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Au contraire du Camembert traditionnel, la pré-acidification du Camembert stabilisé est limitée par l’inoculation des bactéries lactiques thermophiles à une température inférieure à celle de leur croissance optimale (35-39 °C). Geotrichum candidum est une levure essentielle pour l’affinage du fromage Camembert grâce à ses activités biochimiques. En outre, quelques études ont rapporté que cette levure a été détectée dans les fèces humaines suite à la digestion du Camembert. Cette présence pourrait être due soit à la résistance intrinsèque des souches de G. candidum ou en lien avec les propriétés protectrices de la matrice fromagère. L’objectif de notre étude était d’examiner l’effet protecteur procuré à la souche G. candidum LMA-1028, par les propriétés de la matrice du fromage Camembert pendant la digestion statique in vitro. Afin d’y parvenir, deux matrices liquides (i.e. lait 3,25 % matières grasses et un milieu de culture) ainsi que deux matrices fromagères (i.e. Camembert traditionnel et Camembert stabilisé) ont été analysées. La survie de G. candidum et la désintégration de matrices étudiées ont été évaluées à différents temps de digestion aux étapes buccale, gastrique et duodénale. La désintégration du lait et du milieu de culture était plus élevée que celle des matrices fromagères en raison de leur structure liquide. La désintégration du Camembert stabilisé est plus importante que celle du Camembert traditionnel, ceci pourrait être attribué entre autres à une composition en lipides plus élevée. Globalement, la teneur en matière grasse des matrices laitières contrôle la progression de la désintégration. Lors de la digestion in vitro, la survie de G. candidum a été évaluée. Les résultats sur la viabilité de G. candidum LMA-1028 ont montré que cette souche est hautement résistante. La composition, la structure et les propriétés physicochimiques des matrices laitières n’ont pas amélioré la viabilité de G. candidum LMA-1028 pendant le transit gastro-intestinal. / Compared to traditional Camembert-type cheese, stabilized Camembert’s pre-acidification is limited using thermophilic lactic acid bacteria that are inoculated and used under their optimal growth temperature (35-39 °C). Geotrichum candidum is an essential ripening yeast of Camembert cheese due to its biochemical activities. Incidentally, it has been detected in human feces after Camembert consumption. However, this observation could be due either to the intrinsic G. candidum resistance to the gastrointestinal condition or to the protective properties of the Camembert cheese matrix. This study examines the putative protective effect of the cheese matrix on G. candidum LMA-1028 viability during static in vitro digestion. For this purpose, two liquid matrices (i.e. culture medium and pasteurized whole milk (3.25 %fat)) and two Camembert-type cheese variety (i.e. traditional and stabilized) were analyzed. G. candidum LMA-1028 survival under digestive stress was investigated at five digestion times (oral: 2 min, gastric: 60 and 120 min and duodenal: 60 and 120 min), while matrix disintegration was evaluated at three times (oral: 2 min, gastric: 120 min and duodenal: 120 min). Milk and culture medium matrices displayed higher disintegration than cheese matrices due to their liquid nature. The lowest measured disintegration of traditional Camembert compared to stabilized cheese matrix could be attributed to the higher fat content. Overall, dairy matrices disintegration was significantly modulated by the matrix fat content. The structure of the casein networks of milk and Camembert cheeses appears to modulate the accessibility of digestive juice into these matrices during gastric digestion. The difference in the original structure of both Camembert cheese matrices led to different rates of gastric disintegration and resulted in different rates of fat release. When comparing viability counts, G. candidum LMA-1028 showed a high intrinsic resistance to simulated gastrointestinal stresses. Camembert cheese matrices as well as milk didn’t bring additional protection to the studied strain LMA-1028.

