Optimization Of Culture Condition, The Selenium Speciation Anlysis And A Primary Study On Selenium-enriched Mechanism Of Selenium-enriched Aspergillus Oryzae

Selenium is an essential micronutrient element for human beings. The lack of selenium is related to many diseases and has a serious impact on human health. So more and more people from all sectors pay attention on supplement selenium. However, the selenium content of natural foods is generally low, can not meet the body’s demand for selenium. And inorganic selenium could not be used in food because of its high toxicity and low bioavailability, so it has a good prospect in the studying on the conversion inorganic selenium to organic selenium by microorganism.In this study, we first mutagenized Aspergillus oryzae supplied by our lab with UV irradiation and then gained 4 selenium-resistant mutants of Aspergillus oryzae strains through experiments. Next we had the 4 mutants fermentated with selenium, compared their selenium content, screened a selenium-resistant and high selenium-enriched strain. This strain was used for subsequent research as the original strain.Using response surface methodology to optimize the three factors that affect the culture conditions of selenium-enriched Aspergillus oryzae, such as inoculation, medium amount and the rotational speed. And 10.64% inoculation,85.4 mL medium amount and 185 r/min was determined as the best culture conditions. In this case, the selenium content was maximum of 2.349mg/g, comparing with the first strain had a 5.8 percent increase. And then we made the verification testing in the best culture conditions, the average of three test results was 2.325mg/g, a 1.02% lower than prediction value. The result showed that the model could well reflect the actual situation of fermentation.The reverse-high performance liquid chromatography detection methods for organic selenium compounds in selenium-enriched Aspergillus oryzae is established. Which utilizes 6mol/L HC1 solution for organic selenium compounds extraction with a constant temperature of 50℃and 48 h extraction time, 4-chlorine-3,5-dinitrobenzotrifluoride (CNBF) for derivatizaiton, C18 reverse column (4.6 mm×250 mm i.d,5 μm) for separation, acetonitrile-water and pH 6.8 Acetic Acid-Sodium acetate buffer solution for gradient elution, UV detector:240 nm for determination. Results:organic selenium compounds in selenium-enriched Aspergillus oryzae was mainly selenocystine(SeCys2). The LOD(S/N=3) was 0.68 mg/L and the LOQ(S/N=10) was 2.244 mg/L. The linear range was within 5.0-100 mg/L. The recovery was 94.8%-105.8% and contented for 1.250 mg/g selenocystine in it. This method is simple, sensitive and accurate, and could be used in the determination of organic selenium compounds for selenium enriched Aspergillus oryzae and relating products.We analyzed the relation of the selenium-rich ability and the intracellular glutathione in the 4 mutants of Aspergillus oryzae by Spearman coefficient(r). The r was 0.8, it indicated that the selenium-rich ability is in directly proportion to the intracellular glutathione concentration and had a preliminary exploration of the selenium mechanism in Aspergillus oryzae.