Both Candida albicans and lactobacilli are common colonizers of carious lesions in children and adolescents. The purpose of this study is to compare the velocity of acid production between C. albicans and several Lactobacillus species at different pH levels and concentrations of glucose. Washed, pure resting-cell suspensions were obtained by culturing a total of 28 oral isolates comprising the species C. albicans, Lactobacillus rhamnosus, Lactobacillus paracasei paracasei, Lactobacillus paracasei tolerans and Lactobacillus delbrueckii lactis. Acid production from glucose was determined at a constant pH of 7.0, 5.5, 5.0 and 4.0 by repeated titrations with NaOH in an automated pH-stat system. Acid formation rates of yeast and lactobacilli proved to be similar at both neutral and low pH, while in a moderately acidic environment C. albicans produced less acid than the lactobacilli. Ion chromatographic analysis of the cell-free medium after titration revealed pyruvate to be the predominant organic acid anion secreted by C. albicans. The proportion of organic acids to overall acid production by the yeast was below 10% at neutral conditions, in contrast to 42–66% at pH 4.0. Compared to lactobacilli, yeast required a concentration of glucose that was about 50 times higher to allow acid production at half the maximum speed. Considering the clinical data in the literature about the frequency and proportions of microorganisms present in early childhood caries lesions, the contribution of oral lactobacilli as well as C. albicans to overall microbial acid formation appears to be important. / Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG-geförderten) Allianz- bzw. Nationallizenz frei zugänglich.

Background: Probiotic products are used to improve health, cure disease and in a prophylactic aim. However, there is limited research about its general effects on health in children, especially on oral health.  Aim: This study aimed to evaluate potential effects on oral health and otitis in 3-year- old children consuming probiotic Lactobacillus reuteri 17938 and Lactobacillus reuteri 5289.  Methods: This was a quantitative cross-sectional study where questionnaires concerning health and lifestyle of children (n=169) participating in the longitudinal study NorthPop Child, at the age of 4, 9, 18 and 36 months old, were compared to clinical parameters from a visit at the Public Dental Health Care at 3 years of age. Saliva samples from 142 children were analyzed by PCR. Results: This study showed that children consuming Lactobacillus reuteri 17938, had prevalence of the bacteria in saliva. Prevalence of Lactobacillus reuteri 5289 in saliva was not indicated. The prevalence of caries disease tended to be lower in probiotic- consuming children, though not statistically significant. Children consuming probiotics showed a higher prevalence of otitis and consumption of antibiotics compared to non- eaters.  Conclusion: Any conclusions about consumption and frequency required to gain and maintain the bacteria in the oral microbiome could not be made. Contradictory to other studies, this study found that children who consumed probiotics had more occasions of otitis media and antibiotic use. Future studies with a higher number of participants would be of interest to confirm these potential findings.

Durante la mia tesi di dottorato ho selezionato nuovi potenziali ceppi probiotici, combinando l'approccio convenzionale con l’utilizzo di piattaforme di nuova concezione. Mi sono concentrata inizialmente sul cosiddetto "screening tradizionale" finalizzato all'isolamento di nuovi ceppi batterici di lattobacilli e bifidobatteri e alla valutazione della loro sicurezza per il consumo umano e della loro efficacia. I ceppi selezionati sono stati quindi sottoposti ad uno screening più specifico, a seconda dell'applicazione a cui sarebbero stati destinati. In questa fase sono stati utilizzati sia test in vitro che modelli animali, al fine di valutare l'applicabilità dei ceppi di recente selezionati come probiotici per la promozione della salute umana e il loro possibile impiego in campo alimentare. Ho valutato il potenziale utilizzo dei ceppi nell’integrazione alimentare di soggetti che seguono diete ad alto contenuto proteico per ridurre il rischio di accumulo intestinale di ammine biogene, e ho individuato un ceppo di Lactobacillus con presunta attività ammino-degradativa. L'obiettivo principale di un’altra attività di screening è stato lo studio degli effetti immunomodulanti di nuovi ceppi: combinazioni di probiotici sono risultati buoni candidati per il trattamento delle