滨海白首乌蛋白和多糖的提取纯化及其活性功能的研究
发布时间：2018-11-26 15:54
【摘要】：滨海白首乌中蛋白质和多糖含量丰富,是其具有生物理活性的重要物质基础,具有较好的研究和开发的价值。本研究分别提取滨海白首乌中这两种重要物质,对其功能性进行全面的分析,以期为其在食品工业中的应用提供理论依据。本研究对白首乌中的蛋白质进行提取,评价了其理化性质和加工性质;然后采用响应面法优化白首乌多糖的提取条件,并对所得粗多糖进行纯化并分析了白首乌多糖的理化性质和体外抗氧化性。具体研究内容及实验结果如下:首先本研究比较不同溶剂提取法对白首乌可溶性蛋白得率的影响,通过电泳法分析了蛋白的提取效果,同时对白首乌可溶性蛋白进行初步表征;采用液相色谱串联质谱法对提取得到的白首乌蛋白进行鉴定:大豆分离蛋白作为对照,对白首乌蛋白的加工特性进行分析。结果表明:PBS法提取白首乌蛋白优于传统的碱液提取和Tris-HCl法提取;白首乌蛋白的分子量主要分布在348 kDa左右,另一条相对迁移率较低、分子量在700 kDa附近的条带推测为其聚集体,白首乌蛋白分子主要由一种亚基(分子量为28 kDa)构成:白首乌蛋白具有良好的加工性能,特别是持油性、乳化性及乳化稳定性和起泡性,均好于大豆分离蛋白,白首乌蛋白的溶解性与大豆分离蛋白的溶解性随pH变化趋势基本一致。实验选择对料液比、提取温度和提取时间3个因素进行单因素试验。根据单因素试验结果设计中心组合试验,以多糖得率为响应值,采用响应面分析法获得白首乌多糖的最优工艺参数。结果表明,白首乌多糖的最优提取工艺为料液比1:17、提取温度为91℃、提取时间为3 h。在此条件下,白首乌多糖得率为1.58%,与模型的预期值1.61%基本相符。白首乌多糖提取液经过酶法淀粉、Sevge法去蛋白、95%的乙醇沉淀后,再经过透析和冻干后得到不含有核酸和蛋白质粗多糖。经过DEAE-Sepharo Fast Flow阴离子交换色谱和葡聚糖G-150凝胶色谱柱色谱对白首乌多糖进行了分级分离,最终得到一个纯化组分。通过HPGPC测定其相对分子质量为830.93 KDa。采用PMP柱前衍生化法分析白首乌多糖的单糖组成,结果表明白首乌多糖主要是由半乳糖醛酸、半乳糖、阿拉伯糖、鼠李糖、葡萄糖醛酸和甘露糖组成,其摩尔比为甘露糖:鼠李糖:葡萄糖醛酸:半乳糖醛酸:半乳糖:阿拉伯糖=0.058:0.280:0.071:1.455:0.918:0.469。采用4种抗氧化活性测定的方法,对滨海白首乌抗氧化清除自由基能力进行分析和研究,结果表明:白首乌多糖具有一定的清除DPPH自由基、超氧自由基、ABTS+·自由基能力,并且随着多糖的浓度增加,清除率逐渐增高。清除DPPH自由基、超氧自由基、ABTS+·自由基能力的IC_(50)分别为0.5543mg/mL、0.5881mg/mL和0.1232 mg/mL,其中清除ABTS+·自由基能力较强,与VC相接近;白首乌多糖的ORAC值为109.94μmol Trolox/g,表明白首乌多糖具有较强的抗氧化能力。最后,通过建立TNF-α诱导人脐静脉血管内皮细胞产生氧化应激反应模型,来研究白首乌多糖的抗氧化性。结果发现,白首乌多糖可显著提高由TNF-α诱导的人脐静脉血管内皮细胞的活力和SOD酶活性,显著抑制细胞中活性氧(ROS)含量,这表明白首乌多糖能够减少血管内皮细胞受到氧化损坏。
[Abstract]:The content of protein and polysaccharide in the coastal white fleece-flower root is rich, it is the important material foundation of its physical activity, and has good value for research and development. In this study, the two important substances in the coastal white fleece-flower root are respectively extracted, and the function of the two important substances is comprehensively analyzed, so as to provide a theoretical basis for the application of the two important substances in the food industry. In this study, the extraction of protein from Radix Cynanchi Auriculati was carried out, and its physical and chemical properties and processing properties were evaluated. Then, the extraction condition of the polysaccharide was optimized by the response surface method, and the obtained crude polysaccharide was purified and the physical and chemical properties and the in vitro oxidation resistance of the polysaccharide were analyzed. The contents and experimental results are as follows: First, the effect of different solvent extraction method on the soluble protein of Radix Polygoni Multiflori is compared, and the effect of protein extraction is analyzed by electrophoresis, and the soluble protein of Radix Cynanchi Auriculati is initially characterized. The obtained white fleece-flower root protein was identified by liquid chromatography-tandem mass spectrometry: as a control, the processing characteristics of the white fleece-flower root protein were analyzed. The results showed that the extraction of the white fleece-flower root protein by the method of PBS was better than that of the conventional alkaline solution extraction and the Tris-HCl method, the molecular weight of the white fleece-flower root protein was mainly about 348 kDa, the other relative mobility was low, and the band with the molecular weight near 700 kDa was presumed to be an aggregate, The white fleece-flower root protein is mainly composed of a subunit (molecular weight of 28 kDa), the white fleece-flower root protein has good processing performance, in particular to