Résumé confidentiel / Résumé confidentiel

O impacto causado pelo aumento da quantidade de substâncias químicas descartadas no meio ambiente está presente na maioria dos ecossistemas. Poluentes industriais contendo metais são frequentemente transportados para a água, o solo e o ar, podendo-se acumular nas cadeias tróficas e apresentar toxicidade para a biota. Em ambientes aquáticos, a biodisponibilidade e destino dessas substâncias xenobióticas podem ser influenciadas por vários fatores, entre eles a matéria orgânica dissolvida e outros compostos quelantes, os quais tem capacidade de aprisionar ou liberar íons para o ambiente. Os exopolissacarídeos, substâncias excretadas pelo fitoplâncton, podem interagir com diversas substâncias, interferindo na toxicidade dos compostos para os organismos ou comunidades biológicas aquáticas e, conseqüentemente, subestimando o verdadeiro valor tóxico das substâncias. Este trabalho foi conduzido para determinar a influência dos exopolissacarídeos da Clorofícea Pseudokirchniriella subcapitata, e da Cianofícea Anabaena spiroides na toxicidade dos elementos traço cádmio e cromo em Daphnia similis (Cladocera). Os metais foram escolhidos pela afinidade por quelantes orgânicos. Foram realizados testes ecotoxicológicos agudos para verificar a sensibilidade do cladócero Daphnia similis em diferentes concentrações dos metais cádmio (cloreto de cádmio) e cromo (dicromato de potássio), adicionando algas e exopolissacarídeos. Nos testes com os exopolissacarídeos foram utilizadas diferentes frações (excretado total, 10000D). Para obtenção das frações de exopolissacarídeos foram realizadas filtrações tangenciais em cartucho oco de celulose com bomba peristáltica. Nos testes com a adição das algas foram usados números conhecidos de células obtidas do concentrado de algas. Observou-se redução da toxicidade de 20 a 30% nos testes com a adição de excretado total, e reduções menores para as frações 10KD para as clorofícea e cianofícea. Com os resultados deste trabalho, foi possível avaliar a capacidade dessas substâncias em quelar e indisponibilizar compostos tóxicos e avaliar a toxicidade das substâncias quando testadas nos organismos planctônicos. / Chemical substances have been exerting increasing impact on ecosystems. Industrial pollutants containing metals frequently reach water bodies, soil, and air, wherein they may accumulate on the trophic chain, resulting in toxicity to the biota. In aquatic environments, the bioavailability and the destination of these xenobiotics are influence by several factors, such as the amount of solved organic matter and other chelating compounds, since these substances can either bind or liberate ions to the environment. Exopolysaccharides, in particular, are excreted by phytoplankton and, once in water, can interact with several substances altering the toxicity of compounds to aquatic organisms and biological communities. As a consequence, the real toxic potential of these xenobiotics is underestimated. The aim of this work was to determine the influence of the exopolysaccharides produced by the Chloroficeae Pseudokirchniriella subcapitata and by the Cyanophiceae Anabaena spiroides on the toxicity of the trace-elements cadmium and chromo over the primary consumer Daphnia similis. These metals were chosen due to their high affinity towards organic chelators. Tests for acute ecotoxicity were performed to verify the sensitivity of the Cladocera Daphnia similis exposed to different cadmium (cadmium chloride) and chromo (potassium dichromate) concentrations, with and without algae and/or exopolysaccharides. Three fractions of exopolysaccharides were tested (total excreted, < 1000D, and > 1000D), which were obtained by tangencial filtration through a cellulosis membrane using a peristaltical pump. On tests using algae, a fixed number of cells were obtained from an algae concentrate. The toxicity of Daphnia similis to the metals was 20-30% reduced when the total excreted from algae was added, while the reduction in toxicity was lower to the fraction than 10KD. The results demonstrated that exopolysaccharides chelate toxic compounds rendering them unavailable for exerting their effects on planktonic organisms.

