Assessing the Safety and Probiotic Characteristics of Lacticaseibacillus rhamnosus X253 via Complete Genome and Phenotype Analysis

Abstract

Lacticaseibacillus rhamnosus is a generalist that can adapt to different ecological niches, serving as a valuable source of probiotics. The genome of L. rhamnosus X253 contains one chromosome and no plasmids, with a size of 2.99 Mb. Both single-copy orthologous gene-based phylogenetic analysis and average nucleotide identity indicated that dairy-derived L. rhamnosus X253 was most closely related to the human-intestine-derived strain L. rhamnosus LOCK908, rather than other dairy strains. The adaptation of L. rhamnosus X253 and the human-intestine-derived strain L. rhamnosus GG to different ecological niches was explained by structural variation analysis and COG annotation. Hemolytic assays, API ZYM assays, and antimicrobial susceptibility tests were performed to validate risk-related sequences such as virulence factors, toxin-encoding genes, and antibiotic-resistance genes in the genomes of L. rhamnosus X253 and GG. The results showed that L. rhamnosus GG was able to use L-fucose, had a higher tolerance to bile salt, and adhered better to CaCo-2 cells. In contrast, L. rhamnosus X253 was capable of utilizing D-lactose, withstood larger quantities of hydrogen peroxide, and possessed excellent antioxidant properties. This study confirmed the safety and probiotic properties of L. rhamnosus X253 via complete genome and phenotype analysis, suggesting its potential as a probiotic.

Keywords: Lacticaseibacillus rhamnosus; adaptation; genome; phenotype analysis; safety and probiotic characteristics.

Figure 1

Comparative genomic analysis of L. rhamnosus X253 with other L. rhamnosus strains: (A) Venn diagram illustrating the number of genes in the core genome and unique genes of the pan-genome of 10 L. rhamnosus strains. (B) Phylogenetic tree constructed on single-copy orthologous genes to reveal the genetic distance of 10 L. rhamnosus strains. (C) Heatmap of ANI analysis of the genomes of 10 L. rhamnosus strains. (D) Genomic comparison between L. rhamnosus X253 and L. rhamnosus GG.

Figure 2

PCoA of the predicted functional capacity of 10 L. rhamnosus strains based on mapping of all orthogroups to the eggNOG database. Distance matrices based on orthogroup abundance profiles were calculated with the R package vegan (version 2.4.4) and visualized with ggplot2 (version 3.3.5). Each letter represents a different functional category, and L. rhamnosus X253, L. rhamnosus GG, and the other eight strains are denoted by red circles, green triangles, and blue squares, respectively.

Figure 3

Hemolytic ability of L. rhamnosus GG and X253: As a positive control, S. aureus ATCC 6538 generated a clearly visible zone of β-hemolytic activity.

Figure 4

Artificial gastric fluid and intestinal fluid tolerance of L. rhamnosus X253 and GG: Survival rate of L. rhamnosus X253 and GG (10⁸ CFU/mL) in artificial gastric fluids at pH 3.0, followed by artificial intestinal fluids at pH 8.0.

Figure 5

Comparative analysis of L. rhamnosus X253 and L. rhamnosus GG for hydrophobicity, auto-aggregation and adhesion to CaCo-2 cells; *: p < 0.05, **: p < 0.01. (A) Hydrophobicity and auto-aggregation. (B) Adhesion to CaCo-2 cells.

Figure 6

Comparative analysis of hydrogen peroxide tolerance and antioxidant capacity of L. rhamnosus X253 and L. rhamnosus GG: (A,B) Growth of L. rhamnosus X253 (A) and L. rhamnosus GG (B) in MRS broth containing different concentrations of hydrogen peroxide. (C,D) Scavenging rates of DPPH (C) and hydroxyl radicals (D) by bacterial suspensions with different densities (10⁴, 10⁶, and 10⁸ CFU/mL) of strains X253 and GG. *: p < 0.05, **: p < 0.01.