刺梨果多糖提取过程中脱蛋白和脱色方法研究

Study on the Method of Deproteinization and Decolorization in the Process of Extracting Polysaccharides from Rosa roxburghii Fruit

[目的]筛选出梨果粗多糖水提醇沉过程中最佳的蛋白质和色素的脱除方法。[方法]以蛋白质脱除率、多糖保留率以及数据加权平均法构成的综合评分为评价指标,选用三氯乙酸法、三氯乙酸-Sevage法、三氯乙酸-正丁醇法、Sevage法、木瓜蛋白酶法、木瓜蛋白酶-Sevage法、木瓜蛋白酶-三氯乙酸法、木瓜蛋白酶-三氯乙酸-正丁醇法和盐酸法9种脱蛋白方法对刺梨果粗多糖水提物的脱蛋白效果进行对比;同时在单因素试验的基础上,通过正交试验优化三氯乙酸-正丁醇法对刺梨果粗多糖水提物的脱蛋白工艺条件。并以脱色率和多糖保留率为评价指标,比较过氧化氢溶液与活性炭粉末对刺梨果多糖水提物的脱色效果。[结果]三氯乙酸-正丁醇法脱蛋白效果最佳,三氯乙酸-正丁醇法脱蛋白的最佳工艺条件为样液与试剂体积比为1∶2、三氯乙酸与正丁醇体积比为1∶10、振荡时间为60 min,此时脱蛋白率为78.64%,多糖保留率为86.59%。活性炭粉末对刺梨果粗多糖的脱色素和多糖保留率均优于过氧化氢溶液。[结论]该试验优选出的方法具有实际可操作性,可有效脱除刺梨果粗多糖中的蛋白质和色素,最终确定三氯乙酸-正丁醇法和活性炭粉末作为刺梨果多糖初步纯化方法,为进一步研究多糖的色谱分析、结构鉴定、构效关系和生物活性研发提供数据支持。

[Objective]To screen out the best method for removing protein pigment in the process of water extraction alcohol precipitation of Rosa roxburghii fruit crude polysaccharide.[Method]Taking protein removal rate,polysaccharide retention rate and data weighted average method as the evaluation index,9 kinds of deproteinization methods(trichloroacetic acid method,trichloroacetic acid-Sevage method,trichloroacetic acid-n-butanol method,Sevage method,papain method,papain-Sevage method,papain-trichloroacetic acid method,papain-trichloroacetic acid-n-butanol method hydrochloric acid method)were selected to compare the deproteinization effects of crude polysaccharide water extracts from Rosa roxburghii fruit.At the same time,on the basis of single factor experiment,orthogonal experiment was used to optimize the deproteinization process conditions of the water extract of Rosa roxburghii fruit crude polysaccharide by trichloroacetic acid-n-butanol method.And the decolorization rate and polysaccharide retention rate were used as evaluation indicators to compare the decolorization effect of hydrogen peroxide solution and activated carbon powder on the water extract of prickly pear fruit polysaccharide.[Result]The deproteinization effect of the trichloroacetic acid-n-butanol method was the best,the best process conditions for the deproteinization of the trichloroacetic acid-n-butanol method were that the volume ratio of sample solution to reagent was 1∶2,the volume ratio of trichloroacetic acid to n-butanol was 1∶10,and the shaking time was 60 min.At this time,the deproteinization rate was 78.64%,the polysaccharide retention rate was 86.59%.The powdered activated carbon had better depigmentation polysaccharide retention rate of the crude polysaccharides from Rosa roxburghii fruit than the hydrogen peroxide solution.[Conclusion]The method selected in this test has practical operability,and can effectively remove the protein and pigment in the crude polysaccharide of Rosa roxburghii fruit.Finally,the trichloroacetic acid-n-butanol method and activated carbon powder are determined as the preliminary purification method of Rosa roxburghii fruit polysaccharide,which provides data support for further research on chromatographic analysis,structure identification,structure-activity relationship and biological activity research and development of polysaccharides.