Isolation, identification and characterization of regional indigenous Saccharomyces cerevisiae strains

b a z i l i a n j o u n a l o m i c o b i o l o g y 4 7 (2 0 1 6) 181–190 h p://www.bjmic obiol.com.b / Food Mic obiology Isola ion, iden ifica ion and cha ac e iza ion o egional indigenous Saccha omyces ce e isiae s ains Hana ˇ Su anská∗, Dana V áno á, Jiˇ ina Omelko á B no Uni e si y o Technology, Facul y o Chemis y, Depa men o Food Science and Bio echnology, Pu kyˇ no a 464/118, 612 00 B no, Czech Republic a i c l e i n o A icle his o y: Recei ed 29 July 2014 Accep ed 19 May 2015 Associa e Edi o : Ma icê Noguei a de Oli ei a Keywo ds: Saccha omyces genus Oenological p ope ies ITS-PCR-RFLP In e del a PCR yping Species-specific p ime s a b s a c In he p esen wo k we isola ed and iden ified a ious indigenous Saccha omyces ce e isiae s ains and sc eened hem o he selec ed oenological p ope ies. These S. ce e isiae s ains we e isola ed om be ies and spon aneously e men ed mus s. The g ape be ies (Sau i- gnon blanc and Pino noi ) we e g own unde he in eg a ed and o ganic mode o a ming in he Sou h Mo a ia (Czech Republic) wine egion. Mode n geno yping echniques such as PCR-finge p in ing and in e del a PCR yping we e employed o di e en ia e among indige- nous S. ce e isiae s ains. This combina ion o he me hods p o ides a apid and ela i ely simple app oach o iden ifica ion o yeas o S. ce e isiae a s ain le el. In o al, 120 isola es we e iden ified and g ouped by molecula app oaches and 45 o he ep esen a i e s ains we e es ed o selec ed impo an oenological p ope ies including e hanol, sul u dioxide and osmo ic s ess ole ance, in ensi y o floccula ion and desi able enzyma ic ac i i ies. Thei abili y o p oduce and u ilize ace ic/malic acid was examined as well; in addi ion, H2S p oduc ion as an undesi able p ope y was sc eened. The oenological cha ac e is ics o indigenous isola es we e compa ed o a comme cially a ailable S. ce e isiae BS6 s ain, which is commonly used as he s a e cul u e. Finally, some indigenous s ains coming om o ganically ea ed g ape be ies we e chosen o hei p omising oenological p ope - ies and hese s ains will be used as he s a e cul u e, because applica ion o a selec ed indigenous S. ce e isiae s ain can enhance he egional cha ac e o he wines. © 2015 Sociedade B asilei a de Mic obiologia. Published by Else ie Edi o a L da. This is an open access a icle unde he CC BY-NC-ND license (h p://c ea i ecommons.o g/licenses/by-nc-nd/4.0/). In oduc ion The quali y o e men ed oods and be e ages is pa ially de e mined by he mic oo ganisms used o hei p oduc ion. ∗Co esponding au ho . E-mail: [email p o ec ed] (H. ˇ Su anská). Fo ins ance, he seconda y cha ac e o wine is de e mined by senso y cha ac e is ics ha a ise om he di ec ac ion o mic oo ganisms on he subs a e. The e men a ion o g ape mus in o wine is an ecologically complex p ocess, in which bac e ia and o he mic oo ganisms, especially yeas s, play a h p://dx.doi.o g/10.1016/j.bjm.2015.11.010 1517-8382/© 2015 Sociedade B asilei a de Mic obiologia. Published by Else ie Edi o a L da. This is an open access a icle unde he CC BY-NC-ND license (h p://c ea i ecommons.o g/licenses/by-nc-nd/4.0/). 