Modulation of the glycerol and ethanol syntheses in the yeast Saccharomyces kudriavzevii differs from that exhibited by Saccharomyces cerevisiae and their hybrid

Abstract

In the last years there is an increasing demand to produce wines with higher glycerol levels and lower ethanol contents. The production of these compounds by yeasts is influenced by many environmental variables, and could be controlled by the choice of optimized cultivation conditions. The present work studies, in a wine model system, the effects of temperature, pH and sugar concentration on the glycerol and ethanol syntheses by yeasts Saccharomyces cerevisiae T73, the type strain of Saccharomyces kudriavzevii IFO 1802^T, and an interspecific hybrid between both species (W27), which was accomplished by the application of response surface methodology based in a central composite circumscribed design. Results show that carbon flux could be especially directed towards glycerol synthesis instead of ethanol at low pH, high sugar concentrations and low temperatures. In general, the non-wine yeast S. kudriavzevii produced higher glycerol levels and lower ethanol content than wine strains S. cerevisiae T73 and the hybrid W27, with specific and different glycerol production profiles as a function of temperature and pH. These results were congruent with the higher glycerol-3-phosphate dehydrogenase activities estimated for this species, chiefly at low temperatures (14 °C), which could explain why S. kudriavzevii is a cryotolerant yeast compared to S. cerevisiae.

Introduction

Microorganisms, mainly yeasts, play a prominent role in determining the chemical composition of wine and their effects on flavor response are of primary importance (Querol and Fleet, 2006). Many species of yeasts are found in the musts, but only Saccharomyces yeasts, primarily Saccharomyces cerevisiae, are responsible for alcoholic fermentation (Pretorius, 2000). Other species of the Saccharomyces genus like Saccharomyces bayanus var. uvarum, whose specific ecological niche is winemaking at low temperature, or Saccharomyces paradoxus, that has been described as present in Croatian vineyards (Redzepović et al., 2002), can also participate in fermentative processes. Recently, interspecific hybrid strains between Saccharomyces species have been described as involved in winemaking. González et al. (2006) reported the presence of hybrid strains between S. cerevisiae and Saccharomyces kudriavzevii and between S. cerevisiae and S. bayanus during wine fermentations. Several strains selected as commercial wine yeasts also resulted to be Saccharomyces hybrids (Bradbury et al., 2006, González et al., 2006), for example the hybrid strains S. cerevisiae x S. kudriavzevii Lalvin W27 and W46.

Ethanol, glycerol and CO₂ are among the major compounds produced by yeasts during wine fermentation. Production of glycerol is a wasteful process that is associated with ATP expenditure and loss of a reduced three-carbon compound that cannot be re-utilized under anaerobic conditions. The physiological reason for the importance of glycerol synthesis in yeast metabolism resides in its involvement in osmoregulation and in its capacity to serve as a sink for reducing equivalents (Ansell et al., 1997, Nevoigt and Stahl, 1997). Moreover, glycerol seems to play also an important role in low-temperature tolerance in yeasts (Izawa et al., 2004). This way, cryotolerant wine strains produce more glycerol than non-cryotolerant yeasts (Castellari et al., 1994, Bertolini et al., 1996). The amount of glycerol produced by S. cerevisiae is around one tenth of the amount of ethanol, and its concentrations in wine normally range between 4 and 9 g l⁻¹, with average values approximately of 7 g l⁻¹ (Ough et al., 1972, Scanes et al., 1998). It is widely known the importance of both compounds for winemakers. Ethanol directly affects wine aroma and flavor, while the glycerol significantly contributes wine quality by providing slight sweetness, smoothness and fullness (Remize et al., 2000b).

In the last years, there is an increasing demand to produce wines with higher glycerol levels, due to its positive effects on wine's sensory properties, and lower ethanol contents, due to its negative effects on health. Therefore, modulation of the ethanol/glycerol ratio during wine fermentation would be interesting for industrial purposes. This way, three strategies have been used to increase the glycerol synthesis, which normally also reduce levels of ethanol (Michnick et al., 1997). One approach has been to improve wine-producing strains by classical breeding techniques in laboratory conditions (Eustace and Thornton, 1987, Prior et al., 1999). The enological characterization of diverse natural hybrid strains between the species S. cerevisiae x S. kudriavzevii isolated from wine fermentations opens new alternatives on this aspect, due to their interesting properties according to the new trends in winemaking (González et al., 2007) and because hybrids are not considered genetically manipulated organisms (GMOs). In addition, hybrid strains appear as well adapted to the stress conditions (low pH, high sugar concentration and ethanol content) occurring during wine fermentations (Belloch et al., 2008). A second approach has been the molecular manipulation of yeast, either by overexpressing GPD1 or GPD2 genes or by deleting the acetaldehyde dehydrogenase or the pyruvate decarboxylase genes (Remize et al., 1999, Remize et al., 2000a, Remize et al., 2001). Although this second approach allowed a better understanding of the glycerol and ethanol synthetic pathways, the use of GMO in the wine industry is rejected due to current political and social settings. The third approach simply consists in modifying the fermentation conditions to increase glycerol production. In this way, many factors have been found to influence glycerol production by S. cerevisiae, including aeration, temperature, pH, sugar concentration and sulfite content (Gardner et al., 1993, Remize et al., 2000b, Yalcin and Ozbas, 2008), but scarce information is available about these effects on other Saccharomyces species or their hybrids (González et al., 2007). Finally, another additional reason to study the effects of environmental variables on yeast metabolite production is provided by climatic change, which affects wine quality (Jones et al., 2005). This global process is generating musts with higher fermentable sugar contents and higher pH, which promote the necessity to study the influence of such factors on yeast metabolite production.

The present work focuses in the study of the effects of temperature, pH and sugar concentration on glycerol and ethanol syntheses by yeasts S. cerevisiae T73, S. kudriavzevii IFO 1802^T, and the S. cerevisiae x S. kudriavzevii hybrid strain W27, accomplished by the application of response surface methodology. The results obtained could be used to determine the environmental conditions that favour the carbon flux towards glycerol synthesis instead of ethanol.