Evolution of the population of Saccharomyces cerevisiae from grape to wine in a spontaneous fermentation

ARTICLE IN PRESS FOOD MICROBIOLOGY Food Microbiology 23 (2006) 709–716 www.elsevier.com/locate/fm

Evolution of the population of Saccharomyces cerevisiae from grape to wine in a spontaneous fermentation Christine Le Jeune, Claude Erny, Catherine Demuyter, Marc Lollier Laboratoire Vigne Biotechnologie et Environnement de l’Universite´ de Haute-Alsace, 33 rue de Herrlisheim, 68000 Colmar, France Received 22 August 2005; received in revised form 21 January 2006; accepted 13 February 2006 Available online 17 April 2006

Abstract To determine the grape or winery origin of the Saccharomyces cerevisiae involved in spontaneous fermentation, musts were collected at different stages of wine-making process and fermented. First, grapes were collected in two different vineyards and crushed at the laboratory. Second, musts were collected after crushing and clarification in the cellar. Third, musts collected in the cellar were sterilized and inoculated with tartar deposit collected in the vats. The fourth fermentation was in the cellar. For the two vineyards, two hundred of S. cerevisiae clones were isolated for each of the four fermentations, driving to a library of 1600 clones. All the library was analysed by inter-d PCR with a basic set of primers and about 20% of the library was further analysed by inter-d PCR with an improved set of primers. Six, and more than 30 different PCR patterns were obtained from basic- and improved-PCR analysis, respectively. The amounts of each family were analysed at the different stages of wine making. Our study demonstrates that the two vineyards present different S. cerevisiae populations. Moreover the S. cerevisiae strains involved in spontaneous fermentation in the cellar originate partly from the vineyard and partly from the winery, in amounts varying with the must. r 2006 Elsevier Ltd. All rights reserved. Keywords: S. cerevisiae; Wine; Spontaneous fermentation; d PCR

1. Introduction The fermentation of grape must into wine is a complex process, involving bacteria and other micro-organism species, especially yeasts. Indigenous yeasts belonging to the genera Kloeckera, Hanseniaspora, Candida and Pichia grow in the early stages of fermentation but are soon replaced by the more ethanol-tolerant Saccharomyces cerevisiae species, which complete the fermentation process. In the past 30 years, strains of S. cerevisiae have been selected for their enological properties and are used as starters in wine-making process to ensure the success of alcoholic fermentation. Yet the strains of S. cerevisiae involved in fermentation play an important part in the characteristics of the final product (Pe´rez-Coello et al., 1999) and the diversity of S. cerevisiae strains present in spontaneous fermentation contribute to the chemical Corresponding author. Tel.: +33 3 89 20 31 36.

E-mail address: [email protected] (C. Le Jeune). 0740-0020/$ - see front matter r 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.fm.2006.02.007

composition and sensory qualities of the resulting wine (Lurton et al., 1995). Several studies support the hypothesis that active dried yeasts reduce the variability of strains that appear in spontaneous fermentation (Frezier and Dubourdieu, 1991; Beltran et al., 2002) and, possibly, the complexity of resulting wine. For these reasons, winemakers looking for original flavours prefer spontaneous fermentation with indigenous yeasts. In this case, indigenous yeast may come from the vineyard itself or from the winery equipment. The question of the origin of indigenous yeasts is of some importance because this could influence the cultural and/or enological practices of the wine-makers. Now, there is still a lack of agreement concerning the relative contribution of S. cerevisiae originating from the vineyard compared to that originating from the winery. On the one hand, spontaneous alcoholic fermentation is possible in sterilized vessels (Lopes et al., 2002) or in a newly built winery where S. cerevisiae has never been introduced (Beltran et al., 2002). On the other hand, though it has been found on damaged berries (Mortimer