182 b a z i l i a n j o u n a l o m i c o b i o l o g y 4 7 (2 0 1 6) 181–190 c ucial ole. The s ains o Saccha omyces ce e isiae in ol ed in e men a ion play an impo an pa in he cha ac e is ics o he final p oduc and he di e si y o S. ce e isiae s ains p esen in spon aneous e men a ion con ibu e o he chem- ical composi ion and senso y quali ies o he esul ing wine.1 The e o e, one o he mos impo an echnological ad ances in wine-making was he inocula ion o g ape juice wi h selec ed cul u es o S. ce e isiae.2This app oach is based on he e idence ha mic obiological con ol o he e men- a ion p ocess allows be e managemen o his alcoholic e men a ion. I is known ha selec ed s ains o S. ce e isiae supp ess indigenous non-Saccha omyces species and domina e he e men a ion p ocess.3–5 Nowadays, no el bio echnological app oaches in wine- making a e used in se e al aspec s o he e men a ion indus y. Apa om he moni o ing o he mic obial popu- la ions and he con ol o he spoilage yeas s, a en ion is ocused also on he selec ion and u iliza ion o he s a e cul- u es coming om one’s own ineya d, which can enhance he egional cha ac e o he wine.6,7 The me abolic peculia i- ies and he physiological p ope ies o S. ce e isiae yeas may lead o he o ma ion o me aboli es and he ans o ma ion o g ape subs ances ha may en ich he wine fla o .7Ce ain c i e ia need o be me in o de o gua an ee he desi able ea- u es o he yeas s ains selec ed. The mos impo an ones a e: ole ance o e hanol; g ow h a high suga concen a ions; esis ance o sul u dioxide; low p oduc ion o hyd ogen sul- fide; p oduc ion o kille oxins o some enzyma ic ac i i ies.8 Fu he mo e, only eliable and apid iden ifica ion o he yeas species du ing he p ocess and he quali y con ol enables enologis s o assess he ole o yeas s as a main p o agonis o alcoholic e men a ion o as a con aminan . The u iliza ion o molecula me hods enabled apid and p ecise iden ifica ion o he yeas s a he species o s ain le el.1Me cado e al.9 epo ed ha Saccha omyces popula- ions a e ep esen ed by mul iple s ains, e en in inocula ed e men a ions. The e o e, i is impo an o ha e simple and app op ia e me hods ha allow disc imina ion a he s ain le el. This s udy is ocused on indigenous S. ce e isiae s ain (i) selec ion, (ii) iden ifica ion and (iii) echnological cha ac- e iza ion. Yeas s we e isola ed om g apes and mus s du ing he p oduc ion o wines. We selec ed wo ypes o wine a i- e ies – Sau ignon blanc and Pino noi coming om an o ganic and in eg a ed ea ed ineya d si ua ed in Sou h Mo a ia, Czech Republic. Ou objec i e was also ocused on he selec- ion o he iden ifica ion app oaches ha will be simple and sui able o apid and eliable s ain iden ifica ion. The e- o e, isola es o S. ce e isiae we e iden ified and g ouped by se e al molecula app oaches such as ITS-PCR-RFLP, PCR- finge p in ing, species-specific p ime s and in e del a PCR yping. The combina ion o hese echniques enabled apid de ec ion, iden ifica ion and yping o di e en S. ce e isiae s ains. Finally, he isola ed s ains we e sc eened o selec ed echnological p ope ies impo an in he winemaking p o- cess and o u he applica ion as he s a e cul u es. To sum up, his s udy has demons a ed he impo ance o selec ion o an app op ia e and apid iden ifica ion ech- nique and also de e mina ion o some impo an oenological p ope ies. Me hods Yeas species isola ion and cul i a ion Au och honous (indigenous) s ains belonging o Saccha- omyces genus we e isola ed om g ape be ies and also om spon aneously e men ed mus s in di e en s ages o he e - men a ion p ocess. G apes we e collec ed a he I aˇ n ineya d si ua ed in he Sou h Mo a ia egion and belong o he Mikulo wine egion, Czech Republic. Red wine Pino Noi (Pn) and whi e wine Sau ignon blanc (Sg) cul i a s o Vi is ini e a we e chosen. Bo h a ie ies, ypical cul i a s o whi e and, espec i ely, ed Mo a ian wine p oduc ion, we e cul i a ed using he o ganic (O) and also in eg a ed (I) a ming p ocedu e. G ape be ies (heal hy and undamaged) we e collec ed be o e ha es (in Sep embe 2009, 2010, 2011) in o s e ile glasses. Immedia ely a e anspo a ion o he labo a o y, 15–20 