Membrane Lipid Variability in Saccharomyces cerevisiae Wine Strains

Article pubs.acs.org/JAFC

Membrane Lipid Variability in Saccharomyces cerevisiae Wine Strains Rehydrated in the Presence of Metabolic Activators Patricia Díaz-Hellín,† Sergio Gómez-Alonso,† Anna Borrull,§ Nicolas Rozès,§ Ricardo Cordero-Otero,§ and Juan Ú beda*,† †

Tecnologı ́a de Alimentos, IRICA, Universidad de Castilla-La Mancha, Edificio Marie Curie, Avenida Camilo José Cela 10, 13071 Ciudad Real, Spain § Departament de Bioquı ́mica i Biotecnologia, Universitat Rovira i Virgili, Marcel·lı ́ Domingo s/n, 43007 Tarragona, Spain ABSTRACT: Slight variations in lipid composition of wine yeast membranes can alter some essential functions including selective nutrient transport and ion permeability. The absence of oxygen during alcoholic fermentation inhibits fatty acid desaturation and sterol biosynthesis, thereby reducing the stress resistance of yeast cells. In this work, membrane lipids in two commercial active dry yeast strains rehydrated in the presence of three activators (ergosterol, tetrahydrofolic acid, and manganese) were studied. Each was assayed at three different concentrations. The effect of these activators on the phospholipid, neutral lipid, and fatty acid contents in cell membranes was assessed. Also, cell viability and fermentation kinetics were determined. Ergosterol was found to shorten the lag phase and improve cell viability and membrane lipid composition; tetrahydrofolic acid raised neutral lipid levels; and manganese(II) increased cell viability and modified phospholipid composition and linoleic acid concentration. All activators interacted with yeasts in a strain-dependent way. KEYWORDS: Saccharomyces cerevisiae, membrane lipids, fatty acids, active dry yeasts, metabolic activators

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these modifications includes reducing membrane fluidity and altering some essential functions (particularly selective transport of sugars and amino acids).8 Sterols take part in the construction and maintenance of eukaryotic membranes by regulating their permeability and fluidity in addition to their ethanol resistance and plasma H+ATPase activity.9 It should be pointed out that ergosterol plays a different role depending on its concentration in the medium.10 Thus, trace contents of this sterol help trigger cell replication, whereas high levels enable active growth and membrane synthesis. The remarkable enrichment of the plasma membrane in ergosterol parallels the preferential location of cholesterol in the plasma membrane of animal cells.11 Secretory vesicles are second highest in ergosterol concentration, suggesting that these vesicles contribute to the flow of ergosterol from the endoplasmic reticulum to the plasma membrane.12 On the other hand, Soubeyrand et al.13 found the in situ addition of sterols during rehydration enhances the fermentation capacity of yeasts. Several lipids, which have an important rol in membrane fluidity, exhibit dramatic differences in membrane concentration in a temperature-dependent manner.14 Phospholipids (particularly phosphatidylcholine, phosphatidylethanolamine, and, to a lesser extent, phosphatidylserine and phosphatidylglycerol) improve membrane fluidity.15 The unsaturation degree of fatty acids largely influences the physical properties of the cell membrane.14,16 Oleic (C18:1), palmitoleic (C16:1), palmitic (C16:0), and stearic (C18:0) are

INTRODUCTION Wine yeasts suffer osmotic, thermal, nutritional, anaerobic, and toxic stress during alcoholic fermentation. Nutritional and osmotic stresses typically occur in high-sugar musts with poorly available nitrogen, whereas anaerobic−toxic stresses take place in the presence of ethanol and short- and medium-chain fatty acids. Those stressing conditions can lead to stuck or sluggish fermentations. Inoculation with active dry yeasts (ADYs) is a common winemaking practice. On rehydration, ADYs recover their ability to ferment sugars in the must. The dehydration− rehydration process is cell-damaging; thus, cells lose nucleotides, ions, and most soluble cellular components.1 However, most rehydrated cells keep their reproductive ability if they are grown under optimum conditions after rehydration.2 ADY manufacturers address this problem by marketing “fermentation activators”, including growth factors, which affect cell replication, and survival factors, molecules that yeasts are unable to produce under anaerobic conditions.3 The plasma membrane of Saccharomyces cerevisiae is about 7.5 nm thick and exhibits cytoplasmic invagination in some regions. The lipid composition of this membrane strongly influences its tolerance to various types and/or levels of stress, as well as its resistance to toxic compounds such as ethanol, acetic acid, acetaldehyde, and medium-chain fatty acids.4 In addition, lipid composition of the plasma membrane plays a prominent role in the adaptive response of cells.5 Small changes in it can alter some essential functions such as ion permeability or transport.6,7 Some environmental factors (e.g., temperature, oxygen availability), growth rate, and the presence of sterols can modify the lipid bilayer and/or unsaturated fatty acid composition of yeast cell membranes during alcoholic fermentation. The effect of © 2014 American Chemical Society

