Omegawax capillary column

The lipid extract was methylated before analysis. The FAs were converted to fatty acid methyl esters
(FAMEs) according to the method described by Ben Moumen et al. (2015) using BF3 at 14 % weight in
methanol. The separation of the FAMEs was performed on an Agilent gas chromatograph (GC) (HP6890
series, Agilent Technologies, USA) equipped with an Omega wax capillary column
( $\mathrm{30}\mathrm{m}\times \mathrm{0.25}\mathrm{mm}$ , 0.25  $\mathrm{m}$ film thickness) from Supelco (Bellefonte, PA,
USA) and a flame ionization detector (FID). Helium (99.999 %, Air Liquide, Liege, Belgium) was
used as the carrier gas, at a flow rate of 1.7  $\mathrm{mL}{\min }^{-\mathrm{1}}$ . The temperatures of the
injector and detector were set at 150 and 250  ${}^{\circ }\mathrm{C}$ , respectively, and the oven
temperature was 210  ${}^{\circ }\mathrm{C}$ . The injection volume was 1  $\mathrm{\mu }\mathrm{L}$ , in split-less
mode. A FAME standard, containing 37 components (Supelco, Bellefonte, PA, USA), was used to identify
the individual peaks. The average amount of each fatty acid was used to calculate the sum, ratios,
and lipid indices of the meat.

The sample preparation for fatty acids analysis was performed following the procedure as described in our previous study [19 (link)]. Briefly, each sample (Ca.10 g) was weighed and homogenized with 150 mL of chloroform: methanol (2:1, v/v) solvent mixture at 300×g for 3 min using a homogenizer (Polytron, PT-MRC 2100, Littau, Switzerland). After filtration through Whatman filter paper, the filtrate was added with approximately 20 g of Na₂SO₄, thoroughly mixed for 1 min, and then the upper lipid layer was transferred into an Erlenmeyer flask. After drying at 55°C using a rotary evaporator, the lipids layer was reconstituted with 1 mL tricosanoic acid and 1 mL of 0.5 N NaOH. Finally, the lipid was converted to fatty acid methyl esters (FAMEs). Approximately 1.0 mL of FAMEs was taken and placed into auto-sampler vials, sealed, and used for fatty acids analysis. The separation of FAMEs was achieved using a gas chromatography/flame ionization detector (GC-FID; Varian Technologies, Palo Alto, CA, USA) equipped with an Omegawax capillary column (30 m×0.25 mm×0.25 μm film thickness; Supelco, Bellefonte, PA, USA) as described in our previous study [19 (link)]. Identification of fatty acids in the samples was carried out by comparing their retention times with those obtained from standard fatty acids. Individual fatty acids were expressed as relative percent (%) of total fatty acids.

The method for preparing the fatty acid methyl esters (FAMEs) from oils was according to Commission Regulation (EC) No 796/2002 (Annex XB) [41 ]. Analysis of methyl esters with GC-FID was carried out according to a modification of the method described by Lalas et al. [42 (link)]. An Agilent Technologies (Santa Clara, CA, USA) Gas Chromatograph model 7890A, equipped with an Omegawax capillary column (30 m × 320 μm × 0.25 μm) (Supelco, Bellefonte, PA, USA), was used. Helium was the carrier gas at a flow rate of 1.4 mL/min. The column temperature program was: initially isotherm for 5 min at 70 °C, ramped to 160 °C at a rate of 20 °C/min, then increased to 200 °C at a rate of 4 °C/min and increased up to 240 °C at a rate of 5 °C/min. The injector and flame ionization detector (FID) temperatures were maintained at 240 and 250 °C, respectively. The flow rate for hydrogen was 50 mL/min, for air 450 mL/min, and the makeup flow of helium 50 mL/min. Samples of 1.0 μL were injected in split mode (1:100). The individual peaks were identified by comparison with reference standards from FAME Mix C8–C24 (Sigma-Aldrich, St. Louis, MO, USA). The percentage composition of the samples was computed from the GC peak areas using the normalization method (without correction factors). The component percentages were calculated as mean values from triplicate GC-FID analysis.

The sub-samples from sections in each the belly were combined, comminuted, well-mixed and used for fatty acids analysis. The lipids in the samples were extracted using a solvent mixture of chloroform: methanol (2:1, v/v) and then were converted to fatty acid methyl esters (FAME) as described in our previous study [14 (link)]. Approximately 1.0 mL of FAMEs were transferred to auto-sampler vials and was sealed. The separation of FAMEs was achieved using a gas chromatography/flame ionization detector (GC-FID, Varian Technologies, Palo Alto, CA, USA) equipped with an Omegawax capillary column (30 m×0.25 mm×0.25 μm film thickness; Supelco, Bellefonte, PA, USA). The GC oven temperature was maintained at 50°C for 1 min, and ramped at a rate of 25°C/min to 200°C, and further raised at a rate of 5°C per min to 230°C.
The injection port and detector temperatures were set at 250°C and 260°C respectively. Identification of fatty acids in samples was carried out by comparing their retention times with those obtained from standard fatty acids. Individual fatty acids were expressed as relative percent (%) of total fatty acids FAMEs.

To quantify the production of bioethanol, 1-μL filtered sample was subjected to GC analysis; the peak area was compared with the standard (10%, 1%, 0.1% ethanol) to determine the concentration of ethanol using the Omegawax capillary column (I.D. 30 m × 0.32 mm × 0.25 μm; Supelco, Sigma-Aldrich) with nitrogen as the carrier gas. GC was performed at a flow rate of 2 mL/min, with the injector temperature maintained at 220 °C, a split ratio of 20:1, and a GC-FID temperature of 240 °C. The oven temperature was maintained at 60 °C for 10 min.