Reassessment of phenotypic traits for Saccharomyces bayanus var. uvarum wine yeast strains

International Journal of Food Microbiology 139 (2010) 79–86

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International Journal of Food Microbiology j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / i j f o o d m i c r o

Reassessment of phenotypic traits for Saccharomyces bayanus var. uvarum wine yeast strains Isabelle Masneuf-Pomarède b,⁎, Marina Bely a, Philippe Marullo c, Aline Lonvaud-Funel a, Denis Dubourdieu a a b c

Université de Bordeaux, UMR 1219, INRA, ISVV, 210, Chemin de Leysotte, CS 50008, 33882 Villenave d'Ornon cedex, France ENITA de Bordeaux, 1 Cours du Général de Gaulle, CS 40201, 33175 Gradignan cedex, France SARCO, Laffort, 33072 Bordeaux, France

a r t i c l e

i n f o

Article history: Received 8 October 2009 Received in revised form 18 January 2010 Accepted 28 January 2010 Keywords: S. bayanus var. uvarum Biometric study Low-temperature adaptation Wine fermentation Viability

a b s t r a c t Among Saccharomyces yeast, S. cerevisiae and S. bayanus var. uvarum are related species, sharing the same ecosystem in sympatry. The physiological and technological properties of a large collection of geneticallyidentified S. bayanus var. uvarum wine strains were investigated in a biometric study and their fermentation behavior was compared at 24 °C and 13 °C. The variability of the phenotypic traits was considered at both intraspecific and interspecific levels. Low ethanol tolerance at 24 °C and production of high levels of 2-phenylethanol and its acetate were clearly revealed as discriminative technological traits, distinguishing the S. bayanus var. uvarum strains from S. cerevisiae. Although some S. bayanus var. uvarum strains produced very small amounts of acetic acid, this was not a species-specific trait, as the distribution of values was similar in both species. Fermentation kinetics at 24 °C showed that S. bayanus var. uvarum maintained a high fermentation rate after Vmax, with low nitrogen requirements, but stuck fermentations were observed at later stages. In contrast, a shorter lag phase compared with S. cerevisiae, higher cell viability, and the ability to complete alcoholic fermentation at 13 °C confirmed the low-temperature adaptation trait of S. bayanus var. uvarum. This study produced a phenotypic characterization data set for a collection of S. bayanus var. uvarum strains, thus paving the way for industrial developments using this species as a new genetic resource. © 2010 Elsevier B.V. All rights reserved.

1. Introduction Spontaneous fermentation of grape must is a complex microbial process, in which Saccharomyces species play the key role, with Saccharomyces cerevisiae as the dominant strain. In some specific ecological niches, Saccharomyces bayanus var. uvarum contributes to alcoholic fermentation in a mixed population with S. cerevisiae. In the past, S. bayanus var. uvarum was frequently isolated and, in a few cases, was even the predominant species, in spontaneous fermentations of must obtained from grapes cultivated in northern French vineyards such as Burgundy, Alsace, Champagne, and Val de Loire (Massoutier et al., 1998; Naumov et al., 2000, 2001; Demuyter et al., 2004). S. bayanus var. uvarum was also isolated from natural fermentations of botrytized grape must (Naumov et al., 2000, 2001; Sipiczki et al., 2001; Antunovics et al., 2005). Both S. cerevisiae and S. bayanus var. uvarum were also identified in natural fermentations of Recioto and Amarone wines (Torriani et al., 1999; Tosi et al., 2009). This specific ecological situation, where two species share the same microbial ecosystem, may lead to the emergence of natural interspe-

⁎ Corresponding author. Tel.: + 33 5 57 57 58 34; fax: + 33 5 57 57 58 13. E-mail address: [email protected] (I. Masneuf-Pomarède). 0168-1605/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.ijfoodmicro.2010.01.038

cies hybrids in winery environments (Masneuf et al., 1998; Le Jeune et al., 2007; González et al., 2006). Among the Saccharomyces yeast, S. cerevisiae and S. bayanus var. uvarum are related species (Naumov, 2000) . The synteny between these two genomes is very well conserved, with 98% of the genes in S. bayanus var. uvarum retaining the same neighboring relationships as in S. cerevisiae (Bon et al., 2000; Fischer et al., 2001). S. bayanus var. uvarum exhibits homogeneous, well-defined genetic characteristics, with a specific electrophoretic karyotype and stable number of chromosomes (Naumov et al., 1992; Cardinali and Martini, 1994; Louis et al., 1994; Rainieri et al., 1999). Publication of a nearly complete sequence of S. bayanus var. uvarum has facilitated in-depth genetic studies of this species (Cliften et al., 2003; Kellis et al., 2003). Genetic identification of S. bayanus var. uvarum at the strain level is possible using karyotypes and microsatellite marker analysis (Demuyter et al., 2004; Masneuf-Pomarède et al., 2006). Many articles describe the intraspecific genetic diversity of S. cerevisiae, associated with a well-documented variation in technological traits (Bidenne et al., 1992; Ness et al., 1993; Legras et al., 2005). For example, several publications have described the distribution of acetic acid and H2S production, or differences in nitrogen and oxygen demand among S. cerevisiae strains (Romano et al., 1985; Giudici and Zambonelli, 1992; Julien et al., 2000; Marullo et al., 2004). The ability to produce glycerol and succinic acid is a stable, hereditary

