INTERACTIONS BETWEEN SACCHAROMYCES CEREVISIAE CELL WALL AND AFB1

PEREYRA C.M ; CAVAGLIERI L.R; ROSA C.A.R; CHIACCHIERA S.M; DALCERO A.M

Congreso; VI Congreso Latinoamericano de Micotoxicología; 2010

Background. The contamination of animal feed with mycotoxins represents a worldwide problem for farmers. Mycotoxin containing feed can cause serious diseases in farm animals resulting in suffering and even death and thus can cause substantial economic losses (Huwig et al. 2001). Several strategies are available for the mycotoxins detoxification and can be classified as physical, chemical, physicochemical and (micro) biological approaches (Varga and Toth, 2004). One of the most effective methods for controlling mycotoxin hazards in animal husbandry is based on the use of yeast cell wall (YCW) that adsorb mycotoxins, which can be used to reduce the bioavailability of mycotoxins in the digestive tract and alleviate their adverse effects on animals.   Aim. To evaluate the AFB1 adsorption capacity of a commercial preparation of S. cerevisiae YCW using adsorption isotherms.   Materials and methods. Interaction assays between AFB1 and YCW were performed at pH 2 (50 mL of potassium chloride 0.2 M and 13 mL of hydrochloric acid 0.2 M) and pH 6 (100 mL of potassium phosphate bi acid 0.1 M and 11.2 mL of sodium hydroxide 0.1 M) at 37°C. An aliquot of 500 µL (2 µg/mL) YCW was added to each Eppendorf tube containing 500 µL of 2; 5; 7,5; 10 and 15 µg/mL AFB1 solution. Each Eppendorf was introduced into a centrifugem Labor 2K15 centrifuge (Sigma) at 37°C with mechanical agitation for 30 min. Eppendorfs were then centrifuged for 10 min at 14.000 rpm, the supernatant was taken and evaporated to dryness under gentle stream of nitrogen gas and analyzed by HPLC using the methodology described by Trucksess et al. (1994). Each adsorption test was performed in duplicate and controls were performed. Curves representing the amount of bound AFB1 as a function of the amount of added AFB1 were plotted according to the mathematical expressions proposed by three theoretical models (Langmuir, Frumkin-Fowler-Guggenheim and Hill), selected according to the isotherms form.   Results and Discussion. All adsorption isotherms were found to be clearly sigmoid can be adjusted by both, the model of Hill as the FFG. These mathematical models explain situations in which cooperative phenomena occur. The Hill model arises from an extrapolation of the theory that explains enzymatic reactions and therefore assumes that the adsorbent surface there is “n” binding sites. The association of the first adsorbato triggers the immediate association to the remaining sites. The FFG model takes into account the existence of interactions between adsorbed molecules considers that the energy of adsorption depends on the degree of coating of the adsorbent. There were no appreciable changes of isotherms at the studied pH. The YCW adsorption capacity at pH 2 and pH 6 was 1.40 and 2.83 (g g-1) with an adsorption constant of 0.18 x 10-6 (M-1) and 0.12 x 10-6 (M-1), respectively, using the Hill model (R2= 0.964 and 0.996). Similar values of adsorption capacity were obtained when the FFG model was applied. However adsorption constants were lower; at pH 2 and pH 6 constant values of 0.04 x 10-6 M-1 in both cases (R2= 0.975 and 0.953) were obtained. The Hill model has been proposed to explain the shape of adsorption isotherms on yeast cell wall and extracts derived from them (Yianninkouris y col., 2003). This model showed that interaction between the AFB1 and the YCW is cooperative and supports the hypothesis that the three-dimensional conformation mobility of YCW is probably important in the adsorption event.   Conclusion. The YCW was able to adsorb AFB1 in appreciable quantities, through a cooperative interaction mechanism. This mode of data representation is more informative of physical and chemical mechanisms involved in the linkage between YCW and AFB1.   References   Huwig A, Freimund S, Kappeli O, Dutler H. 2001. Mycotoxin detoxication of animal feed by different adsorbents. Toxicology Letters 122, 179–188. Trucksess, M.W., Stack, M.E., Nesheim, S., Albert, R.H., and Romer, T.R. Multifunctional column coupled with liquid chromatography for determination of aflatoxins B1, B2, G1, G2 in corn, almonds, Brazil nuts, peanuts and pistachionuts: collaborative study. Journal of AOAC International 1994. Int 6, 1512-1521. Varga J and Toth B, 2004. Strategies to control mycotoxins in feeds. Acta Veterinaria Hungara 53, 189–203. Yianninkouris A. Poughon L, Cameleyre X, Dussap CG, Francois J, Bertin G and Jouany JP (2003). A novel technique to evaluate interactions between Saccharomyces cerevisiae cell wall and mycotoxins: application to zearalenone. Biotechnology Letters 25, 783- 789.