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PubMed1989

Production and partial characterization of the extracellular polysaccharides from oral Streptococcus salivarius.

The production of polysaccharides from sucrose by extracellular enzymes from oral Streptococcus salivarius isolates and the physico-chemical properties of water-insoluble products (IPs) were investigated. Extracellular enzymes from all the 18 strains tested produced insoluble alpha-D-glucans (IGs) as well as soluble beta-D-fructans, and formed adhering deposits on glass. Generally, the IPs (mostly IGs) of S. salivarius strains differed from the S. sobrinus IPs by (a) containing significant proportions of alpha-D-(1----4)-, in addition to alpha-D-(1----3)- and alpha-D-(1----6)-glucosyl linkages, and much higher proportions of alpha-D-(1----3) than alpha-D-(1----6) linkages, (b) being more susceptible to hydrolysis by mutanase than by dextranase, (c) possessing low or no streptococcal cell-agglutinating ability, and (d) showing weaker adhesion to a glass surface. The degree of the polysaccharide adherence differed greatly among the S. salivarius strains and, therefore, they were divided into three groups of adherence producers; heavy, moderate, and slight. The IPs of the three groups contained, generally in descending order, a higher proportion of higher-molecular-weight fractions, and consisted of higher proportions of IG containing higher proportions of -(1----6)-alpha-D and -(1----4)-alpha-D glucosyl linkages and (1----3,6) branches, but showed higher susceptibility to hydrolysis by mutanase as well as dextranase. Thus, the production and the properties of extracellular insoluble alpha-D-glucans from sucrose differ considerably between oral S. salivarius and cariogenic S. sobrinus.

Agglutination↗

PubMed1989

Genetic and antigenic comparison of Streptococcus mutans fructosyltransferase and glucan-binding protein.

The genes for fructosyltransferase (ftf) and glucan-binding protein (gbp) of Streptococcus mutans strain Ingbritt have been cloned in E. coli using bacteriophage and plasmid vectors. A single ftf gene appears to be responsible for the appearance of several FTF of different electrophoretic mobilities. Despite the fact that both ftf and gbp proteins synthesise fructans from sucrose in S. mutans and shown closely similar electrophoretic mobilities and isoelectric points, the restriction maps of the two genes are distinct, their DNAs do not hybridise and there is no immunological cross-reaction between the two proteins.

Antigens, Bacterial↗

PubMed1995

Characterization of levJ, a sucrase/fructanase-encoding gene from Actinomyces naeslundii T14V, and comparison of its product with other sucrose-cleaving enzymes.

A library of Actinomyces naeslundii T14V DNA was constructed in plasmid pUC18 and from this several sucrose-positive clones were isolated. Evidence was obtained that all these clones contained the same gene. One clone, which carried a plasmid that was named pPNG102, was chosen for further study. It was found that the enzyme specified by this plasmid hydrolyzed sucrose, raffinose, inulin and levan, but not dextran, and did not synthesize fructan or glucan from sucrose. The sequence of the insert in pPNG102 was determined and was found to contain a large ORF that specifies a polypeptide of 99,319 Da with similarity to other sucrases. This gene was named levJ. The deduced amino acid (aa) sequence contained both a potential signal sequence and potential C-terminal cell envelope attachment domain. Alignments revealed an internal 331-aa domain not present in other levanases and sucrases. A neighbour-joining tree showed that sucrases of eukaryotic origin form a cluster with eubacterial sucrase/fructanases, and this cluster does not include other eubacterial sucrases. It is postulated that certain eukaryotic sucrase-encoding genes are of eubacterial origin.

Actinomyces↗

PubMed2004

Structure of polysaccharide from Polygonatum cyrtonema Hua and the antiherpetic activity of its hydrolyzed fragments.

A neutral polysaccharide named PD was isolated from the traditional Chinese medicinal herb, Polygonatum cyrtonema Hua. Five fragments were isolated by Bio-Gel P4 chromatography from hydrolysates of PD. Using assays of cytopathic effect inhibition, neutral red dye uptake and plaque forming inhibition, it was proved that the fragments with degree of polymerization (DP) of 4 and 5 were the shortest ones which retained the activity against herpes simplex virus type 2 (HSV-2) in vero cell culture. The structures of PD and one of its activity-retaining fragments, B3, were determined by permethylation followed with reductive cleavage, mass spectrometry and nuclear magnetic resonance spectrometry. It was shown that PD was a branched fructan with average DP of 28. There was one two-residue side chain composed of (2 --> 6)-linked beta-d-fructofuranosyl (Fruf) residues every three (2 --> 1)-linked beta-d-Fruf residues in the backbone of PD, whereas B3 was a mixture containing 1-kestose and neokestose series of oligosaccharides of DP 3-5 without branches.