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trait of S. cerevisiae (Rainieri et al., 1998). These technological traits depend on the genetic background of the strain considered and are quantitative (Marullo et al., 2004). Previous studies reported that certain technological traits associated with S. bayanus var. uvarum differed significantly from those of S. cerevisiae. In the past, before molecular methods for genetic identification became available, S. bayanus var. uvarum was recognized by its cryotolerance. Indeed, in winemaking, they were known as cryophilic or cold-resistant strains, in contrast to thermotolerant S. cerevisiae (Kishimoto and Goto, 1995; Giudici et al., 1998). However, growth tests at different temperatures were inadequate for identifying the exact taxonomic position of Saccharomyces wine isolates (Antunovics et al., 2005). Many authors reported that S. bayanus var. uvarum strains exhibited a specific fermentation profile, producing less acetic acid and higher levels of glycerol, succinic acid, and higher alcohols (particularly 2-phenylethanol) than S. cerevisiae (Castellari et al., 1994; Giudici et al., 1995; Bertolini et al., 1996; Rainieri et al., 1999; Antonelli et al., 1999). Their ethanol yield was lower than that of S. cerevisiae and they also synthesized malic acid instead of degrading it (Castellari et al., 1994; Giudici et al., 1995). However, in most previous research concerning phenotypic traits, the taxonomic position of the strains studied was not clearly genetically defined and the number of strains considered was limited. In this study, the physiological and technological properties of a large collection of genetically-identified S. bayanus var. uvarum wine strains were investigated. Phenotypic traits related to ethanol tolerance, as well as acetic acid, glycerol, and aroma production during alcoholic fermentation were compared in a biometric study. The fermentation kinetics at different temperatures were also established. The intraspecific phenotypic variance in S. bayanus var. uvarum was compared to the phenotypic data set for S. cerevisiae previously obtained in our laboratory under the same experimental conditions. The link between certain phenotypic traits and the S. bayanus var. uvarum species is discussed. 2. Materials and methods 2.1. Yeast strains The S. bayanus var. uvarum wine strains used in this study are listed in Table 1. They were isolated from different geographical areas (Sancerre, Jurançon, Sauternes, Alsace, and Tokaj). Strain CBS 7001,

Table 1 Saccharomyces var. uvarum strains tested in this study. Strain number Original

This study

P3, PJS3, PJS9, PJS11, LC1 Saμ1 GM14, PM12

S1, S2, S3, S4, S5 S6 J1, J2

TB3C28, DDII4

Sau1, Sau2

RP1-16, RP1-21, RP2-2, RP2-32, RP2-20 RC3-79, RC4-5, RC4-15, RC1-19, RC2-20 RC2-10, RRI3

A1, A2, A3, A4, A5

Source of isolation

References

Fermenting grape must, Sancerre Grapes, Sancerre Fermenting grape must, Jurançon Fermenting grape must, Sauternes Press juice, Alsace

Masneuf-Pomarède et al. (2006)

