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 Limosilactobacillus reuteri. 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 Lmb. reuteri and Lacticaseibacillus rhamnosus, with some products containing additional species such as Lactiplantibacillus plantarum and Lactobacillus acidophilus. 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 Salmonella contamination were detected. Furthermore, virulence-related genes such as hdc, tdc, odc, hyl, cylA, and ace 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.