探討pH值對Agaricus blazei 液態發酵生產一次及二次代謝產物之影響

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黃崇凱(Chung-Kai Huang) 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pH值對Agaricus blazei 液態發酵生產一次及二次代謝產物之影響
(The influence of pH on production of first andsecondary metabolites formation by Agaricus blazeiin submerged culture)

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摘要(中)

Agaricus blazei原產於巴西，為一種食藥用菇，近年來被廣泛地做研究，包括其代謝產物的生物活性等。
隨著A. blazei的療效及生物活性一一被探討出來，快速且大量的提升發酵的產量以及產率也隨之來的重要，隨著各產物的特性不同，各產物的生產條件也不同，本研究為了增加A. blazei液態培養之一次代謝產物粗多醣、麥角固醇和二次代謝產物Blazeispirol A的產量以及探討各產物間的相互關係，藉由改變環境因子pH值來探討，分別討論搖瓶實驗之不同起始培養基pH值為4、5、6、7及發酵槽實驗之控制發酵全程培養基pH值為4、5、6、7四組實驗，在搖瓶及發酵槽實驗中可以得到一致的趨勢，發酵槽實驗中結果發現隨著pH值的提高對粗多醣的產量有著隨之增加的趨勢，從pH值為4時的0.42g/L到pH值=6時有最大的粗多醣產量為0.53g/L；而在pH值為7時粗多醣產量又降為0.41g/L。對麥角固醇而言，則是隨著pH值下降而產量隨之增加。在pH值為7時，麥角固醇的產量為29.45mg/L，隨著pH值下降到pH值為4時有最大產量為43mg/L。但對於二次代謝產物Blazeispirol A而言，卻是隨著pH值的上升而有上升的趨勢，
在pH值為4時有最低產量為0.61mg/L，隨著pH值上升到7時達到了最大產量16.80mg/L，這意味著雖然在低pH值下有利於生產麥角固醇但卻不利於轉換成其它衍生物，如Blazeispirol A。
另外也探討發酵過程全程控制pH值為7以及只控發酵的起始pH值為7之試驗，研究發現在全程控制與不控發酵過程pH值的試驗中，胞外多醣產量分別為0.41以及0.31g/L;而麥角固醇產量則為29.45以及28.33mg/L;至於Blazeispirol A則是為16.80以及12.69mg/L，此外在發酵過程pH值控制為7時發酵所需時間為9天，而只控發酵起始pH值則需為12天，綜合上述無論在產量及產率而言皆是以全程控制發酵過程pH為7之試驗結果較佳，故發酵過程控制pH值是有其必要性的。
利用本研究可以了解培養基的pH值對於A. blazei液態發酵生產多醣、麥角固醇、Blazeispirol A等產物之影響，以提供日後大量生產所需要的產物時之參考。

摘要(英)

Higher fungi are abundant sources of a wide range of useful natural products and new products with interesting biological activities . Agaricusl blazei Murill has been traditionally used as a health food source in Brazil for the prevention of cancer, diabetes, hyperlipidaemia, arteriosclerosis and chronic hepatitis.
The effects of the controlled and uncontrolled pH conditions, as well as of the value of initial pH=7 and controlled pH=7, on production of the valuable bioactive metabolites by A. blazei were investigated in a stirred tank fermenter. The experimental result shows, controlled pH operation was more favourable than the uncontrolled pH operations.
The effects of controlled culture pH ranging from pH 4.0 to 7.0 on cell growth, exopolysaccharide biosynthesis, relationship between ergosterol, and blazeispirol A biosynthesis of A. blazei were examined both in shake flask culture and in a stirred tank fermenter. While the set point of the culture pH during fermentation was controlled from 4.0 to 7.0, the specific growth rate (μ) increased monotonically, and the cell production rate had the same trend; Higher the pH control from 4.0 to 6.0 gradually led to a higher production of production of Polysaccharides. At lower the pH control (pH = 4) was optimum for ergosterol production; however, At higher the pH control (pH = 7) was optimum for blazeispirol A generation.
The work is useful for further optimization of this fungal fermentation process highly efficient production of the valuable bioactive metabolites on a bioreactor scale.

