Study On The Separation And Purification Of Grifola Frondosa Polysaccharide And Its Antioxidant Activity Based On Ethanol-ammonium Sulfate Two-phase System

Grifola frondosa,also known as Maitake,belongs to Polyporaceae and Ramalina.It is a precious edible medicinal fungus.Grifola frondosa riched in polysaccharide,protein,dietary fiber and other nutrients in mycelium and fruiting bodies and polysaccharides is the main bioactive substance.Recently,aqueous two-phase system(ATPS)extraction has been widely used to separate nature products such as polysaccharide and protein as a novel technology.The process is mild and will not cause inactivation or denaturation of bioactive substances and no organic solvent residues.At present,the separation of polysaccharides from Grifola frondosa(GFP)has not been reported by ATPS.Therefore,the polysaccharides were separated from Grifola frondosa by ATPS and optimized the extraction condition using response surface method;the chemical structures were determined by FT-IR,GC-MS and other methods;the antioxidant of pokysaccharides which purified by ATPS were analysed through chemical and cell antioxidant experiments.It provided a theoretical basis for the study and application for polysaccharide separated from Grifola frondosa by ATPs.The main contents and results are as follows:Part one.Study on the separation of GFP by aqueous two-phase systemThe effects of ammonium sulfate concentration,ethanol concentration,temperature and pH on the yield of GFP were studied by single factor method and response surface method(RSM).The results of single factor experiment showed that the optimum conditions were as follows:the concentration of ammonium sulfate was 18%,the concentration of ethanol was 30%,the temperature was 30?and the pH was 4.The results of RSM at 30?showed that the optimum technological conditions were as follows:the concentration of ammonium sulfate was 17%,the concentration of ethanol was 27.9%and pH was 3.22.Under the condition,the yield of the polysaccharide was 90.21%and the purity was 66.7%,which was close to the predicted value.Part two.Structure identification and preliminary characterization of GFPThe preliminary characterization of GFP was determined by FT-IR,GC-MS,CD,SEM,AFM and Congo red experiment.The results of FT-IR showed that GFP purified by ATPs held the configuration with glycosyl ring.GC-MS showed that the GFP was composed of Arabia,mannose,glucose and galactose,and the molar ratio was 1.3:1.0:5.1:1.0.The results of Congo red experiment showed that there was a three helix structure of GFP and the change of external conditions could affect its triple helix structure.The CD experiment showed that there was asymmetric structure in GFP and it would also change with the external conditions.The GFP was observed in an interwoven net structure by SEM.The AFM showed that the GFP was interwoven with a network structure and existed in the form of agglomerate.In a summary,GFP purified by ATPs was three helical asymmetric structrue.Part three.Determination of antioxidant activity of GFPThe antioxidant activity of GFP was investigated by the chemical oxidation and cell oxidation experiments.The results showed that GFP could effective scavenge ABTS~+,DPPH·and·OH and the scavenging rate was 70.43±2.01%,47.17±1.98%and 38.49±1.07%,respectively,and it could resist lipid peroxidation(55.39±1.79%).Compared with the model group,GFP could significantly decreased intracellular ROS content(P<0.05),MDA level(P<0.01)and LDH release(P<0.01)in the range of experiment concentration.When the concentration of GFP was over 100?g/mL,the content of SOD(P<0.05)was increased significantly;when the concentration of GFP was over 600?g/mL,the content of GSH-PX(P<0.05)was increased significantly;when the concentration of GFP was 1000?g/mL,the content of CAT(P<0.05)was increased significantly in cells.In a word,GFP purified by ATPs had strong antioxidant activities in vitro.In a summary,the work studied the separation and purification of polysaccharide from Grifola frondosa and antioxidant activities based on the ethanol-ammonium sulfate aqueous two phase system.The optimum separation process was investigated by the response surface method.The purified polysaccharide was preliminary characterized by physical and chemical methods.The antioxidant activity of the polysaccharide was analyzed by chemical oxidation resistance and cell antioxidant method,which provided a reference for the study and application of the separation of the polysaccharide of the Grifola frondosa...