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PubMed2004

Crystallization and preliminary X-ray diffraction study of fructan 1-exohydrolase IIa from Cichorium intybus.

Fructan 1-exohydrolase IIa (1-FEH IIa), a plant enzyme involved in fructan breakdown, has been crystallized using the hanging-drop vapour-diffusion method at 277 K. The crystals are tetragonal, belonging to space group P4(1)2(1)2 or P4(3)2(1)2, with unit-cell parameters a = 139.83, b = 139.83, c = 181.94 A. Calculation of the Matthews coefficient indicates there to be two or three molecules in the asymmetric unit. Synchrotron radiation was used to collect a complete native data set to a resolution of 2.35 A.

Cichorium intybus↗

PubMed1994

Fermentation of fructans by epiphytic lactic acid bacteria.

A total of 712 strains of lactic acid bacteria isolated from forage grasses were studied for their ability to ferment fructans of phlein- as well as inulin-type. Only 16 strains utilized phlein and eight of these also fermented inulin. They were identified as Lactobacillus paracasei subsp. paracasei, Lact. plantarum, Lact. brevis and Pediococcus pentosaceus. In the species Lact. paracasei subsp. paracasei, all strains gave positive results, whereas the other positive strains possessed unique properties within their own species. In all but two cases (strains of the species Lact. plantarum), the phlein was more intensively fermented than the inulin, as indicated by a lower pH and a higher lactic acid concentration. On the basis of the outcome of this study it seems worthwhile to inoculate grasses of low sugar content before ensiling with an active strain that can ferment fructans.

Fermentation↗

PubMed2000

Purification and characterization of 2,6-beta-D-fructan 6-levanbiohydrolase from Streptomyces exfoliatus F3-2.

Streptomyces exfoliatus F3-2 produced an extracellular enzyme that converted levan, a beta-2,6-linked fructan, into levanbiose. The enzyme was purified 50-fold from culture supernatant to give a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The molecular weights of this enzyme were 54,000 by SDS-PAGE and 60,000 by gel filtration, suggesting the monomeric structure of the enzyme. The isoelectric point of the enzyme was determined to be 4.7. The optimal pH and temperature of the enzyme for levan degradation were pH 5.5 and 60 degrees C, respectively. The enzyme was stable in the pH range 3.5 to 8.0 and also up to 50 degrees C. The enzyme gave levanbiose as a major degradation product from levan in an exo-acting manner. It was also found that this enzyme catalyzed hydrolysis of such fructooligosaccharides as 1-kestose, nystose, and 1-fructosylnystose by liberating fructose. Thus, this enzyme appeared to hydrolyze not only beta-2,6-linkage of levan, but also beta-2,1-linkage of fructooligosaccharides. From these data, the enzyme from S. exfoliatus F3-2 was identified as a novel 2,6-beta-D-fructan 6-levanbiohydrolase (EC 3.2.1.64).

Disaccharides↗

PubMed1979

Nature of the fructan of Streptococcus mutans OMZ 176.

The fructan of Streptococcus mutans OMZ 176 was shown to have a levan structure by comparing the chromatographic mobilities of the saccharides produced by partial acid hydrolysis of ghe fructan and known levan and inulin. This was confirmed by using concanavalin A as a lectin in a double-diffusion gel technique.

Chemical Phenomena↗

PubMed1984

Clinical isolates of Streptococcus mutans serotype c with altered colony morphology due to fructan synthesis.

Streptococcus mutans MT6801 , MT6861 , and MT6879 , which form large mucoid colonies on mitis salivarius agar, were isolated from a mother and her two daughters. These isolates were identified as serotype c by immunodiffusion with serotype-specific antisera. The large colonies formed on sucrose-containing agar were found to contain water-soluble fructan . The cell-free fructosyltransferase ( FTase ) activity of the strains which formed large colonies was five to eight times higher than that of serotype c S. mutans which produced small, rough colonies typical of this serotype. Furthermore, greater quantities of fructan were synthesized from sucrose by growing cells of MT6801 when compared with MT8148 , a typical serotype c S. mutans. Glucosyltransferase and FTase could be isolated by chromatofocusing from culture supernatants of MT6801 and MT8148 . The FTase activity of both strains was eluted at pH 4.5, and glucosyltransferase was released by elution with an NaCl linear gradient. The eluted FTase activity of MT6801 was significantly higher than that of MT8148 . Strains MT6861 and MT6879 were also found to possess a similar property in terms of FTase activity. These results suggest that formation of large mucoid colonies by these strains is a consequence of high FTase activity.

Adult↗

PubMed2001

Roles of fructosyltransferase and levanase-sucrase of Actinomyces naeslundii in fructan and sucrose metabolism.

