In vitro release of aflatoxin M1 during enzymatic digestion of milk proteins: evaluation and analysis

Cow's milk is one of the most widely consumed foods worldwide due to its high nutritional value and rich content of biologically beneficial proteins. However, aflatoxin M1 (AFM1), a mycotoxin recognized for its carcinogenic potential, has been detected in milk, where it can bind to proteins. This study compared three methods for extracting AFM1 from commercial milk samples before and after enzymatic digestion with pepsin and pancreatin.

Initial AFM1 concentrations and those following in vitro enzymatic digestion were measured using high-performance liquid chromatography. Extraction method 3 proved the most effective, combining acetonitrile with ultrasound-assisted extraction. AFM1 was detected in all milk samples at concentrations ranging from 0.25 to 12 μg L^-1, exceeding national (0.5 μg L^-1) and international (0.05 μg L^-1) regulatory limits.

Enzymatic digestion increased AFM1 levels 3.66-fold in four liquid milk samples, suggesting that raw milk measurements might underestimate consumer exposure. Thus, evaluating AFM1 bioaccessibility is essential for accurate toxicological risk assessment of milk and dairy products. © 2025 Society of Chemical Industry.