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研究生:廖國森
研究生(外文):Liao Guo Sen
論文名稱:納豆菌生產果糖聚合物之研究
論文名稱(外文):Production of Levan by Bacillus subtilis Natto
指導教授:吳建一施英隆施英隆引用關係
指導教授(外文):Jane Yii WuIng Lung Shih
學位類別:碩士
校院名稱:大葉大學
系所名稱:生物產業科技學系
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
論文出版年:2007
畢業學年度:95
語文別:中文
論文頁數:182
中文關鍵詞:Bacillus subtilis natto發酵生產聚果醣levan固定化
外文關鍵詞:Bacillus subtilis nattofermentationfructanlevanimmobilization
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Levan為fructofuranosidic殘基以β-(2→6) 形式鍵結之果糖聚合物,可以利用微生物來生產,且能夠應用在化妝品、藥品、藥物釋放、農業與食品工業上。雖然已經有許多關於levan之研究被發表,但其所提到關於levan之產量不是過低,就是生產過程中會有其他產物的產生,而造成純化上的困難。因此,使得levan之價格較高,進而使得levan的應用性受到限制。為了更進一步了解利用Bacillus subtilis natto來大量生產levan之可能性,本研究探討各項影響此B. subtilis natto生產levan時的環境因子,並進行產物之純化與鑑定其結構。B. subtilis natto於含有20% (w/v) sucrose之培養基中,經過24 h培養後,可生產40-50 g/L的levan聚果醣。經膠體過濾層析法 (Gel permeation chromatography, GPC) 分析levan產物時,可發現本實驗之產物是具有兩種不同之分子量 (2 × 107 and 9 × 103 Da),且容易利用乙醇沈澱法來進行純化,並且將此產物利用FTIR (Fourier transform infrared) 與NMR (Nuclear magnetic reasonance) 進行特性分析後,已證實多醣聚合物即為levan。
利用無菌水清洗後之B. subtilis natto菌株以各種氮源基質之條件來進行levan的發酵生產,氮源包括yeast extract、peptone、urea、NH4Cl、NaNO3與玉米漬液。結果顯示B. subtilis natto是能夠代謝無機氮源 (例如,NH4Cl與NaNO3) 來進行levan生產,但是卻無法有效利用有機氮源(尤其是yeast extract與玉米漬液)。另外,蔗糖濃度也是影響levan產量與levan生產速率 (VL) 的原因之一。實驗結果顯示levan之合成是需以蔗糖作為碳源,並且將B. subtilis natto菌株培養於含有250 g/L蔗糖基質的培養基中,是能得到最大的levan產量 (71 g/L) 與levan生產速率。並且探討影響levan生產之各項環境因子 (例如,初始pH與培養溫度),結果顯示pH 7與溫度為37℃為最適合levan的生產條件。另外,本研究亦探討利用PVA固定化菌體顆粒來進行生產levan的可行性,並發現此固定化菌體顆粒確實是能用來大量生產levan 聚果醣。
Levan is a polymer of fructose linked by β-(2→6) fructofuranosidic bonds present in many plants and microbial products. Levan offers a variety of industrial applications in the fields of cosmetics, foods and pharmaceuticals. Although many investigations on levan formation have been reported, they suffered the disadvantages of low yields and the contamination of impure products. Thus, levan has great potential if it can be produced less expensively. To further investigate the possibility of the use of Bacillus subtilis natto for the efficient production of the levan product, we studied and described in this study the factors affecting the production of levan by this Bacillus sp.; the purification and characterization of the products were also investingated. After cultivation for 24 h, 40-50 g/L of levan was produced in medium containing 20% (w/v) sucrose by B. subtilis natto. The product consisted of two fractions with different molecular masses (2 × 107 and 9 × 103 Da), and the high molecular mass product (2 × 107 Da) predominated during fermentation process (~ 24 h), which were easily separated by fractionation using an ethanol gradient. The products were well characterized by Gel permeation chromatography (GPC), Fourier transform infrared (FTIR), and Nuclear magnetic reasonance (NMR).
B. subtilis natto, which was washed with sterile water, was used to produce levan from a variety of nitrogen substrates, including yeast extract, peptone, urea, NH4Cl, NaNO3 and sweet corn extract. The B. subtilis natto strain was able to assimilate the two inorganic nitrogen substrates examined (NH4Cl and NaNO3), whereas it grew less efficiently in organic nitrogen substrates (esp., yeast extract and sweet corn extract). The levan concentration and production rate (VL) was also influenced by the sucrose concentration. The sucrose was utilized preferentially for levan synthesis. Maximum levan concentration (71 g/L) and VL (2.383 g/L/h) were attained at 25% (w/v) sucrose concentration. Levan production from B. subtilis natto was affected by various environmental factors, such as pH and temperature; initial pH 7 and 37℃ were favorable for levan production. Additionally, levan production by PVA immobilized-cell beads was tested. PVA with immobilized bacteria can be used as the general producer of levan.
1. 前言 1
2. 文獻回顧 4
2.1 Levan之介紹 4
2.1.1 化學結構與特性 6
2.1.2 生理功能 9
2.2 Levan之合成機制 10
2.3 Levan之生化降解 14
2.4 利用微生物發酵生產levan 16
2.4.1 生產levan微生物種類 16
2.4.2 碳源對levan產量的影響 22
2.4.3 環境因子對levan產量之影響 24
2.4.4 葡萄糖抑制levan含成之作用 28
2.5 利用純酵素反應 (不含菌體) 合成levan 30
2.5.1 糖類基質濃度對純酵素合成levan之影響 32
2.5.2 額外添加物對純酵素合成levan之影響 34
2.5.3 環境因子對純酵素合成levan之影響 36
2.6 固定化技術生產levan 38
2.7 Levan之應用 41
2.7.1 藥物與製藥方面 41
2.7.2 農業 44
2.7.3 食品工業 44
3. 材料與方法 51
3.1 實驗材料 51
3.1.1 藥品 51
3.1.2 儀器設備 52
3.2 菌株培養 53
3.2.1 菌株來源 53
3.2.2 菌株活化 54
3.2.3 Levan生產培養 54
3.3 去除初始氮含量之方法 55
3.3.1 前培養殘留氮含量之去除 55
3.3.2 不同前培養方式之製備 56
3.4 PVA顆粒製備的方法 56
3.5 粗酵素液的製備與分析 57
3.6 分析方法 58
3.6.1 醣類分析 58
3.6.2 黏度 61
3.6.3 Levan之分析 61
3.7 Levan之純化 64
4. 結果與討論 65
4.1 不同Bacillus subtilis菌株之比較 65
4.2 培養時間對各菌株之levan分子量的影響 69
4.3 培養基組成 73
4.3.1 生成levan過程中氮源之需求 73
4.3.2 不同氮源的影響 79
4.3.3 不同NH4Cl濃度的影響 85
4.3.4 不同前培養之培養基組成及方式的影響 94
4.3.5 不同蔗糖濃度之影響 101
4.4 環境因子的探討 111
4.4.1 不同初始pH值之影響 111
4.4.2 不同培養溫度之影響 121
4.4.3 不同離子強度之影響 131
4.5 發酵液中粗levansucrase酵素之活性探討 144
4.6 固定化B. subtilis natto菌體顆粒生產levan之可行性評估 147
4.7 純化後之levan的NMR分析 158
5. 結論 160
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