*2.9. Free Fatty Acids*

Free fatty acid was detected by a method previously published according to [22], with slight modifications. Determination of fatty acid methyl esters (FAME) was performed using a GC/MS system equipped with a GC-7 Agilent HP-88 capillary column (60 m × 0.25 mm × 0.2 μm). The temperature profile of the oven was 100 ◦C for 13 min followed by increasing at 10 ◦C/min to 180 ◦C for 6 min, then increasing at 1 ◦C/min to 200 ◦C for 20 min, and then increasing at 4 ◦C/min to 230 ◦C for 10.5 min. Injector and detector temperatures were set to 270 and 280 ◦C, respectively. The conditions applied for gas chromatography were nitrogen as the carrier gas at a flow of 1.0 μL/min. Fatty acids were identified and quantified based on chromatographic retention times using reference standard Supelco 37 component FAME mix (Sigma Aldrich Chemical Co., St. Louis, MO, USA). Results obtained were presented as percentage of total fatty acids.
