Three-phase Partitioning For Extraction,Purification,Identifying Structure And Activity Of Polysaccharides From Flammulina Velutipes Fruiting Body

Flammulina velutipes（Curtis Singer）is the world’s third-selling edible fungus.It contains not only fat,protein and amino acids,vitamins,minerals and other nutrients,but also active substances which have a variety of biological activities,such as polysaccharides,lipids,glycoproteins,phenols.Three-phase partitioning（TPP）is a new protein and edible oils separation and purification technology,which combines salting out,isoelectric point precipitation and solvent precipitation processes.TPP is often formed by adding water miscible aliphatic alcohol and salt to a slurry of protein,forming an alcohol-rich upper phase,solid middle phase of protein and salt-rich lower phase.The pigments,lipids,and hydrophobic materials are concentrated in the upper phase,the protein is concentrated in the middle phase,saccharides and other polar components are concentrated in the lower phase.This TPP technique selectively separates the desired protein into the mesophase,with any remaining free protein distributed between the other two phases.This approach has been widely used for the isolation of commercially valuable proteins and oil.TPP have been applied in the fields of plant,animal and microorganism.However,its use for the separation of polysaccharides from edible and medicinal fungi has not yet been reported.1.Optimization of TPP extraction parameters from F.velutipes fruiting bodyThe yield of polysaccharides from Flammulina velutipes fruit body was extracted by TPP.Firstly,the key factor of the yield of mycelium Flammulina velutipes on amount of t-butanol（mL）,mass fraction of（NH₄）₂SO₄（w/v）,time（min）,temperature（°C）,pH and solid-liquid ratio.On the basis of the above single factor experiment,the three factors that have the most significant influence on the yield were designed and screened:mass fraction of（NH₄）₂SO₄,amount of t-butanol（mL）and temperature;the extraction yield of polysaccharides from Flammulina velutipes was determined by central combination design and response surface methodology.The optimal process:material to liquid ratio 1g:12mL,tert-butanol volume ratio 1:1.9,ammonium sulfate mass fraction 37%,temperature 40°C,time 30min,pH=7.Under the above conditions,the yield of Flammulina velutipes polysaccharide was 2.3%.At the same time,the predicted value was approached in the verification test（P<0.005）.It is shown that the established response surface model is suitable for optimizing the process of extracting polysaccharides from Flammulina velutipes fruit body by TPP,which provides a new idea for the extraction of Flammulina velutipes polysaccharides.2.Physicochemical properties and activities in vitro of polysaccharides from Flammulina velutipes fruiting through TPP and traditional alcohol precipitation were compareThree-phase parttioning（TPP）is an innovative method Flammulina velutipes polysaccharides which has been used for the purification of protein to extracting.The yield,purity,chemical properties and in vitro activity of crude polysaccharides obtained by TTP and traditional alcohol precipitation were compared.The results indicated that the TPP extraction of F.velutipes polysaccharides had a higher yield（2.30%）and a purity of 40.53%.The molecular weight distribution of the 60%ethanol-precipitated and TTP extraction are similar by HPLS-MALLS-RI.The overall ratio of monosaccharide components obtained by different purification methods is close,but the TPP extraction of F.velutipes polysaccharides contains Glucuronic acid（conventional alcohol precipitation does not contain or litter）.Three different assays involving the DPPH radical scavenging activity,Trolox equivalent antioxidant capacity（TEAC）assay,and ferric reducing ability of plasma（FRAP）assay were used to determine the antioxidant activities of in vitro F.velutipes polysaccharides,and it was found to have better antioxidant capacity.Moreover,the TPP extraction of F.velutipes polysaccharides possessed strong free-radical scavenging abilities and antioxidant activity in vitro.This study preliminarily explored the TTP extraction method of Flammulina velutipes polysaccharides,which provided a new idea for the extraction and purification of Flammulina velutipes polysaccharides.3.Further purification and physicochemical properties of Flammulina velutipes polysaccharide extracted by TTPThe polysaccharides from F.velutipes fruit extracted by TTP used as raw materials,and the homopolysaccharides FVPT1 and FVPT2 were further separated and purified by S-300 gel column.The molecular weight of polysaccharides was analyzed by HPLS-MALLS-RI:2.06×10⁴,1.64×10⁴.At the same time,the in vitro activity of homopolysaccharide was determined FVPT1 and FVPT2 had lower antioxidative ability,and FVPT1 had good immunological activity.High performance anion chromatography analys monosaccharide of FVPT1,FVPT2 monosaccharide composition:fucose:galactose:glucose:mannose=1:3.5:1:1.4;fucose:galactose:glucose=1:3:10.The chemical structure of homogenous polysaccharides from Flammulina velutipes was studied by methylation experiments,GC-MS and nuclear magnetic resonance spectroscopy（NMR）.