As exigências dos consumidores por produtos de qualidade são cada vez maiores e a indústria avícola está tendo que se adequar a estas mudanças. Um exemplo é a proibição do uso de antibióticos na alimentação animal devido a possível indução de resistência bacteriana. Contudo, a busca de alternativas a estes produtos está sendo alvo de muitas pesquisas e os probióticos se destacam com promessas de equilíbrio da microbiota intestinal e diminuição do estresse imunológico, impedindo a mobilização de nutrientes para atividades que não estejam relacionadas com a produção, promovendo desta forma, uma melhor resposta do sistema imune e conseqüente melhora no desempenho zootécnico. Devido à escassez de estudos relacionando o efeito dos probióticos com a imunidade das aves, o presente estudo foi realizado com o objetivo de verificar a influência de um tipo de probiótico em alguns parâmetros da resposta imune e hematológicos associados ao desempenho de frangos de corte. Foram utilizados 1200 pintos de corte, criados até 42 dias de idade, em um delineamento inteiramente casualizado, com 3 tratamentos (antibiótico, probiótico e controle) e 10 repetições. Considerando-se o período total de criação e nas condições em que o experimento foi conduzido, não foi possível mostrar a influência dos aditivos testados nos parâmetros zooténicos avaliados. Também não foi observada influência na resposta imune de macrófagos, linfócitos T e alteração de peso de órgãos linfóides nas idades avaliadas e com os métodos utilizados. Entretanto, foi possível observar resposta positiva do probiótico em relação ao antibiótico no que diz respeito à produção de anticorpos em resposta à vacina de Newcastle, porém, sem diferir do controle. Para os parâmetros hematológicos, apesar das diferenças encontradas entre os tratamentos, os valores hematológicos do presente estudo encontraram-se dentro da normalidade quando comparados a outros estudos / The demand from consumers for good-quality products has been increasing and the poultry industry has to adjust to it. An example that shows this is the prohibition of the use of antibiotic in animal feeding, given the fact it could induce bacterial resistance. Therefore, the search for alternatives to these products has been the major goal of many researches and the probiotics stand out with promises to balance the intestinal microbial and to reduce immunologic stress, hindering the mobilization of nutrients for activities that are not related with the production, promoting, this way, a better response from the immune system and a consequential improvement in its performance. Due to the lack of studies that relate the effect of the probiotics with the poultry immunity, the present study has been carried through with the objective of verifying the influence of a type of probiotic in some parameters of the immune and hematology response associated to the performance of broilers. 1200 broilers were used, bred up for 42 days, in a completely randomized design with 3 types of treatments (antibiotic, probiotic and control) and 10 repetitions. Considering the total period of creation and the conditions in which the experiment was led, it was not possible to show the influence of additives tested in the evaluated parameters of performance. It was not observed the influence on the immunity of macrophages, T lymphocytes and alteration of weight of lymphoid organs in the evaluated ages and with the used methods either. However, it was possible to observe a positive response from the probiotic - comparing it to the antibiotic - regarding the production of antibodies in response to the vaccine of Newcastle, still, without differing from the control. For the hematological parameters, despite the differences found among the treatments, the hematological values of the present study are found to be within normality when compared to other studies

Probiotic bacteria are live microorganisms, which manifest health benefits in humans. The goal of this work was to characterize rheological properties of probiotic bacteria (Lactobacillus reuteri) formulation in which maltodextrin and sucrose used as excipients. To fulfil the goal, first, thermal, structural and rheological properties of maltodextrin, sucrose solutions and probiotic bacteria slurry were investigated. Probiotic bacteria formulations were prepared by adding probiotic bacteria slurry to maltodextrin and sucrose solutions at different mass fractions. Finally, rheological properties of probiotic bacteria formulations were evaluated. From TGA, the water content of PB slurry including intracellular water found 81%. In DLS, three different types of aggregations of maltodextrin were observed and characteristic size of Probiotic bacteria found 1μm. The optical microscopy results indicate that at the liquid - air interface and in dehydrated state the bacteria are birefringent and arranged in an ordered fashion resembling a nematic phase. Most of the MD, SU and MD+SU solutions show Newtonian behaviour. MD and SU solutions show strong increase of viscosity with increasing concentration. This dependence can be described by using the Spurlin–Martin–Tennent’s model. The viscosities of MD+SU solutions increase with increasing proportion of maltodextrin. The oscillation data of MD and MD+SU solutions can be described by Maxwell model. The viscosities of MD, SU, MD+SU mixed solutions decrease with increasing temperature. This temperature dependency can be described by Arrhenius model of viscosity. At very high concentrations of MD, a deviation from this behaviour is observed. The probiotic bacteria slurry shows shear thinning behaviour at low shear stress and Newtonian behaviour at higher stresses. All probiotic formulations in which probiotic bacteria dispersed in maltodextrin and sucrose solutions show Newtonian behaviour. The viscosities of maltodextrin solutions and MD+SU mixed solutions decrease by addition of probiotic bacteria whereas mixed effects of probiotic bacteria addition on the viscosity of sucrose solutions were observed. The viscosity of probiotic bacteria slurry decreases with increasing temperature, although deviations from this behavior are seen at certain conditions.

