An investigation was conducted to standardize conditions for laccase and lipase activity required to maximize trichothecene degradation. The analytical methods were suitable and validated in model solutions for detection of five analytes (T-2 toxin, deoxynivalenol, nivalenol, 3-acetyldeoxynivalenol and 15-acetyldeoxynivalenol), the methods were then applied in the degradation tests of these trichothecenes. The method of salting-out assisted liquid-liquid extraction (SALLE), using three different NaCl concentration levels, obtained recoveries in the range of 47.4-103.4% at pH 5.0 and 36.6-106.8% at pH 7.0, with an intra-day relative standard deviation under 15% for the majority of the compounds. Quantification limits remained in the region of 0.07 µg mL-1 for 15-acetyldeoxynivalenol and 0.3 µg mL-1 for nivalenol. Finally, the suitable analytical methods were applied in a study of trichothecene degradation by enzymatic action, resulting in a reduction of 12.3% for T-2 toxin, 68.4% for deoxynivalenol, 50.2% for 3-acetyldeoxynivalenol and 45.4% for 15-acetyldeoxynivalenol.
ABSTRACT: Mycotoxin levels in Fusarium head blight (FHB) infections can be difficult to quantify. The relationship between mycotoxin and disease is not consistent and it is not clear if wheatpathogen interaction is of significance in regions where more than one Fusarium species with distinct trichothecene production ability co-exists. This study aimed to investigate whether a set of eight Brazilian wheat genotypes, varying in resistance according to classification by the breeder, exhibit a common or differential resistance to Fusarium graminearum (Fgra) (deoxynivalenolproducing) and Fusarium meridionale (nivalenol-producing) (Fmer) using full-spike and central spikelet inoculation (type II resistance). Fgra was generally more aggressive than Fmer based on the percentage of diseased spikes (99 and 84 %, respectively) and number of diseased spikelets (mean 2.8 and 2.0, respectively) below the central spikelet. The genotype-pathogen species interaction was not significant, but there were differences between the genotypes, with BRS 194 and BRS 327 being the least and most resistant, respectively, based on severity ratings. The incidence of Fusarium-damaged kernel (FDK, %) was not affected by species, but two genotypes (BRS Parrudo and BRS 327) showed a lower incidence of FDK. There were substantial variations in the accumulation of deoxynivalenol and nivalenol among the genotypes, reaching maxima of 691.2 µg g−1 and 355.2 µg g−1, respectively, suggesting that Fgra is a more potent producer of trichothecene. Our data confirm prior resistance classifications by the breeders and suggest that the use of a single highly aggressive Fgra isolate may be sufficient for effective screening for FHB resistance. However, further studies are needed to elucidate the accumulation of resistance to trichothecene.
This study established the conditions for the extraction of the enzyme peroxidase (PO) from soybean meal (SBM). An experimental design methodology was carried out in order to evaluate the effects of stirring rate time, pH and extracting solvent volume on the enzyme extraction. By using 5 g SBM and 50 mL phosphate buffer 10 mmol L-1 pH 4.7, 60 min stirring rate at 100 rpm, an enzyme with specific activity of 100 U mg-1 for SBM was obtained. Two techniques of purification were tested and compared for purification of peroxidase from soybean meal: triphasic partition (TPP) and molecular exclusion chromatography. TPP showcased a greater efficiency with 50% recuperation and a purification factor of 13.6. Peroxidase in crude and pure forms was characterized for kinetics, thermodynamics and biochemistry. The parameter of thermal inactivation indicates high stability to exposure time and temperature increase, showing that enzyme activity is not altered by the presence of constituents of the reaction medium. Peroxidase in crude form represented a greater upkeep in activity, keeping 50% activity for 114 days at 0 °C. Peroxidase in pure form had greater affinity for substrate and reduced Deoxynivalenol levels by 80%, 20% more than the crude form.