臺灣博碩士論文加值系統

由各種研究研究結果顯示，樟芝所含活性物質具有抗生素的生理活性特性，因此利用此特性來篩選對樟芝菌絲體生長具有助益的助益菌，配合菌絲體液態發酵培養的培養基組成及培養條件，以提高菌絲體多醣體產率為此研究目的，並探討助益菌對樟芝菌絲產生多醣之影響。
實驗結果顯示，在溫度25℃、初始pH值4.5、轉速100 rpm，並添加助益菌Pantoea dispersa菌液10 ％V的條件下培養20天，胞外和胞內多醣濃度可達41.02 mg/mL及7.32 mg/mL，菌絲體濃度72.80 mg/mL，和一開始使用基礎MEB培養基培養條件下所產生的胞外及胞內多醣濃度提升7倍及1倍，而菌絲體濃度也提升將近1倍左右。
在大鼠毒性測試中，每天不間斷餵食1.5 × 106 CFU/mL助益菌菌液，經過21天後並沒有任何一隻大鼠死亡，而對照組及實驗組的肝、腎及脾臟經解剖後外觀組織和顏色一切正常，切片比較各組織也無明顯差異，且血液中的肝及腎功能指數GOP、GPT、BUN和Cre等在實驗組與對照組之間也相似，故可初步判斷助益菌Pantoea dispersa對動物體應無傷害。

Previous studies have shown that the bioactive compounds derived from Antrodia camphorata has inherent property of antibiosis. Firstly, we took this advantage to screen out the beneficial bacteria which had the facilitating tendency in growing the mycelium of Antrodia camphorata. Accommodating to the appropriate culture medium and conditions in submerged culture of the mycelium, the main purpose of this study is to improve the polysaccharides production of the cultural mycelium, by co-culturing with the specific beneficial bacterium of Pantoea dispersa.
The results showed that the optimize conditions for the submerged culture of Antrodia camphorata were at the temperature of 25°C, initial pH 4.5, 100 rpm in rotating speed, 20 days culturing period, and by adding Pantoea dispersa 10 %V of the beneficial bacterium suspension. The concentrations of the intracellular and extracellular polysaccharides were up to 41.02 mg/mL and 7.32 mg/mL respectively, the concentration of the mycelium was also up to 72.80 mg/mL. Comparing to the culture in general used MEB medium without adding beneficial bacterium, the results showed this study could enhance 8 and 2 times of intracellular and extracellular polysaccharides production, and 2 times of mycelium weight also.
In order to verify whether the bacterium Pantoea dispersa harms to animals or not, several Rats were set in toxicity tests. The results showed there were no deaths happen after 21 days for continuing feeding with 1.5 × 106 CFU/mL of bacterium suspension. The appearances of livers, kidneys, and spleens tissues seem to be all normal. Also, there were no obvious difference between control and experimental groups through histological sections. In blood testing, the values of GOP, GPT, BUN, and Cre of livers and kidneys were similar between both groups. Thus, we can safely say that Pantoea dispersa would not cause injury to the animals.

第一章 緒論 .....................................1
1-7 前言 .....................................1
1-8 高等擔子菌菇類 ............................3
1-9 研究目的 .....................................7

第二章 文獻回顧 .....................................8
2-1 樟芝的介紹 ............................8
2-2 樟芝的型態 ............................9
2-2-1 樟芝子實體外型構造 ............................9
2-2-2 樟芝子實體菌絲構造 ............................11
2-3 樟芝成分分析 ............................12
2-3-1 ㄧ般成分 .....................................13
2-3-2 微量金屬含量分析 ............................16
2-4 生理活性分析 .....................................16
2-4-1 抗氧化能力 .....................................20
2-4-2 保肝功能 .....................................20
2-4-3 抗腫瘤能力 .....................................21
2-4-4 抗菌能力 .....................................21
2-5 菇類多醣介紹 .....................................22
2-5-1 多醣形式種類 ............................23
2-5-2 （1→3）-β-D-葡聚醣 ............................24
2-5-2-1 （1→3）-β-D-葡聚醣抗腫瘤活性 ..........24
2-5-2-2 （1→3）-β-D-葡聚醣主鏈鍵結形式與生理活性關係
..............................................27
2-5-2-3 多醣分支度對活性關係的引響 ...................28
2-5-2-4 多醣分子量對腫瘤抑制活性關係的引響 ..........31
2-5-2-5 多醣構型對生理活性影響 ...................33
2-6 Analine blue螢光染色法對（1→3）-β-D-葡聚醣的定量 .....................................34
2-6-1 Aniline blue對（1→3）-β-D-葡聚醣的專一性 .35
2-6-2 Aniline blue對（1→3）-β-D-葡聚醣的應用 .37
2-7 多醣檢測處理 ............................42
2-7-1 菌體的移除 ......................................42
2-7-2 相的分離 ......................................42
2-8 細菌對真菌的影響 .............................43


第三章 實驗藥品及分析方法 .............................44
3-1 菌株 ......................................44
3-2 實驗藥品 ......................................44
3-3 實驗儀器 ......................................45
3-4 分析方法 ......................................46
3-4-1 pH值測定 ......................................46
3-4-2 菌絲濃度測定 .............................47
3-4-3 多醣濃度測定 .............................47
3-4-3-1 多醣分離→乙醇沉澱法 ....................47
3-4-3-2 多醣量測定→酚－硫酸比色法（Phenol–sulfuric acid method）........................................48
3-4-3-3 （1→3）-β-D-葡聚醣定量→Aniline blue method
...............................................49

第四章 實驗方法 ......................................53
4-1 集菌法篩選助益菌菌株 ....................53
4-2 樟芝菌絲在培養皿平面培養 53
4-3 基礎固態和液態培養基成分及多醣體產率之液態培養基成份 ...............................................54
4-4 樟芝菌絲接菌量 .............................56
4-5 助益菌菌種篩選 .............................56
4-6 助益菌鑑定 .............................58
4-7 藉由助益菌添加以檢測多醣濃度變化 ...........59
4-8 最適產生樟芝多醣培養基配方配合助益菌添加以檢測多醣濃度變化 ................................................60
4-9 HPLC圖譜分析 ..............................65
4-10 助益菌毒性測試 ..............................65

第五章 實驗結果與討論 ..............................67
5-1 助益菌菌種篩選 ..............................67
5-2 助益菌鑑定 ..............................71
5-3 藉由助益菌添加以檢測多醣濃度變化 ............72
5-4 最適產生樟芝多醣培養基配方配合助益菌添加以檢測多醣濃度變化 ................................................74
5-5 HPLC圖譜分析 ..............................89
5-6 助益菌毒性測試 ..............................91

第六章 結論與未來展望----------------------------------98
6-1 結論 .......................................98
6-2 未來展望 .......................................99
6-3 參考文獻 ......................................100
附錄A ...............................................115
附錄B ...............................................116
附錄C ...............................................117

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