Ann Microbiol (2013) 63:371–379 DOI 10.1007/s13213-012-0484-x

ORIGINAL ARTICLE

Study of yeast populations and their enological properties in Guijoso Appellation of Origin ()

Monica Fernández-González & Ana Isabel Briones

Received: 30 January 2012 /Accepted: 2 May 2012 /Published online: 26 May 2012 # Springer-Verlag and the University of Milan 2012

Abstract The aim of the present study was to select Introduction strains of yeast with good enological qualities which were adapted to the ecological surroundings of Guijoso In enology, yeasts play an important role in forming the Appellation of Origin (A.O). For this, 11 white and red characteristics of a wine independently of other factors. vats from different grape varieties and stages of fermen- Among other technological aspects, the selection of a tation were studied, making a total of 28 samples, with a suitable yeast strain is required in order to obtain qual- selection of 370 isolated yeasts. Yeast cells of the ity wines. Saccharomyces genus were analysed by DNA mitochon- Spontaneous alcoholic fermentation, that is, fermentation drial restriction for discrimination at the strain level, carried out without the addition of commercial dry yeast, is obtaining a total of 23 different molecular patterns. still typical for certain wine cellars, such as the Guijoso The pattern most frequently found was of G04, with Appellation of Origin (A.O), and it is necessary to establish 56 % of the isolated yeasts, followed by G02 with whether fermentation is to be carried out by one or several 15 % and G07 with 9 %. Other patterns found showed predominant strains. A succession of different strains has percentages close to 3 %, such as G01, G03, G05 and been used over the course of winemaking. G06, while the remaining patterns were limited one or Selected yeasts are being used in winemaking with two isolated yeasts. Microfermentations at 25 and 15 °C excellent results in many countries. Usually, the final product were performed using a synthetic must, and the rate of is of a better quality than the wine produced via traditional fermentation, SH2 and foam production and the capacity spontaneous fermentation (Fleet and Heard 1993). to consume sugars from the medium were studied. For strain selection, it is essential to establish the enolog- Furthermore, the killer phenotype, flocculation capacity ical properties of yeast (Degré 1993). For this purpose, there and phenolic off-flavour (POF) characteristics were also are many different selection criteria that can be divided into analysed. Natural musts from and Cabernet positive properties (for example, ethanol tolerance, good Sauvignon varieties were fermented using preselected performance in the transformation of sugars into ethanol, strains and the wines obtained were analysed and tasted. abilitytogrowinhighconcentrationsofsugar)and

