*2.6. Crude Fat and Fatty Acid Determination*

Samples were extracted with a mixture of chloroform and methanol (2:1). The mixture was allowed to stand overnight and the lower lipid layer, transferred into a pretreated and weighed flask, was dried off. The difference in the two weights established the weight of the fat [24].

The fatty acid analysis was performed by gas chromatography after transmethylation with 2% H2SO<sup>4</sup> in methanol at 80 ◦C. The separation and quantification of fatty acid methyl esters were conducted with a Dani Master GC 1000, equipped with an FID detector (Dani Instruments, Milan, Italy) and a capillary column Supelco SLB-IL100 60 m × 0.25 mm, film 0.20 µm(Merck KGaA, Darmstadt, Germany), using the following experimental conditions: injector temperature 220 ◦C; oven temperature from 120 ◦C to 200 ◦C (10 min hold) at a rate of 1 ◦C/min; detector temperature 240 ◦C; carrier gas He at a constant velocity rate of 34 cm/sec; and a split ratio of 1:50.

Fatty acids were identified by comparing the samples with reference standards, using Supelco 37 component Fatty Acid Methyl Esters (FAME) mix in methylene chloride. All samples were analyzed in triplicate.

All chemicals were provided by Merk Life Science (Merck KGaA, Darmstadt, Germany).
