O objetivo deste estudo foi avaliar a presença de fungos e micotoxinas [aflatoxinas e ácido ciclopiazônico (ACP)] em amostras de castanha-do-Brasil coletadas em diferentes estados da região Amazônia brasileira: Acre, Amazonas, Amapá e Pará. Um total de 200 amostras de casca e 200 amostras de amêndoa foram semeadas em ágar Aspergillus flavus-parasiticus para pesquisa da micobiota. As micotoxinas foram analisadas por cromatografia líquida de alta eficiência. A morfológica clássica e a análise molecular revelou a presença dos seguintes fungos, em ordem decrescente de frequência: Phialemonium spp., Penicillium spp., Fusarium spp., Phaeoacremonium spp. e Aspergillus spp. A presença de aflatoxinas e ACP foi detectada em amostras de amêndoa, na frequência de 11% e 22%, respectivamente. Aspergillus nomius e Aspergillus flavus foram as espécies mais isoladas de Aspergillus. A presença de fungos não implica necessariamente na contaminação por micotoxinas, porém a amêndoa da castanha-do-Brasil parece ser um bom substrato para o crescimento de fungos. / The aim of this study was to evaluate the presence of fungi and mycotoxins [aflatoxin and cyclopiazonic acid (ACP)] in Brazil nut samples collected in different States of the Brazilian Amazon: Acre , Amazonas , Amapá and Pará. A total of 200 almond and 200 husk samples were plated on agar Aspergillus flavus - parasiticus for mycobiota isolation. Mycotoxins were analyzed by high performance liquid chromatography. The classical morphological and molecular analysis revealed the presence of the following fungi , in decreasing order of frequency: Phialemonium spp., Penicillium spp., Fusarium spp., Phaeoacremonium spp. and Aspergillus spp. The presence of aflatoxins and ACP was detected in almonds samples at a frequency of 11% and 22% , respectively. Aspergillus nomius and Aspergillus flavus species were the most isolated from Aspergillus. The presence of fungi does not necessarily imply the mycotoxin contamination, but the almond samples of Brazil nut seem to be a good substrate for the growth of fungi.

O amendoim é freqüentemente invadido por fungos toxigênicos antes da colheita. Este trabalho teve como objetivos: determinar a micoflora do solo, ar, sementes plantadas, flores, ginóforo e grãos e cascas de amendoim em diferentes fases de maturação e após a secagem; analisar aflatoxinas B1, B2, G1 e G2, ácido ciclopiazônico e fumonisinas B1 e B2 nas sementes plantadas, grãos e cascas de amendoim em diferentes fases de maturação e após a secagem. Os fungos mais freqüentes nos grãos e cascas de amendoim foram Fusarium spp. e Aspergillus flavus. A espécie A. flavus também foi encontrada em amostras do solo e do ar. Aflatoxinas e ácido ciclopiazônico foram detectados em 32 % das amostras de grãos de amendoim analisadas em concentrações que variaram de 4,20<font face=\"symbol\">mg/kg a 198,84 <font face=\"symbol\">mg/kg e de 260 <font face=\"symbol\">mg/kg a 600 <font face=\"symbol\">mg/kg, respectivamente. Nas cascas somente aflatoxinas foram detectadas em concentrações que variaram de 5,76 <font face=\"symbol\">mg/kg a 218,52 <font face=\"symbol\">mg/kg. Fumonisinas não foram detectadas. Boas práticas agrícolas são indicadas para região. / Peanuts are contaminated frequently by toxigenic fungi before harvest. the present study aimed to: identify the mycoflora of the soil, air, flower, pegs, peanut kernels and hulls in the different maturation stages and after drying; determinate the occurrence of aflatoxins B1, B2, G1 and G2, cyclopiazonic acid (CPA) and fumonisins B1 and B2 in peanut kernels and hulls in different maturation stages and after drying. Fusarium spp. Aspergullus flavus were the most frequent fungi isolated in peanuts hulls and kernels. A. flavus was isolated, also, in air and soil samples. Aflatoxins and cyclopiazonic acid were detected in 32% of the kernels samples in concentration from 4.20 <font face=\"symbol\">mg/kg to 198.84 <font face=\"symbol\">mg/kg and from 260 <font face=\"symbol\">mg/kg to 600 <font face=\"symbol\">mg/kg, respectively. In the peanut hulls only aflatoxins were detected in 24 % of the samples in concentration from 5.76 <font face=\"symbol\">mg/kg to 218.52 <font face=\"symbol\">mg/kg. Fumonisins were not detected. Good agriculture practices are indicated to region.