The goal of this work was a study the influence of food and beverages on the viability and growth of probiotic bacteria. The influence of food and beverages was tested on monocultures of Lactobacillus acidophilus, Bifidobacterium breve and mixed culture of probiotic microorganisms. In the experimental part, probiotic cultures were incubated in selected foods and beverages. After that they were tested in a model conditions of digestive tract. In some probiotic cultures, growth of viable cells during incubation in the digestive tract was observed. The increase of probiotic cells was showed predominantly in foods that contained higher levels of sugars and fats or a suitable combination. Their increase was up to four times in some cases. Based on the results, mixed probiotic cultures aren’t surprisingly exhibited better survival and maintain sufficient amount of viable cells even during the digestive process. Moreover, probiotic microorganisms could be recommended to consumption during meals better than just with a beverage.

Plus de 50 ans après son approbation par les agences réglementaires, le cyclophosphamide (CTX) reste une drogue aux propriétés variées et aux effets pléiotropiques couramment utilisée en clinique. Cet agent cytotoxique, administré en cancérologie, possède des propriétés immuno-modulatrices et stimule les réponses immunitaires anti-tumorales. A doses métronomiques, le CTX induit notamment une polarisation des splénocytes CD4+ vers un profil Th1 et Th17, caractérisés par la sécrétion d’IFNet d’IL-17, nécessaire à l’activité tumoricide du CTX. Comme tout agent cytotoxique, le CTX cible les cellules en prolifération, qu’elles soient normales ou cancéreuses. Le CTX compromet ainsi l’intégrité de la barrière intestinale et l’homéostasie du tractus digestif. Nous avons démontré que l’individu sous CTX a une fragilisation de la barrière intestinale qui permet la rupture de la tolérance de celui-ci à sa flore commensale et son immunisation contre certaines espèces bactériennes. L’immunisation anti-bactérienne est composée de lymphocytes effecteurs CD4+, appelés « Th17 pathogéniques » et producteurs d’IL-17 et d’IFN, qui aident les lymphocytes anti-tumoraux à endiguer la croissance de tumeurs chez la souris. Nous avons mis en évidence que la stérilisation des animaux avec des antibiotiques à large spectre ou ciblant certaines populations bactériennes comme la vancomycine (ciblant les Gram+) et la colistine (ciblant les Gram-), abrogent l’efficacité anti-tumorale du CTX. Par ailleurs, nous avons identifié deux bactéries, une bactérie Gram+ Enterococcus hirae, capable de restaurer l’efficacité de cette chimiothérapie en induisant la polarisation de réponses Th1 et pTh17 stimulant la mise en place de réponses lymphocytaires T CD4 et T CD8 dirigées contre des antigènes tumoraux et une bactérie Gram- Barnesiella intestinihominis, impliquée dans la mise en place de réponses mémoires induites par la combinaison CTX+vaccin. Ces travaux démontrent ainsi l’importance de la flore intestinale dans la réponse à la chimiothérapie par CTX. / More than 50 years after its approval by the Food and Drug Administration, cyclophosphamide (CTX) remains a drug with miscellaneous properties currently used in anti-cancer chemotherapy. This cytotoxic agent has immuno-modulatory properties and stimulate anti-tumoral immune responses. At metronomic doses, CTX induces the polarisation of splenocytes toward a Th1 and Th17 profile, characterized by the secretion of IFN et IL-17, both mandatory for the tumoricidal activity of this drug. CTX, as cytotoxic agent, targets proliferating cells, either normal or tumoral. Indeed, CTX is responsible for disrupting the gut barrier integrity as well as intestinal homeostasis. We have shown that people treated with CTX have a weaker intestinal barrier which breaks the tolerance toward the intestinal microbiota and leads to its immunization against some bacterial strains. This immunization is composed of CD4+ effector lymphocytes called « pathogenic Th17 » producing IFN and IL-17, which helps tumor-infiltrating lymphocytes to control the tumor growth in mice. Broad spectrum antibiotics as well as vancomycin (which mainly kills Gram positive bacteria) and colistin (which mainly eliminates Gram negative bacteria) all compromised the full-blown anticancer activity of CTX in vivo. Moreover, we have identified two bacteria, Enterococcus hirae and Barnesiella intestinihominis, able to rescue the efficacy of CTX abolished with antibiotics. E. hirae, a Gram+ bacterium, elicits Th1 immune responses and pathogenic Th17 cells capable of enhancing tumor-specific CD4+ and CD8+ T cell responses against candidate tumor antigens associated with tumor control. B. intestinihominis, a Gram- bacterium, was able to rescue the long term cognate responses lost with broad spectrum antibiotics or colistin treatment. Our data underscore the role of the gut microbiota in the efficacy of chemotherapy by CTX.