Received: Revised: Accepted: Published: 8679

February 20, 2014 July 4, 2014 July 9, 2014 July 9, 2014 dx.doi.org/10.1021/jf500895y | J. Agric. Food Chem. 2014, 62, 8679−8685

Journal of Agricultural and Food Chemistry

Article

the main fatty acids in cell membranes.17 Besides, fluidity is also governed by the packing degree of these fatty acids. Thus, the less unsaturated and long-chained a fatty acid is, the tighter it packs and the less disordered and fluid is the resulting structure. By contrast, the more unsaturated the fatty acid is, the greater is the fluidity of the membrane and the more easily it can adapt to adverse conditions such as a high ethanol concentration or low temperature during fermentation.18 Consequently, the correlation of unsaturated fatty acid content with ethanol tolerance is potentially due to increased membrane fluidity. S. cerevisiae yields saturated and monounsaturated fatty acids of 16 and 18 carbon atoms, but it is unable to produce polyunsaturated acids (PUFAs).19 Nonpolar lipids such as triacylglycerides (TAGs) provide an energy pool for the synthesis of membrane lipids20 and also serve as precursor pool for phospholipids. The absence of oxygen during alcoholic fermentation suppresses fatty acid desaturation and sterol biosynthesis, thereby reducing the ability of cells to adapt to the dramatic environmental changes involved in the process.21 Dı ́az-Hellı ́n et al.22 found rehydration of some commercially available yeast strains with different metabolic activators, such as ergosterol or ascorbic acid, to increase their fermentation capacity and sugar consumption. Ergosterol is a cell membrane component that enhances membrane plasticity under hyperosmotic conditions and thus favors nutrient transport;23 tetrahydrofolic acid plays a role in energy-generating reactions through the biochemical oxidation of carbohydrates, fats, and proteins; and finally manganese(II) is a component of certain ATP-dependent coenzymes and reactions. The main purpose of this work was to study the membrane lipid composition of two commercial active dry wine yeast strains following rehydration in the presence of three metabolic activators: ergosterol, tetrahydrofolic acid (FH4), and Mn. The last one was selected instead of other oxidation states because it is involved in specific metabolic cycles. To this end, phospholipid (PL), neutral lipid (NL), and fatty acid (FA) compositions of yeasts cell membranes were determined. Yeast viability and fermentation kinetics were also measured.

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amino acids, 3 g/L malic acid, 2 g/L tartaric acid, and 0.5 g/L citric acid, at pH 3.7). The initial concentration of viable cells was 5 × 106 cells/mL. A calibration curve was obtained for cell counts using a Thoma chamber versus optical density (OD) (absorbance at 600 nm) using a spectrophotometer (V-530, JASCO, Tokyo, Japan). Tests were performed in microtitration plates and involved reading the OD at 600 nm at 20 min intervals until stationary growth was reached. Plates were read with a Polar Star Omega ELISA instrument (BMG Labtech GmbH, Ortenberg, Germany). Each determination was performed in octuplicate. Lipid Extraction from Rehydrated Yeast Cells. Prior to lipid extraction, a solution of 100 μL of methanol and 20 μL of 0.1 mM EDTA was added to the pellet of rehydrated yeast cells with 1 g of 0.5 mm glass beads (Biospec Products, Bartlesville, OK, USA) in Eppendorf tubes and then mixed for 5 min in a Mini-Beadbeater-8 (Qiagen, Hilden, Germany).24 Lipid extraction was performed in four steps: The first one involved extracting the organic layer with 200 μL of methanol and 600 μL of chloroform under stirring on an Orbit M60 vortexer for 1 h, followed by centrifugation at 5000 rpm for 1 min. This procedure was repeated three times with different extractant combinations: 300 μL of methanol plus 600 μL of chloroform, 450 μL of methanol plus 450 μL of chloroform, and 600 μL of methanol plus 300 μL of chloroform. The volume of organic layer extracted in each run was placed in a glass tube that was cleaned with one-fourth of the total extract volume of an 0.88% (w/v) KCl solution. This was followed by vortexing, cooling at 4 °C for 10 min, and centrifugation at 3000 rpm for 5 min. The organic phase was collected and finally concentrated to dryness under a nitrogen stream in a Supelco solidphase evaporator. The resulting residue was dissolved in a 2:1 (v/v) chloroform/methanol mixture and stored in an amber-colored vial with a glass insert. Separation and Quantification of Phospholipids (PLs) in Cell Membrane by High-Performance Thin Layer Chromatography (HPTLC). Phospholipids (phosphatidic acid (PA), phosphatidylethanolamine (PE), phosphatidylcholine (PC), and phosphatidylinositol plus phosphatidylserine (PI + PS)) were separated by one-dimensional HPTLC on silica gel plates (10 × 20 cm, 200 μm) (Merck, Darmstadt, Germany) with chloroform, acetone, methanol, glacial acetic acid, and water (50:15:10:10:5, v/v/v/v/v). Samples were loaded by using a Linomat semiautomatic applicator, and the mobile phase was held in a Camag semiautomatic cell. After the plate had been charred with 10% CuSO4 in 8% H3PO4 and heated at 180 °C for 4 min,25 PLs were identified using known standards: phosphatidic acid, phosphatidylethanolamine, phosphatidylcholine, phosphatidylinositol, and phosphatidylserine (Sigma). Calibration curves using standards of every phospholipid at a concentration of 0.5, 1, 2, or 4 μg/ μL were constructed. An image of the plate was acquired with an image scanner from Amersham Biosciences (Piscataway, NJ, USA). Each spot of the image was quantified with the software ImageJ (National Institutes of Health, Bethesda, MD, USA). All phospholipids were determined in triplicate. Separation and Quantification of Neutral Lipids (NLs) in Cell Membrane by Thin Layer Chromatography (TLC). NLs (squalene, sterol esters, lanosterol, ergosterol, and free fatty acids) were separated by one-dimensional TLC on silica gel plates (10 × 20 cm, 250 μm) from Merck. Each plate was developed in three steps with (1) 50:50 (v/v) methyl tert-butyl ether (MTBE)/glacial acetic acid, (2) 80:20:1 (v/v/v) hexane/MTBE/glacial acetic acid, and (3) pure hexane. Plates were processed as described in under Separation and Quantification of Phospholipids (PLs) in Cell Membrane by High-Performance Thin Layer Chromatography (HPTLC), and Sigma standards (squalene, cholesteryl oleate, diolein, triolein, lanosterol, ergosterol, and ethyl oleate) were used to quantify NLs: calibration curves were constructed from the application of standards to every plate at a concentration of 0.5, 1, 2, or 4 μg/ μL. Quantification of Fatty Acids (FAs) in Cell Membrane by Gas Chromatography (GC). Following rehydration, saturated fatty acids (SFAs; C10:0, C12:0, C14:0, C16:0, and C18:0) and unsaturated fatty acids (UFAs; C14:1, C16:1, C18:1, C18:2. and C18:3) were measured. Also, mean fatty acid chain length (ChL) was determined to ease the interpretation of the results. ChL was calculated according to the

