| Cordyceps militaris, a kind of traditional precious medicinal and edible fungi in China, has the functions of antibacterial, anti-tumor, improving immunity, anti-oxidation and scavenging free radicals and so on. It can be used as raw materials of some functional foods to improve and regulate the sub-health status of humans, and as the natural antibacterial ingredients of food and cosmetics to instead preservatives. Therefore, it has important practical values to exploit the Cordyceps militaryis resources.Firstly, a strain was isolated from fresh rigid tussah. The sequences analysis of rDNA ITS fragment gene of the isolates(XD-YCC-2) was aligned with other sequences in the GenBank database. The results was showed that it was members of Cordyceps militaris. Thus, the isolate was designed as C. militaris XD-YCC-2.Secondly, since carbon sources have a great influence on the growth and metabolism of microorganisms, we carried out the researches of GC-MS metabonomics of three different carbon resources, i.e. glucose, sucrose and starch, of C. militaris XD-YCC-2. The objectives of the studies were to determine both the differences in metabolites and differences in metabolic pathways of mycelia and zymotic fluid in liquid fermentation of C. militaris XD-YCC-2.Moreover, we still studied the antibacterial activities of fermentation broth under the different oxygen concentration.The main results are as followed:1. Glucose, a kind of monosaccharide, could be most easily utilized by C. militaris XD-YCC-2 and made it grow fastest in the logarithmic period among three carbon sources. Starch, a kind of polysaccharides, could be slowly utilized by C. militaris XD-YCC-2 at the first instance, while could greatly improve the biomass of mycelium in the later period. After 25 d fermentation, the biomass of mycelia were from high to low in order as: carbon source of starch > glucose > sucrose.2. In the liquid fermentation experiment of GC-MS analysis of ethyl acetate extract from mycelium, the results showed that 49 known metabolites were detected, including 13 kinds of alcohols, 9 acids, 10 alkanes, 5 sugar, 5 esters, 2 ketones, 2 ethers, as well as amino acids, amines, other substances, and adenosine. The metabolites of all three carbon sources showed the biggest difference at the stage of 7-13d(the initial stage of the logarithmic phase), during which the growth and metabolism activity of the fungi strains was exuberant, and a large number of metabolites were consumed, transformed and utilized.3. In the liquid fermentation experiment of GC-MS analysis, the results showed that 15 known metabolites were detected. There were 6 metabolites were common metabolites of mycelium and fermentation broth of the C. militaris XD-YCC-2, which mainly were the products of carbon metabolism. Under the glucose and sucrose as a monosaccharide condition, the growth of the mycelium was synchronized with the material exchange. In other words, the changing obviously of exchange of substances between the fermentation broth and mycelia were occurred mainly in the periods of rapid growth of mycelia. The significant difference from the aforementioned was, under the starch as a monosaccharide condition, the mycelium was exchange and accumulate with culture medium for materials in advance, and the growth of the mycelium was subsequently occurred.4. The metabolites accumulated in mycelium were distinct due to the influence of carbon sources. When Glucose was used, the carbohydrate metabolites were Glycerol and Propanoic acid, while sucrose as carbon source, metabolites was mainly D-fructose, fatty acid and unsaturated fatty acids. In addition, when Starch was used, D-fructose was mainly transformed into rare sugar(D-Tagatose and D-Psicose) and D- mannitol, which were the effective components of novel functional foods.5. The biosynthesis of unsaturated fatty acids and Steroid biosynthesis were the most important differential metabolic pathways of C. militaris XD-YCC-2, which were not affected by the type of carbon source. The metabolisms of Glycine, serine and threonine were related to the metabolisms of Methane, Porphyrin and chlorophyll, Porphyrin, chlorophylland Cyanoamino acid, as well as the biosynthesis of Aminoacyl-tRNA. Strains use Glucose and sucrose as carbon sources performed much more active in the metabolism pathways of glycine, serine and threonine than those use starch.6. The fermentation broth of C. militaris XD-YCC-2 that use glucose as carbon source showed the strongest antibacterial effect on E. coli, S. aureus and B. subtili, and the concentration of the fermentation broth was positively correlated with antibacterial effect. Besides, the inhibition effects were significantly influenced by the oxygen concentration in the bacterial culture. Furthermore, the hyphal fermentation broth showed a stronger antibacterial effect on Gram-positive bacteria than that on Gram-negative bacteria. |
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