Puberty is a critical period of development characterized by rapid physiological changes and significant brain reorganizing and remodeling. These rapid changes render the developing brain particularly vulnerable to stress and immune challenge. In mice, exposure to an immune challenge (lipopolysaccharide; LPS) during puberty causes enduring effects on stress reactivity, cognitive functioning, and depression- and anxiety-like behaviors later in life. However, the mechanisms underlying these effects are unknown. The gut microbiome can profoundly influence the immune system. There is also close bidirectional communication between the gut microbiome and the central nervous system (CNS) through neural, endocrine and immune signaling pathways, which can alter brain chemistry and emotional behaviour. Thus, we hypothesized that altering microbial composition during puberty could mitigate acute immune responses and prevent enduring outcomes later in life. The current thesis examined the effect of gut manipulation with probiotics during puberty on LPS-induced immune responses and enduring anxiety- and depression-like behaviours, and stress-reactivity in adulthood, in male and female CD1 mice (Article 1). Next, we examined age and sex differences in gut microbial composition before and after exposure to an immune challenge. We also examined the effects of consuming a single strain probiotic bacterium (Lactobacillus Reuteri) during puberty on the immune response and the long-term changes in memory, anxiety-like behavior, and stress reactivity in adulthood (Article 2). Lastly, we examined how microbial colonization between pubertal and adult mice can alter acute peripheral and central inflammatory responses to LPS (Article 3). The current dissertation has addressed sex-specific vulnerabilities to an immune challenge during pubertal development and the moderating influence of the gut microbiome. These studies have demonstrated that manipulating the gut microbiome during puberty can mitigate acute immune responses and prevent enduring neurobehavioural outcomes later in life.

Abstract available in the Current Developments in Nutrition.