Geotrichum candidum est une levure dimorphique qui occupe plusieurs rôles importants au sein de l’écosystème microbien de la surface des fromages à croute fleurie et à croute lavée. Ses différentes activités, notamment celles d’alcalinisation et de production de composés aromatiques, sont toutefois reconnues pour être souche-dépendantes. Peu d’études ont été menées sur la génétique de l’espèce G. candidum, bien que celle-ci mériterait d’être exploitée dans l’optique d’optimiser la production de ferments d’affinage utilisés dans les fromages affinés en surface. Dans le cadre de ce travail, les génomes de 11 souches de Geotrichum/Galactomyces spp. isolées de produits laitiers ou de l’environnement, ont été séquencés et comparés au niveau génomique. Également, afin d’avoir une caractérisation complète des souches, leur morphologie, leur capacité à croître en présence de différentes sources de carbone et à 35 °C, ainsi que leur capacité à se développer dans des caillés modèles ont été évaluées. Des divergences ont été observées entre les séquences génomiques des souches de Geotrichum spp. et Galactomyces spp., conduisant à la distinction des différentes espèces étudiées et à la formation de différents sous-groupes au sein de l’espèce G. candidum qui sont similaires à ceux établis par une étude préalablement réalisée par « Multilocus Sequence Typing » (MLST). Des différences ont également été observées entre les souches de l’espèce Geotrichum spp. en ce qui concerne leur morphologie, leur capacité à assimiler différentes sources de carbone ainsi que leurs propriétés alcalinisantes et protéolytiques lors de l’affinage de caillés modèles. La caractérisation génomique et phénotypique de souches de Geotrichum spp. fournit ainsi de nouvelles informations sur leurs caractéristiques et leur comportement dans différents milieux. Ce travail est donc d’une grande importance pour favoriser une meilleure sélection des souches de Geotrichum candidum pour le développement et l’amélioration de fromages affinés en surface. / Geotrichum candidum is a dimorphic yeast which plays several important roles within the surface microbial ecosystem of mold- and smear-ripened cheeses. Its various abilities, in particular those of alkalinization and production of aromatic compounds in cheeses are, however, recognized to be strain-dependent. Few studies have been carried out on the genetics of the species G. candidum, although this kind of information would be very useful in order to optimize the production of ripening cultures used in different types of surface ripened cheeses. In this project, the complete genomes of 11 Geotrichum/Galactomyces spp. strains isolated from dairy products or the environment were sequenced. Furthermore, in order to have a complete characterization of the strains, their morphology and their ability to grow in presence of different carbon sources, at 35°C, and in soft-cheese model curds were tested. Our results showed divergence between the genomic sequences of Geotrichum spp. and Galactomyces spp. strains, leading to the distinction of the different species studied and to the identification of different sub-groups among the G. candidum species that are consistent with those established in a previous study using Multilocus Sequence Typing (MLST). Differences were also observed between Geotrichum spp. strains for their morphology, their ability to metabolize different carbon sources, and their alkalinisation and proteolytic activities on soft-cheese model curd. The genome sequencing and the phenotypic characterization of the Geotrichum spp. strains provide new information about their characteristics and behavior in different media. This work is therefore of great importance to perform a better selection of G. candidum strains for the development and improvement of surface ripened cheeses.

En France, la récente apparition et implantation de l’espèce Fusarium langsethiae sur les orges de brasserie est devenue une source d’inquiétude. Ce champignon étant connu comme producteur de toxines T-2 et HT-2 cela pose le problème de la présence éventuelle de ces mycotoxines dans la bière. Des études ont proposé la levure Geotrichum candidum comme possible outil de contrôle du niveau de contamination par ces molécules. Par contre, le mécanisme d’action est encore inconnu. L’objectif de ce travail était de comprendre l’interaction entre F. langsethiae et G. candidum pour évaluer l’effet sur le développement de F. langsethiae et sur sa production de la toxine T-2. Nous avons évalué l’interaction entre ces micro-organismes en utilisant des cultures séquentielles et des co-cultures. Dans ce dernier cas la différentiation des deux micro-organismes a été faite par DOPE-FISH et par quantification de l’ADN. Les résultats ont montré que la croissance de F. langsethiae n’est pas inhibée dans un milieu pré-fermenté par G. candidum. Par contre, la présence d’un agent actif thermorésistant produit lors du développement de G. candidum affecte la concentration de la toxine T-2. Uneréduction supérieure à 90% est observée en comparaison au témoin. Dans les co-cultures on obtient le même pourcentage de réduction de la concentration de la toxine par rapport au témoin. Dans ce cas les deux micro-organismes se développent mais en moindre quantité que dans leur culture pure, F. langsethiae semblant plus inhibé que G. candidum. / F. langsethiae has been recently installed and detected in French barley used for malting. This species is known as a T-2 and HT-2 mycotoxin producer and for this reason, the possible presence of these mycotoxins in beer is a concern. G. candidum has been suggested to have a control activity in this issue, however the mechanisms is not yet described. The aim of this work was to understand the interaction between F. langsethiae and G. candidum to identify any effect in F. langsethiae growth and the T-2 toxin production. In this work, the interaction was evaluated using sequential cultures and co-cultures. Differentiation of these microorganisms in co-culture was achieved using DOPE-FISH and DNA quantification. Results showed that in the medium prefermented by G. candidum F. langsethiae growth was not inhibited but a thermoresistant active agent affected the T-2 toxin concentration during F. langsethiae development. A reduction over 90% of the T-2 toxin was observed in comparison to the concentration detected in the control. For mixed cultures the same toxin concentration reduction was observed while both microorganisms grew but at a lesser extent compared to pure cultures. In this case the growth of F. langsethiae was more affected than the one of G. candidum.

