Structural studies of gum arabic, the exudate polysaccharide from Acacia senegal

A 25.182-MHz ¹³C-n.m.r. spectrum of gum arabic allows unambiguous characterisation of all the C-1 resonances. These assignments have been confirmed by correlation of the modification of the intensities of these signals after controlled acid hydrolysis and characterisation of the released fragments. The resonances of the other carbons have been assigned through partial relaxed T₁ spectra of the polysaccharides obtained by graded degradation of the gum. These results indicate gum arabic to consist mainly of a (1→3)-β- -galactan core with (1→6)-β- -galacto-pyranosyl branches and with - -arabinofuranosyl-(1→3)-- -arabinofuranosyl and - -rhamnopyranosyl-(1→4)-β- -glucopyranosyluronic acid groups attached to positions 3 and 6, respectively, of the branch units.     

Exopolysaccharide of the gellan family: prospects and potential

The use of microbial polysaccharides in the food, pharmaceutical and chemical industries has increased steadily during the past decade. The biopolymer gellan is a more recent addition to the family of microbial polysaccharides that is gaining much importance due to its novel property of forming thermo-reversible gels when heated and cooled. It is produced and marketed by some companies of Europe, USA, etc under trade names such as Gelrite, Phytagel and Kelcogel. It has applications in diverse fields in the food, pharmaceutical and many other industries. Further research and development in biopolymer technology is expected to expand its use. This article presents a critical review of the available published information on the gellan exopolysaccharide synthesized by Pseudomonas species. In particular information on its structure, physico-chemical properties and the rheology of its solutions etc. is critically assessed. Emphasis has also been paid to characterization of gellan. A brief historical background of the polymer and the biochemical and physiological characteristics of several different existing bacterial isolates which secrete gellan and related polysaccharides are discussed. An attempt has also been made to review the potential and future prospects, highlighting some novel techniques adopted to overcome the mass transfer problems associated with the fermentative production of gellan gum. The efficient downstream processes used for obtaining purified gellan are also highlighted. Attention has also been drawn to the problem associated with the fermentation processes due to the highly viscous nature of gellan gum and effect of different impeller systems on gellan fermentation kinetics and rheological properties.     

C-n.m.r. spectroscopic investigation of methylated and charged agarose oligosaccharides and polysaccharides

The ¹³C-n.m.r. signals of agarose oligomers with various substituted repeating units have been assigned. Enzymic hydrolysis of agaroses gave 2¹-O-methylagarobiose, 6²-O-methylagarobiose, the agarobiose biological precursor, agarobiose 4²-sulfate, 2¹-O-methylagarobiose 4²-sulfate, and pyruvylated agarobiose. The chemical shift data of the oligomers and the parent polymers were compared, and indicated the distribution of the substituents in hybrid polymers.     

Microbial polysaccharides with actual potential industrial applications

The microbial polysaccharides reviewed include xanthan gum, scleroglucan, PS-10, PS-21 and PS-53 gums, polysaccharides from Alcaligenes sp., PS-7 gum, gellan gum, curdlan, bacterial alginate, dextran, pullulan, Baker's Yeast Glycan, 6-deoxy-hexose-containing polysaccharides and bacterial cellulose. Factors limiting the commercial potential of certain microbial polysaccharides such as availability, rheological properties, and polyvalency are outlined. The polysaccharides are classified according to their uses as viscosity-increasing agents and as gelling agents. A third category includes polysaccharides with specific applications such as tailor-made dextran and pullulan and polysaccharides used as substrates for the preparation of rare sugars. The difficulties encountered in development of a polysaccharide at the industrial level are pointed out.     

Rapid purification of commercial gellan gum to highly soluble and gellable monovalent cation salts

Commercial gellan gum contains divalent cation contaminants (mainly Ca²⁺ and Mg²⁺) in levels sufficient to neutralize over one-third of its car☐yl groups. Consequently, in order to dissolve gellan gum in water, the mixtures must be heated to more than 90°C. This has prevented applications of gellan gum to such uses as the immobilization of viable enzymes and cells in beads. A rapid two-step method is described here to purify commercial gellan gum to the monovalent cation salts in an overall yield of 85%, through the intermediate free acid form. The gellan monovalent cation salts were highly soluble at temperatures as low as 5°C, and readily gelled upon exposure to solutions of divalent cations. Laboratory-scale preparations of 100 g of gellan monovalent cation salts were readily achieved in a day.     

