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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 蘇南維 | |
dc.contributor.author | Chun-Jen Fang | en |
dc.contributor.author | 方俊仁 | zh_TW |
dc.date.accessioned | 2021-06-14T16:55:39Z | - |
dc.date.available | 2010-08-06 | |
dc.date.copyright | 2008-08-06 | |
dc.date.issued | 2008 | |
dc.date.submitted | 2008-07-30 | |
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E. and B. V. Milborrow. Retention of the 4-Pro-R hydrogen-atom of mevalonate at C-2,2' of bacterioruberin in Halobacterium halobium. Biochemical Journal 1980, 187: 261-264. Szabo, I., E. Bergantino and G. M. Giacometti. Light and oxygenic photosynthesis: energy dissipation as a protection mechanism against photo-oxidation. Embo Reports 2005, 6: 629-634. 石谷孝佑,食品の品質保質技術開發動向,食品と開發,1993,28: 16-21。 行政院衛生署,食品添加物使用範圍及限量暨規格標準,2005。 吳淑靜,柯文慶及賴滋漢,食品添加物,華香園出版社,1997。 蘇遠志,紅麴製品介紹及國內研究現況,機能性發酵製品研討會,中國農業化學會,2001。 | |
dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/40678 | - |
dc.description.abstract | Haloferax spp.為一極端嗜鹽古菌,必須生長於含有1.5-4.5 M NaCl之環境中。過去之研究顯示該菌株會產生一類C50類胡蘿蔔素,主要為菌紅素(bacterioruberin),其作用為保護細胞免於強烈光照的致命傷害。本研究即利用Haloferax spp.進行兩階段生產菌紅素相關之C50類胡蔔素,並探討最適之生產條件。第一階段使用ATCC 1176培養基作為基礎培養基進行菌體的生產,第二階段則是利用第一階段生成之菌體,以靜止細胞培養方式進行C50類胡蔔素之生產。藉由改變第二階段培養之NaCl濃度、鎂離子(Mg2+)添加量、碳源種類、通氣量及培養時間等,探討對C50類胡蘿蔔素產量之影響。結果顯示第一階段使用含1%葡萄糖之ATCC 1176培養基,以37℃、150 rpm、1vvm培養至對數期中期時將培養液轉出離心,得到之菌體利用含5% NaCl、0.1% CH3COONa並添加8% MgSO4.7H2O之全合成培養基,以37℃、120 rpm震盪培養24 hr可得到合理之最高C50類胡蘿蔔素產量(0.604±0.005 Abs/mL broth)。
色素粗萃物之組成分析與鑑定主要利用TLC及UV-VIS光譜分析。結果顯示色素粗萃物中主要可能存在有3種C50類胡蘿蔔素(Spot 1-3)及1種C45類胡蘿蔔素 (Spot 4)。Spot 1為菌紅素約佔了紅色色素之70%,Spot 2為單縮水菌紅素,約佔紅色色素之20%,Spot 3為雙縮水菌紅素,約佔紅色色素之15%。Spot 4為C45類胡蘿蔔(2-isopentenyl-3,4-dehydrorhodopin)。 色素粗萃物以silica gel進行管柱區分,收集40% EtOAc frc.、EtOAc frc.及MeOH frc.三個區分,經TLC分析比對可知40% EtOAc frc.之主要成分對應為Spot 3;EtOAc frc.對應為Spot 2;而MeOH frc.對應為Spot 1。 色素粗萃物及經管柱區分得到之三個區分物分別進行抗氧化能力測定之結果如下:還原能力由強至弱依序為:Crude extract≒MeOH frc.>40% EtOAc frc.≒EtOAc frc.≒BHA>β-carotene>α-tocopherol;DPPH自由基清除能力由強至弱依序為:Crude extract≒MeOH frc.≒BHA>β-carotene≒40% EtOAc frc.>EtOAc frc.>α-tocopherol。 | zh_TW |
