Impact of formulation and food matrix on probiotic functionality: a multi-analytical study on Lactobacillus acidophilus LA-5®

The increasing use of probiotics in functional foods and dietary supplements highlights the need to better understand how formulation and food matrices influence probiotic performance under human gastrointestinal conditions. In this study, four commercial formulations containing Lactobacillus acidophilus LA-5® were evaluated using a multi-analytical approach combining culture-dependent and culture-independent methods, including plate counting, flow cytometry (FC), whole-genome sequencing (WGS), digital droplet PCR (ddPCR), functional assays, in vitro gastrointestinal digestion, and metabolomic profiling. Flow cytometry revealed that viable cells accounted for 59–88% of total fluorescent units, exceeding culturable counts suggesting the presence of viable but non-culturable populations. Functional assays demonstrated formulation- and dose-dependent antimicrobial activity against selected enteropathogens, with up to 40–50% growth inhibition at higher probiotic cell densities. Simulated gastrointestinal digestion highlighted marked differences in probiotic survival depending on formulation and carrier matrix. Fresh cells and fermented matrices showed improved resistance compared to lyophilized formulations, which were strongly affected by gastric conditions. 1H-NMR metabolomic profiling further revealed matrix-dependent metabolic responses, with distinct signatures observed in apple juice and yogurt systems. These findings demonstrate that probiotic functionality is strongly influenced by formulation and food matrix, highlighting their key role in determining probiotic performance and potential health relevance.