Significantes quantidades de maçãs produzidas no Rio Grande do Sul são usadas para a produção de suco destinado à exportação. As frutas que são utilizadas pela indústria são aquelas que não atingem o padrão exigido para o consumo in natura. Estas maçãs constituem fator de risco quanto à contaminação por fungos, que podem produzir micotoxinas, como a patulina. O Brasil não tem legislação que estabeleça níveis permitidos de patulina em alimentos. Mas muitos países têm adotado a recomendação do Codex Alimentarius que indica um nível máximo permitido de 50 μg/L de patulina em suco de maçã. Esse fato estimula países exportadores, como o Brasil, a pesquisar a qualidade micotoxicológica dos seus produtos, a fim de se tornarem competitivos no mercado internacional e de fornecerem alimentos seguros. Nesse contexto, os objetivos desse trabalho foram: (1) avaliar a contaminação fúngica de maçãs destinadas à produção de suco, bem como a possível presença de patulina nos sucos produzidos a partir destas frutas; (2) adaptar metodologicamente a técnica de cromatografia em camada delgada à quantificação de patulina em maçãs e sucos de maçã; (3) analisar os níveis de patulina nas fases do processamento de suco de maçã; e (4) pesquisar fungos termorresistentes nos sucos em estudo. As amostras de maçãs e de sucos foram fornecidas pela Golden Sucos Ltda de Farroupilha, RS. Penicillium expansum foi a espécie isolada predominantemente, sendo que 94,44% dos isolados pertencentes a esta espécie se mostraram produtores de patulina nas condições testadas. A prevalência de fungos produtores de patulina nas maçãs processadas resultou em altas concentrações da micotoxina nessas maçãs, variando entre 254,62 e 653,37 μg/kg e também nos sucos concentrados produzidos, com níveis de patulina nestes entre 56,04 e 231,44 μg/L. O método desenvolvido para extração e quantificação da patulina foi eficaz. O detector de carga acoplada forneceu boa sensibilidade, precisão, linearidade e recuperação para a determinação quantitativa de patulina. Todos os estágios do processamento do suco contribuíram para a redução dos níveis de patulina. Desde a maçã até o suco a redução foi de 75,2%. A presença de Byssochlamys nivea em uma das amostras de suco indica que a patulina poderia ser produzida por este fungo durante o armazenamento. / Significant amounts of apples produced in the state of Rio Grande do Sul, Brazil, are used to juice production destined to exportation. Fruits used by the industry show some defects and are not accepted for fresh consumption. These apples are a risk factor considering the contamination by fungus, which can produce mycotoxins, such as patulin. Brazil does not have legislation for patulin in food. But many countries have adopted the recommendation of the Codex Alimentarius, which indicates a maximum permitted level of 50 μg/L of patulin in apple juice. This concern stimulates exporting countries, like Brazil, in the research of mycotoxigenic quality of apples and by-products, to become competitive and provide safe foods. In this context, the objectives of this work were (1) evaluate mould contamination of apples used to juice production and possible presence of patulin in apples; (2) methodologically adapt the thin-layer chromatographic technique for quantification of patulin in apples and apple juice; (3) analyze patulin levels in some processing stages of apple juice production; and (4) investigate heat-resistant moulds in juices. The apples and juice samples were obtained from Golden Sucos Ltda, located in Farroupilha city, in Rio Grande do Sul state, Brazil. Penicillium expansum was the most frequently isolated fungus, among these isolates, 94.44% were able to produce patulin under the tested conditions. The prevalence of patulin-producing fungi in apples resulted in high patulin levels in these apples, ranging from 254.62 to 653.37 μg/Kg and in juices ranging from 56.04 to 231.44 μg/L. The method developed to patulin extraction and quantification was effective. The charged coupled device imaging system provides good sensitivity, precision and linearity for the quantitative determination of patulin. All stages of apple juice production contributed to patulin reduction. The overall loss of patulin through processing from apple to apple juice was 75.2%. The presence of Byssochlamys nivea in a juice sample indicates that this fungus could produce patulin during storage.

