The paper describes in vitro model for evaluation of natural mineral adsorbents ability to adsorb mycotoxins. Bentonite, diatomite and zeolite were prepared in the Institute for Technology of Nuclear and Other Mineral Raw Materials, Belgrade. A total of six mycotoxins: aflatoxin B1 (AFL), ochratoxin A (OTA), deoxynivalenol (DON), zearalenone (ZON), diacetoxyscirpenol (DAS) and T-2 toxin were tested in vitro. For adsorption experiments crude extracts of mycotoxins, produced in the Department of Microbiology of Bio-ecological Center in Zrenjanin, were used. The ability for binding mycotoxins was evaluated in the electrolyte 0.1 M K2HPO4 which pH value was adjusted to 3.0 and 6.9, respectively. Mass ratio of individual mycotoxin and natural mineral adsorbent was 1:5000. The experimental mixtures were incubated for 1 hour on a rotary shaker (185 rpm) at room temperature (22-25oC). After incubation the extractions of unadsorbed mycotoxins from the filtrates were performed with organic solvents, and their quantifications were done by thin-layer chromatography (TLC). By the use of TLC method it was noted that bentonite, diatomite and zeolite binded more than 95% of applicated AFL. In the case of OTA only diatomite adsorbed that toxin - adsorption index was 66.67%. Binding of DON has been observed only at pH 3.0 of electrolyte. Its adsorption index varied from 25.00 to 50.00% depending on the type of mineral adsorbent. Effect of electrolyte pH value on the binding of ZON was not so expressed. Its adsorption index ranged from 12.20 to 37.00%. In the case of type A trichothecenes (DAS and T-2 toxin) bentonite, diatomite, and zeolite binded only T-2 toxin. The amount of adsorbed T-2 toxin ranged from 16.66 to 33.33%. The obtained results point out the need for activation or processing of natural mineral adsorbens, especially bentonite and zeolite, in order to increase the efficiency of adsorption of the wider spectrum of mycotoxins.

Potential for the biosynthesis of aflatoxin B1 (AFLB1), ochratoxin A (OTA), diacetoxyscirpenol (DAS), T-2 toxin (T2), and zearalenone (ZON) was investigated in different fungal species belonging to the genera: Aspergillus, Fusarium and Penicillium. The majority of investigated isolates originated from cereal grains, crushed oil soybean seed and fodder mixtures. The simple screening method developed by Filtenborg et al. (1983) was applied with few modifications concerning the type of the medium and cultivation temperature. In order to optimise the biosynthetic conditions for different mycotoxins, the following control cultures, known as mycotin producers were used: OTA - A. ochraceus CBS 108.08, DAS - F. semitectum (SL-B i SL-C), T2 - F. sporotrichioides (ITM-391, M-1-1, R-2301) and ZON - F. graminearum (GZ-LES). The fungi were cultivated on the standard medium (YESA - 2% yeast extract, 15% sucrose and 2% agar, pH 6.5), three modifications of the basic medium (YESAZn - the standard medium supplemented with 0.23 mg/l ZnSO4 x 5 H2O; PPSA - the medium in which yeast extract was replaced with peptone-1; PPSAZn - the medium in which yeast extract was replaced with peptone-1 and supplemented with 0.23 mg/l ZnSO4 x 5 H2O), and the potato-dextrose agar (PDA). The earlier biosynthesis of tested mycotoxins was recorded under the following cultivation conditions of fungal species: AFLB1 - after 14 days on PDA at 27±1°C, OTA - after 10 days on YESA and YESAZn at 27±1°C, DAS - after 10 days on PPSA and PPSAZn at 27±1°C, T2 - after 7 days on PPSAZn and PPSA at room temperature (20-24°C), and ZON - after 1 week on YESA and YESAZn at room temperature (21-24°C)

