fromotherisolates, ITS-PCR-RFLP was employed. Theinternal transcribed spacers (ITS) (ITS1andITS2)and5.8SrDNAgeneregionswereamplifiedby usingspecificprimersITS1(5-TCCGTAGGTGAACCTGCGG-3) andITS4(5-TCCTCCGCTTATTGATATGC-3).11_DNA

0.2mM of dNTP, 0.5␮L of each primer 100pmol␮L−1_,

1×PCRreactionbufferand1UofTaqDNApolymerase(Kapa Biosystems, USA). PCR conditions were as follows: initial denaturationcycle at94◦C for4minfollowed by25 cycles ofamplification,denaturationat94◦Cfor1min,annealingat 48◦Cfor30s,andextensionat72◦Cfor1min;finalextension at72◦Cfor10min.

For RFLP, PCR products were purified by ethanol pre-cipitation and digested by restrictionendonucleases HaeIII (Fermentas,USA)followingthemanufacturer’sinstructions.

Species-specificprimers

ForS.cerevisiaespeciesidentification,asetofpairsof species-specific primers ScerF2 (5 -GCGCTTTACATTCAGATCCCGAG-3) and ScerR2 (5-TAAGTTGGTTGTCAGCAAGATTG-3) were used.12_{DNAamplificationwascarriedoutinafinalvolume}

of25␮Lcontaining0.2mMofdNTP(Invitek,Germany),0.5␮L ofeachprimer100pmol␮L−1_{(GeneriBiotech,CzechRepublic),}

1×PCRreactionbuffer,1.5mMMgCl2and1.25UTaqDNA

poly-merase(BioRad,USA)and0.5␮LoftemplateDNA.PCRcycling conditionsusedwere:initialdenaturationcycle at94◦Cfor 4minfollowedby30cyclesofamplification,denaturationat 94◦Cfor1min,annealingat55◦Cfor1min,andextensionat 72◦Cfor1min;finalextensionat72◦Cfor2min.

PCR-fingerprintingandinterdeltaPCRtyping

In order to distinguish different S. cerevisiae strains, PCR-fingerprinting by using M13 primer and interdelta primers were used. DNA amplification was carried out in a final volume of 25␮L containing 0.2mM of dNTP (Invitek, Ger-many), 0.5␮L of each primer 100pmol␮L−1 _{(VBC Biotech,}

Germany),1×PCRreactionbufferand1UTaqDNApolymerase (Kapa,USA)and0.5␮LoftemplateDNA.

PCR-assaysbyusingM13primer(5 -GAGGGTGGCGGTTCT-3):13_{initialdenaturationcycleat95}◦_{Cfor5min,followedby}

40cyclesofamplificationfollowedbydenaturationat93◦Cfor 0.75s,annealingat50◦Cfor1min,andextensionat72◦Cfor 1min;finalextensionat72◦Cfor6min.ThesecondPCR-assay forM13primer:initialdenaturationcycleat94◦Cfor4min, followedby35cyclesofamplificationfollowedbydenaturation at94◦Cfor30s,annealingat36◦Cfor45s,andextensionat 72◦Cfor45s;finalextensionat72◦Cfor7min.

The conditions for interdelta PCR typing by ␦1 (5-CAAAATTCACCTATATTCTCA-3) and ␦2 (5 -GTGGATTT-TTATTCCAACA-3); ␦2 (5-GTGGATTTTTATTCCAACA-3) and ␦12(5-TCAACAATGGAATCCCAAC-3)14,15_{wereasfollows:}

ini-tialdenaturationcycleat94◦Cfor4min,followedby30cycles ofamplification–denaturationat94◦Cfor30s,annealing at 49◦C for 1minand extensionat 72◦C for 2min. Thefinal extensionwasat72◦Cfor10min.

DetectionofPCRproducts

PCR products and restriction fragments were separated and detected by electrophoresis on 2% (w/v) agarose gel in 1× TBE buffer at 5Vcm−1 for 2h. DNA amplified by single repetitive primers and by delta primers were sepa-ratedon1.5%agarosegelfor3–4h.Thegelswerestainedby

ethidiumbromide(10mgL−1),visualizedunderUVlight(Ultra Lum. Inc., USA) and documented by ScionImage software (Scion, India). Finally, electrophoreograms were processed bythe BioNumerics6.5softwareemployingUPGMA cluster analysis.

