O aumento na demanda por alimentos saudáveis e minimamente processados impulsiona a busca por novos agentes antimicrobianos. As bacteriocinas são peptídeos produzidos via ribossomo por algumas espécies de bactérias, podendo ser usadas na conservação e garantia da inocuidade de alimentos, não apresentando as possíveis ações tóxicas de conservadores clássicos amplamente utilizados na indústria alimentícia. Carnobacterium maltaromaticum C2 foi isolado de peixe defumado brasileiro (Surubim, Pseudoplatystoma sp.), e apresenta grande capacidade de inibir a multiplicação de Listeria monocytogenes, demonstrando seu potencial para aplicação na bioconservação de alimentos. Em estudos anteriores, foi demonstrado que essa linhagem bacteriana produz compostos antimicrobianos de origem proteica. Neste trabalho, foram avaliados aspectos gerais da produção de bacteriocinas por C. maltaromaticum C2, assim como sua purificação e caracterização. C. maltaromaticum C2 produz bacteriocinas entre 5 e 25ºC, com ótimo entre 20 e 25ºC. Do mesmo modo, a produção desses compostos foi maior em caldo APT (All purpose Tween), entretanto para as etapas de purificação foram utilizados o caldo BHI (Brain heart infusion) e CAA (Casamino acids), por causarem menos interferência no processo. Lactobacillus sakei e L. monocytogenes foram inibidos pelas bacteriocinas parcialmente purificadas produzidas por C. maltaromaticum C2, e seus peptídeos antimicrobianos apresentaram moderada estabilidade térmica quando expostos a 100ºC por 30 minutos. Foram utilizadas duas técnicas para extração e purificação das bacteriocinas, a técnica de adsorção-dessorção às células produtoras, e a purificação com a resina XAD-16, baseada em interações hidrofílicas e hidrofóbicas com os peptídeos, seguida de extração em fase sólida, sendo que este último processo de purificação resultou em um extrato com alto teor de pureza, como observado durante as análises por cromatografia líquida de alta eficiência em coluna de fase reversa. Com o auxílio de técnicas de espectrometria de massas, foi detectado nos extratos obtidos a presença das carnobacteriocinas BM1 e B1, assim como o peptídeo antimicrobiano CbnX. Este trabalho é pioneiro na purificação de CbnX, pois anteriormente havia somente a descrição de seu gene, mas não havia sido descrita a purificação do peptídeo. Neste sentido, a linhagem estudada é única até o momento e poderá favorecer estudos de expressão gênica de bacteriocinas, bem como a otimização de processos de bioconservação. / The high demand for healthy and minimally processed foods has increased the search for new antimicrobial agents. Bacteriocins are ribosomally synthesized peptides produced by some bacteria, and are useful for biopreservation and food safety, without the possible toxic effects of classical preservatives widely used in food industry. Carnobacterium maltaromaticum C2 was isolated from Brazilian smoked fish (Surubim, Pseudoplatystoma sp.), and it inhibits Listeria monocytogenes, an important foodborne pathogen. In previous studies, it was demonstrated that this bacterial strain produces bacteriocins. In this study, general aspects of the production of bacteriocins by C. maltaromaticum C2 were evaluated, as well as their purification and characterization. C. maltaromaticum C2 produces bacteriocins between 5 and 25ºC, with the optimum incubation temperature between 20 and 25ºC. Similarly, the production of these compounds was higher in APT (All-purpose Tween) broth. However, for the purification steps, BHI (Brain heart infusion) broth and CAA (Casamino acids) broth were used due to their low interference with the processes. Lactobacillus sakei and L. monocytogenes were inhibited by the partially purified bacteriocins produced C. maltaromaticum C2, and their antimicrobial peptides showed moderate thermal stability when tested at 100ºC by 30 minutes. Two techniques for extraction and purification of the antimicrobial peptides were used, the adsorption-desorption of bacteriocins to the producer cells, and the purification with XAD-16 resin, based on hydrophilic and hydrophobic interactions with the peptides, followed by a step of solid phase extraction. The latter resulted in an extract with high purity, as observed by the analysis with reverse-phase high performance liquid chromatography. With mass spectrometry techniques, carnobacteriocins BM1 and B1 were detected, as well as the antimicrobial peptide CbnX. This is an innovative work because the purification of CbnX had never been reported, except its gene. In this respect, this C. maltaromaticum strain is unique until this moment, and may promote researches on gene expression, as well the optimization of biopreservation processes.

