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系統識別號 U0002-2107201013182500
中文論文名稱 Paenibacillus sp. TKU023生產胞外多醣之條件及特性分析
英文論文名稱 Production and characterization of exopolysaccharides from Paenibacillus sp. TKU023.
校院名稱 淡江大學
系所名稱(中) 生命科學研究所碩士班
系所名稱(英) Graduate Institute of Life Sciences
學年度 98
學期 2
出版年 99
研究生中文姓名 黃梓晃
研究生英文姓名 Tzu-Huang Huang
學號 697180387
學位類別 碩士
語文別 中文
口試日期 2010-07-09
論文頁數 70頁
口試委員 指導教授-王三郎
委員-陳佑汲
委員-梁慈雯
委員-王三郎
中文關鍵字 Paenibacillus sp.  胞外多醣  烏賊軟骨粉 
英文關鍵字 Paenibacillus sp.  exopolysaccharide  squid pen powder 
學科別分類 學科別醫學與生命科學生物學
中文摘要 TKU023係以烏賊軟骨粉為唯一碳/氮源,篩選自台灣北部土壤之一株胞外多醣及生物界面活性劑生產菌,經鑑定為Paenibacillus sp.。TKU023生產胞外多醣之較適培養條件為含有1% 烏賊軟骨、0.1% K2HPO4及0.05% MgSO4.7H2O之50 mL液態培養基(pH 7.32)於37℃振盪培養5天。
TKU023發酵所得上清液經加熱(121℃、20 min)、脫色後可得粗胞外多醣(1.7631 g),再將粗胞外多醣利用Sevag reagent去蛋白、酵素水解、透析後,可將胞外多醣分解成寡醣類物質,利用核磁共振(NMR)光譜分析水解後之寡醣結構,發現其1H、13C譜與麥芽糖之1H、13C譜相似,可推測胞外多醣經酵素水解後可得部分麥芽糖之單體。
此外以烏賊軟骨粉做為發酵碳/氮源 ,利用TKU023在不同培養體積下培養1∼6天,發現培養體積50 mL在培養第5天所得發酵上清液具有較高的DPPH清除能力(77%)及較佳的總酚含量、還原力、螯合亞鐵離子能力。
英文摘要 In this study, an exopolysaccharide (EPS) and biosurfactant producing strain, was isolated from the soil in the northern Taiwan by using squid pen powder (SPP) as the sole carbon/nitrogen source and identified as Paenibacillus sp.. The optimal conditions for the production of the EPS were cells used to inoculate into 50 mL medium containing 0.5% squid pen powder (SPP), 0.1% K2HPO4 and 0.05% MgSO4.7H2O (pH 7.32)followed by culture at 37℃ for 5 days.
The crude EPS (1.7631 g) was obtained from the culture supernatant by autoclave (121℃ , 20 min) and bleaching and then the crude EPS followed by deproteinization with Sevag reagent. The deproteinized EPS was hydrolyzed to oligosaccharides by enzyme and dialyzed with water. The oligosaccharides were analyzed by using nuclear magnetic resonance (NMR). The 1H and 13C NMR spectrometric results suggest that EPS hydrolyzates contained maltose as oligomeric building subunit.
Additionally, Paenibacillus sp. TKU023 was cultivated with various culture volumes for 1~6 days by using squid pen powder as the sole carbon/nitrogen sources. The results indicated that the maximum DPPH free radical scavenging ability and better total phenolic contents, reducing activity, and Fe2+ chelating ability was found at the 5th day by using the medium volume of 50 mL.
論文目次 目錄

簽名頁
授權書
中文摘要...................................................I
英文摘要..................................................II
誌謝......................................................IV
目錄.......................................................V
圖目錄....................................................XI
表目錄..................................................XIII

