臺灣博碩士論文加值系統

本實驗目的在於利用Lactobacillus helveticus CCRC 14092、Lactobacillus helveticus CCRC 14100、Saccharomyces cerevisiae CCRC 21443發酵及酵母菌酵素水解牛乳，以探討牛乳中潛在性血管收縮素轉化酶抑制物 (ACE-I) 之活性，期望能得到有效之抑制物，以提高牛乳之利用性並提供國人預防高血壓之發生。試驗Ⅰ為ACE-I產生方式之探討，以酵素及微生物混合或單獨發酵 (水解) 牛乳，測定其pH值、可溶性蛋白質含量、肽濃度及血管收縮素轉化酶抑制物之活性；試驗Ⅱ為血管收縮素轉化酶抑制物之純化，利用高效能液相層析法純化，並測定其血管收縮素轉化酶抑制物之活性，計算出比活性 (specific activity) 和純化倍數 (purification fold)，以瞭解純化之效果。
結果顯示：L. helveticus CCRC 14100與S. cerevisiae CCRC 21443混合組之蛋白質水解和產肽能力最高，且於發酵20小時時有最高之ACE-I活性 (55%)；以酵母菌酵素水解牛乳比以含乳酸菌發酵之處理組，可得較高之肽濃度 (71.78μM) 和血管收縮素轉化酶抑制率 (86%)；L. helveticus CCRC 14100與S. cerevisiae CCRC 21443 (pH 4.2) 混合組時之ACE-I之IC50為148.54μM ，L. helveticus CCRC 14092與S. cerevisiae CCRC 21443 (pH 4.2) 混合組之ACE-I之IC50為746.20μM，酵母菌酵素水解牛乳4 hr (濃度40 mg/ml) 之ACE-I之IC50則為120.06μM；膠體過濾 (Waters Ultrahydrogel TM 120 , 7.8 × 300 mm) 和逆相層析 (Waters Nova-Pak® C-18 , 3.9× 150 mm) 均可以有效純化ACE-I達41.57倍。

The purposes of this study are to ferment (hydrolyze) milk by Lactobacillus helveticus CCRC 14100, Lactobacillus helveticus CCRC 14092, Saccharomyces cerevisiae CCRC 21443 and enzymes of Saccharomyces cerevisiae and to discuss the activity of hydrolysate which contains angiotensin-I converting enzyme inhibitor (ACE-I) to increase the utilization and healthy value of milk. In the first experiment, the preparation of biopeptides from milk fermented by microorganism or enzyme included the measurement of pH value, soluble protein concentration, peptide content and the activity of ACE-I. In the second experiment, the ACE-I was purified by high performance liquid chromatography (HPLC) to assay the specific activity and purification fold of ACE-I.
The results indicated that the mixed starins of L. helveticus CCRC 14100 and S. cerevisiae CCRC 21443 had the strongest ability in soluble protein and peptide forming and the maximum ACE-I activity was 55% while fermenting for 20 hrs. The peptide content (71.78μM) and ACE-I activity (86%) of milk hydrolyzed by enzyme of S. cerevisiae was higher than those fermented with Lactobacillus. The IC50 of milk fermented by L. helveticus CCRC 14100 and S. cerevisiae CCRC 21443 (pH 4.2) or L. helveticus CCRC 141092 and S. cerevisiae CCRC 21443 (pH 4.2) were 148.54μM and 746.20μM. Then The IC50 of milk hydrolyzed by enzyme of S. cerevisiae (4 hr, 40 mg/ml) was 120.06μM. Gel filtration (Waters Ultrahydrogel TM 120 , 7.8 × 300 mm) and reversal phase chromatography (Waters Nova-Pak® C-18 , 3.9× 150 mm) increased ACE-I activity and improved to 41.57 fold.

壹、中文摘要--------------------------------------------------------------------01
貳、前言--------------------------------------------------------------------------02
參、文獻檢討--------------------------------------------------------------------04
一.乳酸菌及酵母菌-------------------------------------------------------------04
1.乳酸菌的種類--------------------------------------------------------------04
2.酵母菌的種類--------------------------------------------------------------05
二.發酵乳中成分之變化-------------------------------------------------------06
1.蛋白質之變化--------------------------------------------------------------06
2.乳糖之水解-----------------------------------------------------------------06
3.乳酸之生成-----------------------------------------------------------------07
4.其他有機酸-----------------------------------------------------------------07
5.脂肪之水解-----------------------------------------------------------------08
6.微生物之變化--------------------------------------------------------------08
7.礦物質之變化--------------------------------------------------------------09
8.風味物質之生成-----------------------------------------------------------09
三.乳製品的生理活性----------------------------------------------------------09
1.類嗎啡肽--------------------------------------------------------------------10
2.抗癌與免疫調節活性-----------------------------------------------------11
3.抗血栓活性-----------------------------------------------------------------11
4.礦物質結合特性-促進鈣、磷吸收------------------------------------12
5.抗菌活性--------------------------------------------------------------------12
6.抗高血壓之作用-----------------------------------------------------------13
7.促進雙叉桿菌屬之生長--------------------------------------------------14
四.高血壓的簡介----------------------------------------------------------------14
1.定義--------------------------------------------------------------------------14
2.分類--------------------------------------------------------------------------16
3.高血壓的治療及預防-----------------------------------------------------17
五.血管收縮素轉化酶----------------------------------------------------------18
1.血管收縮素轉化酶之生化特性--------------------------------18
2.ACE在腎素-血管收縮素系統與激肽釋放酶-激酶系統中所扮演之
角色---------------------------------------------------------------------------19
六.血管收縮素轉化酶抑制物------------------------------------------------20
1.作用機制--------------------------------------------------------------------20
2.天然性來源-----------------------------------------------------------------21
肆、材料與方法----------------------------------------------------------------27
伍、結果與討論----------------------------------------------------------------35
一.菌株之發酵------------------------------------------------------------------35
二.酵母菌酵素之水解---------------------------------------------------------45
三.活性肽之區分---------------------------------------------------------------54
陸、結論-------------------------------------------------------------------------85
柒、參考文獻-------------------------------------------------------------------86
捌、英文摘要-------------------------------------------------------------------96

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