Analysis of Fatty Acid Composition

The composition of fatty acids (FAs) of QA23 and TdB strains was analyzed in cells which were untreated and treated with 5 μM of MEL in absence of stress (Control and MEL) and 18 h after the oxidative stress (2 mM H₂O₂) was applied to allow the cells to respond to this stress (MEL H₂O₂ and H₂O₂). Yeast cells homogenates were obtained from 50 mL of total cells pellets using glass beads and a Disruptor Genie^® (Scientific Industries, Inc., NY, United States) for 10 min at 4°C. Proteins from the homogenates were then precipitated with 10% (v/v) trichloroacetic acid and quantified with de Folin phenol reagent (Lowry et al., 1951). The total lipids were extracted from cell fractions corresponding to 1 mg of total cell protein according to the method by Folch et al. (1957). The FA composition was determined by gas liquid chromatography (GLC) according to Rußmayer et al. (2015). In brief, the total FAs from lipid extracts were first converted to methyl esters by methanolysis with sulfuric acid (2.5% in methanol (v/v); 80°C for 90 min) and then extracted twice with light petroleum and water (3:1; v/v) by shaking on a Vibrax orbital shaker^® (IKA, Staufen, Germany) for 30 min. Finally, FAs were separated by GLC on a Hewlett-Packard 6890 gas-chromatograph (Agilent Technologies, CA, United States) using an HP-INNOWax capillary column (15 m × 0.25 mm × 0.50 μm film thickness) with helium as a carrier gas. Identification was done by comparison with a commercial FA methyl ester standard mix (NuCheck, Inc., MN, United States) and quantification using the pentadecanoic acid (C15:0, Sigma-Aldrich) as an internal standard. Two biological replicates were set up for each strain and two analytical replicates were performed for each biological replicate.