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研究生:林秀憲
研究生(外文):LIN, HSIU-HSIEN
論文名稱:維生素A的發酵技術改進
論文名稱(外文):Improvement of Vitamin A Fermentation Technology
指導教授:黃志宏黃志宏引用關係
指導教授(外文):HUANG, CHIH-HUNG
口試委員:黃姿雯陳月茸
口試委員(外文):HUANG, TZU-WENCHEN, YUEH-JUNG
口試日期:2020-07-30
學位類別:碩士
校院名稱:國立臺北科技大學
系所名稱:化學工程與生物科技系生化與生醫工程碩士班
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:2020
畢業學年度:108
語文別:中文
論文頁數:55
中文關鍵詞:維生素A大腸桿菌兩相培養
外文關鍵詞:Vitamin AEscherichia colitwo-phase culture
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類維生素A (retinoids)為脂溶性的維生素,有視黃醇(retinol)、視黃醛(retinaldehyde)、視黃酸(retinoic acid)和視黃酯(retinyl esters)等不同型式存在於自然界中,其在人體是不可或缺的,參與多種生理功能的運作,包括維持正常視覺、骨組織生長、免疫系統調節等作用。此外,維生素A在皮膚上也有些特定的作用,例如塗抹在皮膚上,持續使用可減少肌膚皺紋生成以及促進真皮層彈性纖維及膠原蛋白之分泌,且也有助於改善黑色素形成,因此現今在皮膚保養品方面也廣泛被添加使用。市面上添加於皮膚保養品的高純度維生素A皆以化學合成為主要來源,原因是由生物純化的維生素A常有參雜容易引起皮膚過敏的生物源刺激物,但化學合成複雜及純化過程使生產成本提高,故本論文以大腸桿菌透過兩相培養的方式直接生產高純度的維生素A,藉由遺傳工程改質的菌株,能夠大量生成並外泌維生素A,使其在發酵過程中直接溶入油相層之中,以便後續在皮膚保養品中直接添加應用。在搖瓶小量培養測試時加入純氧使氧氣擴散在培養箱當中,其油相中的維生素A濃度已逼近商業生產的合理產值,此數值也達到了此油相可溶解維生素A的最大極限。放大到4公升發酵槽後試量產,其油相中維生素A濃度只達搖瓶培養時的53%,顯示未來放大製程,還有可持續改善之空間。
Retinoid is a lipophilic vitamin. There are different nature types of retinol, including retinaldehyde, retinoic acid, and retinyl esters. They are indispensable in the human body and participate in a variety of physiological functions, including maintaining normal vision, bone tissue growth, immune system regulation, etc. Besides, vitamin A also has some specific effects on the skin. For example, it can reduce the formation of skin wrinkles and can promote the secretion of elastic fibers and collagen in the dermis layer of skin. It also helps to reduce the formation of melanin. Therefore, it is widely used in skincare products, in which the used high-purity vitamin A are all chemically synthesized. The reason is that the biologically purified vitamin A often contains different irritants that easily induce skin allergies, but the complex processes of chemical synthesis and purification increase the production cost. This study was focused on the production of high-purity vitamin A through engineered E. coli using two-phase cultivation. The employed strain was genetically engineered to produce and to secret vitamin A, which directly dissolved into the oil phase during the two-phase cultivation. Therefore, this vitamin A-contained oil may directly apply to skincare products without further extraction. We found the increased oxygen concentration in shaking flasks would gather more vitamin A in the oil of two-phase cultivation. The yield of vitamin A in the oil phase was very close to reasonable commercial production, and its concentration also closed to the maximum amount of soluble vitamin A in this oil. We applied a similar condition to a 4-liter fermentation tank, the preliminary result showed the concentration of vitamin A in the oil phase was only 53% that of the flask result. It indicated that the scale-up process may need to improve in the future.
摘要 i
ABSTRACT iii
誌 謝 v
目錄 vi
表目錄 viii
圖目錄 ix
第一章 緒論 1
1.1 維生素A對人類的重要性 1
1.2 維生素A之應用-由抗皺、淡斑達到全面性抗老 5
1.3 維生素A之市場價值 10
1.4 維生素A之結構與性質 11
1.5 微生物之反應途徑 14
1.6 發酵過程中維生素A之收集-兩相培養 16
1.7 利用大腸桿菌生產維生素A 17
第二章 材料與方法 18
2.1 菌種與質體 18
2.2 藥品與儀器 18
2.3 培養基 19
2.4 菌種保存 20
2.5 大腸桿菌之轉型 20
2.6 菌種培養 20
2.7 菌液菌量及pH值測定 21
2.8 菌液中油相分離及retinoids含量測定 21
第三章 實驗結果 22
3.1 比較Isopropyl Palmitate不同添加比例對維生素A萃取濃度之影響 22
3.2 透過不同pH值之MR改動Fx medium初始pH值後對於retinaldehyde萃取總量之影響 24
3.3 Isopropyl Palmitate與Dodecane萃取維生素A能力之差異 26
3.4 透過在恆溫震盪培養箱中供應純氧以增加維生素A之產量 29
3.5 添加純氧培養後調整萃取相添加比例 33
3.6 發酵槽培養過程中通氣量不同對菌株生長及生產維生素A之影響 35
3.6.1 高通氣量培養測試 35
3.6.2 低通氣量培養測試 36
3.7 於發酵槽進氣中混合純氧培養 38
3.7.1 改變供氧的時間 38
3.7.2 降低發酵槽內通氣量 40
第四章 討論 43
參考文獻 48
附 錄 52
Appendix 1. Electroporation transformation 52
Appendix 2. all-trans-Retinaldehyde檢量線 54
Appendix 3. HPLC分析圖譜之標準品與樣品比對 55


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