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系統識別號 U0002-1807201308464000
中文論文名稱 Paenibacillus mucilaginosus TKU032生產生物界面活性劑與胞外多醣之條件與特性分析
英文論文名稱 Production and characterization of biosurfactant and extracellular polysaccharide from Paenibacillus mucilaginosus TKU032
校院名稱 淡江大學
系所名稱(中) 化學學系碩士班
系所名稱(英) Department of Chemistry
學年度 101
學期 2
出版年 102
研究生中文姓名 曾詩純
研究生英文姓名 Shih-Chun Tseng
學號 600180052
學位類別 碩士
語文別 中文
口試日期 2013-07-04
論文頁數 127頁
口試委員 指導教授-王三郎
委員-梁慈雯
委員-陳佑汲
中文關鍵字 Paenibacillus mucilaginosus  胞外多醣  生物界面活性劑  烏賊軟骨粉 
英文關鍵字 Paenibacillus mucilaginosus  EPS  Biosurfactant 
學科別分類 學科別自然科學化學
中文摘要 本研究初始是以篩選生物界面活性劑生產菌為主,然而於篩菌過程中,意外發現P. mucilaginosus TKU032這株細菌發酵烏賊軟骨所得上清液,除了生產界面活性劑之外,置於室溫中會逐漸形成膠狀物質,而且顏色會漸漸加深,初步實驗發現此膠狀物為一多醣類物質,故將研究方向分為P. mucilaginosus TKU032這株細菌所產生生物界面活性劑及胞外多醣之研究。
以烏賊軟骨粉( SPP)、蝦頭殼粉( SHP)作為碳/氮源,依不同比例 (0.5%-2%)之添加濃度,於25℃、30℃、37℃培養0-5天,發現以2%烏賊軟骨、37℃培養3天之發酵上清液能得到最低的表面張力,相同條件培養四天可得到最多之胞外多醣,至第五天觀察發酵上清液顏色變黑導致不易測量,因此及選用上述條件作為較佳培養條件培養P. mucilaginosus TKU032。

TKU032發酵所得上清液,其表面張力約為36.3 mN/m經過NaOH調整p H值至12後,置放於4℃、24小時,待其沉澱並以離心方法去除沉澱物,將其上清液冷凍乾燥,再以甲醇溶出可溶物質,減壓濃縮至乾可得棕褐色油狀物,即為純化後之生物界面活性劑(1 g/50 mL)。
TKU032發酵所得上清液經過加熱(121℃、20min)脫色後可得粗胞外多醣( 14.8 g/L),再將粗胞外多醣利用Sevag reagent 去蛋白,得到較純的多醣物質;使用酵素水解與另外取用三種不同酸水解胞外多醣,也可得到水解後結構較小之醣類,利用核磁共振( NMR)光譜及基質輔助雷射脫附游離飛行時間質譜儀(MALDI-TOF)分析水解後之寡糖結構。
此外以烏賊軟骨作為發酵碳/氮源,TKU032在培養第1天所的上清液有較高的DPPH清除能力(80%)及較佳的總酚含量、還原力。
表單編號 :ATRX-Q03-001-FM0030-01
英文摘要 This study is based on an initial option of screening of biosurfactant-producing bacteria,after we choose thebacteria which produce biosurfactant,we found the strain of P. mucilaginosus TKU032 have some gell-like substance in the supernatant,and the color of supernatant will become deeper gradually.Preliminary experiments found that this gum is a polysaccharide.This study will be into two part:P. mucilaginosus TKU032 producing bio-surfactants and study of extracellular polysaccharides.
With squid pen powder (SPP), head of the prawn shell powder (SHP) as a carbon / nitrogen source, according to different proportion (0.5% -2%) of the added concentration, at 25 ℃, 30 ℃, 37 ℃ cultured 0-5 days found to be 2% squid cartilage, 37 ℃ fermentation supernatant of cultured for 3 days to get the lowest surface tension.