A6, A7, A8, A9, A10

Fermenting grape must, Alsace

A11, A12

T18/1

T1

Fermenting grape must, Alsace Grapes, Tokaj

B9/2, B2/22, B2/14, B10/38, B2/15 CBS 7001

T2, T3, T4, T5, T6 –

Fermenting grape must, Tokaj Mesophylax adoperus

from which the sequenced strain CLIB 533 derived, was also included in the study. Seven winemaking strains, five industrial (Zymaflore VL1, Zymaflore VL3c, Zymaflore F10, VIN13 and BO213) and two indigenous (JSc1 and SSc1), were used as S. cerevisiae references in the fermentation tests. JSc1 and SSc1 were isolated in Jurançon and Sancerre areas, from the same grape must samples as for the S. bayanus var. uvarum wine strains. Genetic and physiological tests: standard ascus dissection methods were used. Sporulation was induced by incubating cells on acetate medium (1% CH3COONa, 0.5% KCL, 2% agar) for two days at 25 °C. Following preliminary digestion of the ascus walls with cytohelicase (Sigma), adjusted to 2 g/L, ascospores were isolated using a Singer MSM Manual micromanipulator and incubated at 25 °C. Homothallism was scored by microscopic examination of the individual spore clones' ability to sporulate. Melibiose fermentation was examined in YPM (1% yeast extract, 1% peptone, 2% melibiose) with Durham tube for gas detection. Growth at 37 °C was evaluated using the procedure described by Antunovics et al. (2005). 2.2. Biometric study Fermentation experiments were carried out in triplicate in 200 mL sterile Erlenmeyer flasks containing 160 mL Model Synthetic Medium (KP) simulating a standard grape juice at 24 °C (Marullo et al., 2006). The KP medium, buffered to pH 3.3, contained (g/L): L+ tartaric acid (3); citric acid (0.3); L-malic acid (0.3); mineral salts (mg/L): KH2PO4 (2000), MgSO4·7H2O (200), MnSO4·H2O (4), ZnSO4·7H2O (4), CuSO4·5H2O (1), KI (1), CoCl2·6H2O (0.4), (NH4)6Mo7O24·4H2O (1), H3BO3 (1); vitamins (µg): myoinositol (300), biotin (0.04), thiamin hydrochloride (1), pyridoxine hydrochloride (1), nicotinic acid (1), calcium panthothenate acid (1), para-amino benzoic acid (1); nitrogen source: 190 mg/L available nitrogen provided by 300 mg/L (NH4)2SO4 (corresponding to 63.6 mg nitrogen) and a mixture of 18 amino acids (mg/L), corresponding to 126.4 mg/L nitrogen. Different amounts of sugars (an equimolar mix of glucose and fructose) were added: 210 g/L (KP210) and 300 g/L (KP300). For experiments to quantify acetic acid, glycerol, and volatile compounds, KP210 was used, supplemented with anaerobic growth factors: ergosterol (15 mg/L), sodium oleate (5 mg/L), and 0.5 mL Tween 80/ethanol 1:1, v/v (Marullo et al., 2006). Ethanol tolerance was considered in KP300, by measuring the maximum capacity of yeast to ferment in an unlimited sugar medium at 24 °C (Marullo et al., 2004). Ethanol tolerance (vol.%) corresponded to the maximum amount of ethanol in the medium. Fatty acids, prepared in ethanol and fixed by drying on cellulose (0.5 g/L) to obtain 200 NTU (Nephelometric Turbidity Unit), were added to the KP300 instead of anaerobic growth factors. After three days' fermentation, 6 mg/L oxygen was added by air bubbling. The prepared KP media were sterilized by filtration (nitrate cellulose membrane 0.45 μm, Millipore, FRANCE) and supplemented with sulfur dioxide (20 mg/L), in accordance with enological treatments. Yeast was pre-cultured in KP diluted 1/1 with milli-Q water for 24 h at 24 °C and Erlenmeyer flasks were inoculated with 3.5 · 106 cells/mL. 2.3. Measuring fermentation kinetics and estimating cell viability

Demuyter et al. (2004) Naumov et al. (2002) Antunovics et al. (2005) –

To check the adaptation to low-temperature, fermentation progress in 200 mL sterile Erlenmeyer flasks was tested at 13 °C by determining the weight loss due to CO2 release and the kinetic parameters were measured. The lag phase was defined as the time between inoculation and the beginning of CO2 release. This parameter reflects the time necessary for a specific strain to adapt to the must. Mid-fermentation time (T50) was the time necessary to reach 50% of the total CO2 expected. In some experiments, the amount of CO2 released was determined by automatically measuring the bioreactor weight loss every 20 min (El Haloui et al., 1988; Bely et al., 1990). The numerous acquisitions

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and weighing accuracy (0.01 g) made it possible to calculate the fermentation rate with a good level of precision. The CO2 production rate (dCO2 / dt), expressed in g/L. h, was calculated by polynomial smoothing of the last eleven bioreactors weight loss measurements. As fermentation conditions may generate cell mortality, the number of viable cells V (%) was estimated in a 100-μL sample diluted with water to obtain a final 1:1 dilution with methylene blue (0.02% wt/vol in a citrate buffer; pH = 4.2, 65 mM). Blue cells were counted as dead and non-colored cells were counted as alive. A minimum of 150 cells was counted for each time point. 2.4. Chemical analysis Ethanol concentrations (vol.%) were measured by the infrared reflectance method (infra Analyser 450 Technicon, Bran+Luebbe, Plaisir, France). Volatile acidity (extrapolated as acetic acid) and residual sugars were analyzed after distillation by colorimetry (A460 nm) in continuous flux (Sanimat, Montauban, France). Glycerol was analyzed by the enzymatic method (Roche-Biopharm kit). Higher alcohol and ester concentrations were determined by gas chromatography coupled with a flame ionization detector (FID) (CARBOWAX 20 M capillary column type BP20, length 50 m, internal diameter 0.25 mm, film thickness 0.50 µm; VARIAN 3400 gas chromatograph, Merck D-2500 chromato-integrator). 2.5. Statistical analysis The residual sugar values obtained in the fermentation test at 13 °C were subjected to a two-factor ANOVA analysis (temperature:strain) according to the following model: Zij = μ + Ti + Sj Ti × Sj + εij where Z is the residual sugar, T is the temperature effect (i = 1, 2), S is the strain effect (j = 1 to 12), and Yi × Sj is the pairwise interaction effects. A Principal Component Analysis (PCA) was performed using Statbox Software. PCA is a multivariate procedure used to reduce the dimensionality of a data set while retaining as much information as possible. It computes a compact, optimal description of the data set. The principle of the method is to rotate the data so that maximum variabilities are projected onto new axes. PCA was applied to the data obtained in the biometric study. 3. Results 3.1. Genetic and physiological characteristics of the collection The strains tested in this study were previously genetically identified as S. bayanus var. uvarum using different molecular methods (karyotyping, PCR-RFLP of the MET2 gene, and microsatellite multilocus typing). To complete these data, the 28 S. bayanus var. uvarum strains were characterized for their ability to sporulate and produce viable ascospores, ferment melibiose, and grow at 37 °C. All the strains tested were able to sporulate on acetate medium. The proportion of viable ascospores was high: 85–100%. Sporulation of the monosporic clones isolated from one complete tetrad indicated that all the strains were homothallic and homozygous for the HO gene. The Mel+ and thermosensitivity phenotypes had previously been described as specific characteristics to differentiate S. bayanus var. uvarum from S. cerevisiae and S. bayanus (Rainieri et al., 1999). All the strains tested in our study were Mel+. The increase in optical density at 37 °C after 10h was less marked for the S. bayanus var. uvarum, compared to the six S. cerevisiae strains tested (average value for final OD/initial OD) of 5.1 (SD ± 3.4) and 33.1 (SD ± 5.9) for S. bayanus var. uvarum and S. cerevisiae, respect-

