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PubMed2000

Involvement of sucrose synthase in sucrose synthesis during mobilization of fructans in dormant Jerusalem artichoke tubers.

The relative contribution of sucrose synthase and sucrose-phosphate synthase to sucrose synthesis in dormant tubers of Jerusalem artichokes was determined. Feeding dormant tubers alternatively with mixtures of [14C]glucose and unlabeled fructose, and [14C]glucose and [14C]fructose has shown that sucrose synthase contributes ca. 95-97% to sucrose synthesis. This is the first report of sucrose synthesis in Jerusalem artichokes dormant tubers.

Journal Article↗
PubMed2005

Effects of inulin-type fructans on lipid metabolism in man and in animal models.

Studies in rodents show that inulin and oligofructose can reduce the plasma levels of cholesterol and triacylglycerols (TG). In addition, they can oppose TG accumulation in liver and have favourable effects on hepatic steatosis. The hypotriglyceridaemic effect is due to a reduction in hepatic re-esterification of fatty acids, but mainly in the expression and activity of liver lipogenesis, resulting in lower hepatic secretion rate of TG. This repression of lipogenesis is not observed in adipose tissue. The effect on liver lipogenesis can be explained by reduced insulin/glucose levels or by a selective exposure of the liver to increased amounts of propionic acid produced in the large intestine during fermentation of non-digestible carbohydrates. The decrease in plasma cholesterol could also be due to inhibition of cholesterol synthesis by propionic acid or to modifications in the bile acid metabolism. Studies in man yield more conflicting results with a decrease or no effects on plasma lipid levels, and, when a decrease is observed, more marked effects on TG than on cholesterol and more consistent action of inulin than of oligofructose. Besides the difference in the dose of inulin or oligofructose used, differences in metabolic status could play a role in this discrepancy between man and animals since reduction in plasma TG is observed in man mainly in a situation of increased liver lipogenesis (high-carbohydrate diet, obesity, hypertriglyceridaemia). The effects on plasma cholesterol appear also more marked in hyperlipidaemic subjects than in healthy controls, suggesting that inulin and oligofructose have beneficial effects in these types of subjects.

Animals↗
PubMed2005

Methodological considerations in measuring human calcium absorption: relevance to study the effects of inulin-type fructans.

During the last 50 years, a variety of methods have been developed to estimate Ca absorption in man. Mass balances were initially used, but these were unable to accurately measure fractional Ca absorption because they cannot distinguish unabsorbed dietary Ca from endogenous faecal Ca excretion (excretion of previously absorbed Ca back into the gut). A number of isotopic methods have been developed that can measure true fractional Ca absorption, employing radioisotopes, stable isotopes, or both. Different methods involve collection of urine, faecal or plasma samples. Of the currently available methods, the dual isotope tracer method with a timed urine collection is probably the most precise and reliable. It is also relatively straightforward to carry out and avoids the need for a faecal collection. The purpose of the present paper is to discuss the general advantages and disadvantages of the different methods of Ca absorption. In addition, the limitations the different methods have in examining the possible effects of non-digestible oligosaccharides on Ca absorption will be discussed.

Calcium↗
PubMed1994

Differential localization of the Streptococcus mutans GS-5 fructan hydrolase enzyme, FruA.

Streptococcus mutans GS-5 synthesizes an exo-beta-D-fructosidase, FruA, capable of degrading levans, inulins, sucrose and raffinose, with the greatest activity on levans. A previous analysis of the deduced amino acid sequence of the FruA protein revealed the presence of a C-terminus with an LPXTGX membrane sorting sequence and membrane spanning domain, characteristic of many Gram-positive cocci surface proteins. Here it is demonstrated that FruA, which had been previously shown to exist almost exclusively as an extracellular enzyme, can be detected in significant proportions at the surface of S. mutans cells. Moreover, growth of S. mutans GS-5 at steady state in continuous culture at pH values of 7.0, 6.0, or 5.0 revealed that the amount of cell-associated enzyme increased with decreasing pH values, such that roughly 50% of the total fructanase activity of pH 5.0-grown organisms was cell-associated. This result was confirmed using anti-recombinant-FruA antisera in Western blotting of culture supernate and cell-associated enzyme preparations from chemostat-grown cells. Incubation of S. mutans at pH values of 5.0, 6.0 or 7.0 in buffered media yielded results similar to those observed in the chemostat experiments. The release of FruA from S. mutans was also shown to be inhibitable by copper, which is known to interfere with the release of the surface adhesin, P1, from intact cells and protoplasts of S. mutans. These data provide evidence for a unique post-translational mechanism for the regulation of the catabolism of polysaccharides by bacteria.(ABSTRACT TRUNCATED AT 250 WORDS)

