Xanthan–galactomannan interactions as related to xanthan conformations

The influence of xanthan conformation on the physicochemical behaviour of their mixtures with galactomannan from Schizolobium parahybae mannose:galactose ratio (M/G=3), was studied by viscoelastic measurements, differential scanning calorimetry (DSC) and chiroptical (circular dichroism) methods. The results suggested a more effective interaction of the galactomannan with disordered xanthan segments, which are more abundant in low salt concentrations but are still present in lower proportion at temperatures lower than the temperature of xanthan conformational transition (T_m). The dependence of ellipticity with temperature in a circular dichroism (CD) spectra suggested an ordering of the xanthan chains induced by galactomannan at the temperature of gel formation (T_g≈25°C), under conditions where xanthan alone exhibits a disordered conformation. The lower T_g value found (≈25°C) compared with that (60°C) usually described in the literature is certainly related to the M/G ratio and the galactosyl unit distribution along the mannan main chain.     

Associative phenomena in galactomannan-deacetylated xanthan systems

The interaction between mesquite seed galactomannan (MSG; D-mannose to D-galactose ratio (M/G) approximately 1.1) and deacetylated xanthan (DX) in 5 mM NaCl leading to synergistic gel formation at 25 degrees C was investigated and compared with the far more studied system made of xanthan and locust bean gum (LBG; M/G approximately 3.5). Rheology and differential scanning calorimetry were used to measure temperatures of gel formation and transition enthalpy as a function of polymer composition, while circular dichroism was used to probe the conformation of DX in the LBG-DX system. MSG and DX associate at 25 degrees C with a well defined stoichiometry of 0.6:1.0 (w/w) at low ionic strength favouring the disordered coil state of DX. When LBG was used in place of MSG in water or 5 mM NaCl, two types of mechanisms of interpolymeric association are envisaged.     

Interaction properties of d-galactose-depleted guar galactomannan samples

The interaction of galactose-depleted guar galactomannan with agarose has been studied by optical rotation, and with xanthan using an Instron Materials Tester and a Rheometrics Mechanical Spectrometer. Samples were prepared by treatment of guar galactomannan with highly purified α-d-galactosidase. Modified guar galactomannans with a d-galactose content of 19–25%, in admixture with agarose, showed similar optical rotation changes on heating and cooling as did mixtures of agarose and carob galactomannan (23% d-galactose content). Modified samples with 13–16% d-galactose, in the presence of agarose, showed more pronounced optical rotation changes on heating and cooling, but samples with less than 13% d-galactose were only sparingly soluble even on autoclaving. The degree of interaction of galactose-depleted guar galactomannan samples with xanthan, as measured rheologically, increased as the d-galactose content decreased, paralleling the optical rotation changes with galactomannan/agarose mixtures. In the presence of xanthan, samples with a d-galactose content of 25% or less formed firm rubbery gels.     

Effect of galactose-substitution-patterns on the interaction properties of galactomannas

A range of galactomannans varying widely in the contents of d-galactose have been compared for self-association and their interaction properties with agarose and xanthan. Whereas, in general, the most interactive galactomannans are those in which the (1→4)-β-d-mannan chain is least substituted by α-d-galactosyl stubs, evidence is presented which indicates that the distribution of d-galactosyl groups along the backbone (fine structure) can have a significant effect on the interaction properties. For galactomannans containing <30% of d-galactose, those which contain a higher frequency of unsubstituted blocks of intermediate length in the β-d-mannan chain are most interactive. For galactomannans containing >40% of d-galactose, those which contain a higher frequency of exactly alternating regions in the β-d-mannan chain are most interactive. This selectivity, on the basis of galactomannan fine-structure, in mixed polysaccharide interactions in vitro could mimic the selectivity of binding of branched plant-cell-wall polysaccharides in biological systems.     

