| Bacillus amyloliquefaciens equipped with strong resistance to stress and abilities of producing metabolites, has the potential to be used as probiotics and has been applied to biological control. Previous studies about bacillus amloliquefaciens were mainly focused on antimicrobial mechanisms of plant pathogens and fungus. With the studying of bacillus amyloliquefaciens, more reports about the inhibitions of animal pathogens and promoting the growth performance of livestock are published. An the other hands, with the advance of experimental technologies, especially the development of omics, researchers can investigate the effects of diseases, environment and nutrients on the animals systematically. The present study were conducted to investigate the stress resistance and antimicrobial mechanism of bacillus amyloliquefaciens, the effects of bacillus amyloliquefaciens on the structure of cecal microflora and metabolites in broilers, which intend to provide the theoretical foundation for the application of bacillus amyloliquefaciens used into livestocks.Trial 1:The stress resistance, adhesive ability and bacteriostatic spectrum of Bacillus amyloliquefaciensThe studies about stress resistance of bacillus amyloliquefaciens were conducted to investigate the influence of heat, simulated gastric fluid and artificial intestinal juice; the gram staining was conducted to study the strains’ adhesion to Caco-2 cells; the Oxford cup test was performed to map the bacteriostatic spectrum. Data showed that bacillus amyloliquefaciens had outstanding stress resistance:1) the survival rate of strain is 57.4% under 85℃; 2) the survival rate of strain is 36% under the treatment of simulated gastric fluid pH 2.0 after 1 h; 3) have a good tolerance for the 0.1% salt bile; 4) the survive rate is 80% under trypsin after 3 h. The number of strains attached to cells is 7.02 ± 0.83 cell-1 after co-cultured with Caco-2 cell for 2 h, which indicated that bacillus amyloliquefaciens has a favourable adhesion to cells. The colony analyzer indicated that the fermentation of bacillus amyloliquefaciens had a spectrum inhibition of Salmonella, Staphylococcus aureus, Escherichia coli and Proteus vulgaris; while had no bacteriostatic effects on Lactobacillus acidophilus, Bifidobacterium and Lactobacillus rhamnosus.Trial 2:The antibacterial mechanism of Bacillus amyloliquefaciensThe crude extraction of lipopeptides obtained by acid precipitation from the fermentation of bacillus amyloliquefaciens had a benign antibacterial effect, which confirmed by the Oxford cup. The scanning electron microscope and transmission electron microscope showed that the membrane of pathogens were broken down by the crude lipopeptides; the antibiotics of aureomycin and neomycinsulphate had the semblable sterilization. High performance liquid chromatography-mass spectrum showed that the main antibacterial components about the stains’ fermentation are Iturin A and Surfactin.Data of the trial of pathogens’ adhesion showed that bacillus amyloliquefaciens prevent the Caco-2 cells from the harmful bacteria by competitive and exclusive inhibition, while no significant result were found both of the expulsive test and the competitive test about fermentation. Flow cytometry showed that both of the strain and fermentation of bacillus amyloliquefaciens didn’t decreased the apoptosis rate of Caco-2 cells infected with Salmonella typhimurium. Transmission electron microscope showed that bacillus amyloliquefaciens didn’t improve the morphology of infected cells, which the membrane was fulling apart and the intracellular bubble emerged. Confocal microscopy images indicated that bacillus amyloliquefaciens didn’t decrease the fluorescence intensity of Ros in the apoptosis cells infected with salmonella typhimurium. Based on the above tests, we hypothesis that bacillus amyloliquefaciens cannot prevent the Caco-2 cells from the infection of salmonella.Trial 3:Effect of Bacillus amyloliquefaciens on the growth performance, small intestinal development, serum biochemical indices and cecal microflora in broilersFive hundred and forty 1-day-old Ross 308 broilers were randomly divided into 3 treatments (control group, antibiotics group and bacillus amyloliquefaciens group),6 replicates for each treatment, and 30 birds per replicate. On d 1,7,14,21,28,35 and 42, birds were killed to collect the samples for further analysis. Data indicated that the dietary bacillus amyloliquefaciens significantly increased the body weight from d 14 to d 42 and average body gain from d 8-14 and d 1-42 (P< 0.05) compared with control birds. Birds fed bacillus amyloliquefaciens had higher relative ileum length on d 28 and the small intestinal relative weight on d 14 and 28 (P< 0.05) than control birds. Bacillus amyloliquefaciens significantly increased the concentrations of serum total protein on d 42 (P< 0.05) and immunoglobulin G and immunoglobulin M on d 7 and d 42 compared with