臺灣博碩士論文加值系統

中文摘要

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。

Abstract

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.

目次

頁次
中文摘要……………………………………………………… i
英文摘要……………………………………………………… ii
壹、 前言 …………………………………………………… 1
貳、 文獻整理 ……………………………………………… 2
一、 蛋白酶…………………………………………… 2
(一) 蛋白酶的來源………………………………… 2
(二) 蛋白酶的分類………………………………… 2
(三) 蛋白酶的應用………………………………… 2
二、 大豆………………………………………………… 6
(一) 大豆之成分與營養價值……………………… 6
(二) 大豆蛋白的種類……………………………… 9
(三) 大豆中的過敏蛋白與抗營養因子…………… 14
(四) 大豆過敏蛋白的去除方法…………………… 14
三、 大豆粕……………………………………………… 16
(一) 大豆粕的製造過程…………………………… 16
(二) 大豆粕的應用………………………………… 16
參、 材料與方法 …………………………………………… 20
一、 實驗材料…………………………………………… 20
(一) 細菌菌株……………………………………… 20
(二) 藥品與培養基…………………………………… 20
(三) 儀器設備………………………………………… 21
(四) 電腦軟體………………………………………… 22
二、 實驗方法 ………………………………………… 25
(一) 大豆蛋白之製備……………………………… 25
(二) 篩選具有分解大豆蛋白之菌種……………… 25
(三) 蛋白質含量之測定…………………………… 25
(四) 菌數之測定…………………………………… 27
(五) NB-10菌株之鑑定…………………………… 27
(六) Bacillus subtilis NB-10之protease存在位置
探討…………………………………………… 28
(七) 菌株生長條件探討…………………………… 28
(八) 蛋白酶活性之測定…………………………… 29
(九) 蛋白酶最適作用條件測定…………………… 29
(十) 大豆蛋白液之大豆蛋白分解情形…………… 30
(十一) 豆粕發酵……………………………………… 30
(十二) 蛋白質電泳樣品製備………………………… 33
(十三) 蛋白質電泳條件……………………………… 33
(十四) 西方轉漬法…………………………………… 33
肆、 結果與討論…………………………………………… 35
一、 不同種類樣品之篩菌結果……………………… 35
二、 篩選具有分解大豆蛋白之菌種………………… 35
三、 NB-10菌株之鑑定……………………………… 36
四、 B. subtilis NB-10之protease存在位置………… 36
五、 B. subtilis NB-10生長條件之探討……………… 43
六、 B. subtilis NB-10之protease特性……………… 44
七、 B. subtilis NB-10之protease對大豆蛋白液之大豆
蛋白分解效果……………………………………… 52
八、 B. subtilis NB-10於大豆粕之發酵試驗………… 52
九、 西方轉漬法………………………………………… 59
伍、 結論…………………………………………………… 70
陸、 參考文獻……………………………………………… 72
柒、 附錄…………………………………………………… 82









表目次

表次 頁次
表ㄧ、蛋白酶的分類………………………………………………… 4
表二、大豆之營養成分表…………………………………………… 7
表三、大豆之胺基酸組成表………………………………………… 8
表四、大豆蛋白組成份……………………………………………… 10
表五、大豆中的過敏蛋白…………………………………………… 15
表六、大豆粕之營養成分表………………………………………… 18
表七、大豆粕之胺基酸組成分表…………………………………… 19
表八、分離菌株之來源……………………………………………… 23
表九、不同來源樣品分離出可分解大豆蛋白之細菌株…………… 24
表十、不同菌株接種於大豆水蛋白液的菌數生長變化與大豆蛋白
的分解情形…………………………………………………… 38
表十一、B. subtilis NB-10胞外與胞內粗酵素液之蛋白酶活性比較
………………………………………………………………… 42
















圖目次

圖次 頁次
圖一、市售酵素比例分配表………………………………………… 3
圖二、大豆蛋白沉降片段…………………………………………… 11
圖三、大豆蛋白之SDS-PAGE電泳圖……………………………… 12
圖四、大豆蛋白glycinin的結構圖………………………………… 13
圖五、實驗架構圖…………………………………………………… 26
圖六、利用洋菜平板篩選可分解大豆水溶性蛋白質之細菌菌株… 37
圖七、不同菌株的蛋白酶活性比較………………………………… 39
圖八、B. subtilis NB-10於顯微鏡下的圖像………………………… 40
圖九、B. subtilis NB-10之16S ribosomal DNA gene序列………… 41
圖十、B. subtilis NB-10之不同接菌量(A) 1% (v/v) (B) 2% (v/v)
(C) 5% (v/v)於30℃培養條件下之生菌數、培養基pH與
蛋白酶活性之變化…………………………………………… 45
圖十一、B. subtilis NB-10於(A) 30℃及(B) 37℃的菌數生長、培養
基pH與蛋白酶活性之變化………………………………… 46
圖十二、B. subtilis NB-10蛋白酶活性之最適作用pH……………… 48
圖十三、B. subtilis NB-10蛋白酶活性之最適作用溫度…………… 49
圖十四、B. subtilis NB-10蛋白酶之熱穩定性……………………… 50
圖十五、pH對B. subtilis NB-10蛋白酶穩定性之影響……………… 51
圖十六、大豆蛋白液經B. subtilis NB-10粗酵素液處理之蛋白質電
泳圖………………………………………………………… 53
圖十七、大豆蛋白液經B. subtilis NB-10發酵後之蛋白質分解情形
……………………………………………………………… 54
圖十八、不同接菌量的B. subtilis NB-10對大豆粕蛋白的分解情形
……………………………………………………………… 56
圖十九、不同含水量對於大豆粕發酵的影響……………………… 57
圖二十、 不同發酵時間對於大豆粕蛋白的蛋白質變化…………… 58
圖二十一、B. subtilis NB-10於豆粕發酵過程中的生菌數、豆粕pH
及蛋白酶活性變化……………………………………… 61
圖二十二、豆粕經B. subtilis NB-10發酵30 hr的大豆蛋白分解情形
…………………………………………………………… 62
圖二十三、豆粕發酵過程中的粗蛋白含量變化…………………… 63
圖二十四、豆粕發酵過程中的胺基態氮含量變化………………… 64
圖二十五、西方轉漬法分析發酵豆粕之α’-subunit分解情形……… 65
圖二十六、西方轉漬法分析發酵豆粕之α-subunit分解情形……… 66
圖二十七、西方轉漬法分析發酵豆粕之β-subunit分解情形……… 67
圖二十八、西方轉漬法分析發酵豆粕之acidic polypeptide分解情形
…………………………………………………………… 68
圖二十九、西方轉漬法分析發酵豆粕之basic polypeptide分解情形
…………………………………………………………… 69

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