羅漢果皂苷分析與利用靈芝及酵母菌對其進行生物轉換

羅漢果皂苷為一群具有高度甜味的三萜類糖苷分子，已被證實具有調節血糖之生理功能，分子結構上以帶有五個β-D-葡萄糖基的mogroside V是主要天然羅漢果的皂苷形式。由於此類皂苷沒有特殊紫外光及-可見光吸收波長以及含有多種同分異構物，因此在分析上的困難高，本試驗使用液相層析串聯式電灑游離多次質譜 (HPLC/ESI tandem MS) 方法來克服羅漢果皂苷在分析上的困難。羅漢果皂苷在電灑游離狀態下，以使用0.01% 甲酸修飾劑相較於醋酸、三乙基胺及氨水有最佳的離子穩定度。羅漢果皂苷在離子阱中使用碰撞誘導游離 (CID) 的質譜斷裂模式，可觀察到依序減少葡萄醣基的質譜特徵，配合管柱的滯留時間，所獲得分子結構資訊可做為定性分析的依據。Mogroside V在市售羅漢果乾燥果實與濃縮萃取粉末產品中的含量分別為1.5~1.8與25.9%。另外分析7種宣稱含羅漢果之加工產品的含量範圍為0.3~1.8%，其中有一種膏狀飲品與二種糖果未檢出有羅漢果苷。為選擇性的轉換mogroside V為其他較少醣基數目的皂苷型態，於靈芝菌絲培養液中添加羅漢果水萃液，可顯著的增加菌絲生長；即使在只含羅漢果水萃液，而無其他營養的情況下，21天後靈芝菌絲生長量也與培養於麥芽萃取物培養基之控制組無顯著差異。顯示靈芝菌絲可利用β-glucosidase水解mogroside V結構上的葡萄糖，主要轉換成帶有三個葡萄糖基的mogroside IIIE；當環境中缺乏碳源時，靈芝菌絲則會進一步的利用mogroside IIIE上的葡萄糖基，轉成帶有二個葡萄糖基的mogroside II A，而mogroside IIA分子上所帶之β-(1,2) 鍵結的葡萄糖基對靈芝菌絲所分泌的β-glucosidase有較強的耐受性。進一步利用酵母菌 (Saccharomyces cerevisiae) 探討對羅漢果皂苷上醣基型態之轉換機制實驗中，顯示野生型 (wild type) 酵母菌可將羅漢果皂苷萃取物進行生物轉換，將mogroside V的羅漢果皂苷轉換產生具四醣及三醣的羅漢果皂苷，siamenoside I、mogroside IV及mogroside IIIE。為進一步了解酵母菌中主要轉換羅漢果皂苷之葡萄糖水解酵素，利用酵母菌單一非必需基因 (non-essential gene) 剔除突變基因庫 (deletion sets)，篩選出exg1Δ及kre6Δ可影響羅漢果皂苷降解途徑，其中kre6Δ酵母菌突變菌株可快速產生較高專一性之羅漢果皂苷mogroside IIIE；而Exg1則具有選擇性水解羅漢果皂苷之β-1, 6-葡萄糖鍵結的能力。本論文利用HPLC/ESI tandem MS開發一個可例行性檢測羅漢果皂苷分子的分析，利用此分法觀察到靈芝與酵母菌的β-glucosidase對羅漢果皂苷分子上醣基的水解具有選擇性，可應用為製備不同醣苷型態的方法。

關鍵字

羅漢果皂苷靈芝；酵母菌；生物轉換；液相層析串聯式電灑游離多次質譜

並列摘要

The sweetness of Siraitia grosvenori fruit comes from a mixture of cucurbitane-type triterpene saponins, mogrosides I-V. mogroside V, a triterpene pentaglycoside,is the major component of mogrosides.A HPLC-electrospray ionization- tandem mass spectrometry (MSn) was established to analyze contents of mogrosides, compounds without UV-absorption, in S. grosvenorii containing products. Using 0.01% of formic acid affiliated the ionization of mogroside on electrospray interface. It is a superior modifier to acetic acid, triethylamine, and ammonia. The mogroside V molecule sequentially lost its glucosyl unit as the mass spectrometry being operated at collision-induced dissociation mode. This fragmentation pattern provided structural information to identify the compound in addition to retention time on column and the molecular weight. The dried fruit purchased at Chinese herb store in Taipei had morgroside content about 1.5%. The content of an extract powder was 25.6% which was the highest value among the selected commercial samples. Most of examined products contained mogroside V in the range of 0.3~1.8%, whereas some candy and syrup products could not find any contents. The purpose of this study was to investigate the structural modification of mogrosides by Ganoderma lucidum mycelium and yeast. During the growth of the G. lucidum, the mycelium showed significant activity of beta-glucosidase and able to hydrolyze the glucosyl residues of mogroside V. Adding water extract of the fruit did not impact the growth of mycelia in a malt extract medium. The major metabolite of mogroside was tritepene triglucoside, mogroside IIIE. The mycelia of G. lucidum were able to further utilize one glucosyl residue on Mogroside IIIE and converted it to triterpene diglycoside, Morgroside II A when the carbon source was limited. According the results of high-performance liquid chromatography coupled withelectrospray ionization tandem mass spectrometry, the beta-(1,2) linkage of glucosyl were more resistant to hydrolysis of beta-glucosidase of G. lucidum. In the last part of this research, we attempt to selectively convert the major saponin mogroside V, a mogrol pentaglucoside, into mogroside III E, a triglucoside, via the beta-glucosidases of the budding yeast Saccharomyces cerevisiae. We report that the beta-glucopyranosyl and beta-glucopyranosyl-(1,2)-beta-D- glucopyranosyl attached on C-3 and -24 of mogrol, respectively, were resistant to hydrolysis by yeast beta-D-glucosidases. We further screened 16 mutants bearing single defective glucanase or glucosidase genes, thereby demonstrating that Exg1 is a major enzyme of the initiation of mogroside V conversion. Deletion of the KRE6 gene unexpectedly facilitated the production of mogroside III E in yeast culture. This paper demonstrates that yeast knockout mutants are a valuable tool for saponin modification and for studying the specificity of glucosidase function.