Molecular structure of carrageenans and kappa oligomers: a Raman spectroscopic study

The Raman spectra of kappa and iota carrageenan, a desulphated furcellaran and a series of oligomers have been compared in the region 700-1500 cm-1. Spectral differences depending on the amount and the location of the sulphate group on the ring, the chain length, the nature of the counterion and the conformation are discussed. Indications that the ionic interactions in the Na+ salts of the oligomers are different from those in K+ and Rb+ salts are given. On the macromolecular level it is found that the vibrational movements of the skeleton are related to the chain flexibility and the conformation. In gels of K+ and Rb+ kappa carrageenan spectral evidence is given for the existence of structural order.     

Synergistic interactions between the genetically modified bacterial polysaccharide P2 and carob or konjac mannan

Rheological studies have confirmed that the bacterial polysaccharide P2, a genetically modified variant of the Acetobacter xylinum polysaccharide acetan, undergoes synergistic gelation with either of the plant polysaccharides carob or konjac mannan. X-ray fibre diffraction data shows that P2 can form a 5-fold helical structure of pitch 4.7nm and an axial rise per disaccharide repeat of 0.92nm. Optical rotation data demonstrate that P2 undergoes a coil-helix transition in solution and that deacylation enhances the stability of the helical structure in solution. Studies made on mixtures prepared at different temperatures and ionic strengths suggest that denaturation of the P2 helix favours interaction and gelation. Deacetylation of P2 enhances gelation. X-ray diffraction data for oriented fibres prepared from deacetylated P2-konjac mannan mixed films reveal a 6-fold helical structure of pitch 5.54nm with an axial rise per disaccharide repeat also of 0.92nm. This mixed helix provides direct evidence for binding between the two polysaccharides. P2 contains two sites of acetylation: one on the backbone and one on the sidechain. The former site of acetylation inhibits helix formation for P2. It is suggested that this site of acetylation also inhibits formation of the mixed helix, explaining the enhanced gelation of mixtures on deacetylation.     

Salt dependence and ion specificity of the coil–helix transition of furcellaran

The conformational transition and the cation-binding properties of aqueous furcellaran (a gel-forming, low-sulfated polysaccharide of the carrageenan family) in various salts and salt mixtures was studied by optical rotation and by ¹³³Cs-nmr. The results were compared with theoretical predictions based on the Poisson–Boltzmann cell model (PBCM). The conformational transition of furcellaran occurs in a single step, which implies a nonblocklike distribution of sulfate groups along the polymer chain. The chloride salts of sodium, lithium, and tetramethylammonium are equally potent in inducing helix formation of furcellaran, indicating that these ions act by nonspecific electrostatic interactions. In contrast, the potassium and cesium ions specifically promote helix formation and aggregation (gelation) of furcellaran. The divalent calcium and magnesium ions are nonspecific, but more potent than the nonspecific monovalent ions in inducing helices. Anions differ in their capacity to stabilize the furcellaran helix in the sequence Cl^? < NO < Br^? < SCN^? < I^?. The iodide and thiocyanate anions impede aggregation and gel formation. ¹³³Cs-nmr chemical shifts indicate specific binding of cesium ions to the furcellaran helix. Thus, with respect to its ion specificity and ion-binding properties, furcellaran, with 0.6 sulfate group per repeating disaccharide, resembles κ-carrageenan (1 sulfate/disaccharide) but differs from ι-carrageenan (2 sulfates/disaccharide). The conformational transition temperatures of furcellaran are, however, generally higher than those of κ-carrageenan under comparable conditions, and in mixtures of the two polysaccharides, separate transitions still occur, indicating that no mixed helices are formed. The observed ion sensitivity and cation-binding properties of furcellaran agree with predictions, by the PBCM, for a K-carrageenan with a reduced charge density.     

Computer modelling of kappa carrageenam-mannan interactions

Molecular modelling has been used as a theoretical approach to investigate the kappa carrageenan structure and its interaction with mannan chains. Calculations revealed the existence of six minima for the kappa carrageenan structure in solution. Two of them were very close to the structure found in the solid state. The methodology allowed the calculation of the theoretical counterpart of the structures based on x-ray fibre diffractions studies. In the second step of this study, we have shown that there is the possibility of interactions between kappa carrageenan double helices and mannan chains. This interacting process is allowed by the flexibility of the mannan chains and structural changes of the kappa carrageenan double helices. The calculations suggest that the disaccaride mannan fragment might be required for recognition. The result of our investigation are in good agreement with a model of gel structure based on experimental data. This approach could be applied to simulate and predict other associations in molecular assemblies.     

