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研究生:葉政霖
研究生(外文):Yeh, Zheng Lin
論文名稱:鰺魚水解物中胜鑑定與降高血壓的效果
論文名稱(外文):Identification and Antihypertensive Effct of Peptide Derived from Horse Mackerel (Decapterus macarellus) Hydrolysate
指導教授:蔡震壽蔡震壽引用關係
指導教授(外文):Tsai, Jenn Shou
學位類別:碩士
校院名稱:國立海洋大學
系所名稱:食品科學系
學門:農業科學學門
學類:食品科學類
論文種類:學術論文
論文出版年:2002
畢業學年度:90
語文別:中文
論文頁數:72
中文關鍵詞:胜降高血壓血管升壓素轉換酶
外文關鍵詞:Horse MackerelPeptideAntihypertensiveACE
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以本省產鰺魚為原料,經採肉均質(33 % w/w, wet base)並預煮熟後,懸浮液分別加入Flavorzyme、Prozyme 6與Protease N,於50℃進行2與5小時水解,探討水解物中胜對血管升壓素轉換酶的抑制及降高血壓的生理效果。水解產物分子量以1,500 Da以下為主,以Prozyme 6水解2小時者,得可溶性蛋白質含量51.69 mg/ml。以Prozyme 6水解5小時者,得最高胜與游離胺基酸的總量78.9 mg/ml。而以Flavorzyme水解者則較不具苦味。
鰺魚肉熱水抽出物,對血管升壓素轉換酶(ACE)的抑制能力之影響,測得IC50值分別為7.14 mg/ml(nonpreincubation type)與8.73 mg/ml(preincubation type)。魚肉經過酵素水解後,水解物皆可有效提高對ACE的抑制能力。其中以Prozyme 6水解5小時者抑制能力為最高,IC50值為分別為1.02 mg/ml(nonpreincubation)與3.17 mg/ml(preincubation)。將此水解物進一步利用膠過濾法進行劃分,可得三個主要劃分物,其中以分子量1,500-580 Da的劃分物I相對抑制能力最高達49.6﹪。
將水解物3 ml(濃度3 g/kg B.W.)灌食18週齡之原發性高血壓老鼠,灌食後3小時內具有顯著降高血壓的效果(p<0.05)。其中以Prozyme 6水解5小時之水解物於灌食1小時降血壓效果最佳,收縮壓下降18 mmHg,平均血壓下降12 mmHg,舒張壓下降9 mmHg;而Protease N水解者,則於灌食2小時具最佳降血壓效果,收縮壓下降19 mmHg,平均血壓下降19 mmHg,舒張壓下降20 mmHg。利用線性回歸老鼠灌食後心跳速率與血壓間並無相關性。
Mackerel scad (Decapterus macarellus) was caught in Taiwan. After cooking and homogenizing, the muscle protein was hydrolyzed by commercial proteases (Prozyme 6, Protease N, and Flavorzyme). Enzymatic hydrolysis was done at 50°C for 2 and 5 hours, respectively. The effect of hydrolysate on inhibitory activity against angiotensin I converting enzyme (ACE) and antihypertensive effect were investigated. The molecular weight of different hydrolysate was all below 1,500 Da. The maximal soluble protein content was obtained when mackerel scad muscle protein was hydrolyzed by Prozyme 6 for 2 hours. The maximal free amino acids and peptide content was obtained when mackerel scad muscle protein was hydrolyzed by Prozyme 6 for 5 hours. Flavorzyme hydrolysis resulted in hydrolysate of little bitterness.
The IC50 value of ACE for the hot water extract was 7.14 mg/ml (nonpreincubation type) and 8.73 mg/ml (preincubation type), respectively. The ACE inhibitory activity of all hydrolysate was increased by enzymatic hydrolysis. The hydrolysate using Prozyme 6 hydrolysis for 5 hours showed the highest inhibitory activity, and the IC50 value was 1.02 mg/ml (nonpreincubation type) and 3.17 mg/ml (preincubation type), respectively. The hydrolysate was separated into three fractions by gel filtration eluted with deionized water. The fraction I (M.W. = 1,500 — 800 Da) showed the highest inhibitory activity, and the relative inhibitory was 49.6%.
Oral administration of hydrolysate (3 ml, conc. = 3 g/kg B.W.) to spontaneously hypertensive rats (SHR) showed a hypotensive effect after 1 hour oral administration. The hydrolysate using Prozyme 6 hydrolysis for 5 hours showed maximal hypotensive effect at 1 hour after oral administration, and systolic blood pressure (SBP), diastolic blood pressure (DBP) and mean blood pressure (MBP) decreased by 18, 9 and 12 mmHg, respectively. The hydrolysate using Protease N for 5 hours showed the most potent hypotensive effect at 2 hour after oral administration, and systolic blood pressure (SBP), diastolic blood pressure (DBP) and mean blood pressure (MBP) decreased by 19, 20 and 19 mmHg, respectively. No relationship between heart beat and blood pressure was shown.
