2.1 Strains, media and reagents
All strains used in this study are listed in Table 1. The strains ofMycobacterium were cultured at 30 ℃ and 200 rpm in the MYC/01 seed medium containing glycerol 20 g/L, sodium citrate 2.8 g/L, NH4Cl 2.7 g/L, K2HPO4⋅3 H2O 0.5 g/L, MgSO4⋅7 H2O 0.5 g/L and ammonium ferric citrate 0.05 g/L for 2 days. Then the seeds were inoculated with 5% (v/v) into MYC/04 broth containing glucose 25 g/L , sodium citrate 2.8 g/L, corn steep powder 3 g/L, (NH4)2HPO4 5 g/L, starch 2 g/L, K2HPO4⋅3 H2O 0.5 g/L, MgSO4⋅7 H2O 0.5 g/L, ammonium ferric citrate 0.05 g/L, PS 0.1 g/L and cultured at 30 ℃ and 200 rpm for 3 days. The initial pH of the medium was adjusted to 8.0.
The reagents described in the media components were purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). PS (purity of 95%, the main components are stigmasterol, campesterol, and β-sitosterol) was obtained from Shanxi Sciphar Natural Products Co., Ltd. (Shangluo, China). The 9-OH-AD standard was purchased from Sigma (Shanghai, China). HP-β-CD was from Zhiyuan Biotechnology Co., Ltd. (Binzhou, China). The organic solvent was purchased from Bohr Chemical Reagent Co., Ltd. (Shanghai, China).