Resumo: Exopolissacarídeos (EPS) são polímeros produzidos por uma grande variedade de microrganismos e possuem diferentes propriedades estruturais, físicas e químicas. A investigação das propriedades reológicas de suas soluções é importante devido ao crescente interesse na aplicação comercial de polissacarídeos, principalmente na indústria de alimentos. Assim, o objetivo deste trabalho foi investigar as características reológicas dos exopolissacarídeos R1, R2, R3 e R4 produzidos por diferentes linhagens de Rhizobium e Mesorhizobium. Análises quantitativas mostraram que o teor de ácido urônico encontrado em R3 (8,4 %) foi maior que em R1 (2,4 %), R2 (1,7 %) e R4 (0,8 %). A cromatografia de filtração em gel mostrou que R2 e R3 são mais homogêneos e menos polidispersos. Hidrólise ácida total e análise por HPAEC/PAD mostrou glucose como constituinte básico majoritário dos EPS, além de galactose e manose. Todos os polissacarídeos apresentaram comportamento não-Newtoniano, com características de soluções pseudoplásticas nas concentrações de 2, 5 e 10 g/L. O modelo reológico de Ostwald-de-Waele (Lei da Potência) foi utilizado para representar os dados experimentais de tensão de cisalhamento versus taxa de deformação. Os EPS R1, R2 e R4 demonstraram pequeno aumento na viscosidade em presença de NaCl, e apresentaram comportamento viscoelástico de gel, sendo R1 o que apresenta características de gel mais forte. O EPS R3 manteve-se como o menos viscoso, tanto em meio aquoso quanto em solução salina, provavelmente devido ao maior percentual de ácidos urônicos em sua estrutura. Além disso, R3 exibiu comportamento de solução diluída a baixas concentrações, e viscoelástico de gel fraco em concentrações mais elevadas. A análise da influência da temperatura sobre o comportamento viscoelástico das soluções polissacarídicas mostrou... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Exopolysaccharides (EPS) are polymers produced by a great variety of microorganisms and possess different structural, physical and chemical properties. Investigation of rheological properties of these solutions is important due to an increasing interest in polysaccharides commercial applications, mainly in food sectors. The objective of this work was investigate rheological characteristics of exopolysaccharides R1, R2, R3 and R4 produced by different Rhizobium and Mesorhizobium strains. Quantitative analysis showed that uronic acid component found in R3 (8,4 %) was higher than R1 (2,4 %), R2 (1,7 %) and R4 (0,8 %). Gel filtration chromatography indicated that EPS R2 and R3 are more homogeneous and less polidisperse. Acid hydrolysis and HPAEC/PAD analysis revealed that glucose was the main monosaccharide, beyond galactose and mannose. All exopolysaccharides had non-Newtonian behavior, with pseudoplastic characteristics at concentrations of 2, 5 and 10 g/L. The Ostwald-de-Waele (Power Law) was the rheological model used to represent the experimental data of the shear stress versus shear rate. EPS R1, R2 and R4 demonstrated a slight increase in viscosity in presence of NaCl, and viscoelastic behavior, R1 had strong gelling characteristics. The EPS R3 was less viscous, in water solutions and presence of salt, probably due to a high percentual of uronic acids on its structure. Moreover, R3 exhibited diluted solution behavior at low concentration, and viscoelastic weak gelling at high concentrations. Analysis of temperature influence over polysaccharide solutions viscoelastic behavior showed that EPS R1, R2 e R4 had strong gelling characteristics, at concentrations of 5 g/L. / Orientador: Maria de Lourdes Corradi Custódio da Silva / Coorientador: Ana Lúcia Barretto Penna / Banca: Eliana Gertrudes de Macedo Lemos / Banca: Javier Telis Romero / Mestre

Resumo: Os exopolissacarídeos (EPS) são polímeros produzidos por uma grande variedade de microrganismos e podem possuir propriedades físicas, estruturais e químicas diferentes. Considerando o interesse crescente das indústrias na busca de polissacarídeos que apresentem comportamento reológico compatível com sua aplicação, tanto nos setores de alimentos como farmacêutico, cosmético e têxtil, pesquisas vêm sendo realizadas com a esperança da descoberta de novas moléculas promissoras para tais aplicações. O objetivo deste trabalho foi determinar as características reológicas do exopolissacarídeo (botriosferana) produzido pelo Botryosphaeria rhodina MAMB-05 em três fontes de carbono: glucose (EPSGLU), sacarose (EPSSAC) e frutose (EPSFRU). A análise da composição monossacarídica, após hidrólise ácida, mostrou que todos os polímeros são constituídos exclusivamente por glucose e a cromatografia de filtração em gel indicou baixo grau de polidispersividade. Experimentos com o corante Congo Red evidenciaram a presença de tripla hélice nos três polímeros produzidos pelo fungo B. rhodina MAMB-05. Os três polissacarídeos comportaram-se como fluidos não-Newtoniano com características de soluções pseudoplásticas e tixotrópicas e com diminuição