Effects of fermentation temperature on the strain population of Saccharomyces cerevisiae

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Abstract

The influence of fermentation temperature (from 15 to 35 °C) on a mixed strain population was studied. Mitochondrial DNA analysis was used to differentiate Saccharomyces cerevisiae strains and the frequency of each strain during the alcoholic fermentation was determined. The chemical analyses of resulting wines were carried out. The temperature determined how Saccharomyces strains developed and how effectively they fermented. Some strains performed better at high temperatures and others at low temperatures. The maximal population size was similar at all temperatures. At low temperatures, however, it was reached later though it remained constant throughout the alcoholic fermentation. On the other hand, viable cells decreased at high temperatures, especially at 35 °C. Obviously, the composition of the wines changed as the temperature of fermentation changed. At low temperatures, alcohol yield was higher. Secondary metabolites to alcoholic fermentation increased as the temperature increased. Glycerol levels were directly affected by temperature.

Introduction

Alcoholic fermentation is a combination of complex interactions involving must variety, microbiota and winemaking technology. Obviously, some factors strongly affect alcoholic fermentation, and as a consequence, the quality of the wine. The most important ones are the clarification of grape juice, the levels of the sulphur dioxide, the temperature of fermentation, the composition of the grape juice, inoculation with selected yeasts and the interaction with other microorganisms (Ribéreau-Gayon et al., 2000). One of these factors, the temperature of fermentation, directly affects the microbial ecology of the grape must and the biochemical reactions of the yeasts (Fleet and Heard, 1993).

Several authors have suggested that some species of non-Saccharomyces have a better chance of growing at low temperatures than Saccharomyces Sharf and Margalith, 1983, Heard and Fleet, 1988 because they can increase their tolerance to ethanol (Gao and Fleet, 1988). Also the number of different species, as well as their endurance during alcoholic fermentation, is conditioned by both the temperature of the must and the temperature during fermentation. These changes determine the chemical and organoleptic qualities of the wine (Fleet and Heard, 1993). Temperature is also known to affect yeast metabolism, and as a result, the formation of secondary metabolites such as glycerol, acetic acid, succinic acid, etc. (Lafon-Lafourcade, 1983).

In a previous ecological study (Torija et al., 2001), we isolated several strains from a Saccharomyces cerevisiae “natural” population, which were used in the present study. This population had a high diversity of strains, probably related to the absence of killer phenotype, control of temperature and lack of previous inoculation with commercial strains. Very little is known about how fermentation temperature affects the dynamics of the Saccharomyces strain population. Competition during alcoholic fermentation carried out at different temperatures could therefore be a way of testing the natural endurance of indigenous strains. This could be used as a criterion for rapidly selecting one of several strains and at the same time, studying resistance to temperature in a controlled situation, i.e. under laboratory conditions. Inoculating selected yeasts to ensure that alcoholic fermentation begins properly has become common practice in enology, and new molecular biology techniques for identifying strains have made it easier to confirm the imposition of inoculated yeasts (Querol et al., 1992a).

The aim of this paper is therefore to evaluate how temperature affects the dynamics of a known population of Saccharomyces during alcoholic fermentation. We used the 20 most consistent strains present in natural vinifications. We report how Saccharomyces strains isolated from the same wine-producing area interact during alcoholic fermentations at different temperatures and how this affects the composition and characteristics of the wine.

Section snippets

Yeast strains

The strains had previously been isolated and identified in an ecological study at wineries in the Priorat region (Spain) (Torija et al., 2001). Twenty strains of S. cerevisiae with killer sensitive phenotype were used. These strains were cultured for 48 h at room temperature on fresh YPD medium [Glucose 20 g l−1; Peptone Bacteriological (Cultimed, Panreac, Barcelona, Spain) 20 g l−1; Yeast extract (Cultimed) 10 g l−1] before they were used in the fermentations.

Fermentation experiments

The medium was made from

Effect of temperature on yeast growth

Temperatures grouped in three different profiles of fermentation kinetics (see Fig. 1 and Table 1). Fermentations at 15 and 20 °C began more slowly, as we can see by their longer lag phase and slower rate of maximal fermentation, especially at 15 °C. This caused a delay in reaching the maximal population (108 cfu ml−1), but once it was reached it remained at these high values for the whole process, i.e. there was a long stationary phase and no declining phase.

Fermentations at 25 and 30 °C

Discussion

Few studies have analysed how temperature affects the population dynamics of Saccharomyces strains during alcoholic fermentation (Epifanio et al., 1999). Nearly all focused on different species of yeasts Sharf and Margalith, 1983, Heard and Fleet, 1988, Mateo et al., 1991.

As expected, the growth in yeast varied according to temperature. The usual growth curve, with a series of short-lag, exponential, stationary and decline phases, was observed at 25 and 30 °C, whereas at 35 °C a high amount of

Acknowledgments

This work has been supported by grants 2FD1997-1854 and AGL2000-0205-P4-03 from the Dirección General de Investigación of the Ministerio de Ciencia y Tecnologı́a, Spain.

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