3.7. Preparation of Fatty Acid Methyl Esters and GC-MS Analysis

Approximately 25 mg lyophilized yeast powder and 1 mL H₂SO₄-methanol solution (4%, v/v) were mixed in a test tube. After charging with nitrogen gas, the bottle was stirred and heated at 85 °C for 1 h. 1 mL distilled water and 1 mL hexane was added into the tube, mixed by vortexing and then centrifuged at 5500 rpm for 10 min. The supernatant was transferred into another tube, concentrated by bubbling nitrogen and stored at 4 °C for gas chromatography-mass spectrometry (GC-MS) analysis (Agilent Technologies, Wilmington, DE, USA).

The fatty acid methyl ester derivatives were subjected to GC-MS on a very polar column HP-88 (88%-cyanopropyl) aryl-polysiloxane (60 m × 250 μm × 0.2 μm) connected to a MS Engine quadrupole mass spectrometer. The column temperature was held at 70 °C for 1 min, increasing to 210 °C (held for 0 min) and 220 °C (held for 0 min) respectively at a rate of 10 °C·min⁻¹, and then heated to 235 °C at a rate of 10 °C·min⁻¹ and held for 8 min. The injection volume was 1 μL without split. Helium was used as the carrier gas at a flow rate of 0.6 mL·min⁻¹, and the pressure was programmed at a constant flow mode. The mass spectrometer was operated in electron impact mode at ionization energy of 70 eV [36]. Fatty acids were identified based on the comparison of their mass spectra with those stored in NIST 08 MS libraries.