Bpx 70

The fatty acid composition was qualitatively and quantitatively determined using a TurboMass gas chromatography mass spectrometer (PerkinElmer, Massachusetts, United States) with a capillary column (BPX-70, 30 m × 0.25 mm × 0.25 μm) using the method described by Kattner and Fricke [48 (link)] and Song et al. [49 (link)]. Briefly, cellular fatty acid was extracted from 50 mg of C. ellipsoidea powder in 3 mL of 7.5% (w/v) potassium hydroxide in methanol for saponification at 70°C for 3 hours. After the pH was adjusted to 2.0 with hydrochloric acid, the fatty acids were subjected to methyl esterification with 2 mL of 14% (w/v) boron trifluoride in methanol (Beijing Chemical Works, Beijing, China) at 70°C for 1.5 hours. Then 1 mL of 0.9% (w/v) sodium chloride was added and was mixed well. Subsequently, fatty acid methyl esters (FAMEs) were extracted with 4 mL of hexane (Beijing Chemical Works, Beijing, China). The upper phase was removed to a second tube, dried under N₂ and dissolved in acetic ether. FAMEs were analyzed and identified by the comparison of their peaks with a known internal standard 17:0 FAME (Sigma Aldrich, St. Louis, MO, United States).

Cellular fatty acids were extracted by incubating 10 mg of dried seeds of control and transformed plants or 50 mg of yeast powder and freeze-dried algae powder in 3 mL of 7.5% (w/v) KOH in methanol for saponification at 70 °C for 4 h. After the pH was adjusted to 2.0 with HCl, the fatty acid were subjected to methylesterification with 2 mL of 14% (w/v) boron trifluoride in methanol at 70 °C for 1.5 h. A phase separation was produced by adding 1 mL of 0.9% (w/v) NaCl and 4 mL of hexane. The upper phase was dried under a nitrogen gas flow and resuspended in 0.3 mL of acetic ether prior to GC analysis. An analysis of fatty acid methyl esters (FAME) was performed by GC-MS (gas chromatography–mass spectrometry, TurboMass, PerkinElmer, USA) equipped with a capillary column (BPX-70, 30 m × 0.25 mm × 0.25 μm). Hydrogen was used as the carrier gas at a flow rate of 1.0 mL/min. The injector and detector temperatures were held at 250 °C. The column oven was temperature-programmed from 100 to 190 °C at 15 °C/min, where the temperature was held for 1 min increased to 220 °C at 10 °C/min, and then held for 4 min. The total FA content was quantified using heptadecanoic acid (C17:0) (Sigma) as an internal standard added to samples prior to extraction.