The ability of Actinomyces naeslundii to convert sucrose to extracellular homopolymers of fructose and to catabolize these types of polymers is suspected to be a virulence trait that contributes to the initiation and progression of dental caries and periodontal diseases. Previously, we reported on the isolation and characterization of the gene, ftf, encoding the fructosyltransferase (FTF) of A. naeslundii WVU45. Allelic exchange mutagenesis was used to inactivate ftf, revealing that FTF-deficient stains were completely devoid of the capacity to produce levan-type (beta2,6-linked) polysaccharides. A polyclonal antibody was raised to a histidine-tagged, purified A. naeslundii FTF, and the antibody was used to localize the enzyme in the supernatant fluid. A sensitive technique was developed to detect levan formation by proteins that had been separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the method was used to confirm that the levan-synthesizing activity of A. naeslundii existed predominantly in a cell-free form, that a small amount of the activity was cell associated, and that the ftf mutant was unable to produce levans. By using the nucleotide sequence of the levanase gene of a genospecies 2 A. naeslundii, formerly Actinomyces viscosus, a portion of a homologue of this gene (levJ) was amplified by PCR and inserted into a suicide vector, and the resulting construct was used to inactivate the levJ gene in the genospecies 1 strain WVU45. A variety of physiologic and biochemical studies were performed on the wild-type and LevJ-deficient strains to demonstrate that (i) this enzyme was the dominant levanase and sucrase of A. naeslundii; (ii) that LevJ was inducible by growth in sucrose; (iii) that the LevJ activity was found predominantly (>90%) in a cell-associated form; and (iv) that there was a second, fructose-inducible fructan hydrolase activity produced by these strains. The data provide the first detailed molecular analysis of fructan production and catabolism in this abundant and important oral bacterium.

Actinomyces↗

PubMed2005

Inulin-type fructans and bone health: state of the art and perspectives in the management of osteoporosis.

If the primary role of diet is to provide sufficient nutrients to meet the metabolic requirements of an individual, there is an emerging rationale to support the hypothesis that, by modulating specific target functions in the body, it can help achieve optimal health. Regarding osteoporosis prevention, since Ca is most likely to be inadequate in terms of dietary intake, every strategy targeting an improvement in Ca absorption is very interesting. Actually, this process may be susceptible to manipulation by fermentable substrates. In this light, inulin-type fructans are very interesting, even if we need to gather more data targeting bone metabolism before health professionals can actively advocate their consumption to prevent senile osteoporosis. Besides targeting the prevention of postmenopausal osteoporosis, inulin-type fructans still remain a source for putative innovative dietary health intervention. Indeed, given in combination with isoflavones, they may have a potential for maintaining or improving the bone mass of human subjects, by modulating the bioavailability of phyto-oestrogens.

Adolescent↗

PubMed1998

Phloem Transport of Fructans in the Crassulacean Acid Metabolism Species Agave deserti

Four oligofructans (neokestose, 1-kestose, nystose, and an un-identified pentofructan) occurred in the vascular tissues and phloem sap of mature leaves of Agave deserti. Fructosyltransferases (responsible for fructan biosynthesis) also occurred in the vascular tissues. In contrast, oligofructans and fructosyltransferases were virtually absent from the chlorenchyma, suggesting that fructan biosynthesis was restricted to the vascular tissues. On a molar basis, these oligofructans accounted for 46% of the total soluble sugars in the vascular tissues (sucrose [Suc] for 26%) and for 19% in the phloem sap (fructose for 24% and Suc for 53%). The Suc concentration was 1.8 times higher in the cytosol of the chlorenchyma cells than in the phloem sap; the nystose concentration was 4.9 times higher and that of pentofructan was 3.2 times higher in the vascular tissues than in the phloem sap. To our knowledge, these results provide the first evidence that oligofructans are synthesized and transported in the phloem of higher plants. The polymer-trapping mechanism proposed for dicotyledonous C3 species may also be valid for oligofructan transport in monocotyledonous species, such as A. deserti, which may use a symplastic pathway for phloem loading of photosynthates in its mature leaves.

Journal Article↗

PubMed1990

Fructan precipitation from a water/ethanol extract of oats and barley.

Fructan was precipitated from a water and ethanol extract of oat (Avena sativa L.) and barley (Hordeum vulgare L.). The degree of polymerization and response on a differential refractometer, based on peak area and height, was compared to fructan collected from a lead-based HPLC column and to commercially available inulin. Statistically significant differences are discussed.

