bzternational Journal of Food Microbiology, 21 (1994) 315-323 1994 Elsevier Science B.V. All rights reserved 0168-1605/94/$07.

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FOOD 677

315

Population dynamics of natural Saccharomyces strains during wine fermentation

Amparo Querol a,**, Eladio Barrio b,*** and Daniel Ram6n
c,.

a Departamento de Microbiolog[a and ~ Departamento de Gen~tica, Facultad de Ciencias Biol6gicas, Universidad de Valencia, 46100 Burjassot, Spain; c Unidad de Bioingenier{a, Instituto de Agroqu{mica y Tecnolog{a de Alirnentos, Consejo Superior de Investigaciones Cient{ficas, Jaime Roig 1l, 46100 Valencia, Spain
(Received 16 April 1993; accepted 9 October 1993)

Using mitochondrial DNA restriction endonuclease analysis, the dynamics of the natural Saccharomyces cereuisiae strains present in spontaneous wine fermentations have been studied. We observed a sequential substitution of Sacch. cerevisiae strains along fermentation agreeing with different fermentation phases. When the restriction pattern's similarity (measured as the fraction of shared restriction fragments) was high, a clear sequential substitution of the strains was seen. However, when the similarity was low, although a sequential substitution could be also observed between secondary strains, a clearly predominant strain was present along the whole fermentation process. Key words: Saccharomyces cereuisiae; Wine yeast; Population dynamics; mtDNA; Wine fermentation

Introduction

The fermentation of grape juice into wine is a complex microbiological reaction involving the sequential development of various yeast and lactic acid bacteria species. Among these microorganisms, only yeasts are primarily responsible for the alcoholic fermentation. Traditionally, wines have been produced by natural fermentation caused by the development of yeasts originating from the grapes and winery. Yeasts of the genera Kloeckera, Hanseniaspora, Candida and Pichia grow during the early stages of fermentation but eventually die off, leaving Sacch. cereuisiae as the dominant species to complete the fermentation (Amerine et al., 1982; Lafon-Lafourcade, 1983; Querol et al., 1990). The microbiological process of must fermentation has been subject of numerous studies and the variability of wine yeast populations is well-known.
* Corresponding author. Tel: 34-6-3690800; Fax: 34-6-3930001. Present addresses." ** Department of Molecular Biology and Biochemistry and *** Department of Ecology and Evolutionary Biology, University of California, lrvine. Irvine, CA 92717, USA.

SSDI 0 1 6 8 - 1 6 0 5 ( 9 3 ) E 0 0 9 3 - 7

316

Given the difficulty of distinguishing different Sacch. cerevisiae strains, the intra-specific variability of this species during wine fermentation is still unknown. Molecular methods have recenyly been employed for characterization of yeast strains. These comprise analyses of DNA sequence polymorphisms, chromosomal DNA patterns and restriction patterns (for a comparative study see Degr6 et al., 1989 and Querol et al., 1992a). A high polymorphism has been observed within industrial strains belonging to the species Sacch. cerevisiae. This polymorphism constitutes a powerful tool for analysing the indigenous microflora present during spontaneous wine fermentation. Nevertheless, only one ecological survey on the wine yeast populations has been carried out up until now (Vezinhet et al., 1992). In that study, Sacch. cerevisiae clones isolated in two vineyards during several years were characterized by electrophoretic chromosomal patterns or mitochondrial DNA restriction profiles, but only one strain per fermentation was analysed. This previous work constitutes preliminary evidence for the occurrence of specific native strains representative of an ecological area. Restriction endonuclease analysis of mitochondrial DNA (mtDNA) has also been used to distinguish between different strains (Dubordieu et al., 1984; Lee and Knudsen, 1985; Vezinhet et al., 1990). Purification of mtDNA is complex (Gargouri, 1989; Querol and Barrio, 1990), and its application has been limited to characterizing brewery (Amerine et al., 1982; Lee and Knudsen, 1985) and wine (Dubordieu et al., 1984; Hallet et al., 1988; Degr6 et al., 1989; Vezinhet et al., 1992; Querol et al., 1992b) yeast strains. However, the recent development of a simple method of yeast mtDNA restriction analysis (Querol et al., 1992a) has allowed us the rapid characterization of a high number of wild yeast strains, and to monitor molecularly inoculated wine fermentations (Querol et al., 1992c). In the present work, and by means of this new technique, we analyse the dynamics of the Saccharomyces strains during spontaneous wine fermentation.

