臺灣博碩士論文加值系統

纖維素完全水解之產物為具有醱酵性之葡萄糖，為可供動物利用之能量來源。對於單胃動物而言，由於本身無法分泌纖維素酶，因此在纖維素利用率受到限制，本研究目的為篩選纖維素酶生產菌株並針對其酵素生產條件、特性及應用上分析之，藉以評估作為於纖維性飼料添加劑之開發。以酵素擴散圈法自白蟻腸道、牛隻瘤胃液及排泄物中篩選出具有水解纖維素能力之菌株九株，其中具生產纖維素酶能力以牛隻瘤胃液R3菌株為最佳，於以1% 羧甲基纖維素鈉(CMC)為基質，於37℃、pH 5.0，培養7天後，其纖維素酶活性可達356 mU/ml，β-葡聚醣酶活性為20.2 U/ml，甘露聚醣酶活性為612 mU/ml。將R3菌株進行核酸定序與NCBI GenBank基因庫比對鑑定菌種為Bacillus sp.。以纖維性原料麩皮與百慕達草為基質，進行固態醱酵及粗酵素液體外培養試驗，評估對纖維之降解能力。結果試驗顯示，將菌液與麩皮以(1：0.75 v/w)混合，進行固態醱酵處理5天，其中洗纖維降解率達 48.2%。而以液態醱酵試驗，培養48小時後，麩皮之乾物質及中洗纖維降解率分別達24.8及34.7%，其降解效果較相同力價之商業化纖維素酶為佳。以百慕達草為原料進行液態醱酵試驗，R3粗酵素液處理於48小時具有最大之乾物質及中洗纖維降解率(17.4%及16.4%)，商業酵素處理組對在中洗纖維降解率(6.8%)則顯著低於R3粗酵素處理組。綜合上述結果，R3菌株具有生產纖維水解酵素之能力，可降解飼料原料之麩皮及百慕達草纖維素成分，此可提高纖維性原料營養價值及利用率。

Complete hydrolysis of fiber yields the easily fermented glucose, which were the source of energy for animals. Non-ruminant animals can not secrete the cellulose result in the utilization of the fiber is restricted. The purpose of this study was examined the cellulose-producting strains which are isolated from the termites, the rumen liquid of dairy cows, and ruminants waste through the enzyme radial diffusion method. One of the best strain R3 isolsted from the rumen liquid of dairy cows according to the fiber degradability. After the 7 day cultivation period, the carboxymethyl cellulase activity of R3 under the optimal cultural condition (37℃ and pH 5.0) was 356 mU/ml. The 16S rDNA of R3 was extracted, amplified, sequenced and compared with the data base of NCBI GenBank was finally identified as Bacillus sp. The wheat bran was used as substrate in solid fermentation and in vitro fiber degradation determination to evaluate the fiber degradability. The R3 broth from 7-day cultivation was blended with the wheat bran in the ratio in 0.75 ml/g for the solid fermentation. After the 5-day fermentation period, the neutral detergent fiber (NDF) degradability of wheat bran was 48.2%. In the in vitro fiber degradation determination, the nylon bag contained wheat bran was soaked into the suspension which was collected from the 7-day cultivation by centrifugation. The dry matter (DM) and NDF degradability of wheat bran was 24.8 and 34.7%, respectively. Overall, Bacillus sp. R3 has the ability to produce the cellulase to hydrolyze the cellulose and improve the nutrient value of feedstuffs.

目錄 I
圖目錄 III
表目錄 IV
摘要 1
Abstract 3
第一章、前言 5
第一節、研究背景 5
一、現今能源與動物飼糧問題 5
二、找尋具有效分解纖維素之物種 7
三、植物木質纖維素 10
四、常用破壞木質纖維素前處理方法 13
五、纖維素酶 15
六、酵素作為飼料添加劑之效益 17
第二節、研究目的 19
第三節、預定工作項目 21
第二章、材料與方法 21
第一節、實驗材料 21
一、試樣來源： 21
二、藥品及酵素樣品 23
三、培養基 25
四、酵素試劑與緩衝溶液 27
第二節、實驗方法 28
一、菌株分離及篩選 28
二、菌株之保存及培養 29
三、菌種鑑定 30
四、菌株最佳生長條件之探討 30
五、酵素活性及生化特性分析 31
六、酵素水解酵素之生化特性分析 33
七、纖維素水解酵素對纖維性原料降解率之評估 35
第三章、實驗結果與討論 36
一、菌株篩選 36
二、菌株最佳生長條件之探討 38
三、酵素活性測定及其生化特性分析 47
四、纖維素水解酵素對纖維性原料降解率之評估 51
第四章、結論 55
參考文獻 57

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