酪蛋白ACE抑制肽的制备及分离
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摘要
制备具有降血压活性的血管紧张素转换酶(angiotensinⅠ-converting enzyme ,ACE,EC 3.4.15.1)抑制肽是生物活性肽领域最热门的研究方向之一。乳和乳制品作为人类最主要的动物蛋白来源之一,它们不仅具有丰富的营养价值,而且还是许多生物活性肽的重要来源。本论文通过模拟胃肠道消化酶解酪蛋白,制备具有高ACE抑制活性和吸收稳定的酶解产物;然后利用大孔吸附树脂和葡聚糖凝胶层析对酶解产物进行初步分离,并通过两步半制备反相高效液相色谱法(semi- Reverse Phase High Performance Liquid Chromatgraphy, semi-RP-HPLC)对高活性组分进行分离纯化,最终获得具有较高ACE抑制活性的多肽。本研究提供了一种利用酪蛋白制备具有高抑制活性、高稳定性ACE抑制肽的方法,为乳酪蛋白的利用和降压肽产品的开发提供了新的思路和途径,并为进一步研究降压肽的构效关系提供基础。本研究的主要内容如下:
通过第一步胃蛋白酶水解条件的优化获得具有高ACE抑制活性的肽,然后通过第二步胰蛋白酶和胰凝乳蛋白酶的水解条件优化获得具有较高ACE抑制活性的小分子肽。结果表明:第一步水解最优条件为: [E]/[S]=6%,pH=1.8, T=37℃,t=2 h,水解上清液10倍稀释后的ACE抑制率为84.5%,其分子量集中在200D~3000 D;第二步水解最优条件为:胰蛋白酶(6%):胰凝乳蛋白酶(3%)=2:1,pH=7.8,T=48℃,t=5 h,水解上清液10倍稀释后的ACE抑制率为85.9%,分子量集中在100 D~1000 D。研究表明通过模拟胃肠道消化分步酶解可获得具有较高ACE抑制活性的小分子肽。
通过单因素实验确定DA201-C大孔吸附树脂的最佳吸附和洗脱条件,结果表明:吸附过程最佳工艺参数为:大孔吸附树脂用量30 g/100mL多肽溶液,吸附时间t=4 h。依次用25%、50%、75%、100%(体积分数)的乙醇进行梯度洗脱,洗脱过程最佳工艺参数为:100 mL洗脱液/30 g树脂,各浓度的乙醇洗脱时间t=2 h。ACE抑制活性测定结果表明:75%的乙醇洗脱组分具有最高的抑制活性,在0.5 mg/mL的浓度时该组分的ACE抑制率为89.16%,与其他浓度的乙醇洗脱组分的ACE抑制活性差异显著。
通过Sephadex G-10对75%的乙醇洗脱组分进行继续分离,结果表明:组分F2具有较高的ACE抑制活性,在0.2 mg/mL的浓度时其ACE抑制率为42.7%。通过两步semi-RP-HPLC对组分F2进行纯化,结果表明:组分7-3的ACE抑制活性最高,其IC50=26.5μg/mL。
利用液相-电喷雾电离-质谱联用(LC-ESI/MS/MS)对组分7-3进行质谱分析,研究表明,其可能含有的三个肽分别为TFLW、NGGDECDTPT和FPYGTAPDSW。
Now the ACE inhibitory peptide, one kind of the most popular biologically active peptides, is attracting more and more the scientists’attention. Milk and the diary product contain all kinds of essential amino acid which make them one of the most nutrient food, meanwhile some ACE inhibitory peptides have been developed from it. Here, the new ACE inhibitory peptides were obtained from casein by simulating the gastrointestinal digestion. At first, the macroporus adsorption resin DA201-C and Sephadex G-10 were applied to separated the enzyme hydrolysate. Then, the fraction named 7-3 possessed the highest ACE inhibitory rate was obtained after two-step RP-HPLC. The method to develop ACE inhibitory peptide from casein was proved to be effective, and new thought and approach were provided for the utilizing of milk and development of ACE inhibitory peptides. Meanwhile, some basis was provided for the structure-effect relationship. The mainly content of this research is displayed as follows:
Casein was hydrolyzed by pepsin at first, then by trypsin andα-chymotrpsin as the second step, simulating the mammal gastrointernal digestion process. The optimum condition of pepsin hydrolysis was: [E]/[S]=6%, pH=1.8, T=37℃, t=2 h. The ACE inhibitory activity assay revealed an value of 84.5% when the supernatant was diluted 10 times, the molecular weight(MW) distributed between 200 D~3000 D, the next enzyme hydrolysis process with trypsin andα-chymotrpsin accomplished under the following conditions: trypsin(6%):α-chymotrpsin(3%)=2:1, pH=7.8, T=48℃, t=5h, The ACE inhibitory activity assay revealed an value of 85.9% when the supernatant was diluted 10 times, the molecular weight distributed between 100 D~1000 D. The result proved that ACE inhibitory peptides with high activity of low MW could be yielded by simulating gastrointernal digestion.
