| Objective: Improve the method of isolate SW from Rhizoctonia leguminicola fermentation broth;Study the bacteriostasis in vitro of fermentation broth extract from different polar solvent, widen the use scope of Rhizoctonia leguminicola fermentation broth,improve its utilization value;Study the effect to chicken's immunologic function of 3.2871A Rhizoctonia leguminicola fermentation broth。Method:1.Use the potato dextrose agar(PDA) to preserve regularly and graft the Rhizoctonia leguminicola ,fermented culture use the improved Czapek's medium,temperature 25℃,Thermostate culture 14d,collect the fermentation broth and pretreat.Use the method of Reduced pressure concentration,stage extraction,D101macroporous resin adsorption,Silica gel column chromatography,freeze drying,Decompression sublimation etc. to isolate SW from fermentation broth,use the thin layer chromatography to detect timely,use the thin-layer chromatography and gas chromatograph to identify and analyse the isolate product.2.Use the Bauer test and tube dilution method,choose common representative bacteria of GN and GP to test the bacteriostasis of fermentation broth extract from different polar solvent in vitro qualitatively,then on the basis of the results of qualitative bacteriostatic experiment to do the quantitative bacteriostatic experiment,test the MIC of each abstract.3.Study the effect to chicken's immunologic function of Rhizoctonia leguminicola,inject the Rhizoctonia leguminicola fermentation broth (the content of SW were 1,2,4 mg·kg-1) before and after the chikens was vaccinated Newcastle disease vaccine three days ,through test the chikens' antibodytiter,Immune organs index,Peripheral leukocyte count and Spleen lymphocyte proliferation to confirm the using dosage and time.Result:1.The growth state of each Rhizoctonia leguminicola that was preserved regularly and grafted was good;isolated the white powder from the 3.2871A Rhizoctonia leguminicola fermentation broth, and identified that there was the SW in it through the thin-layer chromatography and gas chromatograph.2.Four part extraction from fermentation broth have the different bacteriostatic efficacy.the bacteriostatic efficacy of the extraction from the n-butanol parts was obvious,Reinecke's salt extraction also had the good bacteriostatic efficacy,because of the low concentration of the SW Standard liquids the bacteriostatic efficacy didn't display obviously.Through the further tests we found that the MIC of the extraction from the n-butanol parts to escherichia coli was 50mg/mL,to staphylococcus aureus,Listeria monocytogenes were all 25mg/mL,to the rest two kinds of bacteria was 12.5mg/mL;the MIC of Reinecke's salt extraction to Streptococcus agalactiae,Bacillus cereus were all 50mg/mL,to the rest three kinds of bacteria was 25mg/mL.3.Inject the the Rhizoctonia leguminicola fermentation broth(the content of SW was 2 mg·kg-1) after the chickens was vaccinated Newcastle disease vaccine three days,use it as a kind of adjuvant,could improve the chickens' specific serum antibody titer obviously,the number of peripheral blood was increase,has the definite facilitation effect to the upgrowth of chickens' immune organs;when the Rhizoctonia leguminicola secondary metabolite(the content of SW was 2 mg·kg-1)stimulate the lymphocyte independently or synergeticly, lymphocyte has the obvious proliferation.Conclusion:Improved the method of isolate SW from 3.2871A Rhizoctonia leguminicola fermentation broth,confirmed that there was SW through the thin-layer chromatography and gas chromatograph,but didn't obtain SW puregrad,the experiment need the further study and improvment;four part extraction from fermentation broth have the different bacteriostatic efficacy,the bacteriostatic efficacy of the extraction from the n-butanol parts was most obvious,illustrated that SW had the definite bacteriostatic efficacy;Use the regular dose of Rhizoctonia leguminicola fermentation broth can improve the chickens' humoral immunity and cellular immunity function. |
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