research article characterization and dynamic behavior of...

Hindawi Publishing CorporationBioMed Research InternationalVolume 2013 Article ID 540465 13 pageshttpdxdoiorg1011552013540465

Research ArticleCharacterization and Dynamic Behavior of Wild Yeast duringSpontaneous Wine Fermentation in Steel Tanks and Amphorae

Cecilia Diacuteaz1 Ana Mariacutea Molina2 Joumlrg Naumlhring1 and Rainer Fischer1

1 Molecular Biology Division Fraunhofer Institute for Molecular Biology and Applied Ecology 57392 Schmallenberg Germany2 Facultad de Ingenierıa y Tecnologıa Universidad San Sebastian 4030000 Concepcion Chile

Correspondence should be addressed to Cecilia Dıaz ceciliadiazimefraunhoferde

Received 22 January 2013 Revised 28 March 2013 Accepted 9 April 2013

Academic Editor George Tsiamis

Copyright copy 2013 Cecilia Dıaz et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

We studied the dynamic behavior of wild yeasts during spontaneous wine fermentation at a winery in the Valais region ofSwitzerland Wild yeasts in the winery environment were characterized using a PCR-RFLP method Up to 11 different yeastspecies were isolated from the vineyard air whereas only seven were recovered from the grapes surface We initially investigateda cultureindependent method in pilot-scale steel fermentation tanks and found a greater diversity of yeasts in the musts from twored grape varieties compared to three white grape varieties We found that the yeasts Metschnikowia pulcherrima Rhodotorulamucilaginosa Pichia kluyveri P membranifaciens and Saccharomyces cerevisiae remained active at the end of the fermentation Wealso studied the dynamic behavior of yeasts in Qvevris for the first time using a novel highlysensitive quantitative real-time PCRmethodWe found that non-Saccharomyces yeasts were present during the entire fermentation process with R mucilaginosa and Panomala the most prominent species We studied the relationship between the predominance of different species and the output ofthe fermentation process We identified so-called spoilage yeasts in all the fermentations but high levels of acetic acid accumulatedonly in those fermentations with an extended lag phase

1 Introduction

Low-intervention winemaking methods based on sponta-neous fermentation are becoming more popular amongwine producers and consumers [1 2] Some wine producersand viticulturists have readopted traditional winemakingmethods to generate unique attributes that differentiate theirproducts improve wine quality and increase the variety ofcomplex flavors that characterize regional vineyards

Spontaneous fermentation is a complex process influ-enced by many factors including the endogenous microbialflora the grape variety climatic conditions and thewinemak-ing process [3ndash7] The outcome of the fermentation processcan therefore be difficult to predict and can differ fromyear to year The natural yeast flora found on grapes andin wineries play a significant role during fermentation andare particularly important during spontaneous fermentation

because no additional wine yeasts are introduced into theprocess

Wine flavor is influenced by the large number ofyeast species present during spontaneous fermentation [38ndash11] including those from the genera HanseniasporaMetschnikowia and Candida and more occasionally Toru-laspora and Pichia Most of these non-Saccharomyces yeastsgrow during the early fermentation stages whereas theprocess is eventually completed by Saccharomyces cerevisiaebecause it can tolerate higher levels of alcohol and lower levelsof oxygen [9ndash15]

Previous studies have shown that non-Saccharomycesyeasts can be detected throughout the fermentation process[15] They influence the course of fermentation and the char-acteristics of the resulting wine by producing extracellularenzymes and metabolites of oenological significance thatmodify the sensory and organoleptic properties of wine

2 BioMed Research International

introducing a broader spectrum of aromas and flavors [16ndash18]

Microbiology techniques are often used to isolate andidentify wild yeasts but this requires different types ofmedia and different culture protocols that influence specieswhich are recovered The metabolic status of the cells alsoresults in the presence of viable but nonculturable (VBNC)microbes whose influence on fermentation can be underesti-mated because the population dynamics cannot be evaluatedaccurately Quantitative real-time PCR (qPCR) is a fasterand more reliable alternative to identify and quantify yeastsduring fermentation [19] and is particularly advantageousfor VBNC yeasts because of its sensitivity [20] Althoughthe technique cannot distinguish living cells from intactdead cells it remains the most widely used method forthe evaluation of wild yeast dynamics during fermentationbecause VBNC cells continue to influence wine flavor andpalatability regardless of their actual status [21]

We compared microbiology methods (viable counts)and novel molecular biology techniques polymerase chainreactionrestriction fragment length polymorphism (PCR-RFLP) and qPCR for the identification of yeast species andwe characterized their dynamic behavior during spontaneouswine fermentation in the Valais region of Switzerland inthe 2008ndash2011 harvests We used these new methods toidentify the predominant species present during spontaneousfermentation establishing a standard for the semiquantitativedetection of yeasts with antibodies in a biochip format Such adevicewould allowwinemakers tomake early decisions aboutthe suitability of grapes and the likely success of spontaneousfermentation Also for the first time we studied the dynamicbehavior of wild yeasts during spontaneous fermentation inQvevris (amphora-like clay vessels) the use of which is anemerging trend among European winemakers

2 Materials and Methods

21 Samples Grapes and must samples were collected fromthe vineyards of the winery Albert Mathier et Fils SA inSalgesch Valais Switzerland during the 2008ndash2011 harvestseasons The grape varieties we studied are listed in Table 1Samples were collected in situ and frozen at minus20∘C duringtransport prior to analysis

22 Isolation of Yeasts from the Winery Environment andFermentation Samples During 2008 and 2009 we screenedyeasts present in the winery environment (ie the vineyardwinery facilities and cellar) and in the fermentingwinemustsIn the vineyard grape berries were placed in direct contactwith plates containing Rose Bengal Chloramphenicol Agar(RBCA) a selective medium for yeasts and molds (15 g Lminus1agar 10 g Lminus1 glucose 5 g Lminus1 papain-digested soybean meal1 g Lminus1 KH

2PO4 05 g Lminus1 MgSO

4times 7H2O 005 g Lminus1 Rose

Bengal and 10 g Lminus1 chloramphenicol) We pumped 1000 L ofvineyard air surrounding the grapes through a Millipore MAir Tester T (Millipore USA) and plated the collected residueon RBCA as above We also sampled environmental yeastflora from the winery facilities Contact samples were taken

Table 1 Grape varieties used for the analysis of dynamic wildyeast populations during spontaneous fermentation in stainless steeltanks and Qvevris

Harvest2008-2009 (steel tank) 2010-2011 (Qvevris)

Pinot Noir (R) Chardonnay (W) Resi (W)Grapevariety Cornalin (R) Petit Arvine (W) Ermitage (W)

Gutedel (W)(R) red variety (W) white variety

from the inner surface of clean fermentation tanks beforefilling them with grape juice and 1000 L of air inside thecellar was also filtered and the collected residues were platedas above All the environmental samples were collected intriplicate

Five different grape varieties from the 2008-2009 harvests(Table 1) were processed by spontaneous fermentation todetermine the predominant yeast species at the differentfermentation stages Prefermentation steps such as harvestingand pressing were carried out according to routine wineryprocedures Pressed berries were fermented with the skinto make red wine or were clarified before fermentation toproduce white wine Duplicate fermentations were carriedout in the winery cellar using new 110-L stainless steel tankswithout starting yeast cultures Liquid samples (50mL induplicate) from the fermenting musts were collected dailyfrozen immediately at minus20∘C and stored in the dark priorto analysis Immediately after defrosting liquid samples werecentrifuged at 4000 rpm for 5min The supernatant wastested for chemical parameters and the pellet was resus-pended in 100 120583L distilled water plated on RBCA mediumand incubated at 30∘C for 3ndash7 days and then stored at 4∘Cprior to analysis

23 Fermentation Parameters The fermentations were mon-itored by measuring glucosefructose consumption andethanol formation during fermentation and the acetic acidcontent at the end of fermentation The parameters weredetermined by spectrophotometry at 20 plusmn 1∘C using the D-GlucoseD-Fructose Ethanol andAcetic Acid enzymatic kitsprovided by R-Biopharm (Germany) according to themanu-facturerrsquos instructions Standards and controls were providedin the kits All measurements (duplicate fermentations) weretaken in triplicate

24 Identification of the Predominant Yeast Species A ter-minal restriction fragment length polymorphism (T-RFLP)methodwas developed and optimized for yeast identificationbased on restriction patterns generated from the genomicregion spanning the internal transcribed spacers (ITS1 andITS2) and the 58S rRNA gene These regions show lowintraspecific polymorphism and high interspecific variabilityand have previously been shown to distinguish 26 yeastspecies found on grapes in cellars andor in wine musts[22 23]

