Studies On Technics Of Submerged Culture And Extraction & Purification Of Polysaccharide For Lasiosphaera Fenzlii Reich

Puffball, belonging to Agaricomycetidae, Agaricales, Lycoperdaceae, is a big-type fungus, which included Lasiosphaera Fenzlii Reieh., Calvatia gigantea (Batsch : pers.)Lloyd, Calvatia lilacina(Mont. et Berk.)Lloyd, and so on. It is a rare edible and medicinal mushroom which has great development value and broad application prospects. Modern scientific research has proved that puffball polysaccharide has a distinctive pharmacological effect.This study was focusing on submerged culture, extraction and purification of puffball polysaccharide. With the mycelia biomass as index, it is suitable for puffball submerged culture by screening the ingredient of culture medium and culture condition. The research of extraction and purification for the polysaccharide was made. And by adding the traditional Chinese medicine into medium so as to study the effect on mycelia biomass of puffball submerged culture.Carbon source tests showed that the corn flour was the best carbon for producing mycelia biomass in all the tested carbon sources. Nitrogen source tests showed that the bean powder is superior to the other nitrogen sources. Inorganic salts and growth factors tests showed KH2PO4, MgSO4 can promote the yield of mycelia biomass. The optimal medium was determined was corn flour 2.0%, powder of bean 1.0%, KH2PO4 0.1%,MgSO4 0.1% through the orthogonal test. Response-plane of Carbon source and Nitrogen source was: z=1.7950x+1.1967y-0.1967x2-0.1267y2-0.1850xy-2.9722 z: mycelia biomass, x: Carbon source, y: Nitrogen sourceThe culture condition factors including temperature, initial pH value, loading capacity of culture medium, rotating speed, inoculating amount and culture duration, the results showed that the optimal culture conditions were culture temperature 26℃, initial pH 6.7, rotating speed 130 rpm, the culture duration 96 hours, the inoculating amount 15%, containing 100 ml culture medium in the 250 ml Erlenmeyer flasks.The extraction condition of puffball polysaccharide was combination with time, temperature, times, and solvent proportion. The optimal extraction condition was determined that the best of extraction condition were 1.5 hours, 75℃, 3 times, and solvent proportion 1:22. The experiment, isolating protein from polysaccharide by Sevag, was made, which is orthogonal test of three factors and three levels. The result was shown that optimal conditions of isolating protein were what volume ratio of chloroform to butanol in Sevag solution is 10:3, volume ratio of sample to Sevag solution is 1:1, and surge time is 15 min.In this study, the firstly puffball polysaccharide was purified with macroporous resin and DEAE-cellulose, after the primary separation, the two polysaccharide substances, R-A and R-B, were obtained. For the further separation, basing on the amount of molecular, R-A and R-B were puritied by Sephadex G-100, obtained R-A1 was 92.80%, R-B1 was 96.31%. So as to we know the puffball polysaccharide was purified and classification well by Sephadex G-100.By adding different types and different concentrations of traditional Chinese medicine into media, so as to study the growrh condition of Lasiosphaera fenzlii Reich. It is well-combined our traditional Chinese medicine with the mushroom and it also provide a theoretical basis for its further study in depth.