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and Polsinelli, 1999), S. cerevisiae is extremely rare on grape and vineyard (Martini, 1993; Pretorius, 2000; Sabate et al., 2002) while it can be found colonising the surface of the winery equipment (Vaughan-Martini and Martini, 1995; Sabate et al., 2002; Sangorrin et al., 2002; Beltran et al., 2002). Some commercial or indigenous strains are even found in the winery over several years and are therefore called resident strains (Rosini, 1984; Frezier and Dubourdieu, 1992; Beltran et al., 2002). The use of molecular biology methods allows the rapid and precise identification of yeasts at the species or strain level. These methods include karyotyping by pulsed field gel electrophoresis (Frezier and Dubourdieu, 1991; Izquierdo Canas et al., 1997; Povhe Jemec et al., 2001), RFLP analysis on mitochondrial DNA (Vezinhet et al., 1990; Querol et al., 1994; Sabate et al., 1998; Pramateftaki et al., 2000; Torija et al., 2001; Lopes et al., 2002; Beltran et al., 2002) or on amplified ribosomal genes (Sabate et al., 2002). Alternatively, PCR amplification of S. cerevisiae sequences such as rDNA internal transcribed spacer region (Granchi et al., 1999) or d sequences (Ness et al., 1993; Versavaud et al., 1995; Lopes et al., 2002) was also used. Some times ago, karyotyping was considered to be the reference method. Recently, PCR amplification of d sequences has been improved (Legras and Karst, 2003), and yet can be considered as discriminant as karyotyping or mitochondrial DNA restriction analysis (Schuller et al., 2004). Molecular biology methods have been used to study the S. cerevisiae populations in a given environment (grape or winery) or the dynamic of S. cerevisiae population during the fermentation process. But few studies (Demuyter et al., 2004; Ciani et al., 2004) have focused on the evolution of the S. cerevisiae population from grape to wine. In this study we analyse the populations of S. cerevisiae present on grapes in two different vineyards of the same domain and the evolution of these populations at different stages of wine-making process. S. cerevisiae were characterized by PCR amplification of the d sequences, with a basic set of primers in a first analysis and with an improved set of primers as a confirmation. 2. Materials and methods 2.1. Grape sampling and wine fermentations The two vineyards concerned by this study, called Hengst and Windsbuhl (H and W, respectively), belong to the winery Zind-Humbrecht. Located in central Alsace, near Colmar, they are distant from 15 km one of the other. Each vineyard covers a surface of about 1 ha and is planted with Vitis vinifera variety Gewurztraminer. H vineyard is composed of four different plots of land from various ages while W vineyard is made of one unique homogeneous plot. For each vineyard, four different fermentations were carried out. In the first one, 12 kg of grapes were collected aseptically in the vineyard, the very day of the harvest (the

