Probiotic Potential and Genome-Based Characterization of Lactiplantibacillus plantarum M2, a Promising Isolate Obtained from Spontaneous Fermentation of Humiria balsamifera Pulp

Abstract

Background/Objectives: The growing demand for functional foods and alternative therapeutic strategies has intensified the search for novel probiotic strains from underexplored ecosystems. This study aimed to isolate and phenotypically characterize lactic acid bacteria (LAB) from spontaneously fermented fruits found in the Legal Amazon (Ananas comosus, Humiria balsamifera, Manilkara zapota, and Platonia insignis) and to perform genome-based analysis of the most promising isolate to evaluate its probiotic potential. Methods: The isolates were identified by MALDI-TOF-MS and screened for tolerance to low pH, bile salts, lysozyme, growth at 39 °C, and antimicrobial activity against five enteric pathogens. The most promising isolate was evaluated by coaggregation and biofilm assays, in silico proteome and CAZyme analysis, bacteriocin cluster mining, and in vivo efficacy testing using Tenebrio molitor larvae. Results: Three isolates from H. balsamifera were identified as Lactiplantibacillus plantarum (M1, M2, M4) by MALDI-TOF-MS. These isolates exhibited high resilience to all tested physiological stressors. Antimicrobial activity was contact-dependent, with no inhibition by cell-free supernatants. M2 showed the strongest pathogen exclusion, moderate biofilm formation, and high coaggregation with S. enterica and E. faecalis. Genome analysis of M2 revealed a 3.40 Mb chromosome, absence of acquired resistance or virulence genes, two plantaricin gene clusters, and 93 CAZymes, including GT families linked to exopolysaccharides biosynthesis. SignalP predicted secretion signals in 10 CAZymes. M2 significantly improved larval survival against E. coli and S. enterica, especially under prophylactic treatment. Conclusions: L. plantarum M2 combines safety, stress tolerance, genomic features, and in vivo efficacy, positioning it as a promising probiotic candidate adapted to tropical niches. These findings highlight H. balsamifera as a reservoir of novel probiotic strains.