臺灣博碩士論文加值系統

本研究利用致突變劑N-methyl-N''-nitro-N-nitroso-guanidine (NTG)誘導X. dendrorhous CBS 6938及P. rhodozyma BCRC 21346等野生菌株發生突變，進而用含b-ionone的YM培養基篩選出蝦紅素高產量的菌株，高蝦紅素生產變異株X. dendrorhous NCHU-FS701之總類胡蘿蔔素產量為1,508 ug g-1 yeast，其中色素的87%為蝦紅素，其產量較X. dendrorhous CBS 6938或P. rhodozyma BCRC 21346皆高出3倍以上，變異株NCHU-FS701在YM 培養基的培養條件與原始菌株CBS 6938並無差異，然其生長速率較為緩慢。
利用Box-Behnken design的實驗設計，探討廉價碳源、氮源與金屬離子之最適化蝦紅素生產條件；結果顯示於三角瓶規模中，當以糖蜜43.4 g L-1、尿素2.49 g L-1及氯化鈣0.73 g L-1為培養基時有最大蝦紅素之生產(10.97 mg L-1)。於相同培養基且在固定溶氧及pH 6.5調控下的5 L發酵槽中培養120 hr，則可獲得13.58 mg L-1蝦紅素；若碳源提升2倍時，蝦紅素的生產可進一步提升至25.61 mg L-1。
X. dendrorhous NCHU-FS701所生產的果寡糖以neokestose為主，1-kestose次之，其果寡糖生產酵素屬胞內酵素。本研究利用珠磨式組織研磨機進行菌體破碎，以0.5 mm的玻璃珠處理菌液(30 g-DCW L-1)20分鐘能獲得97%的菌體破碎，再以有機溶劑充分混合破碎細胞懸浮液，於有機層中可回收72%的蝦紅素，水層中則可回收果寡糖生成酵素(446 U mg-1 protein)，此一步驟可同步獲得蝦紅素及果寡糖生成酵素。
X. dendrorhous NCHU-FS701所萃取的果寡糖生成酵素在60oC與pH 5.0時有最大比活性(446 U mg-1 protein)，而金屬離子如K+與Fe3+的添加能提升酵素活性，鎂離子則能提升neokestose於所生產總果寡糖中之比例。當果寡糖生成酵素之粗酵素液以Tris (hydroxymethyl) phosphine (THP)為交聯劑，與幾丁聚醣共交聯進行酵素固定化後，可提升果寡糖生成酵素的熱穩定性，且固定化的酵素經10次的重覆利用後，仍保有70%的酵素活性。

In this study, the wild strains of X. dendrorhous CBS 6938 and P. rhodozyma BCRC 21346 were treated with mutagenic agent N-methyl-N''-nitro-N-nitroso-guanidine (NTG) and plated on yeast malt (YM) agar containing b-ionone as a selective medium. One of mutant isolate (X. dendrorhous NCHU-FS701) was found to contain more than threefold of astaxanthin content compared with the parental strain X. dendrorhous CBS 6938. The total carotenoids content of the mutant NCHU-FS701 was 1,508 ug g-1 yeast, and 87% of the total carotenoids was astaxanthin. The mutant NCHU-FS701 could produce higher astaxanthin concentration, but it grew slower than parental strain CBS 6938 in YM broth.
The cost-down medium including molasses, urea, and CaCl2 were selected and optimized by using Box-Behnken design under shaker flask cultivation of X. dendrorhous NCHU-FS701. Maximum astaxanthin production of 11 g ml-1 was obtained with the medium composition of 43.4 g L-1sugar as glucose equivalent in molasses, 2.49 g L-1 urea, and 0.73 g L-1 CaCl2. Based on the media composition, cell growth and astaxanthin production were enhanced when scale up of astaxanthin production was conducted in a 5-L stirred tank reactor with continuous air supply and pH control.
Cell disruption was needed because the astaxanthin and transfructosylating enzyme are intracellular. The optimal setting for 30 g-DCW L-1 yeast cell disruption was found to consist of 0.5-mm-diameter glass bead at 10°C by using a bead beater for 20 min, in which more than 97% yeast disruption could be observed under microscopy. The disrupted cell culture was then extracted with hexane/ethyl acetate, and 72% total carotenoids in the organic solvent phase and 446 U mg-1 protein of transfructosylating enzyme activity in the water phase was obtained, indicating a simple step for simultaneously obtaining astaxanthin and transfructosylating enzyme.
For the production of fructooligosaccharides (FOS), the transfructosylating enzyme was immobilized onto chitosan using Tris (hydroxymethyl) phosphine (THP) as coupling agents. The optimal pH was 5.0 and the optimal temperature was 60oC for both free and immobilized enzyme. The THP-immobilized enzyme had better thermal stability, with 70% residual activity even after 10 repeated runs at 60oC. Metal ions, such as K+ and Fe3+ had positive effect on the enzyme activity.