Screeningofoenologicalproperties

Flocculationpropertiesofselectedstrainsweretested accord-ing to Bony et al.16 _{with slight modification. Yeasts were}

culturedfor3daysat26◦Cintubescontaining10mLofYPD (2%peptone,1%yeastextractand2%glucose)mediumunder permanent shaking (150rpm). Cells were collected by cen-trifugationandwashedwithdeionizedwater.Afterthat,cells weresuspendedinto10mLof50mMacetatebuffer(pH4.5) enrichedby3mMCaSO4.Thetubesofsuspendedcellswere

mixedfor30sandtheturbidityofyeastsuspensionwas eval-uatedbythenakedeye.Flocculationdegreewasdetermined usingasubjectivescalewhichmeansthatthesedimentation wasobserveddependingonthetime.Thefollowing evalua-tionwasused:+thecellsflocculateandsedimentafter15min (partiallyclearsolution);++immediateflocculation;w– floc-culationafter1h.

Ethanoltolerancewastestedin5mLofYPDmedium sup-plementedby12,14,15,16and17%(v/v)ethanolandthetubes wereinoculatedby100␮Lofcellsuspension(cell concentra-tionapprox.5logCFUmL−1).Inoculatedtubeswerecultivated at26◦Cfor10days.Theculturedensitywasmeasureddaily byDEN-1Bdensitometer(Biosan,Latvia).Specificgrowthrate (h−1)andthelengthofthelagphaset(h)wereestimated.

Osmotoleranceofselected strainswastested in5mLof YPDmediumwith40%(w/v)and50%(w/v)glucose.Thecells densitywasmeasuredasdescribedabove.

H2SproductionbyselectedstrainswastestedonBiggyagar

(Himedia,India)whichcontainsbismuthasanindicator.After incubation at26◦Cfor3–5 days, the zone surrounding the colonywasevaluatedasthefollows:−noproduction;+white colonies;++lightbrown;+++brown;++++darkbrown/black.5

Malicand acetic acidutilization byselectedstrains was tested on 0.67% yeast nitrogen base (YNB, Himedia) agar plates containing 0.5%(w/v) malicacid,respectively, 0.25% (w/v)aceticacid.Thegrowthofcolonieswasscreened.Acetic acidproductionwasscreenedonCaCO3agarplates(Custer’s

chalkmedium)containing0.5%yeastextract,5%glucose,0.5% CaCO3and2%agar.Theaceticacidproductionenabled

solu-bilityofCaCO3whichresultedasaclearzonesurroundingthe

colonies.5

Theisolatedyeastswerealsotestedforsomeenzymatic activitiessuchas␤-glucosidaseandglycosidaseactivities.The activitiesweredetermined byagarplating. Theplateswere incubatedat26◦Cfor3–5days.17

␤-Glucosidase activity was screened onto selective mediumcontaining0.67% yeastnitrogenbase(YNB, Hime-dia),0.5%arbutinand2%agar.ThepHofthemediumwas adjusted to 5 before autoclaving. Two milliliters ofa filter sterilized 1%ferric ammonium citrate solution was added to 100mL media before plates pouring. Colonies showing activity were identified by a dark brown halo around the colonies.17

Table1–NumberofSaccharomycessp.isolates.

Grapevariety Vintage NumberofSaccharomycessp.isolates

Integrated(I) Organic(O)

Grapes(B)/must(M) Grapes(B)/must(M)

Sauvignonblanc(Sg) 2009 1/14 1/11

Pinotnoir(Pn) 2010 0/14 0/17

Sauvignonblanc(Sg) 2011 0/20 0/17

Pinotnoir(Pn) 2011 1/14 0/10

Total 120

Totalofyeastsisolates 524

Glycosidaseactivitywasdeterminedusingtheplateswith theselectivemediumcontaining0.67%yeastnitrogenbase (YNB,Himedia),0.2%rutinand2%agar.Theglycosidase activ-itywasdetectedasaclearzonearoundthecolonies.5,17

Results

and

discussion

SelectionandidentificationofS.cerevisiaestrains

In total, we isolated 120 single colonies of autochthonous Saccharomycessp.strainsfrom524totalyeasts.Yeastswere isolatedfromSauvignonblanc(Sg)andPinotnoir(Pn)coming fromorganic(O)andintegrated(I)farming.Theyeastspecies wereisolatedfromgrapeberriesaswellasfromspontaneously fermentedmust duringthe vintage2009(09),2010(10)and 2011(11).ThelistoftheSaccharomycesisolatesandthesources oftheirisolationareshowninTable1.