20th and the 23rd of October 1997, for H and W, respectively), crushed in the laboratory in a sterilized vertical minipress and clarified overnight at low temperature. The 6 l of resulting juice were incubated in a sterilized glass vessel. In the second fermentation, 6 l of must were aseptically collected after crushing and clarification in the cellar, and fermented at the laboratory in sterilized vessels. In the third fermentation, 1 l of the must collected in the cellar was sterilized by autoclaving and inoculated with tartar deposit collected in the vat before it was filled with must. To collect tartar deposit, the operator entered the vat and scraped the entire inner surface with three sterilized gauze compresses which were then introduced in 5 ml sterile water. After vigorous shaking the sterilized must was inoculated with the resulting liquid. In all three fermentations, musts were supplemented with sulphur dioxide (50 mg l
1), and incubated at 20 1C under anaerobic conditions to the end of fermentation. The fourth fermentation was in the cellar. Temperature of the cellar was about 13 1C, once in the vat the must was not warmed but after the fermentation begun the temperature was maintained between 18 and 22 1C. The evolution of alcoholic fermentation was followed up, in the laboratory, by measuring the loss of weight resulting from CO2 release and, in the cellar, by measuring the must density with a mustimeter. 2.1.1. Isolation of yeasts of enological interest Samples were taken at the beginning (3% (w/w) loss of weight) and at the end (stabilization of the weight loss or of the density loss) of the four fermentations and diluted in sterile water. 0.2 ml of several dilutions were spread onto plates of YPG (Yeast extract 10 g l
1, bactopeptone 20 g l
1, glucose 20 g l
1, agar 15 g l
1) supplemented with biphenyl (15 mg l
1). Plates were incubated at 18 1C for 48 h. For each sample, 100 independent colonies were randomly chosen, resuspended in 1 ml of YPG and frozen at
80 1C after addition of glycerol at 15% final concentration. 2.1.2. Extraction of total DNA from yeasts and PCR amplification After preculture on YPG plates, yeast cultures were grown at 20 1C for 48 h in 2 ml of YPG. Samples of 1.5 ml were centrifuged (5 min, 5000 rpm, 4 1C) and the resulting pellets were resuspended in 1 ml sterile distilled water. The pellets resulting from a second centrifugation (5 min, 5000 rpm, 4 1C) were resuspended in 300 ml extraction buffer (Tris–HCl 10 mM pH 8, EDTA 1 mM, NaCl 100 mM, Triton X100 2%, SDS 1%, pH 8). The mixtures were shaken 3 min with 250 ml small glass beads (diameter 0.5 mm) and 300 ml phenol–chloroform–isoamylic alcohol (25:24:1). After 5 min centrifugation at 12,000 rpm, 250 ml of the upper phases were collected and added to 500 ml cold ethanol for DNA precipitation. After centrifugation (10 min, 12,000 rpm, 4 1C), the pellets of DNA were washed

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with 1 ml ethanol 70%, dried, resuspended in 50 ml sterile water and added with 150 U ml
1 ribonuclease A. Primers used for PCR amplification were specific from the S. cerevisiae species d sequences. Primers d1 (50 CAAAATTCACCTATA/TTCTCA30 ) and d2 (50 GTGGATTTTTATTCCAACA30 ) (Ness et al., 1993) were used for the so-called ‘‘basic’’ d amplification, primers d12 (50 -TCAACAATGGAATCCCAAC-30 ) (Legras and Karst, 2003) and d2 for ‘‘improved’’ d amplification. The PCR amplification was performed with a Perkin Elmer GenAmp PCR System 2400 (Applied Biosystems, Foster City, CA, USA). Each PCR mixture contained 1 unit Taq polymerase (Invitrogen), 5 ml Taq polymerase 10X buffer, 200 mM each dNTP, 1 mM each primer and 5 ml extracted DNA (corresponding to 0.4–2 mg DNA) in a total volume of 50 ml. Amplification was performed for 30 cycles under the following conditions: after 4 min of initial denaturation at 97 1C, each cycle consisted of 30 s denaturation at 94 1C, 1 min primer annealing at 45 1C and 2 min primer extension at 72 1C, followed by a 10-min final extension step at 72 1C. PCR products were analysed by electrophoresis in 1.2% (w/v) agarose gel in 1X TBE buffer (0.09 M Tris–borate, 0.002 M EDTA) and detected, after ethidium bromide staining, over a short UV light source. 3. Results 3.1. Genetic characterization of 1600 S. cerevisiae isolates by basic PCR d amplification For each vineyard, a library of 800 isolates was made up, i.e. one hundred both at the beginning and at the end of four fermentations. The four modalities differed by the origin of yeasts involved in the fermentation: in the first modality, yeasts could only have originated from grapes, in the second one they could have come either from grapes, from harvest equipment or from press, in the third modality collected yeasts were present in tanks before their filling and in the fourth modality, yeasts could have originated from each step of the traditional wine-making process, from the vineyard to the winery. The whole collection of 1600 clones had been genetically characterized by basic PCR amplification of the d interspersed sequences, highly specific for the identification of S. cerevisiae strains amongst other yeasts (Ness et al., 1993). PCR amplification was successfully obtained for 95% of the 1600 yeast isolates. Six different amplification patterns were observed (Fig. 1) and clones showing the same profiles in basic PCR had been clustered in six different families called C1 to C6. The 5% of clones that could not be characterized by inter-d PCR did not belong to the S. cerevisiae species. Because our goal was to analyse the evolution of S. cerevisiae population from grape to wine these clones were not further analysed, but in other experiments made in 1998 in the same winery we found that non-S. cerevisiae strains isolated in similar conditions did all belong to the S. uvarum species.

