透明質酸(Hyaluronic acid，HA)又稱為玻尿酸，其由D-葡萄糖醛酸和N-乙醯胺基葡萄糖相互交替鍵結而成之直鏈多醣體，平均分子量104~107 Dalton，目前已被廣泛應用於醫學及化妝品等。本研究探討獸疫鏈球菌(Streptococcus zooepidemicus) BCRC15414在37℃、150rpm及控制pH值約在7.0之環境下培養，探討通氣因子對透明質酸產量之影響。通氣因子分為空氣及二氧化碳，通氣量為1vvm，在此二種氣體中比較出較佳之通氣因子，再分別以不同通氣量(0.5vvm、1.5vvm及2.0vvm) 進行試驗，以期能找出更適透明質酸生產之通氣條件。結果顯示，以二氧化碳為通氣因子，通氣量1vvm培養至第六小時，透明質酸產量可達最高產量為0.256 g/L，明顯高於以空氣為通氣因子，通氣量1vvm(0.210 g/L, 4hr)。因此後續再以二氧化碳為醱酵培養之主要通氣成分，發現透明質酸產量於通氣量1vvm時可得最高產量之透明質酸，接著依序為2.0vvm(0.234 g/L, 10hr)、1.5vvm(0.187 g/L, 24hr)、0.5vvm (0.176 g/L, 8hr)。由以上數據得知，獸疫鏈球菌在生產透明質酸時，會因為不同進氣條件及培養時間而影響產量。由不同通氣因子及通氣量之實驗結果，可得知最適通氣條件為二氧化碳，於通氣量1vvm下在第六小時時可產出0.256 g/L之透明質酸。

Hyaluronic acid (HA) is a linear polysaccharide composed of repeating disaccharide units of D-glucuronic acid and N-acetylglucosamine linkaged by β(1-4) and β(1-3) glycosidic bond. It has been widespread in medical application and cosmetics. In this study, Streptococcus zooepidemicus BCRC15414 was used for hyaluronic acid production. The fermentation condition was set at 37°C, 150rpm and the pH value at about 7.0, and the effects of aeration factor on hyaluronic acid production was investigated. The aeration factors were divided into two sections, the air and the carbon dioxide, where the aeration rate is 1vvm for comparisons. Then different aeration rate (0.5vvm, 1.5vvm and 2.0vvm) experiments,were carried out to discover the most suitable aeration condition for hyaluric acid production. The result showed that 1vvm of carbon dioxide as aeration factor for 6 hr would give a hyaluronic acid concentration of 0.256 g/L, which is greater than air (0.210 g/L, 4hr). Carbon dioxide was therefore used as main aeration ingredient for fermentation, this study discovered that the hyaluronic acid production had the maximum yield when 1vvm aeration rate was applied.. This study shows that when using Streptococcus zooepidemicus for producing hyaluronic acid, different aeration conditions and culturing time would highly affect the production.