YeastsofthegenusSaccharomycesweredistinguishedfrom the other isolates (fromnon-Saccharomyces species) by ITS-PCR-RFLP(thelengthofPCRampliconwas880bp).Further, forS.cerevisiaespeciesidentification weemployed species-specificprimersScerF2andScerR2.12_{Species-specificprimers}

enableustoidentifyanddistinguishS.cerevisiaespeciesfrom otherspeciesbelongingtotheSaccharomycessensustricto com-plex,whichincludesthespecieswhichcanalsobefoundin fermentedmust(for.ex.Saccharomycesbayanus,Saccharomyces pastorianus,Saccharomyceskudriavzevii).Basedonourresults, all the isolates belonging to the Saccharomyces genus were identifiedbyspecies-specificprimersasS.cerevisiae(thelength ofthePCRproductswas150bp)(datanotshown).

Further,twodifferentPCR-assaysbyusingM13primerwere used.Theseassaysdifferedinannealingtemperature(50◦C vs.36◦C).Theisolatesweredividedintothreegroupsbythe first assay withannealing temperature 50◦C and into four groupsbythe secondPCR-assay(36◦C).Dataare presented aspartofthedendrogram(Fig.1).Twoisolates(markedasU, T)exhibitedadifferentfingerprintingprofilethantherestof theisolates;theseisolatesmaybehybridsofS.cerevisiaeand anotherspeciesbelongingtotheSaccharomycesgenus.Hence, PCR-fingerprintingtechniquesusingM13 primerare ableto groupthespeciesmembersofSaccharomycesgenusbutthey arenotsuitableforresolving.

In order to distinguish various S. cerevisiae strains, we employed ␦1–␦2 and ␦12–␦2 primers amplifying inter-delta sequences. These delta elements are described as appro-priategeneticmarkers foridentification ofpolymorphisms becausethenumberandlocationhaveacertainintraspecific

variability.18_{Theprimerpair␦1–␦2generatedfromthreeto}

eightdifferentfragmentsperstrainwithonecommonband of the size of approximately 1000bp. On the other hand, primer pair ␦12–␦2 provided significantly more fragments per sample and some of them were of low intensity. The lownumberoffragmentspersampleisascribedtotheweak homology exhibitedbytheprimer␦1–␦2towardsthewhole sequence ofS.cerevisiaegenom.19 _{However,bycombination}

ofthesetwosetsofdeltaprimers,weidentified45differentS. cerevisiaestrains.Themajorityoftheisolatedandidentified S.cerevisiaestrainscamefromspontaneousfermentedmusts andbelongedtogroupA(strainA).Despitethefactthatsome authors20,21 _{reported thatit isalmost impossible toisolate}

Saccharomyces sp.populations fromberries andinitialmust by standarddirect agar platingprocedure due totheir low counts (>10–100CFUcm−2) we isolated three strains from grapeberries(seeTable1).Theelectrophoreticpatternsand finaldendrogramshowingthegeneticssimilarityofvarious identifiedS.cerevisiaestrainsareshowninFig.1.

The novel combination of the modern molecular approaches used in this study for strain identification andtypingseemstobesuitableforrapid,reliable,simpleand reproducible identification of S. cerevisiae strains. Schuller etal.,22_Maquedaetal.23_orOrtizetal.24_{reportedapplication}

ofmitochondrialDNArestrictionanalysisorkaryotypingin ordertodistinguishvariousstrainsofS.cerevisiae.However, thesemethodsarelaborintensiveandtheresultsare influ-encedbycomplexity ofdatainterpretation duetothe high number ofgenerated fragmentsaftermtDNA RFLP.On the contrary,weemployeddifferentapproachestothegroupand distinguishSaccharomycesatstrainlevelbasedonconsequent employment of PCR-fingerprinting. For instance Schuller etal.22 _{reportedthatinterdeltaPCRtypinghadverysimilar}

resolvingpower atstrain levelas mtDNAand karyotyping, therefore,theuniquecombinationofthemethodsutilizedin thisworkcanbeconsideredasasimpleandrapidalternative tomtDNAorkaryotyping.