Light scattering studies of tetramethyl ammonium gellan

Tetramethyl ammonium (TMA) gellan does not gel. Light scattering studies suggest that in solutions of TMA gellan, in tetramethyl ammonium chloride (TMACI), the gellan molecules assemble end to end to produce elongated fibrous structures. Such fibrils are envisaged as resulting from double-helix formation between the ends of neighbouring gellan molecules. Fibrils with molecular weights ranging from (1.06 ± 0.06) × 10⁵ to (4.5 ± 0.1) × 10⁶ have been observed. The molecular weights obtained depended upon the pore size of the filters used to clarify the solutions. The formation of strong gels, in the presence of gel promoting cations, is attributed to a localized ordered lateral association, or crystallization of regions of these fibrils. It is suggested that such a model for gelation may be of general applicability to a number of polysaccharide systems.     

X-Ray fibre diffraction studies of members of the gellan family of polysaccharides

X-Ray fibre diffraction studies are reported for gellan gum and the family of related polysaccharides S-130, S-198, S-88 and S-194. Whereas the linear gellan molecules yield highly crystalline patterns, the branched polysaccharides yield well-aligned but poorly crystalline patterns. These patterns are consistent with the proposed 3-fold double helical structure of gellan with small changes in pitch dependent upon type and position of branches.     

Effects of group I cations on the gelation of iota carrageenan

N.m.r. and rheological measurements have been used to study the gelation of iota carrageenan. Gelation has been found to occur only at polymer concentrations above the critical entanglement concentration. The high temperature sol state above the gel-sol transition appears to be an entangled polymer network. Although Li⁺ and Na⁺ ions are less effective at gelling the polymer than K⁺, Rb⁺ and Cs⁺ all cationic forms studied gel at sufficiently high polymer concentration and ionic strength. ⁷Li⁺, ²³Na, ³⁹K, ⁸⁷Rb and ¹³³Cs n.m.r. studies have been made as a function of temperature. The lithium salt form (2.2% w/w concentration) formed a viscoelastic solution at room temperature. The other salt forms gelled on cooling. The spectra of Li, Na and Cs carrageenan showed little change on heating whereas K and Rb spectra showed marked changes in apparent intensity. The nature of the cation interaction with the juntion zones is discussed.     

Deproteinization of gellan gum produced by Sphingomonas paucimobilis ATCC 31461

Deproteinization is a technical bottleneck in the purification of viscous water-soluble polysaccharides. The aim of this work is to provide an appropriate approach to deproteinize crude gellan gum. Several methods of deproteinization were investigated, including Sevag method, alkaline protease, papain and neutral protease. The results revealed that Sevag method had high deproteinization efficiency (87.9%), but it showed dissatisfactory recovery efficiency of gellan gum (28.6%), which made it less advisable in industrial applications. The deproteinization by alkaline protease was demonstrated in this work for the first time, indicating alkaline protease was preferred in the deproteinization of crude gellan gum with high polysaccharide recovery (89.3%) and high deproteinization efficiency (86.4%).     

Different effects of gellan gum and agar on change in root elongation in Arabidopsis thaliana by polyploidization: the key role of aluminum

Journal of Plant Research - Agar and gellan gum have been considered to have different effects on polyploidy-dependent growth in plants. We aim to demonstrate that agar and gellan gum differently...     

Imaging polysaccharides by atomic force microscopy

Techniques have been developed for the routine reliable imaging of polysaccharides by atomic force microscopy (AFM). The polysaccharides are deposited from aqueous solution onto the surface of freshly cleaved mica, air dried, and then imaged under alcohols. The rationale behind the development of the methodology is described and data is presented for the bacterial polysaccharides xanthan, acetan, and the plant polysaccharides 1-carrageenan and pectin. Studies on uncoated polysaccharides have demonstrated the improved resolution achievable when compared to more traditional metal-coated samples or replicas. For acetan the present methodology has permitted imaging of the helical structure. Finally, in addition to data obtained on individual polysaccharides, AFM images have also been obtained of the network structures formed by κ-carrageenan and gellan gum. © 1996 John Wiley & Sons, Inc.     

Response time and electrorheology of semidiluted gellan, xanthan and cellulose suspensions

Response times with electrical fields of gellan and xanthan dry powder suspensions of 25, 32 and 53 μm average diameter and concentrations of 1.0, 1.5 and 2.0% (w/w) dispersed in commercial corn oil were optically measured through a specifically designed set up. In all cases, the delay time was proportional to 1/E^a, where E is the applied field and a is an adjustable parameter. The values of parameter a were very different from the typical value of some known electrorheolgical fluids. Response time of gellan suspensions was shorter than the one obtained for xanthan and it is comparable to the time found by using silica particles in silicon oil. Response times for cellulose were very large and the fibrillation phenomenon was negligible for E<1.0 kv/mm.

