Introduction:

Aflatoxin M1 (AFM1) is a carcinogenic mycotoxin commonly found in dairy products from livestock consuming contaminated feed. Its thermal stability renders pasteurization ineffective, necessitating alternative detoxification strategies. This study evaluated the AFM1-binding capacity of exopolysaccharides (EPS) from kefir grains using two extraction methods.

Methods:

EPS were produced by fermenting semi-skimmed milk with 10% kefir grains. After grain removal and casein elimination, EPS were extracted via (1) cold ethanol precipitation and (2) hot aqueous extraction followed by ethanol precipitation. EPS were dialyzed (14 kDa), lyophilized, and analyzed for sugar and protein content. AFM1 retention (1 μg/L) by 1% EPS was assessed in phosphate buffer (pH 6.8, 30°C) over 3 hours with hourly sampling, ultrafiltration (30 kDa), and HPLC-fluorescence quantification.

Results:

Cold extraction yielded higher solids (9,400 mg) with balanced sugar-to-protein ratios, while hot extraction reduced protein content by 69% and increased sugar concentration eight-fold. Cold-extracted EPS maintained >80% AFM1 retention throughout the assay period. Hot-extracted EPS exhibited lower, time-dependent retention. Method-dependent differences were statistically significant (p < 0.05).

Conclusions:

Extraction methodology significantly affects EPS composition and AFM1-binding efficiency. Cold-extracted EPS, retaining protein components, demonstrated superior AFM1 sequestration, suggesting protein–mycotoxin interactions are crucial for binding. These findings highlight the potential of kefir EPS as a natural mycotoxin mitigation strategy in dairy processing, warranting further investigation for industrial applications.