The same conditions can be obtained up to four days of training extracellular polysaccharide, to observe the fifth day of fermentation supernatant color black lead difficult to measure, and therefore a better choice of culture conditions as the above conditions are cultured P. mucilaginosus TKU032.
TKU032 fermentation resulting supernatant, the surface tension of about 36.3 mN / m after NaOH to adjust pH values through 12, placed at 4 ℃, 24 hours, wait until the precipitate and the precipitate removed by centrifugation, the supernatant lyophilized and then eluted with methanol-soluble substance was concentrated under reduced pressure to dryness to obtain tan oil which is purified of the bio-surfactant.
TKU032 fermentation supernatant obtained after heating (121 ℃, 20min) after bleaching crude extracellular polysaccharide (14.8g / L), then the use of crude extracellular polysaccharide Sevag reagent to protein, enzymatic hydrolysis, dialysis, it can be oligosaccharide structure into smaller substances; another access three different acid hydrolysis extracellular polysaccharide can be obtained in the structure of the carbohydrate is small, the use of nuclear magnetic resonance (NMR) spectra and matrix-assisted laser desorption ionization time of flight mass Instrument (MALDI-TOF) analysis after hydrolysis of oligosaccharide structures.
In addition to fermentation squid pen as carbon / nitrogen source, in the first day of TKU032, the culture supernatant have higher DPPH scavenging ability (80%), and preferably the total phenolic content, reducing power.

表單編號 :ATRX-Q03-001-FM0030-01
論文目次 目錄
頁次
簽名頁
授權書
中文摘要 I
英文摘要 III
致謝 V
目錄 VI
圖目錄 XII
表目錄 XVII
第一章 緒論 1
1.1生物界面活性劑 4
1.2胞外多醣(exopolysaccharide;EPS) 4
第二章 文獻回顧 7
2.1 類芽孢桿菌 ( Paenibacillus)之簡介 7
2.2 界面活性劑 7
2.3 界面活性劑之種類 8
2.3.1 陰離子界面活性劑 8
2.3.2 陽離子界面活性劑 9
2.3.3 非離子界面活性劑 9
2.3.4 兩性離子界面活性劑 10
2.4 生物界面活性劑 11
2.5生物界面活性劑之種類 12
2.6 胞外多醣 15
2.7 抗氧化 17
2.7.1 活性氧與氧自由基 17
