Isolation, Identification, and Validation of Strains from Commercial Probiotics: Do We Get What We Expect?

This study evaluated the viability, microbiological composition, functional traits, and safety of probiotic bacteria isolated from commercial products marketed as containing <i>Limosilactobacillus reuteri</i>. Viable cell counts, biochemical characterization, strain-level identification, functional properties, gastrointestinal tolerance, and safety attributes were assessed. Among the evaluated products, only four presented colony-forming units (CFU) counts consistent with label claims (products E, F, G, and H), while two showed no detectable viable microorganisms (products B and L). All isolates were Gram-positive, catalase-negative, and predominantly rod-shaped. rep-PCR analysis revealed strain homogeneity in most products, whereas others (products A and K) exhibited heterogeneous microbial compositions. Molecular identification based on 16S rRNA sequencing showed a predominance of <i>Lmb. reuteri</i> and <i>Lacticaseibacillus rhamnosus</i>, with some products containing additional species such as <i>Lactiplantibacillus plantarum</i> and <i>Lactobacillus acidophilus</i>. Functional assays demonstrated strain-dependent proteolytic and diacetyl-producing capacities, as well as variable tolerance to simulated gastrointestinal conditions. Most strains preferentially produced L-lactate, although some generated substantial amounts of D-lactate. All isolates were susceptible to antibiotics recommended by EFSA, except for intrinsic vancomycin resistance, and no transferable virulence markers, biogenic amine production, or <i>Salmonella</i> contamination were detected. Furthermore, virulence-related genes such as <i>hdc</i>, <i>tdc</i>, <i>odc</i>, <i>hyl</i>, <i>cylA</i>, and <i>ace</i> were not identified. Overall, the results highlight pronounced discrepancies between label claims and microbiological quality among commercial probiotic products and reinforce the importance of strain-level characterization to ensure safety, functional performance, and regulatory compliance.