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

Os grãos de milho são expostos, no campo e no armazenamento, à ação de fatores físicos, químicos e biológicos, que interagem entre si, favorecendo a contaminação fúngica, principalmente pelos gêneros Aspergillus, Fusarium e Penicilllium, considerados os mais importantes do ponto de vista toxigênico. O presente trabalho teve como principal objetivo investigar a eficácia do agente de biocontrole Afla-guard® no controle de fungos e de micotoxinas em grãos de milho, no campo, em diferentes estágios de maturidade da planta cultivada no município de Cássia dos Coqueiros, Estado de São Paulo. Visou, também, como objetivos adicionais, a determinação da atividade de água das amostras de milho, a pesquisa de fungos do ar, do solo e do milho, e a influência dos fatores climatológicos (temperatura e precipitação pluvial), no crescimento fúngico e na produção de micotoxinas. A micobiota do milho foi determinada pela técnica da semeadura direta utilizando meio de Ágar Dicloran Rosa Bengala Cloranfenicol (DRBC) e a determinação de micotoxinas por Cromatografia Líquida de Alta Eficiência. A identificação dos fungos foi realizada utilizando metodologia clássica (morfológica) e molecular (sequenciamento do DNA ribossomal). Nas amostras de milho, constatou-se a predominância de Fusarium verticillioides, em todas as coletas. Entretanto, a presença de Aspergillus flavus foi detectada, nas áreas tratadas com produto, a partir da 4ª coleta. No ar atmosférico, os fungos mais frequentes foram: Cladosporium spp., Trichoderma spp. e Fusarium solani. A. flavus foi isolado, principalmente, na 3ª coleta, do tratamento. No solo, os gêneros Penicillium, Trichoderma e Rhizopus foram os mais isolados em todas as coletas. A. flavus foi isolado a partir da 3ª coleta, somente nas áreas tratadas. Das amostras analisadas, a presença de fumonisinas foi detectada em 33,3 % das amostras tratadas com o agente de biocontrole e em 66,6% das amostras do grupo controle (não tratadas). Em relação às aflatoxinas, a presença da toxina foi detectada em apenas 2 amostras (1 tratamento e 1 controle) na 4ª coleta. Quanto à análise do potencial aflatoxigênico determinado, encontramos em 4 dos 13 isolados de A. flavus, aflatoxinas B1 e B2. Os demais isolados não foram potencialmente produtores, podendo ser, portanto, oriundos do Afla-guard®. Já em relação à temperatura, 26 °C foi a média encontrada e 6,3 mm foi a média detectada em relação à precipitação pluvial. Embora não tenha apresentado resultados estatisticamente significativos, o emprego do agente de biocontrole foi relevante no que diz respeito a diminuição da contaminação por Fusarium verticillioides e fumonisinas. Entretanto, outros estudos são necessários para uma melhor avaliação do produto / Maize grains are exposed, in the field and in storage, the action of physical, chemical and biological agents that interact with each other, encouraging fungal contamination, mainly by Aspergillus, Fusarium and Penicillium, considered the most important from the point of view toxigenic. This study aimed to investigate the effectiveness of Afla-guard® biocontrol agent in control of fungi and mycotoxins in corn grain in the field, at different stages of maturity of the plant cultivated in the city of Cassia dos Coqueiros, state São Paulo. Aimed also as additional objectives, determining the water activity of the maize samples, the research air fungi, soil and maize, and the influence of climatic factors (temperature and rainfall) in fungal growth and mycotoxin production. The mycobiota maize was determined by the technique of direct seeding using means of Dichloran Rose Bengal Chloramphenicol Agar (DRBC) and the determination of mycotoxins by high-performance liquid chromatography. The identification of fungi was carried out using classical methods (morphological) and molecular (sequencing of ribosomal DNA). In samples of maize, there was a predominance of Fusarium verticillioides in all samplings. However, the presence of Aspergillus flavus was detected in the areas treated with product samplings from the 4th. In atmospheric air, the most common molds were Cladosporium spp, Trichoderma spp.. Fusarium solani and. A. flavus was isolated mainly in 3rd samplings, treatment. On the ground, Penicillium, Trichoderma and Rhizopus were the most isolated in all samples. A. flavus was isolated from the 3rd collects only in the treated areas. Of the samples tested, the presence of fumonisin