Aus E. coli bestehende probiotische Produkte wie Mutaflor (Ardeypharm, Herdecke) und Symbioflor 2 (SymbioPharm, Herborn) werden seit Jahrzehnten erfolgreich für die Behandlung gastroenterologischer Erkrankungen verwendet. Die Probiotika gelten aufgrund der langjährigen Erfahrung als sicher. Seit ca. 20 Jahren werden zunehmend Studien ins Leben gerufen, welche sowohl die Wirkung der Produkte klinisch bestätigen als auch die bisher unbekannten Wirkmechanismen aufklären sollen. Das in Mutaflor enthaltene Bakterium E. coli Nissle 1917 wurde bereits erfolgversprechend in klinischen Studien zur Remissionserhaltung bei Colitis ulcerosa getestet und wird seither als therapeutische Alternative zur Standardmedikation eingesetzt. Auch die Wirkung von Symbioflor 2 bei Erwachsenen und Kindern mit Reizdarmsyndrom konnte in ersten klinischen Studien belegt werden. Es gibt bereits zahlreiche Forschungsarbeiten mit E. coli Nissle 1917, die sich mit der molekularen Charakterisierung des Stamms befassen. Auch das Genom des Stamms wurde sequenziert. Dennoch fehlen schlüssige Argumente, welche Gene, Genprodukte und molekularen Mechanismen den probiotischen Effekt von EcN bewirken. Im Rahmen dieser Arbeit wurde nun das Produkt Symbioflor 2 näher untersucht. Es besteht aus den sechs E. coli-Genomotypen G1/2, G3/10, G4/9, G5, G6/7 und G8, die ursprünglich aus dem Habitat eines Spenders isoliert wurden. Alle sechs Genome inklusive der insgesamt zwölf natürlich enthaltenen Plasmide wurden sequenziert, annotiert und manuell nachbearbeitet. Die sechs E. coli-Genomotypen repräsentieren zusammen das im Produkt Symbioflor 2 enthaltene Pangenom. Somit konnten genomisch kodierte Virulenz- und Fitnessfaktoren analysiert werden. Ein Vergleich mit einer Vielzahl anderer bisher sequenzierter E. coli ermöglichte eine Einordnung der Symbioflor 2 E. coli in das Cluster der apathogenen E. coli. Unter Verwendung eines in vitro Testsystems mit humanen intestinalen Epithelzellen konnte gezeigt werden, dass die probiotischen Stämme E. coli Nissle 1917 und E. coli G3/10 im Gegensatz zu Kontrollstämmen die Adhärenz enteropathogener E. coli signifikant hemmen. In weiteren Versuchen konnten dann kleine ribosomal synthetisierte und antibakteriell wirksame Moleküle, in EcN die Mikrozine M und H47, für diesen Effekt verantwortlich gemacht werden. In der Folge wurde auch in E. coli G3/10 ein neues, bisher unbeschriebenes Mikrozin detektiert, welches Mikrozin S genannt wird. Zudem konnten vier Gene auf dem Plasmid pSYM1 lokalisiert werden, die unterschiedliche Funktionen bei der Produktion von Mikrozin S haben. Zwei der Gene kodieren am Transport beteiligte Proteine. Ein kleiner Leserahmen konnte als das Mikrozin S-kodierende Gen mcsS identifiziert werden. Ein weiteres Gen vermittelt eine Immunität gegenüber Mikrozin S. Seine Expression in einem zuvor sensitiven Stamm macht diesen resistent gegenüber der Wirkung von Mikrozin S. Erst im September 2011 erfolgte ein erster Eintrag in die NCBI-Datenbank, in dem die Gensequenz von mcsS plasmidkodiert in einer Shigelle annotiert als hypothetisches Protein aufgeführt ist. Dem Gen wurde keine Funktion zugewiesen. Wird die Expression von mcsS in dem E. coli-Laborstamm MDS42 in Abwesenheit eines Immunitätsproteins induziert, wirkt das Peptid toxisch auf die bakteriellen Zellen. Mikrozin S kann zudem anhand seiner Aminosäuresequenz und der genetischen Organisation in die Mikrozine-Klasse IIa eingeordnet werden. Mikrozine können vielfältig verwendet werden, haben jedoch im Vergleich zu Bakteriozinen Gram-positiver Bakterien bisher zu wenig Beachtung gefunden. Anwendungsmöglichkeiten liegen in der Lebensmittelindustrie, der Human- und Veterinärmedizin, wo Mikrozin S nah verwandte Gram-negative Bakterien im Wachstum hemmen bzw. abtöten könnte. Zum Beispiel wird im Rahmen dieser Arbeit die Wirkung von E. coli Nissle 1917 und E. coli G3/10 auf enterohämorrhagische E. coli in vitro getestet. Es wird gezeigt, dass das von E. coli G3/10 gebildete Mikrozin S eine Adhärenzminderung aller verwendeten EHEC-Stämme an humane intestinale Epithelzellen vermittelt. Da EcN nur einen der vier getesteten EHEC-Stämme inhibiert, wurden Untersuchungen begonnen, die die Ursache dafür thematisieren. Die in dieser Arbeit generierten Ergebnisse bilden die Grundlage für weitere Studien zu den in dem Produkt Symbioflor 2 enthaltenen E. coli. Zudem können umfangreiche Analysen von Mikrozin S, wie die Reinigung des Proteins, seine Produktion in großem Maßstab und die Testung von Anwendungsmöglichkeiten fortgeführt werden.

