This study investigated the ability of selected probiotic strains to bind aflatoxin M1 (AFM1), a hydroxylated metabolite of aflatoxin B1 classified as a Group 2B carcinogen by the International Agency for Research on Cancer (IARC). Human breast milk samples collected from lactating mothers in Diyarbakır, Türkiye, were experimentally contaminated with AFM1 and treated with live or heat-treated Lactobacillus acidophilus, Lactobacillus rhamnosus, and Enterococcus faecalis. All strains were applied at 108 CFU/mL, a concentration commonly used in AFM1-binding studies to ensure adequate bacterial cell density for toxin adhesion. Samples were incubated at 37 °C for 30 min, 1 h, 4 h, and 24 h, and for 4 h at 37 °C followed by 20 h at 4 °C. AFM1 levels were quantified using ELISA, and pH values were recorded. No measurable binding was detected at 30 min or 1 h, whereas clear binding emerged after 4 h, with the highest binding observed under the 4 h (37 °C) + 20 h (4 °C) condition. At this stage, live E. faecalis bound more than 95 % of AFM1, while L. acidophilus and L. rhamnosus achieved 87.51 % and 82.15 % binding, respectively. Heat-treated strains showed measurable AFM1 binding only at 4 h and completely lost binding at longer incubation times. These findings demonstrate that AFM1 binding depends on strain type, viability, incubation time, and temperature. The strong performance of live E. faecalis highlights strain-specific differences. Biological binding by probiotic microorganisms represents a promising strategy for reducing AFM1 levels in breast milk and dairy products; however, further in vivo and clinical studies are required to confirm safety, stability, and practical implementation.
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