oil-holding, emulsifying and emulsifying stability and foaming property, The solubility of the white fleece-flower root protein and the solubility of the soybean protein isolate are basically consistent with the change of pH. The single factor test was carried out for three factors, such as the ratio of liquid to liquid, the extraction temperature and the extraction time. Based on the results of single factor test, the optimal process parameters of the polysaccharide of Cynanchum multiflorum were obtained by using the response surface analysis method. The results showed that the optimum extraction process of the polysaccharide was 1: 17, the extraction temperature was 91 鈩，
本文编号：2358993
[Abstract]:The content of protein and polysaccharide in the coastal white fleece-flower root is rich, it is the important material foundation of its physical activity, and has good value for research and development. In this study, the two important substances in the coastal white fleece-flower root are respectively extracted, and the function of the two important substances is comprehensively analyzed, so as to provide a theoretical basis for the application of the two important substances in the food industry. In this study, the extraction of protein from Radix Cynanchi Auriculati was carried out, and its physical and chemical properties and processing properties were evaluated. Then, the extraction condition of the polysaccharide was optimized by the response surface method, and the obtained crude polysaccharide was purified and the physical and chemical properties and the in vitro oxidation resistance of the polysaccharide were analyzed. The contents and experimental results are as follows: First, the effect of different solvent extraction method on the soluble protein of Radix Polygoni Multiflori is compared, and the effect of protein extraction is analyzed by electrophoresis, and the soluble protein of Radix Cynanchi Auriculati is initially characterized. The obtained white fleece-flower root protein was identified by liquid chromatography-tandem mass spectrometry: as a control, the processing characteristics of the white fleece-flower root protein were analyzed. The results showed that the extraction of the white fleece-flower root protein by the method of PBS was better than that of the conventional alkaline solution extraction and the Tris-HCl method, the molecular weight of the white fleece-flower root protein was mainly about 348 kDa, the other relative mobility was low, and the band with the molecular weight near 700 kDa was presumed to be an aggregate, The white fleece-flower root protein is mainly composed of a subunit (molecular weight of 28 kDa), the white fleece-flower root protein has good processing performance, in particular to oil-holding, emulsifying and emulsifying stability and foaming property, The solubility of the white fleece-flower root protein and the solubility of the soybean protein isolate are basically consistent with the change of pH. The single factor test was carried out for three factors, such as the ratio of liquid to liquid, the extraction temperature and the extraction time. Based on the results of single factor test, the optimal process parameters of the polysaccharide of Cynanchum multiflorum were obtained by using the response surface analysis method. The results showed that the optimum extraction process of the polysaccharide was 1: 17, the extraction temperature was 91 鈩，
本文编号：2358993
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