探討通氣量與光照度對巴西蘑菇活性物質生產之影響

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林克融(Ko-Jung Lin) 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

探討通氣量與光照度對巴西蘑菇活性物質生產之影響
(Effects of aeration rate and light intensity on the production of bioactive materials by Agaricus blazei in batch cultures)

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摘要(中)

巴西蘑菇是食藥用菇的一種，因其具有許多活性物質而廣受大家注目，尤其是抗腫瘤多醣體。文獻中對其活性物質之功能已有相當多的研究報告，然而對利用液態深層發酵生產的操作條件，文獻卻相對較少，為了提高巴西蘑菇發酵生產的產量，本實驗室過去曾對培養基組成、pH值、攪拌速率、溫度等操作條件進行探討並發表研究報告，因此本研究針對通氣量及光照度對巴西蘑菇液態深層發酵生產活性物質的影響來作探討，本研究探討之主要產物包括多醣體、beta-(1,3)-glucan、beta-(1,3)-glucanase酵素、麥角固醇及二次代謝物blazeispirol A，此外也利用體外測試(in vitro)之動物細胞實驗，評估不同發酵條件所得到的巴西蘑菇多醣體、麥角固醇與blazeispirol A之生物活性或抗腫瘤能力測定。
第一部分的實驗為通氣量對巴西蘑菇活性物質生產之影響，分別進行搖瓶實驗及5 L攪拌式發酵槽實驗。搖瓶震盪培養係利用不同工作體積分別為50、100及150 mL三組搖瓶實驗，探討對巴西蘑菇液態發酵生產上的影響，實驗結果發現，隨著工作體積的增加，單位體積之液面表面積隨之減少；菌體形態隨之由菌球變成絲狀；beta-(1,3)-glucanase酵素活性與blazeispirol A之yield值(YP3/X、YP3/S)隨之下降；但是麥角固醇之yield值(YP2/X、YP2/S)卻是隨之上升的結果。另外，最適菌體、多醣體、beta-(1,3)-glucan、大分子量多醣生產之工作體積為100 mL。
5 L攪拌式發酵槽培養實驗係探討五組不同通氣量分別為0.1-0.5 vvm，對巴西蘑菇液態深層發酵的影響，實驗結果顯示隨著通氣量的增加，氧氣質傳速率係數kLa隨之上升；菌體形態隨之由絲狀變菌球；最適菌體利用氧氣效率之通氣量為0.4 vvm。而最適beta-(1,3)-glucan、大分子量多醣、麥角固醇生成之通氣量為較低之0.2 vvm，此通氣量下生產之多醣體生物活性最高；另外最適菌體生長、多醣體生成、beta-(1,3)-glucanase酵素活性生成之通氣量則為較高之0.4 vvm；但最適blazeispirol A生成及麥角固醇轉化成blazeispirol A之通氣量則介於中間為0.3 vvm。
第二部分的實驗為光照度對巴西蘑菇活性物質生產上的影響，同樣分別進行搖瓶實驗及5 L攪拌式發酵槽實驗。在搖瓶震盪培養分別進行光照度0、300、500與1000 lux四組搖瓶實驗，實驗結果發現，隨著光照度的增加，單位基質之菌體產量(YX/S)、多醣體之yield值(YP1/X、YP1/S)、多醣體中beta-(1,3)-glucan含量、多醣體平均分子量、beta-(1,3)-glucanase酵素活性隨之上升。而最適麥角固醇與blazeispirol A生成之光照度為500 lux。
5 L攪拌式發酵槽培養實驗分別進行光照度0、500、1000與1500 lux四組發酵槽實驗，實驗結果顯示隨著光照度的增加，菌體產率隨之上升。而最適麥角固醇、blazeispirol A、麥角固醇轉化成blazeispirol A之光照度為較低之500 lux，與搖瓶實驗結果一致；另外最適菌體生長、多醣體、beta-(1,3)-glucan、大分子量多醣、beta-(1,3)-glucanase酵素活性生成之光照度為較高之1000 lux，此光照度下生產之多醣體生物活性最高。
此外，對於麥角固醇與二次代謝物blazeispirol A之抗腫瘤能力的測試，在體外試驗(in vitro)動物細胞實驗的結果發現，對Hepa-1c1c7與HeLa兩種癌細胞所得到之IC50皆低於市售抗癌藥物，顯示巴西蘑菇麥角固醇與blazeispirol A具有抗腫瘤之潛力。

