The inoculation of Aspergillus flavus spores into a culture of Streptococcus lactis in LTB medium resulted in none or little aflatoxin production even though growth of the fungus was not hindered. The drop in pH and reduced nutrients in the medium as the result of S. lactis growth were not the cause of the observed inhibition. The inhibition was not eliminated by the addition of carbohydrate equal to the amount utilized by the bacterium prior to the inoculation with the fungus. Aflatoxin production was also inhibited when S. lactis was inoculated after A. flavus had grown. In addition to inhibiting the synthesis of aflatoxin, S. lactis also degraded pre-formed toxin. Aflatoxin, on the other hand, not only reduced the growth of S. lactis but also affected the morphology of the bacterial cell--the cells became elongated and formed long chains. / S. lactis produced and excreted the inhibitor into the medium during the early stage of growth (4 h). The inhibitor was a heat stable low molecular weight compound (MW (LESSTHEQ) 500). Neither volatile (acetic) nor non-volatile (succinic and lactic) acids which were detected in extracts containing the inhibitor were responsible for this inhibition. Lactic acid was found in larger quantities in mixed cultures and its addition to mono fungus culture was found to stimulate aflatoxin production. Chloroform: methanol extraction of the S. lactis culture filtrate removed all the activity to the organic phase. Further, the active compound was insoluble in hexane, not extracted by sodium bicarbonate and was soluble in acetone, indicating a polar lipid. Autoradiographic studies showed that the inhibitor was a product of glucose metabolism. Further characterization indicated that the inhibitor was a phosphoglycolipid containing an aromatic ring structure. / Filtrate extracts of A. flavus grown in presence of S. lactis were toxic to Bacillus megaterium but did not exhibit mutagenic or carcinogenic activity in the Salmonella/Mammalian microsome mutagenicity test.

The malty flavor defect produced by Streptococcus lactis var. maltigenes has been the cause of considerable economic distress to various segments of the dairy industry. This study was conducted in order to develop a more thorough understanding of the chemical nature of this defect, and to formulate a synthetic malty flavor preparation. An 18 hour malty culture and an acidified heated skim milk control were steam distilled using a specially designed, low temperature, reduced pressure glass apparatus fitted with ground glass ball or standard taper joints. After subsequent ethyl ether extractions, the aqueous distillates yielded flavor concentrates which were suitable for gas-liquid chromatographic (GLC) and mass spectrometric analysis. Flavor component identifications were made on both a tentative and positive basis. Tentative identifications were made using the technique of GLC relative retention times. Identifications were considered positive when GLC retention data could be coupled with mass spectral data. Compounds positively identified as being present in the malty culture included acetaldehyde, 3-methylbutanal, phenylacetaldehyde, ethanol, butanol, 2-methylpropanol, 3-methylbutanol, 2-furfurol, phenethyl alcohol, acetone, butanone, 2-pentanone, 2-heptanone, 2-nonanone, ethyl formate, ethyl acetate, ethyl butyrate, ethyl isovalerate, ethyl octanoate, isoamyl acetate and toluene. Compounds tentatively identified included 2-methylpropanal, pentanal, benzaldehyde, 2-furfural, 2-undecanone, 2-tridecanone, ethyl hexanoate, ethyl decanoate, methyl acetate, γ-octalactone, δ-octalactone, formic acid and acetic acid. In the heated skim milk control, acetaldehyde, benzaldehyde, 2-furfural, 2-furfurol, 2-pentanone, 2-heptanone, 2-nonanone, 2-undecanone, 2-tridecanone, ethyl acetate and methyl chloride were positively identified while pentanal, hexanal, octanal, nonanal, 2-hexanone, 2-octanone, ethyl formate, ethyl octanoate, methyl acetate, γ-octalactone and δ-octalactone remained as tentative identifications. Quantitative evaluations of the volatile constituents present in each of four strains of the malty culture were conducted using a gas entrainment, on-column trapping, GLC technique. From the quantitative data obtained from a 24 hour S. lactis var. maltigenes L/M-20 culture, a synthetic malty flavor preparation, suitable for use in baked foods, was developed. This investigation used biscuits as a model system for the baking studies. The biscuits were prepared using the General Mills' Bisquick mix and a malty milk preparation replaced the normal milk requirement. The milk contained 1.70 p.p.m. acetaldehyde, 34.20 p.p.m. 3-methylbutanal, 17.90 p.p.m. 2-methylpropanolr 90.10 p.p.m. 3-methylbutanol and 10.00 p.p.m. diacetyl. / Graduation date: 1968

Graduation date: 1994

Thesis (M.App.Sc.)--University of Melbourne, Gilbert Chandler College, Institute of Land and Food Resources, 2003. / Typescript (photocopy). Includes bibliographical references (leaves 90-103).

Thèse (M.Sc.)--Université Laval, 2007. / Bibliogr.: f. 70-78. Publié aussi en version électronique dans la Collection Mémoires et thèses électroniques.

