Screening And Characterization Of Zearalenone Anaerobic Degrading Bacteria

Zearalenone(ZEN),also known as F-2 toxin,is produced by Fusarium sp.and is widely found in moldy wheat,corn and other cereals.ZEN and its derivatives are strongly toxic,and the consumption of ZEN-contaminated agricultural products by humans and animals can lead to organ,reproductive system,immune system diseases and estrogenic syndrome,which seriously affects human and animal health;at the same time,ZEN contamination to agricultural products has also brought huge economic losses to countries around the world.Grain that is lightly contaminated with ZEN is generally used as feed for animal breeding and therefore poses a significant health risk to livestock.The aim of this study was to isolate ZEN anaerobic degrading bacteria,and to study its degradation characteristics and degradation mechanism to provide new ideas for the degradation and detoxification of ZEN in feeds under anaerobic environment.The main results obtained in the study were as follows:1)Screening and systematic identification of ZEN anaerobic degrading bacteria: Ten anaerobic strains capable of degrading ZEN were isolated from fresh pig manure,among which F39 achieved 87.36% degradation rate of 5.0 mg/L ZEN within 48 h.F39 was selected for further study.The single colonies of F39 on GAM plates had an uneven surface,with uneven petal-like and pseudoroot-like edges spreading outward,opaque colonies,white to yellowish,not easy to pick up,with a pungent odor,and positive Gram stain;under the electron microscope,the bacteria are rod-shaped,without flagella,and can produce spores.The 16 S r DNA and rpo B gene sequences of F39 were amplified by PCR and sequenced,then were compared with related genes of other strains in NCBI,which showed high similarity of 99% with Clostridium botulinum and Clostridium sporogenes;determination of the whole genome sequence of F39,in which no gene sequence related to botulinum toxin production was aligned,proving that F39 is not Clostridium botulinum.Therefore,F39 was finally identified as Clostridium sporogenes based on the phylogenetic status analysis of the 16 S r DNA and rpo B gene sequences of the strain and its morphological observations and physiological and biochemical experiments.2)Study the growth characteristics,the ZEN degradation characteristics and the effect of air exposure of ZEN anaerobic degrading bacteria F39: The growth of F39 was measured under different incubation time,temperature,initial p H and carbon source culture conditions.The results showed that F39 could grow well in the range of 27℃-45℃ and initial p H in the range of7.0-11.0.The optimal growth temperature was 37℃,the optimal initial p H was 7.0,and the most suitable carbon source was beef extract.The degradation effect of ZEN by F39 was measured under different conditions(incubation time,temperature,initial p H,medium,and ZEN concentration),and the results showed that the optimal conditions for the degradation of ZEN by F39 were: degradation temperature 37℃,p H 7.0;the best degradation effect was achieved in GAM medium;ZEN could be effectively degraded in the concentration range of 1.0-15.0 mg/L,with relative degradation speed faster at low concentrations of 1.0 mg/L and slower at high concentrations of 15.0 mg/L which the absolute amount of ZEN reduction was the highest.Air exposure had some effect on F39: the bacterium could not grow in aerobic environment,but had some oxygen tolerance,and could continue to grow after a short time(0-3 d)exposure to air and then transferred to anaerobic environment;the microscopic morphology of F39 was not significantly affected by air exposure in a short time(1 d).3)The localization of ZEN-degrading active substances in F39 was determined,the effect of air exposure on F39’s degradation active substances was studied,ZEN degradation products were isolated and identified,and the detoxification effect of F39 on ZEN was evaluated: By measuring the degradation effect of supernatant and cell crushing solution of F39 on ZEN,it was clear that the degradation active substances in F39 were mainly located inside the cells,and the cell crushing solution could degrade 77.30% of ZEN(5 mg/L)within 6 h;after the F39 cell crushing solution was treated with boiling water bath for 20 min,it no longer had degradation effect on ZEN,and it was presumed that the cell’s degradation active substances were enzymes;the degradation of 5 mg/L ZEN using F39 cell crushing solution exposed to air for 1 d was only able to degrade 20.69% for 6 h,indicating that the degrading active substance of the bacterium cannot effectively maintain its degrading activity under aerobic conditions.During the degradation of ZEN by F39,most ZEN was completely degraded,and a very small amount of α-zearalenol(α-ZEL)and β-zearalenol(β-ZEL)were produced;the cytotoxicity of ZEN degradation products produced by F39 were evaluated,and the results showed that the toxicity of ZEN degradation products to human hepatocellular carcinoma cells Hep G2 was significantly reduced compared with ZEN,and there was no significant estrogenic effect on human breast cancer cells MCF-7,thus ZEN is detoxified by the degradation of F39.4)The degradation effect of ZEN-degrading bacteria F39 on ZEN in different feedstuffs was studied and the factors influencing degradation were identified: Six different feed samples were selected and the degradation effect on ZEN in different feeds by F39 was measured.The results showed that F39 was more effective in degrading ZEN in wheat flour with the degradation rate of 48.65% for 5 mg/L ZEN.The degradation rate of ZEN by F39 in all six feedstuffs was lower than that in GAM medium.By adjusting the p H of the feed to the optimum degradation p H7.0 of F39,the degradation rate of ZEN in feed A could be increased from 25.39% to 43.21%,which showed that the p H of the feed had a significant effect on the degradation rate of ZEN.The effects of degradation time and ZEN concentration in the feed on degradation rate were also measured,and the results showed that the ZEN degradation rate of F39 remained stable after 4days in the feed,and the effect of ZEN concentration on its degradation rate has no significant difference in the range of 0.5-5.0 mg/L.