Conselho Nacional de Desenvolvimento Científico e Tecnológico / This study was conducted to investigate the effect of aflatoxins consumption (AFL), in broiler breeders of different strains. To evaluate reproductive and productive aspects of broiler breeders intoxicated with AFL were used 660 females and 60 males, distributed in a completely randomized design with factorial arrangement with three levels of AFL (0.0, 0.50 and 1.0 mg/kg diet) and two strains, Cobb 500® (LA) and Hybro PG® (LB), totalizing six treatments with five replicates pens of 22 females and 2 males each. The period of intoxication was from 24th to the 64th weeks of age, from 65th to the 70th weeks; all birds were fed a AFL free diet, with the objective of assessing the residual effects. To evaluate the progenies, three experiments were performed from 1 to 21 days. Each one used 600 day old males, from eggs produced by the breeders of the experiment above, at 32, 48 and 64 weeks of age. For each experiment, the same design used in the evaluation of breeders was used, in a total of six treatments with 10 replicates of 10 males each. The laying rate was depreciated by the addition of AFL in the diet. This negative effect was more pronounced when it was added 1mg of AFL/kg, indicating a dose-dependent effect. LB breeders had higher eggs production than LA. Breeders from LA, intoxicated with 0.5mg AFL/kg diet, showed a greater reduction in egg production than birds LB, intoxicated with the same level of AFL. The hatching was not influenced by the AFL in the breeders diets. LB breeders had a poorest fertility and hatching. The hatchability of eggs from breeders intoxicated with 1mg AFL/kg of diet was the lowest, caused by an increase in embryo mortality in the third week of incubation. Evaluating the interaction among AFL and strains, there was a negative effect on the fertility rate only in the LB, when 0.5 or 1.0mg AFL/kg were added to the diets. The progeny from breeders intoxicated with 1mg AFL/kg diet had the lowest body weight at birth and 21 days, in the three experiments. LA breeders produced the heaviest chicks at birth and at 21 days. Studying the interaction AFL and strain, it was observed that AFL intoxication in LB did not affect progeny s initial weight, but in LA, there was a decrease in day old chicks weight produced by intoxicated breeders. The studied levels of AFL influenced negatively the performance of breeders and their progeny. The two strains showed different responses in some of the parameters studied, when they were fed with diets containing AFL. / Este estudo foi conduzido para investigar o efeito do consumo de aflatoxinas (AFL) por matrizes de corte de diferentes linhagens. Para avaliar aspectos produtivos e reprodutivos de matrizes de corte intoxicadas com AFL, foram utilizadas 660 fêmeas e 60 machos, distribuídas em delineamento inteiramente casualizado em arranjo fatorial, com três níveis de AFL (0,0; 0,50 e 1,0 mg/kg de dieta) e duas linhagens, Cobb 500® (LA) e Hybro PG® (LB), totalizando seis tratamentos com cinco repetições de 22 fêmeas e 2 machos cada. O período de intoxicação foi da 24ª à 64ª semana de idade, da 65ª à 70ª semana, todas as aves receberam dietas isentas de AFL, com a finalidade de avaliar efeitos residuais. Para avaliar as progênies, foram realizadas três avaliações de 1 a 21 dias. Cada uma utilizou 600 pintos machos de um dia, provenientes de ovos produzidos por matrizes do experimento acima, na 32ª, 48ª e 64ª semanas de idade. Para cada avaliação, foi adotado o mesmo delineamento utilizado na avaliação das matrizes, totalizando seis tratamentos com 10 repetições de 10 machos cada. A taxa de postura foi depreciada pela adição de AFL na dieta. Esse efeito negativo foi mais acentuado quando foi adicionado 1mg de AFL/kg, indicando um efeito dose dependente. Aves LB apresentaram uma produção de ovos superior a da LA. Matrizes LA, intoxicadas com 0,5mg AFL/kg de dieta, apresentaram uma redução maior na produção de ovos que aves LB, intoxicadas com o mesmo nível de AFL. A eclosão não foi influenciada pelas AFL na dieta das matrizes. Aves da LB apresentaram pior fertilidade, reduzindo a taxa de eclosão da mesma. A eclodibilidade de ovos provenientes de aves intoxicadas com 1mg AFL/kg de dieta foi inferior às demais, devido a um aumento da mortalidade embrionária na terceira semana de incubação. Avaliando a interação entre AFL e linhagens sobre a taxa de fertilidade, verificou-se uma redução desta na LB, quando 0,5 ou 1,0mg AFL/kg foram adicionadas às dietas. A progênie de matrizes intoxicadas com 1mg AFL/kg de dieta apresentou um peso corporal inferior às demais, no primeiro e 21º dia, nas três avaliações realizadas. Matrizes da LA produziram pintos mais pesados ao nascer e aos 21 dias. Estudando a interação AFL e linhagens, observou-se que a intoxicação por AFL em matrizes da LB não afetou o peso inicial da progênie, porém, na LA, observou-se um decréscimo no peso de um dia de pintos produzidos por matrizes intoxicadas. Os níveis de AFL estudados prejudicaram o desempenho das matrizes e de suas progênies. As duas linhagens apresentaram respostas distintas em alguns dos parâmetros estudados, quando as mesmas foram submetidas à dietas contendo AFL.

