利用基因剔除的酵母菌進行人蔘皂苷的生物轉化作用

Abstract

人蔘皂苷（ginsenosides）是人蔘的主要活性成分之一，然而人蔘皂苷的生物可利用率低，本研究目的為利用酵母菌（Saccharomyces cerevisiae）進行人蔘皂苷生物轉換（Biotransformation），以期獲得生物轉換率較高的小分子人蔘皂苷。本實驗將固相萃取管柱（Solid phase extract column）分離純化的1000 μg/mL人蔘精粹物與完整營養培養基（YPD）混合培養野生型酵母菌，超音波細胞破碎儀破碎酵母菌細胞，萃取胞內外物質後利用液相層析串聯式電灑游離多次質譜（HPLC�ESI tandem MS）鑑定酵母菌胞內外人蔘皂苷分子碎片。結果發現培養第6天時偵測到人蔘皂苷Rb1濃度自0.69 ± 0.018 μM顯著降低至0.49 ± 0.038 μM（p＜0.001），人蔘皂苷Rd濃度自0.19 ± 0.006 μM顯著上升至0.44 ± 0.029 μM（p＜0.001），人蔘皂苷Rg3濃度自1.01 ± 0.087 μM顯著降低至0.70 ± 0.036 μM（p＜0.001），並且偵測到胞內具有人蔘皂苷Rg3，顯示酵母菌可能具有將人蔘皂苷進行生物轉換的能力並可能具有人蔘皂苷Rg3的運輸子基因，有潛力做為人蔘皂苷運輸子的篩選平臺。

Ginsenosides are one of the primary bioactive substrates in ginseng. However, ginsenosides have low bioavailability. The purpose of this research is to investigate biotransformation of ginsenosides in yeast, Saccharomyces cerevisiae. We incubated wild-type yeast cells in YPD with 1000 μg/mL commercial ginseng essence product. Yeast cellswere sonicated to disrupt cell walls and ginsenosides were analyzed using HPLC/ESI tandem MS. The results indicated a significant decrease in ginsenosides Rb1 from 0.69 ± 0.018 μM to 0.49 ± 0.038μM（p＜0.001）, an increase in Rd from 0.19 ± 0.006 μM to 0.44 ± 0.029 μM（p＜0.001）, and an decrease in Rg3 from 1.01 ± 0.087 μM to 0.70 ± 0.036 μM （p＜0.001）in the YPD medium after 6 days fermentation. Intracellular ginsenoside Rg3 was also detected. We therefore propose that Saccharomyces cerevisiae may have the ability to biotransform and uptake ginsenosides, and has the capacity to function as a ginsenosides transporter genes screening platform.