Optimization Of Fermentation Conditions Of Zearalenone-degrading Strain H6 And Transcriptome Sequencing Analysis

Zearalenone is the most common mycotoxins found in corn,wheat,cereal foods and their derivatives.A large number of studies have confirmed that zearalenone can directly infect crops such as wheat and corn,or contaminated animal foods such as meat and milk,into the human or animal body,posing a great threat to human and animal health Bacillus amyloliquefaciens is a probiotic that is widespread in nature.At present,research on Bacillus amyloliquefaciens in various countries in the world mainly focuses on biological control,production of industrial enzymes,and maltogenic amylase the action of related metabolites.Many researchers at home and abroad have successfully screened zearalenone-degrading bacteria,but few people studied the metabolic pathways and degrading genes of the degrading bacteria.In this experiment,the single-factor test and orthogonal test were used to optimize the composition of the fermentation medium and the culture conditions of Bacillus amyloliquefaciens H6,a highly efficient zearalenone that had been selected in the previous period,in order to select the best fermentation conditions and provide a reference for the later industrial production of Bacillus amyloliquefaciens.The zearalenone-treated bacillus amyloliquefaciens was sequenced using the transcriptome sequencing technology.By comparing with the untreated group,the differentially expressed genes between the two groups were screened,and their functions and pathways were enriched for analysis,Trying to find genes related to toxin degradation,providing ideas for follow-up research.Study 1:Optimization of fermentation conditions of Bacillus amyloliquefaciens H6The glycerol bacteria kept in the laboratory were purified,and the strains were identified by 16 S r RNA gene sequencing analysis.After the identification result was correct by BLAST,the composition of the fermentation medium of the strain and the fermentation conditions were optimized by single factor method and orthogonal experiment method.The main test results were as follows:1.The results of rrna gene sequencing showed that the homology of H6 with HYM 25(accession no.KT961125.1)reached 99% The strain was identified as Bacillus amyloliquefaciens,which met the requirements of the experiment.2.The best carbon source for B.amyloliquefaciens H6 was soluble starch,and the best nitrogen source was yeast dipping powder.The best inorganic salt was MgSO4.The results of orthogonal experiment showed that the optimum composition of H6 fermentation medium was: soluble starch 3%,yeast extract 2%,MgSO4 0.4%.The concentration of Yeast powder had the greatest influence on the growth of H6,followed by MgSO4 and soluble starch.3.Optimal growth conditions for strain H6: The initial p H is 6;the temperature is 37°C;the speed is 210 r/min;the bottle volume is 50 m L/250 m L.Study 2: Transcriptome sequencing analysis of B.amyloliquefaciens H6The strain H6,which can efficiently degrade zearalenone,was identified in Experiment.The fermentation broth of 200 ml/500 ml was inoculated in 6 bottles with 6% inoculation volume,and the average was divided into two groups,three bottles in each group.After 24 h was cultured,1000 ug/m L of zearalenone 200 u L was added to the experimental group.The same amount of methanol was added to the control group.after co culture 10 h,12000 r/min centrifugation 6min was used to collect the bacteria and extract RNA,and then construct transcriptome library.The libraries were sequenced using the Illumina Hi Seq 2500 platform.After filtering the original sequencing data,the clean reads were mapped to the reference genome,and proceeding the Go and KEGG enrichment analysis of differentially expressed genes,trying to looking for the genes that related to degrading toxins.The results of this study are as followed:1.Six Bacillus amyloliquefaciens transcriptome libraries were successfully constructed,and a total of 49257626 Reads were obtained by sequencing.The clean reads ratio for each sample was above 96%,the Q20 value was above 97%,the Q30 value was above 94%,and the GC content was around 50%.Through the data filtering,mapping to reference genome and the correlation analysis between the samples showed that the overall quality of RNA-Seq was high-quality,which reached the requirement of further analysis.2.Through analysis of the differences between groups,a total of 77 differential genes were screened out,16 up-regulated and 61 down-regulated compared to the control group.The GO and KEGG enrichment analysis of the differential genes revealed that the differential genes were annotated to 109 GO terms.There are 70 notes in the molecular function Term;32 in the biological process(BP)functional term;and 7 in the cellular component term.In the results of the KEGG enrichment pathway,77 differential genes were annotated to KEGG pathway metabolism,genetic information processing,environmental information processing,cellular processes,and human diseases in 5 first-order tiers,especially biosynthesis of type II polyketide substances,Carbapenem biosynthesis pathway.3.Most of the genes that may be screened for toxin degradation are related to basal metabolism and sporulation.Among them,BAMF RS30105,BAMF RS22430,BAMF RS33370 and other genes related to basal metabolism were down-regulated;Genes related to sporulation,such as BAMF RS21465,BAMF RS40385,BAMF RS40385,and BAMF RS28605,were up-regulated.Furthermore,we found BAMF RS30125(hypothetical protein)is rewarding of further study.