Extraction And Identification Of Immunomodulatory Proteins Of Flammulina Velutipe

Immunomodulatory proteins of Flammulina velutipe is a kind of protein extracted from Flammulina velutipes, which can promote the synthesis of nucleic acids and proteins, inhibit allergic reactions, accelerate metabolism, and enhance immunity. Immunomodulatory proteins of Flammulina velutipe is a high quality protein with much function of immune regulation, anti-allergy, anti-cancer, enhance producing cytokine. The aim of this research is to investigate the extraction methord, anti-allergy activity, physical properties of immunomodulatory proteins of Flammulina velutipe. The results of this research can provide important data for application of Immunomodulatory proteins of Flammulina velutipe in food industry, especially in desensitization milk productions. In this study, immunomodulatory proteins of Flammulina velutipe was isolated and purified by gel filtration chromatography and ion exchange chromatography, identified by SDS-polyacrylamide gel electrophoresis and hemagglutination test, clarified by determination anti-allergy activity. The physical properties of isolate were also analysed. The main results were as follows:1) Complex of immunomodulatory proteins of Flammulina velutipe was extracted by5%acetic acid, through cell disruption, salting out (90%(NH4SO4). The yield of extract was0.304%, content of protein was53.5%.2) The result of hemagglutination test of the complex indicated that immunomodulatory proteins of Flammulina velutipes was in the extract. Three fractions K1, K2and K3were separated from the extract by gel filtration Chromatography (pH6.0phosphate buffer,0.8ml/min). The result of hemagglutination test of the three fractions showed immunomodulatory proteins of Flammulina velutipes was in K2.Gel filtration chromatography of K2showed only one peak with26KDa molecular weight (standard protein: Lysozyme, trypsin, ovalbumin, bovine serum albumin). SDS-SDS-polyacrylamide gel electrophoresis showed two bands with13KDa and26KDa. This result indicated that the fraction of K2contains a dimer with subunits of13KDa and a monomer with26KDa. The results indicated that the Immunomodulatory Proteins of Flammulina velutipes was not purified. 3) The immunomodulatory proteins of Flammulina velutipes was isolated by Anion exchange chromatography. HiPrep16/10DEAE-FF ion exchange column was Balanced by10mmol/L Tris-HCl (pH8.0) buffer, eluted by0-0.5mol/L NaCl buffer (the flow rate of1.5ml/min). Seven fractions named F1to F7were separated by ion exchange chromatography. The result of hemagglutination test of the seven fractions showed that only F4was positive indicating that the immunomodulatory proteins of Flammulina velutipe may be presented in F4. SDS-polyacrylamide gel electrophoresis of F4showed only one band of13KDa corresponded with the relative molecular weight of immunomodulatory proteins of Flammulina velutipes. Ion exchange chromatography of F4showed one peak still. This results indicated that fraction F4might be regarded as immunomodulatory proteins of Flammulina velutipes.4) Effect of anti-allergy of F4was investigated. Animal model of food allergy was established, using eight weeks of BALB/C female mice. These mice were orally inoculated with milk beta-lactoglobulin (negative control, PBS). The IgE levels in serum of F4group was decreased than control group significantly (P<0.001). Symptoms of anaphylaxis experimental group decreased significantly than the control group. Histology detection of intestines did not show inflammation as edema、ecclasis. These results indicated that the F4fraction was corresponded to Immunomodulatory Proteins of Flammulina velutipes with anti-allergy activity.5) The physical properties of immunomodulatory proteins of Flammulina velutipe was also analysed for the immunomodulatory proteins being applied in the food industry, especially in desensitization milk production. The results of the physical properties assay of immunomodulatory proteins of Flammulina velutipe showed the solubility、emulsification、emulsion stability and thermal stability closed to milk,Dispertion of immunomodulatory proteins of Flammulina velutipe was better than milk.