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研究生:陳雯賢
論文名稱:不同碳源對樟芝液態培養生產菌絲體及胞外多醣之影響
論文名稱(外文):Influence of different carbon source on mycelium growth and exopolysaccharide in submerged culture of Antrodia camphorata
指導教授:龍明有
學位類別:碩士
校院名稱:明新科技大學
系所名稱:化學工程研究所
學門:工程學門
學類:化學工程學類
論文種類:學術論文
論文出版年:2006
畢業學年度:94
語文別:中文
論文頁數:89
中文關鍵詞:樟芝深層液態培養碳源生物質量多醣體抗氧化DPPH
外文關鍵詞:Antrodia camphoratasubmerged culturecarbon sourceexopolysacchsrideantioxidant activityDPPHbiomass
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樟芝(Antrodia camphorata),是台灣特有的真菌,也是最貴藥用真菌之一,具有抗癌、抗腫瘤及保肝之效果。樟芝子實體生長緩慢,以目前的技術仍無法由人工大量栽培成功,而盜採情形嚴重,為保護牛樟樹不再被砍伐,且避免樟芝滅種的問題,以生物技術利用液體醱酵培養樟芝菌絲體為是目前最經濟、符合環保的有效人工培育法。因此,本研究主要目的是探討各種碳源對樟芝液態培養菌絲體及胞外多醣之影響,進而利用碳源之調控以達增加多醣產量之目的。
實驗結果顯示:在搖瓶培養各種碳源(Glucose、Sucrose、Fructose、Ribose、Xylose、Maltose、Mannose、Galactose)在10 g/L濃度之下對樟芝生物質量及胞外多醣之影響,與控制組(10 g/L Glucose)比較,Maltose及Fructose分別有利於樟芝生物質量及胞外多醣之生合成,而使生物質量和多醣產量增加。進ㄧ步實驗利用各種濃度Fructose (10 g/L、20 g/L、30 g/L、40 g/L、50 g/L、60 g/L)在搖瓶培養下對樟芝生物質量和胞外多醣之影響,控制組為10 g/L之Fructose。與控制組比較,Fructose濃度的增加有助於菌體成長,在60 g/L Fructose時,生物質量最大值為6.11 g/L,胞外多醣亦會隨Fructose濃度增加而增加直到50 g/L為止,當Fructose濃度為60 g/L時將抑制多醣產生,最大胞外多醣產量125.99 mg/L是在Fructose濃度50 g/L。
由醱酵動力曲線圖中顯示,不同濃度的果糖於氣舉式醱酵槽培養之比成長速率以 3 %果糖為最大值(0.32 d-1),隨著果糖濃度增加,生物質量及胞外多醣產率也隨之增加,生物質量及胞外多醣最大值分別為5.02 g/L及164.87 mg/L,而生物質量產率(QX)及胞外多醣產率(QP)以6 %果糖培養為最大值,分別為0.3349 g L-1d-1及10.9912 g L-1d-1。當碳源濃度增加時,將有利於樟芝菌絲體及胞外多醣體生合成,且胞外多醣之生產動力模式為一次代謝物與菌體成長有關。結果顯示饋料批次醱酵生產其菌絲體及胞外多醣產量高於批次醱酵培養生產之菌絲體及胞外多醣產量,3 %果糖的饋料批次醱酵較3 %果醣批式醱酵培養分別提高了36.7 %和44.6 %的生物質量及胞外多醣產量,與6 %果糖批式醱酵培養比較,則分別提高了18.5 %及16.7 %的生物質量及胞外多醣產量。
不同碳源生產樟芝胞外多醣對抗氧化力之影響,結果顯示,胞外多醣於亞麻油酸系統之抗氧化性(antioxidant activity),不同碳源其胞外多醣(200 ppm)之抗氧化力以蔗糖有最佳之抗氧化力(63 %),而不同濃度果糖之多醣於搖瓶試驗中以1 %果糖之抗氧化力最佳(56.5 %),醱酵槽培養也以1 %果糖最好(72 %, 12天),此外,對於2,2-diphenyl-1-picrylhydrazyl(DPPH)自由基清除能力(scavenging activity)測定顯示,以添加蔗糖有最大之清除DPPH能力(76 %),而搖瓶培養不同濃度果糖則是以6 %果糖生產之胞外多醣較高(65%)。醱酵槽培養也以1 %果糖最好(79 %)。不同碳源生產胞外多醣對分子量之影響,結果顯示其分子量Mw為1.82×106~9.70×106 Da。不同碳源之試驗中,以麥芽糖培養之胞外多醣體之重量平均分子量較高(9.70×106 Da),搖瓶試驗中,不同果糖濃度,以6 %果糖培養之胞外多醣體之重量平均分子量最高(7.10×106 Da),醱酵槽培養不同的濃度果糖對胞外多醣之重量平均分子量之影響,以3 %果糖培養之胞外多醣體之重量平均分子量最高(3.09×106 Da)。多醣體之中性單醣成分分析顯示,不同碳源於搖瓶試驗之樟芝多醣體出現的組成為半乳糖及果糖單醣。搖瓶試驗中,不同濃度果糖培養之樟芝多醣體出現的組成為甘露糖、木糖及果糖單醣,而不同濃度果糖於氣舉式醱酵槽培養生產胞外多醣體的中性單醣組成為麥芽糖、蔗糖、葡萄糖及半乳糖單醣。
Antrodia camphorata (also known as Zang-Zhi), it is the peculiar fungi in Taiwan, and it was also one of the most expensive fungi medical mushroom with effect of remedy of anticancer, antitumour and protecting the liver. The fruiting body of A. camphorata grows slow; it is still unable to be successful by a large amount of artificial culture with present technology. In addition, to avoid Cinnamomum micranthum stolen to culture