da viscosidade quando em presença de NaCl. As soluções aquosas de EPSGLU, EPSSAC e EPSFRU apresentaram comportamento viscoelástico, com características de gel forte em 5 e 25°C, não sofrendo mudanças no comportamento, mesmo em presença de NaCl, a 25°C. As soluções polissacarídeos mantiveram as características de géis fortes, com propriedades termoestáveis e termorresistentes, entre 5 a 95°C de temperatura. Esses resultados permitiram concluir que os EPS produzidos pelo fungo B. rhodina MAMB-05 possuem propriedades químicas e físicas, passíveis de utilização nas indústrias de alimentos, farmacêutica, cosmética e biomédica. / Abstract: Exopolysaccharides (EPS) are polymers produced by a great variety of microorganisms and can possess different physical and chemical properties, and structural features. Considering the growing interest in commercial applications of polysaccharides that show rheological compatible behavior within the application, as in the food, pharmaceutical, cosmetic and textile sectors, research have been developed in attempts to discover new promising biomolecules for such applications. The objective of the work presented was to determine the rheological characteristics of the exopolysaccharide botryosphaeran produced by Botryosphaeria rhodina MAMB-05 on three different sole carbon sources: glucose (EPSGLU), sucrose (EPSSUC) and fructose (EPSFRU). The analysis of monosaccharide composition, after acid hydrolysis, showed glucose as the only component and the gel permeation chromatography indicated that the EPSs have an excellent purity grade. All three biopolymers produced by B. rhodina MAMB-05 were found to exist in the triple helix conformational state. The three exopolysaccharides showed non-Newtonian behavior, with pseudoplastic and thixotropic characteristics, and showed a slight decrease in viscosity in presence of NaCl. The three biopolymer gels showed viscoelastic behavior, with characteristics of strong gelling at 5 ºC and 25 ºC, without suffering behavioral changes, even in the presence of NaCl at 25 ºC. The polysaccharide solutions kept the strong gel characteristics possessing thermo-stable and thermo-resistant properties when evaluating the influence of temperature (5 - 95 ºC) over the viscoelastic behavior of solutions. The results concluded that the EPS's produced by fungus B. rhodina MAMB-05 have physical and chemical properties amenable for use in the food, pharmaceutical, cosmetic and biomedical industries. / Orientador: Maria de Lourdes Corradi Custódio da Silva / Coorientador: Ana Lúcia Barretto Penna / Banca: Joana Lea Meira Silveira / Banca: Roberto da Silva / Mestre

O impacto causado pelo aumento da quantidade de substâncias químicas descartadas no meio ambiente está presente na maioria dos ecossistemas. Poluentes industriais contendo metais são frequentemente transportados para a água, o solo e o ar, podendo-se acumular nas cadeias tróficas e apresentar toxicidade para a biota. Em ambientes aquáticos, a biodisponibilidade e destino dessas substâncias xenobióticas podem ser influenciadas por vários fatores, entre eles a matéria orgânica dissolvida e outros compostos quelantes, os quais tem capacidade de aprisionar ou liberar íons para o ambiente. Os exopolissacarídeos, substâncias excretadas pelo fitoplâncton, podem interagir com diversas substâncias, interferindo na toxicidade dos compostos para os organismos ou comunidades biológicas aquáticas e, conseqüentemente, subestimando o verdadeiro valor tóxico das substâncias. Este trabalho foi conduzido para determinar a influência dos exopolissacarídeos da Clorofícea Pseudokirchniriella subcapitata, e da Cianofícea Anabaena spiroides na toxicidade dos elementos traço cádmio e cromo em Daphnia similis (Cladocera). Os metais foram escolhidos pela afinidade por quelantes orgânicos. Foram realizados testes ecotoxicológicos agudos para verificar a sensibilidade do cladócero Daphnia similis em diferentes concentrações dos metais cádmio (cloreto de cádmio) e cromo (dicromato de potássio), adicionando algas e exopolissacarídeos. Nos testes com os exopolissacarídeos foram utilizadas diferentes frações (excretado total, 10000D). Para obtenção das frações de exopolissacarídeos foram realizadas filtrações tangenciais em cartucho oco de celulose com bomba peristáltica. Nos testes com a adição das algas foram usados números conhecidos de células obtidas do concentrado de algas. Observou-se redução da toxicidade de 20 a 30% nos testes com a adição de excretado total, e reduções menores para as frações 10KD para as clorofícea e cianofícea. Com os resultados deste trabalho, foi possível avaliar a capacidade dessas substâncias em quelar e indisponibilizar compostos tóxicos e avaliar a toxicidade das substâncias quando testadas nos organismos planctônicos. / Chemical substances have been exerting increasing impact on ecosystems. Industrial pollutants containing metals frequently reach water bodies, soil, and air, wherein they may accumulate on the trophic chain, resulting