The High-throughput Fermentation Of Hirsutella Sinensis G67 And The Structure-activity Relationship Of Its Polysaccharides

Along with the limitation of wild Cordyceps sinensis and the increase of the market demand, the contradiction between its supply and demand is being aggravated. The method of using liquid fermentation to produce mycelia and extracellular polysaccharides (EPS) of Cordyceps sinensis has the advantages of short incubation period, high yield, and stable quality. The culture conditions of microorganism differ from each other. The yields of EPS and mycelia, which usually have potential biological activities, could be improved by optimizationof the culture conditions. In this paper, the high-throughput fermentation for EPS and mycelia, as well as the structure-activity relationship of polysaccharides produced by Hirsutella sinensis G67, which has been acclimated for long time,are studied. With the metjod of grading-alcoholic precipitation, three fragments of EPS and IPS are obtained, respectively. High Performance Gel Permeation Chromatography (HPGPC), Gas Chromatography (GC), Fourier Transform Infrared Spectroscopy (FTIR), and chemical method are employed to analyse the characteristics of polysaccharides. In vitro moisture retention and absorption abilities, antioxidant activity, and antifungal activity of EPS and intercellular polysaccharide (IPS) are evaluaed. In addition, statistical methods, including analysis of variance (ANOVA), correlation analysis, and linear analysis are used to analyse the structure-activity relationship. The main results are as follows:1. The fermentation conditions of EPS and mycelia are optimized using single factor experiments and response surface methodology (RSM) design. The optimized media is:sucrose 29.05 g/L, yeast extract 5.31 g/L, sodium nitrate 1.33 g/L, dipotassium phosphate 2.10 g/L, magnesium sulfate 0.78 g/L, and 200 g/L potato (boiled). The optimum cultivation conditions, including initial pH value, medium volume, inoculum size, and temperature are 6.0,100 mL/250 mL,10%, and 25℃, respectively. Under this conditions, the maximum yield of EPS and mycelia appear after 72 h, which are 5.27 g/L and 15.92 g/L (dry weight),respectively. An experiment of 30 liter scaled fermentation is performed with the yield of 5.82 g/L (EPS) and 15.90 g/L (mycelia) in 60 h, indicating the strain could be amplified towards industry scale in the coming future.2. The EPS and IPS extraction technology are optimized. The volume of the fermentation liquor is concentrated to 1/6 and pH is adjusted to 8.0 for the extraction of EPS. Thawed mycelia is added 8 multiple of water, and extracted 60 min once at 100℃, then the mycelia extracts is concentrated to 1/6 and pH is adjusted to 8.0 for the extraction of IPS.1,2, and 4 volumes of 95% ethanol is added successively, and three fractions of EPS and IPS are obtained, respectively. The extraction rate of EPS and IPS are 89.7% and 17.5% (dry weight), and the proportion of EPS-3 and IPS-3 is the largest, respectively.3. The characteristics of EPS and IPS. The polysaccharide fractioins precipitated in lower ethanol concentration (EPS-1 and IPS-1) have higher protein content (19.86% and 33.97%), lower neutral sugar content (70.71% and 52.71%), and larger molecular weight (43.0 kDa and 23.1 kDa), while the polysaccharide obtained in higher ethanol concentration (EPS-3 and IPS-3) have lower protein content (2.00% and 1.90%), higher neutral sugar content (2.64% and 85.55%), and smaller molecular weight (4.7 kDa and 10.4 kDa). The ethanol concentration also could influence the monosaccharide constituents of polysaccharide. EPS-1, EPS-2, IPS-1, and IPS-2 are composed of glucose (Glu), galactose (Gal), and mannose (Man), while galactose is not detected in EPS-3 and IPS-3. With the increase of ethanol concentration, the mannose content of IPS declined from 59.0% to 27.2%, while the content of glucose increased from 8.5% to 72.8%. All of the fractions have uronic acid, and there is no significant difference. The result of FTIR indicated that these fractions have high similar characteristics.4. Evaluation of the bioactivity. In vitro moisture retention and absorption abilities, antioxidant activities and antifungal activity of EPS and IPS produced by Hirsutella sinensis G67 are studied. The moisture retention ability of the EPS and IPS is found to be superior to glycerol and only a little inferior to hyaluronic acid (HA). The polysaccharides have ferrous ion chelating ability and radical scavenging activity, showing dose-effect relationship. In addition, EPS and IPS could against gram-positive bacterium, gram-negative bacterium, and candida albicans.5. The characteristics of polysaccharide have great influence on their bioactivities. Polysaccharides with larger molecular weight have better moisture retention ability, while the polysaccharides with smaller molecular weight have better moisture absorbtion ability. Polysaccharides with higher mannose content show stronger hydroxyl radical scavenging activity and the polysaccharides with higher galactose content have stronger superoxidant radical scavenging activity, while the polysaccharides with smaller molecular weight show better ferrous ion chelating abilitv.