was detected in 33.3% of the samples treated with the biocontrol agent and 66.6% of the control group samples (untreated). Regarding the aflatoxins, the presence of the toxin was detected in just 2 samples (1 treatment and 1 control) in 4th collect. The analysis of the potential aflatoxigenic, found in 4 of 13 isolates of A. flavus, aflatoxins B1 and B2. The remaining isolates were not potentially producers and can therefore be derived from the Afla-guard®. Regarding the temperature 26 ° C was found to average 6.3 mm and the average was detected in relation to rainfall. Although not shown statistically significant results, the use of biocontrol agent is relevant as regards the reduction of contamination by Fusarium verticillioides and fumonisins. However, other studies are needed to better evaluate the product

Os grãos de milho são expostos, no campo e no armazenamento, à ação de fatores físicos, químicos e biológicos, que interagem entre si, favorecendo a contaminação fúngica, principalmente pelos gêneros Aspergillus, Fusarium e Penicilllium, considerados os mais importantes do ponto de vista toxigênico. O presente trabalho teve como principal objetivo investigar a eficácia do agente de biocontrole Afla-guard® no controle de fungos e de micotoxinas em grãos de milho, no campo, em diferentes estágios de maturidade da planta cultivada no município de Cássia dos Coqueiros, Estado de São Paulo. Visou, também, como objetivos adicionais, a determinação da atividade de água das amostras de milho, a pesquisa de fungos do ar, do solo e do milho, e a influência dos fatores climatológicos (temperatura e precipitação pluvial), no crescimento fúngico e na produção de micotoxinas. A micobiota do milho foi determinada pela técnica da semeadura direta utilizando meio de Ágar Dicloran Rosa Bengala Cloranfenicol (DRBC) e a determinação de micotoxinas por Cromatografia Líquida de Alta Eficiência. A identificação dos fungos foi realizada utilizando metodologia clássica (morfológica) e molecular (sequenciamento do DNA ribossomal). Nas amostras de milho, constatou-se a predominância de Fusarium verticillioides, em todas as coletas. Entretanto, a presença de Aspergillus flavus foi detectada, nas áreas tratadas com produto, a partir da 4ª coleta. No ar atmosférico, os fungos mais frequentes foram: Cladosporium spp., Trichoderma spp. e Fusarium solani. A. flavus foi isolado, principalmente, na 3ª coleta, do tratamento. No solo, os gêneros Penicillium, Trichoderma e Rhizopus foram os mais isolados em todas as coletas. A. flavus foi isolado a partir da 3ª coleta, somente nas áreas tratadas. Das amostras analisadas, a presença de fumonisinas foi detectada em 33,3 % das amostras tratadas com o agente de biocontrole e em 66,6% das amostras do grupo controle (não tratadas). Em relação às aflatoxinas, a presença da toxina foi detectada em apenas 2 amostras (1 tratamento e 1 controle) na 4ª coleta. Quanto à análise do potencial aflatoxigênico determinado, encontramos em 4 dos 13 isolados de A. flavus, aflatoxinas B1 e B2. Os demais isolados não foram potencialmente produtores, podendo ser, portanto, oriundos do Afla-guard®. Já em relação à temperatura, 26 °C foi a média encontrada e 6,3 mm foi a média detectada em relação à precipitação pluvial. Embora não tenha apresentado resultados estatisticamente significativos, o emprego do agente de biocontrole foi relevante no que diz respeito a diminuição da contaminação por Fusarium verticillioides e fumonisinas. Entretanto, outros estudos são necessários para uma melhor avaliação do produto / Maize grains are exposed, in the field and in storage, the action of physical, chemical and biological agents that interact with each other, encouraging fungal contamination, mainly by Aspergillus, Fusarium and Penicillium, considered the most important from the point of view toxigenic. This study aimed to investigate the effectiveness of Afla-guard® biocontrol agent in control of fungi and mycotoxins in corn grain in the field, at different stages of maturity of the plant cultivated in the city of Cassia dos Coqueiros, state São Paulo. Aimed also as additional objectives, determining the water activity of the maize samples, the research air fungi, soil and maize, and the influence of climatic factors (temperature and rainfall) in fungal growth and mycotoxin production. The mycobiota maize was determined by the technique of direct seeding using means of Dichloran Rose Bengal Chloramphenicol Agar (DRBC) and the determination of