MATERIALS AND METHODS

Yeast Strains and Metabolic Activators. Two different commercially available active S. cerevisiae ADYs designated L2 and L4 and three activators were selected because they improved the behavior of the studied yeasts.22 Half a gram of each yeast strain was rehydrated in 4.5 mL of physiological solution (0.9% w/v) at 35 °C for 30 min in the presence of 4.5 × 10−2 g/L ergosterol plus 3.0 × 10−2 g/L Tween 80 (used as emulsifier), 0.56 g/L tetrahydrofolic acid (FH4), or 1.51 g/L manganese(II) sulfate 1 Hidrate (Sigma). The activators were used at normal dose (N) in addition to half-dose (0.5N) and double dose (2N) for kinetics studies and viability. For lipid membrane assays, normal dose (N) and half-dose (0.5N) were used. Rehydrated cells in the presence of Tween 80 or 0.9% (w/v) physiological solution were used as controls for each treatment. Flow Cytometry Analysis. Following rehydration, viable cell counts were carried out in duplicate using flow cytometry on CyFlow SPACE equipment (Partec GmbH, Münster, Germany) fitted with a 22 mW ion laser for excitation (488 nm) while monitoring with a single emission channel (575 nm band-pass filter). FloMax software (Quantum Analysis GmbH, Münster, Germany) was used for instrument control, data acquisition, and data analysis. As control of full viabililty (99% by propidium iodide stain), an overnight YPD culture of each reference strain was used. Growth Kinetics. Growth kinetics was examined using a sterile synthetic must (125 g/L glucose, 125 g/L fructose, 4.15 g/L YNB with 8680

dx.doi.org/10.1021/jf500895y | J. Agric. Food Chem. 2014, 62, 8679−8685


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following equation: ChL = Σ (CinC,i)/ΣCi, where Ci corresponds to the “i” fatty acid concentration and nC,i is the number of carbon atoms in the mentioned acid. Sedimented cells (108 cells/mL) in sealed tubes with a Teflon-lined screw cap were saponified using 1 mL of 5% NaOH in 50% methanol/ water26 and placed in a dry bath (100 °C) for 30 min. After that, the saponified material was cooled at room temperature, and 2 mL of 6 M HCl and 1 mL of BCl3 were added to obtain methylated free fatty acids. Then, samples were heated at 80 °C in the water bath for 10 min. At that point, fatty acid methyl esters were extracted with 1 mL of hexane; samples were vortexed for 30 s, and the organic phase was collected after centrifugation (3000 rpm, 3 min). The extraction step was repeated again, and then the combined organic phase extract was washed with 5 mL of Milli-Q water to remove excess HCl. Finally, the organic extract was dried with anhydrous sodium sulfate (Na2SO4). Fatty acid methyl esters were separated on a gas chromatograph model 6890 equipped with a 7863 autosampler (Agilent, Santa Clara, CA, USA), a split/splitless injector, and a flame ionization detector (FID). The capillary column used (50 m long × 0.25 mm i.d.) was coated with a 0.25 μm thick film of SGL-1000 (acidified polyethylene glycol) stationary phase, (Sugelabor, Madrid, Spain). The carrier gas was helium at 1 mL/min, the injection volume 3 μL, and the split ratio 8:1. The injector and detector temperatures were both 250 °C. The temperature program was as follows: 100 °C (held for 3 min), 15 °C/min ramp to 210 °C, and a hold for 20 min. Heptanoic acid (20 mg/mL) and heptadecanoic acid (40 mg/mL) were used as internal standards. The amounts of fatty acids found were related to cell dry weights and expressed as percentages of total fatty acids. The determinations were performed in triplicate. Statistical Analysis. Changes in membrane lipid composition upon rehydration of the ADYs in the presence of the metabolic activators were checked by one-way analysis of variance (ANOVA) at the 95% significance level (p ≤ 0.05), using Duncan’s test (SPSS v. 17.0). Data were processed in triads (i.e., each sample was compared with its corresponding control).