Propionibacterium freudenreichii est une bactérie Gram-positive bénéfique, traditionnellement utilisée comme levain d’affinage fromager, qui bénéficie du statut GRAS (Generally Recognized As Safe). P. freudenreichii a révélé un effet immunomodulateur qui a été confirmé in vivo par la capacité à protéger des souris d’une colite aigüe induite. L’effet anti-inflammatoire est cependant hautement souche-dépendant. Il est dû, au moins en partie, à des composés de surface clés qui favorisent ces effets probiotiques. Les bactéries Gram-positives, y compris P. freudenreichii, peuvent être recouvertes d’une couche extérieure protéique, appelée « surface-layer », paracristalline, et formée par l’autoassemblage de protéines dites de S-layer (Slps). Les Slps, dans différentes bactéries, sont impliquées dans plusieurs caractéristiques probiotiques, telles que l’adhésion aux cellules de l’hôte et au mucus, la persistance dans l’intestin, ou encore l’immunomodulation. Le but de cette étude est d’étudil’immunomodulation. Le but de cette étude est d’étudier, chez une souche probiotique de P. freudenreichii, la protéine de surface qui joue le principal rôle dans les interactions probiotiques avec l’hôte. La souche P. freudenreichii CIRM-BIA 129, récemment reconnue comme immunomodulatrice prometteuse, possède plusieurs protéines de surface Slps, y compris SlpB. Dans la présente étude, l’inactivation du gène correspondant, dans la souche mutante CB129¿slpB, a provoqué une baisse drastique de l’adhésion aux cellules intestinales épithéliales HT-29, confirmant le rôle clé des Slps dans l’adhési / Propionibacterium freudenreichii is a beneficial Gram-positive bacterium, traditionally used as a cheese ripening starter, with the GRAS status (Generally Recognized As Safe). P. freudenreichii has revealed an immunomodulatory effect confirmed in vivo by the ability to protect mice from induced acute colitis. The anti-inflammatory effect is however highly strain-dependent and due, at least in part, to key surface compounds favouring probiotic effects. Gram-positive bacteria, including P. freudenreichii, can be covered with an external proteinaceous layer called a surface-layer paracrystalin layer and formed by the self-assembly of surface-layer-proteins (Slps). Slps were shown, in different bacteria, to be involved in several probiotics traits, such as adhesion to host cells and mucus, persistence within the gut, or immunomodulation. The aim of this study is to investigate, in a P. freudenreichii probiotic strain, the surface protein that plays the main role in the probioticinteraction with the host. The P. freudenreichii CIRM-BIA 129 strain recently revealed promising immunomodulatory properties and possesses several Slps, including SlpB. In the presented work, inactivation of the corresponding gene, CB129¿slpBa mutant strain, caused a drastic decrease in adhesion to intestinal epithelial HT-29 cells, further evidencing the key role of Slps in cell adhesion. we investigated immune response of HT-29 cells towards P. freudenreichii CIRM-BIA 129 and CB129¿slpB. The wild type strain mainly induced expression of the immunomodulatory IL-10 by the cells. Interestingly, th

The discovery of antibiotics have revolutionized modern medicine, facilitating the treatment of a variety of bacterial infections, enabled surgeries otherwise impossible to perform and increased life expectancy in all countries. However, the rapid development of resistance among microorganisms and the increasing numbers of clinical outbreaks caused by multiresistant bacteria have accelerated the need for new alternatives to antibiotics. Probiotic bacteria armed with defense systems have been studied as potential substitutes of antibiotics. These probiotic competitors can still contribute to the spread of resistance genes among microorganisms through horizontal gene transfer. The aim of this study was to investigate whether constructed CRISPR-Cas9 systems have the potential to protect probiotic bacteria against horizontal transfer of antibiotic resistance genes. Transformation, transduction and conjugation assays in strains carrying or not carrying a plasmid-bourne CRISPR-Cas9 system were performed in order to compare the frequencies of transfer of the most common resistance genes. The transformation and transduction assays demonstrated that the constructed CRISPR-Cas9 system entails a decrease in efficiency of transfer for targeted resistance genes. Moreover, it can be concluded that potentially increasing Cas9 levels by reducing its degradation results in increased prevention of horizontal gene transfer through transformation and transduction. Finally, we state that the CRISPRCas9 system does not result in protection against antibiotic resistance genes entering the cells through conjugation.

Ice cream mix (12% fat, 11% milk solids nonfat, 12.5% sugar, and 4.5% corn syrup solids) was fermented with Lactobacillus acidophilus and Bifidobacterium bifidum. Half of the mix was heat treated at 82°C for 30 minutes and cooled to 40-41°C. The other half was warmed to 40-41°C and inoculated with the starter cultures. Both were made into ice cream and stored at -29°C. Survival of L. acidophilus and B. bifidum and of β-galactosidase activity were monitored during 17 weeks of frozen storage. Reinforced clostridial medium was used to enumerate culture bacteria. Colony counts, after fermentation, for both L. acidophilus and B. bifidum were about 5 x 108. The population of cultures decreased less than one log cycle after initial freezing. After 17 weeks storage the bacterial counts were 1 x 107 for B. bifidum and were 4 x 106 for L. acidophilus. During the same period, β-galactosidase activity decreased only 31%. Therefore, frozen fermented dairy products provide a good vehicle to supply β-galactosidase enzymes to people who are lactose maldigestors. Frozen fermented ice cream was prepared at four different pH's (5.0, 5.5, 6.0, 6.5) by blending fermented mix with unfermented mix and then was frozen to produce samples for sensory evaluation. All samples were strawberry flavored. These were then evaluated by 88 judges. The preferred pH, based on overall acceptance, was 5.5. A second sensory evaluation was conducted to compare heat-treated with non-heat-treated ice cream. There were no significant differences in appearance, texture, flavor, and overall acceptance between the two samples. Our study shows that ice cream is a suitable vehicle for delivering these beneficial microorganisms and enzymes to consumers.