dc.description.abstract | Haloferax spp., an extremely halophilic archaea bacterium, must grow in the environment containing 1.5-4.5 M NaCl. It can produce a group of 50-carbon (C50) carotenoids, mainly bacterioruberin. According to the previous reports, the physiological role of bacterioruberin, the major pigment in halobacteria, was suggested to protect the cells against severely solar irradiation. The aim of this study was to investigate the cultural conditions for the production of C50-carotenoids by Haloferax spp. using a two-stage strategy. At the 1st stage culture, we used ATCC 1176 medium as the basal medium and studied on increasing the production of Haloferax spp. biomass. The 2nd stage culture was simulated as resting cells culture of the 1st stage harvested cells to conduct the bioconversion of C50-carotenoids and studied on the influence of nutrient factors and cultural conditions for C50-carotenoids production at the stage. The results indicated that the optimum conditions for C50-carotenoids production by Haloferax spp. were to use ATCC 1176 medium contained 1% glucose as the initial medium and incubated the cells to the mid-log phase of growth under 37℃, 150 rpm of agitation and 1 vvm of aeration at the 1st stage culture. Successively, the 2nd stage was proceed to the bioconversion of C50-carotenoids by means of incubating the 1st stage harvested cells in the synthetic salts medium (briefly contained at 5% NaCl, 0.1% CH3COONa and 8% MgSO4.7H2O) under 37℃,120 rpm of agitation for 24 hr. The yield of C50-carotenoids was reached 0.604±0.005 Abs/mL broth.
After the chromatographic fractionations, the main components of crude pigment extracts were analyzed and identified according to the TLC profiles and the corresponding UV-VIS spectra. The results revealed that crude extracts were mainly composed of three C50-carotenoids (Spot 1-3) and a C45-carotenoid (Spot 4). Spot 1 was the major component of crude extracts (70% around of total intensity) and was identified as the compound of bacterioruberin. Spot 2 and Spot 3 were respectively conducted to monoanhydrobacterioruberin and bisanhydrobacterioruberin. Additionally, Spot 4 was suggested as the compound of 2-isopentenyl-3, 4-dehydrorhodopin (a C45-carotenoid) according to the information of UV-VIS spectrum. Furthermore, Fraction containing Spot 1, Spot 2 or Spot 3 was successfully separated from the crude extracts by silica gel chromatography and was respectively corresponded with 40% EtOAc frc. (bisanhydrobacterioruberin major), EtOAc frc. (monoanhydrobacterioruberin major) and MeOH frc. (bacterioruberin major). Antioxidative activities of the crude extracts and the three fractions were determined using the methods of reducing power analysis and DPPH radical scavenging ability. The results of reducing power analysis were shown in the order of crude extract≒MeOH frc.