酶法制备2-O-α-D-吡喃葡萄糖基-L- 抗坏血酸的分离纯化工艺及抗氧化活性分析

Separation and Purification Processes of 2-O-α-D-Glucopyranosyl- L- Ascorbic Acid Prepared Enzymatically and Analysis of Its Antioxidant Activity

摘要:

该研究以L-抗坏血酸和β-环糊精为底物，在海洋环糊精葡萄糖基转移酶MY20的催化下制备了2-O-α-D-吡喃葡萄糖基-L-抗坏血酸（AA-2G），研究了其分离纯化工艺和抗氧化活性。通过使用阳离子离子交换树脂SK1B和阴离子离子交换树脂WA30，重点优化了阴离子交换树脂WA30分离AA-2G的工艺参数，通过响应面试验确定了最佳工艺参数为：洗脱液浓度0.15 mol/L、洗脱液流量3 mL/min、洗脱液pH值为7，在该条件下纯化AA-2G的纯度为91.23 wt.%，回收率89.92 wt.%；在此基础上，建立了多步纯化工艺，使AA-2G的纯度由最初的50.31 wt.%提高到了95.62 wt.%，纯度提高了1.90倍，回收率达到85.12 wt.%；对制备的AA-2G进行体外抗氧化性分析发现，AA-2G对超氧阴离子、羟自由基和DPPH自由基的清除率分别为91.63%、90.21%和95.20%，验证了酶法制备的AA-2G具有抗氧化活性。该研究可为高纯度AA-2G的开发提供理论依据。

Abstract:

In the study, 2-O-α-D-glucosyL-L-ascorbic acid (AA-2G) was prepared using L-ascorbic acid and β-cyclodextrin as substrates under the catalytic action of marine cyclodextrin glucosyltransferase MY20. The separation and purification process and antioxidant activity of AA-2G were examined. By using the cationic ion exchange resin SK1B and the anionic ion exchange resin WA30, the process parameters for separating AA-2G from anion exchange resin WA30 were optimized. The optimal process parameters were determined through response surface experiments: eluent concentration, 0.15 mol/L; eluent flow rate, 3.00 mL/min; eluent pH value 7. Under these conditions, the purity of AA-2G purified was 91.23 wt.%, with a recovery rate of 89.92 wt.%. On this basis, a multi-step purification process was established, in which increased the purity of AA-2G from the initial 50.31 wt.% to 95.62 wt.%, resulting in a 1.90-fold increase of purity and a recovery rate of 85.12 wt.%. In vitro antioxidant analysis of the prepared AA-2G revealed that the scavenging rates for superoxide anion, hydroxyl radical and DPPH radical were 91.63%, 90.21% and 95.20%, respectively, indicating that the enzymatically prepared AA-2G possessed antioxidant activities. The study provides a theoretical basis for the development of high-purity AA-2G.