酪蛋白源降胆固醇肽的酶解制备工艺优化

以酪蛋白为原料，采用中性蛋白酶、碱性蛋白酶以及胰蛋白酶对酪蛋白进行水解，确定制备降胆固醇肽的最佳蛋白酶；通过单因素实验和响应面试验，研究水解pH、水解温度、酶与底物比、底物浓度和水解时间对酪蛋白水解度和胆固醇胶束溶解度抑制率的影响，确定最佳水解条件；而后通过超滤和凝胶过滤层析确定降胆固醇肽的初步分离工艺。结果表明:制备酪蛋白源降胆固醇肽的最佳水解工具酶是中性蛋白酶，其最佳酶解条件为反应温度51.3 ℃，酶与底物浓度比6.47%，pH6.34，底物浓度5 g/100 mL，反应时间3.5 h，胆固醇抑制率为58.25%±0.59%；Sephadex G-10分离酪蛋白降胆固醇肽条件为上样浓度80 mg/mL，上样体积2.5 mL，洗脱速度3.5 mL/min；经酶解、超滤及层析后制备的酪蛋白源降胆固醇肽峰1和峰2样品在100 μg/mL的胆固醇溶解度抑制率为24.2%±0.24%和4.3%±0.16%。经酶解制备分离后，获得具有抑制降固醇胶束溶解活性的降胆固醇肽，为降胆固醇肽的开发提供理论研究基础。

Optimization of Preparation of Casein-derived Cholesterol-lowering Peptide by Enzymatic Hydrolysis

Casein was hydrolyzed by neutral protease, alkaline protease and trypsin to determine the best protease for preparation of cholesterol-lowering peptide. The effects of hydrolysis pH value, hydrolysis temperature, enzyme to substrate ratio, substrate concentration and hydrolysis time on casein hydrolysis degree and cholesterol micelle solubility inhibition rate were investigated by single factor and response surface experiments, and the optimal hydrolysis conditions were determined. Then the separation process of cholesterol-lowering peptide was determined by ultrafiltration and gel filtration chromatography. The results showed that the optimal enzyme was neutral protease, and the optimal hydrolysis conditions were as follows: Reaction temperature 51.3 ℃, enzyme to substrate concentration ratio 6.47%, pH6.34, substrate concentration 5 g/100 mL, reaction time 3.5 h , and cholesterol inhibition rate 58.25%±0.59%; The separation conditions of casein cholesterol-lowering peptide by Sephadex G-10 were as follows: Loading concentration 80 mg/mL, loading volume 2.5 mL, elution rate 3.5 mL/min; The inhibition rates of cholesterol solubility of peak 1 and peak 2 samples were 24.2%±0.24% and 4.3%±0.16% at 100 μg/mL after ultrafiltration and chromatography.