Animals↗

PubMed2005

Inhibition of the expression and activity of cyclooxygenase-2 by chicory extract.

Chicory is a major source of fructans with reported prebiotic-bifidogenic properties. In the present study, the potential anti-inflammatory activities of chicory were investigated. Ethyl acetate chicory root extract produced a marked inhibition of prostaglandin E(2) (PGE(2)) production in human colon carcinoma HT29 cells treated with the pro-inflammatory agent TNF-alpha. Two independent mechanisms of action were identified: (1) a drastic inhibition of the induction by TNF-alpha of cyclooxygenase 2 (COX-2) protein expression and (2) a direct inhibition of COX enzyme activities with a significantly higher selectivity for COX-2 activity. The inhibition of TNF-alpha-dependent induction of COX-2 expression was mediated by an inhibition of NF-kappaB activation. A major sesquiterpene lactone of chicory root, the guaianolide 8-deoxylactucin, was identified as the key inhibitor of COX-2 protein expression present in chicory extract. Altogether, the data presented strongly support chicory root as a promising source of functional food ingredient, combining prebiotic and anti-inflammatory properties.

Anti-Inflammatory Agents↗

PubMed2005

Mutational analysis of the active center of plant fructosyltransferases: Festuca 1-SST and barley 6-SFT.

The active center of the glycoside hydrolase family 32 contains the three characteristic motifs (N/S)DPNG, RDP, and EC. We replaced the N-terminal region including the (N/S)DPNG motif of barley 6-SFT (sucrose:fructan 6-fructosyltransferase) by the corresponding region of Festuca 1-SST (sucrose:sucrose 1-fructosyltransferase). The chimeric enzyme, expressed in Pichia, retained the specificity of 6-SFT. Attempts to replace a larger piece at the N-terminus including also the RDP motif failed. A point mutation introduced in the RDP motif of 1-SST abolished enzymatic activity. Interestingly, point mutations of the EC-motif resulted in an enzyme which had lost the capability to form 1-kestose and glucose from sucrose but still accepted 1-kestose, producing fructose and sucrose as well as nystose.

Binding Sites↗

PubMed2004

Diurnal O2 and carbohydrate levels in wheat kernels during embryony.

In vitro zygotic and somatic embryogenesis procedures for wheat have been improved by simulating in ovulo nutritional, hormonal and dissolved oxygen (dO2) conditions. However, diurnal fluctuations in these conditions during early embryony are not well characterized. In this study, dO2 and water-soluble carbohydrate Levels in wheat kernels were determined after 8 h of light and 8 h of dark at approximately 6, 12 and 18 day post anthesis (DPA). Clark style O2 microelectrodes, having a tip diameter of approximately 115 microm, were inserted into intact kernels immediately distil to the developing embryo, and dO2 levels were recorded at 50 microm intervals into the center of kernels. High-performance anion exchange chromatography with pulsed amperometric detection was used to quantify carbohydrate levels in endosperm sap. dO2 levels in the chlorophyllous layer of the pericarp reached 190 mmoLm(-3) during the day, which probably represents, because of photosynthesis, a supersaturated O2 condition relative to the external environment (21% O2). At the embryo surface, dO2 levels at 6 DPA ranged from 135 to 170 mmolm(-3). At 12 and 18 DPA, dO2 levels at the embryo axis ranged from 100 to 150mmolm(-3). At all three stages, dO2 levels in the center of the endosperm were below 13 mmolm(-3). Extreme fluctuations in carbohydrate levels were observed diurnally during rapid seed fill (12DPA). Levels of sucrose and short-chain fructans were much higher during the day than during the night. In contrast, fructose, glucose, and myo-inositol levels were much higher during the night than during the day. By 18DPA (hard dough stage), carbohydrate levels tended to be similar during the day and night. These dynamic fluctuations may assist in regulating embryony in ovulo, and their simulation might improve the development of somatic and zygotic embryos in vitro.

Carbohydrate Metabolism↗

PubMed2004

A mini exon in the sucrose:sucrose 1-fructosyltransferase gene of wheat.