O aumento na demanda por alimentos saudáveis e minimamente processados impulsiona a busca por novos agentes antimicrobianos. As bacteriocinas são peptídeos produzidos via ribossomo por algumas espécies de bactérias, podendo ser usadas na conservação e garantia da inocuidade de alimentos, não apresentando as possíveis ações tóxicas de conservadores clássicos amplamente utilizados na indústria alimentícia. Carnobacterium maltaromaticum C2 foi isolado de peixe defumado brasileiro (Surubim, Pseudoplatystoma sp.), e apresenta grande capacidade de inibir a multiplicação de Listeria monocytogenes, demonstrando seu potencial para aplicação na bioconservação de alimentos. Em estudos anteriores, foi demonstrado que essa linhagem bacteriana produz compostos antimicrobianos de origem proteica. Neste trabalho, foram avaliados aspectos gerais da produção de bacteriocinas por C. maltaromaticum C2, assim como sua purificação e caracterização. C. maltaromaticum C2 produz bacteriocinas entre 5 e 25ºC, com ótimo entre 20 e 25ºC. Do mesmo modo, a produção desses compostos foi maior em caldo APT (All purpose Tween), entretanto para as etapas de purificação foram utilizados o caldo BHI (Brain heart infusion) e CAA (Casamino acids), por causarem menos interferência no processo. Lactobacillus sakei e L. monocytogenes foram inibidos pelas bacteriocinas parcialmente purificadas produzidas por C. maltaromaticum C2, e seus peptídeos antimicrobianos apresentaram moderada estabilidade térmica quando expostos a 100ºC por 30 minutos. Foram utilizadas duas técnicas para extração e purificação das bacteriocinas, a técnica de adsorção-dessorção às células produtoras, e a purificação com a resina XAD-16, baseada em interações hidrofílicas e hidrofóbicas com os peptídeos, seguida de extração em fase sólida, sendo que este último processo de purificação resultou em um extrato com alto teor de pureza, como observado durante as análises por cromatografia líquida de alta eficiência em coluna de fase reversa. Com o auxílio de técnicas de espectrometria de massas, foi detectado nos extratos obtidos a presença das carnobacteriocinas BM1 e B1, assim como o peptídeo antimicrobiano CbnX. Este trabalho é pioneiro na purificação de CbnX, pois anteriormente havia somente a descrição de seu gene, mas não havia sido descrita a purificação do peptídeo. Neste sentido, a linhagem estudada é única até o momento e poderá favorecer estudos de expressão gênica de bacteriocinas, bem como a otimização de processos de bioconservação. / The high demand for healthy and minimally processed foods has increased the search for new antimicrobial agents. Bacteriocins are ribosomally synthesized peptides produced by some bacteria, and are useful for biopreservation and food safety, without the possible toxic effects of classical preservatives widely used in food industry. Carnobacterium maltaromaticum C2 was isolated from Brazilian smoked fish (Surubim, Pseudoplatystoma sp.), and it inhibits Listeria monocytogenes, an important foodborne pathogen. In previous studies, it was demonstrated that this bacterial strain produces bacteriocins. In this study, general aspects of the production of bacteriocins by C. maltaromaticum C2 were evaluated, as well as their purification and characterization. C. maltaromaticum C2 produces bacteriocins between 5 and 25ºC, with the optimum incubation temperature between 20 and 25ºC. Similarly, the production of these compounds was higher in APT (All-purpose Tween) broth. However, for the purification steps, BHI (Brain heart infusion) broth and CAA (Casamino acids) broth were used due to their low interference with the processes. Lactobacillus sakei and L. monocytogenes were inhibited by the partially purified bacteriocins produced C. maltaromaticum C2, and their antimicrobial peptides showed moderate thermal stability when tested at 100ºC by 30 minutes. Two techniques for extraction and purification of the antimicrobial peptides were used, the adsorption-desorption of bacteriocins to the producer cells, and the purification with XAD-16 resin, based on hydrophilic and hydrophobic interactions with the peptides, followed by a step of solid phase extraction. The latter resulted in an extract with high purity, as observed by the analysis with reverse-phase high performance liquid chromatography. With mass spectrometry techniques, carnobacteriocins BM1 and B1 were detected, as well as the antimicrobial peptide CbnX. This is an innovative work because the purification of CbnX had never been reported, except its gene. In this respect, this C. maltaromaticum strain is unique until this moment, and may promote researches on gene expression, as well the optimization of biopreservation processes.

