| Hirsutella sinensis, isolated from natural Cordyceps, is one of the Cordyceps sinensis, which has the similar biological functions with nature Cordyceps. It is a new and important source in developing products, such as modern Chinese medicine and protective foods. What’s more, as one of the main active components of H. sinensis, polysaccharide plays a significant role in the pharmacological activity, which has super application value. The mycelia of H. sinensis were gained after fermentation by addition of citrus peel in the culture medium. The purified intracellular polysaccharide was obtained by separation and purification from fermentation mycelia of H. sinensis (HS001). This intracellular polysaccharide was further proved to have higher antioxidant activity in vitro, and its chemical structure was also characterized. The main process and results of research are listed as follows:Part â… :Study on separation, purification and antioxidant of intracellular polysaccharide from H. sinensis. (1) The mycelia of H. sinensis (HS001) were gained after fermentation by addition of citrus peel in the culture medium. A yield of 4.6% of crude intracellular polysaccharide was gained from mycelium of H. sinensis by extraction with hot water and precipitation with alcohol. Then the crude polysaccharide was purified to get the pure fraction (HSIPS2) by a set of process, such as deproteinization, depigmentation and the further purification of DEAE-Sepharose Fast Flow chromatography and Sephacryl S-100 gel filtration chromatography. (2) The information of molecular and composition of monosaccharide were measured by a size exclusion chromatography combined with a multi-angle laser light scattering system (SEC-MALLS) and a high performance liquid chromatography (HPLC). Results showed that the macromolecule of HSIPS2 has relatively narrow distribution and comparatively homogeneous molecular weight, and the average molecular weight (Mw), purity, radius of gyration (<S2>z1/2) and hydrodynamic radius (Rh) are 1.463×104 g/mol,99.82%,0.5 nm and 2.3 nm, respectively. The HPLC characterized that HSIPS2 is composed of glucose, galactose, mannose and ribose with a molar ratio of 48.52:6.58:5.17:1.0. It was undoubtedly demonstrated that HSIPS2 is a novel polysaccharide. (3) Further researches on antioxidant in vitro revealed that HSIPS2 exhibited strong antioxidant properties. The oxygen radical absorbing capacity (ORAC) of HSIPS2 is 872.80 μmol TE/g. At the concentration of 1.0 mg/mL, the reducing power of HSIPS2 is 0.99. Besides, the IC50 of DPPH radical scavenging rate, hydroxyl radical scavenging rate and ABTS scavenging rate are 0.73 mg/mL,0.75 mg/mL and 0.47 mg/mL, respectively.Part II:It was the first time to thoroughly study the structure of HSIPS2 from mycelium of H. sinensis. The high performance liquid chromatography (HPLC), infrared spectroscopy (IR), methylation analysis and NMR analysis were carried out to determine the structural features of HSIPS2. The results suggested that HSIPS2 has a backbone of 1,4-linked a-Glcp. The branches composed of 1,5-linked β-Galf and α-Glcp-(1â†' are linked to O-6 of the backbone residues, and the a-Glcp-(1â†' are terminal residues linked to O-6 of 1,5-linked β-Galf.There are a little of 1,3-linked α-Ribf,4-linked a-Manp and 1,2-linked Galp. (2) The conformation of HSIPS2 was analyzed by size-exclusion chromatography combined with multi-angle laser light scattering, refractive index and online viscometry (SEC-MALLS-IR-ViscoStar â…¡) and atomic force microscopy (AFM). The structural parameters of a, v, ML, q and d were 0.67,0.56,334.05 nm-1,0.70 nm and 0.69 nm, respectively, which indicted that HSIPS2 existed as a flexible chain conformation with a coil-like structure in 0.1 mol/L NaNO3 solution at 25℃. Furthermore, this flexible chain conformation was also proved by AFM. The present study provides the basis of theory for elucidating the relationship between structure and activity of HSIPS2. |
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