We previously reported the cloning of a wheat sucrose:sucrose 1-fructosyltransferase (1-SST) cDNA, designated wft2. Wft2 proteins have fructosyltransferase enzyme activity and initiate fructan synthesis (Biosci. Biotechnol. Biochem. 66 (2002) 2297). In the current study, we cloned a genomic DNA fragment carrying the full-length 1-SST gene from winter wheat (Triticum aestivum). The genomic 1-SST gene is 3326 bp in length and contains four exons and three introns. Exon 2 has only 9 bp. This sequence encodes a part of a beta-fructosidase motif (NDPNG), a highly conserved motif found in plant invertases. This is the first report of a mini exon, one of the smallest exons known in plants, being found in a genomic 1-SST gene.

Consensus Sequence↗

PubMed2005

Molecular properties and prebiotic effect of inulin obtained from artichoke (Cynara scolymus L.).

A high molecular weight inulin has been prepared from artichoke (Cynara scolymus L.) agroindustrial wastes using environmentally benign aqueous extraction procedures. Physico-chemical analysis of the properties of artichoke inulin was carried out. Its average degree of polymerization was 46, which is higher than for Jerusalem artichoke, chicory, and dahlia inulins. GC-MS confirmed that the main constituent monosaccharide in artichoke inulin was fructose and its degradation by inulinase indicated that it contained the expected beta-2,1-fructan bonds. The FT-IR spectrum was identical to that of chicory inulin. These data indicate that artichoke inulin will be suitable for use in a wide range of food applications. The health-promoting prebiotic effects of artichoke inulin were demonstrated in an extensive microbiological study showing a long lasting bifidogenic effect on Bifidobacterium bifidum ATCC 29521 cultures and also in mixed cultures of colonic bacteria.

Bifidobacterium↗

PubMed1977

Structure of the D-galactan isolated from garlic (Allium sativum) bulbs.

Hot-water extraction of defatted garlic-bulbs yielded a mixture of polysaccharides containing a D-galactan, a D-galacturonan, an L-arabinan, a D-glucan, and a D-fructan. A trace of L-rhamnose was also detected in the polysaccharide hydrolyzate. The pectic acid was partially removed by precipitation with aqueous calcium chloride; from the remaining polysaccharide mixture, a pure D-galactan containing 97.3% of D-galactose was isolated by fractional precipitation and repeated chromatography through a column of DEAE-cellulose. Methanolysis and hydrolysis of the permethylated D-galactan yielded 2,3,4,6-tetra-, 2,3,6-tri-, and 2,3,di-O-methyl-D-galactose in the molar proportions of 1:2:1. On periodate oxidation, the D-galactan reduced 1.18 molar equivalents of the oxidant per D-galactosyl residue, and liberated one molar equivalent of formic acid per 4.13 D-galactosyl residues. Smith degradation of the D-galactan was also conducted. From these results, a structure has been assigned to the repeating unit of the D-galactan.

Galactose↗

PubMed2000

Determination of inulin in meat products by high-performance liquid chromatography with refractive index detection.

Inulin is a naturally occurring carbohydrate with beneficial nutritional and technological properties. A high-performance liquid chromatographic (HPLC) method was developed for the quantitative determination of these beta-fructans in meat products, containing this type of additive. The method includes extraction of inulin with hot water, followed by hydrolysis with inulinase enzyme, and determination of the released fructose by HPLC with refractive index detection. An internal standard of rhamnose was used to quantify fructose. The method incorporates a sample blank (without inulinase hydrolysis) for each specimen to subtract contributions of free fructose and fructose from sucrose. The results showed good precision with average RSDs of 2.4% for repeatability and 5.2% for reproducibility. Analytical recovery ranged from 102 to 106%. Satisfactory linearity (r=0.999) was obtained.

Chromatography, High Pressure Liquid↗

PubMed1996

Dietary oligofructose modifies the impact of fructose on hepatic triacylglycerol metabolism.

The aim was to investigate if chronic feeding with oligofructose (OFS), a nondigestible fructan that decreases triacylglycerol-very-low-density lipoproteins (TAG-VLDLs) in the serum of rats by reducing hepatic de novo lipogenesis, could counteract the impact of fructose on TAG metabolism. Male Wistar rats fed a standard diet supplemented or not with 10% OFS for 30 days received either tap water or a 10% fructose drinking solution for 48 hours. TAG, phospholipids (PLs), cholesterol, and free fatty acids were assayed both in serum and in liver. Fatty acid de novo synthesis, esterification, and beta-oxidation were assessed in the liver by measuring the activity of key enzymes: fatty acid synthase (FAS), phosphatidate phosphohydrolase (PAP), glycerol-3-phosphate acyltransferase (GPAT), and carnitine palmitoyltransferase-I (CPT-I), respectively. The acute load of fructose increased (1) both liver and serum TAG without affecting other lipids, and (2) de novo fatty acid synthesis and esterification, through induction of FAS and PAP without affecting CPT-I. Long-term feeding with OFS protected rats against liver TAG accumulation induced by fructose. The lower lipogenic capacity of the liver could be the key event in this protection, since even after the fructose load FAS activity remained significantly lower in OFS-fed rats. However, despite its protective effect on the liver, OFS was not able to prevent fructose-induced hypertriglyceridemia, suggesting that OFS feeding could not counteract the fructose-induced defect in TAG-VLDL clearance.