in toxicity to the biota. In aquatic environments, the bioavailability and the destination of these xenobiotics are influence by several factors, such as the amount of solved organic matter and other chelating compounds, since these substances can either bind or liberate ions to the environment. Exopolysaccharides, in particular, are excreted by phytoplankton and, once in water, can interact with several substances altering the toxicity of compounds to aquatic organisms and biological communities. As a consequence, the real toxic potential of these xenobiotics is underestimated. The aim of this work was to determine the influence of the exopolysaccharides produced by the Chloroficeae Pseudokirchniriella subcapitata and by the Cyanophiceae Anabaena spiroides on the toxicity of the trace-elements cadmium and chromo over the primary consumer Daphnia similis. These metals were chosen due to their high affinity towards organic chelators. Tests for acute ecotoxicity were performed to verify the sensitivity of the Cladocera Daphnia similis exposed to different cadmium (cadmium chloride) and chromo (potassium dichromate) concentrations, with and without algae and/or exopolysaccharides. Three fractions of exopolysaccharides were tested (total excreted, < 1000D, and > 1000D), which were obtained by tangencial filtration through a cellulosis membrane using a peristaltical pump. On tests using algae, a fixed number of cells were obtained from an algae concentrate. The toxicity of Daphnia similis to the metals was 20-30% reduced when the total excreted from algae was added, while the reduction in toxicity was lower to the fraction than 10KD. The results demonstrated that exopolysaccharides chelate toxic compounds rendering them unavailable for exerting their effects on planktonic organisms.

Bakterielle Exopolysaccharide (EPS) tragen bei in situ-Bildung durch die Immobilisierung von Wasser maßgeblich zur Erhöhung der Viskosität fermentierter Milchprodukte bei. Die Wirkung ist grundsätzlich mit der kommerzieller Hydrokolloide auf pflanzlicher oder Algenbasis vergleichbar, wird jedoch auf Grund der komplexen Wechselwirkungen mit der Lebensmittelmatrix noch immer kontrovers diskutiert. Ziel der Arbeit war es, nach Kultivierung ausgewählter Milchsäurebakterien EPS in entsprechenden Mengen zu isolieren, um die makromolekularen Eigenschaften zu analysieren und in Beziehung zur chemischen Struktur und Funktionalität zu setzen. Zunächst konnte die in situ-EPS-Bildung durch Batch-Kultivierungen von Milchsäurebakterien im Bioreaktor derart gesteigert werden, dass eine anschließende Isolierung verschiedener EPS-Fraktionen mit einer Reinheit von bis zu 89% (Hetero-EPS) bzw. 99% (Dextrane) möglich wurde. Dies ermöglichte die erstmalige Beschreibung oder Bestätigung der chemischen Strukturen aller ausgewählten EPS. Der Verzweigungsgrad der Dextrane war über Temperatur und pH während der mikrobiellen Synthese steuerbar. Die Einzelschritte der Isolierung wurden außerdem so angepasst, dass makromolekulare Eigenschaften der EPS wie Molekülmasse oder intrinsische Viskosität durch die Isolierung nicht beeinflusst wurden. Die umfassende Untersuchung der makromolekularen Eigenschaften der EPS in wässriger Lösung bildete die Basis für die Erklärung ihrer phänomenologischen Eigenschaften und Funktionalität in fermentierten Produkten. Es zeigte sich, dass fadenziehende EPS höhere intrinsische Viskositäten als nichtfadenziehende EPS aufwiesen. Die intrinsische Viskosität war weiterhin vom Isolierungsverfahren und damit von der Isolatreinheit unabhängig. Durch die damit verbundenen Zeit- und Kosteneinsparungen während der Isolierung eröffnet dies die Möglichkeit, mikrobielle EPS ökonomisch sinnvoll einzusetzen. Durch Scherbehandlung wässriger EPS-Lösungen wurde, unabhängig vom Schersystem, ein linearer Zusammenhang zwischen Viskosität und Molekülmasse nachgewiesen und so das Potential zur gezielten Modifizierung von EPS aufgezeigt. Kinetische Untersuchungen mit ultraschallbehandelten EPS-Lösungen ermöglichten eine Bewertung der Scherempfindlichkeit, die im Einklang mit Untersuchungen zur thermischen und chemischen Belastung von EPS stand. Zur Beurteilung der Funktionalität wurden EPS-Isolate zu rekonstituierter Magermilch vor chemischer Säuerung mit Glucono-δ-lacton zugesetzt. Die resultierende Festigkeit der Modellmilchgele korrelierte mit der absoluten EPS-Konzentration und war somit unabhängig von der Isolatreinheit. Gescherte EPS besaßen eine geringere Molekülmasse und intrinsische Viskosität, was zu geringeren Gelfestigkeiten führte. Die in der Literatur bisher wenig untersuchten kapsulären, zellgebundenen EPS konnten mittels Rasterelektronenmikroskopie visualisiert und ihr Effekt auf die Eigenschaften der Zelloberfläche analysiert werden. Die gewonnenen Erkenntnisse zeigten, dass kapsuläre EPS die Hydrophobizität der Zelloberfläche verringern sowie die Wasserbindung und Gelfestigkeit bei Zusatz zu Modellmilchgelen im Vergleich zu Zellen ohne kapsuläre EPS erhöhen.

