- 學位論文
以酵母菌發酵提高苦瓜甲醇萃取物中功效性成分3β, 7β, 25-trihydroxycucurbita-5, 23 (E)-dien-19-al之探討
Increasing 3β, 7β, 25-trihydroxycucurbita-5, 23 (E)-dien-19-al content in methanolic extract of Momordica charantia L. by yeast fermentation
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摘要
苦瓜為亞洲常見之瓜果類蔬菜，具有許多功效性成分，使之成為歷史悠久之藥用植物。皂苷為苦瓜中重要的活性成分之一，其中，3β, 7β, 25-trihydroxycucurbita-5, 23 (E)-dien-19-al (THC) 被發現對誘導引發有醣尿病之小鼠具有降血醣之功效。因此本實驗希望透過酵母菌發酵山苦瓜以提高具降血醣功效成分3β,7β,25-trihydroxycucurbita-5,23(E)-dien-19-al之含量。實驗先期透過TLC及HPLC-MS/MS分析篩選具皂苷水解能力之菌株，結果發現酵母菌 YCL-L-019 及 YCL-L-020 具轉換 momordicoside L及 momordicoside K 之能力，同時可顯著提升 THC 之含量，並且YCL-L-020之轉換速度比YCL-L-019快。透過滲透壓、剛果紅及 zymolyase 敏感性試驗觀察到YCL-L-020對於滲透壓及剛果紅有較高之敏感性，並於使用SDS-PAGE分析不同酵母菌株之胞外蛋白時亦觀察到YCL-L-020 相對於YCL-L-019有較為複雜之組成，可初步推測 YCL-L-019 及 YCL-L-020 細胞壁存在有差異，為導致其胞外蛋白組成較為複雜及轉換苦瓜皂苷速度較快速的可能原因之一。欲進一步探討是何種酵素作用造成苦瓜皂苷之轉換，本實驗透過分析 YCL-L-020 之胞外蛋白質體及資料庫搜尋推測可能之作用酵素，並以建構質體之方式使 S. cerevisiae 表現推測之酵素，且將表現有該酵素之菌株培養於含苦瓜甲醇萃取物之基質中，觀察該菌株轉換苦瓜皂苷之效果。最終於表現YCL-L-020 BGL1之菌株中觀察到轉換苦瓜皂苷之效果且轉換效果與YCL-L-020之轉換效果相似，後續可再針對該酵素之活性優化及應用進行探討。
並列摘要
Momordica charantia L., also known as bitter gourd, belongs to family Cucurbitaceae and has been used as herbal medicine for a long time in Asia and Africa. Previous studies indicated that saponins play important roles in the bioactivity of Momordica charantia. Particularly, 3β, 7β, 25-trihydroxycucurbita-5, 23 (E)-dien-19-al (THC) is specific saponin that has been reported to have hypoglycemic effects in the diabetes-induced male ddY mice. The aim of this study is to find a unique yeast can enhance the content of THC. In the analysis of thin layer chromatography and HPLC-MS/MS, we found that YCL-L-019 and YCL-L-020 are both able to transform momordicoside L into THC while YCL-L-020 convert more rapidly compared to YCL-L-019. By the test of osmotic sensitivity, congo red sensitivity, Zymolyase sensitivity, and SDS-PAGE analysis, we propose that the difference of cell wall between YCL-L-019 and YCL-L-020 might be a reason why YCL-L-020 have a higher speed in saponin transformation than YCL-L-019. To find out which enzyme is critical for YCL-L-020 to transform the saponins of Momordica charantia further, we use secretome analysis and database searching to screen candidates and clone these enzyme in plasmid and transform into Saccharomyces cerevisiae BY4741 mutants that can’t convert bitter melon saponin in previous experiment. Successfully, we found that the S. cerevisiae BY4741 expresses the BGL1 of YCL-L-020 can specifically convert momordicoside L into THC.
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