USE OF DEATH CONIDIA OF Aspergillus niger AGGREGATE AS ZEARALENONE AND AFLATOXIN B1 ADSORBENT

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

Congreso; VI Congreso Latinoamericano de Micotoxicología; 2010

Background. Mycotoxins are well-know natural contaminants in foods and feeds. In animal husbandry, the consumption of mycotoxin contaminated diet may induce acute and long term chronic effects resulting in a teratogenic, carcinogenic (mainly for liver and kidney), estrogenic or immunosuppressive impact, in addition, poor feed conversion, diminished body weight gain, increased disease incidence due to immune suppression, and interference with reproductive capacities (Mellor, 2001). One of the most effective methods for controlling mycotoxin hazards in animal husbandry is based on the use of specific materials that adsorb mycotoxins, thus limiting their bioavailability in the body (Miazzo et al. 2005). Conidia of black aspergilli as bioadsorpbents are potentially useful in reducing the toxic effects of mycotoxins in animal production. Physicochemical properties of the surface of conidia make them potential candidates as adsorbents of mycotoxins.   Aims. To evaluate the zearalenone (ZEA) and aflatoxin B1 (AFB1) adsorption capacity of death conidia from two non-toxicogenic A. niger aggregate strains.   Materials and methods. Conidia were obtained from two strains of A. niger aggregate (RC84 and RC104). Strains were grown in Czapek yeast extract agar at 28ºC for 7 days in dark. After the incubation period, a loop of conidia was harvested from the colony surface and placed in a tube. Consecutive 3 washes were done with distilled water to remove any impurities and mycelium. Dead conidia were obtained through boiling living spores in distilled water for 15 min and centrifuged at 5000 rpm, discarding the supernatant. A suspension of dead conidia was inoculated on MEA and incubated at 25°C for 7 days and used as negative control. Concentration of dead conidia (1x107 conidia mL-1) was performed using a Neubauer chamber. Conidia were resuspended in solution 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) for the subsequent use in the adsorption test. The adsorption test was performed using a concentration of 1x107 conidia mL-1 at pH 2 and pH 6, as proposed by Bejaoui et al. (2005). An aliquot of 500 mL of conidia concentration was added to each Eppendorf containing 500 µL of 0.5, 5, 10, 20 and 50 µg/mL of ZEA and 0.1, 0.25, 0.5, 1; 2.5 and 5 µg/mL of AFB1, respectively. 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. Each adsorption test was performed in duplicate and controls were performed. Extracts were quantified by high-pressure liquid chromatography (HPLC). Curves representing the amount of bound ZEA or AFB1 as a function of the amount of added ZEA and 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. Differences among isotherm forms were observed when the pH waschanged. For RC104, the highest maximum adsorption (Gmax) was observed at pH 2 (2.8 x 10-6 g conidia-1), meanwhile, the situation was reversed when RC84 was used as adsorbent (2.23 x 10-6 g conidia-1) at pH 6. The efficiency of ZEA adsorption depended on the strain from which the conidia were obtained and could be attributed to differences in their chemical composition. Considering that conidia are hydrophobic structures, since they have deposits of fat and a hydrophobic proteins, their interaction with ZEA might be due to the lipophylic nature of this toxin. The conidia isolated from the black aspergilos strain RC104, were able to adsorbed AFB1 at pH 2, but did not show any considerable adsorption at pH 6. The maximum adsorption of AFB1 by conidia was 0.175 x10-6 g conidia-1. Conidia of black aspergilos strain RC84 were unable to adsorb the AFB1 in all conditions studied. Since there are not published works on the uptake of mycotoxins by conidia analyzed by mathematical models, this report interprets the results using three different mathematical models. The Hill model is applied to sigmoid isotherms as type L. Its mathematical expression includes the dissociation of the constant (KD), the maximum adsorption (Gmáx) and the minimum number (n) of binding sites required for cooperative adsorption. The inverse of KD is the constant of adsorption called (b). Besides comparable adjustments are obtained with the different models, for purposes of comparison, Hill’s model was chosen to explain the results of this report study. The values of Gmáx and b obtained using the Hill model could be used together with the affinity to define and describe the entire phenomenon. The value n was considered more as an indication of the occurrence of a cooperative interaction than as a number of active sites.   Conclusion. This study shows a novel tool to be applied in the adsorption of ZEA with importance in livestock production.   References. Mellor S (2001). Mycotoxins in feed: a global challenge. Feed Mix; 9: 26-28. Miazzo R, Peralta M F, Magnoli C, Salvano M, Ferrero S, Chiacchiera S M, Carvalho E C Q, Rosa C A R and Dalcero A (2005). Efficacy of Sodium Bentonite as a Detoxifier of Broiler Feed Contaminated with Aflatoxin and Fumonisin. Poultry Sci. 84:1-8.