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研究生:張建裕
研究生(外文):Jan-Yi Chang
論文名稱:微生物生產新果寡糖
論文名稱(外文):MICROBIAL PRODUCTION OF NEOFRUCTOOLIGOSACCHARIDES
指導教授:許垤棋
指導教授(外文):Dey-Chyi Sheu
口試委員:許垤棋
口試委員(外文):Dey-Chyi Sheu
口試日期:2014-07-10
學位類別:博士
校院名稱:大同大學
系所名稱:生物工程學系(所)
學門:工程學門
學類:生醫工程學類
論文種類:學術論文
論文出版年:2014
畢業學年度:102
語文別:英文
論文頁數:86
中文關鍵詞:醱酵高效液相層析新果寡糖法夫酵母
外文關鍵詞:neofructooligosaccharideshigh performance liquid chromatographyfermentationXanthophyllomyces dendrorhous
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果寡糖是一種益生源,可以促進腸道中比菲德氏菌的繁殖而增進人體的健康。目前市面上的果寡糖主要是菊竽型果寡糖(蔗果三糖、蔗果四糖和蔗果五糖),而法夫酵母(Xanthophyllomyces dendrorhous)的6G -呋喃果糖苷酶可以生產新果寡糖。與菊竽型果寡糖比較,由於糖苷的鍵結方式,新果寡糖的耐熱性和耐酸性較佳,另外,新果寡糖促進比菲德氏菌的效果也較好,因而受到重視。法夫酵母的酵素可將蔗糖轉變成新蔗果三糖、新蔗果四糖以及少量的蔗果三糖。新果寡糖的產物可以用HPLC分析,其中使用Hydrosphere C18管柱(移動相為純水)對於這些寡糖的解析程度優於amino型的管柱(移動相為75%乙晴),而利用ODS-AQ半製備級管柱(移動相為純水)可以純化新果寡糖。
利用6G -呋喃果糖苷酶活性較低的法夫酵母BCRC 22367於五公升醱酵槽進行液態醱酵可以生產高純度新果寡糖。每公升含有250公克的蔗糖和少量氮源,在醱酵過程蔗糖被酵素慢慢轉變成寡糖、葡萄糖和果糖,同時單糖逐漸被細胞吸收代謝,於20℃醱酵70小時,形成純度87%的果寡糖。
利用法夫酵母BCRC 21346於五公升醱酵槽,培養基成分含3%酵母萃取物和6%的蔗糖,溫度20℃、通氣量2 vvm、攪拌速度300 rpm,pH控制於6.9附近,醱酵36小時可以得到總計每公升約37,000單位的6G-呋喃果糖苷酶活性,其中胞內酵素占95.6%,其餘為胞外酵素。於30℃和pH 6-7此酵素在法夫酵母的細胞內非常安定,而且在pH 6.5有最佳的酵素反應。利用細胞和每公升600公克的蔗糖於30℃以及pH 6.5的條件,反應三小時可得到新果寡糖的產物,每公升含有374公克的果寡糖,回收細胞重覆使用10次,寡糖的生產速率都維持在首次的90%以上。
Fructooligosaccharides (FOS), an important prebiotics, can stimulate the proliferation of bifidobacteria in the intestine thus improve human health. The currently available FOS is primarily inulo-type FOS, which comprises 1-kestose, nystose and fructosyl nystose. Neo-FOS can be produced from sucrose through the catalytic action of 6G-fructofuranosidase (6G-FFase) from Xanthophyllomyces dendrorhous. This neo-FOS product consisted of neokestose, neonystose and small amount of 1-kestose. Neo-FOS has received increasingly interest due to its superior heat- and acid-resistance, and bifidogenetic activity compared to inulo-type FOS. All FOS can be analyzed by HPLC, using amino-type columns (75% acetonitrile as the mobile phase) or Hydrosphere C18 column (water as the mobile phase), which provided better resolution compared to amino-type column. Purification of neo-FOS can be performed by HPLC on a semipreparative ODS-AQ column.
A high-purity neo-FOS was produced by submerged culture of Xanthophyllomyces dendrorhous BCRC 22367. The fermentation medium consisted of 250 g/L of sucrose and small amount of nitrogen source. During the fermentation, sucrose was converted into neokestose, neonystose, 1-kestose, glucose and fructose through the catalytic action of intracellular 6G-FFase. Concurrently, glucose and fructose were consumed by the yeast cells. After 70 h of fermentation at 20oC, a neo-FOS product composed of 87% FOS on a dry weight basis was obtained.
Production of 6G-FFase was performed by submerged culture of Xanthophyllomyces dendrorhous BCRC 21346 using a 5-L fermentor. The culture medium consisted of 3% yeast extract and 6% sucrose. The fermentation was carried out at 20oC, 2 vvm, 300 rpm and the pH of the culture broth was controlled around 6.9. After 36 h of fermentation, total 6G-FFase of 37,000 U/L was obtained. Intracellular enzyme accounted for 95.6% of total enzyme acticity and other was extracellular enzyme. Because intracellular 6G-FFase located in the cell wall was very stable at 30oC and pH 6-7, and was most active at pH 6.5. Neo-FOS was produced by free-whole-cell biotrasformation at 30oC and pH 6.5, using 600 g/L of sucrose as the substrate. After 3 h of reaction, a neo-FOS mixture consisted of 374 g/L of FOS was obtained. Yeast cells were recovered and reused. In 10 cycles of repeated batch operations, more than 90% of FOS productivity as that obtained in the first batch reaction was always achieved.
ACKNOWLEDGEMENTSⅠ
ABSTRACTⅡ
CHINESE ABSTRACTⅣ
TABLES OF CONTENTSVI
LIST OF TABLESVIII
LIST OF FIGURESⅨ
CHAPTER 1 INTRODUCTION1
1.1. Background1
1.2. Scope of the work2
CHAPTER 2 LITERATURE REVIEW4
2.1. Functional properties of FOS4
2.2. Microbial production of FOS10
2.3. Properties of fructosyl transferase17
CHAPTER 3 HPLC ANALYSIS OF NEO-FOS22
3.1. Introduction 22
3.2. Materials and Methods24
3.3. Results and Discussion26
3.4. Conclusions35
CHAPTER 4 PRODUCTION OF HIGH-PURITY NEO-FOS BY CULTURE OF X. DENDRORHOUS36
4.1. Introduction 36
4.2. Materials and Methods40
4.3. Results and Discussion42
4.4. Conclusions46
CHAPTER 5 PRODUCTION OF 6G-FRUCTOFURANOSIDASE BY CULTURE OF X. DENDRORHOUS47
5.1. Introduction47
5.2. Materials and Methods 49
5.3. Results and Discussion52
5.4. Conclusions53
CHAPTER 6 PRODUCTION OF NEO-FOS BY FREE-WHOLE-CELL BIOTRANSFORMATION55
6.1. Introduction 55
6.2. Materials and Methods 55
6.3. Results and Discussion57
6.4. Conclusions68
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