Fig. 1. Basic d-amplification patterns of the six families of Saccharomyces cerevisiae. M: 1 Kbp DNA ladder, lanes 1–6: basic PCR-patterns observed for clones belonging to the S. cerevisiae families C1, C2, C3, C4, C5 and C6.

3.2. Further characterization of 298 S. cerevisiae isolates by improved PCR d amplification When analysed by pulsed field gel electrophoresis, some clones from the family C5 presented different patterns (data not shown), suggesting that the families determined by basic PCR d amplification could be heterogeneous. In order to investigate the homogeneity of these families, 298 clones, representing 11%, 27% and 26% of the families C1, C5 and C6 respectively, were further characterized by improved PCR d amplification with the primers described by Legras and Karst (2003). The clones were chosen at the three stages of wine making, both at the beginning and at the end of fermentation. Amplifications with the improved primers generated more polymorphic patterns than amplification with the basic primers. We verified that the template amount had no effect on the resulting pattern in the range of amounts that we used (0.4–2 mg) unlike elsewhere described (Fernandez-Espinar et al., 2001). According to their improved PCR patterns the clones were grouped into sub-families. There were common features between the different patterns obtained from a given family (Fig. 2), and we never found clones sharing a common

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together 65% of the tested clones (Fig. 3). Finally, when tested with improved primers, the 50 clones of the C6 family presented seven different profiles called C6-1 to C67. Despite this variability the family C6 was considered homogeneous since 86% of the clones exhibited the C6-1 dominant profile while the six other profiles were observed only once or twice (Fig. 3). Because improved d PCR amplification is as discriminant as karyotyping we consider that all the clones sharing the same improved d PCR amplification pattern belong to the same strain. The strains were named according to the improved d PCR amplification patterns, e.g. C1-1, C5-8y.

improved PCR pattern while exhibiting different basic PCR patterns. When tested with improved primers, the 64 clones of the family C1 presented two different profiles called C1-1 and C1-2 (Fig. 2, lanes 1 and 2), representing 22% and 78% of the population, respectively (Fig. 3). The 184 clones of the family C5 presented as much as 26 different profiles called C5-1 to C5-26. Among them, 17 patterns were observed only once or twice and were therefore defined as rare patterns. The four profiles C5-1, C5-8, C5-14 and C5-15 were dominant, and represented

3.3. Biodiversity of S. cerevisiae of enological interest on the vineyards On the grapes, the two populations of S. cerevisiae of enological interest were different. In vineyard H, S. cerevisiae population was quite homogeneous since 99% of the clones belonged to the C5 family (Fig. 4, panel A). When tested by improved PCR the C5 clones presented only two different patterns, namely C5-1 and C5-2 (Table 1). Hence, the population of S. cerevisiae on vineyard H was made of three strains, C5-1, C5-2 and C6 representing 89%, 10% and 1% of the clones, respectively. The population was more heterogeneous in vineyard W where the five families C1 to C5 were found (Fig. 4, panel B). Moreover, while the C1 clones did all belong to the C1-1 strain, the C5 clones corresponded to seven different

Fig. 2. Improved d-amplification patterns of some strains of Saccharomyces cerevisiae. M: 1 Kbp DNA ladder. Lanes 1–10: improved PCR patterns observed for strains C1-1 and C1-2 (lanes 1 and 2) and for strains C5-1, C5-2, C5-7, C5-8, C5-14, C5-15, C5-21 and C5-10 (lanes 3–10).

