Cold-Resistant Lactic Acid Bacteria from Zamorano-Leonesa Donkey Milk: Isolation, Functional Screening, and Genome-Based Insights for Technological Applications

Donkey milk is an underexplored biological niche with distinctive nutritional and microbiological properties, potentially harboring lactic acid bacteria (LAB) with technological or probiotic value. In this study, raw milk from the endangered Zamorano-Leonesa donkey breed was stored at 4 °C for 24 h to simulate realistic cold-chain conditions and favor the recovery of cold-tolerant microorganisms. Fourteen isolates were obtained, eight of which belonged to LAB or species with potential technological interest and were selected for functional evaluation. Phenotypic screening showed that most isolates tolerated acidic conditions (pH 2.5) and that four also resisted 0.3% bile salts. Acidification assays in pasteurized donkey milk revealed variable fermentation performance, with L. mesenteroides subsp. mesenteroides B8 and Lacticaseibacillus paracasei subsp. tolerans B19 displaying the most favorable profiles. These two strains were selected for genome sequencing. Genomic analysis revealed genes associated with acid and bile resistance, adhesion, cold and environmental stress responses, and carbohydrate metabolism. Both genomes also encoded biosynthetic gene clusters linked to secondary metabolites, including β-lactones, lincosamides, and RiPP-like compounds. No acquired antimicrobial resistance genes were detected. To our knowledge, this is the first study combining isolation, phenotypic screening, and genome-based characterization of cold-tolerant LAB from Zamorano-Leonesa donkey milk. Our findings highlight this milk as a valuable reservoir of safe, cold-adapted microorganisms with promising applications in functional dairy products and food biotechnology.