酵母源葡萄糖耐量因子GTF的纯化及其铬价态分析

摘要

Glucose tolerance factor (GTF) can effectively regulate sugar metabolism,improve the affinity of insulin to receptor,and improve fat metabolism.Up to now,its chromic value in GTF is unclear,which limited its application in reducing sugar health food.In this paper,extraction of GTF with ammonia and ethanol method from high chromium yeast was optimized.And then glucose tolerance factor (GTF) with high purity was obtained by two stages of gel chromatography.HPLC-ICP-MS was used to analyze chromium valence of GTF.The high chromium yeast was treated with 0.1 mol/L of ammonium for 4 h and then 30％ of ethanol for 20 min.The total chromium in this extraction was 133 μg.Compared with ammonia extraction alone,the ratio of chromium to protein was increased by 6 times in this way.After extraction,glucose tolerance factor was purified by tow chromatography consecutively.The purified GTF was analyzed by HPLC-ICP-MS.Its chromatography showed that there was only one peak of chromium in this sample.It appeared in range from 31 s to 90 s,which was in agreement with standard of trivalent chromium.There-fore,it could be concluded that chromium in GTF exist in the state of trivalent.This will provide basis for research and development of high chromium yeast for dietary supplement.%酵母源葡萄糖耐量因子(glucose tolerance factor,GTF)可有效调节糖代谢、提高胰岛素与受体的亲和力、改善脂肪代谢.但GTF中的铬的价态不清严重限制了它在辅助降糖保健食品的中应用.实验优化了氨水联合有机溶剂提取GTF的方法.通过两级分子筛联用对GTF进行纯化,得到纯度较高的GTF.采用高效液相色谱(high performance liquid chromatography,HPLC)联用电感耦合等离子体质谱(inductively coupled plasma mass spectrometry,ICP-MS)技术对GTF中铬价态进行分析鉴定.结果表明,富铬酵母粉经0.1 mol/L氨水振荡提取4h后,上清液再经30％乙醇静置20 min,离心收集上清液.该粗提液中铬含量为133 μg,铬蛋白比为5.015;相比与氨水提取法(铬蛋白比0.804),氨水联合有机溶剂法的铬蛋白比提高了6倍,提取效率提高极大.GTF粗提液再经两级葡聚糖凝胶层析柱分离纯化,得到了一个与铬峰重合的蛋白峰.利用HPLC-ICP-MS对其进行铬价态分析,图谱显示GTF样品中铬出峰的时间为31 ～90 s,出峰时间与Cr(Ⅲ)的出峰时间一致,有且只有一个铬峰,GTF中的铬是以Cr(Ⅲ)形态存在,无Cr(Ⅵ)存在.该结果为GTF保健食品或安全膳食铬补充剂的开发提供了理论依据.