Thesis (M.S.)--University of Wisconsin-Madison, 1972. / Typescript. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references.

Induction of extracellular lipases from Pseudomonas fragi CRDA 037 and Geotrichum candidum was used to increase lipase production in a shorter period of time. Induction was performed using edibles oils such as olive oil, canola oil, fish oil and butter fat. Fermentation trials of the microorganisms in the selected media were done in order to optimize the production of lipases. Optimal lipase activity was obtained in the presence of butter fat (1%, v/v) for P. fragi which was cultivated at 15°C during 48 h, and fish oil (0.75%, v/v) for G. candidum incubated at 27°C for 56 h. The induced lipase extracts of P. fragi and G. candidum obtained after these fermentation trials were purified by 3.7 and 5.9-fold respectively using ultrafiltration, while the non-induced fractions were purified by 2.3 and 5.1-fold, respectively. Activities were evaluated using p-nitrophenyl esters such as p-nitrophenyl laurate and p-nitrophenyl palmitate. (Abstract shortened by UMI.)

No description available.

Geotrichum candidum est une levure couramment utilisée comme ferment d'affinage pour la fabrication des fromages à croûte fleurie et à croûte lavée. Cette espèce, pour laquelle très peu d'études génétiques ont été réalisées, présente un intérêt technologique pour les industries fromagères et productrices de ferments. Les membres de l'espèce arborent une diversité de propriétés morphologiques et physiologiques qui sont souche-dépendantes. Afin d'exploiter cette diversité de façon optimale et de fabriquer industriellement des fromages affinés en surface à saveur et à texture plus traditionnelles, il est donc impératif de pouvoir identifier les souches de G. candidum et de les caractériser. Il existe actuellement un débat concernant l'utilisation de la région ITS de l'ADN ribosomique, tel un code-barres, pour l'affiliation des mycètes. Il était donc important de déterminer si les bases du DNA barcoding s'appliquaient à G. candidum. Le premier objectif a été d'augmenter l'information phylogénétique pour cette espèce en séquençant pour la première fois la région complète du 18S-ITS1-5.8S-ITS2-26S de l'ADNr et ce pour 18 souches. Des hétérogénéités intra-espèce et intra-génomique inhabituelles au niveau de l'ADNr ont été révélées amenant un questionnement sur la robustesse de cette région pour l'affiliation des isolats de cette espèce. Les résultats obtenus suggèrent que les techniques de profilage des communautés de mycètes basées sur l'ADNr doivent être utilisées avec précaution. La diversité des propriétés technologiques des souches de G. candium nécessite une méthode de génotypage discriminante générant des profils génétiques fiables pour la caractérisation des souches. Le premier schéma de Multilocus Sequence Typing (MLST) a donc été développé avec 18 souches en identifiant, puis séquençant au complet, six nouveaux gènes domestiques. Le schéma fut ensuite validé sur 22 souches supplémentaires. Le MLST a permis de révéler au sein de l'espèce G. candidum l'existence probable d'une sous-espèce. Les résultats de l'ADNr et du MLST combinés aux résultats publiés sur la plasticité du génome de G. candidum et la variabilité morphologique des souches révèlent la fragilité des frontières entourant cette espèce. L'affiliation des isolats à cette espèce est un véritable défi phylogénétique qui nous rappelle que finalement les frontières des espèces ne sont pas hermétiques et que la nature ne fait pas de "sauts" dans son processus d'évolution.