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ively, except for one S. bayanus var. uvarum strain, T1 (final OD/initial OD:21.5). 3.2. Biometric study A data set of technological traits for a large collection of S. cerevisiae strains was previously established in our laboratory using fermentation tests (Marullo et al., 2004, 2006). The same method (fermentation using KP medium, in triplicate at 24 °C) was used to compare the properties of the S. bayanus var. uvarum strains with the data obtained for S. cerevisiae. When alcoholic fermentation was completed, the KP210 was analyzed for acetic acid, glycerol, 2-phenylethanol, and esters. The KP300 samples were assayed for ethanol content and residual sugars. The action of yeast on malic acid (synthesis or degradation) was not analyzed in this study, due to the low level of malic acid in the KP medium (0.3 g/L), which did not reflect the typical concentrations in grape must. No significant differences were found concerning ethanol yield calculated in KP210 and obtained for 9 S. bayanus var. uvarum and 53 S. cerevisiae strains. A box plot analysis of the biometric study data is presented in Fig. 1. The difference observed within strains or species was estimated by one-factor analysis of variance (Table 2). Concerning the ethanol tolerance trait, 28 S. bayanus var. uvarum strains were screened and compared with data previously obtained for 71 S. cerevisiae strains (Marullo et al., 2004, 2006 and this study). The average values for ethanol production in an unlimited sugar medium at 24 °C were 13.24 vol.% and 9.70 vol.% for S. cerevisiae and S. bayanus var. uvarum, respectively. The maximum ethanol tolerance was 12.16 vol.% for S. bayanus var. uvarum and 16.93 vol.% for S. cerevisiae. The interquartile range was higher in the S. cerevisiae data set than in that of S. bayanus var. uvarum for both species, a strong intraspecific diversity was observed (Table 2, Fig. 1). The acetic acid production values obtained for the S. bayanus var. uvarum strains were compared with previous results for 65 S. cerevisiae strains (Marullo et al., 2004, 2006). The average values for both species were similar: 0.37 g/L and 0.35 g/L for S. bayanus var. uvarum and S. cerevisiae, respectively. Although statistically significant, the species only explains 4.3% of the total variance observed. Intraspecies variation in the data sets was similar for both groups, but some S. bayanus var. uvarum strains (4 out of 28 strains studied) produced less than 0.1 g/L acetic acid. Acetic acid production value for strains CBS 7001 was 0.35 g/L. The glycerol production data for 28 S. bayanus var. uvarum and 6 S. cerevisiae strains are presented in Fig. 1. The average value for the S. bayanus var. uvarum strains was 6.51 g/L (6.4 g/L for CBS 7001), compared with 5.42 g/L for the S. cerevisiae. The interquartile range in the S. bayanus var. uvarum data set was not significatively different than of S cerevisiae. The last technological traits analyzed in the biometric study concerned the volatile compounds produced by the yeast in KP210 during alcoholic fermentation. We assayed the isoamyl acetate, phenylethyl acetate, ethyl hexanoate, ethyl octanoate, and ethyl decanoate compounds (given in Fig. 1 as “the sum of esters”), as well as 2-phenylethanol in the media fermented by 26 S. bayanus var. uvarum and 6 S. cerevisiae strains. The intraspecific variation among S. bayanus var. uvarum was high compared with that of S. cerevisiae. While the mean value for the “sum of esters” was similar for both groups (2.57 mg/L and 1.85 mg/L for S. bayanus var. uvarum and S. cerevisiae, respectively and 1.15 mg/L for the strain CBS 7001), the mean value for 2-phenylethanol differed significantly (155 mg/L and 64 mg/L for S. bayanus var. uvarum and S. cerevisiae, respectively and 183 mg/L for the strain CBS 7001). The higher values for the “sum of esters” in S. bayanus var. uvarum were mainly due to the large amount of phenylethyl acetate, which correlated well with its related alcohol (Antonelli et al., 1999). Principal Component Analysis (PCA) was applied to the results obtained for the three parameters measured (glycerol, acetic acid, and

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Fig. 1. Box plot analysis of the biometric study parameters in KP210 at 24 °C, except for ethanol tolerance (KP300).