L'utilisation de la bactérie C. maltaromaticum dans l'industrie alimentaire n'est pas autorisée en raison de faible connaissance et des pathologies qu'il peut provoquer chez le poisson. Un des objectifs de ce travail est de renforcer la connaissance par la séquence génomique d'une souche fromagère et d'évaluer le devenir de la bactérie après ingestion par le consommateur. L'autre objectif est de mieux connaitre la taxonomie de la bactérie par MLST. La séquence complète de C. maltaromaticum LMA 28 a révélé une taille génomique de 3,8 Mpb qui est atypique dans le genre Carnobacterium. L'analyse génomique indique qu'il contiendrait des gènes d'adaptation à l'environnement intestinal. Il a été montré que la bactérie est capable de survivre le transit gastro-intestinal chez la souris et d'adhérer à des cellules intestinales humaines qui présenteraient des propriétés neutres voire anti-inflammatoires. Le résultat suggère qu'elle est capable de survivre le transit GIT et d'interagir avec l'hôte. Il a été montré que la MLST chez C. maltaromaticum permet d'atteindre un haut niveau de résolution. MLST suggèrent que le lait et les fromages à pâte molle sont peu sélectifs pour C. maltaromaticum. De plus, deux CC majoritaires, principalement représentés par des souches laitières, ont été identifiés. Leur existence suggère qu'une lignée de l'espèce est particulièrement bien adaptée à l'environnement laitier ou qu'un phénomène de domestication est en cours. Un grand nombre de singletons ont été identifiés suggérant que la diversité chez cette espèce est sous-estimée et qu'elle reste à explorer / The bacterium Carnobacterium maltaromaticum is not used in industry due to limited knowledge about this organism and its virulence in fish. One objective of this thesis was to strengthen the body of knowledge by determining the complete genome sequence of the cheese strain and to evaluate the fate of this bacterium after ingestion by the consumer. Another objective was to improve the taxonomic knowledge within the species C. maltaromaticum through the development of a MLST scheme. The complete sequence of the strain C. maltaromaticum LMA 28 revealed a genome size of approximately 3.8 Mbp, which is unusually high in the genus Carnobacterium. The genome analysis of this strain indicates the presence of genes conferring the adaptation to the intestinal environment. The bacterium is able to survive during the gastro-intestinal transit in mice. Moreover, this strain is able to adhere to human intestinal epithelial cell lines and would have neutral or anti-inflammatory properties. These data suggest that C. maltaromaticum LMA 28 is adapted to the digestive tract of mammals. At the taxonomical level, it was shown that MLST is highly discriminatory for the species C. maltaromaticum. In addition, the MLST results suggest that milk and soft cheeses are poorly selective for strains of this species. In addition, two major clonal complexes suggest that a sub-population within this species is well adapted to the dairy environment or that a sub-population is submitted to a domestication process. A high proportion of singletons was obtained suggesting that the diversity was under-estimated and remains to be explored

La souche Carnobacterium maltaromaticum LMA 28, isolée d’un fromage à pâte molle, possède des propriétés physiologiques non conventionnelles pour une bactérie lactique. Sa croissance en TSB-YE et en lait traduisent son exigence nutritionnelle en facteurs de croissance facilement assimilables et sa faible vitesse de production d’acide lactique à partir de glucose, lactose, fructose et saccharose. Le galactose n’est pas métabolisé et lors de l’hydrolyse du lactose n’est pas excrété dans le milieu de culture. Les caillés lactiques sont obtenus après des durées d’incubation non compatibles avec les cadences industrielles. De plus, ils présentent une texture très friable. La numération et l’identification de cette souche, en vue de suivre son comportement dans une matrice fromagère, ont été optimisées par la mise au point du milieu de culture sélectif CM, à l’aide de plan d’expériences, et par la technique de PCR. Le comportement de C. maltaromaticum LMA 28 a été comparé à ceux de deux souches lactiques d’intérêt technologique Lc. lactis DSM 20481 et S. thermophilus INRA 302, dans une large gamme de températures (3 à 37 °C) et de pH (5,2- 8,0). Des essais en co-culture, associant cette souche avec Lc. lactis DSM 20481 ou avec S. thermophilus INRA 302, ont montré que la production d’acide lactique était due à la croissance de la souche lactique traditionnelle. Cependant C. maltaromaticum LMA 28, souche lente, n’est pas inhibée par cette acidification. L’aptitude fromagère de C. maltaromaticum LMA 28 a été testée lors de deux fabrications de fromages à pâte molle. Inoculée à différents niveaux de population, elle a été mise en évidence