第一部分 利用酵素水解明膠製備抗高血壓胜肽
養豬是台灣主要的畜產事業，其副產物之豬皮產量多，善加利用，可提高經濟效益，也可增加飼主收入。從豬皮萃取的明膠(Gelatin)應用於食品、醫藥、工業等方面。但用量有限，經濟價值有待提升。Gelatin的胺基酸序列單純，含有多重再現之規則排序(Pro — X — Gly -Pro )，本研究利用合適的酵素水解可獲得抗高血壓抑制胜肽，將之應用於添加飲料或食物中，因而製備具有保健效果之食物。初步選用五種蛋白酵素：collagenase、trypsin、chymotrypsin、esperase和alcalase水解明膠反應，在初期二小時內，trypsin的效果優於其他蛋白酵素；在四到八小時之水解反應，則esperase最好，其次是collagenase。以時間及價格考量下，選用trypsin及esperase作進一步研究。在不同esperase濃度及水解時間的動力學研究中，添加E / S = 0.2% (1/500 w/w) esperase用量及水解是最經濟及省時的。選用的條件為2 %明膠(pH 8.0)先在37 ºC下以0.2 % Trypsin水解1 hr，再以60 ºC添加0.2 % esperase水解2 hr。在此條件下的水解產物具有60 % ACE抑制作用。水解液顏色淡黃，總生菌數為TFTC，無苦味而有淡淡異味，其冷凍乾燥後的粉末呈淡黃色。L、a、b值為96.58、1.79及5.27。經超過濾處理的分區產物，MWCF 3000其抑制率為77.3 %。水解液經胃、胰液酶之消化作用後，抑制活性可提高至62.7及74.8 %。
第二部分 利用酵素水解卵白蛋白製備抗高血壓胜肽
雞蛋的營養價值高，所含的蛋白質擁有平衡胺基酸組成，故常被用來當做蛋白質品質評估的標準品。因為蛋黃含有較高的膽固醇，因此全蛋的食用大受限制。若將蛋白加工製成機能性食品，如：雞精、營養補充品等，將可提高雞蛋的附加價值及促進銷售量，並提高飼主收入。本研究的主要目的是篩選合適的蛋白質酵素水解雞蛋卵白蛋白(ovalbumin)，得到之水解液再經過連續的超過濾(ultra — filtration)，製備出具有抗高血壓效果的小分子胜肽片段，將之添加在食品中，除了作為保健食品外，另可提升副產品的經濟效益。實驗結果顯示：最佳的水解條件為使用E / S = 0.2 % (1 / 500 w / w) thermolysin水解1 % ovalbumin，在60℃，pH 8，反應4 hr中，其IC50 為33 μg / ml，產率為87 %。水解液色澤呈淺黃色。L、a及b值為99.27、— 0.94 及5.82。無微生物生長。以1 % (w/v)添加入牛乳中，經官能評估結果無苦味，整體接受度高。水解液對胃蛋白酶有良好安定性，再經胰液酶之消化作用，能提高其抑制活性(約104 %)。而水解液經過超過濾不同分子量膜限值分離(MWCF 10000, 3000及1000)，IC50分別為58、43、25 及14 μg / ml。

Part I Production of angiotensin I—converting enzyme
inhibitory peptides from gelatin by protease
Growing pigs is a major animal industry in Taiwan and the yield of porcine skins is excess. If we take good advantage of them, breeders will increase incomes and get high economy value. The gelatins from porcine skins have been used in food, medicine and industry. However its applicable quantity and value is still contains needed to be promoted. The amino acid sequence of gelatin is simple and regular repetition sequences (Pro — x — Gly — Pro). The objective of this investigation is to prepare Angiotensin I — converting enzyme inhibitory peptide hydrolysate from gelatin by using suitable proteases. And the hydrolysates could be added to drink or food as health food. Five commercial proteases have been tested in studying production of ACE inhibitory peptide from porcine skin gelatin. Trypsin showed much better than others in production of ACE inhibitor in first two-hour hydrolysis and esperase turned out to be the best one and collagenase next in following four — eight hours hydrolysis. Base on the cost of enzyme and time consumption in hydrolysis, trypsin and esperase were chose in production of ACE inhibitory peptide. Results of kinetic study indicated that optimal condition to hydrolyze 2 % gelatin solution (pH 8) was digested first by 0.2 % trypsin for 1hr at 37 ºC, then 0.2 % esperase for 2 hr at 60 ºC to have inhibition activity around 60 %. The final hydrolysate is pale yellow in color, blend in taste, and no microbial contamination. L , a , and b of hydrolysate were found to be 96.58 , 1.79 and 5.27 respectively. Freeze — dried powder is pale yellow in color. Further fractionation of hydrolysate by ultra-filtration, ACE inhibition of fractions of MWCF 3000 was found to be 77 % ACE inhibition. Inhibition of activity hydrolysate was stable to pepsin digestion, and increased by pancreatin digestion.
