超声辅助复合酶法提取小龙虾虾壳蛋白及其性质研究

目的 为了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，以实现低值虾壳的高值化利用，为其进一步在食品药品领域的应用提供数据依据。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12000 U，复合比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90.41%，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(1，1-diphenyl -2-picrylhydrazyl，DPPH)和2，2-联氮-二(3-乙基-苯并噻唑-6-磺酸)二铵盐（2，2''-azinobis-(3-ethylbenzthiazoline-6-sulphonate)，ABTS）的EC50值分别为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，并可为虾壳功能活性肽的制备及在食品中的开发应用提供理论基础。

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

Objective To improve the extraction rate of protein from crayfish shells as a by-product and to investigate the functional activity of shrimp shell proteins in order to achieve high value utilization of low value shrimp shells and to provide data for their further application in the food and drug sector. Methods The optimal process for the extraction of crayfish shell proteins by ultrasound-assisted complex enzymatic method was optimized using a response surface Box-Behnken experimental design with the amount of enzyme addition, the proportion of complex enzyme, enzymatic digestion time and ultrasound time as the response variables and the protein extraction rate as response values on the basis of single factors. The antioxidant activity, the ACE inhibition ability and amino acid composition of the peptides prepared by complex enzymatic digestion and single enzyme enzymatic digestion were further compared to investigate the functional properties of the enzymatically digested crayfish shell protein peptides. Results The results showed that the protein extraction under this optimal condition was 90.41% with a relative error of 1.2% from the predicted value by adding enzyme amount of 12000 U, complex ratio 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 DPPH and ABTS showed EC50 values of 4.38 mg/mL and 3.66 mg/mL, respectively,the largest ACE suppression rate is (44.93±0.89%), and there is a competitive suppression mode. The amino acid fraction analysis revealed that the enzymatically prepared crayfish shell peptides 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 This study effectively improved the extraction rate of crayfish shell protein and could provide a theoretical basis for the preparation of functional active peptides from crayfish shells and their development and application in food.