No description available.

No description available.

Submitted by Maike Costa (maiksebas@gmail.com) on 2017-02-01T15:00:05Z No. of bitstreams: 1 arquivo total.pdf: 1370224 bytes, checksum: 70866254562076b628edd3331cf9b3bc (MD5) / Made available in DSpace on 2017-02-01T15:00:05Z (GMT). No. of bitstreams: 1 arquivo total.pdf: 1370224 bytes, checksum: 70866254562076b628edd3331cf9b3bc (MD5) Previous issue date: 2015-04-10 / Minimally processed vegetables are ready-to-eat products that undergo to a physical modification processes such as cutting, peeling and slicing, but preserve characteristics of a fresh food. When processed under unsatisfactory sanitary conditions, these products may represent microbiological risk and, therefore, sanitation becomes a critical step processing. In the present study the effects of the application of the essential oils (EOs) from Origanum vulgare L. – oregano (OVEO) and Rosmarinus officinalis L. – rosemary (ROEO), alone or combined at sub-inhibitory concentrations were assessed against the pathogenic bacteria contaminants of minimally processed vegetables, Listeria monocytogenes, Escherichia coli and Salmonella Enteritidis. The identification of the EOs’ constituents was performed by gas chromatography–mass spectrometry (GC-MS). The inhibitory effects were evaluated by determination of minimum inhibitory concentration (MIC), fractional inhibitory concentration index (FICI) and assessment of viable cell counts in vegetable broth and artificially infected vegetable over time. Thymol and eucalyptol were the major compounds identified in OVEO and ROEO, respectively. MIC value of OVEO was 0.6 μL/mL against the test strains either in single and mixed inoculum. MIC value of ROEO was 5 μL/mL against L. monocytogenes and E. coli and 10 μL/mL against S. Enteritidis in single inocula, while it was 10 μL/mL against the mixed inoculum. FICI of the combined EOs was 0.5 against the mixed bacterial inoculum, suggesting synergic interaction. The incorporation of OVEO and ROEO alone (MIC) or at different sub-inhibitory concentrations in vegetable broth resulted in decrease of viable cell counts of all test strains along the 24 h. A ≥ 3 log cycles reduction in viable cell count of L. monocytogenes, E. coli and S. Enteritidis was observed after exposure to OVEO at MIC. When exposed to ROEO at MIC, the same reduction was observed for cells of L. monocytogenes and E. coli, while S. Enteritidis presented smaller decrease (up to 1.7 log cycles) in viable cell counts. The incorporation of OVEO and ROEO combined at sub-inhibitory concentrations resulted in a decrease in initial viable counts of all strains tested, although the time requested to stablish this decrease varied according to the strain and amount of each EO in combination. Likewise, the EOs applied alone or in combination reduced the viable cell counts of all test strains when essayed in artificially infected vegetables. However, the exposure of vegetables to EOs for 10 minutes caused a greater reduction in viable cell counts compared to results obtained after 5 minutes of exposure. In both essays was observed that the inhibitory effects of the EOs varied according to the time and strain. These findings reinforce the rational use of OVEO and ROEO combined at sub-inhibitory concentrations to guarantee the safety and extend the shelf-life of fresh vegetables. It is also suggested carrying out scientific researches aimed to study the toxicity in animals and /or human