Research On Polysaccharides Of Tianma Fungus Germination HL-003

Tianma is a perennial herb belonging to Orchidaceae Gastrodia elata.Tianma seed as small as dust,and without endosperm and other nutrients reserve,so that it can not germinate,in the absence of exogenous nutrition conditions.Tianma Germination fungus were selected as research subjects of the research,as it play a crucial role in the process of seed germination.Germination fungus were separation,purification and identification,then polysaccharide of mycelium and fermentation brothwere isolated and purified,finally,the antioxidant activity of polysaccharides been investigated.The reason for these studies,only to provide a theoretical basis for the study of germination fungus.(1)Germination fungus were separated from the Tianma original bulbs,it was identified as the germination fungus of Tianma,as it can promote germination of Tianma seed by seed germination experiment.Germination fungus has a typical clamp connection structure of Basidiomycetes observed through a microscope.Germination fungus was identified as Mycena purpureofusca using molecular and bioinformatics identification methods include BLAST and phylogenetic tree.(2)In order to determine polysaccharides of germination fungus HL-003,parameters of the phenol-sulfuric acid method were optimized.Phenol concentration and volume of concentrated sulfuric acid is the independent variable,the maximum absorption wavelength and absorbance were detected under different concentrations of phenol and concentrated sulfuric acid volume.Immediately,the color of time,precision,linearity and recovery were measured in order to determine the optimum parameters.The results show that phenol concentration of 5%,concentrated sulfuric acid content of 3.5 m L,the maximum absorption wavelength of 488 nm,coloration time 15 min.The methods have a high precision and recovery results are reliable,and consistent with the true value.The concentration of glucose in the range of 0~200(mg/m L)have a good linearity.Intracellular and fermentation of polysaccharide contents were 2.028% and 2.998 g / L.,by detecting with optimized method.(3)Response surface methodology was used to optimize main process parameters for the extraction of polysaccharides from HL-003's Intracellular broth based on polysaccharide extraction rate of one-factor tests.A regression model equation was fitted.Experimentally determined that the optimum fermentation alcohol concentration was determined by analysis of different ethanol concentration univariate.The optimum extraction conditions were found to be ultrasound time 19 min,solid-liquid ratio 42 m L/g,extraction temperature 72 ? and extraction time 2.2 h.The extraction rate of polysaccharides under these conditions was up to 2.03 %.The fitted regression model had excellent goodness of fit and showed lower prediction error.Fermentative polysaccharides best alcohol concentration of 75%.(4)Mycelia and fermentation broth polysaccharide were extracted by the method of water extraction and alcohol precipitation.Free protein present in the polysaccharide be removed using Sevag method,so MP and FP without protein were obtained.Mycelium and fermentation broth pure polysaccharide MPP and FPP were obtained from crude polysaccharide using Millipore labscale TFF ultrafiltration system and Sephadex G-100 column chromatography mehods.Monosaccharide composition of polysaccharide,which was after the sulfuric acid hydrolysis,were detected by HPLC and TLC.It was shown that mycelium polysaccharides containing galactose and rhamnose,of which the content of galactose is more than 90%,and fermentative polysaccharides containing galactose and arabinose,of which the content of galactose is more than 80%.As Congo red method can detect the spiral structure of polysaccharides,it was found that the fermentation broth and mycelium polysaccharide molecules exist in a helical structure,and broth contains large amounts of polysaccharide molecules helical structure.(5)Polysaccharide antioxidant activity was evaluated by reducing power assay,and scavenge hydroxyl radicals,superoxide negative radical,DPPH radical and ABTS radical assay.It was found that mycelium and fermentation polysaccharide both have a certain antioxidant activity.Purified polysaccharide have a stronger antioxidant activity than the corresponding crude polysaccharide.Antioxidant capacity of mycelium polysaccharide was stronger than fermentative polysaccharides.Through the above research show that germination fungus polysaccharide have antioxidant activity,in particular mycelium polysaccharide.It has a potential value of development and utilization.