Isolation and In Vitro Evaluation of Yeasts from Mare’s Milk and Fermented Mare’s Milk for Probiotic Potential

Mare’s milk and fermented mare’s milk are potential sources of yeasts for probiotic screening. This study evaluated the in vitro probiotic potential of 23 yeast isolates from these sources. MALDI-TOF MS preliminarily identified 15 isolates as Kluyveromyces marxianus, four as Pichia manshurica, two as Pichia fermentans, one as Saccharomyces cerevisiae and one as Yarrowia lipolytica. Stress tolerance, solvent affinity, autoaggregation, coaggregation, hemolytic activity, and cell-free supernatant-mediated antibacterial activity were assessed. Relative viable counts, calculated against corresponding controls at the same 4-h endpoint, averaged below 6% under 0.3% bile salt exposure and below 1% at pH 2. Responses at pH 3 and elevated temperatures were variable. Solvent affinity ranged from 2.50% to 95.07%, with differences among isolates within each solvent (all p < 0.001). Autoaggregation ranged from 7.50% to 95.40% after 4 h. Coaggregation reached approximately 55% with Escherichia coli, 49% with Salmonella Enteritidis, and 50% with Staphylococcus aureus. Supernatants from 10 isolates inhibited at least one indicator bacterium, and six inhibited all three. R3-4 produced the numerically largest mean inhibition zone against S. aureus (19.3 ± 1.2 mm). No detectable hemolysis was observed. An exploratory five-domain score highlighted R3-4 (putative Y. lipolytica) and four putative K. marxianus isolates, without indicating superiority across all traits. These phenotypic profiles inform further screening, molecular confirmation, comprehensive safety assessment, and equine-relevant functional validation remain necessary.