à tous les stades de la fabrication. Présente à une concentration très faible dans le lait de fabrication, elle devient une flore lactique dominante après l’affinage et le stockage en réfrigération. Cette aptitude technologique est en relation avec son caractère psychrotrophe et sa faculté à se développer activement à des pH alcalins. Son « alimentarité », testée par la production d’amines biogènes, a montré des niveaux nuls ou très faibles en tyramine et en histamine, comme avec S. thermophilus INRA 302 et avec Lc. lactis DSM 20481. L’optimisation de sa production de flaveurs maltées a été abordée sur milieu TSB-YE et sur lait, supplémentés avec de la leucine, de l’isoleucine ou de la valine. La production de 3-méthylbutanal est la plus importante. Les analyses sensorielles des fromages contenant des niveaux de population importants (108-109 ufc.g-1) de C. maltaromaticum LMA 28 n’ont pas permis de mettre en évidence cet arôme. Présente dans de nombreux fromages français AOC ou non AOC, cette espèce opportuniste, de statut GRAS, pourrait être considérée comme un auxiliaire de fabrication intéressant, car elle permet un ralentissement du vieillissement des fromages, en évitant notamment l’apparition de flaveurs désagréables. Cette flore lactique psychrotrophe pourrait être retenue comme flore bactérienne d’affinage / The C. maltaromaticum LMA 28 bacteria strain, isolated from soft cheese, was observed to possess non conventional lactic bacteria physiological properties. Its growth in TSB YE medium and milk was found to be characterised by the requirements for easily assimilated growth nutrients and a low kinetic rate of lactic acid production from glucose, lactose, fructose and sucrose. In addition, it was found to not metabolise galactose or not excrete it during the hydrolysis of lactose. In the process of milk fermentation, it not only took an unusually long duration but produced products of fragile texture. In order to eventually determine the behaviour of this strain in the process of cheese-making, a selective culture medium CM was developed using an experimental design and PCR techniques for its isolation and identification. The behaviour of C. maltaromaticum LMA 28 was compared with that of two strains of lactic bacteria of technological interest namely Lc. lactis DSM 20481 and S. thermophilus INRA 302, within a wide temperature range (3 to 37°C) and of pH (5.2 – 8.0). Tests carried out in co-culture associating this strain with Lc. lactis DSM 20481 or with S. thermophilus INRA 302 showed that the lactic acid production was due mainly to the growth of the traditional lactic strain. In the process, the C. maltaromaticum LMA 28 slow strain was observed not to be inhibited by acidification. The cheese-making potential of C. maltaromaticum LMA 28 was evaluated in the process of two soft cheese manufactures. Inoculated at various levels of population, it was observed to be present at all manufacturing stages. Generally present at very weak concentrations in the starting milk, it becomes a dominant lactic flora following ripening and refrigeration storage. This technological aptitude is in relation with its psychrotrophic character and its ability to actively develop in alkaline medium. Its “alimentarity”, tested by its ability to produce biogenic amines, showed zero or very low levels in tyramine and histamine, as in the case of S. thermophilus INRA 302 and Lc. lactis DSM 20481. The optimization of its malted flavour production capacity was carried out on a TSB-YE medium and on milk supplemented with leucine, isoleucine or valine. In this process the production of 3-méthylbutanal was observed to be the most abundant product while cheese containing high levels (108-109 ufc.g-1) of C. maltaromaticum LMA 28 did not exhibit this flavour. This notwithstanding, the presence of this species of GRAS status in many French AOC and non AOC cheeses could be considered as an interesting auxiliary in cheese manufacturing process since it tends to slow down the aging process and thereby retard the development of unpleasant flavours. In this respect this strain of psychotrophic lactic bacteria could be retained as a flora for cheese ripening process