mycotoxins by high-performance liquid chromatography. The identification of fungi was carried out using classical methods (morphological) and molecular (sequencing of ribosomal DNA). In samples of maize, there was a predominance of Fusarium verticillioides in all samplings. However, the presence of Aspergillus flavus was detected in the areas treated with product samplings from the 4th. In atmospheric air, the most common molds were Cladosporium spp, Trichoderma spp.. Fusarium solani and. A. flavus was isolated mainly in 3rd samplings, treatment. On the ground, Penicillium, Trichoderma and Rhizopus were the most isolated in all samples. A. flavus was isolated from the 3rd collects only in the treated areas. Of the samples tested, the presence of fumonisin was detected in 33.3% of the samples treated with the biocontrol agent and 66.6% of the control group samples (untreated). Regarding the aflatoxins, the presence of the toxin was detected in just 2 samples (1 treatment and 1 control) in 4th collect. The analysis of the potential aflatoxigenic, found in 4 of 13 isolates of A. flavus, aflatoxins B1 and B2. The remaining isolates were not potentially producers and can therefore be derived from the Afla-guard®. Regarding the temperature 26 ° C was found to average 6.3 mm and the average was detected in relation to rainfall. Although not shown statistically significant results, the use of biocontrol agent is relevant as regards the reduction of contamination by Fusarium verticillioides and fumonisins. However, other studies are needed to better evaluate the product

Made available in DSpace on 2015-05-14T12:59:24Z (GMT). No. of bitstreams: 1 arquivototal.pdf: 819195 bytes, checksum: 00bb6e156c9bc61f631332e47253375e (MD5) Previous issue date: 2011-06-22 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / Due increase of incidence of fungal infections for Candida albicans and Aspergillus spp. in immunocompromised patients, and emerging resistant strains to conventional antifungals, it is accentuated the need for new antifungal. Essential oils have been tested for antimycotic activity and possess potential as antifungal agents. This work investigated the activity of essential oil of Cymbopogon winterianus against Candida albicans, Aspergillus flavus and A. fumigatus, by MIC and MFC; time-kill methods and morphological changes in the C. albicans, inhibition of the fungal mycelial growth and analysis of conidia germination of Aspergillus flavus and A. fumigatus when exposed to several concentrations of oil and by cellular leakage and assessed the bind of essential oil with ergosterol. The results shown that essential oil had concentrationdependent antifungal activity, alters the morphology in C. albicans, inhibits conidia germination and the radial mycelia growth of Aspergillus flavus and Aspergillus fumigatus, causes cellular leakage of all strains of fungal species tested and binds to ergosterol. These results make essential oil of Cymbopogon winterianus a potential agent in controlling fungi growth that cause infections, like hospital-acquired infections. / Devido ao aumento na incidência de infecções fúngicas por Candida albicans e Aspergillus spp. em pacientes imunocomprometidos, e ao surgimento de cepas resistentes aos antifúngicos convencionais, é crescente a necessidade de novos antifúngicos. Óleos essenciais têm sido testados contra fungos, e possuem potencial como agentes antifúngicos. Este trabalho investigou a atividade do óleo essencial de Cymbopogon winterianus contra Candida albicans, Aspergillus flavus e A. fumigatus, através da Concentração Inibitória Mímima (CIM), da Concentração Fungicida Mínima (CFM), do tempo de morte e de mudanças morfológicas em C. albicans; da inibição do crescimento radial fúngico e da análise da germinação dos conídios de Aspergillus flavus e A. fumigatus quando expostos a várias concentrações do óleo e através da lise celular e da ligação do óleo essencial com o ergosterol. Os resultados mostraram que o óleo essencial tem atividade antifúngica dependente da concentração, altera a morfologia de C. albicans, inibe a germinação de conídios e o crescimento micelial radial de Aspergillus flavus e Aspergillus fumigatus, causa lise celular de todas as cepas dos fungos testados e se liga ao ergosterol. Estes resultados fazem do óleo essencial de Cymbopogon winterianus um potencial agente no controle do crescimento de fungos que podem causar infecções, como aquelas que acontecem no ambiente hospitalar.

