Probiotics and prebiotic are becoming increasingly important to consumers to alleviate issues surrounding gut health, despite the lack of definitive efficacy studies to support health claims. The addition of both probiotics and prebiotics to foods is challenging due to the harsh environmental conditions within the food itself and during transit through the gastrointestinal (GI) tract. To circumvent these challenges encapsulation technology is being explored to protect sensitive ingredients and to control their release within the lower intestines thereby maximizing the health benefiting effects. The overall goal of this research was to design a protein delivery capsule using phase separated pea protein isolate (PPI)-alginate (AL) mixtures for the entrapment of the synbiot which includes the probiotics, Bifidobacterium adolescentis, and the prebiotic, fructooligosaccharides (FOS), such that the capsule design provides highly effective protection and release within the GI tract. Research was carried out in three studies.<p> In study 1, PPIn (native isolate) and AL interactions were studied in dilute aqueous solutions as a function of pH and biopolymer mixing ratio. Turbidimetric analysis and electrophoretic mobility during an acid titration was used to determine conditions where phase separation occurred. Critical structure forming events associated with the formation of soluble and insoluble complexes in a 1:1 PPIn-AL mixture were found to occur at pH 5.00 and 2.98, respectively, with optimal interactions occurring at pH 2.10. As the PPIn-AL ratio increased, critical pH values shifted towards higher pH until a mixing ratio between 4:1 and 8:1was reached, above which structure formation became independent of the ratios through to ratios of 20:1. Electrophoretic mobility measurements showed a similar trend, where the isoelectric point (pI) shifted from pH 4.00 (homogeneous PPIn) to pH 1.55 (1:1 PPIn-AL). As the ratio increased towards 8:1 PPIn-AL, net neutrality values shifted to higher pHs (~3.80) before becoming constant at higher ratios. Maximum coacervate formation occurred at a mixing ratio of 4:1. Based on these findings, capsule design by segregative phase separation was only used in future studies, due to the acidic nature associated with associative phase separation.<p> In study 2, capsule formation using a native and commercial PPI was studied, and showed no difference between the two formulations during challenge experiments in simulated gastric juice (SGJ). As a result study 3 focused on optimization and characterization of capsules prepared using the commercial PPI. Capsule designs were investigated as a function of protein concentration, prebiotic level, and extrusion conditions (20 vs. 27 G needle) in order to determine protective ability for B. adolescentis within SGJ. Capsule designs were also measured in terms of protein and prebiotic retention during the encapsulation process, geometric mean diameter and size distribution, swelling behaviour and release characteristics within simulated intestinal fluids (SIF). All capsules provided adequate protection over the 2 h duration within SGJ. Capsule breakdown and release was similar for all designs within SIF, with a release mechanism believed to be tied to enzymatic degradation of the PPI material within the wall matrix and/or the amount of excessive Na+ present in the SIF. Capsule size was found to be dependent only on the needle gauge used in the extrusion process. Swelling behaviour of the capsules with SGJ was also found to be dependent only on the protein concentration, where capsules shrank once immersed in SGJ.<p> A 2.0% PPI-0.5% AL capsule without FOS and extruded through a 20 G needle represents the best and most cost effective design for entrapping, protecting and delivering probiotic bacteria. Future work to establish the role FOS could play post-release as the entrapping probiotics colonize the GI tract, and the protective effect of the capsules wall on FOS structure during transit is recommended.

