Evaluation of Probiotic Properties and Safety of Lactobacillus helveticus LH10 Derived from Vinegar through Comprehensive Analysis of Genotype and Phenotype

Abstract

The probiotic potential of Lactobacillus helveticus LH10, derived from vinegar Pei, a brewing mixture, was assessed through genotype and phenotype analyses. The assembled genome was comprised of 1,810,276 bp and predicted a total of 2044 coding sequences (CDSs). Based on the whole genome sequence analysis, two bacteriocin gene clusters were identified, while no pathogenic genes were detected. In in vitro experiments, L. helveticus LH10 exhibited excellent tolerance to simulated gastrointestinal fluid, a positive hydrophobic interaction with xylene, and good auto-aggregation properties. Additionally, this strain demonstrated varying degrees of resistance to five antibiotics, strong antagonistic activity against four tested pathogens, and no hemolytic activity. Therefore, L. helveticus LH10 holds great promise as a potential probiotic candidate deserving further investigation for its beneficial effects on human health.

Keywords: Lactobacillus helveticus; cereal vinegar; phenotypic analysis; safety and probiotic properties; whole-genome sequencing.

Figure 1

Circular map of the LH10 chromosome and functional annotation of the LH10 genome with different databases. (A) From the outside to the inside, the circles represent the coding genes, gene function annotation results (including the annotation information of COG, KEGG, and GO databases), ncRNA, GC content, and the GC skew value, respectively. (B) KEGG database classification statistics, (C) COG database classification statistics, (D) GO database classification statistics, (E) Phylogenetic tree of L. helveticus LH10 based on 16s rRNA sequences. Approximately 1500 bp were included in the 16S rRNA analysis and 500 bootstrap replications were employed in generating the tree.

Figure 1

Circular map of the LH10 chromosome and functional annotation of the LH10 genome with different databases. (A) From the outside to the inside, the circles represent the coding genes, gene function annotation results (including the annotation information of COG, KEGG, and GO databases), ncRNA, GC content, and the GC skew value, respectively. (B) KEGG database classification statistics, (C) COG database classification statistics, (D) GO database classification statistics, (E) Phylogenetic tree of L. helveticus LH10 based on 16s rRNA sequences. Approximately 1500 bp were included in the 16S rRNA analysis and 500 bootstrap replications were employed in generating the tree.

Figure 1

Circular map of the LH10 chromosome and functional annotation of the LH10 genome with different databases. (A) From the outside to the inside, the circles represent the coding genes, gene function annotation results (including the annotation information of COG, KEGG, and GO databases), ncRNA, GC content, and the GC skew value, respectively. (B) KEGG database classification statistics, (C) COG database classification statistics, (D) GO database classification statistics, (E) Phylogenetic tree of L. helveticus LH10 based on 16s rRNA sequences. Approximately 1500 bp were included in the 16S rRNA analysis and 500 bootstrap replications were employed in generating the tree.

Figure 2

ANI heatmap of 16 Lactobacillus helveticus strains. Each cell represents the OrthoANI values between the row and the corresponding genomes of the column.

Figure 3

Locally collinear blocks between L. helveticus R0052 and L. helveticus LH10. Each contiguously colored region is a locally collinear block (LCB), a region without a rearrangement of homologous backbone sequences. LCBs below a genome’s center line are in the reverse complement orientation relative to the reference genome. Lines between genomes trace each orthologous LCB through every genome. Regions outside blocks lack detectable homology among the input genomes.

Figure 4

Hemolytic activity analysis of L. helveticus LH10 with blood agar plates.

Figure 5

Tolerance of L. helveticus LH10 to gastrointestinal fluids. Simulated food digestion process with simulated gastric fluid treatment for the first 3 h and simulated intestinal fluid treatment for the last 8 h.

Figure 6

Inhibitory effect of Lactobacillus helveticus LH10 against Staphylococcus aureus (a), Salmonella enteritidis (b), Escherichia coli (c), and Bacillus cereus (d). Each bright circle which was marked with arrows represents the corresponding inhibitory zone.

Figure 7

The bacteriocin gene cluster predicted from L. helveticus LH10. (A) The main Enterolysin_A protein-encoding gene clusters, (B) the main helveticin J protein-encoding gene clusters searched for through the BAGEL4 bacteriocin database.