中文摘要iv
英文摘要v
誌謝vi
目錄viii
圖目錄xi
表目錄xiv

1. 前言1
2. 文獻回顧2
2.1 獸疫鏈球菌之簡介2
2.2 透明質酸的結構2
2.2.1 透明質酸一級與二級結構2
2.2.2 透明質酸的三級結構3
2.3 透明質酸的分佈7
2.4 透明質酸之性質7
2.4.1 透明質酸之膨潤性7
2.4.2 透明質酸之黏彈性7
2.4.3 透明質酸之分子量10
2.5透明質酸之官能基10
2.6 透明質酸之生化合成13
2.7 透明質酸之分離與純化17
2.7.1 動物組織中之透明質酸萃取17
2.7.2 微生物醱酵萃取17
2.8 影響透明質酸之因子19
2.8.1 溫度之影響19
2.8.2 pH值之影響19
2.8.3 攪拌速率與通氣量之影響20
2.9 透明質酸之應用20
2.9.1 透明質酸於眼科方面之應用21
2.9.2 透明質酸於關節疾病方面之應用21
2.9.3 透明質酸於藥物釋放方面之應用22
2.9.4 透明質酸在傷口癒合方面之應用22
2.9.5 透明質酸於化妝品方面之應用23
3. 材料與方法24
3.1實驗藥品24
3.2儀器設備24
3.3 試驗菌株26
3.4培養基26
3.5菌株活化26
3.6菌株保存27
3.7實驗方法27
3.7.1 搖瓶試驗27
3.7.2 5L發酵槽27
3.7.3 環境因子之影響29
3.7.3.1 pH值之影響29
3.7.3.2 氣體因子之影響29
3.8 分析方法29
3.8.1菌液濃度29
3.8.2 菌體生質量(Biomass)29
3.8.3 乙醇沉澱30
3.8.4 咔唑法檢測透明質酸含量30
3.8.5 透明質酸之純化31
3.8.6 透明質酸分子量檢測(Gel-permeation chromatography，GPC)32
3.8.7 傅立葉紅外線光譜分析儀(FTIR)檢測樣品官能基32
4. 結果與討論34
4.1 搖瓶試驗34
4.2 不同進氣因子對S. zooepidemicus生質量與透明質酸之影響37
4.2.1 pH值無控制下37
4.2.2 pH值控制於7.037
4.3 二氧化碳之不同通氣量對S. zooepidemicus生質量與透明質酸之影響46
4.4 透明質酸分子量檢測(GPC)51
4.5 傅立葉紅外線光譜分析儀(FTIR)檢測官能基65
5.結論72
參考文獻73
圖目錄
圖2.1 透明質酸之一級結構4
圖2.2 透明質酸之二級結構5
圖2.3 透明質酸於水溶液中之三級結構6
圖2.4 透明質酸在不同流體液中之黏彈性質9
圖2.5 透明質酸合成酶之作用示意圖14
圖2.6 透明質酸生化合成之機制15
圖2.7 透明質酸在鏈球菌中的合成路徑16
圖3.1 實驗流程圖28
圖4.1 不同體積之培養基對於透明質酸產量之影響36
圖4.2 S. zooepidemicus於pH值無控制，分別以空氣及二氧化碳為通氣因子，通氣量1 vvm下pH值之變化39
圖4.3 S. zooepidemicus於pH值無控制，分別以空氣及二氧化碳為通氣因子，通氣量各1 vvm下，對菌體生長之影響40
圖4.4 S. zooepidemicus於pH值無控制，分別以空氣及二氧化碳為通氣因子，通氣量各1 vvm下，對菌體乾重之影響41
圖4.5 S. zooepidemicus於pH值無控制，分別以空氣及二氧化碳為通氣因子，通氣量各1 vvm下，對HA產量之影響42
圖4.6 S. zooepidemicus於控制pH值為7.0，分別以空氣及二氧化碳為通氣因子，通氣量各1 vvm下，對菌體生長之影響43
圖4.7 S. zooepidemicus於控制pH值為7.0，分別以空氣及二氧化碳為通氣因子，通氣量各1 vvm下，對菌體乾重之影響44
圖4.8 S. zooepidemicus於控制pH值為7.0，分別以空氣及二氧化碳為通氣因子，通氣量各1 vvm下，對HA產量之影響45
圖4.9 S. zooepidemicus於控制pH值為7.0，以CO2不同通氣量在對菌液濃度吸光值之影響48
圖4.10 S. zooepidemicus於控制pH值為7.0，以CO2 不同通氣量對菌體乾重之影響49
圖4.11 S. zooepidemicus於控制pH值為7.0，以CO2 不同通氣量對HA產量之影響50
圖4.12 GPC標準品圖譜53
圖4.13 S. zooepidemicus於控制pH值為7.0，以1.0 vvm空氣為通氣因子所生產HA之分子量分佈54
圖4.14 S. zooepidemicus於控制pH值為7.0，以0.5 vvm CO2為通氣因子所生產HA之分子量分佈56
圖4.15 S. zooepidemicus於控制pH值為7.0，以1.0 vvm CO2為通氣因子所生產HA之分子量分佈58
圖4.16 S. zooepidemicus於控制pH值為7.0，以1.5 vvm CO2為通氣因子所生產HA之分子量分佈60
圖4.17 S. zooepidemicus於控制pH值為7.0，以2.0 vvm CO2為通氣因子所生產HA之分子量分佈62
圖4.18 S. zooepidemicus於控制pH值為7.0，不同通氣條件所生產HA之分子量分佈圖64
圖4.19 S. zooepidemicus於控制pH值為7.0，以1vvm空氣為通氣因子所生產HA之FTIR圖譜66
圖4.20 S. zooepidemicus於控制pH值為7.0，以0.5 vvm CO2為通氣因子所生產HA之FTIR圖譜67
圖4.21 S. zooepidemicus於控制pH值為7.0，以1.0vvm CO2為通氣因子所生產HA之FTIR圖譜68
圖4.22 S. zooepidemicus於控制pH值為7.0，以1.5vvm CO2 為通氣因子所生產HA之FTIR圖譜69
圖4.23 S. zooepidemicus於控制pH值為7.0，以2.0vvm CO2為通氣因子所生產HA之FTIR圖譜70
圖4.24 S. zooepidemicus於控制pH值為7.0，不同通氣條件所生產HA之FTIR圖譜71
表目錄
表2.1 不同分子量SH的FTIR特性吸收峰波長及其歸類12
表2.2 動物組織萃取與微生物醱酵生產透明質酸之比較18
表3.1 培養基組成成分26
表4.1 S. zooepidemicus於控制pH值為7.0，以1.0 vvm空氣為通氣因子所生產HA之分子量分佈55
表4.2 S. zooepidemicus於控制pH值為7.0，以0.5 vvm CO2為通氣因子所生產HA之分子量分佈57
表4.3 S. zooepidemicus於控制pH值為7.0，以1.0 vvm CO2為通氣因子所生產HA之分子量分佈59
表4.4 S. zooepidemicus於控制pH值為7.0，以1.5 vvm CO2為通氣因子所生產HA之分子量分佈61
表4.5 S. zooepidemicus於控制pH值為7.0，以2.0 vvm CO2 為通氣因子所生產HA之分子量分佈63

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