臺灣博碩士論文加值系統

微生物的酵素在生物轉換上一直扮演著重要的角色，大多數的微生物酵素可以將複雜的大分子分解成較簡單的小單元，有利於其吸收利用。梔子苷是由梔子的果實萃取出來的。而梔子苷經酵素轉換並與胺基酸反應形成梔子素-胺基酸或梔子酸-胺基酸的複合物，呈現藍色或紅色，常被當成食用色素使用。近年來，食品安全的觀念與日俱增，因此，天然的食品色素在工業上的需求量，也越來越高。在這個研究中，我們利用生物轉換技術，篩選具有β-葡萄醣苷酶或羰酸酯解酶活性的菌株，然後優化其酵素產量，以供生物轉換之用。我們由環境分離的50株微生物中，Lactobacillus casei JB-3表現良好的β-葡萄醣苷酶活性。利用培養時間以及培養條件，使酵素活性最佳化，我們發現deMan-Rogosa-Share（MRS）培養基在pH =7時最適合JB-3菌株生長，並且在培養18小時之後，菌量會到達最高峰。然而，因為生長在MRS培養基中該菌之酵素活性並不佳，因此，我們利用MRS培養基放大菌量18小時後，更換成添加4% tryptone的Luria-Bertani（LB）培養基（pH=7），以促進細菌的β-葡萄醣苷酶的表現。而分離株Bacillus subtilis NB-1於pH 7的MRS培養基，以37℃培養18小時，可得最佳菌量與最高羰酸酯解酶產量。再利用超音波裂解法以及分子篩過濾法初步純化β-葡萄醣苷酶和羰酸酯解酶，發現此二酵素之分子量均大於100kDa。僅利用β-葡萄醣苷酶可將梔子苷轉換成梔子藍，加入羰酸酯解酶反應後更可形成梔子紅。我們建立了一種便宜而且安全的方法生產天然藍色色素，這種生物轉換成的色素可以應用於各種不同的工業上，尤其是食品以及飲料上更可提升產品價值。

Bacterial enzymes play important roles in biotransformation. Most of them can biodegrade complex compounds into simpler ones. Geniposide is extracted from gardenia fruit which is an oriental folk medicine. And the biodegraded genipin is very easily to become blue and red when reacts with amino acids. The blue and red pigments are used to being natural food colorants in Japan and Asia. Recently, the concept of food safety has been increased by days. The requirement of natural food colorants is getting more and more in industries. In order to produce natural food colorants, in this study, we screened 50 bacteria strains for the best β-glucosidase or esterase activities and then optimize them. An isolate Lactobacillus casei JB-3 showed the best β-glucosidase activity. The maximum enzyme activity was adjusted by growth time and culture conditions. Our data showed that deMan-Rogosa-Share（MRS）medium at pH=7 was the best culture condition for JB-3 strain to grow. After 18 hours, the cell amount could reach the maximum. However, the enzyme activity was not good when bacteria grew in MRS medium. Therefore the MRS medium was replaced by Luria-Bertani（LB）medium with 4% tryptone. In which the enzyme activity was induced. MRS medium was used to amplify bacteria for 18 hours and was replaced by LB medium with 4% tryptone to maximize microbial β-glucosidase activity. The best enzyme was showed while the pH of medium was around 7. The isolate Bacillus subtilis NB-1 showed the best esterase activity. The best enzyme activity was achieved when B. subtilis NB-1 grew in MRS medium （pH=7） at 37℃ for 18 hours. Sonication and filtration were applied to purify enzymes. The molecular weights of both enzymes are higher than 100kDa. Appling β-glucosidase could produce blue pigment and apply both β-glucosidase and esterase could produce red pigment. A cheaper and safer way to produce natural blue pigment has been established in this study. This bioconversional pigment could be applied in various industries, and it could rised the volue of product, especially in food and beverage industry.

目錄
中文摘要············································································I
Abstract·············································································II
誌謝···················································································III
目錄···················································································IV
圖目錄················································································V
表目錄················································································VII
第一章 前言········································································1
第一節 研究緣起·························································1
第二節 文獻回顧·························································5
第二章 研究材料與方法·························································18
第一節 研究材料·························································18
第二節 研究方法·························································23
第三章 研究結果··································································34
第一節 環境分離菌株之鑑定結果···································34
第二節 分離株之纖維素酶活性分析································39
第三節 分離株之澱粉酶活性分析···································41
第四節 分離株之β-葡萄醣苷酶活性分析··························43
第五節 L. casei JB-3表現β-葡萄醣苷酶之最適化條件··········45
第六節 分離株之羰酸酯解酶活性分析·····························54
第七節 B. subtilis NB-1表現羰酸酯解酶之最適化條件········58
第八節 酵素之純化與應用············································66
第四章 討論········································································72
參考文獻·············································································78

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