To date, a total of 13 probiotic Bacillus species are considered as a Generally Recognized as Safe organism (GRAS) approved by the US Federal Food, Drug, and Cosmetic Act (FDCA), which are used for food and feed additives. However, Bacillus velezensis is not considered as a probiotic candidate in swine farming due to a lack of genetic basis of probiotic action-related traits. Therefore, the present study was undertaken to exploit the genetic basis underlying the probiotic traits of B. velezensis VTX9. The genome sequencing of B. velezensis VTX9 was performed on a PacBio Sequel platform. The probiotic properties including biosafety, antioxidative capacity, and riboflavin and exopolysaccharide production were evaluated by using genotypic and phenotypic analysis. The secondary metabolite potentials were also predicted. Strain VTX9 isolated from swine feces proved some probiotic properties including resistance to 3 mM H2O2, 0.6 mM bile salt, low pH, and antipathogenic activity. The complete genome of B. velezensis VTX9 consists of a 3,985,800 bp chromosome that housed 3736 protein-coding genes and 5 plasmids with the size ranging from 7261 to 20,007 bp. Genome analysis revealed no functional genes encoding enterotoxins and transferable antibiotic resistance, which confirmed the safety of VTX9. A total of 82 genes involved in gastrointestinal stress tolerance were predicted, which has not been reported previously. The maximum production of riboflavin reached 769 ± 7.5 ng/ml in LB medium after 72 h, which was in agreement with the complete de novo riboflavin biosynthetic pathway exploited for the first time in the B. velezensis genome. Antagonistic activity against pathogenic bacteria was attributed to 10 secondary metabolites clusters. The presence of a large gene cluster involved in biosynthesis of exopolysaccharides underscored further the adhesion and biofilm-forming capabilities of VTX9 in swine intestines. Our results revealed for the first time that B. velezensis VTX9 has the potential to be a probiotic candidate. The information provided here on the genome of B. velezensis VTX9 opens new opportunities for using B. velezensis as a feed additive for swine farming in the future.

中文翻译：

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支持益生菌作用的基因的表型特征和分析揭示了贝氏芽孢杆菌 VTX9 作为猪潜在饲料添加剂的潜在前景

迄今为止，共有 13 种益生菌芽孢杆菌属被美国联邦食品、药品和化妆品法案 (FDCA) 批准为公认的安全生物体 (GRAS)，用于食品和饲料添加剂。然而，由于缺乏益生菌作用相关性状的遗传基础，贝氏芽孢杆菌不被认为是养猪业中的益生菌候选物。因此，本研究旨在利用 B. velezensis VTX9 益生菌特性的遗传基础。B. velezensis VTX9 的基因组测序在 PacBio Sequel 平台上进行。通过使用基因型和表型分析评估了益生菌特性，包括生物安全性、抗氧化能力以及核黄素和胞外多糖的产生。还预测了次生代谢物的潜力。从猪粪便中分离的菌株 VTX9 证明了一些益生菌特性，包括对 3 mM H2O2、0.6 mM 胆汁盐的抗性、低 pH 值和抗病原体活性。B. velezensis VTX9 的完整基因组由一个 3,985,800 bp 的染色体组成，其中包含 3736 个蛋白质编码基因和 5 个大小从 7261 到 20,007 bp 的质粒。基因组分析显示没有编码肠毒素和可转移抗生素抗性的功能基因，这证实了 VTX9 的安全性。总共预测了 82 个参与胃肠道应激耐受的基因，这是以前未报道过的。72 小时后，LB 培养基中核黄素的最大产量达到 769 ± 7.5 ng/ml，这与首次在 B. velezensis 基因组中开发的完整的从头核黄素生物合成途径一致。对病原菌的拮抗活性归因于 10 个次级代谢物簇。参与胞外多糖生物合成的大基因簇的存在进一步强调了 VTX9 在猪肠道中的粘附和生物膜形成能力。我们的结果首次表明 B. velezensis VTX9 有可能成为益生菌候选物。此处提供的关于 B. velezensis VTX9 基因组的信息为将来使用 B. velezensis 作为养猪饲料添加剂开辟了新的机会。velezensis VTX9 有可能成为益生菌候选者。此处提供的关于 B. velezensis VTX9 基因组的信息为将来使用 B. velezensis 作为养猪饲料添加剂开辟了新的机会。velezensis VTX9 有可能成为益生菌候选者。此处提供的关于 B. velezensis VTX9 基因组的信息为将来使用 B. velezensis 作为养猪饲料添加剂开辟了新的机会。