Improvement And Production Optimization Of Mycelia And Cordycepin By A Newly Isolated Paecilomyces Hepiali

As a traditional Chinese medicine,Cordyceps sinensis has a broad application prospect in the world.But the damage of geographical environment and overexploitation lead to the demand of Cordyceps sinensis exceeds supply.Modern research shows that the submerged fermentation of Cordyceps fungal can be able to replace Cordyceps sinensis.Paecilomyces hepiali is an endogenous symbiotic bacterium of Cordyceps sinensis,which can use as a substitute for Cordyceps sinensis.Cordycepin can be obtained by the submerged fermentation of Paecilomyces hepiali.As the first natural nucleoside antibiotic isolated from fungal mycelia,cordycepin plays an indispensable role in the treatment of a variety of diseases.Cordycepin is also one of the secondary metabolites of Paecilomyces hepiali.Increasing the cordycepin content in Paecilomyces hepiali is beneficial to enhance the competitiveness of Paecilomyces hepiali as a substitute for wild Cordyceps sinensis.In this paper,we use Paecilomyces hepiali strains which was isolated and identified from Cordyceps sinensis to produce cordycepin.In order to get higher yield of the cordycepin and the dry weight of the mycelium,the original strain was mutagenesis and breeding by the combination of ultraviolet and ⁶⁰Co-gamma radiation.Also the fermentation conditions were optimized to improve the yield of the cordycepin and the dry weight of the mycelium.The main research results are as follows:Firstly,a new strain was isolated from wild Cordyceps sinensis which was collected from YuShu,Qinghai province.Morphological identification of the isolated white colonies was carried out by aseptic operation,including general observation,optical microscope observation and scanning electron microscope?SEM?observation and physiological and biochemical identification.The results of 18S rDNA molecular identification were used to construct the phylogenetic tree.All this analysis show that the new strain was Paecilomyces hepiali,numbered OR-1.Real-time monitoring was conducted on the submerged fermentation of Paecilomyces hepiali OR-1,and the dry weight of Paecilomyces hepiali OR-1 was 11.771 g/L after 5 day,and the content of cordycepin was 0.264 mg/g.Then mutagenesis breeding of Paecilomyces hepiali OR-1 was carried out.OR-1 was treated with ⁶⁰Co-gamma radiation,ultraviolet irradiation and compound mutagenesis of both.The optimal dose for the mutagenesis of ⁶⁰Co-gamma radiation was 600 Gy,and the optimal exposure time for the ultraviolet mutagenesis was 20min.After the compound mutagenesis,a mutagenesis strain with excellent properties was obtained,which was numbered ZJB18001.After the strain was submerged fermentation for 5 days,the dry weight of mycelium could reach 13.275 g/L,and the content of cordycepin was 0.608 mg/g.Finally,the fermentation conditions of the mutant ZJB18001 were optimized.In this study,we used 9 different carbon sources,8 different nitrogen sources,7 different metal ions and 9 precursors to optimize the composition of the medium,so that after optimization the medium is more conducive to the production of cordycepin.After the signal factor optimization,the best medium was carried out by response surface design?RSM?.The results showed that Paecilomyces hepiali strains had the best fermentation yield of mycelium and cordycepin production when peanut oil?21 g/L?was used as carbon source,yeast extract powder?10 g/L?was used as nitrogen source,and 1 g/L KH2PO4,0.5 g/L MgSO4,4.5 mmol/L metal ions and 2.5 g/L adenosine were added.After fermentation for 5 days,the dry weight of mycelium reached18.605 g/L,and the yield of cordycepin was 0.940 mg/g of the mutant strain ZJB18001.Through the wild Cordyceps sinensis,the original Paecilomyces hepiali strain OR-1 was isolated and identification.After mutagenesis,the mutagenesis strain ZJB18001 was obtained followed by medium optimization.Compared with the original strain OR-1,the dry weight of the mycelium of the mutagenesis strain ZJB18001 was increased by 54.65%,and the yield of cordycepin was increased by256.1%.This makes it more competitive to use the submerged fermentation of Paecilomyces hepiali to produce cordycepin and has a good prospect of industrial application.