Lipid Extraction and Fatty Acid Analysis

Total lipids were extracted using the modified method of Bligh and Dyer (51), with some modifications (38). Dried samples (200 mg) were mixed with a total of 15 ml solvent added in this sequence: 6 ml of chloroform: methanol (2:1), 6 ml of chloroform: methanol (2:1) and 3 ml KCl (0.88%). The samples were shaken for 15 s after the addition of each solvent and centrifuged at 5,140 × g for 5 min. The lower phase was set aside, and the upper phase was subjected to further extraction with a solution of chloroform: methanol (2:1, 1 vol.). The lower phase was isolated and added to the first one, and mixed with a solution of methanol: water (1:1, 14 volume). In this case, the lower phase was put aside, dried in the presence of nitrogen flux, and analyzed for lipid composition. Then, fatty acid methyl esters (FAMEs) were obtained using boron trifluoride (BF₃) according to Leone et al. (38), with some modifications. A part of the total lipid extract (200 μl in hexane) was saponified at 90°C for 20 min with 0.5 M KOH in methanol (3 ml). Sixty-six micrograms of the internal standard (methyl-tricosanoate, Sigma-Aldrich, Darmstadt, Germany) was added before saponification. The fatty acids were methylated by adding 2 ml of 14% BF₃ in methanol (Sigma-Aldrich, Darmstadt, Germany). The samples were evaporated under a stream of nitrogen and dissolved in 50 μl of hexane, and 1 μl was analyzed by gas chromatography-mass spectrometry (GC-MS). GC–MS analyses were performed using an AGILENT 5977E gas chromatograph (Agilent Technologies, Santa Clara, CA, United States). Separation of compounds was performed on a VF-WAXms (60 m, 0.25 mm i.d., 0.25 mm film thickness, Agilent Technologies, Santa Clara, CA, United States). GC parameters were as follows: the column temperature was maintained at 160°C for 1 min, programmed at 4°C/min to 240°C for 30 min. Helium was used as a carrier gas at a constant flow rate of 1 ml/min. A mass spectrometer was operated in electron impact mode with a scan range of 50–700 m/z. The temperature of MS source and quadrupole was set at 230 and 150°C. Analyses were performed in full-scan mode. Compounds were identified by comparing the retention times of the chromatographic peaks with those of authentic standards (F.A.M.E. Mix C8-C24, Sigma-Aldrich, Darmstadt, Germany) analyzed under the same conditions. MS fragmentation patterns were compared with those of pure compounds, and a mass spectrum database search was performed using the National Institute of Standards and Technology (NIST) MS 98 spectral database. Fatty acid composition was expressed as a percentage of the total fatty acids in each sample, while the total lipid content was expressed both as yield of lipid extraction in 100 g of fresh JF sample (mg/100 g FW) and as a percentage of dry weight (% DW).