Probiotics and prebiotic are becoming increasingly important to consumers to alleviate issues surrounding gut health, despite the lack of definitive efficacy studies to support health claims. The addition of both probiotics and prebiotics to foods is challenging due to the harsh environmental conditions within the food itself and during transit through the gastrointestinal (GI) tract. To circumvent these challenges encapsulation technology is being explored to protect sensitive ingredients and to control their release within the lower intestines thereby maximizing the health benefiting effects. The overall goal of this research was to design a protein delivery capsule using phase separated pea protein isolate (PPI)-alginate (AL) mixtures for the entrapment of the synbiot which includes the probiotics, Bifidobacterium adolescentis, and the prebiotic, fructooligosaccharides (FOS), such that the capsule design provides highly effective protection and release within the GI tract. Research was carried out in three studies.<p> In study 1, PPIn (native isolate) and AL interactions were studied in dilute aqueous solutions as a function of pH and biopolymer mixing ratio. Turbidimetric analysis and electrophoretic mobility during an acid titration was used to determine conditions where phase separation occurred. Critical structure forming events associated with the formation of soluble and insoluble complexes in a 1:1 PPIn-AL mixture were found to occur at pH 5.00 and 2.98, respectively, with optimal interactions occurring at pH 2.10. As the PPIn-AL ratio increased, critical pH values shifted towards higher pH until a mixing ratio between 4:1 and 8:1was reached, above which structure formation became independent of the ratios through to ratios of 20:1. Electrophoretic mobility measurements showed a similar trend, where the isoelectric point (pI) shifted from pH 4.00 (homogeneous PPIn) to pH 1.55 (1:1 PPIn-AL). As the ratio increased towards 8:1 PPIn-AL, net neutrality values shifted to higher pHs (~3.80) before becoming constant at higher ratios. Maximum coacervate formation occurred at a mixing ratio of 4:1. Based on these findings, capsule design by segregative phase separation was only used in future studies, due to the acidic nature associated with associative phase separation.<p> In study 2, capsule formation using a native and commercial PPI was studied, and showed no difference between the two formulations during challenge experiments in simulated gastric juice (SGJ). As a result study 3 focused on optimization and characterization of capsules prepared using the commercial PPI. Capsule designs were investigated as a function of protein concentration, prebiotic level, and extrusion conditions (20 vs. 27 G needle) in order to determine protective ability for B. adolescentis within SGJ. Capsule designs were also measured in terms of protein and prebiotic retention during the encapsulation process, geometric mean diameter and size distribution, swelling behaviour and release characteristics within simulated intestinal fluids (SIF). All capsules provided adequate protection over the 2 h duration within SGJ. Capsule breakdown and release was similar for all designs within SIF, with a release mechanism believed to be tied to enzymatic degradation of the PPI material within the wall matrix and/or the amount of excessive Na+ present in the SIF. Capsule size was found to be dependent only on the needle gauge used in the extrusion process. Swelling behaviour of the capsules with SGJ was also found to be dependent only on the protein concentration, where capsules shrank once immersed in SGJ.<p> A 2.0% PPI-0.5% AL capsule without FOS and extruded through a 20 G needle represents the best and most cost effective design for entrapping, protecting and delivering probiotic bacteria. Future work to establish the role FOS could play post-release as the entrapping probiotics colonize the GI tract, and the protective effect of the capsules wall on FOS structure during transit is recommended.

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.

