探討牛樟芝在液態中與酵母菌共培養並利用兩階段式操作以增加三萜產量

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吳介人(Chieh-Jen Wu) 查詢紙本館藏

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化學工程與材料工程學系

論文名稱

探討牛樟芝在液態中與酵母菌共培養並利用兩階段式操作以增加三萜產量

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摘要(中)

牛樟芝為實用藥菇中的一種，食用藥菇最吸引人的地方在其抗癌、抗腫瘤的效果，而牛樟芝所含有的特殊三萜類及胞外多醣，為其他植物所沒有，其最主要的功能為：抑制組織胺釋放、防止過敏、促進肝功能、促進血小板凝集、以及降血脂，防止中老年人發生心血管疾病，因此具有相當潛力被開發為未來醫療用藥品，或作為國人近年來最熱門的保健食品。
牛樟芝只生長在特有的牛樟樹內，屬於無褶菌目，多孔菌科。由於其子實體生長非常緩慢，因此難以廣泛應用於醫療用藥品，而樟芝在自然界中也非常稀有，在市面上價格相當昂貴，因此液態培養具有相當潛力，其優點為：菌絲體生長的速度較快、所需空間較小、汙染的機率較低，因此研究將利用液態培養的方式，增加總三萜的產量。
過去增加總三萜的方法，大部分著重於培養基的改變，以及環境因子的操控，近年有學者探討兩階段式發酵，由於三萜的生產是因為牛樟芝菌(Antrodia cinnamomea)受到環境限制而產生的二次代謝物，因此第一階段讓菌體快速生長，第二階段在菌體生長到最高峰時改變環境因子增加總三萜產量。
因此，本計畫嘗試開發兩階段發酵製程系統。在第二階段加入酵母菌進行共培養並以循環式操作間歇性供氧來提升總三萜的產量，並探討共培養機制。實驗結果顯示，第二階段以靜置操作及酵母菌接菌量25%(v/v)能產出最高三萜量46.5 mg/g DW，由實驗結果可發現，酵母菌所產生的酒精為三萜產量增加之主因。

摘要(英)

Antrodia cinnamomea is one of the medicinal mushrooms. The most attractive characteristic of the medicinal mushrooms is its anti-tumor effect. Some bioactive constituents from the fruiting bodies of
A. cinnamomea have been isolated and characterized as a series of polysaccharides, steroids, triterpenoids, and sesquiterpene lactone.
Polysaccharide components extracted from A. cinnamomea mycelia have been shown exhibiting an anti-hepatitis B virus surface antigen effect, having pronounced anti-tumor effects on both in vitro and in vivo model, and displaying strong immunomodulatory and anti-inflammatory effects. The triterpenoids extracted from A. cinnamomea have anti-cholinergic and anti-serotonergic activities activation of alternative-pathway complement and plasma clotting activity. In the results of its perceived health benefits, A. cinnamomea has gained wide popularity as a health food and became the most valuable mushroom in Taiwan.
However, because of its host specificity, slow growth rate and rarity in nature, the fruiting bodies of A. cinnamomea have become the most expensive mushrooms in Taiwan in recent years. Thus, investigators have exerted their efforts to prepare this mushroom from submerged culture. Submerged culture gives rise to potential advantages of higher mycelial production in a compact space and shorter time with lesser chances of contamination.
In previous research, determining how to increase the production of triperpenoids is by the control of cultivating conditions or modification of media compositions. Recently, some researchers investigate the two-stage submerged fermentation. Since the production of triperpenoids is the bioactive metabolites in the result of limitation of culture condition, we let the A. cinnamomea grow in high speed, following by investigating when the biomass of A. cinnamomea reach the highest content and the fungus might have entered into the idiophase, the growth period that secondary metabolites are produced.
Thus, the main objectives of this proposal are to develop two-stage fermentation system. In first stage, we make the A. cinnamomea grow in high speed for 12 days, and Saccharomyces cerevisiae was added to the fermentation broth in second stage with cycling or static process to enhance triterpenoids production. The amount of triterpenoid reached 46.5 mg/g DW by using static operation with 25% of inoculation level of the Saccharomyces cerevisiae in 250 ml shake flask.

