摘 要
為比較靈芝子實體與液態發酵液中（1→3）-β-D-葡聚醣的差異，使用二種赤芝(Ganoderma lucidum)菌株：GL 4及GL 11為原料，以不同的生產方式製得樣品。結果顯示每克子實體熱水可萃取的粗多醣含量，GL4與GL11分別為6.07 mg及11.32 mg，而其中所測得的（1→3）-β-D-葡聚醣又分別為粗多醣之12.0﹪及7.9 ﹪。GL4菌株之發酵液粗多醣的含量中，以5升及20升發酵槽發酵培養分別為0.89 mg/mL及0.51 mg/mL，其中所含的（1→3）-β-D-葡聚醣又分別為粗多醣之10.5 ﹪及32.6 ﹪，GL11菌株之發酵液粗多醣的含量中，以5升及20升發酵槽發酵培養分別為0.67 mg/mL及0.63 mg/mL，其中所含的（1→3）-β-D-葡聚醣又分別為粗多醣之9.3 ﹪及5.8 ﹪，顯示不同菌株與不同培養方式與環境所得粗多醣及（1→3）-β-D-葡聚醣的含量有明顯的差異。
利用分子篩層析檢測赤芝粗多醣之分子量分佈，發現樣品中之（1→3）-β-D-葡聚醣均以大分子存在。子實體具有最大之分子量，但菌種間差異很大，GL 4之分子量約為306 kDa， GL 11分子量約為160 kDa；5升發酵槽培養所得多醣的分子量最小，GL 4及GL 11之分子量為97 kDa及55 kDa，而20升發酵槽產生之多醣菌種間分子量差異小，均約為100 kDa。
利用exo-與endo-(1,3)- β-glucanase將具(1,6)-β-D-glucosyl分支的（1→3）-β-D-葡聚醣完全水解為葡萄糖(glucose)及龍膽雙醣(gentiobiose)，配合高效陰離子交換層析法( High-performance anion exchange chromatography / HPAEC)的檢測方式分析靈芝多醣的分支度。由結果顯示，子實體培養產生之（1→3）-β-D-葡聚醣，GL 4及GL 11分支度分別為0.12及0.11。不同發酵情況所得之（1→3）-β-D-葡聚醣的分支度也明顯受到發酵環境不同的影響，以 5L發酵槽產生之多醣，GL 4及GL 11之分支度分別為0.17及0.13 ，而20 L發酵槽產生之GL 4及GL 11之分支度分別為0.38及0.22。
利用螢光染劑aniline blue對（1→3）-β-D-葡聚醣的專一結合性發展出檢測（1→3）-β-D-葡聚醣含量的螢光呈色法，可以直接測定子實體萃取物與發酵液中的（1→3）-β-D-葡聚醣含量，但因為使用已知的（1→3）-β-D-葡聚醣: Laminarin為標準品，因此以Laminarin equivalent表示檢測量。5﹪H2O2可作為漂白劑去除子實體萃取物或菌絲培養液中的深暗顏色，以減少對螢光檢測的干擾。

ABSTRACT
Two Ganoderma lucidum strains, GL4 and GL11, were selected to study the structural difference of branched （1→3）-b-D-glucans, a potential immune regulated polysaccharide, from fruiting body and mycelium culture. The contents of hot-water extractable crude polysaccharides of GL4 and GL11 were 6.07 and 11.32mg per gram of fruiting body, respectively. The extracted crude polysaccharides of GL4 and GL11 contented 12.0 and 7.9% of the （1→3）-b-D-glucans, respectively. The contents of crude polysaccharides in G4 mycelium culture broth were 0.89 and 0.51mg per ml from 5- and 20-liter fermentator, respectively. The crude polysaccharides of broth from 5- and 20-liter fermentor contented 10.5 and 32.6% of the （1→3）-b-D-glucans, respectively. The contents of crude polysaccharides in G11 mycelium culture broth were 0.67 and 0.63mg per ml from 5- and 20-liter fermentor, respectively. The crude polysaccharides of broth from 5- and 20-liter fermentor contented 9.3 and 5.8% of the （1→3）-b-D-glucans, respectively. The results indicated that differences of polysaccharides and the （1→3）-b-D-glucans contents were significant both in strains and cultivation conditions.