Two strains were selected (G01 and G04) to be used as unfavourable properties (such as H2S production, foam starters in Guijoso A.O. production or volatile acidity). Furthermore, there are some aspects that are usually considered as favourable Keywords Spontaneous fermentation . Wine . Yeast . properties, such as the killer phenotype and malic acid Saccharomyces cerevisiae . Starter degradation, which can be included in a third group called neutral properties (Esteve-Zarzoso et al. 2000). : M. Fernández-González (*) A. I. Briones Although commercial yeasts are available for fermenta- Instituto de Investigación Científica Aplicada de Castilla La tion, the use of pure yeast cultures from the area where Mancha (IRICA), they will be used for wine production, known as local Edificio Marie Curie, Avda. Camilo José Cela, s/n., 13071 Ciudad Real, Spain yeast selection, could be more effective. Local yeasts e-mail: [email protected] are presumed to be more competitive because they are 372 Ann Microbiol (2013) 63:371–379 better acclimated to the environmental conditions (Martini and between Saccharomyces and non-Saccharomyces yeasts, Martini 1990; Regodon et al. 1997) and, therefore, they will lysine agar (Oxoid Unipath, UK) was used on which only be better able to dominate the fermentation (Ciani et al. 2004; non-Saccharomyces yeasts can grow. Capece et al. 2010; Rodriguez et al. 2010). Many yeast strains are selected from different local Spanish Mitochondrial DNA restriction analysis wine regions, for example Alicante (Querol et al. 1992b), (Regodon et al. 1997), Castilla-La Mancha DNA extraction and determination of mitochondrial DNA (Briones et al. 1995) and Cataluña (Esteve-Zarzoso et al. restriction patterns of the strains were carried out as previ- 2000; Torija et al. 2001). ously described (Querol et al. 1992a) with the restriction The Guijoso A.O. is located in the municipality of El endonuclease HinfI (Boehringer Mannheim, Germany). The Bonillo, in the (Spain), and was rec- restriction fragments were separated on a horizontal 0.9 % ognized as an A.O. in a publication of the Official State agarose (Ecogen, Barcelona, Spain) gel in TBE 1X buffer Bulletin (BOE 98 of 25.04.2005) and the Official Journal of and compared against the standards (1-kb DNA ladder; the European Union (OJ C041 of 17.02.2006), the latter Biotools, Madrid, Spain). being an essential legal requirement for the full recognition and protection afforded to an A.O. at the European level. PCR amplification and restriction of rDNA Guijoso is distinguished by the development of high quality wines with grapes from their own vineyards, which are In order to identify the isolates to species level, PCR situated at 1,000 m altitude; the climate is continental and (ITS-5,8 S rDNA)-RFLP was used. The amplification and the low temperatures at night are offset by a high number of restriction of rDNA was carried out as previously described hours of sunshine per year. The floor is made of stone, or by Fernández et al. (2000). The primers used to amplify the gravel, as it is known locally, hence the name “Guijoso”. region were ITS1 (5’-TCCGTAGGTGAACCTGCGG-3’) The Vitis vinifera varieties grown here are Cabernet and ITS4 (5’-CCTCCGCTTATTGATATGC-3’), as previous- Sauvignon, , , , Chardonnay and ly described (White et al. 1990). The amplified DNAs were . The musts in the only winery of the digested with four restriction endonucleases: HinfI, CfoI, Guijoso A.O, called Sanchez Muliterno, have never been HaeIII and HpaII, (Boehringer). The restriction frag- inoculated with active dry yeasts. ments were separated on a horizontal 2 % agarose gel The aim of the present work was to study the population in TBE 1× buffer and compared against the standards of Saccharomyces yeasts in different musts during sponta- (100-bp DNA ladder; Biotools).The isolates were iden- neous alcoholic fermentation and to select one or two strains tified to species level on the basis of the restriction with good enological qualities, and which are ecologically bands obtained, according to the yeast ID database adapted to the surroundings of the cellar, to be used as (http://www.yeast-id.com/). starter cultures for developing wines from this A.O. Enological characteristics of Saccharomyces strains

Materials and methods In order to study the fermentation kinetics, the capacity to consume sugars from the medium and the potential

Sampling and isolation of yeasts SH2 and foam production, a synthetic must was pre- pared [250 g/l sucrose, 20 g/l malt extract, 20 g/l yeast

Spontaneous fermentations were performed on Sánchez extract, 6 g/l tartaric acid, 2 g/l malic acid, 1 g/l (NH4)2SO4, Muliterno cellar, using red (Merlot, Tempranillo, Cabernet 1g/l(NH4)3PO4,0.5g/lcitricacidand100mg/lSO2; Sauvignon and Syrah) and white (Chardonnay and the pH was adjusted to pH 3.5]. Individual fermenta- Sauvignon Blanc) grape varieties from vineyards located tions were performed with S. cerevisiae strains in 200- in the Guijoso A.O in stainless steel vats, except for a ml bottles filled with 150 ml of media and covered with Chardonnay that was partially fermented in barrels, at Müller valves. The fermentations were inoculated with a temperatures of 14–19 °C for white wines and 25–30 °C for population of 106 cells/ml and incubated at 25 and 15 °C red ones. Samples were taken at different stages of fermenta- without shaking, in duplicate. tion for the isolation and enumeration of yeast strains. The fermentation kinetics were followed by monitoring The samples, or their adequate dilutions, were spread the loss of weight in the bottles. The rate was expressed as onto YPD agar (1 % yeast extract, 2 % peptone, 2 % the loss of CO2 (g/l) within 24 and 72 h, using the method- glucose, 2 % agar) and incubated at 28 °C for 48–72 h. ology of Briones et al. (1995). Paper saturated with lead From each sample, 15 yeast colonies were randomly taken, acetate solution was used to perform a qualitative control on isolated and purified on YPD agar. In order to distinguish the possible SH2 production by certain strains, and foam Ann Microbiol (2013) 63:371–379 373 production was determined by visual evaluation (a scale of 0 The organoleptic characteristics of all of the wines pro- to 3). The concentration of reducing sugars at the end of duced with preselected yeasts were tested by a panel of fermentation was measured using the Rebelein Method cellar experts. The tests were conducted in order to select (viniKit, Panreac, Spain). the yeast strains that maintained the typical wines produced The ability to flocculate was measured either directly in in the A.O. the culture medium or in Helm’s acetate buffer (Hussain et al. 1986). Statistical analysis The capacity to decarboxylate the phenol acids p- coumaric acid and ferulic acid was established (Grando ThedatawereanalysedusingSPSS17.0softwarefor et al. 1993). Each strain was inoculated in culture media Windows, using one-factor ANOVA. The differences were with 0.5 mM p-coumaric acid or 0.5 mM ferulic acid evaluated using Duncan’stestatp ≤0.05. The data are added and incubated for 48 h at 30 °C. The production of 4- presented as means and standard deviations. vinyl phenol and 4-vinyl guaiacol was determined by olfacto- ry and spectrophotometric analysis, respectively The killer factor was determined by the method described Results and discussion by Jacobs et al. (1988). CECT 1415 as the sensitive control and CECT 1891 as the killer phenotype were used. Yeast isolation and identification of Saccharomyces isolates