2-phenylethanol), the “sum of esters”, and the strains tested in the biometric study (Fig. 2). The projection conserved 74% of the information with two components, explaining 21% and 53% of the total inertia. Axis 1 clearly separates two groups of S. bayanus var. uvarum strains. The first group, located to the right of axis 1, corresponds to the high acetic acid values, similar to those produced by S. cerevisiae strains. The second group, to the left of axis 1, corresponds to high glycerol, 2-phenylethanol, and “sum of esters” values. 3.3. Fermentation kinetics Fermentation tests were carried out in duplicate in KP210 at 24 °C. A quantitative methodology was used to monitor alcoholic fermentation kinetics (Bely et al., 1990). Five S. bayanus var. uvarum strains were compared to two commercial S. cerevisiae strains: Actiflore BO213 and Zymaflore VL1. Actiflore BO213 was selected for its excellent fermentation characteristics, including strong kinetics, low nitrogen requirement, and extremely high alcohol tolerance (Marullo et al., 2006). In contrast, Zymaflore VL1 was selected for its slow fermentation rate, low alcohol tolerance, and high nitrogen requirement (Marullo et al., 2004, 2006). The overall fermentation kinetic profiles, i.e. the variation in CO2 rate versus fermentation progress, are shown in Fig. 3. The data represent the average value of two independent experiments. The rate curves exhibited a similar behavior in the first part of the fermentation. The lag phase (LP) did not vary from one yeast strain to another under these conditions, lasting about 10 h at 24 °C for all strains (data not shown). The maximum CO2 production rate (Vmax), reached at 10 g/L CO2, varied from 1.45 to 1.55 g/L .h, with no significant interspecies difference. After Vmax, two groups were observed. In the S. bayanus var. uvarum group and S. cerevisiae BO213, fermentation rates were maintained between 1.4 and 1.2 g/L. h up to 30 g/L CO2 released. In contrast, a continuous decrease in fermentation rate was observed for S. cerevisiae VL1. In the last stage of fermentation, several linear phases were observed, with considerable variations in extent and slope. Finally, only the two S. cerevisiae yeasts, BO213 and VL1, were able to complete fermentation, producing the

Table 2 Analysis of variance of fermentation by-products. The effect of yeast strain and species on the final productions of ethanol, glycerol, acetic acid, 2-phenylethanol and the sum of esters were analyzed by one-factor analysis of variance. Data presented are the percent of variance explain by the factor analyzed. Levels on significance are given as following: NS not statistically different, *P b 0.01, **P b 0.001, ***P b 0.0001. Factor

Ethanol

Acetic acid

Glycerol

Sum of esters

2-phenylethanol

Strain Species

94.0*** 64.4***

95.4*** 4.3*

32.7*** NS

92.5*** NS

45.95*** 13.5**

expected 94 g/L CO2, with fermentation times (data not shown) of 120 h and 200 h, respectively. In contrast, stuck fermentations were observed for all S. bayanus var. uvarum strains, confirming the low fermentation capacity of this species at 24 °C. 3.4. Adaptation to low-temperature fermentation Cryotolerant (formerly known as cryophilic) or cold-resistant strains are capable of fermenting well between 6 °C and 30 °C, rather than in the 12–36 °C range of thermotolerant strains (Castellari et al., 1994). In further fermentation experiments in KP210 medium, under the same conditions as those used in the biometric study, the temperature was maintained at 13 °C. Nine S. bayanus var. uvarum strains were compared with one indigenous, JSc1, and two industrial S. cerevisiae strains, VL1 and VIN13, the latter considered to be cryotolerant. All strains completed alcoholic fermentation and residual sugars were below 2 g/L, except for strain T1 (data not shown). The average lag phase was estimated at 41 h (SD ±8.1) for S. bayanus var. uvarum but at 69 h (SD ± 8.9) for the three S. cerevisiae strains. Mid-fermentation time (T50) was estimated at 160 h (SD ± 12.2) and 228 h (SD ± 33) for S. bayanus var. uvarum and S. cerevisiae strains, respectively. Finally, the fermentation was completed in 36 days for all strains tested. Kishimoto et al. (1993) showed that fermentation of cryophilic strains ceased prematurely at intermediate temperatures and their ethanol yields were reduced. Our results, presented in Fig. 4, are in agreement with these observations. Residual sugars after alcoholic fermentations at 13 °C and 24 °C were analyzed by a two-factor analysis of variance. The temperature effect explained 32% of the total variation observed: at 13 °C all the S. bayanus var. uvarum strains except T1 completed alcoholic fermentation, leaving less than 2 g/L residual sugars. In contrast, most strains did not complete alcoholic fermentation at 24 °C. S. bayanus var. uvarum also exhibited a significant strain effect, explaining 28% of the total variance (α = 0.001). The behavior of each strain varied according to temperature, explaining the high interaction effect observed (36% of total variance). In contrast to S. bayanus var. uvarum, all S. cerevisiae strains completed fermentation at both temperatures. The phenomenon of abrupt arrest of fermentation at intermediate temperatures for cryophilic strains, described by Kishimoto et al. (1993), was remarkably illustrated by T2 (Fig. 4). To obtain a deeper insight into this strain's behavior, we compared its fermentation kinetics in KP210 medium at 17 and 24 °C, in duplicate (Fig. 5 and Table 3). At 24 °C the fermentation with T2 stopped after 69 g/L CO2 was released, leaving 52 and 53 g/L residual sugars in the media in the duplicate experiments. In contrast, at 17 °C, fermentation was complete (less than 1 g/L residual sugars). These results confirmed those obtained above in Erlenmeyer flasks. The CO2 production rate curves differed markedly according to temperature. The increase in temperature was reflected in the global fermentation and maximum

