Genomic Characterization and Probiotic Potency of Bacillus sp. DU-106, a Highly Effective Producer of L-Lactic Acid Isolated From Fermented Yogurt

Abstract

Bacillus sp. DU-106, a newly isolated member of Bacillus cereus group, exhibits the predominant ability to produce L-lactic acid. The probiotic potency of test strain revealed its survivability at acidic pH, bile salts and viability in simulated gastric juice in vitro. The acute oral toxicity test indicated its no toxicity to laboratory mice in vivo. We further determined the complete genome of strain DU-106 to understand genetic basis as a potential probiotic. It has a circular chromosome and three plasmids for a total genome 5,758,208 bp in size with a G + C content of 35.10%. Genes associated with lactate synthesis were found in the DU-106 genome. We also annotated various stress-related, bile salt resistance, and adhesion-related domains in this strain, which likely provide support in exerting probiotic action by enabling adhesion to host epithelial cells and survival under gastrointestinal tract. Moreover, strain DU-106 genome lacks the virulence genes encodes cereulide synthetase, enterotoxin FM, and cytotoxin K. These phenotypic and genomic probiotic potencies facilitate its potential candidate as probiotic starter in food industry.

Keywords: Bacillus sp. DU-106; complete genome sequence; lactic acid; probiotics; virulence gene.

FIGURE 1

Colonial morphology (a), Gram stain image (b), and phylogenetic analysis (c) of strain DU-106. Strain DU-106 is grew on MRS plate after 48 h cultivation at 37°C. Neighbor-joining phylogenetic tree is based on the 16S rRNA gene sequences of strain DU-106 and representative strains from GenBank. Significance of each branch is indicated by a bootstrap value calculated for 1000 replicates. Numbers at branching points are bootstrap values >50%. GenBank accession numbers are given in parentheses. Bar, 0.005 substitutions per nucleotide position.

FIGURE 2

High performance liquid chromatography (HPLC) chromatograms of fermentation culture (A) of strain DU-106 after 48 h fermentation at 37°C in 200 mL MRS broth. Time course of lactic acid production and pH (B) during the fermentation by DU-106 in MRS medium at 37°C. The pH was declined from initial 5.58 to 3.35 after 72 h fermentation at 37°C.

FIGURE 3

The potential probiotic properties of Bacillus sp. DU-106 in vitro. Survival rates of Bacillus sp. DU-106 in different pH (A) and the simulated gastric fluid, 0.3% bile salt, and simulated intestinal fluid (B) after 2 h treatment.

FIGURE 4

Toxicological evaluation of Bacillus sp. DU-106 in vivo with laboratory mice. Mean body weights (A) and D-lactic acid levels of blood (B) of BALB/c mice after continuous gavage for 7 days (3.0 × 10⁸ cfu d⁻¹). Mean body weights of male and female KM mice during acute oral toxicity (C).

FIGURE 5

Circular representation (A) and features of the genome of Bacillus sp. DU-106 (B). From the outer to inner circle: (1) predicted protein-coding sequences (CDSs); (2) predicted CDSs related to COG categories; (3) predicted CDSs related to KEGG categories; (4) predicted CDSs related to GO categories; (5) tRNA, rRNA and ncRNA distribution; (6) GC content; and (7) GC skew.

FIGURE 6

Comparative genome analyses of Bacillus sp. DU-106 and other Bacillus strains. Venn diagram of the genome comparison of Bacillus sp. DU-106 with other B. cereus strains (A) Venn diagram displays the orthologous genes between B. cereus ATCC 14579, B. thuringiensis ATCC 10792, B. toyonensis BCT-7112, and B. mycoides ATCC 6462. The maximum likelihood phylogenetic tree was constructed with PhyML based on SNP differences across the whole genome (B) Bootstrap support values were calculated from 100 replicates.