发芽糙米中粗多糖的纯化及分子量测定
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摘要
本文对发芽糙米粗多糖进行纯化,通过比较活性炭吸附法、过氧化氢氧化法、大孔树脂吸附法三种方法的脱色效果,以及Sevage法、三氯乙酸法、酶与Sevage结合方法三种方法的脱蛋白效果,筛选出发芽糙米粗多糖脱色、脱蛋白的最佳方法。分别比较了DEAE-Sepharose CL-6B、DEAE Fast Flow、DEAE Sepharose 52三种柱层析填料对糙米粗多糖的层析纯化效果,筛选出最优填料。并对纯化后的发芽糙米多糖各组分进行分子量的测定。结果表明:大孔树脂AB-8对发芽糙米粗多糖脱色效果最佳,脱色率为86.57%,多糖损失率为28.96%。酶-Sevage法脱蛋白效果较好,且多糖损失率低,脱蛋白率为74.36%,多糖损失率为14.09%。对发芽糙米多糖进行柱层析的最佳填料为DEAE-Sepharose CL-6B。根据线性回归方程计算其平均分子量,水洗多糖组分的平均分子量为1.47×10~5Da,盐洗多糖组分的平均分子量分别为9.62×10~5、5.59×10~6、3.15×10~5Da。结论:确定了针对发芽糙米粗多糖的去除蛋白质和脱色的方法,并筛选出最佳的柱层析填料,分离出四个组分发芽糙米多糖,为发芽糙米粗多糖的提取、纯化、分离逐渐转变为工业化生产提供了理论基础。
In this paper,the crude polysaccharides from germinated brown rice were purified. The decolorization effects of activated carbon adsorption,hydrogen peroxide oxidation and macroporous resin adsorption were compared.The Sevage method,trichloroacetic acid method,enzyme and Sevage method were compared. These method was to screen out the best method for decolorization and deproteinization of germinated brown rice polysaccharide.The chromatographic purification effects of DEAESepharose CL-6B,DEAE Fast Flow and DEAE Sepharose 52 on crude polysaccharides from brown rice were compared and the optimal fillers were screened.The molecular weight of each component of the purified germinated brown rice polysaccharide was measured.The results showed that macroporous resin AB-8 had the best decolorization effect on germinated brown rice polysaccharide,the decolorization rate was 86.57%,and the polysaccharide loss rate was 28.96%.The enzyme-Sevage method had good deproteinization effect and low polysaccharide loss rate,the deproteinization rate was 74.36%,and the polysaccharide loss rate was 14.09%.The best filler for column chromatography of germinated brown rice polysaccharide was DEAE-Sepharose CL-6B.The weight average molecular weight was calculated according to the linear regression equation.The average molecular weight of the washed polysaccharide component was 1.47 × 10~5 Da,and the average molecular weight of the salt washed polysaccharide component was 9.62 × 10~5,5.59 × 10~6,3.15 × 10~5 Da,respectively.Conclusion: The method for removing protein and decolorization of crude polysaccharide from germinated brown rice was determined,and the best column chromatography packing was selected.Four components of germinated brown rice polysaccharide were isolated to extract,purify and separate the crude polysaccharide from germinated brown rice,which provided a theoretical basis for the transformation of polysaccharides into industrial production.