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Fig. 2. Principal Component Analysis (PCA) for the parameters measured in the biometric study: glycerol, acetic acid, 2-phenylethanol, and the “sum of esters”. Data were obtained for 25 S. bayanus var. uvarum strains and 6 S. cerevisiae strains (VL1, F10, VL3, BO, JSc1, SSc1).

CO2 production rates (Vmax) (Bely et al., 1990; Sablayrolles and Barre, 1993). Under these conditions, up to 62 g/L CO2 release, temperature had no effect on viability. Conversely, in the second stage of fermentation, V% differed significantly between the two conditions. At 17 °C, cell viability decreased continuously up to 49% at the end of fermentation. At 24 °C, a drop in cell viability was observed between 62 and 69 g/L CO2 release, corresponding to alcohol concentrations of 8 and 9 vol.%, respectively, explaining the abrupt arrest of fermentation.

Fig. 3. Variations in the CO2 production rate versus fermentation progress in KP210 at 24 °C (max. CO2 production expected: 94 g/L) for 5 S. bayanus var. uvarum (S2, T3, A7, T2, A8) and 2 S. cerevisiae strains (VL1, BO213). Data represent the average value of two independent experiments, max. SD ± 0.05.

4. Discussion A collection of 28 S. bayanus var. uvarum strains, isolated from wine environments of different origins, was tested for several genetic, physiological, and technological properties. These strains tested are homothallic, with a high sporulation ability and spore germination close to 100%, in agreement with previous publications (Naumov et al., 1993; Rainieri et al., 1999). We confirmed that the poor ability to grow at 37 °C, reported to be a characteristic feature of S. bayanus and S. bayanus var. uvarum by various authors, was a physiological trait associated with S. bayanus var. uvarum (Castellari et al., 1992; Vaughan-Martini and Martini, 1993; Kishimoto and Goto, 1995; Antunovics et al., 2005). The 28 strains tested were able to ferment melibiose. However, the Mel+ phenotype is no longer used as a discriminating characteristic, as a few S. cerevisiae strains with a Mel+ phenotype and few S. bayanus var. uvarum strains with Mel− phenotype have been found in wine (Naumov et al., 1993; Antunovics et al., 2005).

Fig. 4. Effect of temperature on residual sugars after alcoholic fermentation in KP210 for 9 S. bayanus var. uvarum (T1, T2, J2, S1, A8, T3, A7, S5, T6) and 3 S. cerevisiae strains (VL1, JSc1 and VIN13). Data represent the average value of three independent experiments.

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Fig. 5. Variations in the CO2 production rate versus fermentation progress in KP210 at 17 and 24 °C (max. CO2 production expected: 94 g/L). Data represent the average value of two independent experiments, max. SD ± 0.05.

The biometric study clearly indicated that S. bayanus var. uvarum species had a lower capacity to ferment at unlimited sugar concentrations at 24 °C, and is thus considered less ethanol tolerant than S. cerevisiae, under these experimental conditions. These results are in accordance with previously published data suggesting that cryotolerant (formerly “cryophilic”) wine strains had low ethanol resistance at 25 °C and low ethanol production at intermediate temperatures (Kishimoto, 1994). It would be useful to determine the ethanol tolerance of both species at low temperatures (e.g. 13 °C) to complete our data set. Cryotolerant strains have been shown to produce low levels of acetic acid but this trait was reportedly strain dependent (Castellari et al., 1992, 1994; Antonelli et al., 1999). Rainieri et al. (1999) identified low acetic acid production as one of the clearlydefined fermentation profile parameters of the S. bayanus sub-group uvarum. Our results demonstrate that, under these experimental conditions, low acetic acid production is not species-specific. Although some S. bayanus var. uvarum strains produced very small amounts of acetic acid, the distribution of values was similar in both groups of strains studied. Our data concerning glycerol production in the biometric study confirm that a high level production of this compound is an enological trait common to thermotolerant strains, as reported by several authors (Castellari et al., 1992, 1994; Rainieri et al., 1998). However, in view of the small panel of S. cerevisiae strains studied, this result requires further confirmation. Results concerning aromatic compounds confirm previous reports that high concentrations of fermentative volatile compounds, especially 2-phenylethanol, are produced by S. bayanus var. uvarum strains (Bertolini et al., 1996; Antonelli et al., 1999; Massoutier et al., 1998; Tosi et al., 2009). Production of high levels of 2-phenylethanol and its acetate is