g ape be ies we e placed in o 150 mL o Mal ex ac medium MEM (Himedia; 2% mal ex ac , 0.1% pep one, 2% dex ose, 2% aga ) and cul i- a ed o 10 days a labo a o y empe a u e (app ox. 23◦C). A e ha , cul u e media (300 ␮L) inocula ed on o MEM aga pla es supplemen ed wi h 250 mg L−1s ep omycin sul a e (Himedia, India) we e incuba ed a 26◦C o 3–5 days. The single colonies we e ob ained by Koch’s dilu ion me hod. The e men a ion p ocess was pe o med in he cella in I aˇ n (du ing he in age 2009, 2010, 2011), which was sepa a ed om common e men a ions o comme cial wine p oduc ion. The e men a ion p ocess ollowing he s anda d p ocedu e was conduc ed in a 1000 L ba el. The cella em- pe a u e was app ox. 10◦C and he empe a u e o he e men a ion was app ox. 18◦C. The mus was spon aneously e men ed and he samples we e collec ed 3 imes pe week du ing he whole e men a ion p ocess. Yeas popula ions om mus we e isola ed as desc ibed p e iously10 and cul i a ed on mal ex ac medium (MEM) supplemen ed wi h 250 mg L−1s ep omycin sul a e (Hime- dia, India). The single colonies (pu e cul u e) we e ob ained by Koch’s dilu ion me hod. In o al, 120 Saccha omyces sp. s ains we e isola ed and iden ified. Pu e cul u es we e p ese ed on MEM aga unde pa a fin oil. The mos p omising s ains will be deposi ed in he yeas cul u e collec ion CCY B a isla a (s ain S. ce e isiae 1-09 has al eady been deposi ed he e). DNA isola ion Genomic DNA was isola ed om single colonies using he comme cial ki Ul aCleanTM Mic obial DNA Isola ion Ki (MoBio, USA) acco ding o he manu ac u e ’s p o ocol. ITS-PCR-RFLP To dis inguish Saccha omyces sp. om o he isola es, ITS-PCR- RFLP was employed. The in e nal ansc ibed space s (ITS) (ITS1 and ITS2) and 5.8S DNA gene egions we e amplified by using specific p ime s ITS1 (5-TCCGTAGGTGAACCTGCGG-3) and ITS4 (5-TCCTCCGCTTATTGATATGC-3).11 DNA amplifica- ion was ca ied ou in he final olume o 50 ␮L con aining b a z i l i a n j o u n a l o m i c o b i o l o g y 4 7 (2 0 1 6) 181–190 183 0.2 mM o dNTP, 0.5 ␮L o each p ime 100 pmol ␮L−1, 1× PCR eac ion bu e and 1 U o Taq DNA polyme ase (Kapa Biosys ems, USA). PCR condi ions we e as ollows: ini ial dena u a ion cycle a 94◦C o 4 min ollowed by 25 cycles o amplifica ion, dena u a ion a 94◦C o 1 min, annealing a 48◦C o 30 s, and ex ension a 72◦C o 1 min; final ex ension a 72◦C o 10 min. Fo RFLP, PCR p oduc s we e pu ified by e hanol p e- cipi a ion and diges ed by es ic ion endonucleases HaeIII (Fe men as, USA) ollowing he manu ac u e ’s ins uc ions. Species-specific p ime s Fo S. ce e isiae species iden ifica ion, a se o pai s o species- specific p ime s Sce F2 (5-GCGCTTTACATTCAGATCCCGAG- 3) and Sce R2 (5-TAAGTTGGTTGTCAGCAAGATTG-3) we e used.12 DNA amplifica ion was ca ied ou in a final olume o 25 ␮L con aining 0.2 mM o dNTP (In i ek, Ge many), 0.5 ␮L o each p ime 100 pmol ␮L−1(Gene iBio ech, Czech Republic), 1× PCR eac ion bu e , 1.5 mM MgCl2and 1.25 U Taq DNA poly- me ase (BioRad, USA) and 0.5 ␮L o empla e DNA. PCR cycling condi ions used we e: ini ial dena u a ion cycle a 94◦C o 4 min ollowed by 30 cycles o amplifica ion, dena u a ion a 94◦C o 1 min, annealing a 55◦C o 1 min, and ex ension a 72◦C o 1 min; final ex ension a 72◦C o 2 min. PCR-finge p in ing and in e del a PCR yping In o de o dis inguish di e en S. ce e isiae s ains, PCR- finge p in ing by using M13 p ime and in e del a p ime s we e used. DNA amplifica ion was ca ied ou in a final olume o 25 ␮L con aining 0.2 mM o dNTP (In i ek, Ge - many), 0.5 ␮L o each p ime 100 pmol ␮L−1(VBC Bio ech, Ge many), 1× PCR eac ion bu e and 1 U Taq DNA polyme ase (Kapa, USA) and 0.5 ␮L o empla e DNA. PCR-assays by using M13 p