• Yeast;
  • Saccharomyces cerevisiae;
  • Commercial yeast strains;
  • Mitochondrial DNA RFLP;
  • Spontaneous fermentation;
  • Vineyard


  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results
  6. 4Discussion
  7. Acknowledgements
  8. References

One thousand six hundred and twenty yeast isolates were obtained from 54 spontaneous fermentations performed from grapes collected in 18 sampling sites of three vineyards (Vinho Verde Wine Region in northwest Portugal) during the 2001–2003 harvest seasons. All isolates were analyzed by mitochondrial DNA restriction fragment length polymorphism (mtDNA RFLP) and a pattern profile was verified for each isolate, resulting in a total of 297 different profiles, that all belonged to the species Saccharomyces cerevisiae. The strains corresponding to seventeen profiles showed a wider temporal and geographical distribution, being characterized by a generalized pattern of sporadic presence, absence and reappearance. One strain (ACP10) showed a more regional distribution with a perennial behavior. In different fermentations ACP10 was either dominant or not, showing that the final outcome of fermentation was dependent on the specific composition of the yeast community in the must. Few of the grape samples collected before harvest initiated a spontaneous fermentation, compared to the samples collected after harvest, in a time frame of about 2 weeks. The associated strains were also much more diversified: 267 patterns among 1260 isolates compared to 30 patterns among 360 isolates in the post- and pre-harvest samples, respectively. Fermenting yeast populations have never been characterized before in this region and the present work reports the presence of commercial yeast strains used by the wineries. The present study aims at the development of strategies for the preservation of biodiversity and genetic resources as a basis for further strain development.


  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results
  6. 4Discussion
  7. Acknowledgements
  8. References

Traditionally, wine fermentation is carried out in a spontaneous way by indigenous yeast either present on the grapes when harvested or introduced from the equipment and cellar during the vinification process. All recent research agrees that the predominant species on healthy grapes are apiculate yeasts like Hanseniaspora uvarum (and its anamorph form Kloeckera apiculata) and oxidative species such as Candida, Pichia, Kluyveromyces and Rhodotorula[1]. Contrarily, fermentative species of the genus Saccharomyces, predominantly Saccharomyces cerevisiae, occur in extremely low number on healthy undamaged berries or in soils [2–4], while damaged grapes are believed to be an important source of S. cerevisiae[5]. The prevalence of strains belonging to this species is well documented among the wineries resident flora [6–10]. The grape's yeast flora depends on a large variety of factors such as climatic conditions including temperature and rainfalls, geographic localization of the vineyard [4,9], antifungal applications[11], grape variety and the vineyard's age [12–14], as well as the soil type[15]. Several ecological surveys, using molecular methods of identification, report a large diversity of genetic patterns among the enological fermentative flora. S. cerevisiae strains seem to be widely distributed in a given viticultural region [16–19], can be found in consecutive years [20,21] and there are also strains predominant in the fermenting flora [2,22], which support the notion that specific native strains can be associated with a terroir.

Selected yeast starters are nowadays widely used since they possess very good fermentative and oenological capabilities, contributing to both standardization of fermentation process and wine quality. In the years following the publication of the S. cerevisiae genome sequence[23], enough evidence was provided showing substantial genetic differences among wine yeast strains [24–26]. Therefore, exploring the biodiversity of indigenous fermentative strains can be an important contribution towards the understanding and selection of strains with specific phenotypes.

The genetic diversity of S. cerevisiae strains has been analyzed by several methods such as karyotyping by pulse field gel electrophoresis[27], mitochondrial DNA restriction analysis (mtDNA RFLP) [28–31], fingerprinting based on repetitive delta sequences [32,33] and microsatellite genotyping [34–36]. Schuller et al.[37] have recently shown that microsatellite typing, using 6 different loci[36], an optimized interdelta sequence analysis[33] and RFLP of mitochondrial DNA generated by the enzyme Hinf I had the same discriminatory power. In the present work mtDNA RFLP analysis using Hinf I was applied as genetic marker for the distinction of S. cerevisiae strains.

The aim of the present work was to assess the biodiversity of the fermenting flora found in vineyards belonging to the Vinho Verde Region in order to define strategies for future wine strain selection programs. Another goal was the establishment of a strain collection contributing to the preservation of S. cerevisiae genetic resources.

