Degradation Mechanism And Safety Evaluation Of Zearalenone Detoxification By Bacillus Velezensis A2

Zearalenone(ZEN)is a secondary metabolite of Fusarium fungi with estrogenic characteristics.The pollution of cereal crops by ZEN is very common,and its residue in cereal produce has done great harm to animals.In the detoxification study of mycotoxin,physical and chemical methods have some limitations concerning cost of production,nutritional value and palatability.In recent years,biological detoxification has become a hot research topic for ZEN decontamination,because it has the advantages of high specificity,high efficiency,and non-toxic metabolites.The aim of this study was to find a strain that can effectively degrade ZEN.The degradation characteristics,degradation products and degradation mechanism of ZEN were discussed,and the potential application value of the strain was evaluated by animal safety test.The main research contents and results are as follows:Experiment ?.Screening and identification of high efficient degrading strains of ZENIn this study,using ZEN as the sole carbon source,nine strains with ZEN degradation rate of more than 50% were screened out from the soil by HPLC detection.The degradation rate of ZEN by A2 strain was up to 100% when the strain A2 was fermented in LB medium for 3 days.Analysis of A2 strain by morphological observation and 16 S r RNA gene sequence alignment.The results showed that strain A2 belonged to Bacillus velezensis.Experiment ?.The detoxification effect of Bacillus velezensis A2 on ZEN in Feed and its preliminary study on the degradation products of ZEN150g ZEN contaminated fodder(ZEN content 60mg/kg),150 m L sterile distilled water and 5m L A2 strain(Culture in LB medium for 24h)were mixed.The treated samples were incubated at 37? for 5 days in an orbital shaker at 150 rpm.The concentration of the ZEN toxin during A2 fermentation was 60?g/m L.No residue of ZEN was detected by HPLC.The results showed that Bacillus velezensis A2 was also suitable for fermentative detoxification of ZEN in feed.The different components of the fermentation broth of A2 strain cultured for 24 h were incubated with ZEN for 48 h respectively,and then the functional substances of A2 strain which can degrade ZEN were localized by HPLC.The A2 strain resuspended by PBS was incubated with ZEN,and the incubation samples were collected at different time intervals.The ZEN degradation product was analyzed by the Agilent 1100 series LC/MSD capture system.The results showed that extracellular solution,intracellular fluid and living bacteria had higher detoxification effect on ZEN,but the inactivated strain had no detoxification effect on ZEN.At the same time,the hydrolytic decarboxylic product of ZEN lactone ring with molecular weight m/z=295.0[292+3H]+ was detected.The preliminary results showed that the active substance secreted by Bacillus velezensis A2 could effectively degrade ZEN,ZEN lactone ring hydrolytic decarboxylic,which was the main mechanism of detoxification.Experiment ?.To evaluate the detoxification effect of Bacillus velezensis A2 on ZEN based on mouse experimentIn this study,the mice(n=40)were randomly assigned to control group(K),ZEN toxin group(ZEN),A2 strain(A2)and detoxification group(A2+ZEN).Use A2 LB-fermentation solution as antidote of ZEN(40mg/kg BW)to manage mice.The test period was 28 days.After the experiment,collect the blood,organs and testes for the corresponding detection.Histopathological analysis showed that arterial feeding of A2 fermentation broth reduced the degree of renal injury induced by ZEN.The results of kit test showed that A2 fermentation broth also decreased the serum levels of creatinine(CRE),uric acid(UA),and blood urea nitrogen(BUN)induced by ZEN.In addition,the A2 fermentation broth also significantly inhibited the increase of malonaldehyde(MDA)content,and reversed the decreases of total superoxide dismutase(T-SOD)and glutathione peroxidase(GSH-Px)activities caused by ZEN.The results of RT-PCR and Western Blot analysis showed that ZEN diet significantly increased the expression of endoplasmic reticulum stress key gene(Bi P),pro-apoptotic gene(JNK,Caspase-12,CHOP)m RNA and protein in the kidney of mice(p<0.05).In addition,the diet of ZEN reduced the expression of m RNA and protein of Bcl-2 and induced the expression of P-JNK and Anti-DDIT3 protein in the kidney of mice.However,oral administration of A2 fermentation broth significantly reversed the abnormal expression of these genes in renal tissue.The results showed that oral Bacillus velezensis A2 fermentation broth could alleviate the oxidative damage and apoptosis of mouse kidney induced by ZEN.Conclusion: Bacillus velezensis A2 screened in this study is an unpublished strain of ZEN degrading bacteria.The mechanism of the degradation of ZEN by Bacillus velezensis A2 is that the active substance secreted by the strain catalyzes the hydrolysis decarboxylation of ZEN lactone ring.At the same time,the oral administration of fermentation broth of A2 strain could relieve the damage of ZEN on the kidney of mice.At the same time,the oral administration of A2 fermentation broth could relieve the damage of ZEN on the kidney of mice.The application of Bacillus velezensis A2 to detoxification of mycotoxin has good development potential.