ime (5-GAGGGTGGCGGTTCT- 3):13 ini ial dena u a ion cycle a 95◦C o 5 min, ollowed by 40 cycles o amplifica ion ollowed by dena u a ion a 93◦C o 0.75 s, annealing a 50◦C o 1 min, and ex ension a 72◦C o 1 min; final ex ension a 72◦C o 6 min. The second PCR-assay o M13 p ime : ini ial dena u a ion cycle a 94◦C o 4 min, ollowed by 35 cycles o amplifica ion ollowed by dena u a ion a 94◦C o 30 s, annealing a 36◦C o 45 s, and ex ension a 72◦C o 45 s; final ex ension a 72◦C o 7 min. The condi ions o in e del a PCR yping by ␦1 (5-CAAAATTCACCTATATTCTCA-3) and ␦2 (5-GTGGATTT- TTATTCCAACA-3); ␦2 (5-GTGGATTTTTATTCCAACA-3) and ␦12 (5-TCAACAATGGAATCCCAAC-3)14,15 we e as ollows: ini- ial dena u a ion cycle a 94◦C o 4 min, ollowed by 30 cycles o amplifica ion–dena u a ion a 94◦C o 30 s, annealing a 49◦C o 1 min and ex ension a 72◦C o 2 min. The final ex ension was a 72◦C o 10 min. De ec ion o PCR p oduc s PCR p oduc s and es ic ion agmen s we e sepa a ed and de ec ed by elec opho esis on 2% (w/ ) aga ose gel in 1× TBE bu e a 5 V cm−1 o 2 h. DNA amplified by single epe i i e p ime s and by del a p ime s we e sepa- a ed on 1.5% aga ose gel o 3–4 h. The gels we e s ained by e hidium b omide (10 mg L−1), isualized unde UV ligh (Ul a Lum. Inc., USA) and documen ed by ScionImage so wa e (Scion, India). Finally, elec opho eog ams we e p ocessed by he BioNume ics 6.5 so wa e employing UPGMA clus e analysis. Sc eening o oenological p ope ies Floccula ion p ope ies o selec ed s ains we e es ed acco d- ing o Bony e al.16 wi h sligh modifica ion. Yeas s we e cul u ed o 3 days a 26◦C in ubes con aining 10 mL o YPD (2% pep one, 1% yeas ex ac and 2% glucose) medium unde pe manen shaking (150 pm). Cells we e collec ed by cen- i uga ion and washed wi h deionized wa e . A e ha , cells we e suspended in o 10 mL o 50 mM ace a e bu e (pH 4.5) en iched by 3 mM CaSO4. The ubes o suspended cells we e mixed o 30 s and he u bidi y o yeas suspension was e al- ua ed by he naked eye. Floccula ion deg ee was de e mined using a subjec i e scale which means ha he sedimen a ion was obse ed depending on he ime. The ollowing e alua- ion was used: + he cells floccula e and sedimen a e 15 min (pa ially clea solu ion); ++ immedia e floccula ion; w – floc- cula ion a e 1 h. E hanol ole ance was es ed in 5 mL o YPD medium sup- plemen ed by 12, 14, 15, 16 and 17% ( / ) e hanol and he ubes we e inocula ed by 100 ␮L o cell suspension (cell concen a- ion app ox. 5 log CFU mL−1). Inocula ed ubes we e cul i a ed a 26◦C o 10 days. The cul u e densi y was measu ed daily by DEN-1B densi ome e (Biosan, La ia). Specific g ow h a e  (h−1) and he leng h o he lag phase (h) we e es ima ed. Osmo ole ance o selec ed s ains was es ed in 5 mL o YPD medium wi h 40% (w/ ) and 50% (w/ ) glucose. The cells densi y was measu ed as desc ibed abo e. H2S p oduc ion by selec ed s ains was es ed on Biggy aga (Himedia, India) which con ains bismu h as an indica o . A e incuba ion a 26◦C o 3–5 days, he zone su ounding he colony was e alua ed as he ollows: − no p oduc ion; + whi e colonies; ++ ligh b own; +++ b own; ++++ da k b own/black.5 Malic and ace ic acid u iliza ion by selec ed s ains was es ed on 0.67% yeas ni ogen base (YNB, Himedia) aga pla es con aining 0.5% (w/ ) malic acid, espec i ely, 0.25% (w/ ) ace ic acid. The g ow h o colonies was sc eened. Ace ic acid p oduc ion was sc eened on CaCO3aga pla es (Cus e ’s chalk medium) con aining 0.5% yeas ex ac , 5% glucose, 0.5% CaCO3and 2% aga . The ace ic acid p oduc ion enabled solu- bili y o CaCO3which esul ed as a clea zone su ounding he colonies.5 The isola ed yeas s we e also es ed o some enzyma ic ac i i ies such as ␤-glucosidase