Proving the Efficacy of Mycotoxins Counteraction Products

Tests which are necessary to evaluate a proper and wide-spectrum product for mycotoxin counteraction, comprise in vitro evaluation of binders in case adsorption is the claimed strategy, or, in case of biotransformation it is crucial to evaluate the toxicity of metabolization products. In vivo trials are indispensable to test the efficacy of the products in practical conditions. Biomin has been always ahead in the counteraction of mycotoxins in animal feeds thus all these steps represent the routine that all possible binders and/or microorganisms must go through when being tested for their Mycotoxin Risk Management potential. 

Finding the best applicable mycotoxin adsorbent for inclusion in the three-strategy based Mycofix® Product Line has been intensely researched. In vitro tests were deployed for screening several bentonites in order to evaluate their ability to bind AfB₁, their specificity and adsorption mechanism. The specificity of the binding materials was tested by performing AfB₁ adsorption tests in various media at different pH levels (2.0, 5.0 and 7.0), as well as in real gastric juice and in a vitamin mix solution.

At a neutral pH, the detoxification efficacy was equal or higher to the one observed at pH 5.0 with a concentration of the adsorbent of 0.02% (w/v), equivalent to 200 g of adsorbent per tonne. At pH 2.0 results indicated 100% AfB1-adsorption for all binders tested. This was found to be due to the conversion of AfB₁ into AfB_2α at acidic pH. pH levels were than adapted to pH 3 to avoid this occurrence.

For those binders that showed an adsorption rate higher than 63% at pH 5.0, with 0.5% (w/v) binder (5 kg/tonne) and 4ppm AfB₁contamination, the chemisorption index (Cα) was measured. Amongst these, two binders presented very good chemisorption index values of 0.98 and 0.92, respectively. A chemisorption index value of 1 indicates no desorption of AfB1 from the binder material. In general, results reveal a tight binding of AfB1 to the tested bentonites.

Moreover, to simulate in vivo application, 47 binders were tested in real gastric juice and the adsorption capacities were compared to the results in the buffer solutions at the same pH. The effectiveness of nearly all binders was affected by the use of real gastric juice.

Subsequently, the potential adsorption of selected vitamins was examined in buffer solutions at pH 3.0 and pH 6.5. Results confirmed that bentonite binders neither adsorb vitamin B5 nor vitamin H. The adsorption of vitamin B12 ranged from 7 to 47%. Still, despite some vitamin adsorption by some of the tested materials AfB₁ was preferably adsorbed.

Therefore, the bentonite incorporated in the Mycofix® product line was extensively tested and scientifically approved (Vekiru, et al., 2007)

It is known that biotransformation is the strategy that leads to less- or even non-toxic metabolites of mycotoxins which adsorption by binders is not possible. For that reason, enzymes and microorganisms have been a subject of research for more than 40 years.

Trichothecenes toxicity can mainly be attributed to their 12,13-epoxide ring. The reductive de-epoxidation by a new strain of Eubacterium isolated from bovine rumen contents (Binder et al., 2000) results in a significant loss of toxicity.  The latter bacterium referred to as BBSH 797 is actually the first bacterial strain cultured, produced and stabilized in order to be applied as feed additive to counteract the negative effects of trichothecenes by biotransformation. Its application in practical studies (Fuchs et al., 2000; Fuchs et al., 2002) identified non-toxic metabolites after the microbial degradation of type A and type B trichothecenes. 

The toxicity of these compounds must be tested using a chicken lymphocyte proliferation assay (LPA). In the case of deoxynivalenol (DON) biotransformation into de-epoxy- deoxynivalenol (DOM-1), this assay shows that at a concentration of 0.63 μg/mL DON the growth of lymphocytes stopped. In the case of DOM-1 only a concentration of 116 μg/mL inhibited proliferation of lymphocyte cells completely. Those different concentrations allowed a toxicity comparison, being concluded that DOM-1 toxicity is approximately 200 times lower than the parent compound DON. These results are in accordance with previous scientific study carried out by Kollarczik et al. (1994). For the use of BBSH 797 as a feed additive, the fermentation and stabilization processes were optimized with respect to fast growth of the microbe and high biotransformation activity of the resulting product. For enhancement of stability during storage, within the gastrointestinal tract and during pelleting, a three-step encapsulation process was implemented.

Ochratoxin A is rapidly degraded by micro-organisms in the rumen to ochratoxin a (OTa) and phenylalanine (Hult et al., 1976; Kiessling et al., 1984).

Several experiments were carried out to find a suitable microorganism for its detoxification. However a suitable mycotoxin-degrading microorganism with practical application in mycotoxin-contaminated feeds was only later on discovered. Following a screening of more than 20 OTA-cleaving microorganisms, which involved the incubation of this mycotoxin with several OTA-degrading isolates, the yeast strain associated with the hindgut of lower termites, Trichosporon mycotoxinivorans MTV was isolated and described (Schatzmayer et al., 2003; Molnar et al., 2004). The stabilization and lyophilization of the yeast enables it to have a practical application in the feed.

Lyophilized cells grown in an optimized culture medium were able to degrade OTA (200 μg/L) completely into OTα within 1 hour of incubation. In an assay conducted with the objective of ascertaining the metabolite's toxicity (macrophage activation assay) it was proved that growth of macrophages was depressed from 0.741 to 2.222 μg/mL of OTA. At concentrations above 6.667 μg/mL of OTA their growth was completely inhibited. Contrarily, concentrations up to 20 μg/mL of OTα did not affect macrophages growth.

