現在位置首頁 > 博碩士論文 > 詳目
論文中文名稱:利用大腸桿菌H10-pink的兩相培養液醱酵以增產維生素A [以論文名稱查詢館藏系統]
論文英文名稱:Enhancing the production of retinoids by two-phase culture of engineered Escherichia coli H10-pink [以論文名稱查詢館藏系統]
院校名稱:臺北科技大學
學院名稱:工程學院
系所名稱:化學工程與生物科技系化學工程碩士班
畢業學年度:107
畢業學期:第一學期
出版年度:107
中文姓名:潘宏展
英文姓名:Hung-Chan Pan
研究生學號:105738504
學位類別:碩士
語文別:中文
口試日期:2018/11/09
論文頁數:61
指導教授中文名:黃志宏
指導教授英文名:Chin-Hung Huang
口試委員中文名:侯劭毅;楊千金
口試委員英文名:SHAO-YI HOU;Chien-Chin Yang
中文關鍵詞:維生素AA醛兩相培養
英文關鍵詞:Vitamin Aretinaldehyderetinoids
論文中文摘要:維生素A(retinoids)為脂溶性維生素,也是人體必需維生素之一,有多種構型,如視黃醇(retinol)、視黃醛(retinal)、視黃酸(retinoic acid)及視黃酯(retinyl ester)等。維生素A對視力、眼睛的保健,以及骨骼發育、免疫系統調節與傷口癒合等重要影響性,已有多的報導與實際應用。此外,一些研究也發現將高純度維生素A稀釋後塗抹在皮膚上,持續使用下能中斷自由基損傷細胞的過程,抑制皮膚皺褶凹陷部分之膠原蛋白分解酶的活性,有助於減少皺紋、增加真皮層膠原蛋白及彈性纖維之分泌,因此在皮膚保養品方面也開始被廣泛的添加使用。本論文以大腸桿菌經兩相培養方式直接生產高純度維生素A,進行醱酵製程改良。藉由遺傳工程改質的E. coli H10-pink菌株,能大量合成維生素A,在醱酵過程中直接被提取溶入油相層中,以方便後續的皮膚保養品的添加應用。此醱酵製程的優點是免去破菌,能減少產物中所含細菌過敏原的數量;此外,視黃醛在油脂包覆下,能減低受到光照的降解。經由醱酵槽放大培養後,在混合純氧並連續饋入甘油(碳源)之醱酵槽培養方式,能有效提高油相中視黃醛產率至700.06 μg/mL (49小時)。經由HPLC以及UPLC/MS的分析,顯示油相中的維生素A,其視黃醛大約占80%,視黃醇18~20%,視黃酸少於2%,故能減少對皮膚的刺激性,具有應用在皮膚保養之價值,未來會繼續朝向增加產率的方向進行。
論文英文摘要:Vitamin A (retinoids) is a lipophilic and antioxidant vitamin which is one of the essential vitamins for human. Vitamin A includes four similar structure compounds with different side chains, such as retinol, retinal, retinoic acid and retinyl ester. Vitamin A is well known in several physiological functions, such as eye care, bone development, immune system regulation, and wound healing. Besides, high purity vitamin A has shown functions in skin care, including reduce the free radical damage, inhibit collagenases to reduce the formation of wrinkles, and stimulate the production of collagen and elastic fibers in the dermis. This study was focused on vitamin A production by improvement the fermentation process of two-phase culture system. An engineered Escherichia coli H10-pink strain was employed to increase the production rate of vitamin A, which would be directly extracted into an oil phase during fermentation. Then, the oil containing enriched vitamin A may become an additive of skin care products. One advantage of this fermentation system is significantly reduced the skin allergen from the debris of E. coli because of skipping the process of cell lysis, the other is that the oil provides protection of vitamin A from light damage. By a series of modifications of fermenter condition, the yield of vitamin A was up to 700.06 μg/mL in the oil phase. That vitamin A was analyzed the composition ration by the HPLC and UPLS/MS, and the results showed that detected components were about 80% retinal, 18 to 20% retinol, and retinoic acid less than 2%. Due to the few amount of retinoic acid in oil, the product would reduce the irritation of skin, and that is an important indicator of evaluation of vitamin A to apply in skin-care products. The next step will keep going on increasing the production rate of vitamin A in this two-phase culture system.
