Screening Of High Production Strains Of Cordyceps Militaris And Optimizing Its' Fermentation Process

Since cordyceps processes many pharmacological activities and biological activities such as anti-tumor, anti-fungi, anti-inflammatory, anti-fatigue, anti-aging, regulating immune function and et al. and possesses good therapy and hygienical function, it was greatly interested by people. However, the sources of wild cordyceps were great limited. The development of the research in the domestication and cultivated and cordyceps were slow. Cordyceps militaris has similar efficacy of cordyceps and some biologically active substances has a higher contents than that of cordyceps. It can be used as the alternative of cordyceps. The domesticated, solid culture and submerged fermentation develop well. However, As a long fermentation period, the low yield of mycelium and low contents of active components in mycelium and et al, the growing needs of cordyceps still can be solved. Therefore, the research in cordyceps militaris fermentation and its optimization is great significant. In this paper, the cordyceps militaris fermentation was elevated by the strain improvement and optimization of fermentation process. Satisfied results were obtained.Screening high-production of cordyceps militaris strain:Cordyceps militaris CGMCC5.699 has been treated by nitrosoguanidine (NTG) and laser. The growth speed and colony size were used as criterion for preliminary screening. Comprehensive considering of biomass, adenosine, polysaccharides and cordyceps acid content, the high-production mutants were screened using shake flask method. The genetic stability of the screened mutants were study by using the method of continuous inoculation. The yields of biomass, adenosine, polysaccharide, protein and cordyceps acid of screened mutant were higher than that of original strain, which reached 12.490 g/L,70 mg/g,0.780 g/L,6.482 g/L and 1.391 g/L respectively.The optimization of cordyceps militaris fermentation medium:The single-factor test method was firstly applied to screen mutant the carbon sources, nitrogen sources and inorganic ions in the fermentation medium of high-production cordyceps militaris. The concentrations of the selected components, ammonium salt ions and olive oil a have been preliminary optimized. Plackett-Burman design experiments have been implemented. The content of adenosine has been used as response value. A muti-linear model combined with F test method was employed to identify the significant components in fermentation medium. Yeast extraction powder, sucrose and vitamin B1 have been selected and used as the casual factors for further optimization. Box-behnken design experiments were implemented. The content of adenosine has also been used as response value. muti-quadratic regression model has been employed to model the data and optimum fermentation medium was obtained (g/L):sucrose 30, peptone 10, yeast extraction powder 18, KH2PO4 3, MgSO4 3, vitamin B1 0.235, ZnCl2 0.011, (NH4)2SO4 10.Optimization of cultural conditions of cordyceps militaris and its' mass-cultural:Comprehensive considering of biomass, adenosine, polysaccharides and cordyceps acid content, the fermentation conditions of high-production mutants were optimized using single factor test method in 5 L fermentor. The optimum fermentation conditions were obtained which were as follows:volume of inoculation 6%, the initial pH of fermentation media 7, rotate speed 250 rpm, temperature 26℃, air flow speed 250 L/h. The validation experiments were implemented under the optimum conditions. The results shown that the yields biomass, adenosine, polysaccharides and cordyceps acid were stable. The mass-cultural of the high-production mutant was studied in 15 L fermentor and 100 L fermentor. The results shown that the yield of biomass, adenosine, polysaccharides and cordyceps acid were elevated during the mass-cultural and the culture time reduced.