| Lepista sordida is a kind of delicious edible fungi which owns higher diatery and medicinal value. Though it can be cultivated artificially, the technology is not developed and lower yield prevented this precious mushroom from commercial production. Polysaccharides are the best known and most potent mushroom-derived substances with antitumor and immunomodulating properties. The data on mushroom polysaccharides were summarized for approximately700species of higher basidiomycetes. In contrast, the research on L. sordida polysaccharides has just begun. In the present thesis, high production cultivating pattern of L. sordida was explored and high yield formula was found, which provided theory basis for cultivating commercially. In the meantime, the extracting technique of L. sordida polysaccharides was studied and the optimal extraction parameter was obtained. Then polysaccharides were extracted and fractioned from the fruiting bodies of wild L. sordida, and the preliminary analysis of the polysaccharides structure was made. The contents are as follows:Firstly, the ecology of wild Lepista spp. growing in the Shuanghe farm of Qiqihaer city, Jingpo-lake natural park and Sandaoguan natural park of Mudanjiang city, which are major production areas of Lepista spp. in Heilongjiang province, was investigated and sporocarps were collected. Subsequently, S1(Qiqihaer), M1(Sandaoguan) and J1(Jingpo-lake) strains were isolated. Based on the initial identification by morphological characteristics of the three unknown strains, the sequence alignment was made and a phylogenetic tree was established with15Lepista spp. strains and one Trametes spp. strain coming from Genebank by using the internal transcribed spacer (ITS) region. The result showed that S1was nearer to L, sordida in genetic distance and clustered together with them, whereas M1and J1were nearer to L. nuda and clustered together with them. So, the strain S1was L. sordida, and the other two strains were L. nuda.Secondly, comparasion test of bionics wild cultivation and greenhouse cultivation of L. sordida were carried out. The result showed that the budding time was the same after covering the soil, and the temperature of fruiting was near to each other. The fruiting bodies were thick, strong and had no sweet smell in greenhouse. But bionics wild sporocarps were thin and similar to wild ones in appearance, and some had special fragrant odor. Comparing with greenhouse cultivation, the bionics wild cultivation appeared higher yield and its biological efficiency was36.8%, yet the former is only17.38%. In the meantime, the effect of formulation on L. sordida mycelia growth and output was investigated. The data showed that the hypha growing rate is positively correlated with the addition of corncobs. The mycelia grew fast in the formula6with the maximum supplementation of corncobs (3.95mm/d), however, the formula3(sawdust48%, corncob32%, wheat bran18%, gypsum powder1%, lime dust1%) appeared the highest biological efficiency,28.4%.Thirdly, the strain of artificial L. sordida was investigated for carbon and nitrogen nutrition by ergosterol contents and the production of polysaccharide in liquid media. The result showed that fructose, mannose, cellobiose, sucrose, glucose and maltose can be used as a carbon source for L. sordida strain by analyzing ergosterol contents. And the former three carbon materials were better for mycelia growth in contrast with sucrose, glucose and maltose. The highest polysaccharide yield could be obtained with fructose as a carbon source. Alanine, glycine, soybean powder and peptone can be used as a nitrogen source. Alanine is best for mycelia growth and bean powder can produce the highest polysaccharide.Forthly, the polysaccharide was extracted from wild L. sordida sporocarps by using hot water extraction method. Effects of four single factors-, different extraction temperature, the ratio of raw material and water, different extraction time, and extraction times, on the yield of polysaccharide were determined. And then, one orthogonal test was conducted so as to obtain the optimal extraction technique by evaluating the yield. The influence of three factors on extraction efficiency decreased in the following order:reflux temperature>material to water ratio> extraction time. Based on2extraction times, the optimal extraction technique for polysaccharides from wild L. sordida sporocarps was extraction temperature65℃, material to water ratio1:40(g/mL) and extraction time was five hours. And under these conditions, the extraction rate of wild L. sordida sporocarp polysaccharides could reach10.39%, and RSD is only1.45%. So, this technique is stable and reliable.Fifthly the polysaccharide was extracted from artificial L. sordida mycelia by using hot water extraction method. Effects of four single factors-, different extraction temperature, the ratio of raw material and water, different extraction time, and extraction times, on the yield of polysaccharide were determined. And then, one orthogonal test was conducted so as to obtain the optimal extraction technique by evaluating the yield. The influence of three factors on extraction efficiency decreased in the following order:reflux temperature> extraction time>. material to water ratio. Based on2extraction times, the optimal extraction technique for polysaccharides from L. sordida mycelia was extraction temperature95℃, material to water ratio1:40(g/mL) and extraction time was three hours. Under these conditions, the extraction rate of artificial L. sordida mycelia polysaccharides could reach9.41%, and RSD is only1.33%. So, this technique is stable and reliable.Sixthly, the crude polysaccharides were obatained from the fruiting bodies of wild L. sordida by hot water extraction method. The different ethanol concentrations were used step-by-step(30%â†'60%â†'80%) to precipitate carbohydrates and got three extracting samples of LSP-30, LSP-60and LSP-80. Their total sugar contents were64.0%,41.4%and17.2% respectively. LSP-30was composed of glucose (Glu), mannose (Man) and galactose (Gal), their contents were23.0%,49.2%and27.8%. LSP-30was then purified on a column of Sephrose CL-6B, resulting in producting two purified polysaccharides (LSP-30-a and LSP-30-b) and their average molecular weight were approximately600KDa and lOKDa. LSP-60was mainly composed of Glc, Man and Gal, and their contents were31.6%,46.4%and22.0%respectively. The molecular weight range of LSP-60was wide and not well-distributed, approximately1KDa. LSP-80was mainly composed of Glc, Man, Gal, and Rha (rhamnose), and their contents were17.4%,68.0%,11.6%and3.0%respectively. LSP-80was then purified on a column of Bio-Gel P-2, resulting in producting three purified carbohydrates (LSP-80-a, LSP-80-b and LSP-80-c) and their average molecular weight were approximately1KDa,700Da and400Da. LSP-80-a was mainly composed of Glc, Man and Gal in a ratio of44.9%,34.4%and20.7%, LSP-80-b was mainly composed of Glc, Man, Gal and Rha in a ratio of20.5%,60.3%,16.4%and2.8%. And LSP-80-c was mainly composed of Glc, Man, Gal and Rha with a ratio of12.2%,50.7%,10.1%and27.0%respectively. |
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