A. camphorata causing its extinction, it is need to culture mycelium of A. camphorata by using the most economy and environmental protection methods, solid and liquid fermentation. Nowadays, the submerged culture of Antrodia camphorata to produce the valuable metabolites has aroused an extensive attention of researches in Taiwan. Thus, the main purpose of this research was to investigate the influence of different carbon sources on the production of mycelium and exopolysaccharide in submerged culture of Antrodia camphorata, to understand the metabolic pathway of exopolysaccharide, and to increase production of exopolysaccharide by regulation of different carbon sources.
The effect of the different carbon sources including glucose, sucrose, fructose, ribose, xylose, maltose, mannose and galactose on production of biomass and EPS are conducted in this study. The result exhibited that maltose and fructose will stimulate biomass and EPS production, respectively, comparing with control 10 g/L of glucose. In addition, the effect of various concentration of fructose (i.e. 10, 20, 30, 40, 50, 60 g/L) on biomass and EPS production of A. camphorata is conducted in shake flask cultures, indicating that biomass production increased with fructose concentration ranging from 10 to 60 g/L. The biomass concentration reaches to 6.11 g/L as fructose concentration is at 60 g/L. EPS production rose when fructose concentration increased until 50 g/L. Too high level of fructose as 60 g/L will inhibit EPS production. The optimal EPS production (125.99 g/L) is found in 50 g/L of fructose.
The fermentation kinetic results showed that effect of the different concentration fructose in air-lift fermentor culture on specific growth rate (μ) has a optimal value (0.32 d-1) at 3% fructose. Production of both biomass and EPS increases with fructose concentrations, giving maximum value in 5.02 g/L and 164.87 mg/L for biomass and EPS, respectively. At 6% fructose, productivity for biomass has optimal value of 0.3349 g L-1 d-1 and productivity for EPS has optimal value of 10.9912 mg L-1 d-1. Increasing fructose concentration will favor production of both biomass and EPS; the kinetic model of EPS production belongs to growth-associated products. Fed-batch culture is conducted in this study to improve production of both biomass and EPS. The result exhibited that both biomass and EPS were enhanced by 36.7% and 44.6%, respectively, due to 3% fructose fed-batch culture comparing with 3% fructose batch culture. Besides, both biomass and EPS by 3% fructose fed-batch culture were enhanced by 18.5% and 16.7%, respectively, comparing with 6% fructose batch culture.
