Behaviour of Saccharomyces cerevisiae wine strains during adaptation to unfavourable conditions of fermentation on synthetic medium: Cell lipid composition, membrane integrity, viability and fermentative activity

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International Journal of Food Microbiology 121 (2008) 84 – 91 www.elsevier.com/locate/ijfoodmicro

Behaviour of Saccharomyces cerevisiae wine strains during adaptation to unfavourable conditions of fermentation on synthetic medium: Cell lipid composition, membrane integrity, viability and fermentative activity Ilaria Mannazzu a,1 , Daniele Angelozzi a , Simona Belviso b,c , Marilena Budroni c , Giovanni Antonio Farris c , Paola Goffrini d , Tiziana Lodi d , Mario Marzona b , Laura Bardi e,⁎ a

Dipartimento di Scienze degli Alimenti, Università Politecnica delle Marche, Via Brecce Bianche, 60131, Ancona, Italy Dipartimento di Chimica Generale Organica e Applicata, Università di Torino, Via Pietro Giuria, 7, 10125 Torino, Italy c Dipartimento di Scienze Ambientali Agrarie e Biotecnologie Agroalimentari, Sezione di Microbiologia Generale ed Applicata, Università degli Studi di Sassari, Viale Italia 39, 07100 Sassari, Italy d Dipartimento di Genetica, Antropologia, Evoluzione, Università di Parma, Parco Area delle Scienze 11/A, 43100, Parma, Italy e C.R.A. Istituto Sperimentale per la Nutrizione delle Piante, S.O.P. Torino, Via Pianezza 115, 10151, Torino, Italy b

Received 13 October 2006; received in revised form 3 May 2007; accepted 2 November 2007

Abstract During must fermentation wine strains are exposed to a variety of biotic and abiotic stresses which, when prevailing over the cellular defence systems, can affect cell viability with negative consequences on the progression of the fermentative process. To investigate the ability of wine strains to survive and adapt to unfavourable conditions of fermentation, the lipid composition, membrane integrity, cell viability and fermentative activity of three strains of Saccharomyces cerevisiae were analysed during hypoxic growth in a sugar-rich medium lacking lipid nutrients. These are stressful conditions, not unusual during must fermentation, which, by affecting lipid biosynthesis may exert a negative effect on yeast viability. The results obtained showed that the three strains were able to modulate cell lipid composition during fermentation. However, only two of them, which showed highest viability and membrane integrity at the end of the fermentation process, reached a fatty acid composition which seemed to be optimal for a successful adaptation. In particular, C16/TFA and UFA/TFA ratios, more than total lipid and ergosterol contents, seem to be involved in yeast adaptation. © 2007 Elsevier B.V. All rights reserved. Keywords: Wine yeast; Saccharomyces cerevisiae; Viability; Lipid composition; Membrane integrity; Fatty acids

1. Introduction During wine production yeast strains are subjected to a variety of biotic and abiotic stresses which, when prevailing over the cellular defence systems, can affect cell viability, with negative consequences on the progression of the fermentative process (Attfield, 1997; Zuzuarregui and del Olmo 2004a;

⁎ Corresponding author. Tel./fax: +39 011 7399714. E-mail address: [email protected] (L. Bardi). 1 Present address: Dipartimento di Scienze Ambientali Agrarie e Biotecnologie Agroalimentari, Sezione di Microbiologia Generale ed Applicata, Università degli Studi di Sassari, Viale Italia 39, 07100 Sassari, Italy. 0168-1605/$ - see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.ijfoodmicro.2007.11.003

Zuzuarregui et al., 2005). Thus, to avoid stuck or sluggish fermentations, wine strains should be able to counteract the effects exerted by environmental stressors through the activation of an adequate stress response (Ivorra et al., 1999; Trabalzini et al., 2003; Zuzuarregui and del Olmo 2004a; Zuzuarregui and del Olmo 2004b). Accordingly, the existence of a correlation between fermentative behaviour and stress resistance has been shown in Saccharomyces cerevisiae wine strains (Ivorra et al., 1999; Querol et al., 2003; Zuzuarregui and del Olmo 2004b). Among the environmental factors that influence the progression of must fermentation, oxygen availability and ethanol accumulation are of primary importance due to their effect on composition and functional properties of cell membranes.