2Materials and methods

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results
  6. 4Discussion
  7. Acknowledgements
  8. References


The sampling plan included a total of 18 sites in three vineyards surrounding a winery, located in northwest Portugal (Região Demarcada dos Vinhos Verdes). In each vineyard, six sampling points were defined according to vineyard geography, and the distance between winery and the sampling sites varied between 20 and 400 m, as shown in Fig. 1. Two sampling campaigns were performed before (early stage) and after (late stage) harvest, in a time frame of about 2 weeks, in order to assess the diversity among fermentative yeast communities during the last stage of grape maturation and harvest. This experiment was repeated in three consecutive years (2001–2003). Samples were not always collected from the same rootstock, but from the same area (±1–2 m). The grapevine varieties sampled were Loureiro (vineyard A), Alvarinho (vineyard P) and Avesso (vineyard C), being all white grapes used in the Vinho Verde Region.


Figure 1. Geographic location of the three vineyards A, C and P in the Vinho Verde Wine Region with indication of the wineries and the corresponding sampling sites PI–PVI, AI–AVI and CI–CVI.

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2.2Fermentation and strain isolation

From each sampling point, approximately 2 kg of grapes were aseptically collected and the extracted grape juice was fermented at 20 °C in small volumes (500 ml), with mechanical agitation (20 rpm). Fermentation progress was monitored by daily determinations of the musts mass loss. When a reduction by 70 g/l was observed, corresponding to the consumption of about 2/3 of the sugar content, diluted samples (10−4 and 10−5) were spread on YPD plates (yeast extract, 1% w/v, peptone, 1% w/v, glucose 2% w/v, agar 2%, w/v), and 30 randomly chosen colonies were collected after incubation (2 days, 28 °C). The isolates obtained from all fermentations throughout this work were stored in glycerol (30%, v/v) at −80 °C.

2.3DNA isolation

Yeast cells were cultivated in 1 ml YPD medium (36 h, 28 °C, 160 rpm) and DNA isolation was performed as described[28] with a modified cell lysis procedure, using 25 U of Zymolase (SIGMA). Cell lysis was dependent on the strain and lasted between 20 min and 1 h (37 °C). DNA was used for mitochondrial RFLP.

2.4Mitochondrial DNA RFLP

Restriction reactions were preformed as described[37]. The attributed designations for observed distinct patterns were A1–A93, C1–C62 and P1–P135, corresponding to isolates from vineyard A, C and P, respectively. Pattern designation ACP10 refers to a strain common to all vineyards and C69P77 and C42P80 were assigned to strains common to vineyard C and P. Pattern profiles that are identical to commercial starter yeasts used by the wineries are designated S1–S6. One representative strain of each of the 297 patterns was withdrawn and tested for growth in a medium containing lysine as sole nitrogen source[38].

2.5Analytical methods

Sugar concentration was determined by a previously described dinitrosalicylic method[39].


  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results
  6. 4Discussion
  7. Acknowledgements
  8. References

In the present work, three vineyards, situated in the Vinho Verde Wine Region, in northwest Portugal, were sampled during the 2001–2003 harvest seasons (Fig. 1). In order to obtain a more detailed picture of fermenting yeast temporal distribution, two sampling campaigns were performed, one before and another after the harvest, in a time frame of about two weeks. A total of 108 grape samples have been planned (six sampling points × two sampling campaigns × three vineyards × three years), from which 54 started a spontaneous fermentation, 36 were not able to start fermentation after 30 days of incubation, whereas 18 samples were not collected due to unfavorable weather conditions and a bad sanitation state of the grapes in 2002. From the 54 fermentations 1620 yeast isolates were obtained. All the isolates were analyzed by their mtDNA RFLP (Hinf I) and a pattern profile was attributed to each isolate, resulting in a total of 297 different profiles.

The total yeast count (cfu in YPD medium) ranged between 1.0 × 106 and 8.0 × 107 cfu/ml must, corresponding to values generally described for grape must fermentations. All isolates belonged to the species S. cerevisiae due to their inability to grow in a medium containing lysine as sole nitrogen source and by their capacity to amplify six S. cerevisiae specific microsatellite loci. None or only 1 of the loci was amplified in other Saccharomyces species occurring in wine such as S. paradoxus and S. bayanus, respectively. No amplification was observed for species that are generally present at initial stages of fermentation, such as Candida stellata, Pichia membranifaciens and Kloeckera apiculata (not shown).

