| As one of notable edible-medicinal basidiomycete, Tricholoma mongolicum Imai enjoys a worldwide reputation. Mycelia and polysaccharide of Tricholoma mongolicum are produced on a large scale by liquid fermentation to be potential for protecting and exploring this precious resource in severe danger, because its domestication and manned planting is difficult and lacks of substantive breakthrough, as well as its natural habitats are destroyed to result in its wild resource reducing sharply.It was investigated intensively in this study that effects of nutrients including carbon source, nitrogen source and inorganic salts, as well as seed age, inoculation volume, temperature, rotate rate, dissolved oxygen, pH value and aeration on yields of mycelia and polysaccharide during the submerged fermentation. Also, fermentation process, extraction and separation process of polysaccharide were determined and optimized. Mycelia and polysaccharide of Tricholoma mongolicum were produced by high-density fermentation using the fermentor, which establishes a base for the large scale production. The main results and conclusions are as follows.1. Based on RAPD fingerprint technique, DNAs of the strains were amplified using the effective arbitrary primer screened by agarose gel electrophoresis. Maps of amplified DNAs obtained by microchip capillary electrophoresis were analyzed by statistical software. The results of cluster analysis show that three strains are all pure cultures of isolates from fruiting body of Tricholoma mongolicum, and Tricholoma mongolicum from Xilingol grassland has no any regionally genetic diversity with that from Hulunbeier grassland.2. A comparison in growth characteristics of three strains was carried out using PDA medium. The strain Q97 was select and used in this study, and its optimal incubation temperature was determined to be 25℃based on growth rate under various temperatures.3. Carbon and nitrogen sources, media ingredients and fermentation technics of the strain Q97 were studied with single factor test and orthogonal test. The experimental results show that Q97 can utilize most of carbon sources and organic nitrogen sources, and the optimum medium formula for shake-flask fermentation was determined as follows: bean cake powder 30g/L, sucrose 50g/L, K2HPO4 1g/L, CaCO3 1g/L, and pH non-adjusted.4. The optimum fermentation period for shake-flask fermentation was 13 days, and maximum biomass was up to 30g/L with 100mg/L of exopolysaccharide.5. The optimum medium formula for the fermentor fermentation was determined based on the experimental results using 5L of fermentor. Fermentation period was shortened and yield was increased by the optimization of fermentation process.6. Fermentation kinetics including strain growth, product formation and substrate consumption kinetics was studied using 20L of fermentor, and the appropriate kinetic models were proposed. Experimental data were in agreement with calculated data according to kinetic models established, which suggests that models appear to provide a reasonable description for the fermentation process of Tricholoma mongolicum in the fermentor.7. Fermentation medium formula for 250L of fermentor was as follows: bean cake powder 30g/L, sucrose 80g/L, K2HPO4 1g/L, and CaCO3 1g/L. The maximum biomass reached 45g/L with 300mg/L of maximum polysaccharide yield after 7~8 days'fermentation.8. The polysaccharide was extracted, separated and purified from fermentation liquid, and its molecular weight was measured with high-performance gel-permeation chromatography (HPGPC) equipped with refractive index detector. The results show that polysaccharide of Tricholoma mongolicum includes at least five varieties of components, and molecular weight average of its main component is 3919.
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