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Oxygen deprivation negatively affects lipid biosynthesis either directly, by blocking oxygen-dependent enzymes (e.g. Δ9-desaturase, squalene epoxydase, the lanosterol demethylation complex), or indirectly, by causing the accumulation of saturated fatty acids (SFAs) and precursors of ergosterol, which regulate the expression of acetyl-CoA carboxylase and hydroxymethylglutaryl-CoA reductase, respectively (Bloomfield and Bloch, 1960; Parks, 1978; Henry, 1982; Wakil et al., 1983; Ratledge and Evans, 1989; Hammond, 1993). Thus, under anaerobiosis, yeast cells are unable to complete the biosynthesis of unsaturated fatty acids (UFAs) and ergosterol, and accumulate intermediates of lipid metabolism (Bardi et al., 1998; Bardi et al., 1999; Belviso et al., 2004). In these conditions, if lipid nutrients are not available, S. cerevisiae cells progressively change the composition of their lipid fractions, reducing the surface area of organelle membranes and diluting their lipid content until the limit of viability (Henry, 1982). It thus follows that S. cerevisiae viability is low during growth in the absence of oxygen and lipid nutrients (Fornairon-Bonnefond et al., 2002) and that the number of generations produced by wine strains may depend on the initial sterol content (Deytieux et al., 2005). Lipid composition of yeast cell membranes and ethanol tolerance are strictly related (Thomas et al., 1978; Piper, 1995). In particular, the ability to operate acyl chain unsaturation (Thomas et al., 1978, Chi and Arneborg, 1999; You et al., 2003) and ergosterol biosynthesis (Shobayashi et al., 2005) seems to be essential for ethanol tolerance, particularly during grape must fermentation, a process that yeasts carry out under hypoxic conditions and increasing ethanol concentrations. These conditions compromise the biosynthesis of sterols and fatty acids thus causing variations in the amount and composition of the lipid fraction of cell membranes. The adaptive response to produced ethanol was evaluated by Arneborg et al., (1995) in chemostat grown cells of S. cerevisiae. A part from that work, most of the data regarding the correlation between ethanol tolerance and cell lipid composition derived from the analysis of cells subjected to ethanol shock (Thomas et al., 1978; You et al., 2003; Aguilera et al., 2006) while, to our knowledge, no attempt has been made to assess changes in the lipid composition of several S. cerevisiae strains, with different ethanol tolerances, during the adaptation to self-produced ethanol in batch fermentations. In the present study we investigated the ability of three strains of S. cerevisiae to adapt to unfavourable conditions of fermentation, in terms of cell lipid composition, membrane integrity, viability and fermentative activity. The rationale was that, during hypoxic growth in a sugar-rich medium lacking lipid nutrients, the ability to modulate cell lipid composition may be one of the factors involved in yeast survival and adaptation to stressful conditions of fermentation. 2. Materials and methods 2.1. Strains and culture conditions The following S. cerevisiae strains were used: BY4743, a laboratory strain (S. cerevisiae MATa/MATαΔ his3Δ1/his3Δ1

