以酵素輔助高效能液相串聯質譜法分析不同發酵程度金萱茶中阿拉伯半乳聚醣結構特徵

Abstract

茶飲主要是從茶葉 (Camellia sinensis) 和茶芽所萃取而得的飲品，因茶飲具有許多保健功能，因此越來越受到歡迎。其中茶葉多醣被認為是茶飲所含活性成分之一，茶葉多醣（TPS）主要由異質聚醣所含的果膠多醣組成（約90％），由於茶葉多醣為一結構複雜且分子量龐大的化合物，因此我們利用酵素水解結合液相層析串聯質譜建立一種分析策略來克服茶葉多醣結構的複雜性以獲得分子特徵。茶葉多醣中的主要單醣成分是半乳糖，阿拉伯糖，鼠李糖和半乳糖醛酸。此外，茶葉多醣包含鼠李糖半乳糖醛酸聚醣及其分支，像是阿拉伯聚醣、I型阿拉伯半乳聚醣（AG-I）和II型阿拉伯半乳聚醣（AG-II）。根據骨架的種類可以把AG分成兩種型式，AG-Ⅰ為以1,4半乳醣為骨幹且在三號碳的位置帶有阿拉伯半乳寡醣的側支；AG-Ⅱ則以1,3-半乳醣為骨幹，且在六號碳的位置上帶有阿拉伯半乳寡醣的側支。為了鑑定茶葉多醣中的複雜結構，使用內聚半乳醣醛酸酶（endo-polygalacturonase），β-1,4-半乳糖苷酶，α-1,5-阿拉伯糖苷酶和β-1,6-半乳糖苷酶將阿拉伯半乳聚醣水解成具有結構特徵性的阿拉伯寡醣、半乳寡醣和阿拉伯半乳寡醣，複雜程度會隨著發酵程度降低。接著利用超高效液相層析串聯質譜分析寡醣異構物和鍵結斷裂模式。我們根據層析圖和質譜圖的結果可以發現（1→4），（1→6）和（1→3）醣苷鍵是AG中的主要鍵結組成，(1→5) 醣苷鍵為阿拉伯聚醣的主要鍵結組成，並使用單株抗體鑑定茶葉多醣的結果表明，它具有RG-I，AG-I、AG-II和阿拉伯聚醣的抗原表位特徵結構。

Tea, water extraction of leaves, and buds of the tea plant (Camellia sinensis L.), are popular beverages. Tea polysaccharides (TPS) in the drink are mainly composed of heteroglycan, including pectic polysaccharides. It needs an analysis strategy to overcome the complexity of tea polysaccharides structure to obtain molecular characteristics. We developed an analytic approach by combining enzyme-assisted liquid chromatography-tandem mass spectrometry. The main monosaccharide components in TPS were galactose, arabinose, rhamnose, and galacturonic acid. Our results of sugar linkage analysis indicated that TPS was pectic polysaccharides composed of rhamnogalacturonan and its branches, arabinan, type I arabinogalactan (AG-I), and type II arabinogalactan (AG-II). We selected endo-polygalacturonanase (PGs), arabinogalactan endo--1,4-galactanase, endo-1,5--L-arabinanase, endo--1,6-galactanase and their combination to release the branches form pectic polysaccharides for further analysis on ultra-high-performance liquid- chromatography-tandem mass spectrometry. The structural characteristics of released arabino-oligosaccharides, galacto-oligosaccharides, and arabino-galacto-oligosaccharides were elucidated according to the fragmentation pattern of each oligomer group. The results showed that (1 → 4), (1 → 6), and (1 → 3) linkages were the main glycosidic structures found in AGs. The complexity decreased as the fermentation degree increased. We further used monoclonal antibodies used for embryogenic cell clusters to verify the epitopes of RG- I, AG-I, and AG-II, which consist of the results obtained from newly developed enzyme-assisted liquid chromatography-tandem mass spectrometry. The developed analytic platform facilitates the routine analysis of pectic polysaccharides from various tea products.