Geotrichum candidum est une levure dimorphique utilisée en fromagerie pour l’affinage des fromages de spécialité. Elle s’implante à la surface des fromages et utilise les constituants de la matrice fromagère (protéines, lipides, acides organiques, etc.), ce qui confère aux fromages leurs propriétés sensorielles typiques. Ces capacités technologiques dépendent toutefois de la souche. Les études récentes basées sur l’analyse phylogénétique et transcriptomique de souches de G. candidum ont révélé une diversité génétique importante au sein de cette espèce, ce qui pourrait expliquer la variabilité des capacités technologiques. Cependant, peu d’informations sont disponibles quant aux caractéristiques génétiques des souches responsables de leurs propriétés aromatisantes et fonctionnelles lors de l’affinage des fromages. La compréhension fine des activités de G. candidum est prioritaire tant pour les artisans-fromagers que pour les grandes fromageries industrielles afin de sélectionner les souches les plus performantes pour leur produit. L’objectif principal de cette étude était de déterminer la diversité génétique entre les souches de la levure Geotrichum candidum par utilisation de la génomique comparative et de proposer une méthode moléculaire pour l’identification et la caractérisation rapide des souches. Les génomes de huit souches de G. candidum d’origine laitière ont été séquencés. Les génomes obtenus avaient une taille moyenne de 24,5 Mb et 5 230 gènes prédits. L’homologie des séquences de chaque souche a permis de les regrouper en trois groupes phylogénétiques distincts pour lesquels des gènes uniques ont pu être identifiés. Sur la base de l’analyse des séquences génomique, une méthode optimisée de génotypage par MLST a été développée et validée sur 41 souches de G. candidum. Cette méthode a permis de reproduire les résultats obtenus avec l’analyse génomique et permet une identification rapide des souches et de leurs groupements phylogénétiques. Les résultats générés dans ce projet permettront de développer des outils pour la sélection optimale des ferments d’affinages basés sur leurs capacités technologiques spécifiques. Ils serviront également à améliorer notre compréhension de la physiologie de cette levure et de décrire et optimiser l’utilisation des souches de G. candidum lors de l’affinage afin d’ultimement contrôler davantage la qualité des fromages. / The yeast Geotrichum candidum is used in several specialty cheeses varieties, such as mold and smear-ripened cheeses, and plays several roles during cheese ripening. Its ability to metabolize proteins, lipids and organic acids enables its growth on the cheese surface and participate to the development of organoleptic properties. By alkalinizing the surface duringi ts growth, it also establishes suitable conditions for the growth of other ripening microorganisms. Yet, several technological abilities of G. candidum are strains dependent. However, little information is available related to the genetic characteristics that define the flavoring and functional properties of this yeast during the ripening of cheeses. A detailed understanding of G. candidum metabolic activities is a priority for both artisanal cheese makers and large industrial cheese factories in order to detect the most efficient strains for their product. The main objective of this study was to determine the genetic diversity within the G. candidum species by comparative genomic and to propose a rapid molecular method for the identification and characterization of the strains. Eight strains of G. candidum of dairy origin was sequenced. The genomes obtained had an average of 24.5 Mb and 5,230 putative genes. The sequence homologies show that the strains divide into three distinct groups for which each contains unique genes. On the basis of the genomic sequences, a MLST method was optimized and validated for 41 G. candidum strains. This method reproduces the results obtained for the genomic analysis and allows a rapid identification of the strains and their grouping.The results generated in this project will improve our understanding of the physiology and the utility of the G. candidum strains during the ripening of cheese to ultimately be able to better control it.