Die Verbraucherakzeptanz von fermentierten Milchprodukten wie Joghurt oder Frischkäse ist von ihrer Textur abhängig. Zur Einstellung der produktspezifischen Textur ohne Zusatz von spezifischen Milchproteinen zur Basismilch oder milchfremden Hydrokolloiden werden in der Milchindustrie spezielle Starterkulturen eingesetzt, die während der Fermentation Exopolysaccharide (EPS) bilden. Diese EPS werden in der Regel als freie EPS ins Serum abgegeben oder verbleiben als kapsuläre EPS an der Bakterienzelle. Freie EPS lassen sich weiter anhand ihres Effektes im Produkt in fadenziehend und nicht-fadenziehend unterteilen. Aufgrund der hohen Biodiversität der EPS sowie des komplexen Zusammenhanges zwischen EPS-Eigenschaften, Substratzusammensetzung und Verfahrensbedingungen existieren jedoch widersprüchliche Ergebnisse zu ihrer Funktionalität im Produkt. Ziel dieser Arbeit war es, die wichtigsten Einflussfaktoren durch Einsatz verschiedener EPS von Lactococcus lactis und Streptococcus thermophilus in definierten Modellsystemen für stichfeste, gerührte und konzentrierte fermentierte Milchprodukte zu identifizieren und die Textureigenschaften mit den strukturellen und makromolekularen Eigenschaften der EPS in Verbindung zu bringen. Neunundzwanzig Einzelstämme von verschiedenen Starterkulturherstellern wurden auf Basis von der Fadenlänge als Indikator für den Fadenzug und der EPS-Menge in sechs Cluster eingeteilt. Aus jedem Cluster wurden Vertreter zur Herstellung von Modellsystemen genutzt, die Textur und Synärese der Produkte instrumentell erfasst und statistisch bewertet, um Einflussfaktoren auf die Eigenschaften der Modellprodukte zu identifizieren. Stichfester Modelljoghurt mit fadenziehenden EPS wies eine höhere Steifigkeit auf als jener mit nicht-fadenziehenden EPS, welche nicht mit der EPS-Menge oder der Fermentationszeit korrelierte. Unabhängig von der Art der freien EPS war beim Vorhandensein von kapsulären EPS die Gelsteifigkeit zusätzlich erhöht. Für gerührten Modelljoghurt ergab eine Komponentenanalyse zur EPS-Menge, zur Säuerungskinetik und zu Produkteigenschaften, dass fadenziehende EPS texturrelevante Eigenschaften wie Partikelgröße und Scherviskosität signifikant beeinflussen. Dies wurde auf die höhere intrinsische Viskosität für fadenziehende EPS im Vergleich zu nicht-fadenziehenden EPS zurückgeführt. Experimente mit einem Dehnrheometer ergaben, dass die Dehnviskosität stärker als die Scherviskosität mit der Fadenlänge zunahm. Ein Grund dafür sind intensivere Wechselwirkungen von fadenziehenden EPS mit Proteinen oder untereinander, was anhand höherer Relaxationszeiten in Dehnung bei höherer Fadenlänge geschlussfolgert wurde. Fadenziehende EPS erhöhten zudem die Fließgrenze von Modellfrischkäse, was hauptsächlich auf EPS-Wechselwirkungen und nicht auf die EPS-Konzentration oder Partikelgröße zurückgeführt wurde. Eine niedrigere Synärese und höhere Steifigkeit wurde entweder beim Vorhandensein von kapsulären EPS oder freien EPS beobachtet, die in Experimenten mit dynamischer Wasserdampfsorption eine höhere Feuchtebeladung aufwiesen. Fadenziehende EPS und kapsuläre EPS besaßen auch im Doppelrahmfrischkäse nach moderater Bruchhomogenisierung (0,05 MPa oder 15 MPa) eine hohe Funktionalität, was mit der höheren intrinsischen Viskosität dieser EPS korrelierte. Die Bruchhomogenisierung bei einem Druck von 30 MPa führte jedoch zu einer niedrigeren Funktionalität aufgrund der scherinduzierten Reduzierung der molekularen Masse der EPS und Umlagerungen in der Mikrostruktur der Käse. Die Ergebnisse zeigen, dass die Technofunktionalität nicht von der Konzentration der in situ produzierten EPS abhängt. Schlüsselfaktoren sind insbesondere die EPS-Lokalisierung (frei oder kapsuläre), intensivere Wechselwirkungen der fadenziehenden EPS mit Proteinen oder untereinander und ein hohes Wasserbindungsvermögen der EPS. Jedoch sollte der Energieeintrag bei der postfermentativen Verarbeitung so niedrig wie möglich sein, um die Funktionalität der fadenziehenden EPS und kapsulären EPS weitestgehend zu erhalten. Dieses Wissen ist für die Auswahl von EPS-bildenden Starterkulturen für den industriellen Maßstab entscheidend, um die gewünschten Produkteigenschaften zu erhalten.