Animals↗

PubMed1996

Salt tolerance in plants and microorganisms: toxicity targets and defense responses.

Salt tolerance of crops could be improved by genetic engineering if basic questions on mechanisms of salt toxicity and defense responses could be solved at the molecular level. Mutant plants accumulating proline and transgenic plants engineered to accumulate mannitol or fructans exhibit improved salt tolerance. A target of salt toxicity has been identified in Saccharomyces cerevisiae: it is a sodium-sensitive nucleotidase involved in sulfate activation and encoded by the HAL2 gene. The major sodium-extrusion system of S. cerevisiae is a P-ATPase encoded by the ENA1 gene. The regulatory system of ENA1 expression includes the protein phosphatase calcineurin and the product of the HAL3 gene. In Escherichia coli, the Na(+)-H+ antiporter encoded by the nhaA gene is essential for salt tolerance. No sodium transport system has been identified at the molecular level in plants. Ion transport at the vacuole is of crucial importance for salt accumulation in this compartment, a conspicuous feature of halophytic plants. The primary sensors of osmotic stress have been identified only in E. coli. In S. cerevisiae, a protein kinase cascade (the HOG pathway) mediates the osmotic induction of many, but not all, stress-responsive genes. In plants, the hormone abscisic acid mediates many stress responses and both a protein phosphatase and a transcription factor (encoded by the ABI1 and ABI3 genes, respectively) participate in its action.

Drug Tolerance↗

PubMed1997

Effects of elevated CO2, nitrogen supply and tropospheric ozone on spring wheat-II. Nutrients (N, P, K, S, Ca, Mg, Fe, Mn, Zn).

CO(2) enrichment is expected to alter leaf demand for nitrogen and phosphorus in plant species with C(3) carbon dioxide fixation pathway, thus possibly causing nutrient imbalances in the tissues and disturbance of distribution and redistribution patterns within the plants. To test the influence of CO(2) enrichment and elevated tropospheric ozone in combination with different nitrogen supply, spring wheat (Tritium aestivum L. cv. Minaret) was exposed to three levels of CO(2) (361, 523, and 639 microl litre(-1), 24 h mean from sowing to final harvest), two levels of ozone (28.4 and 51.3 nl litre(-1)) and two levels of nitrogen supply (150 and 270 kg ha(-1)) in a full-factorial design in open-top field chambers. Additional fertilization experiments (120, 210, and 330 kg N ha(-1)) were carried out at low and high CO(2) levels. Macronutrients (N, P, K, S, Ca, Mg) and three micronutrients (Mn, Fe, Zn) were analysed in samples obtained at three different developmental stages: beginning of shoot elongation, anthesis, and ripening. At each harvest, plant samples were separated into different organs (green and senescent leaves, stem sections, ears, grains). According to analyses of tissue concentrations at the beginning of shoot elongation, the plants were sufficiently equipped with nutrients. Elevated ozone levels neither affected tissue concentrations nor shoot uptake of the nutrients. CO(2) and nitrogen treatments affected nutrient uptake, distribution and redistribution in a complex manner. CO(2) enrichment increased nitrogen-use efficiency and caused a lower demand for nitrogen in green tissues which was reflected in a decrease of critical nitrogen concentrations, lower leaf nitrogen concentrations and lower nitrogen pools in the leaves. Since grain nitrogen uptake during grain filling depended completely on redistribution from vegetative pools in green tissues, grain nitrogen concentrations fell considerably with severe implications for grain quality. Ca, S, Mg and Zn in green tissues were influenced by CO(2) enrichment in a similar manner to nitrogen. Phosphorus concentrations in green tissues, on the other hand, were not, or only slightly, affected by elevated CO(2). In stems, 'dilution' of all nutrients except manganese was observed, caused by the huge accumulation of water soluble carbohydrates, mainly fructans, in these tissues under CO(2) enrichment. Whole shoot uptake was either remarkably increased (K, Mn, P, Mg), nearly unaffected (N, S, Fe, Zn) or decreased (Ca) under CO(2) enrichment. Thus, nutrient cycling in plant-soil systems is expected to be altered under CO(2) enrichment.