Carnobacterium est un genre bactérien présent dans une grande diversité d’environnements et de produits alimentaires. L’objectif de ce travail est d’approfondir les connaissances sur C. maltaromaticum (C. m) par une étude génomique. Neuf génomes de Carnobacterium, dont 5 de C. m, ont été analysés. C. m DSM20342 MX5 possède un génome de 3,85 Mbp, le plus grand génome parmi les LAB connus. Les souches de C. m et C. divergens présenteraient une surface cellulaire caractérisée par une grande diversité moléculaire. Les propriétés de surface qui en découlent seraient à relier à la capacité de ces deux espèces à coloniser différents habitats. Les souches de C. m isolées de différents produits laitiers se caractérisent par une grande diversité génomique. Afin de caractériser le niveau de diversité, les gènes impliqués dans les voies d’utilisation du lactose (voie Tagatose-6Phosphate, gènes lac) et du galactose (voie Leloir, gènes gal) ont été caractérisés au sein du genre Carnobacterium et recherchés par PCR chez 42 souches de C. m. Les deux voies sont organisées différemment chez les différentes souches de C. m. L’analyse au niveau de la population révèle une dissémination des deux voies au sein des 4 lignées de cette population et suggère que les gènes lac et gal ont évolué selon un schéma complexe, reflet du haut niveau de diversité génétique de la population. La recherche de ces gènes au sein des 237 génomes de LAB indique qu’ils présentent un niveau de diversité élevé parmi les différents genres bactériens constituant le groupe des LAB, avec une dominance de la présence de la voie de Leloir. Ce niveau de diversité génétique est retrouvé à l’échelle de l’espèce chez C. m. / Carnobacterium bacteria are present in a wide variety of environments and foods. The objective of this thesis is to deepen the knowledge on C. maltaromaticum (C. m) by a comparative genomic approach. Nine Carnobacterium genomes, including 5 C. m, available on the platform MicroScope were analyzed and compared. C. m DSM20342 MX5 is the largest genome among LAB. Detailed analysis the presence of a high molecular diversity of cell surface proteins in C. m and C. divergens, while the three other Carnobacterium species present only few proteins on their surfaces. This can be related to the ability of these two species to colonize different habitats. Furthermore, C. m strains isolated from various dairy products are characterized by a large genomic diversity. In order to characterize this diversity, genes involved in lactose (Tagatose-6Phosphate pathway, lac genes) and galactose (Leloir pathway, gal genes) metabolic pathways were identified within Carnobacterium genus and detected by PCR in 42 C. m strains. Both pathways are present and organized differently in the C. m strains. The analysis of these genes at the population level shows a spread of the two pathways within the 4 lineages of this population and suggest that the lac and gal genes have evolved in a complex pattern, which reflects the high level of genetic diversity of the population. These genes were characterized in 237 LAB genome and exhibit a high degree of diversity among the different bacterial genera of the LAB group, with a dominance of the presence of the Leloir pathway. This level of genetic diversity is found at the species level for C. m.

Carnobacterium maltaromaticum est une bactérie lactique atypique isolée de fromages à pâte molle. Les caractéristiques majeures de cette bactérie sont i) sa capacité à produire un arôme de type malté ou chocolaté, le 3-méthylbutanal à partir du catabolisme de la leucine ii) sa capacité à produire des bactériocines, peptides à activité antibactérienne vis-à-vis de flores pathogènes et/ou d'altération fréquemment rencontrées en industries alimentaire, telle que Listeria monocytogenes. Une caractérisation phénotypique et génotypique réalisée sur six souches de C. maltaromaticum isolées du même biotope a montré que celles-ci ne sont pas phylogénétiquement identiques. Même si ces souches n'ont pas la capacité à coaguler rapidement le lait, elles sont acidotolérantes. Elles n'influent pas sur la capacité ni la rapidité de coagulation des starters, Lactococcus lactis et Streptococcus thermophilus utilisés en industrie laitière. L'étude de l'impact de C. maltaromaticum sur la flore bactérienne du fromage a révélé que sa présence provoque la diminution de la concentration de Psychrobacter, germe pouvant être responsable d'une accélération du phénomène de vieillissement du fromage. Un plan d'expérience a été réalisé pour mettre en évidence ces inhibitions et les éventuelles interactions entre différents facteurs (concentration cellulaire initiale, concentration en NaCl, pH, durée d'incubation). Deux modèles ont été choisis i) Psychrobacter sp. et ii) L. monocytogenes. La concentration cellulaire de C. maltaromatiucm est le facteur le plus important pour inhiber les bactéries testées. Cette espèce opportuniste pourrait être considérée comme un auxilliaire de fabrication intéressant et pourrait être retenue comme flore bactérienne d'affinage. Toutes les souches de C. maltaromaticum testées sont capables de produire du 3-méthylbutanal pouvant conférer une flaveur maltée au fromage. Les études menées sur la biosynthèse du 3-méthylbutanal ont montré la présence et la fonctionnalité de deux voies métaboliques; la voie directe impliquant l'alpha-cétoacide décarboxylase et la voie indirecte passant par l'alpha-cétoacide déshydrogénase. L'oxygénation du milieu de culture a un impact positif sur la formation du 