Material and Methods

Wine fermentations The two wines studied were produced under commercial conditions during the 1991 vintage in the Valencia region (Spain). Red wine A was produced from Bobal variety grapes, characteristic in Utiel-Requena appellation. Red wine B was produced from Monastrell variety grapes, characteristic in Alicante appellation. Sulfur dioxide (30 mg/liter) was added to the musts of both wines. One fermentation per winery was conducted in tanks of 15 000 liter at 20-25C for a variable time (18-21 days). Microbiological analysis Samples were taken periodically during fermentations for the isolation and enumeration of yeasts. Yeasts were enumerated by spreading 0.1-ml samples of several dilutions (from 10 -1 to 10 -6) onto plates of malt extract agar (Oxoid).

317 Plates were incubated at 28C for 5 days. Yeast colonies were identified according to Barnett et al. (1990). As in previous works we demonstrated the predominant role of Saccharomyces in the fermentation of Alicante wines (Querol et al., 1990 1992b), only those clearly corresponding to Saccharomyces species were analysed.

mtDNA restriction analysis

At each time point when a sample was taken, 50 independent isolates obtained as randomly chosen colonies on plates from the highest dilutions were analysed for mtDNA. This number is statistically significant in accordance with Snedecor and Cochram (1956). A total of 450 colonies were analysed, and their mtDNA patterns were determined by the method of Querol et al. (1992a) using the restriction endonucleases HinfI and RsaI (Boehringer Manheim, Manheim, Germany).

Estimates of similarities between electrophoretic patterns

mtDNA restriction electrophoregrams were scanned and digitized using a LKB 2202 Ultroscan Laser Densitometer (Pharmacia-LKB, Uppsala, Sweden). The similarities between patterns were determined by the fraction of shared bands (Dice coefficient as Sneath and Sokal, 1973). Clustering was calculated by the unweighted pair group method using average linkage (UPGMA; Sneath and Sokal, 1973) included in the NTSYS package (Numerical Taxonomy and Multivariate Analysis System, Exeter Publishing Ltd., Setauket, NY, USA).

Results

The mtDNA restriction patterns of the 450 colonies of Saccharomyces species isolated from natural fermentations in both wineries correspond to thirty-nine different restriction patterns, that is to say, thirty-nine different Saccharomyces strains (Table I). The table represents the frequencies of all mtDNA patterns obtained in wineries A (19 patterns) and B (20 patterns), respectively. The present study of natural fermentations was carried out simultaneously to a study of inoculated fermentations previously reported (Querol et al., 1992c). In order to maintain the same nomenclature, the enumeration of the patterns in the present work correspond to that of the previous study, so, the patterns not shown correspond to those present only in the inoculated fermentation. Figure 1 shows the ratio of occurrence for the most frequent strains during fermentation in winery A. In this winery we observed a sequential substitution of strains along the fermentation, associated to the different fermentation phases. Classically these phases are divided according to the sugar degradation during fermentation, in prefermentation (high sugar content and slow sugar degradation), tumultuous fermentation (high fermentation rate), and slow fermentation or postfermentation (the remaining low sugar content is slowly degradated). In prefermentation, two strains (patterns A v and Ave) were present at significant frequencies, 20.5 and 11.4%, respectively. At the beginning of the tumultuous fermentation, the most frequent pattern was the Atv (37.2%) followed by patterns An, AvI

