**2.6. Data analysis**

different cereals, i.e. maize, oat, wheat, barley and triticale, were chosen based on the results obtained using the ELISA technique (<LOD) and spiked with the T-2 and HT-2 standard at the level of 25 μg/kg. Then, both spiked and pseudo-blank samples were analysed, and the difference in signal abundances and the standard deviation was calculated. The obtained data were used for the calculation of the LOD and the LOQ. Linearity was tested in the range of 5–100 ng/mL, using the standard solution of each mycotoxin. The recovery was determined by virtue of analysing six blank maize samples spiked with T-2 and HT-2 toxin at 50 μg/kg, while the trueness was tested using six replicates, making use of oat flour as the CRM, and

Three samples, which were found to be the most contaminated with T-2 and HT-2 toxins, underwent thermal processing in terms of cooking, roasting and extrusion. These samples and the concentrations of T-2/HT-2 toxins in them were as follows: a maize sample (summary toxin concentration 384 μg/kg that of T-2128 μg/kg and that of HT-2256 μg/kg), an oat sample (summary toxin concentration 267 μg/kg that of T-2107 μg/kg and that of HT-2160 μg/kg) and a triticale sample (summary toxin concentration 151 μg/kg, that of T-2 47.0 μg/kg, and that of HT-2104 μg/kg). From each sample, three parallels were used during processing, and after

The above described contaminated maize, oat and triticale samples were cooked in boiling water (96°C) for 10, 20 and 30 min. After that, cereals were filtered and the samples were left to dry overnight. As for roasting, the contaminated cereals were roasted in an oven (LV9/11/P 320, Nabertherm, Germany) at three different temperatures (180°C, 200°C and 220°C) for 30 min (at each temperature). Once cooked and roasted, the cereals were milled into a fine powder having a particle size of 1.0 mm using an analytical mill (Cyclotec 1093, Tecator, Sweden), intended to be analysed for the levels of T-2 and HT-2 toxin using the LC-MS/MS method.

Before the extrusion cooking, all samples were milled using an IKA MF10 laboratory mill (IKA Werke GmbH, Staufen, Germany) having a 2-mm sieve. The blend preparation was performed based on 1 kg d. m. The samples were conditioned at 25% moisture by spraying an adequate amount of distilled water, while continuously mixed using a laboratory mixer (Kenwood KMM020, JVC Kenwood, Uithoorn, The Netherlands). The prepared mixtures were then put into plastic bags (one bag per sample) and stored overnight in the refrigerator at 4°C in order to equilibrate the moisture. Before the extrusion, the samples were brought to room temperature. The prepared samples were extruded in a single-screw laboratory extruder (Brabender GmbH, Model 19/20DN, Duisburg, Germany) at three different temperature profiles: 135/150/150°C; 135/170/170°C and 135/190/190°C (extruder's dosing/compression/ejection zone). Other constant extrusion parameters were as follows: screw: 4:1; die: 4 mm; screw speed: 100 rpm; and

dosing speed: 40 rpm. After the extrusion, the samples were air-dried overnight.

according to the 712:2009 ISO standard [24].

In all cereal samples, the moisture content was measured before and after thermal processing by taking a 5-g sample and heating it in an oven at 105 ± 2°C. Moisture was determined

later compared with the values assigned for each mycotoxin by the manufacturer.

46 Fusarium - Plant Diseases, Pathogen Diversity, Genetic Diversity, Resistance and Molecular Markers

that three replicates of each were used for the analyses.

**2.5. Thermal processing**

Concentrations (μg/kg) obtained by the ELISA assay are expressed as mean summary values (T-2/HT-2) ± standard deviation (SD), while in toxin-positive samples, the concentrations are given as individual concentrations of each mycotoxin (T-2 or HT-2) obtained by the LC-MS/ MS. Statistical analysis was performed using the Statistica ver. 10.0 software (StatSoft Inc. 1984-2011, Tulsa, OK, USA) and made use of the analysis of variance (ANOVA), the statistical significance thereby being set at 95% (p = 0.05).
