膜分离蚕豆蛋白酶解产物的理化性质及生物活性Physicochemical properties and biological activity of broad bean protein hydrolysate obtained by membrane separation technology

Abstract

以新鲜蚕豆为原料，采用碱溶酸沉法提取蚕豆蛋白，采用4种不同蛋白酶对蚕豆蛋白进行单酶或双酶酶解，通过比较蚕豆蛋白水解度和多肽得率筛选出最优的两种酶复合酶解蚕豆蛋白，将复合酶解液通过膜分离技术分离得到BBPHs-Ⅰ(10 kDa)5个不同分子质量的组分，对5个组分的氨基酸组成、紫外光谱、红外光谱进行分析，同时通过测定体外抗氧化活性及α-葡萄糖苷酶抑制率表征其活性。结果表明：选用菠萝蛋白酶和木瓜蛋白酶对蚕豆蛋白进行复合酶解；与膜分离前比较，膜分离后10 kDa以下的蚕豆蛋白酶解产物总氨基酸含量增加，BBPHs-Ⅱ、BBPHs-Ⅲ、BBPHs-Ⅳ的疏水氨基酸含量较高，此外BBPHs-Ⅲ的总氨基酸、必需氨基酸、疏水氨基酸、芳香氨基酸含量均为最高，分别为65.304%、19.222%、20.762%、8.769%。不同分子质量的蚕豆蛋白酶解产物表现出一定体外抗氧化能力，当质量浓度为10 mg/mL时，BBPHs-Ⅳ的ABTS自由基清除率可达（27.89±0.01）%，BBPHs-Ⅱ的DPPH自由基清除率可高达（57.70±0.00）%；当质量浓度在2～32 mg/mL范围内，不同分子质量蚕豆蛋白酶解产物的α-葡萄糖苷酶抑制活性呈剂量依赖关系，BBPHs-Ⅱ、BBPHs-Ⅲ、BBPHs-Ⅳ表现出良好的α-葡萄糖苷酶抑制活性，质量浓度为32 mg/mL时BBPHs-Ⅲ的α-葡萄糖苷酶活性抑制率最佳，达到（86.56±1.23）%。因此，通过膜分离技术得到的小分子质量的蚕豆蛋白酶解产物具有更好的抗氧化活性和α-葡萄糖苷酶抑制活性，具有良好的开发及应用前景。 Fresh broad beans were used as raw materials, broad bean protein was extracted by alkali solution and acid precipitation. Four different proteases were used for single enzyme or double enzyme hydrolysis of broad bean protein, and the best two enzymes were selected for the complex enzymatic hydrolysis of broad bean protein by comparing the hydrolysis degree and polypeptide yield of broad bean protein. Then the broad bean protein hydrolysate (BBPHs) were fractionated by membrane separation into five fractions of BBPHs-Ⅰ(10 kDa).The amino acid composition, UV and IR spectra of the five fractions were analyzed, and their biological activities were characterized by in vitro antioxidant activity and α-glucosidase inhibition rate. The results showed that pineapple protease and papain were selected for the complex enzymatic hydrolysis of broad bean protein.The total amino acid content of broad bean protease hydrolysate below 10 kDa after membrane separation increased compared with that without membrane separation,and the hydrophobic amino acid contents of BBPHs-Ⅱ, BBPHs-Ⅲ and BBPHs-Ⅳ were higher.In addition, BBPHs-Ⅲ had the highest content of total amino acid, essential amino acid, hydrophobic amino acid and aromatic amino acid with 65.304%, 19.222%, 20.762% and 8.769% respectively.Protein hydrolysate components with different molecular weights showed certain in vitro antioxidant capacity.The ABTS free radical scavenging rate of the BBPHs-Ⅳ could reach (27.89±001)%,DPPH free radical scavenging rate of the BBPHs-Ⅱ could reach (57.70±0.00)% at 10 mg/mL mass concentration.When the mass concentration ranged from 2 mg/mL to 32 mg/mL, the α-glucosidase inhibitory activities of different molecular weight broad bean hydrolysates showed a dose-dependent relationship. BBPHs-Ⅱ, BBPHs-Ⅲ and BBPHs-Ⅳ showed good α-glucosidase inhibitory activities,and BBPHs-Ⅲ possessed the best α-glucosidase inhibition rate 〔(86.56±123)%〕 at 32 mg/mL mass concentration. Therefore, the small molecular weight broad bean protein hydrolysate obtained by membrane separation had higher antioxidant activity and α-glucosidase inhibitory activity，and had good development and application prospects.

Keywords

• 蚕豆蛋白；酶解产物；膜分离；体外抗氧化；α-葡萄糖苷酶
• broad bean protein; enzymatic hydrolysate; membrane separation; in vitro antioxidation; α-glucosidase