Breeding Of Cordyceps Militaris Strain By Its DNA Demethylation With High Yield Of Cordycepic Acid And Optimum Fermentation Process

Genomic DNA demethylation can activate silencing genes and change the secondary metabolites profile,which is a new way to strain improvement.In this study,DNA methylase inhibitor 5-aza C was used to treat Cordyceps militaris strains,which reduced the level of genomic DNA methylation,activated the overexpression of related genes,and increased the yield of Cordyceps acid.This study provided a new way to construct high yield Cordyceps acid production strains by Cordyceps militaris.The purpose of this study was to determine the effect of DNA demethylation of Cordyceps militaris strain on genetic stability and biological activity of Cordyceps militaris strain.DNA methylase inhibitor 5-azacytidine?5-aza C?was used to induce Cordyceps militaris strain CM-L1,to screen out positive mutant strains with high Cordyceps acid content and to study genetic stability and fruiting property.Finally,the mutated strain D-2-7 was obtained.The formula of liquid culture medium of Cordyceps militaris was optimized by single factor test Box-Behnken design and response surface method?RSM?.The concentration of carbon and nitrogen sources in liquid fermentation medium of Cordyceps militaris mutated strain D-2-7 was optimized by single factor experiment.The optimum carbon source is glucose 30.0 g·L^-1,the best nitrogen source is peptone yeast extract 10.0 g·L^-1,and the optimal concentration of inorganic salt is KH₂PO₄ 2.0 g·L^-1,Mg SO₄ 1.5 g·L^-1.The optimum liquid fermentation medium component was used as the test variable and the Cordyceps acid content in mycelium as the response value.The four factors and three levels of Box-Behnken test design were carried out.The multivariate quadratic regression model is used to fit the test data.The experimental results fit well with the model and can reach the highest yield in the course of the experiment.It shows that the response surface model is feasible.The conditions of liquid fermentation of D-2-7 mycelium were optimized.The initial p H value,culture temperature,inoculation amount,liquid loading and shaking speed were investigated respectively.The optimum culture conditions were as follows:initial p H was 6.5 and culture temperature was 26?.The biomass of mycelium and the content of cordyceps acid reached the maximum values of 17.25 g·L^-1 and 13.46 g·L^-1,respectively,when the inoculation amount was 10%and the liquid volume was 150 m L,and the rotational speed was 150 r·min^-1.Mannitol was added to Cordyceps militaris solid medium as a culture additive.When the mannitol content was 1.5%,the content of cordycepin acid in the fruiting body of strain D-2-7 was the best,reaching 69.596 mg·g^-1.Compared with the original strain CM-L1,the content of cordycepin acid in the fruiting body of strain D-2-7 was increased by 13.99%.The content of oxalic acid increased by 54%,and the effect was remarkable.The cultivation experiment showed that the mycelium of strain D-2-7 showed excellent genetic stability through subculture.With the increase of culture cycle,the accumulation of Cordyceps acid was also increased,and finally showed an upward trend of Cordyceps acid content.