BioMed Research International 3

Total DNA from the isolated colonies was extractedusing the First-Beer Magnetic DNA kit (GEN-IAL GmbHTroisdorf Germany) and amplified using primers ITS-5 (51015840-GGA AGT AAA AGT CGT AAC AAG G-31015840) and ITS-4 (51015840-TCC TCC GCT TAT TGA TAT GC-31015840) followed by a secondround of amplificationwith the nested primers ITS-1 (51015840-TCCGTA GGT GAA CCT GCG G-31015840) and ITS-2 (51015840-GCT GCGTTC TTC ATC GAT GC-31015840) [24] The first reaction mixturecomprised 200120583M of each dNTP 10x PCR buffer 05120583M ofeach primer 1 120583L of extracted yeast DNA and 125UHot StartPolymerase (5 Prime Hamburg Germany) in a total volumeof 25120583L The samples were amplified in a thermocycler(VRW Pennsylvania USA) by denaturing at 95∘C for 3minfollowed by 15 cycles of denaturing at 95∘C for 30 s annealingat 57∘C for 30 s and extension at 72∘C for 1min and a finalextension step at 72∘C for 5min The nested amplificationmixture comprised 200120583M of each dNTP 10x PCR buffer05 120583M of each primer (labeled if necessary for product sizedetermination see below) 25U Hot Start Polymerase (5PrimeHamburg Germany) and 05 120583L template DNA (fromthe first-round PCR) in a total volume of 50120583L The mixturewas denatured at 95∘C for 3min then amplified by 20 cyclesof denaturing at 95∘C for 30 s annealing at 62∘C for 30 s andextension at 72∘C for 1min followed by a final extensionat 72∘C for 5min The products were digested with BstYI(New England BioLabs Ipswich) at 60∘C for 1 hThe length ofthe terminal fragment was determined using a 3130 GeneticAnalyzer (Applied Biosystems Darmstadt Germany) priorto the purification of the samples using the Cycle Pure Kit(Omega Bio-tek USA)

25 Primer Design and Real-Time PCR Primers specificfor the 10 predominant yeasts found in the winery andin fermentation samples during the 2008-2009 harvestseason were designed to anneal within the 26S rDNAregion and amplify products 150ndash200 bp in length (Table 2)Each primer pair was designed by processing availablesequences using CLC Combined Workbench 3 Software(CLC-Bio Denmark) and the properties of each primerwere verified using Primer Tool (Sigma-Aldrich USA httpwwwsigmagenosyscomcalcDNACalcasp) The specificityof each primer pair was controlled by searching GenBankusing BLAST (httpwwwncbinlmnihgov BLAST)

Real-time PCR was carried out using an ABI 7300 Real-Time PCR System (Applied Biosystems Hitachi Japan)Each reaction comprised 75 120583L Platinum SYBR Green qPCRSuperMix-UDG (Bio-Rad Hercules CA USA) 200 nM ofeach primer (Metabion Germany) and 03120583L template DNAextracted from must in a total volume of 15120583L The mixturewas heated to 50∘C for 2min and then 95∘C for 2minfollowed by 40 cycles of denaturation at 95∘C for 15 s andannealingextension at 60ndash63∘C (depending on the primers)for 45 sThe cycling temperature was then increased by 03∘Cevery 10 s from 63 to 95∘C to obtain the melting curve TheDNA concentration in the samples was limited to 50 ng peranalysis except for standard curves prepared from samplescontaining a known number of yeast cells All yeast specieswere cultivated in Yeast Extract Peptone Dextrose (YPD)

agar (10 g Lminus1 yeast extract 20 g Lminus1 peptone 20 g Lminus1 glucoseand 01mg Lminus1 chloramphenicol) at 27∘C for 24 h The cellswere counted using a Neubauer chamber DNAwas extractedusing the First-Beer Magnetic DNA kit and serially diluted(1 10) from 107-108 down to 1 cell mLminus1 Each point on thecalibration curve was measured in duplicate Conventionaland real-time PCR was carried out using a range of yeastspecies to verify the specificity of each primer set

26Wild Yeast Dynamics during Spontaneous Fermentation inQvevris Spontaneous fermentation in Qvevris was studiedduring the 2010 and 2011 harvest seasons The white grapevarieties Resi and Ermitage (Table 1) were harvested crushedand fermented in 1500-L Qvevris without clarification Wetook 50-mL samples in triplicate at 2-3-day intervals through-out fermentation that is every time the Qvevris were openedto stir the must The samples were frozen immediately atminus20∘C and stored in the dark prior to analysis DNA wasextracted from the must using a modified CTAB method[25] in which 10mL samples were centrifuged for 1min at3000 rpm to sediment the skin and seeds before the standardprotocol was applied The extracted DNA was then testedby qPCR to identify the wild yeast species present duringspontaneous fermentation as discussed above

3 Results

31 Establishing a PCR-RFLP Method for Yeast IdentificationYeast genomic DNA was amplified using primers ITS4and ITS5 (first round) and the products were amplifiedwith the nested primers ITS1 and ITS2 The sizes of boththe digested and undigested PCR products are unique toparticular yeast genera and also allow the differentiation ofcertain species resulting in the unambiguous identificationof up to 28 species (Table 3) There were only three cases inwhich we were unable to distinguish two different species(1) Hanseniaspora guilliermondii and H uvarum (2) Saccha-romyces bayanus and S pastorianus (3) Dekkera bruxellensisand Cryptococcus flavus The method was optimized usingspecies obtained from theDeutsche Sammlung vonMikroor-ganismen und Zellkulturen GmbH (DSMZ) BraunschweigGermany Even so wild yeast species in wineries are oftenlocal subspecies that are subject to different environmentalselection conditions and their sequences and PCR productsizes can differ slightly from purchased strains Thereforeand in order to validate the method we selected 4 isolatedyeasts and sequenced the first-round PCR products (NCBIaccession numbers KC869927 KC869928 KC869929 andKC869930) to compare these empirical sequences to those inGenBank by using the empirical sequences as BLAST queries

32 Natural Flora in Vineyard and Cellar EnvironmentsYeasts naturally present in the vineyard environment wereisolated from the grape surface and from the air around thegrapes using culture-dependent methods (see Section 22)During 2008 and 2009 up to 11 different yeast species couldbe isolated from the vineyard air although Bulleromyces albusand Sporidiobolus pararoseus were the only species recovered


Table 2 Specific primers used for qPCR analysis

Yeast species Primer name Primer sequence Product size Annealing temperature ∘C

Candida zeylanoides CZ-5fw 51015840-CGATGAGATGCCCAATTCCA-31015840 191 bp 58CZ-3bw 51015840-GAAGGGAACGCAAAATACCAA-31015840

Zygosaccharomyces florentinus ZF-5fw 51015840-CTTGAGCTCCTTGTAAAGC-31015840 256 bp 55ZF-3bw 51015840-CTAGGTTTTCTGCTGCCG-31015840

Metschnikowia pulcherrima MP-5fw 51015840-CAACGCCCTCATCCCAGA-31015840 253 bp 60MP-3bw 51015840-AGTGTCTGCTTGCAAGCC-31015840

Williopsis saturnus WS-5fw 51015840-GGGTGTCCAGTGCTTTG-31015840 199 bp 56WS-3bw 51015840-CCCAAGAAGGGAAGATAATCAC-31015840

Pichia kluyveri PK-5fw 51015840-AGTCTCGGGTTAGACGT-31015840 169 bp 55PK-3bw 51015840-GCTTTTCATCTTTCCTTCACA-31015840

Rhodotorula mucilaginosa RM-5fw 51015840-GCGCTTTGTGATACATTTTC-31015840 169 bp 54RM-3bw 51015840-CCATTATCCATCCCGGAAAA-31015840

Pichia angusta PANG-5fw 51015840-GTGTCCATTTCCGTGTAAGA-31015840 175 bp 56PANG-3bw 51015840-AGCCCACCCACAAG-31015840

Pichia anomala PA-5fw 51015840-ACGTCATAGAGGGTGAGAAT-31015840 197 bp 57PA-3bw 51015840-AAACACCAAGTCTGATCTAATG-31015840

Candida glabrata CG-5fw 51015840-GAGGGTGTCAGTTCTTTGT-31015840 224 bp 56GC-3bw 51015840-GTGAGCTGCGAGAGTC-31015840

