| Pholiota dinghuensis Bi is a new species in Pholiota family which is only found in Guangdong Province of China since 1985.Through modern medical research,medicine fungi polysaccharides possess the bioactivities of immunomodulatory,anti-tumor,antioxidant,hypoglycemic,cholesterol-lowering,anti-inflammatory,anti-bacterial,hepatoprotectivity,anti-virus and intestinal prebiotics,etc.In recent years,polysaccharides of Pholiota dinghuensis Bi,the main active components of Pholiota dinghuensis Bi,have attracted increasing attention,and mycelial polysaccharides from Pholiota dinghuensis Bi(PDP)shows significant anti-tumor activities.However,current research regarding immunomodulation activity of PDP is still limited.Therefore,separation and purification,determination of physical properties,static rheological property and immunomodulation activity of mycelial polysaccharides from Pholiota dinghuensis Bi were mainly studied in the thesis.The main results are as follows:1 The production of submerged fermentation and rheological property of PDPThe mycelia of Pholiota dingensis Bi were produced by the submerged fermentation.Crude polysaccharides(PDP)were obtained by hydrothermal extraction and ethanol precipitation.It was cultivated in a fermentation medium with the composition of 45 g/L glucose,6 g/L yeast extract,1.5 g/L KH2PO4 and 0.5 g/L MgSO4,the potato 100 g/L,malt powder 2.5 g/L,inoculation amount to 15%,under the condition of 28℃and 120 rmp.Ten days later,production of mycelial polysaccharides was measured about 429 mg/L.Effects of concentration,temperature,pH,sucrose,NaCl and CaCl2 on the static rheological property of crude PDP were investigated.The result showed that the apparent viscosity of crude PDP increased with the increase in the crude PDP concentration,while decreased significantly with the increase of shear rate,indicating that the crude PDP solution was a non-Newtonian pseudoplastic fluid.The apparent viscosity of crude PDP decreased when the temperature increased.It’s apparent viscosity remained stable under the PH of 6-10,while increased significantly with a strong acid treatment.The addition of low concentration of sucrose made an increase of the viscosity.The apparent of crude PDP viscosity gradually increased with concentration of CaCl2.2 Isolation,purification and physical properties of PDPThe crude PDP was separated preliminarily through a DEAE fast flow anion exchange chromatography column,and each component were further purified with the Sephadex G-100 gel filtration chromatography column,resulting in three fractions of PDP-1,PDP-2 and PDP-3,with a recovery rate of 11.8%,44.7%and 1.04%respectively.The homogeneity and molecular weights of PDP-1,PDP-2 and PDP-3 were analyzed by HPGPC.Results showed that PDP-1,PDP-2 and PDP-3 were homogenerous polysaccharide and their average molecular weights were 1.50×106,1.42× 106,1.01 ×106 Da,respectively.The content of carbohydrate,uronic acid,protein and sulfuric radical in crude PDP,PDP-1,PDP-2 and PDP-3 were determined according to the reported methods of sulfuric acid-phenol coloration,meta-hydroxydiphenyl method,coomassie brilliant blue coloration and barium chloride-gelatin method,respectively.The results show that the carbohydrate contents of crude PDP,PDP-1,PDP-2 and PDP-3 were 84.53%,93.48%,92.57%and 64.86%respectively;Uronic acid content were 4.47%,2.06%,4.35%and 15.70%respectively;Protein content were 1.69%,0.01%,0.04%and 3.49%;Sulfuric acid content were 1.92%,0.81%,1.78%and 2.25%.In the polysaccharide components measured,PDP-2 contains higher uronic acid and sulfuric acid content,while PDP-3 contains higher protein,uronic acid and sulfuric acid content.The monosaccharide compositions of crude PDP and its purified fractions were determined by high-performance liquid chromatography(HPLC).However,rhamnose was contained in the PDP-3,and was not detected in PDP-1 and PDP-2;galactose and fucose was contained in the PDP-2 and PDP-3,and was not detected in the PDP-1,while PDP-3 was relatively higher than those in PDP-2.3 Immunomodulatory activity of PDPBy using the RAW264.7 model,the immunomodulatory activity in vitro of crude PDP and the three fractions(PDP-1,PDP-2 and PDP-3)were evaluated.The results showed that PDP-2 and PDP-3 could significantly stimulate the proliferation of macrophages,enhance the capability of macrophage phagocytosis and increase the activity of acid phosphatase,while PDP-3 could promote the production of nitric oxide,suggesting that they have potent immunomodulating effect.The immunomodulatory activity of PDP-2 was further evaluated by using Cyclophosphamide-induced immunosuppression mice models.It was found that PDP-2 can increase the spleen index,hemolysin level,lysozyme content and the activities of T-AOC,T-SOD,CAT,GSH-Px,and reduce the MDA level. |
