一株芽孢杆菌胞外多糖的分离纯化及其抗氧化性测定

摘要

基于实验室从新疆罗布泊沙漠筛选到一株芽孢杆菌,研究了该菌胞外多糖的分离纯化工艺及其抗氧化性质.发酵液经离心,抽滤等预处理后,使用Sevag试剂除蛋白,并以无水乙醇作提取溶剂,通过正交实验确定最佳提取条件为:pH为7.0,温度为4℃,时间为1.5 h,料液比为1:4,粗多糖溶解后上活性炭柱(1.5 cm×24 cm),用蒸馏水、60%乙醇及95%乙醇洗脱,分离得到主要部分,再经Sephadex G-100凝胶柱,用0.2 mol/L的NaCl溶液洗脱,硫酸苯酚法和考马斯亮蓝法分别检测洗脱多糖和蛋白质,结果表明,该产物为均一组分的糖蛋白(EPS).在体外抗氧化性实验显示EPS具有很强的抗氧化性,能清除羟基自由基(·OH)、超氧阴离子自由基(·O_2~-),并且对脂质过氧化有抑制作用,是一种很好的天然抗氧化剂.%Based on the Bacillus sp., isolated from Lop Nur Desert, the technology of separation and purification and the antioxidant effect were studied. After centrifugation and vacuum filtration, the deproteiniza-tion of supernatant was operated with Sevag reagent. The crude exopolysaccharide (EPS) was obtained by precipitation with ethanol. The optimum conditions for the isolation were as follow: pH 7.0, temperature 4?, time 1.5 h, and material to ethanol ratio 1: 4. Dissolved in water, the crude EPS was fractional separated on activated carbon column (1.5 cm×24 cm), eluted with distilled water, 60% ethanol, 95% ethanol, and the main fraction was collected. Then the EPS was purified on Sephadex G-100 gel column, eluted with NaCl (0.2 mol/L). Fractions (4 mL, each) were also combined according to total sugar by phenol-sulfuric acid method and protein content was determined by Coomassie brilliant blue. The results showed that EPS was relatively homogeneous glycoprotein. The data of antioxidation in vitro showed that the EPS had a high antioxidant activity, which could quench hydroxyl radical, superoxide radical and had antilipid peroxidation activity. All of these indicated that EPS was a good natural antioxidant.