Part II Production of angiotensin I-converting enzyme
inhibitory peptides from ovalbumin by protease
Eggs are high nutritive food with balance amino acids. Because of high cholesterol content, eating whole eggs is limited. The objective of this work is to prepare Angiotensin I — converting enzyme inhibitory peptide from egg white by suitable proteases. And the hydrolysates can be added on drink or food to promote its economy benefit. The experiment result showed the optimal condition for hydrolysis was that 1 % ovalbumin solution (pH 8) was digested by 0.2 % thermolysin for 4 hr at 60 ºC to have IC50 value 33 μg / ml (yield of 87 %). IC50 of hydrolysate filtrate from further fractionation with MW cut — off 1000, 3000, and 1000 were found to be 58、43、25 and 14 μg/ml respectively. Hydrolysate is pale yellow in color and bland in taste with high acceptability. Inhibition activity of hydrolysate was found stable to pepsin digestion, and pancreatin digestion. The two substrates are prepared ACEI. And the result shows that ACEI from ovalbumin is better.

目錄
頁次
中文摘要．．．．．．．．．．．．．．．．．．．．．．．．．． I
英文摘要．．．．．．．．．．．．．．．．．．．．．．．．．．III
壹、前言．．．．．．．．．．．．．．．．．．．．．．．．．． 1
貳、文獻整理．．．．．．．．．．．．．．．．．．．．．．．． 3
一、基質之介紹．．．．．．．．．．．．．．．．．．．．．．． 3
1. 明膠簡介．．．．．．．．．．．．．．．．．．．．．．．． 3
2. 卵白蛋白簡介．．．．．．．．．．．．．．．．．．．．．． 4
二、酵素水解蛋白質．．．．．．．．．．．．．．．．．．．．． 7
1. 蛋白質水解目的．．．．．．．．．．．．．．．．．．．．． 7
2. 良好水解系統之建立．．．．．．．．．．．．．．．．．．． 7
3. 基質濃度．．．．．．．．．．．．．．．．．．．．．．．． 8
4. 蛋白酶．．．．．．．．．．．．．．．．．．．．．．．．． 8
5. 酵素與基質之比例．．．．．．．．．．．．．．．．．．．． 9
6. pH值與溫度．．．．．．．．．．．．．．．．．．．．．．． 9
7. 水解反應時間．．．．．．．．．．．．．．．．．．．．．． 9
8. 水解系統之控制．．．．．．．．．．．．．．．．．．．．． 10
三、蛋白質水解物之抗高血壓作用(anti—hypertension) ．．．． 10