40% C5 family 30% 20% 10%

100%

C5-26

C5-25

C5-24

C5-23

C5-22

C5-21

C5-20

C5-19

C5-18

C5-17

C5-16

C5-15

C5-14

C5-13

C5-12

C5-11

C5-9

C5-10

C5-8

C5-7

C5-6

C5-5

C5-4

C5-3

C5-2

C5-1

0%

100% C1 family

C6 family

80%

80%

60%

60%

40%

40%

20%

20% 0%

0% C1-1

C1-2

C6-1

C6-2

C6-3

C6-4

C6-5

C6-6

C6-7

Fig. 3. Distribution of the strains (C1-1 to C1-2, C5-1 to C5-26 and C6-1 to C6-7) into the C1, C5 and C6 families.

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Hengst 100%

100% G

P

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C2

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C6

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C5

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C4

C5

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C4

C5

C6

C4

C5

C6

100%

100% Tr

Tk

80%

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20%

0% (A)

C3

0% C1

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C4

C5

C6

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C2

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Windsbuhl 100%

100% G

P

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C2

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100% Tk

Tr 80%

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40%

20%

20%

0% (B)

C3

0% C1

C2

C3

C4

C5

C6

C1

C2

C3

Fig. 4. Composition of the populations of Saccharomyces cerevisiae found at various stages of wine making, for vineyards H (panel A) and W (panel B). Distribution of the families C1 to C6 was analysed at the beginning (grey) and at the end (black) of alcoholic fermentations conducted either at the laboratory, from grape juice (G), must crushed in the cellar (P) or sterilized must inoculated with tartar deposit (Tr), either in the cellar (Tk).

strains namely C5-1, C5-10, and C5-12 to C5-16 (Table 2). Hence, at least 11 different strains were found on vineyard W. This variability is maybe even under-evaluated since the families C2, C3 and C4 have not been tested by the improved d PCR method. The only strain found on both vineyards was C5-1 which was dominant on vineyard H but only minor on vineyard W.

3.4. Evolution of the S. cerevisiae populations during wine making In order to know if new strains of S. cerevisiae were introduced in the musts during the wine-making process, we studied the composition of the S. cerevisiae populations in press, tartar deposit and final fermentation. We made the first analysis by basic PCR d amplification and

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Table 1 Evolution of the composition of the S. cerevisiae C5 family from vineyard Hengst to wine C5-strain

Number of clones found in Grape

C5-1 C5-2 C5-3 C5-4 C5-5 C5-6 C5-7 C5-8 C5-9 C5-10 C5-11

18 2

Total

20

Press

Tank

2 3 1 3 1 1 7 2

5 2 1 2

20

20

1 4 3 1 1

Table 2 Evolution of the composition of S. cerevisiae C5 family from vineyard Windsbuhl to wine C5strain

Number of clones found in Grape

Press

C5-1 C5-10 C5-12 C5-13 C5-14 C5-15 C5-16 C5-2 C5-8 C5-17 C5-18 C5-19 C5-4 C5-7 C5-20 C5-21 C5-22 C5-23 C5-24 C5-25 C5-26

6 2 2 4 12 17 1

19 1

9 1

1 5 2

3

Total

44

1 9 1 1 1

Tank

3 8

3 2 1 1 1 2 3 1 1 41

39

tried to gain more information with improved PCR d amplification. 3.5. Evolution of S. cerevisiae population from vineyard Hengst (H) The S. cerevisiae population isolated after crushing and clarification consisted in six families called C1 to C6 (Fig. 4A, block P). The C1 to C4 families, which were not