Hanseniaspora uvarum HU-5fw 51015840-GGCGAGGGATACCTTTTCTCTG-31015840 172 bp 59HU-3bw 51015840-GAGGCGAGTGCATGCAA-31015840

Pichia fermentans PF-5fw 51015840-TTGCCTATGCTCTGAGGCC-31015840 170 bp 61PF-3bw 51015840-TCCATGTCGGGCGCAAT-31015840

Saccharomyces cerevisiae SC-5fw 51015840-AGGAGTGCGGTTCTTTCTAAAG-31015840 215 bp 59SC-3bw 51015840-TGAAATGCGAGATTCCCCCA-31015840

Torulaspora delbrueckii TD-5fw 51015840-GTGGCGAGGATCCCAG-31015840 186 bp 58TD-3bw 51015840-CTATCGGTCTCTCGCAA-31015840

in both years (Table 4)We recovered seven yeast species fromthe grape surface and species appear to be dependent onthe variety and the year of harvest (Table 4) Aurebasidiumpullulans Cryptococcus magnus Rhodotorula mucilaginosaand Zygosaccharomyces florentinus were the only speciesisolated from both the air and the grape surface Most ofthe yeasts isolated from the vineyard air were also presentin the grape juice at the beginning of fermentation In thecellar environment yeasts were isolated from the surface ofclean and empty barrels (ie before filling the fermentationtanks with the grape must) and from the air inside the cellarroom In 2008 four different species were isolated fromthe cellar air and three from the clean fermentation tank(Table 4) whereas in 2009 only one species (R mucilaginosa)was isolated from the clean fermentation tank All the speciesin the cellar environmentwere also found in the vineyard andall species identified in the cellar environment were also laterfound in the fermenting must

33 Yeast Flora in Steel-Tank Fermentations Changes in thecomposition of the yeast population during spontaneousfermentation in steel tanks were measured using culture-dependent methods We investigated the musts of five grapevarieties during the 2008 and 2009 harvest seasons the red

varieties Pinot Noir and Cornalin which contained 12 and9 different yeast species respectively and the white varietiesGutedel Chardonnay and Petite Arvine which contained 1212 and 7 yeast species respectively (Table 5)

Most of the yeast species we identified were presentin more than one of the musts (Table 5) M pulcherrimaS cerevisiae S bayanus and T delbrueckii were foundin all five musts at some point during fermentation Sixyeast species were only found in one type of must andonly at the beginning of fermentation Four yeast speciesfound in Gutedel musts did not grow in any of the othermustsBulleromyces albusCandida zeylanoidesCryptococcusflavusDekkera bruxellensis (the latter could not be distin-guished on the basis of their PCR-RFLP patterns) and Filoba-sidium floriforme Similarly Pichia burtonii and P holstii onlyfound in Cornalin musts (Table 5) There were no speciesassociated exclusively with red or white grape varieties

The composition of the yeast populations also changedsignificantly during fermentation Initially 7ndash12 differentspecies were found in the musts (depending on the variety)but this declined to 1ndash5 species by themidfermentation whennitrogen becomes limiting and the ethanol concentrationbegins to increase rapidly (Table 5) By the end of fermenta-tion only six different yeast species could be recovered fromthe musts The ethanol-resistant strain S bayanus made up


Table 3 Sizes of digested and undigested nested PCR productsrepresenting different yeast species derived using the T-RFLPmethod

Yeast species Size (bp)Nested PCR Digested

Aureobasidium pullulanslowast 254 mdashBotryotinia fuckelianalowast mdash 223Bulleromyces albus 195 164Candida glabrata 478 224Candida zemplinina 200Candida zeylanoides 269 238Cryptococcus flavus 169 134Cryptococcus magnus 211 mdashDekkera bruxellensis 168 134Epicoccum nigrumlowast 216 mdashFilobasidium floriforme 237 204Hanseniaspora guilliermondii 364 66Hanseniaspora uvarum 365 66Hyphopichia burtonii 159 124Issatchenkia orientalis 178 148Kluyveromyces lactis 305 273Metschnikowia pulcherrima 140 107Metschnikowia sp 348 mdashPichia angusta 379 351Pichia anomala 258 228Pichia fermentans 156 122Pichia holstii mdash 248Pichia kluyveri 162 128Pichia membranifaciens 165 130Rhodotorula mucilaginosa 229 198Saccharomyces bayanus 435 405Saccharomyces cerevisiae 441 410Saccharomyces pastorianus 435 405Sporidiobolus pararoseus 222 197Torulaspora delbrueckii (wild yeastisolate) 370 340

Williopsis saturnus 252 134Zygosaccharomyces florentinus 244 213lowastMolds

a substantial proportion of the yeasts in all musts (Figure 1)and was the only strain detected in Gutedel musts during themid- and late fermentation stages In contrast S cerevisiaewas found in the Chardonnay Pinot Noir and Petite Arvinemusts at the end of fermentation and M pulcherima waspresent in the Chardonnay Pinot Noir and Cornalin mustsat the end of fermentation The other species retrieved atthe end of the fermentation were P klyveri (Chardonnay andCornalin musts) P membranifaciens (Chardonnay must)and R mucilaginosa (Pinot Noir must) (Figure 1)

The progress of fermentation was monitored by measur-ing sugar consumption and ethanol production (Figure 2)

0102030405060708090

100

Yeas

t con

trib

utio

n (

)

Pinot Noir Cornalin Chardonnay Petit Arvine Gutedel

Metschnikowia pulcherrima Saccharomyces bayanusSaccharomyces cerevisiae Pichia klyveriRhodotorula mucilaginosa Pichia membranifaciens

Figure 1 Yeast population recovered at the end of spontaneousfermentation in steel tanks during the 2008 and 2009 harvestseasons

as well as acetic acid production (Table 6) The red varietiesPinot Noir and Cornalin reached dryness (less than 4 g Lminus1 oftotal sugar) 6ndash11 days after pressing (Figure 2) The lag phaseof the PinotNoir fermentations in 2008 (ie the period beforeglucose consumption increases rapidly) was relatively longcompared to the Cornalin fermentations in the same year (2-3 days) (Figure 2) In contrast the white grape varieties failedto reach dryness in fermentations during 2008 and 2009 andthe Petite Arvine and Chardonnay vessels contained highlevels of residual sugar at the end of fermentation (Figure 2)In 2008 the fermentation of Gutedel grapes was delayed atthe midexponential phase (days 6ndash13) whereas Petite Arvinewas characterized by sluggish fermentation from the lateexponential phase (day 11) onwards (Figure 2)

34 Real-Time PCR Thirteen pairs of specific primers weredesigned for the rapid identification and quantification ofthe yeast species we detected The primers designed for Zflorentinus C glabrata and P fermentans showed evidenceof nonspecific annealing and were therefore eliminated fromthe study The sequences and annealing temperatures of theremaining primers are summarized in Table 2Themelt curveanalysis for each PCR showed a single peak (data not shown)Standard curveswere established for each pair of primersThereaction efficiencies ranged between 7254 (P anomala) and9868 (S cerevisiae) with high reproducibility The lowestdetection limit was 102 cells Lminus1

35 Yeast Flora inQvevri Fermentations Thedynamic behav-ior of the yeast populations in Qvevri spontaneous fermen-tations was monitored by qPCR during the 2010 and 2011harvest seasons There was a slight tendency towards higheryeast diversity in the Resi variety compared to Ermitage with10 and 8 different yeast species respectively (Table 7) Mostof the species were present in varieties and M pulcherimaR mucilaginosa P anomala H uvarum S cerevisiae and


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Figure 2 Spontaneous fermentation profile expressed in g Lminus1 for the white grape varieties in stainless steel tanks during the 2008 and 2009harvest seasons (a) Pinot Noir (b) Cornalin (c) Chardonnay (d) Petit Arvine (e) Gutedel


Table 4 Yeasts isolated from the winery environment during the 2008 and 2009 harvest seasons

Yeast species Air vineyard Air cellar Tank surface Grape surfacelowast

2008 2009 2008 2009 2008 2009 2008 2009Aureobasidium pullulans + Gu GuBulleromyces albus + +Candida zemplinina +Cryptococcus magnus + GuFilobasidium floriforme Ch Ch Pa GuHanseniaspora uvarum + +Metschnikowia pulcherrima + +Pichia angustaPichia anomala + + CoPichia kluyveri + +Rhodotorula mucilaginosa + + + Pn Pa GuSporidiobolus pararoseus + +Williopsis saturnus PaZygosaccharomyces florentinus + + + Ch ColowastVarieties of grapes and musts Pinot Noir (Pn) Cornalin (Co) Chardonnay (Ch) Petite Arvine (Pa) Gutedel (Gu)

