酶解马氏珠母贝肉制备降血压肽的研究
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摘要
高血压是引发心血管疾病如心力衰竭、中风、冠心病和心肌梗塞等的重要危险因子。血管紧张素转化酶抑制剂(ACEI)是目前用于预防治疗高血压的重要药物之一，但化学合成的ACEI易引起恶心、呕吐、腹泻等副作用。因此，安全性高、副作用小、易吸收等优点的食源性ACEI尤其引人注目，已成为生物活性肽研究领域的热点之一。
本文以马氏珠母贝肉为原料，选用6种蛋白酶在各自适宜的条件下进行酶解，采用HPLC法分析酶解产物对血管紧张素转换酶(ACE)的抑制活性，筛选出碱性蛋白酶为其制备的最佳酶种类，并确定了水解度为21％时，产物具有较好的抑制活性。在此基础上，分别考察了加酶量，底物浓度，酶解温度及pH等对水解度的影响，结果表明：加酶量为0．175 g·L~(-1)、底物浓度为35 g·L~(-1)、温度为45℃、pH为9．5及水解时间240 min为最适酶解工艺条件。通过SDS-聚丙烯酰胺凝胶电泳测定相对分子质量分布，发现酶解产物中主要为14．4 kD以下的多肽分子。
根据实验数据和经验公式推导出碱性蛋白酶酶解马氏珠母贝肉的动力学模型分别为DH=5．757ln[1+(44．023E_0／S_0-0．0448)t]和r=(253．44E_0-0．2576S_0)exp[-0．1738(DH)]，并求得酶失活常数k_d为51．737min~(-1)，水解活化能△E_A为57．05 kJ·mol(-1)，酶失活活化能△E_D为63．30 kJ·mol(-1)。理论值与实际值进行对比，动力学模型与实验结果非常吻合，说明它具有很好的实际应用价值。
利用10 KD超滤膜对ACEI进行初步分离，确定了最适工艺参数：压力0．4 MPa，初始料液浓度25 mg·mL~(-1)，pH 7．0，操作温度35℃。通过对超滤前后溶液各部分抑制活性的检测发现，超滤能使较高ACEI组分获得有效富集。
Hypertension is a major risk factor of cardiovascular diseases, such as heart failure, apoplexy, coronary disease and myocardial infarction. Angiotensin I-converting enzyme inhibitory (ACEI) is one kind of important drugs for prevention and treatment of hypertension, but chemical synthesis of ACEI is extremely easy to cause queasiness, vomit, diarrhea and other side effects. Therefore, angiotensin I-converting enzyme inhibitor peptides from food protein has to be especially noticeable because of its advantages which includes safty, little side effects, easily absorbed, and so on, and become one of the focuses in the bioactive peptides research fields.
In this article, six different proteolytic enzymes were employed to hydrolyze Pinctada martensii meat to produce the hydrolysates under their suitable condition, and the high performance liquid chromatography method was developed for determination of the inhibitory activity of angiotensin I-converting enzyme (ACE) in hydrolysates. The result indicated that hydrolysis of Pinctada martensii meat with alkaline proteinase produced the highest ACE inhibitory activity. Therefore, alkaline proteinase was established for further study on optimization of hydrolysis conditions. And the degree of hydrolysis was determined on 21% when hydrolysate has the highest ACE inhibitory activity. On this basis, the factors, including the enzymatic quantity, concentration of substrate, temperature, pH and reaction time, were inspected respectively on DH. The result showed that the optimum conditions of alkaline proteinase to hydrolyze Pinctada martensii meat was: enzymatic concentration at0.175 g·L~(-1), concentration of substrate at 35 g·L~(-1), temperature at 45℃, pH on 9.5 and hydrolysis time at 240 min. Detected by SDS-polyacrylamide gel electrophoresis, the result about weight of molecular indicated that lots of peptides from hydrolysate was less than 14.4 kD.
Based on the experimental data and empirical formula, the dynamics model about hydrolyzing of Pinctada martensii meat by alkaline proteinase was deduced respectively. And the results of general kinetic equation for this hydrolysis process were suggested: DH=5.757ln[1+(44.023E_0/S_0-0.0448)t] and r=(253.44E_0-0.2576S_0)exp[-0.1738(DH)]·Meanwhile, some kinetic parameters were obtained respectively: Enzymic inactivation constant k_d is 51.737 min~(-1), the hydrolysis activation energy△E_A is 57.05 kJ·mol~(-1) and theinactivation energy△E_D is 63.30 kJ·mol~(-1). Compared theoretical value with actual value, thedynamic model was proved very good agreement with experimental result, and showed its good practical value.