The aim of the study in this thesis was to assess the stability of the mycotoxin, cyclopiazonic acid (CPA) in milk and dairy products processed from contaminated milk. A method was developed to detect CPA in milk and milk products using micellar electrokinetic capillary chromatography (MEKC), a technique of capillary electrophoresis (CE), which was rapid and non-labour-intensive. The quantifying efficiency of CE in detecting CPA was compared to Reverse Phase Liquid Chromatography. Heat-stability of CPA in milk was assessed under different conditions. A longer heat treatment of 60 degrees centigrade for 30 minutes led to a 10% decrease in the level of CPA. The results from this thesis demonstrate that CPA in milk at concentrations found in naturally contaminated milk could not be eliminated by the heat-treatment during milk processing, storage, processing and manufacture of dairy products. Occurrence of CPA in cheese curd, butter or cream following manufacture with contaminated milk was demonstrated. CPA is left in milk despite UV-visible radiation treatment with or without hydrogen peroxide and/or riboflavin. Chemical treatment, which is capable of completely eliminating CPA, is prohibited and impractical to use for milk treatment. Stability of CPA in milk and milk products confirms the potential of the toxin to reach consumers of dairy products. / Doctor of Philosophy (PhD)

O amendoim é freqüentemente invadido por fungos toxigênicos antes da colheita. Este trabalho teve como objetivos: determinar a micoflora do solo, ar, sementes plantadas, flores, ginóforo e grãos e cascas de amendoim em diferentes fases de maturação e após a secagem; analisar aflatoxinas B1, B2, G1 e G2, ácido ciclopiazônico e fumonisinas B1 e B2 nas sementes plantadas, grãos e cascas de amendoim em diferentes fases de maturação e após a secagem. Os fungos mais freqüentes nos grãos e cascas de amendoim foram Fusarium spp. e Aspergillus flavus. A espécie A. flavus também foi encontrada em amostras do solo e do ar. Aflatoxinas e ácido ciclopiazônico foram detectados em 32 % das amostras de grãos de amendoim analisadas em concentrações que variaram de 4,20<font face=\"symbol\">mg/kg a 198,84 <font face=\"symbol\">mg/kg e de 260 <font face=\"symbol\">mg/kg a 600 <font face=\"symbol\">mg/kg, respectivamente. Nas cascas somente aflatoxinas foram detectadas em concentrações que variaram de 5,76 <font face=\"symbol\">mg/kg a 218,52 <font face=\"symbol\">mg/kg. Fumonisinas não foram detectadas. Boas práticas agrícolas são indicadas para região. / Peanuts are contaminated frequently by toxigenic fungi before harvest. the present study aimed to: identify the mycoflora of the soil, air, flower, pegs, peanut kernels and hulls in the different maturation stages and after drying; determinate the occurrence of aflatoxins B1, B2, G1 and G2, cyclopiazonic acid (CPA) and fumonisins B1 and B2 in peanut kernels and hulls in different maturation stages and after drying. Fusarium spp. Aspergullus flavus were the most frequent fungi isolated in peanuts hulls and kernels. A. flavus was isolated, also, in air and soil samples. Aflatoxins and cyclopiazonic acid were detected in 32% of the kernels samples in concentration from 4.20 <font face=\"symbol\">mg/kg to 198.84 <font face=\"symbol\">mg/kg and from 260 <font face=\"symbol\">mg/kg to 600 <font face=\"symbol\">mg/kg, respectively. In the peanut hulls only aflatoxins were detected in 24 % of the samples in concentration from 5.76 <font face=\"symbol\">mg/kg to 218.52 <font face=\"symbol\">mg/kg. Fumonisins were not detected. Good agriculture practices are indicated to region.

