Detoxifacation By Bacillus Licheniformis CK1 Of Zearalenone In Feed For Tibetan Piglets And Its Potential Mechanisms For Detoxification

Zearalenone(ZEA),produced by Fusarium genus,is an estrogenic mycotoxin,which widely presents in foods,feedstuffs and raw materials.Intake of ZEA by humans and animals can lead to hyperestrinism and cause the reproductive disorders.Pigs are the most sensitive animal to ZEA.In animal production,ZEA-contaminated feed can cause harm to the health of animals,and thus bring huge economic losses to the animal husbandry.Compared with the traditional methods of removing ZEA,microbial degradation of ZEA has the advantages of strong specificity,high efficiency,and no pollution to feed and environment.In this study,Bacillus licheniformis CK1 isolated from soil was first shown to have an ability to degrade ZEA.Then,the the degradation of ZEA-contaminated feed by CK1 on piglets were studied.In order to investigate the potential mechanism of ZEA degradation by strain CK1,the gene for degrading ZEA was cloned and expressed successfully,which lays a foundation for application of the enzyme in the industry.The major experiment contents and results are as follows.Experiment 1: The aim of this experiment was to study the ability of strain CK1 to degrade ZEA-contaminated feed.Gram staining showed that strain CK1 was a gram-positive bacterium.16 S rRNA sequencing and phylogenetic tree results showed that the CK1 strain was Bacillus licheniformis.Strain CK1 did not form hemolytic rings on the blood plate,and its genome did not contain the genes of hemolysin and enterotoxin,so CK1 could be used to ferment feed safely.Fourty five mg purified ZEA was fully mixed with the basic diet,fermented by strain CK1 in vitro,and then the concentration of ZEA in the feed was determined by high performance liquid chromatography.The results showed that the strain CK1 had a good ability to degrade ZEA-contaminated feed and the rate of degradation was 61.06%.Experiment 2: The purpose of this experiment was to study whether the strain CK1 could alleviate the toxicity of ZEA in feed to piglets.A total of 18 female weaning Tibetan piglets(average body weight: 6.48 ± 0.74 kg)were randomly divided into three groups and each group had 6 replicates.The experimental period was 21 days.The control group(C)was fed with a basic diet(ZEA-free),treatment group 1(T1)was fed a ZEA-contaminated diet,and treatment group 2(T2)was fed the diet treated with strain CK1.The results showed that there was no significant difference on the performance of piglets among three groups.The vulva size and the relative weight of genital organs of piglets were significantly higher in T1 group than those in control group,while those in T2 group was significantly lower than those in T1 group.The levels of luteinizing hormone(LH),follicle stimulating hormone(FSH),progesterone(P)and serum oestradiol(E2)in T1 group were significantly lower than those in control group,the levels of LH,P and E2 in T2 group were significantly lower than those in control group,and the level of testosterone(T)was not significant difference among three groups.In T1 group,histopathology examination found that the morphology of oocytes was abnormal,the outer membrance of uterus abscised,the inflammatory cell infiltration appeared in liver.These symptoms in T2 group were reduced and the morphology was similar to that of the control group.In T1 group,the expression level of ER? in uterus and ovary and the expression level of ER? in vagina were significantly higher than those in T2 group and control group,while these indicators were not significant difference between T2 group and control group.In vagina,the expression levels of ABCB1 and ABCb4 in T1 group were significantly higher than those in T2 group and control group,and the expression level of ABCG2 was significant difference between T1 and T2 group,while the expression levels of ABCA1 and ABCD3 were not significant difference among three groups.In uterus,the expression levels of ABCA1 and ABCb4 in T1 group were significantly higher than those in T2 group and control group,while the expression levels of ABCB1,ABCD3 and ABCG2 were not significant difference among three groups.In ovary,the expression levels of ABCB1,ABCb4,ABCD3 and ABCG2 in T1 group were higher than those in T2 group and control group.In vagina,uterus and ovary,the expression levels of ABC transporter were not significant difference between T2 and control group(except ABCG2 in vaginal).The results showed that strain CK1 could alleviate the harm of ZEA in feed to piglets.Experiment 3: This experiment was designed to understand the potential mechanism of ZEA degradation by strain CK1.Using bioinformatics to analyze known ZEA degradation enzymes,Prx31 gene for degrading ZEA was successfully amplified from the genome of strain CK1.The fragment size was 564 bp,encoding 187 amino acids.NCBI BLAST analysis revealed that the peroxidase PRX31 in B.licheniformis CK1 belonged to typical 2-Cys Peroxiredoxin.The three-dimensional structure shows that PRX31 is a toroid-shaped complexes.The gene was ligated with the expression vector pET-28a(+)and transformed into E.coli BL21(DE3).An engineered PRX31 expression strain was constructed and heterologous expression of the recombinase was achieved.The purified protein with high specificity was obtained by Ni-affinity chromatography at a concentration of 1.45 mg/mL.The degradation rate of the recombinant peroxidase PRX31 was 68.99%.The optimum temperature,time and pH for ZEA degradation by the recombinant peroxidase PRX31 were 40?,12 h and 8.0,respectively.In addition,ZEA degradation products could significantly reduce the proliferation rate of MCF-7 cells.The results showed that PRX31 was,at least partially,responsible for ZEA degradation by strain CK1.In summary,the strain CK1 could degrade ZEA-contaminated diet,protect the piglets from the toxicity of ZEA and eliminate the spread of ZEA in the food chain completely,which guarantees food security and provides the basis and theory for the application of ZEA biodegradation in feed.In addition,we also studied the molecular mechanism of ZEA degradation by strain CK1,which provides new idea and method for the development of biological enzyme preparation.