Evaluation of mycotoxin sequestering agents for aflatoxin and deoxynivalenol: an in vitro approach
© Kong et al.; licensee Springer. 2014
Received: 9 April 2014
Accepted: 26 June 2014
Published: 8 July 2014
An experiment was conducted to determine the efficacy of mycotoxin sequestering agents for binding or degrading aflatoxin B1 (AFB1) and deoxynivalenol (DON) by an in vitro method. Ten toxin binder products including 5 bentonite clays (bentonite A, B, C, D, and E), 2 cellulose products (cellulose A and B), a yeast cell wall, an activated charcoal, and a mixture product containing minerals, microorganisms, and phytogenic substances were used in this experiment. An in vitro procedure was used to mimic the digestive process in pigs. The binding ability for AFB1 of the cellulose products was less compared with the values of other sequestering products (p < 0.05). The percent adsorption of AFB1 by bentonite clays, cellulose products, yeast cell wall product, activated charcoal product, and the mixture product were 92.5 (average of 5 bentonite products), −13.5 (average of 2 cellulose products), 92.7, 100.2, and 96.6, respectively. The respective values for DON were 3.24, 11.6, 22.9, 14.4, and 4.3. In conclusion, most toxin sequestering agents used in the present study had potential to bind AFB1 rather than DON based on the in vitro study which simulated the pH condition of the gastrointestinal tract of pigs.
Dietary mycotoxins have been shown to cause detrimental effects in swine health and production. Aflatoxin and deoxynivalenol (DON) are produced by molds such as Aspergillus and Fusarium, respectively, and are frequently found in feedstuffs in swine diets (Council for Agricultural Science and Technology, 2003). Recent studies employing meta-analytical approach indicated that aflatoxin and DON depressed the growth performance of pigs (Andretta et al. 2012; Mok et al. 2013).
Several methods have been used to overcome detrimental effects of mycotoxins from contaminated feedstuffs. These include the thermal inactivation and irradiating as physical method, treatment with acid/base solutions, ozonation, and ammoniation as chemical method, and degradation of toxins by microorganisms as biological method (Diaz and Smith 2005). In feed industry, toxin sequestering agents have been frequently used because of its economic feasibility and suitability for nutritional perspective.
Several in vitro methods have provided a good idea of binding affinity and capacity, consequently have been used as a screening method for potential mycotoxin sequestering agents (Diaz et al. 2002; Marroquin-Cardona et al. 2009). However, these methods may not be directly applicable to pig diets because they did not use the successive incubation at different pH conditions similar to the intestinal environment of pigs. Thus, the objective of this experiment was to determine the binding efficacy of various sequestering agents to mycotoxins by an in vitro method which mimicked the gastrointestinal condition of pigs.
Materials and methods
Ten toxin binder products including 5 bentonite clays (bentonite A, B, C, D, and E), 2 cellulose products (cellulose A and B), a yeast cell wall product, an activated charcoal product, and a mixture product consisted of minerals, microorganism, and phytogenic substances were used in this experiment.
The standard solution of AFB1 (2 μg/mL) and DON (100.2 μg/mL) in acetonitrile (Romer Labs Diagnostic GmbH, Tulln, Austria) were diluted to 10 and 250 ng/mL using distilled water, respectively. The quantification ranges of enzyme-linked immunosorbent assay (ELISA) kit used for analysis on AFB1 and DON were from 2 to 50 and 250 to 5,000 ng/mL, respectively.
In vitro procedure
An in vitro procedure was modified from suggested in vitro digestion procedure which simulates the digestion procedure of pigs (Boisen and Fernández 1997). Each sample consisted of 2.5 mL of phosphate buffer (0.1 M, pH 6.0) and 0.5% suspension of each sequestering agent was transferred to 50 mL conical tube and 5 mL of diluted mycotoxin standard solution was added to the conical tube. For control treatment, 5 mL of phosphate buffer added. The pH was adjusted to pH 2.0 by adding 300 μL 1 M HCl for simulating pH in the stomach. Then each sample was incubated for 2 h in shaking incubator at 39°C. The incubation of samples was conducted in triplicates of each sequestering agent sample. After 2-h incubation, 1 mL of phosphate buffer (0.2 M, pH 6.8) was added to the conical tube. For simulating the conditions in the small intestine, 300 μL of 1 M NaOH was also added and incubated at pH 6.8 for 4 h. After incubation, the mixture was centrifuged and the supernatant was obtained for analysis of residual unbound AFB1 and DON. The AgraQuant® Aflatoxin B1 (COKAQ8000) or Deoxynivalenol (COKAQ4000) ELISA test kits (Romer Labs Inc., Singapore) were used to detect the residual unbound AFB1 or DON concentration, respectively.
Calculations and statistical analyses
where IMT (ng/mL) is the initial amount of mycotoxin (AFB1 or DON) in the digestion conical tube; UMT (ng/mL) is the residual amount of unbound mycotoxin (AFB1 or DON) in the conical tube after digestion procedure.
Data were analyzed by MIXED procedure of SAS (SAS Inst. Inc., Cary, NC, USA). The model included the sequestering agent as a fixed variable. Differences among least squares means were determined by the PDIFF option with the Tukey’s adjustment. The significance was declared at an alpha-level of 0.05.
Results and discussion
In vitro percent adsorption of aflatoxin by various sequestering agents*
Amount of aflatoxin, ng/mL
Aflatoxin adsorption, %
Yeast cell wall
In vitro percent adsorption of deoxynivalenol by various sequestering agents*
Amount of deoxynivalenol, ng/mL
Deoxynivalenol adsorption, %
Yeast cell wall
In conclusion, the present study showed that most sequestering agents tested had sufficient potential to bind AFB1 rather than DON based on the in vitro experiment which mimicked the pH condition of the gastrointestinal tract of pigs.
The authors are grateful for the support by Rural Development Administration (Republic of Korea; PJ008405). The paper resulted from the Konkuk University research support program.
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