論文目次:摘要 i
ABSTRACT iii
誌謝 v
目錄 vi
表目錄 ix
圖目錄 x
第一章 緒論 1
1.1 維生素A對人體的重要性 1
1.2 維生素A基本結構與性質 5
1.3 維生素A之市場價值 6
1.4 維生素A之應用- 皮膚抗皺、美容 7
1.5 微生物之反應途徑 10
1.6 醱酵過程之維生素A收集 12
1.7 利用大腸桿菌生產維生素A 13
第二章 材料與方法 14
2.1 菌種與質體 14
2.2 藥品與儀器 14
2.3 培養基 15
2.4 菌種保存 16
2.5 大腸桿菌之轉型 16
2.6 菌種培養 16
2.7 生長菌量測定 17
2.8 Retinoids含量測定 17
2.9 葡萄糖含量測定 18
2.10 Retinoids成分分析 19
第三章 實驗結果 20
3.1 比較Dodecane與Isopropyl Palmitate對維生素A產量影響 20
3.2 探討Isopropyl Palmitate不同添加比例對維生素A之提取影響 23
3.3 搖瓶培養下比較不同甘油濃度的影響 25
3.4 搖瓶培養下比較加鋼圈的影響 27
3.5 探討避光對搖瓶培養的維生素A產量影響 30
3.6 探討不同溫度對搖瓶培養下的維生素A產量影響 31
3.7 醱酵槽批次培養 33
3.8 醱酵槽以混合純氧方式之批次培養 35
3.9 醱酵槽混合純氧並連續饋入甘油之批次培養 37
3.10 醱酵槽混合純氧、連續饋入甘油及調控pH之批次培養 39
第四章 結果與討論 41
參考文獻 47
附錄 52
Appendix 1. Electroporation transformation 52
Appendix 2. all-trans-retinal檢量線 54
Appendix 3. Glucose檢量線 54
Appendix 4. UPLC- Synapt G2 HDMS之樣品分析結果 55
Appendix 5. HPLC分析圖譜之標準品與樣品比對 61
論文參考文獻:Abdelmalek, M., & Spencer, J. (2006). Retinoids and wound healing. Dermatol Surg, 32(10), 1219-1230. doi:10.1111/j.1524-4725.2006.32280.x
Anja Thielitz, & Gollnick, H. (2008). Topical Retinoids in Acne Vulgaris. American Journal of Clinical Dermatology, 9(6), 369-381.
Bempong DK, Honigberg IL, & NM., M. (1995). Normal phase LC-MS determination of retinoic acid degradation products. J Pharm Biomed Anal., 13(3), 285-291.
Blomhoff, R., & Blomhoff, H. K. (2006). Overview of retinoid metabolism and function. J Neurobiol, 66(7), 606-630. doi:10.1002/neu.20242
C. Mercier, & Chabardes, P. (1994). Organometallic chemistry in industrial vitamin A and vitamin E synthesis Pure &Appl. Chem., 66(7), 1509-1518.
Creidi, P., Vienne, M.-P., Ochonisky, S., Lauze, C., Turlier, V., Lagarde, J.-M., & Dupuy, P. (1998). Profilometric evaluation of photodamage after topical retinaldehyde and retinoic acid treatment. J Am Acad Dermatol, 39(6), 960-965.
