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研究生:王志宏
研究生(外文):Chih-Hung Wang
論文名稱:異質半乳寡醣之製備: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 galacto-oligosaccharides :β-galactosidase reaction condition、product compositions and microbial activity
指導教授:賴鳳羲
指導教授(外文):Phoency Lai
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:2007
畢業學年度:95
語文別:中文
論文頁數:75
中文關鍵詞:半乳醣&半乳醣&半乳醣&半乳醣&半乳醣&半乳醣&
外文關鍵詞:galacto-oligosaccharides?-galactosidase
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中文摘要
本實驗旨在以乳糖與其它單糖混合經β-galactosidase (Aspergillus oryzae EC 3.2.1.23)作用合成新型半乳寡糖並探討反應條件、產物成分及微生物活性。實驗結果顯示,在寡醣合成上寡醣產量與半乳糖的比例成正相關性,乳糖與半乳糖比例1:4組在0.5 小時即有比乳糖組高出一倍的最大寡醣產量的出現,是所有混合組別中最高者。乳糖於0~30 %乙醇反應系統中與酵素反應,無論是水解或合成作用均因乙醇的存在而有下降的現象,濃度越高效果越差。合成的寡醣以不同比例活性炭和矽藻土吸附分離,結果發現30%乳糖組(L30)經單活性炭組吸附即有96.3 %的寡醣吸附力,是所有活性炭與矽藻土混合組別中最高者,再經10 %與20 %乙醇沖提畫分後可得13.5 %的收率,其寡醣純度更高達97.9 %。相同條件下乳糖與木糖混合組(LX)也有5.4 %寡醣收率及92.0 %的純度,乳糖與果糖混合組(LF)有6.1 %的寡醣收率與93.9 %的純度。純化的寡醣經酸水解及陰離子交換層析分析,結果顯示L30組單糖成分為葡萄糖約30%與半乳糖約70%,LX及LF組單糖成分皆為葡萄糖31%與半乳糖69%。將純化的寡醣以取代葡萄糖碳源的方式,進行微生物B. longum或P. freudenreichii單獨及共同培養的總生菌數並無明顯提升作用,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異。B. longum及P. freudenreichii單獨與混合培養於含寡醣的培養液中,72小時後的發酵液對Escherichia coil、 Propionbacterium acnes、 Clostridium perfringens等病原菌均具有抑菌效果出現,但對 Salmonella typhimurium 則無任何抑菌作用。
ABSTRACT

This study was to investigate the reaction condition, product compositions and microbial activity of new galacto-oligosaccharides produced from the mixtures of lactose and other sugars by -galactosidase (Aspergillus oryzae EC 3.2.1.23). The results of saccharide composition analysis indicate that the oligosaccharide yield was positively proportional to the galactose ratio applied and reached a maximum at a lactose:galactose ratio of 1:4, double the case of pure lactose system. For the 30% lactose system in the presence of 10-30% ethanol, the oligosaccharide yields reduced with increasing the ethanol concentration, regardless of hydrolysis or synthesis reaction. By separating the crude oligosaccharide products with various ratios of activated charcoal and Celite, the results indicate that the products from 30% lactose (L30) had an adsorption percentage of 96.3%, the highest one among all mixtures of activated charcoal and Celite. The following fractionation by eluting successively with 10% and 20% ethanol aqueous solutions gave an oligosaccharide yield of 13.5% and a purity of 97.9%. Under the same separation and fractionation conditions, the products from lactose-xylose mixtures (LX) had an oligosaccharide yield of 5.4% and 92.0% purity and those form lactose-fructose mixtures (LF) showed 5.4% oligosaccharide yield and 92.0% purity. By acid-catalyzed hydrolysis and anion-exchange chromatographic analysis, the monosaccharide compositions were 30% glucose and 70% galactose for the L30 products and 31% glucose and 69% galactose for both LX and LF products. The purified oligosaccharides instead of glucose as a carbon source did not increase significantly the total counts of B. longum or P. freudenreichii either in pure culture or co-culture. However, the resulting fermented supernatants showed some inhibiting effects on pathogenic microorganisms, depending on the microorganisms examined. The fermented supernatants of B. longum and P. freudenreichii cultured alone or co-cultured with the obtained oligosaccharides for 72 hr fermentation broths inhibition effects on Escherichia coli, Propionbacterium acnes, and Clostridium perfringens, but negligible effects on Salmonella typhimurium.
目錄
中文摘要 I
Abstract II
目錄 IV
圖目錄 VI
表目錄 VIII
壹、前言 1
貳、文獻回顧 2
一、寡醣 2
二、酵素方式合成寡醣 2
三、糖苷鍵水解酶催化的寡醣合成作用 9
四、有機溶劑對寡醣合成的影響 11
五、半乳糖苷酶 15
六、半乳寡醣合成 16
七、寡醣鑑定 17
八、寡木醣的純化與脫色 19
九、寡醣類之分離 19
十、寡醣之保健功效 22
參、材料與方法 23
一、材料 23
A.酵素與糖質 23
B.化學試劑 23
C.儀器 23
二、方法 24
A.寡醣合成實驗流程 24
B.添加乙醇有機溶劑對寡醣合成影響實驗 25
C.活性炭與矽藻土劃分實驗 26
D.活性炭純化寡醣實驗 26
E.純化實驗 27
三、組成鑑定 28
四、益菌生試驗 29
A.測試菌株及來源 29
B.培養基配置 29
1.厭氧菌生長試驗用培養基 29
2.厭氧菌培養基之製備 30
C.益(抑)菌生實驗流程 30
1.益菌生試驗 30
2.抑菌生試驗 31
D.實驗方法 31
1.雙叉桿菌及丙酸菌增殖培養 31
2. 雙叉桿菌及丙酸菌之培養 32
3.雙叉桿菌及丙酸菌發酵GOS發酵液對病源性細菌之抑菌活性試驗 32
肆、結果與討論 35
一、酵素反應條件 35
二、混合不同單糖之反應系統 38
三、添加乙醇有機溶劑之反應系統 48
四、活性炭與矽藻土吸附寡醣實驗 51
五、寡醣純化 52
六、單醣組成分析 57
七、益菌生實驗 61
A、益菌生試驗 61
B、抑菌生試驗 62
八、結論 69
伍、參考文獻 70
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