IN VITRO AND IN VIVO EFFICACY ASSESSMENT OF A NEW BENTONITE BASED MATERIAL ACTING AS A MULTI-MYCOTOXIN BINDER

Background Bentonite, a mineral authorized in Europe as feed additive for reducing mycotoxin contamination in feed (EU Reg. No. 1060/2013), is selective in adsorbing aflatoxins AFB1, but ineffective for other mycotoxins of zootechnical interest. This work intends to develop and assess the efficacy of an innovative bentonite-based material (bio-organoclay) acting as a multi-mycotoxin binder, by performing in vitro and in vivo experiments. Methods The efficacy of the bio-organoclay in sequestering aflatoxins B1 (AFB1), fumonisin B1 (FB1), ochratoxin A (OTA), and zearalenone (ZEA) was firstly tested in vitro. Then, its efficacy in reducing the urinary excretion of mycotoxins was tested in rats by using the biomarker approach. For each toxin, two groups of rats (0.3 kg initial body weight) were considered: control animals received the mycotoxins without the detoxifier, and treated animals received the mycotoxins with the bio-organoclay at 0.5% w/w of feed consumption. Mycotoxins were administered by a singular intragastric oral bolus. Samples of urine were collected at different time points (4-72 h for AFB1, ZEA and FB1; 4-320 h for OTA). AFB1, ZEA and its metabolites of phase I biotransformation (?-ZOL, ?-ZOL and ?-ZAL), OTA and FB1 were analyzed in urine by in-house validated UPLC methods. Mycotoxin content in urine was normalized to urinary creatinine concentration. Toxicokinetic parameters, including area under the curve (AUC) and maximal mycotoxin concentration (Cmax), were calculated and used to compare control and treated groups. Results A bentonite containing Na-smectite as major mineral was modified and functionalized. Acid-activation and functionalization processes were optimized at lab level, and optimal conditions were identified. In in vitro studies, at low dosages (0.25-0.5% w/v), the new produced bio-organoclay sequestered more than 95% of AFB1, FB1, OTA, and ZEA, in a large range of pH values (3-9). Mycotoxin adsorption occurred simultaneously with high capacity and affinity as determined by equilibrium isotherms. In rats, the bio-organoclay reduced urinary excretion of AFM1, ZEA, FB1 and OTA respect to the control group, by reducing the AUC and Cmax values. AUC0?72 was significantly reduced by -94% for AFM1, -54% for OTA, and -40% for FB1 (p<0.05). Cmax value of ZEA was significantly reduced by -65% (p<0.05) Conclusion The use of the bio-organoclay as a feed additive can be considered a valid approach to reduce mycotoxins bioavailability in animals exposed to the main mycotoxins, answering to the great demand of these kind of products by animal feed producers. This product can be considered safe, as it has been obtained using reagents that are listed in the European Union Register of Feed Additives (EC Regulation, No. 1831/2003). This research was financially supported by the European Union's Horizon 2020 Research and Innovation Programme under Grant Agreement No. 678781 (MycoKey).