Table 3 Cell viability estimation (%) during S. uvarum T2 fermentation, at 17 and 24 °C. KP210 (max. CO2 production expected: 94 g/L). Fermentation progress (CO2 g/L) 0

22

32

42

52

62

69

78

90

94

Viability (%) at 17 °C Viability (%) at 24 °C

89 92

86 92

86 Nd

87 91

84 83

83 7

78 –

65 –

49 –

90 92

Data represent the average value of two independent measurements, max. SD ± 0.5.

clearly a discriminant technological trait for distinguishing S. bayanus var. uvarum from S. cerevisiae. Moderate amounts of these volatile compounds have a pleasant rose-like odor, contributing positively to wine aroma, but at high concentration, they mask varietal aromas. Finally, phenotypic traits reported for the CBS 7001 strain (acetic acid, glycerol, sum of esters and 2-phenylethanol production) are concordant with data obtained for the S. bayanus var. uvarum strains. The HO/HO genotypes, the high percentage of first generation spore germination, and the low number of heterozygous microsatellite loci previously reported compared to S. cerevisiae are concordant results, indicating that homozygous genes are probably more frequent in natural diploid S. bayanus var. uvarum strains than in S. cerevisiae strains (Masneuf-Pomarède et al., 2006). We thus expected to obtain less intraspecific variability in the phenotypic traits compared to data obtained for S. cerevisiae. However, for example, the distribution of acetic acid production is similar for both species. The fermentation kinetics of 5 S. bayanus var. uvarum and 2 control S. cerevisiae strains at 24 °C were investigated using an automatic monitoring device. Both species exhibited similar performance at the beginning of fermentation i.e. lag phase duration and Vmax. This parameter is reached during the cell-growth phase when nitrogen depletion occurs in the medium and is closely related to the amount of available assimilable nitrogen (Bely et al., 1990). This result suggested that these species had the same nitrogen utilization profile in the first stage in the process. Later in fermentation, after Vmax, substantial differences were observed in the rate curve trends. The S. bayanus var. uvarum and the high-performance S. cerevisiae, BO213, maintained high fermentation rates. In contrast, a drastic decrease was observed for S. cerevisiae VL1. Salmon et al. (1993) showed that this decrease in fermentation rate was due to a loss of sugar transport activity, triggered by protein synthesis arrest. Recently Julien et al. (2000), comparing the nitrogen requirements of different commercial S. cerevisiae yeasts in the stationary phase, reported that the VL1 strain required high nitrogen levels, in contrast to BO213. In order to determine whether the high performance of S. bayanus var. uvarum species correlated with low nitrogen demand, we performed constant-rate fermentations by adding nitrogen, using the protocol described by Julien et al. (2000). The S. bayanus var. uvarum nitrogen requirements were low, like those of BO213, until mid-fermentation but abrupt arrests were observed later (results not shown). These results confirmed those obtained during experiments without nitrogen supplementation, as well as the low ethanol tolerance of this species at 24 °C. S. bayanus var. uvarum strains were reported by several authors to be cryotolerant, i.e. they have a short generation time, as well as good growth and fermentability at low temperatures (7–13 °C) (Walsh and Martin, 1977; Castellari et al., 1994; Kishimoto and Goto, 1995; Serra et al., 2005). By measuring the fatty acid composition in cells at 8 and 30 °C, Kishimoto et al. (1994) showed that “cryophilic” and “mesophilic” wine strains had different temperature adaptation mechanisms. Recent research provides insight into the effect of growth temperature on differential responses in laboratory and wine strains of S. cerevisiae (Pizarro et al., 2008). Low-temperature growth alters biomass composition and yield as well as the sugar uptake rate, reducing fermentation efficiency. Our results showed that the nine S. bayanus var. uvarum strains tested had similar fermentation low-temperature profiles, unlike those of S. cerevisiae strains. The shorter lag phase and mid-fermentation time of S. bayanus var. uvarum illustrated their cold-resistant properties. In this study, a comparison of T2's fermentation kinetics at 17 and 24 °C revealed an abrupt arrest of fermentation at 24 °C, with high residual sugar levels. This stuck fermentation was due to a drastic decrease in yeast viability at 8 vol.% ethanol, thus confirming the results previously suggested by Kishimoto et al. (1994). Further investigation is required to clarify the role of the ethanol/temperature couple in this mortality mechanism: does it act by decreasing plasma membrane H+-ATP activity or modifying the change in the fatty acid and sterol

