Multi-Analytical Approach for the Quality and Functional Evaluation of Probiotic Formulations: The Case of...

The increasing use of probiotics in dietary supplements and functional foods highlights the need for robust analytical strategies to assess microbial viability, functional performance, and safety. Conventional culture-based methods may underestimate probiotic potential, particularly in multi-strain formulations. In this study, a multi-analytical framework was applied to evaluate four commercial formulations containing Lactobacillus acidophilus LA-5^®, differing in composition and delivery technology. A combination of culture-dependent and culture-independent approaches was employed, including plate counting, flow cytometry (FC), whole-genome sequencing (WGS), digital droplet PCR (ddPCR), antimicrobial activity assays, in vitro gastrointestinal digestion, and metabolomic profiling. WGS confirmed the taxonomic identity of all isolates as L. acidophilus and the absence of genes of concern. FC revealed that viable cells accounted for 59-88% of total fluorescent units, exceeding culturable counts and indicating the presence of viable but non-culturable populations. ddPCR enabled strain-level quantification of taxonomic and selected functional genes. Functional assays demonstrated formulation- and dose-dependent antimicrobial activity against selected enteropathogens, with up to 40-50% growth inhibition at higher probiotic cell densities. In vitro digestion experiments showed marked differences in gastrointestinal resistance, with lyophilized products undergoing reductions of up to 5-6 log units during the gastric phase, whereas fresh cells and fermented matrices retained viable populations after intestinal digestion. ¹H-NMR-based metabolomic analysis of post-digestion samples further revealed formulation-dependent metabolic fingerprints. This integrated approach provides a biologically relevant framework linking probiotic viability, functionality, and metabolic behavior, supporting improved quality assessment of commercial products and highlighting the role of formulation and matrix effects in determining probiotic performance.