Journal Article↗

PubMed1999

Biochemical and structural characterization of the glucan and fructan exopolysaccharides synthesized by the lactobacillus reuteri wild-type strain and by mutant strains

Lactobacillus reuteri LB 121 cells growing on sucrose synthesize large amounts of a glucan (D-glucose) and a fructan (D-fructose) with molecular masses of 3,500 and 150 kDa, respectively. Methylation studies and 13C or 1H nuclear magnetic resonance analysis showed that the glucan has a unique structure consisting of terminal, 4-substituted, 6-substituted, and 4,6-disubstituted alpha-glucose in a molar ratio of 1.1:2.7:1.5:1.0. The fructan was identified as a (2-->6)-beta-D-fructofuranan or levan, the first example of levan synthesis by a Lactobacillus species. Strain LB 121 possesses glucansucrase and levansucrase enzymes that occur in a cell-associated and a cell-free state after growth on sucrose, raffinose, or maltose but remain cell associated during growth on glucose. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of sucrose culture supernatants, followed by staining of gels for polysaccharide synthesizing activity with sucrose as a substrate, revealed the presence of a single glucansucrase protein of 146 kDa. Growth of strain LB 121 in chemostat cultures resulted in rapid accumulation of spontaneous exopolysaccharide-negative mutants that had lost both glucansucrase and levansucrase (e.g., strain K-24). Mutants lacking all levansucrase activity specifically emerged following a pH shiftdown (e.g., strain 35-5). Strain 35-5 still possessed glucansucrase and synthesized wild-type glucan.

Journal Article↗

PubMed2003

A biologically active fructan from the roots of Arctium lappa L., var. Herkules.

From the roots of Arctium lappa L., var. Herkules a low-molecular-weight fructofuranan of the inulin-type has been isolated by water extraction and ethanol precipitation, followed by ion-exchange chromatography and gel filtration of the crude precipitate. The methods employed in structural determination were methylation analysis and 1H and 13C NMR spectral measurements. In tests for antitussive activity in cats the fructan was found to be equally active as some non-narcotic, synthetic preparations used in clinical practice to treat coughing, and in mitogenic and comitogenic tests its biological response was comparable to that of the commercial Zymosan immunomodulator.

Animals↗

PubMed2006

Glucan and fructan production by sourdough Weissella cibaria and Lactobacillus plantarum.

After a large screening on sourdough lactic acid bacteria, exopolysaccharide (EPS)-forming strains of Weissella cibaria, Lactobacillus plantarum, and Pediococcus pentosaceus were selected. After 6 days of incubation at 30 degrees C, the synthesis of EPS in MRS-based broth ranged from 5.54 to 7.88 mg mL-1. EPS had an apparent molecular mass of ca. 104 Da. As shown by carbohydrate consumption, the synthesis of EPS was found from sucrose only. Two types of homopolysaccharides were synthesized: glucans simultaneously with growth and fructans after 1 day of incubation. Two protein bands of ca. 180-200 kDa were in situ detected on SDS-PAGE gels incubated with sucrose. PCR products of ca. 220 bp were found for L. plantarum PL9 (100% of identity to putative priming glycosyltransferase of L. plantarum WCFS1) and W. cibaria WC4 (80% of identity to putative glycosyltransferase, epsD, of Bacillus cereus G9241) by using hybrid primers for the priming gtf genes. Degenerated primers DexreuR and DexreuV showed a unique PCR product, and the predicted amino acid sequences were identical for W. cibaria WC4 and L. plantarum PL9. The sequence had similarity with polysaccharide biosynthesis glycosyltransferases. W. cibaria WC4 or L. plantarum LP9 synthesized ca. 2.5 g kg-1 EPS during sourdough fermentation with sucrose added. Compared to the sourdough started with an EPS-negative strain, the sourdough started with W. cibaria WC4 or L. plantarum LP9 increased the viscosity, and the resulting bread had higher specific volume and lower firmness. The synthesis of EPS by selected sourdough lactic acid bacteria could be considered as a useful tool to replace the additives for improving the textural properties of baked goods.

Bacteria↗

PubMed2006

A fructan, from Radix ophiopogonis, stimulates the proliferation of cultured lymphocytes: structural and functional analyses.

A fructan, Opaw-2, with molecular mass of about 14 kDa, was isolated from the tuberous roots of Ophiopogon japonicus. Opaw-2 comprises fructose and glucose with a molar ratio of 30:1. Linkage and NMR analyses indicated that Opaw-2 has a backbone structure of beta-(1-->2)-Fruf and beta-(2-->6)-Fruf residues that branches at O-6 of beta-(1-->2)-Fruf residues with alpha-1-linkage to the Glcp residues and terminates with Fruf residues. In cultured lymphocytes, the application of Opaw-2 significantly stimulated the proliferation of lymphocytes in a dose-dependent manner. By using atomic force microscopy, Opaw-2 showed a morphological change from globular to helical fibrous shape at increasing concentrations.

Animals↗

PubMed2002

Experimental evidences on the potential of prebiotic fructans to reduce the risk of colon cancer.