M.Tech. / Fungi and their mycotoxins are major contaminants of cereals and maize which is the major staple food for Angolan population. The consumption of fungal and mycotoxins contaminated maize can induce health problems as these mycotoxins are known to induce immune suppression and other chronic diseases known as mycotoxicoses. In addition, to these mycotoxins also induce chronic diseases such as cancers of various types (liver, oesophageal, brain). Mycotoxins also immensely affect organs such as fragility and haemorrhage in tissues, liver-necrosis, bile-duct proliferation, caustic effects, intestinal haemorrhage and diarrhoea, adenomatosis, tremors, inco-ordination, mania, coma, photosensitization, sloughing of the extremities, nephrosis, uremia, infertility, prolonged oestrus, huge implications and infections in people with Human immunodeficiency virus (HIV)- AIDS. There is a very little known about the incidence of mycotoxins in Angola, which are associated with Food, Environment and Health. Therefore, the aim of this scientific novel study in history of Angola was to analyse maize samples obtained from major markets in Angola and to establish their quality with regard to mycotoxins and fungi and well as to establish possible links to the prevalent diseases as well as chronic diseases recorded among Angolan populations. To attain these aims, 60 samples of maize were randomly selected and purchased from open rural and nonregulated informal (dusty and food exposed to open air) markets around Luanda, Angola. Luanda, being the capital city of Angola was found to be the best place to sample, because of the presence of huge markets in which food products as well as maize produced in different provinces are delivered and sold to its high population. Thus, sampling in these markets was representative of the whole country and the results obtained give an approximate the status of the quality of maize consumed in different parts of Angola. The result from this study approximates the status of possible health risks that consumers can be exposed due to consumption of these mycotoxins. The samples were analysed in the Food Environmental and Health Research Laboratories at the University of Johannesburg for fungi and mycotoxins contamination using fungal screening methodologies. In terms of mycotoxins extraction, a multi-mycotoxin (solvent-solvent) extraction, solid phase extraction using SAX column for fumonisins (FBs) and immuno-affinity column (VICAM) were used for confirmation of results. Normal thin layer chromatography (TLC), reversed phase thin layer chromatography (RPTLC) (both for FBs confirmation), fluorometer “VICAM” and high performance liquid chromatography (HPLC) were techniques used for mycotoxins detection, identification and quantification. From the results obtained in this study, fungal isolation revealed contamination of the three most important toxigenic species: Aspergillus, Penicillium, Fusarium spp.