Submitted by Maike Costa (maiksebas@gmail.com) on 2017-09-13T12:15:49Z No. of bitstreams: 1 arquuivototal.pdf: 3577415 bytes, checksum: 0e9d2a60e77718e50720d15b7cad6fb1 (MD5) / Made available in DSpace on 2017-09-13T12:15:49Z (GMT). No. of bitstreams: 1 arquuivototal.pdf: 3577415 bytes, checksum: 0e9d2a60e77718e50720d15b7cad6fb1 (MD5) Previous issue date: 2016-02-18 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / In recent decades fungal infections have increased, and their high rates of morbidity and mortality have brought them much attention. Immunocompromised patients are more susceptible to microorganism infections; in particular, fungal species of the genus Aspergillus spp. Among the current infectious filamentous fungi they have a rather high incidence. Antifungal treatments have been subjected to numerous studies, and the increasing number of resistant fungal species has been highlighted. Thus, we see the importance of seeking new, more effective, and less toxic therapeutic sources. The aim of this study was to evaluate the in vitro antifungal activity and structure activity relationships of coumarin compounds against Aspergillus species. For this the MIC of 24 coumarin compounds was determined and subsequent SAR studies were performed with computer software. 12 of the 24 tested coumarin derivatives have antifungal activity, with 5 has shown excelent activity. Thus two derivatives, 7-hydroxy-6-nitrocoumarin (Cou-UNO2), and 4-acetoxycoumarin (Cou-UMB16), with better MIC values were evaluated via inhibition of mycelial growth and conidial germination, and by mechanism of action testing. The products were evaluated in combination with antifungals standards. The results showed that 12 of the 24 tested coumarin derivatives have antifungal activity, with MIC values ranging from 1024-16μg/ml. SAR studies have showed that the presence of a short aliphatic chain, and/or electronegative groups like ring substituents are favorable for antifungal activity. The coumarin derivatives with better MIC values (16μg/ml); Cou-UNO2 and Cou-UMB16 were capable of inhibiting both the mycelial growth and conidial germination of Aspergillus spp. Their activity is on the structure of the fungal cell wall. Cou-NO2, in a sub-inhibitory concentration, enhanced the in vitro effects of azoles, and in combination with azoles (voriconazole and itraconazole) there was an additive effect. Cou-UMB16 in combination with azoles (voriconazole and itraconazole) had synergistic or additive effects depending on the strain used. Thus this study concludes that coumarin derivatives have antifungal activity against the species A. flavus and A. fumigatus. The coumarin Cou-UMB16 and Cou-UNO2 have able to inhibit the mycelial growth and conidio germination and that this activity is due to its action on the fungal cell wall and the presence of electronegative groups as substituents of the benzopyrone ring favors this activity. / As infecções fúngicas, nas últimas décadas, têm se destacado devido ao seu crescente aumento e suas elevadas taxas de morbidade e mortalidade. Pacientes imunocomprometidos são os que possuem maior susceptibilidade a estes micro-organismos. Dentre estas infecções fúngicas, as espécies pertencentes ao gênero Aspergillus spp. possuem alta incidência entre os fungos filamentosos. A terapêutica antifúngica tem sido alvo de numerosos estudos e nestes tem sido destacado o crescente aumento de espécies fúngicas resistentes. Dessa forma, destaca-se a importância da busca de novas fontes terapêuticas que se apresentem mais eficazes e com menos tóxicas ao hospedeiro. Assim o objetivo deste estudo foi avaliar a atividade antifúngica in vitro e relação estrutura atividade (SAR) dos compostos cumarínicos frente às espécies do gênero Aspergillus. Para tal foi determinada a CIM de 24 compostos cumarínicos e posteriormente realizado estudos SAR com softwares computacionais. Dos 24 compostos ensaiados, 12 demonstraram possuir atividade antifúngica e 5 obtiveram forte atividade antifúngica. Destes derivados cumarínicos com forte atividade antifúngica, dois merecem destaque, 7-hidroxi-6-nitrocumarina (Cou-UNO2) e 4-acetóxicumarina (Cou-UMB16), com melhores valores de CIM, foram avaliados via testes de inibição do crescimento micelial, germinação dos conídios e testes de determinação do mecanismo de ação. Por fim os produtos foram avaliados em combinação com antifúngicos padrões. Os resultados demonstraram que 12 derivados cumarínicos dos 24 ensaiados possuem atividade antifúngica, com valores de CIMs variando entre 1024-16μg/mL. A SAR demonstra que a adição de grupos eletronegativos, como substituintes do anel benzopirona, favorece a atividade antifúngica destes compostos. Os derivados cumarínicos com melhores CIM (16μg/mL), 7-hidroxi-6-nitrocumarina (Cou-UNO2) e 4-acetóxicumarina (Cou-UMB16) foram capazes de inibir o crescimento micelial e a germinação dos conídios de Aspergillus spp. E estes possuem ação sobre a estrutura da parede celular fúngica. Em uma concentração subinibitória, Cou-UNO2 potencializou a ação in vitro dos derivados azólicos e em combinação com os derivados azólicos (voriconazol e itraconazol) observou-se um efeito aditivo. Já Cou-UMB16 em combinação com os derivados azólicos (voriconazol e itraconazol) obteve um efeito sinérgico a aditivo, dependendo da linhagem utilizada. Logo este estudo conclui que os derivados cumarínicos possuem atividade antifúngica contra as espécies de A. flavus e A. fumigatus. As cumarinas Cou-UNO2 e Cou-UMB16 foram capazes de inibir o crescimento micelial e a germinação dos conídios e que esta atividade seja devido a sua ação sobre a parede celular fúngica. E que esta atividade é favorecida pela presença de moléculas eletronegativas no anel básico da cumarina (1,2-benzopirona).