Table 5 Yeasts found during spontaneous fermentation in stainless steel tanks during the 2008 and 2009 harvest seasons

Yeast species Grape varietyPinot Noir Cornalin Chardonnay Petit Arvine Gutedel

Bulleromyces albus aCandida zemplinina a a b a aCandida zeylanoides aCandida Krusei or Issatchenkia orientalis a aCryptococcus flavus or Dekkera bruxellensis aFilobasidium floriforme aHanseniaspora uvarum a b a aMetschnikowia pulcherrima a b c a b c a b c a aPichia anomala a a aPichia burtonii aPichia holstii aPichia kluyveri a a c a aPichia membranifaciens a b c aRhodotorula mucilaginosa c a a aSaccharomyces bayanus a b c a b c a b c a b c a b cSaccharomyces cerevisiae a b c a a b c a c aSporidiobolus pararoseus b aTorulaspora delbrueckii a a a a aZygosaccharomyces florentinus a aa detected at the beginning of the fermentation b detected during log phase c detected during stationary phase

T delbrueckii were also found at every fermentation stage(Figures 3 and 4) In contrast C zemplinina and P angustawere found only in the Resi variety and although P kluyveriwas found in both varieties it was present only at certainfermentation stages during the 2010 harvest and was notdetected in 2011 (Table 7)

R mucilaginosa was the dominant species in the 2011Ermitage fermentations whereas P anomala was the domi-nant species in the Resi fermentations in both harvest yearsUp to eight yeast species were detected in the Ermitage

fermentations during 2010 and 2011 (Table 7) and in bothcases the dominant species in the must before fermentationwere R mucilaginosa and P anomala although they weremore abundant in 2011 (Figure 3) The less-abundant specieswere H uvarum S cerevisiae T delbrueckii and C zem-plinina although all of them were present throughout thefermentation These species were 10 times more abundantin 2010 than in 2011 except R mucilaginosa which wasmore abundant in 2011 S cerevisiae was the most abundantspecies at the beginning of the 2010 fermentations and it


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Figure 3 Dynamic behavior of wild yeast populations during the spontaneous fermentation of Ermitage grapes in Qvevris measured byqPCR (a) 2010 harvest (b) 2011 harvest

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Figure 4 Dynamic behavior of wild yeast populations during the spontaneous fermentation of Resi grapes in Qvevris measured by qPCR(a) 2010 harvest (b) 2011 harvest

proliferated rapidly reaching its maximum concentration(1 times 106 cellsmLminus1) by day 6 In contrast R mucilaginosawas the most abundant species in the 2011 fermentations(1 times 106 cellsmLminus1) and S cerevisiae proliferatedmore slowlyreaching its maximum concentration after 14 days In 2010the yeast population declined slowly during fermentationwhereas in 2011 the P anomala S cerevisiae and R mucilagi-nosa populations remained high (Figure 3)

The Resi fermentations during 2010 and 2011 beganrapidly (before 5 days in both cases) with P anomaladominating throughout fermentation and H uvarum and Tdelbrueckii present at lower levels (Figure 4) The concentra-tion of yeast including S cerevisiae was slightly higher in

2010 than 2011 although the onset of fermentation in 2011was more rapid beginning after 1 day (Figure 4) In 2010the highest concentration of S cerevisiae (1 times 106 cellsmLminus1)was achieved 2 days after fermentation began whereas in2011 the concentration increased rapidly during the first dayand remained high until the end of the fermentation (1 times106 cellsmLminus1)

The progress of the Ermitage and Resi fermentations wasmonitored and compared The onset of the Ermitage fer-mentation took longer in 2010 but was nevertheless completeafter 14 days in both 2010 and 2011 (Figure 5) The Ermitagefermentations did not reach dryness by day 14 in 2011 andthe ethanol content was lower than in the 2010 fermentation


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Figure 5 Spontaneous fermentation profile in Qvevris during the 2010 and 2011 harvests (a) Ermitage (b) Resi

Table 6 Acetic acid production in the spontaneous fermentations

Fermentation vessel Variety Harvest season Acetic acid (mg Lminus1)

Pinot Noir 2008 6561 plusmn 25

2009 15469 plusmn 2348

Cornalin 2008 15071 plusmn 1862

2009 1450 plusmn 707

Steel tanks Chardonnay 2008 15464 plusmn 9143

2009 37915 plusmn 0

Petite Arvine 2008 13164 plusmn 7688

2009 2800 plusmn 0

Gutedel 2008 2493 plusmn 0

2009 940 plusmn 390

QvevriResi 2010 1800 plusmn 0

2011 2700 plusmn 100

Ermitage 2010 1600 plusmn 300

2011 650 plusmn 200

concomitant with the production of significant amounts ofacetic acid (Table 6) No measurements were taken beyondday 14 because the Ermitage and Resi wines were blendedat this stage The Resi fermentations become more rapidly in2011 than in 2010 beginning on the same day (or shortly after)the Qvevris were filled However the fermentation processreached dryness in both yearsTheErmitagemust took longerto begin fermentation than Resi starting 2 and 10 days laterin 2010 and 2011 respectively

4 Discussion

41 Isolation and Identification of Predominant Yeast SpeciesThe isolation media we used enabled us to select different

yeasts that were present in the winery environment and inwine musts undergoing spontaneous fermentation in steeltanks thus favoring the detection and proliferation of someyeast species over others Rose Bengal Chloramphenicol Agar(RBCA) medium was chosen instead of Sabouraud mediumbecause the latter favored mold growth over yeasts (data notshown) Freezing the samples prior to analysis may havereduced the viability of the yeast although it is thought thatthis is a minor effect [26 27] Therefore we acknowledgethat yeast species present in low numbers are unlikely to bedetected using this method whereas abundant species aremore likely to be recognized Thus only seven yeast specieswere isolated from the grape surface (A pullulansCmagnusF floriformeRmucilaginosaW saturnus andZ florentinus)


Table 7 Yeasts identified by qPCR during spontaneous fermentations in Qvevris during the 2010 and 2011 harvest seasons

Yeast species Resi Ermitage2010 2011 2010 2011

Candida zemplinina a b c bMetschnikowia pulcherima a b c a b c a b c a b cWilliopsis saturnus a c a b cPichia kluyveri a bRhodotorula mucilaginosa a b c a b c a b c a b cPichia angusta b cPichia anomala a b c a b c a b c a b cHanseniaspora uvarum a b c a b c a b c a b cSaccharomyces cerevisiae a b c a b c a b c a b cTorulaspora delbrueckii a b c a b c a b c a b ca detected at the beginning of the fermentation b detected during log phase c detected during stationary phase

with A pullulans and R mucilaginosa previously reportedas colonizers of the grape surface [28] A further 11 yeastspecies were isolated from vineyard air samples all ofwhich had previously been detected in winery environmentalsamples [28 29] The anamorphic yeast Kloeckera apiculatapreviously reported as the predominant yeast species on thegrape surface and in air samples [28] was not found in ourinvestigation but instead the teleomorphic speciesH uvarumwas found in our environmental samples

We found that differences in yeast diversity were oftendependent on the grape variety This phenomenon can beattributed to several factors including the different stagesof berry ripening at harvest physical damage to the grapesurface and pest management practices [29] Although westudied different grape varieties grown in the same area andprocessed at the same winery microclimatic conditions andviticultural practices may have influenced the yeast diversitywe detected

Most of the yeasts isolated from the vineyard air were alsopresent in the grape juice at the beginning of fermentationAll the yeasts identified in the cellar were also found laterin the fermenting must R mucilaginosa was found in airsamples from both the vineyard and the cellar and on thegrape surface but not on the tank surface During 2008 Zflorentinus was the only species found in all environmentalsamples (air and contact samples from both the vineyard andthe cellar)

