本研究為利用培養基組成份不同，誘導Bacillus subtilis YJ-3生產選擇性胞外酵素水解大豆溶液，併用乳酸菌Lactobacillus plantarum BCRC 10069發酵24小時，探討發酵過程中大豆蛋白水解情形，進一步分析可溶性蛋白質、胜?及總游離胺基酸含量，並探討發酵產物經不同分子量劃分後與金屬鈣離子螯合情形，藉以研發富含機能性螯合金屬離子胜?飲品。結果顯示Bacillus subtilis YJ-3 在複合培養基 (含skim milk 1.0%、soya meal 1.0%、sucrose 1.0%、NaCl 0.5%、K2HPO4 0.25%) 於37oC、150 rpm 震盪培養3天後，中、鹼性蛋白?活性為680.6、567.9U/mL. min。利用該酵素液於50oC 水解大豆蛋白溶液6小時，pH由 7.38降至5.81，加入 0.1% 蔗糖經乳酸菌 (LAB) 發酵24小時後，其可溶性蛋白質由23.9 mg/mL增加至 24.6 mg/mL、胜?含量由 5.1 mg/ml上升到 12.3 mg/ml及總游離胺基酸含量由 2.6 mg/mL上升至 4.4 mg/mL，顯示乳酸菌在發酵過程中可降解蛋白質。大豆蛋白經胞外液水解6小時及併用Lactobacillus plantarum BCRC 10069 發酵24小時，再利用切向流式超過濾系統將濾液依分子量大小劃分MW為 <1 kDa、1-5 kDa及 >5 kDa，進行噴霧乾燥，則每克可溶性蛋白質其鈣含量分別為 139.03、71.60及 50.91mg，噴乾後胜?鈣粉分子量小於 1 kDa的抗氧化能力為最強，其清除 DPPH 能力最高為94%，總抗氧化能力 (TEAC) 為 3.50 mM 且還原力最高，顯示胜?鈣粉具抗氧化機能性。根據上述實驗結果顯示大豆蛋白經 Bacillus subtilis YJ-3 酵素液水解及併用乳酸菌發酵後，能有效使蛋白質水解，以 <1 kDa 以下的胜?其螯合鈣離子能力最佳且抗氧化能力亦最強。

Abstract
The purpose of this study was to investigate the optimal media for Bacillus subtilis YJ-3 to produce proteases and its application in the hydrolysis of soybean proteins, which could consequently enhance their functionalities. The optimal medium for Bacillus subtilis YJ-3 was investigated based on the composition of tryptic soy broth (TSB). The highest neutral (680.6 U/mL•min) and alkaline (567.9 U/mL•min) proteases activities were observed in that incubated in modified medium containing 1% skim milk, 1% soybean meal, 1% sucrose, 0.5% NaCl, 0.25% K2HPO4 after 72 hr incubation at 37oC, 150 rpm. The pH of hydrolysates of soybean decreased from 7.38 to 5.81 after 6 hr hydrolysis using selective enzymes of 3 days incubated Bacillus subtilis YJ-3 at 50oC. During 6 hr hydrolysis, peptides and total free amino acids significantly increased, compared with those without hydrolysis. The soluble proteins and peptides increased from 23.9 to 24.6 mg/mL and 5.1 to 12.3 mg/mL, respectively (P<0.05), while free amino acids increased from 2.6 to 4.4 mg/mL after 24 hr LAB fermentation at 37oC. The peptides with MW less than 1000 Da after 24 hr LAB fermentation could chelate calcium (139.3 mg/g), while those between 1000 and 5000 Da could only chelate 71.60 mg/g. The peptides with MW higher than 5000 Da could only chelate 50.91 mg of calcium/g of soluble proteins or peptides. According to the results obtained from this study, the peptides with MW <1000 Da showed the highest DPPH scavenging ability, calcium chelating activity, TEAC and reducing power.