Viscosity measurements of semidiluted xanthan, gellan and cellulose suspensions (1.0 and 1.5% w/w) under the influence of electrical fields, were performed in a parallel plates rheometer. Results in the range of stress <70 Pa showed that viscosity values of gellan suspensions were larger than those obtained with xanthan or cellulose under the same applied electric field at shear rates higher than 10 s⁻¹. However, cellulose suspensions showed larger viscosity values compared with the ones measured with xanthan and gellan suspensions at very low shear rates. Dielectric measurements of cellulose, xanthan and gellan 1.5% w/w suspensions were performed in the range 10⁰–8×10⁴ Hz. Results agree with a Maxwell–Wagner type relaxation model.     

Release Mechanisms Behind Polysaccharides-Based Famotidine Controlled Release Matrix Tablets

Polysaccharides, which have been explored to possess gelling properties and a wide margin of safety, were used to formulate single-unit floating matrix tablets by a direct compression technique. This work has the aim to allow continuous slow release of famotidine above its site of absorption. The floating approach was achieved by the use of the low density polypropylene foam powder. Polysaccharides (κ-carrageenan, gellan gum, xyloglucan, and pectin) and blends of polysaccharides (κ-carrageenan and gellan gum) and cellulose ethers (hydroxypropylmethyl cellulose, hydroxypropylcellulose, sodium carboxymethyl cellulose) were tried to modulate the release characteristics. The prepared floating tablets were evaluated for their floating behavior, matrix integrity, swelling studies, in vitro drug release studies, and kinetic analysis of the release data. The differential scanning calorimetry and Fourier transform infrared spectroscopy studies revealed that changing the polymer matrix system by formulation of polymers blends resulted in formation of molecular interactions which may have implications on drug release characteristics. This was obvious from the retardation in drug release and change in its mechanistics.     

Amphiphilic derivatives of sodium alginate and hyaluronate: synthesis and physico-chemical properties of aqueous dilute solutions

This paper reports on the synthesis and the physico-chemical characterisation of various amphiphilic derivatives of two natural polysaccharides, sodium alginate and sodium hyaluronate, in which a rather small proportion of the carboxylic groups (≤10% mol) was esterified by long alkyl chains (C₁₂H₂₅ or C₁₈H₃₇).

The derivatives thus prepared were characterised by gas chromatography, ¹H and ¹³C n.m.r. spectroscopy and size exclusion chromatography coupled to a multi-angle laser light scattering detection. The tendency of these water-soluble compounds to hydrophobic association in aqueous solutions was evidenced firstly in dilute regime using capillary viscometry as well as fluorescence spectroscopy in the presence of a molecular probe, 1,1-dicyano-(4′-N,N-dimethylaminophenyl)-1,3-butadiene.     

X-ray and computer modeling studies on gellan-related polymers: molecular structures of welan, S-657, and rhamsan

The primary structures of the four bacterial polysaccharides gellan, welan, S-657, and rhamsan are the same with respect to their backbones, but have different side-chains. This difference has a profound influence on their behavior in aqueous media. Solutions of gellan gum form stable aqueous gels under appropriate ionic conditions. By contrast, welan, S-657, and rhamsan do not gel but give very viscous solutions over a wide range of thermal, pH, and salt conditions. X-Ray fiber diffraction analysis and computer modeling of these branched polysaccharides demonstrate that they all have the same half-staggered, double-helical conformations as in the unbranched gellan, suggesting, therefore, that the side chains are responsible for diminishing gelling behavior. Depending on the size and location, the side chains shield the carboxylate groups to varying degrees; this shielding is substantial in welan and S-657, but less in rhamsan. In all cases, side-chain-main-chain interactions within the double helix prevent the carboxylate-mediated aggregation of double helices that is necessary for the gelation.     

Structure of the exudate gum from Meryta sinclairii

A gum that exudes from the wounded trunk of the New Zealand native tree Meryta sinclairii has been isolated. The gum was completely precipitated by the β-glucosyl Yariv reagent and was thus determined to be an arabinogalactan-protein (AGP). It contained >95% w/w carbohydrate and only 2% w/w protein with a high level of hydroxyproline. SEC-MALLS showed that the gum had a weight-average molecular weight of 4.45×10⁶ Da compared with 6.02×10⁵ Da for gum arabic. Constituent sugar and linkage analyses were consistent with polymers comprised of a highly branched backbone of 1,3-linked galactopyranosyl (Galp) residues, with side-chains made up of arabinofuranose- (Araf) containing oligosaccharides, terminated variously by rhamnopyranosyl (Rhap), arabinopyranosyl (Arap), Galp and glucuronopyranosyl (GlcpA) residues. Analysis by one-dimensional and two-dimensional ¹H and ¹³C NMR experiments confirmed the linkage analyses. The structure of the gum is discussed in comparison with the structure of gum arabic and other AGPs.     

