探討pH 對Lactobacillus kefiranofaciens產胞外多醣及乳
酸之影響

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范峻勝(Chun-sheng Fan) 查詢紙本館藏

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化學工程與材料工程學系

論文名稱

探討pH 對Lactobacillus kefiranofaciens產胞外多醣及乳酸之影響
(The effect of pH value on the production of the exopolysaccharide and lactic acid by Lactobacillus kefiranofaciens )

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摘要(中)

Lactobacillus kefiranofaciens所生產的胞外多醣是一種水溶性的多醣，名為克弗蘭多醣(Kefiran)，因為結構與其他物理、化學性質特殊，將可以廣泛的被應用於食品、醫藥工業方面。
本研究目的在探討不同在pH 值下液態發酵培養對乳酸菌L.
kefiranofaciens生產胞外多醣及乳酸之影響，與產生之多醣生物活性的影響。
實驗結果發現控制pH 5.5時，最有利於菌體的生長，不論是單位基
值對菌體轉化率Y(x/s)或比生長速率(μ)都是最高的，分別為104 mg/g及0.063 1/hr；而控制pH 5的時候最有利於胞外多醣的生成，單位基質及單位菌體的多醣產率分別為19.4 mg/g及195 mg/g。而在乳酸轉化率方面，發現低pH值有助於乳酸的產生。
在克弗蘭多醣的生物活性測定上，在不同初始pH之血清瓶實驗中，
發現多醣活性並沒有差異，可能是由於分子量並無明顯差異；而在控不同pH值發酵槽條件下培養，發現於pH 5的多醣其生物活性最高。發現多醣活性隨著分子量成正比的關係，分子量較大，其生物活性也隨之較高。

摘要(英)

Exopolysaccharide(EPS) which is produced by Lactobacillus
kefiranofaciens is a kind of water-soluble
exopolysaccharide, named kefiran.
It is widely applied to food and pharmaceutical industry due to its unique structure and physical, chemical properties.
The purpose of this study is to discuss the impact of fermentation on L. kefiranofaciens when it produce exopolysaccharide and lactic acid under different pH value.
Research has shown that pH 5.5 is the most favorable environment for cell growth, both Y (x / s), which is 104 mg / g, and μ, which is 0.063 1/hr, are the highest. Also, pH 5 is most favorable for the formation of EPS, with
Yp/s 19.4 mg/g and Yp/x 195 mg/g and low pH is good for the generation of lactic acid.
With EPS biological activity measuring, no difference was found in TNF released between different initial pH in Serum bottle, no significant difference in Molecular Weight might be the reason. And different pH values
in the fermenter culture conditions, the EPS found in the pH 5 the highest biological activity. Proportional with the molecular weight polysaccharide found in the relationship between activity, molecular weight, the higher the biological activity also followed.

關鍵字(中)

★ 乳酸
★ 胞外多醣
★ 酸鹼值
★ 乳酸菌

關鍵字(英)

