Detection And Application Of Water-soluble Active Substances In Bt Bacteria Fermentation Broth

At present,Zwittermicin A?ZwA?is an antibiotic with both antibacterial and antibacterial functions.Although its chemical structure and certain chemical properties have been elucidated,the separation and purification of this substance has not been completely solved.With the wide application of Bt preparations,the application potential of ZwA in synergistic activity is also expanding.The way to get the ZwA high-purity standard and the detection of its content in the sample are especially important.In order to broaden and realize the greater application of ZwA in agric?lture,the support of ZwA quantitative method is urgently needed.The purpose of this study is to solve the above bottleneck problem.Process for purifying samples with a purity of 84%?91%.The ZwA purification process:?1?The fermentation broth is concentrated,alcohol-deposited,and centrifuged to obtain a crude extract;?2?extracted with absolute ethanol;?3?purified using a 500D dialysis bag,and dialyzed after repeated dialysis;?4?using a solid phase extraction col?mn for elution,collecting 15%methanol eluate fraction;?5?using high-performance liquid-phase hydrophilic col?mn HILIC ternary mobile phase elution,double-effect bacteriocin will be at 3.1?3.2min.A sample with a purity of 84%to 91%is finally obtained.Established a high performance liquid chromatography method for the determination of ZwA in Bt fermentation broth.The composition of Bt fermentation broth is complex,High-performance liquid phase detection of ZwA,selection of high performance liquid chromatography col?mns and appropriate elution methods are key.In this experiment,HILIC col?mn?4.6×250mm;3?m?was used,with 1.0mL/min acetonitrile-methanol?25:75?as mobile phase and 15min acetonitrile-methanol-water?25:72:3?as mobile phase.35?,UV detection wavelength 210nm.The res?lts showed that the R² was 0.9986 in the linear range of0?100.0?g/mL,and the recoveries of ZwA in the addition level of 32.78?g/mL were 93.2%?97.5%,and the RSD was 1.28%.The precision of this method was good.It can be used to detect the content of ZwA in various fermentation broths.A method for quantitatively detecting the ZwA content in Bt fermentation broth by spectrophotometry was established.Method:According to the structure of ZwA?NH₂CONH₂?,it can react with diacetyl-anthracene under acidic conditions to form a red substance has a special absorbance at 480nm.Res?lts:optimal parameters for the establishment method were 300 mL/L of phosphoric acid,1000?g/mL of diacetyl-hydrazine,15 min of water in a 75? water bath,and cooling at room temperature in the dark,using a spectrophotometer.The absorbance was measured at 480nm.When the ZwA content is in the range of 0?200?g/mL,the regression equation of the standard curve is y=0.0114x+0.07082,R²=0.99182,indicating that the correlation is good,and the experiment is not interfered by other substances,and the specificity is strong.Biological activity of ZwA and thiazoline on root-knot nematode after compounding.The relationship between the concentration of thiazosin and the inhibition of root-knot nematode was determined by using root-knot nematode as test insect.The inhibitory effect of different addition amounts of ZwA and thiazolyl on root-knot nematode was determined.The res?lts showed that under the addition of 0.2mg synergistic ability of 30%concentration of thiazophos to nematodes reached 2.711 times.