關鍵字(中)

★ 牛樟芝
★ 共培養
★ 兩階段式發酵

關鍵字(英)

論文目次

目錄
第一章、緒論 1
1.1 研究動機 1
1.2 研究目的 2
第二章、文獻回顧 3
2.1 菇類介紹 3
2.2 樟芝介紹 3
2.3 牛樟芝效用 5
2.4 牛樟芝成分分析 5
2.5 樟芝之藥理作用文獻考察 6
2.6 真菌三萜類的介紹 9
2.6.1 合成萜類的單體 9
2.6.2 萜類的重要性質 10
2.6.3 三萜的合成機制 10
2.6.4 菇類三萜的基本結構 14
2.6.5 樟芝之三萜 17
2.7 菇類多醣的介紹 23
2.7.1 多醣的分類 23
2.7.2 影響多醣活性的因素 23
2.7.3 菇類多醣的生理活性 24
2.7.4 樟芝之多醣 26
2.8 液態發酵培養 27
2.9 影響發酵的物理化學因子 27
2.9.1 培養基組成 27
2.9.2 攪拌速率 31
2.9.3 溫度 32
2.9.4 pH值 32
2.9.5 黏度(viscosity) 33
2.9.6 溶氧值(DO) 33
2.9.7 接種量 33
2.9.8 發酵槽的形式 34
2.10 共培養 34
2.10.1 Rhodotorula glutinis 與Spirulina platensis共培養 [51] 34
2.10.2 Chlorella sp. 與Saccharomyces cerevisiae共培養 [52] 35
2.10.3 Trichoderma reesei與Aspergillus niger共培養 35
2.10.4 Saccharomyces cerevisiae 與Pichia stipites [55] 36
第三章、材料與方法 37
3.1 實驗材料 37
3.1.1 實驗菌株 37
3.1.2 實驗藥品 38
3.1.3 實驗儀器及其他設備 39
3.2 實驗方法 41
3.2.1 菌株保存 41
3.2.2 培養基組成 41
3.2.3 種菌操作條件 44
3.2.4 單培養實驗 45
3.2.4 共培養實驗 46
3.2.5 Saccharomyces cerevisiae共培養之接菌量 46
3.2.6 共培養機制探討 46
3.2.7 大型發酵槽 47
3.4 分析方法 48
3.4.1 發酵液處理流程 48
3.4.2 菌重分析 49
3.4.3 葡萄糖殘量分析(還原醣測定) [56] 49
3.4.4 胞外多醣體分析 51
3.4.4.1 粗多醣之分離 51
3.4.4.2 酚-硫酸呈色法分析(總多醣) [56] 51
3.4.5 胞內三萜之分析[57] 52
第四章、結果與討論 54
4.1 共培養對三萜產量的影響 54
4.1.1 共培養菌種探討 54
4.1.2 共培養探討 56
4.2 兩階段式操作 58
4.2.1 兩階段式共培養對菌重的影響 58
4.2.2 兩階段式共培養對三萜的影響 59
4.3 酵母菌接菌量 60
4.4 共培養機制探討 61
4.4.1 酵母菌對樟芝的影響 61
4.4.2 酒精添加對樟芝的影響 62
4.5 循環式操作探討 63
4.6氣泡塔反應器 64
4.6.1 單培養發酵動力曲線 64
4.6.2 兩段段式共培養發酵對菌重之影響 65
4.6.3 兩段段式共培養發酵對三萜之影響 66
第五章、結論 68
5.1 結論 68
5.2 建議 69
第六章、參考文獻 70

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指導教授

徐敬衡(Chin-Hang shu)

審核日期

2014-8-26

推文