四、高血壓(Hypertension) ．．．．．．．．．．．．．．．．． 11
1. 定義．．．．．．．．．．．．．．．．．．．．．．．．．． 11
2. 分類．．．．．．．．．．．．．．．．．．．．．．．．．． 13
3. 治療高血壓．．．．．．．．．．．．．．．．．．．．．．． 13
五、血管收縮素轉化酶．．．．．．．．．．．．．．．．．．． 16
1. 簡介．．．．．．．．．．．．．．．．．．．．．．．．．． 16
2. 腎素 — 血管收縮素系統．．．．．．．．．．．．．．．．． 17
六、血管收縮素轉化酶抑制劑．．．．．．．．．．．．．．．． 19
1. ACE的抑制原理．．．．．．．．．．．．．．．．．．．．． 19
2. ACEI的來源．．．．．．．．．．．．．．．．．．．．．．． 19
第一部分 利用酵素水解明膠製備抗高血壓胜肽．．．．．．．．． 29
參、材料與方法．．．．．．．．．．．．．．．．．．．．．． 29
一、材料與試藥．．．．．．．．．．．．．．．．．．．．．． 29
二、儀器設備．．．．．．．．．．．．．．．．．．．．．．． 30
三、實驗步驟．．．．．．．．．．．．．．．．．．．．．．． 32
1. 酵素成品中蛋白質含量分析．．．．．．．．．．．．．．．． 32
2. 酵素水解反應．．．．．．．．．．．．．．．．．．．．．． 32
3. 血管收縮素轉化酶抑制活性之測定．．．．．．．．．．．．． 34
4. 高效能液相層析之分析條件．．．．．．．．．．．．．．．． 36
5. 總氮(Total nitrogen)之測定．．．．．．．．．．．．．．． 36
6. 色澤分析(Color)．．．．．．．．．．．．．．．．．．．． 36
7. 總生菌數測定(Total plate count)．．．．．．．．．．．． 37
8. 官能評估(Sensory Evaluation)．．．．．．．．．．．．．． 37
9. 超過濾區分水解物(Ultra—filtration)．．．．．．．．．． 37
10. 分子篩試驗(Gel filtration chromatography)．．．．．．． 38
11. 水解物經胃、腸道酵素之消化模擬試驗．．．．．．．．．． 38
肆、結果與討論．．．．．．．．．．．．．．．．．．．．．． 39
一、明膠水解物最適條件之建立．．．．．．．．．．．．．．． 39
二、水解液之性質分析．．．．．．．．．．．．．．．．．．． 51
三、超過濾區分物對ACE之抑制活性作用．．．．．．．．．．．． 56
四、超過濾區分物之分子量分佈．．．．．．．．．．．．．．． 57
五、胃腸酵素對水解物之ACE抑制活性作用．．．．．．．．．．． 59
第二部分 利用酵素水解卵白蛋白製備抗高血壓胜肽．．．．．．． 61
參、材料與方法．．．．．．．．．．．．．．．．．．．．．． 61
一、材料與試藥．．．．．．．．．．．．．．．．．．．．．． 61
二、儀器設備．．．．．．．．．．．．．．．．．．．．．．． 61
三、實驗步驟．．．．．．．．．．．．．．．．．．．．．．． 62
1. 酵素水解反應．．．．．．．．．．．．．．．．．．．．． 62
2. 半抑制濃度(IC50)之測定．．．．．．．．．．．．．．．． 63
3. 分子量分布之測定．．．．．．．．．．．．．．．．．．． 63
4. 統計分析．．．．．．．．．．．．．．．．．．．．．．． 64
肆、結果與討論．．．．．．．．．．．．．．．．．．．．．． 65
一、卵白蛋白水解之最適條件．．．．．．．．．．．．．．．． 65
二、水解液一般性質之測定．．．．．．．．．．．．．．．．． 74
三、超過濾區分物對ACE之抑制活性作用．．．．．．．．．．． 76
四、區分物分子量之分佈分析．．．．．．．．．．．．．．．． 78