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leu2Δ0/leu2Δ0 met15Δ0/MET15 LYS2/lys2Δ0 ura3Δ0/ura3Δ0 purchased from Euroscarf, Frankfurt, Germany); L2056, a commercial enological strain (Lallemand, Montreal, Canada); M25, a flor wine strain commonly utilized for must fermentation at the industrial level, deposited with the Culture Collection of DiSAABA (Dipartimento di Scienze Ambientali Agrarie e Biotecnologie Agroalimentari, University of Sassari, Sardinia, Italy). Bench-scale fermentations were carried out in triplicate in SJ medium (2 g l− 1 YNB without amino acids, 7 g l− 1 ammonium sulphate, 120 g l− 1 glucose, 120 g l− 1 fructose, 30 mg l− 1 leucin, 20 mg l− 1 histidine, 20 mg l− 1 uracil, pH 4.4). Briefly, yeast strains were pre-cultured aerobically in YEPD (20 g l− 1 glucose, 10 g l− 1 yeast extract, 20 g l− 1 peptone) (liquid:air ratio, 1:10), and 5 × 105 cells ml− 1 were inoculated in 100 ml flasks containing 75 ml SJ medium (liquid:air ratio, 7.5:10) equipped with glass capillary stoppers and incubated statically at 20 °C for 25 days. A flask for each sampling time was inoculated, and both the culture broth and cells underwent the analyses described below. Yeast growth was determined by viable plate counting. All experiments were carried out at least in triplicate from independent pre-cultures. 2.2. Analytical determinations of fermented SJ medium The 10139106035 and 10176290035 enzymatic kits (RBiopharm Boehringer–Mannheim, Germany) were used for the determination of the residual glucose and fructose contents and for the production of ethanol, respectively, at the time points indicated during fermentation. 2.3. Cell lipid extraction Cells were collected at days 1, 3, 7 and 20 by centrifugation (5 min at 625 × g), washed in sterile water and freeze dried. The pellets were powdered in a mortar, weighted and subjected to lipid extraction. This was performed according to Taylor and Parks (1978) modified as described by Belviso et al., (2004). Pentadecanoic acid in chloroform was added as internal standard. The lipid extract was dried in a rotary evaporator (Rotovapor, Laborota 4000, Heidolph Instruments GMBH & Co KG, Schwabach, Deutschland) and dissolved in 5 ml chloroform for storage at − 25 °C for no longer than 2 weeks. Fatty acids and sterols contents were referred to the dry weight of freeze dried cells. 2.4. Determination of cellular fatty acid content The methyl esters of the fatty acids contained in lipid extract prepared as described above, were obtained according to Christie (1982), and then analyzed by gas chromatography with a DANI GC 1000 DPC, equipped with an FID detector (DANI Instruments s.p.a., Milan, Italy) on a DB-5 capillary column (30 m length, 0.25 mm i.d., 0.25 μm film thickness) (J&W Scientific Inc., Folson, CA, USA). The operating conditions were: temperature from 80 °C to 120 °C at 4 °C min− 1, from 120 °C to 220 °C at 5 °C min− 1, from 220 °C to

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280 °C at 7 °C min− 1, 280 °C for 10 min; injector temperature: 290 °C; detector temperature: 290 °C; carrier gas: nitrogen; carrier gas flow: 1.0 ml min− 1; injection volume: 2 μl. The results were expressed as mg (g dry weight of cells)− 1.

2.7. Chemicals and reagents

2.5. Determination of cellular sterols

3. Results and discussion

Sterols contained in lipid extract prepared as described above, were analysed according to Xu et al., (1988). One milliliter of each lipid extract was dried in a rotary evaporator (Rotovapor) and then dissolved in 1 ml toluene. The lipids were saponified with 4 ml 10% KOH in 90% ethanol at room temperature overnight. After the addition of 10 ml water, the unsaponified fraction was extracted three times by gently shaking for 3 min with 15 ml diethyl ether. The organic phase was dried in a rotary evaporator (Rotovapor, Laborota 4000, Heidolph Instruments GMBH & Co KG, Schwabach, Deutschland), and then dissolved in isopropanol. Ergosterol, lanosterol and squalene were detected by HPLC (Jasco PU-980, equipped with a Jasco UV-2075 Plus detector, Jasco International CoLtd, Tokyo, Japan) on a Supelco LC-18 column (15 cm × 4.6 mm; particle size 5 μm) (Supelco, Sigma–Aldrich, St Louis, MO, USA). The operating conditions were: methanol:water mobile phase of 96:4 (v/v), 1 ml min− 1 flow rate, 20 μl injection volume, and detection at λ 205 nm. The results were expressed as mg (g dry weight of cells)− 1.

