白芨多糖的酶法精制工艺条件研究

Enzymatic Purification of Crude Polysaccharides Extracted from Bletilla striata Roots

进行白芨多糖的酶法精制工艺研究,初步考察5种蛋白酶对白芨多糖的精制效果,并选择木瓜蛋白酶进一步研究,考察加酶量、pH值、温度和酶解时间对白芨多糖纯度和蛋白残余率的影响,确定了适宜的酶解条件:加酶量8～10mg/g底物,pH7.0～7.5,温度45～50℃,酶解时间120～150min。精制白芨多糖中葡甘聚糖最高含量可达97.0%,蛋白含量由粗多糖的0.91%下降到0.32%。实验同时进行了酶法随程处理研究,获得的多糖纯度达92.0%。该法提供了一种更为简单的白芨多糖提取和精制工艺。

Generally, the purity of crude polysaccharide extracted from Bletilla striata roots applied in Chinese traditional medicine is no higher than 80%, which seriously restricts its further utilization as an additive or a medicine material. In order to purify the crude polysaccharide extracted from Bletilla striata roots using 80% ethanol, this study employed the enzymatic hydrolysis technology to remove protein impurities from the crude polysaccharide extract. Papain was selected for further study because it presented the most outstanding efficiency of protein removal compared to neutral protease, chymocotrypsin, Amano protease, papain and alkaline protease. Four parameters affecting the efficiency of protein removal of papaln including enzyme concentration, pH value, temperature and enzymolysis duration were investigated by one-factor-at-a-time （OFAT） experiments. The optimum ranges of the four parameters for protein removal were as follows： enzyme concentration 8 - 10 mg/g substrate, pH 7.0 - 7.5, temperature 45 - 50℃, and enzymolysis duration 120 - 150 min, and in verification experiments repeated three times a highest purity of 97.0% of the polysaccharide was observed, as well as a decrease of protein content in the crude polysaccharide from 0.91% to 0.32%. When papain was directly added to the extraction solution, a purity of 92.0% could be achieved at 4 h of extraction. The one-step extraction - purification procedure may be a more simple and rapid technique for the extraction and purification of polysaccharide from Bletilla striata roots.