Biology Of A New Type Of Cordyceps And Its Active Substances

Cordyceps sp. was collected from Changbai mountain. The anamorph species, the selection of the optimal fermentation medium and conditions for polysaccharides production, the optimal artificial cultivation, chemical compounds analysis, the extraction methods, characterization, components and bioactivities of polysaccharides are studied.(1) Hirsutella beakdumountainsis, collected from Baekdu Mountain, China, is described as a new anamorph species. The analysis of ITS sequence shows that Hirsutella beakdumountainsis is clustered together with Cordyceps sinensis and Hirsutella sinensis in the phylogenetic tree. Conidiophoras has A and B two types. They are single or branching with two phialides, erect or nodding at the top. Vegetative hyphae twist to be coremium. Conidia are hyaline, aseptata, laevia, fusiform, ovoid, produced in group of 2-6 in a dense slimy ball at the end of the phialide. These characters are distinguished from Hirsutella sinensis and supported the establishment of this new species.(2) The fermentation condition and medium for mycelia growth and exopolysaccharides production were optimized. The optimized conditions and medium were as follows:initial pH 5.5, potato extract 20% (w/v), sucrose 2.5%, peptone 0.5%, K2HPO4 0.2%, MgSO4 0.05% and fermentation 4 days. The highest production of EPS and mycelium biomass yield reached 2.17g/L and 10.06g/L, respectively.The optimized condition for the artificial cultivation was strength of the light 1000 lx, the ratio of rice and nutrient liquid (g:mL) 30:45, silkworm pupa powder 30g and temperature 15℃.We can get the fruit bodies after 45d cultivation.(3) Compare the compositions of natural fruiting bodies of Cordyceps sinensis (NFCS), mycelia from shake culture (SCHS) and the fruiting bodies from artificial cultivation (ACHS) of Hirsutella sp. The crude protein of NFCS accounted for 22.97% obviously lower than those of SCHS (27.99%) and ACHS (31.89%). The contents of total and essential amino acids were in the following descending order:SCHS>ACHS>NFCS, respectively. The total content of five nucleosides in SCHS (16.82mg/g) was significantly higher than those of NFCS (5.08mg/g) and ACHS (4.45mg/g). The content of D-mannitol in NFCS, SCHS and ACHS was 8.9%,11.5% and 10.2%, respectively.(4) The suitable procedure for extraction exo-polysaccharides is determined as follows: the filtrate is concentrated to 1/6 volume at 70℃, adjusted to pH 7.0, and then precipitated by 3 volume of 95% ethanol. The suitable procedure for extraction intra-polysaccharides is determined as follows:the dried mycelium is added 30 multiple of water, and extracted 150min twice at 100℃. In such suitable procedure for extraction, the water-soluble intra-polysaccharides (IPS) and exo-polysaccharides (EPS) are obtained from the mycelium and filtrate.The active peaks of IPS1 and EPS1 are obtained when IPS and EPS are purified by anion-exchange chromatography on Q-Sepharose FF and gel chromatography on Sephacryl S-300HR, which was proved by Sephacryl S-300HR chromatography. The average molecular weight of IPS is determined to be 42kDa. IPS is composed of mannose, glucose and galactose in molar ratio of 2.8:3.0:1.0,4.5% uronic acid,5.3% protein. The average molecular weight of EPS is determined to be 78kDa. EPS is composed of mannose, glucose and galactose in molar ratio of 4.5:8.01.0,6.0% uronic acid,2.47% protein.(5) The antibacterial effect of EPS was observed that they were most effective against gram-positive bacterium, especially Bacillus subtilis and Micrococcus tetragenus. The MIC of exopolysaccharides to Bacillus subtilis and Micrococcus tetragenus was 1mg/mL and 2.5mg/mL, respectively.The in vitro antioxidant activities are further investigated. The results show that IPS and EPS have effective antioxidant activities in various systems. EPS exhibits strong hydroxyl radical, superoxide radical and hydrogen peroxide scavenging activities; Although IPS can not scavenge hydroxyl radical, it exactly shows similar effects with EPS in other antioxidation systems.The studies of this paper provide a theoretic foundation for further industrialization and clinic application.