Figure 1. Percentage of viability of L2 (black bars) and L4 (gray bars) strains after rehydration with ergosterol (Erg.) + Tween 80, FH4, and Mn(II) at half (0.5N), normal (N), and double (2N) doses. Controls: Tween 80 (rehydration with Tween 80) and control (rehydration with physiological solution). Values are means of n = 2 ± standard deviations. (∗) Significantly different from each control (p ≤ 0.05, n = 2).

L2 reached the stationary growth phase after 38 h of incubation and L4, after 83 h (data not shown). As viability, the lag time (T-Lag) was strain-dependent. Thus, L2 rehydrated in 0.5N ergosterol exhibited a shorter lag phase than both the control and the samples rehydrated with ergosterol at the other two doses (Table 1). It also affected the duplication rate, which was higher than the control and the other evaluated samples. Similar differences were observed in L4 rehydrated with a 0.5N or N dose of ergosterol. As can be seen from Table 1, μmax and Nmax for L4 (0.44 h−1 and 0.12, respectively) doubled the values for L2 (0.22 h−1 and 0.06, respectively), and none of the activators succeeded in increasing them in either strain. Rehydration in the presence of the tested activators had an effect on either μmax or Nmax; thus, the strain that started cell division later (L4) grew faster. This could be a result of the fact that the slower the yeast adaptation to the medium, the LAG phase, the better the fermentation kinetic in the LOG phase. It is noticeable that double doses (2N) did not give remarkable results compared to the other dosages, so it was decided to reject them for further assays. Phospholipids and Neutral Lipids. Overall, PL contents were higher in L2 than in L4 irrespective of the particular rehydration treatment (Figure 2). On the other hand, the NL content was invariably higher in L4 (Figure 3). The ergosterol treatment significantly increased the content in phosphatidylcholine and phosphatidylinositol−phosphatidylserine in the L2 membrane (Figure 2A), as well as those in all neutral lipids except lanosterol (Figure 3A). Worthy of special note is the accumulation of ergosterol in L2 treated with this activator and also the increase in sterol esters resulting from esterification of sterols. On the contrary, ergosterol had no influence on either the composition or the concentration of the L4 strain phospholipids, except for PA at 0.5N dose, for which concentration diminished significantly (Figure 2B). FH4 increased the contents in phospholipids and some neutral lipids (squalene, sterol esters, and lanosterol) of L2. By contrast, it caused a slight decrease in the PL concentration of L4 at half-dose (0.5N) and increased it at the normal dose (N), but never overcoming significantly the value of the control. Manganese(II) affected significantly neutral lipid content in L2 and FA, lanosterol, and squalene for L4. On the other hand, it decreased the contents in all PLs of L4 and only PA in L2 (Figures 2 and 3).

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RESULTS AND DISCUSSION Cell Viability. In a previous work, Dı ́az-Hellı ́n et al.22 found that rehydration of L2 in the presence of glutathione and glycerol increased cell viability in this yeast strain after 15 days of fermentation. Likewise, L4 exhibited an increase of viability after rehydration in the presence of tetrahydrofolic acid (FH4) and ascorbic acid. Some extrinsic variables in relation with the transport and conservation of the dry yeasts could influence this parameter. Dehydrated yeast can lose up to 30% of soluble cell compounds when rehydrated, which proves the nonfunctionality of the cell membrane.1 A faster reduction in nucleotides and trehalose leakage are therefore beneficial for the vitality of rehydrated yeast cells.2 As can be seen in Figure 1, L2 exhibited higher viability than L4 in all studied cases. This could be a consequence of a specific strain factor because viability percentages hardly vary compared with controls in both strains. By exception, ergosterol and manganese increased L2 viability in a statistically significant manner, whereas L4 exhibited significantly increased viability when rehydrated in the presence of manganese irrespective of its dose, contrary to what happened with ergosterol, which produced a significant decrease. Growth Kinetics. Besides the lag period and the asymptotic value, another valuable parameter of the growth curve is the maximum specific growth rate (μmax). Because the logarithm of the absorbance is used, μmax is given by the slope of the line when the organisms grow exponentially.27 In this work, the growth kinetics of the two yeast strains in terms of initial population (N0), maximum population (Nmax), specific growth rate (μmax), lag time (T-Lag), and duplication time (T-d) were examined. 8681