★ pH
★ exopolysaccharide
★ lactic acid
★ Lactobacillus kefiranofaciens

論文目次

目錄
摘要 i
Abstract ii
誌謝 iii
目錄 iv
圖目錄 ix
表目錄 xi
第一章緒論 1
1-1研究背景 1
1-2研究目的 2
第二章文獻回顧 3
2-1乳酸菌 3
2-1.1 乳酸菌介紹 3
2-1.2 乳酸菌分類 6
2-1.3 乳酸菌應用 7
2-1.4 乳酸菌對於人體益處 8
2-1.5 Lactobacillus kefiranofaciens之介紹 11
2-2多醣體 12
2-2.1 多醣體介紹 12
2-2.2 乳酸菌多醣體 12
2-2.3 克弗蘭多醣 14
2-2.3.1 克弗蘭多醣介紹 14
2-2.3.2 克弗蘭多醣應用 15
2-3 人體免疫系統與抗腫瘤介紹 16
2-3.1 人體免疫系統簡介 16
2-3.2 細胞激素(cytokines)的調節 17
2-3.3 腫瘤壞死因子 (TNFα) 17
2-3.4 ?-D-葡聚糖的抗腫瘤機制 18
2-3.5 影響多醣體活性因素 20
2-3.5.1 多醣體活性與分子量的關係 20
2-3.5.2 (1→3) 鍵結的βD葡聚醣主鏈與活性的關係 20
2-3.5.3 多醣體分支度與活性的關係 21
2-3.5.4 多醣體構形與活性的關係 21
2-3.6 多醣體抗腫瘤生物活性之動物細胞實驗 22
2-4 乳酸(Lactic acid)介紹 23
2-4.1 乳酸介紹 23
2-4.2 乳酸應用 23
2-5 深層液態發酵 24
2-6 影響發酵的物理化學等環境生長因子 24
2-6.1培養基組成 25
2-6.1.1碳源 25
2-6.1.2氮源 25
2-6.1.3碳氮比 25
2-6.2 攪拌速率 26
2-6.3 pH值 26
2-6.4 溫度 28
2-6.5接種量 28
2-6.6 發酵槽形式 29
2-6.7 共培養系統 29
第三章實驗材料與方法 30
3-1 實驗規劃 30
3-2 實驗材料 31
3-2.1 實驗菌株 31
3-2.2 實驗藥品 31
3-2.3 實驗儀器及其他設備 32
3-2.4 實驗裝置 34
3-3 實驗方法 35
3-3.1 菌種保存 35
3-3.2 培養基組成 35
3-3.3 發酵培養 37
3-3.3.1 血清瓶實驗 37
3-3.3.2 發酵槽實驗 38
3-3.4 實驗分析方法 38
3-3.4.1實驗分析流程 38
3-3.5 乳酸菌多醣體之生物活性測定 44
3-3.5.1 細胞株 44
3-3.5.2 細胞株保存 44
3-3.5.3 細胞株解凍 45
3-3.5.4 細胞株繼代培養 45
3-3.5.5 細胞株培養液組成 45
3-3.5.6 動物細胞實驗流程 46
3-3.6 格蘭氏染色法 (Gram Stain) 47
第四章結果與討論 49
4-1 菌體型態 49
4-1.1 顯微鏡下觀察 49
4-1.2 格蘭氏染色法 49
4-2 血清瓶實驗 50
4-2.1 血清瓶實驗之L. kefiranofaciens 菌體的比較 51
4-2.2 血清瓶實驗之產物粗多醣的比較 53
4-2.3 血清瓶實驗之產物乳酸的比較 54
4-3 發酵槽培養實驗 57
4-3.1發酵槽實驗之發酵過程控pH值(controlled-pH)及不控pH值(uncontrolled-pH)對L.kefiranofaciens影響比較 57
4-3.1.1 控制pH 值對於發酵時間的影響 57
4-3.1.2 控制pH 值對於菌體生長的影響 59
4-3.1.3 控制pH 值對於生產粗多醣的影響 61
4-3.1.4 控制pH 值對於生產乳酸的影響 62
4-3.1.5 控制pH 與否對於粗多醣與乳酸產率比較 62
4-3.2不同pH 值之發酵槽實驗對L. kefiranofaciens 生長影響 65
4-3.2.1 不同pH值之發酵槽實驗對於菌體生長的影響 68
4-3.2.2 不同pH值之發酵槽實驗對於粗多醣的影響 69
4-3.2.2 不同pH值之發酵槽實驗對於乳酸的影響 70
4-4 L. kefiranofacien多醣體之生物活性測試 72
4-4.1 pH值對多醣活性及分子量的比較之探討 72
4-4.2 多醣分子量及濃度對生物活性的影響 76
4-4.3 克弗蘭多醣與市售乳酸菌飲料多醣之比較 77
第五章結論 78
5-1 結論 78
第六章參考文獻 81

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指導教授

徐敬衡(Chin-hang Shu)

審核日期

2010-7-20

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