This study simulates in vitro the effects of (i) rumen acidity and (ii) change in rumen protozoa numbers on the recovery of aflatoxins (AFs). Two 24-h fermentation experiments were carried out using the same batch in vitro fermentation systems and substrate (dried corn meal) containing 11.42, 2.42, 7.65 and 1.70 μg/kg of AFB1, AFB2, AFG1 and AFG2 respectively. In Experiment 1, two buffer concentrations (normal salts dosage or lowered to 25%) were tested. Buffer reduction decreased gas production (730 vs. 1101 mL, p < 0.05), volatile fatty acids (VFA) and NH3 concentrations in the fermentation liquid (39.8 vs. 46.3 mmol/L, and 31.7 vs. 46.5 mg/dL respectively, p < 0.01). Recovery of all four AFs types was higher (p < 0.01) in the reduced buffer fermentation fluid, both as a percentage of total AF incubated (73.6% vs. 62.5%, 45.9% vs. 38.1%, 33.6% vs. 17.9% and 18.9% vs. 6.24% for AFB1, AFB2, AFG1 and AFG2 respectively) and as amounts relative to VFA production (163.4 vs. 123.5, 22.1 vs. 15.7, 48.8 vs. 22.5 and 6.16 vs. 1.86 ng/100 mmol of VFA, for AFB1, AFB2, AFG1 and AFG2 respectively). In Experiment 2, Stevia rebaudiana Bertoni extracts (S) or a Camphor essential oil (Cam) were added to fermenters and compared to the control (no additives, C). S and Cam addition resulted in a 25% reduction (p < 0.05) and a 15% increase (p < 0.05) in protozoa counts respectively, when compared to C. Both plant additives slightly reduced (p < 0.05) AFB1 recovery as a percentage of total AFB1 incubated (68.5% and 67.7% vs. 74.9% for S, Cam and C respectively). Recoveries of all other AFs were unaffected by the additives. In conclusion, the rumen in vitro AFB1 recovery (63%–75%) was higher than other AFs (3%–46%) and the acidic fermentation environment increased it. In our conditions, changes in protozoa numbers did not affect AFs recovery.