The viable counts of the environmental samples showedthe presence of only non-Saccharomyces species AlthoughS cerevisiae and related species such as S bayanus arepredominantly responsible for fermentation they representonly a small fraction of the diversity we identified whichis consistent with other reports showing that S cerevisiae israrely isolated from natural sources such as berry and leafsurfaces when using viable countmethods [30ndash33]The smallnumber of species isolated from the cellar environment (airand tank surface) during 2009 compared to 2008 may havebeen caused by the sanitary conditions adopted by the wineryafter the sampling results in 2008 The dynamic behavior of

the yeast populations through the different stages of fermen-tation in steel tanks also differed among grape varieties Thedetection of some yeast species only during the later stagesof fermentation probably reflects their proliferation to cellnumbers above the detection threshold of our assay ratherthan their genuine absence at the beginning of fermentationThe relative greater diversity of yeast species in red comparedto white wines is consistent with the higher pH of red winesproviding favorable conditions for yeast growth [34] In whitewines yeasts isolated from the grape skin were not found inthemust probably because they remained in the skin fractionduring clarification and thismay also have contributed to thelower species diversity we observed

The higher yeast diversity during the early stages offermentation predominantly reflects the low ethanol toler-ance of non-Saccharomyces species [3 9 10 17 35 36]Nevertheless we found that non-Saccharomyces yeasts suchas P klyveri P membranifaciens R mucilaginosa and Mpulcherima were active in the late fermentation stages insome must varieties This is consistent with previous reportsof ethanol tolerance in M pulcherima [10 35 37] but Rmucilaginosa is usually found during the early stages offermentation and its presence along with the Pichia specieslater in fermentation could add complexity but also reducethe wine quality [34 38]

Considering the results from the 2008 and 2009 harveststogether we observed that the generally higher yeast diver-sity in the must at the beginning of the fermentation wascoincident with the rapid onset of the exponential phaseWe evaluated the interrelation between the yeast speciesand the success of fermentation We found that despite thediversity of yeasts in red and white varieties white mustsgenerally contained higher residual sugar levels than redmusts and that sluggish fermentation was more likely Suchfermentationswere characterized by the initial predominanceof C zemplinina and S bayanus as well as lower levels ofM pulcherima and S cerevisiae contrasting with the redwine musts The impact of these properties on fermentationreflects the better performance of S cerevisiae compared with


the lower fructose uptake capacity of S bayanus [39] whichis consistent with our results

42 Dynamic Behavior of Wild Yeasts during SpontaneousFermentation in Qvevris We developed a novel qPCRmethod for the rapid sensitive and culture-independentdetection of yeast species throughout fermentation revealingthat the non-Saccharomyces yeast R mucilaginosa and Panomala dominated the final stages of spontaneous fermen-tation in Qvevris These results are important because non-Saccharomyces yeasts can influence the flavor and quality ofwine in both positive and negative ways [40ndash42] despite theirmetabolic activity and abundance [19 20 43]

The diversity of the yeast species was variety dependentand vintage dependent with C zemplinina and P angustapresent only in the variety of Resi and P kluyveri presentin both wines but only during the 2010 harvest H uvarumhas previously been identified as the predominant speciesduring the early stages of fermentation [9ndash11 35] but wefound no evidence for this species on the grape surface(viable cell count method) and found it was less prevalentduring amphora fermentations (qPCR method) In con-trast R mucilaginosa was found to be abundant in boththe amphora and steel-tank fermentations using qPCR andculture-dependent methods respectively

The Resi fermentations commenced almost immediatelyin 2011 even though similar numbers of yeast cells werepresent at the beginning of fermentation in both yearsand S cerevisiae was less abundant in 2011 than 2010 Theminimal lag phase and rapid fermentation (completed in 3days) could be explained by the climatic conditions in theweeks prior to harvest which increased the temperature ofthe berries and the must after crushing (data not shown)favoring the rapid proliferation of S cerevisiae This sug-gests that berry temperature before pressing could play akey role in the success of spontaneous fermentation Wetested this hypothesis by studying parallel fermentationsErmitage fermentations underwent a longer lag phase in2011 (11 days) than 2010 (5 days reaching dryness by day14) During 2011 R mucilaginosa and P anomala were thepredominant species throughout fermentation and theseare considered spoilage yeasts [44] Several previous studieshave shown that longer lag phases provide an opportunityfor non-Saccharomyces yeasts and other microorganisms tooutcompete beneficial microbes and produce toxic andornoxious compounds causing spoilage [45ndash49] Accordinglywe found that 065 g Lminus1 acetic acid was produced in thisfermentationwhich is above the upper range in normalwinesand is considered undesirable [8 50 51]

Despite the presence of spoilage yeast the success ofspontaneous fermentation seems to correlate with the lengthof the lag phase since fermentations with a longer lagphase were more likely to fail The onset of fermentationalso depended on the temperature of the must so this isa key factor to consider when predicting the outcome of aspontaneous fermentation

Our integration of novel analytical methods with tra-ditional winemaking using Qvevris provides the basis for

further experiments to determine the influence of Qvevris onspontaneous fermentation Comparative studies with steel-tank fermentations using the same raw materials (grapevariety and harvest year) should be carried out to investigatethe impact of Qvevris in more detail

5 Conclusions

The predominant yeasts found in the winery (ieMetschnikowia pulcherima Rhodotorula mucilaginosaand some Pichia species) were used as a basis for thedevelopment of an antibody chip for the identification andsemiquantitative detection of wild yeast The effect of theinitial yeast concentration and the berrymust temperatureon the length of the fermentation lag phase and thus thequality of spontaneous fermentation will be investigatedin more detail to improve the performance of this deviceWe have also provided the first quantitative evidencedescribing the dynamic behavior of yeast populations duringspontaneous fermentation in amphora vessels

Conflict of Interests

The authors declare that there is no conflict of interests

Acknowledgments

The authors thank Albert Mathier et Fils winery especiallythe owner (Amedee Mathier) and oenologist (Fadri Kuonen)for kindly providing the samples for testing This work wasfunded by the Fraunhofer Institute

References

[1] M R Provenzano H El Bilali V Simeone N Baser DMondelli and G Cesari ldquoCopper contents in grapes and winesfrom a Mediterranean organic vineyardrdquo Food Chemistry vol122 no 4 pp 1338ndash1343 2010

[2] W Kaltzin ldquoNatural wines als Trendrdquo 2012 httpwwwder-winzeratid=25002C51097872C2C2CY2Q9NDg3D

[3] G H Fleet ldquoYeast interactions and wine flavourrdquo InternationalJournal of Food Microbiology vol 86 no 1-2 pp 11ndash22 2003

[4] A Cuadros-Inostroza P Giavalisco J Hummel A EckardtL Willmitzer and H Pena-Cortes ldquoDiscrimination of wineattributes by metabolome analysisrdquo Analytical Chemistry vol82 no 9 pp 3573ndash3580 2010

[5] J Marais ldquoEffect of different wine-making techniques on thecomposition and quality of pinotage wine I Low-temperatureskin contact prior to fermentationrdquo South African Journal ofEnology and Viticulture vol 24 no 2 pp 70ndash75 2003

[6] P Hernandez-Orte M Cersosimo N Loscos J Cacho EGarcia-Moruno and V Ferreira ldquoAroma development fromnon-floral grape precursors by wine lactic acid bacteriardquo FoodResearch International vol 42 no 7 pp 773ndash781 2009

[7] P Romano C Fiore M Paraggio M Caruso and A CapeceldquoFunction of yeast species and strains in wine flavourrdquo Interna-tional Journal of FoodMicrobiology vol 86 no 1-2 pp 169ndash1802003


[8] M G Lambrechts and I S Pretorius ldquoYeast and its importanceto wine aromamdasha reviewrdquo South African Journal of Enology andViticulture vol 21 special issue pp 97ndash129 2000

[9] M Combina A Elıa L Mercado C Catania A Ganga and CMartinez ldquoDynamics of indigenous yeast populations duringspontaneous fermentation of wines fromMendoza ArgentinardquoInternational Journal of Food Microbiology vol 99 no 3 pp237ndash243 2005

[10] E DiMaro D Ercolini and S Coppola ldquoYeast dynamics duringspontaneous wine fermentation of the Catalanesca graperdquoInternational Journal of Food Microbiology vol 117 no 2 pp201ndash210 2007

[11] A Rementeria J A Rodriguez A Cadaval et al ldquoYeastassociated with spontaneous fermentations of white wines fromthe rdquoTxakoli de Bizkaiardquo region (BasqueCountryNorth Spain)rdquoInternational Journal of Food Microbiology vol 86 no 1-2 pp201ndash207 2003

[12] G H Fleet Wine Microbiology and Biotechnology Taylor ampFrancis 1993

[13] E H Hansen P Nissen P Sommer J C Nielsen and NArneborg ldquoThe effect of oxygen on the survival of non-Saccharomyces yeasts during mixed culture fermentations ofgrape juice with Saccharomyces cerevisiaerdquo Journal of AppliedMicrobiology vol 91 no 3 pp 541ndash547 2001