318 TABLE I Frequencies (%) of the m t D N A patterns obtained in each sampling of wineries A and B Patterns a Winery A A2 (2) A3 (4) A4 (6) 0.00 2.13 24.19 4.88 41.46 2.44 0.00 12.19 0.00 0.00 0.00 7.32 4.88 0.00 0.00 2.44 0.00 0.00 0.00 Bn Bin Blv Bv Bx Bxl Bxu Bxm Bxvl B xvu Bxw n Bx~ x Bxx B xx]x Bxx x Bxxx~ BxxxI l Bxxxm Bxxxl v Bxxxv Patterns ~ Winery B BI (0) 32.50 12.50 0.00 10.00 0.00 0.00 17.50 2.50 0.00 2.50 7.50 0.00 0.00 2.50 2.50 2.50 2.50 2.50 0.00 0.00 B2 (3) 36.36 9.119 0.00 9.09 0.00 4.54 2.72 9.09 0.00 4.54 6.82 11.36 2.72 0.00 0.00 0.00 0.00 0.00 2.72 2.72 B3 (5) 38.09 9.52 0.00 21.43 2.38 0.00 2.38 0.00 2.38 9.52 11.91 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 B4 (8) 47.62 11.9 0.00 14.28 0.00 0.00 2.38 1.14 0.0/) 7.14 9.52 0.00 I)./10 0.00 0.00 0.00 0.00 0.00 0.00 0.01/ B5 118) 64.28 30.95 0.00 0.00 0.00 0.00 0.110 0.00 /).00 0.00 0.00 0.00 4.76 0.00 0.00 0.00 0.00 0.1/0 0.00 0.00

Samples: A1 Time (days): (01 Au Am Air Av Avl Avl I Avn I A~x Ax A x~ Axn Axi n Axi v A xv Axv I A xvn A xvm A xxxn A xH 4.54 6.82 6.82 20.45 11.36 2.27 4.54 4.54 4.54 2.27 4.54 2.27 2.27 2.27 2.27 4.54 4.54 0.00 0.00

16.28 0.00 0.00 0.00 37.21 20.51 2.32 5.13 1 3 . 8 5 43.59 0.00 0.00 0.00 0.00 0.00 20.51 2.32 0.00 0.00 0.00 0.00 0.00 1 8 . 6 0 10.26 2.32 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 4.65 0.00 2.32 0.00

a Pattern nomenclature corresponds to that used in the previous study (Querol at al., 1992c).

and Axl n (16.3, 13.9 and 18.6%, respectively). Finally, along the tumultuous fermentation process, patterns A u and Axm were clearly replaced by patterns Alv and Avl, these two patterns being the most frequent (24.2 and 41.5%, respectively) at the end of the fermentation process.

45

3o

25 20
15

j/

10
0

-----~--~

3 Time (days)

Fig. 1. The Sacch. cerevisiae strains most frequently present in winery A (111 A n , [] AIV , * A v , <>Avl , AIX , A AxII1 ) The frequency of appearance of each isolate was defined as the percentage of the colonies analysed corresponding to the selected isolate versus the total number of colonies.

319

70
60

50
30

10 1
0

)4
0 2 4 6 8 10 12 14 16 18

Time (days) Fig. 2. The Sacch. cerevisiae strains most frequently present in winery B. (11 Bn, [] BIII, , Bv, ~ Bxu , Bxm, zx B x v n , B x v n i ) The frequency of appearance of each isolate was defined as the percentage of the colonies analysed corresponding to the selected isolate versus the total number of colonies.