摘要(英)

In oriental culture, mushrooms, attracted attention because of their biochemical composition and medicinal characteristics, have been used as health foods and home remedies for thousands of years. The Basidiomycete fungus Agaricus blazei Murill, originally a native of Brazil, is one such species. The chemical components isolated from its fruiting bodies have been widely studied, especially its polysaccharides with antitumor activity. Besides, ergosterol and blazeispirol A are important bioactive components of A. blazei. However, yields and functionality of these bioactive metabolites produced by fermentation in submerged culture are highly dependent on the culture conditions, such as medium composition and environmental parameters including pH, temperature, agitation rate and aeration rate. Nevertheless, relatively few efforts have been focused on the influence of these operational conditions on the production of these metabolites. In previous studies, we reported the effects of medium composition, pH, temperature and agitation rate on the production of bioactive polysaccharides by A. blazei in batch cultures.
Therefore, the aim of this study was to propose a fermentation process of A. blazei with highly bioactive polysaccharides, ergosterol and blazeispirol A by investigating the effects of aeration rate and light intensity on their yields, the molecular weight distribution of polysaccharides, beta-(1,3)-glucan content in polysaccharides, beta-(1,3)-glucanase activity and TNF-alpha release capability on macrophage cells, to investigating the antitumor activity of ergosterol and blazeispirol A against the mouse hepatoma cell lines, Hepa-1c1c7, and human cervical epithelioid carcinoma cell line, HeLa, and to propose a novel approach to optimizing fermentation process of blazeispirol A using the conversion ratio of ergosterol into blazeispirol A.
First part of this research is to investigate the effects of aeration rate on the production of bioactive metabolites by A. blazei in batch cultures. The flask experiments were carried out by investigating three working volumes, 50, 100 and 150 mL. The results of flask experiments show that as the working volume increased, the value of surface of liquid per volume, beta-(1,3)-glucanase activity, and the yields of blazeispirol A (YP3/X, YP3/S) decreased, and the morphology of mycelia was changed from pellet to filamentous; however, the yields of ergosterol (YP2/X, YP2/S) enhanced. In addition, the optimal working volume for biomass, polysaccharides and beta-(1,3)-glucan formation was at 100 mL.
5 L stirred tank experiments were carried out by investigating five aeration rates, 0.1 to 0.5 vvm. The results of stirred tank experiments show that as the aeration rate increased, the oxygen transfer rate coefficient, kLa, enhanced, and the morphology of mycelia was changed from filamentous to pellet. Additionally, the optimal aeration rate for beta-(1,3)-glucan, high molecular weight fraction of polysaccharides and ergosterol formation was at 0.2 vvm, while that for biomass, polysaccharides and ?-(1,3)-glucanase activity formation was at 0.4 vvm, and the maximal bioactivity of polysaccharides was at the aeration rate of 0.2 vvm. However, the optimal aeration rate for blazeispirol A formation was at 0.3 vvm.
Second part of this research is to investigate the effects of light intensity on the production of bioactive metabolites by A. blazei in batch cultures. The flask experiments were carried out by investigating four light intensities, 0, 300, 500, 1000 lux. The results of flask experiments show that as the light intensity increased, the yields of biomass (YX/S) and polysaccharides (YP1/X, YP1/S), beta-(1,3)-glucan content, the average molecular weight of polysaccharides and beta-(1,3)-glucanase activity enhanced. Besides, the optimal light intensity for ergosterol and blazeispirol A formation was at 500 lux.
5 L stirred tank experiments were carried out by investigating four light intensities, 0, 500, 1000 and 1500 lux. The results of stirred tank experiments show that the optimal light intensity for ergosterol and blazeispirol A formation was at 500 lux, while that for biomass, polysaccharides, beta-(1,3)-glucan, high molecular weight fraction of polysaccharides and beta-(1,3)-glucanase activity formation was at 1000 lux, and the maximal bioactivity of polysaccharides was at the light intensity of 1000 lux.
Based on the result of the lower IC50 values of ergosterol and blazeispirol A against the proliferation of Hepa-1c1c7 and HeLa cells presented in this study, the contents of ergosterol and blazeispirol A in A. blazei play important roles in antitumor effects besides polysaccharides.