The effect of EPS (200 ppm) produced from different carbon sources in flask cultures on both antioxidant activity and 2,2-diphenyl-1-picrylhydrazyl(DPPH) scavenging effect is examined. The result indicated that EPS (200 ppm) obtained from sucrose have largely effect on antioxidant activity (63%) and DPPH scavenging activity (76%). Besides, the effect of EPS (200 ppm) produced from different concentration fructose in flask cultures on antioxidant activity is also investigated and found that fructose at 10 g/L has an optimal antioxidant activity (56.5%) and 60 g/L for DPPH scavenging activity (65%). In air-lift fermentor cultures, the effect of EPS (200 ppm) produced from different concentration fructose on antioxidant activity and DPPH scavenging activity exhibited that an optimal antioxidant activity (56.6%) and a maximum DPPH scavenging activity (76%) are is at 1% fructose.
The effect of EPS produced from different carbon sources in flask cultures on molecular weight (Mw) is examined. The result indicated that Mw of EPS from all carbon sources tested range from 1.82×106 to 9.70×106 Da. EPS produced from maltose has a maximum Mw (9.70×106 Da). In EPS from different carbon sources tested, the maximum Mw of EPS is 7.1×106 Da at 6% fructose. In air-lift fermentor cultures, EPS produced from 3% fructose has higher Mw of 3.09×106 Da. The result of carbohydrate composition analysis revealed that composition of EPS produced different carbon sources in flask culture consists of galactose and fructose; however, composition of EPS produced different concentration fructose in flask culture composes of fructose, xylose and mannose. Composition of EPS produced different concentration fructose in air-lift fermentor cultures consists of glucose, sucrose, maltose and galactose.
目錄

中文摘要 ---------------------------------------------------------------------------------- I
英文摘要 -------------------------------------------------------------------------------- III
目錄 --------------------------------------------------------------------------------------- V
圖次索引 ------------------------------------------------------------------------------ VIII
表次索引 --------------------------------------------------------------------------------- X
第一章 緒論 --------------------------------------------------------------------------- 1
1.1 研究動機與背景 -------------------------------------------------------------- 1
1.2 研究目的 ----------------------------------------------------------------------- 1
第二章 文獻回顧 --------------------------------------------------------------------- 3
2.1 樟芝介紹 ----------------------------------------------------------------------- 3
2.2 樟芝的藥理特性與功用 ----------------------------------------------------- 5
2.3 真菌多醣體的介紹 ---------------------------------------------------------- 14
2.3.1 何謂多醣體 ------------------------------------------------------------ 14
2.3.2 菇類多醣的生理活性 ------------------------------------------------ 17
2.3.3 樟芝之多醣 ------------------------------------------------------------ 18
2.4 醱酵培養技術介紹 ---------------------------------------------------------- 19
2.4.1 深層醱酵培養及傳統固態培養的比較 --------------------------- 19
2.4.2 深層醱酵培養的定義及培養基的成分和功能 ------------------ 19
2.4.3 影響深層醱酵培養的其他物理化學因子 ------------------------ 24
第三章 材料與方法 ----------------------------------------------------------------- 28
3.1 實驗架構 ---------------------------------------------------------------------- 28
3.2 實驗材料 ---------------------------------------------------------------------- 29