Gel permeation chromatography with laser-light scattering was exerted to measure the molecular weight of the（1→3）-b-D-glucans. Comparing the molecular size of（1→3）-b-D-glucans from fruiting body, the size (306 kDa) from GL4 was larger than the size (106 kDa) from G11. The molecular size of the （1→3）-b-D-glucans from fruiting body was larger than the size of those from mycelium culture. The degree of branching of the （1→3）-b-D-glucans was assayed by enzymatic hydrolysis with high-performance anion-exchange chromatography. The branched （1→3）-b-D-glucans were converted to glucose and gentiobiose (from branching points) by using endo- and exo-（1→3）-b-D-glucanase and degree of branching was calculated from the molar ratio of glucose and gentiobiose. The average degree of branching of the β-D-glucans from GL4 and GL11 fruiting body were 0.12 and 0.11, respectively. The degree of branching of the β-D-glucans from mycelium culture was in the range of 0.13 to 0.38 and was significantly affected by both strains and cultivation conditions.
The contents of the （1→3）-b-D-glucans in the hot-water extracts could be estimated by using a fluorescence method developed recently in our laboratory. The results should presented as laminarin equivalents (LE), because the laminarin, a known （1→3）-b-D-glucan, was used as standard. The method was based on the selective reaction between aniline blue, a fluorescence dye, and （1→3）-ABSTRACT
Two Ganoderma lucidum strains, GL4 and GL11, were selected to study the structural difference of branched （1→3）-b-D-glucans, a potential immune regulated polysaccharide, from fruiting body and mycelium culture. The contents of hot-water extractable crude polysaccharides of GL4 and GL11 were 6.07 and 11.32mg per gram of fruiting body, respectively. The extracted crude polysaccharides of GL4 and GL11 contented 12.0 and 7.9% of the （1→3）-b-D-glucans, respectively. The contents of crude polysaccharides in G4 mycelium culture broth were 0.89 and 0.51mg per ml from 5- and 20-liter fermentator, respectively. The crude polysaccharides of broth from 5- and 20-liter fermentor contented 10.5 and 32.6% of the （1→3）-b-D-glucans, respectively. The contents of crude polysaccharides in G11 mycelium culture broth were 0.67 and 0.63mg per ml from 5- and 20-liter fermentor, respectively. The crude polysaccharides of broth from 5- and 20-liter fermentor contented 9.3 and 5.8% of the （1→3）-b-D-glucans, respectively. The results indicated that differences of polysaccharides and the （1→3）-b-D-glucans contents were significant both in strains and cultivation conditions.
Gel permeation chromatography with laser-light scattering was exerted to measure the molecular weight of the（1→3）-b-D-glucans. Comparing the molecular size of（1→3）-b-D-glucans from fruiting body, the size (306 kDa) from GL4 was larger than the size (106 kDa) from G11. The molecular size of the （1→3）-b-D-glucans from fruiting body was larger than the size of those from mycelium culture. The degree of branching of the （1→3）-b-D-glucans was assayed by enzymatic hydrolysis with high-performance anion-exchange chromatography. The branched （1→3）-b-D-glucans were converted to glucose and gentiobiose (from branching points) by using endo- and exo-（1→3）-b-D-glucanase and degree of branching was calculated from the molar ratio of glucose and gentiobiose. The average degree of branching of the β-D-glucans from GL4 and GL11 fruiting body were 0.12 and 0.11, respectively. The degree of branching of the β-D-glucans from mycelium culture was in the range of 0.13 to 0.38 and was significantly affected by both strains and cultivation conditions.
The contents of the （1→3）-b-D-glucans in the hot-water extracts could be estimated by using a fluorescence method developed recently in our laboratory. The results should presented as laminarin equivalents (LE), because the laminarin, a known （1→3）-b-D-glucan, was used as standard. The method was based on the selective reaction between aniline blue, a fluorescence dye, and （1→3）-β-D-glucans. Five percent of hydrogen peroxide was used, as bleaching agent, to reduce the inference of brown substances existed in the extracts.

