Extraction and Analysis of Sterols

Sterol extraction was performed as previously described (Guo et al., 2018). Yeast cells were harvested by centrifugation at 12,000 rpm for 2 min after fermentation process and boiled in 3 N HCl for 5 min to break the cell wall. Cells were pelleted and washed by distilled water to remove the remaining HCl. After neutralizing by NaOH, saponification reaction of cells was carried out in 3 M NaOH-methanol solution at 60°C for 4 h. n-Hexane and silica sand were added for sterol extraction with vortex. The n-hexane phase was collected and dried by a centrifugal vacuum evaporator. N-methyl-N-(trimethylsilyl) trifluoroacetamide (MSTFA) was used to derivatize the obtained sterols (30°C for h) and samples were ready for GC-MS analysis.

Sterols were separated and analyzed by GCMS-QP2020 (SHIMADZU, Japan) using a DB-5 fused-silica capillary column (30 m × 0.25 mm i.d., film thickness 0.25 μm, J&W Scientific, CA). Mass spectra ranged at 50–800 m/z, and helium was used as the carrier gas. Operating conditions were inlet temperature 260°C, initial temperature 70°C for 2 min then ramp 30°C/min to 250°C then ramp 10°C/min to 280°C and held for 15 min. Finally, the temperature increased to 290°C at 5°C/min and was held for 5 min. Sterol standards (squalene, A1, A2, B3, B1, 7-DHC, etc.) were purchased from Sigma-Aldrich (United States).