The production of H2S was determined on BiGGY agar at 25 °C for 48 h and acetic acid production on calcium Sampling was performed on 11 red and white fermentations carbonate agar at 25 °C for 7 days (Caridi et al. 2002). from different grape varieties (Chardonnay, Sauvignon In order to test the tolerance of the strains for different Blanc, , Tempranillo, Merlot, Syrah), ethanol concentrations (8, 10 and 12 %v/v) a modified resulting in a total of 28 samples from different stages of methodology of Parish and Carroll (1987) was followed. fermentation: beginning (BF), middle (MF) and end (EF).

The CO2 was collected under Durham bells; the test was The molecular analysis methods described above were considered positive if the bell was filled up to one-third of applied to a total of 370 isolates randomly selected from all its capacity within a maximum of a 3-day incubation period of the colonies obtained. Of the 370 isolates from different at 30 °C. stages of fermentation, 358 were classified as the Saccharomyces genus because we were unable to grow Microvinification assays them using lysine as the only nitrogen source; the rest were non-Saccharomyces isolates. Selected strains that passed the preliminary screening We obtained 23 different mtDNA restriction patterns were used for the microvinification assays with natural from a total of 358 Saccharomyces colonies isolated from white and red musts: 1.8 l of white must from the throughout the alcoholic fermentation process using HinfIas Chardonnay grape variety and 3.5 kg of red crushed the restriction endonuclease. Figure 1 shows 18 of these grapes from the Cabernet Sauvignon variety with patterns. The degree of variability was a good parameter

50 ppm SO2 were used, following traditional winemak- with which to evaluate the number of strains actively in- ing methods. The samples were inoculated with 106 volved in fermentation. The average variability found in this cells/ml. The white musts were fermented at a con- study (6.4 %) was similar to values found in some previous trolled temperature of 18–20 °C until the alcoholic studies: 8.6 % (Querol et al. 1994); 2.2–4.2 % (Schütz and fermentation was complete. The red musts were fer- Gafner 1994) and clearly lower than the corresponding mented at 24–25 °C in contact with the skin until final results of 22 % (Torija et al. 2001) and 20.7 % (Nadal et fermentation. All experiments were performed in dupli- al. 1996) 32, 42 and 38 % and 23, 23 and 22 % from three cate. The wines were analysed for conventional param- different cellars and two consecutive years (Izquierdo et al. eters according to the OIV recommendations. 1997), respectively. The major volatiles were analysed using 3-pentanol as The pattern most frequently found was of G04, with 56 % the internal standard. A Perkin Elmer gas chromatograph of the isolated yeasts, followed by G02 with 15 % and G07 equipped with a VINICOL packed column was used with 9 %. Other patterns found showed percentages close to (Gonzalez and Gonzalez 1994). The conditions were as 3 %, such as G01, G03, G05 and G06, while the remaining follows: detector temperature: 225 °C; injector temperature: patterns were limited one or two isolated yeasts. The distri-

200 °C; carrier gas: N2; flow: 15 ml/min; injection volume: bution of isolates according to their patterns and the samples 1 μl. The oven programme was as follows: initial tempera- from which they came are shown in Fig. 2. ture 40°C/3 min; 6°C/min up to 60 °C, constant for 16 min; From these results, it appears that the G04 corresponded and 6°C/min up to 126 °C, constant for 12 min. to a well-established genetic pattern in the winery, which 374 Ann Microbiol (2013) 63:371–379

