Youjun Deng

In vitro and in vivo characterization of mycotoxin-binding additives used for animal feeds in Mexico.

The study was conducted to characterize and compare twelve different additives distributed in Mexico as mycotoxin binders utilizing: (1) equilibrium isothermal analysis for aflatoxin B1 (AFB1) adsorption, (2) a variety of mineralogical probes, and (3) Hydra toxicity bioassay. The test additives Milbond-TX® (MLB), Mycoad® (MCA), Volclay FD181® (VOL), Fixat® (FXT), Toxinor® (TOX), Mexsil® (MEX), Mycosil® (MYC), Klinsil® (KLS), Zeotek® (ZEO), Duotek® (DUO), Mycosorb™ (MSB), and Mycofix® Plus 3.0 (MIX) were compared with NovaSil™ Plus (NSP). Isotherms for AFB1 adsorption were conducted at pH 2 and pH 6.5, mimicking pH conditions in the stomach and small intestine. Mineralogical analysis included determination of swelling volume, X-ray diffraction analysis, and fractionation procedures. A Hydra vulgaris toxicity study was performed to evaluate the potential safety of the additives. Computer-generated isotherm data were fit using the Langmuir model, and parameters of Q max and K d were estimated. The most effective additives for AFB1 at both pH conditions were NSP, MLB, MCA and VOL, while the least effective was MSB. The amounts of sand, silt and clay fractions varied among the additives. Nine of the additives showed the presence of smectite. Most of the additives were found to be non-toxic to Hydra except for the organoclays (ZEO, DUO) and MSB. In general, NSP demonstrated the highest sorption capacity in the bulk material and the different fractions. Studies to characterize these binding additives further and to evaluate their multiple mycotoxin sorption claims are ongoing.

THE DETERMINATIVE ROLE OF THE EXCHANGE CATION AND LAYER-CHARGE DENSITY OF SMECTITE ON AFLATOXIN ADSORPTION

Using bentonites to adsorb aflatoxin is an effective method of minimizing the toxicity of aflatoxin to animals and humans. Early studies indicated a more than 10-fold difference in aflatoxin adsorption capacity among different bentonites. The determining mineralogical and chemical properties of the clays in aflatoxin adsorption are still poorly understood. The objective of this study was to test the hypothesis that a bentonite’s selectivity and adsorption capacity for aflatoxin is mainly determined by the ‘size matching’ requirement, on a nm scale, between the non-polar interlayer surface domains and the aflatoxin molecules. The non-polar surface domain size of smectites was varied by (1) selecting smectites with different charge densities; and (2) changing the valence and the size of exchange cations to control the amount of water in the hydration shells of the cations. Infrared spectroscopy and X-ray diffraction were also used to characterize the aflatoxin-smectite complexes to investigate if layer-charge density would affect the bonding strength between aflatoxin and the minerals. A large aflatoxin adsorption capacity and high selectivity for aflatoxin were achieved by selecting smectites that had low charge density as represented by their <110 meq/100 g cation exchange capacity. An individual smectite’s selectivity and adsorption capacity for aflatoxin could be enhanced or weakened by replacing the exchange cation. When the smectite was saturated with divalent cations that have smaller hydrated radius (e.g. Ba2+), the smectite’s adsorption capacity and affinity for aflatoxin were enhanced. Aflatoxin entered the interlayer of all six smectites tested. The strength of its bonding to the smectites was not affected by the layer-charge density of the smectites. The results confirmed the importance of nm-scale polarity and size match between aflatoxin molecules and the adsorbing sites on smectite. The high selectivity for aflatoxin can be achieved by selecting a smectite with adequate charge density or by replacing the exchange cations with divalent cations that have low hydration energy.

Development of High Capacity Enterosorbents for Aflatoxin B1 and Other Hazardous Chemicals

Previously, a calcium montmorillonite clay (NovaSil) included in the diet of animals has been shown to bind aflatoxin B1 (AfB1) and reduce the symptoms of aflatoxicosis. To investigate and improve the capacity and efficacy of clay-based materials as aflatoxin sorbents, we developed and tested calcium and sodium montmorillonite clays amended with nutrients including l-carnitine and choline. Also, we determined the sorption of AfB1 by isothermal analysis and tested the ability of these amended sorbents to protect adult hydra from AfB1 toxicity. The results showed that exchanging montmorillonite clays with l-carnitine and choline inhibited swelling of the clays and increased the sorption capacity and efficacy of clay surfaces for AfB1. Results from dehydroxylated and heat-collapsed clays suggested that AfB1 was primarily adsorbed in the clay interlayer, as predicted from thermodynamic calculations and computational modeling. The hydra bioassay further indicated that the modified clays can significantly protect adult hydra from AfB1 with as low as 0.005% clay inclusion. This enterosorbent therapy may also be applied to screen hazardous chemicals such as pesticides and PAHs based on similar sorption mechanisms. Taken together, enterosorbent therapy could be delivered in nutritional supplements, foods that are vulnerable to aflatoxin contamination, flavored liquids and animal feeds during emergencies and outbreaks of acute aflatoxicosis, and as a screening model for hazardous environmental chemicals.

TRANSFORMATION OF ADSORBED AFLATOXIN B1 ON SMECTITE AT ELEVATED TEMPERATURES

Aflatoxins cause liver damage and suppress immunity. Through adsorption, smectites can be used to reduce the bioavailability of aflatoxins. To further reduce the toxicity of aflatoxins and to eliminate the treatments of aflatoxin-loaded smectites, the ability to degrade the aflatoxin adsorbed to non-toxic or less toxic compounds is desirable. The objective of the present study was to investigate the effects of temperature and the exchange cation on the transformation of adsorbed aflatoxin B1 on smectite. An AfB1-Ca-smectite (sm) complex was synthesized. To enhance the Lewis acidity of the complexes, the exchanged calcium in the complex was replaced with Mn and Cu to obtain AfB1-Mn-sm and AfB1-Cu-sm complexes, respectively. The aflatoxin-sm complexes and pure aflatoxin B1 were dried at 60°C in aluminum cups, and heated from 100 to 200°C in 25°C steps. Aflatoxin B1 and its transformation products were extracted with methanol after the heat treatment. The extracts were analyzed using UV spectroscopy, high performance liquid chromatography (HPLC)-fluorescence/UV, ultra-performance liquid chromatography (UPLC)-photodiode array (PDA), and electrospray ionization-tandem quadrupole-mass spectrometry (ESI-TQDMS). The solid residues were analyzed using Fourier-transform infrared spectroscopy (FTIR). The UV and FTIR spectra of the AfB1-sm clay residue extracts obtained after heating had decreased AfB1 peak intensities and shifted peak positions with increased heating temperature. Significant shifts in band positions and changes in the shape of the UV spectra were observed in the extracts from the AfB1-Ca-sm complex heated at 175°C, the AfB1-Cu-sm complex heated at 150°C, and the AfB1-Mn-sm complex heated at 125°C. The HPLC and UPLCMS analyses of AfB1-sm complex extracts indicated a gradual decrease in AfB1 concentration with increased heating temperature and the formation of aflatoxins B2, B2a, M1, M2, and other unidentified compounds. No new compound was observed in the extracts of pure aflatoxin B1 after a comparable heating experiment. These results suggest that smectite can effectively convert aflatoxin to other less toxic forms at elevated temperatures. Smectite ion exchange with Cu or Mn transition-metal cations and heat treatment induced more efficient conversion of the adsorbed aflatoxin B1 molecules to other compounds.