malattie infiammatorie e autoimmuni (miastenia grave e sclerosi multipla) nel modello di topo. Infine, i ceppi sono stati sottoposti a screening per una potenziale applicazione in campo alimentare, finalizzata ad indagare la possibilità di produrre formulazioni di probiotici “atomizzati” da utilizzare insieme alla base commerciale del gelato. / During my PhD thesis I screened new potential probiotic strains by combining conventional approach to newly designed platforms. I focused first on the so-called "conventional screening” aimed to the isolation of new bacterial strains of lactobacilli and bifidobacteria and to assess their safety for human consumption and their efficacy by in-vitro methods. Selected strains were then submitted to a targeted screening by specifically designed models, depending on the application to which they would be destined. These models combined in vitro tests and animal models in order to assess the applicability of newly selected strains as probiotics for the promotion of human health and their possible use in food field. I evaluated the potential use of strains in the food supplementation of subjects that follow high-protein diets to reduce the risk of intestinal accumulation of biogenic amines, and I identified a strain of Lactobacillus with alleged amino degradative activity. The main objective of another screening activity was the study of the immunoregulatory effects of new strains: combinations of probiotics resulted good promising candidates for the treatment of inflammatory and autoimmune diseases (myasthenia gravis and multiple sclerosis) in rat model. Finally, strains were screened for a potential application in food field, in order to investigate the possibility of producing spray-dried probiotic formulations to be used together with commercial ice-cream bases.

A RMN é uma das principais técnicas usadas no estudo de perfis metabólicos pois é uma técnica quantitativa, altamente reprodutível e não-seletiva. Desta forma, o objetivo desta tese foi a aplicação da RMN no estudo metabólico em diferentes processos biológicos. A primeira aplicação foi na análise do perfil metabolômico e metabonômico dos agentes causadores da leishmaniose. Para tal, foram analisados extratos celulares das espécies Leishmania major e Leishmania donovani e extratos destas espécies crescidas com os fármacos miltefosina e paromomicina. Os espectros de RMN de 1H foram analisados pelo programa ChenoMX e os espectros de RMN 2D pelo software TopSpin e as bases de dados HMDB e BMRB. Cerca de 50 metabólitos foram identificados, como a adenina, arginina, glutamina, glutamato, manose, isoleucina, leucina e tirosina. Técnicas quimiométricas como PCA, PLS-DA e heatmaps foram utilizadas para investigar os metabólitos responsáveis pela diferenciação das duas espécies bem como entre as culturas com e sem fármacos. O segundo estudo foi com o composto metilglioxal. A presença de metilglioxal em queijos contribui com o escurecimento e deterioração do sabor, mesmo em baixas temperaturas. Por esta razão, foram estudados os produtos da adição de metilglioxal em extrato de queijo parmesão e o comportamento de diferentes espécies de lactobacilos na presença e na ausência de metilglioxal em extrato de queijo parmesão. Os resultados permitiram um entendimento maior das reações envolvidas entre esta molécula e os componentes do queijo, o que ajudará encontrar alternativas para a resolução do problema e aumentar o tempo de prateleira do queijo. O terceiro estudo realizado foi sobre a produção de etanol a partir de biomassa lignocelulósica. O objetivo deste estudo foi modificar geneticamente uma espécie de lactobacilo para a produção de etanol e analisar a vinhaça de planta de etanol pós-fermentação com levedura. Os espectros de RMN de 1H e 1H-13C mostraram que as bactérias modificadas são eficientes na produção de etanol e que dois açucares foram erroneamente identificados por cromatografia como maltose e maltotriose quando na verdade eram trealose e arabinose.  Por meio destes destes estudos mostrou-se parte das inúmeras aplicações que a RMN tem na metabolômica e que existem diversas ferramentas estatísticas disponíveis para auxiliar no estudo metabolômico. / NMR is one of the main techniques used in metabolic profile studies because it is a quantitative, highly reproducible and non-selective technique. Thus, the aim of this thesis was the application of NMR in metabolic studies of different biological processes. The first application was the analysis of the metabolomic and metabonomic profile of the causative agents of leishmaniasis. Cell extracts of the species Leishmania major and Leishmania donovani and extracts of these species grown with miltefosine and paromomycin drugs were analyzed. 