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.

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.

A aplicação de substâncias naturais com efeito antifúngico é motivada pela necessidade de alternativas aos métodos existentes que nem sempre são aplicáveis, eficientes ou sem risco de danos ao consumidor ou meio ambiente. Além disso, estudos para elucidar o comportamento de espécies toxigênicas mediante fungicidas naturais tornam-se necessárias, contribuindo dessa forma com a segurança alimentar. Neste trabalho, extrato fenólico de Spirulina foi utilizado para avaliar a atividade antifúngica sobre 12 cepas toxigênicas de Fusarium graminearum, isoladas de cevada e trigo. As rotas metabólicas que poderiam ser afetadas foram avaliadas através da determinação de compostos estruturais (glicosamina e ergosterol) e da atividade de enzimas do metabolismo primário dos micro-organismos. Os resultados indicaram que os extratos fenólicos reduziram a velocidade de crescimento das espécies toxigênicas estudadas. O IC50 foi obtido na concentração de 3% a 8% (p/p) de extrato fenólico. A utilização desse fungicida natural foi promissora para a inibição da multiplicação fúngica, em especial pela inativação dos sistemas enzimáticos (amilase e protease) de Fusarium graminearum.

The application of natural antifungal substances is motivated by the need for alternatives to existing methods that are not always applicable, efficient, or that do not pose risk to consumers or the environment. Furthermore, studies on the behaviour of toxigenic species in the presence of natural fungicides have enabled their safe application in the food chain In this study, Spirulina LEB-18 phenolic extract was assessed for its antifungal activity on 12 toxigenic strains of Fusarium graminearum isolated from barley and wheat. The susceptible metabolic pathways were assessed through the determination of structural compounds (glucosamine and ergosterol) and enzyme activity of the microorganisms' primary metabolism. The results indicate that phenolic extracts reduced the growth rate of the toxigenic species investigated. The IC50 was obtained by applying 3 to 8% (p/p) of phenolic compounds in relation to the culture medium. The use of this natural fungicide proved promising for the inhibition of fungal multiplication, especially in terms of the inactivation of enzymatic systems (amylase and protease) of Fusarium graminearum.

Este estudo comparou quatro procedimentos de extração para a determinação simultânea das micotoxinas citrinina e ocratoxina A em amostras de arroz. Os procedimentos de Soares e Rodriguez-Amaya, Tanaka e os métodos de extração QuEChERS (Quick, Easy, Cheap, Effective, Rugged and Safe) e por ultrassom foram comparados em termos de geração de resíduos, limite de detecção, limite de quantificação e recuperação simultânea de micotoxinas. O procedimento de Soares e Rodriguez-Amaya e a extração por ultrassom apresentaram as menores recuperações, 83 e 55% para a ocratoxina A (OTA) e 55 e 48% para citrinina (CIT), respectivamente. O procedimento de Tanaka e a extração QuEChERS tiveram recuperações de 98 e 105% para OTA e 64 e 78% para CIT, respectivamente. Este último permitiu a extração simultânea das duas micotoxinas com uma redução de até 25 vezes na quantidade de solventes.

This study compared four extraction procedures for the simultaneous determination of mycotoxins ochratoxin A and citrinin in samples of rice. Soares and Rodriguez-Amaya and Tanaka procedures and the extraction methods QuEChERS (Quick, Easy, Cheap, Effective, Rugged and Safe) and by ultrasound were compared in terms of residue generation, limit of detection, limit of quantification and simultaneous recovery of mycotoxins. Soares and Rodriguez-Amaya and the method by ultrasound presented the lowest recoveries, 83 and 55% for ochratoxin A (OTA) and 48 and 55% for citrinin (CIT), respectively. Tanaka procedure and QuEChERS method presented the highest recoveries, 98 and 105% for OTA and 64 and 78% for CIT, respectively. The latter enabled the simultaneous extraction of the two mycotoxins, with a reduction of up to 25 times of the amount of the involved solvent.

This study validated a simple and applied method for determining mycotoxins aflatoxin B1, aflatoxin B2, ochratoxin A, zearalenone and deoxynivalenol, in water from the rice production chain. Five solvent combinations for extraction were tested, with quantification performed by TLC/HPTLC and confirmation by LC-MS/MS. Mycotoxins in water from field and rice industries were evaluated. Mycotoxin recovery levels were around 90%. Two samples from rice parboiling waste were contaminated (deoxynivalenol/aflatoxin B1, 110/9 ng mL-1; and deoxynivalenol, 100 ng mL-1). Zearalenone, deoxynivalenol and ochratoxin A (36, 30 and 28%) were carried to soaking water during parboiling.

The present study was carried out to establish the optimal conditions for performing ochratoxin A (OTA) and citrinin (CIT) extraction using the QuEChERS method in rice. Employing the factorial experimental design, variables that significantly influenced the extraction stages were determined. The following variables were analyzed: addition of water, acidification of acetonitrile with glacial acetic acid, as well as amounts of magnesium sulfate, sodium acetate, sodium citrate and diatomaceous earth. The best combining procedure resulted in a predictive model using more water and less diatomaceous earth. Recoveries of CIT and OTA were 78-105%.