Made available in DSpace on 2017-07-21T18:53:21Z (GMT). No. of bitstreams: 1 Jaster Henrique.pdf: 1499767 bytes, checksum: 4be6aed7c051fea889ec07d6c395c8ac (MD5) Previous issue date: 2014-01-23 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / The Reblochon, one of the most important cheeses in France, presents peculiar aroma and flavor characteristics due to the ripining process by the yeast Geotrichum candidum, and has attracted interest in Brazilian cheese sector. However, there is just a little information about technology and quality characteristics of Reblochon processing. Adjusting the product to consumers in Brazil covers several steps, from the composition of the milk used, until the protocol definition of fabrication and quality standards that must be defined during the ripining process. Thus, the present study aimed to evaluate and define some quality parameters of type Reblochon cheese during the ripining process. Bovine milk Holstein and Jersey were used as raw materials for the production of Reblochon cheese. The results show that the humidity content remained on average 50% during the whole process of ripining (21 days). The total ash or mineral showed the highest concentrations at the cheese surface after 14 days ripining. The fat was on average 5% higher for the cheeses made with milk from Jersey cows than from Holstein. The color parameters showed a reduced brightness (L) along the ripining and decreased the a* values at the surface and increased in the center of the cheese. The proteolysis was high on in the cheeses made with milk from Holstein, and also on the surface of both cheeses. Texture analysis showed that the hardness, gumminess and chewiness showed an increase in the first 7 days of ripining and decreased from the 14th day of ripining. The cohesiveness and elasticity decreasedin cheeses after 21 days of ripining. A strong correlation between ripining time and NPN (r = 0.87), cohesiveness (r = -0.86) and resilience (r = -0.92) was observed. Elasticity x NPN (r = -0.96; regarding the physical characteristics correlated with the physicochemical, were found significant values for Hardness x protein (r = 0.85), tirosina x elasticity (r = -0.89), tirosina x cohesiveness (r = -0.86), cohesiveness x NPN (r = -0.88), and Resilience x NPN (r = -0.89). Therefore, the results indicate the quality parameters for the Reblochon type cheese. Changes after 14 days of ripining modify some analyzed attributes and the product begins to have identity of Reblochon cheese indicating the beginning consumption. / O queijo Reblochon, um dos queijos mais importantes na França, apresenta características de aroma e sabor peculiares devido ao processo de maturação pela levedura Geotrichum candidum, e vem despertando interesse pelo setor de processamento de queijos finos brasileiros. Porém, existe pouca informação sobre a tecnologia de processamento e características de qualidade desse queijo. Adequar esse produto ao consumidor brasileiro abrange diversas etapas, desde a composição do leite utilizado até a definição de protocolo de fabricação e padrões de qualidade que devem ser definidos no processo de maturação. Desta forma, o presente trabalho teve como objetivo avaliar e definir alguns parâmetros de qualidade de queijos tipo Reblochon, durante o processo de maturação. Foram utilizados como matéria prima os leites bovinos da raça Holandesa e Jersey. Os resultados demonstram que a umidade permaneceu com teor médio de 50% durante todo o processo de maturação (21 dias). As cinzas ou minerais totais apresentaram concentrações maiores na superfície dos queijos após 14 dias de maturação. A gordura foi em média 5% maior para os queijos elaborados com leite da raça Jersey. Os parâmetros de cor apresentaram uma redução na luminosidade (L) ao longo da maturação e os valores a* diminuíram na superfície e aumentaram no centro do queijo. A proteólise foi mais acentuada para os queijos da raça Holandesa, e também na superfície de ambos os queijos. A análise de textura mostrou que a dureza, gomosidade e mastigabilidade apresentaram um aumento nos primeiros 7 dias de maturação, decrescendo a partir do 14º dia de maturação. A coesividade e elasticidade apresentaram um decréscimo nos valores em queijos com 21 dias de maturação. Foi observada uma forte correlação entre o tempo de maturação e o NPN (r=0,87), a coesividade (r=-0,86) e a resiliência (r=-0,92). Em relação as características físicas correlacionadas com as físico-químicas, foram encontrados valores significativos para dureza x proteína (r= 0,85), elasticidade x tirosina (r=-0,89); elasticidade x NPN (r=-0,96), coesividade x tirosina (r=-0,86), coesividade x NPN (r= -0,88), e resiliência x NPN (r= -0,89). Desta forma, os resultados indicam parâmetros de qualidade para o queijo tipo Reblochon. Fica notório que transformações após os 14 dias de maturação modificam alguns atributos analisados e o produto começa a ter identidade de queijo Reblochon indicando o início do seu consumo.

