Optimizetion Of Liquid Fermentation Process Of Tremella Fuciformis And Identify Oosporein From Fermentation Broth

Polysaccharide is the main active component of the Tremella fuciformis, which has the function of increasing immunity, lowering blood sugar, anti tumor, antioxidant, etc."Tongjiang"Tremella fuciformis is a edible and medical fungus produced in Sichuan,which is one of the best quality of Tremella fuciformis in china. But over the years, Hypoxylon archeri, a companion fungus of "Tongjiang"7rewella fuciformis is degradation, it is difficult to breed excellent companion fungus. For these reasons the yield of "Tongjiang" Tremella fuciformis can not be increased, it difficult to control the quality of products through the field-cultivation of the fruiting body. Modern bio-fermentation technology provides an efficient way to solve this problem. It can not only keep the quality of active components, but also can shorten the production cycle to save the costs. Currently, the components of liquid medium of Tremella fuciformis is complicated and caused troubles for subsequent proceedings. The fermentation waste water is also harmful to environments. During the concentration of substrate in the fermentation broth changes, a red compound is produced as well as polysaccharide. It is reported that the red or yellow compound is accompanied with the fruiting body of Tremella fuciformis without this compound, there is no fruiting body at all. Unfortunately, as far as we know, the structure and its effects to the fruiting body of Tremella fuciformis are not reported yet. To well understand the fruiting body of Tremella fuciformis so as to improve the industrial application of liquid fermentation, it is important to study the physical-chemical property and bio-activity of the Tremella fuciformis polysaccharides and the structure-property relationship of the red compound produced during the fermentation.Yield of target product in fermentation broth is main purpose, combined with the activity test of the target product, Optimizate the liquid fermentation process of Tremella fuciformis with defined medium and Tremella fuciformis liquid fermentation metabolism to produce red substances, Isolation and structure identification research.1. Optimization a chemical defind medium to improve the yield of mycelium and exopolysaccharides of Tremella fuciformis. Select suitable fermentation cycle for Tremella fuciformis mycelium submerged fermentation, the carbon and nitrogen sources, initial pH, fermentation temperature, inoculum volume, cell age and rotate speed in the5L fermenter to optimize expand the cultured.Optimum fermentation period is6days, the best carbon source is glucose, the best nitrogen source for the beef extract production medium conditions optimized for the maximum amount of mycelium12.32±0.15g/L; exopolysaccharides get the maximum yield of5.94±0.27g/L.Optimal submerged culture of the Tremella fuciformis mycelium synthetic medium carbon source is0.2%glucose, nitrogen,0.6%glutamic acid and glycine1:1combination of amino acids, the addition of0.4%glycine and serine1:1combination of amino acids.2. Separation and Purification water-soluble polysaccharide from the mycelium and fermentation broth of Tremella fuciformis.Optimization of extraction process of polysaccharide from mycelium was investigated by RSM.The optimum extraction conditions were found to be extraction temperature89.19℃, the liquid-solid ratio19.07, the extraction time of5.29h. After removal of protein, IPS was isolated two components named IPS1and IPS2, EPS was also isolated two component named EPS1and EPS2by ion exchange chromatography and molecular size gel filtration chromatography. EPS2is a single peak by HPLC-RID, the relative molecular mass of420602Da, PMP derivatization and HPLC determination the monosaccharide composition, IPS1was composed by mannose, galactose, glucose, rhamnose, the molar ratio is93.7:47.3:37.4:1IPS2was composed by mannose, rhamnose, glucuronic acid, glucose, galactose in the molar ratio:45.8:1:6.4:18.9:32.2; EPS1was composed by mannose, galactose, glucose, rhamnose, the molar ratio of:93.7:47.3:37.1:1, EPS2was composed by mannose, galactose, glucose, rhamnose, glucuronic acid, the molar ratio is2.4:2.7:6.4:1:33.7.3. Produced red compound----oosporein was islated and identified from Tremella fuciformisDuring mycelium of Tremella fuciformis turn to fruiting body, it produced red compound which was isolated and identified its structure, it’s important to reveal the mechanism that mycelium turn to fruiting bodies of Tremella fuciformis, while test the compounds biological activity was useful to develop and utilize. Liquid fermentation technology to select culture conditions to pruduced red compound of Tremella fuciformis, multiple column chromatographic separation mode Tremella fuciformis metabolism of red compound monomer and the monomer was analysis byX-crystal diffraction, to identify its structure was oosporein, According to the structural characteristics of the oosporein and reported in the references, test its activity by animal experiments.The results showed2%maltose as a carbon source,0.5%soybean meal leaching solution as nitrogen source, fermentation time2-3d, Tremella fuciformis began to metabolic oosporein.(2) Optimal purification process of oosporein from Tremella fuciformis by D101macroporous resin adsorption and get compound’s crystal. Oosporein can completely was adsorbed by D101 macroporous resin in1h and was eluted by70%ethanol, eluent after enrichment, was staticed at4℃for24h, then oosporein was get crystals. Through the X-ray diffraction detect crystals; the compound’s structure identification is oosporein.4. Bio-activity of water-soluble polysaccharide from mycelia、extracellular polysaccharides and oosporeinThe results of in vitro antioxidant activity show that water-soluble polysaccharide (IPS) from mycelium was more effective than the extracellular polysaccharide (EPS) on DPPH scavenging activity. IPS2was more effective than IPS1and EPS2was more effective than EPS1, The concentration of IPS and EPS at50mg/mL have strong inhibition ability for HeLa cells in vitro, Effection of EPS on behavior of depression model mice that showed it has antidepressant-like activity.The mice was irrigated stomach with oosporein at50mg/kg and25mg/kg, TST and FST time reduce, The concentration at25mg/kg lowered the numbers of activity,50mg/kg no significant increases the numbers of activity, it showed that oosporein has antidepressant activities.