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.

Orientadores: José Luiz Pereira, Marta Hiromi Taniwaki / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos / Made available in DSpace on 2018-08-19T08:18:04Z (GMT). No. of bitstreams: 1 Calderari_ThaianeOrtolan_M.pdf: 6842829 bytes, checksum: 4d9030fc65c6d69a678e084cd772b7ed (MD5) Previous issue date: 2011 / Resumo: A castanha do Brasil (Bertholletia excelsa) é uma das mais importantes espécies de exploração extrativista da floresta Amazônica, sendo exportada para diversos países devido ao seu alto valor nutritivo. No entanto, os baixos níveis tecnológicos característicos de sua cadeia produtiva, considerada ainda extrativista e as condições inadequadas de manejo da matéria prima, favorecem o aparecimento de contaminação por fungos produtores de aflatoxinas, compostos tóxicos considerados cancerígenos para humanos. Este problema é um entrave para a comercialização do produto, principalmente no mercado externo, dado ao rigoroso controle de países europeus e Estados Unidos em relação aos níveis de toxinas presentes nos alimentos. Nestas condições, o presente trabalho teve por objetivo investigar a incidência de fungos em castanhas do Brasil e avaliar o potencial toxigênico dos isolados Aspergillus section flavi para a produção de aflatoxinas, bem como analisar a presença de aflatoxinas nesta matriz. Um total de 143 amostras provenientes dos Estados do Pará, Amazonas e São Paulo em diferentes etapas da cadeia produtiva da castanha foi analisado. A técnica utilizada para análise da infecção fúngica foi o plaqueamento direto em meio Dicloran 18% Glicerol. Os resultados foram expressos em porcentagem de infecção fúngica. Os isolados suspeitos foram purificados em meio Czapek extrato de levedura ágar e incubados a 25ºC/7 dias em diferentes temperaturas para a identificação das espécies. Para a análise do potencial toxigênico de cada isolado da seção flavi foi utilizada a técnica do ágar plug. Para a análise de aflatoxinas foi utilizada coluna de imunoafinidade para extração e limpeza das amostras e Cromatografia Líquida de Alta Eficiência e detector de fluorescência acoplado ao sistema de derivatização Kobracell para detecção e quantificação das aflatoxinas. Dentre o total de amostras coletadas, aquelas provenientes das florestas foram as que apresentaram maior valor médio de atividade de água, assim como maior porcentagem de infecção fúngica quantificada e biodiversidade de espécies. Considerando todas as amostras avaliadas, foram no total 13.421 isolados de fungos filamentosos, sendo que as espécies mais incidentes foram Aspergillus flavus, Aspergillus nomius, Penicillium citrinum, Aspergillus tamarii, Syncephalastrum racemosum e Penicillium sp. Dentre as espécies encontradas, 450 isolados de Aspergillus nomius e 9 de Aspergillus parasiticus foram identificados e 100% apresentaram capacidade de produção de aflatoxinas AFB1, AFB2, AFG1, AFG2. Dos de 703 isolados de Aspergillus flavus, 63,5% apresentaram a capacidade de produzir aflatoxinas AFB1 e AFB2. A média de contaminação por aflatoxinas totais obtida foi de 7,17 µg/kg (ND-104,2 µg/kg), 1,13µg/kg (ND-7,44µg/kg) e 0,47 µg/kg (ND-0,2 µg/kg) para as amostras dos Estados do Pará, Amazonas e de São Paulo, respectivamente. Das 143 amostras coletadas, apenas 5 amostras excederam o limite máximo de aflatoxinas totais estabelecido pela União Européia e pela ANVISA (10,0ug/kg para castanhas do Brasil sem casca destinadas ao consumo direto para humanos) / Abstract: The Brazil nut (Bertholletia excelsa) is one of the most important species extracted from the Amazon forest, and is exported to several countries due to its high nutritional value. However, the low technological level of its productive chain and inadequate raw material handling favour contamination points for aflatoxin fungi producers aflatoxins. The presence of aflatoxins in Brazil nuts has been a barrier for its marketing, mainly for the export market, due to rigorous control of European countries and the United States. Therefore, the present work had the objective of investigating the incidence of fungi in Brazil nuts and evaluate the toxigenic potential of Aspergillus section flavi isolates to produce aflatoxins, as well as analyzing the presence of aflatoxins in this product. A total of 143 samples from three different states, at different stages of the Brazil nut chain was analyzed. The technique used for fungi infection analized was direct plating in DG18. The results were expressed in percentage of fungal infection. The suspected isolates were purified on Czapek yeast extract agar (CYA) and incubated at different temperature for species identification. For toxin production analysis of each isolatec Aspergillus section flavi the agar plug technique was used. For aflatoxin analysis an immunoafinity column was used for extraction and cleaning of the sample, high performance liquid for aflatoxin detection and quantification in Brazil nuts, chromatography (HPLC) with a fluorescence detector was used, coupled with the Kobracell derivatization system. Among the analyzed samples, the ones collected directly from the forests had the highest water activity, the highest fungal infection and greatest biodiversity of species. A total of 13,421 filamentous fungi were quantificated from all the samples with the most common isolated species were: Aspergillus flavus, Aspergillus nomius, Penicillium citrinum, Aspergillus tamarii, Syncephalastrum racemosum e Penicillium spp. All the 450 strains of Aspergillus nomius and 9 strains of Aspergillus parasiticus, showed 100% capacity of aflatoxin B1, B2, G1, G2 production. Out of 703 species of Aspergillus flavus isolated, 63.5% showed capacity of aflatoxin B1 e B2 production. The average of total aflatoxin contamination was: 7.17µg/kg (ND-104.2 µg/kg), 1.13µg/kg (ND-7.44µg/kg) and 0.47 µg/kg (ND-0.2 µg/kg) for samples from Pará, Amazon and São Paulo, respectively. Out of 143 analyzed samples, only 5 samples exceded the maximum level for total aflatoxins established by the European Union and ANVISA of 10 µg/kg for shelled Brazil nuts intended for direct human consumption / Mestrado / Ciência de Alimentos / Mestre em Ciência de Alimentos