Les champignons du genre Aspergillus sont des moisissures banales de l'environnement. Elles sont présentes dans le sol et sur des végétaux en décomposition. Les Aspergillus se propagent par l'intermédiaire de spores microscopiques en suspension dans l'air. L'Homme et les animaux sont exposés en permanence aux spores aspergillaires mais les défenses immunes empêchent leur développement dans l'organisme. Lorsque ces défenses sont amoindries, une aspergillose est possible. Dans ce cas, Aspergillus fumigatus et A. flavus sont le plus souvent incriminés. Les oiseaux sont beaucoup plus sensibles que les mammifères et l'environnement représenté par les élevages aviaires est propice à la prolifération des moisissures du genre Aspergillus. L'objectif de ce travail de thèse a été de caractériser la diversité génétique et la sensibilité aux antifongiques d'isolats d'Aspergillus provenant d'élevages aviaires dans la province du Guangxi en Chine. La première partie de la thèse est une analyse bibliographique sur les champignons du genre Aspergillus, les aspergilloses et les caractéristiques de l'élevage aviaire en Chine. Une première enquête a été réalisée dans 3 élevages près de la ville de Nanning et dans un élevage (incluant un éclosoir) à proximité de la ville de Guilin. Des écouvillonnages pharyngés et des prélèvements d'air ont été réalisés pendant plusieurs semaines. Des prélèvements ont également été faits sur des œufs dans l'éclosoir. Cette enquête a montré que le niveau de contamination fongique dépendait du type d'élevage. De nombreux isolats fongiques ont pu être collectés : 188 isolats d'A. fumigatus et 159 isolats d'A. flavus. La seconde partie du travail expérimental a porté sur la caractérisation de la diversité génétique d'A. fumigatus et d'A. flavus. Pour cela, la technique MLVA (multiple locus VNTR analysis) a été utilisée. Pour A. flavus, 8 marqueurs VNTR (variable-number tandem-repeat) ont été sélectionnés et une réaction PCR multiplex a été mise au point. Au total, 91 isolats d'A. flavus, incluant 6 souches de référence, ont été caractérisées avec le panel des 8 marqueurs VNTR. Cette analyse a permis de définir 78 génotypes distincts et un index de discrimination de 0,993. L'analyse de 188 isolats d'A. fumigatus avec 10 marqueurs VNTR a permis de définir 142 génotypes distincts. Certains génotypes d'A. flavus ou d'A. fumigatus sont clairement regroupés dans le nuage de point généré par l'analyse MST (minimum spanning tree). La troisième partie du travail expérimental a porté sur la sensibilité aux antifongiques de 177 isolats d'A. fumigatus. Ces isolats ont été récupérés dans des élevages aviaires en Chine et en France. Les isolats de Chine sont pour la plupart sensibles avec des valeurs minimales inhibitrices (vis-à-vis de l'itraconazole) comprises entre 0,38 et 0,75 µg/mL. Les isolats de France sont pour la plupart sensibles avec des valeurs minimales inhibitrices (vis-à-vis de l'itraconazole) comprises entre 0.19 and 1 µg/mL. Quatre souches ont été considérées comme résistantes : 2 souches provenant de deux élevages en Chine et 2 souches provenant de deux élevages en France. Des mutations sur le gène Cyp51A ont été détectées pour 11 isolats (3 résistants et 8 sensibles). Vingt et une mutations nucléotidiques ont été identifiées. Onze de ces mutations sont silencieuses et 9 sont à l'origine d'un changement de la composition de la protéine. Sept substitutions ont déjà été décrites dans la littérature ; les mutations A116R, E130D et Q131H sont originales. / Fungi of the genus Aspergillus are moulds, which occur most frequently in soil, water and decaying vegetation. They sporulate abundantly and the spores are easily dispersed into the environment by air. As a result of this ubiquitous presence, animals and people are constantly exposed to Aspergillus spores. Aspergillus fumigatus and A. flavus are recognized as predominant causes of fungal diseases in humans and wide range of animals. Birds are much more sensitive that mammals and in avian farms, environmental conditions are favorable to the development of many fungal species, including Aspergillus spp. The objective of the present study was to assess the genetic diversity and antifungal susceptibility of Aspergillus isolates from avian farms in Guangxi, China. The