Screening Of Aflatoxin B₁ Degrading Strain And Purification Of Its Active Enzyme

This study isolated bacterial strain of highly active AFB₁ degradation from the soil that combine the 16S rDNA sequence comparison and physiological and biochemical experiments authenticate bacterial strain species,optimize the bacterial strains'growth condition,analysis and purify bacterial strains'main degrading active substances,study the main factors affecting the degradation efficiency of active,and evaluate the safety and effectiveness of the actual action of bacterial liquid and enzyme liquid to provide basis for its actual production application.The main results showed as follows:1)In this study,a highly active samples were screened from soil and sewage using a newly coumarin-anhydrous ethanol medium by gradually increase coumarin concentration in medium and was named A6.The bacteria were identified as pseudomonas aeruginosa by comparative analysis of 16S rDNA sequence and physiological and biochemical identification results.2)Based on the results of single factor experiments of liquid volume,temperature,initial pH and inoculum,optimizing the bacterial growth conditions by orthogonal test.The growth culture conditions were as follows:fermentation temperature was 37°C,initial pH was 7.0,inoculum amount was 8%,loaded liquid was 30mL/100mL.Compared with before optimization,AFB₁ degradation rate increased to 85.69%.3)The main active ingredient of A6 which acts to degrade AFB₁ is a thermostable extracellular enzyme with a molecular weight of 20 kD.The extraction process is:65%ammonium sulfate precipitation,gradient ultrafiltration was performed successively by using30 kD?10 kD?3 kD ultrafiltration tubes,gel chromatography by using Sephadex G-50.This method can purify the protein 71 times.4)The reaction condition of enzyme solution is:degradation time>120 h,the degradation rate of AFB₁ increased to 90.47%;the optimal pH value of 7.0,enzyme solution has the degradation ability in the pH value of 5.0⁸.0;suitable substrate concentrations from100?g/L to 1000?g/L;temperature range is 37°C-70°C,which can maintain 71%degradation rate;Zn²⁺,Mg²⁺and Fe³⁺are major inhibitors of enzyme activity.5)Compared with the AFB₁ standard?86.91%?,the inhibition rate of AFB₁biotransformed extract on HUVEC cells decreased to 28.79%after 72 h,and the cell inhibition rate of the enzyme solution was 15.58%,which had little toxic effect on cells.6)The A6 fermentation broth and enzyme solution did not significantly inhibit the growth of Aspergillus flavus7)The enzyme solution and fermentation broth can also exert high degradation ability in the food matrix.After 5 days of incubation,the enzyme solution degraded 90.48%of AFB₁ in peanut solids.At the same time,the optimal ratio of solid to water in the bacterial liquid is1:1.5,which can degrade 55.34%of AFB₁ after 5 days of incubation.