以液相層析串聯質譜探討羅漢果皂素在酸與酵母菌模式中的結構轉換

Abstract

羅漢果皂素為一群具有甜味與生理功能性的三萜類醣苷分子，為暸解食品加工中此類醣苷分子的轉換結構，本論文以液相層析串聯電灑式多次質譜方法，探討羅漢果皂素在酸與酵母菌模擬模式中結構轉換。使用市售羅漢果果實濃縮萃取物為原料，其中含有帶有五個葡萄醣基之 mogroside V 含量為25.56%，為主要的皂素結構。使用逆相的碳十八固相萃取管，以45-80%甲醇可以自羅漢果水萃物中分離得到此類皂素；使用碳十八逆相層析分析管柱，以45-75%甲醇梯度可以分離含有不同數目醣基結構的羅漢果皂素。在酸性溶液系統的轉化實驗中，使用濃度2N的鹽酸溶液，以溫度60℃加熱三小時，可以由mogroside V酸水解得到十一種帶有不同醣基之羅漢果皂素，以串聯式電噴灑游離(electrospray ionization, ESI)質譜的正離子模式，可由特殊斷裂片段訊號，初步判斷其醣苷鍵結模式，再由核磁共振HSQC圖譜比對，確認帶有四個葡萄醣基的羅漢果皂素質譜特徵和核磁共振結構相符；以負離子模式觀察，可以鑑定依序減少葡萄醣基的質譜特徵。在酵母菌反應的實驗中，將羅漢果萃取物與酵母菌Saccharomyces cerevisiae BY4741 wild type在YPD培養液中反應零至六小時，發現經過四小時後，mogroside V含量明顯下降，而siamenoside I (四個醣基)、mogroside IV (四個醣基)與mogroside III(三個醣基)含量有明顯的上升，顯示酵母菌可選擇水解利用羅漢果皂素的beta-1,6-醣苷鍵結，使siamenoside I、mogroside IV、mogroside III含量上升。依照文獻前述三種水解產物仍具有強於蔗糖195-465倍的甜味，表示以羅漢果萃取物作為酵母菌相關食品的甜味劑，經過酵母菌發酵後仍可保持甜味性質。

Mogrosides are major triterpenoidal saponins in fruits of Siraitia grosvenorii, Lo Han Kuo, with intensive sweetness and many biological activities. To establish an analytical protocol of mogrosides for food quality control, a liquid chromatography- electron spray ionization- tandem mass spectrometric method, LC-ESI-MS/MS, was developed to investigate the structural conversion of the mogrosides in acid and yeast model systems. A commercial extracted and concentrated product was used as studying material. The major component of the mogrosides was found to be the penta-glucosyl saponin, mogrosides V, in the commercial product. Its content was as high as 25.56% of the weight of the product studied. All types of mogrosides could be enriched by using reversed phase C-18 solid-phase-extraction cartridges in the fraction eluted out with 45-80% of methanol and separated by a reversed phase C-18 analytical HPLC column in a water/methanol gradient elution with 45-75% methanol ratio (V/V). Eleven different mogrosides differing in glucosly numbers and glycosidic linkages could be obtained from acid hydrolysis model system using 2N hydrocholoric acid solution with heating in 60°C for 3 hours and be distinguished through their molecular weight and specific fragment patterns in the positive ion mode of electrospray-ionization by a linear ion-trap mass spectrometric analysis, respectively. These glucopyranosyl units could be lost sequentially in the negative collision-induced dissociation (CID) mode on mass spectrometric analysis. In the yeast model system, Saccharomyces cerevisiae BY4741 wild type converted majority of mogrosides V into siamenoside I (with 4 glucosyl moieties), mogroside IV (with 4 glucosyl moieties) and mogroside III (with 3 glucosyl moieties) without substantial amount of mogrosides with one or two glucosyl moieties and aglycone. The structures of mentioned three mogrosides were further confirmed by nuclear magnetic resonance (nmr) analysis. The results indicated that the enzyme system of the yeast used could selectively hydrolyze the (1, 6)-beta-glycosidic linkage of mogrosides but not (1, 2)-beta-glycosidic linkage on the mogrosides. According the literature, the remaining mogrosides, siamenoside I, mogroside IV and mogroside III, still presents 195 ~ 465 times sweetness higher than sucrose. These results imply that the mogrosides can be used as a sweetener in the yeast fermented food system.