响应面试验优化末水坛紫菜多糖除蛋白工艺及其抗氧化活性

目的：研究末水坛紫菜多糖的除蛋白方法及所制备多糖在细胞内的抗氧化作用。方法：响应面法优化末水坛紫菜多糖酶法除蛋白工艺条件；建立H2O2诱导HeLa细胞氧化损伤模型，MTT法测定细胞存活率，2′,7′-二氯荧光素二乙酸酯检测细胞内活性氧水平。结果：样品液中末水坛紫菜多糖酶法除蛋白最佳工艺条件为木瓜蛋白酶用量0.7 mg/mL、酶解温度50 ℃、酶解时间55 min，此时蛋白清除率为61.28%；在此基础上，用4%三氯乙酸继续除蛋白，蛋白清除率可达80.73%，多糖保留率为79.7%。与模型组相比，经末水坛紫菜多糖处理的损伤细胞存活率显著提高（P＜0.01），且细胞内活性氧含量显著下降（P＜0.01）。结论：木瓜蛋白酶-三氯乙酸联用可有效除去末水坛紫菜多糖中的蛋白。末水坛紫菜多糖有较强的抗氧化能力，能清除细胞内过剩的活性氧，修复H2O2对HeLa细胞的氧化损伤。

Optimization of Deproteinization Process of Polysaccharides Extracted from Abandoned Porphyra haitanensis and Their Antioxidant Activity

Objective: This study aimed to investigate the deproteinization process of polysaccharides extracted from abandoned Porphyra haitanensis and to evaluate their cellular antioxidant activity. Methods: Response surface methodology was used to optimize the process conditions for the enzymatic deproteinization of P. haitanensis polysaccharides with papain. An oxidative stress model was built by stimulating HeLa cells with H2O2 and the methyl thiazolyl tetrazolium (MTT) method was used to determine the cell survival rate. The level of cellular reactive oxygen species (ROS) was also measured by 2’,7’-dichlorodihydrofluorescin diacetate (DCFH-DA). Results: The optimized deproteinization conditions were determined as follows: hydrolysis time, 55 min; temperature, 50 ℃; and papain concentration, 0.7 mg/mL; giving a percentage of deproteinization of 61.28%. After further deproteinization with 4% trichloroacetic acid (TCA), the deproteinization rate increased up to 80.73%, and the retention rate of polysaccharide was 79.7%. Compared with the model group, the survival rate of cells treated with the purified polysaccharides from P. haitanensis was significantly increased (P < 0.01), and the level of ROS was significantly decreased (P < 0.01). Conclusions: The combination of papain hydrolysis and TCA treatment was an effective method for the removal of protein from Porphyra haitanensis polysaccharides. Porphyra haitanensis polysaccharides had significant antioxidant activity and could relieve the H2O2-induced injury in HeLa cells by eliminating redundant ROS.