泽泻叶多糖复合酶辅助提取工艺的优化及其益生元活性研究

Optimization of compound enzyme-assisted extraction process and probiotic activity for polysaccharides from Alisma plantago-aquatica leaves

目的优化泽泻叶多糖复合酶辅助提取工艺,并评价其益生元活性。方法在单因素试验基础上,以料液比、酶解温度、酶解时间为影响因素,多糖提取率为评价指标,响应面法优化提取工艺。DEAE-52纤维素柱分离纯化粗多糖,分析其组成,HPGPC法测定其分子量。考察多糖对L.plantarum、L.buchneri、Weissella confuse X1、L.gasseri密度、pH值的影响,以及对菌群失调小鼠肠道的调节作用。结果最佳条件为复合酶(纤维素酶-木瓜蛋白酶)比例1∶2,酶用量1.20%,料液比1∶25,酶解温度55℃,酶解时间60 min,多糖提取率为2.57%。粗多糖中的中性多糖重均分子量为37187 Da,含量为72.64%;酸性多糖重均分子量为33134 Da,含量为62.22%。中性多糖、酸性多糖促进4种乳杆菌增殖,降低培养基pH值。与自然恢复组比较,中性多糖中剂量组、酸性多糖低剂量组LPS水平降低(P<0.05),中性多糖各剂量组SOD活性升高(P<0.05),中性多糖各剂量组及酸性多糖中、高剂量组MDA水平降低(P<0.05)。多糖减少肠道中有害菌数量,增加有益菌数量,降低盲肠、结肠内容物pH值;除酸性多糖低剂量组外,各多糖组结肠氨含量低于自然恢复组(P<0.05)。结论该方法稳定可行,可用于复合酶辅助提取具有益生元活性的泽泻叶多糖,其机制可能为改善肠黏膜通透性和氧化应激反应,调节肠道菌群。

AIM To optimize the compound enzyme-assisted extraction process for polysaccharides from Alisma plantago-aquatica Linn.leaves,and to evaluate their probiotic activity.METHODS With solid-liquid ratio,enzymolysis temperature and enzymolysis time as influencing factors,the extraction rate of polysaccharides as an evaluation index,the extraction process was optimized by response surface method on the basis of single factor test.DEAE-52 cellulose column was adopted in the isolation and purification of crude polysaccharides,after which the composition was analyzed,along with the molecular weight determination by molecular weight.The effects of polysaccharides on densities and pH values of L.plantarum,L.buchneri,Weissella confuse X1 and L.gasseri,and the regulation effects on intestines of flora imbalance mice were investigated.RESULTS The optimal conditions were determined to be 1∶2 for complex enzyme(cellulase-papain)ratio,1.20%for enzyme consumption,1∶25 for solid-liquid ratio,55℃for enzymolysis temperature,and 60 min for enzymolysis time,the extraction rate of polysaccharides was 2.57%.The neutral polysaccharide in crude polysaccharides demonstrated the weight-average molecular weight and content of 37187 Da and 72.64%,while those of acidic polysaccharide were 33134 Da and 62.22%,respectively.Both neutral polysaccharide and acidic polysaccharide promoted the proliferation of four kinds of Lactobacillus and reduced the pH value of culture medium.Compared with natural recovery group,neutral polysaccharide medium-dose group and acidic polysaccharide low-dose group displayed decreased LPS level(P<0.05),various doses of neutral polysaccharide groups exhibited increased SOD activity(P<0.05),and various doses of neutral polysaccharide groups and acidic polysaccharide medium-dose,high-dose groups showed decreased MDA level(P<0.05).The polysaccharides reduced the count of harmful bacteria and elevated the count of beneficial bacteria in intestinal tract,along with the decrease of content pH values in caeca and colons.Except for the acidic polysaccharide low-dose group,various polysaccharide groups demonstrated lower ammonia content in colons than the natural recovery group(P<0.05).CONCLUSION This stable and feasible method can be used for the compound enzyme-assisted extraction of polysaccharides with probiotic activity from A.plantago-aquatica leaves,the mechanisms may contribute to the improvement of intestinal mucosal permeability,oxidative stress and regulation of intestinal flora.