Thesis--University of Florida. / Description based on print version record. Typescript. Vita. Includes bibliographical references (leaves 142-145).

Proefschrift Rijksuniversiteit Groningen. / Auteursnaam op omslag: Henk Bolhuis. Datum laatste controle: 14-11-1996. Met bibliogr., lit. opg. - Met samenvatting in het Nederlands.

Submitted by Marco Antônio de Ramos Chagas (mchagas@ufv.br) on 2017-07-17T16:04:41Z No. of bitstreams: 1 texto completo.PDF: 305457 bytes, checksum: 62f65ddfb7b0cad1f80dadfaef449290 (MD5) / Made available in DSpace on 2017-07-17T16:04:41Z (GMT). No. of bitstreams: 1 texto completo.PDF: 305457 bytes, checksum: 62f65ddfb7b0cad1f80dadfaef449290 (MD5) Previous issue date: 2001-09-21 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Foi isolado um mutante auxotrófico para uracila de Kluyveromyces lactis com potencial para ser hospedeiro em sistemas de expressão heteróloga de proteínas recombinantes. Uma população selvagem de K. lactis foi irradiada com luz ultravioleta e selecionada na presença de ácido 5 fluoro orótico (5FOA). Dez colônias auxotróficas para uracila e resistentes a 5FOA foram selecionadas. Todas cresceram em meio utilizando lactose como única fonte de carbono e exibiram atividade de β galactosidase normal em relação à célula selvagem. Os mutantes apresentaram velocidades específicas de crescimento inferiores a da célula selvagem em soro de queijo ultrafiltrado (SUF), o que indicou ser o soro de queijo um meio limitante em uracila, afetando o crescimento dos mutantes. O mutante M7 foi selecionado por apresentar uma freqüência de reversão baixa (1,7 x 10 -10 ). A complementação da auxotrofia na linhagem M7 mediante transformação com o plasmídeo PTG802 contendo o gene URA3 de S. cerevisiae foi analisada. Melhor eficiência de transformação de K. lactis foi obtida com o método de choque térmico com acetato de lítio, polietilenoglicol e DNA carreador fita simples. Um transformante mostrou viiiintegração do plasmídeo em um fragmento de 6,0 Kb. A velocidade de crescimento da célula mutante (0,04 h -1 ) em SUF foi inferior a da célula selvagem (0,26 h -1 ). Estes resultados sugerem que o SUF é limitante em uracila. Conseqüentemente a transformação dos mutantes, com o gene URA3, fisiologicamente causou aumento na velocidade especifica de crescimento (0,22-0,25h -1 ) quando cultivadas em SUF, atingindo os níveis da célula selvagem e evidenciando a complementação da auxotrofia. / A Kluyveromyces lactis uracil auxotrophic mutant was isolated with potential as host for heterologous recombinant protein expression systems. A wild-type K. lactis population was irradiated with ultraviolet light and mutants selected in the presence of 5-fluoroorotic acid (5FOA). Ten uracil auxotrophic colonies resistant to 5FOA were selected. All grew in medium with lactose as sole carbon source and exhibited β-galactosidase activity comparable to that of the wild-type cell. The mutants presented specific growth rates lower than the wild-type cell when grown in ultrafiltered cheese whey (SUF), indicating that cheese whey is uracil limited, thereby affecting mutant growth. Mutant M7 was selected since it presented a low reversion frequency (1.7 x 10 -10 ). Auxotrophic complementation of the M7 lineage, through transformation with the PTG802 plasmid containing the URA3 gene from S. cerevisiae was analyzed. Better K. lactis transformation efficiency was obtained with the thermal shock method with lithium acetate, polyethylene glycol and single stranded DNA carrier. One transformed cell presented a 6.0 kb fragment integrated into the plasmid. Transformed cells grown in SUF presented specific growth rates (0,22-0,25 h -1 ) similar to that of the wild-type cell. (0,26 h -1 ) and superior to that of the untransformed mutant cell (0,04 h -1 ). Consistent with this observation, transformation of the mutants with the URA3 gene physiologically causes an increase in specific growth, when grown in SUF, reaching levels of the wild-type cell and exhibiting auxotrophic complementation. / Dissertação importada do Alexandria

