“ALL CLAY-BASED ADDITIVES ARE NOT THE SAME”

If a specific clay is believed to possess the necessary properties to be considered a good candidate for adsorbing (binding) one or more mycotoxins the normal procedure is to eventually subject the clay to in-vitro mycotoxin binding tests. If these invitro screening tests prove that a specific clay has a high degree of mycotoxin binding capability it does not necessarily mean that the clay is going to be an effective binder of one or more mycotoxins in-vivo. Follow up studies are required with the more essential and costly in-vivo tests using an animal species that will be consuming the clay once it is added to an animal’s diet. Improved Milbond-TX® (IMTX) has been extensively tested since 1999 for its ability to successfully bind aflatoxin B₁ (AFB₁). In previous issues of Milwhite’s Journal all of the in-vivo safety and efficacy studies with IMTX, conducted in different university trials using different animal species, have been the topics of discussion. Many of the additives in the commercial marketplace that companies claim to be mycotoxin binders are periodically tested in comparison studies to determine, if indeed, their claims are true and they have the necessary properties to bind certain mycotoxins and specifically AFB₁. Such a comparison study was conducted by Marroquin-Cardona and colleagues in 2009. These investigators conducted in-vitro tests that characterized and compared 12 different additives all of which were being sold in Mexico as mycotoxin binders and claiming to have the ability to bind AFB₁. A few of these additives had claims of being able to bind multiple mycotoxins. Names of the additives and the experimental results obtained for each additive, as well as a more detailed description of the tests to which all 12 additives were subjected in this comparison study can easily be found in the cited publication of Marroquin-Cardona et.al. (2009) and Figures 1 and 2 presented here were adapted from their publication.

Using X-ray diffraction analysis to characterize the crystalline structure of these clays, 9 were confirmed to contain Smectite, but the effectiveness of the AFB₁ binding ability among the 9 clays was considerably different. This means that even though clays contain Smectite, it does not necessarily mean that such clays will have a similar ability to bind AFB₁ because there are other factors that are contributing to their AFB₁ binding ability. Or, another way to think about this is that other factors are exerting a negative influence on the ability of a Smectite clay to efficiently bind AFB₁. From the data collected in their study, these authors concluded that two of the major factors contributing to a high AFB₁ binding potential were the particle size in the clay additive and the presence of the clay mineral, montmorillonite, in Smectite. The properties of montmorillonite that contribute to the high AFB₁ binding potential of Smectite clays was discussed in a previous issue of Milwhite’s Journal.

Since kaolinite, mica, silica, and clinoptilolite may have been present in the 12 clay-based additives tested, these investigators decided to determine their ability to bind AFB₁. Reference standards of these 4 minerals were obtained from the Clay Mineral Repository at Purdue University in West Lafayette, Indiana, USA. In order to test their capacity to adsorb AFB₁, 11 initial solutions were developed containing increasing concentrations of AFB₁ ranging from 0.4 to 8.0 ppm in 5 ml and 0.1 mg of each mineral was added to each solution. The pH and temperature of each solution was 6.5 and 25◦ C, respectively. Suspensions of these solutions were formed and following dilution, incubation at 25◦ C for 2 hours and final centrifugation, the amount of AFB₁ remaining in solution was determined. Qmax was determined for each additive. Qmax is simply a term that is used to express the AFB₁1 binding ability of a feed additive. Qmax is expressed as mol/kg (i.e., moles of AFB₁ bound/kg of additive) and can be found on additive labels in the commercial marketplace. Qmax is considered an important required criteria when companies apply for additive licensing. During the past 30 years, research data from numerous studies have shown that the above 4 minerals have a minimal ability to adsorb AFB₁. Confirmation of the inability of these minerals to bind AFB₁ is shown in Figure 1. Only Smectite was shown to have an ability to adsorb AFB₁. This should come as no surprise since Smectite clays that possess the highest AFB₁ adsorptive capacity are also associated with having high concentrations of montmorillonite.

Therefore, a major characteristic of the feed additives tested in this study and having the highest ability to adsorb AFB₁ was to contain a high concentration of montmorillonite and IMTX contains approximately 85% montmorillonite.

Of the 9 clays that contained Smectite only 4 exhibited acceptable abilities to bind AFB₁. In Figure 2, the capacity of each clay to bind AFB₁ is presented. The procedure followed for these 12 samples to determine their adsorptive capability of AFB₁ was the same as that used for the 4 minerals discussed above. It can be noted that of the 12 clays, IMTX had the second highest Qmax which was greater than 0.3 mol AFB₁/kg.

From this comparison study of 12 additives, the authors concluded that the following attributes contributed to a clay’s ability to effectively bind AFB₁. It should be pointed out here that IMTX has all of these attributes. Proper particle size has a major influence on the AFB₁ binding effectiveness of an additive. The active ingredient in a clay with a high AFB₁ binding capability is Smectite (i.e., montmorillonite). The effectiveness of AFB₁ binding in an additive is affected if contamination of Smectite occurs resulting in dilution as a result of sand, silt, organic matter and minerals that have a negative effect on the binding ability of a clay. The capability of a clay to adequately expand or swell in the presence of water is an essential characteristic and montmorillonite is considered an expandable clay. With adequate “swelling volume” (SV), molecules of AFB₁ can easily enter the interlayer spaces of Smectite clays noted for their highly layered structure. In fact, in this comparison study, the additives that had the lowest SV or no SV at all had the lowest ability to bind AFB₁ (i.e., lowest Qmax).

One of the most extensive studies to ever be conducted and reported on the ability of Smectite clays to adsorb AFB₁ is the M.S. Thesis of Kannewischer (2006). In this research, 20 smectite clay samples were tested for their chemical and physical properties and their properties related to the effectiveness to adsorb AFB₁. Discovered was the fact that differences in Smectite composition are responsible for differences in their adsorption capabilities and not so much the relative abundance of a Smectite clay. Among the Smectite samples tested there existed as much as a 10-fold ability to adsorb AFB₁. A major conclusion from this extensive research study involving 20 Smectites was that the greatest ability to adsorb AFB₁ resulted when a Smectite clay was mostly composed of montmorillonite.

Note: A complete description of the experiments mentioned in this issue and all of the data collected in the experiments conducted by the authors can be found in the referenced publication located in the footnote below. The information compiled in this issue of Milwhite’s Journal was compiled by Dr. Orlando Osuna, Director of Health Science at Milwhite, Inc. and Dr. Richard Miles, Professor Emeritus, University of Florida, Gainesville, FL, USA.

Marroquin-Cardona, A., Y. Deng, J.F. Taylor, C.T. Hallmark, N.M. Johnson and T.D. Phillips. 2009. In-vitro and in-vivo characterization of mycotoxin-binding additives used for animal feeds in Mexico. Food Additives and Contaminants, Vol. 26, No. 5, May 733-743.