3.1. A decrease in membrane integrity is observed during fermentation under unfavourable conditions

2.6. Flow cytometry A Coulter Epics XL Flow Cytometer (Beckman Coulter Inc, Miami, FL, USA) was used that was equipped with a 15 mW air-cooled argon-ion laser (emission, 488 nm) and five sensors for the detection of forward and side light scatter, green (525 nm, channel 1), yellow (575 nm, channel 2) and orange-red (620 nm, channel 3) fluorescence. Size calibration was performed using the flow cytometry size-calibration kit F13838 (Molecular Probes, Inc., Eugene, OR, USA). Yeast samples were harvested, washed and re-suspended in PBS (8.00 g l− 1 NaCl, 0.20 g l− 1 KCl, 1.44 g l− 1 Na2HPO4, 0.24 g l− 1 KH2PO4, pH 7.4) to the final concentration of 1–2 × 106 cells ml− 1 (OD600 0.08–0.20). Cell suspensions were stained according to the following procedures, with at least 1.5 × 104 cells for each experiment analysed. Propidium iodide (PI) staining: 10 μl PI stock solution in PBS (1 mg ml− 1) were added to 1 ml cell suspension just prior to the analysis. Fluorescence was detected in fluorescence channel 3. Nile Red (NR) staining: the stock solution (1 mg ml− 1 in acetone, stored in the dark at−20 °C) was diluted to a working solution of 10 μg ml− 1. Ten microliters of this working solution was added to 1 ml cell suspension, and the samples were gently vortexed and analyzed immediately. Nile Red emission was detected in fluorescence channel 2 and related to neutral lipids content as indicated by Greenspan et al., (1985). Data were visualised by means of the WinMDI flow cytometry software (Joseph Trotter, Salk Institute for Biological Studies, La Jolla, CA, USA).

Unless otherwise stated, all chemicals and reagents were from Sigma–Aldrich, St Louis, MO, USA.

The kinetics of growth and fermentation of three strains of S. cerevisiae were analysed during static incubation in SJ, a synthetic medium which lacks lipid nutrients and mimics the composition of grape must for sugar and nitrogen contents. The three strains underwent an early arrest of cell division and reached a maximum of about 2 × 107 CFU ml− 1 (Fig. 1). However, significant differences were observed among them regarding viability. M25 showed a marked decrease in viable plate counting and reached the lowest number of viable cells at day 25 (1 × 104 UFC ml− 1 Fig. 1). L2056 and the laboratory strain BY4743 maintained a similar and higher percentage of viable cells throughout the fermentation process. Moreover, even though all of them underwent stuck fermentation after day 20, differences in fermentative capability were observed (Fig. 2). L2056 was the most efficient both in sugar consumption and ethanol production (Fig. 2). BY4743, as expected from a laboratory strain, yielded the lowest amount of ethanol. M25, notwithstanding the low number of viable cells throughout fermentation, produced more ethanol than BY4743 (Fig. 2). Ethanol is a stress factor for wine strains growing under fermentative conditions (Piper, 1995). It modifies the polarity of membranes and the hydration of polar head-groups of membrane surfaces (plasma membrane and organelles), thus affecting the efficiency of membrane functions, e.g. uptake of nutrients and excretion of ethanol (Ingram, 1976; Thomas and

Fig. 1. Growth kinetics of the yeast strains used: viable plate counting of cells inoculated in SJ medium and incubated statically at 20 °C. Error bars represent standard deviations; where not seen, they lie within the symbol (n = 3).