cellars, where a large number of S. cerevisiae strains, randomly selected, appeared with the same (or very similar) karyotype, showing that strains are highly char- acteristic of wineries. These results disagree with those obtained by Csoma et al. (2010), who reported that the alcoholic fermentation in grape wines is performed by a highly diverse yeast consortia rather than by one or two dominant strains. The G02, although in lower amounts than the G04, wasfoundinalmostallthetanks,especiallyinthered varieties. Cabernet Sauvignon (B) contained a signifi- cant percentage of G07, which appeared in all the Fig. 1 mtDNA-RFLP patterns with Hinf I endonuclease of some of the indigenous Saccharomyces isolates from Guijoso. The identities of the stages, and lower proportions were also found in the strains are indicated by numbers at the top of the gel. G01–G18 (lanes samples of Cabernet Sauvignon (A), Merlot, Syrah and 1–18), lane M 1-kb DNA ladder (Biotools) Tempranillo. The sample Merlot (C3), at the end of fermentation, showed the highest variability in patterns, was isolated from all the tanks sampled, being almost where eight different isolates were found. In contrast, exclusive to the middle fermentation of the Chardonnay the lowest variability was found in Chardonnay (J2) in (J). It is not often that a strain is found which is so well the middle of fermentation, where only colonies belonging to established and adapted to its ecological environment; the G04 pattern were isolated. The G05 was found in several perhaps the idiosyncrasies of the winery, the altitude of deposits, mainly at the end of fermentation. the vineyards and the existence of microclimates make The Saccharomyces and non-Saccharomyces isolates this the typical pattern of the A.O. A similar result was were identified to species level on the basis of the restriction obtained by Briones et al. (1996) in three different bands obtained, and of the ITS-5.8 S rDNA region, digested

100%

90%

80%

70%

60%

50% Yeast strains 40%

30%

20%

10%

0% A1 A2 A3 B1 B2 B3 C1 C3 D1 D3 E2 E3 F1 F2 F3 G1 G2 H1 H2 H3 I2 I3 J2 J3 K1 K2 Vats at different stages of fermentation G01 G02 G03 G04 G05 G06 G07 G18 Others No sacch

Fig. 2 Percentage of yeast strains pattern presented at different fer- Merlot vat2 (D), Merlot vat3 (E), Merlot vat4 (F), Syrah (G), Tempra- mentation stages (1 beginning, 2 middle, 3 end) in samples of Cabernet nillo (H), Sauvignon Blanc (I), Chardonnay vat1 (J), Chardonnay Sauvignon vat1 (A), Cabernet Sauvignon vat2 (B), Merlot vat1 (C), partially barrel fermented (K) Ann Microbiol (2013) 63:371–379 375 with the enzymes HinfI, HaeIII and CfoI. The restriction Fermentations were carried out on synthetic must at 25 enzyme HpaII was used to differentiate between S. cerevi- and 15 °C to determine the speed of fermentation, the siae and S. pastorianus. sugar consumption, and the production of H2Sand The 358 isolates grouped into 23 patterns were identified foam. The results obtained are shown in Table 1. as Saccharomyces cerevisiae, and the non-Saccharomyces Regarding the rate of fermentation at 25 °C, eight statis- isolates found only at the beginning of the fermentation tically different groups were obtained. The first included from a Merlot must (C1) were Debaryomyces polymorphus G19, which was the slowest of all, followed by G18 and and Torulaspora delbrueckii. G09, with a rate of ≤0.8 g CO2/l/h. The fastest were G20 and G22, with values above 1.3 g CO2/l/h, and no signifi- Enological characteristics of the Saccharomyces strains cant differences were found between G14 and G05. The rest showed intermediate rates of fermentation. Regarding the Once the strains have been isolated and identified, it is residual sugar (any that contained more than 10 g/l were not necessary to select those with the best enological prop- included in the analysis), six groups were obtained: G04, erties because not all Saccharomyces strains found in G05 and G01, with the lowest values of residual sugar (≤3g/l), cellars have the same abilities in wine. Whereas genet- and G07 and G02, with more than 7.8 g/l. All the strains ically identical individuals have the same phenotypic (except G18 and G19) showed rates of fermentation above behaviour, it was decided to select a representative of 0.8 g/l/h. Depending on the results for this battery of tests, it each molecular pattern to undergo a battery of tests to was decided to discard patterns G06, G09, G14, G15, G18, provide information about their winemaking skills. G19, G22 and G23 because they left more than 10 g/l of

Table 1 Results of the fermentations at 15 and 25 °C using a synthetic must by the 23S. cerevisiae strains

