| Selenium, which is the trace element of human being, plays an important role in anti-oxidation, anti-stress, immunity improving and cancer prevention, etc., while Sodium selenite, the representative of inorganic selenium, has high-toxicity and low utilization rate. Compared with inorganic selenium, organic selenium has a higher absorption rate, better biological activity, and lower toxicity. Se-enriched PSP is an organic selenium compound formed by PSP and selenium, and inorganic selenium-sodium selenite can be converted into organic selenium by Coriolus versicolor, thus the formation of Se-enriched PSP can further enhance the anti-tumor efficacy and reduce the toxicity of chemotherapy.First,We detected the selenium level of Se-enriched PSP using graphite furnace atomic absorption spectrometry. We explored the microwave digestion conditions: including the chosen digestion reagent, the dosage of reagent digestion, the digestion temperature, the consumption time of acidolysis. Besides we optimize the ashing and atomization temperature, the basal-body modifier of atomic absorption spectroscopy;on the basis of the precision, accuracy, linear, scope of the graphite furnace atomic absorption spectrometry method detecting the organic selenium content of Se-enriched PSP, a suitable detecting way for the selenium content of Se-enriched PSP is established: nitric acid: hydrogen peroxide(5:1) as acid digestion reagent, 3ml of nitric acid, 0.6ml of hydrogen peroxide, the microwave digestion temperature is 180℃and the consumption time of acidolysis with 3h; we also selected palladium acetate as the basal-body modifier, 1200 ℃ /1900 ℃ for ashing and atomization temperature, respectively.We obtained its substance through fruiting bags of Coriolus versicolor, isolated Coriolus versicolor monospore, then cultivated on the PDA plate. Coriolus versicolor protoplasts was obtained after hydrolysis of Coriolus versicolor by 2% of lywallzyme enzymolysised 32℃ in a water bath about 3h, then irradiated with UV rays during 0s, 30 s, 60 s, 90 s, 120 s and 150 s, respectively. 150 mutant strains can be obtained by first screening which had the better selenium tolerance rate after fruiting in a concentration of 80μg/ml sodium selenite PDA plate, as 5.9-30-1,6.26-30-4,9.20-60-5. At last,we obtained three strains of mutagenesis Coriolus versicolor, were fermented in Se-enriched deep liquid which 26 ℃ with 72 h, the Coriolus versicolor strains 5.9-30-1 was chosen by comparing mutant strain of body mycelium yield, PSP obtained rate, mycelium selenium content, which has the best adjustability of selenium,which selenium tolerance rate is 2.2%,mycelium yield is 11.45g/l, selenium content of mycelium is 469mg/kg,PSP rate is 1.26g/l and selenium content of PSP is 401.6mg/kg。According to the method of water extraction and alcohol prec ipitation, we investigated the extraction process for Se-enriched PSP, considered the ratio of water/substance, extraction temperature, extraction time and obtained optimal extraction conditions: the water/substance ratio 30:1, temperature 100℃, extraction time 2h. We also investigated single factor experiment of shake flask Se-enriched fermentation to confirm three key factors including the inoculation, liquid volume, sodium selenite concentration, and the optimized design of experiments could be got according three factors and three levels of response surface experimental by using Design-Expert.8 software: inoculation of 11.18% and liquid volume of 9.39%, sodium selenite concentration of 30μg/ml,, Se-enriched PSP selenium content 259.3mg/kg. Compared with the optimization of the predicted value, and the relative error of about 10%。... |
PDF Full Text Request