*4.8. Fatty Acids*

The extraction of fatty acids was carried out following the procedure reported by Bligh & Dyer [100]. An aliquot (10.0 mg) of each lyophilized pool was homogenized with 1.0 mL of chloroform (CHCl3) and 3.0 mL of methanol (MeOH). This mixture was then added with 1.0 mL of MeOH and 1.0 mL of water, homogenized, let to settle, and filtered through paper. After settling, the filtrate was centrifuged at 3000 rpm for 15 min. Two layers were formed: the bottom one (CHCl3), containing the isolated lipids, was transferred to a rotating evaporator, model P/N Hei-VAP Precision ML/G3 (Heidolph Instruments GmbH & Co., Schwabach, Germany). The upper layer (MeOH/H2O) was subjected again to all the steps above described in order to reach an exhaustive extraction. For the fatty acid methylation, the dried lipidic extract was recovered through addition of 1 mL hexane, then added with reagen<sup>t</sup> (CH3OH/H2SO4, 9:1), and heated at 100 ◦C for 1 h. The hydrocarbon layer was collected and injected into the GC instrumentation. Qualitative analyses were carried out in a GCMS-TQ8030 (Shimadzu, Kyoto, Japan) system equipped with an AOC-20i autosampler and a capillary column Supelco SLB-IL100 (60 m × 0.25 mm, film thickness 0.20 μm). GC conditions were set as follows: injector, 280 ◦C; injection volume: 1.0 mL; head pressure: 26.7 kPa; carrier gas: He, at a linear velocity of 30.0 cm/s (constant); split ratio 1:100; oven temperature program: 50–280 ◦C at 3 ◦C/min, held 10 min. MS conditions: operation mode was in full scan; ion source and interface temperatures were 220 ◦C and 250 ◦C, respectively; and scan mass range was 40 to 400 *<sup>m</sup>*/*<sup>z</sup>*. For FAMEs identification, a triple means methodology was used: (i) spectral matching with Wiley and NIST databases; (ii) co-injection with standards (Supelco 37 component FAME mix, Supelco, St. Louis, MO, USA); and (iii) comparison with literature data [101]. Data handling was performed by GCMS solution software. Quantitative analyses were carried out on a Master GC-DANI system, equipped with a capillary column Supelco SLB-IL100 (60 m × 0.25 mm, film thickness 0.20 μm). Oven temperature program: 120–200 ◦C at 1 ◦C min−<sup>1</sup> (10 min). Injector and FID temperatures were respectively set at 220 and 240 ◦C. Carrier gas was He, at a constant linear velocity of 30.0 cm s<sup>−</sup>1. FID conditions: sampling frequency: 25 Hz; gases: makeup (He), 25 mL min−1; H2, 40 mL min−1; air, 280 mL min−1. Data were processed through the Clarity software (Dani). All determinations were run in triplicate.