五、卵白蛋白水解液、保健機能食品與降高血壓藥對ACE抑制活性
之比較．．．．．．．．．．．．．．．．．．．．．．．．80
六、胃腸酵素對水解物之ACE抑制活性作用．．．．．．．．．．． 83
伍、結論．．．．．．．．．．．．．．．．．．．．．．．．． 85
陸、參考文獻．．．．．．．．．．．．．．．．．．．．．．． 86
表目錄
頁次
表一、蛋白的其他重要成份．．．．．．．．．．．．．．．．．． 6
表二、聯合國委員會在2003年對十八歲以上成人定義及分類標準 ．．12
表三、不同來源的血管收縮素轉化酶抑制劑及其IC50值．．．．．． 21
表四、發酵食品中的ACEI．．．．．．．．．．．．．．．．．．． 28
表五、蛋白質分解酵素之ㄧ般特性．．．．．．．．．．．．．．． 33
表六、明膠水解液之色澤與總生菌數．．．．．．．．．．．．．． 53
表七、明膠水解液及脫脂牛奶之官能評估比較．．．．．．．．．． 55
表八、卵白蛋白水解液之顏色分析．．．．．．．．．．．．．．． 74
表九、卵白蛋白水解液及脫脂牛奶官能評估比較．．．．．．．．． 75
表十、卵白蛋白水解液及經超過濾處理之濾液對ACE抑制活性作用 ． 77
表十一、比較卵白蛋白水解液、保健機能食品及藥物Enalapril抑制
活性作用．．．．．．．．．．．．．．．．．．．．． 81
圖目錄
頁次
圖一、雞蛋的結構．．．．．．．．．．．．．．．．．．．．．． 5
圖二、卵白蛋白的一次結構．．．．．．．．．．．．．．．．．． 5
圖三、腎素 — 血管收縮素系統中血管收縮素轉化酶之催化反應．． 20
圖四、標準品HHL、HA、HL及HA與HL 1:1 共注射之高效能液相層析
圖．．．．．．．．．．．．．．．．．．．．．．．．． 40
圖五、以高效能液相層析法測定蛋白質水解物對ACE抑制活性作用
(A)無抑制劑(未經酵素水解當blank)(B)有抑制劑(酵素水解)
．．．．．．．．．．．．．．．．．．．．．．．．．． 41
圖六、蛋白質微量分析標準曲線．．．．．．．．．．．．．．．． 43
圖七、不同酵素與基質比例水解明膠對ACE之抑制活性作用．．．． 45
圖八、不同酵素水解明膠對ACE之抑制活性作用．．．．．．．．． 46
圖九、不同濃度明膠對ACE之抑制活性作用．．．．．．．．．．． 48
圖十、不同濃度明膠與黏度之關係．．．．．．．．．．．．．．． 49
圖十一、添加兩次不同組合酵素水解明膠對ACE之抑制活性作用 ．． 50
圖十二、水解液濃縮後之ACE抑制活性作用．．．．．．．．．．． 54
圖十三、明膠水解液之分區產物經Sephadex G — 25管柱分離層析
圖．．．．．．．．．．．．．．．．．．．．．．．． 58
圖十四、由消化酵素分解明膠水解液之活性．．．．．．．．．．． 60
圖十五、以高效能液相層析法測定蛋白質水解物對ACE抑制活性作用
(A)無抑制劑(未經酵素水解當blank)(B)有抑制劑(酵素水
解)．．．．．．．．．．．．．．．．．．．．．．． 66
圖十六、不同酵素水解卵白蛋白對ACE之抑制活性作用．．．．．． 70
圖十七、不同酵素與基質比例水解卵白蛋白對ACE之抑制活性作用． 71
圖十八、不同pH水解卵白蛋白對ACE 之抑制活性作用 ．．．．．． 72
圖十九、不同溫度水解卵白蛋白對ACE之抑制活性作用．．．．．． 73
圖二十、卵白蛋白水解物及經過超過濾後得到分區濾液之分子量分佈
情形．．．．．．．．．．．．．．．．．．．．．．． 79
圖二十一、Enalapril 經系列稀釋後對血管收縮素轉化酶之抑制作用
．．．．．．．．．．．．．．．．．．．．．．．．． 82
圖二十二、胃腸消化酵素分解水解物之活性．．．．．．．．．．． 84

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