[14] R B Boulton V L Singleton L F Bisson and R E KunkeePrinciples and Practices of Winemaking Springer 1998

[15] N P Jolly O P H Augustyn and I S Pretorius ldquoThe role anduse of non-saccharomyces yeasts in wine productionrdquo SouthAfrican Journal of Enology and Viticulture vol 27 no 1 pp 15ndash38 2006

[16] M Ciani and F Maccarelli ldquoOenological properties of non-Saccharomyces yeasts associated with wine-makingrdquo WorldJournal ofMicrobiology andBiotechnology vol 14 no 2 pp 199ndash203 1998

[17] C M Egli W D Edinger C MMitrakul and T Henick-KlingldquoDynamics of indigenous and inoculated yeast populations andtheir effect on the sensory character of Riesling andChardonnaywinesrdquo Journal of Applied Microbiology vol 85 no 5 pp 779ndash789 1998

[18] A Soden I L Francis H Oakey and P A Henschke ldquoEffectsof co-fermentation with Candida stellata and Saccharomycescerevisiae on the aroma and composition of Chardonnay winerdquoAustralian Journal of Grape and Wine Research vol 6 no 1 pp21ndash30 2000

[19] N Hierro B Esteve-Zarzoso A Gonzalez A Mas and J MGuillamon ldquoReal-time quantitative PCR (QPCR) and reversetranscription-QPCR for detection and enumeration of totalyeasts in winerdquo Applied and Environmental Microbiology vol72 no 11 pp 7148ndash7155 2006

[20] K Zott O Claisse P Lucas J Coulon A Lonvaud-Funel and IMasneuf-Pomarede ldquoCharacterization of the yeast ecosystem ingrape must and wine using real-time PCRrdquo Food Microbiologyvol 27 no 5 pp 559ndash567 2010

[21] L Cocolin and D Ercolini Molecular Techniques in the Micro-bial Ecology of Fermented Foods Springer 2007

[22] B G Baldwin ldquoPhylogenetic utility of the internal transcribedspacers of nuclear ribosomal DNA in plants an example fromthe compositaerdquo Molecular Phylogenetics and Evolution vol 1no 1 pp 3ndash16 1992

[23] Y C Chen J D Eisner M M Kattar et al ldquoPolymorphicinternal transcribed spacer region 1 DNA sequences identify

medically important yeastsrdquo Journal of Clinical Microbiologyvol 39 no 11 pp 4042ndash4051 2001

[24] T White T Bruns S Lee and J Taylor ldquoAmplification anddirect sequencing of fungal ribosomal RNA genes for phyloge-neticsrdquo in PCR Protocols A Guide to Methods and ApplicationsM A Innis D H Gelfand J J Sninsky and T J White Edspp 315ndash322 Academic Press 1990

[25] M A Lodhi G N Ye N F Weeden and B I Reisch ldquoAsimple and efficientmethod forDNAextraction fromgrapevinecultivars and Vitis speciesrdquo Plant Molecular Biology Reportervol 12 no 1 pp 6ndash13 1994

[26] S Pardo M A Galvagno and P Cerrutti ldquoStudies of via-bility and vitality after freezing of the probiotic yeast Sac-charomyces boulardii physiological preconditioning effectrdquoRevista Iberoamericana de Micologia vol 26 no 2 pp 155ndash1602009

[27] C Alves-Araujo M J Almeida M J Sousa and C LeaoldquoFreeze tolerance of the yeast Torulaspora delbrueckii cellularand biochemical basisrdquo FEMSMicrobiology Letters vol 240 no1 pp 7ndash14 2004

[28] A Martini ldquoOrigin and domestication of the wine yeastSaccharomyces cerevisiaerdquo Journal of Wine Research vol 4 no3 pp 165ndash176 1993

[29] P Raspor D M Milek J Polanc S Smole Mozina and NCadez ldquoYeasts isolated from three varieties of grapes cultivatedin different locations of the Dolenjska vine-growing regionSloveniardquo International Journal of Food Microbiology vol 109no 1-2 pp 97ndash102 2006


[31] A Vaughan-Martini and A Martini ldquoFacts myths and legendson the prime industrial microorganismrdquo Journal of IndustrialMicrobiology vol 14 no 6 pp 514ndash522 1995

[32] M J De La Torre M C Millan P Perez-Juan J Morales and JM Ortega ldquoIndigenous yeasts associated with twoVitis viniferagrape varieties cultured in southern SpainrdquoMicrobios vol 100no 395 pp 27ndash40 1999

[33] I S Pretorius ldquoTailoring wine yeast for the new millenniumnovel approaches to the ancient art of winemakingrdquo Yeast vol16 no 8 pp 675ndash729 2000

[34] T Deak and L R Beuchat ldquoYeasts associated with fruit juiceconcentratesrdquo Journal of Food Protection vol 56 no 9 pp 777ndash782 1993

[35] M J Torija N Rozes M Poblet J M Guillamon and A MasldquoYeast population dynamics in spontaneous fermentationscomparison between two different wine-producing areas overa period of three yearsrdquo International Journal of General andMolecular Microbiology vol 79 no 3-4 pp 345ndash352 2001

[36] P Satora and T Tuszynski ldquoBiodiversity of yeasts during plumWegierka Zwykla spontaneous fermentationrdquo Food Technologyand Biotechnology vol 43 no 3 pp 277ndash282 2005

[37] A Querol M Jimenez and T Huerta ldquoMicrobiological andenological parameters during fermentation of musts from poorand normal grape-harvests in the region of Alicante (Spain)rdquoJournal of Food Science vol 55 no 6 pp 1603ndash1606 1990

[38] V Loureiro and M Malfeito-Ferreira ldquoSpoilage yeasts in thewine industryrdquo International Journal of Food Microbiology vol86 no 1-2 pp 23ndash50 2003


[39] I Magyar and T Toth ldquoComparative evaluation of someoenological properties in wine strains of Candida stellata Can-dida zemplinina Saccharomyces uvarum and Saccharomycescerevisiaerdquo Food Microbiology vol 28 no 1 pp 94ndash100 2011

[40] J Mora J I Barbas and AMulet ldquoGrowth of yeast species dur-ing the fermentation of musts inoculated with kluyveromycesthermotolerans and saccharomyces cerevisiaerdquo American Jour-nal of Enology and Viticulture vol 41 no 2 pp 156ndash159 1990

[41] E Longo J B Velazquez C Sieiro J Cansado P Calo and TG Villa ldquoProduction of higher alcohols ethyl acetate acetalde-hyde and other compounds by 14 Saccharomyces cerevisiaewine strains isolated from the same region (Salnes NW Spain)rdquoWorld Journal of Microbiology amp Biotechnology vol 8 no 5 pp539ndash541 1992

[42] C Lema C Garcia-Jares I Orriols and L Angulo ldquoContribu-tion of Saccharomyces and non-Saccharomyces populations tothe production of some components of Albarino wine aromardquoAmerican Journal of Enology and Viticulture vol 47 no 2 pp206ndash216 1996

[43] I Andorra S Landi A Mas J M Guillamon and B Esteve-Zarzoso ldquoEffect of oenological practices on microbial popula-tions using culture-independent techniquesrdquo Food Microbiol-ogy vol 25 no 7 pp 849ndash856 2008

[44] J I Pitt and A D Hocking Fungi and Food Spoilage Springer3rd edition 2009

[45] G S Drysdale and G H Fleet ldquoThe growth and survivalof acetic acid bacteria in wines at different concentrations ofoxygenrdquo American Journal of Enology and Viticulture vol 40no 2 pp 99ndash105 1989

[46] L F Bisson ldquoStuck and sluggish fermentationsrdquo AmericanJournal of Enology and Viticulture vol 50 no 1 pp 107ndash1191999

[47] C G Edwards R B Beelman C E Bartley and A LMcconnell ldquoProduction of decanoic acid and other volatilecompounds and the growth of yeast and malolactic bacteriaduring vinificationrdquo American Journal of Enology and Viticul-ture vol 41 no 1 pp 48ndash56 1990

[48] C G Edwards K M Haag and M D Collins ldquoIdentificationand characterization of two lactic acid bacteria associated withsluggishstuck fermentationsrdquoAmerican Journal of Enology andViticulture vol 49 no 4 pp 445ndash448 1998

[49] M J Torija G Beltran M Novo et al ldquoEffects of fermentationtemperature and Saccharomyces species on the cell fatty acidcomposition and presence of volatile compounds in winerdquoInternational Journal of Food Microbiology vol 85 no 1-2 pp127ndash136 2003