Significantes quantidades de maçãs produzidas no Rio Grande do Sul são usadas para a produção de suco destinado à exportação. As frutas que são utilizadas pela indústria são aquelas que não atingem o padrão exigido para o consumo in natura. Estas maçãs constituem fator de risco quanto à contaminação por fungos, que podem produzir micotoxinas, como a patulina. O Brasil não tem legislação que estabeleça níveis permitidos de patulina em alimentos. Mas muitos países têm adotado a recomendação do Codex Alimentarius que indica um nível máximo permitido de 50 μg/L de patulina em suco de maçã. Esse fato estimula países exportadores, como o Brasil, a pesquisar a qualidade micotoxicológica dos seus produtos, a fim de se tornarem competitivos no mercado internacional e de fornecerem alimentos seguros. Nesse contexto, os objetivos desse trabalho foram: (1) avaliar a contaminação fúngica de maçãs destinadas à produção de suco, bem como a possível presença de patulina nos sucos produzidos a partir destas frutas; (2) adaptar metodologicamente a técnica de cromatografia em camada delgada à quantificação de patulina em maçãs e sucos de maçã; (3) analisar os níveis de patulina nas fases do processamento de suco de maçã; e (4) pesquisar fungos termorresistentes nos sucos em estudo. As amostras de maçãs e de sucos foram fornecidas pela Golden Sucos Ltda de Farroupilha, RS. Penicillium expansum foi a espécie isolada predominantemente, sendo que 94,44% dos isolados pertencentes a esta espécie se mostraram produtores de patulina nas condições testadas. A prevalência de fungos produtores de patulina nas maçãs processadas resultou em altas concentrações da micotoxina nessas maçãs, variando entre 254,62 e 653,37 μg/kg e também nos sucos concentrados produzidos, com níveis de patulina nestes entre 56,04 e 231,44 μg/L. O método desenvolvido para extração e quantificação da patulina foi eficaz. O detector de carga acoplada forneceu boa sensibilidade, precisão, linearidade e recuperação para a determinação quantitativa de patulina. Todos os estágios do processamento do suco contribuíram para a redução dos níveis de patulina. Desde a maçã até o suco a redução foi de 75,2%. A presença de Byssochlamys nivea em uma das amostras de suco indica que a patulina poderia ser produzida por este fungo durante o armazenamento. / Significant amounts of apples produced in the state of Rio Grande do Sul, Brazil, are used to juice production destined to exportation. Fruits used by the industry show some defects and are not accepted for fresh consumption. These apples are a risk factor considering the contamination by fungus, which can produce mycotoxins, such as patulin. Brazil does not have legislation for patulin in food. But many countries have adopted the recommendation of the Codex Alimentarius, which indicates a maximum permitted level of 50 μg/L of patulin in apple juice. This concern stimulates exporting countries, like Brazil, in the research of mycotoxigenic quality of apples and by-products, to become competitive and provide safe foods. In this context, the objectives of this work were (1) evaluate mould contamination of apples used to juice production and possible presence of patulin in apples; (2) methodologically adapt the thin-layer chromatographic technique for quantification of patulin in apples and apple juice; (3) analyze patulin levels in some processing stages of apple juice production; and (4) investigate heat-resistant moulds in juices. The apples and juice samples were obtained from Golden Sucos Ltda, located in Farroupilha city, in Rio Grande do Sul state, Brazil. Penicillium expansum was the most frequently isolated fungus, among these isolates, 94.44% were able to produce patulin under the tested conditions. The prevalence of patulin-producing fungi in apples resulted in high patulin levels in these apples, ranging from 254.62 to 653.37 μg/Kg and in juices ranging from 56.04 to 231.44 μg/L. The method developed to patulin extraction and quantification was effective. The charged coupled device imaging system provides good sensitivity, precision and linearity for the quantitative determination of patulin. All stages of apple juice production contributed to patulin reduction. The overall loss of patulin through processing from apple to apple juice was 75.2%. The presence of Byssochlamys nivea in a juice sample indicates that this fungus could produce patulin during storage.

Significantes quantidades de maçãs produzidas no Rio Grande do Sul são usadas para a produção de suco destinado à exportação. As frutas que são utilizadas pela indústria são aquelas que não atingem o padrão exigido para o consumo in natura. Estas maçãs constituem fator de risco quanto à contaminação por fungos, que podem produzir micotoxinas, como a patulina. O Brasil não tem legislação que estabeleça níveis permitidos de patulina em alimentos. Mas muitos países têm adotado a recomendação do Codex Alimentarius que indica um nível máximo permitido de 50 μg/L de patulina em suco de maçã. Esse fato estimula países exportadores, como o Brasil, a pesquisar a qualidade micotoxicológica dos seus produtos, a fim de se tornarem competitivos no mercado internacional e de fornecerem alimentos seguros. Nesse contexto, os objetivos desse trabalho foram: (1) avaliar a contaminação fúngica de maçãs destinadas à produção de suco, bem como a possível presença de patulina nos sucos produzidos a partir destas frutas; (2) adaptar metodologicamente a técnica de cromatografia em camada delgada à quantificação de patulina em maçãs e sucos de maçã; (3) analisar os níveis de patulina nas fases do processamento de suco de maçã; e (4) pesquisar fungos termorresistentes nos sucos em estudo. As amostras de maçãs e de sucos foram fornecidas pela Golden Sucos Ltda de Farroupilha, RS. Penicillium expansum foi a espécie isolada predominantemente, sendo que 94,44% dos isolados pertencentes a esta espécie se mostraram produtores de patulina nas condições testadas. A prevalência de fungos produtores de patulina nas maçãs processadas resultou em altas concentrações da micotoxina nessas maçãs, variando entre 254,62 e 653,37 μg/kg e também nos sucos concentrados produzidos, com níveis de patulina nestes entre 56,04 e 231,44 μg/L. O método desenvolvido para extração e quantificação da patulina foi eficaz. O detector de carga acoplada forneceu boa sensibilidade, precisão, linearidade e recuperação para a determinação quantitativa de patulina. Todos os estágios do processamento do suco contribuíram para a redução dos níveis de patulina. Desde a maçã até o suco a redução foi de 75,2%. A presença de Byssochlamys nivea em uma das amostras de suco indica que a patulina poderia ser produzida por este fungo durante o armazenamento. / Significant amounts of apples produced in the state of Rio Grande do Sul, Brazil, are used to juice production destined to exportation. Fruits used by the industry show some defects and are not accepted for fresh consumption. These apples are a risk factor considering the contamination by fungus, which can produce mycotoxins, such as patulin. Brazil does not have legislation for patulin in food. But many countries have adopted the recommendation of the Codex Alimentarius, which indicates a maximum permitted level of 50 μg/L of patulin in apple juice. This concern stimulates exporting countries, like Brazil, in the research of mycotoxigenic quality of apples and by-products, to become competitive and provide safe foods. In this context, the objectives of this work were (1) evaluate mould contamination of apples used to juice production and possible presence of patulin in apples; (2) methodologically adapt the thin-layer chromatographic technique for quantification of patulin in apples and apple juice; (3) analyze patulin levels in some processing stages of apple juice production; and (4) investigate heat-resistant moulds in juices. The apples and juice samples were obtained from Golden Sucos Ltda, located in Farroupilha city, in Rio Grande do Sul state, Brazil. Penicillium expansum was the most frequently isolated fungus, among these isolates, 94.44% were able to produce patulin under the tested conditions. The prevalence of patulin-producing fungi in apples resulted in high patulin levels in these apples, ranging from 254.62 to 653.37 μg/Kg and in juices ranging from 56.04 to 231.44 μg/L. The method developed to patulin extraction and quantification was effective. The charged coupled device imaging system provides good sensitivity, precision and linearity for the quantitative determination of patulin. All stages of apple juice production contributed to patulin reduction. The overall loss of patulin through processing from apple to apple juice was 75.2%. The presence of Byssochlamys nivea in a juice sample indicates that this fungus could produce patulin during storage.

