Sustainable valorization of pomegranate leaf by-products through their functional interaction with Saccharomyces boulardii: an in vitro proof-of-concept study

Introduction Next-generation biotics are redefining microbiota-targeted strategies by combining beneficial microorganisms with bioactive plant-derived substrates. Pomegranate ( Punica granatum L.) leaves, an abundant agri-food by-product, represent a sustainable source of polyphenols with antioxidant, antimicrobial, and potential prebiotic and microbiota-modulating properties. This exploratory in vitro proof-of-concept study aimed to preliminarily characterize a pomegranate leaf extract (PLE), assess its antibacterial activity, evaluate its association with Saccharomyces boulardii growth and viability, and characterize the resulting yeast-derived fractions and heat-inactivated biomass. Methods A pomegranate leaf extract (PLE) was obtained by microwave-assisted extraction using a hydroethanolic solvent. PLE was preliminarily characterized using Folin–Ciocalteu, aluminum chloride, DPPH, and FTIR analyses. PLE was tested against selected Gram-positive and Gram-negative bacteria, including Staphylococcus aureus , Escherichia coli , Salmonella enterica , Yersinia enterocolitica , Aeromonas veronii , and Aeromonas hydrophila . S. boulardii growth and viability were evaluated under glucose-free conditions in the presence of different PLE concentrations. Intracellular and extracellular fractions obtained following yeast–polyphenol interaction, together with heat-inactivated yeast cells, were analyzed for polyphenol content, antioxidant capacity, and soluble protein levels. Results Microwave-assisted extraction yielded an extract with high Folin-reactive estimate and DPPH radical-scavenging capacity that exhibited antibacterial effects against selected pathogenic bacterial strains. PLE increased viable/culturable S. boulardii counts in a dose- and time-dependent manner under glucose-free conditions, with the highest response observed at 5 mg/mL. Yeast–polyphenol interaction was associated with changes in polyphenol content, antioxidant capacity, and soluble protein levels in intracellular and extracellular fractions. Heat-inactivated yeast cells retained detectable bioactive components, suggesting potential advantages in terms of technological stability. Discussion Collectively, these findings support a condition-dependent in vitro interaction between PLE and S. boulardii and provide an exploratory proof-of-concept for further mechanistic and host-relevant investigation. The present data do not establish validated prebiotic, synbiotic, postbiotic, or parabiotic products. Further chromatographic, mechanistic, safety, stability, gastrointestinal, and in vivo studies are required.