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Les bifidobactéries sont des bactéries probiotiques exerçant différents effets bénéfiques sur la santé de l’hôte. Parmi les effets revendiqués, la modulation des différentes fonctions immunitaires demeure l’un des effets les plus intéressants. Plusieurs travaux sont rapportés dans la littérature sur les effets immunomodulateurs de certaines souches probiotiques. Cependant, les résultats sont parfois controversés et les mécanismes impliqués dans cet effet immunomodulateur sont encore très peu élucidés. Le but de ce projet est d’évaluer le potentiel immunomodulateur de certaines souches de bifidobactéries isolées à partir de fèces de bébé, et d’identifier les mécanismes moléculaires par lesquels ces bifidobactéries exercent leurs effets sur les différentes fonctions immunitaires. Trois souches de bifidobactéries d’origine fécale productrices d’exopolysaccharides (B. thermoacidophilum RBL81, RBL82 et RBL64), une souche de Bifidobacterium lactis Bb12 et des souches ATCC (B. animalis, B. breve, B. longum, B. infantis, B. bifidum et B. pseudolongum) ont été utilisées. Les résultats obtenus montrent que la paroi cellulaire induit la plus forte stimulation de la prolifération cellulaire (splénocytes), accompagnée d’une forte production de IFN-γ et d’une augmentation de la sécrétion de IL-10. Une stimulation des fonctions immunitaires a également été observée avec le contenu cytoplasmique mais à un niveau moindre par rapport à la paroi. Par contre, les exopolysaccharides bruts ou fractionnés n’ont induit aucun effet. Une étude plus approfondie utilisant B. lactis Bb12 comme modèle a démontré que l’activité immunomodulatrice du contenu cytoplasmique est associée à la présence de peptides et/ou protéines acides, de poids moléculaire très variable et de faible hydrophobicité. Par ailleurs, le pouvoir immunogène de la paroi de bifidobactéries a été mise à profit pour produire des anticorps monoclonaux spécifiques capables de détecter les espèces du genre Bifidobacterium. Ces anticorps semblent être spécifiques à une protéine majeure d’environ 58 kDa de poids moléculaire (PM) commune à plusieurs bifidobactéries. Une fois purifiés, ces anticorps ont été utilisés pour développer un test d’immuno-culture original permettant la détection spécifique et sensible des bifidobactéries. / Bifidubaktiri d iprubyuteken (probiotiques) i yetwassnen atas assagi, yeεni ţ-ţibaktiryin mara twarnunt i tgulliyin ţţakent ayen yelhan i tdawsa. Tibaktiriyin agi zemrent ad snernint kra n tsuγnin yeţhuddun γef tfekka mgal atanan n uεebbud d izerman n bnadem. D acu kan, ar assa, ur nessin ara amek tibaktiriyin stanent tafekka. Iswi n usenfar agi d anelmud n unezmar n usnerni n ustan n bifidubaktiri. Ayagi i wakken a d-naf isemduyen n kra n tsegrin n tsiluliyin (cellules) am iferdisen (éléments) yellan daxel, s-ufella neγ i d-deggirent ar berra γef tririt n uhuddu. Krad (3) n ccetlat n bifidubaktiri id-yekkan seg izerman llufan ţwalmendent ţwasdemrent ar yiwet n ccetla tamselγut Bifidobacterium lactis Bb12. Agmuden i d-nessufeγ qqaren-d d akken asnerni yelhan n ufadi n tsiluliyin n udihan (rate) n amumad, i d- yettabaε unerni amuqran n IFN-γ yakw d usennerni n usufeγ (sécrétion) n IL-10, yefkaten-id ulesi n tsilulit (paroi cellulaire). Ayen yellan daxel n tsilulit d tazunin-ines (ipeptidiyen yak tiprutiniyin) snernayent drus γef ulesi. Ma d ayen