Lactic acid bacteria (LAB) are of interest because of their potential to modulate immune responses. The effects of LAB range from regulation to stimulation of the immune system. It has been reported that LAB affect health via two main mechanisms: directly through physical interactions between LAB and cells of the immune system, and indirectly through the products of these bacteria. The studies presented in this thesis examine the direct and indirect effects of LAB on the immune system specifically on murine dendritic cells (DCs). Mouse DCs (in form of the DC2.4 cell line) were treated in vitro with a fraction of bovine milk fermented with Lactobacillus helveticus-2 (LH-2) or three synthetic peptides identified within the fermented milk fraction. Cell culture supernatants were analyzed for presence of tumor necrosis factor (TNF)-α and interleukin (IL)-6 to determine the effects of LAB on DC activation. The results of this study showed that the ability of the milk derived fraction and the synthetic peptides to induce DC activation and production of pro-inflammatory cytokines was limited, suggesting that these peptides may induce regulatory immune responses. A series of studies was performed in vitro to investigate the effects of six LAB species and strains, (LH-2), Lactobacillus acidophilus-5 (La-5), Lactobacillus acidophilus-115 (La-115), Lactobacillus acidophilus-116 (La-116), Lactobacillus acidophilus-14 (La-14), and Lactobacillus salivarius, on maturation and activation of DC2.4. Production of TNF-α, IL-6 and IL-10 by DCs was determined after treating cells with live LAB. The expression of DC maturation markers, CD80 and CD40, was also measured using flow cytometry after stimulation with LAB. In addition, the expression of toll-like receptors (TLRs) 2, 4 and 9 by DCs stimulated with LAB was measured. Our results revealed that LAB act differentially on pro-inflammatory and anti-inflammatory cytokine production and induction of co-stimulatory molecules by DCs. Specifically, L. salivarius was found to be the most effective LAB to induce pro-inflammatory cytokine production and expression of co-stimulatory molecules. Moreover, La-14, La-116 and La-5 induced moderate maturation and activation of DCs. On the other hand, LH-2 and La-115 are the least likely lactobacilli to induce DC response. In conclusion, various strains and species of LAB can differentially regulate DC activation and maturation, raising the possibility that these microbes can influence and steer immune responses of the host.

I lactobacilli sono considerati dei microorganismi non-patogeni. Molti di loro appartengono al gruppo batterico GRAS e/o sono nell’elenco QPS. Dal momento che i lactobacilli intenzionalmente aggiunti agli alimenti possono agire come reservoir di geni di resistenza, la valutazione del rischio deve essere continuamente aggiornata. Lo scopo di questa tesi era la valutazione di alcuni metodi usati per testare e caratterizzare le specie del genere Lactobacillus per quanto riguarda la sicurezza e la potenziale attività probiotica. Nella prima parte due metodi di micro diluzione, il metodo ISO e CLSI, soni stati comparati testando la resistenza agli antibiotici di 54 ceppi L. plantarum. Sulla base di risultati ottenuti il metodo ISO era più adatto per valutare la resistenza di questa specie. Il test del limite di sensibilità della PCR per 8 paia di primers specifici per il rilevamento dei lactobacilli e bifido batteri da feci ha confermato i loro diversi livelli di efficacia. La seconda parte della tesi descrive un progetto di ricerca mirato sulla identificazione di nuovi ceppi probiotici fra diversi ceppi di Lactobacillus paracasei e Lactobacillus rhamnosus identificando dei geni o loci responsabili della interazione con l’ospite, immunomodulazione, e l’inibizione della crescita dei patogeni. Le analisi fenotipiche dei 40 ceppi hanno confemato una grande variabilità fra di loro, che può servire per associare delle caratteristiche fenotipiche a quelle genotipiche. Tra i ceppi dello stesso progetto è stato individuato un ceppo di L. mucosae. Dal momento che questa è una specie relativamente nuova, le sue caratteristiche sono state analizzate comparandole con altri 3 ceppi appartenenti alla stessa specie. In questo modo sono state confermate alcune informazioni su L. mucosae, ma soprattutto sono stati forniti dei dati nuovi sulle proprietà di questa specie. / The species of the Lactobacillus genus are generally believed to be microorganisms with no pathogenic potential. Many of them have granted GRAS and QPS status. Non-pathogenic bacteria as lactobacilli-intentionally added or accidentally present in food-are under evaluation, as they could act as reservoir of resistant genes. This thesis was aimed to evaluate some methods used for testing and to characterize some Lactobacillus species, as regards their safety and potential probiotic activity. The first part of the research focused on the comparison of two broth microdilution methods: ISO and CLSI, in order to assess the resistance of 54 L. plantarum strains to antimicrobial agents. The results suggest better performances of the phenotypic assay developed by ISO, at least for strains belonging to L. plantarum species.Then the assessment of the PCR detection limit for 8 sets of primers for the detection of lactobacilli and bifidobacteria from infant faeces confirmed different levels of effectiveness for the primers. Next part of the thesis was the research project aimed at identifying genes or genetic loci of different strains of two Lactobacillus species (i.e. Lactobacillus paracasei and Lactobacillus rhamnosus) involved in the interaction with the host, immune-modulation of host cells and pathogen growth inhibition in order to find new probiotic strains. The phenotypic analysis of 40 selected strains demonstrated large variability between strains of these species, which could serve to the association of phenotypic differences to genome specificities. A strain of Lactobacillus mucosae was found within the framework of the same project. As it is a relatively new species, it was chosen to further investigate its properties, comparing it with three other L. mucosae strains. This study led to confirm some information but first and foremost it has provided new data on the examined species.