L’utilisation de mécanismes anti-phages est l’une des stratégies pour prévenir et contrôler la contamination par les bactériophages qui peuvent causer des pertes importantes pour l’industrie de la transformation laitière. AbiQ est un mécanisme d’avortement de l’infection phagique de type toxine-antitoxine (type III) isolé d’une souche de Lactococcus lactis. Des études précédentes ont démontrées que la toxine ABIQ (protéine) coupe son antitoxine (ARN non codant) in vivo, et que la protéine virale ORF38 du lactophage P008 joue un rôle essentiel dans l’activité anti-phage. Dans cette étude, nous nous sommes intéressés au mode d’action d’AbiQ via trois objectifs spécifiques : caractériser l’effet de différentes mutations de l’antitoxine, analyser le comportement viral et cellulaire en présence d’AbiQ et définir le rôle de la cible/activateur viral ORF38. Nos résultats démontrent qu’il est possible d’optimiser l’efficacité anti-phage par modification de l’antitoxine et suggèrent que l’ORF38 interagit avec l’ARN antitoxine permettant la libération de la toxine. / Antiphage mechanisms represent one of the strategies available to prevent or control contamination by virulent phages, which are a major risk of fermentation failure in the dairy industry. The lactococcal phage abortive infection system AbiQ belongs to type III toxin-antitoxin system. It has been previously demonstrated that the toxin ABIQ (protein) cleaves his antitoxin (non-coding RNA) in vivo, while the protein ORF38 from phage P008 plays an essential role in the antiphage activity. In this study, we investigated the mode of action of AbiQ through three specific objectives: describe the effect of specific modifications in the antitoxin, analyse the viral and cellular behaviours in presence of AbiQ and determine the role of the phage P008 target/activator ORF38. Our results show that we can optimise the antiphage activity of AbiQ through mutation in the antitoxin and suggest that ORF38 can bind the antitoxin RNA to favour the release of the toxin.

Tableau d’honneur de la Faculté des études supérieures et postdoctorales, 2013-2014. / Bactéries et phages sont en perpétuelle compétition dans l’environnement et évoluent conjointement. Plusieurs mécanismes ont été développés de part et d’autre pour maintenir un état d’équilibre. D’un côté, les phages sont des entités qui se répliquent efficacement et rapidement en parasitant leur bactérie hôte. D’un autre côté, les bactéries ont acquis des mécanismes de résistance aux bactériophages tels que les systèmes d’avortement de l’infection (Abi) pour limiter l’infection et la propagation des phages dans les populations bactériennes. Plus spécifiquement, le système antiphage AbiQ a été isolé de la bactérie Lactococcus lactis. Cette dernière est utilisée pour la production de divers produits laitiers fermentés. Malgré cet environnement contrôlé, des phages peuvent perturber les fermentations s’ils sont présents dans le lait et les usines. Il n’est donc pas surprenant que cette bactérie possède des systèmes contre ces envahisseurs. Le système antiphage AbiQ serait similaire à un mécanisme de type toxine-antitoxine (TA), mais son fonctionnement demeure jusqu’à maintenant inconnu. Au cours de ce projet de doctorat, trois objectifs ont été poursuivis afin de comprendre la relation entre la bactérie Lactococcus lactis et ses phages. Dans un premier temps, le génome du phage 949, un membre d’un groupe rare de lactophages portant son nom, a été séquencé et caractérisé. Une comparaison de tous les génomes de phages infectant L. lactis a été réalisée afin de confirmer la classification actuelle. Dans un deuxième temps, le mode d’action toxine-antitoxine du système AbiQ de L. lactis a été approfondi. Pour ce faire, des expériences ont été réalisées afin de démontrer que ce système est un mécanisme de type TA composé d’une antitoxine d’ARN et d’une toxine protéique avec une activitée ribonucléase. Dans un troisième temps, l’effet d’AbiQ sur la réplication des phages a été évalué. En isolant des phages mutants ayant la capacité de contourner le mécanisme, des cibles phagiques potentielles du système AbiQ ont été identifiées. Comprendre la relation phage-hôte est la clé pour le développement de bactéries résistantes aux phages dans un contexte de production industrielle ou pour limiter l’apparition de résistance aux phages lors du traitement des infections bactériennes par la thérapie phagique. / Bacteria and phages are continuously challenging each other and evolve in most ecosystems. Many strategies have been adapted by both of them to reach an equilibrium state. On one side, phages can infect efficiently and rapidly their bacterial host. On the other side, bacteria have acquired antiphage mechanisms to resist phage infection and limit their propagation such as abortive infection mechanisms (Abi). Specifically, the AbiQ antiphage system was isolated from Lactococcus lactis. This bacterium is used to produce an array of fermented dairy products. Even if this environment is tightly controlled, phages are ubiquitous in milk and in factories, and they can affect fermentations. The AbiQ antiphage system ressembles to a toxin-antitoxin (TA) system, but its mode of action is still unknown. In this study, three objectives were persued in order to better understand the relationship between Lactococcus lactis and its phages. First, the phage 949 genome, a member of a rare lactophage group that bears its name, was sequenced and characterized. A genome comparison of all lactococcal phages sequenced to date was done to confirm the current phage classification. In the second objective, we have confirmed that AbiQ is indeed a toxin-antitoxin system. Experiments were performed to demonstrate that this TA mechanism is composed of a RNA antitoxin and a protein toxin with ribonuclease activity. In the third objective, the effect of AbiQ on the phage replication was evaluated. By isolating phage mutants able to escape the mechanism, different phage targets were identified. Understanding the phage-host relationships is the key to develop efficient tools to reduce phage infection in industrial settings or to limit the development of phage resistance in other applications such as in phage therapy.