Supelco 37 fame mix

Larvae were ground to powder with liquid nitrogen. Totally 0.2 g of larvae powder was collected into a glass tube, and 1 μg/mL of C17:0 was added as the internal standard. Total Fatty Acids were extracted by chloroform and dried by N₂. Methyl esterification was carried out with the methanol-H₂SO₄ method; n-hexane was used to extract fatty methyl esters (FAME) [52 ]. The FAME products were measured by an Agilent 6890N Gas Chromatography gas phase analyzer with a DB-23 column (60 m × 250 m × 0.25 m). The sampling parameter was set as follows: detector temperature, 260 °C; inlet temperature, 270 °C. The heating program was started initially at 100 °C for 13 min, then 10 °C/min to 180 °C for 5 min, 1 °C/min to 200 °C for 20 min, and finally heated at 4 °C/min to 240 °C for 10 min [53 ]. Helium was used as the carrier gas. The C4–C24 FAME mixture (Supelco 37 F. A. M. E. Mix, Sigma-Aldrich, St. Louis, MO, USA) was used as the standard solution.

Total lipid was extracted from ~0.5 g of homogenised salmon flesh in 20 volumes of ice-cold chloroform/methanol (2:1 v/v) using an Ultra-Turrax tissue disruptor (Fisher Scientific, Loughborough, UK) and the lipid determined gravimetrically54 (link). Fatty acid methyl esters (FAME) from total lipid were prepared by acid-catalysed transmethylation at 50 °C for 16 h55 . FAME were extracted and purified as described previously56 (link), and separated and quantified by gas-liquid chromatography using a Fisons GC-8160 (Thermo Scientific, Milan, Italy) equipped with a 30 m × 0.32 mm i.d. × 0.25 μm ZB-wax column (Phenomenex, Cheshire, UK), ‘on column’ injection and flame ionisation detection. Hydrogen was used as carrier gas with an initial oven thermal gradient from 50 °C to 150 °C at 40 °C.min⁻¹ to a final temperature of 230 °C at 2 °C.min⁻¹. Individual FAME were identified by comparison to known standards (Supelco™ 37-FAME mix; Sigma-Aldrich Ltd., Poole, UK) and published data56 (link). Data were collected and processed using Chromcard for Windows (Version 1.19; Thermoquest Italia S.p.A., Milan, Italy). Fatty acid content per g of tissue was calculated using heptadecanoic acid (17:0) as internal standard.

We used gas chromatography to analyze fatty acid composition of the total fatty acid extracts of isolated strains. Fatty acid methyl esters (FAMEs) were identified using a “Chromatek-Crystall-5000.2” with a 2D sample injector (Chromatek, Yoshkar-Ola, Russia) gas chromatograph with a flame-ionization detector and a Zebron ZB-FFAP capillary gas chromatographic column. An isothermal column configuration was used. The temperature of detector and evaporator was 240 °C. The internal standard was C 22:0 FA. Chromatek-Analytik-5000.2 software was used for data recording and integration. FAMEs were identified with standard mixtures Supelco 37 FAME mix (Sigma Aldrich, St. Louis, MO, USA) and by comparing the equivalent lengths of carbon chains and table constants according to (Cabrini et al., 1992 (link); Gago et al., 2011 (link)).

Gas chromatography (GC) was applied for analysis of acid profile of acidolysis products (PC-egg, LPC-egg, 3-OMe-CA-PC, 3-OMe-CA-LPC) and standards (PC-egg, 3-OMe-CA). For this purpose purified samples (SPE/column chromatography methods) were derivatized into corresponding to them methyl esters as described before [22 (link)] and analyzed on an Agilent 6890N [20 (link)]. Retention times of products were compared with retention time of a standard (Supelco 37 FAME Mix, Sigma Aldrich).

Fatty acid methyl esters (FAMEs) from total lipid or total neutral and polar lipid fractions were prepared by acid-catalised transesterification of total lipid according to the method of Christie [34 ]. FAMEs were separated and quantified by a GLC (Fisons GC-8160, Thermo Scientific, Milan, Italy) equipped with a 30 mm × 0.32 mm i.d. × 0.25 μm ZB-wax column (Phenomenex, Cheshire, UK), fitted with “on column” injection and flame ionisation detection. Hydrogen was used as the carrier gas and temperature programming was from 50 to 150 °C increasing at 40 °C·min⁻¹, followed by a gradient of 2 °C·min⁻¹ to a final temperature of 230 °C. Individual FAMEs were identified by comparison with known standards (Supelco 37 FAME mix, Sigma-Aldrich Ltd., Poole, UK) and published data [33 (link)]. Data were collected and processed using Chromcard for Windows Version 1.19 (Thermoquest Italia SpA, Milan, Italy).

The fatty acid (FA) composition of GSO, CO or CNO was analyzed by GC-MS as fatty acid methyl esters (FAMEs) [22 (link)], following the method proposed by Villa-Rodríguez et al. [23 (link)] in a GC-MS (VARIAN Saturn 2100D) equipped with a CP7420 column (100 m, 0.25 mm i.d.) using helium ultra-high purity grade (1 mL/min) as the carrier gas. Operating conditions were: oven (T (°C)/time (min)/rate (°C/min)): 160/4/20 and 198/42/1, injector (EFC Type 1) and detector temperatures were 250 °C and 180 °C, respectively. The mass spectrometer was operated in the electron impact (EI) mode at 70 eV in the scan range of 40–500 m/z. FAMEs were identified by comparing the peak’s retention against commercial standards (Supelco 37 FAME Mix; Sigma Chemical Co., St. Louis, MO, USA) and by comparing the respective ion chromatograms with those reported in the NIST 2008 library (NIST/EPA/NIH Mass Spectral Library, Version 2.0). Phytosterol (PST) composition in all three oils was evaluated by direct saponification (KOH 0.5 M) and capillary gas chromatography following the method proposed by Fleteouris et al. [24 (link)] in a 6890 GC System Gas Chromatograph (Hewlett-Packard Development Company, L.P., Houston, TX, USA) equipped with a SP™-2560 Capillary GC Column (L × I.D. 100 m × 0.25 mm, d_f 0.20 μm;) using helium Ultra High Purity grade (1 mL/min) as the carrier gas.