Effluents with high concentration of organic matter and xenobiotic compounds, especially in the case of the agrochemicals, cause environmental concern. In the present work, the adaptative ability of two filamentous fungi, which cause white rot (Geotrichum candidum Link & Pers. and Phanerochaete chrysosporium Burds.), to tolerate high concentration of xenobiotic substances was analyzed. In solid medium, P. chrysosporium and G. candidum presented excellent development under aerated and not aerated conditions (30ºC and dark), in potato-dextrose-agar and malt extract-agar, respectively. In liquid medium, the same was observed in broth-potato-dextrose and malt extract. The fungi also developed well in liquid media deficient in proteins and carbohydrates, but with high concentrations (10 and 25 mg/L) of the agrochemicals Boral, Decanote, Gliphosate, Paraquat and Plenum - all used in fields of sugar-cane, as the ones of the "S.A Usina Coruripe Açúcar e Álcool" (Coruripe - AL). These chemicals are classified as persistent, belong to the toxicological classes II and III, and have different solubilities. The fungi presented tanninolitic activity when inoculated in samples of the crude effluent with supplement of nutrients, under aeration or not. The activity of the laccase of G. candidum in the same medium was higher in the 3rd day of the growth without aeration, occurring a reduction of that in the 5th day. On the other hand, the fungus P. chrysosporium presented a higher activity of laccase in the 7th day of the growth, also without aeration. Both fungi did not interfere in the color, turbidity, concentration of nitrates and nitrites of this medium. G.candidum revealed equal efficiency in the reduction of the concentration of phosphates of the crude effluent, under aeration (78.18%) and not aeration (79.33%) conditions. However, it did not reduce the COD. P. chrysosporium was efficient in the reduction of this COD, and the difference between the aerated growth (26.77%) and the not aerated one (29.55%) was very small. It did not reduce the total tanning bark concentration, but it diminished the concentration of phosphates under aerated (55.55%) and under not aerated (61.65%) condition. The addition of nutrients in the medium of diluted effluent (2 X, 4 X and 8 X) showed that there is a direct effect of the concentration of the COD on its percentage of removal by the inoculated fungi. There is also a direct effect on the remotion of total phenolic compounds and reduced glycids. The conversion of COD in the diluted effluent (8X) was higher in the growth of P. chrysosporium (65.66%) than in the one of G.candidum (57%). The same it can be said regarding to the best rates of phenol degradation (59.66% and 50.33%, respectively). The pH measured in the media supplemented with nutrients was always higher (7,56-7,8) than the initial one (5,5). In the higher dilution of the effluent (8X) plus the supplement of nutrients, the maximum removal of COD and total phenols after the treatment occurred in 7th day of incubation of both fungi. In all the cases of diluted samples plus supplement of nutrients, the discoloration drastically increased after the inoculation of the fungi. Therefore, the use of these microorganisms in the bioremediation of the effluent of sugar-alcohol industry is recommended only