cells, in order to ensure the health safety of these oils when used in food. / Hortaliças minimamente processadas são produtos prontos para consumo que foram submetidos a processos de modificação física como corte, descascamento e fatiamento, mas que preservam as características de um alimento fresco. Quando processados sob condições higiênico-sanitárias insatisfatórias, esses produtos podem representar risco microbiológico e, portanto, a sanitização é uma etapa crítica do processamento. No presente estudo foi avaliado o efeito da aplicação dos óleos essenciais de Origanum vulgare L. - orégano (OEOV) e Rosmarinus officinalis L. - alecrim (OERO), isolados e combinados em concentrações sub-inibitórias, contra as bactérias patogênicas contaminantes de hortaliças minimamente processadas Listeria monocytogenes, Escherichia coli e Salmonella Enteritidis. A identificação dos constituintes dos óleos essenciais (OEs) foi realizada por meio de cromatografia gasosa acoplada a espectrometria de massas (CG-MS). Os efeitos inibitórios foram avaliados pela determinação da concentração inibitória mínima (CIM), índice de concentração inibitória fracional (FICI) e avaliação de contagens de células viáveis em caldo vegetal e em vegetais frescos artificialmente contaminados. Timol e eucaliptol foram os compostos majoritários detectados no OEOV e OERO, respectivamente. O valor da CIM do OEOV foi de 0,6 μL/mL frente às cepas teste em inóculo simples e misto, enquanto o valor da CIM do OERO foi de 5μL/mL frente L. monocytogenes e E. coli e 10 μL/mL frente S. Enteritidis em inóculo simples e 10 μL/mL contra o inóculo misto. O ICIF dos OEs combinados foi de 0,5 frente o inóculo bacteriano misto, sugerindo interação sinérgica. A incorporação do OEOV e OERO isolados (CIM) ou em diferentes concentrações sub-inibitórias no caldo vegetal resultou em diminuição de contagens de células viáveis de todas as cepas de teste ao longo de 24 h. Uma redução ≥ 3 ciclos log na contagem de células viáveis de L. monocytogenes, E. coli e S. Enteritidis foi observada após a exposição a CIM do OEOV. Quando expostas a CIM do OERO, essa redução foi observada para as células de L. monocytogenes e E. coli, enquanto S. Enteritidis apresentou menor redução (até 1,7 ciclos log) na contagem de células viáveis. A incorporação de OEOV e OERO combinados em concentrações sub-inibitórias resultou em redução da contagem inicial de células viáveis de todas as cepas ensaiadas, embora o tempo necessário para estabelecer esta diminuição variou de acordo com a cepa e quantidade de cada OE na combinação. Da mesma forma, OEs aplicados isoladamente ou em combinação reduziram as contagens de células viáveis de todas as cepas teste quando ensaiadas em vegetais frescos artificialmente infectados. No entanto, a exposição dos vegetais aos OEs isolados e em combinação durante 10 minutos causou uma maior redução nas contagens de células viáveis quando comparado aos resultados obtidos após 5 minutos de exposição. Em ambos os ensaios observou-se que os efeitos inibitórios dos OEs variaram de acordo com o tempo e o tipo de cepa. Esses achados reforçam a utilização racional dos OEOV e OERO combinados em concentrações sub-inibitórias para garantir a segurança e prolongar a vida de prateleira de vegetais frescos. Sugere-se ainda a realização de pesquisas científicas voltadas para o estudo da toxicidade em animais e/ou com células humanas, de modo a assegurar a inocuidade destes OEs quando aplicados em alimentos.

