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.     

Structural features of acetylated galactomannans from green Coffea arabica beans

Polysaccharides were extracted from green Coffea arabica beans with water (90 °C, 1 h). Galactomannans were isolated from the water extract using preparative anion-exchange chromatography. Almost all of the galactomannans eluted in two neutral populations, while almost all of the arabinogalactans bound to the column, indicating that these arabinogalactans contain charged groups. Analysis of the molecular weight distribution of the two neutral populations showed that they differ in their molecular weight. Further characterization of these neutral populations by NMR and by MALDI-TOF MS after enzymatic degradation with an endo-mannanase, showed the presence of acetyl groups linked to the galactomannans, a feature not previously described for this type of polysaccharides from coffee beans. It was found that the high molecular weight (ca. 2000 kDa) neutral fraction was highly substituted both with galactose residues and acetyl groups, while the low molecular weight (ca. 20 kDa) population was much less substituted. Based on these results it can be concluded that at least two distinctly different populations of galactomannans are present in green coffee beans. It was also shown that the degradation of the galactomannans from green coffee beans with an endo-mannanase from A. niger is hindered by the presence of acetyl groups.     

Effect of polysaccharide structure on mechanical and thermal properties of galactomannan-based films

Films were prepared from guar gum and locust bean gum galactomannans. In addition, enzymatic modification was applied to guar gum to obtain structurally different galactomannans. Cohesive and flexible films were formed from galactomannans plasticized with 20-60% (w/w of polymer) glycerol or sorbitol. Galactomannans with lower galactose content (locust bean gum, modified guar gum) produced films with higher elongation at break and tensile strength. The mechanical properties of films were improved statistically significantly by decreasing the degree of polymerization of guar gum with mannanase treatments (4 h) of 2 and 10 nkat/g, whereas 50 nkat/g produced films with low elongation at break and tensile strength. Galactomannans with approximately 6 galactose units per 10 mannose backbone units resulted in films with 2 peaks in loss modulus spectra, whereas films from galactomannans with approximately 2 galactose groups per 10 mannose units behaved as a single phase in dynamic mechanical analysis.     

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.     

Nonesterified galacturonic acid sequence homology of pectins

The methyl ester distribution of pectins was studied with a recently developed enzymatic method. Endopolygalacturonase of Kluyveromyces fragilis was used to degrade pectin and the composition of the degradation products was determined with high-performance anion-exchange chromatography at pH 5. Three characteristics indicative for the distribution of nonesterified galacturonic acid residues were obtained: the percentage of nonesterified galacturonic acid residues liberated of the total number of nonesterified galacturonic acid in the undigested polymer, the proportion of nonesterified mono-, di-, and trigalacturonic acid released, and the ratio of the sum of the peak areas of methyl ester containing oligomers divided by the sum of the peak areas of the nonesterified oligomers detected. From these characteristics and the degree of methyl esterification, the mean sequence similarity of the methyl ester distributions was calculated. Computational techniques commonly employed in the determination of the sequence similarity of DNA and proteins were used to discriminate the various types of distributions found and to construct a distance tree. In general, three types of methyl ester distributions could be discerned in pectin: random, high, and blockwise esterified. This report is the first to describe a parametric approach for the comparison of the substituent distribution in polymers. The importance of this novel approach in the study of the methyl ester distribution and the functional properties of pectin is discussed.     

Modes of action of β-mannanase enzymes of diverse origin on legume seed galactomannans

β-Mannanase activities in the commercial enzyme preparations Driselase and Cellulase, in culture solutions of Bacillus subtilis (TX1), in commercial snail gut (Helix pomatia) preparations and in germinated seeds of lucerne, Leucaena leucocephala and honey locust, have been purified by substrate affinity chromatography on glucomannan-AH-Sepharose. On isoelectric focusing, multiple protein bands were found, all of which had β-mannanase activity. Each preparation appeared as a single major band on SDS-polyacrylamide gel electrophoresis. The enzymes varied in their final specific activities, Km values, optimal pH, isoelectric points and pH and temperature stabilities but had similar MWs. The enzymes have different abilities to hydrolyse galactomannans which are highly substituted with galactose. The preparations Driselase and Cellulase contain β-mannanases which can attack highly substituted galactomannans at points of single unsubstituted d-mannosyl residues if the d-galactose residues in the vicinity of the bond to be hydrolysed are all on only one side of the main chain.     