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Table 1. Growth Kinetics of L2 and L4 Strains Rehydrated with Ergosterol + Tween 80, FH4, or Mn(II) at Half (0.5N), Normal (N), and Double (2N) Doses Measured by Means of Optical Density at 600 nma N0b L2

Tween 80 ergosterol

0.5N N 2N

control FH4

0.5N N 2N 0.5N N 2N

Mn(II)

L4

−0.64 −0.70 −0.70 −0.74 −0.68 −0.67 −0.73 −0.69 −0.67 −0.73 −0.74

Tween 80 ergosterol

0.5N N 2N

control FH4

0.5N N 2N 0.5N N 2N

Mn(II)

μmax

Nmaxb

± ± ± ± ± ± ± ± ± ± ±

0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.03

−1.01 ± −1.00 ± −0.10 ± −0.99 ± −1.06 ± −1.04 ± −1.06 ± −1.00 ± −0.95 ± −1.00 ± −0.98 ±

0.01

± ± ± ± ± ± ± ± ± ± ±

0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01

0.12 ± 0.12 ± 0.09 ± 0.09 ± 0.10 ± 0.10 ± 0.11 ± 0.09 ± 0.10 ± 0.10 ± 0.10 ±

0.01

0.06 0.04 0.07 0.04 0.05 0.06 0.06 0.08 0.06 0.07 0.08

0.02 0.01 0.01 0.01 0.01 0.01 0.01 0.02 0.02

0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01

T-Lag

± ± ± ± ± ± ± ± ± ± ±

0.05 0.04 0.04 0.04 0.05 0.04 0.04 0.04 0.04 0.04 0.04

0.44 ± 0.35 ± 0.32 ± 0.44 ± 0.39 ± 0.41 ± 0.44 ± 0.45 ± 0.39 ± 0.40 ± 0.37 ±

0.03

0.21 0.16 0.20 0.23 0.22 0.20 0.22 0.19 0.20 0.22 0.23

6.59 1.82 4.90 5.61 6.03 6.39 5.52 5.22 6.39 6.03 6.13

± ± ± ± ± ± ± ± ± ± ±

10.8 ± 7.95 ± 6.86 ± 10.1 ± 9.24 ± 9.67 ± 10.3 ± 11.8 ± 8.56 ± 10.6 ± 7.59 ±

0.03 0.03 0.02 0.02 0.02 0.03 0.02 0.02 0.02

T-d

0.70 0.48 0.95 0.51 0.60 0.78 0.63 0.15 0.78 0.45 0.28

± ± ± ± ± ± ± ± ± ± ±

0.16 0.20 0.15 0.14 0.15 0.15 0.14 0.16 0.15 0.12 0.11

1.57 ± 1.99 ± 2.17 ± 1.57 ± 1.79 ± 1.69 ± 1.57 ± 1.55 ± 1.78 ± 2.00 ± 1.89 ±

0.04

3.23 4.42 3.43 3.07 3.09 3.44 3.14 3.67 3.44 3.16 2.99

0.3 0.58 0.3 0.29 0.26 0.2 0.2 0.32 0.4 0.36

0.06 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04

a

Controls: Tween 80 (rehydration with Tween 80) and control (rehydration with phisiological solution). N0, initial population, Nmax, maximum population, μmax, maximum specific growth rate (h−1), T-Lag, lag time (h); T-d, duplication time (h). Values are means of n = 8 ± standard deviation. bLogarithmic scale.

Table 2. Percentage Composition of Fatty Acids (C14:1, C16:0, C16:1, C18:0, C18:1, C18:2, and C18:3) in L2 and L4 Strains Rehydrated with Ergosterol + Tween 80 (A1), FH4 (A2), and Mn(II) (A3) at Half (0.5N) and Normal (N) Dosesa C14:1 L2

Tween 80 ergosterol control FH4 control Mn(II)