yεenan EPS (exopolysaccharides) ulac asnnerni i d-yekkan segsen. Ma d tiprutiniyin n ulesi n bifidubakiri (B. animalis, B. breve, B. longum, B. infantis, B. bifidum et B. pseudolongum) banent-ed d timesnerniyin timuqranin n uhareb γef tfekka n umumad (Balb/c) ssutuyent afares amuqran n tfekkamgalin. Tifekkamgalin i d-yeffγen d yiwet n txellalt (IgG) mgal B. longum ţwafursent-ed s trennawet u sknent-ed tasedmirt tanmidagt (réaction croisée) yakw d ccetlat nniden n bifidubakiri. Anecta yekka-d seg yiwet n teprutint (protéine) tamsihart i yezzayen 58 kd. Tulmist n tfekkamgalin i d-yefursen yeţwasentem-ed s usadez (test) ibaw i d- yelummzen mi ţ-nerεed yakkw d cctali nniden n tbaktiriyin. Tufin n bifidubaktiri yeddren s tfekkamgalin i d-yedran yefursen yedra-d s usadez n immuno-culture. Tifekkamgalin tulmisin i-d yekkan seg yiwet n txellayt id-nessenfel zemrent ad ţwaqedcent i tifin n cctali n bfidobktiri di tgwellyin. / Probiotic bifidobacteria are known to have beneficial effects on host health. One of the interesting properties of these bacteria is their capacity to modulate the host immune function. However, the mechanisms by which the bifidobacteria influence the immune response are not well known. This project aimed to evaluate the immunodulatory potential of some bifidobacterial strains isolated from newborn feces. We tried to determine the cellular and molecular mechanisms involved in immune response induced by cytoplasmic content, cell wall and exopolysaccharides from bifidobacteria. Three exopolysaccharide-producing fecal strains of bifidobacteria (B. thermoacidophilum RBL81, RBL82, and RBL64) and a commercial available strain (Bifidobacterium lactis Bb12) were tested using mouse splenocytes. The results demonstrate that a high stimulation of cell proliferation and interferon-gamma (IFN-γ) production were induced by cell wall. In addition, a concomitant stimulation of interleukin-10 (IL-10) secretion was observed. The cytoplasmic content was also shown to be immunostimulating, but less than cell wall. However, the effects observed were dose or strain-species-dependent. B. lactis Bb12 was found to be significantly more immunostimulating than other bifidobacterial strains used. Partially fractionated peptides and acidic fraction from B. lactis Bb12 showed a low hydrophobicity and appeared heat stable and mitogenic. In contrast, no immunostimulating effects were induced by exopolysaccharides. The mitogenic properties of cell wall protein were then explored to develop specific monoclonal antibodies (Mab) able to detect bifidobacteria species in food. Common proteins were revealed in cell wall extracts from bifidobacteria (B. animalis, B. breve, B. longum, B. infantis, B. bifidum et B. pseudolongum). The proteins obtained were found to be immunonogenic in Balb/c mouse. Monoclonal antibodies (IgG) -anti-B. longum- produced cross-reacted with all bifidobacteria tested. The shared antigenicity shown by bifidobacteria was revealed by an epitope supported by a common protein of 58 kDa. This was confirmed by immune-transmission electron microscopy observation, which showed the specific interaction of these antibodies with bifidobacterial cell wall proteins. The Mab produced was also shown to be sensitive (105 cfu/ml) and specific to members of the bifidobacterial genus. The Mab developed allowed detection of viable cells of bifidobacteria using immuno-culture test.