Bacterial Vaginosis (BV) is a disorder of unknown etiology, characterized by a foul smelling vaginal discharge, loss or reduction of the normal vaginal Lactobacilli, and overgrowth of other anaerobic bacteria. Thus, it presents a formidable problem for clinicians as well as microbiologists researching its etiology, clinical course, treatment, and epidemiology. The present work focuses on the unresolved issues of the epidemiology and treatment of BV in order to provide valid methods for treatment studies of this condition and to describe the prevalence of BV in defined populations. The first study validates the use of PAP-stained smears in the diagnosis of BV. The study assesses the methods of Amsel’s clinical criteria and Nugent criteria on Gram-stain smears, against Pap-stained smears and also validates different observers. The result shows that the PAP-staining of vaginal smears is a good method in BV diagnosis; the kappa value is 0.86 (interobserver weighted kappa index) compared to 0.81 for Gram-stained smears, and 0.70 for rehydrated air-dried smears using the mean Nugent score as the criterion standard. This enables population based studies on archived PAP-stained smears from the screening of cervical cancer. In the second study, we use the knowledge gained from study one to investigate the prevalence of BV in a cohort from the population of Åland. The prevalences of BV on the Åland Islands were: 15.6 %, 11.9 %, 8.7 %, and 8.6% in 1993, 1998, 2003, and 2008, respectively. This means that the prevalence of BV decreased between1993-2008 from 15.6% to 8.6%. The confidence intervals are not overlapping, thus indicating a significant decrease in prevalence from 1993 to 2008. The third study is a prospective, double-blind placebo controlled treatment study of BV. After conventional treatment with clindamycin, the patients were treated with adjuvant treatment of Lactobacilli-loaded tampons or placebo. The study showed no differences between the treatment and the placebo group, indicating that the tampon does not work at all. There are a variety of possible explanations for the result, which are analyzed in this thesis. The fourth study aimed to evaluate whether clindamycin is retained for a long time in the vaginal mucosa, thus disturbing the Lactobacilli in an attempt to reimplant Lactobacilli in the probiotic treatment studies. In conventional treatment, it is also useful to know whether clindamycin is retained, especially when considering the pressure from antibiotics on the antimicrobial sensitivity pattern. In the study, we found that the clindamycin disappears rapidly. Conclusion: BV research requires effort from many different scientific disciplines and the riddle of this condition and its treatment can only be resolved by concerted actions in research and treatment. The vision for the future includes, among other factors, better molecular biology based diagnostic tools, and knowledge of population based bacterial floras.

