臺灣博碩士論文加值系統

北蟲草為一種食藥用菇菌，含有許多生物活性物質，如腺苷、蟲草素、多醣體、蛋白多醣、核酸等，其活性物質具有許多保健功效，如抗腫瘤、免疫調節、降血糖、降膽固醇、抗細菌及病毒等。目前許多研究應用液態醱酵培養北蟲草，生產菌絲體及代謝產物，其優勢為具有培養週期短、品質較穩定、易於商業化大規模生產等優點。因此，以液態培養方式進行北蟲草菌絲體及生物活性物質生產，是有利於規模放大與接軌工業化之研究題材。
本論文主要以北蟲草Cordyceps militaris應用發酵槽培養，探討不同類型發酵槽體、酸鹼值、轉速與通氣量對發酵系統之影響。配合添加營養源或添加物並與LED燈照射，對菌絲體生長及代謝產物胞外多醣體(EPS)及蟲草素生產之探討。結果顯示，以攪拌式發酵槽體進行北蟲草培養，發酵槽條件為轉速150 rpm，通氣量1.5 vvm，溫度25 ℃下，蟲草素產量可達至102.22±0.99 mg/L。
進一步探討，當將培養基中酵母萃取粉濃度改為40 g/L、並添加10 g/L甘胺酸可達到最大菌體量為32.3±1.1 g/L；將培養基中酵母萃取粉濃度增加為40 g/L，為生產胞外多醣之最佳培養條件，最大產量為1805.65±15.15 mg/L；培養基中酵母萃取粉濃度改為40 g/L、添加10 g/L甘胺酸並在發酵期間全程經LED白光照射，為生產蟲草素之最佳條件，蟲草素含量、比產率與生產力分別為1098.8±25 mg/L、37.16 mg/g與91.57 mg/L/day。

Cordyceps militaris contains many biological activities, claimed to possess anti-tumor, immune regulation, anti-bacteria and anti-virus activities, etc. Cordyceps militaris was reported to contain a number of bio-active components such as adenosine, cordycepin, exo-polysaccharides, protein polysaccharides and nucleic acids. The application of the submerged culture of mushroom was expected to reduce cultivation time, higher production, and realize the scale-up process.
In this study, the factors including carbon/nitrogen concentration, additives, bioreactor and light source on biomass and metabolites production by C. militaris were investigated. The result indicated that, in flask, the cultivation at 40 g/L glucose, 40 g/L yeast extract and glycine at 10 g/L were the best conditions for cordycepin production. Based on the experiments, the culture conditions were optimized in the stirred-tank bioreactor. The fermentation operated at 40 g/L glucose, 40 g/L yeast extract and 10 g/L glycine were observed to be the most favorable conditions for cordycepin production under white light. Under the optimal conditions, cordycepin production and productivity can reach to 1098.8±25 mg/L and 91.57 mg/L/d, respectively.