The 10 KD ultrafiltration membrane was used to separate ACEI initially. At this time, the optimum process parameters were determined: The pressure△P at 0.4 MPa, the initialconcentration of substrate is 25 mg·mL~(-1), pH on 7.0, operating temperature for 35℃.Meanwhile, compared inhibitory activity of solutions with before and after ultrafiltration, the result showed that ultrafiltration was able to enrich higher ACE inhibitory activity components of hydrolysate effectively.
本文以马氏珠母贝肉为原料，选用6种蛋白酶在各自适宜的条件下进行酶解，采用HPLC法分析酶解产物对血管紧张素转换酶(ACE)的抑制活性，筛选出碱性蛋白酶为其制备的最佳酶种类，并确定了水解度为21％时，产物具有较好的抑制活性。在此基础上，分别考察了加酶量，底物浓度，酶解温度及pH等对水解度的影响，结果表明：加酶量为0．175 g·L~(-1)、底物浓度为35 g·L~(-1)、温度为45℃、pH为9．5及水解时间240 min为最适酶解工艺条件。通过SDS-聚丙烯酰胺凝胶电泳测定相对分子质量分布，发现酶解产物中主要为14．4 kD以下的多肽分子。
根据实验数据和经验公式推导出碱性蛋白酶酶解马氏珠母贝肉的动力学模型分别为DH=5．757ln[1+(44．023E_0／S_0-0．0448)t]和r=(253．44E_0-0．2576S_0)exp[-0．1738(DH)]，并求得酶失活常数k_d为51．737min~(-1)，水解活化能△E_A为57．05 kJ·mol(-1)，酶失活活化能△E_D为63．30 kJ·mol(-1)。理论值与实际值进行对比，动力学模型与实验结果非常吻合，说明它具有很好的实际应用价值。
利用10 KD超滤膜对ACEI进行初步分离，确定了最适工艺参数：压力0．4 MPa，初始料液浓度25 mg·mL~(-1)，pH 7．0，操作温度35℃。通过对超滤前后溶液各部分抑制活性的检测发现，超滤能使较高ACEI组分获得有效富集。
Hypertension is a major risk factor of cardiovascular diseases, such as heart failure, apoplexy, coronary disease and myocardial infarction. Angiotensin I-converting enzyme inhibitory (ACEI) is one kind of important drugs for prevention and treatment of hypertension, but chemical synthesis of ACEI is extremely easy to cause queasiness, vomit, diarrhea and other side effects. Therefore, angiotensin I-converting enzyme inhibitor peptides from food protein has to be especially noticeable because of its advantages which includes safty, little side effects, easily absorbed, and so on, and become one of the focuses in the bioactive peptides research fields.
In this article, six different proteolytic enzymes were employed to hydrolyze Pinctada martensii meat to produce the hydrolysates under their suitable condition, and the high performance liquid chromatography method was developed for determination of the inhibitory activity of angiotensin I-converting enzyme (ACE) in hydrolysates. The result indicated that hydrolysis of Pinctada martensii meat with alkaline proteinase produced the highest ACE inhibitory activity. Therefore, alkaline proteinase was established for further study on optimization of hydrolysis conditions. And the degree of hydrolysis was determined on 21% when hydrolysate has the highest ACE inhibitory activity. On this basis, the factors, including the enzymatic quantity, concentration of substrate, temperature, pH and reaction time, were inspected respectively on DH. The result showed that the optimum conditions of alkaline proteinase to hydrolyze Pinctada martensii meat was: enzymatic concentration at0.175 g·L~(-1), concentration of substrate at 35 g·L~(-1), temperature at 45℃, pH on 9.5 and hydrolysis time at 240 min. Detected by SDS-polyacrylamide gel electrophoresis, the result about weight of molecular indicated that lots of peptides from hydrolysate was less than 14.4 kD.
Based on the experimental data and empirical formula, the dynamics model about hydrolyzing of Pinctada martensii meat by alkaline proteinase was deduced respectively. And the results of general kinetic equation for this hydrolysis process were suggested: DH=5.757ln[1+(44.023E_0/S_0-0.0448)t] and r=(253.44E_0-0.2576S_0)exp[-0.1738(DH)]·Meanwhile, some kinetic parameters were obtained respectively: Enzymic inactivation constant k_d is 51.737 min~(-1), the hydrolysis activation energy△E_A is 57.05 kJ·mol~(-1) and theinactivation energy△E_D is 63.30 kJ·mol~(-1). Compared theoretical value with actual value, thedynamic model was proved very good agreement with experimental result, and showed its good practical value.
The 10 KD ultrafiltration membrane was used to separate ACEI initially. At this time, the optimum process parameters were determined: The pressure△P at 0.4 MPa, the initialconcentration of substrate is 25 mg·mL~(-1), pH on 7.0, operating temperature for 35℃.Meanwhile, compared inhibitory activity of solutions with before and after ultrafiltration, the result showed that ultrafiltration was able to enrich higher ACE inhibitory activity components of hydrolysate effectively.
引文
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