高效液相色谱法测定魔芋精粉中葡甘聚糖的含量(1)
[摘要]目的 建立HPLC方法测定魔芋精粉中葡甘聚糖(konjac glucomannan，KGM)含量，并对高效液相色谱(HPLC)法与比色法测定结果进行比较。方法 魔芋葡甘聚糖用2.5 mol/L的H2SO4溶液110℃水解2 h，生成D-甘露糖、D-葡萄糖，经与1-苯基-3-甲基-5-吡唑啉酮(PMP)反应得到衍生物。HPLC法中采用的色谱柱为TOSOH TSK-GEL ODS，流动相A(乙腈)∶流动相B(0.45 g磷酸二氢钾、0.5 ml TEA、100 ml乙腈、900 ml纯水混合，pH=7.5)进行梯度洗脱，流速为1.0 ml/min，检测波长250 nm，柱温30℃；比色法参照GB/T 18104-2000进行测试。结果 HPLC法测定中D-甘露糖和D-葡萄糖分别在21.2～159.3 μg(r=0.9998)和21.5～154.2 μg(r=0.9999)范围内线性关系良好；KGM平均回收率为98.9%，RSD为2.2%。与比色法比含量少5%～7%。结论 HPLC法与比色法相比测定KGM含量更准确，可更好地用于魔芋精粉质量控制和分级。
[关键词]魔芋；葡甘聚糖；比色法；高效液相色譜法；含量测定
[中图分类号] R927.2 [文献标识码] A [文章编号] 1674-4721(2016)12(c)-0125-04
Determinination of konjac glucomannan in konjac refined powder by HPLC
CHEN Yan-jie CHU Yin-ling HAN Chao-yi ZHANG Jie
ENNOVA Health Science and Technology Co.，Ltd. of ENN Group，Langfang 065001，China
[Abstract]Objective To establish an HPLC method for content determination of konjac glucomannan (KGM) in konjac refined powder，and compare with results of colorimetric assay.Methods KGM was hydrolyzed with H2SO4 (2.5 mol/L) at 110℃ for 2 hours，which reacted with 1-phenyl-3-methyl-5-pyrazolone (PMP) before HPLC separation and formed two monosaccharides，D-mannose and D-glucose.HPLC was performed on the column of TOSOH TSK-GEL ODS with gradient elution of mobile phase A (acetonitrile) and mobile phase B (0.45 g potassium dihydrogen phosphate，0.5 ml TEA，100 ml acetonitrile，900 ml water，pH=7.5).The flow rate was 1.0 ml/min，detection wavelength was 250 nm，and column temperature was 30℃.Colorimetric method was tested with reference to GB/T18104-2000.Results Linearity of D-mannose was good in range of 21.2-159.3 μg (r=0.9998)；while D-glucose in range of 21.5-154.2 μg (r=0.9999).The average recovery of KGM was 98.9% (RSD 2.2%).The contents were less 5%-7% than that by colorimetry.Conclusion HPLC method is more accurate than colorimetry to determine content of KGM in konjac refined powder，and would be better for quality control and grading of konjac refined powder.
