# Safety and probiotic characterization of Lacticaseibacillus rhamnosus SAL2: insights from integrated genomics and functional validation

## TL;DR

This study shows that Lacticaseibacillus rhamnosus SAL2 is a safe probiotic with strong antioxidant properties, making it a promising candidate for functional foods.

## Contribution

The study introduces a novel integrated genomic and functional approach to probiotic characterization, revealing extracellular metabolites as key mediators of antioxidant activity.

## Key findings

- L. rhamnosus SAL2 has a single circular chromosome with genes primarily involved in carbohydrate metabolism and transport.
- The strain exhibited strong antioxidant activity through both intact cells and cell-free supernatants.
- L. rhamnosus SAL2 matched the safety profile of L. rhamnosus GG but showed superior antioxidant performance.

## Abstract

Integrated genomic-phenotypic frameworks provide a more comprehensive approach to probiotic characterization, resolving longstanding ambiguities in strain safety assessment. Crucially, extracellular metabolites–not cell-bound components–emerge as dominant mediators of bacterial antioxidant activity, thereby refining the mechanistic understanding of microbial reactive oxygen species mitigation.

This study evaluated the probiotic potential and safety of Lacticaseibacillus rhamnosus SAL2 using whole-genome sequencing and functional validation. Hybrid Illumina/PacBio sequencing enabled complete de novo assembly, annotation, and pathogenicity assessment of this independently isolated strain. We benchmarked L. rhamnosus SAL2 against the reference strain L. rhamnosus GG through multidimensional assessment of safety and probiotic characterization. Antioxidant capacity was further evaluated through H2O2 tolerance assays and free radical scavenging tests.

The genome of L. rhamnosus SAL2 comprises a single circular chromosome of 2,989,570 bp with no plasmids. Annotation using the KEGG, GO, COG, CAZy, and TCDB databases revealed that the majority of genes are involved in carbohydrate metabolism, cellular nitrogen compound metabolic process, carbohydrate transport and metabolism, glycoside hydrolases, and primary active transporters, respectively. Virulence factors were primarily limited to immune evasion mechanisms. L. rhamnosus SAL2 lacked hemolytic activity and was susceptible to multiple antibiotic classes. The strain exhibited high viability under simulated human gastrointestinal conditions while displaying strong mucosal adhesion potential. Notably, both intact cells and cell-free fermentation supernatants of L. rhamnosus SAL2 exhibited significant antioxidant activity. When comprehensively benchmarked against the classic probiotic reference strain L. rhamnosus GG, L. rhamnosus SAL2 matched its core safety profile and probiotic properties while demonstrating quantitatively superior antioxidant activity.

Lacticaseibacillus rhamnosus SAL2 showed safety and probiotic characteristics comparable to the reference strain, with enhanced antioxidant performance. These findings highlight its potential as a probiotic candidate for developing microecological preparations or functional foods, particularly due to its exceptional gastrointestinal tolerance, adhesive capacity, and free radical scavenging efficacy.