中文摘要 iii
Abstract iv
第一章、緒論 1
一、前言 1
二、研究目的 1
第二章、文獻整理 2
一、蝦紅素 2
(一) 蝦紅素簡介 2
(二) 蝦紅素的功用 6
1. 養殖業之應用 6
2. 抗氧化劑 6
3. 抗癌 7
4. 抗發炎 7
5. 其它保健功能 7
(三) 蝦紅素的來源 10
1. 水產加工廢棄物 10
2. 人工合成 10
3. 微細藻類 10
4. 紅色酵母菌 11
二、紅色酵母菌 14
(一) Phaffia rhodozyma 與 Xanthophyllomyces dendrorhous簡介 14
(二) 色素的生產 17
1. 高蝦紅素生產的突變 17
2. 遺傳工程 17
3. 培養環境因子 18
4. 培養基質控制 20
5. 發酵工程的應用 20
(三) 類胡蘿蔔素之萃取 22
1. 物理法 22
2. 化學法 22
3. 酵素分解法 22
三、果寡糖 23
(一) 果寡糖理化特性 23
(二) 果寡糖之生理特性 23
(三) 果寡糖的結構與生合成 24
(四) 果寡糖的生產模式 28
四、固定化酵素 30
(一) 酵素固定化的方式 30
1. 物理吸附法 30
2. 離子鍵結 30
3. 共價鍵結 30
4. Bio-specific binding 30
5. 交錯聯結架橋 30
6. 包埋法 31
(二) 固定化材質 31
1. 載體 31
2. 交聯劑 31
五、實驗設計 32
(一) 反應曲面法 33
(二) Box-Bohnken design 33
第三章、材料與方法 35
一、實驗設計與流程 35
二、實驗材料 36
(一) 實驗菌株 36
(二) 化學藥劑 36
(三) 儀器及器材 37
三、實驗方法 38
(一) 菌種保存與活化 38
1. 活化 38
2. 保存 39
3. 果寡生產酵素生產之誘導培養 39
(二) 高蝦紅素生產變異株之誘導與分離 39
1. 致突變劑NTG處理 39
2. 紫外線照射處理 39
3. 突變株的篩選與培養 39
(三) 原生質體融合之操作 40
1. 細胞壁的去除 40
2. 原生質體融合 40
(四) 三角瓶規模培養試驗 40
1. 突變株的培養探討 40
2. 廉價培養基的組成試驗 40
3. 最適化培養基條件 41
(五) 酵母菌產孢試驗 41
(六) 廉價培養基之前處理 42
1. 糖蜜溶液之製備 42
2. 米酒蒸餾廢液、豆渣及醬油粕粉末之製備 42
(七) 發酵槽之操作 42
(八) 總類胡蘿蔔素之定量 43
1. 總類胡蘿蔔素之萃取 43
2. 總類胡蘿蔔素之測定 43
3. 蝦紅素之定量 43
(九) 菌體乾重之測定 44
(十) 總醣之測定 44
(十一) 總氮之測定 44
(十二) 組織研磨機對菌體之破碎處理 44
1. 菌體破碎之處理 44
2. 色素及粗酵素的萃取 45
(十三) 果寡糖生成酵素之粗酵素固定化 45
1. 粗酵液之製備 45
2. 交聯劑之配製 45
3. 載體之製備 45
4. 酵素之固定化 46
(十四) 果寡糖生成酵素活性測定 46
1. 酵素活性分析 46
2. 固定化酵素活性分析 46
3. 酵素作用溫度及pH之影響 47
4. 添加金屬離子之影響 47
5. pH耐受性 47
6. 酵素熱穩定性 47
7. 固定化酵素於連續批式之應用 47
(十五) 糖類之測定 47
四、數據分析 48
第四章、結果與討論 49
一、菌種改良 49
(一) 野生菌株之生長特性 49
(二) 致突變劑之變異誘導 52
1. 致突變劑之使用 52
2. 篩選性培養基 52
3. 高蝦紅素生產變異株 52
(三) 紫外線B之照射 53
(四) 原生質體融合 53
(五) 高蝦紅素生產變異株之生長特性 57
1. 不同培養溫度之影響 57
2. 不同起始pH之影響 57
3. 不同碳源之影響 61
4. 產孢試驗之觀察 61
5. 變異株X. dendrorhous NCHU-FS701色素之生產與比較 66
二、廉價培養基之應用 68
(一) 不同氮源之影響 68
(二) 不同無機鹽類之影響 71
(三) 最適培養條件之探討 73
(四) 小型發酵槽之培養 78
1. X. dendrorhous NCHU-FS701在發酵槽中生長及色素生產情形 78
2. 不同pH的調控對X. dendrorhous NCHU-FS701之影響 78
3. 不同碳源濃度對X. dendrorhous NCHU-FS701之影響 79
4. 饋料批式之培養 84
三、細胞壁破碎試驗 86
(一）細胞破碎的較佳條件 86
1. 不同粒徑玻璃珠對細胞破碎之影響 86
2. 細胞懸浮液濃度對細胞破碎之影響 89
3. 玻璃珠使用量對細胞破碎之影響 89
(二）細胞破碎後酵素與蝦紅素之萃取 92
四、果寡糖生成酵素之特性 92
(一) 粗酵素液之特性 92
1. 溫度與pH之影響 93
2. 金屬離子之影響 93
(二) 固定化酵素之熱穩定性與pH耐受性 98
(三) 連續批式利用固定化酵素 98
第五章、結論 102
第六章、未來發展 103
第七章、參考文獻 105
附錄一、專利證書 120
附錄二、縮寫表 121

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