ScreeningofselectedoenologicalpropertiesofvariousS. cerevisiaestrains

Becausenotallyeaststrainsarerelevantforthespecific con-ditionsandcharacteristicsofwine,anumberofcriteriahave beenproposedfortheselectionofnewyeaststrainsforuse inthewinemakingprocess.Oneofthemostimportant cri-terionsforhigh-qualitywineproductionisuseofS.cerevisiae strainwithsuitabletechnologicalproperties.Thus,inorder

Composite 40 60 80 100 M13-50°C M13-37°C d1-d2 d12-d2 W1 Z A3 A2 K1 X1 Y BS6 25-10 E1 F1 A1 V I M J H R Q O N K D F G E C 13-10 27-10 P A B L 1-09 2-09 13-09 5-09 A4 X W 20-09 S 15-09 T U

Fig.1–DendrogrambasedonthesimilarityofPCR-fingerprintingpatternsofS.cerevisiaeisolates.S.cerevisiaeBS6–control strain.ThedescriptionontherightsideofthedendrogramisthelabeloftheisolatedS.cerevisiaestrains.

b r a z i l i a n j o u r n a l o f m i c r o b i o l o g y 4 7 (2 0 1 6) 181–190

Table2–OenologicalpropertiesofdifferentindigenousSaccharomycescerevisiaestrainsandonecommercialS.cerevisiaestrainBS6asacontrol.Pn–PinotNoir;O,I– organic,integrated;M,B–must,grapeberries;09–11–meanyearofisolation(2009–2011).Thegraylabelledfields–thestrainswhichphysiologicalpropertieswere betterthancontrolstrainBS6.

Isolated strain

Sourceof isolation

Desirabletechnologicalproperties

Ethanolresistance(%) Osmotolerance

12%ethanol 14%ethanol Ethanol(%) 40%glucose 50%glucose (h−1₎b _lagphaset (h) (h−1₎b _lagphaset (h) 15 16 17 (h−1₎b _lagphaset (h) (h−1₎b _lagphaset (h) A PnIB-11 0.061 22 0.019 58 + − − 0.037 19 0.029 24 A1 SgOM-11 0.054 22 − − − − − 0.044 19 0.029 24 A2 PnOM-11 0.058 24 0.025 59 + − − 0.049 19 0.021 24 A3 PnIM-11 0.055 24 − − − − − 0.037 28 0.013 24 A4 SgIM-11 0.092 24 0.017 39 − − − 0.052 19 0.022 24 B PnOM-10 0.060 24 0.028 85 − − − 0.055 19 0.023 30 C SgOM-09 0.060 24 0.033 58 − − − 0.057 19 0.020 24 D PnOM-10 0.075 24 0.018 42 − − − 0.054 19 0.014 24 E PnIM-10 0.080 22 0.013 42 − − − 0.051 19 0.020 43 E1 SgOM-11 0.057 24 0.033 80 − − − 0.051 19 0.020 24 F SgOM-09 0.097 24 0.019 42 − − − 0.059 19 0.021 35 F1 SgIM-11 0.073 16 0.017 43 − − − 0.051 19 0.020 24 G PnOM-10 0.058 24 0.018 45 − − − 0.065 19 0.021 28 H SgIM-09 0.056 22 0.014 42 + − − 0.064 19 0.025 67 I SgOM-09 0.090 22 0.063 24 − − − 0.052 19 0.019 51 J PnOM-10 0.055 24 0.046 24 − − − 0.058 19 0.024 52 K PnIM-10 0.061 14 0.034 68 − − − 0.060 19 0.016 43 K1 PnIM-11 0.065 24 0.032 68 − − − 0.060 24 0.022 45 L SgIB-09 0.071 22 0.018 40 − − − 0.071 19 0.019 43 M PnOM-10 0.061 14 0.017 40 + w − 0.051 19 0.023 51 N PnIM-10 0.068 20 0.023 58 − − − 0.055 19 0.022 45 O PnIM-10 0.055 20 0.005 150 − − − 0.061 19 0.028 67 P PnOM-10 0.080 24 0.028 50 − − − 0.068 24 0.020 51 Q SgOM-09 0.078 24 0.014 42 − − − 0.061 19 0.012 43 R SgIM-09 0.060 24 0.013 24 − − − 0.062 19 0.018 43 S SgIM-09 0.063 24 0.011 25 + − − 0.064 19 0.017 43 T SgIM-09 0.070 24 0.010 24 + w − 0.065 19 0.017 43 U SgIM-09 0.062 22 0.013 42 − − − 0.057 24 0.020 35 V SgOM-11 0.049 4 0.010 38 − − − 0.064 19 0.022 51 W SgIM-11 0.060 20 0.013 24 + + − 0.062 19 0.020 43