Dans le tractus gastro-intestinal, l'adhésion des bactéries commensales à l’épithélium permet leur maintien, ce qui aide à contrôler l’implantation de germes indésirables par des mécanismes de concurrence (effets nutritionnels, sites spécifiques d'adhésion ...). Bien que le rôle de la couche du mucus (principalement composée de glycoprotéines à haut poids moléculaire, appelées mucines) recouvrant la muqueuse soit connu et décrit depuis de nombreuses années, notamment pour sa fonction de barrière protectrice, l'intérêt pour décrypter les mécanismes précis d’interaction(s) avec le microbiote (bactéries commensales, pathogènes ou probiotiques) n’a que récemment émergé. Dans ce cadre, l'objectif de cette thèse, menée en collaboration entre le Laboratoire d’Ingénierie des Systèmes Biologiques et des Procédés de Toulouse et le Laboratoire d’Analyse et d’Architecture des Systèmes de Toulouse, est de caractériser in vitro le comportement muco-adhésif de Lactococcus lactis, le modèle des bactéries lactiques, en utilisant des approches de quantification multi-échelles (du niveau moléculaire à l’échelle multicellulaire) sur un large panel de souches naturelles et recombinantes. Une attention particulière est accordée au rôle des mucines, en utilisant le modèle PGM (mucine gastrique de porc ou « Pig Gastric Mucin »).La première partie du travail a porté sur la quantification à l'échelle de la cellule unique des interactions entre L. lactis et une surface abiotique (polystyrène) recouverte de PGM, en utilisant la microscopie à force atomique (AFM). La faisabilité de la méthode a tout d'abord été démontrée sur la souche modèle L. lactis ssp. cremoris MG1820. La couche de PGM a été caractérisée en utilisant des méthodes analytiques complémentaires (AFM, XPS - spectroscopie de photoélectrons induits par rayons X, QCM-D - Microbalance à Quartz à mesure de Dissipation...). En parallèle, les bactéries L. lactis ont été immobilisées sur la pointe AFM et utilisées comme « sonde de force », en considérant la souche naturelle IBB477 (L. lactis ssp. cremoris), d’origine laitière et présentant une forte persistance dans le tractus digestif du rat lors d’essais réalisés in vivo (collaboration avec l’Institut de Biochimie et de Biophysique de Varsovie, Pologne). Comparé aux conditions contrôle (i.e., surface de polystyrène sans PGM), les niveaux de force d'adhésion enregistrés entre L. lactis et PGM sont inférieurs, ceci en raison des répulsions électrostatiques, hydrophiles et stériques s’établissant entre la couche de PGM et la paroi cellulaire. La forme des courbes représentant l’évolution de la force au retrait en fonction de la distance est également différente. Une analyse détaillée souligne la contribution, conjointe et différente selon les souches testées, d’événements (i) non adhésifs, (ii) adhésifs non spécifiques (interactions électrostatiques, hydrophobes, de van der Waals) et (iii) adhésifs spécifiques (liaison de type ligand/récepteur). La contribution spécifique a ensuite été explorée plus finement en termes de constantes cinétiques d’association et de dissociation. Nous avons, par ailleurs, poursuivi notre exploitation de la biodiversité naturelle chez les lactocoques en étudiant la souche TIL448 (L. lactis ssp. lactis) d’origine végétale, en collaboration avec l’Institut MICALIS de Jouy-en-Josas. Nous avons ainsi démontré, pour la première fois chez L. lactis, le rôle combiné des protéines à domaine(s) MUB (« Mucus-Binding ») et des pili, à travers l'analyse approfondie des données AFM (force d'adhésion, répartition des événements adhésifs spécifiques/non spécifiques, distances d'interaction...). Le rôle des pili a été confirmé sur des souches recombinantes piliées (L. lactis ssp. lactis IL1403), toujours en partenariat avec l’Institut MICALIS de Jouy-en-Josas. En parallèle, en collaboration avec l’Unité de Glycobiologie Structurale et Fonctionnelle de Villeneuve d'Ascq, nous avons cherché à identifier les O-glycannes de PGM (fractions neutre et acide), impliqués dans le processus d'interaction avec la surface bactérienne. Pour confirmer l’ensemble des résultats obtenus à l'échelle de la cellule unique et en mode statique par AFM (effet anti-adhésif de PGM, comportement muco-adhésif différent selon les souches de L. lactis), la deuxième partie du travail a été consacrée à des expérimentations à l’échelle de l’ensemble de la population bactérienne, en conditions dynamiques (QCM-D, chambre à écoulement cisaillé). Nous avons évalué par QCM-D chez les souches IBB477 et MG1820 les propriétés viscoélastiques des dépôts bactériens, en relation avec le comportement bio-adhésif vis-à-vis de la couche de PGM. Les données obtenues par AFM et chambre à écoulement cisaillé sur ces mêmes souches ont été confrontées pour accéder