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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 蘇南維(Nan-Wei Su) | |
dc.contributor.author | Kung-Hao Kuo | en |
dc.contributor.author | 郭功浩 | zh_TW |
dc.date.accessioned | 2021-07-10T22:16:53Z | - |
dc.date.available | 2021-07-10T22:16:53Z | - |
dc.date.copyright | 2017-08-31 | |
dc.date.issued | 2017 | |
dc.date.submitted | 2017-08-15 | |
dc.identifier.citation | Andrews, J. F. (1968). A mathematical model for the continuous culture of microorganisms utilizing inhibitory substrates. Biotechnology and Bioengineering, 10(6), 707-723.
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dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/77703 | - |
dc.description.abstract | 異黃酮(isoflavone)是植物中的二次代謝物,其化學結構與人體雌激素(estradiol)相似,因此具有雌激素的活性,被稱為植物雌激素(phytoestrogen)。異黃酮依化學結構不同,可分成四大類: malonyl-glucosides、acetyl-glucosides、glucosides和aglycones。而其中以不帶醣基之異黃酮(aglycones)有較佳之生理活性。近年來,許多文獻指出屬於aglycones類的genistein具有預防骨質疏鬆、抗心血管疾病、乳癌及前列腺癌等多項生理活性。然而,genistein不溶於水的特性,使其生物可利用率(bioavailability)不佳。本研究室先前篩選出Bacillus subtilis BCRC 80517菌株可對genistein進行磷酸酯化修飾,形成genistein 7-O-phosphate (G7P),轉換後產物G7P可大幅提升genistein的水溶性,經動物實驗證實,可增加genistein之生物可利用率。然而,先前並未利用發酵槽進行genistein生物轉換的探討及放大規模純化。因此,本論文即以此為基礎,進行後續的研究。
本研究分為三部分。第一部分,利用5 L通氣攪拌式發酵槽放大發酵製程生產G7P。結果顯示,發酵槽以最適化批次(batch)式發酵生產,最高投入轉換基質genistein濃度為18.52 mM,可於發酵時間30小時,達到100%的完全轉換,生成G7P 18.87 mM,相較搖瓶振盪培養,提高轉換基質genistein濃度達9倍之多。再者,以最佳二次性饋料(fed-batch)式發酵生產,可於發酵時間48小時內,使37.0 mM之轉換基質genistein達100%之完全轉換,生成G7P 38.21 mM,相較搖瓶振盪培養,提高轉換基質濃度達18倍之多。半連續式發酵結果顯示,最適種菌保留量為15%,前兩個批次發酵皆能維持100%之完全轉換,於第三個批次開始轉換率大幅下降。第二部分,建立自發酵液回收G7P之最適化程序。結果顯示,利用微過濾膜進行發酵液之濃縮,濃縮液以三倍體積乙酸乙酯萃,減壓濃縮至乾後,回溶於90%乙醇溶液,再以1 N NaOH溶液調鹼,使G7P-2Na析出,析出產物G7P-2Na經隔夜烘乾後,G7P-2Na之純度及回收率為89% (w/w)與67%;通過微過濾膜之濾液,以HP-20膨脹床管柱進行純化,經過一倍管柱體積之pH 1、5% NaCl水溶夜清洗再以兩倍管柱體積之0.1 N NaOH水溶液進行脫附,脫附液經凍乾,得到產物G7P-2Na之回收率及純度為90%及72%。第三部分,評估G7P-2Na之降解動力學與物理特性。結果顯示,G7P-2Na粉末之降解為一級反應,於常溫下非常安定,幾乎不會降解,G7P-2Na粉末之活化能為24.61 kcal/mole;將G7P-2Na添加於糖水中,G7P-2Na之活化能(Ea)下降至14.06 kcal/mol,推算儲存於23.5 °C下之半衰期(T0.5)及降解20%(T0.8)之值,分別為35天及11天。Genistein、G7P和G7P-2Na在25oC下水中的溶解度分別為4.7、1.2×105 mg/L與1.1×108 mg/L。顯示G7P之溶解度比genistein高出2萬5千倍之多,然而,G7P-2Na之溶解度又比G7P高出1000倍之多。G7P若以鈉鹽形式存在其熔點為289oC,相較G7P之熔點高出57 oC。本研究結果顯示,G7P確實具有潛力放大規模生產,開發為新穎性之保健產品。 | zh_TW |
dc.description.abstract | Genistein, one of the polyphenolic second metabolites in soybeans, has a number of pharmacological and biological activities; however, low water solubility and poor bioavailability limit its use. Our previous study revealed a water-soluble phosphate conjugate of genistein 7-O-phosphate (G7P), generated by biotransformation of Bacillus subtilis BCRC 80517 with genistein. In pharmacokinetics studies, G7P greatly improved the maximum plasma level and bioavailability of its corresponding genistein in vivo. However, the present studies only use shake flasks to produce G7P. This study aimed to scaling the fermentation process up from shake flasks to laboratory-scale bioreactor for the production of G7P, and developing a feasible and promising process for recovering G7P from fermentation broth.
