Genus Bifidobacterium: taxonomy studies and gene expression analysis on folate pathway

Folates (vitamin B9) are essential water soluble vitamins, whose deficiency in humans may contribute to the onset of several diseases, such as anaemia, cancer, cardiovascular diseases, neurological problems as well as defects in embryonic development. Human and other mammals are unable to synthesize ex novo folate obtaining it from exogenous sources, via intestinal absorption. Recently the gut microbiota has been identified as an important source of folates and the selection and use of folate producing microorganisms represents an innovative strategy to increase human folate levels. The aim of this thesis was to gain a fundamental understanding of folate metabolism in Bifidobacterium adolescentis. The work was subdivided in three main phases, also aimed to solve different problems encountered working with Bifidobacterium strains. First, a new identification method (based on PCR-RFLP of hsp60 gene) was specifically developed to identify Bifidobacterium strains. Secondly, Bifidobacterium adolescentis biodiversity was explored in order to recognize representing strains of this species to be screened for their folate production ability. Results showed that this species is characterized by a wide variability and support the idea that a possible new taxonomic re-organization would be required. Finally B. adolescentis folate metabolism was studied using a double approach. A quantitative analysis of folate content was complemented by the examination of expression levels of genes involved in folate related pathways. For the normalization process, required to increase the robustness of the qRT-PCR analysis, an appropriate set of reference genes was tested using two different algorithms. Results demonstrate that B.adolescentis strains may represent an endogenous source of natural folate and they could be used to fortify fermented dairy products. This bio-fortification strategy presents many advantages for the consumer, providing native folate forms more bio-available, and not implicated in the discussed controversy concerning the safety of high intake of synthetic folic acid

Identification of species belonging to the Bifidobacterium genus by PCR-RFLP analysis of a hsp60 gene fragment

Abstract BACKGROUND: Bifidobacterium represents one of the largest genus within the Actinobacteria, and includes at present 32 species. These species share a high sequence homology of 16S rDNA and several molecular techniques already applied to discriminate among them give ambiguous results.The slightly higher variability of the hsp60 gene sequences with respect to the 16S rRNA sequences offers better opportunities to design or develop molecular assays, allowing identification and differentiation of closely related species. hsp60 can be considered an excellent additional marker for inferring the taxonomy of the members of Bifidobacterium genus. RESULTS: This work illustrates a simple and cheap molecular tool for the identification of Bifidobacterium species. The hsp60 universal primers were used in a simple PCR procedure for the direct amplification of 590 bp of the hsp60 sequence. The in silico restriction analysis of bifidobacterial hsp60 partial sequences allowed the identification of a single endonuclease (HaeIII) able to provide different PCR-restriction fragment length polymorphism (RFLP) patterns in the Bifidobacterium spp. type strains evaluated. The electrophoretic analyses allowed to confirm the different RFLP patterns. CONCLUSIONS: The developed PCR-RFLP technique resulted in efficient discrimination of the tested species and subspecies and allowed the construction of a dichotomous key in order to differentiate the most widely distributed Bifidobacterium species as well as the subspecies belonging to B. pseudolongum and B. animalis

Validation of candidate reference genes in Bifidobacterium adolescentis for gene expression normalization

none4noNormalization is an essential prerequisite for producing accurate real-time PCR expression analyses. The objective of this study is the selection of a set of optimal reference genes in Bifidobacterium adolescentis gene expression studies under bile exposure. B adolescentis is a particularly abundant species in the human adults gut microbiota, exerting relevant probiotic activities. In the gastrointestinal tract, bile represents a hard challenge for bacterial survival, because of its toxic effect. The natural exposure to bile in the colonic environment induces cells adaptation and tolerance mechanisms in bifidobacteria, which determines changes in gene expression profile, influencing the expression levels of housekeeping genes. In this context, the stability of 9 putative reference genes (cysS, purB, recA, rpoB-L, GADPH-R, 16S rRNA, glnA1, gyrA2, sdhA) was examined in B. adolescentis exposed to bile extract, using two different software (BestKeeper and NormFinder). Both algorithms identified gyrA2 and sdhA as the most stable genes under our experimental conditions, while 16S rRNA is the least reliable HKGs. To our best knowledge, this is the first attempt to validate reference genes in Bifidobacterium spp. and the results offer an appropriate set of reference genes suitable for qRT-PCR studies on B. adolescentis strains under bile stress.noneStenico, Verena; Baffoni, Loredana; Gaggia, Francesca; Biavati, BrunoStenico, Verena; Baffoni, Loredana; Gaggia, Francesca; Biavati, Brun