and glycosidase ac i i ies. The ac i i ies we e de e mined by aga pla ing. The pla es we e incuba ed a 26◦C o 3–5 days.17 ␤-Glucosidase ac i i y was sc eened on o selec i e medium con aining 0.67% yeas ni ogen base (YNB, Hime- dia), 0.5% a bu in and 2% aga . The pH o he medium was adjus ed o 5 be o e au ocla ing. Two millili e s o a fil e s e ilized 1% e ic ammonium ci a e solu ion was added o 100 mL media be o e pla es pou ing. Colonies showing ac i i y we e iden ified by a da k b own halo a ound he colonies.17 184 b a z i l i a n j o u n a l o m i c o b i o l o g y 4 7 (2 0 1 6) 181–190 Table 1 – Numbe o Saccha omyces sp. isola es. G ape a ie y Vin age Numbe o Saccha omyces sp. isola es In eg a ed (I) O ganic (O) G apes (B)/mus (M) G apes (B)/mus (M) Sau ignon blanc (Sg) 2009 1/14 1/11 Pino noi (Pn) 2010 0/14 0/17 Sau ignon blanc (Sg) 2011 0/20 0/17 Pino noi (Pn) 2011 1/14 0/10 To al 120 To al o yeas s isola es 524 Glycosidase ac i i y was de e mined using he pla es wi h he selec i e medium con aining 0.67% yeas ni ogen base (YNB, Himedia), 0.2% u in and 2% aga . The glycosidase ac i - i y was de ec ed as a clea zone a ound he colonies.5,17 Resul s and discussion Selec ion and iden ifica ion o S. ce e isiae s ains In o al, we isola ed 120 single colonies o au och honous Saccha omyces sp. s ains om 524 o al yeas s. Yeas s we e isola ed om Sau ignon blanc (Sg) and Pino noi (Pn) coming om o ganic (O) and in eg a ed (I) a ming. The yeas species we e isola ed om g ape be ies as well as om spon aneously e men ed mus du ing he in age 2009 (09), 2010 (10) and 2011 (11). The lis o he Saccha omyces isola es and he sou ces o hei isola ion a e shown in Table 1. Yeas s o he genus Saccha omyces we e dis inguished om he o he isola es ( om non-Saccha omyces species) by ITS- PCR-RFLP ( he leng h o PCR amplicon was 880 bp). Fu he , o S. ce e isiae species iden ifica ion we employed species- specific p ime s Sce F2 and Sce R2.12 Species-specific p ime s enable us o iden i y and dis inguish S. ce e isiae species om o he species belonging o he Saccha omyces sensu s ic o com- plex, which includes he species which can also be ound in e men ed mus ( o . ex. Saccha omyces bayanus, Saccha omyces pas o ianus, Saccha omyces kud ia ze ii). Based on ou esul s, all he isola es belonging o he Saccha omyces genus we e iden ified by species-specific p ime s as S. ce e isiae ( he leng h o he PCR p oduc s was 150 bp) (da a no shown). Fu he , wo di e en PCR-assays by using M13 p ime we e used. These assays di e ed in annealing empe a u e (50◦C s. 36◦C). The isola es we e di ided in o h ee g oups by he fi s assay wi h annealing empe a u e 50◦C and in o ou g oups by he second PCR-assay (36◦C). Da a a e p esen ed as pa o he dend og am (Fig. 1). Two isola es (ma ked as U, T) exhibi ed a di e en finge p in ing p ofile han he es o he isola es; hese isola es may be hyb ids o S. ce e isiae and ano he species belonging o he Saccha omyces genus. Hence, PCR-finge p in ing echniques using M13 p ime a e able o g oup he species membe s o Saccha omyces genus bu hey a e no sui able o esol ing. In o de o dis inguish a ious S. ce e isiae s ains, we employed ␦1–␦2 and ␦12–␦2 p ime s ampli ying in e -del a sequences. These del a elemen s a e desc ibed as app o- p ia e gene ic ma ke s o iden ifica ion o polymo phisms because he numbe and loca ion ha e a ce ain in aspecific a iabili y.18 The p ime pai ␦1–␦2 gene a ed om h ee o eigh di e en agmen s pe s ain wi h one common band o he size o app oxima ely 1000 bp. On he o he hand, p ime pai ␦12–␦2 p o ided significan ly mo e agmen s pe sample and some o hem we e o low in ensi y. The low numbe o agmen