Os organismos extremófilos são considerados atualmente reservatórios de novas biomoléculas de interesse biotecnológico. Dentro deste grupo encontram-se os microrganismos que requerem altas concentrações de sal para crescer, denominados halófilos. Também existem os halotolerantes que são aqueles microrganismos que não precisam de sal para proliferar, mas toleram altas concentrações de NaCl. Os ambientes salinos provaram ser uma fonte rica de microrganismos halotolerantes produtores de novos compostos naturais e, portanto, a pesquisa nestes ambientes torna-se de grande importância. No Brasil, na região salina de Areia Branca no bioma caatinga, foram isoladas bactérias halotolerantes que foram estudadas com o objetivo de avaliar a produção de novos biopolímeros de interesse biotecnológico. Acredita-se que os polímeros naturais desses microrganismos extremos podem ter aplicações inovadoras ou características diferentes às tradicionais. Neste estudo, os isolados foram identificados em nível de gênero com base na análise da sequência do gene 16s rRNA. Os isolados foram principalmente bactérias Gram-positivas atribuídas às famílias Bacillaceae, Staphylococcaceae, Microbacteriaceae e uma bactéria Incertae Sedis do filo firmicutes, afiliadas aos gêneros Bacillus, Staphylococcus, Curtobacterium e Exiguobacterium, respectivamente. Apenas um isolado Gram-negativo foi identificado e atribuído como membro da família Pseudomonadaceae, incluso no gênero Pseudomonas. Avaliou-se a tolerância ao sal dos isolados em meio TSB suplementado com 5, 35, 60 e 120 g/L de NaCl. Todos os isolados apresentaram a capacidade de crescer nas quatro concentrações de NaCl avaliadas, com exceção do isolado Exiguobacterium sp. sac36 que não cresceu na concentração de 120 g/L de NaCl no meio. Realizaram-se ensaios de acúmulo de polihidroxialcanoatos (PHA) e evidenciou-se que quatro isolados do gênero Bacillus são capazes de acumular 3-hidroxibutirato (3HB) a partir de glicose, xilose, e alguns destes em glicerol. Adicionalmente, confirmou-se que quando há altas concentrações de NaCl no meio, o acúmulo de 3HB dos isolados produtores diminui. Observou-se também que doze isolados halotolerantes são produtores de exopolissacarídeos (EPS). Testes realizados indicaram que os mesmos podem ter efeitos imunoestimulantes em macrófagos. Finalmente, avaliou-se a produção de ácido hialurônico (AH) pelos isolados halotolerantes. Segundo sugere o método de Alcian blue, todos os isolados foram capazes de produzir AH, mostrando que a maior parte deles acumulou o biopolímero em concentrações maiores ou semelhantes ao controle. Os resultados obtidos evidenciam que os isolados halotolerantes avaliados são uma fonte rica em compostos com atividades promissoras para as diferentes indústrias. O presente trabalho contribui no estudo do potencial biotecnológico de microrganismos isolados no bioma caatinga, destacando sua biodiversidade, versatilidade e a necessidade de continuar explorando esses ambientes extremos pouco estudados. / Extremophile organisms are considered reservoirs of new biomolecules of biotechnological interest. In this group there are microorganisms that require high salt concentration to grow, called halophiles, and halotolerant microorganisms, that do not need salt to proliferate but can tolerate high concentrations of NaCl. Saline environments proved to be a rich source of new natural compounds by halotolerant producers and therefore, research in these environments becomes of great importance. In Brazil, in the saline region of Areia Branca in the caatinga biome, halotolerant bacteria were isolated and studied in order to evaluate the production of new biopolymers of biotechnological interest. It is believed that the natural polymers of those extreme microorganisms could have innovative applications or different characteristics from the traditional biopolymers. In this study, the isolates were identified at the genus level based on 16S rRNA gene sequence analysis. Isolates were mainly Gram-positive bacteria from Bacillaceae, Staphylococcaceae and Microbacteriaceae families, and Bacillus, Exiguobacterium, Staphylococcus and Curtobacterium genera. One of the Gram-negative isolate was identified as member of the Pseudomonadaceae family, genus Pseudomonas. The evaluation of salt tolerance of the bacterial isolates on TSB medium supplemented with 5, 35, 60 and 120 g / L NaCl was performed. All the isolates showed the ability to grow in the four concentrations evaluated, except for Exiguobacterium sp. sac36, that did not grow at 120 g / L NaCl. Polyhydroxyalkanoate (PHA) accumulation assays were performed using glucose, xylose and glycerol as carbon source. The results showed that four strains of the genus Bacillus were able to accumulate 3-hydroxybutyrate (3HB) in all conditions. Additionally, it was confirmed that the presence of high concentrations of NaCl in the medium causes a decrease in 3HB accumulation in the cells. It was observed that twelve halotolerant bacteria produced exopolysaccharides (EPS). Tests performed indicated that those EPS could have immunostimulatory effects on macrophages. Finally, hyaluronic acid (HA) production was evaluated. According to Alcian blue method, all strains were able to produce HA, showing that most of the isolates accumulated the biopolymer in higher or similar concentrations to the control. The results showed that the halotolerant isolates are a rich source of compounds with promising activities for different industries. This study contributes to the knowledge of microorganisms from the caatinga biome and their biotechnological potential, highlighting their biodiversity, versatility and the need to continue exploring these poorly studied extreme environments.