Figure 2 shows the ratio of occurrence for the most frequent strains during fermentation in winery B. In this winery we observed the predominance of strain BII during the whole fermentation process. At the end of the fermentation this strain represented 64.3% of the total Saccharomyces isolates from winery B. The other patterns were always present in lower frequencies along the whole process. Nevertheless, a sequential replacement of secondary strains was observed along the different fermentation phases, similar to that observed in winery A. In the prefermentation phase, the most frequent secondary strain w a s B x i I (17.5%). During the tumultuous fermentation phase this strain was replaced by strain B v (21.4%). At the end of the process, the pattern B i l I w a s the most frequent among the secondary strains (30.1%). In order to determine possible relationships between strains, we performed a cluster analysis of similarity between mtDNA restriction patterns from the most frequent strains present in more than 9% in at least one sample, as well as between those from some representatives (randomly chosen) of the sporadic strains present in less than 5% in wineries A and B. The estimates of similarity between these restriction patterns, given as the fraction of shared fragments (F, Dice coefficient), are shown in Table II, and the phenogram resulting from the cluster analysis by the UPGMA method (Sneath and Sokal, 1973) is depicted in Fig. 3. Three main clusters were observed, one of them grouping all strains isolated from winery A and the other two connecting winery B strains. In the first cluster, a high similarity (76 to 94%) between the patterns of the most frequent strains (A n, A I V , A v , A v I , A t x and A x I I I ) w a s observed. On the contrary, the similarities between patterns of the strains from winery B were much lower (24 to 86%). Thus, they were grouped in two different clusters, one including strains Bn, Bnl , B x I , Bxi1, BXIII, B x v I I and B x I x , which was characterized by lower estimates of similarity, and the other one including the more related strains By, B x v l i I and Bxx.

320 TABLE II

Similarities between mtDNA restriction patterns of the strains studied: similarities were determined as the fraction of shared restriction fragments (Dice coefficient) Strains Winery A
II AII Aw Av Avi A~x Axlll Axv Avn Ax~ Ax Axv I 1.0000 0.8485 0.8387 0.8387 0.9375 0.8125 0.8235 0.7500 0.6471 0.7742 0.7500 IV 1.0000 0.8000 0.8000 0.9032 0.8387 0.8485 0.8387 0.6061 0.7333 0.7097 0.4000 0.5185 0.4828 0.3846 0.5185 0.4615 0.3200 0.4827 0.4138 0.3333 V VI IX XIII XV VII XI X XV1

1.0000 0.7857 0.8965 0.7586 0.7097 0.6207 0.6452 0.7143 0.7587 0.2143 0.4000 0.5185 0.2500 0.3200 0.3333 0.3478 0.2963 0.3704 0.2857

1.0000 0.8965 0.7586 0.7097 0.6896 0.5806 0.9286 0.6897 0.2143 0.5600 0.5926 0.3333 0.4000 0.4167 0.3478 0.4444 0.3704 0.3571

1.0000 0.8667 0.8125 0.7333 0.6875 0.8276 0.8000 0.2759 0.5385 0.5714 0.3200 0.3846 0.4000 0.3333 0.4286 0.4286 0.3448

1.0000 0.8750 0.7333 0.7500 0.8276 0.8667 0.2069 0.5385 0.4286 0.3200 0.3077 0.3200 0.2500 0.5000 0.4286 0.3448

1.0000 0.8125 0.6471 0.7097 0.7500 0.2581 0.4286 0.4000 0.3704 0.3571 0.3704 0.2308 0.4000 0.4000 0.3225

1.0000 0.6250 0.6207 0.6667 0.3448 0.3846 0.3571 0.3200 0.4615 0.4000 0.2500 0.4286 0.4286 0.3448

1.0000 0.6452 0.8750 0.1935 0.2857 0.4000 0.1481 0.2143 0.1481 0.2308 0.3333 0.3333 0.2581

1.0000 0.7586 0.1429 0.5600 0.5185 0.3333 0.3200 0.3333 0.2609 0.5185 0.2963 0.2857

1.0000 0.2759 0.3846 0.5000 0.1600 0.2308 0.1600 0.3333 0.3571 0.4286 0.3448

Bxu 1 0.2581 Bxi 0.5000 BI1 0.5333 Bxx 0.2963 Bxvi11 0.3571 Bv 0.3704 BIt I 0.3077 B xll 0.4000 Bxvtl 0.4000 Bxl x 0.3226

In winery B a dominant strain, Bll , appeared along the whole fermentation process. According to cluster analysis, the closest secondary strain to this dominant one was strain B m (82% of similarity between both m t D N A restriction patterns). As can be seen in Fig. 2, strain B n i followed the same growth dynamics as strain Bu, being the only two strains which appeared at the end of the fermentation.

