Comprehensive in vitro probiotic screening and ITS-based molecular identification and characterization of yeast strains isolated from agro-industrial and rumen sources

Probiotic yeasts, particularly Saccharomyces cerevisiae, are receiving increasing attention for their health-promoting effects. However, identifying safe and efficient strains from diverse and underexplored ecological niches remains challenging. This study addressed this gap by isolating and characterizing yeast strains from agro-industrial byproducts and rumen-derived sources, followed by a comprehensive evaluation of their probiotic attributes and safety profiles. Fifty-four yeast isolates were subjected to a multi-step in vitro screening workflow, including morphological assessment, safety evaluation (hemolytic, gelatinase, and DNase activities), gastrointestinal tolerance tests, cell surface characterization (hydrophobicity, auto-aggregation, co-aggregation), antimicrobial activity, antioxidant capacity, and antibiotic susceptibility. Molecular identification of selected strains was performed using ITS sequencing and phylogenetic analysis. A total of ten strains passed the primary safety and functional criteria. Among them, four top-performing isolates showed exceptional probiotic potential, exhibiting gastrointestinal survival rates > 80%, cell surface hydrophobicity > 80%, and co-aggregation with Escherichia coli ATCC 25922 > 70%. These isolates also produced antimicrobial inhibition zones > 12 mm and demonstrated DPPH radical scavenging activity > 80%. After excluding three antibiotic-resistant strains, nine final candidates were confirmed by PCR, comprising four S. cerevisiae and five Pichia fermentans strains. Yeasts isolated from non-conventional agro-industrial and rumen-derived sources demonstrated strong in vitro probiotic properties and fulfilled essential safety requirements. These findings highlight their potential application as functional ingredients in food and feed formulations. Nevertheless, in vivo studies are required to validate their efficacy and safety in host systems.