Letters in Applied Microbiology 1998, 26, 452–455 Diversity of Saccharomyces strains in wine fermentations: analysis for two consecutive years J. Sabate 1 , J. Cano 1 , A. Querol 2 and J.M. Guillamo ´n 3 1 Unidad de Microbiologia, Facultad de Medicina, Universidad Rovira i Virgili, Reus, Tarragona, 2 Departamento de Biotecnologı ´ a, Instituto de Agroquı ´ mica y Tecnologı ´ a de Alimentos (C.S.I.C.), Paterna, Valencia, and 3 Departamento de Bioquı ´ mica y Biotecnologı ´ a, Facultad de Enologı ´ a, Universidad Rovira i Virgili, Tarragona, Spain 1729/98: received 7 January 1998 and accepted 18 March 1998 J. SABATE, J. CANO, A. QUEROL AND J.M. GUILLAMO ´ N. 1998. An ecological study of Saccharomyces cerevisiae strains in spontaneous alcoholic fermentation has been conducted in the same winery for two consecutive years (1994 and 1995). Yeast cells were identified and characterized using mitochondrial DNA restriction analysis. Although a great diversity of wild strains was observed, a sequential substitution of S. cerevisiae strains during the different phases of fermentation was detected. Furthermore, the most frequent strains were encountered in both years, and the dynamic populations were not influenced by climatic conditions. Finally, the RsaI restriction enzyme produced a species- specific pattern which allowed the identification of all the isolates as S. cerevisiae. INTRODUCTION Traditionally, wines have been produced by natural fer- mentation caused by yeasts from the grapes and winery. Yeasts of the genera Hanseniaspora, Candida and Pichia grow during the early stages of fermentation but eventually die off, leaving Saccharomyces cerevisiae as the dominant species to complete the fermentation. The microbial process of must fermentation has been the subject of numerous studies (for a review see Fleet and Heard 1993). In addition, the dynamics of the S. cerevisiae strains during spontaneous alcoholic fer- mentation contribute to the chemical composition and sen- sory qualities of the resulting wine (Lurton et al. 1995). A number of different strategies, based on the analysis of DNA polymorphism, have been used to differentiate the enological strains of S. cerevisiae (for a review see Querol and Ramo ´n 1996). They are powerful tools, not only for industrial and technological controls, but also for ecological research into the intraspecific diversity of the indigenous microbiota of wines (Versavaud et al. 1995). In previous stud- ies, several authors have examined the evolution of the native population of S. cerevisiae during the course of spontaneous wine fermentation (Querol et al. 1992a, 1994; Vezinhet et al. 1992; Schu ¨tz and Gafner 1993, 1994), i.e. without the seed of selected strains. Many different strains have been observed Correspondence to: Jose M. Guillamo ´n, Departamento de Bioquı ´mica y Biotecnologı ´a, Facultad de Enologı ´a. Universidad Rovira i Virgili. Ramo ´n y Cajal s/n; 43005-Tarragona, Spain (e-mail: jmgn@astor.urv.es). © 1998 The Society for Applied Microbiology generally, but few of these have been dominant in the latter stages of the process. At a Bordeaux winery, Frezier and Dubourdieu (1992) found the same dominant strain in spon- taneous fermentation for two consecutive years and Guil- lamo ´n et al. (1996) found significant correlations between geographical origin and the genetic relationships among strains. In the present work, the dynamics of S. cerevisiae strains in spontaneous fermentation have been studied in the same winery for two consecutive years. The 1994 vintage was very rainy in the last stages of the ripening process, presenting grapes with poor sanitary conditions. The 1995 vintage was harvested under normal conditions. A rapid and simple method of yeast mtDNA restriction (Querol et al. 1992b) has been applied to analyse the influence of climatic factors on the dynamics of natural S. cerevisiae strains. MATERIALS AND METHODS Wine fermentations Two red wine-making processes were studied in the same fermentation vessel in a winery from Porrera (Priorat wine region, Tarragona, Spain) in 1994 and 1995. Dry yeast was never used. The wine making processes were carried out under the same conditions in both years. Samples were taken aseptically at four different stages of the fermentation: the beginning (day 3), the middle (day 5) and the end of the