The results of mtDNA RFLP for the 1620 isolates are summarized in Table 1. Among the total 450 isolates collected in vineyard A, 93 corresponded to unique patterns whereas in C and P a total 450 and 690 strains were isolated, corresponding to 62 and 135 unique patterns, respectively.

Table 1.  MtDNA RFLP analysis of 1620 yeast isolates from fermented must prepared with grapes collected in vineyards A, C and P of the Vinho Verde Region, indicated in Fig. 1, during the harvest of 2001, 2002 and 2003
  SiteNumber of isolatesNumber of distinct patternsNumber of unique patternsCommon patterns
  1. E – early sampling stage; L – late sampling stage; NF – no spontaneous fermentation; NC – not collected.

Vineyard A
  AIV AV AVI    
  AIII 8  
  AV 1  
  AIV AV AVI    
 LAI1801634ACP10 A06 A11
  AII 2 ACP10
  AIII 9 A11 A13
  AIV 6 A06 A13
  AV 9 A13
  AVI 1 
  AIV AV AVI    
 LAI180341ACP10 S3
  AII 1 ACP10 S3
  AIII 9 A06
  AIV 12 
  AV 19 
  AVI 2 
Vineyard C
  CIV9052S1 S2 S3 S4 S5
  CV 4 S4 S5
  CVI 1 S1
  CIII 4 S1 S4 S5
  CIV 2 S3 S5
  CV 4 S1 S3 S4 S5
  CVI 8 S1 S2 S4 S5
  CIV CV CVI    
  CV CVI    
 LCI180832S3 S4 C69P77 C63
  CII 3 C63
  CIII 1 ACP10
  CIV 18 S1, C42P80
  CV 9 
  CVI 2 S4
Vineyard P
  PII 2 P136
 LPI180662S3 S5 S6 P136
  PII 17 S3 S6 ACP10 P03 P136
  PIII 8 
  PIV 21 P03 P24 P50 P136
  PV 15 S3 P24
  PVI 13 S3 S6 P136
  PII 4 ACP10 S6 P136
  PIII 1 ACP10
  PV 10 S3 S6 P50 P136
  PVI 1 ACP10
  PII 1 
  PIII 1 P136
  PVI 2 ACP10
 LPI1801547ACP10, S3 C69P77
  PII 1 
  PIII 9 P136
  PIV 18 S3
  PV 5 C42P80
  PVI 5 

For 11 common patterns, found in more than one fermentation (Table 1 and Fig. 2), and also for six commercial starter yeast strains (S1–S6), a wider geographical and temporal distribution was verified. Patterns S1–S6 corresponded to commercial starter yeasts that had been used in the wineries for the last few years. Perennial strains were associated with more sites of a single vineyard (patterns A06 and S6, P136, P50), but showed also a wider distribution across multiple sampling sites in two or three vineyards (patterns S3, S4, and ACP10). Patterns S1, S2, C63, A11, A13, P03 and P24 were found only in one year but across several sampling sites of a single vineyard, while strain S5 had a wider distribution across several sampling sites of vineyard C and P. Patterns C42P80 and C69P77 appeared only in a single sampling site during 2003 of both vineyards C and P. Pattern ACP10 is the only “regional” isolate with a wider geographical distribution, whereas A06, A11, A13, C63, P03, P27, P50 and P136 can be considered as “vineyard-strains” due to their occurrence in multiple sampling sites and/or years.


Figure 2. Examples of common mitochondrial DNA RFLP (Hinf I) patterns, as listed in Table 1, found in yeast strains isolated from spontaneous fermentations of must collected as described in Section 2.

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The wet weather in the summer 2002 resulted in severe fungal infestations and heavy applications of chemical sprays. This was possibly the reason that merely 12 unique patterns were identified among the 150 strains collected in the late sampling stage in 2002 in vineyard P. In 2003, this relation was again more similar to the one found in 2001 (47 and 62 unique patterns among each 180 isolates from the late sampling stages of vineyard P).

As shown in Fig. 3, onset of spontaneous fermentation was verified in almost all grape samples collected in the late sampling campaign. This was rarely the case for most of the samples collected some days before the harvest. Must prepared from grapes collected in the early sampling stage in vineyard A, never started to ferment spontaneously. An accidental agrichemical over-dosage occurred in 2001, resulting in delayed spontaneous fermentation onset for three of the four post-harvest samples (II, III and VI). In the following two years, fermentation profiles were similar to samples from C and P, suggesting the recovery of the intervenient flora.