Submitted by Juliana Jabur Damiao Polete null (36492619870) on 2016-05-19T02:12:33Z No. of bitstreams: 1 DISSERTAÇÃO - JULIANA WITZLER 28-03-16.pdf: 908490 bytes, checksum: 813f3b334ef44efd2e6b6abb35e2702e (MD5) / Approved for entry into archive by Felipe Augusto Arakaki (arakaki@reitoria.unesp.br) on 2016-05-23T14:22:27Z (GMT) No. of bitstreams: 1 witzler_jjp_me_arafcf.pdf: 908490 bytes, checksum: 813f3b334ef44efd2e6b6abb35e2702e (MD5) / Made available in DSpace on 2016-05-23T14:22:27Z (GMT). No. of bitstreams: 1 witzler_jjp_me_arafcf.pdf: 908490 bytes, checksum: 813f3b334ef44efd2e6b6abb35e2702e (MD5) Previous issue date: 2016-03-28 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / A utilização de microrganismos probióticos para a manutenção da saúde bucal tem despertado o interesse da comunidade científica, tendo em vista que estudos indicam que tais microrganismos podem conferir benefícios como: atividade anticariogênica, tratamento de doenças periodentais e halitose, e redução na população de microrganismos patogênicos relacionados a patologias bucais. Nesse sentido, o objetivo do presente trabalho foi o desenvolvimento e a caracterização de uma pastilha alimentícia diet contendo o microrganismo Enterococcus faecium CRL 183 e a avaliação do potencial da referida cepa probiótica em sobreviver em saliva humana e inibir a multiplicação da cepa patogênica Streptococcus mutans ATCC 25175. O microrganismo E. faecium CRL 183 foi microencapsulado por extrusão e coacervação em matriz complexa. A técnica de coacervação complexa apresentou o melhor desempenho em relação à manutenção da viabilidade durante o período de armazenamento à temperatura ambiente (23ºC), sendo selecionada para a produção das pastilhas potencialmente probióticas. As pastilhas foram processadas em três tratamentos: PC – pastilha controle, sem adição do probiótico; PPP1 – com adição do microrganismo probiótico e PPP2 – pastilha com adição do microrganismo probiótico e de inulina (prebiótico). Análises microbiológicas (viabilidade e segurança), físico-químicas (umidade, atividade de água, pH e cor) e sensoriais (testes de aceitação e intenção de compra) foram conduzidas no tempo inicial e ao longo do tempo de estocagem. A sobrevivência do probiótico à saliva e a inibição da multiplicação da cepa patogênica, foram avaliadas através da inoculação da cepa pura e das pastilhas em saliva e da técnica de difusão em poços, respectivamente. A cepa de E. faecium teve sua viabilidade reduzida (p<0,05) imediatamente após a produção das pastilhas e durante o tempo de armazenamento à temperatura ambiente, em ambos os tratamentos PPP1 e PPP2. Na etapa de análise sensorial, as pastilhas apresentaram médias de aceitação superiores a seis para os atributos aroma, textura e impressão global, sem diferirem significativamente entre si (p<0,05). A adição da cepa probiótica e da substância prebiótica reduziu a aceitação do produto em relação à aparência e cor e melhorou a impressão dos consumidores em relação ao sabor. As análises físico-químicas revelaram que as formulações PC, PPP1 e PPP2 não diferiram entre si em relação aos parâmetros físico-químicos avaliados, com exceção do parâmetro cor. Todos os tratamentos apresentaram características microbiológicas adequadas durante os 28 dias de estudo. A cepa E. faecium CRL 183 inoculada em saliva humana na forma de pastilhas e de células livres teve sua população de células viáveis aumentada após 24 horas de incubação. O teste de difusão em poços evidenciou que a cepa probiótica foi capaz de inibir a multiplicação de S. mutans ATCC 25175 nas condições do estudo. Os resultados obtidos indicam que a associação do probiótico com inulina melhorou a aceitação do produto em relação ao atributo sabor e que a cepa probiótica sobrevive à saliva humana e apresenta potencial para inibir a multiplicação do microrganismo causador de cárie dental – S. mutans ATCC 25175. No entanto, a queda brusca da viabilidade da cepa probiótica durante o armazenamento das pastilhas, indica a necessidade de aprimoramento do processo de obtenção das mesmas, além de adequação da embalagem utilizada à matriz alimentícia. / The interest in the local effect of probiotic microorganisms is increasing among the scientific community, since studies have indicated that such microorganisms can present anticariogenic activity, help on the treatment of