[50] M Vilanova Z Genisheva L Bescansa A Masa and J MOliveira ldquoVolatile composition of wines from cvs Blancolexıtimo Agudelo and Serradelo (Vitis vinifera) grown inBetanzos (NW Spain)rdquo Journal of the Institute of Brewing vol115 no 1 pp 35ndash40 2009

[51] I Mato S Suarez-Luque and J F Huidobro ldquoSimple determi-nation of main organic acids in grape juice and wine by usingcapillary zone electrophoresis with direct UV detectionrdquo FoodChemistry vol 102 no 1 pp 104ndash112 2007

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of


Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology


Molecular Biology International

GenomicsInternational Journal of


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014


BioinformaticsAdvances in

Marine BiologyJournal of



Signal TransductionJournal of


BioMed Research International

Evolutionary BiologyInternational Journal of



Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Genetics Research International


Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational



Enzyme Research



Microbiology


introducing a broader spectrum of aromas and flavors [16ndash18]

Microbiology techniques are often used to isolate andidentify wild yeasts but this requires different types ofmedia and different culture protocols that influence specieswhich are recovered The metabolic status of the cells alsoresults in the presence of viable but nonculturable (VBNC)microbes whose influence on fermentation can be underesti-mated because the population dynamics cannot be evaluatedaccurately Quantitative real-time PCR (qPCR) is a fasterand more reliable alternative to identify and quantify yeastsduring fermentation [19] and is particularly advantageousfor VBNC yeasts because of its sensitivity [20] Althoughthe technique cannot distinguish living cells from intactdead cells it remains the most widely used method forthe evaluation of wild yeast dynamics during fermentationbecause VBNC cells continue to influence wine flavor andpalatability regardless of their actual status [21]

We compared microbiology methods (viable counts)and novel molecular biology techniques polymerase chainreactionrestriction fragment length polymorphism (PCR-RFLP) and qPCR for the identification of yeast species andwe characterized their dynamic behavior during spontaneouswine fermentation in the Valais region of Switzerland inthe 2008ndash2011 harvests We used these new methods toidentify the predominant species present during spontaneousfermentation establishing a standard for the semiquantitativedetection of yeasts with antibodies in a biochip format Such adevicewould allowwinemakers tomake early decisions aboutthe suitability of grapes and the likely success of spontaneousfermentation Also for the first time we studied the dynamicbehavior of wild yeasts during spontaneous fermentation inQvevris (amphora-like clay vessels) the use of which is anemerging trend among European winemakers

2 Materials and Methods

21 Samples Grapes and must samples were collected fromthe vineyards of the winery Albert Mathier et Fils SA inSalgesch Valais Switzerland during the 2008ndash2011 harvestseasons The grape varieties we studied are listed in Table 1Samples were collected in situ and frozen at minus20∘C duringtransport prior to analysis

22 Isolation of Yeasts from the Winery Environment andFermentation Samples During 2008 and 2009 we screenedyeasts present in the winery environment (ie the vineyardwinery facilities and cellar) and in the fermentingwinemustsIn the vineyard grape berries were placed in direct contactwith plates containing Rose Bengal Chloramphenicol Agar(RBCA) a selective medium for yeasts and molds (15 g Lminus1agar 10 g Lminus1 glucose 5 g Lminus1 papain-digested soybean meal1 g Lminus1 KH

2PO4 05 g Lminus1 MgSO

4times 7H2O 005 g Lminus1 Rose

Bengal and 10 g Lminus1 chloramphenicol) We pumped 1000 L ofvineyard air surrounding the grapes through a Millipore MAir Tester T (Millipore USA) and plated the collected residueon RBCA as above We also sampled environmental yeastflora from the winery facilities Contact samples were taken

Table 1 Grape varieties used for the analysis of dynamic wildyeast populations during spontaneous fermentation in stainless steeltanks and Qvevris

Harvest2008-2009 (steel tank) 2010-2011 (Qvevris)

Pinot Noir (R) Chardonnay (W) Resi (W)Grapevariety Cornalin (R) Petit Arvine (W) Ermitage (W)

Gutedel (W)(R) red variety (W) white variety

from the inner surface of clean fermentation tanks beforefilling them with grape juice and 1000 L of air inside thecellar was also filtered and the collected residues were platedas above All the environmental samples were collected intriplicate

Five different grape varieties from the 2008-2009 harvests(Table 1) were processed by spontaneous fermentation todetermine the predominant yeast species at the differentfermentation stages Prefermentation steps such as harvestingand pressing were carried out according to routine wineryprocedures Pressed berries were fermented with the skinto make red wine or were clarified before fermentation toproduce white wine Duplicate fermentations were carriedout in the winery cellar using new 110-L stainless steel tankswithout starting yeast cultures Liquid samples (50mL induplicate) from the fermenting musts were collected dailyfrozen immediately at minus20∘C and stored in the dark priorto analysis Immediately after defrosting liquid samples werecentrifuged at 4000 rpm for 5min The supernatant wastested for chemical parameters and the pellet was resus-pended in 100 120583L distilled water plated on RBCA mediumand incubated at 30∘C for 3ndash7 days and then stored at 4∘Cprior to analysis

23 Fermentation Parameters The fermentations were mon-itored by measuring glucosefructose consumption andethanol formation during fermentation and the acetic acidcontent at the end of fermentation The parameters weredetermined by spectrophotometry at 20 plusmn 1∘C using the D-GlucoseD-Fructose Ethanol andAcetic Acid enzymatic kitsprovided by R-Biopharm (Germany) according to themanu-facturerrsquos instructions Standards and controls were providedin the kits All measurements (duplicate fermentations) weretaken in triplicate

24 Identification of the Predominant Yeast Species A ter-minal restriction fragment length polymorphism (T-RFLP)methodwas developed and optimized for yeast identificationbased on restriction patterns generated from the genomicregion spanning the internal transcribed spacers (ITS1 andITS2) and the 58S rRNA gene These regions show lowintraspecific polymorphism and high interspecific variabilityand have previously been shown to distinguish 26 yeastspecies found on grapes in cellars andor in wine musts[22 23]







3 Results































0102030405060708090

100

Yeas

t con

trib

utio

n (

)








0

50

100

150

200

250

300

0 5 10Day

Total sugar 09

Total sugar 08

Ethanol 09

Ethanol 08

Con

cent

ratio

n (g

Lminus1)

(a)

0

50

100

150

200

250

300

0 5 10 15Day

Total sugar 09

Total sugar 08

Ethanol 09

Ethanol 08

Con

cent

ratio

n (g

Lminus1)

(b)

300

250

200

150

100

50

0

Con

cent

ratio

n (g

Lminus1)

0 5 10 15 20Day



(c)

300

250

200

150

100

50

0

Con

cent

ratio

n (g

Lminus1)

0 5 10 15 20Day



(d)

250

200

150

100

50

0

Con

cent

ratio

n (g

Lminus1)

0 5 10 15 20Day



(e)













Beginning End

1 3 5 7 9 11 13 15Day

1 times 107

1 times 106

1 times 105

1 times 104

1 times 103

1 times 102

1 times 101

Cell

s (m

Lminus1)

M pulcherrima


(a)

Beginning End

Day1 3 5 7 9 11 13 15

1 times 107

1 times 106

1 times 105

1 times 104

1 times 103

1 times 102

1 times 101

M pulcherrima


Cell

s (m

Lminus1)

(b)


Beginning End

0 3 6 9 12 15Day

1 times 107

1 times 105

1 times 103

1 times 101

Cell

s (m

Lminus1)

M pulcherrima


(a)

Day

Beginning End

0 1 2 3 4 5 6

1 times 107

1 times 105

1 times 103

1 times 101

Cell

s (m

Lminus1)

M pulcherrima


(b)







Con

cent

ratio

n (g

Lminus1)

0

25

50

75

100

125

150

0 5 10 15 20Day



(a)

Con

cent

ratio

n (g

Lminus1)

0

25

50

75

100

125

150

0 5 10 15Day



(b)





2009 15469 plusmn 2348


2009 1450 plusmn 707


2009 37915 plusmn 0


2009 2800 plusmn 0


2009 940 plusmn 390


2011 2700 plusmn 100


2011 650 plusmn 200


4 Discussion






















5 Conclusions




Acknowledgments


References






























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology







3 Results































0102030405060708090

100

Yeas

t con

trib

utio

n (

)








