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|標題:||Bacillus subtilis NB-10之蛋白酶特性分析及其在飼料豆粕發酵的應用
Characterization of proteases from Bacillus subtilis NB-10 and their application on soybean meal fermentation
In this study, bacteria with the enzymes for the hydrolysis of soybean antigenic proteins (mainly glycinin and beta-conglycinin) were screened and isolated from natto, animal fecal, soil samples and reference bacteria by using agar plate containing soluble soybean meal proteins. Eighty-three bacterial strains able to hydrolyze soybean soluble proteins were isolated. The protease activities of these isolates were further compared by Bradford protein assay method, and seven isolates and three reference bacteria showed higher enzyme activity. The protease activities of ten strains were further compared by protease assay and NB-10 isolates showing the higher enzyme activity. NB-10 was designated as Bacillus subtilis NB-10 by DNA sequencing and chosen for further study.
An optimum shaken flask cultivation condition for B. subtilis NB-10 was achieved, with the Tryptic Soy Broth as growth medium, the optimum temperature was 30℃. The bacterial cell got into stationary phase and the protease started to be produced after 12hr and showed the highest protease activity after 32hr of incubation at the experimental condition. The protease of B. subtilis NB-10 was extracellular protease and the optimal pH and temperature were 11 and 60℃, respectively. The protease of B. subtilis NB-10 could retain good stability under pH 6~8 and 30~37℃, respectively.
SDS-PAGE analysis results demonstrated that the β-conglycinin and glycinin in soybean meal protein broth were eliminated after 12-hr fermentation with B. subtilis NB-10. Besides this, α'-subunit, α-subunit and β-subunit of β-conglycinin and acidic polypeptide of glicinin within soybean meal were eliminated after a 24-hr solid state fermentation with B. subtilis NB-10. Western blot analysis further proved that B. subtilis NB-10 could eliminate α'-subunit, α-subunit and β-subunit of β-conglycinin and acidic polypeptide of glycinin within soybean meal. For these reasons, B. subtilis NB-10 isolated from natto had the capacity to degrade allergenic proteins and had the potential to be applied in soybean meal fermentation in the future.
本研究利用大豆水溶性蛋白洋菜平板，由納豆、動物糞便、土壤及標準菌株中篩選能夠分解大豆過敏性蛋白質的細菌，共篩選出具有分解大豆水溶性蛋白之細菌83株。利用Bradford蛋白質定量方法比較分離株分解大豆蛋白的能力高低，從中挑選具有高分解力的分離株7株與標準菌株3株，進一步再比較此10株菌株的蛋白酶活性，結果顯示，編號NB-10的分離株其蛋白酶活性表現最高, 經由定序結果將其命名為Bacillus subtilis NB-10並進行後續實驗。
利用搖瓶探討B. subtilis NB-10培養條件的結果顯示，以TSB (Tryptic soy broth)為培養基質，其適合的生長溫度為30℃，培養12hr後菌株的生長開始趨於平緩，蛋白酶也開始生成，在培養32hr後具有最高的蛋白酶活性。B. subtilis NB-10分泌的蛋白酶為胞外蛋白酶，蛋白酶之最適反應條件為pH 11與60℃，在pH 6~8與30~37℃之穩定性較佳，顯示在中性環境及低溫的環境下，蛋白酶可維持較高的穩定性。
接種B. subtilis. NB-10於大豆蛋白液進行12-hr液態培養，由SDS-PAGE分析結果顯示，接菌培養可有效的去除大豆蛋白液中的過敏蛋白β-conglycinin與glycinin；另外，實際應用於大豆粕進行24-hr固態發酵試驗，經SDS-PAGE分析結果顯示，大豆粕的過敏蛋白β-conglycinin與glycinin中acidic polypeptide有被分解的現象，對照西方轉漬法的結果，經過B. subtilis NB-10發酵的豆粕確實不會引起β-conglycinin與glycinin的acidic polypeptide的免疫反應。因此，由納豆中篩選的B. subtilis NB-10具有分解致過敏蛋白β-conglycinin與glycinin的能力，未來應具有應用於飼料豆粕發酵之潛力。
|Appears in Collections:||食品暨應用生物科技學系|
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