after a correction of the ratio C: N: P to about 100:1,6:1. / Efluentes com alta concentração de matéria orgânica e compostos xenobióticos, especialmente no caso dos agroquímicos, causam preocupação ambiental. No presente trabalho, dois fungos filamentosos causadores de podridão branca (Geotrichum candidum Link e Pers. e Phanerochaete chrysosporium Burds.) mostraram-se capazes de se adaptar a meios com elevados teores de matéria orgânica xenobiótica ou não. Em meio sólido, verificou-se que P. chrysosporium e G. candidum, apresentaram ótimo desenvolvimento em condições aeradas e não aeradas, a 30ºC e escuro, em meios batata-dextrose-ágar e extrato de malte-ágar, respectivamente. Em meio líquido, o mesmo foi observado em caldo-batatadextrose e meio extrato de malte. Os fungos também desenvolveram-se bem em cultivos líquidos deficientes em proteínas e carboidratos, e contendo altas concentrações (10 e 25 mg/L) dos agroquímicos Boral, Decanote, Glifosato, Paraquat e Plenum, todos utilizados nas lavouras de cana de açúcar da S.A Usina Coruripe Açúcar e Álcool (Coruripe AL), classificados como persistentes, de classes toxicológicas II e III, e com diferentes solubilidades. Ambos os fungos apresentaram atividade taninolítica quando inoculados em amostras de efluente de agroindústria sucro-alcooleira não diluídas e não acrescida de nutrientes, sob aeração ou não. A atividade da lacase do fungo G. candidum, nesse mesmo meio, foi maior no 3o dia de cultivo sem aeração, ocorrendo uma diminuição no 5o dia. Já o fungo P. chrysosporium, apresentou maior atividade da lacase no 7o dia de cultivo sem aeração. Em relação à cor, turbidez, concentração de nitratos e nitritos, não houve variação desses parâmetros nas amostras inoculadas por ambos os fungos. G. candidum mostrou-se igualmente eficiente na redução da concentração de fosfatos, em cultivos aerados (78,18%) e não aerados (79,33%) do efluente bruto. Todavia, não reduziu a DQO do mesmo. P. chrysosporium mostrou-se eficiente na redução da DQO, sendo pequena a diferença entre os cultivos aerados (26,77%) com relação aos não aerados (29,55%), e não reduziu o teor de taninos totais, mas diminuiu a concentração de fosfatos sob aeração (55,55%) e não aerado (61,65%). A adição de nutrientes no cultivo dos fungos em efluente agroindustrial diluído (2X, 4X e 8X), mostrou que houve um efeito direto da concentração da DQO sobre sua porcentagem de remoção e sobre a redução de fenóis totais e glicídeos redutores. A conversão da DQO em efluente diluído (8X) foi maior no cultivo de P. chrysosporium (65,66%) do que no de G. candidum (57%). O mesmo pode ser dito quanto às melhores taxas de degradação de fenóis (59,66% e 50,33%, respectivamente). Quanto ao pH mensurado nos cultivos suplementados com nutrientes, foi sempre maior (7,56-7,8) que o inicial (5,5). Já nos cultivos utilizando a maior diluição (8X) e o suplemento de nutrientes, a máxima remoção de DQO e de fenóis totais após o tratamento ocorreu no 7º dia do cultivo para ambos os fungos. Em todos os casos de amostras diluídas e acrescidas de nutrientes, a descoloração aumentou drasticamente após o cultivo dos fungos estudados. Portanto, recomenda-se a utilização desses microrganismos na biorremediação de efluentes sucro-alcooleiros desde que haja uma correção da proporção C:N:P de cerca de 100:1,6:1.

