Identification Of Cordyceps Sinensis Anamorph And Study Of Its Growth Characteristics

Cordyceps sinensis is a traditional Chinese Medicine with extremely important pharmacological value. In recent years, because natural Cordyceps sinensis has been over-collected, this resource was on the brink of extinction. The conflict of shortage led to find natural Cordyceps's substitutes. The isolation and culture of Cordyceps sinensis anamorph is one of the effective ways.To that end, in our experiment a Cordyceps sinensis isolate was molecularly identified, growth and culture characteristics were comprehensively investigated, and main active ingredients were determined. These studies laid the foundation for Cordyceps sinensis anamorph's future commercial development, and provided the basis for which artificial cultured Cordyceps mycelia substituted natural Cordyceps sinensis.The rDNA ITS of Cordyceps sinensis isolate and cultured Cordyceps militaris were amplified and sequenced. Between AB067721 (Hirsutella sinensis, Japan) and our isolate the similarity is 100%. Compared with AJ309359 (Hirsutella sinensis, Guizhou University) there was a G at the site of 544. There was a C at the site of 83 which was different from A of AJ488274 (Hirsutella sinensis, Taiwan). So our isolate could be confirmed as Hirsutella sinensis; Between Cordyceps militaris and our isolate the similarity is only 76%, obviously belonging to different species.Solid medium of Hirsutella sinensis was optimized, finding that modified PDA was the best one; and for the first time our study showed strain's temperature adaptability characteristics using photographs, that was,18℃was the most favorable, above 25℃strains can not grow. On micro-morphological observation of mycelia we used environmental scanning electron microscope, clearly showing the conidia size, shape and way of growing. This detailed report on colony characteristics provided a reliable basis for future identification and separation of Cordyceps anamorph.This study discussed the effect of macro-elements, trace-elements and amino acids on Hirsutella sinensis's growth through orthogonal test, finding that MgSO₄·7H₂O, FeCl₃·6H₂O, lysine, methionine can promote growth, and Fe³⁺ was the major influencing factor; In addition, the liquid medium was also optimized and found that in 40 days modified PDB and PPDB got the maximum mycelium, up to 7.84-8.34g/L;Color (black or brown) deeper, growth more vigorous. The accumulated data of solid-liquid culture furthered the research process of Hirsutella sinensis cultivation Biotechnology.Determination of adenosine, cordycepin and polysaccharide contents in H.inensis mycelium were carried out. Cordycepin and adenosine were analyzed by RP-HPLC method. Polysaccharide was analyzed by phenol-sulfuric acid method. The result showed that cordycepin and adenosine could be completely separated with the HPLC method we reported; Cordycepin and adenosine contents in H.sinensis were 342.7±5.20μg/g, 10.41±1.32μg/g, and polysaccharide content was 16.81±0.669%. After comparing these three components between H.sinensis mycelium and C. militaris, we found that cordycepin and adenosine contents were lower than that in C. militaris but polysaccharide contents in H.sinensis were higher than that in C. militaris.