Screening And Identification Of Carbosulfan-degrading Microorganism In Traditional Fermented Food

Carbosulfan is a commonly used carbamate pesticide because it is widely used in fruits and vegetables,and there are pesticide residues.Screening carbosulfan's degrading microorganism can be used to reduce pesticide residues in foods and improve food safety.In this study,soybean meal(Douchi)was used as the separation material,and the degrading microorganism were domesticated and enriched by gradually increasing the concentration of carbosulfan in the liquid medium,and then the enriched liquid broth was separated by slab scribing,determined the degrading rate by high performance liquid chromatography.14 degradation strains with high degradation rate were screened.Four strains of Carbosulfan degrading microorganism with high degradation rate were identified by PCR sequencing and sequence alignment.JJ-1 was identified as Pseudomonas putida,JJ-4 was identified as Pichia.pastoris,JJ-5 was identified as P.Fluorescens,and JJ-8 was identified as Penicillium citrinum.The paper studied the influencing factors of the degradation of the four strains.Compared with the self-degradation rate of carbosulfan,it was found that the degradation rate of strain JJ-4 to carbosulfan at pH 5-9 exceeded its own hydrolysis rate.When pH=8,the degradation rate of strain JJ-4 was 50.87%.At this time,the hydrolysis rate of carbosulfan was only 5.29%,and the degradation rate of strain JJ-4 was observed at ambient temperature of 25??40?.There is 2 to 3 times the hydrolysis rate of carbosulfan.When sucrose is used as a carbon source,the degradation effect of JJ-4 is also slightly improved.The optimal pH of strain JJ-4 was 8,and the optimum temperature was 35?.The best degradation effect was obtained when sucrose was used as an external carbon source.The first-order kinetic equation was fitted to Ct=36.189e0.1009d(R2=0.9912)at pH=7 and 30?.At this time,the carbosulfan had a half-life of 3.20d.Further research on the carbosulfan degrading enzyme of JJ-4(Pichia.pastoris)was carried out.In the high concentration group(50mg/L),the degradation rate of the crude enzyme solution reached 86.59%and the immobilized enzyme reached 92.89%.In the low concentration group(5mg/L),the degradation efficiency of the crude enzyme solution was 99.49%,and the immobilized enzyme could reach 86.24%.The experimental results showed that the enzyme of Pichia pastoris JJ-4 could degrade the carbosulfan to a safe limit of 0.01 mg/L or less at 3 h.In the in vitro simulated digestion experiment,the control group did not hydrolyze the carbofuran after the rapid hydrolysis of carbosulfan into carbofuran in the gastric environment simulation.The experimental group added with the immobilized enzyme was able to hydrolyze carbofuran while rapidly hydrolyzing the carbosulfan within 2.5 hours.The results of GC-MS analysis showed that the strain JJ-4(Pichia.pastoris)could hydrolyze the carbosulfan and carbofuran to form a low toxicity and non-toxic compound.