目錄
頁次
頁次 I
摘要 1
Abstract 3
壹、前言 4
貳、文獻整理 6
一、蛋白質水解產物之製備及應用 6
(A) 蛋白質水解產物的製備方法 6
1. 酸、鹼水解 (化學法) 6
2. 酵素法 7
3. 微生物發酵水解法 8
(B) 蛋白?種類與特性 8
(C) 蛋白?在工業上的應用 9
(D) 蛋白質水解產物之生理活性 9
二、大豆之營養價值 10
(A) 大豆簡介 10
(B) 大豆蛋白水解物 11
三、乳酸菌之特性 12
四、礦物質的生理作用 16
五、鈣的生理功能 20
?、實驗材料與方法 23
實驗設計 23
一、實驗材料 24
(A)原料及菌株 24
1. 原料 24
2. 實驗之菌株 24
(B)主要儀器 24
(C)主要藥品 25
二、實驗方法 27
Ι. 酵素液之製備 27
(A) 菌種的活化與保存 27
(B)培養基組成對 Bacillus subtilis YJ-3 生產蛋白?活性之影響 27
(1)不同氮源培養基組成 27
(2)不同碳源培養基組成 27
(C) 胞外液製備 28
1. 菌數之計數 28
2. pH 值之測定 28
ΙΙΙ. 乳酸菌發酵大豆水解物 29
(一) 大豆水解物之發酵 29
(二) 發酵期間生菌數及pH 值變化 29
(三) 乳酸菌發酵對大豆水解產物之影響 29
1. 可溶性蛋白質含量測定 29
2. 胜胜含量測定 30
3. 游離胺基酸含量測定 31
(四) 水解物及發酵產物之不同分子量劃分 31
1. 水解物及發酵產物的分子量劃分 31
2. 游離胺基酸分析 31
3. 胜?鈣含量測定 32
(五) 胜?電泳分析 32
(六) 膠體過濾 33
1. 膠體過濾層析法 33
2. 分子量標定 34
(七) 發酵產物之抗氧化能力測定 34
1. 清除 a, a-Diphenyl-b-picrylhydrazyl (DPPH)自由基能力測定 34
2. 還原力測定 35
3. Trolox equivalent antioxidant capacity (TEAC) 36
(八) 統計分析 37
肆、結果與討論 38
ㄧ、YJ-3菌株之鑑定 38
二、Bacillus subtilis YJ-3生長曲線及pH變化 38
三、培養基組成對 Bacillus subtilis YJ-3 生產蛋白?活性之影響 39
四、Bacillus subtilis YJ-3 胞外酵素液水解大豆之最適條件 40
五、大豆水解物併用乳酸菌發酵 41
(1) 發酵期間 pH 值及乳酸菌數的變化 41
(2) 乳酸菌發酵對大豆蛋白質之影響 41
六、大豆水解物及發酵產物之劃分 42
a. 不同分子量胜?的鈣螯合能力 42
b. 胜?電泳 (Tricine-SDS-PAGE) 分析 43
c. 游離胺基酸 (Free amino acid, FAA) 分析 43
d. 膠體過濾層析 44
e. 不同分子量胜?溶液的抗氧化能力 45
(1) 清除 DPPH 自由基之能力 45
(2) 還原力 46
(3) Trolox equivalent antioxidant capacity 46
伍、結論 47
陸、參考文獻 48
表目錄
表一、Bacillus subtilis YJ-3之16S rDNA部分鹼基序列 58
表二、Bacillus subtilis YJ-3之微生物脂肪酸鑑定系統分析結果 59
表三、以TSB為基礎培養基改變氮源之中性蛋白?活性 60
表四、以TSB為基礎培養基改變氮源之鹼性蛋白?活性 61
表五、以1 % soymeal 和1% skim milk 為氮源改變碳源之中性蛋白?活性 62
表六、以1 % soymeal 和1% skim milk 為氮源改變碳源之鹼性蛋白?活性 63
表七、以1% soymeal和1% skim milk為氮源改變蔗糖濃度之中性蛋白?活性 64
表八、以1% soymeal和1% skim milk為氮源改變蔗糖濃度之鹼性蛋白?活性 65
表九、大豆蛋白溶液在蛋白?水解0、2、4、6小時後可溶性蛋白質、胜?含量、總游離胺基酸含量及pH值之變化 66
表十、不同蔗糖濃度對大豆蛋白水解物經乳酸菌37oC發酵24小時後菌數及pH值之變化 67
表十一、不同蔗糖濃度對大豆蛋白水解物經乳酸菌37oC發酵24小時後可溶性蛋白質、胜?與總游離胺基酸含量之變化 68
表十二、不同分子量大豆胜?溶液之螯合鈣含量 69
表十三、不同分子量之大豆胜?溶液游離胺基酸含量(mg/100g) 70
表十三、不同分子量之大豆胜?溶液游離胺基酸含量(mg/100g) (續) 71
圖目錄
圖一、Bacillus subtilis YJ-3掃描式電子顯微鏡照片 72
圖二、Bacillus subtilis YJ-3生長曲線及pH變化 73
圖三、不同分子量之大豆胜?溶液胜?電泳圖譜 74
圖四、MW >5 kDa大豆胜?溶液之Sephadex G-25膠體過濾層析圖譜 75
圖五、MW 1-5 kDa大豆胜?溶液之Sephadex G-25膠體過濾層析圖譜 76
圖六、MW <1 kDa大豆胜?溶液之Sephadex G-25膠體過濾層析圖譜 77
圖七、不同分子量之大豆胜?鈣溶液之清除DPPH自由基能力 78
圖八、不同分子量之大豆胜?鈣溶液之還原力 79
圖九、不同分子量之大豆胜?鈣溶液之TEAC值 80

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