Phosphate Low Sensitivity Of Resistant Strains Of Cordyceps Militaris Screening And Optimization Of The Fermentation Conditions

Cordyceps militaris is a medicinal fungus with health care function,It can produce a variety of active substances, which cordycepin has many biological activities such as antibacterial, anti-inflammatory, anti-tumor, improve immunity. In this paper, make the speculated cordycepin metabolic pathway as the theoretical basis, a high cordycepin yield and resistant to high concentrations of KH₂PO₄ militaris resistance mutants was obtained by diethyl sulfate（DES） chemical mutagenesis, and determine the strain’s optimum medium and the best culture conditions by single factor and orthogonal experiment. Meanwhile, preliminary analysis the antibacterial component of Cordyceps militaris fermentation broth. Specific content and results are as follows:（1） Mutagenesis of the high-yielding strains of Cordycepin. Make a high cordycepin yield C.militaris FFCC 5111-C as the original strain, the single spore suspension was diluted to 10-2 times, DES mutagenesis 25 min, used the individually detection method screened four triple auxotrophic strains（Xan-+Gua-+ L-His-）. Numbered C.militaris FFCC 5111-CI、C.militaris FFCC 5111-CII、C.militaris FFCC 5111-CIII and C.militaris FFCC 5111-CIV. Detected the cordycepin content of broth, and compared with original strain C.militaris FFCC 5111-C,cordycepin yield growth rates were 113.24%, 110.29%, 151.47%,134.80%.（2） Test the tolerance of four triple auxotrophic strains to high concentrations of KH₂PO₄. The results showed that, tolerance of C.militaris FFCC 5111-CII to high concentration KH₂PO₄ is better than the others. When the KH₂PO₄ concentration was 2.5 g/L, the highest cordycepin production was 0.758 g/L.（3） Determin the optimum medium and optimization of culture conditions. With a single factor test to determine the best medium of C. militaris FFCC 5111-CII is: glucose 40 g/L, peptone 12 g/L, KH₂PO₄ 2.7 g/L, MgSO4 0.7 g/L, Gua 0.06 g/L, Xan 0.08 g/L, L-His 0.08 g/L, VB1 0.11 g/L. Optimization of Culture Conditions. By orthogonal experiment, determine the optimal culture conditions of C.militaris FFCC 5111-CII is initial pH value is 6, the culture period is 16 d, medium volume is 100 m L and inoculation amount is 5%. Variance analysis, the effect of initial pH value and the culture period are significant, medium volume and the amount of inoculum had no significant effect.（4） Fungistasis testing. Fermentation broth doesn’t inhibit Candida albicans ATCC 90028, while have obvious inhibitory effect to Staphylococcus aureus ATCC 25923、ATCC 29213, Enterococcus faecalis ATCC 29212, Escherichia coli ATCC 25922 、 ATCC 35218,Pseudomonas aeruginosa ATCC 27853 and Klebsiella Pneumoniae ATCC 700603. For Klebsiella Pneumoniae ATCC 700603, antibacterial effect of guanine and broth were significant and similar, inferred that fermentation broth inhibiting the pathogen component was likely to guanine; For Pseudomonas aeruginosa ATCC 27853, hypoxanthine, guanine, xanthine, cytidine, adenosine and cordycepin have antibacterial effect. Wherein, the inhibition strength of adenosine and cordycepin is relatively weak.