中文摘要
本研究擬探討Bacillus subtilis YJ-1生產蛋白之最適培養條件，再利用該酵素液對雞胸肉進行水解，評估所得水解物之抗氧化能力及胜與胺基酸含量。結果顯示Bacillus subtilis YJ-1在含1 % skim milk、1% soya meal、1 % rice、0.5 % Sucrose、0.5% NaCl之最適培養基下，於5L發酵槽培養60小時，納豆激活性達22.5 FU/ml，中、鹼性蛋白在修飾培養基:1% skim milk+3% soy meal+0.5% glucose+1% NaCl+0.05% K2HPO4 +0.05% MgCl•6H2O+0.1% glucoseamine培養72小時分別達27 unit/ml與197 unit/ml，pH 值由12小時為6.74，上升至72小時為8.23。利用該酵素液於50oC水解雞肉0-5小時，結果發現雞肉水解5小時後，產物胜含量由0.527mg/ml 隨水解時間增加至32.867 mg/ml。同時觀察SDS-PAGE 電泳分析雞肉於不同水解時間後蛋白質降解之變化，結果發現雞肉漿水解產物隨著水解時間增加大分子逐漸消失，於5小時雞肉漿中蛋白質分子明顯由大分子降解至小分子。雞肉經水解之游離胺基酸 (free amino acid, FAA) 含量之變化大部份 FAA 之含量皆有增加趨勢，其中以Glutamic acid、Isoleucine、β-Alanine、γ-ABA、Ornithine 之增加量最為明顯。在雞肉水解物中也發現有γ-ABA( γ-aminobutyric acid)(GABA)存在為一神經傳導物質，有降血壓、抗憂鬱之功效其雞肉水解物中以添加失活酵素水解獲得的0.07 mg/100g 經水解5小時後上升達2.49 mg/100g。雞肉水解物之抗氧化活性試驗，雞肉水解物捕捉DPPH自由基的能力隨著水解時間增加而增加，於5小時水解後其清除能力由24% 增加至55%。雞肉漿水解物之還原力，在水解2小時後吸光值為最高達 0.36 nm，有小幅提升還原力的效果。

Abstract
To investigate the optimum conditions for the production of protease by Bacillus subtilis YJ-1, the medium containing 1 % skim milk, 1 % soya meal, 1 % rice, 0.5 % sucrose, 0.5% NaCl seemed to the best medium for Bacillus subtilis YJ-1 fermentation in 5-L fermentor. The Nattokinase activity was 22.5 FU/mL after 60 hrs fermentation. The hightest neutral and alkaline proteases was obtained when the medium was the mixture of 1% skim milk, 3% soy meal, 0.5% glucose, 1% NaCl, 0.05% K2HPO4, 0.05% MgCl•6H2O and 0.1% glucoseamine. Their activies were 27 and 197 unit/mL, respectively, and the pH increased from 6.74 to 8.23 after 72 fermentation at 37oC. The collected enzymes from 72-hr fermented broth were employed to hydrolyze the chicken. The soluble peptides increased from 0.527mg/ml to 32.867 mg/ml after 5 hrs hydrolysis at 50oC. SDS-PAGE indicated the obvious degradation of chicken protein was observed during 5 hrs hydrolysis at 50 oC by enzymes from Bacillus subtilis YJ-1 after 72 hr fermentation. The free amino acids (FAA) of hydrolyzed chicken, especially glutamic acid, isoleucine, β-alanine, γ-ABA and ornithine increased significantly with the duration of hydrolysis. The DPPH scavenging capability and reducing power of the hydrolysate significantly increased after 5 hrs fermentation. This phenomenon indicated that chicken could be substantially hydrolyzed by Bacillus subtilis YJ-1 proteases and subsequently increased the antioxidative ability.