Deoxynivalenol (DON) contamination of grain destined for animal feeds is a major toxicological risk to monogastrics and is suspected of restricting productivity in ruminants. Whereas bacterial additives have been developed that can detoxify DON in the rumen and lower intestine, there are currently no commercial inoculants able to perform this task in crimped grain (CG) silage, a regionally important method of moist grain preservation based on homo- and heterofermentative lactic acid bacteria or chemical additives. Determining whether this ensiling process alongside the action of detoxifying bacteria has the potential to remove DON in CG prior to ingestion, was explored in mini-silo ensiling experiments. CG was heat treated (100 °C, 60 min) or ensiled fresh in triplicate 50 g silos, spiked with 5 mg/kg DON and inoculated with lactic acid bacteria derived from wild birds, natural epiphytic inoculants and commercially sourced silage additives (21 d). DON recovery was only significantly reduced (31.2 ± 14.4% recovery, p < 0.001, n= 30) by heat treatment, as determined by IAC-RP-HPLC-UV. Bacterial assemblage analysis by 16S rRNA PCR-DGGE-SEQ identified Weissella cibaria, Pantoea agglomerans, Bacillus subtilis, B. licheniformis and Hafnia alvei as candidate detoxification agents, of which W. cibaria and H. alvei decreased DON recovery in vitro (11.3 and 6.2% recovery respectively, p < 0.05, n = 18), which translated to inoculated W. cibaria yielding a decrease in DON recovery (67.2± 14.4%, 28 d) in naturally contaminated crimped wheat (13.5 ± 1.0 mg/kg, 35-40% moisture, p < 0.05, n =15). As W. cibaria is a lactic acid bacteria already associated with fermented CG by default it has promise as a novel DON detoxification agent in CG silage. DON is however just one of many hepatotoxic co-contaminants. Retrorsine, a DNA-crosslinking pyrrolizidine alkaloid derived from Ragwort (Senecio sp.) was investigated for interactive toxicity with DON in an in vitro co-exposure experiment. HepG2 cells were exposed to Log10 multifactorial binary exposures for 48 h followed by a suite of assays to elucidate mechanisms of interactive cytotoxicity, genotoxicity and modulation of the proteome. Retrorsine was tentatively confirmed to form DNA/protein crosslinks in the comet, micronucleus and crosslinking assays, whilst DON was found to potently induce cytotoxicity and apoptosis. Co-exposure yielded a complex toxicity response, with low doses yielding antagonistic effects and high doses trending towards additive effects, although DON dose was generally the principle component. The difficulties associated with undertaking an interactive toxicity study where both toxins have multiple metabolic and cellular targets are highlighted.

As condições climáticas presentes nas regiões produtoras de trigo, do Brasil e dos principais países do qual o produto é importado, favorecem o aparecimento de doenças importantes desta cultura, dentre elas a fusariose, causada principalmente pelo fungo Fusarium graminearum Schwabe. Além dos danos diretos causados pela doença, os grãos infectados podem ser tóxicos para o homem e animais devido à presença de micotoxinas especialmente o desoxinivalenol (DON). Um total de 100 amostras de trigo, sendo 50 de trigo nacional (provenientes do Estado de São Paulo, Paraná e Rio grande do Sul) e 50 de trigo importado (Argentina e Paraguai) foram coletadas de empresas que normalmente comercializam ou processam trigo durante o período de maio a dezembro de 2005. Foram avaliados o percentual de freqüência de fungos, especialmente Fusarium graminearum, a contaminação com DON, percentual de grãos giberelados e realizadas correlações entre os parâmetros avaliados. Os resultados indicaram que freqüência média de Fusarium graminearum e F. spp. foram baixas (&#8804;2,6 e &#8804;3,6%, respectivamente), porém foi maior no trigo nacional do que no trigo importado. Do total de amostras avaliadas 94% do trigo nacional e 88% do trigo importado apresentaram-se contaminadas com DON em níveis médios de 332 µg.kg-1 (nacional) e 90 µg.kg-1 (importado). Existiu correlação positiva e significativa entre contaminação com DON e percentual de grãos giberelados (r = 0,83; p<0,0001), freqüência de Fusarium graminearum (r = 0,92; p<0,0001) e freqüência de Fusarium spp. (r = 0,86; p<0,0001). / The climatic conditions present in wheat producing areas from Brazil and main countries from which the product is imported favor the occurrence of important diseases in this crop, among them the Fusarium Head Blight or scab. It is mainly caused by the fungus Fusarium graminearum Besides, direct damages caused by this disease, the infected kernels may be toxic for humans and animals due to presence of mycotoxins (e.g deoxynivalenol). A total of 100 wheat samples, being 50 from national production (São Paulo, Paraná and Rio Grande Do Sul states) and 50 from imported one (Argentina and Paraguay), were collected during the period of May to December 2005 from companies that normally commercialize or process wheat. Frequency (%) of fungi occurrence, specially Fusarium graminearum and Fusarium spp., DON contamination and Fusarium damaged kernels (%) were evaluated. Correlations between the evaluated parameters were carried out. Frequency of Fusarium graminearum and Fusarium spp. were low (&#8804;2.6 and &#8804;3.6%, respectively), however it was higher in Brazilian wheat when compared with imported wheat. Ninety-four percent of national wheat samples and 88% of the imported samples were DON contaminated (mean levels, 332 µg.kg-1 and 90 µg.kg-1, respectively). The occurrence of DON was highly correlated with percentage of Fusarium damaged kernels, (r = 0,83; p<0.0001), percentual frequency of Fusarium graminearum (r = 0,92; p<0,0001) and percentual frequency of Fusarium spp. (r = 0,86; p<0,0001).