Yeast strain Fermentation at 25°C Fermentation at 15°C

a b c c a b c c RF RS Fm H2S RF RS Fm H2S

G01 1.03 ± 0.27 cdefg 3.0 ± 0.4 a 0 1 0.28 ± 0.14 3.8 ± 1.1 ab 0 1 G02 0.99 ± 0.01 cdefg 8.0 ± 0.0 f 0 1 0.54 ± 0.08 c 7.4 ± 0.8 c 0 1 G03 1.01 ± 0.00 cdefg 7.1 ± 0.1 def 0 1 0.50 ± 0.17 bc 6.4 ± 0.9 c 0 2 G04 0.97 ± 0.03 cde 2.2 ± 0.1 a 0 0 0.33 ± 0.16 3.7 ± 1.6 ab 0 0 G05 1.24 ± 0.07 gh 2.8 ± 0.1 a 0 2 0.53 ± 0.06 c 3.9 ± 1.6 ab 0 0 G06 1.01 ± 0.06 cdefg >10 1 3 G07 1.03 ± 0.04 cdefg 7.8 ± 1.2 f 0 1 0.32 ± 0.11 7.4 ± 0.2 c 0 1 G08 1.03 ± 0.03 cdefg 6.7 ± 0.1 cdef 0 2 0.38 ± 0.04 6.7 ± 0.3 c 0 0 G09 0.80 ± 0.15 bc >10 0 3 G10 0.99 ± 0.10 cdefg 7.2 ± 1.2 ef 0 2 0.33 ± 0.13 6.9 ± 2.0 c 0 0 G11 1.13 ± 0.03 cdefg 4.5 ± 0.7 b 0 1 0.47 ± 0.14 bc 2.9 ± 0.6 a 0 1 G12 1.10 ± 0.09 cdefg 5.9 ± 0.8 bcde 0 3 0.28 ± 0.04 6.0+/0.4 bc 0 2 G13 1.01 ± 0.02 cdefg 5.7 ± 0.7 bcd 0 1 0.24 ± 0.07 ab 6.7 ± 0.4 c 0 0 G14 1.23 ± 0.03 fgh >10 0 3 G15 0.99 ± 0.06 cdef >10 1 3 G16 1.00 ± 0.02 cdefg 5.7 ± 0.5 bcd 0 0 0.31 ± 0.09 7.3 ± 0.1 c 0 0 G17 0.94 ± 0.02 cd 5.7 ± 0.7 bcd 0 1 0.40 ± 0.16 7.1 ± 0.3 c 0 0 G18 0.63 ± 0.30 b >10 0 2 G19 0.02 ± 0.0 a >10 0 1 G20 1.35 ± 0.11 h 5.9 ± 0.4 bcde 0 0 0.16 ± 0.07 a 7.5 ± 0.5 0 0 G21 1.06 ± 0.02 defg 5.7 ± 0.1 bcd 0 1 0.31 ± 0.07 6.8 ± 1.6 c 0 2 G22 1.36 ± 0.06 h 5.5 ± 0.2 bc 0 2 0.24 ± 0.08 ab 9.8 ± 1.1 d 0 0 G23 1.20 ± 0.01 efgh >10 0 3 a Rate fermentation (RF) as 24-72 hg/l/h CO2 b Residual sugars (RS) in g/l c Foam (Fm) and H2S production during fermentation at 25 and 15 °C, indicated as 0 non-producing, 1 weak, 2 moderate, 3 high Values not sharing the same letter are significantly different (ANOVA and and Duncan’s test for α00.05) 376 Ann Microbiol (2013) 63:371–379 residual sugar. It should be noted that patterns G06, G09 and Table 2 Phenotypic characteristics of the 23 authoctonous S. cerevi- siae strains G18, in addition to not consuming sugars, also produced H2S. a b c Only two disqualified strains (G06 and G15) of the 23 tested Yeast H2S AA Decarboxylation Killer Flocculation produced foam. strain characterd (%)e Eight of the initial 23 genetic patterns were dis- FA pCA carded; microvinifications at 15 °C were performed G01 0 1 3 3 N 98,1 using the remaining strains. Regarding the rate of fer- G02 0 2 2 1 K 96,2 mentation, three groups were obtained. The slower G03 0 1 2 0 N 97,7 group corresponded to G20 with a ratio below 0.2 in G04 0 1 1 2 N 96,6 contrast to the higher speed at 25 °C, so this yeast was G05 0 2 2 2 N 98,0 greatly affected by the change in temperature. Higher G06 2 1 2 3 N 96,1 speeds were obtained for G02 and G05. According to G07 0 1 0 0 N 95,7 the amount of residual sugar, four groups were obtained, G08 0 1 0 0 K 97,0 with the G11 pattern leaving the lowest amount of G09 2 1 1 3 S 97,1 residual sugar left in the medium, and G22 the most. G10 0 1 1 2 S 97,5 Overall, the G11 strain performed better at lower tem- G11 0 1 1 1 N 97,5 peratures and G05 was the least affected by the change G12 2 1 2 2 S 97,4 in temperature. G13 0 2 1 3 N 97,7 At low temperatures, no yeast produced foam and the G14 0 0 0 0 S 95,4 amount of H2S was generally lower than at a higher G15 0 1 2 2 N 98,3 temperature. The higher levels of H2S in the fermentations G16 0 1 1 2 K 97,1 performed under higher temperatures may not necessarily G17 0 1 1 2 N 98,7 have been due to increased production but to an inabil- G18 0 1 0 0 N 97,3 ity to retain the volatile compound within the cells G19 0 0 0 0 N 91,4 (Rupela and Tauro 1985). G20 0 0 1 2 N 96,4 The only strains capable of leaving an amount of residual G21 0 1 2 3 S 92,1 sugar below 5 g/l in the medium that originally contained G22 0 0 0 1 K 96,8 250 g/l sugar were G01, G04, G05 and G11. It should be G23 0 0 0 0 S 94,0 noted that the yeast strains were subjected to a double stress: a H S production on Biggy agar high levels of sugar and a low temperature. 2 b Acetic acid (AA) on carbonate agar Other parameters of the strains were also studied, such as c Decarboxylation of ferulic acid (FA)orcoumaricacid(pCA)are the production of H2S and acetic acid on solid media, the indicated as 0 non-producing, 1 weak, 2 moderate, 3 high ability to decarboxylate ferulic and p-coumaric acids, the d Killer character as killer phenotype (K), K neutral (N)andK presence of killer toxins, the resistance to this toxin and the sensitive (S) flocculation capacity (Table 2). The behaviour of the 23 e Degree of flocculation in percentage (%) Saccharomyces strains on the screening media was stud- ied. Most of the strains examined were white on BiGGY agar and only three of them were a dark hazel 4 vinyl guaiacol. These compounds are associated with colour, which is consistent with the data obtained from unpleasant odours in wine, such as horse sweat, pharmaceu- the fermentation. However, the strains (G14, G15 and tical and varnish odours. As shown in Table 2,mostofthe