These results were in accordance with those of other scientific studies were OTα was shown to be non-toxic or at least 500 times less toxic than OTA (Chu et al., 1972; Bruinink et al., 1998).

The toxicity of zearalenone (ZON) relies on the molecule's resemblance with the sexual female hormone estradiol, which enables it to couple with the estrogenic receptors acting as an estrogen agonist in the brain resulting in severe effects on the reproductive system (Gromadzka et al., 2008).

The metabolisation of this toxin by Trichosporon mycotoxinivorans MTV lead to a compound that is no longer estrogenic. Also, results of the degradation study did not detect α- and β-zearalenol, which are more estrogenic than ZON itself. The non-estrogenicity of the metabolite has been proven in an in vitro assay (E-screen assay) with a human estrogen-receptor-positive breast cancer cell line, which proliferates in the presence of estrogens  (Schatzmayr et al., 2003).  The sample resulting from ZON incubation with Trichosporon mycotoxinivorans MTV did lack the ability to induce any response indicating an estrogenic activity, thus confirming the stable degradation of zearalenone into a non-estrogenic metabolite.

 

Although crucial for the initial steps of product development, all these in vitro experiments do not substitute the need for in vivo trials. Several successful in vivo scientific trials were done with Mycofix® product line throughout the years. To name a few, trials with female piglets and with poultry were conducted at the Laboratório de Análises Micotoxicológicas (LAMIC). The first had as objective to evaluate the effects of Mycofix® Plus (MPL) on reproduction performance parameters (feed intake, weight gain, feed conversion, body weight), on organ characteristics (weight and size of reproductive tract) and on vulva morphology (vulva volume) of female growing piglets fed ZON-contaminated diets (2 ppm). Results confirmed the protective effect of MPL in performance parameters, organ characteristics and vulva morphology. Another had the aim of evaluating the efficacy of Mycofix® in diminishing the toxic effects of aflatoxins added to the diet of broiler chickens. Once again, the protective and significant (P≤0.05) effect of the inclusion of 0.5 % of Mycofix® was proven in body weight (+ 9.97 %), feed intake and liver relative weight (- 13.93 %) of treated animals when in comparison with animals fed only aflatoxin-contaminated diets.

Binder, E. M., Heidler, D., Schatzmayr, G., Thimm, N., Fuchs, E., Schuh, M., Krska, R., and Binder, J. (2000) Microbial  detoxification of mycotoxins in animal feed, Proceedings of The 10^th  International IUPAC Symposium on Mycotoxins and Phycotoxins, Brasil.
Bruinink, A.; Rasonyi, T.; Sidler, C. (1998) Differences in neurotoxic effects of ochratoxin A, ochracin and ochratoxin-α in vitro. Nat. Toxins (6), pp: 173 - 177.
Chu, F. S.; Noh, I.; Chang, C. C. (1972) Structural requirements for ochratoxin intoxication. Life Sci. (11), pp: 503 - 508.
Fuchs, E., Binder, E.M., Heidler, D. and Krska, R. (2000) Charakterisierung von metaboliten nach dem bakteriellen abbau von A- und B-Trichothecenen durch BBSH 797. Proceeding of the 22^nd Mykotoxin-Workshop, Bonn, Germany.
Fuchs, E., Binder, E.M., Heidler, D. and Krska, R. (2002) Structural characterization of metabolites after the microbial degradation of type A trichothecenes by the bacterial strain BBSH 797. Food Additives and Contaminants (19), pp: 379-386.
Kiessling, K. H., Pettersson H., Sandholm, K. and Olsen, M. (1984) Metabolism of aflatoxin, ochratoxin, zearalenone, and three trichothecenes by intact rumen fluid, rumen protozoa, and rumen bacteria. Applied and environmental microbiology (47), pp: 1070-1073.
Kollarczik, B.; Gareis, M.; Hanelt, M. (1994) In vitro transformation of the Fusarium mycotoxins deoxynivalenol and zearalenone by the normal gut microflora of pigs. Nat. Toxins (2), pp: 105 - 110.
Gromadzka, K.; Waskiewicz, A.; Chelkowski, J.; Golinski, P. (2008) Zearalenone and its metabolites: occurrence, detection, toxicity and guidelines. W. Mycot. J.(1), pp: 209 - 220.
Hult, K., Telling, A. and Gatenbeck, S. (1976) Degradation of ochratoxin A by a ruminant. American Society for Microbiology (32), pp: 443-444.
Molnar, O., Schatzmayr, G., Fuchs, E., Prillinger, H. (2004) Trchosporon mycotoxinivorans sp. nov., a new yeast species useful in biological detoxification of various mycotoxins. Syst. applied microbiology (27), pp: 661-671.
Schatzmayer, G., Heidler, D., Fuchs, E., Mohnl, M., Täubel M., Loibner A. P., Braun R., Binder E. M. (2003) Investigation of different yeast strains for the detoxification of Ochratoxin A. Mycotoxin Research (19), pp: 124 -128.
Vekiru, E., Fruhauf, S., Sahin M., Ottne F., Schatzmayr, G., Krska, R. (2007) Investigation of various adsorbents for their ability to bind Atlatoxin. Mycotoxin Research (23), pp: 27-33