2.7.2 活性氧與氧自由基之傷害 18
2.7.3 抗氧化劑之作用機制 19
2.7.3.1 自由基終止劑 19
2.7.3.2 還原劑或氧清除劑 20
2.8 幾丁質 20
第三章 材料與方法 24
3.1 實驗材料 24
3.1.1 實驗菌株 24
3.1.2 實驗材料 24
3.1.3 實驗儀器 25
3.2實驗方法 27
3.2.1 生產生物界面活性劑之菌株篩選 27
3.2.2 表面張力測量 27
3.2.3 總醣測定 28
3.2.4 還原醣測定 28
3.3 生物界面活性劑較適生產條件探討 30
3.3.1 碳/氮源探討 30
3.3.2 碳/氮源濃度探討 30
3.3.3 培養基體積探討 30
3.3.4 培養溫度探討 31
3.3.5 培基養pH 值探討 31
3.3.6 較適培養時間 31
3.4 生物界面活性劑之特性分析 32
3.4.1熱穩定性 32
3.4.2鹽安定性 32
3.4.3 p H 安定性 33
3.4.4 臨界微胞濃度 33
3.4.5 乳化指數 33
3.5 EPS較適生產條件探討 35
3.5.1 碳/氮源探討 35
3.5.2 碳/氮源濃度探討 35
3.5.3 培養基體積探討 36
3.5.4 培養溫度探討 37
3.5.5 培基養pH 值探討 37
3.5.6 較適培養時間 37
3.6 EPS置備條件探討 38
3.6.1 粗EPS製備 38
3.6.2 EPS去蛋白 38
3.7 EPS水解 41
3.7.1 酵素水解EPS 41
3.7.2 酸水解EPS 41
3.8 抗氧化活性測試 41
3.8.1 DPPH自由基清除能力之測定 41
3.8.2 總酚含量測定 43
3.8.3 還原力測定 43
3.9 NMR測定 44
第四章 結果與討論 45
4.1 生物界面活性劑生產菌之篩選 45
4.2 菌株TKU032之鑑定 47
4.3生物界面活性劑較適生產條件探討 51
4.3.1培養基濃度探討 51
4.3.2 碳/氮源種類探討 52
4.3.3 培養基體積探討 52
4.3.4 培養溫度探討 53
4.3.5 培基養pH 值探討 53
4.3.6 較適培養時間 53
4.3.7較適培養條件探討 54
4.4 生物界面活性劑之純化與結構鑑定 64
4.4.1 生物界面活性劑純化程序 64
4.4.2 生物界面活性劑結構分析 65
4.5生物界面活性劑特性分析 71
4.5.1 熱穩定性分析 71
4.5.2 鹽安定性分析 76
4.5.3 pH 值安定性分析 79
4.5.4 臨界微胞濃度分析 83
4.5.5 乳化能力分析 86
4.7 胞外多醣生產菌之篩選 90
4.8 胞外多醣生產菌較適生產條件探討 90
4.8.1 培養基種類探討 90
4.8.2碳/氮源濃度探討 91
4.8.3 培養基體積探討 92
4.8.4 培養溫度探討 93
4.8.5 培基養pH 值探討 93
4.8.6 較適培養時間 93
4.8.7較適培養條件探討 94
4.9抗氧化分析 103
4.9.1 DPPH自由基清除能力之測定 103
4.9.2 總酚含量測定 104
4.9.3 還原力測定 105
4.9.4綜合結果 106
4.10胞外多醣分析與測定 109
4.10.1 胞外多醣製備方法 109
4.10.2 胞外多醣去蛋白 109
4.10.3 胞外多醣結構分析 111
4.10.4酸水解胞外多醣 115
第五章 結論 118
第六章 參考文獻 119

圖目錄
圖3.1 乳化指數計算之示意圖 34
圖3.2 EPS之去蛋白流程圖 40
圖4.1 P. mucilaginosus TKU032進行NCBI/BLAST比對結果 46
圖4.2 P. mucilaginosus TKU032 16S rDNA部分鹼基序列 48
圖4.3 發酵液之酸鹼值變化 56
圖4.4 不同碳/氮源類對TKU032產生界面活性劑上清液表面張力之影響 56
圖4.5 不同SPP濃度 於50mL錐型瓶中對TKU032生產生物界面活性劑之表面張力影響 57
圖4.6 不同SPP濃度 於100mL錐型瓶中對TKU032生產生物界面活性劑之表面張力影響 57
圖4.7 不同培養體積對於TKU032生產生物界面活性劑之表面張力探討 58
圖4.8 通氣瓶與錐形瓶(通氣量)影響TKU032生產生物界面活性劑之表面張力探討 58
圖4.9 不同培養溫度對於TKU032生產生物界面活性劑之表面張力探討 59
圖4.10培養基經過滅菌前後之酸鹼值變化 59