The objective of this study was to evaluate the reduction on the levels of mycotoxins in wheat products and by-products: deoxynivalenol (DON) in whole wheat flour, wheat bran and the efluent from wet milling of wheat flour, and zearalenone (ZEN) in wheat bran. Firstly, the reduction of DON contamination was studied on whole wheat flour, naturaly contaminated, and considering different moisture levels, as well as in wet milling effluent of wheat flour. Further, the impact of the ozonation process on the rheological properties of the processed flour was evaluated. Secondly, the wheat bran naturally co-contaminated with DON and ZEN was studied, considering the degradation of both mycotoxins and the impact of the ozonation process on the bran phenolic compound content and on the antioxidant capacity. The DON degradation in the whole wheat flour increased with both processing time and moisture content. By changing these process parameters, it was possible to obtain products in accordance with the legal limits of Brazil and the European Union, even starting with concentration 2-4 times higher than the legal limits. However, the rheological properties of the whole wheat flour were affected by the process, probably due to protein modifications. The DON concentration on the wet milling effluent was linearly reduced by the ozonation. In wheat bran naturally contaminated and in its equilibrium moisture, the ozonation reduced both DON and ZEN contamination. The degradation of ZEN was higher and faster than the degradation of DON, which could be explained by their molecular structures. It was also observed that the ozonation process did not negatively affect the phenolic compounds and the antioxidant capacity, which is high desirable from a nutritional point of view. Consequently, this work concludes that the ozonation process was effective in reducing DON and ZEN in different wheat products and efluent. It is noteworthy that the results obtained are promising for future studies and to elucidate the mechanism of action of ozone on mycotoxins and constituents of food. / O objetivo deste estudo foi avaliar a redução nos níveis de micotoxinas em produtos e subprodutos de trigo: desoxinivalenol (DON) em farinha de trigo integral, farelo de trigo e efluente de moagem da farinha de trigo e zearalenona (ZEA) em farelo de trigo. No primeiro momento, a redução da contaminação por DON foi estudada em farinha de trigo integral, naturalmente contaminada e considerando diferentes níveis de umidade, bem como no efluente de moagem úmida da farinha. Além disso, o impacto do processo de ozonização nas propriedades reológicas da farinha processada foi avaliado. Em segundo lugar, estudou-se o farelo de trigo naturalmente co-contaminado com DON e ZEA, considerando a degradação de ambas as micotoxinas e o impacto do processo de ozonização no conteúdo do composto fenólico do farelo e na capacidade antioxidante. A degradação de DON na farinha de trigo integral aumentou tanto com o tempo de processamento quanto com o teor de umidade. Ao alterar esses parâmetros de processo, foi possível obter produtos de acordo com os limites legais do Brasil e da União Européia, mesmo com a concentração 2-4 vezes superior aos limites legais. Contudo, as propriedades reológicas da farinha de trigo integral foram afetadas pelo processo, provavelmente devido a modificações de proteínas. A concentração de DON no efluente de moagem úmida foi linearmente reduzida pela ozonização. Em farelo de trigo naturalmente contaminado e em sua umidade de equilíbrio, a ozonização reduziu a contaminação DON e ZEA. A degradação do ZEA foi maior e mais rápida que a degradação do DON, o que poderia ser explicado pelas suas estruturas moleculares. Observou-se também que o processo de ozonização não afetou negativamente os compostos fenólicos e a capacidade antioxidante, o que é altamente desejável do ponto de vista nutricional. Consequentemente, este trabalho conclui que o processo de ozonização foi efetivo na redução de DON e ZEA em diferentes produtos de trigo e efluentes. Vale ressaltar que os resultados obtidos são promissores para futuros estudos e elucidar o mecanismo de ação do ozônio sobre micotoxinas e constituintes dos alimentos.