Este trabalho teve por objetivo estudar a partição da Lipase de Geotrichum candidum (LGc) em sistemas aquosos bifásicos (SAB) formados por PEG 1500 ou 4000 ou 6000 g mol-¹ + citrato de sódio + água e por copolímero tribloco L35 1900 g mol-¹ + citrato de sódio + água. A lipase extracelular foi produzida pelo microrganismo Geotrichum candidum NRRL Y - 552 por fermentação a 30,0 oC, pH 7,0 e sob agitação de 250 rpm. Os estudos de partição foram conduzidos com o sobrenadante do caldo fermentado, rico em lipase, após filtração para eliminação dos microrganismos. Estudos cinéticos e de eletroforese SDS-PAGE confirmaram a obtenção da lipase na etapa de fermentação. A influência da temperatura, pH, adição de NaCl, massa molar do polímero, hidrofobicidade do sistema e composição de mistura de PEG e do citrato de sódio sob o coeficiente de partição da lipase foram estudadas. Em todos os experimentos realizados a enzima particionou preferencialmente para a fase inferior com valores de KLGc em torno de 0,05-0,6. Medidas da atividade da lipase nas fases dos SAB mostraram que houve significativa perda da atividade enzimática durante o processo. Os valores médios da atividade nas fases superior e inferior foram de 1,5 U/ml e 5,5 U/ml, respectivamente, enquanto que no caldo bruto filtrado a atividade foi de 11,75 U/ml. Parâmetros termodinâmicos do processo de transferência de material entre as fases foram obtidos usando a aproximação de Van't Hoff nos estudos de partição realizados em diferentes temperaturas. O elevado valor negativo obtido para a variação de entalpia de transferência constituem evidência da existência de fortes interações entre a LGc e o PEG, contudo, os baixos coeficientes de partição encontrado nos SAB sugerem que o processo de transferência da LGc nestes sistemas seja entropicamente dirigido. Os SAB formados pela mistura de PEG 1500 g mol-¹ + citrato de sódio + água em pH 7,0 foram otimizados pela metodologia de superfície resposta. As condições que maximizam a partição do LGc na fase inferior foram determinadas nas concentrações extremas de PEG e citrato de sódio dentro das faixas de concentrações estudadas. / The aim of this work was to study the Geotrichum candidum lipase (GcL) partition in aqueous two-phase systems (ATPSs) formed by PEG 1500, 4000 or 6000 g mol-¹ + sodium citrate + water and by triblock copolymer L35 1900 g mol-¹ + sodium citrate + water. Extracellular lipase was produced by the microorganism Geotrichum candidum NRRL Y-552 by fermentation at 30.0oC%, pH 7.0, and stirring at 250rpm. Partition studies were carried out with the surfactant from the fermented, lipase rich broth, after filtration for elimination of microorganisms. Kinetic studies and SDS-PAGE electrophoresis confirmed lipase in the fermentation stage. The influence of temperature, pH, NaCl addition, polymer molar mass, hydrophobicity of the system and compounding of the PEG and sodium citrate blend under the lipase coefficient of partition were studied. In all experiments carried out, the enzyme partitioned preferentially to the lower phase with KGcL values around 0.05-0.6. Measurements of the lipase activity in phases of ATPSs showed that there was relevant loss of the enzymatic activity during the process. Mean activity values in the upper and lower phases were of 1.5 U/ml and 5.5 U/ml, respectively, while activity in the raw broth was of 11.75 U/ml. Thermodynamic parameters of material transfer process between phases were obtained by using Van’t Hoff’s approximation in the partition studies carried out at different temperatures. The high negative value obtained for the enthalpy transfer variation point out the existence of strong interaction between the GcL and the PEG. However, the low coefficients of partition found in the ATPSs suggest that the GcL transfer process in these systems is entropically driven. The ATPSs formed by the blend PEG 1500 g mol-¹ + sodium citrate + water in pH 7.0 were optimized by the response surface methodology. The conditions that maximize the GcL partition in the lower phase were determined in extreme external conditions of PEG and sodium citrate within the concentration bands studied. / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES

Geotrichum candidum est une levure hémiascomycète ubiquitaire longtemps considérée comme un champignon filamenteux. C'est l'une des levures les plus fréquemment trouvées dans les fromages dans les quelles elle contribue à l'affinage. Dans le cadre du projet ANR ALIA Food Microbiomes en partenariat avec des industriels fromagers et producteur de levain, nous avons caractérisé l'espèce G. candidum par une étude phylogénétique et placé de manière non ambigüe G. candidum parmi les levures hémiascomètes. Une analyse MLST a permis de séparer les souches étudiées en deux groupes. Le premier contient essentiellement des souches environnementales tandis que le second ne contient que des souches isolé du fromage. Cela suggère une certaine sélection ou spécialisation d'un groupe de souche dans la fabrication du fromage. Une méthode de typage inter LTR plus discriminante a permis de typer l'ensemble des souches et peut fournir aux industriels un outil robuste pour le suivi d'une souche en production. Le génome de G. candidum CLIB 918 = ATCC 204307 a été séquencé. Les premières analyses ont mis en évidence des discontinuités évolutives parmi les gènes qui le composent. Parmi les 6802 gènes identifiés, 315 gènes présentent des orthologues chez les champignons filamenteux et non chez les levures. Cela suggère que durant l'évolution, G. candidum a conservé un grand nombre de gènes qui a été perdu chez les autres levures ou en a reçu certain par transfert horizontal de gènes. L'existence de ce même type de gènes chez d'autre levure ayant une position basale dans l'arbre des hémiascomycètes, suggère que G. candidum et ces levures ont une position intermédiaire lors de la transition évolutive champignon vers levure. Il est à noter que certains d'entre eux sont impliqués dans le métabolisme et pourraient jouer un rôle dans l'adaptation de cette levure à la fabrication du fromage.

