Studies On The Species And Active Compouds Of Cordyceps From The Low Altitude Area In Chongqing

Cordyceps is a genus of fungi in the family of Clavicipitaceae, class of Pyrenomycetes Sphaeriales, phylum of Ascomycotina, and the fungi parasitize in insects forming a complex with their host. Up to date, there are more than 400 Cordyceps species found, and about100 of those were reported in China. At present, Cordycepts sinensis is investigated mostly in China. It is widely used in traditional Chinese medicine and has great potential in medicine and its requiement is rising that resulted in that Cordycepts sinensis was digged excessively by people. So screening and evaluating new Cordyceps to meet the market are necessary. At present, Cordyceps from high altitude areas are investigated mostly while Cordyceps from low altitude areas are less researched in China. In this paper, a new Cordyceps collected from a low altitude area in Chongqing City of China was researched on its taxonomy and active compounds systematically. The main results were described as followed: 1 Research on the taxonomy of the Cordyceps from a low altitude area in Chongqing20 samples of Cordyceps collected from Chenshi town in Chongqing were identified. It was showed that these samples were the same and was a new Cordyceps named as Cordyceps chongqingensis sp. nov..The host insect for Cordyceps is the larvae of Lepidoptera Hepialus, with a size of 40-50×5 mm. Cordyceps superficies is covered with lutea Mycelia, 1-3 stromata fasciculating on its cephal. Its stipes is arcuation of 25-35×2.5-4 mm. Its breeding part is cylindrical, flavovirens, 25-35×5-7 mm in length. Its perithecium is ampuliform and section implanting into the breeding part, measured 811-1210(-1524)×1127-1343μm. The ascus is cylindrical, 5-7.5×170-200μm; with 4 filiate ascospores, 2-3×30-45μm each. This Cordyceps is mostly similar to Cordyecps taii Liang et Liu except its bigger sizes of stiepes, perithecium, ascus and ascospore.The sclerotium and stroma were selected for the anamorph isolation by random sampling. The same strain CQM1T occurrence with high frequence was obtained. CQM1Twas cultured on rich rice medium at room temperature and some stromata like that of the wild Cordyceps could be observed. It is indicated that this isolated strain CQM1T was confirmed to be the anamorph of Cordyceps chongqingensis sp. nov..In this study, both traditional and modern biological techniques were used to determine the anamorph of Cordyceps chongqingensis sp. nov. Based on colony characteristics of CQM1T on different agar media and morphological characteristics under a microscope, the anamorph of Cordyecps was a new species of Metarhizium. In the end, CQM1T was identified as one of the variants of Metarhizium taii from analysis of its 26S rDNA D1/ D2 and ITS regions, designed as Metarhizium taii var. chongqingense nov.Morphological identification of CQM1T: CQM1T was separately cultured on Sabouraud, Czapek or PDA agar media at 25oC. For the first few days, colonies were white and composed of downy mycelium. Jasper conidium crowded together in the center of colonies during the time of conidium producing. There are three modes. The most common mode is conidiophores on mycelium; the less common one is conidiophores on apex of mycelium; and the least common one is conidiophores on primary phialides. Conidia string together. Some are fusiform and others are oblong with constriction in central region, amph-symmetry or mono-thinning. Compared with the known Metarhizium, this anamorph strain has three characeristics:①three ways for producing conidia, of which conidiophores on mycelium is the most common one.②Conidia are multi-shaped with fusiform and oblong conidia in the same string.③The size of conidium is close to that of M.anisopliae var majus although it is shorter. However, it is longer than those of all other Metarhiziums. So the anamorph of Cordyecps is a new species of Metarhizium.Molecular identification of CQM1T:DNA of ITS and 26S rDNA D1/D2 of anamorph were sequenced. The length of 26S rDNA D1/ D2 region is 601 bp while that of ITS (ITS1, 5.8S rDNA, and ITS2) is 553 bp. Two neighbour-joining phylogenetic trees were generated based on the 26S rDNA D1/ D2 and ITS sequences. In the phylogenetic tree generated with 26S rDNA D1/ D2 region, the closest relative to Metarhizium chongqingensis CQM1T was Cordyceps taiiT. And the percentage of sequence max identity is 100% between Metarhizium chongqingensis CQM1Tand Cordyceps taiiT. In the phylogenetic tree with ITS region, the closest relative to Metarhizium chongqingensis CQM1T was Metarhizium taii G97017T. And the percentage of sequence max identity is 98% between Metarhizium chongqingensis CQM1T and Metarhizium taii G97017T. It was reported that Metarhizium taii was the anamorph of Cordyceps taii. As a consequence, the anamorph of Cordyceps chongqingensis sp. nov. was identified as one of the variants of Metarhizium taii from analyses of its 26S rDNA D1/ D2 and ITS regions, designed as Metarhizium taii var. chongqingense nov.2 Research on mycelia culture conditions of CQM1TFirstly, the growth medium of anamorph was researched. Based