Molecular Identification Of Uncaria Sinensis And Purification Of Metabolites From SGM

This thesis study comprises two parts of contents:1. Molecular identification of Uncaria sinensis;2.The related study of antibacterial composition from SGM.SGM was the recombinant integrated by Serratia Marcescens and Rhodotorula glutinis through parents protoplast fusion protoplast technology. The crude extraction of Serratia Marcescens showed good antibacterial activity in our previous study. In the present study, the SGM strain was identified by 16 S rRNA method. The metabolites of SGM was separated and identified by purification and structure identification method. The mainly research work was followed:1. Molecular identification of Uncaria sinensis. ITS of the Uncaria sinensis rDNA was amplified by universal primers ITS4/ITS5. The amplified sequence of ITS was 99.8% similar to U. sinensis FJ980386.2. Molecular identification of SGM strain. Internal transcribed spacer(ITS) of the SGM rDNA was amplified by universal primers ITS4/ITS5 and 27f/1492 r. The amplified sequence of ITS was 100% similar to Rhodotorula glutinis meanwhile and the amplified 16 S rRNA sequence was 96% similar to Serratia Marcescens.3. Optimization of the culture condition of SGM. The effect of carbon source, nitrogen source, culture temperature and time on the biomass and antibacterial activity was studied. The result showed the optimized condition followed as: sucrose 1.5%, beef extract 0.8%,,culture 60 h at 30℃. The biomass of SGM strain reached 62 g/L with inhibition zone diameter 15.7mm under the aforementioned condition.4. To assay the antibacterial activity and stability of the SGM extract. The screened microorganism was Bacillus subtilis with the concentration of 5x105 cuf/m L and 30μL dosage. The relationship between logarithmic of relative concentration of SGM extracts and inhibition zone diameter was agreed with the classic function equation of antibiotics. The SGM extracts from suspension was relative stable under 30-121℃, 15-75 min and 2-40 times of frozen-thaw cycles.5. Identification of the structure of the antibacterial substances. Four compounds were identified as Adenosine, Dibutyl phthalate(DBP), Ergosterol, Ergosterol peroxide by UV, MS, NMR spectra purified on gel silica column chromatography and sephadex LH20 column chromatography, based on the assay of antibacterial activities of different extraction fractions. The minimum inhibitory concentration(MIC) of DBP against Bacillus subtilis was 10mg/mL, while DBP showed no inhibitory effect on Escherichia Coli and Pseudomonas aeruginosa. Adenosine and Ergosterol showed no inhibitory effect on Bacillus subtilis, Escherichia Coli and Pseudomonas aeruginosa. The MIC of Ergosterol peroxide against Bacillus subtilis and Escherichia Coli were 2.5 mg/m L, 2.5 mg/m L respectively.