Inulin is extracted from the chicory root. It is a set of fructans with its monomers linked by means of beta(2-1) bonds. This linkage cannot be hydrolysed by either pancreatic or by brush border digestive enzymes in the upper intestinal tract of humans. As such the carbohydrates arrive in the colon, where they are fermented by bifidobacteria and other lactic acid producing bacteria, thus enhancing their relative populations in the gut. Recent research in experimental animal models revealed that inulin has significant anticarcinogenic properties. It acts chemopreventively by reducing the incidence of azoxymethane (AOM) - induced aberrant crypt foci and tumours in the colon. These effects may be due to the stimulation of bifidobacteria, which themselves have been shown to act as antigenotoxic in the colon and to reduce AOM-induced tumours. Also fermentation products, including the short-chain fatty acid butyrate, could contribute to the protective effects. In this case a mechanism may be the induction of apoptosis of already transformed cells. The experimental evidence from animal studies and from studies elucidating potential mechanisms strongly supports the possibility that inulin will contribute to reducing risks for colon cancer in humans. In order to obtain more insight into this possibility, human dietary intervention studies relating biomarkers of reduced risk to inulin consumption are needed.

Animals↗

PubMed2005

Lactobacillus paracasei subsp. paracasei 8700:2 degrades inulin-type fructans exhibiting different degrees of polymerization.

Ten strains of lactobacilli were assessed for their capacity to degrade inulin-type fructans, which are well-known prebiotics. Both oligofructose and inulin were tested. The dairy isolate Lactobacillus acidophilus IBB 801 degraded only oligofructose. The human isolate Lactobacillus paracasei subsp. paracasei 8700:2 degraded oligofructose and long-chain inulin and grew rapidly on both energy sources. In both cases, fractions of different degrees of polymerization were fermented. Moreover, large and short fractions of oligofructose were degraded simultaneously. When L. paracasei subsp. paracasei 8700:2 grew on oligofructose-enriched inulin, oligofructose was preferentially metabolized. In all cases, lactic acid was the main metabolic end product. Significant amounts of acetic acid, formic acid, and ethanol were produced when long-chain inulin or oligofructose-enriched inulin was used as the sole energy source.

Culture Media↗

PubMed2004

The large subunit determines catalytic specificity of barley sucrose:fructan 6-fructosyltransferase and fescue sucrose:sucrose 1-fructosyltransferase.

Plant fructosyltransferases are highly homologous in primary sequence and typically consist of two subunits but catalyze widely different reactions. Using functional expression in the yeast Pichia pastoris, we show that the substrate specificity of festuca sucrose:sucrose 1--beta-D-fructosyltransferase (1-SST) and barley sucrose:fructan 6--beta-D-fructosyltransferase (6-SFT) is entirely determined by the large subunit. Chimeric enzymes with the large subunit of festuca 1-SST (LSuB) and the small subunit of barley 6-SFT have the same catalytic specificity as the native festuca 1-SST and vice versa. If the LSuB is expressed alone, it does not yield a functionally active enzyme, indicating that the small subunit is nevertheless essential.

Amino Acid Motifs↗

PubMed1998

Expression of a functional barley sucrose-fructan 6-fructosyltransferase in the methylotrophic yeast Pichia pastoris.

The cDNA encoding sucrose-fructan 6-fructosyltransferase (6-SFT) from barley (Hordeum vulgare) has been expressed in the methylotrophic yeast Pichia pastoris, using a translational fusion into vector pPICZ alphaC, containing the N-terminal signal sequence of Saccharomyces cerevisiae alpha-factor to allow entry into the secretory pathway. Transformed Pichia produced and secreted a functional 6-SFT which had characteristics similar to the barley enzyme, but had a pronounced additional 1-SST activity when incubated with sucrose.

5' Untranslated Regions↗

PubMed2006

Different effects of difructose anhydride III and inulin-type fructans on caecal microbiota in rats.

The effects of different kinds of inulin-type fructans on caecal microbiota were evaluated in rats. Four groups of male Wistar rats were fed either a control diet, or diets containing 5% inulin, 5% fructooligosaccharides (FOS), or 5% difructose anhydride III (DFAIII) for two weeks. In the DFAIII group, caecal propionate, butyrate, counts of bifidobacteria, and total anaerobes were lower than in the inulin group, while caecal propionate, succinate, counts of bifidobacteria, and total anaerobes were lower than in the FOS group. Compared to controls, in the DFAIII group the counts of clostridia in caecum were increased by 3 log units. However, this change was statistically not significant. There were no differences between inulin and FOS groups for the pool of short chain fatty acids in caecum and bacterial counts. Results indicate that DFAIII has different effects on caecal microbiota compared to inulin and FOS and that these differences are most likely due to the alpha(3-->2) bonds in DFAIII.

Animals↗