Discussion A number of different strategies have been used for determining the population dynamics during wine fermentation. Some approaches have used the killer phenotype (Rozi~re et al., 1989; Longo et al., 1990), or the galactose fermentation phenotype (Rozi6re et al., 1989). Nevertheless, these attempts have been restricted due to the difficulty in distinguishing among different wild Saccharomyces strains present in the non-sterile must. The development of a new method for the detection of yeast m t D N A restriction patterns, which does not require m t D N A purification, simplifies the characterization of yeast strains (Querol et al., 1992a).

321

Winery B
XII1 XI II XX XVIII V IIl XII XVII XIX

1.000 0.4000 0.3704 0.1667 0.4000 0.2500 0.3478 0.2963 0.5926 0.5714

1.000 0.6667 0.2857 0.3636 0.3809 0.6000 0.6667 0.4167 0.5600

1.0000 0.3478 0.5000 0.5217 0.8181 0.5385 0.5385 0.5185

1.0000 0.6667 0.8000 0.3158 0.4348 0.1739 0.1667

1.0000 0.8571 0.4000 0.3333 0.2500 0.2400

1.0000 0.4210 0.3478 0.3478 0.2500

1.0000 0.4545 0.4545 0.5217

1.0000 0.3846 0.3704

1.0000 0.5926

1.0000

This method allowed us to analyse a high number of wine strains from fermentations conducted by inoculated active dry yeasts (Querol et al., 1992c). In the present work we analyse the dynamics of the wild Saccharomyces strains present in spontaneous grape must fermentations. We observed a sequential substitution of Sacch. cerevisiae strains along fermentation agreeing with different fermentation phases. When the similarity between strains was low, as occurred in winery B, we observed a clear dominance of one of the strains. When the similarity between strains was high (winery A), we observed a sequential substitution of the predominant strains during each fermentation phase. It is worth noting that most of the patterns were present in both wineries only in the first days of the fermentation (prefermentation phase). During the fermentation phases a reduction of the strain diversity was observed. The importance of determining the yeast variability present in the initial musts could be easily understood from the comparison of the results obtained from the analysis of the population dynamics during natural fermentations (present study), and during inoculated processes (Querol et al., 1992c). As pointed out above, the study of natural fermentations was carried out simultaneously to the study of inoculated fermentations. Both types of fermentations were performed at each

322
O. 132 0-t48

0.,64

O. 80

0.96

I~
I

__~AI

I fiI

AIU AU AXIII AXU AUI AX AUII

AXI

AXUI 8XIII BXUII 8I BXI BXII BII BIll BXX BXUIII BU

Fig. 3. Cluster analysis of similarity between restriction patterns of the most frequent and some sporadic (randomly chosen) haplotypes present in winery A and B. This phenogram was obtained by the UPGMA (Unweighted pair-group method with arithmetic mean) method (Sneath and Sokal, 1973) from the similarity between haplotypes measured as the fraction of shared restriction fragments (F, Dice's coefficient, see the same reference)

winery from the same must and same conditions. In winery A, a high similarity between restriction patterns and a sequential substitution of the predominant strains during the different phases of the natural fermentation were observed. After inoculation of the same musts with a selected Sacch. cerevisiae strain, even though it was predominant along the whole inoculated fermentation, the introduced strain had problems replacing the natural flora due to the presence of different strains in each phase. However, in winery B, a lower similarity and the presence of a dominant natural strain were observed during the spontaneous fermentation. In this case, the inoculated yeast rapidly replaced this predominant natural strain during all the fermentation processes.

Acknowledgements
This work was supported by grant ALI90-0949 from Comisidn Interministerial de Ciencia y Tecnolog~a, Spain. A.Q. and E.B. were recipients of fellowships from Conselleda de Cultura, Educaci6n y Ciencia de la Generalitat Valenciana. The authors are grateful to P. Sanz for critical reading of the manuscript.