3.2.1 實驗菌株 --------------------------------------------------------------- 29
3.2.2 實驗藥品 --------------------------------------------------------------- 29
3.2.3 實驗儀器及設備 ------------------------------------------------------ 31
3.2.4 實驗裝置 ----------------------------------------------------------------32
3.3 實驗方法 ---------------------------------------------------------------------- 32
3.3.1 菌株保存 --------------------------------------------------------------- 32
3.3.2 培養基成分 ------------------------------------------------------------ 32
3.3.3 操作條件 --------------------------------------------------------------- 34
3.4 分析方法 ---------------------------------------------------------------------- 35
3.4.1 醱酵液分析流程 ------------------------------------------------------ 35
3.4.2 菌體乾重測定(Biomass,X) ---------------------------------------- 35
3.4.3 多醣濃度分析 --------------------------------------------------------- 36
3.4.4 殘醣分析 --------------------------------------------------------------- 36
3.4.5 多醣體分子量分析 --------------------------------------------------- 38
3.4.6 多醣體之單醣組成分析 --------------------------------------------- 38
3.4.7 多醣體抗氧化力分析 ------------------------------------------------ 39
3.4.7.1 多醣體於亞麻油酸乳化系統之抗氧化性 ----------------- 39
3.4.7.2 多醣體之DPPH 自由基清除能力之測定 ----------------- 39
第四章 結果與討論 ----------------------------------------------------------------- 41
4.1 樟芝菌絲型態 ---------------------------------------------------------------- 41
4.1.1 固態培養 --------------------------------------------------------------- 41
4.1.2 搖瓶培養 --------------------------------------------------------------- 41
4.1.3 氣舉式醱酵槽 --------------------------------------------------------- 42
4.2 搖瓶培養實驗 ---------------------------------------------------------------- 43
4.2.1不同碳源培養基對於樟芝菌絲生長及胞外多醣之影響 ------- 43
4.2.1.1不同碳源對樟芝菌絲體生長的影響 ------------------------ 43
4.2.1.2不同碳源對樟芝胞外多醣體產量影響 --------------------- 44
4.2.1.3 對樟芝轉化率YP/X、YX/S 和YP/S 的影響 --------------------- 45
4.2.2 不同濃度的果糖培養對於樟芝生長之影響 --------------------- 49
4.2.2.1不同濃度果糖對樟芝菌絲體生長的影響 ------------------ 49
4.2.2.2不同濃度果糖對樟芝胞外多醣體產量影響 --------------- 50
4.2.2.3 不同濃度果糖對樟芝轉化率YP/X、YX/S 和YP/S 的影響 --- 51
4.3 不同濃度果糖於氣舉式醱酵槽實驗 ----------------------------------- 55
4.3.1 不同濃度果糖對樟芝菌絲體生長之影響 ------------------------ 55
4.3.2 不同濃度果糖對μ、QX和YX/S的影響 ----------------------------- 56
4.3.3 不同濃度果糖對樟芝胞外多醣產量影響------------------------- 57
4.3.4 不同濃度果糖對QP、YP/X和YP/S 的影響 -------------------------- 57
4.4 饋料批次醱酵培養於氣舉式醱酵槽實驗 ------------------------------ 62
4.5 多醣體分子量分布的影響 ------------------------------------------------ 63
4.6 多醣體之中性單糖成份測定 --------------------------------------------- 71
4.7 多醣體之抗氧化性質分析 ------------------------------------------------ 74
4.7.1 亞麻油酸系統中多醣體之抗氧化力測定 ------------------------ 74
4.7.2 捕捉DPPH 能力之測定 --------------------------------------------- 74
第五章 結論與建議------------------------------------------------------------------ 79
5.1 結論 --------------------------------------------------------------------------- 79
5.2 建議 --------------------------------------------------------------------------- 80
參考文獻--------------------------------------------------------------------------------- 81
作者簡介 -------------------------------------------------------------------------------- 89




圖次索引

圖2-1、三帖類化合物結構 ---------------------------------------------------------- 8
圖2-2、超氧化物歧化 ---------------------------------------------------------------- 9
圖2-3、人體內之自由基與氧化反應與疾病生成之關係 -------------------- 11
圖2-4、ß-(1®3)-D-葡聚糖結晶結構 --------------------------------------------- 14
圖2-5、多醣體抗腫瘤機制-BRM ------------------------------------------------- 15