Bacterial Outer Membrane Proteins↗
PubMed1999

Regulation of expression of the fructan hydrolase gene of Streptococcus mutans GS-5 by induction and carbon catabolite repression.

The polymers of fructose, levan and inulin, as well as sucrose and raffinose, are substrates for the product of the fruA gene of Streptococcus mutans GS-5. The purpose of this study was to characterize the DNA immediately flanking fruA, to explore the regulation of expression of fruA by the carbohydrate source, and to begin to elucidate the molecular basis for differential expression of the gene. Located 3' to fruA was an open reading frame (ORF) with similarity to beta-fructosidases which was cotranscribed with fruA. A transcriptional initiation site, located an appropriate distance from an extended -10-like promoter, was mapped at 165 bp 5' to the fruA structural gene. By the use of computer algorithms, two overlapping, stable stem-loop sequences with the potential to function as rho-independent terminators were found in the 5' untranslated region. Catabolite response elements (CREs), which have been shown to govern carbon catabolite repression (CCR) by functioning as negative cis elements in gram-positive bacteria, were located close to the promoter. The levels of production of fruA mRNA and FruA were elevated in cells growing on levan, inulin, or sucrose as the sole carbohydrate source, and repression was observed when cells were grown on readily metabolizable hexoses. Deletion derivatives containing fusions of fruA promoter regions, lacking sequences 5' or 3' to the promoter, and a promoterless chloramphenicol acetyltransferase gene were used (i) to demonstrate the functionality of the promoter mapped by primer extension, (ii) to demonstrate that CCR of the fru operon requires the CRE that is located 3' to the promoter region, and (iii) to provide preliminary evidence that supports the involvement of an antitermination mechanism in fruA induction.

Amino Acid Sequence↗
PubMed1994

Accumulation of fructose polymers in transgenic tobacco.

Fructan, a polyfructose molecule, is a storage compound in a limited number of plant species. Usually these species accumulate fructan with a low degree of polymerization (DP) and most of these plants have properties which preclude their use as a fructan source. With the eventual aim of allowing the accumulation of high DP fructans in non-fructan storing plants, we have investigated whether carbohydrate flow in the plant cell can be directed to produce this polymer. For this purpose the SacB gene from Bacillus subtilis, which encodes levansucrase, was modified and introduced into tobacco plants. Transgenic plants containing the sacB gene accumulate fructans. The size and properties of this fructan are similar to fructan produced by Bacillus subtilis, and is stable in plants. Although the level of fructan accumulation in the transgenic tobacco plants ranged from 3-8 percent of the dry weight, no levansucrase mRNA or protein could be detected in these plants. Extension of this work should permit the production of this high molecular weight biopolymer in crop plants for applications in food and non-food products.

Bacillus subtilis↗
PubMed2004

Seasonal dynamics of non-structural carbohydrates in bulbs and shoots of the geophyte Galanthus nivalis.

Seasonal dynamics of non-structural carbohydrates were studied in Galanthus nivalis L. over a 2-year period. The plants were collected in the field and separated into above- and below-ground biomass. The polysaccharide fraction of the bulbs consisted of fructans and starch. Seasonal variations suggest that the polysaccharides were utilized for carbon and energy supply for re-growth and flower development. With the re-sprouting of the bulbs in autumn the fructans within the bulbs were depolymerized and an increase of low degree of polymerization fructans as well as sucrose was observable. Within shoots the major polysaccharides were fructans, the starch content was much lower. Gas liquid chromatography and high-performance, anion-exchange chromatographyanalysis of the fructan fraction revealed that the fructans within the shoots were predominantly those with a low degree of polymerization. In addition to the two polysaccharides the other dominant sugar in shoots was sucrose. During the period of slow re-growth and flowering, fructan and starch pools were depleted to different degrees. Calculation of the difference between the carbohydrate content at the start of visible growth and at the time of lowest content revealed that the starch pool showed a higher depletion than the fructan pool. During the re-growth periods in 1996/97 and 1997/98 fructans were catabolized by 39 and 32% only, whereas the starch pool was depleted by 92% (1996/97) and 79% (1997/98), respectively. During rapid shoot growth and fruiting, the bulbs and above-ground organs appeared to be competing sinks for the photosynthetically fixed carbon. Refilling of the bulbs carbohydrate reserve started in February/March In shoots, the period of refilling the bulbs was characterized by a low content of oligosaccarides and a high content of hexoses.