Orientadores: Pilar Rodriguez de Massaguer, Amauri Rosenthal / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos / Made available in DSpace on 2018-08-08T21:56:14Z (GMT). No. of bitstreams: 1 Sant'Ana_AndersondeSouza_M.pdf: 2935543 bytes, checksum: 8836bcdf5bfa2652ee4e277718b808dc (MD5) Previous issue date: 2007 / Resumo: A patulina é uma micotoxina produzida por fungos pertencentes aos gêneros Penicillium, Aspergillus e Byssochlamys. Penicillium expansum se destaca por ser potencial produtor de patulina nas maçãs, enquanto Byssochlamys nivea e B.fulva se destacam pela potencial produção desta micotoxina em sucos de maçãs pasteurizados. À patulina tem sido atribuídos diversos efeitos agudos e crônicos adversos à saúde humana. No presente estudo determinou-se quantitativamente o risco dos níveis de patulina no suco de maçã ultrapassar o limite de 50ppb estabelecido pela Organização Mundial de Saúde (OMS) e a probabilidade de produção da patulina por bolores termoresistentes sobreviventes à pasteurização do produto. Para isto, i) avaliou-se a ocorrência de bolores termoresistentes e patulina em amostras pertencentes à 5 diferentes lotes de suco de maçã provenientes de uma unidade produtora localizada no sudeste do Brasil; ii) avaliou-se a capacidade de produção da patulina por cepas de B.fulva (IOC 4518) e B.nivea (ATCC 24008 e FRR 4421) em suco de maçã armazenado à 21ºC e à 30ºC (consideradas temperaturas médias anuais das regiões tropicais e subtropicais do Brasil); iii) determinou-se a cepa Byssochlamys produtora de patulina mais termoresistente dentre as três estudadas, em suco de maçã; iv) determinou-se a resistência térmica através do método dos tubos TDT (thermal death tubes) da cepa mais termoresistente; v) Estabeleceu-se o efeito da pasteurização em sistema contínuo (UHT), simulando as condições industriais, sobre a cepa de Byssochlamys spp mais termoresistente produtora de patulina; vi) determinou-se a probabilidade de produção de patulina pela cepa mais termoresistente produtora de patulina em suco clarificado de maçã variando-se a temperatura de estocagem (21ºC e 30ºC), com carga remanescente pós-processo de 10º e 101 esporos / 100 mL e vii) determinou-se quantitativamente o risco da patulina em suco clarificado de maçã a partir de 15 cenários que relacionados a diferentes cargas de esporos sobreviventes e temperatura de estocagem do suco pós-pasteurização, utilizando-se a modelagem de Monte Carlo, feita através do software @RISK versão 4.5 for students com 1 simulação e 10000 iterações. Os resultados revelaram que a ocorrência de bolores termoresistentes nas amostras de suco de maçã é baixa (<10ºesporos/100mL), com a cepa isolada de (Aspergillus carneus ¿ IOC 4519) não sendo confirmada como um bolor termoresistente. As três cepas estudadas (B.nivea FRR 4421, B.nivea ATCC 24008 e B.fulva IOC 4518) foram capazes de produzir a patulina em concentrações que dependeram da carga de esporos inoculada no suco de maçã, temperatura de estocagem e material de embalagem. B.fulva IOC 4518 foi determinada como a cepa mais termoresistente, sobrevivendo ao choque térmico de 95ºC por 5 min. Valores D* à 85ºC, 90ºC, 92ºC e 95ºC iguais a 64,58 min; 16,68 min; 6,31 min e 3,10 min, respectivamente foram obtidos, enquanto o valor Z foi igual a 7,4ºC. O processo de pasteurização do suco de maçã na unidade Microthermics mostrou que há variabilidade com relação ao número de reduções causadas pelo processo equivalente ao industrial quando variações na temperatura de processo da ordem de até 1ºC são observadas. Maiores probabilidades de crescimento de B.fulva IOC 4518 e maior extensão da deterioração no suco de maçã estão relacionadas ao aumento da carga de esporos sobreviventes e temperatura de estocagem pós-processo. A produção de patulina por B.fulva IOC 4518 é influenciada principalmente pela temperatura de estocagem quando a carga de sobreviventes é elevada (101esporos/100mL), com maiores quantidades da micotoxina sendo produzidas à 30ºC do que à 21ºC. Através do modelo de risco para o suco de maçã e patulina pôde-se concluir que a etapa de recepção é sempre a que mais impactou para que níveis elevados de patulina estejam presentes nos sucos de maçã. Mas, a etapa de estocagem após a pasteurização quando há bolores termoresistentes sobreviventes, foi a responsável pelas maiores concentrações finais de patulina, à medida que se aumentava o tempo de estocagem. As etapas de lavagem, filtração e seleção das frutas, respectivamente, são as responsáveis pela redução nos níveis de patulina durante o processamento do suco, enquanto a pasteurização em virtude da elevada resistência térmica desta micotoxina praticamente não apresenta efeito algum para redução dos níveis da micotoxina no produto final. Após 10000 iterações, dentre os 15 cenários avaliados, somente nos de número 1 (sem sobreviventes à pasteurização e concentração de patulina na matéria-prima na faixa baixa), 2 (sem sobreviventes à pasteurização e concentração de patulina na matéria-prima na faixa média), 4 (10ºesporos/100mL de B.fulva IOC 4518 sobreviventes à pasteurização, com temperatura de estocagem de 21ºC e concentração de patulina na matéria-prima na faixa baixa) e 7 (10ºesporos/100mL de B.fulva IOC 4518 sobreviventes à pasteurização, com temperatura de estocagem de 30ºC e concentração de patulina na matéria-prima na faixa baixa) o limite