To determine the fat content of the larvae, a defatting process was carried out. In a screw-capped, amber-glass vial, 1 g of sample was added along with 10 mL of hexane. The mixture was stirred at 600 rpm for 60 min at 40 °C. The mixture was centrifuged at 4500 rpm for 5 min. The supernatant was transferred to a pre-weighted flask and the defatting process was repeated two more times to the solid residue. Each time, the supernatants were transferred to the flask. The solvent was removed using a rotary evaporator and the weight of the extracted oil was calculated.
To determine the fatty acids contained in the extracted oil, fatty acid methyl esters (FAMEs) were prepared according to Commission Regulation (EC) No 796/2002 (Annex XB) [22 ]. The analysis of methyl esters with GC-FID was carried out according to a modified method described by Lalas et al. [23 (link)]. An Agilent Technologies (Santa Clara, CA, USA) Gas Chromatograph model 7890A, equipped with a capillary column Omegawax (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 as follows: initially isotherm for 5 min at 70 °C, an initial programmed rate of 20 °C/min up to 160 °C, then a second rate of 4 °C/min up to 200 °C, and a final rate of 5 °C/min up to 240 °C. 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 was 50 mL/min. Samples of 1 μL were injected into the split mode (1:100). The individual peaks were identified by comparison of reference standards of FAME Mix C8–C24 (Sigma-Aldrich, St. Louis, MO, USA). The percentage composition of the samples was computed from the peak areas using the normalization method (without correction factors). The component percentages were calculated as mean values from triplicate GC-FID analysis [22 ,24 (link),25 (link)].
The calculated oxidizability value (COX) was also measured using the method described by Fatemi and Hammond [25 (link)], as shown below:
where 18:1 represents the % percentage of oleic acid, 18:2 of ω-6, linoleic acid, and C18:3 the percentage of ω-3, linolenic acid.
To determine the fatty acids contained in the extracted oil, fatty acid methyl esters (FAMEs) were prepared according to Commission Regulation (EC) No 796/2002 (Annex XB) [22 ]. The analysis of methyl esters with GC-FID was carried out according to a modified method described by Lalas et al. [23 (link)]. An Agilent Technologies (Santa Clara, CA, USA) Gas Chromatograph model 7890A, equipped with a capillary column Omegawax (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 as follows: initially isotherm for 5 min at 70 °C, an initial programmed rate of 20 °C/min up to 160 °C, then a second rate of 4 °C/min up to 200 °C, and a final rate of 5 °C/min up to 240 °C. 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 was 50 mL/min. Samples of 1 μL were injected into the split mode (1:100). The individual peaks were identified by comparison of reference standards of FAME Mix C8–C24 (Sigma-Aldrich, St. Louis, MO, USA). The percentage composition of the samples was computed from the peak areas using the normalization method (without correction factors). The component percentages were calculated as mean values from triplicate GC-FID analysis [22 ,24 (link),25 (link)].
The calculated oxidizability value (COX) was also measured using the method described by Fatemi and Hammond [25 (link)], as shown below:
where 18:1 represents the % percentage of oleic acid, 18:2 of ω-6, linoleic acid, and C18:3 the percentage of ω-3, linolenic acid.
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