Antioxidant and stress-adaptive properties of putative probiotic bacteria in Pakistani fermented buffalo milk

Introduction Fermented buffalo milk products from South Asia remain an underexplored source of microbial diversity with potential health-promoting benefits. This study investigates the probiotic and industrial suitability of lactic acid bacteria (LAB) and non-LAB isolates from traditional Pakistani dairy, addressing gaps in region-specific probiotic discovery. Methods Forty-seven bacterial isolates were obtained from fermented buffalo milk products (yogurt and cheese). Molecular identification (16S rRNA sequencing) classified isolates into LAB and non-LAB taxa. Probiotic potential was evaluated via in vitro assays for gastrointestinal stress tolerance (pH 2.0, 0.5% bile), antioxidant activity (DPPH scavenging), and industrial adaptability (growth at 4–45°C, 2–6% NaCl). Results Eight strains were prioritized, including Lactobacillus plantarum Y 1 , L. brevis Cc 3 , Streptococcus thermophilus Y 6 /Cc 1 /Cm 5 , and non-LAB Bacillus dendritiformis Y 9 . L. plantarum Y 1 exhibited exceptional acid resistance (>5.0 log10 CFU/mL at pH 2.0) and bile tolerance (6.5 log10 CFU/mL). L. brevis Cc 3 combined high bile resilience (6.0 log10 CFU/mL) with robust antioxidant activity (52% DPPH scavenging), while S. thermophilus Y 6 showed 48% antioxidant capacity. Non-LAB isolates, particularly B. dendritiformis Y 9 , demonstrated unexpected bile stress survival (5.4–5.5 log10 CFU/mL). All strains grew under industrial conditions (4–45°C, 2–6% NaCl), except S. thermophilus Cc 1 , which was heat-sensitive above 40°C. Conclusion This study highlights South Asian buffalo milk as a reservoir of both conventional LAB and novel non-LAB strains with dual stress tolerance and antioxidant functionality. L. plantarum Y 1 and L. brevis Cc 3 emerge as prime candidates for developing culturally tailored functional foods to address regional nutritional challenges. The resilience of non-traditional isolates such as B. dendritiformis Y 9 challenges existing probiotic taxonomical biases, suggesting broader microbial resources for gut-health innovations. These findings advocate for integrating regionally adapted probiotics into functional diets to enhance gastrointestinal health and oxidative stress mitigation in South Asian populations.