酶解法制备阿胶蛋白肽工艺条件优化及抗氧化活性分析

Optimization of Enzymatic Hydrolysis Process for Preparing Peptides from Asini Corii Colla and Its Antioxidant Activity

目的:研究酶解法制备阿胶蛋白肽的工艺条件及阿胶蛋白肽的抗氧化活性。方法:以阿胶为原料,通过酶解实验优化碱性蛋白酶Alcalase 2.4L制备阿胶蛋白肽的酶解工艺条件。以酶解pH、酶与底物比、酶解温度作为考察因素,以阿胶的水解度为指标,在单因素实验的基础上,通过正交试验确定最佳工艺条件;利用高效凝胶色谱法测定酶解前后阿胶溶液的分子量分布情况,并通过检测对ABTS+和DPPH自由基的清除率,考察酶解前后阿胶溶液的抗氧化活性,从而综合分析酶解效果;采用4种不同大小孔径的超滤膜分离阿胶蛋白肽液,研究经超滤膜分离后不同分子量阿胶蛋白肽的抗氧化活性。结果:阿胶溶液的最佳酶解条件为:pH10.5、酶与底物比9%、酶解温度63℃,此条件下测得阿胶酶解液的水解度为15.93%±1.32%;酶解后阿胶溶液的分子量大部分分布在5000 Da以下;当浓度为10 mg/mL时,未酶解和酶解后的阿胶蛋白肽溶液ABTS+自由基清除率分别为75.83%±0.32%和93.16%±0.21%,DPPH自由基清除率为16.93%±2.41%和39.95%±1.27%;经超滤处理获得五组不同分子量的阿胶蛋白肽(>30 kDa、10~30 kDa、3~10 kDa、1~3 kDa和<1 kDa),分析比较发现低分子量的阿胶蛋白肽抗氧化效果较好。结论:碱性蛋白酶Alcalase 2.4L能够有效制备阿胶蛋白肽,且阿胶蛋白肽具有良好的抗氧化活性。试验结果为后续开展阿胶蛋白肽的抗氧化机制研究提供科学依据。

Objective:To investigate the technological conditions and antioxidant properties of Asini Corii Colla(ACC)peptides prepared by enzymolysis.Methods:The enzymatic hydrolysis conditions of ACC protein peptide prepared by Alcalase 2.4L were optimized using ACC as raw material.On the basis of single factor experiments,orthogonal test was carried out to determine the optimum process conditions with pH,enzyme-substrate ratio and enzymolysis temperature as the factors and the degree of hydrolysis(DH)of ACC as the indexes.To comprehensively analyze the enzymatic hydrolysis effect,the molecular weight distribution of ACC solution before and after enzymatic hydrolysis was determined by high performance gel chromatography,and the antioxidant activity of ACC solution before and after enzymatic hydrolysis was investigated by detecting scavenging rates of ABTS+and DPPH free radicals.Four kinds of ultrafiltration membranes with different pore sizes were used to separate the ACC peptide solution,and the antioxidant activity of ACC peptide with different molecular weight was studied. Results: The optimum enzymolysis conditions were pH10.5, enzyme-substrate ratio 9% and enzymolysis temperature 63 ℃. Under these conditions, the DH was 15.93%. After enzymatic hydrolysis, the molecular weight of ACC solution was mostly below 5000 Da. When the concentration was 10 mg/mL, the ABTS+ free radical scavenging rate of the ACC peptide solutions before and after enzymolysis were 75.83%±0.32% and 93.16%±0.21%, respectively. The ability of scavenging DPPH free radicals was 16.93%±2.41% and 39.95%±1.27%. Five groups of ACC peptides with different molecular weight (>30 kDa, 10~30 kDa, 3~10 kDa, 1~3 kDa and <1 kDa) were obtained by ultrafiltration. It was showed that the ACC peptides with small molecular weight had good antioxidant effects. Conclusion: Alcalase 2.4L could be effectively used for preparing CCA peptides, and the ACC peptides possess good antioxidant activities. These results would provide scientific basis for further research on the antioxidant mechanism of ACC peptide.