Submitted by Reginaldo Soares de Freitas (reginaldo.freitas@ufv.br) on 2016-09-02T17:52:28Z No. of bitstreams: 1 texto completo.pdf: 2355892 bytes, checksum: 4032444818648b5fe8bb04d8cb10473d (MD5) / Made available in DSpace on 2016-09-02T17:52:28Z (GMT). No. of bitstreams: 1 texto completo.pdf: 2355892 bytes, checksum: 4032444818648b5fe8bb04d8cb10473d (MD5) Previous issue date: 2016-02-23 / Conselho Nacional de Desenvolvimento Científico e Tecnológico / A podridão azeda causada pelo fungo Geotrichum é uma doença pós- colheita que ocorre em frutas e hortaliças. Visando caracterizar a diversidade genética de Geotrichum sp. para a escolha de estratégias de controle eficientes, este trabalho tem o objetivo de avaliar diferentes marcadores ISSRs e IRAPs para determinação da diversidade intra específica de Geotrichum. No total, 62 isolados de locais geograficamente distintos e diferentes hospedeiros foram utilizados neste estudo. Para a análise de diversidade genética, o DNA genômico foi extraído a partir do micélio. A região parcial dos genes que compreende o ISSR e transposon foram amplificadas usando sete oligonucleotídeos. A partir dos perfis de bandas foi construída uma matriz binária a qual foi adicionada ao software PAST3 onde o índice de similaridade de Jaccard foi calculado para cada combinação de duas amostras. Os dendrogramas foram construídos com base na matriz de similaridade de acordo com o método das médias aritméticas de grupos não ponderados (UPGMA- unweighted pair group method with arithmetic averages). Em todos marcadores moleculares analisados foram encontrados pelo menos um clado representado por dois ou mais isolados que apresentaram perfis idênticos. Existe uma tendência de isolados de um mesmo hospedeiro ou região geográfica serem semelhantes quando foi utilizado diferentes marcadores moleculares. Não houve semelhança entre os isolados quando os oligonucleotídeos IRAPs e microssatélites foram comparados pois o número de clados formados pelos marcadores IRAPS foram menores. Geotrichum possui grande diversidade genética e, portanto, pode permitir a este patógeno uma melhor adaptabilidade às condições ambientais e a diferentes hospedeiros. / The sour rot caused by fungus Geotrichum genus is a disease that occurs in fruits and vegetables. In order to characterize the genetic diversity of Geotrichum sp. for the choice of efficient control strategies, this study aims to evaluate different ISSRs and IRAPs markers for determination of intraspecific diversity of Geotrichum. A total of 62 isolates from geographically different locations and different hosts were used in this study. For the analysis of genetic diversity, genomic DNA was extracted from the mycelium. The partial region of the genes comprising the ISSR and transposon were amplified using seven primers. From the bands profiles was built a binary matrix which were added to PAST3 software where the Jaccard similarity index was calculated for each combination of two samples. The dendrograms were constructed based on the similarity matrix according to the method of arithmetic averages of unweighted groups (UPGMA- unweighted pair group method with arithmetic averages). In all molecular markers analyzed were found at least one clade represented by two or more isolates that showed identical profiles. Strains not grouped according to the town which were isolated. There is a tendency of isolates from the same host or geographic region are similar when we used different molecular markers. There were not similarities between the isolated when IRAPs and microsatellite primer were compared because the number of clades formed by IRAPS markers were lower. Geotrichum has high genetic diversity and therefore may allow the pathogen better adaptability to environmental conditions and the different hosts.