s pe sample is asc ibed o he weak homology exhibi ed by he p ime ␦1–␦2 owa ds he whole sequence o S. ce e isiae genom.19 Howe e , by combina ion o hese wo se s o del a p ime s, we iden ified 45 di e en S. ce e isiae s ains. The majo i y o he isola ed and iden ified S. ce e isiae s ains came om spon aneous e men ed mus s and belonged o g oup A (s ain A). Despi e he ac ha some au ho s20,21 epo ed ha i is almos impossible o isola e Saccha omyces sp. popula ions om be ies and ini ial mus by s anda d di ec aga pla ing p ocedu e due o hei low coun s (>10–100 CFU cm−2) we isola ed h ee s ains om g ape be ies (see Table 1). The elec opho e ic pa e ns and final dend og am showing he gene ics simila i y o a ious iden ified S. ce e isiae s ains a e shown in Fig. 1. The no el combina ion o he mode n molecula app oaches used in his s udy o s ain iden ifica ion and yping seems o be sui able o apid, eliable, simple and ep oducible iden ifica ion o S. ce e isiae s ains. Schulle e al.,22 Maqueda e al.23 o O iz e al.24 epo ed applica ion o mi ochond ial DNA es ic ion analysis o ka yo yping in o de o dis inguish a ious s ains o S. ce e isiae. Howe e , hese me hods a e labo in ensi e and he esul s a e influ- enced by complexi y o da a in e p e a ion due o he high numbe o gene a ed agmen s a e m DNA RFLP. On he con a y, we employed di e en app oaches o he g oup and dis inguish Saccha omyces a s ain le el based on consequen employmen o PCR-finge p in ing. Fo ins ance Schulle e al.22 epo ed ha in e del a PCR yping had e y simila esol ing powe a s ain le el as m DNA and ka yo yping, he e o e, he unique combina ion o he me hods u ilized in his wo k can be conside ed as a simple and apid al e na i e o m DNA o ka yo yping. Sc eening o selec ed oenological p ope ies o a ious S. ce e isiae s ains Because no all yeas s ains a e ele an o he specific con- di ions and cha ac e is ics o wine, a numbe o c i e ia ha e been p oposed o he selec ion o new yeas s ains o use in he winemaking p ocess. One o he mos impo an c i- e ions o high-quali y wine p oduc ion is use o S. ce e isiae s ain wi h sui able echnological p ope ies. Thus, in o de b a z i l i a n j o u n a l o m i c o b i o l o g y 4 7 (2 0 1 6) 181–190 185 Composi e 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 – Dend og am based on he simila i y o PCR-finge p in ing pa e ns o S. ce e isiae isola es. S. ce e isiae BS6 – con ol s ain. The desc ip ion on he igh side o he dend og am is he label o he isola ed S. ce e isiae s ains. 186 b a z i l i a n j o u n a l o m i c o b i o l o g y 4 7 (2 0 1 6) 181–190 Table 2 – Oenological p ope ies o di e en indigenous Saccha omyces ce e isiae s ains and one comme cial S. ce e isiae s ain BS6 as a con ol. Pn – Pino Noi ; O, I – o ganic, in eg a ed; M, B – mus , g ape be ies; 09–11 – mean yea o isola ion (2009–2011). The g ay labelled fields – he s ains which physiological p ope ies we e be e han con ol s ain BS6. Isola ed s ain Sou ce o isola ion Desi able echnological p ope ies E hanol esis ance (%) Osmo ole ance 12% e hanol 14% e hanol E hanol (%) 40% glucose 50% glucose  (h−1)blag phase (h)  (h−1)blag phase (h) 15 16 17  (h−1)blag phase (h)  (h−1)blag phase (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 a z i l i a n j o u n a l o m i c o b i o l o g y 4 7 (2 0 1 6) 181–190 187 Table 2 – (Con inued) Isola ed s ain Sou ce o isola ion Desi able echnological p ope ies E hanol esis ance (%) Osmo ole ance 12% e hanol 14% e hanol E hanol (%) 40% glucose 50% glucose  (h−1)blag phase (h)  (h−1)blag phase (h) 15 16 17  (h−1)blag phase (h)  (h−1)blag phase (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 Con ol 0.064 20 0.012 