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(Fig. 3). This did not seem to be related to the final ethanol concentrations. In fact, L2056 which produced the highest level of ethanol showed the lowest percentage of permeable cells. M25 showed the highest percentage of permeable cells even though producing less ethanol than L2056. BY4743 produced the lowest amount of ethanol and showed an intermediate level of membrane permeability (Fig. 3). Interestingly, permeability to PI , which according to Marza et al., (2002) shows a good correlation with cell death, was generally lower than expected on the basis of viable plate counting. This discrepancy, particularly evident in M25 which maintained the ability to ferment sugars up to day 20, notwithstanding the low viable count, suggests a shift to a viable but un-culturable state, not unusual at the end of wine fermentation (Divol and LonvaudFunel, 2005). However, as the rate of fermentation depends on both the total viable biomass and the rate of sugar utilization of single cells (Monteiro and Bisson, 1991), also a high fermentative capability per viable cell could be involved in ethanol production in M25. 3.2. Fatty acids composition influences cell viability and membrane integrity Changes in the composition of cell lipid fraction can influence the activity of many membrane-associated proteins and transporters thus potentially leading to growth arrest and cell death (Vigh et al., 1998). To assess whether the observed differences in viability and membrane integrity could be related to quantitative and qualitative cell lipid composition, these were analysed in the three strains at different time points (Figs. 4 and 5, Table 1). L2056, whose viability and membrane integrity at day 20 were higher than those of M25 and similar to those of BY4743, showed the lowest Total Fatty Acids (TFAs, sum of palmitic plus stearic plus oleic plus palmitoleic acids) (Fig. 4). Fig. 2. Fermentation kinetics: sugar consumption (A) and ethanol production (B) were evaluated during the progression of fermentation. Error bars represent standard deviations; where not seen, they lie within the symbol (n = 3).

Rose, 1979; Mishra and Prasad, 1988; Alexandre et al., 1994; Mishra and Kaur, 1991). Moreover, at concentrations above 15 g l− 1, it leads to cell death by increasing membrane permeability (Marza et al., 2002). One of the main factors affecting yeast survival in the presence of ethanol is the ability to modulate the lipid composition of cell membranes (Thomas et al., 1978). To assess whether the observed decrease in cell viability of M25 could be related to an increase in membrane permeability caused by ethanol, a flow cytometric analysis of propidium iodide (PI) stained cells sampled during SJ fermentation was carried out (Fig. 3). PI is known to stain nucleic acids in cells characterised by a defective membrane integrity and is widely utilized to measure membrane permeability (Deere et al., 1998; Marza et al., 2002). The three strains maintained a good level of membrane integrity until day 7 but showed a significant increase in the percentage of cells with permeable membranes at day 20

Fig. 3. Cell membrane permeability: PI-stained cells were analysed by flow cytometry during the progression of fermentation. Error bars represent standard deviations (n = 4).

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content of palmitoleic acid in this last strain, suggests that in these strains the steps of fatty acids biosynthesis are differently regulated downstream of palmitic acid. Moreover, these differences are expressed during logarithmic growth; indeed, the observed changes in the qualitative compositions of fatty acids were negligible after day 3. Interestingly, after day 7, when cell division stopped, L2056 slightly increased TFAs

Fig. 4. Cellular fatty acid content during fermentation: (A) TFAs (palmitic + palmitoleic + oleic + stearic acids), (B) C16/TFA ratio [(palmitic + palmitoleic acids) (palmitic + palmitoleic + stearic acid + oleic acid)− 1]; (C) UFA / TFA [(oleic + palmitoleic acids) (palmitic + palmitoleic + stearic acid + oleic acids)− 1]. Error bars represent standard deviations; where not seen, they lie within the symbol (n = 3).

M25, which was the least viable and showed the lowest membrane integrity, harboured a high content of TFAs (Fig. 4). In particular, at day 1 it showed a significant increase in TFAs, mainly due to stearic acid, the main fatty acid in this strain (Table 1). BY4743, which exhibited the highest viability and a membrane integrity higher than M25, harboured the highest TFAs content during active growth (Fig. 4). The high content of SFAs (mainly stearic acid) observed in M25, and the predominance of C16 in L2056 and BY4743, with a very high

Fig. 5. Cellular sterol content during fermentation: (A) Ergosterol; (B) Lanosterol; (C) Squalene. Error bars represent standard deviations; where not seen, they lie within the symbol (n = 3).

