Genomic Insights into the Probiotic Functionality and Safety of Lactiplantibacillus pentosus Strain TBRC 20328 for Future Food Innovation

<i>Lactiplantibacillus</i> species have been historically used for food applications. Although several species are regarded as safe according to their regulatory status, the safety issues and functional roles of these lactic acid bacteria have been given attention. A selected <i>Lactiplantibacillus</i> strain TBRC 20328, with probiotic properties isolated from fermented Isan-style pork sausage (Mam), was evaluated for its safety through whole-genome sequencing and analysis using integrative bioinformatics tools. The metabolic genes were assessed through comparative genome analysis among <i>Lactiplantibacillus</i> species. The genome of the strain TBRC 20328 consisted of one circular chromosome (3.49 Mb) and five plasmids (totaling 0.25 Mb), encoding 3056 and 284 protein-coding genes, respectively. It exhibited an average nucleotide identity (ANI) with other <i>Lactiplantibacillus pentosus</i> strains of over 95%. Whole-genome analysis confirmed the absence of virulence and antimicrobial resistance genes, supporting its safety for food applications. Functional annotation revealed clusters for bacteriocins (plantaricin EF and pediocin) and polyketides, indicating potential roles in biopreservation and host interactions. Genes involved in the biosynthesis of some short-chain fatty acids and exopolysaccharides were also identified. Comparative genomic analysis across 33 other <i>Lactiplantibacillus</i> strains identified 2380 orthogroups, with 94 unique to the <i>Lp. pentosus</i> group. These included gene clusters involved in malonate decarboxylation, leucine biosynthesis, and 5-oxoprolinase activity. Such distinct genomic features emphasize the sustainable biotechnological potential and safety of <i>Lp. pentosus</i> TBRC 23028. Together, the findings highlight its promise as a safe and functional probiotic candidate with broad applications in functional food development and precision fermentation technologies.