Aflatoxin M₁ (AFM₁) has been detected in many parts of the world both in raw milk and many dairy products, causing great economic losses and human disease. Unfortunately, there are few studies dealing with AFM₁ immunotoxicity/interactions with lactic acid bacteria for potential application as a natural preventive agent. The aim of this study was to isolate (from dairy products) food-grade probiotic bacteria able to degrade/bind AFM₁ in vitro and evaluate whether the same organism(s) could impart a protective role against AFM₁-induced immunotoxicity in exposed Balb/c mice. Bacteria (Lactobacillus plantarum MON03 and L. rhamnosus GAF01) were isolated from Tunisian artisanal butter and then tested for abilities to eliminate AFM₁ from phosphate-buffered saline (PBS) and reconstituted milk (containing 0.05, 0.10, and 0.20 µg AFM₁/ml) after 0, 6, and 24 h at 37°C. Results showed that the selected bacteria could ‘remove’ AFM₁ both in PBS and skimmed milk. The binding abilities of AFM₁ by L. plantarum MON03 and L. rhamnosus GAF01 strains (at 10⁸ CFU/ml) in PBS and reconstituted milk ranged, respectively, from 16.1–78.6% and 15.3–95.1%; overall, L. rhamnosus showed a better potential for removal than L. plantarum. ‘Removal’ appeared to be by simple binding; the bacteria/AFM₁ complex was stable and only a very small proportion of mycotoxin was released back into the solution. L. rhamnosus GAF01 had the highest binding capacity and was selected for use in the in vivo study. Those results indicated that use of the organism prevented AFM₁-induced effects on total white and red blood cells, and lymphocyte subtypes, after 15 days of host treatment. These studies clearly indicated that L. rhamnosus GAF01 was able to bind AFM₁ in vitro and—by mechanisms that might also be related to a binding effect—counteract AFM₁-induced immunotoxicity. Moreover, by itself, this bacterium was not toxic and could potentially be used as an additive in dairy products and in biotechnology for mycotoxin detoxification.

Beja is an agricultural area in northwest Tunisia. It contributes to national needs by offering cereals and milk to the market for human and animal consumption. A small number of studies on mycotoxin occurrence in feedstuffs and raw milk from lactating dairy cows in this region are available. Therefore, 226 samples were collected from farms and local markets during November 2008 until April 2010. Samples consisted of 112 raw cow milk, 56 blood from lactating cows and 58 feed destined for dairy cows. Plasma and feed were analysed for aflatoxin B₁ (AFB₁). Milk samples were analysed for aflatoxin M₁ (AFM₁). All samples were treated using a simultaneous methanolic-aqueous extraction, followed by immunoaffinity column clean-ups and were investigated by competitive enzyme-linked immunoabsorbent assay (ELISA). Recoveries were 80%–95% and 81%–92% for AFB₁ and AFM₁, respectively, while the limit of detection (LOD) was 0.01 µg/kg or µg/l for both mycotoxins. Results revealed the presence of AFB₁ in 84.4% of the feed samples (mean 18.7 ± 1.4 µg/kg), and 39.2% of the plasma-examined samples (median 7.1 ± 1.0 µg/l) were found to be contaminated at levels higher than the Tunisian and the European Union (EU) limit for dairy animals, which are 20 and 5 µg/kg in animal feed, respectively. AFM₁ was detected in 60.7% of the cow raw milk samples examined (median 13.6 ± 1.4 µg/l). Contaminated levels were higher than the EU limit of 0.05 µg/l. It was concluded that more precaution should be taken on hygiene controls in order to prevent fungal contamination.