Probiotici e prebiotici nell\u2019alimentazione animale

L\u2019incidenza delle malattie trasmesse da alimenti di origine animale \ue8 in costante ascesa in tutti i Paesi industrializzati; si crea, perci\uf2, la necessit\ue0 di attuare misure di controllo sempre pi\uf9 restrittive negli allevamenti, soprattutto a seguito del divieto sull\u2019uso degli antibiotici come promotori della crescita a scopo sub-terapeutico. I principali patogeni che si ritrovano nei prodotti di origine animale, a volte veicolati nel tratto gastrointestinale degli animali in maniera asintomatica, sono: Campylobacter jejuni, Clostridium perfringens, E. coli 0157:H7, Listeria monocytogenes, Salmonella, Staphylococcus aureus. \uc8 necessario trovare, velocemente, soluzioni alternative per controllare e prevenire la diffusione dei patogeni enterici, al fine di ridurre la loro presenza nel prodotto finito e limitare la minaccia per la salute dell\u2019uomo. La modulazione del tratto gastrointestinale, attraverso l\u2019utilizzo di additivi alimentari come probiotici e prebiotici, offre una valida soluzione per mantenere gli animali in salute, ridurre il rischio di contrarre malattie gastrointestinali e migliorare la qualit\ue0 delle carni destinate al consumo umano. In questo articolo verranno discusse le pi\uf9 recenti applicazioni di probiotici e prebiotici in campo animale, volte alla riduzione della presenza di microrganismi patogeni, potenziali cause di intossicazioni alimentari, nelle carni

Identification of Bifidobacterium spp. using hsp60 PCR-RFLP analysis: An update

A PCR-RFLP technique has been applied on 13 species of Bifidobacterium in order to update a previous study carried out by Baffoni et al. [1]. This method is based on the restriction endonuclease activity of HaeIII on the PCR-amplified hsp60 partial gene sequence, and allows a rapid and efficient identification of Bifidobacterium spp. strains at species and subspecies level

Nutritional and nutraceutical aspects of KAMUT\uae khorasan wheat grown during the last two decades

Recently, organic farming systems have attracted the attention of consumers because of their low environmental impact. Organic agriculture is a valid alternative to conventional farming and ancient wheat, such as KAMUT\uae khorasan wheat (T. turgidum ssp. turanicum), has emerged as an industry leader for its nutritional and functional properties (anti-inflammatory, antioxidant and prebiotic). The aim of the present study was to evaluate environmental effects on the quality of KAMUT\uae khorasan grains harvested in the last two decades (1989\u20132012) on one farm in Montana (USA), through the evaluation of phytochemical accumulation. Results revealed high variability in the amounts of macronutrients and nutraceuticals. In particular, from 1989 to 2012, there was a decreasing trend in starch content (ranging from 70\ub787 to 50\ub754/100 g) and amylose (from 41\ub748 to 31\ub746% of total starch) with a slight increase of insoluble dietary fibre (from 12\ub714 to 17\ub775/100 g). The soluble dietary fibre content varied among the years of cultivation even if the general trend remained constant (4\ub757\u20132\ub782/100 g). High variability of total polyphenols content was observed with the free soluble fraction present at lower levels than bound polyphenols (BP). Moreover, an inverse correlation between free and BP was observed. The results obtained in the present study show that the influence of environmental conditions plays a fundamental role in the accumulation of primary and secondary metabolites in wheat kernels and strongly modulate the nutritional and nutraceutical value of flour