Structural analysis of galactomannans by NMR spectroscopy

The structure of naturally occurring galactomannans was characterized by high resolution NMR spectroscopy involving two-dimensional (2D) NMR measurements of the field gradient DQF-COSY, HMQC, HMBC, and ROESY experiments. Four galactomannans with different proportions of galactose (G) and mannose (M), from fenugreek gum (FG), guar gum (GG), tara gum (TG), and locust bean gum (LG), were investigated. Because these galactomannans had very high molecular weights, hydrolysis by dilute H₂SO₄ was carried out to give the corresponding low molecular weight galactomannans, the structural identities of which were established by comparison of the specific rotations, shape of the GPC profiles, and NMR spectra with those of higher molecular weight galactomannans. The correlation signals GH1-GC4, -GC5, and -MC6 in HMBC and GH1-GH6 in ROESY spectra of FG showed that more than two galactopyranose units with the 1 → 4 linkage were connected at C6 of the mannopyranose main chain. The coupling constant (J_H1,2) of galactose was 3.4 Hz, indicating that galactose has an α-linkage. The main chain mannose was found to connect through the 1 → 4 linkage, because of the appearance of the correlation signals MH1-MC4, and MC1-MH4 in the HMBC spectrum due to the long-range correlation signals between two neighboring mannopyranose residues through the M4-O-M1 bond. Although the main chain mannose J_H1,2 was not observed, probably because of the high molecular weight, the specific rotation of LG with a higher proportion of mannose was low, [α]_D²⁵ = +10.8°, compared with that of FG with a lower proportion of mannose, [α]_D²⁵ = +90.5°, suggesting that the mannose in the main chain had a α-linkage. These results suggest that the galactomannans comprise a (1 → 4)-β-mannopyranosidic main chain connected with more than two (1 → 4)-α-galactopyranosidic side chains, in addition to the single galactopyranose side chain, at C6 of the mannopyranose main chain.     

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.     

Real-time monitoring of enzymatic hydrolysis of galactomannans

Enzymatic hydrolysis was monitored in real-time using time dependent static light scattering (TDSLS) for a variety of galactomannans from native Brazilian flora. alpha-Galactosidase, which strips only the (1-6)alpha-D galactose side groups, and beta-mannanase, which hydrolyses only the (1-4)beta-D mannan main chain into oligosaccharides were investigated separately and in combination. The time-dependent signatures matched those describing side-chain stripping for galactosidase, whereas those resulting from the action of mannanase followed the signature typical of random backbone cleavage. Use of both enzymes together required that the TDSLS theory of polymer degradation be extended to the case where random backbone cleavage sites appear as side chains are stripped by the first enzyme. Whereas galactosidase allowed mannanase to access more backbone cleavage sites as time passes, leading to a higher degree of hydrolysis, there was no increase in rate constants. The distribution of random fragments in the case of mannanase digestion alone followed reasonably well the predictions for random cleavage of a single-strand polymer with a restricted number of cleavage sites. The fragment distributions were evaluated by size exclusion chromatography.     

Isolation and structural characterization of the water-extractable polysaccharides from Cassia obtusifolia seeds

The seed of Cassia obtusifolia is a food or herbal medicine used for improving eyesight, treating constipation and other disorders, and polysaccharides have been implicated in these pharmacological activities. The endosperm of the seeds, Cassia gum, is a commercial thickening or gelling agent, composed mainly of galactomannans. However, the whole seeds of C. obtusifolia, rather than the endosperm, are used in folk medicine or food, which might contain more complex constituents of polysaccharides. In this study, the whole seeds of C. obtusifolia were extracted with boiling water, and from the water extract, three homogeneous fractions were isolated, designated CFAA-1, CFAA-3, and CFBB2, respectively, after treatment with Fehling solution followed by anion-exchange and gel permeation chromatography. Using chemical and spectroscopic methods, CFAA-1, and CFAA-3 were elucidated to be both branched galactomannans with different molecular weights, consisting of 1,4-linked β-d-mannopyranosyl backbone with single-unit α-d-galactopyranosyl branches attached to O-6 of mannose, while CFBB2 was shown to be a linear (1→4)-α-polygalacturonic acid.     

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.     

Substituent distribution and clouding behavior of hydroxypropyl methyl cellulose analyzed using enzymatic degradation

The distribution of substituents along the polymer backbone will have a strong influence on the properties of modified cellulose. Endoglucanases were used to degrade three different batches of hydroxypropyl methyl cellulose (HPMC) derivatives with similar chemical properties. The phase separation of the HPMCs as a function of temperature, i.e., the clouding behavior, was analyzed prior to degradation. The total amount of unsubstituted glucose was determined using total acid hydrolysis followed by high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD). The products after enzymatic degradation were analyzed with size-exclusion chromatography with online multiangle light scattering and refractive index detection and also with reducing end determination. To further characterize the formed products, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry was employed for analysis of short-chained oligosaccharides. The different endoglucanases showed varying degradation capability of HPMC derivatives, depending on structure of the active site. The investigated HPMCs had different susceptibility to degradation by the endoglucanases. The results showed a difference in substituent distribution between HPMC batches, which could explain the differing clouding behaviors. The batch with the lowest cloud point was shown to contain a higher number of non-degradable, highly substituted regions.     

