酵素水解豬血漿中白蛋白以製備高血壓抑制胜肽

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Title:	酵素水解豬血漿中白蛋白以製備高血壓抑制胜肽
Other Titles:	Utilization of porcine blood albumin for production of angiotensin ?-converting enzyme inhibitory peptides by protease
Authors:	江美昭 Chiang, Mei-Chao
Contributors:	蔡正宗 Tsai, Tsun-Chung 東海大學食品科學系
Keywords:	白蛋白;ACE抑制劑;胜肽;酵素水解產物 Albumin;ACE inhibitor;Peptide;Enzymatic hydrolysate
Date:	2003
Issue Date:	2011-05-19T06:20:19Z (UTC)
Abstract:	豬血的營養成分相當豐富，內含有17~18%的蛋白質，若能有效利用豬血，將能製造出具有經濟價值的產品。本研究目的為探討蛋白質分解酵素水解豬血漿中白蛋白(albumin)之最佳條件，並偵測水解物中胜?對血管收縮素轉化酶(angiotensin converting enzyme， ACE)抑制活性之影響，以及建立毛細管電泳之分析方法。實驗中選用四種商業用蛋白質分解酵素：Protease A、Alcalase、Flavourzyme和Umamizyme進行水解反應，利用RP-HPLC進行ACE抑制活性分析。結果顯示，Alcalase之水解物對ACE的抑制作用優於其他蛋白質分解酵素之水解物。在不同Alcalase濃度及水解時間下，0.04% Alcalase之用量及水解一小時可達到極高抑制率。在選用之水解條件下，白蛋白水解物具有89%之抑制率，其IC50為1.51 mg/ml，優於市售之發酵乳A (3.58 mg/ml)，而一顆Enalapril溶於80 ml水中，其IC50為0.03 mg/ml。經超濾膜區分之產物，分子量限值愈小，IC50值有愈低的趨勢，其中分子量限值10 KD、3 KD和1 KD以下的區分產物其IC50分別為1.145、1.065和0.957 mM。水解物色澤淺黃，L、a、b值為71.28、1.19及20.76，經加熱去除Alcalase活性後，沒有微生物生長情形，但產品本身不良氣味導致接受度降低。實驗中開發之毛細管電泳分析方法是一個快速、簡單、敏銳，又能降低試劑用量的分析技術，其分離條件在65 cm熔矽毛細管(有效長度55 cm)、內徑75 μm、40 mM磷酸緩衝液(pH 6.0)、20 kV電壓和偵測波長228 nm下，能有效將Hippuric acid (HA)和Hippuryl-Histidyl-Leucine (HHL)分離，可作為未來測定血管收縮素轉化酶抑制活性的分析方法之一。 Porcine blood is rich in nutrients and contains 17~18% protein. Effective utilization of porcine blood will produce beneficial products. The purpose of this study was to investigate the optimum conditions for protease hydrolysis of porcine blood albumin to produce inhibitor to angiotensin converting enzyme (ACE), and develop capillary electrophoresis for inhibition measurement. Four commercial proteases - Protease A, Alcalase, Flavourzyme and Umamizyme - were choiced for hydrolysis. Reversed-phase (C8) high-performance liquid chromatography (HPLC) was used to determine ACE inhibition activity of the hydrolysates. Results showed that Alcalase was better than others in production of ACE inhibitor. 0.04% Alcalase was found to be effective enough in one-hour hydrolysis of porcine albumin. Under optimum condition (pH 7.5, 55℃), hydrolysate showed 89% ACE inhibition with IC50 1.51 mg/ml, better than fermented milk A (3.58 mg/ml). Whereas, IC50 of one Enalapril pill in 80 ml water is 0.03 mg/ml. IC50 of hydrolysate filtrate from sequential ultrafiltration with MW cut-off 10 KD, 3 KD and 1 KD membrane were found to be 1.145、1.065 and 0.957 mM, respectively. The crude hydrolysate was pale brown in color and its L, a, and b value were found to be 71.28, 1.19 and 20.76 respectively. The hydrolysate was sterile, however, it’s unpleasant in odor resulted in low acceptability. The capillary zone electrophoresis was a rapid, simple and sensitive method. The analytic method have been developed to separate and quantify Hippuric acid (HA) and Hippuryl-Histidyl-Leucine (HHL). Best separation conditions for reaction sample with a fused-silica capillary 65 cm (55 cm to the detector) × 75 μm I.D., was found to use 40 mM phosphate (pH 6.0) as the running buffer, an applied voltage 20 kV and detection wavelength 228 nm.
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