Submitted by Luriany Pompeo Ferraz (luriany@hotmail.com) on 2018-05-10T13:54:38Z No. of bitstreams: 1 Tese Luriany Pompeo Ferraz.pdf: 2121530 bytes, checksum: dbc5ecaf4e4d278a002ba09e3ed9af57 (MD5) / Approved for entry into archive by Alexandra Maria Donadon Lusser Segali null (alexmar@fcav.unesp.br) on 2018-05-10T18:09:10Z (GMT) No. of bitstreams: 1 ferraz_lp_dr_jabo.pdf: 2121530 bytes, checksum: dbc5ecaf4e4d278a002ba09e3ed9af57 (MD5) / Made available in DSpace on 2018-05-10T18:09:10Z (GMT). No. of bitstreams: 1 ferraz_lp_dr_jabo.pdf: 2121530 bytes, checksum: dbc5ecaf4e4d278a002ba09e3ed9af57 (MD5) Previous issue date: 2018-04-10 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / O fenômeno killler é característico de leveduras que produzem e excretam proteínas ou glicoproteínas que são inibidoras de células microbianas sensíveis. A estabilidade e atividade das toxinas killer são altamente sensíveis a fatores como pH, temperatura de incubação das leveduras, composição e propriedades físico-químicas do meio e concentração de células sensíveis. Sporobolomyces koalae é uma nova espécie de levedura com potencial para produção de toxina killer. No intuito de se conhecer mais sobre as funções desse microrganismo no ecossistema, esse trabalho teve por objetivos: (i) detectar a atividade killer do precipitado proteico de S. koalae, (ii) caracterizar bioquimicamente e funcionalmente o precipitado proteico S. koalae, (iii) purificar parcialmente a toxina killer produzida por S. koalae e, finalmente, (iv) verificar sua ação antagônica sobre Geotrichum citri-aurantii e Penicillium digitatum, patógenos que ocorrem na póscolheita de citros. Pelos resultados obtidos neste trabalho, foi possível detectar a presença de atividade killer no precipitado proteico da levedura S. koalae contra células sensíveis da levedura Saccharomyces cerevisiae NCYC 1006. O precipitado proteico da levedura apresenta várias proteínas com diferentes tamanhos moleculares e, parte dessas proteínas é positiva para atividade de β1,3-glucanase, quitinase e protease, podendo ser uma dessas enzimas ou, o sinergismo entre elas, o responsável pelo fator killer da levedura. A funcionalidade de suas proteínas para atividade killer aumenta em pH 4.9 e em uma faixa de temperatura de 22 °C a 27 °C e, o melhor agente precipitante das proteínas da levedura foi o etanol 80%. A purificação parcial das proteínas, que constituem o precipitado proteico de S. koalae, mostrou a existência de aproximadamente quatro bandas proteicas em uma das frações de purificação, Fração 1, com tamanhos aproximados que variaram de 8 a 65 kDa. A atividade killer, presente no precipitado proteico da levedura S. koalae, não apresenta ação antagônica sobre Geotrichum citri-aurantii e Penicillium digitatum. / The killer phenomenon is characterized by yeasts that produce and excrete proteins or glycoproteins that are inhibitors of sensitive microbial cells. The stability and activity of killer toxins are highly sensitive to the factors such as pH, the temperature of incubation of yeasts, composition and physical-chemical properties of the medium and concentration of sensitive cells. Sporobolomyces koalae is a new species of yeast with potential for killer toxin production. In order to know more about the functions of this microorganism in the ecosystem, this work had as objectives: (i) to detect the killer activity of the S. koalae protein precipitate, (ii) to characterize biochemically and functionally the S. koalae protein precipitate, (iii) to partially purify the killer toxin produced by S. koalae, and, finally, (iv) to verify its antagonistic action on Geotrichum citri-aurantii and Penicillium digitatum, pathogens that occur in the postharvest of citrus. By the results obtained in this work, it was possible to detect the presence of killer activity in the protein precipitate of S. koalae against sensitive cells of Saccharomyces cerevisiae NCYC 1006. The protein precipitate from yeast has several proteins with different molecular sizes and some of these proteins are positive for β1,3-glucanase, chitinase and protease activity. It may be one of these enzymes or synergism between them, the responsible for the killer factor. The functionality of their proteins for killer activity increases at pH 4.9 and a temperature range of 22 oC to 27 oC and the best precipitant of protein from yeast was ethanol (80%). Partial purification of the proteins showed the existence of approximately 4 protein bands in one of the purification fractions, Fraction 1, with approximate sizes ranging from 8 to 65 kDa. The killer activity, present in the protein precipitate of yeast S. koalae, did not present antagonistic action on G. citri-aurantii and P. digitatum. / FAPESP: 14/25067-3