A castanha-do-brasil é uma planta de grande importância econômica na região da Amazônia. Os baixos níveis tecnológicos característicos de sua cadeia produtiva e as condições inadequadas de manejo da matéria prima, favorecem o aparecimento de contaminação por fungos produtores de micotoxinas. A fumonisinas B2 (FB2) é uma micotoxina produzida por espécies de Fusarium e Aspergillus section Nigri, e é motivo de preocupação para a saúde humana e de animais. O objetivo do presente trabalho foi verificar o potêncial toxigênico quanto à produção de fumonisina B2 das cepas de A. section Nigri isolados de castanha-do-brasil e verificar a contaminação desse alimento por essa micotoxina. De um total de 200 cepas de A. section Nigri testadas quanto à capacidade de produção de fumonisina B2, apenas 39 cepas (19,5 %) produziram FB2, cento e trinta e oito (69 %) não foram produtoras de fumonisina B2 e 23 cepas (11,5 %) apresentaram picos menores que o limite de detecção. Das 100 amostras de castanha-do-brasil analisadas, nenhuma estava contaminada por fumonisina B2. / Brazil nut is a plant with vast economic importance in the Amazon region and is an important product exported by Brazil. However, low levels of technology, and inadequate management of raw material favor the appearance of contamination by fungi that produce toxic metabolites called mycotoxins. Fumonisin B2 (FB2) is a mycotoxin produced by Fusarium species and Aspergillus section Nigri, and is of concern for human health. The aim of this study was to assess the toxigenic potential for fumonisin B2 production by Aspergillus section Nigri strain isolated from brazil nut and check the contamination of food by this mycotoxin. From a total of 200 strains of Aspergillus section Nigri tested for the ability to produce fumonisin B2, only 39 strains (19.5 %) were producing FB2, 138 (69 %) were not producing fumonisin B2, and 23 strains (11.5 %) showed peaks less than the detection limit. Of the 100 samples of brazil nuts analyzed, none were contaminated with fumonisin B2.

<p>Apstrakt je obrađen tehnologijama za optičko prepoznavanje teksta (OCR)</p><p>Ispitano je prisustvo plesni u začinskim sme&scaron;ama (za proizvodnju mortadele, mesnog nareska, srpske Ijute kobasice, alpske i tirolske salame, vir&scaron;le i &scaron;unkarice), crnom biberu u zrnu i mlevenoj začinskoj paprici. Intenzitet kontaminacije bio je najveći u začinskim sme&scaron;ama (46,13%), zatim crnom biberu (28,95%) i začinskoj paprici (24,92%).<br />Uočena je dominacija grupe kserofilnih plesni iz rodova Aspergillus, Emericella, Eurotium, Paecilomyces, Penicillium i Xeromyces. Konstatovano je da su u začinskim sme&scaron;ama i crnom biberu najbrojniji bili predstavnici rodova Aspergillus i Penicillium, ali su po zastupljenosti u uzorcima (100%) dominirale plesni roda Eurotium {E. herbariorum). U uzorcima začinske paprike najče&scaron;će su detektovane plesni roda Rhizopus (R. stolonifer) (87,5%).<br />Od ukupno 45 vrsta identifikovanih tokom mikolo&scaron;kih ispitivanja začina, 55,5% je bilo potencijalno toksigenih. Prisustvo aflatoksina B1 nije konstatovano ni ujednom od začina, ohratoksin A je pronađen kod dva uzorka crnog bibera (u tragovima; 32,00 &mu;g/kg), a zearalenon kod tri uzorka crnog bibera (192,00 do 288,00 &mu;g/kg).<br />Sposobnost sinteze ohratoksina A utvrđena je kod 4 od 5 testiranih sojeva Aspergillus ochraceus (20,00 do 340,00 &mu;g /1). Većina sojeva A. versicolor i 50% E. herbariorum sintetisali su sterigmatocistin u značajnim količinama (100,00 do 1600,00 &mu;g /1, odnosno l60,00 do 400,00 &mu;g /l).<br />A. versicolor se pokazao prilično tolerantnim na uzlaganje visokim temperaturama, preživev&scaron;i toplotni tretman na 800C (5 i 10 min), ali je naru&scaron;ena fiziolo&scaron;ka aktivnost rezultovala smanjenjem proizvodnje sterigmatocistina.</p><p>Koncentracijom kalijum sorbata od 0,5% u potpunosti je inhibiran rast A. versicolor na čvrstoj hranljivoj podlozi, ali ne i u tečnoj; proizvodnja sterigmatocistina registrovana je samo pri nižim koncentracijama (0,05 i 0,1%).</p> / <p>Abstract was processed by technology for Optical character recognition (OCR)</p><p>The presence of moulds in spice mixtures (for production of mortadella, luncheon meat, &quot;serbian&quot; hot sausage, &quot;alpine&quot; and &quot;tirol&quot; salami, Frankfurter and ham sausage) was investigated. The highest intensity of contamination was in spice mixtures (46,13%), then in black pepper (28,95 %) and in ground red pepper (24,92%).