Fermentation of xylo-oligosaccharides by Bifidobacterium adolescentis DSMZ 18350: kinetics, metabolism, and ß-xylosidase activities.

Xylo-oligosaccharides (XOS) are sugar oligomers of ß-1,4-linked xylopyranosyl moieties which exert bifidogenic effect and are increasingly used as prebiotics. The kinetics and the metabolism of Bifidobacterium adolescentis DSMZ 18350 growing on XOS and xylose were investigated. The growth rate was higher on XOS, but greater biomass yield was attained on xylose. Unlike other prebiotics, XOS oligomers were utilized simultaneously, regardless of their chain length. Throughout XOS utilization, xylose concentration slightly increased, being not neatly consumed and remaining unfermented. During growth on XOS, ß-xylosidase activity was present in the cytosol, but it occurred in the supernatant as well. A ß-1,4-xylolytic enzyme was purified from the supernatant of XOS cultures. The enzyme, a homotetramer of a 39-kDa single protein, was capable of complete XOS hydrolysis and exhibited maximum activity at pH 6.0 and 55 °C. Based on the molecular weight, the protein can be ascribable to the product of the gene BAD_1527, the activity of which has been inferred as an endo-ß-1,4-xylanase, but has not been characterized so far. This ß-1,4-xylolytic enzyme, found to be active in the cultural supernatant, gives a reason for the never explained accumulation of the monosaccharides in the media of bifidobacterial cultures growing on XOS, without excluding the major role of the intracellular hydrolysis of the imported oligomers.

Administration of folate-producing bifidobacteria enhances folate status in Wistar rats.

To develop a probiotic that provides the host with folate, we administered folate-overproducing bifidobacteria (Bifidobacteria adolescentis MB 227, B. adolescentis MB 239, and B. pseudocatenulatum MB 116) to Wistar rats with induced folate deficiency. Four groups of rats were fed a solid, low-folate diet with no supplements, folate-producing bifidobacteria [probiotic (PRO)], oligofructose [prebiotic (PRE)], or PRO plus PRE [symbiotic (SYM)] for 14 d. The SYM group also had a significantly higher (16.4 +/- 3.7 nmol/L) than in the PRO group (9.1 +/- 0.3 nmol/L), which was greater than in the control (4.8 +/- 0.5 nmol/L) and PRE groups (5.3 +/- 1.4 nmol/L). The SYM group also had a significantly higher hepatic folate concentration than in the other groups, whereas the kidney folate concentration did not differ among the groups. In the unsupplemented group, the pH of feces did not change during the trial, whereas diets containing bifidobacteria and/or oligofructose led to significant acidification due to enhanced saccharolytic metabolism. As a consequence of feeding rats PRE, PRO, and SYM diets, lactobacilli and bifidobacteria were significantly greater than in controls, whereas coliforms and enterococci were lower. This experiment showed that B. adolescentis MB 227, B. adolescentis MB 239, and B. pseudocatenulatum MB 116 exert both the beneficial effects of probiotics and produce folate in vivo, positively affecting the folate status of rats. The simultaneous administration of oligofructose and folate-producing bifidobacteria enhance their effectiveness on folate status. This study provides new perspectives on the specific use of probiotics to deliver important vitamins such as folate.

Zinc uptake by lactic Acid bacteria.

The study aims to investigate zinc biosorption by strains of lactobacilli and bifidobacteria with a view to exploit them as organic matrixes for zinc dietary supplementation. Sixteen human strains of Lactobacillus and Bifidobacterium were assayed for zinc uptake. The minimum inhibitory concentration of zinc salts differed among the strains, but was never below 15 mmol L(-1). When cultured in MRS broth containing 10 mmol L(-1) ZnSO4, all the strains were capable of accumulating zinc in the range between 11 and 135 µmol g(-1). The highest amount of cell-bound zinc was obtained in L. acidophilus WC 0203. pH-controlled batch cultures of this strain revealed that zinc uptake started in the growth phase, but occurred mostly during the stationary phase. Pasteurized and viable cultures accumulated similar amount of zinc, suggesting that a nonmetabolically mediated mechanism is involved in zinc uptake. These results provide new perspectives on the specific use of probiotics, since L. acidophilus WC 0203 could function as an organic matrix for zinc incorporation. The bioavailability of Lactobacillus-bound zinc deserves to be investigated to provide a future basis for optimization of zinc supplementation or fortification.

Folate production by bifidobacteria as a potential probiotic property.

The ability of 76 Bifidobacterium strains to produce folate was investigated. In order to evaluate folic acid productivity, bifidobacteria were cultivated in the folate-free semisynthetic medium SM7. Most of the tested strains needed folate for growth. The production and the extent of vitamin accumulation were not a function of species but were distinctive features of individual strains. Six strains among the 17 that grew without folate produced significantly higher concentrations of vitamin (between 41 and 82 ng ml(-1)). The effects of exogenous folate and p-aminobenzoic acid (PABA) concentrations on folate production were evaluated. In contrast to most of the other strains, the folate yield of B. adolescentis MB 239 was not negatively affected by either PABA or exogenous folic acid. Folate production by B. adolescentis MB 239 was studied in the pH range of the colonic environment, and a comparison of folate production on raffinose, lactose, and fructo-oligosaccharides, which belong to three important groups of fermentable intestinal carbon sources, was established. Differences in folate biosynthesis by B. adolescentis MB 239 were not observed as a function either of the pH or of the carbon source. Fecal culture experiments demonstrated that the addition of B. adolescentis MB 239 may increase the folate concentration in the colonic environment.