圖4.11 滅菌後之酸鹼值影響TKU032生產生物界面活性劑之表面張力探討 60
圖4.12 不同培養天數影響TKU032生產生物界面活性劑之表面張力 60
圖4.13 TKU032培養不同天數影響培養基之酸鹼值變化 62
圖4.14 培養天數影響TKU032細菌生產量 63
圖4.15 純化後之TKU032生產生物界面活性劑MALD-TOF MS光譜圖 66
圖4.16 純化後之TKU032生產生物界面活性劑1H NMR 光譜圖 67
圖4.17 純化後之TKU032生產生物界面活性劑13C NMR光譜圖 68
圖4.18 純化後之TKU032生產生物界面活性劑LC-MS光譜圖 69
圖 4.19 TKU032所生產生物界面活性劑之純化流程 70
圖4.20 高溫高壓對生物界面活性劑表面張力之影響 74
圖4.21高溫高壓對生物界面活性劑之乳化能力影響 74
圖4.22 市售界面活性劑與TKU032生產之生物界面活性劑經過高溫高壓前後之表面張力比較 75
圖4.23 市售界面活性劑與TKU032生產之生物界面活性劑經過高溫高壓前後之乳化能力比較 75
圖4.24 鹽度對不同界面活性劑與TKU032生產之生物界面活性劑之表面張力影響 77
圖4.25 不同種類之界面活性劑在不同鹽度下之表面張力 77
圖4.26 TKU032生物界面活性劑於不同酸鹼值環境之表面張力 82
圖4.27 TKU032生產生物界面活性劑之臨界微胞濃度 84
圖4.28 SDS界面活性劑之臨界微胞濃度 84
圖4.29 SDS於不同濃度之乳化能力 87
圖4.30 CTAB於不同濃度之乳化能力 87
圖4.31 TKU032生產生物界面活性劑於不同濃度之乳化能力(缺圖) 88
圖 4.32 TKU032生產生物界面活性劑樣品濃度為5mg/L的乳化能力情形 88
圖4.33 不同培養天數於50毫升培養基對TKU032細菌生長之影響 96
圖4.34 不同培養天數於100毫升培養基對TKU032細菌生長之影 96
圖 4.35 不同培養天數於50毫升培養基對TKU032生長醣類之影響 97
圖 4.36 不同培養天數於100毫升培養基對TKU032生長醣類之影響 97
圖 4.37 不同碳源影響TKU032生產EPS 98
圖4.38 培養體積影響TKU032之EPS生產 98
圖4.39 培養溫度影響TKU032生產EPS 99
圖4.40 烏賊軟骨培養基於滅菌前後酸鹼值變化 99
圖4.41 滅菌前之酸鹼值影響EPS生產 100
圖4.42 TKU032 EPS最適培養時間 100
圖4.43 TKU032上清液對 DPPH自由基清除能力 104
圖4.44 發酵上清液總酚含量之變化與DPPH自由基清除能力關係 105
圖4.45發酵上清液還原力與DPPH自由基清除能力關係 106
圖4.46 胞外多醣分析圖 110
圖4.47 α-澱粉酶水解後之MALDI光譜圖 112
圖4.48發酵液上清液使用乙醇沉澱之多醣進行MALDI光譜 113
圖4.49發酵液上清液使用乙醇沉澱之多醣進行MALDI光譜 114
圖4.50 TKU032胞外多醣之水解能力測量 115
圖4.51酸水解後懸浮液之MALDI光譜(HCl) 116
圖4.52酸水解後懸浮液之MALDI光譜(Acetic Acid) 117

表目錄
表1.1 生物界面活性劑之化學組成及微生物來源分類 3
表2.1 界面活性劑之種類 10
表2.2 微生物及其生產的界面活性劑 14
表2.3 胞外多醣生產菌之生長環境 22
表2.4幾丁質經X-ray繞射雙股螺旋及對稱軸分子的排列方向 23
表4.1 P. mucilaginosus TKU032 16S rDNA部分鹼基序列比對結果 49
表4.2 P. mucilaginosus TKU032生物界面活性劑之較適生產條件 61
表4.3 TKU032與TKU029兩菌生產之生物界面活性劑比較 62
表4.4 生物界面活性劑之化學組成及微生物來源分類 72
表4.5 不同界面活性劑之耐鹽度比較 78
表4.6 生物界面活性劑p H穩定範圍比較 81
表4.7生物界面活性劑之臨界微胞濃度比較 85
表4.8 不同界面活性劑之乳化能力比較 89
表4.9 TKU032胞外多醣之較適生產條件 95
表4.10 不同微生物生產之胞外多醣特徵比較 101
表4.11 TKU032 於不同天數生產物之比較 107
表4.12 微生物來源之DPPH自由基清除能力比較 108
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