<br />The group of xerophyllic moulds of genera Aspergillus, Emericella, Eurotiiim, Paecilomvces, Penicillium and Xeromyces was dominant. It was found out that the representatives of genera Aspergillus and Penicillium were the most numerous, but considering the incidence in samples (100%) the moulds of genus Eurotium (E. herbariourum) were dominant. In samples of ground red pepper the moulds of the genus Rhizopus (R. stolonifer) (87,5%) were the most frequently detected. Among 45 species identified during mycological investigation of spices, 55,5% were possibly toxicogenic. The presence of aflatoxin B1 was not observed in any of spices tested, ochratoxin A was found in two samples of black pepper (from traces to 32,00 &mu;g/kg), and zaeralenone in three samples of black pepper (192,00 to 288,00 &mu;g/kg).<br />The ability for synthesis of ochratoxin A was observed in 4 of 5 tested strains of Aspergillus ochraceus (20,00 to 340,00 &mu;g/1). The majority of A. versicolor strains and 50% of E. herbariorum synthesized sterigmatocystin in significant quantities (100,00 up to 1600,00 &mu;g/1, and l60,00 up to 400,00 &mu;g/l, respectively).</p><p>A. versicolor showed to be rather tolerant to exposure to high temperatures by surviving the heat treatment at 800C (5 and 10 min), but the disturbed physiological activity resulted in reduced production of sterigmatocystin.<br />By concentration of potassium sorbate of 0,5% the growth of A. versicolor on solid medium was totally inhibited, but not on liquid medium; the production of sterigmatocystin was registered only at lower concentrations (0,05 and 0,1%).</p>

Mycotoxins are secondary metabolites of moulds, which attack cereals, for example barley, from which mycotoxins then get to beer. This submitted work is focused on ochratoxin A, deoxynivalenol and zearalenone, which can occur in beer. The first part of this master’s thesis consists of literary research, which describes mycotoxins in general, points out their occurrence, prevention of their formation and delivers information about their physical and chemical properties and toxicity. Furthermore, the research contains basis of malt and beer technology, the occurrence of mycotoxins in beer and raw materials for its production. The research describes changes in concentration of mycotoxins across malt and beer production. The next part deals with possibilities of determination of mycotoxins in barley, malt and beer, compares individual methods of their determination and points out many difficulties of some analyses. The experimental part of this work pursues determination of ochratoxin A, deoxynivalenol and zearalenone in different types of beer with the help of UPLC-FLR, HPLC-MS and ELISA. Instrumental techniques are validated and gathered results are compared with the results in literature. The goal of this master’s thesis is to assess the stability of ochratoxin A and deoxynivalenol in beer over time. The gained results show that there are changes in the concentration of ochratoxin A over time, nevertheless those changes show no pattern. Overall, there was a decrease in concentration in 47 % of the samples and an increase in 28 % of them. In the rest of the samples the concentration did not change. The concentration of deoxynivalenol does not change over time. One of the other goals of this thesis is monitoring of selected mycotoxins in beer. The average concentration of ochratoxin A in the samples was 39 ng/l and deoxynivalenol 9,9 g/l. Zearalenone did not occur in any of the samples when determined by liquid chromatography. All results agree with literature. Next, the thesis compares different analytical methods for determination of ochratoxin A, deoxynivalenol and zearalenone. The screening method ELISA is compared to UPLC-FLR and HPLC-MS. The determination of ochratoxin A by ELISA has shown to be time consuming, nevertheless the results responded to instrumental technique. ELISA overestimated the results of determination of deoxynivalenol in beer by 363–697 % and with zearalenone there were found false positive results.