G23) that produced a large amount of H2Sduring strains isolated from this ecosystem had these properties to a fermentation did not show consistent results on BIGGY agar, greater or lesser degree, similar to the results obtained by thus both methods could be complementary. Almost all Shinohara et al. (2000). the strains showed a slight halo on the calcium carbon- Only 17 % of the strains produced the killer toxin, but ate agar, and three of them did so in moderation (G02, almost 60 % were resistant to its action. Killer yeast strains G05 and G13), so that these strains may not be suitable were isolated at different sampling points and did not prevail for winemaking. However, G05 showed other character- in any of the fermentations, according to Povhe Jemec et al. istics that are highly prized in winemaking, such as a (2001). Csoma et al. (2010)found5strainsofS. uvarum and good rate of fermentation, and little amount of residual 17 of S. cerevisiae that were sensitive to K2, but only 1 S. sugar in the wine, so this strain was not eliminated by cerevisiae and 1 S. uvarum strain showed the killer activity, this test. The presence of polyphenoloxidase (cinnamate indicating that killer strains may be rare in the natural yeast decarboxylase) in wine yeasts decarboxylated phenolic populations studied. Also, killer factor toxins are usually acids and produced volatile phenols such as 4-ethyl and found to be inactive in wines because of their pH level n irbo 21)63:371 (2013) Microbiol Ann

Table 3 Principal enological parameters at the end of fermentations of Chardonnay and Cabernet Sauvignon wines elaborated with the most successful preselected yeasts (G01, G04, G05 and G11) – 379

Chardonnay Cabernet Sauvignon

G01 G04 G05 G11 G01 G04 G05 G11

Alcohol degree %v/v 13.73 ± 0.08 13.74 ± 0.10 13.70 ± 0.10 13.73 ± 0.07 13.10 ± 0.03 a 13.13 ± 0.05 a 12.90 ± 0.08 b 13.08 ± 0.07 a pH 3.37 ± 0.21 3.24 ± 0.04 3.31 ± 0.07 3.26 ± 0.04 3.33 ± 0.18 3.37 ± 0.11 3.25 ± 0.18 3.36 ± 0.08 Volatile acidity g/l AcH 0.25 ± 0.07 0.25 ± 0.03 0.27 ± 0.06 0.36 ± 0.08 0.12 ± 0.04 0.15 ± 0.03 0.07 ± 0.06 0.15 ± 0.01