In the first part of this thesis, a fermentation process for G7P production by Bacillus subtilis BCRC 80517 was successfully scaled up from 500 mL shake flask to 5 L stirred tank bioreactor. At first, through the optimal batch fermentation process, at the end of 30 h incubation time, the bioconversion rate of genistein were 100%, and the biotransformed product G7P in the harvested broth were 18.87 mM, compare to the previous flask studies, the concentration of G7P enhanced up to 9-fold. Second, we have developed a successful fed batch fermentation strategy, which can produce up to 38.21 mM of G7P at the end of 48 h incubation time, enhanced the productivity of G7P by 18 times compare with the previous flask studies. To further increase the G7P productivity, the fed batch fermentation process was repeated 4 times with cell reused. The results showed that repeated fed batch fermentation was effective in improving the fermentation efficiency and maintaining high G7P productivity for the first and second batch fermentation. In the second part of this thesis, we developed a feasible and promising process for recovering G7P from fermentation broth. At first, using micro-filtration for fermentation broth concentrating, the retentate part through EA extraction and pH adjustion, we could obtained G7P with 67% recovery and 89% (w/w) in purity. The permeate from membrane filtration through HP-20 expanded bed adsorption, washing and 0.1N NaOH solution desorption, G7P with 90% recovery and 72% (w/w) in purity was obtained. At the third part of this thesis, according to the Handbook of Pharmaceutical Salts, we chosed sodium ion as salt formers to form G7P-2Na. The results of storage test showed that G7P-2Na’s activation energy (Ea) was 24.61, exhibiting very stable characteristics. When the G7P-2Na was added to the syrup, the G7P-2Na’s activation energy (Ea) was dropped to 14.06 kcal/mol. The water solubility at 25oC of genistein, G7P and G7P-2Na were 4.7, 1.2×105 mg/L, and 1.1×108 mg/L respectively. The water solubility of G7P were 25,000-fold higher than genistein, however G7P-2Na were 1000-fold much higher than G7P. At the end of the studies, we found that the melting point of G7P-2Na were 289 oC higher than the 232 oC of G7P. | en |
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dc.description.tableofcontents | 縮寫對照表 x