b r a z i l i a n j o u r n a l o f m i c r o b i o l o g y 4 7 (2 0 1 6) 181–190

187

Ethanolresistance(%) Osmotolerance

12%ethanol 14%ethanol Ethanol(%) 40%glucose 50%glucose (h−1)b _lagphaset (h) (h−1)b _lagphaset (h) 15 16 17 (h−1)b _lagphaset (h) (h−1)b _lagphaset (h) W1 PnIM-11 0.076 14 0.016 38 − − − 0.077 19 0.020 43 X SgIM-11 0.064 24 0.018 42 − − − 0.072 19 0.022 43 X1 PnOM-11 0.075 25 0.016 25 + + − 0.064 19 0.018 51 Y SgOM-11 0.073 22 0.006 24 − − − 0.065 19 0.022 67 Z PnOM-11 0.064 16 0.007 24 − − − 0.058 19 0.018 43 1-09 SgOB-09 0.067 22 0.009 24 + + − 0.071 19 0.021 52 2-09 SgIM-09 0.058 24 0.011 40 + + − 0.064 19 0.016 55 5-09 SgIM-09 0.062 22 0.010 42 − − − 0.065 19 0.024 46 13-09 SgOM-09 0.058 16 0.008 24 − − − 0.063 19 0.021 51 15-09 SgOM-09 0.067 22 0.013 24 − − − 0.069 24 0.020 46 20-09 SgOM-09 0.059 22 0.006 26 + w − 0.062 19 0.019 46 13-10 PnOM-10 0.062 20 0.009 40 − − − 0.060 19 0.020 60 27-10 PnIM-10 0.065 24 0.003 80 − − − 0.065 19 0.021 44 25-10 PnIM-10 0.063 20 0.014 42 − − − 0.060 19 0.020 26 BS6 Control 0.064 20 0.012 24 − − − 0.070 24 0.018 26 Positive(%) 100 − 96 − 24 9 − − − 100 − Weak(%) − − 4 − − 7 − − − − − Negative(%) − − − − 76 84 100 − − − − Isolated strain Sourceof isolation

Desirabletechnologicalproperties Undesirableproperties

Osmotolerance H2S

production SO2tolerance MAutilization AAutilization AAproduction Glycosidaseactivity Flocculation

0.5% 0.25% A PnIB-11 + + + w − + ++ A1 SgOM-11 + + + w − + ++ A2 PnOM-11 + + + + − + +++ A3 PnIM-11 + + + + − + ++ A4 SgIM-11 + + + + − + ++ B PnOM-10 + w w w − ++++ − C SgOM-09 + w + + − + +++ D PnOM-10 + + + w − + +++ E PnIM-10 + + + w − + ++++ E1 SgOM-11 + + + + − + ++++ F SgOM-09 + + + + − + + F1 SgIM-11 + + + w − + ++

b r a z i l i a n j o u r n a l o f m i c r o b i o l o g y 4 7 (2 0 1 6) 181–190 Isolated strain Sourceof isolation

Desirabletechnologicalproperties Undesirableproperties

Osmotolerance H2S

production SO2tolerance MAutilization AAutilization AAproduction Glycosidaseactivity Flocculation