3-méthylbutanal et du 3-méthylbutanol avec la stimulation des voies directes et indirectes / Carnobacterium maltaromaticum is a lactic acid bacterium isolated from atypical soft cheeses. The major characterstics of this bacterium are i) its ability to produce a malty or chocolate-like flavor 3-methylbutanal from leucine catabolism ii) its ability to produce bacteriocins against pathogenic and spoilage bacteria for instance, Listeria monocytogenes. Phenotypic and genotypic characterization of six strains of C. maltaromaticum isolated from the same habitat showed that they were not phylogenetically identical. Although these strains lacked the ability to coagulate the milk quickly, they were acid tolerant. They did not affect the milk coagulation capacity of startes, Lactococcus lactis and Streptococcus thermophilus used in dairy industry. The study on the impact of C. maltaromaticum on the bacterial flora of cheese showed that its presence resulted in the decrease of the concentration of Psychrobacter, which might be responsible for accelerating the aging phenomenon of cheese. An experimental plan was realized to highlight these inhibitions and possible interactions between factors (cell concentration, NaCl, pH, incubation time) by choosing two models i) Psychrobacter sp. and ii) L. monocytogenes. The cell concentration of C. maltaromatiucm was the factor more significant for the inhibitions of the tested bacteria. Being psychrotrophic, alkalinophilic, malty or chocolate flavor producing and biopreservative agent, this species could play a role as a ripening flora of cheese. All strains of C. maltaromaticum tested were able to produce 3-methylbutanal conferring any malt flavor to cheese. The results on the physiology involved in the biosynthesis of 3-methylbutanal showed the presence and functionality of both metabolic pathways; the direct by alpha-ketoacid dacarboxylase enzyme and indirect comprising alpha-keto acid dehydrogenase enzyme. The oxygenation of culture medium had a positif impact on the formation of 3-methylbutanal and 3-methylbutanol with the stimulation of both direct and indirect metabolic routes

Compétition et diversité sont des phénomènes majeurs en microbiologie. Dans le secteur de l’agroalimentaire, la compétition est à la base de la biopréservation, un procédé dont l’objectif est d’inhiber des microorganismes indésirables par l’utilisation de microorganismes compétiteurs. La diversité microbienne est quant à elle à l’origine de la diversité de produits fermentés et en particulier de la typicité des fromages. Pourtant, l’écologie de la compétition microbienne dans les aliments et le lien avec la diversité microbienne sont très mal connus. L’objectif de ces travaux de thèse était d’une part d’étudier la diversité et d’autre part la compétition chez une bactérie représentative de l’écosystème fromager. Le modèle d’étude choisi est la bactérie lactique Carnobacterium maltaromaticum. Une analyse de diversité de 21 souches de C. maltaromaticum a été réalisée par MultiLocus Sequence Typing (MLST) et a permis de compléter la structure connue de la population de C. maltaromaticum. Cette étude a permis de révéler la présence de 56 génotypes au sein d’une collection de 71 souches, montrant une grande diversité génétique au sein de cette espèce. La compétition par interférence a été étudiée par réalisation de tests de compétition à haut débit. Chaque test de compétition mettait en jeu deux souches de la collection, une souche en situation d’expédition et une souche en situation de réception. Au total 5776 tests ont été réalisés à partir de la collection de 76 souches. Les résultats ont révélé que 60% des souches inhibaient au moins une autre souche de la collection indiquant que la compétition intraspécifique est majeure au sein de l’espèce C. maltaromaticum. Par ailleurs, une grande variabilité de largeur de spectre d’inhibition et de spectre de sensibilité a été observée. Une approche d’analyse de réseau a révélé une architecture « nested » du réseau compétitif suggérant que l’inhibition dépend non seulement des caractéristiques antagonistes des souches inhibitrices mais également du niveau de sensibilité des souches réceptrices. Une analyse génomique de 26 souches de la collection a été réalisée en vue de prédire leur contenu en gènes codant la synthèse de substances antagonistes et a permis d’identifier des gènes codant potentiellement des bactériocines. En conclusion, ces travaux de thèse ont montré que l’espèce Carnobacterium maltaromaticum est d’une part caractérisée par une grande diversité génétique et que d’autre part la compétition par interférence est fréquente au sein de la population / Competition and diversity are major phenomena in microbiology. In the agri-food sector, competition is the very basis of biopreservation, a process which objective is to inhibit undesirable microorganisms