Purification,Characterization,and Bioactivities Of A Polysaccharide From Bjerkandera Fumosa

Bjerkandera fumosa,a basidiomyceye fungus belonging to the genus Bjerkandera in the family of Polyporaceae,is a medicinal fungus and an annual white-rot-fungus.Extraction of the fruiting body from B.fumosa is considered to possess numerous bioactivities,such as immunomodulation and anticancer（especially anti-uterine cancer）.However,up to now,the active components and bioactivities of the fruiting body from B.fumosa have not been reported.Given the limited and unstable supply of wild B.fumosa,the cultivation of the fruiting body requires long time and its product quality is difficult to control,which limit the further development of medicinal fungus,including B.fumosa.In contrast,submerged fermentation has potential advantages for higher mycelial production over a shorter incubation time and availability of convenient control with less chance of contamination.Therefore,submerged fermentation has become a promising alternative for efficient production of mycelial biomass and bioactive compounds.In recent years,with the destruction of natural conditions,wild fungus resources are increasingly scarce,which limit the development and utilization of medicinal fungi.Liquid fermentation method has high mycelia yield,short culture period,easy to control conditions,less pollution,Is a large number of preparation of mycelium and its active substances effective way.Under this background,in order to discover the highly bioactive purified fraction of polysaccharides from B.fumosa,the optimization of culture conditions of submerged fermentation,isolation and purification,physicochemical characterization,structural elucidation and bioactivities of polysaccharides from B.fumosa were carried,which will provide preliminary research and theoretical basis of the deep development of medicines or health-care products.The main contents and results are shown as follows:（1）Optimization of liquid fermentation medium of B.fumosaMycelial biomass as an indicator,the liquid fermentation medium was optimized by single-factor experiment,Plackett-Burman experiment,steepest ascent methed and Box-Behnken response surface experiment,finally,the optimal medium composition was identified as followed（g/L）:corn flour 60,soybean powder 2,K₂HPO₄ 1.5,KH₂PO₄0.75,MgSO₄ 0.05,FeSO₄ 0.005,CuSO₄ 0.002,ZnSO₄ 0.001,CoCl₂ 0.0036,MnSO₄0.004,the mycelial biomass verified 19.303 g/L as increased by 7.114 folds compared to the yield of basal mycelium.（2）Purification of mycelial polysaccharides from B.fumosaThe mycelia of B.fumosa were obtained by liquid fermentation.The cultured mycelia were extracted with distilled water,alcohol precipitation and enzyme-Sevag method,then we acquired the deproteinized polysaccharide（DBFM）,which was light brown powder and soluble.The polysaccharide DBFM was separated by DEAE-32cellulose anion exchange column and obtained neutral polysaccharide DBFN and acid polysaccharide DBFA1 and DBFA2.DBFN、DBFA1 and DBFA2 were further purified by Sepharose CL-6B gel chromatography column respectively,and three fractions,named DBFM1,DBFM2 and DBFM3,were obtained.DBFM1,DBFM2 and DBFM3were identified by high performance gel permeation chromatography（HPGLC）,and results showed that only DBFM3 was homogeneous polysaccharide,so we chose DBFM3 for further study.（3）Physicochemical characterization,structural elucidation of the polysaccharide DBFM3The molecular weight,total sugar content,protein content and uronic acid content of DBFM3 were determined by HPGLC,phenol-sulfuric acid method,Bradford method and m-hydroxydiphenyl colorimetric method,respectively.The results showed that the molecular weight of DBFM3 was 1.8×10⁵Da,the total sugar content was 57.64%,protein content was 1.758%and uronic acid content was 7.29%.The monosaccharide composition of DBFM3 was determined by high performance liquid chromatography（HPLC）.The results showed that DBFM3 was composed of mannose,galacturonic acid and galactose at a molar ratio of 1:18.16:0.702.The UV absorption spectra of DBFM3 suggested that the polysaccharide contained trace protein.The fourier transform infrared spectroscopy（FT-IR）and nuclear magnetic resonance（NMR）analysis showed that the hexose ring form of DBFM3 was pyranose conformation,and the glycosidic linkage wasβ-type,which might possess 1→3,1→3,6 and 1→6glycosidic bond.（4）Biological activity of mycelia polysaccharides from B.fumosaAntioxidant experiments in vitro showed that DBFM and purified fraction DBFM3had DPPH and hydroxyl radical scavenging activity in dose-dependent manner,the scavenging free radicals ability of DBFM3 was significantly stronger than that of unpurified DBFM.DBFM3 had a protective effect on DNA damage induced by H₂O₂,and reduced the damage of DNA by hydroxyl radicals.DBFM and DBFM3 could reduce the SH-SY5Y cells damage induced by H₂O₂,and improve the cell viability,and the activity of DBFM3 was higher than that of unpurified DBFM.The assays of splenocyte proliferation in vitro of DBFM and purified fraction DBFM3 were manipulated to detect the immunological activity.Results showed that DBFM and purified fraction DBFM3 significantly promoted lymphocyte proliferation,and increased the proliferation of lymphocytes in the presence of ConA or LPS as mitogen,and the activity of DBFM3 was higher than that of DBFM.