目錄
頁次
中文摘要……………………………………………………………I
英文摘要……………………………………………………………Ⅲ
目錄…………………………………………………………………Ⅳ
圖目錄………………………………………………………………Ⅷ
表目錄………………………………………………………………Ⅸ
壹、前言…………………………………………………………………1
貳、文獻整理……………………………………………………………2
一、靈芝…………………………………………………………………2
（一）前言…………………………………………………………2
（二）靈芝簡介……………………………………………………3
（三）靈芝的分類與特徵…………………………………………4
（四）靈芝的生長與栽培…………………………………………4
（五）靈芝的一般成分……………………………………………7
（六）靈芝的功效…………………………………………………8
（七）靈芝的活性成分………………………………………… 12
二、（1→3）-b-D-葡聚醣………………………………………………15
（一）（1→3）-b-D-葡聚醣的抗腫瘤活性………………………15
（二）（1→3）-b-的主鏈與活性的關係…………………………16
（三）多醣分子量分佈與活性的關係…………………………17
（四）多醣分支度與活性的關係………………………………18
（五）多醣分子構形與活性的關係……………………………21
三、Aniline blue………………………………………………………23
（一）Aniline blue的介紹………………………………………23
（二）Aniline blue對（1→3）-b-D-葡聚醣的特異性…………24
參、材料與方法…………………………………………………………26
一、實驗材料…………………………………………………………26
（一）靈芝子實體………………………………………………26
（二）靈芝液態發酵液…………………………………………26
二、實驗方法…………………………………………………………27
（一）多醣的萃取………………………………………………27
（二）多醣的粗蛋白的去除……………………………………28
（三）多醣性質的測定………………………………………28
1. 多醣中總醣量的檢測……………………………………28
2. 多醣中蛋白質量的檢測……………………………………29
3. 多醣中uronic acid含量的檢測 …………………………29
4. 多醣中還原醣含量的檢測…………………………………29
（四）靈芝所含的（1→3）-b-D-葡聚醣檢測……………………30
1. （1→3）-b-D-葡聚醣(Aniline blue assay)的測定…30
（1） 標準曲線的建立……………………………………30
（2） 樣品檢測……………………………………………30
（3） 暗色物質的干擾與脫色……………………………31
2. 膠體過濾層析………………………………………………31
3.靈芝多醣分子量之測定……………………………………32
4.靈芝多醣分支度之鑑定……………………………………32
肆、結果與討論…………………………………………………………35
一、葡聚醣之定量分析 ………………………………………………35
（一）總醣及總粗多醣之定量 …………………………………35
（二）（1→3）-b-D-葡聚醣含量分析……………………………37
1. 深暗顏色樣品之脫色處理…………………………………38
2. 脫色處理對葡聚醣的影響…………………………………39
(1) （1→3）-b-D-葡聚醣檢測量之影響
－Aniline blue assay 檢測螢光強度之影響……………39
(2) （1→3）-b-D-葡聚醣之氧化情形
－檢測酸性醣（uronic acid ）之產生 …………………40
(3) （1→3）-b-D-葡聚醣之聚合度(degree of polymerization)變化
－檢測還原糖量之變化………………………………41
二、靈芝所含的（1→3）-b-D-葡聚醣檢測…………………………46
（一）靈芝多醣區分之分子量分佈情形 ……………………46
（二）靈芝多醣區分之分子量 …………………………………48
（三）具（1→3）-b-D-葡聚醣的靈芝多醣區分之分支度………56
伍、結論………………………………………………………………68
陸、參考文獻…………………………………………………………70
圖目錄
頁次
圖一、Aniline blue主要成分之結構………………………………23
圖二、Sirofluor的結構……………………………………………24
圖三、GL 4子實體萃取粗多醣之分子量分佈……………………49
圖四、GL 4小型發酵(5L)培養之發酵液粗多醣之分子量分佈………50
圖五、GL 4大型發酵(20 L)之發酵液粗多醣之分子量分佈…………51
圖六、GL 11子實體萃取粗多醣之分子量分佈……………………52
圖七、GL 11小型發酵(5 L)培養之發酵液粗多醣之分子量分佈……53
圖八、GL 11大型發酵(20 L)之發酵液粗多醣之分子量分佈………54
圖九、單醣標準品以HPAEC檢測之標準迴歸曲線………………57
圖十、雙醣標準品以HPAEC檢測之標準迴歸曲線………………58
圖十一、GL 4子實體萃取純化多醣以HPAEC分析………………61
圖十二、GL 4小型發酵(5 L)之發酵液純化多醣以HPAEC分析……62
圖十三、GL 4大型發酵(20 L)之發酵液純化多醣以HPAEC分析…63
圖十四、GL 11子實體萃取純化多醣以HPAEC分析……………64
圖十五、GL 11小型發酵(5 L)之發酵液純化多醣以HPAEC分析…65
圖十六、GL 11大型發酵(20 L)之發酵液純化多醣以HPAEC分析…66
表目錄
頁次
表一、一些具有明顯抗腫瘤活性的（1→3）-b-D-葡聚醣與其分支度……………………………………………………19
表二、靈芝粗多醣醣類含量分析………………………………36
表三、H2O2脫色處理對（1→3）-b-D-葡聚醣( laminarin) 檢測的
影響…………………………………………………………43
表四、H2O2脫色處理對（1→3）-b-D-葡聚醣(lentinan)檢測的影響…………………………………………………………44
表五、laminarin及lentinan以5﹪H2O2 90℃加熱30分鐘處理的酸性醣及還原醣含量之變化…………………………………………45
表六、靈芝粗多醣膠體過濾層析法上 （1→3）-β-D-葡聚醣之分佈與分子量…………………………………………………55
表七、單醣標準品以HPAEC檢測之迴歸方程式……………………57
表八、雙醣標準品以HPAEC檢測之迴歸方程式……………………58
表九、靈芝多醣分支度分析……………………………………………67

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