| Cordyceps militaris belongs to cordyceps fungi of ascomycetes, has similar medicine pharmacological action with cordyceps sinensis. The growth conditions of cordyceps fungi are not strict, so that the cystocarp could be grown in a variety of artificial medium. Therefore, the development and research of cordyceps militaris become a hot issue in recent years. Most studies had focused on cultivation technique and biological active ingredient, but about the cordyceps militaris hyphae growth, fruiting body differentiation characteristics and its law of development are not clear yet. In the actual cultivation, we can not effectively control the differentiation development condition, bacterial species quality, and the hypha growth will be a very big difference which causes that fruiting body differentiation development is not regular, not even sprout, and causing serious loss. This paper is based on 9 strains of cordyceps militaris bacteria preserved in laboratory as the research object. Firstly, screening good strains by antagonism test, analysis of esterase isozyme and cultivate observation, then optimizing the cordyceps militaris liquid and solid culture conditions by single factor and orthogonal experiment, and then determining the change of major enzyme activity in different growth stages of cultivation matrix, finally, summarizing the physiological characteristics and morphological changes of cordyceps militaris fruiting body differentiation and its growth process. The main results were as follows:⑴By Antagonism test the nine strains of cordyceps militaris bacteria,results show that C1, C2 and C9 have no antagonism phenomenon that the hyphae grew as one and presented tan-white sparsely villous, C3, C4, C5, C6 and C7 have no antagonism phenomenon that the hyphae presented a white circle prostrate, C8 and other bacterial have antagonism phenomenon that the hyphae presented white villous. Esterase isozyme analysis showed that six strains of Cordyceps militaris on enzymes in all detected bands, C4, C3, C7, C8, C5 and C2 were 0.41, 0.45, 0.47, 0, 0.46 and 0.36. It shows that C3, C4, C5 and C7 have affinity, C2 and C8 have no affinity with them. With the fruiting body survy experiment of all kinds of strains, fruiting body of C6 thrived, presented with the color orange and the yield of 8.7 g/bottle, C2 and C8 grew hyphae only, the colour and lustre are poor, did not change color after see the light, and did grew cystocarp. The furitingbody figure survey was basically identical with the results of the antagonism experiment, esterase isozyme analysis. So comprehensive conclusion of nine kinds of cordyceps militaris bacteria are divided into three categories, C1, C2 and C9 for category, C3, C4, C5, C6 and C7 for category, and C8 for category.⑵Based on C6 strains,screened amylaceum and peptone as the slant culture medium carbon source and nitrogen source which suit mycelium growth. The liquid medium carbon and nitrogen source are sugar and peptone, growing conditions are speed of 160r/min and p H value of 6.0. the significant impact of various factors on mycelia dry weight were nitrogen source > bacteria species > carbon and temperature > initial p H > rotation speed. By comparing the different liquid medium formula, we can get the liquid medium formula for the mycelial growth is 1 liter of water containing potatoes for 200 g, glucose for 20 g, peptone for 3g, monopotassium phosphate for 0.5g, magnesium sulfate for 0.2g and VB1 for 10 mg.⑶Optimizing the furiting body cultivation medium, then screened the optimal medium form for the mycelial growth as which contains wheat for 40 g as compost, nutritive medium 60 m L,including 1 liter of water with sugar for 30 g, peptone for 5g, monopotassium phosphate for 0.5g, magnesium sulfate for 0.2g, VB1 for 10 mg, silkworm chrysalis meal for 0.5g, as. Suitable growth condition is the temperature of 22℃, air humidity of 80% and light intensity of 200 lux. Under such condition, cordyceps militaris hyphae turned color fast, fruit body grew robustly, and presented bright orange and have high biologic output.⑷As the research of major enzyme activity and total sugar, reducing sugar, the change of the free amino acid content of cordyceps militaris culture medium in different growth stages, shows that the enzymatic activity of carboxymethyl cellulase, amylase, acid protease is low in the hypha growth period, they were 1.14U、2.6、5.6U,but it had the highest value in fruiting body growth periods, they were 10.71 U, 8.4 U and 44.8 U. With the fruiting body growth, total sugar content、reducing sugar content and free amino acid content are higher at first and then decline. In the hypha growth period, they were 0.347 mg/g、0.131 mg/g、3.2 mg/g, In fruiting body growth periods, they were 0.671mg/g、0.427mg/g、5.6mg/g。⑸It takes several periods for cordyceps militaris hyphae to grow into mature fruiting body, These periods are bacterial species germination period, hyphal growth period, mycelial color turning period, primordium differentiation phase, stroma formation period, entity differentiation period, ascus and ascospore development period, mature fruiting period. The process of vegetative growth to reproductive growth until the mature the entire takes about 50 to 60 days. Hypha germination generally need a week or so, it presents white villous. Microscopic observation results show that the mycelium density assumes the dendritic structure, have the diaphragm, the conidium was born in conidium terrier. The result of slice shows that in the mature fruiting body, stroma perithecia release irregular distribution around a large number of spores. Perithecium in general there are eight ascospores, ascospores generally 5 or 6 arranged between a septum points ascospores Phenomenon. |
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