Study On Organic Selenium Synthesis By Edible Fungi Fermentation

The trace mineral selenium is an essential nutrient of fundamental importance to human biology. With the deeper understanding of selenium, people found selenium has antioxidant, anti-aging, improve immunity, preventing cardiovascular disease and cancer and so on. The blood selenium of many persons is lower than normal blood selenium levels, which need to develop more effective selenium products to meet the demand. More and more evidence was showed that the organic selenium compounds can be more effectively absorbed and converted. In this paper, the effective synthesis of organic selenium was regarded the research target, and the chemical composition and organic selenium synthesis pathway were the theoretical support. Flammulina velutipes mycelia and yeasts were as materials of efficient organic selenium accumulation, by optimizing the fermentation process and determination of selenium content of selenium compounds.At first, a method to obtain the content of total selenium was introduced based on reversed phase high performance liquid chromatography (RP-HPLC). Piazselenol was synthesized by sufficient 1,2-diaminobenzene and selenite in acid medium. The selenium content was obtained by RP-HPLC determining absorption peak area of piazselenol. The minimum detection concentration of selenium and the measurable range were ensured. The method of precision experiments, repeatability and stability experiments and the recovery rate is within the error range (5%). Cr3+, EDTA and ascorbic acid among the ions affected the reaction greatly. Compared with the methods reported previously, the method used here is simple and reliable.The organic selenium compounds were isolated in Flammulina velutipes mycelia and yeasts by extraction separation technology, and the major organic selenium compounds in selenium-enriched Flammulina velutipes mycelia and yeasts were selenium containing proteins and peptides. Selenium-containing polysaccharides in Flammulin velutipes and yeasts were isolated, and selenium content in selenium-containing polysaccharides was less 1% in the total selenium, which means selenium-containing polysaccharides aren't the main organic selenium.The effects of temperature, rotation speed of shaking table, inoculation method and inoculation amount on organic selenium synthesis in fungi were researched, and culture conditions and main medium components were optimized, and the highest organic selenium yield was obtained. Flammulina velutipes mycelia synthesize more organic selenium by spore inoculation than mycelia inoculation. Flammulina velutipes mycelia and yeasts can synthesize proteins using inorganic nitrogen source (ammonium nitrate), and they synthesize more organic selenium using inorganic nitrogen source than organic nitrogen source. Response surface methodology (RSM) and central composite design (CCD) have been applied to realize the maximum soluble organic selenium synthesis in Flammulina velutipes mycelia and yeasts by optimizing the concentrations of carton source, nitrogen source and Se (IV) in fermentation broth. Yeasts have higher ability of synthesizing organic selenium than Flammulina velutipes mycelia and the culture time is shorter. Furthermore, soluble organic selenium of Candida rugosa was highest as far as the unit fermentation broth in three yeasts, which was 4.81±0.18mg/L. The weight percentage of organic selenium in yeasts can be increased by enhancing selenite in broth, however, organic selenium content in the unit fermentation broth decreases. The weight percentage of organic selenium in Candida utilis is the highest in three yeasts, and can reach 1.95±0.089 mg/g by enhance effective components in broth.The growth curve rate fed-batch strategy is an optimal feeding strategy to synthesize soluble organic selenium, which was 4.63±0.24 mg/L(the terminal selenite concentration was 0.10mM) in Flammulina velutipes mycelia, compared with that by batch culture, which was 3.43±0.13 mg/L. The maximum by the feeding strategy and the maximum soluble organic selenium synthesis in Candida rugosa was 5.24±0.19mg/L, which was higher than that by batch culture.The total glutathione in Candida rugosa, Candida utilis and Saccharomyces cerevisiae was determined by oxidized glutathione (GSSG) derived with dansyl chloride, which is highest in Saccharomyces cerevisiae (1.23±0.062mg/g), and lowest in Candida rugosa(0.43±0.022mg/g). The actual substituation ratio of selenium in Saccharomyces cerevisiae is highest, and Candida rugosa lowest. Glutathione plays an important role from inorganic selenium to organic selenium. Organic selenium content in unit dry weight of Candida rugosa increased 30.1% and 22.5% respectively, by adding precursor's amino acids of glutathione and only serine under the experiment conditions.During the process of biosynthesis of organic selenium by Candida rugosa in fermentor, increasing oxygen helped biosynthesis of organic selenium in the exponential phase of Candida rugosa, and organic selenium reached 4.21±0.13mg/L, from 3.52±0.12mg/L, and the difference was significant.