Les Penicilliums sont des champignons filamenteux appartenant au genre Ascomycota. Ces champignons ont été utilisés par l’homme pour la production de nourriture depuis des siècles. Plus récemment, ils ont aussi été utilisés dans l’industrie biotechnologique pour la production de composés chimiques d’intérêts pharmaceutiques. Certaines espèces de Penicillium sont par ailleurs des moisissures contaminants certains aliments, d’autres sont des pathogènes de plantes, y compris de certains fruits. Leur génomique est globalement peut connue. Dans cette étude, nous avons analysé les génomes de deux espèces nouvellement séquencées, Penicillium roqueforti et Penicillium camemberti. Nous reportons ici le développement d’une nouvelle méthodologie pour l’amélioration et la validation d’assemblage de génomes en utilisant une technologie permettant l’observation de molécules d’ADN unique, le Peignage Moléculaire. En utilisant cette méthode, nous avons amélioré l’assemblage de Penicillium roqueforti. Ce manuscrit décrit aussi de multiples occurrences d’un transfert horizontal d’un ilot génomique de plus de cinq cent kilobases entre plusieurs Penicillium. Ce cas de transfert horizontal indique une fréquence d’échange latéral de matériel génétique plus forte qu’attendue. Enfin nous présentons un inventaire préliminaire du potentiel génomique pour la production de métabolites secondaires dans ces importants Penicillium alimentaires. / Penicillium are filamentous fungi belonging to the Ascomycota genus. Penicillium species have been used by Man for centuries in food making processes. More recently they have also been used in the biotechnology industry for the production of compounds of pharmaceutical interest. Some Penicillium species are food spoilage agents, pathogens of plants including fruits. Aspects of their genomics are largely unknown. In this study, we analysed the genomes of two newly sequenced species, Penicillium roqueforti and Penicillium camemberti. Here we report the development of a new methodology for improving and validating genome assembly using an original single DNA molecule technology, Molecular Combing. Using this methodology we were able to produce a high quality genome assembly of Penicillium roqueforti. This work also reports the multiple and recurrent horizontal transfer of a large genomic island of over half a megabase between several Penicillium species. This horizontal transfer indicates a higher frequency of lateral genetic exchange between cheesemaking fungi than previously expected. Finally, we present an early assessment of the genomic potential for secondary metabolite production in these important food associated penicilliums.

Cheddar Cheese samples (good cheese, weak cheese, cheese made with pasteurized milk, cheese made with heat-shocked milk, cheese from production plant A, cheese from production plant B, cheese made with adjunct culture, and cheese made without adjunct culture), were evaluated during the ripening stage. Proteolysis was studied by a fractionation scheme, resulting in an insoluble fraction analyzed by urea polyacrylamide gel electrophoresis (Urea-PAGE), and a soluble fraction which was further investigated through water soluble nitrogen (WSN), trichloroacetic acid soluble nitrogen (TCA-SN) and phosphotungstic acid soluble nitrogen (PTA-SN) analyzed by total Kjeldahl nitrogen content (TKN). Reversed phase high performance liquid chromatography (RP-HPLC) was used to study the peptide profile of the water soluble fraction. Lipolyisis was studied by levels of individual free fatty acids determined through gas chromatography-flame ionization detection (GC-FID) after isolation employing solid phase extraction (SPE). Volatile sulfur compounds were studied using head space solid phase micro-extraction (SPME) coupled with gas chromatography-pulsed flame photometric detection (PFPD). It was found that Urea-PAGE is capable to differentiate samples according their age, but cannot discriminate samples regarding the treatment assessed, quality or origin of the samples. However, measurements of total Kjeldahl Nitrogen (TKN) of the WSN, TCA-SN, and PTA-SN fractions, and the principal component analysis of the RP-HPLC peptide profile of the WSN fraction, revealed differences in the rate and pattern of proteolysis for each one of the manufacturing cases. Good cheese, cheese produce in plant TCCA, cheese made in plant CRP with adjunct culture isolated from plant TCCA cheese, and cheese made with heat-shocked milk developed higher level of total nitrogen for the WSN, TCA-SN and PTA-SN fractions, indicating that primary and secondary proteolysis were faster for these samples. This is supported by a PCA model with three principal components that account for the 80-83% of the variability of the data from the RP-HPLC peptide profile analysis, which discriminates the samples according to age and manufacturing practice. In addition, FFA profiles demonstrated higher levels of low and medium chain free fatty acids for good cheese, cheese produce in plant TCCA, cheese made in plant CRP with adjunct culture, and cheese made with heat-shocked milk samples, which suggest faster lipolysis during ripening. The Volatile Sulfur Compounds (VSC) analysis showed higher levels of DMS and MeSH and lower levels of H2S, suggesting faster catabolism of sulfur containing amino acids in good cheese, cheese produce in plant TCCA, cheese made in plant CRP with adjunct culture, and cheese made with heat-shocked milk. / Graduation date: 2013