Os ésteres são importantes compostos orgânicos, obtidos por síntese química ou extraídos de alguns produtos naturais utilizando-se solvente em meio adequado. Estudos mostram que enzimas, em particular lipases, podem ser aplicadas na síntese de diversos ésteres. O principal objetivo deste trabalho foi a obtenção de acetato de butila, um éster de aroma característico de abacaxi, utilizando lipase de Geotrichum candidum produzida em fermentação submersa e imobilizada em matriz polimérica de alginato de bário e gelatina reticulada com glutaraldeído. A caracterização bioquímica foi realizada tanto para a lipase na forma livre como para a lipase na forma imobilizada. O rendimento em conversão molar de substrato foi determinado por cromatografia gasosa. A enzima apresentou atividade enzimática máxima após 48 horas de fermentação de 37,7 U/mL. Os valores ótimos para pH e temperatura da enzima na forma livre e imobilizada foram pH 6,5 e 40 °C e pH 7,5 e 45 °C, respectivamente. A enzima na forma livre foi estável do pH 6,0 ao 8,0 e à temperatura de 35 a 45 °C, já na forma imobilizada, foi estável do pH 5,5 ao 8,5 e na faixa de temperatura de 30 a 55 °C. A lipase imobilizada teve seus parâmetros cinéticos determinados, e os valores obtidos para o Km e Vmax, foram 0,115 mmol e 0,718 µmol.mL-1.min-1, respectivamente. As melhores condições de síntese do bioaroma para a enzima na forma livre foram: temperatura de 30 °C, 12,5% de enzima em relação à quantidade de butanol utilizada, proporção molar do substrato 60% de acetato de vinila em um período de 24 horas. O rendimento alcançado neste caso foi de 97,2 % de conversão molar em acetato de butila. Para a enzima imobilizada as melhores condições foram: temperatura de 45 °C, 12,5% de enzima em relação à quantidade de butanol utilizada, proporção molar do substrato 60% de acetato de vinila em um período de 24 horas. O rendimento alcançado neste caso foi de 99,1%, que demonstra que lipase produzida por Geotrichum candidum na forma imobilizada apresenta excelente capacidade de sintetizar acetato de butila (bioaroma de abacaxi). / Esters are important organic compounds obtained by chemical synthesis or derived from some natural products using a solvent in appropriate medium. Studies have shown that enzymes, particularly lipases, ca be applied in the synthesis of various esters. The main objective of this study was to obtain butyl acetate, a characteristic ester aroma of pineapple, using lipase from Geotrichum candidum produced in submerged fermentation and immobilized in a barium alginate and gelatin polymer matrix crosslinked with glutaraldehyde. The biochemical characterization was performed for both free and immobilized lipases. The yield in molar conversion of substrate was determined by gas chromatography. The enzyme showed maximum enzymatic activity after 48 hours of fermentation of 37.7 U/mL. The optimum values for pH and temperature of the enzyme in free and immobilize form were pH 6.5 and 40 °C and pH 7.5 and 45 °C, respectively. The enzyme was stable in free form from pH 6.0 to 8.0 and at temperatures from 35 to 45 °C, and in the immobilized form from pH 5.5 to 8.5 and at temperatures from 30 to 55 °C. Kinetic parameters of the immobilized lipase were determined, and the values obtained for Km and Vmax were 0.115 mmol and 0.718 µmol.mL-1.min-1, respectively. The best conditions for the synthesis of flavor by enzyme in free form were: 30 °C of temperature, 12.5% of enzyme for the amount of butanol used, and molar ratio of substrate 60% of vinyl acetate in a 24 hours period. The yield achieved in this case was 97.2% of molar conversion in butyl acetate. For the immobilized enzyme the best conditions were: 45 °C of temperature, 12.5% of enzyme for the amount of butanol used, and molar ratio of substrate 60% of vinyl acetate in a 24 hours period. The yield achieved in this case was 99.1%, demonstrating that lipase produced by Geotrichum candidum in immobilized form has an excellent ability to synthesize butyl acetate (pineapple flavor).