de 50ppb de patulina não seria ultrapassado para nenhuma das iterações, considerando-se valores médios e máximos da concentração final desta micotoxina. A distribuição da concentração de patulina no produto final se ajustou às distribuições do tipo Betageneral, Lognormal e Inversa Gaussiana dependendo da concentração de patulina na recepção das frutas, com a média e a maior parte dos dados se concentrando à esquerda / Abstract: Patulin is a mycotoxin produced by some species from Penicillium, Aspergillus and Byssochlamys ssp genera. Penicillium expansum are known by their potential to produce patulin in apples, while Byssochlamys nivea and B.fulva are recognized by its potential to produce this micotoxin in pasteurized apple juice. Several acute and chronic effects to human health have been attributed to patulin. This study has quantitatively assessed the risk of levels of patulin to exceed the level established by The World Health Organization ¿ WHO (50ppb) and also was determined the probability of patulin being produced by heat-resistant mold which survived the apple juice pasteurization. Therefore, the following items have been analyzed: i) it was evaluated the occurrence of heat resistant mold and patulin in samples belonging to 5 different lots of apple juice from a factory located in the southeast of Brazil; ii) the ability of patulin production by the strains of B.fulva (IOC 4518) and B.nivea (ATCC 24008 and FRR 4421) in apple juice stored at 21°C and 30°C (these are average year temperature in the tropical and subtropical regions of Brazil); iii) it was determined which of the three patulin producer strains, B. fulva and/or B.nivea, was the most heat resistant in apple juice; iv) it was determined the heat resistance (D and Z values) of the most heat resistant patulin producer strain of Byssochlamys using thermal death tubes (TDT); v) the effect of the continuous pasteurization system (UHT), simulating the industrial conditions, has been established over the most heat resistant and patulin producer strain of Byssochlamys spp; vi) the probability of producing patulin by the most heat resistant strain in clarified apple juice stored at 21°C and 30°C with a survival level post pasteurization process of 10º e 101 spores/100 mL, has also been established. vii) it has been quantitatively assessed the risk of patulin in clarified apple juice using Monte Carlo simulation, with @Risk software for students (version 4.5). The simulation was carried out with 10000 iterations. The results showed that the occurrence of heat resistant mold in the apple juice samples examined was low (<10ºesporos/100mL), with the strain Aspergillus carneus ¿ IOC 4519 isolated not confirming their heat resistance. The three mold strains studied (B.nivea FRR 4421, B.nivea ATCC 24008 and B.fulva IOC 4518) were able to produce patulin in concentrations that were dependent of spore inocula in apple juice, storage temperature and package type. B.fulva IOC 4518 was determined as the most heat resistant strain, surviving to heat shock at 95ºC/5 min. D* values at 85ºC, 90ºC, 92ºC and 95ºC of 64,58 min; 16,68 min; 6,31 min and 3,10 min, respectively were obtained, while z value was of 7.4ºC. The apple juice pasteurization process applied in a Microthermics pilot plant showed variability related to the number of decimal reductions caused by the equivalent process when temperature variations were near 1ºC. Higher growth probabilities for B.fulva IOC 4518 and higher extension of spoilage of apple juice are related to the increase of survival spore level and to storage temperature pos-pasteurization. The patulin production by B.fulva IOC 4518 was mainly influenced by storage temperature when survival spores level is elevated (101spores/100mL), with the higher quantities of this mycotoxin being produced at 30ºC than 21ºC. The risk assessment model for the apple juice and patulin showed that fruit reception is always the step that more impacted to higher levels of patulin being found in apple juices. However, storage step after pasteurization, when there are heat resistant survivors was responsible for the higher final concentrations, when the storage time increased. Fruit washing, juice filtration and fruit selection, respectively, are the main responsible steps to reduce patulin levels during apple juice processing, while juice pasteurization due to high heat resistance of patulin practically does not presents effects on mycotoxin reduction in the final product. After 10000 iterations, among the 15 scenarios evaluated, only in the scenario number 1 (without mold survival to pasteurization and with low level of patulin concentration in fruits), 2 (without mold survival to pasteurization and with medium level of patulin concentration in fruits), 4 (10º/100mL of B.fulva IOC 4518 spores survival to pasteurization, with storage temperature at 21ºC and low level of patulin in fruits) and 7 (10º/100mL of B.fulva IOC 4518 spores survival to pasteurization, with storage temperature at 30ºC and low level of patulin in fruits) the limit of 50ppb of patulin would not be exceeded for any of iterations, considering medium and maximum values of the final quantity of this mycotoxin. The final distribuition of patulin concentration in apple juice best fitted to Betageneral, Lognormal and Inverse Gaussian depending on the concentration on fruit reception, with the mean and the most part of data skewed to the left / Mestrado / Mestre em Ciência de Alimentos

