Abstract: In order to explore the separation and purification process of glucose tolerance factor (GTF),the purification steps were simplified in order to obtain GTF with high purity.Previous ammonia extraction experiments were based on organic solvent precipitation to remove protein.In the present study,based on the previous study,the purification process was further optimized with different organic solvents,using the ratio of organic chromium content (μg) and total protein content (mg) as evaluation criteria.The purity of isolated GTF was further verified by high performance liquid chromatography analysis.In addition,the glucose consumptions in insulin resistance type Hep-G2 cells treated with GTF before and after purification were measured for the detection of its glucose metabolism regulating activity.As a result,the optimized purification process was with 30% cold ethanol precipitation for 20 min,10000 ×g centrifugation.Compared with traditional ammonia extraction,the organic chromium content and the content of total protein ratio improved 6 times using the developed method.The extraction rate and purity of GTF were significantly improved and the purified GTF showed a regulating effect on insulin resistance type Hep-G2 cells in glucose metabolism (P<0.01).

Key words: high chromium yeast, glucose tolerance factor, extraction and purification, gel chromatograph