白芸豆中α-淀粉酶抑制剂的提取纯化及活性测定条件优化

Extraction and Purification and Activity Determination Conditions Optimization of α-Amylase Inhibitor in White Kidney Bean

摘要:

为开发一种简单、有效、低成本的α-淀粉酶抑制剂（α-AI）提取纯化工艺及探索其活性测试最佳条件，本研究以白芸豆为原料，采用水提法从中提取α-AI。首先对3,5-二硝基水杨酸（DNS）微孔法测定α-AI活性的条件进行了研究；随后在单因素基础上，通过正交试验确定了α-AI提取的最佳工艺条件，并通过酸沉-醇沉、超滤和透析对其进行了纯化。结果表明，DNS微孔法测定α-AI活性的最佳条件为：测定波长为482 nm、DNS用量为80 μL、反应时间为15 min、静置时间为10 min；α-AI提取的最优工艺为：提取温度为45 ℃、提取时间为2.5 h、提取pH为6.6、液料比为7.5；经纯化后得到了较纯的、活性较高的α-AI，其IC50 为0.89±0.04 mg/mL，提取率为（1.92±0.23）%。该提取纯化工艺可为α-AI的低成本工业化生产提供一定的指导意义。

Abstract:

In order to develop a simple, effective and low-cost extraction and purification process of α-amylase inhibitor (α-AI) and explore the optimal conditions for its activity test, white kidney beans were used as raw materials to extract α-AI by water extraction in this study. After studying the conditions for the determination of α-AI activity by 3,5-dinitrosalicylic acid (DNS) micropore method, the optimal process conditions for α-AI extraction were determined by orthogonal test on the basis of single factor and the α-AI crude extract was purified by acid precipitation-alcohol precipitation, ultrafiltration and dialysis. The results indicated that the optimal conditions for the determination of α-AI activity were: determination wavelength of 482 nm, DNS dosage of 80 μL, reaction time of 15 min, and the rest time was 10 min. And the optimum process for α-AI extraction were: Extraction temperature of 45 ℃, extraction time of 2.5 h, extraction pH of 6.6 and the liquid-solid ratio was 7.5. Moreover, the α-AI with high purity and activity was obtained after purification, which with an IC50 of 0.89±0.04 mg/mL and a yield of 1.92±0.23%. The extraction and purification process may be can provide some guiding significance for the low-cost industrial production of α-AI.