Investigation of the impact of feeding Lactobacillus plantarum CRL 1815 encapsulated in microbially derived polymers on the rat faecal microbiota

Aims

The aim of this study was to evaluate the impact of the administration of microencapsulated Lactobacillus plantarum CRL 1815 with two combinations of microbially derived polysaccharides, xanthan : gellan gum (1%:0·75%) and jamilan : gellan gum (1%:1%), on the rat faecal microbiota.

Methods and Results

A 10‐day feeding study was performed for each polymer combination in groups of 16 rats fed either with placebo capsules, free or encapsulated Lact. plantarum or water. The composition of the faecal microbiota was analysed by fluorescence in situ hybridization and temporal temperature gradient gel electrophoresis. Degradation of placebo capsules was detected, with increased levels of polysaccharide‐degrading bacteria. Xanthan : gellan gum capsules were shown to reduce the Bifidobacterium population and increase the Clostridium histolyticum group levels, but not jamilan : gellan gum capsules. Only after administration of jamilan : gellan gum‐probiotic capsules was detected a significant increase in Lactobacillus‐Enterococcus group levels compared to controls (capsules and probiotic) as well as two bands were identified as Lact. plantarum in two profiles of ileum samples.

Conclusions

Exopolysaccharides constitute an interesting approach for colon‐targeted delivery of probiotics, where jamilan : gellan gum capsules present better biocompatibility and promising results as a probiotic carrier.

Significance and Impact of Study

This study introduces and highlights the importance of biological compatibility in the encapsulating material election, as they can modulate the gut microbiota by themselves, and the use of bacterial exopolysaccharides as a powerful source of new targeted‐delivery coating material.     

Structural study of the extracellular polysaccharide gum from Rhizobium strain CB744

The structure of the extracellular polysaccharide gum from nitrogen-fixing Rhizobium sp. strain CB744 (a member of the slow-growing Cowpea group) has been investigated. Gas-chromatographic analysis of the alditol acetates of the acid hydrolysate showed the gum to be composed of galactose, 4-O-methylgalactose, mannose, and glucose in the molar ratio of 1:2.5:3.5:7.0. The polysaccharide is unusual in that it contains no carbonyl substituent, although such substituents are common amongst polysaccharides produced by the slow-growing group. The native and de-branched polysaccharides were examined by methylation analysis. The anomeric configurations were determined by ¹³C-n.m.r. and oxidation by chromium trioxide. It is concluded that there are two β-(1→4)-linked glycopyranosyl residues for each α-(1→4)-linked mannopyranosyl residue, and that each mannose is substituted at O-6 by a β-galactopyranosyl residue, with 71% of the galactose groups being present as 4-O-methylgalactose.     

Genome sequence of Sphingomonas elodea ATCC 31461, a highly productive industrial strain of gellan gum

The commercial gelling agent gellan gum is a heteropolysaccharide produced by Sphingomonas elodea ATCC 31461. However, the genes involved in the biosynthesis, regulation, and modification of gellan gum have not been fully characterized. Here we describe the draft genome sequence of stain ATCC 31461 and major findings from its annotation.     

High Rate of N2 Fixation by East Siberian Cryophilic Soil Bacteria as Determined by Measuring Acetylene Reduction in Nitrogen-Poor Medium Solidified with Gellan Gum