24 − − − 0.070 24 0.018 26 Posi i e (%) 100 − 96 − 24 9 − − − 100 − Weak (%) − − 4 − − 7 − − − − − Nega i e (%) − − − − 76 84 100 − − − − Isola ed s ain Sou ce o isola ion Desi able echnological p ope ies Undesi able p ope ies Osmo ole ance H2S p oduc ion SO2 ole ance MA u iliza ion AA u iliza ion AA p oduc ion Glycosidase ac i i y Floccula ion 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 − + ++ 188 b a z i l i a n j o u n a l o m i c o b i o l o g y 4 7 (2 0 1 6) 181–190 Table 2 – (Con inued) Isola ed s ain Sou ce o isola ion Desi able echnological p ope ies Undesi able p ope ies Osmo ole ance H2S p oduc ion SO2 ole ance MA u iliza ion AA u iliza ion AA p oduc ion Glycosidase ac i i y Floccula ion 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 Con ol + + + − − + ++ Posi i e (%) 100 91 98 29 − 91/2 9a/20 Weak (%) − 9 2 38 − 7 31/38 Nega i e (%) − − 0 33 100 − 2 No e: no glucosidase and glycosidase ac i i ies o all es ed indigenous s ains and also o comme cial BS6 s ain. aS ong posi i e; o H2S p oduc ion: posi i e (+++ and ++++), weak (+ and ++). bSpecific g ow h a e; he s anda d de ia ions we e no highe han 20%. b a z i l i a n j o u n a l o m i c o b i o l o g y 4 7 (2 0 1 6) 181–190 189 o in es iga e pheno ypic di e ences among 45 indigenous S. ce e isiae s ains, which we e iden ified by molecula me hod as di e en s ains (see abo e), we sc eened hem o he selec ed echnological p ope ies. The s ain S. ce e isiae BS6, which is comme cially a ailable, was used as a con ol s ain. The comple e lis o esul s, summa izing oenologi- cal p ope ies ha a e impo an o s ain selec ion and hei applica ion in he winemaking p ocess, is p o ided in Table 2. The use o local, au och honous, selec ed s ains o S. ce e- isiae as s a e s is a he p e e able, since hese yeas s a e be e acclima ed o pa icula condi ions cha ac e is ics o he specific egion/a ea o wine p oduc ion8and, mo eo e , u iliza ion o he local isola e o S. ce e isiae is likely o aise he egional cha ac e o he wine. Ba ajón e al.25 epo ed ha high e men a ion powe is ob iously ela ed o he capaci y o he s ain o o e come he s ess associa ed wi h wine e men a ion. Fo his pu pose, we es ed osmo ic and e hanol s ess ole ance. The majo a ge o e hanol is he memb ane, al e ing he memb ane o ganiza ion and pe meabili y and consequen ly inhibi ing glucose anspo and e men a ion a e unde enological condi ions.26 As expec ed, he physiological di e - ences among he s ess ole an species S. ce e isiae depended on he s ain. The g ow h pa ame e s o all he es ed s ains including specific g ow h a e and leng h o he lag phase a e lis ed in Table 2. Eigh een isola ed s ains e ealed be e g ow h cha ac e is ics han con ol s ain BS6 in he p es- ence o 12% e hanol and 29 showed highe g ow h a e when exposed o 14% e hanol. Fu he , ou isola ed s ains (W, X1, 2-09, 1-09) we e able o g ow in 16% e hanol bu none o he es ed s ains was able o g ow in he p esence o 17% e hanol. Highe suga con en o he g ape mus can esul in inhi- bi ion o he yeas me abolism and, he e o e, in sluggish e men a ion.24 The e o e, ole ance owa ds osmo ic p es- su e is a desi able p ope y o he yeas s a e cul u e. All he isola ed s ains we e capable o g owing in he p esence o 40% and 50% glucose. Fou s ains (W1, X, L and 1-09) exhibi ed highe g ow h a e han he con ol s ain BS6 in he p es- ence o 40% glucose. In e es ingly, despi e he ac ha To alo e al.26 did no no ice any g ow h o S. ce e isiae s ains in he p esence o 50% glucose, mos o ou isola es we e capable o g owing when cul i a ed in he p esence o 50% glucose. Mo e- o e , he majo i y o he sc eened s ains e ealed a highe g ow h a e on 50% glucose