| Inonotus obliquus, also known as "Chaga", the European people like to call it "Chaga". It belongs to basidiomycotina, mushrooms, Aphyllophorales, thorn leather bacteria families, which was a kind of mystery medicinal fungus from the virgin forest of Siberia. The Inonotus obliquus parasitizes on the birch, and it is extremely cold resistant strains that can even survive under the environment of minus69℃. Its growth period extends to15years. The adult bacteria of wild Inonotus obliquus will blot birchen essence and cause trees death. So its natural resource is of great shortage, and its price in the international market is much higher than that of Phellinus igniarius which is also one kind of precious medicinal fungi. Therefore, artificial cultivation of fruiting body of Inonotus obliquus is very necessary. Liquid culture is an effective way to increase production of Inonotus obliquus. Then it can offer sufficient raw materials for the research and development of Inonotus obliquus. In recent years, people are constantly strengthening the breadth and depth study on liquid culture and mycelium foundation training to shorten the growth cycle and improve its yield. At the same time, with the intensive study of active substance like polysaccharides of medicinal fungi, polysaccharides as main component of Inonotus obliquus, its nutritional value and medicinal value gradually causes people’s attention. So it becomes hot developing field in pharmaceutical industry and health products industry.This paper focus on the research of chemical structure and biological immune activity of polysaccharides which were extracted from the fruiting body and production of a submerged culture with the control medium optimized by the Response Surface Optimization (RSM). The total sugar and protein content of all polysaccharides were determined, including fractions subjected to DEAE-52and sephadex G-200column chromatography. The immune activities were stated by how much the cytokines TNF-α, IFN-γ,1L-1β,1L-2had secreted after the cells were stimulated by polysaccharides fractions. The GC chromatography was utilized to analyze monosaccharide composition. IR and NMR method was used to make clear the glycosidic bond and anomeric carbon configuration.The experimental results showed:1) The total sugar content of EPS and IPS was higer than that of FSPS, while the protein level was much lower. The deproteinization of EPS, IPS and FSPS were termed as DEPS, DIPS, DFSPS, respectively. The sugar content of DIPS was80.9%, which was the highest among them. DEPS1, DEPS2, DEPS3, DEPS4, DIPS1, DIPS2, DIPS3, DFSPSlwere fractions of DEPS, DIPS, DFSPS that had been subjected to DEAE-52column chromatography. DIPS1owned the highest total sugar content of86.1%. The protein content of all the samples from FSPS was higher than20%;2) The fractions of DEAE-52column chromatography were further purified by flowing against to Sephadex G-200, and the collections were named PDEPS1, PDEPS2, PDEPS3, PDEPS4, PDIPS1, PDIPS2, PDIPS3, PDFSPS1, respectivel;3) According to the experiment of cytokines test, all polysaccharide samples can enhance cytokines level secreted by human peripheral blood mononuclear cells (hPBMCs), and the promoting role would be enhanced as the increasing of the concentration. Overall, the activity of crude polysaccharides from fermentation was alike when compared with crude polysaccharides from fruiting body;4) During the cytokine test, there were almost no difference in regulating role among purified fractions of DEPS,DIPS and FSPS at a relatively low concentration. But, when the concentration was high to150μg/mL, the activity of them was significant different and the least effective was mostly PDFSPS1;5) Monosaccharide composition analysis showed that glucose accounted for the largest proportion, the other main components were mannose, galactose and xylose, while the content of galactose was the highest monosaccharide composition of EPS, but there was no much difference in amount of glucose, xylose, galactose, mannose, arabinose and rhamnose;6) IR and NMR were used for structure determination. It suggested that all polysaccharides owned similar structure according to the IR spectrums. The strong and broad peaks at3400cm-1belongs to the stretching vibration absorption peaks of OH. The sharp peak at3000-2800cm-1belongs to stretching vibration absorption peak of C-H. The peaks at1200-1000cm-1belongs to stretching vibration absorption peaks of C-O (including C-O-H and C-O-C). The absorption peaks above are typical peaks of polysaccharides, by which it can be judged whether the sample is polysaccharides or not. From the IR spectrums, it could be found that most polysaccharides had peak at1650-1550Ocm-1which belonged to variable Angle vibration of N-H chemical bond. This suggests that polysaccharides are mostly glycoprotein complex. All the absorption peaks from930cm-1to960cm-1state that polysaccharides of the Inonotus obliquus are mostly pyranose. All the absorption peaks at870cm-1or810cm-1express that mannose is one of the main monosaccharide compositions;7) The molecular weight of polysaccharides was measured by size exclusion chromatography with laser light scattering(SEC-LLS). The molecular weight of EPS fractions is between28to58kDa, the weight-average molecular weight of DFSPS1is32kDa, and molecular weight of IPS fractions is between89to120kDa. Obviously, it can be concluded the fermentation polysaccharides is of higher molecular weight, the molecular weight of each IPS fraction is approximate100kDa;From all the results above, there is no remarkable difference in promoting cytokine secretion among all the polysaccharides. But the activity of fermentation polysaccharides was a little better than polysaccharides of fruiting body when they were at the same concentration. In a word, fermentation polysaccharides of Inonotus nexpandable fermentation production of Inonotus obliquus, and it also provides evidence for its development and application in clinic as a kind of drug. |
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