| Coenzyme Q10 plays an important role in the proton transfer and electron Transfer of mitochondria respiratory chain, which has a broad applications in clinical medicine, nutrition, health care products and cosmetics research and development and so on, as a good biological medicines and natural antioxidants. Currently, coenzyme Q10 production methods are generally divided into direct extraction, chemical synthesis and microbial fermentation. Compared with the other two methods, microbial fermentation method because of the advantage of raw materials cheap and rich, product separation process is relatively simple, the product is natural without the question of chirality, biological activity, easily absorbed, and can achieve scale and industrial production by fermentation tanks and so on, has become the most potential method of coenzyme Q10 production.In recent years endophytes as a new type of microbial resources have become one of the hotspots of microbial resources research, because of great significance and potential value.In this experiment, I take the heart of a healthy individual swine as experimental material. After the treatment with rigorous surface disinfection and sterilization, it was cultured for enrichment for some time with microbial general media, then the broth was applied on the basis medium of beef extract and peptone, single colony was picked continuously for pure culture, in the next period of time, by the scribing method to get plenty of alternate strain.Metabolites were extracted from the fermentation broth by the method of alcohol-alkaline saponification extraction. The qualitative analysis of the product include TLC combine with UV spectrophotometry and leucomethylene blue reaction, which was used to screen microorganisms with the ability of output coenzyme Q10. The method of UV absorption compared with the standard curve was used to do quantitative analysis of the product to filter out target strain.After filtering out target strain, morphological identification, biochemical identification and molecular characterization were used to identify the target cell. Morphological identification include colony morphology, Gram stain, capsule stain, flagella stain, spore stain and scanning electron microscope. Physiological and biochemical tests include urease test, oxidase test, catalase test and sugar fermentation experiments, etc. Molecular identification is through extracting genomic, amplifying 16 S r DNA sequences, then by BLAST sequence alignment and phylogenetic tree of MEGA5.1 for further classification and identification. Finally, a simple analysis of the growth characteristics of this was taken, growth curve was draught, medium was optimized by orthogonal test. The main results are as follows:(1) six kinds of strains are isolated from swine heart;(2) The strain of st-2 and st-6 has the ability of producing coenzyme Q10;(3) The optimal conditions for the extraction of soda saponification: saponification temperature 90 ℃, saponification time 30 min, extracted twice;(4) The preferably ratio of developing solvent in TLC is petroleum ether / ethyl acetate = 5.5: 1;(5) The strain of st-6 gram-positive, encapsulated, without flagella and spores, equal binary fission, tiny in form of 0.2~0.3μm;(6) St-6 has the ability to produce urease, protease, amylase and reduction of nitrate etc;(7) This strain belongs to Kocuria;(8) The best medium for this strain is : peptone 0.5%, glucose 0.3%, yeast extract 0.3%, beef extract 0.25%, sodium chloride 0.6%.(9) Cell production increased by an average 47.8 percent compared with that before optimization. |
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