| Glucose Tolerance Factor (GTF) is a low molecular weight Cr binding polypetide, consisted of glycine, cysteine, glutamic acid and aspartic acid and Cr3+ being its essential active center. GTF has been proven to play an important role in normal glucose homeostasis by many studies and it has become the focus of studies. The aim of this paper is to carry out comprehensive research in the field of a high yield of GTF by yeast and the structure and function of GTF. The main results of this paper are as follows:According to the property of ammonia-solubility of GTF, develop an effective ammonia extraction method of organic chromium and determination method by perfect and optimize its extraction and digestion parameters. 0.1g yeast suspended in 10mL 0.1mol/L NH4OH, shook with 200r/min for 3h at 37℃, collected the supernatant with centrifugation, microwave digested and oxidized the supernatant. Determining the content of the organic chromium of GTF, residual chromium in the yeast after extraction and the total chromium in the yeast with flame atomic absorption spectrometry. The experiment result showed that the theoretical residual chromium content meet with determination result closely. This method is suitable for determining GTF in the yeast.Seven strains yeast is mutagenized by piggyback experiment. Using chromium chloride hexahydrate as the chromium source, A strain of high-biomass, producing glucose tolerance factor yeast strain was screened through gradient concentration of chromium plates cultivation and liquid fermentation cultivation. The biomass of it was 40 g/L. the content of organic Cr was 1287μg/g.dcw and that of total Cr was 1917μg/g.dcw. Compared with the original strain, the organic chromium of YS-3 was improved by 53% with same biomass and pH during cultivation.YPD is fitter for YS-3 to produce more GTF compared with wort. Growth of YS-3 in media with 800μg/mL Cr3+ was strongly inhibited, and was stimulated with 400μg/mL Cr3+. In the presence of Cr3+400μg/mL, the lag phase is prolonged and after initial six hours the concentration of cell increased sharply and exceeded that without Cr3+. The accumulation of chromium ions into yeast cells was mainly completed in the exponential growth(6 h～44 h) and the fastest speed of accumulating chromium mainly occurred in the lag phase(0 h～6 h). The amount of organic chromium doubled through supplementation of chromium, reaching 2644μg/g.dcw without significant decrease in biomass. With pilot-scale fed-batch fermentation, the organic chromium reached 1930μg/g.dcw and biomass 33g/L.Through bioaccumulation and biotransformation, chromium was uptake into the yeast to bind on the protein, which had a great impact on the protein secondary structure. When chromium binding on the big molecule weight protein, the secondary structure changed fromα-helix and random-coil toβ-turn,β-turn increased,α-helix increased, random-coil decreased a little andβ-sheet changed nothing. When chromium binded on the small molecule weight protein, the secondary structure changed fromβ-sheet toα-helix and random-coil,β-sheet decreased greatly, random-coil increased greatly,α-helix increased a little andβ-turn remain intact.The distribution of chromium and protein were analyzed in Peak1 and Peak2 through HPLC-ICP-MS/AES, different purification method was adopted to purify two groups of chromium containing protein. In the case of low molecule weight protein, ammonia extract was purified by ultra-filtration(100kD,50kD,10KD), condense, gel filtration column chromatography (Sephadex G25), gel filtration column chromatography (Sephadex G15), achieving PP1 containing chromium. In the case of big molecule weight protein, ammonia extract was purified by gel filtration column chromatography (Sephadex G75), HPLC, achieving the eighth tube containing chromium.Ultraviolet wavelength scan and amino acid composition analysis showed that PP1 was a polypeptide containing chromium, consisted of glycine, cysteine, glutamic acid and aspartic acid. N-termianl sequence analysis showed that amino acids in PP1 were possibily connected by coordination bond, not by the amide bond. The peptide mass fingerprints produced by PP1 were studied by MALDI-TOF-TOF-MS and PP1 was analyzed by HPLC-ICP-MS/AES. The result showed that protein in peak PP1 belong to clusters of similar protein with high purity. There are only two quasi-molecular ions with high ion abundance in first order mass spectrum, m/z ratio of ions 712.2535, 769.2747. Make a speculation on the proportion of amino acid composition of the two quasi-molecular ions according to its second order mass spectrum and the result of amino acid composition of peak PP1.The peptide mass fingerprints produced by the eighth tube in Peak1 was studied by HPLC-ESI-MS. Data from HPLC-ESI-MS showed that the big molecule weight containing chromium possibly is Glyceraldehyde-3-phosphate dehydrogenase 3 or Ribosomal protein L2A or Protein HBT1.Diabetes mellitus models induced by streptozotocin were treated with high chromium brewer's yeast (250～1000μg/kg.dCr) for a period of 3 months. It showed that organic chromium have an improvement on diabetes mellitus. TCHO decreased very significantly in low group and middle group and TG decreased significantly in low group. Serum insulin increased significantly in low group and high group. GHb decreased significantly in high group; Pathological examination of pancrease islet revealed that organic chromium had a certain protective effect on theβ-cell.Above all, the organic chromium yield of YS-3, mutagenized by space piggyback, reached 1930μg/g .dcw and biomass of it reached 33g/L, through supplement chromium twice. The composition of GTF isolated from YS-3 was in accord with other reports. Furthermore effect of high chromium yeast on diabetes mice showed that it have improvement on blood glucose, blood lipids and impaired islet andβcells. We could draw a conclusion that GTF from yeast should be paid more attention to and studied further.
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