Gel-forming structures and stages of red algal galactans of different sulfation levels

Sol-gel transition processes of algal galactans were studied using cryofixation method in combination with freeze-drying and scanning electron microscopy (SEM) techniques. The structures formed in successive stages of gelling process upon cooling were rapidly frozen at defined temperature points and viewed by SEM. It was established that in the case of both types of gelling galactans investigated, a fine honeycomb-like network exists for a wide range of solution temperatures. The formation and structure of this network depends on the structural type, gelling stage, and concentration of the galactan in solution. The honeycomb suprastructures exist also in carrageenan and agarose sols (at temperatures considerably exceeding the gelling temperatures). An additional helical network formed showed different behaviour in the case of carrageenan and agar-type polysaccharides. In the gel-formation process, tightening of the network takes place in both types of galactan gels; the honeycomb structures persist in carrageenan (furcellaran) but not in agarose gels.     

Screening for synergistic interactions in dilute polysaccharide solutions

A simple viscometric approach has been used to screen for binding interactions between different polysaccharides in very dilute solution where exclusion effects should be negligible. The method involves preparing stock solutions to approximately the same, low, viscosity (η_sp≈1), dialysing to identical ionic conditions, mixing in various proportions, and looking for departures from the initial common viscosity.

Mixtures of xanthan or de-acetylated xanthan with locust bean gum (LBG) or konjac glucomannan (KM) show massive enhancement of viscosity, as anticipated from the formation of synergistic gels at higher concentrations. However, no viscosity changes on mixing with LBG or KM were observed for other conformationally ordered bacterial polysaccharides (welan and rhamsan) or for alginate and pectin with sufficient Ca²⁺ to induce almost complete conversion to the dimeric ‘egg box’ form, demonstrating that conformational rigidity is not, in itself, sufficient for other polysaccharides to form heterotypic junctions with mannan or glucomannan chains.

Interactions of carrageenans with LBG appear to depend on both conformation and the extent of aggregation. Mixtures of LBG with K⁺ kappa carrageenan in 100mM KCl (which is known to promote extensive aggregation of double helices) gave erratic values for rotational viscosity and showed typical gel-like mechanical spectra under low-amplitude oscillation. Disordered carrageenans (K⁺ kappa in water and lambda in 100mM KCl) showed no evidence of interaction with LBG. Negative results were also obtained for iota carrageenan under ionic conditions believed to promote ordering without significant aggregation (100mM KCl). However, under conditions where limited aggregation might be expected (iota carrageenan in 90 mM CaCl₂; Me₄N⁺ kappa carrageenan in 150 mM Me₄NI), significant reductions in viscosity were observed on mixing with LBG, which may indicate some intermolecular association but without the formation of an extended network structure.     

Molecular structure of potassium kappa carrageenate gels: a rheological and pulsed electric birefringence study

Measurements have been made of the shear modulus as a function of biopolymer concentration for pure potassium kappa carrageenate gels. The results have been discussed on the basis of rubber elasticity theory to investigate the free polymer linkages between junction zones within the gels. Pulsed electric birefringence studies have been made on segmented kappa carrageenan as a model for the junction zones of the gel.     

BETA/KAPPA-CARRAGEENANS AS EVIDENCE FOR CONTINUED SEPARATION OF THE FAMILIES DICRANEMATACEAE AND SARCODIACEAE (GIGARTINALES,RHODOPHYTA)1

The Dicranemataceae consists of five species in four genera of macroscopic red algae endemic to the southern half of Australia plus a single species from southern Japan. Investigations of the nonfibrillar wall components of five of the six species show that all are composed mainly of hybrid (or mixed) beta (β)/kappa(κ)-type carrageenans. Detailed studies of Tylotus obtusatus (Sonder) J. Agardh show that it produces the largest dry-weight percentage of β-carrageenan yet recorded. Monosaccharide composition, total sulfate content, sulfation pattern revealed by infrared and ¹³C-nuclear magnetic resonance spectroscopy and a positive specific optical rotation ([α]_D+ 54°) are indicative of a low-sulfate-containing carrageenan with gelling properties similar to those of agar and furcellaran. β-carrageenan is recorded in only five other red algal species belonging to relatively unrelated families, and we conclude that its uniform occurrence in the highly specialized family Dicranemataceae has phylogenetic significance. Chemical and anatomical examination of the genus Sarcodia, which produce lambda-type carrageenan in both its gametophytic and tetrasporophytic phases, suggests that, despite the recent proposal to incorporate the Dicranemataceae into the Sarcodiaceae, the two families should continue to be separated.     