The presented thesis deals with the issue of mycotoxins in brewing materials and beer. Attention was devoted mainly to the selected fusarium mycotoxins (deoxynivalenol, zearalenol, T-2 toxin, and HT-2 toxin) ochratoxin A and aflatoxins B1, B2, G1, and G2. The aim of the thesis was to optimize and validate analytical methods for the determination of the above mentioned mycotoxins in the brewing materials and beer. Analytes were separated using high-performance liquid chromatography with mass – spectrometric detection (HPLC-MS/MS) and ultra-performance liquid chromatography with fluorescence detection (UPLC/FLR). These analytical methods were then applied for mapping the occurrence of fusarium mycotoxins in malting barley crops in the Czech Republic and monitoring the level of contamination with mycotoxins in malting and brewing industries. In addition, experiments studying over-foaming of beer were conducted as primary gushing – over-foaming of beer – is connected, similarly as mycotoxins, with the presence of microscopic filamentous fungi in the raw materials for beer production. Studies describing in detail these methods are part of this thesis (Annex I – V). From all published results, it is evident that the occurrence of mycotoxins in cereals including barley is natural and cannot be completely prevented, not even if all conditions of correct agricultural practice are observed. It is known that some mycotoxins present in contaminated malting barley pass to the final product – beer due to their chemical and physical properties. However, the mycotoxin concentrations found do not mean any significant health risk for consumers.

Les mycotoxines sont de métabolites secondaires produits par de nombreuses espèces fongiques. Les effets sanitaires induits par l’ingestion de ces substances sont bien documentés et certaines mycotoxines font désormais l’objet de réglementations quant à leurs teneurs maximales tolérables dans les aliments. Cependant, d’autres voies d’exposition à ces contaminants sont possibles. Si l’action irritante ou allergisante liée à l’inhalation de spores fongiques ou de fragments mycéliens a été démontrée, l’inhalation de mycotoxines est aussi suspectée d’induire certains troubles respiratoires ou certaines pathologies. En effet, les mycotoxines peuvent être retrouvées dans les spores mais également sur des particules plus fines facilement aérosolisables et donc susceptible d’être inhalées. Cependant, les données concernant le danger associé à l’exposition humaine aux mycotoxines par inhalation sont encore très parcellaires. Dans ce contexte, nos travaux ont eu comme objectif principal la caractérisation de l’aérosolisation des mycotoxines lors de la colonisation de différents matériaux rencontrés dans les environnements intérieurs par des moisissures toxinogènes. Tout d’abord nous avons étudié la croissance et la production de mycotoxines lors de la colonisation de matériaux de construction (papier peint, toile de verre peinte, papier peint vinyle, sapin) par trois espèces fongiques d’intérêt: Aspergillus versicolor, Penicillium brevicompactum, Stachybotrys chartarum. Ces espèces ont été choisies à cause de leur présence fréquente dans les environnements intérieurs et de leur diversité d’organisation mycélienne. De plus, ces trois espèces produisent des toxines différentes: stérigmatocystine, acide mycophénolique et trichothécènes macrocycliques pour A. versicolor, P. brevicompactum et S. chartarum, respectivement. Ces travaux ont démontré que, pendant leur développement sur les matériaux testés, les trois espèces produisent des mycotoxines. Le matériau le plus favorable au développement fongique et à la toxinogénèse est le papier peint. L'acide mycophénolique, la stérigmatocystine et les trichothécènes macrocycliques peuvent ainsi être produits à des niveaux de 1.8, 112.1 et 27.8 mg/m2, respectivement, sur ce matériau. Ces toxines peuvent ensuite être partiellement aérosolisées. Nous avons montré que l’aérosolisation dépend des espèces et de leur structure mycélienne mais aussi des conditions de culture et du flux d’air. Ce transfert dans l'air est observé après des sollicitations aérauliques qui peuvent être rencontrées facilement dans les environnements intérieurs car elles correspondent au mouvement de personne dans une pièce (0.3 m/s), à la vitesse de l'air dans les diffuseurs de plafond (2 m/s), à des coutants d’air ou des claquements de porte (6 m/s). P. brevicompactum est l’espèce la plus facile à aérosoliser. La majeure partie de la charge toxique des aérosols est retrouvée dans des particules dont la taille correspond à celle de spores ou de fragments de mycélium. Cependant, pour les