關鍵字(中)

★ 巴西洋菇
★ 發酵
★ 代謝產物

關鍵字(英)

★ pH
★ Agaricus blazei
★ secondary metabolite

論文目次

目錄...................................................Ⅰ
圖索引.................................................VI
表索引.................................................VIII
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大型真菌類之生理活性.................................3
2.2 A. blazei的介紹.....................................5
2.2.1 A. blazei之發現與由來.............................5
2.2.2 A. blazei的型態特徵及分類.........................6
2.2.3 A. blazei之化學組成...............................7
2.2.4 A. blazei的生理活性...............................9
2.2.5 A. blazei的安全性.................................11
2.3菇類活性成份探討.....................................11
2.3.1菇類多醣的介紹.....................................11
2.3.1.1 A. blazei之多醣體...............................15
2.3.2菇類麥角固醇的介紹.................................18
2.3.2.1 A. blazei之麥角固醇.............................20
2.3.3 A. blazei中所含活性成份blazeispirol A的探討.......21
2.4 深層液態發酵培養....................................25
2.5 影響發酵的物理化學等環境生長因子....................25
2.5.1 培養基組成........................................25
2.5.1.1 碳源............................................25
2.5.1.2 氮源............................................26
2.5.1.3 碳氮比..........................................26
2.5.1.4 無機鹽類........................................26
2.5.2 攪拌速率..........................................27
2.5.3 pH值..............................................27
2.5.4 溶氧值(DO值)......................................29
2.5.5 溫度..............................................30
2.5.6 接種量............................................31
2.5.7發酵槽的形式.......................................31
2.6 發酵動態行為的數學模式..............................32
2.6.1 與生長有關的產物(growth-associated product).......32
2.6.2 與生長無關的產物(nongrowth-associated product)....33
2.6.3 混合生長相關的產物(mixed-growth-associated product)................................................33
第三章材料與方法.......................................34
3.1 實驗材料............................................34
3.1.1 實驗菌株.........................................34
3.1.2 實驗藥品.........................................34
3.1.3 實驗儀器及其他設備................................35
3.1.4 實驗裝置..........................................37
3.2 實驗方法............................................38
3.2.1 菌株保存..........................................38
3.2.2 培養基組成........................................38
3.2.3 操作條件..........................................40
3.2.4 分析方法..........................................41
3.2.4.1 發酵液處理流程...............................41
3.2.4.2 菌重分析.....................................42
3.2.4.3 葡萄糖殘醣分析...............................42
3.2.4.4 多醣體分析...................................44
3.2.4.4.1 粗多醣之分離............................44
3.2.4.4.2 酚-硫酸呈色法分析(總多醣)...............44
3.2.4.5 麥角固醇之分析...............................45
3.2.4.5.1 麥角固醇萃取..................................45
3.2.4.5.1 麥角固醇含量分析..............................46
3.2.4.6 Blazeispirol A 之分析........................47
3.2.4.6.1 Blazeispirol A萃取............................47
3.2.4.6.2 Blazeispirol A含量分析........................47
第四章實驗結果與討論...................................50
4.1 搖瓶培養實驗........................................50
4.1.1 搖瓶實驗之控不同起始pH值對A. blazei生長的影響....50
4.2 發酵槽培養實驗......................................56
4.2.1 發酵槽實驗之pH值(controlled)對A. blazei生長的影響
........................................................57
4.2.1.1 發酵動力曲線的比較..............................57
4.2.1.2 pH值對於菌體生長型態的影響......................60
4.2.1.3 pH值對於發酵時間的影響..........................64
4.2.1.4 pH值對於菌體生長的影響..........................64
4.2.1.5 pH值對於產物粗多醣的影響........................66
4.2.1.6 pH值對於產物麥角固醇的影響......................68
4.2.1.7 pH值對於產物Blazeispirol A的影響................69
4.2.2 發酵槽實驗之發酵過程控pH值(controlled-pH)及不控pH值 (uncontrolled-pH)對A. blazei生長之比較..................75
第五章結論.............................................82
參考文獻................................................85

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指導教授

徐敬衡(Chin-Hang Shu)

審核日期

2007-7-20

推文