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研究生:巫秋燕
研究生(外文):Chiu-yen Wu
論文名稱:新型寡果糖之製備-轉糖酵素反應條件、產物成分及微生物活性
論文名稱(外文):Preparation of new fructose -oligosaccharides -β-fructosyl transferase reaction condition、product compositions and microbial activity
指導教授:賴鳴鳳
學位類別:碩士
校院名稱:靜宜大學
系所名稱:食品營養研究所
學門:醫藥衛生學門
學類:營養學類
論文種類:學術論文
論文出版年:2006
畢業學年度:95
語文別:中文
論文頁數:76
中文關鍵詞:新型質寡醣
外文關鍵詞:New fructose -oligosaccharides
相關次數:
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中文摘要
近年來對果寡醣之生理活性相關的研究及報告多如雨後春筍,而傳統果寡醣之生產多以降解菊苣而來,但反應產物、產量及寡醣分子量不易控制,相較於利用酵素來進行果寡醣之生產,則酵素反應較易受控制;因此本研究採用Aureobasidium pullulans所產生之β–fructosyl transferase進行新型果寡醣之生產及條件探討,反應條件分別利用乙醇及水做為反應介質進行酵素反應,同時利用Glucose oxidase去除反應中葡萄糖之干擾,並經活性炭純化後,進一步以純化物進行腸道益生菌之培養試驗。結果顯示A. pullulans培養96小時可產生10.2U β–fructosyl transferase(水解活性7 U,轉移活性3.2 U ) ,將酵素與20%蔗糖於pH 5.5 、55℃、150 rpm反應5小時,可得聚合度為三糖至五糖之果寡醣。於5~10 %乙醇反應系統中添加木糖及阿拉伯糖(xyl、ara)作為接收基質,雖乙醇濃度提高對於異質果寡醣產率無顯著影響,但其所合成之異質果寡醣產物結構則有所不同,若再添加Glucose oxidase於系統,發現在蔗糖組,5 %乙醇系統較檸檬酸緩衝液反應系統可更有效移除葡萄糖而提高果寡醣產率。在混合醣類系統(suc+xyl及ara)不論在 5 %乙醇系統或檸檬酸緩衝液反應系統添加Glucose oxidase,對於清除葡萄糖皆無較顯著效果。以活性炭與矽藻土混合比例1:2吸附合成寡醣產物,再以30%乙醇進行沖堤劃分則發現其對寡醣吸附力可達 80 %。將純化之果寡醣作為碳源,無論是單獨培養或共同培養對B. longum或P. freudenreichii的總生菌數並無提升作用,對於所測試之病原菌抑菌效果則因發酵菌種及病原菌不同而不同程度之影響。B. longum及P. freudenreichii混合培養於異質果寡醣,其發酵液對Escherichia coil、 Propionbacterium acnes、 Clostridium perfringens等病原菌皆具有抑菌效果,但對 Salmonella typhimurium 無明顯抑菌效果。
Abstract
Recently there have been great interests in prebiotic properties of fructooligosaccharides (FOS). Traditionally, FOS are produced by degradation of chicory inulins, but the composition, yield and molecular weight of the product were not easily controlled, in contrast to enzymatic synthesis. Therefore, this study was to investigate the production condition of new FOS using β–fructosyl transferase from Aureobasidium pullulans. The experiments were conducted for finding the optimal cultivate condition of A. pullulans and the effects of ethanol-water mixture as reaction media and removing glucose by glucose oxidase, and finally using the mixture of activated charcoal and Celite as adsorbants to purify the FOS products for microbial activity analysis. The results showed that the extracellular broth of A. pullulans after 96 h incubation exhibited 10.2 U β–fructosyl transferase activity (hydrolase activity 7 U, transferase activity 3.2 U). The 20% sucrose added with the broth for reaction at pH 5.5, 55oC and 150 rpm for 5 hr could yield the mixtures containing tri- to pentasaccharides. Adding xylose and ababinose as donors in 5-10% ethanol reaction systems resulted in various heterogeneous FOS, despite that elevating ethanol concentration did not significantly enhance the FOS yield. The incorporation of glucose oxidase could effectively remove glucose and increase FOS yield for the pure sucrose system. But, this was not for the case of the sucrose mixtures with xylose or arabinose. After adsorbing by activated charcoal-Celite mixture (1:2 w/w) and followed by eluting with 30% ethanol, the adsorbing rate of the FOS preparation was found up to 80%. Using the purified FOS as a carbon source, the total counts of Bifidobacterium longum and Propionibacterium freudenreichii were not significantly improved in either single culture or co-culture. However, the resultant fermentation broths of B. longum and P. freudenreichii possessed some antimicrobial activities for a certain species of pathogens such as Escherichia coli, Propionbacterium acnes, and Clostridium perfringens, but not for Salmonella typhimurium.