During 2009 and 2010, the microbiological tests of a total of 40 samples of protein feed ingredients (sunflower meal, soybean, soybean cake, soybean grits and soybean meal) originating from Vojvodina were carried out. The most of the samples (57.5%) matched the Serbian regulations on feed. Microbiologically, there was not adequate quality of protein ingredients, which was a consequence of the presence of pathogenic bacteria: Proteus spp. in 12 samples of sunflower meal, 1 sample of soybean meal and 2 samples of soybean cake, and E. coli in 2 samples of soybean meal. The highest total number of bacteria (1 x 107 g-1) and the highest number of yeasts and molds (148.000 g-1) was identified in one sample of sunflower meal. Mycological analysis of protein feed established the dominance of species from the genera Aspergillus (A. flavus, A. fumigatus, A. niger and A. ochraceus), Fusarium (F. solani, F. subglutinans and F. verticillioides) and Mucor (Mucor hiemalis f. hiemalis and M. racemosus f. racemosus). The study of biochemical characteristics of 10 fungal isolates from sunflower meal, soybean grits and cake has established that: a) 2 cultures of Aspergillus spp. possessed antagonistic activity against other fungal species, b) 1 isolate F. solani biosynthesized T-2 toxin, c) 1 culture of F. subglutinans produced zearalenone, d) 4 isolates of Mucor spp. showed the ability to degrade one or both trichothecenes of type A (diacetoxyscirpenol - DAS and T-2 toxin). Mycotoxicological studies that included 24 samples of protein ingredients showed the absence of mycotoxins (aflatoxin B1, ochratoxin A, zearalenone, deoxynivalenol, DAS and T-2 toxin) in all 15 samples of sunflower meal. In the samples of soybean and its products (meal and cake) only T-2 toxin was detected in 3 analyzed samples. The amount of this fusariotoxin did not exceed 375 μg kg-1

Under controlled in vitro conditions the capacity of the Mucor racemosus f. racemosus 1215/09 isolate to degrade type A trichothecenes (diacetoxyscirpenol - DAS and T-2 toxin) was observed in the liquid nutritive medium. According to previously performed experiments it was proved that the selected isolate, originating from sunflower meal, had the ability to degrade these fusariotoxins when growing on the modified Vogel’s agar supplemented with crude extracts of DAS and T-2 toxin. In order to determine biodegradation of fusariotoxins, the liquid nutritive medium - SPY (5% sucrose + 0.1% peptone + 0.1% yeast extract, pH 6.2) was simultaneously inoculated with the isolate M. racemosus f. racemosus 1215/09 and: a) Fusarium semitectum SL-B (DAS producer) or b) F. sporotrichioides R-2301 (T-2 toxin producer). The SPY media, inoculated with single fungal isolates, were used as a control of toxin biosynthesis. The cultures were incubated at room temperature (21-26ºC) on the rotary shaker (175 rpm). After the 3-5-day incubation, the filtration of liquid cultures and the extraction of fusariotoxins from filtrates with ethyl-acetate were performed. Determinations of DAS and T-2 toxin were done by thin layer chromatography using silica gel G. Depending on the incubation duration, M. racemosus f. racemosus in the mixed culture with F. semitectum degraded from 90.0 to 99.97% of DAS present in the medium (40,000- 120,000 µg l-1), while in the mixed culture with F. sporotrichioides it degraded from 95.0 to 96.7% of T-2 toxin present in the medium (240,000 µg l-1). Sterile filtrates of mixed cultures and single culture of M. racemosus f. racemosus, obtained by passing liquid cultures through the 0.45-µm membrane filter and added to the SPY medium, did not affect degradation of type A trichothecenes that had been biosynthesised by isolates F. semitectum SL-B and F. sporotrichioides R-2301 in the liquid medium

Nine isolates of fungi of genera Aspergillus, Fusarium, Paecilomyces and Penicillium were cultured on the modified Vogel’s medium with the addition of crude ochratoxin A (OTA) extract. This crude OTA extract was derived from a natural solid substrate on which Aspergillus ochraceus strain CBS 108.08 was cultivated. OTA was isolated, partially purified, dried by evaporating and dissolved in ethanol (1 mg ml-1), and added to the test medium up to the final concentration of 10 μg ml-1. The presence of OTA residues was determined after 7 and 14 day cultivation of fungi in the test medium at 27±1°C. The Paecilomyces lilacinus isolate (Inf. 2/A), which completely degraded OTA (150 μg) after only seven days, was selected for further studies. Wet sterile rice grains (50 g + 25 ml distilled water) were inoculated with individual isolates of fungi A. ochraceus (strain CBS 108.08) and P. lilacinus (isolate Inf. 2/A), and with their combination. In the case of P. lilacinus monoculture, 0.9 mg of crude OTA was also added into cultivation substrate. Each test was done in three replications. After the four week cultivation of individual and combined fungi at 27±1°C, inoculated rice grains were dried to the constant weight and pulverized. OTA was determined in these samples by the application of standard TLC method for fodder analysis. OTA in the amount of 61.310 μg kg-1 dry matter (DM) was determined only in the samples inoculated with a producer of ochratoxin A (A. ochraceus, strain CBS 108.08). On the other hand, a much smaller amount of OTA (80 μg kg-1 DM) was detected in samples inoculated with combined cultures of A. ochraceus and P. lilacinus isolates. Gained results indicate that P. lilacinus degraded, on average, 99.8% of OTA. After four week cultivation, the same fungal isolate in the samples of wet sterile rice kernels with the addition of 0.9 mg of crude OTA, completely degraded added crude OTA (<8 μg kg-1)