0.5% 0.25% G PnOM-10 + + + w − + ++++ H SgIM-09 + + + + − w ++++ I SgOM-09 + + + ++ − + ++ J PnOM-10 + + + ++ − + ++ K PnIM-10 + w + ++ − + ++ K1 PnIM-11 + w + w − + ++ L SgIB-09 + + + w − + ++ M PnOM-10 + + + ++ − + + N PnIM-10 + + + w − + ++++ O PnIM-10 + + + w − + ++++ P PnOM-10 + + + w − + + Q SgOM-09 + + + − − + + R SgIM-09 + + + w − + + S SgIM-09 + + + + − + + T SgIM-09 + + + − − + ++ U SgIM-09 + + + − − + ++ V SgOM-11 + + + − − + ++ W SgIM-11 + + + − − + + W1 PnIM-11 + + + − − + ++++ X SgIM-11 + + + − − + + X1 PnOM-11 + + + w − + + Y SgOM-11 + + + w − + + Z PnOM-11 + + + + − + + 1-09 SgOB-09 + + + w − + + 2-09 SgIM-09 + + + w − + + 5-09 SgIM-09 + + + − − + ++++ 13-09 SgOM-09 + + + − − w ++ 15-09 SgOM-09 + + + − − + ++++ 20-09 SgOM-09 + + + − − + + 13-10 PnOM-10 + + + − − w +++ 27-10 PnIM-10 + + + − − + ++ 25-10 PnIM-10 + + + − − + ++ BS6 Control + + + − − + ++ Positive(%) 100 91 98 29 − 91/2 9a_/20 Weak(%) − 9 2 38 − 7 31/38 Negative(%) − − 0 33 100 − 2

Note:noglucosidaseandglycosidaseactivitiesforalltestedindigenousstrainsandalsoforcommercialBS6strain.

a _{Strongpositive;forH}

2Sproduction:positive(+++and++++),weak(+and++). b _{Specificgrowthrate;thestandarddeviationswerenothigherthan20%.}

toinvestigatephenotypicdifferencesamong45indigenousS. cerevisiaestrains,whichwereidentifiedbymolecularmethod as different strains (see above), we screenedthem for the selectedtechnologicalproperties.ThestrainS.cerevisiaeBS6, which is commercially available, was used as a control strain. Thecomplete listofresults, summarizing oenologi-calpropertiesthatareimportantforstrainselectionandtheir applicationinthewinemakingprocess,isprovidedinTable2. Theuse oflocal,autochthonous,selectedstrains ofS. cere-visiaeasstartersisratherpreferable,sincetheseyeastsare betteracclimatedtoparticularconditionscharacteristicsfor thespecificregion/areaofwineproduction8_{and,moreover,}

utilizationofthelocalisolateofS.cerevisiaeislikelytoraise theregionalcharacterofthewine.

Barrajónetal.25_{reportedthathighfermentationpoweris}

obviouslyrelatedtothecapacityofthestraintoovercomethe stressassociatedwithwinefermentation.Forthispurpose,we testedosmoticandethanolstresstolerance.

Themajortargetofethanolisthemembrane,alteringthe membraneorganizationandpermeabilityandconsequently inhibiting glucose transport and fermentation rate under enologicalconditions.26_{Asexpected,thephysiological}

differ-encesamongthestresstolerantspeciesS.cerevisiaedepended onthestrain.Thegrowthparametersofallthetestedstrains including specificgrowth rate and lengthofthe lag phase arelistedinTable2.Eighteenisolatedstrainsrevealedbetter growth characteristics than control strain BS6 inthe pres-enceof12%ethanoland29showedhighergrowthratewhen exposedto14%ethanol.Further,fourisolatedstrains(W,X1, 2-09,1-09)wereabletogrowin16%ethanolbutnoneofthe testedstrainswasabletogrowinthepresenceof17%ethanol. Highersugarcontentofthegrapemustcanresultin inhi-bition of the yeast metabolismand, therefore, in sluggish fermentation.24 _{Therefore, tolerance towardsosmotic}

pres-sureisadesirablepropertyoftheyeaststarterculture.Allthe isolatedstrainswerecapableofgrowinginthepresenceof40% and50%glucose.Fourstrains(W1,X,Land1-09)exhibited highergrowth ratethanthe controlstrainBS6inthe pres-enceof40%glucose.Interestingly,despitethefactthatTofalo etal.26_{didnotnoticeanygrowthofS.cerevisiaestrainsinthe}

presenceof50%glucose,mostofourisolateswerecapableof growingwhencultivatedinthepresenceof50%glucose. More-over,themajorityofthescreenedstrainsrevealed ahigher growthrateon50%glucosethanthecontrolstrain.