Changes to the galactose/mannose ratio in galactomannans during coffee bean (<Emphasis Type="Italic">Coffea arabica</Emphasis> L.) development: implications for in vivo modification of galactomannan synthesis

Endosperm was isolated from Arabica Caturra coffee beans 11, 15, 21, 26, 31 and 37 weeks after flowering, and the chemical composition and relative solubility of its component polysaccharides determined at each growth stage. Chemical analysis of the total mannan content of the cell wall material was done after solubilisation of galactomannans by alkaline extraction of the cell wall material followed by enzymatic digestion of the alkali-insoluble residue with a mixture of endo-mannanse and endo-glucanase. Eleven weeks after flowering, galactomannans accounted for approximately 10% of the polysaccharides but were highly substituted, with galactose/mannose ratios between 1:2 and 1:7. As the bean matured, galactomannan became the predominant polysaccharide, until 31 weeks after flowering it accounted for approximately 50% of the polysaccharides. However, it was less substituted, possessing galactose/mannose ratios between 1:7 and 1:40. Early in bean growth, up to 50% of the cell wall polysaccharides were extractable but as the galactomannan content of the bean increased there was a reduction in the extractability of all polysaccharides. The decrease in the galactose/mannose ratio of the galactomannans commenced between 21 and 26 weeks after flowering and was in synchrony with a rise in the concentration of free galactose in the beans. The results indicated that the degree of substitution of the galactomannans in coffee beans is developmentally regulated and may result, in part, from the modification of a primary synthetic product by the action of an alpha-galactosidase.     

Tobacco transgenic lines that express fenugreek galactomannan galactosyltransferase constitutively have structurally altered galactomannans in their seed endosperm cell walls

Galactomannans [(1-->6)-alpha-D-galactose (Gal)-substituted (1-->4)-beta-D-mannans] are major cell wall storage polysaccharides in the endosperms of some seeds, notably the legumes. Their biosynthesis in developing legume seeds involves the functional interaction of two membrane-bound glycosyltransferases, mannan synthase (MS) and galactomannan galactosyltransferase (GMGT). MS catalyzes the elongation of the mannan backbone, whereas GMGT action determines the distribution and amount of Gal substitution. Fenugreek (Trigonella foenum-graecum) forms a galactomannan with a very high degree of Gal substitution (Man/Gal = 1.1), and its GMGT has been characterized. We now report that the endosperm cell walls of the tobacco (Nicotiana tabacum) seed are rich in a galactomannan with a very low degree of Gal substitution (Man/Gal about 20) and that its depositional time course is closely correlated with membrane-bound MS and GMGT activities. Furthermore, we demonstrate that seeds from transgenic tobacco lines that express fenugreek GMGT constitutively in membrane-bound form have endosperm galactomannans with increased average degrees of Gal substitution (Man/Gal about 10 in T(1) generation seeds and about 7.5 in T(2) generation seeds). Membrane-bound enzyme systems from transgenic seed endosperms form galactomannans in vitro that are more highly Gal substituted than those formed by controls under identical conditions. To our knowledge, this is the first report of structural manipulation of a plant cell wall polysaccharide in transgenic plants via a biosynthetic membrane-bound glycosyltransferase.     

“Melt-in-the-mouth” gels from mixtures of xanthan and konjac glucomannan under acidic conditions: A rheological and calorimetric study of the mechanism of synergistic gelation