periodontal and halitosis diseases, and reduction in the population of pathogenic microorganisms associated to oral pathologies. The main purpose of this study was the development and characterization of a diet lozenge containing the probiotic strain Enterococcus faecium CRL 183. Its potential of surviving in the human saliva environment and the inhibition of the pathogenetic strain Streptococcus mutans ATCC 25175, were also evaluated. The probiotic strain E. faecium CRL 183 was microencapsulated through the extrusion and complex coacervation techniques. The last one was selected to the next step of the study (lozenges production), as it showed the best performance in comparison to the extrusion technique, regarding viability maintenance during the storage period at room temperature (23ºC). The lozenges were produced through three different treatments: PC – control formulation, without the probiotic; PPP1 – probiotic formulation; PPP2 – probiotic formulation with inulin addition. Microbiologic (viability and security), physicochemical (moisture, water activity, pH and color) and sensorial (acceptance and purchase intention) analyses were conducted during the storage period. The probiotic survival to human saliva was evaluated through inoculation of the pure probiotic strain and the probiotic lozenges in saliva. The agar diffusion technique was used to evaluate the E. faecium CRL 183 inhibition potential against the pathogenic strain, S. mutans ATCC 25175. The probiotic strain had the viability decreased after the lozenges production and also during the storage period at room temperature for the PPP1 and PPP2 treatments. At sensorial analysis, lozenges showed acceptance averages higher than 6 to flavor, texture and global impression attributes, with no significant difference among then (p<0,05). The addition of the probiotic strain and the prebiotic ingredient reduced the product acceptance regarding appearance and color and improved the flavor impression. The physicochemical analysis revealed that the formulations PC, PPP1 and PPP2 did not differ among themselves concerning the parameters evaluated, except for color. The microbiologic results obtained during the storage were appropriated to the confectionery category. The E. faecium strain inoculated in saliva as lozenges and as free cells had the cell viability increased after 24 h of incubation. The diffusion test showed that the probiotic strain was able to inhibit the growth of S. mutans ATCC 25175 in the study conditions. The results suggested that the association of probiotic microorganism and inulin improved the acceptance of the product, in relation to the flavor attribute, and that the probiotic strain survived in the human saliva and has potential to inhibit the multiplication of dental caries-causing organism – S. mutans ATCC 25175. However, the drastic viability reduction during the storage period indicates the necessity of a process refinement and also the adjustment of the packaging to food matrix.

Made available in DSpace on 2014-06-11T19:32:53Z (GMT). No. of bitstreams: 0 Previous issue date: 2008-12-04Bitstream added on 2014-06-13T19:43:43Z : No. of bitstreams: 1 delfino_tpc_dr_jabo.pdf: 382159 bytes, checksum: 8e7833c07c682a4bbf8eee1668ce058f (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / A avicultura comercial tem como objetivo obter alta produtividade a baixo custo e oferecer ao consumidor produto de qualidade. Uma bactéria patogênica que tem preocupado o setor avícola nos últimos tempos é a Salmonella. Neste sentido, o presente trabalho objetivou caracterizar seis isolados de Lactobacillus acidophilus em combinação com antimicrobianos na inibição de Salmonella Enteritidis e Salmonella Gallinarum. O experimento foi desenvolvido nas dependências da FCAV/UNESP – Campus de Jaboticabal. Para tanto foram conduzidos quatro estudos, sendo que dois foram dedicados em selecionar uma bactéria probiótica produtora de bacteriocina e avaliar a interação de substâncias antimicrobianas como EDTA, àcido acético e Nisina “in vitro” sobre a capacidade inibitória de Salmonella Enteritidis e Salmonella Gallinarum em diferentes tempos e o terceiro e quarto experimento estudam o potencial de aplicação do probiótico e do antimicrobiano em aves. O isolado de Lactobacillus acidophilus C1, demonstrou ser produtor de bacteriocina e inibir a multiplicação