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’autres levures 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. / Geotrichum candidum is a hemiascomycetous yeast frequently found in the environment and foodstuffs. It is one of the main yeasts in cheese and it is widely used as adjunct culture in the maturation of cheese. Within ANR project ALIA Food Microbiomes in partnership with industry, we characterized the species the species G. candidum by a multigene phylogenetic study. MLST analysis allowed us to separate the studied strains into two groups. The first contains mainly environmental strains while the second contains only strains isolated from cheese. This suggests a specialization or a selection of a group of strains within industry. We developed a typing method by inter LTR profiles, which can provide a robust tool for an industrial monitoring of strains. The genome of G. candidum CLIB 918 = ATCC 204307 was sequenced. Preliminary analyses revealed evolutionary discontinuities among genes. 6802 genes where identified in which 315 genes have orthologs in filamentous fungi and not in yeast. This suggests that during evolution, G. candidum has retained a large number of genes which have been lost in other yeasts or has received some by horizontal gene transfer. The existence of this other yeasts also having a basal position in hemiascomycetous tree suggests that G. candidum and these other yeasts have an intermediate position during the evolutionary transition fungus to yeast. It is noteworthy that some of them are involved in the metabolism and may play a role in the adaptation of the yeast to the cheese environment.

Os ésteres são importantes compostos orgânicos, obtidos por síntese química ou extraídos de alguns produtos naturais utilizando-se solvente em meio adequado. Estudos mostram que enzimas, em particular lipases, podem ser aplicadas na síntese de diversos ésteres. O principal objetivo deste trabalho foi a obtenção de acetato de butila, um éster de aroma característico de abacaxi, utilizando lipase de Geotrichum candidum produzida em fermentação submersa e imobilizada em matriz polimérica de alginato de bário e gelatina reticulada com glutaraldeído. A caracterização bioquímica foi realizada tanto para a lipase na forma livre como para a lipase na forma imobilizada. O rendimento em conversão molar de substrato foi determinado por cromatografia gasosa. A enzima apresentou atividade enzimática máxima após 48 horas de fermentação de 37,7 U/mL. Os valores ótimos para pH e temperatura da enzima na forma livre e imobilizada foram pH 6,5 e 40 °C e pH 7,5 e 45 °C, respectivamente. A enzima na forma livre foi estável do pH 6,0 ao 8,0 e à temperatura de 35 a 45 °C, já na forma imobilizada, foi estável do pH 5,5 ao 8,5 e na faixa de temperatura de 30 a 55 °C. A lipase imobilizada teve seus parâmetros cinéticos determinados, e os valores obtidos para o Km e Vmax, foram 0,115 mmol e 0,718 µmol.mL-1.min-1, respectivamente. As melhores condições de síntese do bioaroma para a enzima na forma livre foram: temperatura de 30 °C, 12,5% de enzima em relação à quantidade de butanol utilizada, proporção molar do substrato 60% de acetato de vinila em um período de 24 horas. O rendimento alcançado neste caso foi de 97,2 % de conversão molar em acetato de butila. Para a enzima imobilizada as melhores condições foram: temperatura de 45 °C, 12,5% de enzima em relação à quantidade de butanol utilizada, proporção molar do substrato 60% de acetato de vinila em um período de 24 horas. O rendimento alcançado neste caso foi de 99,1%, que demonstra que lipase produzida por Geotrichum candidum na forma imobilizada apresenta excelente capacidade de sintetizar acetato de butila (bioaroma de abacaxi). / Esters are important organic compounds obtained by chemical synthesis or derived from some natural products using a solvent in appropriate medium. Studies have shown that enzymes, particularly lipases, ca be applied in the synthesis of various esters. The main objective of this study was to obtain butyl acetate, a characteristic ester aroma of pineapple, using lipase from Geotrichum candidum produced in submerged fermentation and immobilized in a barium alginate and gelatin polymer matrix crosslinked with glutaraldehyde. The biochemical characterization was performed for both free and immobilized lipases. The yield in molar conversion of substrate was determined by gas chromatography. The enzyme showed maximum enzymatic activity after 48 hours of fermentation of 37.7 U/mL. The optimum values for pH and temperature of the enzyme in free and immobilize form were pH 6.5 and 40 °C and pH 7.5 and 45 °C, respectively. The enzyme was stable in free form from pH 6.0 to 8.0 and at temperatures from 35 to 45 °C, and in the immobilized form from pH 5.5 to 8.5 and at temperatures from 30 to 55 °C. Kinetic parameters of the immobilized lipase were determined, and the values obtained for Km and Vmax were 0.115 mmol and 0.718 µmol.mL-1.min-1, respectively. The best conditions for the synthesis of flavor by enzyme in free form were: 30 °C of temperature, 12.5% of enzyme for the amount of butanol used, and molar ratio of substrate 60% of vinyl acetate in a 24 hours period. The yield achieved in this case was 97.2% of molar conversion in butyl acetate. For the immobilized enzyme the best conditions were: 45 °C of temperature, 12.5% of enzyme for the amount of butanol used, and molar ratio of substrate 60% of vinyl acetate in a 24 hours period. The yield achieved in this case was 99.1%, demonstrating that lipase produced by Geotrichum candidum in immobilized form has an excellent ability to synthesize butyl acetate (pineapple flavor).

Biomasses of Penicillium camemberti, Penicillium roqueforti and Geotrichum candidum strains were grown and harvested after a culture incubation period that corresponded to the maximal dry weight of mycelium as well as to lipoxygenase (LOX) activity. The crude enzymatic extracts were recovered from the homogenized mycelium cells and the partially purified LOX extracts were obtained by ammonium sulfate precipitation. Using linoleic acid as substrate, the partially purified LOX extracts from P. camemberti, P. roqueforti and G. candidum exhibited a major activity, at pH 6.50, 5.50 and 3.75, respectively, and a minor one at pH 8.00. The partially purified LOX extracts exhibited an overall preferential specificity towards free fatty acids, including linoleic, linolenic and arachidonic acids, than that for fatty acid acylglycerols, including mono-, di- and tri-linolein. The Km and Vmax values for partially purified LOXs were investigated. Normal phase high-performance liquid chromatography (NP-HPLC) and gas-liquid chromatography/mass spectrometry (GC/MS) analyses showed that the LOX activity of the partially purified LOX extracts converted mainly linoleic acid into the corresponding 9- and 13-hydroperoxides (HPODs); however, the production of a relatively important proportion of 10-HPOD, ranging from 4 to 9% of the total HPODs, was also demonstrated with the partially purified LOX extracts from Penicillium sp. The chiral phase HPLC analysis demonstrated the production, by the partially purified LOX extracts from Penicillium sp., of both (R)- and (S)-enantiomers of HPODs, with a slightly higher proportion of the (S)-enantiomers. The crude enzymatic extracts from Penicillium sp. were incubated, at pH 6.50, with individual racemic mixture of 9(R,S)-, 10(R,S)-, 12( R,S)- and 13(R,S)-HPODs, prepared by photooxidation and separated by NP-HPLC. The experimental results indicated that only 10( S)-HPOD isomer was cleaved by a hydroperoxide lyase activity that resulted by the for

No description available.