Low Molecular Weight Fructans Research Articles

Identification of enzymatic activities involved in agave fructan consumption by Bifidobacterium longum subsp. infantis ATCC 15697

Metabolism of complex non-commercial fructans by Bifidobacteria has not been explored. In this work the production of the extracellular, cell-bound and intracellular β-fructofuranosidases by the probiotic Bifidobacterium longum subsp infantis ATCC 15697 in culture medium containing agave fructan and chicory inulin as carbon source was evaluated. The analysis of the profile of products formed in the hydrolysis of agave and inulin fructans by the three enzymatic fractions and the proteomic analysis of B. longum subsp infantis ATCC 15697 indicated the importance of B.longum_l1, an exo-β-fructofuranosidase, in the metabolism of the prebiotics studied. We found that B. longum hydrolyzes inulin and agave fructan through an exo-type mechanism involving B.longum_l1 exclusively and consumes low molecular weight fructans first in spite of their structure, suggesting that B. longum growth in these two structurally different substrates depends more on fructan molecular weight than on its chemical nature.

Freezing Tolerance Attributes during Spring Deacclimation for Three Asparagus Cultivars with Varying Adaptation to Southern Ontario

Winterhardiness in asparagus (Asparagus officinalis) may be related to proper cold acclimation and induction of freezing tolerance in the fall, levels and maintenance of freezing tolerance in the winter, and the timing of deacclimation in the spring. Premature deacclimation and the inability to reacclimate could result in crown damage from spring freeze-thaw cycles. A field experiment was conducted, replicated over 2 years, to determine how three cultivars with varying adaptation to southern Ontario deacclimate in the spring by assessing LT50 (the temperature at which 50% of plants die) and biochemical and physiological parameters associated with freezing tolerance. ‘UC 157’ (UC), the least-adapted cultivar, deacclimated after soil temperatures rose above freezing; LT50 values increased linearly over time and were unaffected by fluctuations in soil temperature. ‘Jersey Giant’ (JG), a cultivar with moderate adaptation, rapidly deacclimated with increased soil temperature but appeared to partially reacclimate as temperatures decreased. For ‘Guelph Millennium’ (GM), the most-adapted cultivar, LT50 values did not change, maintaining the greatest levels of freezing tolerance during the spring sampling period. Although LT50 values did not differ among cultivars on the first spring sampling date, ranking for freezing tolerance at the final sampling in each year was GM>JG>UC, which is consistent with adaptation. Rhizome traits were most associated with freezing tolerance and included high concentrations of low-molecular-weight fructans (LFs), glucose, and proline and low percentage water and sucrose concentration. Overall, data suggest that the timing of deacclimation and loss of freezing tolerance in the spring may significantly affect winterhardiness; cultivars that lose freezing tolerance early and cannot reacclimate could suffer most from late spring freeze-thaw cycles.

Comparison of the water-soluble carbohydrate composition and fructan structures of Agave tequilana plants of different ages

Abstract The composition of water-soluble carbohydrates from Agave tequilana plants of 2, 4 and 6½ years were compared by HPLC, HPAEC-PAD, MALDI-TOF-MS and GC–MS. The plants of 2 years exhibited the highest levels of free monosaccharide and low molecular weight fructans (DP 3–DP 6) with potential application as prebiotics. A maximum of fructan polymerisation was achieved at 4 years with mean DP from 3 to 30, then it was decreased at 6½ years with mean DP from 4 to 24. The linkage analysis showed an increase and decrease in the branching degree from 2 to 6½ years with a maximum at 4 years, correlated to changes in t-β- d -Fruf linkages with increased and decreased synthesis of (2→1) and (2→6)-β- d -Fruf as well as 1,6-di-β- d -Fruf linkages.

Kinetic behaviour and specificity of β-fructosidases in the hydrolysis of plant and microbial fructans

