Metabolismo di oligosaccaridi prebiotici in Bifidobacterium per il potenziale sviluppo di nuovi prodotti alimentari funzionali

The growth and the metabolism of Bifidobacterium adolescentis MB 239 fermenting GOS, lactose, galactose, and glucose were investigated. An unstructerd unsegregated model for growth of B. adolescentis MB 239 in batch cultures was developed and kinetic parameters were calculated with a Matlab algorithm. Galactose was the best carbon source; lactose and GOS led to lower growth rate and cellular yield, but glucose was the poorest carbon source. Lactate, acetate and ethanol yields allowed calculation of the carbon fluxes toward fermentation products. Similar distribution between 3- and 2-carbon products was observed on all the carbohydrates (45 and 55%, respectively), but ethanol production was higher on glucose than on GOS, lactose and galactose, in decreasing order. Based on the stoichiometry of the fructose 6-phosphate shunt and on the carbon distribution among the products, ATP yield was calculated on the different carbohydrates. ATP yield was the highest on galactose, while it was 5, 8, and 25% lower on lactose, GOS, and glucose, respectively. Therefore, a correspondance among ethanol production, low ATP yields, and low biomass production was established demonstrating that carbohydrate preferences may result from different sorting of carbon fluxes through the fermentative pathway. During GOS fermentation, stringent selectivity based on the degree of polymerization was exhibited, since lactose and the trisaccharide were first to be consumed, and a delay was observed until longer oligosaccharides were utilized. Throughout the growth on both lactose and GOS, galactose accumulated in the cultural broth, suggesting that β-(1-4) galactosides can be hydrolysed before they are taken up. The physiology of Bifidobacterium adolescentis MB 239 toward xylooligosaccharides (XOS) was also studied and our attention was focused on an extracellular glycosyl-hydrolase (β-Xylosidase) expressed by a culture of B. adolescentis grown on XOS as sole carbon source. The extracellular enzyme was purified from the the supernatant, which was dialyzed and concentrated by ultrafiltration. A two steps purification protocol was developed: the sample was loaded on a Mono-Q anion exchange chromatography and then, the active fractions were pooled and β-Xylosidase was purified by gel filtration chromatography on a Superdex-75. The enzyme was characterized in many aspects. β- Xylosidase was an homo-tetramer of 160 kDa as native molecular mass; it was a termostable enzyme with an optimum of temperature at 53 °C and an optimum of pH of 6.0. The kinetics parameter were calculated: km = 4.36 mM, Vmax = 0.93 mM/min. The substrate specificity with different di-, oligo- and polysaccharides was tested. The reactions were carried out overnight at pH 7 and at the optimum of temperature and the carbohydrates hydrolysis were analyzed by thin layer chromatography (TLC). Only glycosyl-hydrolase activities on XOS and on xylan were detected, whereas sucrose, lactose, cellobiose, maltose and raffinose were not hydrolyzed. It’s clearly shown that β-Xylosidase activity was higher than the Xylanase one. These studies on the carbohydrate preference of a strain of Bifidobacterium underlined the importance of the affinity between probiotics and prebiotics. On the basis of this concept, together with Barilla G&R f.lli SpA, we studied the possibility to develop a functional food containing a synbiotic. Three probiotic strains Lactobacillus plantarum BAR 10, Streptococcus thermophilus BAR 20, and Bifidobacterium lactis BAR 30 were studied to assess their suitability for utilization in synbiotic products on the basis of antioxidative activity, glutathione production, acid and bile tolerance, carbohydrates fermentation and viability in food matrices. Bile and human gastric juice resistance was tested in vitro to estimate the transit tolerance in the upper gastrointestinal tract. B. lactis and L. plantarum were more acid tolerant than S. thermophilus. All the strains resisted to bile. The growth kinetics on 13 prebiotic carbohydrates were determined. Galactooligosaccharides and fructo-oligosaccharides were successfully utilized by all the strains and could be considered the most appropriate prebiotics to be used in effective synbiotic formulations. The vitality of the three strains inoculated in different food matrices and maintained at room temperature was studied. The best survival of Lactobacillus plantarum BAR 10, Streptococcus thermophilus BAR 20, and Bifidobacterium lactis BAR 30 was found in food chocolate matrices. Then an in vivo clinical trial was carried out for 20 healthy volunteers. The increase in faecal bifidobacteria and lactobacilli populations and the efficacy of the pre-prototype was promising for the future develop of potential commercial products.