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compositions of cell-membranes to adapt their fluidity, as already described for S. cerevisiae (Alexandre et al., 1994; Torija et al., 2003; Beltran et al., 2008). The differential responses of strains from these two species concerning their low-temperature growth adaptation mechanism requires further investigation at both physiological and transcriptional levels. From an ecological point of view, this low-temperature adaptation trait may represent a selective advantage, explaining the specific niche of S. bayanus var. uvarum, frequently isolated from must obtained from grapes harvested in cold climates and fermented at low temperatures. As S. bayanus var. uvarum occurs relatively infrequently in natural wine ecosystems, very few strains have been proposed for industrial development. This study produced a phenotypic characterization data set for a collection of 28 S. bayanus var. uvarum strains. Some of these have relevant oenological traits, such as low acetic acid production, high production of volatile compounds, and low-temperature adaptation, which are complementary to those of S. cerevisiae. These data pave the way for industrial developments using S. bayanus var. uvarum as new genetic resource in breeding strategies or co-inoculation systems, in association with S. cerevisiae. Acknowledgements This work was supported by grants from SARCO (Bordeaux, France). The authors wish to thank Matthias Sipiczki and Christine Le Jeune for kindly supplying strains and acknowledge Anne-Lise Turrin, an engineering student, for her contribution to this project. References Alexandre, H., Rousseaux, I., Charpentier, C., 1994. Ethanol adaptation mechanisms in Saccharomyces cerevisiae. Biotechnology and Applied Biochemistry 20, 173–183. Antonelli, A., Castellari, L., Zambonelli, C., Carnacini, A., 1999. Yeast influence on volatile composition of wines. Journal of Agriculture and Food Chemistry 47, 1139–1144. Antunovics, Z., Irinyi, L., Sipiczki, M., 2005. Combined application of methods to taxonomic identification of Saccharomyces strains in fermenting botrytized grape must. Journal of Applied Microbiology 98, 971–979. Beltran, G., Novo, M., Guillamón, J.M., Mas, A., Rozès, N., 2008. Effect of fermentation temperature and culture media on the yeast lipid composition and wine volatile compounds. International Journal of Food Microbiology 121, 169–177. Bely, M., Sablayrolles, J.M., Barre, P., 1990. Automatic detection of assimilable nitrogen deficiencies during alcoholic fermentation in enological conditions. Journal of Fermentation and Bioengineering 70, 246–252. Bertolini, L., Zambonelli, C., Giudici, P., Castellari, L., 1996. Higher alcohol production by cryotolerant Saccharomyces strains. American Journal of Enology and Viticulture 47, 343–345. Bidenne, C., Blondin, B., Dequin, S., Vezinhet, F., 1992. Analysis of the chromosomal DNA polymorphism of wine strains of Saccharomyces cerevisiae. Current Genetics 22, 1–7. Bon, E., Neuvéglise, C., Casaregola, S., Artiguenave, F., Wincker, P., Aigle, M., Durrens, P., 2000. Genomic exploration of the hemiascomycetous yeasts: 5. Saccharomyces bayanus var. uvarum. FEBS Letters 22, 37–41. Cardinali, G., Martini, A., 1994. Electrophoretic karyotypes of authentic strains of the sensu stricto group of the genus Saccharomyces. International Journal of Systematic Bacteriology 44, 791–797. Castellari, L., Pacchioli, G., Zambonelli, C., Tini, V., Grazia, L., 1992. Isolation and initial characterization of cryotolerant Saccharomyces strains. Italian Journal of Food Sciences 3, 179–186. Castellari, L., Ferruzzi, M., Magrini, A., Giudici, P., Passarelli, P., Zambonelli, C., 1994. Unbalanced wine fermentation by cryotolerant vs. non-cryotolerant Saccharomyces strains. Vitis 33, 49–52. Cliften, P., Sudarsanam, P., Desikan, A., Fulton, L., Fulton, B., Majors, J., Waterston, R., Cohen, B.A., Johnston, M., 2003. Finding functional features in Saccharomyces genomes by phylogenetic footprinting. Science 4, 71–76. Demuyter, C., Lollier, M., Legras, J.L., Le Jeune, C., 2004. Predominance of Saccharomyces bayanus var. uvarum during spontaneous fermentation, for three consecutive years, in an Alsatian wiery. Journal of Applied Microbiology 97, 1140–1148. El Haloui, N., Picque, D., Corrieu, G., 1988. Alcoholic fermentation in winemaking: on-line measurement of density and carbon dioxide evolution. Journal of Food Engineering 8, 17–30. Fischer, G., Neuvéglise, C., Durrens, P., Gaillardin, C., Dujon, B., 2001. Evolution of gene order in the genome of two related yeast species. Genome Research 11, 2009–2019. Giudici, P., Zambonelli, C., 1992. Biometric and genetic study on acetic acid production for breeding of wine yeast. American Journal of Enology and Viticulture 43, 370–374. Giudici, P., Zambonelli, C., Passarelli, P., Castellari, L., 1995. Improvement of wine composition with cryotolerant Saccharomyces strains. American Journal of Enology and Viticulture 46, 143–147.

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