plus finement au mode d’interaction avec PGM (densité de liaisons sur la surface bactérienne). Enfin, nous avons évalué chez IL1403 l’effet des pili sur la dynamique de détachement et d’orientation sous cisaillement contrôlé.En conclusion, la combinaison des échelles d'observation et d’analyse, aussi bien au niveau de la cellule unique qu’à celui de l’ensemble de la population bactérienne, nous permet désormais de disposer de nouvelles connaissances sur les mécanismes diversifiés d'interaction entre L. lactis et PGM, visant à une meilleure compréhension des fonctionnalités de cette bactérie au niveau du tractus gastro-intestinal / In the gastrointestinal tract, adhesion of commensal bacteria to epithelial cells allows their retention, which helps to control the implementation of unwanted germs through mechanisms of competition (nutritional effects, specific sites of adhesion ...). Indeed, bacterial adhesion to the intestinal epithelium seems to be important for the balance of intestinal microbiota. Although the role of the mucus layer lining the mucosa, which is mainly composed of large glycoproteins termed mucins, is known and described for many years, particularly for its protective barrier function, the interest for unraveling precise mechanisms of interaction with bacteria (commensal, pathogens or probiotics) has just recently emerged. In this framework, the aim of the PhD thesis was to characterize in vitro muco-adhesive behavior of Lactococcus lactis, the model of Lactic Acid Bacteria, using multi-scale approaches (from molecular to multicellular levels) on a large set of natural and recombinant strains. A particular attention was paid to the role of mucins, using the PGM model (Pig Gastric Mucin).The first part of the work was focused on the quantification at nanoscale of interactions between L. lactis and adsorbed PGM, using AFM. The feasibility of the method was first demonstrated on the reference strain MG1820. PGM coating was characterized using a complementary set of analytical methods (AFM, XPS, quartz crystal microbalance with dissipation monitoring…). In parallel, L. lactis cells were immobilized onto the AFM tip and used as a living force probe, considering the natural strain IBB477 (L. lactis subsp. cremoris), isolated from Polish artisanal dairy products and previously shown to display in vivo retention in the rat gut (collaboration with the Institute of Biochemistry and Biophysics of Warsaw, Poland). Compared to control conditions (i.e., no PGM coating), adhesion force levels recorded for PGM were lower, due to the interplay of electrostatic, hydrophilic and steric repulsions. The shape of retraction force-distance curves for L. lactis/PGM interactions was also different. The detailed analysis of curve shape highlighted the contribution of non-adhesive, non-specific (electrostatic, hydrophobic, van der Waals interactions) and specific adhesive events (ligand/receptor bonding), depending on the strain under study. Specific forces were analyzed in terms of dissociation/association kinetic constants.We then explored the natural biodiversity among lactococci by studying the natural strain of L. lactis (subsp. lactis) TIL448 from plant origin, in collaboration with MICALIS (Jouy-en-Josas). We demonstrated, for the first time for L. lactis, the combined role played by “MUB-like” domain-containing protein and pili, through the thorough analysis of AFM data (adhesion force, repartition of specific/non-specific adhesive events and distances of interaction…). The role of pili was also confirmed with recombinant piliated strains (L. lactis subsp. lactis IL1403), in partnership with MICALIS (Jouy-en-Josas). In parallel, in collaboration with the “Unité de Glycobiologie Structurale et Fonctionnelle de Villeneuve d'Ascq”, a first attempt was done to identify the O-glycans of PGM (neutral and acid fractions), involved in interactions with the bacterial surface.To confirm these results achieved at single-cell scale and under static mode by AFM (anti-adhesive of PGM, different muco-adhesive properties among several strains of L. lactis), the second part of the work was devoted to experiments at multicellular scale under dynamic conditions (quartz crystal microbalance with dissipation monitoring – QCM-D, shear stress flow chamber). We evaluated by QCM-D, for MG1820 and IBB477 strains, the viscoelastic properties of the cell layers, in relation with the bio-adhesive behavior towards PGM. The data obtained by AFM and shear stress flow chamber were combined to access more precisely to the interaction mode with PGM (density of bonds over the cell surface). Finally, using the recombinant piliated strain (IL1403), we focused on the effect of pili on detachment and re-orientation dynamics under shear flow