Fructosidases, in particular exo-β-fructosidases, may act on fructans such as inulins and levans of plant and bacterial origin to produce fructose. In this paper, the kinetic properties of a commercial preparation (Fructozyme L) and a recombinant exoinulinase (BfrA) from Thermotoga maritima, were studied using fructan polymer substrates from various sources. Both enzymatic preparations preferentially hydrolyzed β2-1 linkages and low molecular weight fructans. We show that chicory inulin is degraded most efficiently by both preparations, followed by bacterial inulin, in spite of its high molecular weight and branching in β2-6 positions. All bacterial levans were more slowly hydrolyzed. Michaelis–Menten kinetics describe the hydrolysis of sucrose and low molecular weight fructans (≤8.3 kDa) by both enzyme preparations, while first order kinetics were observed with respect to bacterial fructans due to the high molecular weight and, therefore, low molar concentrations. Comparison of second order rate constants indicates that bacterial inulin ( Leuconostoc citreum CW28) is hydrolyzed more slowly with both enzyme preparations than chicory inulin by approximately one order of magnitude. For Leuconostoc mesenteroides NRRL B-512F levan, the second order rate constant for Fructozyme L is 200-fold lower than for chicory inulin. However, the second order rate constant for BfrA is only 22-fold lower than for chicory inulin. Taken together, our studies characterize the kinetics of fructan hydrolysis and also suggest that the kinetic parameters may be used to differentiate between fructan structures.

Partitioning of water soluble carbohydrates in vegetative tissues of Lolium multiflorum Lam. ssp. italicum cv. Lema

In temperate grasses, fructans are the major storage polysaccharides, being accumulated mainly in mature leaf sheaths, and also in the roots. The partitioning of carbohydrates within different organs regulates plant growth and development. The aim of the present work was to analyze the partitioning of water soluble carbohydrates in five different parts (elongating leaf blades, expanded leaf blades, upper and lower segments of the stubble, and roots) of plants of L. multiflorum cv. Lema, in order to contribute to an understanding of soluble carbohydrates distribution in these plants. Soluble carbohydrates and total fructose were analyzed in plants cultivated during 4 months in a glasshouse, by colorimetric, TLC and HPAEC-PAD techniques. Results showed that the greatest portion of total soluble carbohydrates was constituted of free and combined fructose, in all parts of the plants. The stubble contained the highest level of carbohydrates, followed by the elongating leaf blades, expanded leaf blades and roots. The leaf sheaths were not analyzed separately from the stubble, which explains the high levels of carbohydrates found in this part of the plant. The high metabolism of the elongating leaf blades, when compared to that of the expanded leaf blades, could explain the increased amounts of fructans stored in those tissues. Analysis by HPAEC-PAD showed that the elongating leaf blades and the roots had the highest proportions of low molecular weight fructans that could be readily mobilized, supplying the demand of growing tissues in other organs.

Tissue-specific changes in remobilisation of fructan in the xerophytic tussock species Festuca novae-zelandiae in response to a water deficit.

Cellular responses of the native New Zealand grass species Festuca novae-zelandiae (Hack.) Cockayne to a water deficit and re-hydration treatment were investigated. Leaf extension ceased after 28 d of withholding water, while mitotic activity within the intercalary meristem still continued. By 35 d, no evidence of mitotic activity could be detected. At the apical meristem, cell division commenced 24 h post re-watering after 49 d of dry-down and was accompanied by synthesis of storage oligosaccharides of the fructan type. Changes in water-soluble carbohydrates over the course of the water deficit were examined in consecutive leaf segments comprising the leaf base (meristem region), elongation zone, the enclosed and the exposed lamina, as well as basal sheath segments from the two next oldest leaves. In fully hydrated leaf tissue, the fructan pool was mainly composed of the low molecular weight fructans of the inulin and neokestose series with higher concentrations towards the leaf base. Fructan concentrations decreased over the course of the water deficit with the leaf base retaining significantly higher concentrations than any other tissue, until tissue water content fell below 45%. Sucrose content increased in each tissue during the course of the dry-down, and was highest at the leaf base, where a concentration of 200 μmol g DW-1 was measured after 49d of dry-down.In 1.5 h after re-watering, levels of 1-kestotriose increased relative to the level of sucrose at the leaf base, indicating re-synthesis of fructans from accumulated sucrose. By 24 h post re-watering, the ratio of sucrose to 1-kestotriose declined from six in non-watered plants to two and higher molecular weight fructans became detectable. The negative correlation between fructan and sucrose content, which indicates an inter-conversion depending on tissue water content, suggests that, in this species, fructans serve as a carbohydrate pool, while sucrose stabilises the meristem during extreme water deficit.

High level fructan accumulation in a transgenic sugar beet.

