The high incidence of aflatoxins (AFs) in chocolates suggests the necessity to create a practical and cost-effective processing strategy for eliminating mycotoxins. The present study aimed to assess the adsorption abilities of activated charcoal (A. charcoal), yeast (Saccharomyces cerevisiae), and the probiotic Lactobacillus rhamnosus as AFs adsorbents in three forms-sole, di- and tri-mix-in phosphate-buffered saline (PBS) through an in vitro approach, simulated to mimic the conditions present in the gastrointestinal tract (GIT) based on pH, time and AFs concentration. In addition, the novel fortification of chocolate with A. charcoal, probiotic, and yeast (tri-mix adsorbents) was evaluated for its effects on the sensory properties. Using HPLC, 60 samples of dark, milk, bitter, couverture, powder, and wafer chocolates were examined for the presence of AFs. Results showed that all the examined samples contained AFs, with maximum concentrations of 2.32, 1.81, and 1.66 µg/kg for powder, milk, and dark chocolates, respectively. The combined treatment demonstrated the highest adsorption efficiency (96.8%) among all tested compounds. Scanning electron microscope (SEM) analysis revealed the tested adsorbents to be effective AF-binding agents. Moreover, the novel combination of tri-mix fortified chocolate had a minor cytotoxicity impact on the adsorptive abilities, with the highest binding at pH 6.8 for 4 h, in addition to inducing an insignificant effect on the sensory attributes of dark chocolate. Tri-mix is thus recommended in the manufacturing of dark chocolate in order to enhance the safety of the newly developed product.
Keywords: Lactobacillus rhamnosus; Saccharomyces cerevisiae; aflatoxin; biocontrol; chocolate contamination; chocolate safety; gastrointestinal tract; natural adsorbent; probiotic.