through the use of microbial competitors. Microbial diversity lies at the origin of the diversity of fermented products and particularly of the typicality of cheeses. However, the ecology of microbial competition in food and the link with microbial diversity are poorly understood. The goal of this thesis was to study the diversity and the competition of a representative model bacterium of the cheese ecosystem. The study model chosen is the lactic acid bacterium Carnobacterium maltaromaticum. An analysis of the diversity of 21 strains of C. maltaromaticum was performed by MultiLocus Sequence Typing (MLST) and allowed to complete the scheme of C. maltaromaticum population structure. This study revealed the presence of 56 genotypes among a collection of 71 strains, showing a high genetic diversity within this species. Interference competition was studied by performing high-throughput competition assays. Each competition assay involved two strains, one in the position of the sender strain and the other in the position of the receiver. In total, 5776 tests were performed on a collection of 76 strains. The results revealed that 60% of strains inhibited at least one other strain of the collection, indicating that intraspecific competition is major in C. maltaromaticum. Moreover, a large variability in the width of inhibition and sensitivity spectra has been observed. A network analysis approach revealed a nested architecture of the competitive network, suggesting that inhibition depends not only on the antagonistic characteristics of the inhibitory strains but also on the level of sensitivity of the receiver strains. A genomic analysis of 26 strains from the collection was performed in order to predict their gene content encoding the synthesis of antagonistic substances, and it allowed the identification of genes potentially encoding bacteriocins. In conclusion, this thesis has shown that the species Carnobacterium maltaromaticum is characterized by a high genetic diversity and that interference competition is frequent in the population

L'industrie alimentaire cherche constamment à améliorer les processus de conservation des aliments. La biopréservation s’impose comme une alternative à l’utilisation de conservateurs dits «chimiques» et fait appel à des micro-organismes sélectionnés en tant que cultures protectrices pour leurs propriétés antimicrobiennes naturels. L'objectif de cette étude était de tirer profit de la diversité de Carnobacterium maltaromaticum afin d’identifier des souches capables d'inhiber la croissance du pathogène alimentaire Listeria monocytogenes. Pour cela, une méthode de typage moléculaire permettant la reconnaissance fine de souches a été établie, puis une méthode de criblage d’activités antibactériennes à robustesse élevée a été mise au point. La méthode de typage moléculaire est une méthode MLVA (Multiple-locus variable-number tandem repeat (VNTR) analysis) avec 3 loci de VNTR (VNTR-A, VNTR-B, VNTR-C) permet de discriminer 15 génotypes différents au sein d’une collection de 24 souches. Cela confirme la grande diversité génotypique et phénotypique présente au sein de la population de C. maltaromaticum et la performance de la méthode. La méthode de criblage est basée sur la réalisation d’essais de compétition à haut débit où chaque membre d’une collection de C. maltaromaticum a été co-cultivé avec une souche bioluminescente de L. monocytogenes. Il a été montré que la production de luminescence est le reflet de la croissance de L. monocytogenes en micro-culture et qu’il est ainsi possible de tester la stabilité du pouvoir antagoniste de chaque membre de la collection dans de multiples conditions de cultures différentes. Cette méthode a ainsi permis d’identifier des souches de C. maltaromaticum dont la propriété anti-L. monocytogenes est peu influencée par les conditions de cultures. Outre l’intérêt fondamental que présente cette méthode pour l’étude des interactions compétitives dans l’aliment, elle a permis d’identifier des souches présentant un fort potentiel de valorisation dans le domaine de la biopréservation alimentaire / The food industry is constantly seeking to improve food preservation processes. Biopreservation imposes itself as an alternative to the use of chemical preservatives and involves micro-organisms selected as protective cultures for their natural antimicrobial properties. The aim of this study was to benefit from the diversity of Carnobacterium maltaromaticum in order to identify strains capable of inhibiting the growth of Listeria monocytogenes, a food-borne pathogen. For this purpose, a molecular typing method that enables fine strain identification has been established, then a method of screening of antibacterial activities with high robustness has been developed. The molecular typing method is a MLVA (Multiple-locus variable-number tandem repeat (VNTR) analysis) with 