>40% EtOAc frc.≒EtOAc frc.≒BHA>β-carotene>α-tocopherol. And the results of DPPH radical scavenging ability were shown in the order of crude extract≒MeOH frc.≒BHA>β-carotene≒40% EtOAc frc.>EtOAc frc.>α-tocopherol. | en |
dc.description.provenance | Made available in DSpace on 2021-06-14T16:55:39Z (GMT). No. of bitstreams: 1 ntu-97-R95623007-1.pdf: 1596107 bytes, checksum: d514720a77fcc256a97bf585fe5a3345 (MD5) Previous issue date: 2008 | en |
dc.description.tableofcontents | 口試委員會審定書 i
誌謝 ii 中文摘要 iv 英文摘要 v 第一章、研究動機 1 第二章、文獻整理 3 第一節、食用色素 3 第二節、類胡蘿蔔素 5 壹、存在與分佈 5 貳、化學結構 7 參、物理、化學特性 10 肆、生理機能與保健功效 11 一、提供顏色來源 11 二、光合作用與光保護作用 12 三、營養與保健機能 13 第三節、C45及C50類胡蘿蔔素 17 壹、存在與分佈 17 貳、化學結構 18 參、生化合成 21 肆、生理機能 23 第四節、菌紅素 24 壹、存在與分佈 24 貳、化學結構 24 參、生物活性 26 肆、分析與鑑定 26 一、光譜法 26 二、薄層層析法 31 三、高效能液相層析法 32 第五節、菌株介紹 37 壹、分類學地位 37 貳、富鹽菌屬之命名由來及特性 39 參、Haloferax spp.之一般特性介紹 41 一、大小、型態 41 二、生長環境 41 三、營養條件 41 四、生化學特性 41 第三章、材料與方法 42 第一節、實驗架構 42 壹、C50類胡蘿蔔素生產條件探討 42 貳、色素之分離與純化 43 參、色素之應用性研究 44 第二節、實驗材料 45 壹、菌株來源 45 貳、化學試藥 45 第三節、儀器設備 46 第四節、實驗方法 47 壹、C50類胡蘿蔔素生產條件探討 47 一、兩階段培養策略 47 二、基礎培養基組成 47 三、菌種保存 48 四、菌種活化 48 五、生長曲線測定 48 六、第一階段培養條件之探討 48 七、第一階段發酵槽放大培養 49 八、第二階段培養條件之探討 49 九、色素之萃取與定量 50 貳、色素之分離與純化 51 一、溶劑分配與再萃取 51 二、管柱區分 51 三、色素組成分析與鑑定 52 參、色素之應用性研究 53 一、β-Carotene標準品之製備 53 二、色素樣品之定量 53 三、還原能力測定 54 四、DPPH自由基清除能力之測定 55 第四章、結果與討論 56 第一節、C50類胡蘿蔔素生產條件探討 56 壹、第一階段培養條件探討 56 一、葡萄糖濃度對菌體量之影響 56 二、發酵槽放大培養 58 貳、第二階段培養條件探討 61 一、氯化鈉與鎂離子濃度對色素產量之影響 61 二、碳源種類對色素產量之影響 65 三、培養時間對色素產量之影響 66 四、通氣量對色素產量之影響 67 第二節、色素之分離與純化 71 壹、溶劑分配與再萃取 71 貳、色素組成分析與鑑定 72 參、管柱區分 78 第三節、色素之應用性研究 86 壹、還原能力 86 貳、DPPH自由基清除能力 87 第五章、結論 94 第六章、參考文獻 95 附錄一、Haloferax spp.菌體外觀型態 98 附錄二、TLC-IIA定量方法 100 附錄三、NaCl與Mg2+濃度對色素產量之影響 101 圖目錄 圖2.1 Fucoxanthin化學結構 5 圖2.2 Astaxanthin化學結構 6 圖2.3 Lycopene化學結構 8 圖2.4 β-carotene化學結構 8 圖2.5 Okenone化學結構 8 圖2.6 Neoxanthin化學結構 9 圖2.7 Pyrrhoxanthin化學結構 9 圖2.8 Azafrin化學結構 9 圖2.9 Bacterioruberin diglucoside化學結構 10 圖2.10葉綠素複合體吸收陽光後的變化 14 圖2.11三重態氧之分子軌域電子組態 15 圖2.12單重態氧之分子軌域電子組態 15 圖2.13葉黃素循環 16 圖2.14非環狀C40H56結構 17 