Studies On Optimization Of Fermentation Conditions, Structure And Antioxidant Activity Of Cordyceps Militaris Polysaccharides

Cordyceps militaris is a model species of the Cordyceps with a wide range of biological activities and medical values. Polysaccharide is one of the important active ingredients of cordyceps militaris. Therefore, this research found the best fermentation conditions to get the polysaccharides of cordyceps militaris by the single factor experiment and response surface method. The crude intracellular polysaccharides and exopolysaccharides which were extracted from the liquid fermentation of cordyceps militaris were separated, purified and analyzed the basic physicochemical properties. The pure intracellular polysaccharides and exopolysaccharides were subjected to compare the chemical structure and antioxidant activities in vitro. The results provided the practical basis for the large-scale and industrialized liquid fermentation of cordyceps militaris. Meanwhile provided the scientific basis for the development and application of cordyceps militaris polysaccharides. The major results are as follows.1. In this study, the research object is cordyceps militaris BYB-08. The optimum medium composition and culture conditions for the polysaccharides of cordyceps militaris were determined by the single factor analysis. On the basis results of the single factor analysis, three significant effective factors were selected by Plackett-Burman factorial experiment design. They were sucrose, yeast extract powder and KH2PO4. The result of Box-Benhnken experimental design showed that the optimal fermentation conditions for polysaccharides of cordyceps militaris were6.41%sucrose,3.79%yeast extract powder,0.072%KH2PO4, fermentation temperature25℃, fermentation time6d, rotate speed150r/min, amount of inoculum10%, loaded liquid100mL/250mL. The experiment measured total polysaccharide content was3.19g/L, Which is very close to3.23g/L predicted from Box-Benhnken experimental design. The result indicated that the model is accurate, reliable and fit.2. Intracellular polysaccharides and exopolysaccharides were extracted from fermentation liquid of Cordyceps militaris by ultrasonic extraction and water extraction, ethyl alcohol deposition, respectively. Deproteinization, dialysis, DEAE-cellulose-52and SepharoseCL-6B were used for separation and purification of the crude polysaccharides. Ultraviolet spectrum, specific rotation and gel permeation chromatography (GPC) were used to identify the purity of IPS-3and EPS-3. The results of ultraviolet spectrometry showed that IPS-3and EPS-3not contain nucleic acid. The results of specific rotation and GPC also suggested that IPS-3and EPS-3were uniform polysaccharide components. Weight-average molecular weight (Mw) of IPS-3and EPS-3were67,920Da and37,350Da. IPS-3and EPS-3had rich polysaccharides, uronic acid and small amounts of glycoprotein. Infrared spectroscopy (IR) and gas chromatography (GC) were used to characterize the primary structure of IPS-3and EPS-3. The results showed that the infrared spectrum of IPS-3and EPS-3were similar and had the characteristic absorption peaks of polysaccharide. IPS-3had both alpha glycosidic bond configuration and beta glycosidic bond configuration, at the same time containing beta-D-mannose. EPS-3had the absorption peaks in811cm-and852cm-1. The results showed that EPS-3containing alpha-pyran mannose glycosidic bond configuration. The gas chromatograph revealed that IPS-3and EPS-3had the same monosaccharide compositions, namely mannose, glucose and galactose, and had no rhamnose, arabinose and xylose. IPS-3was composed of mannose, galactose and glucose at4.56:3.85:1.0. EPS-3was composed of mannose, glucose and galactose at2.66:8.88:1.0.3. The molecular chain conformations of the IPS-3and EPS-3polysaccharide solution were studied by Congo red test, particle size distribution and ultraviolet spectrum. The result showed that IPS-3and EPS-3polysaccharide solutions cannot form compound with Congo red and did not have triple helical structure, but had a random coil chain. Different concentrations (0.01,1.0mg/mL) and stored time (0,24h) of IPS-3and EPS-3effected the average particle size and particle size of dispersion index of IPS-3and EPS-3. The result showed that with the increase of concentration and stored time of IPS-3and EPS-3, polysaccharide molecules became mutual entanglement and generate gathered, and formed different space conformations. So the average particle size of IPS-3and EPS-3increased and particle size of dispersion index of IPS-3and EPS-3changed. Ultraviolet spectrum analysis results showed that temperature and metal ions had little effects on the conformation of cordyceps polysaccharides. pH, urea and dimethyl sulfoxide can destroy hydrogen bonds between or in the molecules of IPS-3and EPS-3and increase the asymmetry of polysaccharide molecules. So these factors can lead to the changes of conformation of polysaccharides.4. Scanning electron microscopy (SEM) showed that IPS-3and EPS-3gathered closely with a random cross chain network structure. IPS-3and EPS-3had branches and showed some flexibility. The solid apparent morphology of IPS-3and EPS-3still presented certain differences. X-ray diffraction confirmed that IPS-3and EPS-3were amorphous state. Differential scanning calorimeter showed that the glass transition temperature of IPS-3and EPS-3were55.6℃and143.8℃, respectively. Under the condition of high temperature, IPS-3and EPS-3can occur endothermic and exothermic reaction.5. The antioxidant activities of IPS-3and EPS-3from Cordyceps militaris were analyzed by scavenging rates of DPPH radical,· OH, O2·-, the ferrous ion chelating capacity and total reducing capacity. The result showed that IPS-3and EPS-3had good scavenging effect on DPPH radical. At the same time, IPS-3and EPS-3had scavenging effect on· OH, O2·-, the ferrous ion chelating capacity and total reducing capacity. But there were some differences in antioxidant activity between IPS-3and EPS-3.