圖2-6、胞外多醣合成的流程圖 -------------------------------------------------- 16
圖3-1、氣舉式生物反應器 -------------------------------------------------------- 32
圖3-2、醱酵液分析流程 ----------------------------------------------------------- 35
圖3-3、多醣檢量線 ------------------------------------------------------------------ 36
圖3-4、DNS法glucose標準曲線 --------------------------------------------------- 37
圖3-5、多醣分子量檢量線 -------------------------------------------------------- 65
圖4-1、 a.樟芝菌絲體平面固態培養(18天)及b.液態搖瓶培養情形
(14 天)------------------------------------------------------------------------------- 41
圖4-2、樟芝菌絲體在氣舉式醱酵槽中生長14天之型態 -------------------- 42
圖4-3、不同碳源對樟芝菌絲體含量之影響 ----------------------------------- 44
圖4-4、不同碳源對樟芝胞外多醣產量之影響 -------------------------------- 45
圖4-5、不同碳源對YP/X之影響 --------------------------------------------------- 47
圖4-6、不同碳源對YX/S之影響 --------------------------------------------------- 47
圖4-7、不同碳源對YP/S之影響 --------------------------------------------------- 48
圖4-8、不同濃度fructose添加對菌絲體含量之影響 ------------------------- 50
圖4-9、不同濃度fructose的添加對胞外多醣產量之影響 ------------------- 51
圖4-10、不同濃度fructose對YP/X的影響 --------------------------------------- 53
圖4-11、不同濃度fructose對YX/S之影響 --------------------------------------- 53
圖4-12、不同濃度fructose對 YP/S之影響 -------------------------------------- 54
圖4-13、醱酵動力曲線圖(10 g/L fructose) -------------------------------------- 59
圖4-14、醱酵動力曲線圖(20 g/L fructose) -------------------------------------- 59
圖4-15、醱酵動力曲線圖(30 g/L fructose) -------------------------------------- 60
圖4-16、醱酵生長曲線圖(40 g/L fructose) -------------------------------------- 60
圖4-17、醱酵生長曲線圖(50 g/L fructose) -------------------------------------- 61
圖4-18、醱酵生長曲線圖(60 g/L fructose) -------------------------------------- 61
圖4-19、饋料批式醱酵生長曲線圖 ---------------------------------------------- 63
圖4-20、不同碳源於搖瓶試驗中之多醣分子量分布圖 --------------------- 66
圖4-21、不同濃度果糖於搖瓶試驗中之多醣分子量分布圖 ---------------- 68
圖4-22、不同濃度果糖於氣舉式醱酵槽之多醣分子量分布圖 ------------- 69
圖4-23、搖瓶試驗生產之多醣體其單醣成分標準曲線 ---------------------- 71
圖4-24、不同碳源於搖瓶試驗生產之多醣體之中性單醣成分 ------------- 71
圖4-25、不同濃度果糖於搖瓶試驗生產之多醣體之中性單醣成分 ------- 72
圖4-26、不同濃度果糖於醱酵槽培養生產之多醣體之中性單醣成分 ---- 72
圖4-27、不同碳源之胞外多醣(200 ppm)於搖瓶試驗中之抗氧化力 ----- 74
圖4-28、不同濃度果糖之胞外多醣(200 ppm)於搖瓶試驗中之抗氧化力
--------------------------------------------------------------------------------------------75
圖4-29、不同濃度果糖之胞外多醣 (200 ppm)於氣舉式醱酵槽之抗氧化力
------------------------------------------------------------------------------------------- 75
圖4-30、不同碳源之胞外多醣(200 ppm)於搖瓶試驗中之捕捉自由基效應
------------------------------------------------------------------------------------------- 76
圖4-31、不同濃度果糖之胞外多醣(200 ppm)於搖瓶試驗中之捕捉自由基
效應 ----------------------------------------------------------------------------------- 76
圖4-32、不同濃度果糖之胞外多醣 (200 ppm)於氣舉式醱酵槽之捕捉自由
基效應 -------------------------------------------------------------------------------- 77






表次索引

表2-1、具有抗腫瘤作用的真菌多醣類 ------------------------------------------ 18
表3-1、實驗藥品全目錄 ---------------------------------------------------------- 29
表3-2、實驗儀器及設備全目錄 -------------------------------------------------- 31
表3-3、樟芝菌固態培養基之組成------------------------------------------------ 33
表3-4、樟芝菌液態前培養基之組成 -------------------------------------------- 33
表4-1、不同碳源於搖瓶培養14天之結果 ------------------------------------ 48
表4-2、不同濃度果糖於搖瓶培養14天之結果 ------------------------------ 55
表4-3、不同初始濃度果糖於氣舉式醱酵槽培養之結果 -------------------- 62
表4-4、多醣體分子量在(a)不同碳源於搖瓶試驗;(b)不同濃度果糖於搖瓶試
驗及 (c)不同濃度果糖於氣舉式醱酵槽中之影響 ----------------------------- 66
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