關鍵字(中)

★ 巴西蘑菇
★ 通氣量
★ 光照度
★ 多醣體
★ 麥角固醇
★ 二次代謝物
★ 腫瘤壞死因子
★ 抗腫瘤

關鍵字(英)

★ aeration rate
★ light intensity
★ antitumor activity
★ TNF
★ blazeispirol A
★ ergosterol
★ polysaccharide
★ Agaricus blazei

論文目次

中文摘要..................................................i
英文摘要................................................iii
謝誌.....................................................vi
目錄....................................................vii
圖目錄.................................................xiii
表目錄..................................................xix
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..........................................4
2-1 菇類的介紹...........................................4
2-1-1 菇類活性物質.....................................5
2-1-2 菇類多醣體.........................................7
2-2 人體免疫系統及抗腫瘤機制............................11
2-2-1 人體免疫系統簡介..................................11
2-2-2 ?-D-葡聚醣(glucan)的抗腫瘤機制...................11
2-2-3 多醣體抗腫瘤生物活性之動物細胞實驗................12
2-3 巴西蘑菇(Agaricus blazei Murill)....................13
2-3-1 巴西蘑菇的化學成分................................14
2-3-2 巴西蘑菇的活性物質................................16
1. 巴西蘑菇之多醣體......................................18
2. 巴西蘑菇之麥角固醇(Ergosterol)........................22
3. 巴西蘑菇之二次代謝物Blazeispirol A....................24
2-4 液態深層培養........................................27
2-5 影響液態深層發酵的環境因子..........................28
2-5-1 培養基組成........................................29
1. 碳源..................................................29
2. 氮源..................................................29
3. 碳氮比................................................30
4. 無機鹽類..............................................30
2-5-2 攪拌速率(Agitation rate)..........................31
2-5-3 溫度..............................................32
2-5-4 pH值..............................................32
2-5-5 溶氧量(Dissolved oxygen)..........................34
2-5-6 黏度(Viscosity)...................................35
2-5-7 通氣量(Aeration rate).............................35
2-5-8 光照度(Light intensity)...........................38
第三章、材料與方法.......................................42
3-1 實驗材料............................................42
3-1-1 實驗菌株..........................................42
3-1-2 實驗藥品..........................................43
3-1-3 實驗儀器及其他設備................................45
3-1-4 發酵實驗裝置......................................47
3-2 巴西蘑菇發酵實驗....................................48
3-2-1 菌株保存..........................................48
3-2-2 培養基組成........................................49
1.固態平面培養基.........................................49
2.前培養之液態培養基.....................................49
3.種瓶與發酵實驗之液態培養基.............................50
3-2-3 實驗方法..........................................51
1.固態平面培養...........................................51
2.前培養之液態培養.......................................51
3.種瓶液態培養...........................................52
4.主發酵培養.............................................52
3-3 分析方法............................................54
3-3-1 發酵液處理流程..................................54
3-3-2 菌重分析..........................................54
3-3-3 葡萄糖殘量分析....................................54
3-3-4 巴西蘑菇多醣體濃度測定............................56
3-3-5 巴西蘑菇?-(1,3)-Glucan濃度測定...................57
3-3-6 巴西蘑菇多醣體分子量測定..........................60