第一章 緒論................................................1
第二章 文獻回顧............................................2
2.1 多黏芽胞桿菌Paenibacillus之簡介.....................2
2.2 生物界面活性劑......................................2
2.3 胞外多醣............................................3
2.4 抗氧化..............................................6
2.4.1 活性氧與氧自由基.................................6
2.4.2 活性氧與氧自由基傷害.............................6
2.4.3 抗氧化劑之作用機制...............................7
2.4.3.1 自由基終止劑...............................8
2.4.3.2 還原劑或氧清除劑...........................8
2.4.3.3 金屬螯合劑.................................8
2.5 幾丁質..............................................9
第三章 材料與方法.........................................10
3.1 實驗材料...........................................10
3.1.1 實驗菌株........................................10
3.1.2 實驗材料........................................10
3.1.3 實驗儀器........................................11
3.2 實驗方法...........................................12
3.2.1 菌株篩選........................................12
3.2.2 液滴擴散法測定..................................12
3.2.3 表面張力測量選..................................13
3.2.4 總醣測定........................................13
3.3 EPS較適生產條件探討................................14
3.3.1 碳/氮源.........................................14
3.3.2 碳/氮源濃度.....................................14
3.3.3 培養體積(通氣量)..............................14
3.3.4 培養溫度........................................15
3.3.5 培養基之酸鹼值..................................15
3.3.6 較適培養時間....................................15
3.4 EPS較適生產條件探討................................15
3.4.1 粗EPS製備.......................................15
3.4.2 EPS去蛋白.......................................16
3.5 EPS水解............................................16
3.6 薄層色層層析.......................................17
3.7 抗氧化活性試驗.....................................17
3.7.1 DPPH自由基清除能力之測定........................17
3.7.2 總酚含量測定....................................18
3.7.3 還原力測定......................................19
3.7.3 螯合亞鐵離子能力測定............................19
3.8 NMR測定............................................20
3.9 胞外多醣促進乳酸菌生長測定定.......................20
3.9.1 不同階段EPS之探討 ..............................20
3.9.2 不同濃度EPS之探討...............................21
第四章 結果與討論.........................................22
4.1 生物界面活性劑/胞外多醣生產菌之篩選................22
4.2 菌株特性...........................................23
4.3 培養條件探討.......................................27
4.3.1 碳/氮源之選擇...................................27
4.3.2 碳/氮源濃度之影響...............................27
4.3.3 培養體積(通氣量)..............................28
4.3.4 培養基之溫度探討................................28
4.3.5 培養基之酸鹼值探討..............................28
4.3.6 較適培養時間....................................29
4.3.7 EPS生產較適條件.................................29
4.4 抗氧化分析.........................................39
4.4.1 DPPH自由基清除能力之測定........................39
4.4.2 總酚含量測定....................................40
4.4.3 還原力測定......................................40
4.4.4 螯合亞鐵離子能力................................41
4.4.5 綜合結果........................................41
4.5 胞外多醣分析與定性.................................42
4.5.1 胞外多醣製備....................................42
4.5.2 胞外多醣去蛋白..................................42
4.5.3 胞外多醣水解....................................43
4.5.4 薄層色層層析....................................43
4.5.5 NMR分析..........................................44
4.6 EPS對於L. paracasei TKU010生長之測定...............44
4.6.1 不同階段之EPS對於L. paracasei TKU010生長之探討..44
4.6.2 不同濃度之去蛋白EPS對於L. paracasei TKU010生長
之探討...........................................44
第五章 結論...............................................62
第六章 參考文獻...........................................63

圖目錄

圖4.1 Paenibacillus sp. TKU023 16S rDNA部分鹼基序列.......24
圖4.2 Paenibacillus sp. TKU023進行NCBI/BLAST比對結果......25
圖4.3 不同碳/氮源對於Paenibacillus sp. TKU023產生胞外多醣的 影響......................................................31
圖4.4 不同SPP濃度對Paenibacillus sp. TKU023產生胞外多醣之影響........................................................32

圖4.5 不同培養體積對於Paenibacillus sp. TKU023產生胞外多醣之
影響................................................33
圖4.6 不同培養溫度對於Paenibacillus sp. TKU023產生胞外多醣之
影響................................................34
圖4.7 不同培養基pH值對於Paenibacillus sp. TKU023產生胞外多醣
之影響..............................................35
圖4.8 Paenibacillus sp. TKU023培養於烏賊軟骨粉培養基所產生胞
外多醣變化情形......................................36
圖4.9 不同培養體積及發酵時間對DPPH清除率的影響............46
圖4.10 總酚含量與DPPH自由基清除能力關係圖.................48
圖4.11 還原力與DPPH自由基清除能力關係圖...................49
圖4.12 亞鐵離子螯合能力與DPPH自由基清除能力關係圖.........50
圖4.13 還原力與總酚關係圖.................................51
圖4.14 亞鐵離子螯合能力與總酚關係圖.......................52
圖4.15 Paenibacillus sp. TKU023所生產胞外多醣製備流程圖...53
圖4.16 胞外多醣分析圖.....................................54
圖4.17 薄層色層分析Paenibacillus sp. TKU023所生產胞外多醣.55
圖4.18 胞外多醣之1H-NMR圖譜...............................56
圖4.19 胞外多醣之13C-NMR圖譜..............................57
圖4.20 麥芽糖之1H-NMR圖譜.................................58
圖4.21 麥芽糖之13C-NMR圖譜................................59
圖4.22 不同胞外多醣對於L. paracasei TKU010生長影響........60
圖4.23 不同濃度之去蛋白EPS對於L. paracasei TKU010生長影響.61

表目錄

表2.1 胞外多醣生產菌之生長環境.............................5
表4.1 Paenibacillus sp. TKU023 16S rDNA部分核苷酸序列比對結果........................................................26
表4.2 TKU023胞外多醣之較適生產條件........................37
表4.3 微生物來源之胞外多醣特性比較........................38
表4.4 微生物來源之DPPH自由基清除能力比較..................47
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