de Salmonella Enteritidis e Salmonella Gallinarum. Dentre os antimicrobianos testados o que apresentou efeito na eliminação de Salmonella Enteritidis foi a combinação de Ácido Acético + nisina e Ácido Acético + Lactobacillus acidophilus C1. O uso do probiótico no estudo em aves reduziu a excreção de Salmonella sp, mas não sua eliminação no trato intestinal das aves, já que é possível constatar sua presença no tratamento controle. A combinação de nisina e ácido acético não levou a redução da multiplicação de Salmonella sp. nas aves. O estudo permitiu verificar que existe um bom potencial de aplicação do probiótico citado, assim como o uso de alguns antimicrobianos na segurança alimentar. / The commercial poultry aims at obtaining high productivity at low cost and offering quality products to the consumer. A pathogenic bacterium which has worried the poultry sector in recent times is Salmonella. Therefore, the present study aimed at characterizing six isolates of Lactobacillus acidophilus in combination with antimicrobials in the inhibition of Salmonella enteritidis and Salmonella gallinarum. The experiment was developed in the dependencies of FCAV/UNESP – Campus of Jaboticabal. In order to do this, four studies were conducted, being two of them dedicated to select a bacteriocin-procucer probiotic bacterium and evaluate the interaction of antimicrobial substances such as EDTA, acetic acid and “in vitro”Nisin on the inhibitory capacity of Salmonella enteritidis and Salmonella gallinarum in different periods and the third and fourth experiment study the potential of application of the probiotic and antimicrobial in poultry. The isolate of Lactobacillus acidophilus C1, showed to be bacteriocin producer and inhibit the multiplication of Salmonella enteritidis and Salmonella gallinarum. Among the antimicrobial tested, the one which presented effect in the elimination of Salmonella enteritidis, was the combination of Acetic Acid + nisin and Acetic Acid + Lactobacillus acidophilus C1. The use of the probiotic in the study of birds reduced the counting of Salmonella sp, but not its elimination in the intestinal tract of the birds, as we can see its presence in the control treatment. The combination of nisin and acetic acid did not show the reduction of the multiplication of Salmonella sp. in birds. The study has shown that there is a good potential of application of the mentioned probiotic, as well as the use of some antimicrobial in food safety.

Made available in DSpace on 2014-06-11T19:32:54Z (GMT). No. of bitstreams: 0 Previous issue date: 2008-03-31Bitstream added on 2014-06-13T18:44:29Z : No. of bitstreams: 1 garcia_gr_dr_jabo.pdf: 313576 bytes, checksum: 3615e7690e51b47d22430c998944cfd2 (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / O presente trabalho objetivou caracterizar um isolado de Bacillus subtilis para utilização como agente probiótico para bovinos. Foram determinadas “in vitro” a capacidade e o tipo de efeito inibitório do isolado de B. subtilis do produto comercial Biotop sobre Salmonella, Escherichia coli e Clostridium perfringens, além de sua estabilidade, viabilidade, resistência a antimicrobianos e potencial de ação em comparação com a Nisina e em combinação com EDTA. Foram avaliados os efeitos da adição do isolado na dieta de 32 bezerros da raça Holandesa, em quatro diferentes tratamentos (controle; 1 g/dia; 2 g/dia e 4 g/dia), sobre o consumo de matéria seca, perímetro torácico, ganho de peso, consistência fecal e incidência de doenças. Também foi realizado um desafio com E. coli e monitoramento do “score” de fezes, temperatura retal (oC) e parâmetros sanguíneos. O isolado de B. subtilis foi mais eficaz contra C. perfringens, principalmente quando associado com a Nisina e EDTA. A adição do probiótico na dieta de bezerros aumentou o consumo de matéria seca, ganho de peso e perímetro torácico. No desafio bacteriano não foram observadas diferenças significativas para presença de Bacillus spp. e E. coli nas fezes. A análise do “score” de fezes, temperatura retal e parâmetros sanguíneos não demonstraram diferenças significativas entre os tratamentos. O produto avaliado demonstrou resultados satisfatórios quanto aos parâmetros de produção animal, sendo recomendada sua utilização para bezerros lactantes, principalmente na dosagem de 4 g/animal/dia. / The present work aimed to characterize