3-3-7 巴西蘑菇beta-(1,3)-Glucanase活性之分析............60
3-3-8 巴西蘑菇麥角固醇之萃取與分析......................61
3-3-9 巴西蘑菇二次代謝物Blazeispirol A之萃取與分析.......63
3-3-10 麥角固醇轉化成Blazeispirol A之轉化率計算.........66
3-4 動物細胞實驗........................................67
3-4-1 細胞株............................................67
3-4-2 細胞培養液組成....................................67
3-4-3 細胞培養..........................................68
3-4-4 細胞株保存........................................69
3-4-5 細胞株解凍........................................69
3-4-6 細胞計數方法....................................70
3-4-7 巴西蘑菇多醣體生物活性實驗方法....................70
3-4-8 麥角固醇與Blazeispirol A抗腫瘤能力測定............71
第四章、通氣量對巴西蘑菇活性物質生產之影響...............73
4-1 前言............................................73
4-2 實驗方法........................................73
4-2-1 搖瓶實驗操作條件................................73
4-2-2 5 L攪拌式發酵槽操作條件.........................74
4-2-3 kLa值與OUR的量測................................75
4-3 搖瓶實驗之結果與討論............................76
4-3-1 單位工作體積之液面表面積........................76
4-3-2 工作體積對巴西蘑菇菌體生長的影響................77
4-3-3 工作體積對巴西蘑菇活性多醣與beta-(1,3)-glucanase酵素活性生產的影響.........................................79
4-3-4 工作體積對巴西蘑菇麥角固醇(Ergosterol)與二次代謝物Blazeispirol A生產的影響.................................85
4-4 5 L發酵槽實驗之結果與討論.......................87
4-4-1 通氣量對巴西蘑菇菌體形態的影響..................88
4-4-2 通氣量對巴西蘑菇發酵動力學的影響................90
4-4-3 通氣量對kLa與SOUR的影響.........................93
4-4-4 通氣量與SOUR對巴西蘑菇菌體生產的影響............95
4-4-5 通氣量與SOUR對巴西蘑菇活性多醣與beta-(1,3)-glucanase酵素活性生產的影響..............................97
4-4-6 通氣量與SOUR對巴西蘑菇麥角固醇(Ergosterol)與二次代謝物Blazeispirol A生產的影響............................103
4-5 麥角固醇與巴西蘑菇二次代謝物Blazeispirol A抗腫瘤能力測試..................................................107
4-6 結論...........................................109
4-6-1 搖瓶實驗結論...................................109
4-6-2 5 L攪拌式發酵槽實驗結論........................110
第五章、光照度對巴西蘑菇活性物質生產之影響..............111
5-1 前言...........................................111
5-2 實驗方法.......................................112
5-2-1 搖瓶實驗操作條件.................................112
5-2-2 5 L攪拌式發酵槽操作條件..........................112
5-3 搖瓶實驗之結果與討論...........................116
5-3-1 光照度對巴西蘑菇菌體生產的影響.................116
5-3-2 光照度對巴西蘑菇活性多醣與beta-(1,3)-glucanase酵素活性生產的影響..........................................117
5-3-3 光照度對巴西蘑菇麥角固醇(Ergosterol)與二次代謝物Blazeispirol A生產的影響................................122
5-4 5 L發酵槽實驗之結果與討論......................124
5-4-1 光照度對巴西蘑菇發酵動力學的影響...............124
5-4-2 光照度對巴西蘑菇菌體生產的影響.................127
5-4-3 光照度對巴西蘑菇活性多醣與beta-(1,3)-glucanase酵素活性生產的影響..........................................130
5-4-4 光照度對巴西蘑菇麥角固醇(Ergosterol)與二次代謝物Blazeispirol A生產的影響................................136
5-5 結論...........................................139
5-5-1 搖瓶實驗之結論...................................139
5-5-2 5 L攪拌式發酵槽之結論..........................140
第六章、總結與建議......................................141
6-1 總結...............................................141
6-2 建議...............................................142
參考文獻................................................143
作者簡歷................................................155
著作目錄................................................156

參考文獻

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指導教授

徐敬衡(Chin-Hang Shu)

審核日期

2011-1-26

推文