trichothécènes macrocycliques, des toxines ont également été trouvées sur des particules plus petites que les spores, qui pourraient être facilement inhalées par les habitants et pénétrer profondément dans les voies respiratoires. Afin de mieux caractériser le danger réel associé à l’inhalation de ces composés, des études de cytotoxicité ont été réalisés en utilisant des cellules pulmonaires et en comparant avec les résultats observés sur cellules digestives. La toxicité sur cellules pulmonaires est comparable à celle observée sur cellules digestives. Les trichothécènes macrocycliques sont beaucoup plus toxiques que les autres toxines testées avec des IC50 de l’ordre du ng/ml. Au final, nous avons évalué la persistance de ces contaminants lors de l’application d’eau de javel, procédé de décontamination le plus fréquemment utilisé. Nous avons montré qu’une procédure de nettoyage normale ne permet qu’une élimination partielle des moisissures. / Mycotoxins are secondary metabolites produced by many fungal species. Health effects induced by the ingestion of these substances are well documented and some mycotoxins are now regulated for their maximum tolerable levels in foods. However, other routes of exposure to these contaminants are possible. Thus, if irritating or allergenic reactions related to the inhalation of fungal spores or mycelial fragments have been demonstrated, inhalation of mycotoxins is also suspected to be causing certain respiratory disorders or certain pathologies. Indeed, mycotoxins can be found in spores but also on finer particles which are easily aerosolized and therefore likely to be inhaled. However, data on the hazard associated with human exposure to mycotoxins by inhalation are still very fragmented. In this context, our main objective was to characterize the aerosolization of mycotoxins during the colonization of different materials encountered in indoor environments by toxinogenic molds. First we studied growth and production of mycotoxins during the colonization of building materials (wallpaper, painted fiberglass wallpaper, vinyl wallpaper, fir, fiberglass) by three fungal species of interest: Aspergillus versicolor, Penicillium brevicompactum, Stachybotrys chartarum. These species were chosen because of their frequent presence in indoor environments and their diverse mycelial organization. In addition, these three species produce different toxins: sterigmatocystin, mycophenolic acid and macrocyclic trichothecenes for A. versicolor, P. brevicompactum and S. chartarum, respectively. These studies have shown that, during their development on tested materials, three species produce mycotoxins. The most favorable material for fungal development and toxinogenesis is wallpaper. Mycophenolic acid, sterigmatocystin and macrocyclic trichothecenes can thus be produced at levels of 1.8, 112.1 and 27.8 mg/m2, respectively, on this material. These toxins can then be partially aerosolized. We have shown that aerosolization depends on species and their mycelial structure, but also on culture conditions and airflow. This transfer to air is nevertheless observed after aeraulic solicitations which can be easily encountered in indoor environments because theycorrespond to the movement of people in a room (0.3 m/s), speed of air in ceiling diffusers (2 m/s), slamming doors or air drafts when opening windows(6 m/s). P. brevicompactum showed to be the easiest to aerosolize. The major part of the aerosols’ toxic charge is found in particles whose size corresponds to that of spores or mycelial fragments. However, for macrocyclic trichothecenes, toxins were also found in particles smaller than spores, which could easily be inhaled by occupants and penetrate deep into the respiratory tract. In order to better characterize the actual hazard associated with inhalation of these compounds, cytotoxicity studies have been performed using lung cells and comparing with results observed on digestive cells. Pulmonary toxicity is comparable to that observed in digestive cells. Macrocyclic trichothecenes are much more toxic than other tested toxins with IC50 in order of ng/ml. In parallel, we analyzed the VOCs specifically produced during active mycotoxinogenesis in order to identify potential biomarkers of the actual production of mycotoxins that could be used as tools for monitoring of indoor environments. Unfortunately, this approach has not, for the moment, led to the identification of specific targets. In the end, we evaluated the persistence of these contaminants during application of bleach, the most frequently used decontamination process. We have shown that a normal cleaning procedure allows only partial removal of mold.