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This study investigated the differential effects of Bacillus subtilis-and Bacillus licheniformis-fermented products (defined as SFP and LFPs, respectively) on microbial antibiotic resistance gene (ARG) and virulence factor gene (VFG) composition in the cecal digesta of 35-day-old broilers by using metagenomic sequencing. First, 160 1-day-old unsexed Arbor Acres broiler chicks were randomly allocated to four treatment groups: basal diet (CON group); basal diet + enramycin (10 mg/kg, ENM group); basal diet + SFPs (108 colony-forming units (CFU) of B. subtilisspores/kg, SFP group); and basal diet + LFPs (108 CFU of B. licheniformisspores/kg, LFP group). Principal coordinate analysis of ARG and VFG composition indicated distinct clustering among the cecal samples of the groups. At the antibiotic resistance class level, LFP treatment increased the expression of peptide resistance genes and decreased the expression of aminocoumarin resistance genes compared with the other groups. Compared with the other groups, LFP treatment promoted bcrA, ugd, and efrBexpression but suppressed parYexpression, whereas SFP treatment inhibited efrAexpression. The abundance of the peptide resistance gene bcrAin Lachnoclostridiumspecies was higher in the CON and LFP groups than in the ENM and SFP groups, whereas the abundance of the peptide resistance gene rpoB2in Bacteroidesspecies was lower in the ENM and LFP groups than in the SFP group. No specific VFGs were regulated only by SFPs or LFPs. SFP and LFP treatment inhibited clpCexpression compared with the other groups. clpCabundance in Bacteroidesspecies was lower in the LFP group than in the CON group, whereas its abundance in Faecalibacteriumspecies was lower in the SFP group than in the CON and ENM groups. These results demonstrated that SFPs and LFPs differentially regulate microbial ARG and VFG composition in the cecal digesta of broilers. LFP supplementation modulated more antibiotic resistance classes and ARGs than did SFP supplementation.

Soybean meal is the main vegetable protein source in animal feed. Soybean meal contains several anti-nutritional factors, which directly affect digestion and absorption of soy protein, thereby reducing growth performance and value in animals. Fermented soybean meal is rich in probiotics and functional metabolites, which facilitates soybean protein digestion, absorption and utilization in piglets. However, the mixed solid-state fermentation (SSF) conditions of soybean meal remain to be optimized. In this study, we investigated the optimal parameters for SSF of soybean meal by Lactobacillusspecies and Clostridium butyricum. The results showed that two days of fermentation was sufficient to increase the viable count of bacteria, lactic acid levels and degradation of soybean protein in fermented soybean meal at the initial moisture content of 50%. The pH value, lowering sugar content and oligosaccharides in fermented soybean meal, was significantly reduced at the initial moisture content of 50% after two days of fermentation. Furthermore, the exogenous proteases used in combination with probiotics supplementation were further able to enhance the viable count of bacteria, degradation of soybean protein and lactic acid level in the fermented soybean meal. In addition, the pH value and sugar content in fermented soybean meal were considerably reduced in the presence of both proteases and probiotics. Furthermore, the fermented soybean meal also showed antibacterial activity against Staphylococcus aureusand Escherichia coli. These results together suggest that supplementation of both proteases and probiotics in SSF improves the nutritional value of fermented soybean meal and this is suitable as a protein source in animal feed.