The effect of acidification on a typical commercial xanthan and on pyruvate-free xanthan (PFX), alone and in gelling mixtures with konjac glucomannan (KGM), has been studied by differential scanning calorimetry (DSC) and small-deformation oscillatory measurements of storage modulus (G′) and loss modulus (G″). For both xanthan samples, progressive reduction in pH caused a progressive increase in temperature of the disorder–order transition in DSC, and a progressive reduction in gelation temperature with KGM. This inverse correlation is interpreted as showing that synergistic gelation involves disruption of the xanthan 5-fold helix, probably by attachment of KGM to the cellulosic backbone of the xanthan molecule (as proposed previously by a research group in the Institute of Food Research, Norwich, UK). Higher transition temperature accompanied by lower gelation temperature for PFX in comparison with commercial xanthan at neutral pH is explained in the same way. However, an additional postulate from the Norwich group, that attachment of KGM (or galactomannans) can occur only when the xanthan molecule is disordered, is inconsistent with the observation that gelation of acidified mixtures of KGM with PFX can occur at temperatures more than 60 °C below completion of conformational ordering of the PFX component (as characterised by DSC). Increase in G′ on cooling for mixtures of commercial xanthan with KGM at pH values of 4.5 and 4.25 occurred in two discrete steps, the first following the temperature-course observed for the same mixtures at neutral pH and the second occurring over the lower temperatures observed for mixtures of KGM with PFX at the same values of pH. These two “waves” of gel formation are attributed to interaction of KGM with, respectively, xanthan sequences that had retained a high content of pyruvate substituents, and sequences depleted in pyruvate by acid hydrolysis. At pH values of 4.0 and lower, gelation of mixtures of KGM with commercial xanthan followed essentially the same temperature-course as for mixtures with PFX, indicating extensive loss of pyruvate under these more strongly acidic conditions. Mixtures prepared at pH values in the range 4.0–3.5 gave comparable moduli at room temperature (20 °C) to those obtained at neutral pH, but showed substantial softening on heating to body temperature, suggesting possible applications in replacement of gelatin in products where “melt-in-the-mouth” characteristics are important for acceptability to the consumer.     

Composition and structure of a galactomannan macromolecules from seeds of Astragalus lehmannianus Bunge

The composition and structure of a galactomannan from seeds of Astragalus lehmannianus, an endemic legume species, is reported for the first time. The purified galactomannan (yield, 4.8%) contained 55% D-mannose and 45% D-galactose and had a molecular weight of 997.03 kDa. Its aqueous solutions were optically active and highly viscous (the specific rotation, [alpha]D, equaled +81.3 degrees; the characteristic viscosity, [eta], 868.4 ml/g). Chemical, chromatographic, and spectral (IR and 13C-NMR spectroscopy) methods were used to demonstrate that the main chain of the molecule is formed by residues of 1,4-beta-D-mannopyranose, 78% of which are substituted at position 6 with single alpha-D-galactopyranose. The distribution of galactose along the chain was calculated from NMR spectra: frequencies of occurrence, per pair of neighboring mannose units, of (1) two substituents, (2) one substituent, and (3) no substituents were 65.3, 31.5, and 3.2%, respectively. The specific rotation of galactomannans was shown to correlate with their content of galactose.     

Galactomannan (from Gleditsia sinensis Lam.) and xanthan gum matrix tablets for controlled delivery of theophylline: In vitro drug release and swelling behavior

Galactomannan (G) from Gleditsia sinensis Lam. and xanthan gum (X), as sustained release materials for controlled delivery of theophylline, were mixed in different ratios of 7:3, 5:5, and 3:7 to yield enhanced release-controlling performance. The polysaccharides content of tablets was 10% (w/w), either alone or in mixtures. From in vitro dissolution test, G10% and X10% matrices released 91.4 and 87.7% of drug within 24 h, respectively. The synergistic interactions between galactomannan and xanthan effectively retarded the drug diffusion, and the most sustained drug release (75.5% at 24 h) was found in formulation GX7:3. The drug release data fitted to the kinetic model indicated the anomalous transport mechanism (0.5 < n < 1.0). Additionally, the swelling behavior and morphological changes of the tablets were investigated. The results illustrated the potential of binary mixtures of G. sinensis galactomannan and xanthan as novel sustained release materials for controlled drug delivery.     

