超声辅助复合酶法提取小龙虾虾壳蛋白及其性质研究

Extraction and characterization of Procambarus clarkii shell proteins by ultrasound assisted complex enzymatic hydrolysis

目的提高副产物虾壳中蛋白的提取率,并探究虾壳蛋白的功能活性。方法以小龙虾虾壳为原料,在单因素的基础上,以加酶量、复合酶比例、酶解时间和超声时间为响应变量,蛋白提取率为响应值,采用响应面Box-Behnken试验设计优化超声辅助复合酶法提取小龙虾壳蛋白的最佳工艺。进一步比较复合酶解与单酶酶解制备的多肽的抗氧化活性、抗血管紧张素转化酶(angiotensin converting enzyme,ACE)活性和氨基酸成分组成,研究酶解虾壳蛋白多肽的功能性质。结果加酶量10200 U,复合酶比例0.65,酶解时间2.8 h,超声时间30 min,该最优条件下的蛋白质提取率为90.30%,与预测值相对误差为1.2%。与单酶酶解液相比,复合酶解液的抗氧化能力和ACE抑制能力均显著增强,清除1,1-二苯基-2-三硝基苯肼和2,2-联氮-二(3-乙基-苯并噻唑-6-磺酸)二铵盐的半最大效应浓度分别为4.38 mg/mL和3.66 mg/mL,最大的ACE抑制率达(44.93±0.89)%,且具有竞争性抑制模式。氨基酸组分分析发现酶解制备的虾壳多肽中必需氨基酸和疏水性氨基酸含量丰富,分别占氨基酸总量的43.06%和33.52%,从而表现出抗氧化活性和抗ACE活性。结论该研究有效提高了虾壳蛋白的提取率,得到的虾壳多肽中富含多种必需氨基酸和疏水性氨基酸,表现出良好的抗氧化活性和ACE抑制活性。

Objective To improve the extraction rate of protein from Procambarus clarkii shells as a by-product,and investigate the functional activity of shrimp shell proteins.Methods Using Procambarus clarkii shell as raw material,the response surface Box-Behnken test design was used to optimize the optimal extraction process of Procambarus clarkii shell protein by ultrasound assisted complex enzyme with enzyme addition amount,ratio of complex enzyme,enzymatic hydrolysis time and ultrasonic time as response variables and protein extraction rate as response values on the basis of single factor.The antioxidant activity,the angiotensin converting enzyme(ACE)inhibition ability and amino acid composition of the proteins prepared by complex enzymatic digestion and single enzyme enzymatic digestion were further compared to investigate the functional properties of the enzymatically digested Procambarus clarkii shell protein proteins.Results The protein extraction under this optimal condition was 90.30%with a relative error of 1.2%from the predicted value by adding enzyme amount of 10200 U,ratio of complex enzyme of 0.65,enzymatic digestion time of 2.8 h and ultrasound time of 30 min.Compared with the single enzyme digestion solution,the antioxidant and ACE inhibition abilities of the complex digestion solution were significantly enhanced,and the scavenging of 1,1-diphenyl-2-trinitrophenylhydrazine and 2,2-diazo-di(3-ethyl-benzothiazole-6-sulfonic acid)diamiammonium salts showed the concentration for 50%of maximal effect of 4.38 mg/mL and 3.66 mg/mL,respectively.The largest ACE suppression rate was(44.93±0.89)%,and there was a competitive suppression mode.The amino acid fraction analysis revealed that the enzymatically prepared Procambarus clarki shell proteins were rich in essential and hydrophobic amino acids,accounting for 43.06%and 33.52%of the total amino acids,respectively,thus exhibiting antioxidant activity and anti-ACE activity.Conclusion proteinsThis study effectively improves the extraction rate of Procambarus clarkii shell protein,and the obtains shrimp shell proteins are rich in many essential amino acids and hydrophobic amino acids,and shows good antioxidant activity and ACE inhibitory activity.