found on grapes (Fig. 4A, block G), were minor families while C5 and C6, already present on grapes, were predominant. The tartar deposit was collected in the vat before its filling and incubated in presterilized must. Ninty-seven percent of the clones isolated from this fermentation belonged to the C1 family, the other few clones belonged to the C2 and C6 families (Fig. 4A, block Tr). Finally, the population collected in the tank contained clones from the six families C1 to C6 (Fig. 4A, block Tk) and was quite similar to the population found after the press (Fig. 4A, block P). So S. cerevisiae population found in the tank seemed to be made of yeasts families originating from vineyard, e.g. C5, of yeasts introduced between vineyard and must such as C2, C3, C4 and C6 families and of yeasts inoculated from tartar deposit i.e. C1. Improved PCR d amplification was used to validate these results. Thirty-five clones belonging to C1 family were tested by improved PCR d amplification and they were all C1-2 strain. The 60 clones of C5 family distribute among 11 different strains. The C5 population evolved, and included from two to nine different strains according to the process stage (Table 1). The six strains C5-3 to C5-8 appeared between the vineyard and the must while the three strains C5-9 to C5-11 appeared in the tank. These last strains were not detected in the tartar deposit before tank’s filling. Both basic and improved PCR d amplification show that the S. cerevisiae population found in the tank was made of yeasts already present on grapes, but also of yeasts introduced between vineyard and press, of yeasts inoculated from the tartar deposit and of strains appeared directly in the tank. 3.6. Evolution of S. cerevisiae population from vineyard Windsbuhl (W) The S. cerevisiae population isolated after crushing and clarification contained yeasts from the six families C1 to C6 (Fig. 4B, block P). The main differences with the population present on grapes (Fig. 4B, block G) were the apparition of C6 family and the quasi-extinction of C1 family. Before the filling of the tank, 98% of the S. cerevisiae clones isolated from the tartar deposit were of the C1 family (Fig. 4B, block Tr). Finally, the must collected in the tank contained clones from the families C1, C2, C3 and C5 already present on grapes, and C6 which looked like being introduced between vineyard and press. C1 could also originate from tartar deposit. Twenty-nine clones from the C1 family were tested by improved PCR d amplification. The 15 C1-clones that were collected on grapes were all C1-1 strain, while the 14 C1-clones collected in tartar deposit or in tank were all C1-2 strain. This indicates that the C1 yeasts found in the tank in the final fermentation were inoculated from tartar deposit. Forty clones from the C5 family were also tested. The results (Table 2) show that five strains, C5-2, C5-8 and

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C5-17 to C5-19 appeared between the vineyard and the must. Furthermore, nine new strains were found in tank. Two of these strains, C5-4 and C5-7, were found on the grapes and tank of vineyard H (Table 1) while the seven strains C5-20 to C5-26 were not found elsewhere. To conclude, and as in vineyard H, some of the strains present in W tank originated from grapes and some strains were introduced during the wine-making process, between vineyard and press or from tartar deposit. Finally some strains appeared in the tank. 4. Discussion In this study we have demonstrated that the S. cerevisiae involved in a spontaneous fermentation can be an addition of cells originating from the vineyard and of cells originating from the cellar. 4.1. Comparison of the basic and improved PCR d amplifications In our study, improved PCR d amplification was more discriminative than basic PCR d amplification, as described elsewhere (Legras and Karst, 2003; Schuller et al., 2004). Especially the family C5 was found to be very heterogeneous, whereas families C1 and C6 were much more homogeneous. Nevertheless, the clustering determined with basic PCR d amplification remained consistent. Improved PCR amplification confirmed the results provided by basic PCR amplification and provided better insights into the biodiversity of the populations. Moreover, it allowed us to prove that the C1 yeasts found in the tank of must W originated from tartar deposit rather than from grape. It also revealed that new strains that were not detected on grapes nor press nor tartar deposit appeared in tanks. Finally, improved PCR showed that the populations found in the W and H tanks were not so similar as suggested by basic PCR d amplification.