A (1→4)-β-mannan-specific monoclonal antibody and its use in the immunocytochemical location of galactomannans

Galactomannan was coupled to a protein carrier for the preparation of monoclonal antibodies. The monoclonal antibodies generated bound to galactomannans from different sources as well as to glucomannan and galactoglucomannan. One monoclonal antibody, BGM C6, was characterised and found to be specific for (1-->4)-beta-linked mannopyranosyl residues; it had a binding affinity estimated at 1x10(-6) M for the (1-->4)-beta-linked mannohexaose. BGM C6 was used in immunogold labelling studies to locate galactomannans in the endosperm walls of normal coconuts (Cocos nucifera L.) and those of the mutant makapuno at two different developmental stages. The pattern and intensity of antibody labelling varied for each type of coconut at the mature and immature stages, indicating differences in the galactomannan composition of the endosperm walls.     

Composition and structure of a galactomannan macromolecule from seeds ofAstragalus lehmannianus bunge

The composition and structure of a galactomannan from seeds ofAstragalus lehmannianus, an endemic legume species, is reported for the first time. The purified galactomannan (yield, 4.8%) contained 55% D-mannose and 45%D-galactose and had a molecular weight of 997.03 kDa. Its aqueous solutions were optically active and highly viscous (the specific rotation [α]_D equaled +81.3°; the characteristic viscosity [η], 868.4 ml/g). Chemical, Chromatographic, and spectral (IR and¹³C-NMR spectroscopy) methods were used to demonstrate that the main chain of the molecule is formed by residues of 1,4-β-D-mannopyranose, 78% of which are substituted at position 6 with single α-D-galactopyranose. The distribution of galactose along the chain was calculated from NMR spectra: frequencies of occurrence per pair of neighboring mannose units of ( 1 ) two substituents, (2) one substituent, and (3) no substituents were 65.3%, 31.5, and 3.2, respectively. The specific rotation of the galactomannans was shown to correlate with their content of galactose.     

Agar/galactomannan gels applied to shoot regeneration from tobacco leaves

This study concerns the efficacy of partial agar substitution by galactomannans as support in plant regeneration media for Nicotiana tabacum. The production of multiple shoots from leaf-derived callus and their rooting were evaluated. The galactomannans applied were obtained from Cassia fastuosa (cassia) and Cyamopsis tetragonolobus (guar gum — a commercial galactomannan) seeds. The results obtained on media solidified with mixtures of agar/galactomannan (3 g dm⁻³ each) gels were compared with those on media gelled with a standard concentration of agar (6 g dm⁻³). The in vitro performance allowed to conclude that the use of galactomannans raised the number of shoots and improved their quality. Furthermore, the length of roots and the size of leaves were significantly higher in the media solidified with agar/guar galactomannan mixtures.     

Pressure cell assisted solution characterization of polysaccharides. 2. Locust bean gum and tara gum

Following the work carried out on guar gum in our first paper of a series, the "pressure cell" solubilization method was applied to two other less highly substituted galactomannans: locust bean gum (LBG) and tara gum. True molecular solution of the polymers was achieved using appropriate temperature, time, and pressure regimes. The technique of capillary viscometry was used to determine the intrinsic viscosity [eta] of the "pressure cell" treated and untreated samples. Molecular weight (M(w)) and radius of gyration (R(g)) were determined by light scattering. The data obtained for LBG and tara gum were compared statistically with reliable data found for guar gum in the literature. The variation in [eta] with M(w) followed the Mark-Houwink-Sakurada relationship, giving the exponent alpha = 0.74 +/- 0.01 for galactomannans consistent with random coil behavior. The characteristic ratio, C(infinity), and the chain persistence length, L(p), were both calculated for LBG and tara gum using the Burchard-Stockmayer-Fixman (BSF) method which is appropriate for flexible to semiflexible chains. A general value of 9 < C(infinity) < 16 and 3 < L(p) < 5 nm can now be estimated with statistical confidence for all galactomannans. According to our statistical analysis, the chain persistence length was found to be insensitive to the degree of galactose substitution.     