Este trabalho teve por objetivo avaliar a degradação de deoxinivalenol por Aspergillus oryzae e Rhizopus oryzae durante fermentação submersa e correlacioná-la com a atividade de enzimas oxidoredutases. O meio submerso foi constituído por água destilada estéril contaminada com 50 μg de DON e inóculo de 4 × 10(6) esporos.mL-1 de meio das espécies fúngicas Aspergillus oryzae e Rhizopus oryzae, separadamente em cada experimento. A amostragem foi realizada a cada 24 horas de processo para medida da atividade específica da enzima peroxidase, e a cada 48 horas para a determinação dos níveis de DON. Os resultados mostraram que as espécies fúngicas avaliadas possuem capacidade de metabolizar DON, acompanhada de aumento na atividade da enzima oxidativa PO. No intervalo de 48 horas de fermentação, ocorreu a maior atividade específica da enzima peroxidase (ΔABS/minute.μg.protein-1), 800 e 198, correspondendo a maior velocidade de degradação de DON de 10.8 e 12.4 ppb/hour, respectivamente para Rhizopus oryzae e Aspergillus oryzae.

This work aims to evaluate deoxynivalenol degradation by Aspergillus oryzae and Rhizopus oryzae in a submerged fermentation system and to correlate it to the activity of oxydo-reductase enzymes. The submerged medium consisted of sterile distilled water contaminated with 50 μg of DON and 4 × 10(6) spore.mL-1 inoculum of Aspergillus oryzae and Rhizopus oryzae species, respectively in each experiment. Sampling was performed every 24 hours for monitoring the peroxidase specific activity, and every 48 hours for determining mycotoxin levels. Results showed that the fungi species were able to decrease DON levels as the peroxidase activity increased. The 48 hours fermentation interval presented the highest peroxidase specific activity (ΔABS/minute.μg.protein-1), 800 and 198, while the highest DON degradation velocity was 10.8 and 12.4 ppb/hour, respectively in both cases for Rhizopus oryzae and Aspergillus oryzae.

O objetivo deste trabalho foi avaliar o efeito de deoxynivalenol e toxina T-2 na atividade de a e b-amilase em malte. A contaminação artificial foi de acordo com planejamento experimental completo nos níveis de 0 a 1000ng/g para cada micotoxina. O aumento da concentração da toxina T-2 inibiu a atividade da a-amilase. Entretanto, a coocorrência a altas concentrações das duas toxinas indicou efeito sinergistíco. Um aumento da atividade enzimática ocorreu na presença de altos níveis das micotoxinas avaliadas (1000ng/g de malte). Os tricotecenos interferiram na performance das enzimas aminolíticas na etapa de malteação, permitindo determinar um modelo preditivo para a atividade da β-amylase.

The objective of this work was to evaluate the effect of DON and T-2 toxin levels on the malt amylase activity. The malt was contaminated in accordance with central composite design experiment with DON and T-2 toxin levels until 1000 ng/g. The activities of the enzymes were evaluated by Bernfeld method. The increase in T-2 toxin concentration inhibited the α-amylase activity. However, the increase of both toxins concentration caused inverse effect. The interaction between toxins indicated synergetic effect on the β-amylase activity. An increased activity occurred when the toxins contamination levels in malt were higher (1000ng/g malt). The trichothecenes interfered with the performance of aminolitic enzymes in the stage of malting, resulting in a significant model for enzymatic activity of β-amylase.

O objetivo foi avaliar a capacidade das espécies fúngicas Aspergillus oryzae e Rhizopus oryzae em degradar desoxinivalenol (DON) em fermentação submersa. O meio submerso utilizado foi água destilada estéril contaminada com 1μg mL-1 DON, inoculado com 4 × 10(6) esporos mL-1 das espécies fúngicas. A amostragem foi realizada a cada 48 h até 240 h de processo, determinando a massa residual de DON, percentual e velocidade de degradação. A massa residual de DON no meio coletado foi extraída por partição líquido-líquido e quantificada por cromatografia gasosa através de derivação com anidrido trifluoroacético. O tempo requerido para a maior degradação de DON foi 96 e 240 h para Aspergillus oryzae e Rhizopus oryzae, respectivamente, e velocidade de degradação de 0,62 e 0,54 μg h-1, respectivamente. Rhizopus oryzae ocasionou a maior diminuição na concentração de DON, aproximadamente 90% em 240 h, enquanto Aspergillus oryzae ocasionou com maior rapidez, 74% de redução em 96 h.

The objective was to evaluate the capacity of Aspergillus oryzae and Rhizopus oryzae to degrade deoxynivalenol (DON) during submerged fermentation. The submerged medium utilized was sterile distilled water with 1μg mL-1 DON, added and inoculated with 4 × 10(6) spores mL-1 of the fungal species. Sampling was performed every 48 h to 240 h. DON analyses included residual mass, percentage and velocity of degradation. Residual mass of DON in the collected medium was extracted by liquid-liquid partition and quantified by gas chromatography through derivation with trifluoroacetic anhydride. The time required for the largest DON degradation was 96 h and 240 h by Aspergillus oryzae and Rhizopus oryzae respectively, and degradation rate were 0.62 and 0.54 μg h-1, respectively. Rhizopus oryzae caused the largest decrease in DON at around 90% in 240 h, while Aspergillus oryzae caused the most rapid degradation with a 74% reduction of DON at 96 h.