Figure 3. Fermentation profile (lines) and sugar content (bars) of must samples collected in the early (open circles and bars) and late (closed circles and bars) sampling campaigns from which yeast strains analyzed in this work were isolated. In each plot, mtDNA RFLP pattern designations of the yeast isolates are inserted. Predominating strains are double (geqslant R: gt-or-equal, slanted50%) or simple (20–50%) underlined. Pattern designations from post-harvest fermentations are bold. Common patterns are highlighted in grey squares.

Fermentation started after six to twelve days being generally accomplished by one to twenty strains. Apparently no correlation between the number of strains involved in a fermentation and sampling site, year or vineyard was found. The wider distributed strain (ACP10) was dominant in six fermentations (AII-2002, AI-2003, AII-2003, CIII-2003, PIII-2002, PVI-2002) contributing to 77–100% (23–30 strains) of the total yeast flora, but was of minor importance in five fermentations (AI-2002, PII-2001, PII-2002, PI-2003, PVI-2003), accounting for only 3–10% (one to three strains), and being accompanied by one to sixteen different strains. The distribution of this strain is not associated with the capability to predominate in fermentation, and competition with accompanying strains seems to play the key role.

Vineyard-specific patterns of samples collected in the early stage did not appear after two weeks at the same site (P, 2001 and 2003, C, 2001) with the exception of the more generalized patterns S1, S2, S3, S4, S5, ACP10 and P136, speaking in favor of a very diversified S. cerevisiae flora.


  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results
  6. 4Discussion
  7. Acknowledgements
  8. References

Biogeographical large-scale surveys and studies on the genetic diversity of S. cerevisiae strains isolated from spontaneous fermentations have documented the dynamic nature of these populations. In the present study, 297 different genetic patterns have been found among 1620 isolates obtained from 54 small scale fermentations performed with grapes from three vineyards located in the Vinho Verde Region, during a three years period. The vast majority of the patterns were unique, demonstrating an enormous biodiversity of S. cerevisiae strains in the Vinho Verde Region. Considering the ratio between the number of isolates and the number of patterns as an approximate biodiversity estimative, our results showed similar values to previously published surveys on genetic diversity of autochthonous oenological S. cerevisiae strains in other regions with viticulture traditions such as Bordeaux[2], Charentes [17,45], Campagne and Loire Valley[21], in France; El Penedèz[46], Tarragona[7], Priorato [20,22] and La Rioja[47] in Spain; Germany and Switzerland[41]; Tuscany, Sicily[48] and Collio[49] in Italy; Amyndeon and Santorini[42] in Greece; Western Cape [16,18,43] in South Africa; and Patagonia[19] in Argentina.

The present study has been carried out in a viticultural region that has never been characterized before and includes aspects that have not been considered in previous works, such as the appearance of several commercial yeast strains, and the comparison of yeast populations that can be found in grape samples before and after the harvest.

The vast majority of the strains did not display a perennial behavior, being the flora of each year characterized by the appearance of many new patterns. This might be attributed to the sampling of only 12 × 2 kg of grapes per vineyard and year, being not enough to grasp the entire biodiversity wealth of a given area. Another reason for the appearance of new patterns could be attributed to recombination and evolutionary forces, but it seems unlikely that such changes occur from one year to another to justify the presence of many distinct patterns in consecutive years. Mitochondrial DNA RFLP patterns are stable when S. cerevisiae cells undergo about five to seven divisions during alcoholic fermentation (our unpublished data).

Among all patterns only ACP10 showed a wide regional distribution with a perennial behavior providing preliminary evidence for a strain representing a “terroir” as described [17,21]. However, the wider distribution of a strain is not necessarily correlated with a better technological fitness. This makes sense from an ecological point of view, since the selective forces that act in a vineyard are completely different from those that yeast may find in a fermenting grape must. Further physiological characterization under wine making conditions is required to evaluate the potentialities of this strain. The appearance of this strain did not obey to a generalized pattern, but rather to sporadic presence, absence and reappearance, due to natural population fluctuations. The perennial appearance of pattern ACP10 is a consequence of its prevalence in the local microflora. In different fermentations, ACP10 was dominant or not, showing that the final outcome of fermentation was dependent on the specific composition of the yeast community in the must, that is influenced by many factors such as the killer effect which depends strongly on the ratio of killer to sensitive cells at the beginning of the fermentation[50].