In this paper,the crude polysaccharides from germinated brown rice were purified. The decolorization effects of activated carbon adsorption,hydrogen peroxide oxidation and macroporous resin adsorption were compared.The Sevage method,trichloroacetic acid method,enzyme and Sevage method were compared. These method was to screen out the best method for decolorization and deproteinization of germinated brown rice polysaccharide.The chromatographic purification effects of DEAESepharose CL-6B,DEAE Fast Flow and DEAE Sepharose 52 on crude polysaccharides from brown rice were compared and the optimal fillers were screened.The molecular weight of each component of the purified germinated brown rice polysaccharide was measured.The results showed that macroporous resin AB-8 had the best decolorization effect on germinated brown rice polysaccharide,the decolorization rate was 86.57%,and the polysaccharide loss rate was 28.96%.The enzyme-Sevage method had good deproteinization effect and low polysaccharide loss rate,the deproteinization rate was 74.36%,and the polysaccharide loss rate was 14.09%.The best filler for column chromatography of germinated brown rice polysaccharide was DEAE-Sepharose CL-6B.The weight average molecular weight was calculated according to the linear regression equation.The average molecular weight of the washed polysaccharide component was 1.47 × 10~5 Da,and the average molecular weight of the salt washed polysaccharide component was 9.62 × 10~5,5.59 × 10~6,3.15 × 10~5 Da,respectively.Conclusion: The method for removing protein and decolorization of crude polysaccharide from germinated brown rice was determined,and the best column chromatography packing was selected.Four components of germinated brown rice polysaccharide were isolated to extract,purify and separate the crude polysaccharide from germinated brown rice,which provided a theoretical basis for the transformation of polysaccharides into industrial production.
引文
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[23]穆文静,杜玲,危瑞平,等.钝顶螺旋藻多糖Sevage法脱蛋白工艺的研究[J].内蒙古石油化工,2011(10):2-5.
[24]郝博慧,杨鑫,马莺.蕨麻叶Sevage法脱蛋白工艺研究[J].食品工业科技,2011(2):254-258.
[25]扈瑞平,张兴夫,杜玲,等.沙葱多糖Sevage法除蛋白工艺的研究[J].内蒙古大学学报:自然科学版,2009,40(6):658-662.
[26]陈宝,张立颖,沈芳,等.迷迭香多糖活性炭脱色工艺研究[J].应用化工,2012,7(41):7-10.
[27]陈健,耿安静,徐晓飞,香菇多糖的过氧化氢脱色工艺研究[J].食品工业科技,2010,31(3):12-15.
[28]付学鹏,杨晓杰.植物多糖脱色技术的研究[J].食品研究与开发,2007,28(11):166-169.
[29]付昊雨,张大伟,吴庆燕,等.红松松子壳多糖分离纯化及对小鼠抗氧化活性的影响[J].吉林农业大学学报,2017,39(2):233-237.
[30]冯孟鑫.人参果多糖的分级分离纯化及理化性质与抗肿瘤活性初探[D].北京:北京中医药大学,2016.
[31]Song C F,Wang S G,Yang J,et al.Optimization of vacuummicrowave radiation pretreatment on extraction of ganoderma polysaccharides[J].Mathematical Problems in Engineering,2015(4):113-116.
[32]安晓娟,冯琳,宋红平,等.植物多糖的结构分析及药理活性研究进展[J].中国药学杂志,2012,47(16):1271-1275.
[33]赵颖,宋新波,张丽娟,等.高效凝胶色谱法测定甘草多糖分子量及其分子量分布[J].天津中医药,2015,32(1):46-48.
[2]林俊.近5年多糖抗肿瘤活性研究进展[J].中国生化药物杂志,2013,21(3):130-131.
[3]Wang H,Shen W Z,Ooi E V,et al.The antiviral activity of polysaccharides extracted from Lobophora Variegata[J].Acta Nutrimenta Sinica,2007,29(3):271-275.
[4]Skeie G,Braaten T,Olsen A,et al.Whole grain intake and survival among scandinavian colorectal cancer patients[J].Nutrition and Cancer,2014,66(1):6-13.
[5]高雅,张春红,韩艳秋,等.糙米的营养价值及加工利用现状[J].农业科技与装备,2013(2):56-58.
[6]吴凤凤.发芽对糙米主要营养成分、生理功效和加工特性的影响[D].无锡:江南大学,2013.
[7]刘俊飞,汤晓智,扈战强,等.超声波辅助酶预处理对糙米发芽及发芽糙米理化特性的影响[J].食品科学,2015,36(4):11-18.
[8]Oh S H,Soh J R,Cha Y S.Germinated brown rice extract shows a nutraceutical effect in the recovery of chronic alcoholrelated symptoms[J].Journal of Medicinal Food,2003,6(2):115-121.
[9]Ohtsubo K,Suzuki K,Yasui Y.Bio-functional components in the processed pre-germinated brown rice by a twin-screw extruder[J].Journal of Food Composition and Analysis,2005,18(4):303-316.
