Ganoderma Isolation, Identification And Culture Study

This article elaborately studied the identification, analyzing the ITS sequence of Ganoderma applanatum, separating, purification and structural analyzing of polysaccharide from the fruiting body of G applanatum from the City of NanJing, JiangSu Province and its cultured mycelia were studied by means of tissue isolation and culture, which include the result showed as below:1. G applanatum was discovered widely by our investigation in Nanjing, Jiangsu. The fruiting body applied in this study was collected from the lower trunk of camphor in Ginling college, Nanjing normal university. The strain was sepreated, identified and cultivated with tissue separating. The fruiting body, mycelia and basidiospores were observed by scanning electron microscope for providing intuitionistic evidence to identify G applanatum as medicinal materials.2. The internal transcribed spacer (including ITS1, ITS2 and 5.8S rDNA gene) of nuclear ribosomal DNA from the fruiting body and mycelia of G applanatum were amplified by means of PCR technology. The internal transcribed spacer(ITS) of Ganoderma applanatum fruiting body and mycelia were determined. T, C, A and G were 30.2%, 22.8%, 21.7% and 25.3% respectively in these sequences. The comparability between the sequence determined and the sequence in GenBank(access number is AJ608709) is 99.81% by aligment. This indicated the isolated mycelia must be G applanatum. Phylogenetic relationship species among Ganoderma was analyzed elementary based on ITS sequences. It lay foundation for the application of G applanatum from wild source.3. The optimum culture medium of G applanatum fruiting body was 78% cottonseed shell, 20% wheat bran, 1% sucrose, 1% gesso, 60%—65% of water. The optimum liquid fermentation culture medium of G applanatum was 9% molasses and 0.4% peptone and optimum culture conditions of G applanatum was investigated by the orthogonal experiment in three factors and four levels, the optimum temperature was 34℃ and the optimum rotation rate was 115 rpm.4. Polysaccharide was obtained from the fruiting body of Ganodermaapplanatum by extraction with hot water and precipitated by alcohol. The protein in it was removed by sevage method(chloroform:isoamyl alcohol was 3:1). The best ratio of hot water and fruiting body was 5: Land the extraction effect for adding water time after time was better than only adding water once under the condition of same volume water. To protect the activity of polysaccharide, We choosed to extract polysaccharide under 70°C for 2.5 hours once. The monosaccharide compositions of Ganoderma applanatum polysaccharide by means of TLC analysis were xylose, mannose, glucose and et al.