目錄
中文摘要………………………………………………………………Ⅰ
英文摘要………………………………………………………………Ⅱ
表目錄…………………………………………………………………Ⅳ
圖目錄…………………………………………………………………Ⅷ
第一章、前言…………………………………………………………1
第二章、文獻回顧
一、果寡醣……………………………………………………………2
二、果寡醣合成酵素種類……………………………………………4
三、果醣轉移酶特定受質……………………………………………8
四、轉移果醣基糖質之接收基質…………………………………15
五、酵素對有機溶劑之耐受性及應用………………………………17
六、寡木醣的純化與脫色……………………………………………20
七、寡醣類之分離……………………………………………………21
八、腸道益生菌試驗…………………………………………………23
第三章、材料與方法
※實驗材料……………………………………………………………25
※實驗流程……………………………………………………………26
※實驗方法……………………………………………………………28
一、Aureobasidium pullulans菌株保存……………………………28
二、A. pullulans 之生長週期及酵素活性測定……………………28
三、 酵素反應最適條件探討…………………………………………29
四、果寡醣劃分產物之純化 …………………………………………30
五、活性炭純化寡醣產物之劃分 ……………………………………30
1.活性炭對低聚果糖最大吸附力之測定.………………………… 30
2.批式分離果寡醣 ………………………………………………… 30
3.單糖組成分析 …………………………………………………… 32
六、微生物試驗………………………………………………………33
一、益菌生試驗 ………………………………………………………32
1.B. longum及P. freudenreichii之活化增殖培養及菌株保存…32
2. B. longum及P. freudenreichii分別對異質果寡醣之利用試驗33
二、抑菌生試驗………………………………………………………33
1. B. longum及P. freudenreichii發酵異質果寡醣發酵液之對病源性細菌抑菌活性試驗……………………………………………33
2. B. longum及P. freudenreichii混合培養對於病原性細菌之抑菌生試驗……………………………………………………………34
第四章、結果與討論
一、Aureobasidium pullulans生長期間之變化 …………………36
1. A. pullulans生長曲線及pH值測定 ……………………………37
二、酵素活性之測定 ……………………………………………… 40
三、最適反應總醣濃度( 10 ~ 30 % )之篩選 ……………………41
四、添加不同基質於含有 5~10 %乙醇有機溶劑之反應系統 ……47
五、加入Glucose oxidase於反應系統中其對產率之影響…………50
六、轉糖酵素反應產物之劃分與純化………………………………55
1.活性炭與矽藻土吸附性質……………………………………55
2.以活性炭與矽藻土批式劃分與純化果寡醣…………………56
3.單糖組成分析…………………………………………………57
七、微生物試驗………………………………………………………62
1.益菌生試驗…………………………………………………………62
a.雙叉桿菌和丙酸菌對新型異質果寡醣之利用其生菌數之變化…62
b.雙叉桿菌和丙酸菌對新型異質果寡醣之利用其pH值之變………62
2.抑菌生試驗 …………………………………………………………63
a.雙叉桿菌及丙酸菌發酵新型異質果寡醣發酵液對於病原性細菌之
抑菌活性……………………………………………………………… 63
b.新型異質果寡醣對雙叉桿菌及丙酸菌混合發酵液對於病原性細菌
之抑菌活性…………………………………………………………… 64
c.抑菌活性之來………………………………………………………65
第五章、結論…………………………………………………………71
第六章、參考文獻……………………………………………………73
第六章、參考文獻
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