Abstract
The probiotic market increases world-wide every year and probiotic interventions improve health and wellbeing. However, the growing scientific and commercial interest on probiotics has not been accompanied by an equal increase in the quality of the products. Thus, definition of detailed guidelines to check the compliance of probiotics with the label claims is crucial. To this extent, the main outcome of this project is the definition of protocols and guidelines for the evaluation of the quality and functionality of probiotics for human consumption. The following quality criteria (Qc) are considered: Qc-1, taxonomy, functionality, and safety of strains; Qc-2, cell viability and core-benefits quantification; Qc-3, functional profiling of the probiotic strain depending on product formulation and production technology; Qc-4, culture-dependent evaluation of probiotic viability and changes in metabolic profiles when administered with foods endowed with their own microbial population.
Results achieved
The PRIN 2022 project “Guidelines and Protocols for Quality and Functional Assessment of Probiotic Formulations (Prob-ID)” was designed to develop standardized methodologies and analytical protocols for evaluating the quality, safety, and functionality of probiotic products intended for human consumption. The rapid expansion of the probiotic market has not been accompanied by equally robust quality-control procedures, leading to concerns regarding strain identification, viable cell counts, functional efficacy, and consistency between product labels and actual composition. The project therefore aimed to establish reliable and reproducible approaches that could support scientific research, industrial quality control, and future regulatory standardization of probiotic products. The project involved four Italian research institutions with complementary expertise in microbiology, genomics, metabolomics, and food biotechnology: the Università Cattolica del Sacro Cuore (project coordinator), the University of Milan, the University of Bologna, and the National Research Council – Institute of Food Production Sciences (CNR-ISPA). Research activities focused on four major quality criteria: (i) taxonomic identification, functionality, and safety of probiotic strains; (ii) quantification of viable cells and evaluation of probiotic “core benefits”; (iii) functional characterization of probiotic formulations according to different production technologies; and (iv) assessment of probiotic functionality in relation to food matrices and simulated gastrointestinal digestion. As a representative model, the consortium selected Lactobacillus acidophilus LA-5®, one of the most widely used probiotic strains worldwide. Four commercial products containing this strain were chosen to represent different formulation types: a multi-strain probiotic supplement, a single-strain lyophilized capsule, a single-strain lyophilized granule supplement, and a multi-strain fermented milk product. The Università Cattolica del Sacro Cuore coordinated the project and performed genomic and taxonomic characterization of LA-5®. Whole Genome Sequencing and bioinformatic analyses confirmed species identity and demonstrated the absence of plasmids, antibiotic resistance genes, and virulence factors. Comparative genome analysis revealed only a single nucleotide polymorphism with no impact on probiotic functions. Six ddPCR assays targeting taxonomic and functional markers were developed and validated, providing reliable tools for strain identification and quality control. The University of Milan evaluated probiotic viability and functional traits by combining culture-dependent methods and flow cytometry. All products contained viable cell levels compatible with recommended probiotic doses. S-layer proteins were detected in all formulations and digestion studies showed that cell-associated S-layer proteins were resistant to gastric digestion, whereas isolated proteins were rapidly degraded. These findings highlighted the importance of cell-associated structures in preserving probiotic functionality. The University of Bologna investigated antimicrobial, antibiofilm, and antiadhesion activities against selected gastrointestinal pathogens. Probiotic-derived supernatants strongly inhibited pathogen growth, biofilm formation, and adhesion to intestinal epithelial cells. Metabolomic profiling by 1H-NMR identified 59 metabolites, and integrated analyses revealed correlations between specific metabolites and biological activities, providing insights into the mechanisms underlying probiotic functionality. CNR-ISPA evaluated probiotic survival and functionality under simulated gastrointestinal conditions using the INFOGEST digestion model and different food matrices. Viable LA-5® cells survived the intestinal phase only in the fermented milk product. Metabolomic analyses revealed formulation- and matrix-dependent differences, particularly between mono-strain and multi-strain products, highlighting the influence of formulation design and food matrix composition on probiotic behavior. Although the project successfully achieved its main objectives, several technical challenges were encountered during implementation. The most significant involved the planned use of the SHIME® dynamic gastrointestinal simulation system. Difficulties in identifying suitable fecal donors, together with instrument availability and procurement issues, prevented completion of the SHIME experiments. Consequently, the consortium relied on the INFOGEST digestion model as the principal experimental platform. Despite this methodological adjustment, the project’s scientific objectives remained fully achievable, and the quality of the results was not compromised. Dissemination and communication activities played a significant role throughout the project. Results were presented at national and international conferences dedicated to probiotics, food microbiology, and microbial biotechnology. To support outreach and stakeholder engagement, the consortium also established a dedicated LinkedIn profile for the project. The project generated substantial scientific output. One manuscript describing the integrated framework for probiotic quality assessment has been published in Probiotics and Antimicrobial Proteins (https://doi.org/10.1007/s12602-026-11100-z). Two additional manuscripts are currently in preparation: one focusing on the correlation between antimicrobial activity and metabolomic signatures, and another investigating the bioavailability and stability of S-layer proteins during gastrointestinal digestion. In conclusion, the Prob-ID project successfully established a comprehensive framework for the quality and functional assessment of probiotic formulations. By integrating genomic, microbiological, functional, and metabolomic approaches, the consortium developed analytical tools and standardized protocols capable of evaluating strain identity, safety, viability, functionality, and metabolic activity. The results provide a valuable foundation for future research, industrial quality-control strategies, and the potential development of internationally recognized standards for probiotic products.Dettagli del progetto
Responsabile scientifico: Beatrice Vitali
Strutture Unibo coinvolte:
Dipartimento di Farmacia e Biotecnologie
Coordinatore:
Università Cattolica del Sacro Cuore(Italy)
Contributo totale Unibo: Euro (EUR) 47.965,00
Durata del progetto in mesi: 24
Data di inizio
12/10/2023
Data di fine:
28/02/2026
