Abstract

ABSTRACT Objective: This study aimed to establish an efficient isolation, culture, and preservation system for lactic acid bacteria from Mongolian horse feces by systematically evaluating the effects of sampling methods, culture conditions, media types, and cryopreservation protocols. Methods: Fecal samples were collected using rectal and natural defecation methods, and bacteria were isolated by the spread plate technique, with isolates subcultured and identified via 16S rRNA gene se-quencing. The performance of five commercial media (A-E), aerobic versus anaerobic conditions, and different glycerol concentrations (10% and 30%), temperatures (-20 and -80 Degree Celsius), and storage dura-tions (7, 15, and 30 days) were compared. Results: Rectal sampling showed significantly higher lactic acid bacterial survival and greater species diversity (12 vs. 8). Anaerobic culture yielded substantially higher counts and diversity (707 strains, 12 species) compared to aerobic conditions (36 strains, 5 species). Medium C (M.R.S. AGAR) exhibited the best isolation performance, producing the highest number of strains (311) and species (11), with selective enrichment for both bacilli and cocci. Glycerol effectively improved strain survival during sample cry-opreservation, with the optimal short-term storage (<15 days) achieved at -20 Degree Celsius with 30% glycerol. Functional screening of randomly selected strains from optimized conditions demonstrated promising growth, acid and bile salt tolerance, and antimicrobial activity. Conclusion: Rectal sampling combined with anaerobic incubation using Medium C is recommended for optimal isolation. For samples that cannot be processed immediately, short-term preservation at -20 Degree Celsius with 30% glycerol prior to isolation is effective. This study provides practical technical references for the acquisition and preservation of equine intestinal lactic acid bacteria resources.