L4

L4


0.5N N 0.5N N 0.5N N

0.5N N 0.5N N 0.5N N

C16:0

C16:1

C18:0

C18:1

C18:2

C18:3

0.11 0.15 0.08 0.06 0.07 0.11 0.06 0.07 0.08

± ± ± ± ± ± ± ± ±

0.04 0.05 0.01 0.01 0.00 0.01b 0.01 0.01 0.01

16.0 16.6 16.5 16.8 16.4 17.0 16.8 17.1 17.0

± ± ± ± ± ± ± ± ±

1.1 0.4 0.3 0.6 0.6 0.5 0.6 0.9 0.1

39.7 37.1 41.8 38.4 39.9 39.5 38.4 37.3 37.7

± ± ± ± ± ± ± ± ±

0.9 1.1 2.0 3.7 1.5 0.8 3.7 0.6 1.7

6.77 7.43 5.82 8.31 7.46 6.79 8.31 8.00 7.65

± ± ± ± ± ± ± ± ±

0.22 0.87 0.56 1.52 0.64 0.59 1.52 0.77 0.85

30.6 31.7 29.1 29.2 28.6 29.2 29.2 30.4 30.5

± ± ± ± ± ± ± ± ±

1.1 0.5 1.3 1.7 0.9 0.5 1.7 0.4 1.0

5.56 5.71 5.53 6.00 6.26 5.99 6.00 6.01 6.01

± ± ± ± ± ± ± ± ±

0.17 0.49 0.21 0.17 0.48 0.31 0.17 0.23 0.20

0.49 0.35 0.43 0.39 0.41 0.48 0.39 0.50 0.27

± ± ± ± ± ± ± ± ±

0.01 0.06b 0.02 0.10 0.07 0.03 0.10 0.00 0.02

0.00 0.28 0.25 0.13 0.16 0.19 0.13 0.06 0.08

± ± ± ± ± ± ± ± ±

0.00 0.01 0.15 0.06 0.01 0.05 0.06 0.02 0.01

21.2 20.5 19.8 19.2 19.4 18.4 19.2 20.4 20.7

± ± ± ± ± ± ± ± ±

2.5 1.0 1.2 1.4 2.2 1.1 1.4 1.0 0.2

55.5 49.2 48.0 48.5 51.3 55.6 48.5 50.2 49.8

± ± ± ± ± ± ± ± ±

8.7 2.8 2,4 3.6 4.9 0.7 3.6 4.5 0.3

6.61 5.30 6.89 6.08 5.84 5.20 6.08 5.82 5.66

± ± ± ± ± ± ± ± ±

0.56 0.33b 0.89 0.36 0.45 1.13 0.36 1.24 0.28

21.7 23.3 23.1 22.5 22.9 21.0 22.5 23.3 23.5

± ± ± ± ± ± ± ± ±

0.5 1.8 1.6 0.2 3.2 0.2 0.2 2.3 0.2

0.32 0.38 0.41 0.03 0.24 0.31 0.03 0.00 0.17

± ± ± ± ± ± ± ± ±

0.05 0.11 0.28 0.05 0.03b 0.05b 0.05 0.00 0.02b

0.26 0.10 0.12 0.23 0.46 0.54 0.23 0.13 0.13

± ± ± ± ± ± ± ± ±

0.11 0.02 0.02 0.11 0.13 0.04 0.11 0.02 0.02

Controls: Tween (rehydration with Tween 80) and control (rehydration with physiological solution). Values are means of n = 3 ± standard deviation. bStatistically significant differences of the treated sample with respect to its control (one-way ANOVA; p ≤ 0.05; n = 3).

a

stated above, this compound was the most efficient activator for both yeast strains. Tween 80 had an unexpected effect: whereas L2 was unaffected by its presence, L4 exhibited increased amounts of all neutral lipids (Figure 3). Fatty Acid Composition. The composition of plasma membrane is especially relevant to the production of industrial

On the basis of the kinetic study results, T-Lag (Table 1), ergosterol (0.5N) was the most efficient activator. Therefore, its presence can have a beneficial effect on both yeasts. Ergosterol has been found to accumulate in cells during the initial fermentation stage;28 also, it is added together with some amino acids and vitamins to some yeast preparations to accelerate fermentation.29 As 8682



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Figure 2. Concentrations (μg/mg dry weight) of phosphatidic acid (PA), phosphatidylethanolamine (PE), phosphatidylcholine (PC), and phosphatidylinositol + phosphatidylserine (PI + PS) determined by TLC in L2 (A) and L4 (B) strains. Treatments are defined in the legend. Controls: (black bars) Tween 80 (rehydration with Tween 80); (slashed bars) control (rehydration with physiological solution). Values are means of n = 3 ± standard deviations. (∗) Significantly different from each control (p ≤ 0.05, n = 3).

Figure 3. Concentration (μg/mg dry weight) of squalene, sterol esters, triacylglycerides (TAG), lanosterol, ergosterol, and fatty acids (FA) determined by TLC in L2 (A) and L4 (B) strains. Treatments are defined in the legend. Controls: (gray bars) Tween 80 (rehydration with Tween 80); (slashed bars) control (rehydration with physiological solution). Values are means of n = 3 ± standard deviations. (∗) Significantly different from each control (p ≤ 0.05, n = 3).