圖2.15 Decaprenoxanthin化學結構 18 圖2.16組成非環狀C45及C50類胡蘿蔔素的主要末端基團及多烯長鏈 19 圖2.17組成環狀C45及C50類胡蘿蔔素的主要末端基團及多烯長鏈 20 圖2.18非環狀C50-類胡蘿蔔素推測之生化合成途徑 22 圖2.19類胡蘿蔔素強化細胞膜結構之示意圖 23 圖2.20菌紅素及其相關衍生物之結構式 25 圖2.21菌紅素(A)及雙縮水菌紅素(B)之UV-VIS光譜圖 27 圖2.22菌紅素與單縮水菌紅素質譜分析之碎裂示意圖 30 圖2.23來自H.salinarum之色素萃取液利用HPLC分析之層析圖譜 33 圖2.24來自鹽田微生物之色素萃取液利用HPLC分析之層析圖譜 35 圖2.25富鹽菌屬相關極性脂質組成及其結構式 40 圖3.1 C50類胡蘿蔔素生產流程圖 42 圖3.2 C50類胡蘿蔔素分離與純化流程圖 43 圖3.3色素抗氧化試驗流程圖 44 圖4.1葡萄糖濃度對Haloferax spp.生長之影響 57 圖4.2第一階段培養中Haloferax spp.之生長曲線 59 圖4.3第一階段培養菌液濁度對數值與時間之關係 60 圖4.4第二階段培養基中NaCl濃度對Haloferax spp.生產C50類胡蘿蔔素之影響 63 圖4.5第二階段培養基中Mg2+濃度對Haloferax spp.生產C50類胡蘿蔔素之影響 64 圖4.6第二階段培養基中NaCl濃度對Haloferax spp.生產C50類胡蘿蔔素之影響 65 圖4.7第二階段培養基中碳源種類及添加量對Haloferax spp.生產C50類胡蘿蔔素之影響 68 圖4.8兩階段培養時間對Haloferax spp.生產C50-carotenoids之影響 69 圖4.9兩階段培養時間對Haloferax spp.生產C50-carotenoids效率之影響 70 圖4.10通氣量對Haloferax spp.生產C50-carotenoids之影響 71 圖4.11色素粗萃物之TLC展開結果 74 圖4.12不同培養條件所得到之色素粗萃液之TLC展開結果 75 圖4.13 不同培養條件所得到之色素粗萃液之TLC展開結果 76 圖4.14 利用silica gel進行色素粗萃物之管柱區分 81 圖4.15管柱區分物之TLC展開結果 82 圖4.16 不同色素樣品復溶於乙醇中UV-VIS光譜 84 圖4.17重疊不同色素樣品復溶於乙醇中UV-VIS光譜 85 圖4.18 色素粗萃物之還原能力 88 圖4.19 三個色素區分物之還原能力 89 圖4.20 色素粗萃物之DPPH自由基清除能力 91 圖4.21 三個色素區分物之DPPH自由基清除能力 92 圖A.1 Haloferax spp.之外觀型態 98 圖A.2 Haloferax spp.以共軛焦顯微鏡拍攝之照片 99 圖A.3 TLC-IIA定量色素樣品之線性評估 100 圖A.4第二階段培養基中NaCl與Mg2+含量對Haloferax spp.生產C50類胡蘿蔔素之影響 101 圖A.5第二階段培養基中NaCl與Mg2+含量對Haloferax spp.生產C50類胡蘿蔔素之影響 102 圖A.6第二階段培養基中NaCl與Mg2+含量對Haloferax spp.生產C50類胡蘿蔔素之影響 103 表目錄 表2.1 一般常見之天然食用色素 4 表2.2 菌紅素及其相關衍生物之吸光係數 28 表2.3 富鹽菌屬中各菌種之生化特性差異 38 表3.1 色素樣品之定量參數 54 表4.1 不同培養條件得到之色素粗萃液中各成分相對組成 77 表4.2 不同色素樣品之UV-VIS光譜吸收波峰 83 表4.3 測試樣品還原能力之EC50值 90 表4.4 測試樣品清除DPPH自由基之EC50值 93 | |
dc.language.iso | zh-TW | |
dc.title | 由Haloferax spp.生產C50類胡蘿蔔素之研究 | zh_TW |
dc.title | Studies on the production of C50 carotenoids by Haloferax spp. | en |
dc.type | Thesis | |
dc.date.schoolyear | 96-2 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 林銘澤,古國隆,王美苓,李敏雄 | |
dc.subject.keyword | 菌紅素,富鹽菌屬,生物轉換, | zh_TW |
dc.subject.keyword | bacterioruberin,Haloferax,bioconversion, | en |
dc.relation.page | 103 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2008-07-30 | |
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 |
文件中的檔案:
檔案 | 大小 | 格式 | |
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ntu-97-1.pdf 目前未授權公開取用 | 1.56 MB | Adobe PDF |
系統中的文件,除了特別指名其著作權條款之外,均受到著作權保護,並且保留所有的權利。