first part of the experimental work related the evolution of fungal contamination in 3 avian farms near the city of Nanning and one farm (including a hatchery) near the city of Guilin. Pharyngeal swabs and air samples were collected during several weeks and 3 cycles of hatching were monitored. The average contamination level with Aspergillus spp. and Mucorales was significantly different according to the farms. The survey allowed to collect a total number of 188 A. fumigatus and 159 A. flavus isolates. The second part of the work was about the genetic diversity of A. fumigatus and A. flavus. For that purpose, the Multiple Locus Variable-number tandem-repeat (VNTR) Analysis was specifically developed and used. For A. flavus, 8 VNTR markers were selected and a multiplex reaction was designed. A total number of 91 A. flavus isolates, including 6 reference strains were typed with the panel of 8 VNTRs. This analysis yielded 78 different genotypes, which corresponds to a combined loci index of 0.993. Among all genotypes, 71 were only found once. The analysis of 188 A. fumigatus isolates using 10 VNTR markers led to the resolution of 142 distinct genotypes. Clusters of A. flavus or A. fumigatus isolates could be defined by using the graphing algorithm Minimum Spanning Tree. The third part of the experimental work was about the antifungal susceptibility of 177 A. fumigatus isolates collected in avian farms in China and France. Most of the isolates from China were susceptible to itraconazole with a Minimum Inhibitory Concentration (MIC) comprised between 0.38 and 0.75 µg/mL. Most of the isolates from birds and avian farms in France were susceptible to itraconazole with a MIC comprised between 0.19 and 1 µg/mL. MIC values of isolates collected in farms with antifungal chemoprophylaxis were not higher than those of isolates collected from birds (that never received antifungal drugs before the sampling). Susceptibility testings demonstrated that 4 isolates should be considered as resistant to itraconazole: (2 isolates from avian farms in Guangxi, China and 2 isolates from avian farms in France). A modification of the Cyp51A sequence was identified in 11 isolates (3 azole-resistant and 8 azole-susceptible isolates). Twenty-one nucleotidic mutations were detected. Eleven of these mutations were silent and 10 yielded to amino acid substitutions. Seven of these substitutions had already been described whereas mutations A116R, E130D and Q131H were original.

L’aflatoxine B1 (AFB1) est une mycotoxine contaminant de nombreux substrats agricoles, notamment les céréales, arachides, pistaches, graines de coton et autres fruits secs. Elle est génotoxique, agit comme un initiateur de la cancérogenèse et elle est classée par le CIRC (Centre International de Recherche sur le Cancer) dans le groupe I des molécules carcinogènes pour les hommes et les animaux. Cette toxine est produite par des espèces fongiques appartenant essentiellement au genre Aspergillus et à la section Flavi ; A. flavus étant l’espèce la plus préoccupante. Cette espèce peut se développer à la fois au champ ou lors du stockage des matières premières après récolte. Plusieurs études montrent qu’un grand nombre de denrées alimentaires destinées à l’alimentation humaine et animale sont contaminées par les aflatoxines mettant en évidence la faible efficacité ou l’insuffisance des stratégies actuelles utilisées pour la maîtrise de cette contamination. L’objectif principal de cette étude est le développement d’une stratégie alternative basée sur l’utilisation de produits naturels et visant à réduire la production de la toxine par les moisissures aflatoxinogènes. Grâce à l’élaboration d’un outil moléculaire regroupant un ensemble de gènes impliqués directement ou indirectement dans la biosynthèse de l’AFB1, ce travail permet de mieux comprendre le mécanisme d’action moléculaire de composés anti-aflatoxinogènes. Des extraits aqueux de plantes méditerranéennes ont été testés pour leur efficacité à inhiber l’aflatoxinogénèse. Ainsi, l’extrait aqueux d’hysope, Micomeria graeca, présente des propriétés anti-aflatoxinogènes importantes sans modification de la croissance fongique. Son effet inhibiteur sur plusieurs souches productrices d’aflatoxines ainsi que son mécanisme d’action ont été