Dawadi, P. B., & Lugtenburg, J. (2010). Synthesis and use of stable isotope enriched retinals in the field of vitamin A. Molecules, 15(3), 1825-1872. doi:10.3390/molecules15031825
Degim, I. T. (2005). Understanding Skin Penetration: Computer Aided Modeling and Data Interpretation. Current Computer-Aided Drug Design, 1(1), 11-19. doi:10.2174/1573409052952242
Didierjean, L., Tran, C., Sorg, O., & Saurat, J. H. (1999). Biological activities of topical retinaldehyde. Dermatology, 199 Suppl 1, 19-24. doi:10.1159/000051373
Dietary Reference Intakes for Vitamin A, Vitamin K, Arsenic, Boron, Chromium, Copper, Iodine, Iron, Manganese, Molybdenum, Nickel, Silicon, Vanadium, and Zinc. (2001). doi:10.17226/10026
Dowling, J. E., & Wald, G. (1958 ). VITAMIN A DEFICIENCY AND NIGHT BLINDNESS. Proc Natl Acad Sci U S A., 44(7), 648-661.
Fernández-García, E., Carvajal-Lérida, I., Jarén-Galán, M., Garrido-Fernández, J., Pérez-Gálvez, A., & Hornero-Méndez, D. (2012). Carotenoids bioavailability from foods: From plant pigments to efficient biological activities. Food Research International, 46(2), 438-450. doi:10.1016/j.foodres.2011.06.007
Fraser, P. D., & Bramley, P. M. (2004). The biosynthesis and nutritional uses of carotenoids. Prog Lipid Res, 43(3), 228-265. doi:10.1016/j.plipres.2003.10.002
Gao, X. H., Zhang, L., Wei, H., & Chen, H. D. (2008). Efficacy and safety of innovative cosmeceuticals. Clin Dermatol, 26(4), 367-374. doi:10.1016/j.clindermatol.2008.01.013
Griffiths CE, Russman AN, Majmudar G, Singer RS, Hamilton TA, & JJ., V. (1993). Restoration of collagen formation in photodamaged human skin by tretinoin (retinoic acid). N Engl J Med., 329(8), 530-535. doi:10.1056/NEJM199308193290803
Jang, H.-J., Yoon, S.-H., Ryu, H.-K., Kim, J.-H., Wang, C.-L., Kim, J.-Y., . . . Kim, S.-W. (2011). Retinoid production using metabolically engineered Escherichia coli with a two-phase culture system. Microb Cell Fact, 10, 59. doi:10.1186/1475-2859-10-59
Jang, H. J., Ha, B. K., Kim, J. W., Jung, K. H., Ahn, J., Yoon, S. H., & Kim, S. W. (2014). Comparison of extraction phases for a two-phase culture of a recombinant E. coli producing retinoids. Biotechnol Lett, 36(3), 497-505. doi:10.1007/s10529-013-1385-1
Jang, H. J., Ha, B. K., Zhou, J., Ahn, J., Yoon, S. H., & Kim, S. W. (2015). Selective retinol production by modulating the composition of retinoids from metabolically engineered E. coli. Biotechnol Bioeng, 112(8), 1604-1612. doi:10.1002/bit.25577
Jang, H. J., Yoon, S. H., Ryu, H. K., Kim, J. H., Wang, C. L., Kim, J. Y., . . . Kim, S. W. (2011). Retinoid production using metabolically engineered Escherichia coli with a two-phase culture system. Microb Cell Fact, 10, 59. doi:10.1186/1475-2859-10-59
Jennifer J. Otten, Jennifer Pitzi Hellwig, & Meyers, L. D. (2006). Dietary Reference Intakes: The Essential Guide to Nutrient Requirements.
Jose David, P. (1989). Studies on the Synthesis of Vitamin-A and Related Compounds. FACULTY OF SCIENCE, APPLIED CHEMISTRY, COCHIN UNIVERSITY OF SCIENCE AND TECHNOLOGY, 1-181.
Kammeyer, A., & Luiten, R. M. (2015). Oxidation events and skin aging. Ageing Res Rev, 21, 16-29. doi:10.1016/j.arr.2015.01.001
Karrer, P. (1937). Carotenoids, Flavins and Vitamin A and B2. In Nobel Lectures, Chemistry (1922-1941), 433-448.
Keilin, D., & Hartree, E. F. (1948). Properties of Glucose oxidase (Notatin). Biochem J., 42(2), 221-229.