目錄
目錄----------------------------------------------------------------------------------I~VII
表目錄-----------------------------------------------------------------------------------IV
圖目錄------------------------------------------------------------------------------------V
中文摘要--------------------------------------------------------------------------------VI
英文摘要-------------------------------------------------------------------------------VII
第一章 前言----------------------------------------------------------------------------1
第二章 文獻回顧----------------------------------------------------------------------2
一、枯草桿菌簡介----------------------------------------------------------------------2
(一)生理特性--------------------------------------------------------------------------2
(二)Bacillus 的分類-----------------------------------------------------------------2
(三) Bacillus subtilis 分泌之酵素及產業上之應用----------------------------3
1、蛋白(Proteases) ---------------------------------------------------------------3
2. 血栓溶解酵素－納豆激(Nattokinase) -------------------------------------5
(一)、納豆激的發現及其特性--------------------------------------------------5
(二)、血液凝固(Blood Clotting) 與血栓溶解之關聯-------------------------5
(三)、納豆激之安定性及功能性----------------------------------------------7
(四)、含有血栓溶解酵素之發酵產品-------------------------------------------7
3、α-澱粉(-Amylase)-----------------------------------------------------------8
4、其他酵素-----------------------------------------------------------------------8
二、微生物之增殖及營養需求------------------------------------------------------9
1、碳源-----------------------------------------------------------------------------9
2、氮源-----------------------------------------------------------------------------10
3. 維生素與微量元素------------------------------------------------------------10
三、蛋白質水解及其水解物利用--------------------------------------------------10
四、抗氧化活性-----------------------------------------------------------------------12
1.自由基---------------------------------------------------------------------------------12
2.生物體之抗氧化防禦系統---------------------------------------------------------13
3 抗氧化劑的作用機制--------------------------------------------------------------13
(1) 自由基終止型(Free radical terminators) -------------------------------------13
(2) 還原劑或氧清除劑者( Reducing agents or oxygen scavengers) ---------13
(3)螯合劑型(Chelating agents) -----------------------------------------------------14
第三章 材料與方法------------------------------------------------------------15
第一節 實驗材料-------------------------------------------------------------------15
第二節 實驗流程-------------------------------------------------------------------17
第三節 實驗方法-------------------------------------------------------------------18
一、 菌株之活化與保存 -----------------------------------------------------------18
二、 菌株培養基組成對菌種生長之影響 --------------------------------------18
1、菌數之計數 -----------------------------------------------------------------18
2、pH值之測定 -----------------------------------------------------18
3、酵素活性測定--------------------------------------------------18
三、Bacillus subtilis 之粗酵素液製備--------------------------------------------20
四、酵素水解物之製備 ------------------------------------------------------------20
五、水解物之成份變化-------------------------------------------------------------20
1、可溶性蛋白質含量測定----------------------------------------------------20
2、胜含量測定----------------------------------------------------------------21
3、游離胺基酸分析- -------------------------------------------------------------21
4、電泳分析----------------------------------------------------------------------22
六、水解物之抗氧化探討 ----------------------------------------------------------22
1. 清除DPPH自由基能力之測定----------------------------------22
2. 還原力測定-------------------------------------------------------------------22
3. Trolox equivalent antioxidant capacity (TEAC)--------------------------23
4. 螯合亞鐵離子能力----------------------------------------------------------23
七、統計分析-------------------------------------------------------------------------23
第四章 結果與討論----------------------------------------------------------24
一、菌種之生理特性探討----------------------------------------------------------24 1.Bacillus subtilis YJ-1之生長情形----------------------------------------------24
二、培養基組成對生產Nkase活性之影響--------------------------------------24
(1)最適碳、氮源-----------------------------------------------------------------------24
(2)以濃縮方式提高Nkase之活性--------------------------------------------------24
三、利用發酵槽擴大量產------------------------------------------------------------25
(1)添加酸與鹼控制pH對其生產Nkase之活性影響---------------------------25
(2)修飾培養基組成對其生產蛋白之活性影響--------------------------------25
(3)修飾培養基對其中、鹼性蛋白活性影響-----------------------------------27
四、雞肉水解物之成份變化---------------------------------------------------------27
(1) 可溶性蛋白及胜含量之變化------------------------------------------------27
(2) 游離胺基酸分析------------------------------------------------------------------28
(3)電泳分析-----------------------------------------------------------------------------29
五、雞肉水解物之抗氧化活性試驗------------------------------------------------29
(1)清除α, α-diphenyl-β-picrylhydrazyl (DPPH)自由基能力--------------------29
(2)還原力--------------------------------------------------------------------------------30
(3) Trolox equivalent antioxidant capacity (TEAC) ------------------------------30
(4)螯合亞鐵離子能力-----------------------------------------------------------------30
第五章 結論--------------------------------------------------------------------------32
參考文獻--------------------------------------------------------------------------------55





表目錄

表一 商業用細菌性鹼性蛋白之商品名、來源及應用--------------------33
表二 以不同碳、氮源培養Bacillus subtilis YJ-1 3天NKase與
pH變化情形-----------------------------------------------------------------34
表三 以表二之E組配方進行濃縮NKase之變化情形----------------------35
表四 Bacillus Subtilis YJ-1於5L發酵槽量產之NKase之變化情形------36
表五 Bacillus Subtilis YJ-1於5L發酵槽量產，發酵第36小時添加
1%黃豆粉之NKase之變化情形------------------------------------------37
表六 水解過程水解物中可溶性蛋白及胜含量之變化-------------------38
表七 雞肉水解產物於及水解期間之游離胺基酸含量(mg/100 g)變化--39

圖目錄

圖一 納豆菌培養液的人工血栓溶解活性-----------------------------------40
圖二 納豆激之組成結構-----------------------------------------------------41
圖三 血塊形成步驟--------------------------------------------------------------42
圖四 水解血栓中Fibrin之機制------------------------------------------------43
圖五 納豆激對血栓溶解的變化情形-------------------------------------44
圖六 Bacillus subtilis YJ-1以TSA在37 oC培養36小時後之菌相------45
圖七 Bacillus subtilis YJ-1以TSB培養3天其菌數、pH變化情形-------46
圖八 不同發酵時間之NKase於人工血纖維蛋白平板試驗---------------------47
圖九 Bacillus subtilis YJ-1以修飾培養基於37 oC培養3天生菌數
、pH變化情形----------------------------------------------------------------48
圖十 Bacillus subtilis YJ-1於修飾培養基培養72hr中、鹼性蛋白
活性之變化------------------------------------------------------------------49
圖十一 利用Bacillus subtilis YJ-1胞外酵素水解雞肉漿之
SDS-PAGE分析圖--------------------------------------------------------50
圖十二 雞肉水解物於50℃水解5小時清除DPPH自由基之變化---------51
圖十三 雞肉水解物於50℃水解5小時之還原力變化-----------------------52
圖十四 雞肉水解物於50℃水解5小時TEAC值之變化---------------------53
圖十五、雞肉水解物於50oC水解5小時之螯合亞鐵離子能力之變化------54

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