323

References
Amerine, M.A., Berg, H.W., Kunkee, R.E., Ough, C.S., Singleton, V.L.U.L. and Webb, A.D. (1982) The technology of wine making. 4th Edn. AVI Publ. Westport, CT. Barnett, J.A., Payne, R.W. and Yarrow, D. (1990) Yeasts: characteristics and identification. 2nd Edn. Cambridge University Press, Cambridge. Degr~, R., Thomas, D.Y., Ash, J., Mailhiot, K., Morin, A. and Dubord, C. (1989) Wine yeast strain identification. Am. J. Enol. Vitic. 40, 309-315. Dubordieu, D., Sokol, A., Zucca, J., Thalouarn, P., Datte, A. and Aigle, M. (1984) Identification des souches de levures isol~es de vins par 1 analyse de leur ADN mitochondrial. Connais Vigne Vin. 21, 267-278. Gargouri, A. (1989) A rapid and simple method for extraction Yeast mitochondrial DNA. Curr. Genet. 15, 235-237. Hallet, J.N., Craneguy, B., Zucca, J., Thalouarn, P., Datte, A., Aigl~, M. (1988) Caracterisation de differentes souches industrielles de levures oenologiques par les profiles de restriction de leur ADN mitochondrial. Prog. Agric. Vitic. 105,328-333. Lafon-Lafourcade, Sacch. (1983) Wine and brandy. In: Biotechnology, Vol. 5. Reed, G. Ed. VerlagChemie. Heidelberg. pp. 81-161. Lee, S.Y., Knudsen, F.B. (1985) Differentiation of brewery yeast strains by restriction endonuclease analysis of their mitochondrial DNA.J. Inst. Brew. 91, 169-173. Longo, E., Velazquez, J.B., Cansado, J., Calo, P. and Villa, T. (1990) Role of killer effect in fermentation conducted by mixed cultures of Saccharomyces cerevisiae. FEMS Microbiol. Lett. 71, 331-336. Querol, A. and Barrio, E. (1990) A rapid and simple method for the preparation of yeast mitochondrial DNA. Nucl. Acids Res. 18, 1657. Querol, A., Jimenez, M. and Huerta, T. (1990) A study on microbiological and enological parameters during fermentation of must from poor and normal grapes harvest in the region of Alicante (Spain). J. Food Sci. 55, 1603-1606. Querol, A., Barrio, E. and Ram6n, D. (1992a) A comparative study of different methods of yeast strain characterization. Syst. Appl. Microbiol. 15, 439-446. Querol, A., Huerta, T., Barrio, E. and Ramdn, D. (1992b) Strain for use as dry yeast in fermentation of Alicante wine: selection and DNA patterns. J. Food Sci. 57, 183-185. Querol, A., Barrio, E. Huerta, T., and Ramdn, D. (1992c) Molecular monitoring of wine fermentations conducted by active dry yeast strains. Appl. Environ. Microbiol. 58, 2948-2953. Rozi~re, C., Raginel, F., Sanchez, C. and Strehaiano, P. (1989) Implantation de levures s~lectionn6es. Etude en site industriel de vinification. Rev. Fr. Oenologie 119, 37-41. Snedecor, G. and Cochram, W. (1956) Statistical Methods, 5th Edn. Iowa State Univ. Press. Ames, Iowa. USA. Sneath, P.H.A. and Sokal, R.R. (1973) Numerical taxonomy. W.H. Freeman and Company. San Francisco. Vezinhet, F., Blondin, B. and Hallet, J.N. (1990) Chromosomal DNA patterns and mitochondrial DNA polymorphisms as tools for identification of enological strains of Saccharomyces cereeisiae. Appl. Microbiol. Biotechnol. 32, 658-671. Vezinhet, F., Hallet, J.N., Valade, M. and Poulard, A. (1992) Ecological survey of wine yeast strains by molecular methods of identification. Am. J. Enol. Vitic. 43, 83-86.