高效液相体积排阻色谱法测定稻米支链淀粉链长的相对分子质量分布

doi:10.3864/j.issn.0578-1752.2013.16.020

中国农业科学 ›› 2013, Vol. 46 ›› Issue (16): 3488-3495.doi: 10.3864/j.issn.0578-1752.2013.16.020

高效液相体积排阻色谱法测定稻米支链淀粉链长的相对分子质量分布

杨小雨12, 刘正辉1, 李刚华1, 王强盛1, 王绍华1, 丁艳锋1

1.南京农业大学农学院/农业部作物生理生态与生产管理重点实验室，南京 210095
2.贵州省亚热带作物研究所，贵州兴义 562400

收稿日期:2012-12-12 出版日期:2013-08-15 发布日期:2013-03-22
通讯作者: 通信作者丁艳锋，E-mail：dingyf@njau.edu.cn。通信作者刘正辉，E-mail：liuzh@njau.edu.cn
作者简介:杨小雨，E-mail：sntstzdaxia@aliyun.com
基金资助:
国家科技支撑计划（2012BAD04B08）、国家自然科学基金项目（31171485）、新世纪优秀人才支撑计划（NCET-10-0472）

Determination of Rice Amylopectin Chain-Length Distribution by Using High Performance Size Exclusion Chromatography

YANG Xiao-Yu-12, LIU Zheng-Hui-1, LI Gang-Hua-1, WANG Qiang-Sheng-1, WANG Shao-Hua-1, DING Yan-Feng-1

1.Agronomy College of Nanjing Agricultural University/Key Laboratory of Crop Physiology and Ecology and Production Management, Ministry of Agriculture, Nanjing 210095
2.Guizhou Institute of Subtropical Crops, Xingyi 562400, Guizhou

Received:2012-12-12 Online:2013-08-15 Published:2013-03-22

摘要/Abstract

摘要： 【目的】建立稻米支链淀粉链长相对分子质量分布的高效液相体积排阻色谱法（high performance size exclusion chromatography, HPSEC）的测定方法。【方法】先采用丁醇沉降法分离提纯稻米支链淀粉，纯化后的支链淀粉经异淀粉酶酶解切开分支糖苷键，然后运用HPSEC测定其相对分子质量分布。建立了稻米支链淀粉链长相对分子质量分布的HPSEC分析方法：Shodex SB-G、Shodex SB-803（1 000—100 000）、Shodex SB-802.5（300—10 000）3根凝胶色谱柱串连，柱温40℃；流动相为0.1 mol•L-1 Tris+0.1 mol•L-1 NaCl，pH=7.40，流速0.5 mL•min-1；采用示差折光检测器（Differential refractive index detector, RI），检测器温度为37℃；待测样品浓度为5 mg•mL-1，进样量20 μL。【结果】本法提纯的稻米支链淀粉：淀粉含量为99.4%、蛋白残余为0.02%、支链淀粉碘复合物最大吸收峰为517 nm，为高纯度支链淀粉。供试大米样品脱分支支链淀粉的平均聚合度（DP）分布在2—120，无杂峰干扰。【结论】试验结果和方法验证表明本方法数据可靠、重复性好、简单易行。

关键词: rice amylopectin , HPSEC , molecular weight distribution

Abstract: 【Objective】The aim of this study is to establish a sensitive high performance size exclusion chromatography with differential refractive index detector (HPSEC-RI) methods for measuring molecular weight distribution of rice amylopectin. 【Method】The proposed method includes three parts: Firstly, separation of amylopectin by butanol precipitation method, Secondly, hydrolysis of rice amylopectin by isoamylase, and Thirdly, qualification of the molecular weight distribution by HPSEC-RI. The proposed parameters of HPSEC-RI are as follows: columns of Shodex SB-G, Shodex SB-803(1 000-100 000) and Shodex SB-802.5(300-10 000) being connected in tandem, column temperature being 40℃, the mobile phase being 0.1 M Tris+0.1 M NaCl (pH=7.40) with a rate of 0.5 mL•min-1, detector temperature being 37℃, sample concentration being 5 mg.mL-1 and the input volume being 20 μL.【Result】The rice amylopectin purified in this study is of high-purity, with the starch content being 99.4%, protein residue being 0.02%, and the peak of rice amylopectin-iodine complex absorption spectrum being 517 nm. The tested rice sample amylopectin chain-length distribution is in the range of DP 2-120. 【Conclusion】The precision and repeatability verification test showed that the proposed method performed well in these aspects including good reproducibility, simple sample preparation, and high efficiency.

Key words: rice amylopectin , HPSEC , molecular weight distribution

杨小雨12, 刘正辉1, 李刚华1, 王强盛1, 王绍华1, 丁艳锋1. 高效液相体积排阻色谱法测定稻米支链淀粉链长的相对分子质量分布[J]. 中国农业科学, 2013, 46(16): 3488-3495.

YANG Xiao-Yu-12, LIU Zheng-Hui-1, LI Gang-Hua-1, WANG Qiang-Sheng-1, WANG Shao-Hua-1, DING Yan-Feng-1. Determination of Rice Amylopectin Chain-Length Distribution by Using High Performance Size Exclusion Chromatography[J]. Scientia Agricultura Sinica, 2013, 46(16): 3488-3495.

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高效液相体积排阻色谱法测定稻米支链淀粉链长的相对分子质量分布

Determination of Rice Amylopectin Chain-Length Distribution by Using High Performance Size Exclusion Chromatography

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