Journal Article↗
PubMed2002

Specific effects of fructo- and gluco-oligosaccharides in the preservation of liposomes during drying.

The fructan family of oligo- and polysaccharides is a group of molecules that have long been implicated as protective agents in the drought and freezing tolerance of many plant species. However, it has been unclear whether fructans have properties that make them better protectants for cellular structures than other sugars. We compared the effects of fructans and glucans on membrane stability during air-drying. Although glucans of increasing chain length were progressively less able to stabilize liposomes against leakage of aqueous content after rehydration, fructans showed increased protection. On the other hand, glucans became more effective in protecting liposomes against membrane fusion with increasing chain length, whereas fructans became less effective. Fourier transform infrared spectroscopy showed a reduction of the gel to liquid-crystalline phase transition temperature (T(m)) of air-dried liposomes by approximately 25 degrees C in the presence of sucrose and maltose. For the respective pentasaccharides, the reduction of T(m) of the lipids was 9 degrees C lower for samples containing fructan than for those containing glucan, indicating increased sugar--membrane interactions for the fructan compared to the glucan. A reduced interaction of the longer-chain glucans and an increased interaction of the respective fructans with the phospholipid head groups in the dry state was also indicated by dramatic differences in the phosphate asymmetric stretch region of the infrared spectrum. Collectively, our data indicate that the fructo-oligosaccharides accumulated in many plant species under stress conditions could indeed play an important role in cellular dehydration tolerance.

Cichorium intybus↗
PubMed2002

Effects of various antiplaque agents on fructosyltransferase activity in solution and immobilized onto hydroxyapatite.

Fructosyltransferases (FTFs) are extracellular enzymes which synthesize fructans from sucrose. Cell free FTFs are found in the dental plaque biofilm as well as in saliva. Fructans play an important role in the progression of dental caries, mainly by serving as an extracellular nutrition reservoir for bacteria. The objective of the present study was to compare the effects of several antiplaque agents on the synthesis of fructans by FTF immobilized on hydroxyapatite (HA) or in solution. The effect of chlorhexidine, cetylpyridinium chloride, sodium lauryl sulfate and Tween on FTF activity was tested using radioactive assays. Their effect on fructan structure was tested using circular dichrosim-optical rotative dispersion (CD-ORD) analysis and Fourier transform infrared (FT-IR) spectroscopy. Our results show that the antiplaque agents tested had an inhibitory effect on FTF activity both in the immobilized phase and in solution, although the inhibitory effect was more pronounced in solution. Structural changes in fructans, due to the presence of the antiplque agents, were recorded as additional C-H or O-H bands demonstrated in FT-IR analysis. However, non-significant changes in peak location were detected in CD-ORD spectrum between fructans synthesized in solution and on HA surfaces, and after treatment with the different antiplaque agents. Our study shows that several antiplaque agents may affect FTF activity and the synthesis of fructans by FTF, immobilized on hydroxyapatite or in solution.

Adsorption↗
PubMed2000

Cloning and functional analysis of sucrose:sucrose 1-fructosyltransferase from tall fescue.