Grape variety of vine A was Loureiro, being Alvarinho and Avesso the cultivars of vineyard P and C, respectively, indicating that the grape variety could contribute to the finding of so many distinctive patterns. Traditional wine-making practices are very similar in A, C and P, and differences in climatic influences seem to be of minor influence since the three vineyards are geographically close. However, one can not exclude microclimatic influences, not recorded in the present study.

A first sampling campaign was performed some days before the harvest; a second was carried out a few days after the end of harvest. This was accomplished in a time frame of about two weeks, in order to obtain a more detailed picture of the temporal distribution of fermenting yeast populations during the harvest. As grapes mature to full ripeness, yeasts become more abundant. The last stage of the grape maturation can favor fermentative yeast proliferation on grape surfaces, due to the decrease of grape skin integrity and must leakage from the berries. Insects are the probable source of yeast on damaged grapes. Yeast colonization of grapes can reach values of about 105–106 cfu/berry[51]. Before vintage, only 5% of the grapes harbor yeasts, while this number is much higher (60%) during vintage[52]. As expected, only 11 of 42 pre-harvest samples (26%) were able to ferment spontaneously compared to 43 of 48 post-harvest samples (90%). The associated strains were also much more diversified in the late sampling campaign (267 patterns among 1260 isolates) compared to the early stage (30 patterns among 360 isolates). With only one exception (pattern P136), autochthonous strain patterns from the early sampling stage did not appear in the late sampling stage, speaking in favor of a succession of S. cerevisiae strains. Alternatively, differences can be attributed to the fact that different grape bunches were harvested, that may have, although in close proximity to each other, a distinct flora. It seems unlikely that the enormous increase in strain variability at harvest time is due to a spreading of winery-resident flora with harvesting equipment.

Spontaneous fermentations were performed by one or more predominating strains accompanied by no, few or many “secondary” strains, or by a very heterogeneous yeast community with no prevalent strain(s). This is in agreement with other studies reporting the presence of one or two predominating strains constituting more than 50% of total biomass, and a varying number of “secondary” strains [7,17,19,29,40,41], or presence of many distinct strains with no prevalence [22,42]. The occurrence of both situations has also been reported [16,18,43].

Considering that the origin of wine yeasts is still controversial [3,5,8,44], our results clearly indicate that S. cerevisiae occurs in vineyard ecosystems belonging to the Vinho Verde Region in sufficient high numbers to conduct a spontaneous fermentation from musts prepared with approximately two kg of grapes. However, some remarks have to be made concerning our experimental approach. Grape must creates selective and very stressful conditions for yeast, totally distinct from the environmental influences in nature. It is therefore clear that our data refer only to S. cerevisiae strains capable to survive the conditions imposed by fermentation, under our experimental circumstances, giving therefore a distorted picture (underestimation) of the kind of strains really occurring in vine. As the detection limit of our experimental approach is 3.3% (one strain in 30 isolates), rare strains, although capable to survive fermentation, might also have not been detected. Searching for S. cerevisiae in 18 sites, in two campaigns and over three years using a direct-plating method from single grape berries, as described[3] would be highly labor-intensive. Therefore we regard our approach as an acceptable compromise, allowing good estimation of population composition, but preventing a precise description in terms of relative strain abundance in nature.

The present work is the first large-scale approach about the vineyard-associated strains from the Vinho Verde Region in Portugal, being a useful approach to obtain a deeper insight into ecology and biogeography of S. cerevisiae strains, even among geographically close regions. We consider these studies indispensable for the developing of strategies aiming at the preservation of biodiversity and genetic resources as a basis for further strain development.


  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results
  6. 4Discussion
  7. Acknowledgements
  8. References

This study was supported by the project ENOSAFE (No. 762, Programa AGRO, medida 8) and the grant no. 657 C2 from the cooperation agreement between the Portuguese Institute for International Scientific and Technological Cooperation (ICCTI) and the French Embassy in Lisbon. The authors appreciate the kind assistance of the enologists Rui Cunha, Anselmo Mendes, Euclides Rodrigues and José Domingues for facilitating sampling campaigns in the three vineyards. Ana Rodrigues, Luis Quintas and Carlos Rocha are acknowledged for support in grape collection and sample processing.


  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results
  6. 4Discussion
  7. Acknowledgements
  8. References
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