0

50

100

150

200

250

300

0 5 10Day

Total sugar 09

Total sugar 08

Ethanol 09

Ethanol 08

Con

cent

ratio

n (g

Lminus1)

(a)

0

50

100

150

200

250

300

0 5 10 15Day

Total sugar 09

Total sugar 08

Ethanol 09

Ethanol 08

Con

cent

ratio

n (g

Lminus1)

(b)

300

250

200

150

100

50

0

Con

cent

ratio

n (g

Lminus1)

0 5 10 15 20Day



(c)

300

250

200

150

100

50

0

Con

cent

ratio

n (g

Lminus1)

0 5 10 15 20Day



(d)

250

200

150

100

50

0

Con

cent

ratio

n (g

Lminus1)

0 5 10 15 20Day



(e)













Beginning End

1 3 5 7 9 11 13 15Day

1 times 107

1 times 106

1 times 105

1 times 104

1 times 103

1 times 102

1 times 101

Cell

s (m

Lminus1)

M pulcherrima


(a)

Beginning End

Day1 3 5 7 9 11 13 15

1 times 107

1 times 106

1 times 105

1 times 104

1 times 103

1 times 102

1 times 101

M pulcherrima


Cell

s (m

Lminus1)

(b)


Beginning End

0 3 6 9 12 15Day

1 times 107

1 times 105

1 times 103

1 times 101

Cell

s (m

Lminus1)

M pulcherrima


(a)

Day

Beginning End

0 1 2 3 4 5 6

1 times 107

1 times 105

1 times 103

1 times 101

Cell

s (m

Lminus1)

M pulcherrima


(b)







Con

cent

ratio

n (g

Lminus1)

0

25

50

75

100

125

150

0 5 10 15 20Day



(a)

Con

cent

ratio

n (g

Lminus1)

0

25

50

75

100

125

150

0 5 10 15Day



(b)





2009 15469 plusmn 2348


2009 1450 plusmn 707


2009 37915 plusmn 0


2009 2800 plusmn 0


2009 940 plusmn 390


2011 2700 plusmn 100


2011 650 plusmn 200


4 Discussion






















5 Conclusions




Acknowledgments


References






























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology





























0102030405060708090

100

Yeas

t con

trib

utio

n (

)








0

50

100

150

200

250

300

0 5 10Day

Total sugar 09

Total sugar 08

Ethanol 09

Ethanol 08

Con

cent

ratio

n (g

Lminus1)

(a)

0

50

100

150

200

250

300

0 5 10 15Day

Total sugar 09

Total sugar 08

Ethanol 09

Ethanol 08

Con

cent

ratio

n (g

Lminus1)

(b)

300

250

200

150

100

50

0

Con

cent

ratio

n (g

Lminus1)

0 5 10 15 20Day



(c)

300

250

200

150

100

50

0

Con

cent

ratio

n (g

Lminus1)

0 5 10 15 20Day



(d)

250

200

150

100

50

0

Con

cent

ratio

n (g

Lminus1)

0 5 10 15 20Day



(e)













Beginning End

1 3 5 7 9 11 13 15Day

1 times 107

1 times 106

1 times 105

1 times 104

1 times 103

1 times 102

1 times 101

Cell

s (m

Lminus1)

M pulcherrima


(a)

Beginning End

Day1 3 5 7 9 11 13 15

1 times 107

1 times 106

1 times 105

1 times 104

1 times 103

1 times 102

1 times 101

M pulcherrima


Cell

s (m

Lminus1)

(b)


Beginning End

0 3 6 9 12 15Day

1 times 107

1 times 105

1 times 103

1 times 101

Cell

s (m

Lminus1)

M pulcherrima


(a)

Day

Beginning End

0 1 2 3 4 5 6

1 times 107

1 times 105

1 times 103

1 times 101

Cell

s (m

Lminus1)

M pulcherrima


(b)







Con

cent

ratio

n (g

Lminus1)

0

25

50

75

100

125

150

0 5 10 15 20Day



(a)

Con

cent

ratio

n (g

Lminus1)

0

25

50

75

100

125

150

0 5 10 15Day



(b)





2009 15469 plusmn 2348


2009 1450 plusmn 707


2009 37915 plusmn 0


2009 2800 plusmn 0


2009 940 plusmn 390


2011 2700 plusmn 100


2011 650 plusmn 200


4 Discussion






















5 Conclusions




Acknowledgments


References






























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


0

50

100

150

200

250

300

0 5 10Day

Total sugar 09

Total sugar 08

Ethanol 09

Ethanol 08

Con

cent

ratio

n (g

Lminus1)

(a)

0

50

100

150

200

250

300

0 5 10 15Day

Total sugar 09

Total sugar 08

Ethanol 09

Ethanol 08

Con

cent

ratio

n (g

Lminus1)

(b)

300

250

200

150

100

50

0

Con

cent

ratio

n (g

Lminus1)

0 5 10 15 20Day



(c)

300

250

200

150

100

50

0

Con

cent

ratio

n (g

Lminus1)

0 5 10 15 20Day



(d)

250

200

150

100

50

0

Con

cent

ratio

n (g

Lminus1)

0 5 10 15 20Day



(e)













Beginning End

1 3 5 7 9 11 13 15Day

1 times 107

1 times 106

1 times 105

1 times 104

1 times 103

1 times 102

1 times 101

Cell

s (m

Lminus1)

M pulcherrima


(a)

Beginning End

Day1 3 5 7 9 11 13 15

1 times 107

1 times 106

1 times 105

1 times 104

1 times 103

1 times 102

1 times 101

M pulcherrima


Cell

s (m

Lminus1)

(b)


Beginning End

0 3 6 9 12 15Day

1 times 107

1 times 105

1 times 103

1 times 101

Cell

s (m

Lminus1)

M pulcherrima


(a)

Day

Beginning End

0 1 2 3 4 5 6

1 times 107

1 times 105

1 times 103

1 times 101

Cell

s (m

Lminus1)

M pulcherrima


(b)







Con

cent

ratio

n (g

Lminus1)

0

25

50

75

100

125

150

0 5 10 15 20Day



(a)

Con

cent

ratio

n (g

Lminus1)

0

25

50

75

100

125

150

0 5 10 15Day



(b)





2009 15469 plusmn 2348


2009 1450 plusmn 707


2009 37915 plusmn 0


2009 2800 plusmn 0


2009 940 plusmn 390


2011 2700 plusmn 100


2011 650 plusmn 200


4 Discussion






















5 Conclusions




Acknowledgments


References






























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology












Beginning End

1 3 5 7 9 11 13 15Day

1 times 107

1 times 106

1 times 105

1 times 104

1 times 103

1 times 102

1 times 101

Cell

s (m

Lminus1)

M pulcherrima


(a)

Beginning End

Day1 3 5 7 9 11 13 15

1 times 107

1 times 106

1 times 105

1 times 104

1 times 103

1 times 102

1 times 101

M pulcherrima


Cell

s (m

Lminus1)

(b)


Beginning End

0 3 6 9 12 15Day

1 times 107

1 times 105

1 times 103

1 times 101

Cell

s (m

Lminus1)

M pulcherrima


(a)

Day

Beginning End

0 1 2 3 4 5 6

1 times 107

1 times 105

1 times 103

1 times 101

Cell

s (m

Lminus1)

M pulcherrima


(b)







Con

cent

ratio

n (g

Lminus1)

0

25

50

75

100

125

150

0 5 10 15 20Day



(a)

Con

cent

ratio

n (g

Lminus1)

0

25

50

75

100

125

150

0 5 10 15Day



(b)





2009 15469 plusmn 2348


2009 1450 plusmn 707


2009 37915 plusmn 0


2009 2800 plusmn 0


2009 940 plusmn 390


2011 2700 plusmn 100


2011 650 plusmn 200


4 Discussion






















5 Conclusions




Acknowledgments


References






























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


Con

cent

ratio

n (g

Lminus1)

0

25

50

75

100

125

150

0 5 10 15 20Day



(a)

Con

cent

ratio

n (g

Lminus1)

0

25

50

75

100

125

150

0 5 10 15Day



(b)





2009 15469 plusmn 2348


2009 1450 plusmn 707


2009 37915 plusmn 0


2009 2800 plusmn 0


2009 940 plusmn 390


2011 2700 plusmn 100


2011 650 plusmn 200


4 Discussion






















5 Conclusions




Acknowledgments


References






























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology




















5 Conclusions




Acknowledgments


References






























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

research article characterization and dynamic behavior of...

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