raffinose and fructooligosaccharide

Fructans, homopolymers of fructose produced by fructosyltransferases (FTs), are emerging as intriguing components in halophiles since they are thought to be associated with osmotic stress tolerance and overall fitness of microorganisms and plants under high-salinity conditions. Here, we report on the full characterization of the first halophilic FT, a levansucrase from Halomonas smyrnensis AAD6

Topics: Amino Acid Sequence; Escherichia coli; Fructans; Fructose; Halomonas; Hexosyltransferases; Kinetics; Oligosaccharides; Raffinose; Recombinant Proteins; Sequence Alignment; Sucrose

We investigated the effects of dietary supplementation of difructose anhydride III (DFA III), raffinose (Raf), and fructooligosaccharides (FOS) on diet-induced obesity development. Male rats were fed normal or high-fat and high-sucrose (HFS) diet, with or without supplementing (3%) DFA III, Raf, or FOS, for 8 or 5 weeks. Supplementing DFA III to the HFS diet decreased energy intake compared to the non-supplemented HFS diet. Accordingly, body weight gain and fat accumulation reduced in DFA III-fed rats. Cecal acetate production and plasma glucagon-like peptide-1 (GLP-1) and peptide-YY (PYY) were elevated in DFA III-fed rats, while Raf and FOS partially affected these parameters. These results demonstrate that DFA III has suppressive effect on excessive energy intake driven by the palatable obesogenic diet, possibly due to combined effects of increased anorexigenic factors such as cecal acetate production and GLP-1/PYY secretion.

Topics: Animals; Cecum; Diet, High-Fat; Dietary Sucrose; Disaccharides; Energy Intake; Fermentation; Hormones; Male; Oligosaccharides; Raffinose; Rats; Rats, Sprague-Dawley

Although diet has an important influence on the composition of gut microbiota, the impact of dietary protein sources has only been studied to a minor extent. In this study, we examined the influence of different dietary protein sources regarding the effects of prebiotic oligosaccharides on the composition and metabolic activity of gut microbiota. Thirty female rats were fed casein and soy protein isolate with cellulose, raffinose (RAF), and fructooligosaccharides (FOS). Microbiota composition was examined by real-time qPCR and denaturing gradient gel electrophoresis. Dietary protein source affected cecum microbiota; acetic acid concentration and Lactobacillus spp. populations were greater with soy protein than with casein. Prebiotic oligosaccharides had distinctive effects on gut microbiota; RAF increased the acetic acid concentration and Bifidobacterium spp. populations, and FOS increased the butyric acid concentration regardless of the dietary protein. Likewise, Bifidobacterium sp., Collinsella sp., and Lactobacillus sp. were detected in microbiota of the rats fed RAF, and Bacteroides sp., Roseburia sp., and Blautia sp. were seen in microbiota of the rats fed FOS. Interactions between dietary proteins and prebiotic oligosaccharides were observed with Clostridium perfringens group populations and cecum IgA concentration. RAF and FOS decreased C. perfringens group populations in casein-fed rats, and the combination of soy protein and RAF substantially increased cecum IgA concentration. These results indicate that dietary proteins can differentially modulate the effects of prebiotic oligosaccharides on gut fermentation and microbiota, depending on the type of carbohydrate polymers involved.

Topics: Animals; Bacteria; Bifidobacterium; Butyric Acid; Caseins; Cecum; Denaturing Gradient Gel Electrophoresis; Diet; Dietary Proteins; Feces; Female; Fermentation; Gastrointestinal Microbiome; Lactobacillus; Microbiota; Oligosaccharides; Prebiotics; Raffinose; Rats; Soybean Proteins

Fructan-exopolysaccharides (fructan-EPS) (inulin and levan) and their oligosaccharides (fructooligosaccharides, FOS) have drawn considerable interest in the food and pharmaceutical industries. EPS-producing lactic acid bacteria have been reported to produce β-fructans (inulin and levan), as well as α-glucans, by the function of sucrase enzymes, i.e., fructansucrase and glucansucrase. A fructansucrase ftfCNC-2(1) gene from Weissella confusa strain MBFCNC-2(1) was previously cloned in Escherichia coli. In this study, we aimed to express the ftf[CNC-2(1)] gene in Bacillus subtilis to obtain the active form of the extracellular recombinant protein FTF[CNC-2(1)]. This cloning was achieved by inserting the gene in-fusion with the signal sequence of the B. subtilis subtilisin E. SDS-polyacrylamide gel electrophoresis analysis and in situ activity assay with Periodic Acid-Schiff staining revealed that the recombinant FTF[CNC-2(1)] was successfully expressed as an extracellular protein from B. subtilis DB403 in its active form, which was confirmed using sucrose and raffinose.