Synthesis of galactomannan sulfates

The experimental conditions for the sulfation of legume galactomannans were found, which allow for the obtainment of polysaccharides with a high degree of substitution. Sulfate esters of four galactomannans of different composition (galactose content, 16.4-47.5%) were synthesized using the SO3-pyridine complex in dimethylformamide as a sulfating agent. The degree of substitution was as high as 1.4-1.8; it did not correlate with the content of galactose in the polysaccharides. It was found that the degree of sulfation depended on the reaction temperature in the range of 19-60 degrees C.     

Cassia grandis Linn. f. seed galactomannan: structural and crystallographical studies

Cassia grandis is a small or medium sized tree, found in abundance throughout India. The seeds contain about 50% endosperm gum and possess the characteristics of becoming a potential source of seed gum. The purified polysaccharide has been characterized as a pure galactomannan having a mannose-galactose ratio of 3.15; molecular weight (Mw) 80,200; polydispersity (Mw/Mn), 1.35 and intrinsic viscosity [eta], 848 mL/g. Methylation, periodate oxidation, Smith degradation and 13C NMR studies confirm that the polysaccharide has the basic structure of legume galactomannans consisting of a beta-(1-->4)-linked main mannan backbone to which galactose units are attached at O-6. The orthorhombic lattice constants of the hydrated gum are as follows: a=9.00, b=24.81, c=10.30 A. The crystallographic data establish that the probable space group symmetry of the unit cell is P2(1)2(1)2. The results are in contradiction to earlier reports (Indian J. Chem. 16B (1978) 966; J. Indian Chem. Soc. 55 (1978) 1216) in which a non-galactomannan polysaccharide structure has been assigned having a main chain of (1-->4)-linked galactose and mannose units in the molar ratio 6:3, where 50% of the galactose units branched with two galactose and one mannose through 1-->3 linkage.     

Synthesis of galactomannan sulfates

The experimental conditions for the sulfation of legume galactomannans were found, which allow for the obtainment of polysaccharides with a high degree of substitution. Sulfate esters of four galactomannans of different composition (galactose content, 16.4–47.5%) were synthesized using the SO₃-pyridine complex in dimethylformamide as a sulfating agent. The degree of substitution was as high as 1.4–1.8; it did not correlate with the content of galactose in the polysaccharides. It was found that the degree of sulfation depended on the reaction temperature in the range of 19–60°C.     

Solution properties of the galactomannans extracted from the seeds of Caesalpinia pulcherrima and Cassia javanica: comparison with locust bean gum.

The galactomannans from the seeds of Caesalpinia pulcherrima and Cassia javanica were extracted from the milled seeds in water at room temperature. Both products, as well as a commercial sample of locust bean gum (LBG), were purified by precipitation in isopropyl alcohol. The intrinsic viscosity determined for LBG, [eta] = 15.2 dl/g, was slightly higher than those for the other two galactomannans. The dependence of the specific viscosity at zero shear rate on the coil overlap parameter, C[eta], revealed a similar behaviour for the three galactomannans. A master curve was obtained with a critical concentration, C*, at C*[eta] = 3.3. The slope of the curve in the concentrated regime is higher than the values in the range of 3.9-6.6, obtained for the generalized behaviour of several random coil polysaccharides. Dynamic experiments showed that, at the concentrations studied, the behaviour of the galactomannans was typical of systems with predominant entanglement networks in the region between the terminal and plateau zones of frequency response. The correlation between dynamic and steady shear properties (Cox Merz rule) was satisfactory for the three galactomannans.     

