酶解鸡血球制备抗氧化肽的工艺优化和分析鉴定

Optimized Preparation, Characterization and Identification of Antioxidative Peptide Derived from Chicken Blood Corpuscle Proteins by Enzymatic Hydrolysis

为挖掘家禽血液潜在的抗氧化特性,以鸡血球为原料,筛选最佳蛋白酶酶解制备抗氧化肽,并对酶解工艺进行响应面优化。采用超滤、阳离子交换色谱、凝胶色谱及高效液相色谱对酶解物进行连续分离纯化,并用质谱进行结构鉴定。结果表明,酸性蛋白酶水解血球蛋白制备的酶解物具有最高的1,1-二苯基-2-三硝基苯肼(1,1-diphenyl-2-picrylhydrazyl,DPPH)自由基清除能力(>80%)。其最佳酶解条件为酶用量2%、酶解温度45℃、酶解时间3.0 h,该条件下的DPPH自由基清除能力、超氧阴离子自由基清除能力和还原力分别为(98.31±0.66)%、(28.89±0.31)%和1.94±0.03。经系列分离纯化获得抗氧化活性最强的组分,其在质量浓度为0.1 mg/mL时,DPPH自由基清除能力和还原力分别为(87.16±1.59)%和0.21±0.01。经高效液相色谱-质谱联用技术鉴定,目标肽的氨基酸序列为MGQKDSYVGDEAQSKRGILT,分子质量为2 182.1 Da。

In order to explore the potential antioxidative properties of poultry blood,several proteases were screened for use in the production of antioxidative peptide from chicken red blood cells and the enzymatic hydrolysis process was optimized using response surface methodology.The hydrolysate was purified sequentially by ultrafiltration,cation exchange chromatography,gel filtration chromatography,and high-performance liquid chromatography.The antioxidative peptide obtained was identified by mass spectrometry.The results showed that the hydrolysate obtained using acidic protease had the highest scavenging activity against 1,1-diphenyl-2-picrylhydrazyl(DPPH)radical(>80%)among 6 proteases used.The optimal hydrolysis conditions were obtained as follows:enzyme/substrate(E/S)ratio 2%,temperature 45℃and time 3.0 h.The DPPH radical scavenging activity,superoxide ion-scavenging activity and reducing power of the hydrolysate obtained using the optimized conditions were(98.31±0.66)%,(28.89±0.31)%and(1.94±0.03),respectively.After purification,the fraction with the highest antioxidative activity was obtained,whose DPPH radical-scavenging activity and reducing power were(87.16±1.59)%and(0.21±0.01)at 0.1 mg/mL,respectively.Finally,the peptide was identified by liquid chromatography-mass spectrometry(LC-MS/MS)as MGQKDSYVGDEAQSKRGILT with a molecular mass of 2 182.1 Da.