on Czapek medium, the effects of carbon sources and nitrogen sources on dry weight of mycelia were studied. It was resulted that the micrmolecule carbon sources of Sucrose and glucose surpass the polycose dextrin and corn flour and the glucose was the best carbon source. In the end, Sucrose was selected for the carbon source with its lower price, and its optimal content was 3%. In the selection of organic nitrogen source, fish flour can produce maximum mycelium, and soyben cake followed that. But they have more nearly effects on producing mycelium. So soyben cake was selected for organic nitrogen source for reducing produce cost, and its optimal content was 0.5%. Considering on the results of above, the compounding of growth medium was NaNO3 0.2%,K2HPO4 0.1%,KCl 0.05%,MgSO4.7H2O 0.05%,FeSO4 0.001%,sucrose 3.0%,silkworm chrysalis flour 0.5%,soybean flour 0.5%. Subsequently, the Effects of different growth temperatures, medium pH and rotation speed of shaking bed on dry weight of mycelia were studied by orthogonal tests, the best conditions included: temperature 28℃, pH 5.0, rotation speed 180 r/min. Under these optimal conditions, the maximum of dry weight of mycelia was obtained at 7th day that was 35.6 g/L.3 Screening on fermentation conditions of active compoundFirstly, the fermentation medium of anamorph was researched. Based on the growth medium, the effects of different abio-salts NaNO3 , K2HPO4 , KCl ,MgSO4.7H2O,FeSO4 on antimicrobial activity were studied, respectively. It was resulted that antimicrobial activity depressed without NaNO3 and KCl while antimicrobial activity rised without K2HPO4. FeSO4 and MgSO4.7H2O have little effects on activity. Subsequently, effects of different C:N on antimicrobial activity were studied. It was showed that the optimal C:N is 30:1 and all the tests using silkworm flour had higher active than that using soybean flour. Above all, the compounding of fermentation medium was NaNO3 0.05%,KCl 0.05%,sugar 3.0%,silkworm chrysalis flour 0.05%. In the end, the effects of fermentation temperature, medium pH and rotation speed of shaking bed on antimicrobial activity were studied by orthogonal tests. And the optimal fermentation conditions included: temperature 25℃, pH6.5, rotation speed 200r/min. Under these conditions, the maximum of antimicrobial activity was obtained at 6thday and the antimicrobial ring was 30.4mm.4 Isolation, purification and structure identification of active compound The technical paths and technological methods were achieved for the isolation, purification and preparation of bio-active compound A and B through researches of decolorization with macroporous resin, sedimentation with acetone, column chromatography, extraction with n-butanol and preparation with HPLC. Compound A and B were confirmed oligosaccharides by some analysis of their physico-chemical properties and spectrum data (UV,IR,EI-MS,1H-NMR,13C-NMR) primarily. And their structures must be confirmed exactly with more analysis of more data. It will offer some scientific proof on the application of Cordyceps chongqingensis sp. nov..5 Analysis of the other active components of CQM1T cultureFive main active components of the mycelia and broth of CQM1T were determined. And the same time, the content of the five components were compared with that of the other Cordyceps. It was showed that the contents of Cordyceps acid (mannitol), Cordyceps polysaccharides, amino acids and protein of the CQM1T were higher than that of the other Cordyceps (Cordyceps sinensis North has only slightly higher crude protein content than that of Cordyceps chongqingensis sp.nov.). In particular, the contents of Cordyceps acid and Cordyceps polysaccharide were significantly higher than that of the other Cordyceps. But the total amount of amino acids of CQM1T was very close to that of the others. All with 17 amino acids, of which glutamate had the highest content; essential amino acids were rich, accounting for 36.45% of the total amino acids. Therefore, CQM1T can fully replace Cordyceps sinensis and can be cultured by man-made based on its active components only.Above all, Cordyceps collected from the low altitude area in Chongqing was identified. It was showed that the Cordyceps was a new Cordyceps named as Cordyceps chongqingensis sp. nov.. The anamorph of Cordyceps chongqingensis sp. nov. was isolated and identified. It was identified as one of the variants of Metarhizium taii, designed as Metarhizium taii var. chongqingense nov. It was showed that Cordyceps chongqingensis sp.nov. have significant superiority over the Cordyceps sinensis:①moderate growth conditions ( its optimal growth temperature is 28℃while Cordyceps sinensis grows at 18-20℃), growing faster (its growth cycle is 7days while Cordyceps sinensis grows for 1 month), easy to industrial culture.②not only having higher ratio of active components than that of the other Cordyceps, but also producing high bio-activity compounds to repress both G+ and G- bacteria. In this paper, two active compounds were prepared and their chemical structures were analized as oligosaccharides. And these studies can promote Cordyceps chongqingensis sp.nov. to serve people.