金钗石斛多糖的酶解工艺优化及其抗氧化活性

Process optimization of enzymatic hydrolysis and antioxidant activity of Dendrobium nobile polysaccharide

摘要:

采用α-半乳糖苷酶酶解技术对金钗石斛多糖进行处理，从而增强了其抗氧化活性。通过单因素和响应面试验确定了最佳酶解条件：α-半乳糖苷酶加酶量20 U/mL、pH 7.0、温度46 ℃、时间11 h，制备得到金钗石斛酶解多糖DNP-30E，并对酶解前后多糖进行了结构表征及抗氧化活性分析。结果表明，酶解后还原糖含量由0.63%升高至48.66%；分子量由7.95×105 Da降至2083 Da；酶解后，多糖中的Glu(1α→6)-Man键发生断裂，并引起葡萄糖端基氢构型翻转。当多糖浓度为2 mg/mL时，DNP-30E对·ABTS清除率从22.92%提升至47.87%，·OH清除率从31.46%提升至56.75%，·O2-清除率从24.52%提升至35.61%，FRAP值从141.67提升至441.06（0.4 mg/mL）。当多糖浓度为5~10 mg/mL时，DNP-30E与DNP-30相比细胞毒性显著降低。在SDS刺激HaCaT细胞氧化应激模型的基础上发现，在多糖浓度0.5 mg/mL时，DNP-30E组比DNP-30组的ROS含量降低了34.21%，MDA含量降低了50.2%，SOD酶活力提升了84.17%。结果表明，金钗石斛多糖经过酶解后具有开发成为新型天然抗氧化剂的应用潜力。

Abstract:

The α-galactosidase enzymatic hydrolysis was applied to enhance the antioxidant activity of Dendrobium nobile polysaccharide. The optimum enzymolysis conditions were determined by single factor and response surface tests: α-galactosidase dosage 20 U/mL, pH 7.0, at 46 ℃ for 11 h. The polysaccharide DNP-30 was further hydrolyzed according to the optimal enzymatic hydrolysis conditions to obtain DNP-30E. The structure and antioxidant activity of DNP-30 and DNP-30E were analyzed. The results showed that the content of reducing sugar increased from 0.63% to 48.66% after enzymolysis, and weight-average molecular weight decreased from 7.95×105 Da to 2083 Da. Enzymolysis may break Glu(1α→6)-Man bond in DNP-30 and reverse the configuration of glucose terminal hydrogen. When the concentration is 2 mg/mL, the scavenging rate of ·ABTS increased from 22.92% to 47.87%, the scavenging rate of ·OH increased from 31.46% to 56.75%, the scavenging rate of ·O2- increased from 24.52% to 35.61%, the FRAP value increased from 141.67 to 441.06 (0.4 mg/mL). When the concentration of polysaccharide was 5~10 mg/mL, the cytotoxicity of DNP-30E was significantly reduced compared with DNP-30. Based on the oxidative stress model of HaCaT cells stimulated by SDS, when the concentration is 0.5 mg/mL, compared with DNP-30 group, ROS content in DNP-30E group decreased by 34.21%, MDA content decreased by 50.20%, and SOD activity increased by 84.17% The results suggested that the enzymatically degraded polysaccharides of Dendrobium nobile has the potential to be developed into a new natural antioxidants.