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transport to the cellar. Even if the first hypothesis is supported by the existence of S. cerevisiae strains associated with the press (Sangorrin et al., 2002) we suppose that the strains were rather introduced during the grape harvest, mainly because they vary with the vineyard. The tartar deposit of both tanks contained C1-2 S. cerevisiae strain, but the involvement of this strain in spontaneous fermentation varied with the must. Indeed, this family became dominant in the fermentation of must W, but not in the fermentation of must H. The presence of resident strains associated with tanks has already been described (Sabate et al., 2002; Beltran et al., 2002; Sangorrin et al., 2002). When these strains are starter strains used in the previous years, they tend to dominate indigenous strains (Rosini, 1984; Frezier and Dubourdieu, 1991; Vaughan-Martini and Martini, 1995; Beltran et al., 2002). In our study, strain C1-2 is not a selected starter strain, but an indigenous strain. Therefore, its part in fermentation probably depends on the equilibrium of S. cerevisiae population and on the enological properties of the various families. The S. cerevisiae populations found in the tanks containing musts from H and W vineyards consisted in at least 14 and 18 strains respectively. The heterogeneity of these populations is probably under-estimated since although we analysed by improved PCR only 10% and 20% of the collected clones for vineyard H and W, respectively, we observed a lot of rare patterns. In each case, population included one or few dominant clones and a number of rare profiles. These results are consistent with the observations of several authors (Nadal et al., 1996; Sabate et al., 1998; Guttierez et al., 1999; Sangorrin et al., 2002). Studies on the dynamic of fermenting populations have often described a sequential replacement of strains. Because of the heterogeneity of the family C5 and possibly of the families C2 to C4 and of the relatively restricted number of clones tested by improved PCR d amplification, we are not able to discuss this aspect of spontaneous fermentation. 4.3. Biodiversity of S. cerevisiae in the vineyard

4.2. Evolution of the population of S. cerevisiae during the wine-making process In this study, the dominant strains involved in spontaneous fermentations in the cellar seem to originate from the winery (press and tartar deposit) rather than from grapes. Nevertheless, some strains originating from the vineyard were still present and the two musts turned to be different one from the other, suggesting an involvement of the initial population in the composition of the final population. For both H and W wines, new families of S. cerevisiae were introduced in the must at the very first stages of wine making, before or during crushing and clarification. The introduction of new strains at these stages of process was already observed in another study (Demuyter et al., 2004), with the S. cerevisiae as well as with the S. uvarum species. The newly introduced cells could have been inoculated in the cellar during crushing, or during grape harvest and

We were able to detect three and at least 11 different families on the grapes of vineyards H and W, respectively. The dominant family from vineyard H (C5-1) was also found in vineyard W and even in other alsatian vineyards and therefore could be considered as a ‘‘regional strain’’ (Pramateftaki et al., 2000; Beltran et al., 2002; Versavaud et al., 1995). The biodiversity observed on vineyard W are consistent with the few studies allowing an estimation of S. cerevisiae biodiversity on the vineyard (Povhe Jemec et al., 2001; Beltran et al., 2002; Ciani et al., 2004). By contrast, and in spite of the heterogeneity of this vineyard, the S. cerevisiae population of vineyard H seems surprisingly homogeneous. Hence, the variable biodiversity of the two vineyards do not seem to be related to the age of grapevine but perhaps to local pedological or climatic variations. In this study, we have analysed the populations of S. cerevisiae of enological properties throughout the

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wine-making process, from grapes to fermentation in the cellar. We were able to demonstrate that, in a winery where selected enological yeasts had never been used, S. cerevisiae dominating the fermentations were partly originating from grapes and partly introduced during the wine-making process, in the early stages or in the vat. Moreover, though the two musts were processed in the same winery, the dominant families involved in their fermentations were not the same. This result refutes the affirmation that S. cerevisiae can be found only in the winery environment (Martini, 1993) or that winery yeasts dominate indigenous yeasts. There is an opposition between our conclusion and that of Ciani et al. (2004) who observed that wineryassociated strains dominated grape-originating strains; however the populations they analysed were far more homogeneous than the ones we obtained. In our case, the populations involved in spontaneous fermentation results from a subtle equilibrium between vineyard and winery strains. Our study has only focused on the presence of the strains during fermentation, yet the part of each strain in the properties of the wine produced has still to be investigated.

Acknowledgements The authors wish to thank Mr Olivier Humbrecht for facilities during the sampling in his winery in Turckheim (Alsace, France).

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