Resultsofflocculationtestsshowedthat,asalsoreported inotherstudies,24,27 _{mostofthe strains(91%)remained in}

suspensionafter10minatrest.Thisisanimportantfeature whenselectingactivedriedyeastsforvinification,whereyeast shouldideallyremaininsuspensionduringfermentation.24

Enzymesplayadefinitiveroleintheproductionofwine. Theenzymaticactivitiesdonotonlyoriginatefromthegrapes itself,butalsofromyeastsandothermicroorganisms.17_Based

on ourresults, tested strains lacked ␤-glucosidase activity. Unliketheotherauthors,5,28_{wedidnotdetectanyglycosidase}

productionbyS.cerevisiaestrainstested.

Toproduceahigh-qualitywine,itisimportanttoobtaina finebalancebetweenthevariouschemicalconstituents, espe-ciallybetweenthesugarandacidcontent.29 _Asreportedby

Suárez-LepeandMorata,7_{yeastsmightbeselectedfortheir}

abilityto produce and degradeacetic and malic acid.Our

resultsshowedthat91%(98%)ofourisolateswereabletogrow ontheagarmediumcontainingmalicoraceticacidassole car-bonsources.Thecapabilityofdegradationofmalicaswellas aceticacidmaybeconsideredasadesirablepropertyofyeast strainsbecauseitleadstothedeacidificationofwine.30 _On

thecontrary,aceticacidproductionwasobservedfor29%of testedstrains.Severalstudieshavelinkedtheproductionof aceticacidtoincreasedglycerolproductionwhichgivesthe wineadesirableproperty.31,32

Hydrogensulfitehasnegativeorganolepticimpactonwine due toformationofoff-flavors.7 _{Fifteenisolates(29%)}

syn-thesizedH2SandremainingstrainsexhibitedonlylowH2S

production.Onlyonestrain(B)didnotproduceH2S,however,

thisstrainisnotsuitableforapplicationasastarterculture duetostrongflocculationproperties.

Yeast selection offers the best way to obtain strains of S. cerevisiae or other oenological species with proper-ties that might improve the sensorial profile, technolog-ical properties or regional character of the wine.7 _The

assays to determine yeast properties that could influ-ence fermentative capacity and the ability to adapt to stressful conditions related to the wine-production pro-cessrevealeddifferencesforsomestrains.Someindigenous strains exhibited better adaption to stressful conditions than the control strain. On the contrary, some of them produced a high amount of hydrogen sulfite, causing off-flavor.

Thus,ifwecompareallthetestedstrains,only15(A,A4, F, F1, I, J, L, M, R, S, X1, Z, 1-09, 2-09, 27-10) of them are suitableforfurthertestingasstartercultures.Basedonour results,thesestrainsshowedpropertechnologicalproperties that areverysimilar tothecontrolcommercialS.cerevisiae BS6strain.Thesestrainscanbechosenforfuturelarge-scale fermentation processes instead of commercially available strains.

Conclusions

The present study demonstrated application of appropri-atemodernmoleculartechniquesthataresuitableforrapid S.cerevisiaestrainidentificationandfurthertestingofvarious strainsfortheirtechnologicalpotential.Thecombinationof usedmodernmoleculartechniquesincludingspecies-specific primers, and interdelta PCR typing enabled us to identify S.cerevisiaeatstrainlevel.Alsoimportantphysiological char-acteristics ofthe yeastsusedin this study are suitablefor rapidselectionofthedifferentS.cerevisiaestrainsthatcanbe appliedinthewinemakingprocess.Applicationoftheselected strainswithsuitabletechnologicalpropertiesinthewine fer-mentationprocess should increasethe qualityofthe wine and enhancetheregionalcharacteroftraditionalMoravian wines.Hence,someisolatedindigenousstrainswillbetested in a large-scale fermentationprocess bya small Moravian winery.

Conflicts

of

interest

Acknowledgments

Thiswork wassupported bya project ofM ˇSMT ˇCR (Grant No.MSM0021630501) andbyaStandard projectofspecific researchNo.FCH-S-13-1912.

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