Penicillium expansum est un contaminant commun des pomaceae (pommes et poires) causant la pourriture bleue. Ce champignon est le principal responsable de la présence de patuline dans les pommes et ses produits dérivés. Actuellement, la voie de biosynthèse de la patuline n’est que partiellement élucidée et le cluster de gènes correspondant n’est décrit que chez Aspergillus clavatus, champignon tellurique incapable de se développer dans les pommes. La caractérisation moléculaire de la voie de biosynthèse de la patuline est la condition sine qua none à toute étude visant à comprendre la régulation de la biosynthèse de la patuline, mais également à toute action permettant de limiter sa synthèse. C’est pourquoi le premier objectif de cette thèse a été de caractériser le cluster de gènes spécifique de la voie de biosynthèse chez Penicillium expansum. Celui-ci est caractérisé par une taille de 40 kb et contient les 15 mêmes gènes qu’Aspergillus clavatus, les seules différences résidant dans l’organisation et l’orientation des gènes. La caractérisation de la seconde étape de la voie de biosynthèse de la patuline a été ensuite entreprise chez Aspergillus clavatus, organisme modèle. Le gène patG code pour l’acide 6-méthylsalicylique décarboxylase responsable de la conversion de l’acide 6-méthylsalicylique en m-crésol. Pour faire suite au premier objectif, la régulation de la voie de biosynthèse de la patuline a été étudiée. Pour cela, une souche mutante pour le facteur de régulation spécifique à la patuline patL a été généré puis la production de patuline ainsi que l’expression des gènes du cluster analysés. Les résultats de cette étude ont montré que le gène patL joue le rôle d’interrupteur au sein du cluster. L’absence de patL conduit à une extinction totale de l’expression des gènes du cluster et à une abscence de production de patuline par Penicillium expansum. Dans cette même étude, des tests de pathogénicité ont été entrepris sur des pommes de différentes variétés démontrant ainsi que la patuline peut être un facteur de virulence facilitant l’infection de certaines variétés de pommes telles que la Golden Delicious ou la Pink Lady. Enfin, l’influence de la lumière a été évaluée en analysant l’impact de différentes longueurs d’ondes sur la croissance et la production de patuline de Penicillium expansum. Que ce soit in-vitro ou in-vivo, la croissance et la production de patuline sont très affectés par les lumières blanche, bleue et rouge. Favoriser le stockage des pommes sous les lumières blanche, bleue ou rouge plutôt qu’à l’obscurité pourrait devenir un moyen de prévention contre la contamination par Penicillium expansum. En conclusion, cette thèse présente un aspect fondamental avec la caractérisation du cluster de gènes chez Penicillium expansum et la caractérisation de la seconde étape de la voie de biosynthèse de la patuline ; mais aussi un aspect appliqué avec l’utilisation des lumières de différentes couleurs comme méthode de prévention contre Penicillium expansum durant le stockage des pommes. / Penicillium expansum is the common contaminant of apples and the causal agent of blue mold rot. This fungus is the main patulin producer in apple based products. Actually, the patulin biosynthesis is partially elucidates and the gene cluster has been elucidated in Aspergillus clavatus, a telluric fungi unable to grow on apples. The molecular characterization of the patulin biosynthetic pathway is the key step for a better understanding of the mechanisms leading to patulin production and will help to define strategies to reduce its presence in apple products. The first objective of this thesis was the characterization of the patulin gene cluster in Penicillium expansum. The latter includes the same 15 genes as in Aspergillus clavatus but in a different order and orientation. Then, the second step of this biosynthetic pathway has been characterized and the patG gene encode for the 6-methylsalicylic decarboxylase involved in the 6- methylsalicylic acid conversion into m-cresol. The second objective consists of the study of the patulin regulation. For that, a patL mutated strain was generated and the patulin production and the patulin gene cluster expression were assessed. The mutation of this gene results in a down-regulation of the rest of the genes in the cluster associated with a lack of patulin production. Pathogenicity tests on apples revealed that patulin could act as a virulence factor in some apple varieties, like Golden Delicious or Pink Lady. In the last part of this thesis, the influence of different wavelength lights on the growth and the patulin production by Penicillium expansum were assessed in vitro and in vivo. In both cases, growth and patulin production were significantly affected under white, blue and red lights. Consequently, the apple storage under these lights could be a good alternative to the storage in the dark. In conclusion, this thesis presents a fundamental aspect that consist in the characterization of the patulin gene cluster in Penicillium expansum and the characterization of the second step of this pathway. An applied aspect is also provided by the use of the different wavelength lights to prevent the Penicillium expansum contamination during apple storage.

