酪蛋白水解物的类蛋白反应修饰产物的分离纯化及其抗氧化活性研究

Separation and Purification of Modified Casein Hydrolysates Using Plastein Reaction and Their Antioxidant Activities

摘要:

本文采用木瓜蛋白酶在pH 6.5的条件下对酪蛋白进行水解2 h，水解产物测得DPPH 35.89%±0.13%，超氧阴离子清除能力为21.39%±0.33%，还原能力为53.00%±2.00%。分别导入Tyr、Phe和Try对水解产物进行修饰，结果表明类蛋白反应时间为6 h时，三种产物的抗氧化性最高，其中Try修饰产物抗氧化性最好，测得DPPH、超氧阴离子清除能力、还原能力分别46.84%±1.16%、34.10%±0.32%、70.00%±2%（p<0.05）。随后，将Try修饰下的产物进行分离纯化，使用装有G-15葡聚糖凝胶的色谱柱，水为洗脱液，上样浓度为20 mg/mL，流速为0.5 mL/min，洗脱效果最佳，得到三个峰，分别测定其抗氧化性，结果表明峰二的抗氧化性最好，测得DPPH、超氧阴离子清除能力、还原能力分别为58.46%±0.57%、38.42%±0.47%和80.00%±0.02%（p<0.05）。将峰二产物通过液相色谱进一步分离鉴定，得到单一峰，证明蛋白纯化下的产物纯度较高。最后，得到了高纯度的抗氧化肽。

Abstract:

Casein was hydrolyzed with papain at pH 6.5 for two hours, and the 2,2-diphenyl-1-picrylhydrazyl (DPPH) scavenging capacity, superoxide anion scavenging capacity, and reducing power of the hydrolysis product (CH) were measured and found to be 35.89%±0.13%, 21.39%±0.33%, and 53.00%±2.00%, respectively. Subsequently, phenylalanine (PHE), tyrosine (TYR), and tryptophan (TRY) were used to modify the casein hydrolysate, respectively. The results showed that the antioxidant activities of the three products (CHPHE, CHTYR, and CHTRY) were highest when the plastein reaction time was six hours. Among them, the highest antioxidant activity was found in CHTRY, whose DPPH radical scavenging capacity, superoxide anion scavenging capacity, and reducing power were 46.84%±1.16%, 34.10%±0.32%, and 70.00%±2%, respectively (p<0.05). CHTRY was separated and purified using a Sephadex G-15 column, and the optimal elution result was obtained when deionized water was used as the elution solution, the concentration of loaded sample was 20 mg/mL, and the flow rate was 0.5 mL/min. Three peaks (P1, P2, and P3) were obtained, and their antioxidant activities were determined. The results showed that P2 had the highest antioxidant activity and its DPPH radical scavenging capacity, superoxide anion scavenging capacity, and reducing power were 58.46% ±0.57%, 38.42%±0.47%, and 80.00%±0.02% (p<0.05), respectively. The P2 product was further separated and identified by liquid chromatography, and a single peak was obtained, confirming a relatively high purity of the product. Finally, antioxidant peptides with a high purity were obtained.