Classificazione su base molecolare dei tumori mammari della cagna mediante metodica immunoistochimica

Background. A new classification system of human breast tumours based on the immunohistochemical characterization has been applied to mammary tumours of the female dog with the aim to verify its association with invasion and grade, and prognostic aid in veterinary medicine. Methods. Forty-five canine mammary carcinomas with a two-year post-mastectomy follow-up were selected from our database, and the following antibodies were applied: anti-cytokeratines 14, 5/6, oestrogen receptor (ER), progesterone receptor (PR), and ERB-B2. . The tumours were grouped for phenotype as: luminal-like (ER+ and/or PR+, CK14-, CK5/6-) type A (ERB-B2-), and B (ERB-B2+); basal-like (ER-, PR-, CK14+ and/or CK5/6+, ERB-B2-); ERB-B2 (ER-, PR-, CK14-, CK5/6-, ERB-B2+). Association with invasion, grade and histotypes were evaluated and Kaplan-Meier survival curves estimated, then compared by survival analysis. Results. Thirty-five cases with luminal pattern (ER+ and PR+) were subgrouped into 13 A type and 22 B type, if ERB-B2 positive or negative . Most luminal-like A and basal-like cases were grade 1 carcinomas, while the percentage of luminal B cases was higher in grade 2 and 3 (Pearson Chi-square P=0.009). No difference in the percentage of molecular subtypes was evidenced between simple and complex/mixed carcinomas (Pearson Chi-square P=0.47). No significant results were obtained by survival analysis, even if basal-like had a more favourable prognosis than luminal-like. Conclusion. The panel of antibodies identified only 3 groups (luminal-like A and B, and basal-like) in the dog. Even though canine mammary tumours may be a model of human breast cancer, the existence of the same types of carcinoma as in the woman need to be confirmed. Canine mammary carcinomas show high molecular heterogeneity, which would benefit from a classification based on molecular differences. However, by multivariate analysis, the molecular classification appears a variable with a dependent value if compared to invasion and grade that are independent, suggesting that, at present, caution should be used in the application of such a classification to the dog, in which invasion and grade supply the most important prognostic information.

Analysis of Two Component Systems in Group B Streptococcus Shows that RgfAC and the Novel FspSR Modulate Virulence and Bacterial Fitness

Group B Streptococcus (GBS), in its transition from commensal to pathogen, will encounter diverse host environments and thus require coordinately controlling its transcriptional responses to these changes. This work was aimed at better understanding the role of two component signal transduction systems (TCS) in GBS pathophysiology through a systematic screening procedure. We first performed a complete inventory and sensory mechanism classification of all putative GBS TCS by genomic analysis. Five TCS were further investigated by the generation of knock-out strains, and in vitro transcriptome analysis identified genes regulated by these systems, ranging from 0.1-3% of the genome. Interestingly, two sugar phosphotransferase systems appeared differently regulated in the knock-out mutant of TCS-16, suggesting an involvement in monitoring carbon source availability. High throughput analysis of bacterial growth on different carbon sources showed that TCS-16 was necessary for growth of GBS on fructose-6-phosphate. Additional transcriptional analysis provided further evidence for a stimulus-response circuit where extracellular fructose-6-phosphate leads to autoinduction of TCS-16 with concomitant dramatic up-regulation of the adjacent operon encoding a phosphotransferase system. The TCS-16-deficient strain exhibited decreased persistence in a model of vaginal colonization and impaired growth/survival in the presence of vaginal mucoid components. All mutant strains were also characterized in a murine model of systemic infection, and inactivation of TCS-17 (also known as RgfAC) resulted in hypervirulence. Our data suggest a role for the previously unknown TCS-16, here named FspSR, in bacterial fitness and carbon metabolism during host colonization, and also provide experimental evidence for TCS-17/RgfAC involvement in virulence.