This project focused on the development and validation of an ultra-performance liquid chromatography tandem mass spectrometer (UPLC-MS/MS) method for the determination of Patulin in apple-based products. Patulin is one of the many mycotoxins that are secondary metabolites from about 60 filamentous fungi. The mold often appears as black or blue on fruit, vegetables or crops. To determine the concentration of Patulin in consumer products is important since it may affect consumer health. The symptoms are often flu-like and can lead to kidney-failure and neurotic damage. The Swedish Food Agency is tasked to analyze consumer products to determine if they are safe to ingest. The European Commission has set maximum residue limits for several toxins that can potentially appear in groceries on the market. Using an UPLC-MS/MS allows for the accurate qualification and quantification of Patulin in apple juice and purees. The method was validated by analyzing several lots of apple juices and a proficiency test from Fapas®. The recovery rate ranged between 70.5-103.8% and were accepted because they met the recovery criteria in Regulation (EC) No. 401/2006 for Patulin.

<p>Sprovedena studija po prvi put izve&scaron;tava o prisustvu patulina, sekundarnog metabolita određenih vrsta plesni, u proizvodima od jabuka, kao i proceni rizika usled unosa patulina od strane odojčadi, dece, adolescenata i odrasle populacije u Republici Srbiji. Ukupno 356 uzoraka sokova i ka&scaron;ica za odojčad i malu decu (48 i 66, redom), sokova za decu (mala pakovanja sa cevčicom, 100) i sokova u porodičnom pakovanju (142), sakupljeno je sa trži&scaron;ta tokom tri godine i analizirano primenom tečne hromatografije sa ultraljubičastom detekcijom, metodom koja je predhodno validirana. Prisustvo patulina je detektovano u 44% i 17% uzoraka sokova i ka&scaron;ica za odojčad i malu decu, redom, u količinama nižim od zakonskog ograničenja od 10 &mu;g/kg (maksimalno 8,3 i 7,7 &mu;g/kg, redom). Udeo kontaminiranih uzoraka među sokovima za decu iznosio je 43%, sa najvi&scaron;om koncentracijom patulina od 30,2 &mu;g/kg ispod maksimalno dozvoljenog nivoa od 50 &mu;g/kg. Patulin je detektovan u 51% sokova u porodičnom pakovanju, sa 0,7% uzoraka iznad zakonske granice od 50 &mu;g/kg (prosečna koncentracija 4,3 &mu;g/kg). Sokovi od jabuka su pokazali značajno vi&scaron;i udeo kontaminiranih uzoraka (74% u odnosu na 28%), kao i vi&scaron;i prosečni sadržaj patulina (6,4 u odnosu na 2,1 &mu;g/kg) u poređenju sa sokovima od me&scaron;anog voća. Procena rizika usled unosa patulina od strane odojčadi, dece, adolescenata i odrasle populacije u Republici Srbiji, sprovedena primenom determinističkog i probabilističkog pristupa, uključujući rezultate biodostupnosti patulina, pokazala je da je odnos između procenjenog dnevnog unosa i toksikolo&scaron;ke referentne doze za patulin od 0,4 &mu;g/kg telesne mase, koji se označava kao &ldquo;hazard quotient&rdquo;, znatno ispod 1, &scaron;to ukazuje na tolerantan nivo izloženosti i nepostojanje razloga za zabrinutost za zdravlje populacije. Dodatno, analiza ostataka pesticida i toksičnih metala potvrdila je bezbednost proizvoda od jabuka na trži&scaron;tu. Međutim, identifikacija vi&scaron;estrukih ostataka pesticida je razlog za aktivan pristup i pažljivo planiranje i sprovođenje monitoringa bezbednosti hrane, posebno hrane za odojčad i malu decu, kao najosetljivije populacione grupe.</p> / <p>This study reports for the first time the occurrence of patulin, a secondary metabolite of certain fungi, in apple-based food, as well as risk assessment related to patulin intake by infants, children, adolescents and adults in Serbia. In total, 356 samples of infant fruit juices (48), infant pur&eacute;e (66), juices for children (small package with straw, 100), and juices in family package (142) were collected from the market over three years (2013&ndash;15) and analysed using validated method based on liquid chromatography with ultraviolet detection. Patulin was found in 44% of infant juices and 17% of infant pur&eacute;e, with all values below the legal limit of 10 &mu;g/kg (maximum 8.3 and 7.7 &mu;g/kg, respectively). The proportion of contaminated samples among fruit juices for children was 43%, with the highest patulin concentration at 30.2 &mu;g/kg, not exceeding the maximum allowed level of 50 &mu;g/kg. Patulin was found in 51% of juices in family package, with 0.7% of the samples in excess of the legal limit of 50 &mu;g/kg (mean 4.3 &mu;g/kg). Apple juices showed significantly higher percentage of contaminated samples (74% versus 28%), as well as higher mean patulin content (6.4 versus 2.1 &mu;g/kg) when compared with the multifruit ones. Risk assessment of patulin intake by Serbian infants, children, adolescents and adults, conducted by deterministic and probabilistic approaches and including the bioaccessibility results, revealed a ratio between exposure and toxicological reference dose for patulin of 0,4 &mu;g/kg body weigth, called hazard quotients, well below 1, indicating a tolerable exposure level and no health concern. Furthermore, analysis of pesticide residues and toxic metals confirmed safety of apple products on the market. However, identification of multiple pesticide residues is a reason for an active attitude and carefully planned and conducted monitoring of food safety, expecialy in the case of food for infants and young children, as they are the most susceptible population group.</p>

To assess the effects of gamma radiation at low doses, spores of Penicillium expansum NRRL 971 and Penicillium patulum NRRL 989 were exposed to doses of 0, 50, 100, 150, and 200 krads. The amount of patulin for each culture was quantitatively determined and growth at each dose was evaluated. The results showed a 31% increase in the amount of patulin synthesis after a dose of 150 krads for Penicillium expansum At 200 krads, both fungi showed low levels of patulin production and inhibited growth. Eight color mutants of P. expansum and eight color mutants of P. patulum were produced by exposing spores of each fungus to doses of 150 and 200 krads of gamma radiation. An evaluation of patulin production and growth for each mutant showed that color mutation could result in mutants with an enhanced ability to grow or produce increased levels of patulin. When grown at a subatmospheric pressure of 320 mm Hg, P. expansum produced 30% more patulin per flask, when compared to control cultures. At 160 mm Hg, P. expansum showed a 75% decrease over the control cultures. P. patulum was greatly affected by the temperature of 15℃ and showed decreases both in growth and patulin production. Effective control of growth and patulin synthesis was found for these fungi at 160 mm Hg.