O objetivo deste estudo foi avaliar a presença de fungos e micotoxinas [aflatoxinas e ácido ciclopiazônico (ACP)] em amostras de castanha-do-Brasil coletadas em diferentes estados da região Amazônia brasileira: Acre, Amazonas, Amapá e Pará. Um total de 200 amostras de casca e 200 amostras de amêndoa foram semeadas em ágar Aspergillus flavus-parasiticus para pesquisa da micobiota. As micotoxinas foram analisadas por cromatografia líquida de alta eficiência. A morfológica clássica e a análise molecular revelou a presença dos seguintes fungos, em ordem decrescente de frequência: Phialemonium spp., Penicillium spp., Fusarium spp., Phaeoacremonium spp. e Aspergillus spp. A presença de aflatoxinas e ACP foi detectada em amostras de amêndoa, na frequência de 11% e 22%, respectivamente. Aspergillus nomius e Aspergillus flavus foram as espécies mais isoladas de Aspergillus. A presença de fungos não implica necessariamente na contaminação por micotoxinas, porém a amêndoa da castanha-do-Brasil parece ser um bom substrato para o crescimento de fungos. / The aim of this study was to evaluate the presence of fungi and mycotoxins [aflatoxin and cyclopiazonic acid (ACP)] in Brazil nut samples collected in different States of the Brazilian Amazon: Acre , Amazonas , Amapá and Pará. A total of 200 almond and 200 husk samples were plated on agar Aspergillus flavus - parasiticus for mycobiota isolation. Mycotoxins were analyzed by high performance liquid chromatography. The classical morphological and molecular analysis revealed the presence of the following fungi , in decreasing order of frequency: Phialemonium spp., Penicillium spp., Fusarium spp., Phaeoacremonium spp. and Aspergillus spp. The presence of aflatoxins and ACP was detected in almonds samples at a frequency of 11% and 22% , respectively. Aspergillus nomius and Aspergillus flavus species were the most isolated from Aspergillus. The presence of fungi does not necessarily imply the mycotoxin contamination, but the almond samples of Brazil nut seem to be a good substrate for the growth of fungi.

Certains champignons, notamment des Ascomycètes, peuvent synthétiser des métabolites secondaires toxiques pour les hommes et les vertébrés, appelés mycotoxines. Étant donné que la présence de ces champignons dans les aliments de base constitue un risque potentiel pour la santé humaine et animale, les aliments de base sont éliminés lorsqu'ils sont contaminés. La section Flavi est un des groupes de champignons les plus importants du point de vue économique et sanitaire car il comprend des espèces productrices de mycotoxines. Parmi les mycotoxines produites par ce groupe se trouvent les aflatoxines (AF), considérées comme une préoccupation majeure en raison de leurs effets délétères chez les vertébrés. Les espèces de la section Flavi se développent principalement dans les régions tropicales et subtropicales car elles bénéficient de conditions environnementales optimales. De plus, les conditions de récolte et de stockage sont souvent inappropriées, favorisant ainsi leur développement. Dans les régions tempérées, ces espèces se rencontrent moins fréquemment. Cependant, le réchauffement climatique pourrait favoriser leur colonisation. L'identification des espèces d'Aspergillus de la section Flavi est un défi, en raison de l'inter- et intra-variabilité des caractères. Par conséquent, l'utilisation d'une seule méthode d'identification (caractérisation morphologique, moléculaire ou du profil des métabolites secondaires) est insuffisante. Inversement, le développement d'outils moléculaires a facilité la tâche. Le but de notre étude était de déterminer les relations entre les espèces d'Aspergillus de la section Flavi à partir de différents marqueurs moléculaires (ITS, benA, cmdA, amdS, préA, perB, ppgA, aflP, gènes Mat1), puis d'identifier ceux qui permettent une classification des espèces par inférence phylogénétique. L'utilisation de l'inférence phylogénétique dans cette étude a montré qu'il s'agit d'une approche robuste pour identifier les espèces d'Aspergillus de la section Flavi, notamment en confirmant certaines hypothèses déjà proposées pour les espèces de la section Flavi. En effet, l'ajout de marqueurs moléculaires a permis de confirmer le placement phylogénétique des espèces dans la section Flavi. De plus, une nouvelle espèce cryptique a pu être décrite : Aspergillus korhogoensis (appartenant au clade A. flavus). Notre étude a également pu mettre en évidence que les marqueurs moléculaires sélectionnés (benA, cmdA, mcm7, rpb1, preB, preA et ppgA) sont de bons candidats pour l'étude d'autres sections d'Aspergillus. L'utilisation de l'inférence phylogénétique est une méthode élégante permettant d'identifier de façon précise les espèces. Sur la base de nos résultats, il est recommandé d'utiliser des matrices