指導教授：呂廷璋臺灣大學：食品科技研究所王鐘凰Wang, Chung-HuangChung-HuangWang2014-11-272018-06-292014-11-272018-06-292014http://ntur.lib.ntu.edu.tw//handle/246246/262451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.摘要 I Abstract III 目錄 V 圖目錄 IX 表目錄 XI 附表附圖目錄 XII 壹、前言 1 貳、文獻回顧 3 一、食用菇蕈簡介 3 (一)生物分類 3 (二)營養價值 3 (三)食用菇蕈之活性多醣及其生理功效 5 二、靈芝簡介 6 (一)靈芝之活性多醣及其生理功效 7 (二)靈芝菌絲液態發酵於生產靈芝多醣的相關研究 8 三、(1,3)-β-D-葡萄聚醣簡介 10 (一)生物來源及基本結構 10 (二)(1,3)-β-D-葡萄聚醣與真菌細胞壁的關係 11 (三)環境條件對(1,3)-β-D-葡萄聚醣高級結構的影響 13 (四)真菌(1,3;1,6)-β-D-葡萄聚醣之生物活性及分子結構對活性表現的影響 15 四、(1,3)-β-D-葡萄聚醣定量檢測方法 18 (一)酵素水解結合呈色法或層析法 18 1 endo-與exo-(1,3)-β-D-Glucanase 19 2 Lyticase與β-glucosidase, exo-1,3-β-glucanase 20 3 exo-1,3-β-Glucanase, β-glucosidase與amyloglucosidase, invertase 23 (二)染劑錯合呈色法 23 1 Aniline blue 螢光測定法 23 2 Congo red 可見光測定法 25 (三)抗體識別結合酵素聯結免疫分析法 25 五、巨噬細胞與其免疫刺激反應 26 (一)巨噬細胞及其功能 26 (二)脂多醣誘導巨噬細胞產生之免疫刺激 27 (三)免疫刺激評估指標 29 1 iNOS與NO 29 2 COX-2 29 3 Tumor necrosis factor-alpha (TNF-alpha) 29 參、研究目的與實驗架構 31 一、研究目的 31 二、實驗架構 32 肆、材料與方法 33 一、實驗材料及其製備 33 (一)食用菇蕈子實體及其凍乾粉末製備 33 (二)食用菇蕈水溶性膳食纖維 (熱水可萃多醣) 製備 33 (三)食用菇蕈鹼可萃多醣製備 37 (四)靈芝菌絲液態培養發酵產物 37 (五)靈芝菌絲液態發酵產物水溶性多醣的製備及區分 38 (六)具報導基因細胞殖株平台 40 二、化學藥品 41 (一)化學試劑 41 (二)標準品 41 (三)酵素 42 三、儀器設備 42 四、實驗方法 43 (一)粗多醣含量 43 (二)(1,3;1,6)-β-D-葡萄聚醣含量及其(1,6)-β-D-glucosyl分支比例 43 (三)β-D-葡萄聚醣含量-Megazyme分析套組 47 1 Mushroom and yeast beta-glucan assay 47 2 Enzymatic yeast beta-glucan 49 (四)單醣組成 50 (五)分子量分佈 51 (六)巨噬細胞免疫刺激活性檢測 52 伍、結果與討論 55 一、不同酵素水解法測定菇蕈子實體樣品中葡萄聚醣的比較 55 二、不同萃取溶液對菇蕈子實體中(1.3;1,6)-β-D-葡萄聚醣可萃出量及其分支比例的影響 64 (一)鹼萃條件的評估 64 (二)不同萃取方式所得(1,3;1,6)-β-D-葡萄聚醣可萃出量的比較 65 (三)不同萃取方式所得(1,3;1,6)-β-D-葡萄聚醣分支度的比較 67 三、菇蕈子實體中水溶性及非水溶性膳食纖維之(1,3;1,6)-β-D-葡萄聚醣的分佈 68 四、利用食用菇蕈水溶非消化性多醣 (水溶性膳食纖維) 評估具報導基因細胞殖株平台的應用 72 (一)報導基因細胞殖株平台 (iNOS-及COX-2-directed luciferase) 適用性確認 72 (二)具報導基因細胞殖株平台篩檢菇蕈水溶非消化性多醣的活性表現 73 (三)菇蕈水溶非消化性多醣對刺激老鼠巨噬細胞分泌TNF-alpha及NO的釋放量 78 (四)報導基因細胞殖株平台與其他檢測免疫調節活性之測定方法結果的比較 79 五、靈芝菌絲發酵產物中可溶性(1,3;1,6)-β-D-葡萄聚醣的含量濃度及分子結構特性 82 (一)可溶性(1,3;1,6)-β-D-葡萄聚醣濃度的變異性 82 (二)可溶性多醣中(1,3;1,6)-β-D-葡萄聚醣所占比例及其單醣組成 85 (三)可溶性(1,3;1,6)-β-D-葡萄聚醣的分子特徵 88 (四)可溶性(1,3;1,6)-β-D-葡萄聚醣的分離純化及其免疫活性的確認 92 陸、結論 95 柒、參考文獻 974120977 bytesapplication/pdf論文公開時間：2019/03/09論文使用權限：同意有償授權(權利金給回饋學校)(1,31,6)-β-D-葡萄聚醣分支度可食用菇靈芝RAW 264.7iNOSCOX-2酵素水解螢光酵素分析thesishttp://ntur.lib.ntu.edu.tw/bitstream/246246/262451/1/ntu-103-D97641002-1.pdf