37 component fame mix

The fatty acid composition of the extracted seed oil was analyzed using gas chromatograph (Agilent GC-6890, Los Angeles, CA, USA) equipped with a flame-ionization detector (FID) and a capillary column (DB-FFAP, 100 m × 0.25 mm, film thickness 0.20 µm) according to the National Standard of the People’s Republic of China GB5009.168-2016 [21 ]. The GC settings were as follows: nitrogen was used as a carrier gas with a flow rate of 1 mL/min and a split ratio of 1:100. The temperatures of the injection block and the detector were 270 °C and 280 °C, respectively. The column temperature was maintained at 100 °C for 13 min; then, it was programmed to increase to 180 °C at a rate of 10 °C/min, maintained at 180 °C for 6 min, raised to 200 °C at a rate of 1 °C/min, and maintained at 200 °C for 20 min; finally, it was raised to 230 °C at a rate of 4 °C/min and maintained at 230 °C for 10.5 min. GC chromatogram peaks were identified by comparing the retention time from the mixture of the standard FAME (fatty acid methyl esters, 37-component FAME Mix from Sigma-Aldrich, St. Louis, MO, USA). Each fatty acid peak was quantified using an internal standard concentration equivalent. Fatty acids were expressed as percentage of the total fatty acids content. Each sample was independently analyzed in three replicates.

Fatty acids were determined after the extraction and transesterification procedures described by Pojić et al. [16 (link)]. Briefly, lipids were extracted with chloroform/methanol mixture (2 : 1 v/v), and the extracts obtained were dried by vacuum evaporation at 40°C. Further, methyl esters were prepared from the extracted lipids by transesterification with 14% boron trifluoride in methanol. The fatty acid profile was analysed with an Agilent 7890A gas chromatograph system (Santa Clara CA, United States) with a flame ionization detector equipped with a fused silica capillary column (DB-WAX 30 m, 0.25 mm, 0.50 µm). The carrier gas was helium with a flow rate of 1.26 ml min⁻¹. The fatty acid peaks were identified by comparing relative retention times of FAME from the sample with retention times of standards from Sigma-Aldrich Chemical Co. 37 component FAME mix (St Louis, MO, USA) and with data from an internal data library. The results were expressed as a weight percentage of crude fat.

Fatty acid methyl esters (FAME) were obtained from intramuscular fat (previously extracted) by methylation using. KOH (85% in MetOH). Separation of FAMEs was carried out using a gas chromatograph (model 4890 Series II; Hewlett-Packard, Palo Alto, CA, USA) fitted a Carbowax™ fused silica capillary column (30 m × 0.25 mm id; 0.25 μm film thickness; (Ohio Valley, Marietta, OH, USA). For the identification of individual FAME was used by a standard mixture of 37 Component FAME Mix (Sigma–Aldrich, Supelco 37 Component FAME Mix- CRM47885, St. Louis, MO, USA).

FAMEs were extracted via a one-step transesterification method, where a known amount of freeze-dried biomass was treated, using sulfuric acid (95%; analytical grade from Sigma-Aldrich) and methanol solution (H₂SO₄:CH₃OH = 1:10), and sonicated for 10 min. This step was followed by heat treatment at 80 °C for 2 h. Further, the mixture was transferred to a tube containing 1 mL of distilled water and 3 mL of hexane: chloroform (4:1; hexane and chloroform from Sigma-Aldrich, St. Louis, MO, USA) mixture and centrifuged. The top layer containing FAME fractions was filtered and analyzed, using GD-FID (Shimadzu 2010 plus, Kyoto, Japan) [29 (link)]. Then, 2 µL of sample was injected into gas chromatography set at 100–240 °C/5 min holding time. The sample was separated by using 100 m column with He as a carrier gas. FAMEs were identified based on retention time observed, using Supelco standards obtained from Sigma-Aldrich (37 Component FAME Mix: Cat # Number 200-838-9, St. Louis, MO, USA).

The FAMEs were prepared based on the AOCS method Ce‐1b 89 (2007). A 37‐component FAME mix from Sigma‐Aldrich was used as the FAME external standard, which was run in parallel with the samples.
The FAMEs were analyzed using an Agilent 7890B GC system with a flame ionization detector (Agilent Technologies) and a fused‐silica capillary column (Sp‐2560, 100 m × 0.25 mm × 0.2 μm, Supelco Inc.). The injection port and detector temperatures were both set at 250°C. The column temperature was initially ramped to 210°C at a rate of 3°C/min and held for 1 min, then ramped to 219°C at a rate of 0.5°C/min and held for 1 min, and finally ramped to 240°C at a rate of 10°C/min and held for 13.5 min. The final temperature was 250°C, which was held for 1 min. The GC peaks were identified by comparing the retention times with those of the corresponding standards, with the relative contents then calculated, and expressed in mol%.
The PA‐EE was analyzed under the same GC conditions. The concentrations of PA‐EE were calculated from the peak areas from GC using the corresponding linear regression equation in an external standard calibration method.

Fat was extracted using chloroform and methanol as described by Bligh and Dyer (1959) and used for determination of the fatty acid profile. To prevent lipid oxidation during and after analysis, 0.02% butyl hydroxy toluene was added to the chloroform used. Fatty acid composition was determined by gas chromatography. Fat was saponified in methanolic KOH solution and then esterified in methanolic H₂SO₄ solution. The fatty acid methyl esters (FAME) were analyzed using an Agilent Technologies gas chromatograph (HP 6890) fitted with a capillary column DB-23 (50% cyanopropyl-methylpolysiloxane, 60 m × 0.25 mm × 0.25 μm) and flame ionization detection. The temperature of the injector port and the detector was set at 250 °C, and the carrier gas was nitrogen (0.6 ml/min). After injection (1 μl, split ratio 50:1), the oven temperature was held at 120 °C for 5 min, increased to 240 °C at a rate of 4 °C min^− 1, and held at this temperature for 10 min. Standard fatty acid methyl esters (37-component FAME Mix and PUFA no. 2 from Sigma, Saint Louis, MO, USA) were run under the same conditions and the subsequent retention times were used to identify the fatty acids. Fatty acids were expressed as percentage of the total fatty acids content [25 (link)].