concaténées pour effectuer une inférence phylogénétique dans cette section, et la meilleure combinaison inclut les gènes benA, cmdA, et l'inclusion d'un autre gène : mcm7, rpb1, preB, preA ou ppgA. A l'inverse, l'utilisation du gène ITS chez Aspergillus peut conduire à une sous-estimation de la diversité car le gène est très fortement conservé. L'étude des gènes du loci Mat1 dans la section est utile pour accroître les connaissances sur la reproduction sexuée chez les ascomycètes. De plus, plusieurs fonctions de la machinerie biologique fongique sont liées aux gènes du loci Mat1. La caractérisation du profil métabolique secondaire chez les souches d'Aspergillus de la section Flavi doit être utilisée, non seulement comme outil d'identification, mais également pour discriminer les souches toxinogènes et atoxinogènes. La section Flavi renferme des espèces capables de produire à la fois de mycotoxines et de composés bénéfiques. Parmi les mycotoxines qui devraient faire l'objet d'une attention particulière figurent les AF, l'acide cyclopiazonique, les versicolorines a et b, la stérigmatocystine. Une étude plus approfondie du métabolisme secondaire sera également utile pour la recherche de nouveaux composés bénéfiques. / Some fungi, mostly Ascomycota, are able to synthesize secondary metabolites that are toxic to humans and vertebrates, called mycotoxins. Since the presence of these fungi in staples represents a potential risk to human and livestock health, staples are eliminated when they are contaminated. The section Flavi is one most important group of fungi from an economic and public health point of view because it comprises several mycotoxin producer species. Amongst the mycotoxins produced by this group are aflatoxins (AFs), considered a main concern because of their deleterious effects on humans and vertebrates. Species from section Flavi grow mainly in tropical and subtropical regions where environmental conditions are optimal, and harvest and storage conditions are not always appropriate to avoid production of mycotoxins, which enhance their growth. In temperate regions, these species are less frequent; however, climate changes can favor their colonization. Species identification in Aspergillus section Flavi is challenging because of inter- and intra- variability of traits. Therefore, the use of one identification method (morphological, molecular or secondary metabolite profile characterization) is futile. Conversely, the development of molecular tools has facilitated the task. The aim of this study was to screen the species relationships in Aspergillus section Flavi based on different molecular markers (ITS, benA, cmdA, amdS, preA, preB, ppgA, aflP, Mat1 genes), and subsequently identify which ones allow a fine species classification in the section Flavi by phylogenetic inference. The use of phylogenetic inference in the present study showed that it is a robust approach to identify Aspergillus section Flavi species. The use of this technique confirmed some of the hypotheses proposed in the Flavi section, since more genetic information was added, thus strengthening the placement of the species in the Flavi section. In addition, we described a new cryptic species in this section Aspergillus korhogoensis that is nested in A. flavus clade as the sister taxon of A. parvisclerotigenus. Likewise, the molecular markers (benA, cmdA, mcm7, rpb1, preB, preA or ppgA) were good candidates for studying other sections in Aspergillus. The use of phylogenetic inference is a good method for fine-scale species identification; however, it should be used carefully, and the morphological approach and characterization of secondary metabolites should also be carried out. Based on our results, concatenated matrices are recommended to perform phylogenetic inference in this section, and the best combination includes benA, cmdA, and the inclusion of at least one another gene (preB, mcm7, rpb1, preA or ppgA). Conversely, the use of ITS in Aspergillus may lead to an underestimation of the diversity because the gene is highly conserved. Studying mating type MAT1 loci in the section is helpful to increase the knowledge of sexual reproduction in ascomycetes. In addition, several functions of fungal biological machinery are linked to Mat1 loci genes. Secondary metabolic profile characterization of Aspergillus section Flavi strains should be performed, not only as an identification tool, but also to discriminate toxinogenic and atoxinogenic strains. Section Flavi encloses species able to produce a mixture of mycotoxins and beneficial compounds. Amongst mycotoxins that should be screened are AFs, cyclopiazonic acid, A and B versicolorin, sterigmatocystin, tenuazonic acid. An exhaustive study of the secondary metabolism can also be useful to investigate novel beneficial products.