A novel synergistic galactomannan-based unit dosage form for sustained release of acarbose

In the current study, the potential of a novel combination of a galactomannan with acarbose (100 mg) was evaluated for attaining a desired hypoglycaemic effect over a prolonged period of time. Three major antidiabetic galactomannans viz., fenugreek gum, boswellia gum, and locust bean gum were selected in order to achieve a synergistic effect in the treatment alongwith retardation in drug release. In vitro studies indicated that batches containing various proportions of fenugreek gum (AF40-60) were able to control drug release for a longer duration of approximately 10–12 h. In contrast, the matrices prepared using boswellia and locust bean gum were able to sustain the release for relatively shorter durations. Drug release mainly followed first-order release kinetics owing to the highly soluble nature of the drug. In vivo study depicted a significant reduction (p < 0.001) in the postprandial blood glucose and triglyceride levels in the diabetic rats on treatment with formulation AF40. Thus, the developed system provides a better control of the postprandial glycaemic levels and it also obviates the need of conventional multiple dosing of acarbose. Furthermore, it also reduces the occurrence of side effects like diarrhea and loss of appetite.     

FAB CIDMS/MS analysis of partially methylated maltotrioses derived from methylated amylose: a study of the substituent distribution

Amylose was methylated with CH3I in alkaline aqueous suspension, yielding methylated amylose (MeAl) with a degree of substitution of 1.44 (s < 0.01). Determination of the monomer composition showed that HO-6 and HO-2 were highly substituted in contrast to HO-3 (7:2:5.5, HO-2:HO-3:HO-6). By using partial acid hydrolysis, oligomers were prepared that varied both in degree of polymerisation and in methyl-content. Studies on the distribution of substituents in trimers showed large deviations from random distributions. By using CID tandem mass spectrometry, the substituent distribution in these trimers was determined in more detail. Various sets of trimers with equal amounts of methyl-groups but differing in substituted positions were quantified. From the monomer composition of MeAl, the probability of each trimer was calculated and compared to the outcome of the measured distributions. It was concluded that trimers with terminal tri- or non-substituted glucose monomers at the non-reducing end were formed preferentially during partial hydrolysis and that partial hydrolysis of MeAl yielded oligomers in a non-random way. This is the first study that describes the partial hydrolysis of MeAl in such detail.     

高效液相色谱法分析猪屎豆种子胶多糖中的单糖组成

建立了一种采用SUGAR SP-G 0810(Pb型)糖分析色谱柱,以纯水为流动相,利用示差折光检测器的高效液相色谱外标法直接分离分析半乳甘露聚糖胶中单糖组成的方法。木糖、葡萄糖、半乳糖和甘露糖的分离在20 min内完成,检出限分别达到2.0μg,20μg,1.0μg和20μg,线性范围为2～10 mg/mL。该方法简便、快速、重现性好,用于猪屎豆种子胶多糖中单糖组分的测定,并进行回收率试验,结果5次测定的半乳糖和甘露糖的回收率分别为95.83%和103.68%,RSD分别为1.53%和1.50%。     

Affinity interactions between agarose and β-1,4-glycans: a model for polysaccharide associations in algal cell walls

This paper reviews the extensive and previously unpublished work on the interactions between agarose and 1,4-linked β-d-glycans carried out at Unilever Research, Colworth Laboratory, UK. The effect of the following variables is discussed: (i) galactose content of galactomannans; (ii) substitution patterns in the agarose molecule; (iii) structural variations in the 1,4-β-d-glycan main chain; and (iv) molecular size of the 1,4-β-d-glycans.Double helices of agarose, a non-substituted regular polysaccharide comprising 1,3-linked β-d-galactose and 1,4-linked 3,6-anhydro-α-l-galactose, bind in an ordered cooperative fashion to an extended ribbon ordered conformation of sequences of 1,4-linked β-d-mannopyranose residues in plant galactomannans to give mixed gelling systems. This interaction survives, in a modified form, substitution along the agarose molecule by O-methyl ether and O-sulphate esters at O6 of the d-galactose and O2 of the 3,6-anhydro-l-galactose, and 4,6-linked pyruvic acid ketal groups on the d-galactose. The higher the level of substitution on the agarose, the weaker the interaction with galactomannan.In general, the higher the level of galactose substitution in the galactomannan the lower the extent of interaction with agarose. Evidence is presented, however, which indicates that the fine structural distribution of galactose along the galactomannan molecule is also an important determinant for the co-gelling interaction. Substituted 1,4-linked β-d-glucomannans, β-d-glucans and β-d-xylans which can form closely similar extended ribbon order conformations to the galactomannans also participate in co-gelling interactions with agarose. These β-d-glycans are similar in structure to important skeletal polysaccharides such as hemicelluloses and cellulose. This suggests that the binding between agars and β-d-glycans might mimic biological cohesion between skeleton and gel phases in natural red seaweed cell walls. The sensitivity of the interactions studied to fine details of agar and β-d-glycan structure is what might be expected on biological grounds, since the wide and subtle variations of natural polysaccharide structure are presumed to represent a mechanism for control of their intermolecular interactions.