Total acidity g/l TH2 7.26 ± 0.88 6.78 ± 0.41 6.25 ± 0.51 7.05 ± 0.50 9.06 ± 1.14 8.56 ± 0.93 10.09 ± 1.07 9.02 ± 0.79 Tartaric acid g/l 3.49 ± 0.35 3.52 ± 0.21 3.38 ± 0.34 3.46 ± 0.22 3.50 ± 0.35 3.34 ± 0.37 3.72 ± 0.38 3.48 ± 0.49 Malic acid g/l 2.04 ± 0.14 2.01 ± 0.17 1.85 ± 0.07 2.05 ± 0.03 3.06 ± 0.55 2.93 ± 0.41 3.36 ± 0.34 3.04 ± 0.21 Lactic acid g/l 0.31 ± 0.01 a 0.28 ± 0.04 a 0.28 ± 0.04 a 0.45 ± 0.03 b 0.86 ± 0.12 0.90 ± 0.27 1.33 ± 0.15 1.10 ± 0.18 Reducing sugar g/l 1.37 ± 0.16 a 1.22 ± 0.06 a 3.36 ± 0.20 b 1.66 ± 0.23 a 1.63 ± 0.25 1.77 ± 0.34 1.78 ± 0.25 1.20 ± 0.59 Glycerol g/l 5.93 ± 0.21 b 5.42 ± 0.13 ab 4.94 ± 0.37 a 6.93 ± 0.44 c 9.84 ± 0.19 a 9.04 ± 0.22 b 9.98 ± 0.26 a 9.93 ± 0.38 a Colour intensity –– – – 29.9 ± 1.3 29.0 ± 1.3 29.2 ± 2.7 29.6 ± 2.1 Tonality –– – – 0.41 ± 0.04 0.40 ± 0.04 0.44 ± 0.06 0.41 ± 0.02 Methanol mg/l 3.81 ± 0.40 a 4.40 ± 0.27 a 4.94 ± 0.69 a 2.10 ± 0.11 b 4.34 ± 0.47 a 6.71 ± 0.95 b 4.07 ± 0.16 a 6.70 ± 0.66 b 1-Propanol mg/l 2.81 ± 0.40 a 6.33 ± 0.79 b 5.59 ± 0.59 b 2.64 ± 0.18 a 2.70 ± 0.38 a 4.58 ± 0.54 b 2.72 ± 0.24 a 4.93 ± 0.55 b Isobutanol mg/l 1.80 ± 0.31 a 2.88 ± 0.27 b 3.69 ± 0.34 c 1.34 ± 0.18 a 2.46 ± 0.35 a 4.89 ± 0.41 b 2.78 ± 0.25 a 5.52 ± 0.57 b Ethyl acetate mg/l 3.82 ± 0.34 a 7.49 ± 1.07 c 5.84 ± 0.54 bc 4.94 ± 0.20 ab 2.10 ± 0.11 a 3.33 ± 0.21 b 1.08 ± 0.31 c 4.13 ± 0.08 d 2-methyl-1butanol mg/l 3.46 ± 0.49 a 9.18 ± 0.81 b 7.51 ± 0.90 b 2.60 ± 0.28 a 14.79 ± 1.67 a 38.87 ± 4.09 b 18.26 ± 1.87 a 55.90 ± 5.15 c 3-methyl-1butanol mg/l 6.24 ± 0.88 a 21.46 ± 1.33 b 20.22 ± 1.14 b 5.28 ± 0.61 a 18.68 ± 2.64 a 50.01 ± 3.62 b 23.70 ± 4.77 a 70.86 ± 6.03 c

All values are the mean±SD of duplicate fermentations. Values not sharing the same letter are significantly different (ANOVA and Duncan’s test for α00.05). – Not detected 377 378 Ann Microbiol (2013) 63:371–379