I. Mannazzu et al. / International Journal of Food Microbiology 121 (2008) 84–91 Table 1 Fatty acid composition of cells Day

M25

L2056

BY4743

0 1 3 7 20 0 1 3 7 20 0 1 3 7 20

C16:0

C16:1

C18:0

C18:1

Mean ± SD

Mean ± SD

Mean ± SD

Mean ± SD

5.15 ± 1.63 10.49 ± 3.32 7.80 ± 2.47 5.72 ± 1.81 4.40 ± 1.39 1.12 ± 0.28 1.39 ± 0.00 2.20 ± 0.78 1.91 ± 0.01 2.03 ± 0.09 9.21 ± 1.22 9.63 ± 4.30 9.49 ± 2.01 9.57 ± 0.00 1.66 ± 0.11

12.92 ± 5.70 8.96 ± 2.71 3.85 ± 1.16 4.71 ± 1.43 1.83 ± 0.30 6.52 ± 0.26 4.56 ± 0.00 1.73 ± 0.47 1.76 ± 0.04 2.29 ± 0.24 23.26 ± 3.15 25.01 ± 9.74 17.49 ± 3.01 15.77 ± 0.00 3.33 ± 0.56

3.63 ± 1,02 27.79 ± 7.84 12.53 ± 3.53 8.85 ± 2.50 9.22 ± 2.78 0.32 ± 0.05 0.23 ± 0.00 0.46 ± 0.18 0.52 ± 0.11 0.87 ± 0.00 4.22 ± 0.22 4.85 ± 2.46 5.49 ± 0.98 8.41 ± 0.00 1.29 ± 0.12

7.83 ± 3.49 3.84 ± 1.03 1.27 ± 0.34 2.69 ± 0.73 1.77 ± 0.17 3.28 ± 0.17 1.38 ± 0.00 0.97 ± 0.46 0.66 ± 0.02 1.09 ± 0.01 15.06 ± 1.52 7.88 ± 3.06 5.53 ± 1.08 5.06 ± 0.00 1.40 ± 0.22

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was significantly different in the three strains. In particular, it was higher and comparable in L2056 and BY4743 and significantly lower in M25 starting from day one. The impact of UFA/TFA ratio on ethanol tolerance is rather controversial. del Castillo Agudo (1992) reported that ethanol tolerance correlates with a decrease in fatty acids unsaturation index while other authors demonstrated that a high UFA/TFA correlates with ethanol tolerance (Mishra and Kaur, 1991; Alexandre et al., 1994; Chi and Arneborg, 1999; You et al., 2003). Our data, which were obtained on cells which gradually develop ethanol tolerance during fermentation, agree with the second hypothesis and suggest that the low unsaturation index shown by M25 during the fermentation process could have contributed to the decrease in viability and membrane integrity. Thus the observed changes in C16/TFA and UFA/TFA ratios could play a main role in the differences in viability and membrane integrity observed in the three strains.

Data are expressed as mg (g dry weight)− 1; SD = standard deviation (n = 3).

3.3. Ergosterol content is not essential for cell viability content while BY4743 decreased it dramatically. The two strains reached a comparable final level of TFAs at day 20, which was significantly lower than that of M25. Thus, a high final content of TFAs was not useful for cell viability and membrane integrity. The three strains modulated TFAs content but only L2056 and BY4743 were able to reach a fatty acids level which might be optimal for a successful adaptation to increasing ethanol concentrations. Thomas et al., (1978) have reported that in the presence of ethanol cell viability is related to the presence of ergosterol and of specific fatty acids in plasma membranes. Oleic acid seems to be the most important UFA in counteracting the toxic effects of ethanol through its effect on plasma membrane fluidity (You et al., 2003). However, also the presence of shorter (C16) monounsaturated fatty acids seems to lead to ethanol tolerance due to the possibility to allocate ethanol molecules in the hydrophobic core of the membranes (Thomas and Rose, 1979). Thus, to elucidate the role of different fatty acids on viability and membrane integrity, their relative abundance on TFAs were analyzed. As shown in Fig. 4 while L2056 and BY4743 increased the C16/TFAs ratio in the first 72 h and maintained it unvaried and at similar levels until the end of fermentation, M25 exhibited the opposite behaviour and took this ratio at a significantly lower level during active growth and at the end of the fermentation process. Thomas et al. (1978) hypothesized that the increase in the relative content of C16 increases ethanol tolerance. Accordingly L2056 and BY4743 were more viable and their membrane were significantly less permeable of that of M25 at day 20. Thus, the higher C16/TFA ratio observed in L2056 and BY4743 could be involved in a better adaptation of these two strains to stressful conditions of fermentation. The impact of the unsaturation index (UFA/TFA ratio) on ethanol tolerance was also analysed. Possibly due to a progressive oxygen depletion, the three strains showed a decrease of the unsaturation index in the first three days after which it remained unvaried. However, the unsaturation index