1H spectra were analyzed by ChenoMX software and 2D spectra by TopSpin software and HMDB and BMRB databases. About 50 metabolites were identified, such as adenine, arginine, glutamine, glutamate, mannose, isoleucine, leucine and tyrosine. Chemometric techniques were used to investigate the metabolites responsible for the differentiation of the two species and between cultures with and without drugs. The second study was with methylglyoxal molecule. The presence of methylglyoxal in cheese contributes to browning and deterioration of flavor, even at low temperatures. For this reason, products of the addition of methylglyoxal in Parmesan cheese extract and the behavior of different species of lactobacilli in the presence and absence of methylglyoxal in Parmesan cheese extract were studied. The results allowed a better understanding of the reactions involved between this molecule and the components of the cheese, which will help find alternatives to solve the problem and increase the shelf life of the cheese. The third study was about the production of ethanol from lignocellulosic biomass. The objective of this study was to genetically modify a lactobacillus species to produce ethanol and analyze the post-fermentation thin stillage. NMR spectra showed that the modified bacteria are efficient in production of ethanol and that two sugars were erroneously being identified by chromatography as maltose and maltotriose when they actually were trehalose and arabinose. These studies showed part of the numerous applications that NMR has in metabolomics and that there are several statistical tools available to assist in metabolomic studies.

A RMN é uma das principais técnicas usadas no estudo de perfis metabólicos pois é uma técnica quantitativa, altamente reprodutível e não-seletiva. Desta forma, o objetivo desta tese foi a aplicação da RMN no estudo metabólico em diferentes processos biológicos. A primeira aplicação foi na análise do perfil metabolômico e metabonômico dos agentes causadores da leishmaniose. Para tal, foram analisados extratos celulares das espécies Leishmania major e Leishmania donovani e extratos destas espécies crescidas com os fármacos miltefosina e paromomicina. Os espectros de RMN de 1H foram analisados pelo programa ChenoMX e os espectros de RMN 2D pelo software TopSpin e as bases de dados HMDB e BMRB. Cerca de 50 metabólitos foram identificados, como a adenina, arginina, glutamina, glutamato, manose, isoleucina, leucina e tirosina. Técnicas quimiométricas como PCA, PLS-DA e heatmaps foram utilizadas para investigar os metabólitos responsáveis pela diferenciação das duas espécies bem como entre as culturas com e sem fármacos. O segundo estudo foi com o composto metilglioxal. A presença de metilglioxal em queijos contribui com o escurecimento e deterioração do sabor, mesmo em baixas temperaturas. Por esta razão, foram estudados os produtos da adição de metilglioxal em extrato de queijo parmesão e o comportamento de diferentes espécies de lactobacilos na presença e na ausência de metilglioxal em extrato de queijo parmesão. Os resultados permitiram um entendimento maior das reações envolvidas entre esta molécula e os componentes do queijo, o que ajudará encontrar alternativas para a resolução do problema e aumentar o tempo de prateleira do queijo. O terceiro estudo realizado foi sobre a produção de etanol a partir de biomassa lignocelulósica. O objetivo deste estudo foi modificar geneticamente uma espécie de lactobacilo para a produção de etanol e analisar a vinhaça de planta de etanol pós-fermentação com levedura. Os espectros de RMN de 1H e 1H-13C mostraram que as bactérias modificadas são eficientes na produção de etanol e que dois açucares foram erroneamente identificados por cromatografia como maltose e maltotriose quando na verdade eram trealose e arabinose.  Por meio destes destes estudos mostrou-se parte das inúmeras aplicações que a RMN tem na metabolômica e que existem diversas ferramentas estatísticas disponíveis para auxiliar no estudo metabolômico. / NMR is one of the main techniques used in metabolic profile studies because it is a quantitative, highly reproducible and non-selective technique. Thus, the aim of this thesis was the application of NMR in metabolic studies of different biological processes. The first application was the analysis of the metabolomic and metabonomic profile of the causative agents of leishmaniasis. Cell extracts of the species Leishmania major and Leishmania donovani and extracts of these species grown with miltefosine and paromomycin drugs were analyzed. 