Journal Article↗

PubMed1999

Production of modified polymeric carbohydrates.

The cloning of a gene responsible for the phosphorylation of glucans has made it possible to genetically engineer the phosphorylation level of starches in higher plants. Through the manipulation of starch synthase activity, it is now also possible to genetically tailor the chain-length distribution in the amylopectin. Both findings will lead to the development of novel starches utilized as a renewable resource. The production of fructans on a large scale can also be envisioned for the near future.

Biopolymers↗

PubMed2001

Inulin determination for food labeling.

Inulin and oligofructose exhibit valuable nutritional and functional attributes, so they are used as supplements as soluble fiber or as macronutrient substitutes. As classic analytical methods for dietary fiber measurement are not effective, several specific methods have been proposed. These methods measure total fructans and are based on one or more enzymatic sample treatments and determination of released sugars. To determine inulin for labeling purposes, we developed an easy and rapid anion-exchange high-performance liquid chromatography (HPLC) method following water extraction of inulin. HPLC conditions included an Aminex HPX- 87C column (Bio-Rad), deionized water at 85 degrees C as the mobile phase and a refractive index detector. The tested foods included tailor-made food products containing known amounts of inulin and commercial products (cookies, milk, ice creams, cheese, and cereal bars). The average recovery was 97%, and the coefficient of variation ranged from 1.1 to 5% in the food matrixes. The obtained results showed that this method provides an easier, faster and cheaper alternative than previous techniques for determining inulin with enough accuracy and precision for routine labeling purposes by direct determination of inulin by HPLC with refractive index detection.

Animals↗

PubMed2001

Contrasting responses of photosynthesis and carbon metabolism to low temperatures in tall fescue and clovers.

Growth, photosynthesis and carbohydrate metabolism in plants of two grassland species, clover (Trifolium subterraneum L. cv. Areces and Gaitan) and tall fescue (Festuca arundinacea Schreb.), shifted from 25 to 12 degrees C for 1 day or developed at 12 degrees C were compared with controls kept at 25 degrees C. Cold development produced a larger inhibition of growth in fescue than in clovers. In contrast, transferring plants from high to low temperature inhibited photosynthesis to a lesser extent in fescue than in clovers, this difference being associated with an increase in the activation state of Calvin cycle enzymes in fescue, but not in the clovers, a decreased cytosolic fructose-1,6-bisphosphatase (cFBPase, EC 3.1.3.11) activity in clovers, and an accumulation of hexose phosphates only in fescue. Development at 12 degrees C partly relieved the inhibition of photosynthesis in clovers, in contrast with fescue, which correlated with increases in total ribulose-1,5-bisphosphate carboxylase oxygenase (Rubisco, EC 4.1.1.39) activity only in clovers, and with greater increases in total stromal FBPase (sFBPase) activity in clovers than in fescue. The activity of sucrose synthesis enzymes was increased in the two clovers and fescue developed in the cold, while carbohydrate accumulation was much bigger in cold-developed fescue than in clovers because of a 5-fold increase in fructan contents in the former. The contents of phosphorylated intermediates increased in clovers but decreased in fescue grown at 12 degrees C. Our results suggest that restricted ribulose-1,5-bisphosphate (RuBP) regeneration limited the recovery of photosynthetic capacity in cold-developed fescue.

Journal Article↗

PubMed2005

Effects of oligofructose on glucose and lipid metabolism in patients with nonalcoholic steatohepatitis: results of a pilot study.

OBJECTIVE: In experimental animals, recent results suggest that the addition of inulin-type fructans such as oligofructose (OFS) in the diet decreases triacylglycerol accumulation in the liver tissue. Therefore, we have investigated the effect of daily ingestion of OFS in seven patients with nonalcoholic steatohepatitis (NASH), confirmed by liver biopsies. DESIGN: They received 16 g/day OFS or maltodextrine (placebo) for 8 weeks in a randomized double-blind crossover design. Energy intake, body composition, liver steatosis and blood parameters were analysed after 4 and 8 weeks of dietary supplementation. RESULTS: Compared to placebo, OFS decreased significantly serum aminotransferases, aspartate aminotransferase after 8 weeks, and insulin level after 4 weeks, but this could not be related to significant effect on plasma lipids. CONCLUSION: This pilot study supports the putative interest of OFS in the management of liver diseases associated with abnormal lipid accumulation in humans.

Adult↗