Keith P. West, J. (2003). Vitamin A deficiency disorders in children and women. Food and Nutrition Bulletin, 24(4 (supplement)), S78-S90.
Kim, Y. S., Park, C. S., & Oh, D. K. (2010). Retinal production from beta-carotene by beta-carotene 15,15-dioxygenase from an unculturable marine bacterium. Biotechnol Lett, 32(7), 957-961. doi:10.1007/s10529-010-0239-3
Lee, J. H., Choi, J. G., Kim, Y. S., Kim, K. R., Kim, S. W., & Oh, D. K. (2012). Enhancement of retinal production by supplementing the surfactant Span 80 using metabolically engineered Escherichia coli. J Biosci Bioeng, 113(4), 461-466. doi:10.1016/j.jbiosc.2011.11.020
McCaffery, P., Evans, J., Koul, O., Volpert, A., Reid, K., & Ullman, M. D. (2002). Retinoid quantification by HPLC/MSn. Journal of Lipid Research, 43(7), 1143-1149. doi:10.1194/jlr.D200012-JLR200
Mukherjee S, Date A, Patravale V, Korting HC, Roeder A, & G., W. (2006). Retinoids in the treatment of skin aging: an overview of clinical efficacy and safety. Clin Interv Aging., 1(4), 327-348.
Olivier Sorg, Stéphane Kuenzli, Gürkan Kaya, & Saurat., J.-H. (2005). Proposed mechanisms of action for retinoid derivatives in the treatment of skin aging. Journal of Cosmetic Dermatology, 4(4), 237-244. doi:10.1111/j.1473-2165.2005.00198.x
Oroshnik, W. (1978). Synthesis of Vitamin A, intermediates and conversion thereof to Vitamin A. USA Patent, No. 4,092,366, 1-9.
Parker, G. L., Smith, L. K., & Baxendale, I. R. (2016). Development of the industrial synthesis of vitamin A. Tetrahedron, 72(13), 1645-1652. doi:10.1016/j.tet.2016.02.029
Poon, F., Kang, S., & Chien, A. L. (2015). Mechanisms and treatments of photoaging. Photodermatol Photoimmunol Photomed, 31(2), 65-74. doi:10.1111/phpp.12145
Rigopoulos, D., Ioannides, D., Kalogeromitros, D., & Katsambas, A. D. (2004). Comparison of topical retinoids in the treatment of acne. Clin Dermatol, 22(5), 408-411. doi:10.1016/j.clindermatol.2004.03.013
Ross, A. C., & Restori, K. H. (2013). Vitamin A and the immune system. 221-243. doi:10.1533/9780857095749.2.221
Saurat, J. H., Didierjean, L., Masgrau, E., Piletta, P. A., Jaconi, S., Chatellard-Gruaz, D., Siegenthaler, G. (1994). Topical Retinaldehyde on Human Skin: Biologic Effects and Tolerance. Journal of Investigative Dermatology, 103(6), 770-774. doi:10.1111/1523-1747.ep12412861
Shao, Y., He, T., Fisher, G. J., Voorhees, J. J., & Quan, T. (2017). Molecular basis of retinol anti-ageing properties in naturally aged human skin in vivo. Int J Cosmet Sci, 39(1), 56-65. doi:10.1111/ics.12348
Thielitz, A., Abdel-Naser, M. B., Fluhr, J. W., Zouboulis, C. C., & Gollnick, H. (2008). Topical retinoids in acne--an evidence-based overview. J Dtsch Dermatol Ges, 6(12), 1023-1031. doi:10.1111/j.1610-0387.2008.06741.x
Valla, A. R., Cartier, D. L., & Labia, R. (2003). Recent Progress in Retinoid Chemistry. 28, 69-107. doi:10.1016/s1572-5995(03)80139-4
WHO. (2009). Global prevalence of vitamin A deficiency in populations at risk 1995-2005. In WHO global database on vitamin a deficiency.
論文全文使用權限:不同意授權