Topics: Bacillus subtilis; Cloning, Molecular; Extracellular Space; Fructans; Inulin; Oligosaccharides; Protein Sorting Signals; Raffinose; Recombinant Proteins; Subtilisins; Sucrase; Sucrose; Weissella

Pseudomonas syringae pathovars possess multiple levansucrases with still unclear specific roles for bacteria. We have cloned and expressed three levansucrase genes, lsc1, lsc2 and lsc3, from P. syringae DC3000 in Escherichia coli. Levansucrases synthesize a high molecular weight fructan polymer, levan, from sucrose and in the case of some levansucrases, fructooligosaccharides (FOS) with potential prebiotic effects are also produced. The ability of purified Lsc3 protein of DC3000 to synthesize FOS was tested using prolonged incubation time and varied concentrations of sugar substrates. Thin-layer chromatography (TLC) analysis of reaction products disclosed formation of FOS from both sucrose and raffinose, revealing a new catalytic property for P. syringae levansucrases. In order to analyze Lsc3-produced FOS in underivatized form, we optimized a novel method recently introduced in carbohydrate research, based on fully automated chip-based nanoelectrospray ionization (nanoESI) high-capacity ion trap mass spectrometry (HCT-MS). Uding chip-based nanoESI MS in negative ion mode, FOS, with degrees of polymerization up to five, were detected in reaction mixtures of Lsc3 with sucrose and raffinose. For confirmation, further structural analysis by tandem mass spectrometry (MS/MS) employing collision-induced dissociation at low energies was performed. To validate the method, commercial inulin-derived FOS preparations Orafti P95 and Orafti Synergy1, which are currently used as prebiotics, were used as controls. By chip-based nanoESI HCT-MS, similar FOS distribution was observed in these reference mixtures. Thereby, the obtained data allowed us to postulate that FOS produced by the Lsc3 protein of P. syringae DC3000 may be prebiotic as well.

Topics: Bacterial Proteins; Chromatography, Thin Layer; Escherichia coli; Gene Expression Regulation, Bacterial; Hexosyltransferases; Microchip Analytical Procedures; Molecular Structure; Oligosaccharides; Pseudomonas syringae; Raffinose; Spectrometry, Mass, Electrospray Ionization; Sucrose; Tandem Mass Spectrometry

Lactobacillus sanfranciscensis is a key organism of the lactic microflora in traditional and industrial sourdough fermentations. In this paper we provide evidence for the formation of heterooligosaccharides (HeOS) by L. sanfranciscensis during growth in sourdough. To identify the HeOS based on HPAEC-PAD analysis, HeOS standards were synthesized by enzymatic reactions with L. sanfranciscensis levansucrase in a chemically defined system in the presence of raffinose, maltotriose, maltose, xylose, or arabinose as acceptor carbohydrates. The oligosaccharides known to originate from the corresponding acceptor reactions, 1(F)-beta-fructosylraffinose, 1(F)-beta-fructofuranosylmaltotriose, erlose (1(F)-beta-fructofuranosylmaltose), xylsucrose, 1(F)-beta-fructosylxylsucrose, and arabsucrose, were identified by HPAEC-PAD. Evidence for the formation of further tri-, tetra-, and pentasaccharides was provided. Wheat doughs with sucrose were fermented with L. sanfranciscesis TMW 1.392 or the isogenic, levansucrase-negative strain TMW 1.392Deltalev, and the analysis of dough extracts or invertase-treated dough extracts provided evidence for the formation of arabsucrose and erlose in sourdough in addition to 1-kestose and nystose.

Topics: Arabinose; Bread; Fermentation; Lactobacillus; Maltose; Oligosaccharides; Raffinose; Sucrose; Trisaccharides; Triticum; Xylose

An Ussing chamber technique was used to determine the effects of six indigestible saccharides on net Ca absorption from the luminal side to the basolateral side of isolated preparations of rat jejunal, ileal, cecal and colonic epithelium in vitro. The concentrations of Ca in the Tris buffer solution on the serosal side and on the mucosal side were 1.25 and 10 mmol/L, respectively. After a 30-min incubation, the Ca concentration in the serosal medium was determined and the net transepithelial Ca transport was calculated. The addition of 0.1-200 mmol/L maltitol, difructose anhydride (DFA)III, DFAIV, raffinose, fructooligosaccharide (FOS) or polydextrose (PD) to the mucosal medium increased the net Ca absorption dose-dependently in the jejunum, ileum, cecum and colon preparations. The threshold concentration required to enhance Ca transport and the extent of enhancement of Ca transport differed among the saccharides tested and among the portions of the intestine examined. Among the saccharides tested, DFA IV had the strongest effect on Ca absorption in the jejunum and cecum. We conclude that indigestible carbohydrates directly affect the epithelial tissue and promote Ca absorption in both the small and large intestine in vitro.

Topics: Animals; Biological Transport; Calcium; Cecum; Colon; Digestion; Epithelium; Fructose; Glucans; Ileum; Intestinal Mucosa; Intestine, Large; Intestine, Small; Jejunum; Male; Maltose; Oligosaccharides; Polysaccharides; Raffinose; Rats; Rats, Sprague-Dawley; Sugar Alcohols