For evaluating N₂ fixation of diazotrophic bacteria, nitrogen-poor liquid media supplemented with at least 0.5% sugar and 0.2% agar are widely used for acetylene reduction assays. In such a soft gel medium, however, many N₂-fixing soil bacteria generally show only trace acetylene reduction activity. Here, we report that use of a N₂ fixation medium solidified with gellan gum instead of agar promoted growth of some gellan-preferring soil bacteria. In a soft gel medium solidified with 0.3% gellan gum under appropriate culture conditions, bacterial microbiota from boreal forest bed soils and some free-living N₂-fixing soil bacteria isolated from the microbiota exhibited 10- to 200-fold-higher acetylene reduction than those cultured in 0.2% agar medium. To determine the N₂ fixation-activating mechanism of gellan gum medium, qualitative differences in the colony-forming bacterial components from tested soil microbiota were investigated in plate cultures solidified with either agar or gellan gum for use with modified Winogradsky''s medium. On 1.5% agar plates, apparently cryophilic bacterial microbiota showed strictly distinguishable microbiota according to the depth of soil in samples from an eastern Siberian Taiga forest bed. Some pure cultures of proteobacteria, such as Pseudomonas fluorescens and Burkholderia xenovorans, showed remarkable acetylene reduction. On plates solidified with 1.0% gellan gum, some soil bacteria, including Luteibacter sp., Janthinobacterium sp., Paenibacillus sp., and Arthrobacter sp., uniquely grew that had not grown in the presence of the same inoculants on agar plates. In contrast, Pseudomonas spp. and Burkholderia spp. were apparent only as minor colonies on the gellan gum plates. Moreover, only gellan gum plates allowed some bacteria, particularly those isolated from the shallow organic soil layer, to actively swarm. In consequence, gellan gum is a useful gel matrix to bring out growth potential capabilities of many soil diazotrophs and their consortia in communities of soil bacteria.In 1967, Schöllhorn and Burris discovered that nitrogenase from an N₂-fixing rhizobium of soybean can reduce acetylene to produce ethylene (C₂H₄) (32), a reaction analogous to the conversion of the natural substrate N₂ into ammonia. Shortly afterwards, it was shown that this acetylene reduction activity parallels N₂ reduction by nitrogenase (13), and since then, acetylene reduction assays have been widely used in the evaluation of biological N₂ fixation. An acetylene reduction assay is generally performed under the following conditions: precultured bacterial cells are suspended into N-free or -deficient liquid medium containing a carbon source, usually d-glucose or d-mannitol (35) at 0.5 to 2.0%, and exposed for 24 h or less at a representative room temperature, e.g., 25°C (2). However, this method is not applicable to free-living, microaerobic N₂-fixing bacteria, which have been regarded as notoriously difficult to culture. To solve this problem, Döbereiner and her group developed a soft gel method (7), which used 0.2% agar as a gel matrix for the medium. Due to a vertical gradient of dissolved oxygen concentrations, these microaerobes formed a thin layer at the particular depth of the medium that contained an ideal level of dissolved oxygen (10). Also, significant activities in acetylene reduction assays were observed for N₂-fixing microaerobes, particularly those from the rhizoplane of monocotyledonous crop plants (e.g., Azospirillum and Herbaspirillum spp.) (1, 9, 40). To date, these soft gel media solidified with 0.2% agar have been widely used as the most basic method for the screening of free-living or difficult-to-culture N₂-fixing bacteria (2, 16).In an agar composed of soft gel, however, the layer formation of highly transparent colony-forming bacteria is often obscured and is more difficult to observe than comparable layer formation in water due to the higher turbidity of the agar gel, and some members of the soil bacterial community do not show any positive response in acetylene reduction assays under these conditions. These drawbacks to the usage of agar as a soft gel matrix delayed the recognition that free-living N₂ fixers make a potent contribution to the support of ecosystems under adverse soil conditions. Hashidoko et al. developed an improved soft gel medium for growth of N₂-fixing bacteria in 2002 (15). In their study, 0.2% agar was replaced with 0.3% gellan gum, a bacterial extracellular polysaccharide (EPS) produced by Sphingomonas elodea (a synonym of Sphingomonas paucimobilis) ATCC 31461 (12, 17, 18). Initially, gellan gum was used for the purpose of preparing a highly transparent soft gel medium that was better for culturing microaerobic N₂-fixing rhizobacteria. It had other favorable physical properties: when 0.3% gellan gum containing Winogradsky''s mineral mixture was autoclaved, the medium remained in a liquid form over a period of several hours while cooling to room temperature. Even after the gellan gum had been solidified, the soft gel was easily liquefied upon mechanical agitation. The liquefied medium was able to resolidify after a short period of time, so it was easy to uniformly disperse inoculants into the soft gel medium. The outstanding transparency (14) and other properties of this gel matrix enable easy visualization of transparent colony-forming N₂-fixing bacteria and also allow observation of their responses to various concentrations of dissolved oxygen and cell motilities (15).In many preliminary experiments, nitrogen-poor gellan gum media allowed high growth of diazotrophs, but this study was needed to compare gellan gum with agar as a gel matrix for N₂ fixation. Because Siberian boreal forest soils have been noted for their low N₂-fixing capability (3), we first cultured bacterial microbiota from the eastern Siberian Taiga forest bed in gellan gum medium. A quantitative comparison of N₂ fixation behaviors of free-living soil bacteria was attempted to investigate gellan gum as a potential N₂ fixation-promoting soft gel matrix. We here first report on the efficacy of gellan gum as a soft gel matrix for monitoring acetylene reduction by the use of free-living N₂-fixing soil bacteria.