Enzymes of grasses involved in fructan synthesis are of interest since they play a major role in assimilate partitioning and allocation, for instance in the leaf growth zone. Several fructosyltransferases from tall fescue (Festuca arundinacea) have previously been purified (Lüscher and Nelson, 1995). It is surprising that all of these enzyme preparations appeared to act both as sucrose (Suc):Suc 1-fructosyl transferases (1-SST) and as fructan:fructan 6(G)-fructosyl transferases. Here we report the cloning of a cDNA corresponding to the predominant protein in one of the fructosyl transferase preparations, its transient expression in tobacco protoplasts, and its functional analysis in the methylotrophic yeast, Pichia pastoris. When the cDNA was transiently expressed in tobacco protoplasts, the corresponding enzyme preparations produced 1-kestose from Suc, showing that the cDNA encodes a 1-SST. When the cDNA was expressed in P. pastoris, the recombinant protein had all the properties of known 1-SSTs, namely 1-kestose production, moderate nystose production, lack of 6-kestose production, and fructan exohydrolase activity with 1-kestose as the substrate. The physical properties were similar to those of the previously purified enzyme, except for its apparent lack of fructan:fructan 6(G)-fructosyl transferase activity. The expression pattern of the corresponding mRNA was studied in different zones of the growing leaves, and it was shown that transcript levels matched the 1-SST activity and fructan content.

Amino Acid Sequence↗
PubMed1988

Diurnal Growth of Tall Fescue Leaf Blades : II. Dry Matter Partitioning and Carbohydrate Metabolism in the Elongation Zone and Adjacent Expanded Tissue.

The spatial distributions of net deposition rates of water soluble carbohydrate-free dry matter (WSC-free DM) and WSC were evaluated within and above the elongation zone of tall fescue (Festuca arundinacea Schreb.) leaf blades during light and darkness. Imported DM used for WSC-free DM synthesis during darkness (67% of the total in experiment I and 59% in experiment II) was greater than during light (47% in both experiments), suggesting that the 65% higher leaf elongation rate during darkness was accompanied by higher rates of synthesis of cellular structural components. Deposition rates of WSC in the basal and central part of the elongation zone (0-20 mm from the ligule) were similar during light and darkness, but above 20 millimeters WSC deposition occurred during light and WSC loss occurred during darkness. WSC deposition and loss throughout the elongation zone and the recently expanded tissue were mostly due to net synthesis and degradation of fructan. Fructan was predominantly low molecular weight and contributed about 50% of the total osmotic partial pressure of WSC. In the most actively growing region, where fructan synthesis was most rapid, no diurnal change occurred in molecular weight distribution of fructan. WSC solute concentrations were diluted in the most actively growing tissue during darkness because net monosaccharide and fructan deposition were unaltered and sucrose deposition was decreased, but growth-associated water deposition was increased by 77%. Net rates of fructan synthesis and degradation were not related to tissue sucrose concentration, but appeared to respond to the balance between assimilate import and assimilate use in synthesis of cellular structural components (i.e. WSC-free DM) and deposition of monosaccharides. Fructan synthesized in tissue during most active elongation was degraded when the respective tissue reached the distal limit of the elongation zone where assimilate import in darkness was insufficient to maintain synthetic processes associated with further differentiation of cells.

Journal Article↗
PubMed1998

Differences in chain length distribution of inulin from Cynara scolymus and Helianthus tuberosus are reflected in a transient plant expression system using the respective 1-FFT cDNAs.

A newly isolated cDNA clone, Cy3, encoding the fructan fructan 1-fructosyltransferase (1-FFT) from artichoke was expressed using tobacco protoplasts as expression system. Analysis of the inulin molecules synthesized upon incubation of protoplast extracts with a mixture of oligofructans (DP3-5) shows the production of inulins with a degree of polymerization (DP) of up to 23, whereas parallel experiments performed using a 1-FFT cDNA from Jerusalem artichoke led to the production of fructans with a DP of up to only 12. The results of in vitro fructan synthesis catalyzed by transiently expressed enzymes therefore reflect the difference of in vivo fructan composition of Jerusalem artichoke (M(DP) = 8-10) and artichoke (M(DP) = 65). These data suggest that the fructan pattern in a given species is mainly defined by the enzymatic characteristics of 1-FFT.

Amino Acid Sequence↗
PubMed2002

Assessment of the fructanolytic activities in the rumen bacterium Treponema saccharophilum strain S.