han he con ol s ain. Resul s o floccula ion es s showed ha , as also epo ed in o he s udies,24,27 mos o he s ains (91%) emained in suspension a e 10 min a es . This is an impo an ea u e when selec ing ac i e d ied yeas s o inifica ion, whe e yeas should ideally emain in suspension du ing e men a ion.24 Enzymes play a defini i e ole in he p oduc ion o wine. The enzyma ic ac i i ies do no only o igina e om he g apes i sel , bu also om yeas s and o he mic oo ganisms.17 Based on ou esul s, es ed s ains lacked ␤-glucosidase ac i i y. Unlike he o he au ho s,5,28 we did no de ec any glycosidase p oduc ion by S. ce e isiae s ains es ed. To p oduce a high-quali y wine, i is impo an o ob ain a fine balance be ween he a ious chemical cons i uen s, espe- cially be ween he suga and acid con en .29 As epo ed by Suá ez-Lepe and Mo a a,7yeas s migh be selec ed o hei abili y o p oduce and deg ade ace ic and malic acid. Ou esul s showed ha 91% (98%) o ou isola es we e able o g ow on he aga medium con aining malic o ace ic acid as sole ca - bon sou ces. The capabili y o deg ada ion o malic as well as ace ic acid may be conside ed as a desi able p ope y o yeas s ains because i leads o he deacidifica ion o wine.30 On he con a y, ace ic acid p oduc ion was obse ed o 29% o es ed s ains. Se e al s udies ha e linked he p oduc ion o ace ic acid o inc eased glyce ol p oduc ion which gi es he wine a desi able p ope y.31,32 Hyd ogen sulfi e has nega i e o ganolep ic impac on wine due o o ma ion o o -fla o s.7Fi een isola es (29%) syn- hesized H2S and emaining s ains exhibi ed only low H2S p oduc ion. Only one s ain (B) did no p oduce H2S, howe e , his s ain is no sui able o applica ion as a s a e cul u e due o s ong floccula ion p ope ies. Yeas selec ion o e s he bes way o ob ain s ains o S. ce e isiae o o he oenological species wi h p ope - ies ha migh imp o e he senso ial p ofile, echnolog- ical p ope ies o egional cha ac e o he wine.7The assays o de e mine yeas p ope ies ha could influ- ence e men a i e capaci y and he abili y o adap o s ess ul condi ions ela ed o he wine-p oduc ion p o- cess e ealed di e ences o some s ains. Some indigenous s ains exhibi ed be e adap ion o s ess ul condi ions han he con ol s ain. On he con a y, some o hem p oduced a high amoun o hyd ogen sulfi e, causing o -fla o . Thus, i we compa e all he es ed s ains, only 15 (A, A4, F, F1, I, J, L, M, R, S, X1, Z, 1-09, 2-09, 27-10) o hem a e sui able o u he es ing as s a e cul u es. Based on ou esul s, hese s ains showed p ope echnological p ope ies ha a e e y simila o he con ol comme cial S. ce e isiae BS6 s ain. These s ains can be chosen o u u e la ge-scale e men a ion p ocesses ins ead o comme cially a ailable s ains. Conclusions The p esen s udy demons a ed applica ion o app op i- a e mode n molecula echniques ha a e sui able o apid S. ce e isiae s ain iden ifica ion and u he es ing o a ious s ains o hei echnological po en ial. The combina ion o used mode n molecula echniques including species-specific p ime s, and in e del a PCR yping enabled us o iden i y S. ce e isiae a s ain le el. Also impo an physiological cha - ac e is ics o he yeas s used in his s udy a e sui able o apid selec ion o he di e en S. ce e isiae s ains ha can be applied in he winemaking p ocess. Applica ion o he selec ed s ains wi h sui able echnological p ope ies in he wine e - men a ion p ocess should inc ease he quali y o he wine and enhance he egional cha ac e o adi ional Mo a ian wines. Hence, some isola ed indigenous s ains will be es ed in a la ge-scale e men a ion p ocess by a small Mo a ian wine y. Conflic s o in e es The au ho s decla e no conflic s o in e es .