Genetic characterization and phenotypic variability in Torulaspora delbrueckii species: Potential applications in the wine industry Philippe Renault a,b , Cecile Miot-Sertier a , Philippe Marullo a,b , Purificacion Hernández-Orte c , Laure Lagarrigue a , Aline Lonvaud-Funel a , Marina Bely a, ⁎ a Université Bordeaux, UMR 1219, INRA, ISVV, Villenave d'Ornon, 33140, France b SARCO, LAFFORT Bordeaux, 33072, France c University Zaragoza, Faculty of Sciences, Zaragoza, 50009, Spain abstract article info Article history: Received 14 April 2009 Received in revised form 16 June 2009 Accepted 20 June 2009 Keywords: Non-Saccharomyces Torulaspora delbrueckii Wine fermentation Selection Volatile acidity Genetic characterization In this study, several strains of Torulaspora delbrueckii yeast species were evaluated in the laboratory for their enological properties. In a preliminary step, the ability of different molecular methods to discriminate among T. delbrueckii strains was compared. A combination of 7 PCR methods was able to separate 21 strains into 18 groups, while an REA-PFGE method allowed, in one experiment, the separation into 19 groups. The T. delbrueckii strains used presented a wide phenotypic variability in fermentation behaviour, e.g. Lag Phase (LP) duration, T50 parameter (time necessary to ferment half the sugar), and ethanol production. These 3 parameters have to be considered for industrial selection, particularly the LP duration. The majority of T. delbrueckii strains produced 8 to 11% and 7 to 10% ethanol vol. at 17 °C and 24 °C, respectively, with a maximum ethanol concentration of 12.35 at 17 °C and 10.90% vol. at 24 °C. The phenotypic variability of this species was also reflected in volatile acidity, glycerol, and aroma production. These experiments confirmed the low volatile acidity and glycerol production of this species and revealed a difference in osmotic stress response, compared to Saccharomyces cerevisiae. T. delbrueckii presented high fermentation purity and produced low levels of undesirable volatile compounds, such as hydrogen sulphide and volatile phenols. © 2009 Elsevier B.V. All rights reserved. 1. Introduction Spontaneous alcoholic fermentation of grape must is a complex process, involving the sequential action of several yeast genera and species, found on grapes and in must. The early stages in fermentation are dominated by the growth of non-Saccharomyces yeasts, character- ized by low fermentative capacity. After the first few days' fermentation, they die off, due to the increasing concentration of ethanol (Heard and Fleet, 1985, 1986). Subsequently, Saccharomyces cerevisiae, an ethanol- tolerant species, takes over the fermentation. Quantitative studies of wine-making ecology have shown that significant levels of certain non- Saccharomyces species survive for longer periods than previously thought (Cabrera et al., 1988; Egli et al., 1998; Fleet, 2003; Heard and Fleet, 1985; Herraiz et al., 1990; Lema et al., 1996; Moreno et al., 1991; Pardo et al., 1989; Pina et al., 2004; Zott et al., 2008). Indeed, several authors have reported the influence of non-Saccharomyces yeast species on wine quality (Cabrera et al., 1988; Ciani and Ferraro, 1998; Ciani and Picciotti, 1995; Fleet, 2003, 2008; Herraiz et al., 1990; Lema et al., 1996; Romano et al., 2003) and evaluated the biotechnological interest of their enzymatic activities (e.g. esterases, β-glucosidase, and proteases), assumed to enhance fruit aromas in wine (Fernández-González et al., 2003; Fleet, 2008; Rosi et al., 1994; Strauss et al., 2001). Recent metabolic and analytical profiles of non-Saccharomyces yeasts showed that Torulaspora delbrueckii species (formerly Saccharomyces rosei) had a positive impact on the flavour of alcoholic beverages (Ciani and Maccarelli, 1998; Ciani and Picciotti, 1995; Herraiz et al., 1990; Moreno et al., 1991). Indeed this species exhibits low production of undesirable compounds, such as acetaldehyde, acetoin, acetic acid, and ethyl acetate (Cabrera et al.,1988; Ciani et al., 2006; Ciani and Maccarelli, 1998; Ciani and Picciotti, 1995; Herraiz et al., 1990; Martinez et al., 1990; Peynaud, 1956; Plata et al., 2003; Viana et al., 2008). In view of its high fermentation purity, T. delbrueckii, in mixed or sequential culture with S. cerevisiae, was proposed as a way of minimising acetic acid production in wine under standard or high-sugar conditions (Bely et al., 2008; Ciani et al., 2006; Lafon-Lafourcade et al., 1981). The aromatic potential of this species has also been investigated: Viana et al. (2008), Plata et al. (2003), and Ciani and Maccarelli (1998) showed the low capacity of T. delbrueckii to produce esters, while Hernández-Orte et al. (2008) suggested that this species significantly modulated the levels of various varietal aroma compounds (nor-isoprenoids, terpenols, benzenoids, volatile phenols, vanillin, and lactones), by hydrolysing their respective precursors. Nevertheless, despite increasing interest in T. delbrueckii species in wine applications, most studies to date only focused on a few strains, so no satisfactory overview of the wine-making properties of this International Journal of Food Microbiology 134 (2009) 201–210 ⁎ Corresponding author. Tel.: +33 5 5757 5866; fax: +33 5 5757 5813. E-mail address: marina.bely@univ-pau.fr (M. Bely). 0168-1605/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.ijfoodmicro.2009.06.008 Contents lists available at ScienceDirect International Journal of Food Microbiology journal homepage: www.elsevier.com/locate/ijfoodmicro