AIMS: To characterize the fructose polymer degrading enzymes of rumen bacterium Treponema saccharophilum strain S. METHODS AND RESULTS: Conventional methods were used to examine bacterial growth and enzyme activities. Electrophoretic zymogram under native conditions, and thin layer chromatography, were applied to identify and characterize the enzymes. Treponema saccharophilum utilized Timothy grass fructan, inulin and sucrose but not free fructose. Timothy grass fructan was degraded at a significantly higher rate than sucrose and inulin. Two fructanolytic enzymes were found in the soluble, and one in the membrane fraction of bacterial cell extract. The first degraded each mentioned carbohydrate to monosaccharides. The second released oligosaccharides only from Timothy grass fructan. CONCLUSIONS: The bacterium T. saccharophilum strain S is capable of synthesizing non-specific beta-fructofuranosidases and 2,6-beta-D-fructan fructanohydrolase. The enzymes are of constitutive character. SIGNIFICANCE AND IMPACT OF THE STUDY: It has been stated for the first time that the 2,6-beta-D-fructan fructanohydrolase is synthesized by the rumen bacterium T. saccharophilum. This organism appears to be responsible for grass fructan degradation in the rumen.

Animals↗
PubMed2000

Transgenic potato (Solanum tuberosum) tubers synthesize the full spectrum of inulin molecules naturally occurring in globe artichoke (Cynara scolymus) roots.

The ability to synthesize high molecular weight inulin was transferred to potato plants via constitutive expression of the 1-SST (sucrose:sucrose 1-fructosyltransferase) and the 1-FFT (fructan: fructan 1-fructosyltransferase) genes of globe artichoke (Cynara scolymus). The fructan pattern of tubers from transgenic potato plants represents the full spectrum of inulin molecules present in artichoke roots as shown by high-performance anion exchange chromatography, as well as size exclusion chromatography. These results demonstrate in planta that the enzymes sucrose:sucrose 1-fructosyltransferase and fructan:fructan 1-fructosyltransferase are sufficient to synthesize inulin molecules of all chain lengths naturally occurring in a given plant species. Inulin made up 5% of the dry weight of transgenic tubers, and a low level of fructan production also was observed in fully expanded leaves. Although inulin accumulation did not influence the sucrose concentration in leaves or tubers, a reduction in starch content occurred in transgenic tubers, indicating that inulin synthesis did not increase the storage capacity of the tubers.

Asteraceae↗
PubMed2005

Inulin and fructo-oligosaccharides differ in their ability to enhance the density of cancellous and cortical bone in the axial and peripheral skeleton of growing rats.

Although dietary fructans improve calcium absorption and bone mineral content (BMC) in rats, their effect on calcium bioavailability and bone density may vary with their degree of polymerization. Therefore, for a 3-month period, growing rats received either a control diet or a diet enriched with either oligofructose (OLF) or inulin. At sacrifice, body weight, lean body mass and appendicular bone length were similar in the 3 groups. Rats fed fructans had a similar increase in cecal wall weight (30%), but the relative increase in cecal levels of calbinding-9 K was 2 in the OLF group and 4 in the inulin group. Further, the significant decrease in serum levels of type I collagen C-telopeptide was greater in the inulin group (30%) than in the OLF group (16%). The increase in whole-body bone mineral content (BMC) as measured by DXA was greater in the inulin group than in the OLF group but DXA detected an increase in the BMC of excised femurs only in the inulin group. In contrast, pQCT conducted ex vivo detected a significant increase in the area and mineral density (BMD) of the cancellous bone of both the proximal tibia and vertebra in rats fed fructans and the effect of inulin was greater (P < 0.01) than that of OLF (P < 0.05). Further, inulin but not OLF significantly enhanced the BMD of the cortical bone in both appendicular and peripheral sites (P < 0.01) as well as the polar stress/strain index of femurs (P < 0.01). These observations suggest that, although both inulin and OLF both have a positive effect on BMD, the greatest effect of inulin is related to the higher capacity of this fructan to reduce bone resorption. The different anti-resorptive capacity of the 2 fructans might be related to their different impact on calcium absorption and bioavailability since the increase in cecal amounts of calbindin-9 K, a protein known to play an important role in calcium absorption, was greater in rat fed inulin than in rats fed OLF. Although cecal wall hyperplasia may be of concern, it remains to establish whether the positive effect of fructans observed on calcium absorption in humans is also associated with a positive effect on bone mass and/or mineral density.

Absorptiometry, Photon↗