Duas formulações de queijo de leite cru e fermento endógeno foram produzidas e avaliadas após 30, 60 e 180 dias de armazenamento em câmaras de maturação de dois laticínios do Sudoeste do Paraná foram estudadas para avaliar as características, como uma das etapas de desenvolvimento de um queijo típico regional. A produção foi acompanhada desde a elaboração do fluxograma de processamento, coleta das amostras de queijo para realização de análises de proteína, lipídeos, umidade, cinzas, carboidratos, extrato seco total, gordura no extrato seco, calorias; atividade de água, textura instrumental (dureza, adesividade, elasticidade, coesividade, mastigabilidade e resiliência), cor (CIE Lab), análises microbiológicas (contagem de coliformes totais, contagem de coliformes termotolerantes e contagem de Staphylococcus coagulase positiva e pesquisa de Salmonella spp.); teste de aceitação relacionada às características sensoriais (cor, aparência, odor, textura, sabor) e intenção de compra. Essa pesquisa contribuiu com informações relevantes ao processo produtivo, tais como, a constatação da viabilidade do fermento lático liofilizado elaborado a partir das bactérias ácido láticas isoladas do leite da mesorregião do Sudoeste do Paraná e cujos resultados das análises dos queijos indicaram similaridade entre as formulações, no que se refere a caracterização física e físico-química, além de boa qualidade microbiológica, onde as discrepâncias entre as amostras dos laticínios não foram percebidas sensorialmente pelos provadores. Ajustes na padronização relacionados ao controle de qualidade tecnológica serão um fator de extrema importância para o sucesso das empresas e pequenos produtores envolvidos no projeto e que se dispõem a produzir o Santo Giorno, um queijo fino, com o grande diferencial de agregar características da região, com elevado padrão de qualidade higiênico-sanitária e com indicativos de grande aceitação pelo consumidor. / Two cheese formulations made from raw milk and endogenous yeasts with 30, 60 and 180 days of maturation in two dairy Paraná Southwest were studied to evaluate their quality through physical, physical-chemical, microbiological and sensorial characteristics, as one of the stages of development of a typical regional cheese. The production was accompanied from designing the flowchart processing, where the samples were collected to perform the analysis of proteins, lipids, moisture, ash, carbohydrates, total solids, fat in dry matter, calories; water activity, instrumental texture (hardness, adhesiveness, springiness, cohesiveness, resiliency and chewiness), instrumental color (CIE Lab); microbiological quality was assessed searching for total and thermotolerant coliforms, coagulase positive staphylococci and Salmonella spp.; the acceptance related to sensory characteristics of color, appearance, flavor, texture, taste and purchase intent was evaluated through the structured hedonic scale. This research contributed information relevant to the production process, such as the realization of the viability in freeze-drying lactic acid bacteria yeast isolated from milk in the Southwestern Mesoregion of Parana and the results of the analysis of the cheese showed similarity between formulations, regarding the physical, physical-chemical characterization, moreover good microbiological quality, where the differences between samples of dairy products were not perceived by sensory panelists. Adjustments in standardization related to technological quality control is an extremely important factor for the success of dairy companies and small producers involved in the project and that they have the option of producing the Santo Giorno, a fine cheese, with the great advantage of adding features of region, with high standard of sanitary conditions and with great consumer acceptance of indicative.

Duas formulações de queijo de leite cru e fermento endógeno foram produzidas e avaliadas após 30, 60 e 180 dias de armazenamento em câmaras de maturação de dois laticínios do Sudoeste do Paraná foram estudadas para avaliar as características, como uma das etapas de desenvolvimento de um queijo típico regional. A produção foi acompanhada desde a elaboração do fluxograma de processamento, coleta das amostras de queijo para realização de análises de proteína, lipídeos, umidade, cinzas, carboidratos, extrato seco total, gordura no extrato seco, calorias; atividade de água, textura instrumental (dureza, adesividade, elasticidade, coesividade, mastigabilidade e resiliência), cor (CIE Lab), análises microbiológicas (contagem de coliformes totais, contagem de coliformes termotolerantes e contagem de Staphylococcus coagulase positiva e pesquisa de Salmonella spp.); teste de aceitação relacionada às características sensoriais (cor, aparência, odor, textura, sabor) e intenção de compra. Essa pesquisa contribuiu com informações relevantes ao processo produtivo, tais como, a constatação da viabilidade do fermento lático liofilizado elaborado a partir das bactérias ácido láticas isoladas do leite da mesorregião do Sudoeste do Paraná e cujos resultados das análises dos queijos indicaram similaridade entre as formulações, no que se refere a caracterização física e físico-química, além de boa qualidade microbiológica, onde as discrepâncias entre as amostras dos laticínios não foram percebidas sensorialmente pelos provadores. Ajustes na padronização relacionados ao controle de qualidade tecnológica serão um fator de extrema importância para o sucesso das empresas e pequenos produtores envolvidos no projeto e que se dispõem a produzir o Santo Giorno, um queijo fino, com o grande diferencial de agregar características da região, com elevado padrão de qualidade higiênico-sanitária e com indicativos de grande aceitação pelo consumidor. / Two cheese formulations made from raw milk and endogenous yeasts with 30, 60 and 180 days of maturation in two dairy Paraná Southwest were studied to evaluate their quality through physical, physical-chemical, microbiological and sensorial characteristics, as one of the stages of development of a typical regional cheese. The production was accompanied from designing the flowchart processing, where the samples were collected to perform the analysis of proteins, lipids, moisture, ash, carbohydrates, total solids, fat in dry matter, calories; water activity, instrumental texture (hardness, adhesiveness, springiness, cohesiveness, resiliency and chewiness), instrumental color (CIE Lab); microbiological quality was assessed searching for total and thermotolerant coliforms, coagulase positive staphylococci and Salmonella spp.; the acceptance related to sensory characteristics of color, appearance, flavor, texture, taste and purchase intent was evaluated through the structured hedonic scale. This research contributed information relevant to the production process, such as the realization of the viability in freeze-drying lactic acid bacteria yeast isolated from milk in the Southwestern Mesoregion of Parana and the results of the analysis of the cheese showed similarity between formulations, regarding the physical, physical-chemical characterization, moreover good microbiological quality, where the differences between samples of dairy products were not perceived by sensory panelists. Adjustments in standardization related to technological quality control is an extremely important factor for the success of dairy companies and small producers involved in the project and that they have the option of producing the Santo Giorno, a fine cheese, with the great advantage of adding features of region, with high standard of sanitary conditions and with great consumer acceptance of indicative.