La pourriture bleue causée par Penicillium expansum est l'une des maladies les plus dommageables des fruits pomaceae (pommes et poires). Outre des dégâts directs, cette maladie pose un problème de santé publique car l'agent pathogène produit des mycotoxines nocives pour l'homme et les animaux dont la plus sérieuse est la patuline. La croissance du champignon pathogène et la production de patuline requièrent des conditions physico-chimiques particulières. Les informations existantes à ce propos demeurent cependant modestes et insuffisantes pour envisager de développer des moyens de lutte contre l'apparition du champignon. Par ailleurs, la patuline reste avec l'ochratoxine A, les seules toxines dont la voie de biosynthèse n'a pas encore été complètement établie, tant sur le plan chimique que moléculaire. Cette étude apporte dans un premier temps des données complémentaires sur les facteurs physico-chimiques (température, pH….) qui conditionnent la croissance de P. expansum de même que sa capacité à produire la patuline. La connaissance de ces besoins et de ces conditions conduit en pratique à lutter et contrôler la contamination par la patuline tout le long de la chaine alimentaire. Dans un deuxième temps, cette thèse apporte des améliorations spectaculaires sur le plan fondamental, en termes d'élucidation de la voie de biosynthèse de la patuline. Le cluster des gènes impliqués dans la biosynthèse de cette mycotoxine chez l'espèce la plus préoccupante P. expansum a été entièrement identifié et caractérisé. Pour lever encore plus le voile sur la biosynthèse de cette mycotoxine, la caractérisation du facteur de régulation spécifique de cette voie (patL) a été également établie. Une perturbation de ce gène a provoqué une incapacité de production de patuline et une sévère diminution de l'expression des gènes Pat. De même, grâce à ce mutant déficient, il a été montré que la patuline pourrait agir comme facteur de virulence lors du développement de la moisissure dans les pommes. La caractérisation de la dernière étape de la voie de biosynthèse de la patuline a ensuite été entreprise par mutagenèse dirigée du gène patE du cluster de la patuline, chez la même espèce. Ce dernier code pour une Glucose méthanol Choline (GMC) oxydoréductase responsable de la conversion de l'ascladiol en patuline. L'ascladiol est également une molécule clé de la dégradation de la patuline par diverses espèces bactériennes ou de levures et plus particulièrement lors de la fermentation alcoolique. La non-toxicité de l'ascladiol accumulé chez le mutant ∆patE a été démontrée sur une lignée cellulaire intestinale humaine (Caco-2), suggérant que la patuline perd sa toxicité avec l'ouverture du deuxième cycle. Finalement, un système de détection et de quantification de P. expansum par PCR en temps réel a été développé en ciblant un gène hautement spécifique de la voie de biosynthèse de la patuline, patF. Cette approche préventive nous a ainsi permis d'avoir une estimation rapide de la contamination en patuline dans les pommes à partir de la quantification d'ADN de P. expansum. En conclusion, l'ensemble de ces travaux qui s'inscrivent dans le cadre de la gestion du risque « patuline » dans la filière fruit a permis d'amener des réponses tant sur le plan fondamental que sur le plan appliqué avec le séquençage du cluster, le développement d'un outil de diagnostic et la démonstration que l'ascladiol ne présentait aucune cytotoxicité. / Among diseases affecting apples, blue mold caused by Penicillium expansum is a major concern causing yield and quality losses due to the production of mycotoxins, of which patulin is the most alarming one. This mycotoxin was proven to be harmful for humans and animals. The pathogen growth and the patulin production occur under specific physico-chemical conditions (temperature, pH…). However, the description of these conditions in literature remains largely insufficient for the development of strategies to fight the development of the fungus. Furthermore, patulin remains, along with ochratoxin A, the only toxins for which the biosynthetic pathway is not fully established yet at both chemical and molecular levels. Firstly, this study provides supplementary data on the physico-chemical factors that modulate P. expansum growth and its ability to produce patulin. The acquaintance of these conditions leads, in practice, to the control of the patulin contamination along the food chain. Secondly, significant improvements were brought on the fundamental level, especially by elucidating the patulin biosynthetic pathway. The cluster of genes involved in the biosynthesis of this mycotoxin was fully identified and characterized in the species of greatest concern P. expansum. In order to reveal additional info on the biosynthesis of this mycotoxin, the specific factor of the pathway (patL) was characterized. The disruption of this gene has led to failure in patulin production and an important decrease in Pat genes expression. Furthermore, pathogenesis studies, using this same deficient strain showed that patulin potentially acts as a virulence factor during P. expansum development on apples. The last step of the patulin biosynthetic pathway was later characterized by site-directed mutagenesis of the patE gene in the same species. This gene encodes a Glucose Methanol Choline (GMC) oxidoreductase that is responsible for the conversion of ascladiol to patulin. Ascladiol is not only the last intermediate in the patulin pathway but also the main product of patulin degradation during the alcoholic fermentation of apple juice. The non-toxicity of ascladiol accumulated by the ΔpatE strain was proved against the human Caco-2 cell line. Finally a Real time PCR assay was developed to specifically detect and quantify P. expansum. This was done by targeting a highly specific gene from the patulin gene cluster in P. expansum, patF. This predictive approach allowed the quick estimation of the patulin content via the quantification of the P. expansum DNA in apples. To conclude, this thesis is part of the patulin's risk management study in the fruit sector; it provides significant improvements on both fundamental and practical levels. These advances are mainly characterized by the sequencing of the patulin gene cluster, the development of a molecular diagnostic tool and the demonstration of the non-cytotoxicity of ascladiol.

