"菇蕈中活性多醣(1,3;1,6)-β-D-葡萄聚醣的分析與功能評估"

Abstract

真菌的細胞壁組成中具(1,6)-β-D-葡萄糖基分支之(1,3)-β-D-葡萄聚醣為一可做為生物反應修飾劑（biological response modifiers）的活性多醣，本研究利用可專一性檢測的酵素水解 [endo-與exo-(1,3)-β-D-glucanase] 結合陰離子交換層析方法量化台灣常見食用菇及靈芝菌絲發酵產物內該種葡萄聚醣的含量及分支度特性。利用0.5 M NaOH鹼液可提高(1,3;1,6)-β-D-葡萄聚醣的萃出量，為熱水萃取的4.4 – 16.4倍，除木質化程度較高的靈芝以外，不同萃取方式所得的食用菇可溶性(1,3;1,6)-β-D-葡萄聚醣其分支度並無明顯差異。靈芝子實體含有不同分支比例的β-D-葡萄聚醣，熱水萃取可得到較高分支比例的部份 (分支度0.24)，鹼液萃取因使低分支比例的葡萄聚醣溶出，使平均分支比例降低至0.13。而食用菇蕈子實體內非水溶(1,3;1,6)-β-D-葡萄聚醣含量為每克菇體乾重5.11 – 202.50 mg，水溶性(1,3;1,6)-β-D-葡萄聚醣含量則為每克菇體乾重0.18 – 15.36 mg，以白木耳最低，杏鮑菇最高。由食用菇蕈水溶非消化多醣刺激含有報導基因轉殖質體的老鼠巨噬細胞株RAW 264.7，檢測報導基因 (luciferase) 的活性表現及腫瘤壞死因子 (tumor necrosis factor-alpha, TNF-alpha)、一氧化氮 (nitric oxide, NO) 的釋放量，發現黑木耳、巴西洋菇、白木耳及珊瑚菇有高度的免疫刺激效果，顯示食用菇蕈多醣中具免疫刺激活性者並非只有(1,3;1,6)-β-D-葡萄聚醣。另外，也證實報導基因luciferase活性增加所代表誘導型一氧化氮合成酶 (inducible nitric oxide synthase, iNOS) 和誘導型環氧化酶 (cyclooxygenase-2, COX-2) 基因promoter的活化，與TNF-alpha釋放量有高度正向相關，顯示以此平台取代TNF-alpha檢測的可行性，以及做為一高通量篩檢食材成分免疫刺激效果之快速平台的可應用性。 靈芝為一重要食藥兩用之菇蕈，在現代生技工業中常用液態發酵方式培養菌絲體以獲得高量的多醣，因此選用Ganoderma lucidum BCRC36123菌株12個批次培養的菌絲發酵產品，研究此類產品中多醣產量與(1,3;1,6)-β-D-葡萄聚醣含量的變異。結果顯示發酵液中可溶性(1,3;1,6)-β-D-葡萄聚醣之濃度有大輻的變動，變異範圍在1.3 – 79.9 mg/dL，但在有顯著產量的條件下，該多醣的分支度及分子量輪廓相似，分別介於0.21 – 0.36及10^5 – 10^6 g/mol，並利用(1,3;1,6)-β-D-葡萄聚醣易互相絮集的特性，成功地以35%乙醇劃分沈降的方式將其自原多醣樣品中分離純化出，也藉由刺激老鼠巨噬細胞株RAW 264.7釋放TNF-alpha的活性證實，該多醣為一適切的活性指標。

The bioactive polysaccharides, (1,3)-β-D-glucans with (1,6)-β-D-glucosyl branches, are components of structural polysaccharides of fungal cell walls and have been classified as biological response modifiers (BRM). In this study, we used enzymatic-high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD) method to determine the amount and degree of branching (DB) of (1,3;1,6)-β-D-glucans in samples including the fruity bodies of edible mushrooms cultivated in Taiwan and the mycelium products of Ganoderma lucidum by submerged cultifation. Alkaline solution (0.5 M NaOH) extraction could increased the yield of (1,3;1,6)-β-D-glucans to 4.4 – 16.4 folds compared with the one from hot water extraction. Except the fruiting body of Ganoderma lucidum, which exhibits ligniform, the DB of edible mushrooms reveal no difference by various extraction methods. The (1,3)-β-D-glucans of G. lucidum comprised of water-soluble branching (DB 0.24) component and essential linear component that occurred in the alkaline solution extraction. Moreover, the content of insoluble dietary fiber (IDF)-(1,3;1,6)-β-D-glucans contents were 5.11 – 202.50 mg/g (dry basis), and soluble dietary fiber (SDF)-(1,3;1,6)-β-D-glucans were 0.18 – 15.36 mg/g (dry basis). The results indicated that the majority of (1,3;1,6)-β-D-glucans occurred in the insoluble dietary fiber of mushroom. We also investigated bioactivity of non-digestible polysaccharides (ND-PS) from various mushroom species by two reporter cell platforms (RAW 264.7 containing constructed plasmid, iNOS promoter-luciferase or COX-2 promoter-luciferase), and the TNF-alpha and NO concentrations in medium were determined as well. The results indicated that the immune-stimulation activities of (1,3;1,6)-β-D-glucans are mild, and some other bioactive polysaccharides may also contribute to the immune modulation activity. Moreover, we confirmed the strong positive correlations between iNOS or COX-2-directed luciferase reporter platform and the ELISA-based assay for medium TNF-alpha through this data set. This suggested that the promoter-luciferase assays successfully reflect the TNF-alpha concentration levels and the platform is applicable as a high throughput screening for the detection of mushroom polysaccharides with immune-modulatory activities. We further analyzed twelve cultivation products of Ganoderma lucidum mycelium samples. Although the results display that the amount of (1,3;1,6)-β-D-glucans significantly varied in different fermentation conditions, the DB and molecular weight of (1,3;1,6)-β-D-glucans restrict to a narrow range. For the high aggregating tendency of (1,3;1,6)-β-D-glucans, we successfully purified (1,3;1,6)-β-D-glucans by 35% ethanol precipitation method. We further confirmed the purity of (1,3;1,6)-β-D-glucans and demonstrated its bioactivity by TNF-alpha releasing assay in RAW 264.7 cells.