control birds and antibiotic birds. The dietary bacillus amyloliquefaciens significantly increased the diversity of cecal microflora compared with control birds. Compared with control and antibiotics birds, dietary bacillus amyloliquefaciens significantly increased the concentration of butyrate on d 7 and methylacetic on d 21 in the cecal content. On d 42, birds fed with dietary bacillus amyloliquefaciens and antibiotic had higher concentration of acetic acid, methylacetic and butyrate than the control birds (P< 0.05).Trial 4:Effects of Bacillus amyloliquefaciens on the diversity of cecal jnicroflora in broilers through high-throughput sequencingThe present test was conducted to investigate the change of structure and diversity of cecal microflora based on high-throughput sequencing. A total of 3601-d-old broilers Ross 308 were randomly divided into 3 treatments (control group, antibiotics group and bacillus amyloliquefaciens group),6 replicates for each treatment, and 20 birds per replicate. On d 1,7,21 and 42, birds were killed to collect the cecum samples for further analysis, which six birds’cecum were combined into one replicate in case of the individual difference. After detection, the quality of sequencing were calculated, meanwhile the OTUs number of single sample and the diversity of cecal microflora within muti-samples were compared with each other. Results indicated that:1) the OTUs number of antibiotic birds was less than bacillus amyloliquefaciens and control birds (P < 0.05), while no difference was found between later two group; the sequencing depth almost cover all species of samples and satisfy the further analyses.2) distinctive OTUs were found between the birds in different treatments.3) the heatmap about differential birds were clustered with each group at different time points.4) the OTUs rank curves of birds’ cecal microflora were nearly flattened out on d 21, which means of the homogeneous distribution of cecal microflora.5) the broiler’s cecum contained of Firmicutes, Proteobacteria, Bacteroides and Tenericutes in the phylum level; Faecalibacterium, Lactobacillus, Ruminococcus, Bacteroides and Proteus in the genus level.6) the addition of antibiotic significantly decreased the abundance and diversity of cecal microbial in early stage broilers (from d 1 to 21), while no significantly difference in bacillus amyloliquefaciens and control broilers.7) compared with control birds, the dietary bacillus amyloliquefaciens significantly increased the population of predominant bacteria (Oscillosporia 10.2%, Lachnospira 10.9%), and the antibiotic significantly increased the population of Blautia (3.8%) and Lactobacillus (1.2%) on d 7; either the population of Faecalibacterium (16.4%) and Ruminococcus (12.6%) in bacillus amyloliquefaciens birds or the population of Dorea (3.0%) in antibiotic birds was significantly higher on d 21; the population of Faecalibacterium (22.4%) and Bacteroides (3.4%) were significantly higher than control birds, while the population of Ruminococcus (7.1%) was significantly fewer than control birds on d 42.Trial 5:Studies about the effects of antibiotics and Bacillus amyloliquefaciens on the metabolites of plasma and cecal content of broilers in early stageThe aim of present trial was to investigate the effects of antibiotic and bacillus amyloliquefaciens on the metabolites of plasma and cecal content in early stage broilers. On d 1,7,14 and 21, birds were killed to collect the plasma and cecum samples for the further analysis. Results found that:1) the PCA scores showed that individual groups were clustered together at each time point, which means that the addition of antibiotics and probiotics changed the metabolites.2) the distinguishing metabolites were screened out by the PLS-DA and OPLS-DA modeling:on d 21,20 plasma metabolites involved amino acid metabolism were significantly changed by the addition of antibiotics, which the concentration of valine, hydroxyproline, galactonic acid, uric acid, oxalic acid, methyl palmitoleate, monopalmitin et al. were increased; 13 plasma metabolites involved amino acid metabolism and fatty acid metabolism were found in bacillus amyloliquefaciens broilers, which the concentrations of dihydroxy-benzoic acid, a-glucosamine-D-phosphate, aspartic acid, valine, methionine et al. were increased.3) on d 21,9 cecal metabolites involved carbohydrate metabolites and amino acid metabolism were significantly changed by the antibiotics, which only the concentration of maltotriose and glycerol a-monopalmitate were increased; 11 cecal metabolites involved amino acid metabolism and glyceride metabolism were significantly changed by the supplementation of bacillus amyloliquefaciens, which the concentration of N-Acetyl-β-D-mannosamine, gentiobiose, quinate, glycine,3,7, 12-trihydroxycoprostane,5-Hydroxyindole-3-acetic acid et al. were significantly increased. |
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