On the chain flexibility of arabinoxylans and other beta-(1-->4) polysaccharides

The recent paper by Dervilly-Pinel and co-workers (Carbohydr. Res. 2001, 330, 365-372) presents a complete macromolecular characterisation of a series of de-esterified arabinoxylans extracted and fractionated from wheat flour. From their measurements, they were able to extract parameters related to chain flexibility, such as the Mark-Houwink exponent a and the chain persistence length. However, the estimate they obtain for the latter parameter is rather larger than would be expected, since the arabinoxylan backbone is beta-(1-->4) like cellulose and the galactomannans. By treating their data in an alternative, but well accepted manner, we are able to obtain a lower value of persistence length, which agrees well with estimates for this parameter for cellulose, in the literature, and our own recent measurements for a series of differently substituted galactomannans. These results suggest that the parameters obtained may be applicable to other beta-(1-->4) polysaccharides.     

Viscoelastic and light scattering studies on thermally induced sol to gel phase transition in fish myosin solutions

Viscoelastic (VE) and dynamic light scattering (DLS) analyses of fish (white croaker) myosin solutions were performed at myosin concentrations of 30 mg/mL for VE and 0.1 mg/mL for DLS at 0.6M KCl and pH 7.0 to clarify thermally induced gelation. The hydrodynamic radius R(h) considerably decreased around 30-35 degrees C. The shear modulus G was constant below 25 degrees C and increased by incubating the sample at 30 degrees C. G further increased as the temperature of the incubated sample decreased. The curves of G vs T for different time courses showed a sharp peak around 35 degrees C and a moderate peak around 60 degrees C in the heating process, while a stepwise increase in G was observed around 30 degrees C in the cooling process when the temperature was elevated to not more than 60 degrees C. No distinct stepwise change was observed once the temperature of the sample exceeded 60 degrees C. The absolute value of G strongly depended on the maximum elevated temperature and the incubation time at that temperature. The corresponding behavior of the viscosity eta was observed for each time course. Based on these results, the mechanism of thermally induced gelation of myosin solutions is discussed in view of S-S bridge formation in the head and tail portions and unwinding/rewinding of coiled-coil alpha-helices in the tail portion.     

On the galactosyl distribution of commercial galactomannans

A simple method was developed that enabled the enzymatic determination of the galactose distribution in galactomannans. endo-Mannanase of Aspergillus niger was used to degrade the galactomannan polymers and the degradation products were determined with high-performance anion-exchange chromatography. A whole range of commercial high-to-low substituted galactomannans was analyzed in this way. It was found that differences in the anion-exchange chromatograms reflected dissimilarities in the distribution of galactose and could be used directly to discern these dissimilarities. The differences among the various elution profiles were used to construct a similarity distance tree. In addition to this approach, the absolute amount of non-substituted mannose released by the enzyme was found to be a good discriminating factor. In this way, galactomannans with regular, blockwise, and randomly distributed galactose could be discerned. All guars and the highly substituted gum of Prosopis juliflora were found to have a blockwise distribution of galactose. For different batches of tara gum both random and blockwise distributions were found. Among batches of locust bean gum the greatest variation was observed: both random, blockwise, and ordered galactose distributions were present. Cassia gum was found to have a highly regular distribution of galactose.     

Structural characterization of a galactomannan from the cyanolichen Leptogium azureum

A galactomannan was isolated from the cyanolichen Leptogium azureum via successive alkaline extraction and precipitation with Fehling solution. The structure of the polysaccharide was investigated using NMR spectroscopy, methylation analysis, Smith degradation, and HPSEC-MALLS. As galactomannans from other lichens species, the polymer obtained presents a (1→6)-linked main chain of -mannopyranose, substituted preferentially at O-2 by -Manp or β-Galp non-reducing ends. As observed in previous investigations, the C-1 region of the ¹³C-NMR of these heteropolysaccharides are typical of some lichens species, and can be used as fingerprints in chemotaxonomy. However, in despite of the general structure in common, the substitution level of this structure and their content of mannose is higher than of the others galactomannans obtained of lichenized fungi contained the green alga of the genus Trebouxia.