第一章 序論……………………………………………………………... 1
第二章 文獻回顧………………………………………………………... 3
一、胜的生理活性…………………………………………………... 3
1. 類鴉片…………………………………………………………… 3
2. 幫助營養素吸收………………………………………………… 4
3. 免疫防護………………………………………………………… 5
4. 抗凝血…………………………………………………………… 5
5. 酵素調節………………………………………………………… 6
6. 抗菌……………………………………………………………… 7
7. 抗氧化…………………………………………………………… 8
二、高血壓簡介………………………………………………………... 9
1. 定義……………………………………………………………… 9
2. 分類……………………………………………………………… 11
3. 降血壓藥物的作用原理………………………………………… 12
三、血管升壓素轉換酶之介紹………………………………………... 14
1. ACE對RAS之作用……………………………………………… 14
2. ACE的生化特性…………………………………………………. 15
3. ACE的抑制………………………………………………………. 17
四、原發性高血壓老鼠簡介…………………………………………... 19
第三章 材料與方法……………………………………………………... 21
一、實驗材料………………………………………………………….. 21
二、實驗方法…………………………………………………………... 22
1. 鰺魚肉酵素水解物的製備……………………………………… 22
2. 可溶性蛋白質含量的測定……………………………………… 22
3. 胜與游離胺基酸含量的測定………………………………… 23
4. 對ACE抑制能力的測定………………………………………... 23
5. IC50值的測定…………………………………………………….. 25
6. Superdex膠體過濾………………..……………………………… 26
7. Sephadex膠體過濾…………………………………….………… 26
8. 高效液相層析…………………………………………………… 26
9. 實驗動物飼養條件…………..………………………………….. 27
10. 老鼠血壓的測定………………...……………………………... 27
11. 統計分析……………………………………………………….. 28
第四章 結果與討論……………………………………………………... 29
一、蛋白酶種類與水解時間對鰺魚肉蛋白質水解產物之影響…….. 29
1. 可溶性蛋白質含量……………………………………………… 29
2. 胜與游離胺基酸含量方……………………………………… 29
3. 苦味性…………………………………………………………… 30
4. 分子量分布……………………………………………………… 31
二、鰺魚肉蛋白質水解物濃度對ACE之抑制能力………………... 32
1. 熱水抽出物……………………………………………………… 32
2. 酵素水解物……………………………………………………… 33
3. 各劃分物………………………………………………………… 34
三、鰺魚肉蛋白質水解物對ACE之IC50值………………………… 36
1. 熱水抽出物……………………………………………………… 36
2. 酵素水解物……………………………………………………… 37
四、鰺魚肉蛋白質水解物主要胜純化……………………………. 38
五、鰺魚肉蛋白質水解物對原發性高血壓老鼠的影響……………. 39
1. 收縮壓…………………………………………………………… 39
2. 平均血壓………………………………………………………… 40
3. 舒張壓…………………………………………………………… 40
4. 心跳速率………………………………………………………… 41
第五章 結論……………………………………………………………... 43
參考文獻…………………………………………………………………. 44
圖表………………………………………………………………………. 52
圖目錄
頁次
圖一、鰺魚肉蛋白質以Flavorzyme水解不同時間的快速蛋白質層析圖譜……………………………………….……………………………… 52
圖二、鰺魚肉蛋白質以Protease N水解不同時間的快速蛋白質層析圖譜………………………………………………….……………………… 53
圖三、鰺魚肉蛋白質以Prozyme 6水解不同時間的快速蛋白質層析圖譜…………………………………………………………………………. 54
圖四、鰺魚肉熱水抽出物與抑制ACE能力之關係…………………... 55
圖五、鰺魚肉經Prozyme 6水解之產物濃度與抑制ACE能力的關係 56
圖六、鰺魚肉經Protease N水解之產物濃度與抑制ACE能力的關係 57
圖七、鰺魚肉蛋白質以Prozyme 6與Protease N水解5小時的膠體層析圖譜……………………………………………………………………. 58
圖八、鰺魚肉蛋白質以Prozyme 6水解5小時的高效液相層析圖譜.. 59
圖九、鰺魚肉蛋白質以Prozyme 6水解5小時主要產物的高效液相層析圖譜……………………………………………………………………. 60
圖十、鰺魚肉蛋白質以Prozyme 6水解5小時主要產物的質譜儀圖譜 61
圖十一、原發性高血壓老鼠灌食鰺魚肉蛋白質水解物之收縮壓的變化情形…………………………………………………………………… 62
圖十二、原發性高血壓老鼠灌食鰺魚肉蛋白質水解物之平均血壓的變化情形………………………………………………………………… 63
圖十三、原發性高血壓老鼠灌食鰺魚肉蛋白質水解物之舒張壓的變化情形…………………………………………………………………… 64
圖十四、原發性高血壓老鼠灌食鰺魚肉蛋白質水解物之心跳速率與血壓的關係……………………………………………………………… 65
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