Identification by MALDI-TOF MS and probiotic characterization of Enterococcus faecium strains isolated from equine milk

Developing innovative functional foods requires selecting robust, biologically active probiotic microorganisms capable of tolerating processing conditions and gastrointestinal transit. Non-bovine milk serves as a unique repository of underexplored lactic acid bacteria (LAB) with promising health-promoting properties.

This study aimed to isolate, identify, and evaluate LAB strains from raw Algerian equine milk for their technological, safety, and probiotic characteristics.

Nine isolates were screened for environmental adaptability under technological stresses (2–6.5% NaCl, 10–63.5°C, pH 9.8) and subjected to a rigorous in vitro simulated gastrointestinal transit model, including sequential exposure to salivary α-amylase, gastric fluid (pH 2), intestinal fluid (0.05 g/L bile salts, 0.1 g/L lysozyme), and pancreatic enzymes. DPPH radical scavenging capacity, and antagonistic action against five foodborne pathogens, using agar well diffusion method, were also quantified. High-throughput proteomic profiling via MALDI-TOF MS ensured accurate taxonomic classification.

Nine isolates showed strong physiological tolerance, growing in up to 6.5% NaCl and surviving alkaline conditions (pH 9.8). Strains S5 and S8 exhibited high survival rates (>70%) under simulated gastric conditions (pH 2.0, 120 min), exceeding the survival of the reference probiotic strain Bb12. Both strains maintained viability under simulated intestinal conditions containing lysozyme (0.1 g/L) and bile salts (0.05%). MALDI-TOF MS identified S5 and S8 as Enterococcus faecium with high-confidence identification scores of 2.40 and 2.36, respectively. Strain S8 showed antimicrobial activity against Staphylococcus aureus (15.33 mm) and Bacillus cereus (16.33 mm) and demonstrated antioxidant activity of 67.74 ± 1.0%, comparable to the reference strain Bb12 (63.84%).

These findings indicate that equine-milk-derived E. faecium S5 and S8 exhibit promising technological and bioactive properties. However, given the opportunistic nature of this species and the potential risk of antibiotic resistance gene transfer, extensive safety profiling, including virulence gene screening and in vivo validation is mandatory before considering these strains for functional food applications.