總目錄
中文摘要 i
ABSTRACT ii
總目錄 iii
圖目錄 v
表目錄 vii
第一章 緒論 1
1.1 前言 1
1.2生化反應器之簡介 4
1.2.1 氣舉式醱酵槽與網管式醱酵槽 4
1.3研究動機 9
第二章 文獻回顧 10
2.1 北蟲草菌 (Cordyceps militaris) 10
2.2 北蟲草代謝產物 13
2.2.1 蟲草多醣體 13
2.2.2 腺苷、蟲草素 14
2.3 影響北蟲草發酵培養之因素 15
2.4 北蟲草固態培養 21
2.5 Light-Emitting Diode (LED) 之簡介 22
2.6 光接收器介紹 23
2.6.1 光敏素 (phytochromes) 23
2.6.2 隱花色素 (cryptochromes) 23
2.6.3 植物向光素 (phototropins) 24
2.7 光照對微生物的影響與應用 24
第三章 實驗方法與材料 25
3.1 實驗藥品 25
3.2 實驗儀器 26
3.3 實驗菌株與培養 28
3.3.1 實驗菌株來源 28
3.3.2 固態平板菌株培養與繼代 28
3.3.3 液態菌種發酵 28
3.3.4 發酵槽培養 30
3.3.5 搖瓶發酵實驗 32
3.3.6 發酵槽實驗 32
3.4 LED燈及光強度計 33
3.5 分析方法 34
3.5.1 發酵液取樣分析之流程圖 34
3.5.2菌絲體(mycelia biomass)乾重之測定 34
3.5.3發酵液殘糖之測定 34
3.5.4 發酵液中胞外多醣(EPS)濃度之測定 36
3.5.5 發酵液中腺苷、蟲草素濃度之分析[22, 25, 55] 37
第四章 結果與討論 40
4.1 碳氮源濃度對北蟲草液態培養之影響 41
4.1.1 碳氮源濃度對北蟲草液態發酵生產菌絲體與發酵碳源消耗之影響 41
4.1.2 碳氮源濃度對北蟲草液態發酵生產胞外多醣之影響 43
4.1.3 碳氮源濃度對北蟲草液態發酵腺苷與蟲草素含量之影響 45
4.2 添加物種類及濃度對北蟲草液態培養之影響 48
4.2.1 添加物種類對北蟲草液態發酵生產菌絲體、碳源消耗與胞外多醣含量之影響 48
4.2.2 添加物種類對北蟲草液態發酵腺苷與蟲草素含量之影響 51
4.2.3 甘胺酸濃度對北蟲草液態發酵菌絲體與蟲草素含量之影響 53
4.3 發酵槽培養對北蟲草液態發酵之影響 56
4.3.1 發酵槽體培養對北蟲草液態發酵之影響 57
4.3.2 酸鹼值調控對北蟲草在發酵槽培養下之影響 60
4.3.3 不同攪拌速率對北蟲草在發酵槽培養下之影響 63
4.3.4 不同通氣量對北蟲草在發酵槽培養下之影響 65
4.4 LED光照下對北蟲草於發酵槽培養之影響 68
4.4.1 LED光照下對北蟲草菌體生成、碳源消耗與胞外多醣生成於發酵槽培養之影響 68
4.4.2 LED光照下對北蟲草腺苷消耗與蟲草素生成於發酵槽培養之影響 71
4.5 最佳培養條件對北蟲草於發酵槽體培養之影響 74
4.5.1 最佳培養條件對北蟲草菌體生長於發酵槽體培養之影響 75
4.5.2 最佳培養條件對北蟲草菌體生長於發酵槽體培養之影響 77
4.6 重複批次發酵對北蟲草於發酵槽體培養之影響 79
4.6.1 重複批次發酵對北蟲草菌體生長與胞外多醣於發酵槽體培養之影響 79
4.6.2 重複批次發酵對北蟲草生產蟲草素於發酵槽體培養之影響 81
第五章 結論與未來展望 82
5.1 結論 82
5.2 未來展望 86
參考文獻 87
圖目錄
圖1-1 氣舉式及網管式發酵槽示意圖 6
圖1-2 攪拌通氣式發酵槽示意圖 7
圖2-1 野生冬蟲夏草子實體 13
圖2-2北蟲草子實體 13
圖2-3 腺苷(Adenosine)結構圖 14
圖2-4 蟲草素(Cordycepin)結構圖 14
圖3-1 發酵液取樣分析之流程 34
圖3-2 葡萄糖標準曲線 35
圖3-3 胞外多醣標準曲線 37