3 VNTR loci (VNTR-A, VNTR-B, VNTR-C). It allowed to discriminate 15 different genotypes among a collection of 24 C. maltaromaticum strains. These results confirmed the high strain diversity within this species and showed the high performance of this method. The screening method is based on high throughput competition assays where each member of a collection of C. maltaromaticum strains was co-cultivated with a bioluminescent strain of L. monocytogenes. It was established that the bioluminescence of this strains is highly correlated to bacterial growth in micro-cultures, indicating that it is possible to investigate the stability of antagonistic properties of each collection member under multiple different culture conditions. The use of this method allowed to identify C. maltaromaticum strains with bioprotection properties that are poorly influenced by culture conditions. Besides the fundamental interest of this method for investigations of competitive interactions in food, it allowed to identify strains with high potential for bioprotection applications

Seafood permits the transmission of many bacterial pathogens. In order to reconcile consumer demands with important safety standards, traditional means of regulating microbial spoilage and safety hazards in foods are combined with novel technologies. These include biological antimicrobial systems, such as the use of lactic acid bacteria (LAB) and/or their bacteriocins, such as Carnobacterium maltaromaticum CS526 and its bacteriocin piscicocin CS526. The aims of this study were to investigate the presence of Listeria monocytogenes in temperate seafood, namely fresh and smoked salmon, fresh and smoked haddock, and fresh mussels and oysters. Additionally, there was an aim to recover, characterise and use bacteriocin-like-substance to control Listeria monocytogenes in cold smoked haddock. Vibrio spp., Enterobacteriaceae representatives, total aerobic heterotrophic counts and Listeria monocytogenes were isolated from commercially prepared smoked and fresh Atlantic salmon, smoked and fresh haddock, live mussels and oysters using selective media and tryptone soya agar (TSA). Vibrio spp. occurred in high densities (>106 CFU gˉ1) in mussels and Enterobacteriaceae representatives were recorded at >106 CFU gˉ1 in fresh salmon. Total aerobic heterotrophic counts in fresh salmon, live mussels and oysters reached 107, > 107, and > 106 CFU gˉ1, respectively. Listeria monocytogenes was recorded at 5.0 x 104 CFU gˉ1 in mussels. In total sixty one bacterial isolates were recovered from the seafood examined. The results revealed 19 genera of bacteria, i.e. Acinetobacter, Aerococcus, Aeromonas, Bacillus, Brochothrix, Carnobacterium, Citrobacter, Corynebacterium, Enterobacter, Escherichia coli, Moraxella, Micrococcus, Pseudomonas, Psychrobacter, Serratia, Shewanella, Staphylococcus, Vibrio and Listeria. The prominent characteristics of fish spoilage isolates were demonstrated by the ability of the isolates to reduce trimethylamine oxide (TMAO) to trimethylamine, and to produce H₂S. Sh. baltica OS185, Aeromonas spp. HB-6, Sh. baltica, Sh. putrefaciens, A. hydrophila HX201006-3, A. salmonicida subsp. achromogenes, A. hydrophila, C. freundii, Enterobacter cloacae were strong producers of TMA and H₂S. The spoilage microorganisms were tested for potential pathogenicity. The result revealed that 6/15 of the spoilage microorganisms produced proteolytic, lecithinase, blood (β and α haemolysin) and elastinase activity, respectively, whereas 7/15 of the spoilage microorganisms showed lipolytic activity. Cell free supernatants, ammonium sulphate precipitated supernatants and semi-purified bacteriocin-like substances of Carnobacterium maltaromaticum MMF-32 and KOPRI 25789 producing strains isolated from commercially prepared smoked salmon were investigated for their potential antimicrobial activity against potentially pathogenic and food spoilage microorganisms. Generally, a broad spectrum of activity was revealed against potentially pathogenic and food spoilage microorganisms in vitro. Cold-smoked haddock treated with bacteriocin producing C. maltaromaticum MMF-32, C. piscicola A9b bacˉ phenotype nonbacteriocin producing strain a mutant of C. piscicola A9b bac+, cell free supernatants, ammonium sulphate precipitated supernatants and semi-purified bacteriocin-like substances was challenged with L. monocytogenes ATCC 19114 up to 103 CFU gˉ1, respectively. Samples were stored at 4 °C for 10 days. L. monocytogenes and total bacterial counts were determined along with changes in total volatile base nitrogen (TVBN) and biogenic amines production as well as texture, colour and odour. Although the study on anti-listerial effects of C. maltaromaticum MMF-32 was not successful, this organism did have a positive effect on retention of firmness and sensory perception in cold smoked haddock.