Ergosterol, the main sterol in yeast, is involved in membrane integrity and fluidity and the activity of membrane bound enzymes (Parks and Casey, 1995). Its biosynthesis is reduced in the presence of ethanol and under oxygen depletion (Shobayashi et al., 2005). The three strains differed markedly for ergosterol content (Fig. 5). In particular, BY4743 was very rich in ergosterol throughout the fermentation process, while L2056 was characterized by the lowest ergosterol content from day 3 on. In agreement with the repressive effect of ethanol on ergosterol biosynthesis (Shobayashi et al., 2005) ergosterol content was inversely related to the final ethanol concentration. However, similarly to what observed for TFAs, a high content of ergosterol was not essential for cell viability and membrane integrity during fermentation.

Fig. 6. Flow cytometry of NR-stained cells. Cells sampled at the indicated times were stained with NR and analysed by flow cytometry. Fluorescence measured in channel 2 was normalized for Forward Scatter to minimize the effect of cell size. Fluorescence intensity provides an indirect measure of the neutral lipids content. Data are representative of two independent experiments.

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The three strains differed also for de novo sterol biosynthesis. In fact, after a transient increase of ergosterol at day 1, M25 and L2056 decreased it dramatically during exponential growth and kept it at low levels until the end of fermentation. The impairment of ergosterol biosynthesis observed in these two strains was supported by an increase in squalene and lanosterol contents, respectively. On the contrary, BY4743 maintained the ability to synthesise ergosterol until the end of fermentation. Accordingly, L2056 and M25 showed a rapid increase of neutral lipid associated fluorescence (Fig. 6). Fluorescence intensity of NR stained cells provides an indirect measure of the neutral lipid contents (Greenspan et al., 1985). Neutral lipids are mainly steryl esters and triacylglycerols accumulated in form of lipid granules (Zweytick et al., 2000). These last represent a cellular depot for sterols and fatty-acids that can be recycled for membrane biogenesis during growth resumption (Valachovich et al., 2001). Moreover, the accumulation of lipid granules may be considered as a survival mechanism involved in the detoxification of excess sterols, sterol precursors and/or fatty acids, which could cause membrane perturbations (Müllner and Daum, 2004). The delayed increase in neutral lipid associated fluorescence in BY4743 suggests that this strain, which needs to activate the accumulation of lipid granules by the end of fermentation, is capable of a balanced lipid biosynthesis for a longer time. This might be due to a better utilization of oxygen dissolved in the growth medium. In conclusion, a comparison of M25 with L2056 and BY4743 leads to hypothesise that C16/TFA and UFA/TFA ratios influence viability and membrane integrity, that conversely do not depend from TFAs and/or ergosterol content. Thus, even though the three strains were able to modulate TFAs composition during SJ fermentation, only L2056 and BY4743 reached a fatty acids composition which might be optimal for a successful adaptation to increasing ethanol concentrations. M25, that showed the lowest viability and membrane integrity at day 20, was characterized by the lowest UFA/TFA and C16/TFA. This seemed the most unfavourable lipid composition for survival during growth under unfavourable conditions. Acknowledgments The authors wish to thank Sandro Annese, Eleonora Bertolone and Giacomo Zara for their useful discussion and critical reading of the manuscript. The work was financially supported by MURST PRIN Anno 2003-Prot. N 2003077174. D.A. received a grant from Enologica Fenocchio s.n.c., Grottammare (AP). S.B. received a grant from MURST PRIN Anno 2003-Prot. N 2003077174. References Aguilera, F., Peinado, R.A., Millàn, C., Ortega, J.M., Mauricio, J.C., 2006. Relationship between ethanol tolerance, H+-ATPase activity and the lipid composition of the plasma membrane in different wine strains. International Journal of Food Microbiology 110 (1), 34–42.

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