酶解缫丝蚕蛹蛋白抗氧化肽的分离与稳定性研究

Separation and Stability of Antioxidant Peptides from Silkworm Pupa Protein by Enzymatic Hydrolysis

目的:研究酶解缫丝蚕蛹蛋白抗氧化肽的分离和稳定性,为其开发应用提供基础数据。方法:超滤分离酶解缫丝蚕蛹蛋白抗氧化肽,比色法测定其抗氧化能力。结果:超滤分级后得到分子质量为200～3 000 D的酶解缫丝蚕蛹蛋白抗氧化肽对O2-?、?OH、1,1-二苯基-2-三硝基苯肼(1,1-diphenyl-2-picrylhydrazyl,DPPH)自由基的IC50分别为18.85 mg/mL、3.13 mg/mL、35.23 μg/mL。该抗氧化肽经过4 h胃蛋白酶+胰蛋白酶处理前后对DPPH自由基清除率分别为81.05%、82.26%;在pH 4、8条件下处理1 h,对DPPH自由基清除率分别为98.02%、11.80%(100 μg/mL);在温度95 ℃条件下处理1 h,对DPPH自由基清除率为87.11%(100 μg/mL);用浓度为1.0 mol/L的NaCl处理6 h,相比对照组,对DPPH自由基清除率由51.58%下降到22.68%(60 μg/mL)。结论:超滤后得到的分子质量为200～3 000 D的酶解缫丝蚕蛹蛋白抗氧化肽的抗氧化能力比酶解原液有一定提高;且在酸性、高温、脱盐处理后对DPPH自由基的清除能力保持较好;胃肠道消化酶对其DPPH自由基清除能力的影响不显著(P>0.05)。

Objective: To study the separation and stability of antioxidant peptides produced by enzymatic hydrolysis of silkworm pupa protein with a commercial enzyme preparation. Methods: The antioxidant peptides derived from silkworm pupa protein were separated and purified by ultrafiltration. Their antioxidant activity was determined by colorimetry.Results: The antioxidant peptides with molecular weights of 200–3 000 D were obtained by ultrafiltration and their IC50 for superoxide anion, hydroxyl, 1,1-diphenyl-2-picrylhydrazyl hydrate (DPPH) free radical were 18.85 mg/mL, 3.13 mg/mL,and 35.23 μg/mL, respectively. The DPPH free radical scavenging rate of the ant ioxidant peptides after treatment with both pepsin and trypsin for 4 h was increased from an initial level of 81.05% to 82.26%. The DPPH free radical scavenging rate of the antioxidant peptides at a concentration of 100 μg/mL was 98.02% and 11.80% after treatment at pH 4 and 8 for 1 h,and 87.11% after heat treatment at 95 ℃ for 1 h, respectively. The DPPH free radical scavenging rate at a concentration of 60 μg/mL was reduced from an initial level of 51.58% to 22.68% after 6 h of incubation in the presence of 1.0 mol/L NaCl. Conclusion: The 200–3 000 D antioxidant peptides generated by enzymatic hydrolysis of silkworm pupa protein were capable of effectively scavenging superoxide anion and DPPH free radical and had reducing power; their DPPH free radical scavenging activity in an acidic environment was higher than in an alkaline environment, and was effectively maintained by desalination but not significantly affected by digestive enzymes or temperature.