|Tipo di tesi||Tesi di dottorato di ricerca|
|Titolo||Apprendere le differenze funzionali del microbiota intestinale umano in normali condizioni di salute e in presenza di patologie.|
|Titolo in inglese||Capturing the functional differences of the human gut microbiota in health and disease|
|Settore scientifico disciplinare||CHIM/11 - CHIMICA E BIOTECNOLOGIA DELLE FERMENTAZIONI|
|Corso di studi||Scuola di D.R. in SCIENZE, TECNOLOGIE E BIOTECNOLOGIE AGRO-ALIMENTARI|
|Data inizio appello||2016-03-18|
|Disponibilità||Accessibile via web (tutti i file della tesi sono accessibili)|
Gli argomenti di questo progetto di tesi hanno riguardato il rapporto fra il microbiota intestinale e funzioni associate allo stato di salute dell’uomo.
The main topics of this thesis project concerned the relationship between the intestinal microbiota and the functions associated to the human health status. In an in vitro model system of intestinal microbiota it was determined the inhibition of the Enterobacteriaceae growth caused by the Bifidobacterium breve B632 strain, previously selected for its specific probiotic activities. Two groups of fecal cultures were carried out in parallel. One of them is composed by the fermentative processes employing the fecal sample of a 2 months old newborn as inoculum. The second one was performed adding to the fecal culture the probiotic strain. The concentrations of the different microbial groups have been compared through FISH and qPCR. Based on the RAPD-PCR results concerning the bifidobacteria isolated on RB selective medium, B. breve B632 colonized the intestinal microbiota cultures representing the 64% of the total bifidobacteria at the steady state. After 18 h of fermentation the Enterobacteriaceae concentration, determined through FISH and qPCR, was of 0.42 and 0.44 order of magnitude lower into the cultures supplemented with the probiotic than the controls ones (p < 0.05). This results are promising and suggest the possibility to proceed to the probiotic validation in vivo. The second part of the project regarded the characterization of the proteolytic bacterial population in healthy adults. The proteins fermentation produces, in addition to ammonium and organic acids, also some toxic metabolites known as uremic toxins: indole and p-cresol. In this study, in an in vitro model system, fecal fermentations have been carried out in a medium containing only proteins and peptides as carbon source. The proteolytic activity has been confirmed by the accumulation of the ammonium, indole and p-cresol and by the organic acid profile produced. The identification of the proteolytic component of the microbiota has been determined through the sequencing of the amplicones of the gene encoding for the 16S RNA. The fermentations have been carried out using different concentrations of the initial inoculum in order to give a competitive advantage to the bacteria present in higher concentration or to those present at lower concentration. Comparing the culture relative abundances at the beginning of the process and after 6 and 12 h it was observed an increase of the bacteria belonging to the Clostridiales order and to the Alphaproteobacteria, Deltaproteobacteria and Gammaproteobacteria class particularly. It is important to observe that the majority of the bacterial groups is identified at genus or cluster level. For this reason this study needs further functional validation carried out on pure cultures that can be reported to precise species. In the last part of this project it was studied the chemical and microbiological composition of the feces of two cohorts composed of 10 healthy subjects and 8 patients affected by chronic kidney disease. The pH was significantly higher into the patients groups than the healthy ones, while for the ammonium, indole and p-cresol differences were not observed. The carbohydrates concentrations were higher in the feces of the patients than in those of the volunteers. On the same samples related to the healthy volunteers cohort, the transformation of the indole and p-cresol and the transformation of their precursors (tryptophan and tyrosine) into indole and p-cresol through experiments with resting cells have been carried out. The p-cresol is recalcitrant and it is barely degraded, while the indole transformation seems to be induced by the presence of the higher concentrations of the same metabolite. Both metabolites were quickly accumulated in the presence of the precursors tryptophan and tyrosine.