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P. roqueforti est une moisissure connue comme contaminant des ensilages et des aliments, mais aussi utilisée comme ferment pour la production de fromages à pâte persillée. Cette espèce est également connue pour son potentiel de production de métabolites secondaires à impact positif, comme les arômes, mais aussi à impact négatif, tels que des mycotoxines (dont la roquefortine C -ROQC- et l’acide mycophénolique -MPA-). Dans la première partie de ce travail, l’occurrence de la ROQC, du MPA et de l’aflatoxine M1 (seule mycotoxine réglementée dans les produits laitiers), dans une collection de 86 fromages à pâte persillée de différentes variétés et origines géographiques (15 pays), a été réalisée. Il apparaît que, si l’aflatoxine est inférieure au seuil de détection (LOD) de la méthode, les concentrations en ROQC et MPA sont quant à elles très variables et qu’une co-occurrence de ces mycotoxines existe dans 51% des échantillons testés. Dans la seconde partie, la toxicité de ces mycotoxines sur modèles cellulaires intestinal ou monocytaires a été évaluée. L’étude de cytotoxicité en mono-exposition a permis d’établir des valeurs de référence (CI50); d’autre part, un effet synergique des deux mycotoxines aux plus fortes concentrations testées a été révélé sur la lignée cellulaire intestinale (Caco-2). L’implication du mécanisme apoptotique après une exposition précoce (3 et 6 heures) à la ROQC et/ou au MPA a aussi été mise en évidence sur le modèle THP-1 ainsi qu’une absence d’implication de l’apoptose après 24 heures de mono ou de co-exposition sur modèle intestinal Caco-2. Enfin, dans la troisième partie, une étude de facteurs biotique ou abiotiques pouvant potentiellement moduler la production de ROQC et de MPA a été réalisée. L’évaluation du potentiel mycotoxigénique de 96 souches de P. roqueforti a mis en évidence que le milieu cheese-agar (mimant la composition physico-chimique du fromage) était beaucoup moins favorable que le milieu YES pour la production des deux mycotoxines et une grande variabilité dans la capacité de production des souches a été observée. L’étude de l’influence des facteurs abiotiques a montré qu’une température de 12°C ou une concentration en NaCl de 8% ou encore une atmosphère contenant 5% d’O2 diminuait significativement la production de ROQC et de MPA indépendamment de la croissance, alors que les autres facteurs (pH et présence de précurseurs de la ROQC) n’avaient pas d’effet dans les conditions testées. / P. roqueforti is a mould associated with silage and food contamination but also used as a ripening culture for the production of blue-veined cheese. Moreover, this species is also known for its potential to produce secondary metabolites. These metabolites can have a positive impact (e.g. aromas) or a negative impact (e.g. mycotoxins, including roquefortine C -ROQC- and mycophenolic acid -MPA-). In the framework of this work, we first studied the occurrence of MPA, ROQC and aflatoxin M1 (the only regulated mycotoxin in dairy products) within an 86 blue-veined cheese collection (representing 15 countries). Aflatoxin contents were always below the method detection limit. For MPA and ROQC, concentrations were highly variable and a co-occurrence of both mycotoxins was observed in 51% of the tested samples. Then, the toxicity of the 2 mycotoxins was established on various cell models (intestinal and monocytic). Mono-exposure studies allowed IC50 determination. Moreover, a synergistic effect was observed on Caco-2 cells when MPA and ROQC were used for co-exposure at the highest tested concentrations. While an apoptotic mechanism was observed at early THP-1 exposure stages (3 and 6h), no apoptose occurred for Caco-2 cells after 24 h of mono or co-exposure. Finally, a study of the biotic and abiotic factors potentially modulating P. roqueforti MPA and ROQC production was performed. Mycotoxigenic potential evaluation of 96 de P. roqueforti strains highlighted that a cheese-agar medium (mimicking cheese physico-chemical composition) was less favourable than the synthetic YES medium for mycotoxin production. A large variability in terms of mycotoxin production was also observed among the tested strains. Besides, while a significant effect of the temperature (12°C) and NaCl (8%) and O2 (5%) concentrations was observed on ROQC and MPA production, no significant effect of pH and ROQC amino-acid precursor addition could be detected on the production of both mycotoxins.