1H spectra were analyzed by ChenoMX software and 2D spectra by TopSpin software and HMDB and BMRB databases. About 50 metabolites were identified, such as adenine, arginine, glutamine, glutamate, mannose, isoleucine, leucine and tyrosine. Chemometric techniques were used to investigate the metabolites responsible for the differentiation of the two species and between cultures with and without drugs. The second study was with methylglyoxal molecule. The presence of methylglyoxal in cheese contributes to browning and deterioration of flavor, even at low temperatures. For this reason, products of the addition of methylglyoxal in Parmesan cheese extract and the behavior of different species of lactobacilli in the presence and absence of methylglyoxal in Parmesan cheese extract were studied. The results allowed a better understanding of the reactions involved between this molecule and the components of the cheese, which will help find alternatives to solve the problem and increase the shelf life of the cheese. The third study was about the production of ethanol from lignocellulosic biomass. The objective of this study was to genetically modify a lactobacillus species to produce ethanol and analyze the post-fermentation thin stillage. NMR spectra showed that the modified bacteria are efficient in production of ethanol and that two sugars were erroneously being identified by chromatography as maltose and maltotriose when they actually were trehalose and arabinose. These studies showed part of the numerous applications that NMR has in metabolomics and that there are several statistical tools available to assist in metabolomic studies.

The rapid and accurate identification of the bacterium of the genus Lactobacillus, which are an important part of the normal gastrointestinal microflora and fermented dairy products are currently mainly used amplification methods. The aim of the study was to analyze the possibility of resolution of selected bacterial strains of the genus Lactobacillus, using the metod of polymerase chain reaction in the real time combined with high resolution melting curve analysis (qPCR HRM). It was tested five primers designed for qPCR-HRM analysis of lactic acid bacteria. The specificity of the primers was also verified simultaneously using bioinformatic analysis. On the basis of analysis of the DNA were selected as the most appropriate primers P1V1/P2V1, V3F/V3R and V6F/V6R. The suitability of the primers V3F/V3R and V6F/V6R was verified on a complex sample of food supplement from which the DNA was isolated using magnetic particles. The presence of bacteria of the genus Lactobacillus was performed using high resoluting melting analysis curves. The obtained results were in agreement with the information given by the manufacturer.

Inflammatory bowel diseases, (IBD), are a group of chronic disorders of the gastro-intestinal tract, and include Crohn’s disease (CD) and Ulcerative Colitis (UC). The pathogenesis is not known, but involves at least in part a loss of tolerance towards the commensal colonic microbiota. In this thesis, we show in animal models of CD and UC that the colonic mucosal blood flow increased compared to healthy animals. This blood flow increase is due to an up regulation of endothelial nitric oxide synthase (NOS). Further, we show in the UC model that the thickness of the firmly adherent colonic mucus layer increased compared to healthy animals. This increase is due to an up regulation of inducible NOS in the epithelium. Both the blood flow and mucus thickness increase appear to be protective mechanisms.  We demonstrate that the firmly adherent colonic mucus layer acts as a partial barrier towards luminal bacteria. In the UC model, this barrier is destroyed, causing increased bacterial translocation. The adhesion molecule P-selectin was up regulated in the UC model, leading to increased interactions between leukocytes and the endothelium, but also increased interactions between platelets and the endothelium. This indicates that not only leukocytes, but also platelets are involved in colonic inflammation. The addition of the probiotic bacterial strain Lactobacillus reuteri prevented disease by normalizing P-selectin levels and endothelial interactions with leukocytes and platelets. Lactobacillus reuteri also decreased bacterial translocation over the epithelium. In summary, this thesis highlights the importance of colonic barrier functions, and investigates the role of the microbiota in experimental IBD.