specific strain factor. Numerous extrinsic variables in relation with the transport and conservation of the dry yeasts could influence this parameter, too. In general, rehydration in the presence of the tested molecules caused no substantial change in cell viability, which suggests that maybe yeasts require a longer exposure time. Nevertheless, ergosterol was proved to be the most efficient of the three activators assayed. On the basis of the kinetic results, 0.5N dose reduced the lag time of both yeast strains. It also improved cell viability and membrane lipid composition; tetrahydrofolic acid raised neutral lipid levels, and manganese(II) increased cell viability and modified phospholipid composition and linoleic acid concentration. All activators interacted with yeasts in a strain-dependent way. As is already known, the content of membrane fatty acids is strain-dependent; the activators had little impact on such proportions. L2 cell membranes had higher contents in C18 fatty acids (stearic, oleic, linoleic, and linolenic) than L4 cell membranes. Ergosterol treatment resulted in cellular accumulation of this lipid but only in L2. A quantitative lipid analysis revealed the presence of not only free but also esterified ergosterol (as sterol esters). This would make yeasts more robust and efficient in industrial processes under osmotic and hydric stress conditions than they are in alcoholic fermentation.13 It should be noted that the target parameters were assessed on freshly rehydrated yeasts. Taking into account that the

yeast strains in submerged stirred cultures; thus, enriching cell membranes with linoleic acid (C18:2) and ergosterol has been found to improve their ethanol tolerance.30 Table 2 lists the proportions of membrane fatty acids in the yeasts after rehydration with the different activators. Overall, the activators had little impact on such proportions; nevertheless, L2 membranes had higher contents in C18 fatty acids (stearic, oleic, linoleic, and linolenic) than L4 membranes irrespective of the particular treatment; therefore, this variable is strain-dependent because already it is known. By contrast, L4 contained greater amounts of C16:0 and C16:1. Rehydrating with manganese(II) (N) or FH4 at any dose significantly increased the content in C18:2 of L4. In the same manner, the N dose of FH4 increased C14:1 of L2. By contrast, the presence of ergosterol decreased the content in C18:3 of L4. As can be seen in Table 3, the contents in SFAs and UFAs were similar (i.e., treatment-independent) in all samples, with no significant differences among activators or doses. Nevertheless, L2 showed a lesser SFA and a higher UFA content than L4. Moreover, mean chain lengths (ChL) were similar in both strains. Besides, only ergosterol (N dose) caused a statistically significant change (a decrease) in ChL and exclusively in L2. In summary, metabolic activator treatment modified (increased or decreased) the production of fatty acids in both yeasts, but failed to alter their proportions. Both strains showed different viabilities. L2 exhibited the highest viability in all cases. This could be a consequence of a 8683



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(7) Hazel, J. R.; Williams, E. E. The role of alterations in membrane lipid composition in enabling physiological adaptation of organisms to their physical environment. Prog. Lipid Res. 1990, 29, 167−227. (8) Henschke, P. A.; Rose, A. H. Plasma Membranes in the Yeasts, 2nd ed.; Academic Press: London, UK, 1991; pp 297−435. (9) Deytieux, C.; Mussard, L.; Biron, M. J.; Salmon, J. M. Fine measurement of ergosterol requirements for growth of Saccharomyces cerevisiae during alcoholic fermentation. Appl. Environ. Microbiol. 2005, 68, 266−271. (10) Rodríguez, R. J.; Low, C.; Bottema, C.; Parks, L. W. Multiple functions for sterols in Saccharomyces cerevisiae. Biochim. Biophys. Acta 1985, 837, 336−343. (11) Lange, Y.; Swaisgood, M. H.; Ramos, B. V.; Steck, T. L. Plasma membranes contain half the phospholipid and 90% of the cholesterol and sphingomyelin in cultured human fibroblasts. J. Biol. Chem. 1989, 264, 3786−3793. (12) Zinser, E.; Sperka-Gottlieb, C. D.; Fasch, E. V.; Kohlwein, S. D.; Paltauf, F.; Daum, G. Phospholipid synthesis and lipid composition of subcellular membranes in the unicellular eukaryote Saccharomyces cerevisiae. J. Bacteriol. 1991, 173 (6), 2026−2034. (13) Soubeyrand, V.; Luparia, V.; Pascale, W.; Doco, T.; Vernhet, A.; Ortiz-Julien, A.; Salmon, J. M. Formation of micelle containing solubilized sterols during rehydration of active dry yeasts improves their fermenting capacity. J. Agric. Food Chem. 2005, 53, 8025−8032. (14) Henderson, C. M.; Zeno, W. F.; Lerno, L. A.; Longo, M. L.; Block, D. E. Fermentation temperature modulates phosphatidylethanolamine and phosphatidylinositol levels in the cell membrane of Saccharomyces cerevisiae. Appl. Environ. Microbiol. 2103, 79, 5345− 5356. (15) Walker, G. M. Yeast Physiology and Biotechnology; Wiley: West Sussex, UK, 1998; pp 19−21. (16) Avery, S. V.; Howlett, N. G.; Radice, S. Copper toxicity towards Saccharomyces cerevisiae: dependence on plasma membrane fatty acid composition. Appl. Environ. Microbiol. 1996, 62, 3960−3966. (17) Daum, G.; Lees, N. D.; Bard, M.; Dickson, R. Biochemistry cell biology and molecular biology of lipids of Saccharomyces cerevisiae. Yeast 1998, 14, 1471−1510. (18) Guilloux-Benatier, M.; Fur, Y. L.; Feuillat, M. Influence of fatty acids on the growth of wine microorganisms Saccharomyces cerevisiae and Oenococcus oeni. J. Ind. Microbiol. Biotechnol. 1998, 20, 144−149. (19) Yazawa, H.; Iwahashi, H.; Kamisaka, Y.; Kimura, K.; Uemura, H. Production of polyunsaturated fatty acids in yeast Saccharomyces cerevisiae and its relation to alkaline pH tolerance. Yeast 2009, 26, 167−184. (20) Grillitsch, K.; Connerth, M.; Köfeler, H.; Arrey, T. N.; Rietschel, B.; Wagner, B.; Karas, M.; Daum, G. Lipid particles/ droplets of the yeast Saccharomyces cerevisiae revisited: lipidome meets proteome. Biochim. Biophys. Acta 2011, 1811, 1165−1176. (21) Rodríguez- Vargas, S.; Sánchez- García, A.; Martínez- Rivas, J. M.; Prieto, J. A.; Randez- Gil, F. Fluidization of membrane lipids enhances the tolerance of Saccharomyces cerevisiae to freezing and salt stress. Appl. Environ. Microbiol. 2006, 73, 110−116. (22) Díaz-Hellín, P.; Ú beda, J.; Briones, A. Improving alcoholic fermentation by activation of Saccharomyces sp. during rehydration stage. LWT−Food Sci. Technol. 2013, 50, 126−131. (23) Dupont, S.; Beney, L.; Ferreira, T.; Gervais, P. Nature of sterols affects plasma membrane behavior and yeast survival during dehydration. Biochim. Biophys. Acta−Biomembranes 2011, 1808, 1520−1528. (24) Tronchoni, J.; Rozès, N.; Querol, A.; Guillamón, J. M. Lipid composition of wine strains of Saccharomyces kudriavzevii and Saccharomyces cerevisiae grown at low temperature. Int. J. Food Microbiol. 2012, 155 (3), 191−198, DOI: 10.1016/j.ijfoodmicro.2012.02.004. (25) Redón, M.; Guillamón, J. M.; Mas, A.; Rozès, N. Effect of active dry wine yeast storage upon viability and lipid composition. World J. Microbiol. Biotechnol. 2008, 24, 2555−2563.