Penicillium roqueforti é um fungo deteriorante que ocasiona perdas principalmente em queijo parmesão, devido alterações no sabor e na cor do produto final. O controle biológico com leveduras do gênero Saccharomyces tem capacidade antifúngica e algumas têm efeito probiótico. Desta forma, o objetivo do presente trabalho foi utilizar leveduras como biocontrole do fungo P. roqueforti em queijo parmesão. Inicialmente, realizou análises in vitro para verificar a capacidade das leveduras S. cerevisiae UFT 5962, UFT 5992, UFT 5976, YEF 186 e S. boulardii em reduzir a quantidade de esporos produzidos por P. roqueforti e como também investigar o potencial de substâncias GRAS (bicarbonato de sódio, carbonato de sódio, carbonato de cálcio, cloreto de cálcio nas concentrações 1, 3 e 5% e cloreto de potássio nas concentrações 0,1; 0,5 e 1%) na inibição deste fungo. Foi realizada a segunda etapa do experimento através de testes em queijos parmesão utilizando o controle biológico clássico aplicando as leveduras e o controle biológico integrado onde utilizou a substância GRAS selecionada associado às leveduras antagonistas. Os resultados foram calculados pela área de contaminação e realizado o tratamento estatístico com teste Scott-Knott. Além disso, foi averiguado o tempo de permanência das leveduras na superfície dos queijos parmesão, através da inoculação e re-isolamento das leveduras no queijo. Nos resultados in vitro a S. cerevisiae UFT 5992 reduziu 92% dos esporos no 7o dia, e não foi observado modificações nas estruturas dos esporos. Das cinco substâncias GRAS testadas o bicarbonato de sódio a 1% obteve 100% de inibição do crescimento fúngico, e as leveduras antagonistas (S. boulardii e S. cerevisiae UFT 5992) sobreviveram por 60 dias nesta solução. Os resultados obtidos dos ensaios de controle biológico clássico constataram que a levedura S. cerevisiae YEF 186, reduziu em 17,5% o crescimento do P.roqueforti comparado com o controle no 13º dias. No controle integrado (S. cerevisiae UFT 5992+ BS), a redução fúngica foi de 18%. Nos dois processos, as leveduras sobreviveram por 50 dias demostrando uma boa adaptação das leveduras em queijo parmesão e podendo agregar valor probiótico ao produto final, estendendo sua vida de prateleira por tempo considerado. / Penicillium roquefortiis a filamentous fungus that can to produce loses, mainly in parmesan cheese, due the alterations on organoleptic characteristics. The biological control, using yeasts from Saccharomycesgenus can to control this pathogen and some species from this genus are considered probiotic. In this way, the objective of this work was to use yeasts to control P. roqueforti in parmesan cheese. Initially, were performed in vitro assays to verify the S. cerevisiae, strains UFT 5962, UFT 5992, UFT 5976, YEF 186 and S. boulardii capacity in to reduce the spore production by P. Roqueforti. At the same time, were verified the activities of some GRAS substances (sodium bicarbonate, calcium carbonate and calcium chloride at 1, 3, and 5 % and potassium chloride at 0.1, 0.5, and 1 %) over this pathogen. After this, were realized a classical biological control (using only the yeasts to control the pathogen) and integrated biological control (using yeast plus the selected GRAS substances) assays in the cheese. The results were obtained measuring the growing area of pathogen and the values were analyzed by Scott-Knott test. The time of endurance of the yeasts in the parmesan surface was obtained by re-isolation of the yeasts inoculated on cheese surface. S. cerevisiae UFT 5992 reduced 92 % of spore production after 7 days, but were not observed morphological alterations in the spores. Sodium bicarbonate at 1 % reduced totally the pathogen growth, and the antagonistic yeasts (S. boulardii e S. cerevisiae UFT 5992) keep alive in sodium bicarbonate (1 %) by 60 days. In the classical biological control, S. cerevisiae YEF 186, reduced the pathogen growth in 17.5 %after 13 days. The integrated control (S. cerevisiae UFT 5992+ BS) reduced the pathogen in 18 %.In both process, the yeasts keep alive by 50 days in parmesan surface, showing a good possibility of their use as probiotic and increasing the shelf life of this product.