caractérisés. Ce dernier semble impliquer des gènes pivots dans la régulation des métabolismes fongiques primaire et secondaire ainsi que des voies impliquées dans la réponse cellulaire au stress oxydatif. Ces travaux permettent de valider l’utilisation d’extraits naturels comme stratégie alternative de lutte contre la contamination des aliments par les aflatoxines. / Aflatoxin B1 (AFB1) is a mycotoxin contaminating numerous agricultural commodities such as cereals, peanuts, pistachios, cottonseed and other dried fruits. It is a genotoxic initiating carcinogenesis and is classified by the IARC (International Agency for Research on Cancer) in the group I of molecules that are carcinogenic for humans and animals. This toxin is produced by fungal species mainly belonging to the Flavi section of the Aspergillus genus, A. flavus being the most preoccupying species. This species can develop both in the field and during storage of commodities following harvest. Several studies show that a significant number of food and feedstuffs are contaminated by aflatoxins, highlighting the feeble efficiency or the shortfall of the strategies currently used to prevent such contamination. The main objective of this study is the development of an alternative strategy based on the use of natural products and aiming the reduction of toxin production by aflatoxinogenic fungi. Through the elaboration of a molecular tool grouping a number of genes directly or indirectly linked to AFB1’s biosynthesis, this work allows a better understanding of the molecular mechanism of action of anti-aflatoxinogenic compounds. Aqueous extracts of Mediterranean plants were tested for their ability to inhibit aflatoxinogenesis. Thus, hyssop’s aqueous extract - Micomeria graeca – is shown to present significant anti-aflatoxinogenic properties without reducing fungal growth. Its inhibiting effect in other AFB1-producing species and its mechanism of action were both characterized. The latter seems to involve pivotal genes in fungal primary and secondary metabolisms as well as genes implicated in the fungal cellular response to oxidative stress. These works allocate the use of natural extracts as an alternative strategy to counteract aflatoxin contamination of food and feed.

Stonecats (Noturus flavus, Rafinesque 1818) are designated as a Vermont state-listed endangered species because their known distribution is limited to two systems, the LaPlatte and Missisquoi rivers. The restricted distribution and lack of knowledge on abundance in either river is cause for concern in the continued survival of these populations. Based on the capture numbers and large size range of individuals, we predicted that the population in the LaPlatte River, which provides quality benthic habitat, is stable. However, the Missisquoi River population has the potential for increased intermittent mortalities from two sources, lampricide (3-trifluoromethyl-4-nitrophenol) treatment every four years and dewatering during drought conditions. In 2012, 2013, and 2014 we captured, PIT tagged (> 90 mm total length), and VIE marked all Stonecats collected using backpack electrofishing and minnow traps in the LaPlatte and Missisquoi rivers. A total of 1252 were PIT tagged in the LaPlatte River and 125 in the Missisquoi River. First we estimated survival and seniority of Stonecats in the LaPlatte River, using the Pradel model in Program Mark, and derived an average annual lambda of 0.9826. The population estimates from the LaPlatte River were modeled in a population viability analysis (PVA). Few Stonecats were captured in the Missisquoi River, so we used the PVA model to estimate the extinction rates with increased intermittent mortalities on 4-, 6-, and 8-year cycles to predict the long-term viability of the population. With an initial number of 2000 individuals, the population became extinct 100% of the time with an increase in mortality of 0.1 on a 4-year cycle. Our results indicate that the LaPlatte River population is stable, but the Missisquoi River population, in the area affected by lampricide, is not. These results are informative for developing future management scenarios, however, our approach has uncertainty that can only be addressed through obtaining more data on the Missisquoi River population.