(between pH 3–3.5). In fact, strains that are sensitive to the followed by G04. Significant differences in 3-methyl-1-buta- killer phenotype may dominate alcoholic fermentations, con- nol and 2-methyl-1-butanol were found in the same samples firming that this is not a prerequisite for selecting a strain of G05 and G04 with the other two, which means that the yeast (Mas et al. 2002). biosynthesis of both higher alcohols showed a linear relation- The flocculation ability of yeast strains is an inter- ship (Wondra and Berovi 2001). esting property for the production of sparkling wines, For red wines, the degree of alcohol was very similar in but it is not desirable in table wine; it would result in all cases, ranging from 12.9 for G05 to 13.1 for the rest, the as little homogeneous fermentation because ferment in pH was around pH 3.3 in all cases and the values of the base deposits. All isolates had results above 90 %, methanol, volatile acidity and ethyl acetate were very low meaning that they remained in suspension and, there- in all samples, although samples G04 and G11 showed fore, were not all flocculent strains. significantly higher values of methanol and ethyl acetate On the basis of these results, we preselected four compared to G01 and G05. No significant differences were strains: G01, G04, G05 and G11, for which the princi- found for tartaric, malic or lactic acid. The glycerol content pal characteristics were: fast implantation in must, the in G04 was significantly lower than in the rest. All the low production of H2S or foam, and a low concentra- strains studied completely finished fermentation, leaving tion of residual sugars (<5 g/l) in the wines produced the wines with less than 2 g/l of residual sugar. Sample by these strains at 15 and 25 °C. G11 showed the highest values for all higher alcohols tested, closely followed by G04. Winemaking trials with the selected strains in natural must The finished wines were subjected to a sensory analysis by expert tasters, and the results revealed that the most Using the four selected strains that showed the best suitable strain for use as a starter culture in the fermentation enological properties (G01, G04, G05 and G11), micro- of musts of the A.O. was the G04 strain for red wines and vinifications of Chardonnay (22.6° Brix, pH 3.31 and Chardonnay partially barrel-fermented, but G01 would be total acidity 7.66 g/l) and Cabernet Sauvignon (22.4° suitable for young white wines. Brix, pH 3.44 and total acidity 9.15 g/l) musts were The following year, these two selected strains were used performed. The resulting wines were analysed to deter- as starter cultures for industrial fermentations at 1×106 mine their physico-chemical parameters, higher alcohols cells/ml of each strain for Chardonnay (G01) and (1-propanol, isobutanol, 3-methyl-1-butanol and 2- Cabernet Sauvignon (G04) musts in the Sanchez methyl-1-butanol), methanol and ethyl acetate contents. Muliterno winery. Samples were taken at 72 h of inoc- TheresultsareshowninTable3. The white wines ulation, and 20 independent isolates, obtained as colo- contained a very similar degree of alcohol, 13.7°, pH nies on YPD agar, were analysed for their mtDNA 3.3, and high levels of total acidity, whereas a higher restriction patterns using endo-nuclease Hinf I. When acidity is beneficial. The only significant differences the fermentation was performed using the autochthonous were found for lactic acid and glycerol, for which G11 strain G04, a 95 % recovery of the inoculated strain showed higher values, and G05 left more residual sugar was observed in red wine, and 90 % of G01 was in the wine than the others. The concentrations of ethyl observed in white wine. These selected strains are cur- acetate and methanol were not high in any of the cases. rently being used as starter cultures in the fermentation Higher alcohol contents are mainly produced by yeasts of wines from this cellar. during alcoholic fermentation. These alcohols, together with their esters, play an important role in the aroma of wine. A Acknowledgments The present work was financed by the project low alcohol concentration contributes to the aromatic com- 2004 COB-73 from the Sanchez Muliterno winery. Mónica Fernández González wants to thank the Albacete Science & Technology Park for plexity of wine, whereas high concentrations mask the aro- the award of an INCRECYT contract. ma of wine aroma due to their penetrating odours (Ribereau- Gayon et al. 2000). The highest concentration of propanol, 6.3 mg/l, was found in sample G04, and the lowest, References 2.6 mg/l, in sample G01. 2-Methyl-1-butanol, 3-methyl- 1-butanol and isobutanol are produced by yeast from Briones A, Úbeda J, Grando S (1996) Differentiation of Saccharomyces their respective amino acid precursors (isoleucine, leucine cerevisiae strains isolated from fermenting must according to their and valine, respectively) via their corresponding ketoacids karyotype patterns. Int J Food Microbiol 28:369–377 (Fleet and Heard 1993).Theproductionofsuchalcoholsis Briones AI, Ubeda JF, Cabezudo MD, Martin-Alvarez P (1995) dependent on the cellular growth and level of dissolved oxy- Selection of spontaneous strains of Saccharomyces cerevisiae as starters in their viticultural area. In: Charalambous G (ed) Food gen in the medium. The concentration of isobutanol was flavours: generation, analysis and process influence. Elsevier, highest in the Chardonnay wines inoculated with G05, Amsterdam, pp 1597–1622 Ann Microbiol (2013) 63:371–379 379

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