Table 3. Percentage (Micrograms per Milligram Dry Weight) of Saturated (SFA; C16:0 and C18:0) and Unsaturated (UFA; C14:1, C16:1, C18:1, C18:2, and C18:3) Fatty Acids and Chain Length [ChL = Σ(CinC,i)/ΣCi] of L2 and L4 Strains after Rehydration with Ergosterol + Tween 80, FH4, and Mn(II) at Half (0.5N) and Normal (N) Dosesa SFA L2


L4


0.5N N 0.5N N 0.5N N

0.5N N 0.5N N 0.5N N

UFA

ChL

23.5 25.0 23.2 25.9 24.6 24.7 25.9 25.9 25.5

± ± ± ± ± ± ± ± ±

1.1 0.9 0.9 2.1 0.9 1.2 2.1 0.1 0.9

76.5 75.0 76.8 74.1 75.4 75.3 74.1 74.1 74.6

± ± ± ± ± ± ± ± ±

1.1 0.9 0.9 2.1 0.9 1.2 2.1 0.1 0.9

16.8 16.9 16.8 16.9 16.8 16.8 16.9 16.9 16.9

± ± ± ± ± ± ± ± ±

0.0 0.0 0.0b 0.1 0.0 0.0 0.1 0.0 0.0

28.5 26.8 28.1 25.4 25.3 23.6 25.4 26.3 26.4

± ± ± ± ± ± ± ± ±

3.2 1.1 1.0 1.1 2.0 2.0 1.1 2.3 0.2

71.5 73.2 71.9 74.6 74.7 76.4 74.6 73.7 73.6

± ± ± ± ± ± ± ± ±

3.2 1.1 1.0 1.1 2.1 2.0 1.1 2.3 0.2

16.4 16.5 16.5 16.6 16.6 16.5 16.6 16.6 16.6

± ± ± ± ± ± ± ± ±

0.2 0.0 0.1 0.1 0.1 0.0 0.1 0.1 0.0

a

Controls: Tween 80 (rehydration with Tween 80) and control (rehydration with physiological solution). Values are means of n = 3 ± standard deviation. bStatistically significant differences of the treated sample with respect to its control (one-way ANOVA; p ≤ 0.05; n = 3).

above-described changes occur within the first 24 h of cell growth, it would therefore be interesting to perform another subsequent sampling during lag phase to ensure the effect of the different treatments tested.

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AUTHOR INFORMATION

Corresponding Author

*(J.Ú .) Phone: +34 926 295300, ext. 4324. Fax: +34 926 295318. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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REFERENCES

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Membrane Lipid Variability in Saccharomyces cerevisiae Wine Strains

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