Isolation And Identification Of Compounds From Fermented Mycelia Of Hirsutella Sinensis

Cordyceps sinensis, one of the most valued traditional Chinese medicine, means "winter-worm and summer-grass" in China. C. sinensis is commonly used to replenish the kidney, soothe the lung, boost and maintain overall body health; other treatments such as for fatigue, night sweating, hyperglycemia, renal dysfunction and arrhythmias also have been claimed. Due to the unique medicinal value, the natural shortage and increasing demand, mycelia fermentation of C. sinensis fungal species is a feasible and sustainable means for producing the medicinal fungus and its bioactive compounds. On the basis of morphological and molecular biology evidence, Hirsutella sinensis is currently considered as the correct anamorph of C. sinensis.This paper is focusing on the mycelia fermentation from H. sinensis. The fermented mycelia were washed3times by pure water and subjected to a dry oven at60℃. Then the dried mycelia were ground into powder (approximately60mesh) and stored at4℃for further researches.Firstly, the total protein content of H. sinensis fermented mycelia was determined by Kjeldahl determination. The total protein in fermented mycelia and waste were383.4mg/g and10.804mg/mL respectively. The hydrolysates of protein were filtered by amino acid analyzer, results show that total amino acid content in fermented mycelia was306.28mg/g and histidine, arginine, glutamic acid contents were40.93,36.13,28.25mg/g respectively. Cordyceps acid was detected by UV detector at412nm, the content in fermented mycelia was10.99%. The optimized extraction conditions were:extracted in ultrapure water (m/v,l/30) on40℃for1hour by3times, the highest cordyceps acid content was11.37%on these conditions. Further more, uridine, guanosine, adenine, adenosine and cordycepin contents were0.123mg/g,0.308mg/g,0.551mg/g,0.823mg/g and0.308mg/g severally. Trace minerals were determined using a flame atomic absorption spectrophotometer. Na, K, Mg, Ca, Fe, Zn, Cu, Mn, Si, Ni, Pb, Co, Hg were all detected, however the highest content were Na (1582.9mg/g) and K (9247mg/g) respectively.In addition, we used Soxhlet extraction method to extract crude fat from H. sinensis fermented mycelia, and then the volatile components were detected by gas chromatography-mass spectrometry method. The crude fat content in fermented mycelia was5.66%. The volatile components mainly included oleic acid, linoleic acid and hexadecanoic acid. Two compounds EP-1and EP-2were obtained by extraction and purification. Their structures were determined by MS,1H NMR,13C NMR and IR, EP-1was hexadecanoic acid and EP-2was ethyl oleate.Finally, water-soluble polysaccharides were extracted from mycelia of H. sinensis by hot water. By using the phenol-sulfuric acid assay, the total content of polysaccharides was determined to be3.848%. The polysaccharides were deproteinized with Sevag reagent followed by ethanol precipitation, then purified by ion-exchange column (DEAE-Sephadex-25) and gel-filtration column (Sephacryl S-100HR), giving two polysaccharide fractions termed HSP-1and HSP-2. The components of HSP-1and HSP-2, identified by GC-MS, were D-mannose, D-glucose and D-galactose, with different molar ratios of0.99:0.61:1and0.12:0.71:1respectively. The molecular weights of them were3.5×103kDa and3.4×103kDa respectively. The NMR and IR spectrums show that monosaccharides of HSP-1and HSP-2were mainly P-pyranoses, however HSP-1had small number of a-pyranose.