Kinetics and metabolism of Bifidobacterium adolescentis MB 239 growing on glucose, galactose, lactose, and galactooligosaccharides.

The kinetics and the metabolism of Bifidobacterium adolescentis MB 239 growing on galactooligosaccharides (GOS), lactose, galactose, and glucose were investigated. An unstructured unsegregated model for growth in batch cultures was developed, and kinetic parameters were calculated with a recursive algorithm. The growth rate and cellular yield were highest on galactose, followed by lactose and GOS, and were lowest on glucose. Lactate, acetate, and ethanol yields allowed the calculation of carbon fluxes toward fermentation products. Distributions between two- and three-carbon products were similar on all the carbohydrates (55 and 45%, respectively), but ethanol yields were different on glucose, GOS, lactose, and galactose, in decreasing order of production. Based on the stoichiometry of the fructose-6-phosphate shunt and on the carbon distribution among the products, the ATP yield was calculated. The highest yield was obtained on galactose, while the yields were 5, 8, and 25% lower on lactose, GOS, and glucose, respectively. Therefore, a correspondence among ethanol production, low ATP yields, and low biomass production was established, demonstrating that carbohydrate preferences may result from different distributions of carbon fluxes through the fermentative pathway. During the fermentation of a GOS mixture, substrate selectivity based on the degree of polymerization was exhibited, since lactose and the trisaccharide were the first to be consumed, while 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 beta(1-4) galactosides can be hydrolyzed before they are taken up.

Fermentative production of superoxide dismutase with Kluyveromyces marxianus.

This work sought to develop a fermentative process for the microbial production of superoxide dismutase (SOD), to overcome extraction from animal tissues. Twenty-eight wild-type yeast strains were screened for SOD productivity. Kluyveromyces marxianus L3 showed the highest SOD activity (62 U mg(-1)) and was used for process development. Oxidative stress conditions and parameters affecting oxygen transfer rate were exploited to improve production. The effects of dilution rate (0.067 vs 0.2 h(-1)), aeration pressure (0.3 vs 1.2 bar) and H(2)O(2) (0 vs 50 mM) were studied during chemostat experiments. Low dilution rate, high pressure and H(2)O(2) resulted in an increase in CuZn-SOD up to 475 U mg(-1). When a regulation of oxygen saturation was applied during batch cultures, CuZn-SOD was progressively higher at 60, 80 and 90% dissolved oxygen tension (DOT) (250, 330 and 630 U mg(-1), respectively). Furthermore, the highest growth rate and biomass yield were achieved at 90% DOT, this being therefore the best DOT condition for high overall productivity. Growth and productivity on different carbon sources were compared. Specific activity was higher on glycerol than on lactose or glucose (496, 454 and 341 U mg(-1), respectively). The highest biomass yield was achieved on lactose. It may be therefore the best substrate for SOD production.

Substrate preference of Bifidobacterium adolescentis MB 239: compared growth on single and mixed carbohydrates.

The utilization of mono-, di-, and oligosaccharides by Bifidobacterium adolescentis MB 239 was investigated. Raffinose, fructooligosaccharides (FOS), lactose, and the monomeric moieties glucose and fructose were used. To establish a hierarchy of sugars preference, the kinetics of growth and sugar consumption were determined on individual and mixed carbohydrates. On single carbon sources, higher specific growth rates and cell yields were attained on di- and oligosaccharides compared to monosaccharides. Analysis of the carbohydrates in steady-state chemostat cultures, growing at the same dilution rate on FOS, lactose, or raffinose, showed that monomeric units and hydrolysis products were present. In chemostat cultures on individual carbohydrates, B. adolescentis MB 239 simultaneously displayed alpha-galactosidase, beta-galactosidase, and beta-fructofuranosidase activities on all the sugars, including monosaccharides. Glycosyl hydrolytic activities were found in cytosol, cell surface, and growth medium. Batch experiments on mixtures of carbohydrates showed that they were co-metabolized by B. adolescentis MB 239, even if different disappearance kinetics were registered. When mono-, di-, and oligosaccharides were simultaneously present in the medium, no precedence for monosaccharides utilization was observed, and di- and oligosaccharides were consumed before their constitutive moieties.

Fermentation of fructooligosaccharides and inulin by bifidobacteria: a comparative study of pure and fecal cultures.

The utilization of fructooligosaccharides (FOS) and inulin by 55 Bifidobacterium strains was investigated. Whereas FOS were fermented by most strains, only eight grew when inulin was used as the carbon source. Residual carbohydrates were analyzed by high-performance anion-exchange chromatography with pulsed amperometric detection after batch fermentation. A strain-dependent capability to degrade fructans of different lengths was observed. During batch fermentation on inulin, the short fructans disappeared first, and then the longer ones were gradually consumed. However, growth occurred through a single uninterrupted exponential phase without exhibiting polyauxic behavior in relation to the chain length. Cellular beta-fructofuranosidases were found in all of the 21 Bifidobacterium strains tested. Four strains were tested for extracellular hydrolytic activity against fructans, and only the two strains which ferment inulin showed this activity. Batch cultures inoculated with human fecal slurries confirmed the bifidogenic effect of both FOS and inulin and indicated that other intestinal microbial groups also grow on these carbon sources. We observed that bifidobacteria grew by cross-feeding on mono- and oligosaccharides produced by primary inulin intestinal degraders, as evidenced by the high hydrolytic activity of fecal supernatants. FOS and inulin greatly affected the production of short-chain fatty acids in fecal cultures; butyrate was the major fermentation product on inulin, whereas mostly acetate and lactate were produced on FOS.