We have transformed sugar beet into a crop that produces fructans. The gene encoding 1-sucrose:sucrose fructosyl transferase (1-SST), which was isolated from Helianthus tuberosus, was introduced into sugar beet. In H. tuberosus, 1-SST mediates the first steps in fructan synthesis through the conversion of sucrose (GF) into low molecular weight fructans GF2, GF3, and GF4. In the taproot of sugar beet transformed with the 1-sst gene, the stored sucrose is almost totally converted into low molecular weight fructans. In contrast, 1-sst expression in the leaves resulted in only low levels of fructans. Despite the storage carbohydrate having been altered, the expression of the 1-sst gene did not have any visible effect on phenotype and did not affect the growth rate of the taproot as observed under greenhouse conditions.

Fructan contents and dry matter deposition in different tissues of the wheat grain during development

ABSTRACTThe role of fructan metabolism in the assimilate relations of the grain of wheat (Triticum aestivum L.) was investigated by determination of the dry matter and fructan content of grain components at short intervals during grain filling. During the initial phase of rapid expansion, most of the assimilates entering the grain were partitioned to the outer pericarp. A large fraction of these assimilates were used for the synthesis of fructan. Dry matter deposition and fructan synthesis in the outer pericarp ceased at about 5d after anthesis. At the same time, the endosperm and the inner pericarp and testa started to accumulate dry matter at a fast rate. This was also associated with significant fructan synthesis in the latter tissues. The outer pericarp lost about 45% of its former maximum dry weight between 9 and 19 d after anthesis. This loss was due almost entirely to the near complete disappearance of water‐soluble carbohydrates, most of which was fructan. The inner pericarp and testa accumulated dry matter until about mid‐grain filling. The fructan contents of the inner pericarp and testa and the endosperm decreased slowly towards the end of grain filling. Most of the fructans in the inner pericarp and testa and the endosperm had a low molecular weight, whereas higher molecular weight fructans predominated in the outer pericarp. The embryo did not contain fructan. The presence of low molecular weight fructans in the endosperm cavity at mid‐grain filling was confirmed. It is suggested that fructan synthesis is closely linked to growth‐related water deposition in the different tissues of the wheat grain and serves to sequester the surplus of imported sucrose.

Cereal Fructans: In Vitro and In Vivo Studies on Availability in Rats and Humans

The bioavailability of cereal fructans (fructooligosaccharides) was investigated both in vitro and in vivo. In vitro studies indicated very slow hydrolysis by human gastric juice and by homogenate of the intestinal mucosa (rat). After intubation of fructans into the stomachs of rats, the recovery of fructans in the small intestine and colon was approximately the same as that of an unabsorbed marker (polyethylene glycol), indicating no or very low disappearance of fructans in the small intestine. In vivo studies of the small intestine in rats showed that the rate of disappearance of fructans was lower than that of mannose, which is known to be absorbed through passive diffusion. In addition the cariogenic effect of cereal fructans was compared to that of glucose. Acid formation from low molecular-weight fructans was found in human dental plaque in vitro. A mouth rinse with unfractionated fructans, containing some quantities of sucrose, fructose and glucose, resulted in relatively low pH values in human plaque in vivo, even if the decrease in pH was somewhat less pronounced when compared with a mouth rinse with glucose.

Carbohydrate Metabolism of Salt-tolerant Fructan Grasses as Exemplified with Puccinellia peisonis

Summary The physiological role of carbohydrates, particularly of water-soluble fructose polymers (fructans), in metabolism was studied in Puccinellia peisonis, a salt-tolerant, caespitose grass growing at the banks of salty pools (“Lacken”) in the Seewinkel area (Burgenland, Austria). High resolution steric exclusion chromatography (SEC) made it possible to determine the molecular-weight distribution of the homologous fructan series and to quantify the individual components. The seasonal changes in the concentration of free monosaccharides, sucrose, oligo- and polyfructans (in 4 fractions with increasing molecular weight) and starch, which always occurred in small quantities, were studied in relation to the plant's phenology. Results showed that high-molecular fructans (with a degree of polymerization, DP of more than 18) mainly served as storage components, whereas fructose oligomers, similar to the raffinose series in many dicotyledonous species, provided resistance to freezing. Fructose and sucrose contributed much to the osmotic potential of the plant, which as most monocotyledonous species is highly efficient in excluding excess ions from the substrate. The resulting osmotic values, however, are too low to bridge dry periods in summer. In such cases decomposition of the water-soluble fructans may lead to a rapid increase of the osmotic potential. Moreover, the possible role of low molecular-weight fructans (with DP 7 to 10) as effectors of osmoregulation is discussed. The multiform functions of a polymeric series of simple carbohydrates may help to explain the success of Poaceae in growing on dry biotopes.