A Green Strategy to Produce Potential Substitute Resource for Bear Bile Using Engineered Saccharomyces Cerevisiae

BackgroundBear bile powder is a precious natural material characterized by high content of tauroursodeoxycholic acid (TUDCA) at a ratio of 1.00–1.50 to taurochenodeoxycholic acid (TCDCA).ResultsIn this study, we use the crude enzymes from engineered Saccharomyces cerevisiae to directional convert TCDCA from chicken bile powder to TUDCA at the committed ratio in vitro. This S. cerevisiae strain was modified with heterologous 7α-hydroxysteroid dehydrogenase (7α-HSDH) and 7β-hydroxysteroid dehydrogenase (7β-HSDH) genes. S. cerevisiae host and HSDH gene combinatorial optimization and response surface methodology was applied to get the best engineered strain and the optimal biotransformation condition, respectively, under which 10.99 ± 0.16 g/L of powder products containing 36.73±6.68 % of TUDCA and 28.22±6.05 % of TCDCA were obtained using 12.00 g/L of chicken bile powder as substrate.ConclusionThis study provides a healthy and environmentally friendly way to produce potential alternative resource for bear bile powder from cheap and readily available chicken bile powder, and also gives a reference for the green manufacturing of other rare and endangered animal-derived valuable resource.

Study on the Determination of Tauroursodeoxycholic Acid in Bear Bile Powder by HPLC-MS/MS

Objective — To establish a high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method for the rapid determination of tauroursodeoxycholic acid (TUDCA) in bear bile powder. Method — The chromatographic conditions were optimized. A Hypersil GOLD chromatographic column (Thermo Fisher, 100 mm×2.1 mm) was used with the mobile phase of methanol:water (0.1mmol/l ammonium acetate + 0.1% formic acid) = 75:25 for isocratic elution; the column temperature is 30.0℃; the flow rate is 0.2mL/min; the injection volume is 5uL; the negative ion scanning mode and the select reaction monitoring are used. Through the compound optimization function of the instrument, the two characteristic ion pairs of TUDCA are m/z: 498.5/124.0 and m/z: 498.5/79.8 (quantitative ion pair). Results — The detection limit of this method was 5ng/mL, and the linear range was 20~1000ng/mL (r=0.9993). The same sample was measured in parallel with the high performance liquid chromatography (HPLC/UV) method, and the analysis results were basically the same. Conclusion — Compared with HPLC/UV, HPLC-MS/MS is simple, more sensitive and accurate for the determination of TUDCA in bear bile powder. The method can be used in the quality assessment and process control of bear bile powder.