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研究生:魏秀真
研究生(外文):Hsin-Chen Wei
論文名稱:利用獸疫鏈球菌生產透明質酸之研究
論文名稱(外文):The Study of Production of Hyaluronic acid Using Streptococcus zooepidemicus
指導教授:吳建一
指導教授(外文):Jane-Yii Wu
學位類別:碩士
校院名稱:大葉大學
系所名稱:生物產業科技學系
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
論文出版年:2007
畢業學年度:95
語文別:中文
論文頁數:200
中文關鍵詞:獸疫鏈球菌透明質酸固定化氧氣質傳係數
外文關鍵詞:Streptococcus zooepidemicushyaluronic acidimmobilizationoxygen mass transfer coefficient
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本研究主要是研究Streptococcus zooepidemicus BCRC 15414在批次發酵下,探討不同培養條件對生產hyaluronic acid (HA)之分子量及特性影響。這些不同培養條件包含不同葡萄糖濃度 (0-40 g/L)、攪拌速度 (50 and 150 rpm)、曝氣速率 (0.5 and 2 Lmin-1)、絕對厭氧及添加不同NaCl濃度 (0-5%, w/v)。實驗結果顯示出在pH為9.0的條件下,當攪拌速率為150 rpm且葡萄糖濃度為20 g/L時,HA產量可達最大值(1.79 g/L)且分子量為1.76×106 Da;當曝氣速率為2.0 Lmin-1時,HA產量可達2.05 g/L且分子量為1.97×106 Da。因此,攪拌速度和曝氣速率對於細胞生長和HA生產是很重要的影響。另外,在利用固定化S. zooepidemicus PVA (polyvinyl alcohol)顆粒進行批次發酵生產HA方面,實驗結果顯示出利用固定化S. zooepidemicus PVA顆粒進行批次發酵生產,最好的HA產量約為1.0 g/L。除此之外,本實驗將純化後的HA樣品以NMR (nuclear magnetic resonance)、GPC (gel permeation chromatography)及EA (elemental analyzer)進行分析,確定產物之結構及分子量範圍,結果顯示此產物確實為 HA。
在HA發酵過程中,由於HA累積而使發酵液黏度增加。因此,氧氣質傳速率會明顯地減少。在培養期間,氧氣質傳係數是扮演 重要角色。所以,本研究主要探討攪拌速度、曝氣速率及HA溶液黏度對kLa值(oxygen mass transfer coefficient)的影響。實驗結果顯示出攪拌速度和曝氣速率增加時,kLa值也會隨之增加。當攪拌速度為300 rpm,kLa值為最大,其值約為0.3787 min-1;當曝氣速率為2.0 Lmin-1,kLa值為最大,其值約為0.1328 min-1。
此外,本研究亦探討在批次發酵系統下,用S. zooepidemicus生產胞外多醣HA之發酵動力學。本實驗所用simple model是以Logistic equation模擬菌體生長、Luedeking-Piret equation來模擬HA生產、而葡萄糖和氧氣消耗則是利用Luedeking- Piret- like equation模擬得知。
The effect of different culture variables on the molecular weight and properties of hyaluronic acid (HA) by Streptococcus zooepidemicus BCRC 15414 was investingated in the batch culture. The culture conditions contained different glucose concentrations (0-40 g/L), agitation rate (50 and 150 rpm), aeration rate (0.5 and 2 Lmin-1), obligate anaerobic and added various concentrations of NaCl (0-5%). The experimental results showed that HA production was maximum (1.79 g/L) and molecular weight of HA was 1.76×106 Da when agitation rate was controlled at 150 rpm and glucose concentration was 20 g/L at pH 9. When aeration rate was 2 Lmin-1, HA concentration reached 2.05 g/L and molecular weight of HA was 1.97×106 Da. Therefore, different agitation speeds and aeration rates were found to be an important effect on cell growth and HA production. Additionally, HA production using immobilized S. zooepidemicus beads in batch fermentation, the maximum HA production by immobilized-cell beads was 1.0 g/L. Moreover, the purified samples were characterized by a nuclear magnetic resonance (NMR), gel permeation chromatography (GPC) and elemental analyzer (EA). The results showed that the purified sample from the fermentation broth was HA.
During HA fermentation, the viscosity of culture broth increased because of the accumulating HA, which produced obviously decrease of oxygen mass transfer. For culture, the oxygen mass transfer coefficient (kLa) played an important role. Hence, the effects of agitation rate, aeration rate and viscosity of HA solution on kLa were studied. The results exhibited that kLa increased with the increase of aeration rate and agitation rate. The maximum kLa values was 0.3787 min-1 at agitation rate of 300 rpm; the maximum kLa values was 0.1328 min-1 at aeration rate of 2.0 Lmin-1.
Furthermore, the fermentation kinetics of an extracellular polysaccharides by S. zooepidemicus was investigated in a batch system. The study used a simple model induced Logistic equation for growth, the Luedeking-Piret equation for HA production and Luedeking- Piret- like equations for glucose and oxygen consumptions to simulate HA fermentation.