第一章、前言 1 第二章、文獻整理 2 第一節 大豆異黃酮 2 1.類黃酮簡介 2 2.化學結構與生理活性 2 3.吸收與代謝 3 4.異黃酮之生物可利用率 8 4-1 BCS分類系統 8 4-2異黃酮之吸收與代謝 8 4-3.異黃酮於人體之口服生物可利用率 9 第二節 異黃酮之微生物轉換 17 1.類黃酮之微生物轉換 17 2.異黃酮之微生物轉換 18 2-1 去醣基化 (Deglycosylation) 18 2-2 羥基化 (Hydroxylation) 18 2-3 醣基化 (Glycosylation) 19 2-4 琥珀酰化 (Succinylation) 20 2-5 甲基化 (O-methylation) 20 2-6 氯化 (Chlorination) 20 2-7 磷酸化 (Phosphorylation) 20 第三節 前驅藥物 26 1.前驅藥物定義與介紹 26 2.磷酸酯前驅藥物 26 3.金雀異黃酮磷酸酯衍生物 27 第四節 生物工程技術 36 1.發酵槽 36 2.發酵培養策略 36 2-1 批次式(batch)發酵培養 36 2-2 饋料批次式(fed batch)發酵培養 37 2-3 連續式(continuous)發酵培養 38 2-4 半連續式(semi-continuous)發酵培養 39 第五節 發酵液的菌體分離技術 39 1.離心 39 2.過濾 39 第六節 生物分離與純化 41 1.萃取分離技術 41 2.利用膜過濾系統搭配溶劑萃取技術 41 第三章 材料與方法 43 第一節 實驗大綱 43 第二節 材料與方法 44 1.實驗材料 44 1-1 菌株 44 1-2金雀異黃酮 (genistein) 44 1-3 培養基 45 1-4 試藥與溶劑 46 1-5 實驗儀器設備 46 2.實驗方法 47 2-1 B. subtilis BCRC 80517對genistein之生物轉換 47 2-2 限制性基質探討 48 2-3 以最適基礎培養基發酵生產G7P 49 2-4 以饋料式(fed-batch)發酵培養策略生產G7P 49 2-5 半連續式發酵 50 2-6自發酵液中回收G7P之程序 51 2-7 HPLC定量分析及分光光度計掃描分析 52 2-8以加速試驗評估其安定性 53 2-9 模擬市售飲品添加G7P-2Na評估其安定性 53 2-10 溶解度測定 53 2-11 熔點測定 53 3.分析方法 54 3-1高效液相層析儀分析異黃酮之條件 54 3-2 B. subtilis BCRC 80517對異黃酮之生物轉換 54 3-3大豆異黃酮含量計算方式 55 3-4總醣測定-酚硫酸法 56 3-6統計分析 56 4.動力學資料分析 56 4-1反應速率常數 56 4-2半衰期(T0.5)及活化能 57 第四章 結果與討論 58 第一節 利用5 L通氣攪拌式發酵槽探討最適生產G7P條件 58 1.不同種菌培養時間對延滯期之影響 58 1-1 B. subtilis BCRC 80517在基礎培養基之生長曲線 58 1-2不同種菌培養時間對發酵延滯期之影響 59 2.限制性基質探討 60 2-1以發酵槽批次式(batch)發酵生產G7P 60 2-2碳源測試 63 2-3基礎培養基之碳源濃度 64 2-4氮源測試 66 2-5基礎培養基中氮源濃度 68 2-6金屬離子測試 71 3.以饋料式(fed-batch)發酵培養策略,生產G7P 76 3-1一次性饋料策略 76 3-2二次性饋料策略 79 4.半連續式發酵 83 4-1最適發酵液置換量 83 4-2以半連續式發酵,探討不同批次間之轉換效率 86 第二節 探討自發酵液中回收G7P程序 88 1.以微過濾膜進行發酵液之濃縮 90 2.乙酸乙酯萃取濃縮液中G7P之效率 91 3.G7P之滴定曲線 92 4.利用溶解度差異使G7P-2Na析出 92 5.利用HP-20膨脹床管柱純化 93 5-1濾液對HP-20膨脹床管柱之吸附貫穿曲線 94 5-2以鹼液進行脫附 95 第三節 評估G7P-2Na之降解動力學與物理特性 98 1.HPLC定量分析及分光光度計掃描分析 98 2.以加速試驗評估其安定性 101 2-1比較G7P-2Na與G7P之降解速率 101 3.溶解度試驗 108 4.熔點特性 108 第五章 結論 109 第六章 參考資料 111 | |
dc.language.iso | zh-TW | |
dc.title | 經由微生物轉化程序探討金雀異黃酮磷酸酯之生產與回收 | zh_TW |
dc.title | Studies on the Production and Recovery of Genistein 7-O-phosphate via a Biotransformation Process | en |
dc.type | Thesis | |
dc.date.schoolyear | 105-2 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 李敏雄,賴進此,陳錦樹(CHIN-SHUH CHEN),胡紹揚 | |
dc.subject.keyword | 發酵槽,枯草桿菌,生物轉化,異黃酮磷酸酯衍生物,膨脹床吸附,鈉鹽製備,膜過濾, | zh_TW |
dc.subject.keyword | Bacillus subtilis,isoflavone,biotransformation,bioreactor,expanded bed adsorption,genistein 7-O-phosphate,sodium salts,membrane filtration, | en |
dc.relation.page | 118 | |
dc.identifier.doi | 10.6342/NTU201703264 | |
dc.rights.note | 未授權 | |
dc.date.accepted | 2017-08-15 | |
dc.contributor.author-college | 生物資源暨農學院 | zh_TW |
dc.contributor.author-dept | 農業化學研究所 | zh_TW |
顯示於系所單位: | 農業化學系 |
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