<p>Maize is a major calorie source for people living in Sub-Sahara Africa. In this region, <i>Aspergillus flavus</i> causes ear rot diseases in maize, contributing to food insecurity due to aflatoxin contamination. The biological control principle of competitive exclusion has been applied in both the United States and Africa to effectively reduce aflatoxin levels in maize at harvest by introducing atoxigenic strains that out-compete toxigenic strains. The goal of this study was to determine if the efficacy of preharvest biocontrol treatments carry over into the drying period, which is often delayed in Sub-Sahara Africa by the complexities of postharvest drying practices and lack of modern drying machinery. Maize was collected from fields in Texas and North Carolina that were treated with commercial biocontrol, and control fields that were untreated. To simulate moisture conditions similar to those experienced by farmers during drying in Sub-Sahara Africa, we adjusted the grain to 20% moisture content and incubated it at 28 ℃ for 6 days. Although the initial number of infected kernels in most samples were high, less than 24% of kernels were infected with <i>Aspergillus flavus</i> and aflatoxin levels were low (<4ppb). Both toxigenic and atoxigenic strains increased and spread through the grain over the incubation period, and aflatoxin levels increased, even in samples from biocontrol-treated fields. Our molecular analysis suggests that applied biocontrol strains from treated fields migrate to untreated fields. The results also indicate that the population of toxigenic <i>A. flavus</i> in the harvested grain will grow and produce aflatoxin during the drying period when moisture is high. Therefore, any potential postharvest reduction in aflatoxin accumulation will depend on how effective the biocontrol strain was at displacing the toxigenic populations prior to harvest.</p>

Orientador: Vera Aparecida de Oliveira Tiera / Banca: Dayane Batista Tada / Banca: Eduardo Alves de Almeida / Resumo: O presente trabalho descreve a síntese, caracterização e estudo das propriedades antimicrobianas de derivados anfifílicos de quitosana contra o fungo Aspergillus flavus. A quitosana utilizada nas sínteses foi obtida a partir da reação de desacetilação heterogênea de quitosana comercial. O grau médio de desacetilação foi determinado por Ressonância Magnética Nuclear de Hidrogênio, cujo resultado foi de 97,3%. As massas moleculares das quitosanas comercial e desacetilada foram determinadas por cromatografia de permeação em gel e os resultados obtidos foram 338,46 kDa e 137,12 kDa, respectivamente. Os derivados anfifílicos foram sintetizados em um processo de duas etapas: inicialmente realizou-se a alquilação da quitosana desacetilada pela inserção de uma proporção fixa de grupos dodecila (4%) na cadeia polimérica com posterior redução da base de Schiff empregando-se borocianohidreto de sódio. Em seguida foram inseridas proporções crescentes do grupo quaternário pentiltrimetilamônio. Os graus de substituição com grupos dodecila (GSDD) e grupos pentiltrimetilamônio (GDPE) foram determinados por RMN de 1H. O GSDD obtido foi de 4,0%, enquanto que os GDPE foram de 4,8; 12,7 e 46,3%. Todos os derivados sintetizados foram testados in vitro contra o fungo Aspergillus flavus em concentrações crescentes. Os ensaios microbiológicos mostraram que os derivados modificados com grupos dodecila e pentiltrimetilamônio são mais eficientes em inibir o crescimento do fungo Aspergillus flavus quando comparado à quitosana desacetilada e que a presença de grupos hidrofóbicos na cadeia do polímero proporciona uma interação mais forte com a membrana celular. Os resultados dos estudos utilizando modelos de membranas corroboraram com os dos ensaios microbiológicos, evidenciando que a presença de grupos dodecila na cadeia da quitosana aumenta a interação com as vesículas, resultando em... / Abstract: In this work is described the synthesis, characterization and study of antimicrobial properties of amphiphilic derivatives of chitosan against Aspergillus flavus. Deacetylated chitosan was obtained from the heterogeneous deacetylation of the commercial chitosan. The degree of deacetylation was determined by Hydrogen Nuclear Magnetic Resonance and the result was 97.3%. Molecular weights of commercial and deacetylated chitosans were determined by gel permeation chromatography and the obtained values were 338.46 kDa and 137.12 kDa, respectively. The hydrophobic derivatives were synthesized by a reductive amination reaction with dodecylaldehyde followed by reduction with sodium cyanoborohydride. Next increasing proportions of the quaternary group pentyltrimethylammonium was inserted on hydrophobic derivative. The degrees of substitution by dodecyl (4.0%) and pentyltrimethylammonium groups (4.8, 12.7 and 46.3%) were determined by Hydrogen Nuclear Magnetic Resonance. All synthesized derivatives were tested in vitro against Aspergillus flavus at increasing concentrations. The results showed that the modified derivatives with pentyltrimethylamonnium and dodecyl groups are more effective in inhibiting the growth of the fungus Aspergillus flavus when compared to deacetylated chitosan and the presence of hydrophobic groups in the polymer chain provides a stronger interaction with the cell membrane. The results of studies with model membranes agreed with the results of microbiological tests, showing that the presence of hydrophobic groups in chitosan backbone increases the interaction with lipid vesicles resulting in an increase in vesicles diameter and zeta potential. At first, it can be postulated that the mechanism of chitosan action against fungi is favored by hydrophobic interactions between chitosan derivatives and membrane lipids and may involve the adsorption on the cell wall and cell membrane disruption... / Mestre