an isolated of Bacillus subtilis to be used as a probiotic for calves. Were determinate in vitro its inhibition capacity and the type of effect of the isolated, that is present in the commercial product Biotop against Salmonella, Escherichia coli and Clostridium perfringens. It also was tested its stability, viability, resistance to antibiotics and its potential action in comparison with Nisin and in combination with EDTA. They were appraised the effects of the addition of the isolated in the diet of 32 calves Holstein Frisian in four different treatments (control; 1 g/day; 2 g/day and 4 g/day), over the intake of dry matter, thoracic perimeter, gain of weight, faecal consistence and incidence of diarrhoea. Also a challenge was accomplished with E. coli and the score of faeces monitored, rectal temperature (oC) and sanguine parameters. The isolated of B. subtilis was more effective against C. perfringens, mainly when associated with Nisin and EDTA. The addition of the probiotic in the diet of calves increased the dry matter intake, weight enhance and thoracic perimeter. In the bacterial challenge significant differences were not observed in the counting of Bacillus spp. and E. coli in the faeces. The analysis of the score of faeces, rectal temperature and sanguine parameters didn't demonstrate significant differences between the treatments. The appraised product demonstrated satisfactory results as for the parameters of animal production, being recommended to be use for nursing calves, mainly in the dosage of 4 g/animal/day.

Remyelination describes the regeneration of myelin sheaths, and is considered one of the most promising strategies for improving the prognosis of demyelinating diseases such as multiple sclerosis. Data from animal models and human studies have shown that remyelination can occur extensively in the central nervous system (CNS), leading to functional recovery and axonal protection. However, remyelination does not always proceed to completion, and its failure is associated with progressive neurological disability. Thus, there is clinical need for interventions that can optimise the conditions for remyelination. Recent advances in genomics and animal husbandry have kindled an interest in the microbiome as a means to influence processes throughout the body. Our commensal microbes communicate with host cells at epithelial barriers, stimulate neural and endocrine axes and directly produce a plethora of long-range signalling molecules. Critically, the development and maintenance of the immune system depend on signals from the microbiota, and we know that a well-coordinated immune response is a key determinant of the success of remyelination. This thesis explores how the microbiome can influence CNS remyelination. To do so, I have studied remyelination in three murine models of microbiome alteration. Firstly, long-term oral administration of an antibiotic cocktail was used to deplete the microbiota of adult mice. Following focal demyelination, these mice had deficits in their inflammatory response, clearance of myelin debris and differentiation of new oligodendrocytes from oligodendrocyte progenitor cells (OPCs). Faecal microbial transplant was able to rescue aspects of the inflammatory response and phagocytosis, but not OPC differentiation. Secondly, I looked at remyelination in germ-free (GF) mice following cuprizone-induced demyelina- tion. As with the antibiotics-treated mice, there were deficits in inflammation following demyelination, which tended to peak later than in control mice. Finally, I investigated the potential of a therapeutic probiotic (VSL#3) to improve remyelination in aged mice. In contrast to antibiotic treatment, probiotic administration caused a slight enhancement in the onset of inflammation following focal demyelination. However, there was no significant improvement in OPC differentiation or toluidine blue rank analysis, suggesting these changes in inflammation were not sufficient to positively modulate remyelination. The results from these three studies introduce a significant but previously unconsidered environmental influence on remyelination in the CNS. Whilst the effects are subtle relative to more direct interventions, the microbiome can be manipulated simply and non-invasively, which may provide a useful adjunct to other strategies for optimising remyelination.