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研究生:鄭明福
研究生(外文):Ming-Fu Jeng
論文名稱:以納豆菌生產γ-聚麩胺酸之研究
論文名稱(外文):Study of production of poly(γ-glutamic acid) by Bacillus subtilis (natto)
指導教授:李順來李順來引用關係許孟博
指導教授(外文):Shun-Lui LeeMeng-Bor Hsu
學位類別:碩士
校院名稱:南台科技大學
系所名稱:生物科技系
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:67
中文關鍵詞:γ-聚麩胺酸發酵納豆
外文關鍵詞:poly(γ-glutamic acid)fermentationBacillus subtilis (natto)
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聚麩胺酸[poly(γ-glutamic acid);γ-PGA]為日本傳統發酵食品納豆中,水溶性微生物高分子聚合物,可作為環境保護、保健食品、醫藥、化妝品等用途。本研究以市售納豆菌N1為生產菌株,期望經由培養參數之篩選,探討γ-PGA產率增高可行性。一般而言,發酵液黏度増加,代表γ-PGA產量提高。納豆菌37℃、150 rpm搖瓶培養實驗,得知下列結果: 一. 採用菌種活化二次能提升菌量60%。二. 納豆菌N1培養於LB培養基中( Yeast extract 15g/L、Tryptone 5g/L、Sodium chloride 4g/L、Glucose 30g/L ),菌量成長較高。三. 納豆菌N1發酵於修飾之M2培養基中( Monosodium glutamate monohydrate 171.7g/L、Glucose 80g/L、Peptone 15g/L、Yeast extract 5g/L、Urea 3g/L、K2HPO4 2g/L ),獲得較高γ-PGA產量。 四. 發酵期間使用修飾過M2培養基饋料,能増加黏度值63.1%。接續以五公升發酵槽培養納豆菌N1,探討溫度、轉速、通氣量、pH及饋料對γ-PGA產量影響。當發酵條件為37℃、350 rpm、0.8vvm及pH=7.0培養,可得最高之發酵黏度。結論顯示: 納豆菌N1於五公升發酵槽比搖瓶培養能増加黏度 432.0%,同時縮減50.2%發酵時間; 五公升發酵槽最適發酵條件確能明顯提高γ-PGA產量。
Poly(γ-glutamic acid) (γ-PGA) is a water-soluble biopolymer that can be found in Japanese traditional fermented food, natto, which has potential applications in the environmental protection, health food, medicine and cosmetics. This study has focused on the screening of cultural parameters for high γ-PGA production by B. subtilis (natto) N1. In general, the increase in broth viscosity implies the increase ofγ- PGA content. In this study, B. subtilis (natto) was incubated at 37℃ and 150 rpm by flask culture. The following results were obtained: 1, The biomass of the inoculum increased up to 60% by a two-step activation process. 2, N1(natto) generated higher biomass as cultured in LB medium ( Yeast extract 15g/L, Tryptone 5g/L, Sodium chloride 4g/L and Glucose 30g/L ). 3, Higher γ-PGA production was obtained when N1(natto) was fermented in modified M2 medium ( Monosodium glutamate monohydrate 171.7g/L, Glucose 80g/L, Peptone 15g/L, Yeast extract 5g/L, Urea 3g/L and K2HPO4 2g/L ). 4, Feeding modified M2 medium during fermentation raised 63.1% of the broth viscosity. Next, we investigated the effect of temperature, agitation, aeration, pH and feeding stratagy on γ-PGA production of natto N1 in a 5L jar fermenter. The highest broth viscosity were obtained under the cultural conditions of 37℃ , 350 rpm , 0.8vvm and pH=7.0. In conclusion:The natto N1 which was cultured in the 5L jar fermenter could not only increase 432.0% of the broth viscosity, but also reduce 50.2% of the fermentation time than that in the shaker flask. Thus, the optimal fermentation conditions could significantly increaseγ- PGA production.
中文摘要 iv
英文摘要 v
誌謝 vi
目次 vii
表目錄 ix
圖目錄 x
第一章 前言 1
第二章 文獻回顧 2
2.1 聚麩胺酸之發現 2
2.2 聚麩胺酸之特性及應用 9
2.3 研究動機及目的 11
第三章 材料與方法 13
3.1 實驗架構 13
3.2 使用菌株 14
3.3 培養基 14
3.3.1 菌株保存培養基 14
3.3.2 菌種生長培養基 14
3.3.3 γ-PGA生產培養基 15
3.4 培養方法 15
3.4.1 搖瓶培養方法 15
3.4.2 五公升發酵槽培養方法 17
3.5 分析方法 20
3.5.1 菌體生長量之測定(OD600nm) 21
3.5.2 黏度之測定 21
3.5.3 總醣類分析 22
3.5.4 麩胺酸鈉分析方法 23
3.5.5 γ-PGA含量及分子量分佈之測定 25
第四章 搖瓶之結果與討論 29
4.1 基本實驗條件之選擇 29
4.1.1 測量細菌量之UV波長選擇 29
4.1.2 菌種之活化方式 30
4.2 菌種生長培養基之探討 30
4.2.1 納豆菌菌株之篩選 30
4.2.2 菌種生長培養基之篩選 33
4.3 γ-PGA生產培養基之探討 36
4.3.1 γ-PGA生產培養基之篩選 36
4.3.2 修飾生產培養基配方 38
4.3.3 修飾M2生產培養基之饋料 42
4.4 搖瓶模式之討論 43
第五章 5L發酵槽之結果與討論 45
5.1 確認培養基搭配組合 45
5.2 溫度對γ-聚麩胺酸產率的影響 47
5.3 攪拌速率對γ-聚麩胺酸產率的影響 49
5.4 通氣量對γ-聚麩胺酸產率的影響 50
5.5 饋料對γ-聚麩胺酸產率的影響 51
5.6 篩選五公升發酵槽最佳參數組合 53
5.6 1 生產γ-PGA篩選出最佳參數組合 53
5.6 2 γ-PGA之分子量分佈 54
5.6 3 五公升發酵槽與搖瓶培養結果之比較 58
5.6 4 探討發酵液最高黏度的產生 58
5.7 五公升發酵槽模式之討論 60
第六章 總結 61
參考文獻 63
作者簡介 67
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