Parmi les maladies affectant les pommes, la moisissure bleue causée par Penicillium expansum est une préoccupation majeure. Elle cause des pertes de rendement et de qualité dues également à la production de mycotoxines telles que la patuline (PAT) et la citrinine (CIT). La PAT est la plus alarmante en raison de ses propriétés cytotoxiques, génotoxiques et immunosuppressives.L'Union européenne (UE) a établi des réglementations spécifiques pour protéger la santé des consommateurs et des niveaux maximaux de 50 g / kg sont fixés pour les jus de fruits et les produits dérivés, 25 g / kg pour les purées de pommes et les compotes et 10 g / kg pour les aliments destinés aux bébés et aux jeunes enfants. En dépit de ces mesures, la PAT continue à être présente dans les aliments et / ou les boissons commerciaux, dépassant parfois les limites maximales. Des recherches supplémentaires sont par conséquent nécessaires pour minimiser la contamination des produits alimentaires par cette mycotoxine et son champignon producteur. Bien que la plupart des études sur P. expansum soient essentiellement centrées sur la PAT, le génomede ce champignon présente d'autres clusters de métabolites secondaires (SM) prédits dont certains peuvent être associés à des métabolites potentiellement toxiques. Afin de contrôler la synthèse de SM, l'étude des facteurs de transcription globaux régulant leur production est essentielle. Dans une première partie, le gène veA, appartenant à la famille des protéines du complexe velvet, a été caractérisé et son impact sur le développement du champignon, sa virulence et son métabolisme secondaire a été élucidé. La délétion de ce gène a conduit à une réduction de la production de PAT et de CIT et à une diminution de l'expression de leurs clusters de gènes. VeA a également un impact global sur le métabolisme secondaire, puisque 15 des 35gènes de structure présentent une régulation différentielle sur les milieux testés. Dans une deuxième partie, l’influence de l’acide ascorbique (AA) sur la concentration de PAT dans le jus de pomme trouble a été étudiée à la fois en laboratoire et en milieu semi-industriel. Une méthodologie analytique séparant la PAT et d'autres composés générés au cours de la réaction aété optimisée. Les conditions optimales d'action de l’AA sur la PAT ont été analysées. De plus,nous avons identifié des produits de dégradation moins toxiques que la PAT et résultant du traitement par l’AA. Pour conclure, cette thèse se rattache à la gestion des risques de la PAT dans le secteur des fruits ; elle apporte des connaissances et des améliorations significatives tant sur le plan fondamental que sur le plan pratique. Ces avancées résident principalement dans la description d'une souche mutée de P. expansum moins toxique que celle naturellement retrouvée dans la nature, et décrivant un additif alimentaire améliorant les qualités de nombreux produits transformés et diminuant la concentration de PAT en générant des composés moins toxiques. / Among diseases affecting apples, blue mould caused by Penicillium expansum is a major concern. It causes yield and quality losses, as well as food safety issues due to the production of mycotoxins such as patulin (PAT) and citrinin (CIT). PAT is the most worrying one and has cytotoxic, genotoxic and immunosuppressive properties. The European Union (EU) has established specific regulations to protect the consumer’s health and maximum levels of PAT of 5 g/kg is set for fruit juices and derived products, 25 g/kg for apple purees and compotes and 10g/kg for food intended for babies and young children. However, PAT is still found in commercial food and/or beverage products, sometimes exceeding the maximum limits and more research is needed to minimize contamination of food products by this mycotoxin and its fungus. Even though most studies on P. expansum have focused on PAT itself, the genome of this fungus exhibits other predicted secondary metabolite (SM) clusters, some of which may be associated with potentially toxic metabolites. In order to control the synthesis of SMs, the study of global transcription factors regulating their production is essential. In a first part, the veA gene, belonging to the velvet family, was characterised and its impact on the development of the fungus, its virulence and its secondary metabolism was elucidated. The disruption of this gene led to the failure in PAT and CIT production and a decrease in the expression of their gene cluster. It also revealed a global impact on the secondary metabolism, as 15 of 35 backbone genes showed differential regulation on the media tested. In a second part, the influence of ascorbic acid (AA) on the concentration of PAT in cloudy apple juice was studied on both lab and semi-industrial scale. An analytical methodology separating PAT and other compounds generated during the reaction was optimized. Optimal conditions of action of AA on PAT were studied. In addition, degradation products less toxic than PAT and resulting from AA treatment were identified. To conclude, this thesis is part of the risk management of PAT in the fruit sector; it provides significant improvements at both fundamental and practical levels. These advances are mainly characterized by the description of a mutated strain of P. expansum that is less toxic than that naturally occurring in nature, and the description of a food additive that improves numerous products qualities and affects PAT concentration, thusgenerating less toxic compounds