圖3-4 腺苷(Adenosine)標準曲線 38
圖3-5 蟲草素(Cordycepin)標準曲線 39
圖4-1 不同碳氮源濃度對北蟲草菌絲體生長之時間歷時圖 42
圖4-2 不同碳氮源濃度對北蟲草碳源消耗之時間歷時圖 43
圖4-3 不同碳氮源濃度對北蟲草生產胞外多醣之時間歷時圖 44
圖4-4 不同碳氮源濃度對北蟲草腺苷消耗之時間歷時圖 46
圖4-5 不同碳氮源濃度對北蟲草生成蟲草素之時間歷時圖 46
圖4-6 不同添加物對北蟲草菌絲體生長之時間歷時圖 49
圖4-7 不同添加物對北蟲草碳源消耗之時間歷時圖 49
圖4-8 不同添加物對北蟲草生產胞外多醣之時間歷時圖 50
圖4-9 不同添加物對北蟲草腺苷消耗之時間歷時圖 51
圖4-10 不同添加物對北蟲草生成蟲草素之時間歷時圖 52
圖4-11 甘胺酸濃度對北蟲草菌絲體生長之時間歷時圖 54
圖4-12 甘胺酸濃度對北蟲草生成蟲草素之時間歷時圖 54
圖4-13 發酵槽體對北蟲草菌絲體生長之時間歷時圖 58
圖4-14 發酵槽體對北蟲草碳源消耗之時間歷時圖 58
圖4-15 發酵槽體對北蟲草發酵結果之比較 59
圖4-16 發酵液中pH值之時間歷時圖 61
圖4-17 不同pH值對北蟲草菌絲體生長與碳源消耗之時間歷時圖 61
圖4-18 不同pH值對北蟲草消耗腺苷與生成蟲草素之時間歷時圖 62
圖4-19 不同攪拌速率對北蟲草菌絲體生長與碳源消耗之時間歷時圖 64
圖4-20 不同攪拌速率對北蟲草生成蟲草素之時間歷時圖 64
圖4-21 不同攪拌速率對北蟲草發酵結果之比較 65
圖4-22 不同通氣量對北蟲草菌絲體生長與碳源消耗之時間歷時圖 66
圖4-23 不同通氣量對北蟲草消耗腺苷與生成蟲草素之時間歷時圖 67
圖4-24 不同通氣量對北蟲草發酵結果之比較 67
圖4-25 LED光照對北蟲草菌絲體生長於發酵槽之時間歷時圖 69
圖4-26 LED光照對北蟲草碳源消耗於發酵槽之時間歷時圖 70
圖4-27 LED光照對北蟲草腺苷消耗於發酵槽之時間歷時圖 72
圖4-28 LED光照對北蟲草生產蟲草素於發酵槽之時間歷時圖 72
圖4-29 不同培養條件對北蟲草菌體生長之歷時圖 76
圖4-30 不同培養條件對北蟲草胞外多醣生產之影響 76
圖4-31 不同培養條件對北蟲草蟲草素生產之影響 78
圖4-32 重複批次發酵對北蟲草菌體於發酵槽體中之影響 80
圖4-33 重複批次發酵對北蟲草生產胞外多醣於發酵槽體中之影響 80
圖4-34 重複批次發酵對北蟲草生產蟲草素於發酵槽體中之影響 81
表目錄
表1-1在日本已有臨床使用的菇菌類多醣體抗腫瘤藥劑 2
表1-2 蟲草屬所含之低分子量二次代謝產物 3
表2-1北蟲草分類學資料 10
表2-2 發酵培養模式比較 20
表2-3 光譜顏色波長及範圍 22
表3-1 實驗藥品總覽 25
表3-2 實驗儀器總覽 26
表3-3 固態培養基 29
表3-4 液態培養基 29
表4-1 各碳氮源濃度培養北蟲草菌絲體之產量及產率 42
表4-2 各碳氮源濃度培養北蟲草之菌量、多醣產率、比產率及最大產量 44
表4-3 各碳氮源濃度培養北蟲草代謝蟲草素之產量、比產率及生產力 47
表4-4 各添加物對北蟲草代謝蟲草素之產量、比產率及生產力 52
表4-5 甘胺酸濃度對北蟲草菌絲體生長、蟲草素產量、比產率及生產力 55
表4-6 發酵槽之特性整理 56
表4-7 不同種類發酵槽之操作條件 57
表4-8 發酵槽體對北蟲草之蟲草素產量、比產率及生產力 59
表4-9 不同pH值條件下發酵結果 62
表4-10 LED光照對北蟲草菌絲體、碳源與胞外多醣於發酵槽培養下之實驗結果 70
表4-11 LED光照對北蟲草菌絲體、碳源與胞外多醣於發酵槽培養下之實驗結果 73
表4-12 不同培養條件對北蟲草生產蟲草素於發酵槽培養下之實驗結果 78
表4-13 重複批次對北蟲草生產胞外多醣於發酵槽培養下之實驗結果 79
表5-1 各研究團隊與本實驗室目前發酵生產蟲草素之統整表 84

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