[Key words]Konjac；Glucomannan；Colorimetry；HPLC；Content determination
魔芋精粉是从多年生草本植物魔芋(Amorphophallus rerieri)块根中经干燥、粉碎、风选等步骤后获得。从营养学角度看，魔芋精粉是一种低热、低脂、低糖和高膳食纤维的食品。随着研究的深入，人们对膳食纤维的认识逐渐从“无营养物质”到今天具有重要的生理作用[1-2]，并被营养学界认定为第7类营养素。研究表明富含膳食纤维的魔芋精粉具有降血脂、降血糖、通便和保护肝脏等作用[3-6]。
魔芋葡甘聚糖(konjac glucomannan，KGM)是魔芋精粉主要功效成分，也是评价魔芋精粉和相关产品质量的关键指标[7-8]，其中GB/T 18104-2000中就是依据KGM的含量对魔芋精粉进行分级。KGM属于可溶性半纤维素，结构包含由D-葡萄糖和D-甘露糖通过β-1，4糖苷键聚合而成的主链，以及在主链甘露糖上由β-1，3糖苷键聚合的支链[9]。KGM具有亲水性、增稠性、稳定性好和口感平滑细腻等多种特征，同时又具有多种保健功能，广泛应用于普通食品、保健食品甚至药品中[10-12]。KGM含量测定方法包括比色法[7]、气相色谱法[8]和高效液相色谱(HPLC)法[13-15]等。HPLC法中有采用糖分析柱或者氨基柱结合示差检测器测定含量，不需要衍生的步骤，但是对色谱柱和检测器要求较高；也有采用PMP衍生化后用C18色谱柱结合紫外检测器测定含量。本实验通过优化PMP衍生条件建立反相高效液相色谱测定魔芋精粉中KGM的含量；同时参照GB/T18104-2000用比色法测定魔芋精粉中KGM含量，分析比较两种测定方法的优缺点。 (陈彦杰 褚银玲 韩超轶 张洁)
[关键词]魔芋；葡甘聚糖；比色法；高效液相色譜法；含量测定
[中图分类号] R927.2 [文献标识码] A [文章编号] 1674-4721(2016)12(c)-0125-04
Determinination of konjac glucomannan in konjac refined powder by HPLC
CHEN Yan-jie CHU Yin-ling HAN Chao-yi ZHANG Jie
ENNOVA Health Science and Technology Co.，Ltd. of ENN Group，Langfang 065001，China
[Abstract]Objective To establish an HPLC method for content determination of konjac glucomannan (KGM) in konjac refined powder，and compare with results of colorimetric assay.Methods KGM was hydrolyzed with H2SO4 (2.5 mol/L) at 110℃ for 2 hours，which reacted with 1-phenyl-3-methyl-5-pyrazolone (PMP) before HPLC separation and formed two monosaccharides，D-mannose and D-glucose.HPLC was performed on the column of TOSOH TSK-GEL ODS with gradient elution of mobile phase A (acetonitrile) and mobile phase B (0.45 g potassium dihydrogen phosphate，0.5 ml TEA，100 ml acetonitrile，900 ml water，pH=7.5).The flow rate was 1.0 ml/min，detection wavelength was 250 nm，and column temperature was 30℃.Colorimetric method was tested with reference to GB/T18104-2000.Results Linearity of D-mannose was good in range of 21.2-159.3 μg (r=0.9998)；while D-glucose in range of 21.5-154.2 μg (r=0.9999).The average recovery of KGM was 98.9% (RSD 2.2%).The contents were less 5%-7% than that by colorimetry.Conclusion HPLC method is more accurate than colorimetry to determine content of KGM in konjac refined powder，and would be better for quality control and grading of konjac refined powder.
[Key words]Konjac；Glucomannan；Colorimetry；HPLC；Content determination
魔芋精粉是从多年生草本植物魔芋(Amorphophallus rerieri)块根中经干燥、粉碎、风选等步骤后获得。从营养学角度看，魔芋精粉是一种低热、低脂、低糖和高膳食纤维的食品。随着研究的深入，人们对膳食纤维的认识逐渐从“无营养物质”到今天具有重要的生理作用[1-2]，并被营养学界认定为第7类营养素。研究表明富含膳食纤维的魔芋精粉具有降血脂、降血糖、通便和保护肝脏等作用[3-6]。
魔芋葡甘聚糖(konjac glucomannan，KGM)是魔芋精粉主要功效成分，也是评价魔芋精粉和相关产品质量的关键指标[7-8]，其中GB/T 18104-2000中就是依据KGM的含量对魔芋精粉进行分级。KGM属于可溶性半纤维素，结构包含由D-葡萄糖和D-甘露糖通过β-1，4糖苷键聚合而成的主链，以及在主链甘露糖上由β-1，3糖苷键聚合的支链[9]。KGM具有亲水性、增稠性、稳定性好和口感平滑细腻等多种特征，同时又具有多种保健功能，广泛应用于普通食品、保健食品甚至药品中[10-12]。KGM含量测定方法包括比色法[7]、气相色谱法[8]和高效液相色谱(HPLC)法[13-15]等。HPLC法中有采用糖分析柱或者氨基柱结合示差检测器测定含量，不需要衍生的步骤，但是对色谱柱和检测器要求较高；也有采用PMP衍生化后用C18色谱柱结合紫外检测器测定含量。本实验通过优化PMP衍生条件建立反相高效液相色谱测定魔芋精粉中KGM的含量；同时参照GB/T18104-2000用比色法测定魔芋精粉中KGM含量，分析比较两种测定方法的优缺点。 (陈彦杰 褚银玲 韩超轶 张洁)