目錄

封面內頁
簽名頁
授權書………………………………………………………………iii
中文摘要……………………………………………………………iv
英文摘要……………………………………………………………vi
誌謝…………………………………………………………………viii
目錄…………………………………………………………………ix
圖目錄………………………………………………………………xiv
表目錄………………………………………………………………xviii
符號說明……………………………………………………………xx

1. 前言………………………………………………………1
2. 文獻回顧…………………………………………………5
2.1 HA簡介……………………………………………………5
2.1.1 HA結構……………………………………………………5
2.1.2 HA來源及生產……………………………………………9
2.1.3 HA生化合成機制…………………………………………12
2.2 HA之物性…………………………………………………20
2.2.1 HA之黏彈性………………………………………………20
2.2.2 HA之分子量………………………………………………20
2.3 HA之應用…………………………………………………22
2.3.1 化妝品之應用……………………………………………22
2.3.2 醫學之應用………………………………………………23
2.3.3 其他………………………………………………………26
2.4 HA流變學行為……………………………………………27
2.4.1 HA流變學行為之影響因子………………………………29
2.4.2 牛頓流體…………………………………………………32
2.4.3 非牛頓流體………………………………………………33
2.4.4 黏度與分子量之關係……………………………………35
2.5 氧氣質傳係數之影響因子………………………………35
2.5.1 攪拌速率…………………………………………………38
2.5.2 曝氣速率…………………………………………………40
2.5.3 黏度………………………………………………………41
2.5.4 固定化顆粒之填充比例…………………………………42
2.5.5 額外添加物………………………………………………44
2.6 基質在固定化菌體顆粒擴散行為之影響因子…………47
2.6.1 擴散行為…………………………………………………47
2.6.2 基質在固定化菌體顆粒擴散行為之影響………………49
3. 材料與方法………………………………………………54
3.1 實驗材料…………………………………………………54
3.1.1 實驗藥品…………………………………………………54
3.1.2 儀器設備…………………………………………………56
3.2 菌株活化…………………………………………………58
3.2.1 菌株來源…………………………………………………58
3.2.2 菌株活化…………………………………………………58
3.2.3 HA生產培養………………………………………………58
3.3 影響HA生產之因子探討…………………………………59
3.3.1 環境因子之影響…………………………………………59
3.3.2 添加物之影響……………………………………………60
3.3.3 緩衝溶液配製……………………………………………60
3.4 固定化微生物顆粒之製備及基本性質測定……………61
3.4.1 批次發酵大量培養:收集菌體細胞準備固定化………61
3.4.2 固定化微生物顆粒之製備………………………………61
3.4.3 固定化顆粒基本性質……………………………………62
3.4.4 固定化菌體顆粒之SEM觀察……………………………64
3.4.5 以固定化菌體顆粒進行HA生產……………………… 64
3.5 氧氣質傳(kLa)實驗……………………………………65
3.5.1 生物反應器………………………………………………65
3.5.2 模擬固定化顆粒在HA溶液之氧氣質傳實驗……………65
3.5.3 氧氣質傳係數的測量……………………………………66
3.6 固定化顆粒之擴散實驗…………………………………67
3.6.1 擴散理論…………………………………………………67
3.6.2 基質在固定化顆粒之擴散實驗…………………………70
3.7 發酵槽實驗………………………………………………72
3.7.1 懸浮系統…………………………………………………72
3.8 分析方法…………………………………………………73
3.8.1 醣類分析…………………………………………………73
3.8.1.1 DNS法……………………………………………………73
3.8.1.2 高效能液相層析儀(HPLC)分析………………………74
3.8.2 黏度………………………………………………………74
3.9 HA之分析…………………………………………………74
3.9.1 膠體滲透層析(GPC) …………………………………74
3.9.2 奧士瓦黏度計……………………………………………75
3.9.3 莢膜染色…………………………………………………76
3.9.4 高效能液相層析儀(HPLC)分析…………………………77
3.9.5 核磁共振(NMR)分析……………………………………78
3.9.6 元素分析…………………………………………………78
3.9.7 蛋白質定量分析…………………………………………79
3.10 HA之純化…………………………………………………80
3.11 HA生產動力學解析………………………………………80
3.12 統計分析…………………………………………………80
4. 結果與討論………………………………………………81
4.1 環境因子之影響…………………………………………81
4.1.1 Buffer對HA產量之影響……………………………… 81
4.1.2 在pH 7條件下,不同葡萄糖濃度對HA產量之影響… 89
4.1.3 在pH 9條件下,不同葡萄糖濃度對HA產量之影響… 94
4.1.4 不同碳源對HA產量之影響………………………………98
4.1.5 攪拌速率與曝氣量對HA產量之影響……………………102
4.1.6 不同NaCl濃度對HA產量之影響…………………………109
4.1.7 不同前培養方式及時間對HA產量之影響………………115
4.2 影響氧氣質傳(kLa)之因子…………………………… 120
4.2.1 攪拌速率對kLa之影響……………………………………120
4.2.2 曝氣量對kLa之影響………………………………………127
4.2.3 同時攪拌速率和曝氣量對kLa之影響……………………131
4.3 固定化顆粒實驗……………………………………………134
4.3.1 基質在固定化顆粒之擴散實驗……………………………134
4.3.2 固定化顆粒之BET(Brunauer-Emmett-Teller)分析…136
4.3.3 固定化菌體顆粒生產HA……………………………………136
4.3.4 固定化顆粒之SEM觀察…………………………………… 140
4.4 HA生產動力學解析…………………………………………142
4.4.1 微生物生長…………………………………………………143
4.4.2 產物生成……………………………………………………146
4.4.3 基質分解 (以葡萄糖為例) ………………………………150
4.5 批次發酵槽實驗……………………………………………153
4.5.1 懸浮系統……………………………………………………153
4.6 純化確定HA結構之分析……………………………………156
4.6.1 純化HA之元素分析…………………………………………156
4.6.2 純化HA之NMR分析…………………………………………157
4.6.3 純化HA之inherent viscosity…………………………………161
4.6.4 解凍復凍對HA分解之影響…………………………………165
5. 結論…………………………………………………………167
參考文獻…………………………………………………………………169
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