[10]Cheng Z,Zhang Y,Song H,et al.Extraction optimization,characterization and antioxidant activity of polysaccharide from Gentiana scabra bge[J].International Journal of Biological Macromolecules,2016,93:369-380.
[11]汪艳群,孟宪军,李冬男,等.五味子多糖脱蛋白工艺的研究[J].食品工业科技,2011,32(3):280-284.
[12]欧文,黄榕彬,曹轩,等.荠菜多糖提取液的除蛋白工艺研究[J].化工技术与开发,2012,41(11):7-10.
[13]赵艳,王白娟,杨青松,等.红雪茶多糖过氧化氢脱色工艺优化[J].南方农业学报,2016,47(5):710-714.
[14]秦亚东,汪荣斌,周娟娟.白芍多糖脱色工艺研究[J].中成药,2015,37(12):2783-2787.
[15]王书华,饶娜,安芳.金莲花多糖的分离纯化及其抗氧化作用研究[J].中成药,2013,35(11):2384-2389.
[16]胡飞,何艳克.苦瓜多糖的分离纯化及抗氧化性的研究[J].食品工业科技,2009,30(8):247-249.
[17]付学鹏,杨晓杰.植物多糖脱色技术的研究[J].食品研究与开发,2007(11):166-169.
[18]李丹丹,金征宇.牛蒡菊糖脱色工艺的研究[J].农业工程学报,2007(8):241-244.
[19]夏玮,吕庆,张文清,等.大孔吸附树脂脱色桑叶多糖的研究[J].食品与发酵工业,2007(2):141-144.
[20]马永强,张静,王鑫,等.酶法提取甜玉米芯多糖的对比研究[J].哈尔滨商业大学学报,2015(4):162-165.
[21]Ross K A,Godfrey D,Fukumoto L.Purification and characterization of water soluble polysaccharides from sweet cherries,raspberries,and ginseng:Chemical composition and bioactivity[J].Research in Health and Nutrition,2015,29(3):1-13.
[22]王艾平,周丽明.考马斯亮蓝法测定茶籽多糖中蛋白质含量条件的优化[J].河南农业科学,2014,43(3):150-153.
[23]穆文静,杜玲,危瑞平,等.钝顶螺旋藻多糖Sevage法脱蛋白工艺的研究[J].内蒙古石油化工,2011(10):2-5.
[24]郝博慧,杨鑫,马莺.蕨麻叶Sevage法脱蛋白工艺研究[J].食品工业科技,2011(2):254-258.
[25]扈瑞平,张兴夫,杜玲,等.沙葱多糖Sevage法除蛋白工艺的研究[J].内蒙古大学学报:自然科学版,2009,40(6):658-662.
[26]陈宝,张立颖,沈芳,等.迷迭香多糖活性炭脱色工艺研究[J].应用化工,2012,7(41):7-10.
[27]陈健,耿安静,徐晓飞,香菇多糖的过氧化氢脱色工艺研究[J].食品工业科技,2010,31(3):12-15.
[28]付学鹏,杨晓杰.植物多糖脱色技术的研究[J].食品研究与开发,2007,28(11):166-169.
[29]付昊雨,张大伟,吴庆燕,等.红松松子壳多糖分离纯化及对小鼠抗氧化活性的影响[J].吉林农业大学学报,2017,39(2):233-237.
[30]冯孟鑫.人参果多糖的分级分离纯化及理化性质与抗肿瘤活性初探[D].北京:北京中医药大学,2016.
[31]Song C F,Wang S G,Yang J,et al.Optimization of vacuummicrowave radiation pretreatment on extraction of ganoderma polysaccharides[J].Mathematical Problems in Engineering,2015(4):113-116.
[32]安晓娟,冯琳,宋红平,等.植物多糖的结构分析及药理活性研究进展[J].中国药学杂志,2012,47(16):1271-1275.
[33]赵颖,宋新波,张丽娟,等.高效凝胶色谱法测定甘草多糖分子量及其分子量分布[J].天津中医药,2015,32(1):46-48.