Then the enzyme hydrolysate was separated with macroporus adsorption resin DA201-C.
The peptide was absorbed on the resin and eluted in turns by ethanol of different concentration: 25%, 50%, 75% and 100%. The optimum condition of absorbance was described as below: when the peptide concentration was 20 mg/mL, resin weight was 30 g per 100 mL peptide solution, t=4 h; elution condition parameter: the volume of ethanol was the same as the peptide solution, t=2 h. Elution fraction of ethanol of 75% shows the highest ACE inhibitory activity. It was 89.16% when the concentration was 0.5 mg/mL.
Next, the elution fraction of ethanol of 75% was separated further by sephadex G10. Two peaks was obtained and F2 showed higher ACE inhibitory activity. The ACE inhibitory rate of F2 was 42.7% when the concentration was 0.2 mg/mL.
Next, the component 7-3 with the highest ACE inhibitory activity was purified through two step semi-RP-HPLC and IC50 was 26.5μg/mL.
The component 7-3 may contain TFLW、NGGDECDTPT and FPYGTAPDSW after analysed with LC-ESI/MS/MS.
通过第一步胃蛋白酶水解条件的优化获得具有高ACE抑制活性的肽,然后通过第二步胰蛋白酶和胰凝乳蛋白酶的水解条件优化获得具有较高ACE抑制活性的小分子肽。结果表明:第一步水解最优条件为: [E]/[S]=6%,pH=1.8, T=37℃,t=2 h,水解上清液10倍稀释后的ACE抑制率为84.5%,其分子量集中在200D~3000 D;第二步水解最优条件为:胰蛋白酶(6%):胰凝乳蛋白酶(3%)=2:1,pH=7.8,T=48℃,t=5 h,水解上清液10倍稀释后的ACE抑制率为85.9%,分子量集中在100 D~1000 D。研究表明通过模拟胃肠道消化分步酶解可获得具有较高ACE抑制活性的小分子肽。
通过单因素实验确定DA201-C大孔吸附树脂的最佳吸附和洗脱条件,结果表明:吸附过程最佳工艺参数为:大孔吸附树脂用量30 g/100mL多肽溶液,吸附时间t=4 h。依次用25%、50%、75%、100%(体积分数)的乙醇进行梯度洗脱,洗脱过程最佳工艺参数为:100 mL洗脱液/30 g树脂,各浓度的乙醇洗脱时间t=2 h。ACE抑制活性测定结果表明:75%的乙醇洗脱组分具有最高的抑制活性,在0.5 mg/mL的浓度时该组分的ACE抑制率为89.16%,与其他浓度的乙醇洗脱组分的ACE抑制活性差异显著。
通过Sephadex G-10对75%的乙醇洗脱组分进行继续分离,结果表明:组分F2具有较高的ACE抑制活性,在0.2 mg/mL的浓度时其ACE抑制率为42.7%。通过两步semi-RP-HPLC对组分F2进行纯化,结果表明:组分7-3的ACE抑制活性最高,其IC50=26.5μg/mL。
利用液相-电喷雾电离-质谱联用(LC-ESI/MS/MS)对组分7-3进行质谱分析,研究表明,其可能含有的三个肽分别为TFLW、NGGDECDTPT和FPYGTAPDSW。
Now the ACE inhibitory peptide, one kind of the most popular biologically active peptides, is attracting more and more the scientists’attention. Milk and the diary product contain all kinds of essential amino acid which make them one of the most nutrient food, meanwhile some ACE inhibitory peptides have been developed from it. Here, the new ACE inhibitory peptides were obtained from casein by simulating the gastrointestinal digestion. At first, the macroporus adsorption resin DA201-C and Sephadex G-10 were applied to separated the enzyme hydrolysate. Then, the fraction named 7-3 possessed the highest ACE inhibitory rate was obtained after two-step RP-HPLC. The method to develop ACE inhibitory peptide from casein was proved to be effective, and new thought and approach were provided for the utilizing of milk and development of ACE inhibitory peptides. Meanwhile, some basis was provided for the structure-effect relationship. The mainly content of this research is displayed as follows:
Casein was hydrolyzed by pepsin at first, then by trypsin andα-chymotrpsin as the second step, simulating the mammal gastrointernal digestion process. The optimum condition of pepsin hydrolysis was: [E]/[S]=6%, pH=1.8, T=37℃, t=2 h. The ACE inhibitory activity assay revealed an value of 84.5% when the supernatant was diluted 10 times, the molecular weight(MW) distributed between 200 D~3000 D, the next enzyme hydrolysis process with trypsin andα-chymotrpsin accomplished under the following conditions: trypsin(6%):α-chymotrpsin(3%)=2:1, pH=7.8, T=48℃, t=5h, The ACE inhibitory activity assay revealed an value of 85.9% when the supernatant was diluted 10 times, the molecular weight distributed between 100 D~1000 D. The result proved that ACE inhibitory peptides with high activity of low MW could be yielded by simulating gastrointernal digestion.
