臺灣博碩士論文加值系統

細菌素是由細菌所產生具有抑菌效果的蛋白質，可以殺菌或者抑制他種細菌的生長，目前被認為在食品工業上極具發展潛力。本研究室自臺北縣市各地採集約 80 個樣品進行篩選分離，經初步篩選得到 24 個可生成具有抑菌活性的樣品。同時，使用購買自食品工業發展研究所的菌株 Bacillus subtilis BCRC 10447 建立檢測抑菌活性的條件。再將篩選所得具有抑菌活性的菌株單株分離後，進行檢測。菌株 6-1-1 所生產之細菌素於檢測中發現具有較廣泛的抑菌範圍，可抑制格蘭氏陽性菌 ( Micrococcus luteus 和 Bacillus subtilis ) 及陰性菌 ( Escherichia coli )，因此選擇其作為本研究之篩選菌株。根據細胞染色、生化測試及 16S rDNA 序列分析比對結果，初步鑑定菌株 6-1-1 屬於 B. subtilis。選擇 tryptic soy broth ( TSB ) 為基礎培養基，探討本菌株之最適生產條件。三角瓶培養結果：於 TSB 培養基中加入 2% 之 KH2PO4 及 0.25% 之 tryptone，同時起始 pH 值控制在 7.0，進行 30 oC 下的培養，可以有效將菌體量 ( OD600 ) 從 6.0 提升至 8.2，抑菌活性也由 640 A.U./mL 提高至 2560 A.U./mL。

Bacteriocins are proteinaceous compounds lethal to bacteria other than the producing strain. As a group, they are considered having potential application on the food industry. The purposes of this study were collecting of Bacillus subtilis ATCC 6633 ( BCRC 10447 ) from BCRC to set up the antimicrobial detection model and using the model to screen and isolate the bacteria from 80 samples including waters, soils, and sands around Taipei city. First, there were 24 bacteria selected by initial screening. Finally we chose strain 6-1-1 which has the broader activity spectrum, including Micrococcus luteus, Bacillus subtilis G(+), and Escherichia coli G(-). Beside antimicrobial activity analysis, we had accomplished bacteria identification according to its morphological characters and biochemical activities. Strain 6-1-1 is classified to Bacillus subtilis. Then we tried to optimize the growth conditions. The optimal medium compositions are adding 0.25% tryptone, and 2% KH2PO4 in TSB, controlled pH at 7.0. Furthermore, the optimal growth conditions are culturing with 50 mL medium in a 250 mL Erlenmeyer flask at 30 oC. Under these conditions, biomass concentrations expressed as OD600 were increased from 6.0 to 8.2, and the antimicrobial activity were increased from 640 A.U. mL-1 to 2560 A.U. mL-1.

第一章 緒 論…………………………………………………………………………1
一、細菌素…………………………………………………………………………1
1-1 細菌素之起源及分類……………………………………………………1
1-2 影響細菌素生成之因子…………………………………………………5
1-3 細菌素之應用……………………………………………………………7
二、研究動機與目的……………………………………………………………11
三、研究架構……………………………………………………………………12
第二章 材料與方法…………………………………………………………………13
一、藥品試劑……………………………………………………………………13
二、實驗菌株……………………………………………………………………13
2-1 指標菌株…………………………………………………………………13
2-2 篩選菌株…………………………………………………………………13
三、培養方法……………………………………………………………………16
3-1 三角瓶搖瓶培養…………………………………………………………16
3-2 最適條件的探討…………………………………………………………17
四、分析方法……………………………………………………………………18
4-1 菌體濃度測試……………………………………………………………18
4-2 抑菌活性測試……………………………………………………………18
4-3 篩選菌株初步鑑定………………………………………………………20
五、儀器設備……………………………………………………………………22
六、引子…………………………………………………………………………22
第三章 結果與討論…………………………………………………………………23
一、 進行採樣及採樣地點資料整理……………………………………………23


二、以生產具有抑菌活性的細菌素 subtilin 之 Bacillus subtilis BCRC 10447建立檢測抑菌活性的條件…………………………………………………24
2-1 建立確定具有抑菌活性菌株流程………………………………………24
2-2 建立抑菌物質抑菌活性量化流程………………………………………27
三、野生菌株分離及篩選………………………………………………………29
四、分離菌株初步鑑定…………………………………………………………34
五、分離菌株之最適生長條件…………………………………………………40
5-1 最適培養基的選擇………………………………………………………40
5-2 磷酸鹽類添加濃度之探討………………………………………………40
5-3 碳源添加型式之探討……………………………………………………40
5-4 氮源添加型式之探討……………………………………………………45
5-5 最適起始 pH 值之探討…………………………………………………49
5-6 最適培養溫度之探討……………………………………………………49
5-7 最適通氣量之探討………………………………………………………52
第四章 結 論………………………………………………………………………54
第五章 參考文獻……………………………………………………………………56

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