The Studies On Growth Characteristics Of Hirsutella Sinensis In Solid And Liquid Culture Condition

The article reviewed the biological characteristics of Ophiocordyceps sinensis and Hirsutella sinensis, and their research progress. The key point was summarized the asexual conclusive evidence of Ophiocordyceps sinensis, morphology and growth characteristics of Hirsutella sinensis, the extraction and determination of active material in hypha, then begin the experiment and research on this basis. Hirsutella sinensis as the research object of the experiment, after the culture medium optimization experiment, cultivated in solid and liquid conditions respectively, by determine:1) Under the liquid culture, the content of hypha biomass, polysaccharide, marnnitol, protein and other valid medical composition and the dynamic changes of the catalase, chitinase, urease, protease and related enzyme activity was determination.2)Under the solid culture of Hirsutella sinensis, the hypha morphological relevant features (including hyphae, conidia features and formation mechanism) and hypha biomass, hypha polysaccharide, mannitol dynamic changes of the content.The experimental results are as follows:1. Through the single factor experiment of carbon source, nitrogen source, additive and L934multiple factor orthogonal test, medium for Hirsutella sinensis composition and ratio was optimized. The results show: for Hirsutella sinensis, the best carbon sources, nitrogen source, additive are glucose, hydrolysis dairy protein and yeast powder, milk, the best ratio in order as2%:0.5%:0.1%:20%.2. Under the liquid culture, through detect hypha biomass, anthracene ketone-sulfuric acid method to detect hypha polysaccharide, sodium periodate colorimetric detect hypha mannitol content, Coomassie blue staining to detect hypha protein content. The results show: the hypha biomass reach maximum on the16d, the highest rate is2.74mg/mL, hypha polysaccharide, mannitol, protein content are positive correlated with hypha biomass, their biggest content is4.73%,3.54%and20.99%respectively. Under18℃, hypha biomass and the contents of polysaccharide are more than the other temperature conditions in the same period, but the content of mannitol is higher under20℃.3. Under the liquid culture, use Potassium Permanganate titration method to detect the hydrogen peroxide enzyme activity, N-acetyl glucosamine colorimetric detect chitin enzyme activity, Indophenol-blue colorimetric detect urease activity, Ninhydrin colorimetric detect protease activity. The results show:the activity of catalase and protease increase gradually with time; urease activity increase gradually within the4-8d, and reach the highest in the8d, then fall after; chitin enzyme increase gradually in the8-20d. 4.Under the solid culture, by the optical microscope detect and record bacteria body structure, features and growth mode, hypha asexual spores (conidia) formation mechanism, blood count edition detect conidia quantity change. The results show:the hyphae of Hirsutella sinensis breed with budding growth mode, conidia is formed by conidia sporangium longitudinal crack formation opposite conidia mainly. Conidia can be detected obviously when trained in20d, the quantity in the30d is maximum, and different temperature condition does not influence the form time of conidia, but can effects the number of conidia, with14℃conditions the number of conidia is the highest.5. Under the solid culture, hypha biomass, polysaccharide, mannitol content increase gradually with the augment of the training time, in the training prophase (0to20d)the difference is not obvious, with the accumulation of nutriment the difference is obvious increasingly. Under18℃culture conditions the hypha biomass, polysaccharide and mannitol content are higher than the other, variable temperature conditions (14to18℃/12h) is less than constant temperature conditions (18℃) in the way of hypha growth promoting effect.Through the above experiment, the results can provide the theory basis and the reference for the industrial production of the succedaneums of Ophiocordyceps sinensis-liquid fermentation hypha, at the same time, provide bacteria vaccination, growth, reproduce, preservation for Ophiocordyceps sinensis and provide the basis for finally realize the artificial cultivation of Ophiocordyceps sinensis.