Then the enzyme hydrolysate was separated with macroporus adsorption resin DA201-C.
The peptide was absorbed on the resin and eluted in turns by ethanol of different concentration: 25%, 50%, 75% and 100%. The optimum condition of absorbance was described as below: when the peptide concentration was 20 mg/mL, resin weight was 30 g per 100 mL peptide solution, t=4 h; elution condition parameter: the volume of ethanol was the same as the peptide solution, t=2 h. Elution fraction of ethanol of 75% shows the highest ACE inhibitory activity. It was 89.16% when the concentration was 0.5 mg/mL.
Next, the elution fraction of ethanol of 75% was separated further by sephadex G10. Two peaks was obtained and F2 showed higher ACE inhibitory activity. The ACE inhibitory rate of F2 was 42.7% when the concentration was 0.2 mg/mL.
Next, the component 7-3 with the highest ACE inhibitory activity was purified through two step semi-RP-HPLC and IC50 was 26.5μg/mL.
The component 7-3 may contain TFLW、NGGDECDTPT and FPYGTAPDSW after analysed with LC-ESI/MS/MS.
引文
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2.夏瑜,胡一河,王临池. 2010年苏州市高血压现状之调查分析[J].中国卫生检验杂志, 2011, 21(5): 1259-1261
3. Ferreira S H. A brandynin-potentiating factor (bpf) present in the venom of bothrops jararacal[J]. Br J Pharmacol Chemother, 1965, 24(1): 163-169
4. Ondetti M A, Williams N J, Sadoa E F, etc. Angiotensin-converting Emzyme inhibitory from the venom of Brotrops Jararaca: Isolation elucidation of structure and synthesis[J]. Biochemistry, 1971, 19(22): 4033-4039
5. Ondetti M A, Rubin B, Cushman D W. Design of specific inhibitors of angiotensin converting enzyme: new class of orally active antihypertentive angents[J]. Science, 1977, 196: 441-444
6. Oshima G. Shimabukuro H. Nagasawa K. Peptide inhibitors of angiotensin-converting enzyme in digests of gelatin by bacterial collagenase[J]. Biochimica et Biophysica Acta, 1979, 566: 128-137
7. Maeno M, Yamamoto N, Takano T. Identification of an antihypertensive peptide from casein hydrolysates produced by a proteinase from Lactobacillus helveticus CP790[J]. J Dairy Sci, 1996, 19: 1316-1321
8.姜瞻梅.酶解酪蛋白生产ACE抑制肽[D]: [硕士学位论文].哈尔滨:东北农业大学食品科学与工程系, 2003
9.张国胜,孔繁东,祖国仁,等.大豆蛋白抗高血压活性肽的研究[J].中国乳品工业, 2004, 32(8): 12-14
10. Saiga A, Okumura T, Makihara T, etc. AngiotensinⅠ-converting enzyme inhibitory peptides in a hydrolyzed chicken breast muscle extract[J]. J Agri Food Chem, 2003, 51: 1741-1745
11.王宇.乳酸菌发酵乳抑制血管紧张素转化酶活性的研究[D]: [硕士学位论文].无锡:江南大学食品学院, 2008
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