Introduction Probiotic lactic acid bacteria derived from agro-food sources play a pivotal role in promoting human health and advancing functional food development, however the genomic determinants underlying their adaptive versatility and probiotic functionality remain insufficiently characterized. Methodology The current study employs a comparative genomics framework to evaluate the genetic diversity, metabolic plasticity, and evolutionary trajectories of Lactiplantibacillus plantarum (HMX2 and NMGL2) and Pediococcus acidilactici (BCB1H), aiming to elucidate the molecular determinants and functional mechanisms governing their probiotic efficacy. Various genomic analysis, including whole genome sequencing, genome annotation, average nucleotide identity (ANI), and Pan Genome analysis, was performed to assess the mechanism of adaptations and genomic variations. Results NMGL2 exhibited the largest genome of 3.46 Mb among these strains, while having a total of 3,402 genes together with plasmids, which enhanced metabolic flexibility. A total of 59 genes were found which are likely linked with the functions of probiotic-related traits such as adhesion, immune modulation, and stress response. Although 59 genes were identified to be linked to probiotic characteristics like adhesion, immunomodulation, and stress resistance, the identified genomic features reflect the potential functionality and do not necessarily result in phenotypes. Discussion The effectiveness of a probiotic is assessed based on more than just the availability of these genes; their regulation and expression are crucial. L. plantarum HMX2 and NMGL2 are found to be the same species but differ slightly at the genetic level, as indicated by the ANI between the two bacteria, which stands at 99.80%. On the other hand, the ANI between P. acidilactici BCB1H and L. plantarum is only around 68%, which is way less than 95% and thus indicates that they are not of different species. This shows that the two genera diverged at an early stage in their evolutionary path. Through Pan-Genome analysis, gene clusters were identified for varying levels of adaptability. The genetic features of L. plantarum NMGL2 suggest potential adaptability for industrial and probiotic use, though these predictions require experimental validation. The combined studies on transcriptomics and metablomics are required for the validation of functional potential in the genomics of these studies.
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