Cation specificity of the order—disorder transition in iota carrageenan: effects of kappa carrageenan impurities

The temperature dependence of the optical rotation of carrageenan segments (iota, kappa and iota/kappa mixtures) in different salt solutions (tetramethylammonium or potassium chloride) has been studied. In solutions of pure iota carrageenan (90% iota structure) no evidence of any pronounced cation specificity nor of any thermal hysteresis in the iota carrageenan order-disorder transition was found. However, mixtures of iota and kappa carrageenan displayed a cation specificity similar to that previously observed in iota samples of lesser chemical purity, indicating that kappa impurities in iota samples may be located in regular sequences, rather than randomly in the carrageenan primary structure. Our findings strongly suggest that the order-disorder transitions of iota and kappa sequences in mixed samples occur independently.     

Rheological studies of synergistic kappa carrageenan-carob galactomannan gels

The mechanical properties, Young's modulus and yield stress of different carob galactomannan-kappa carrageenan blends have been examined using an Instron testing machine as a function of the following parameters:

1. (1) Constant total polysaccharide concentration.

2. (2) Galactomannan concentration for a given carrageenan concentration.

3. (3) Carrageenan concentration for a given galactomannan concentration.

4. (4) Galactomannan molecular weight.

5. (5) Carrageenan molecular weight.

The properties of the binary gels were compared to the kappa carrageenan gels. From the results, two types of gel structure could occur: a coupled network defined by specific junction zones (and involving unsubstituted mannose residues of the galactomannan chains) and also a gel structure with both polysaccharides associating independently.     

The extraction,structure, and gelling properties of hybrid galactan from the red alga <Emphasis Type="Italic">Furcellaria lumbricalis</Emphasis> (Baltic Sea,Estonia)

The structure and composition of galactan from Furcellaria lumbricalis (furcellaran) were investigated in connection with rheological specificities, gel structure, and extraction conditions. The polysaccharide was characterized by ¹³C nuclear magnetic resonance (¹³C-NMR) and Fourier transform infrared spectroscopy, inductively coupled plasma-optical emission spectrometry, electrothermal atomization atomic absorption spectrometry, and gel permeation chromatography methods. The microstructure of polymer gels was studied using a cryofixation method in combination with freeze-drying and scanning electron microscopy (SEM) techniques. The undersulfated furcellaran backbone consists mainly of 3,6-anhydro-d-galactose (28.5–30.1%) and galactose residues, the latter being partly sulfated in positions 4 and 6, which give rise to some specific properties of the gel. Also, residues of 6-O-methyl-d-galactose as a minor component are found to be present. The water-extracted furcellaran with the average molecular weight about 290 kDa is rich in nitrogen, calcium, magnesium, and potassium, while the sodium content is rather low. The low sulfur content (5.3%) and ¹³C-NMR spectra refer to an undersulfated nature of this galactan. The extraction of seaweeds in low concentration alkaline solutions (instead of water) leads to a significant increase of the minimum size of the galactan particles and the value of gel strength (more than 12 times for Rb-containing gels). The properties of the gel are dependent on specific tentacle-like structure units present in furcellaran gels established by a high-resolution SEM.     

Competitive inhibition of interchain interactions in polysaccharide systems

The scope and validity of competitive inhibition to explore specific intermolecular association in polysaccharide systems is demonstrated, using alginate, carrageenan and xanthan-galactomannan gels as models. Alginate gelation can be abolished almost completely in the presence of an equimolar concentration of short poly-l-guluronate chain segments, and weakened by short mixed sequences, while poly-d-mannuronate has little effect; this is consistent with previous evidence of the relative importance of these three block-types in interchain association. Carrageenan gels are weakened by the presence of short homologous segments only under conditions of limited helix-helix aggregation, where it is understood that direct interchain association through double helices contributes appreciably to total crosslinking. Gel formation by association of the ordered conformation of xanthan with poorly substituted mannan backbone regions of galactomannans is significantly inhibited by the addition of a galactomannan having fewer unsubstituted chain sequences, and which can therefore bind to xanthan without significant network formation. In general, therefore, gel formation through ordered junction zones of fixed stoichiometry is inhibited by chains that are capable of participation in one stable junction, but not in two or more, so that they occupy binding sites without contributing to the development of the network.     

Rheology of kappa-carrageenan in mixtures of sodium and cesium iodide: two types of gels

Recent studies on dilute solutions (Borgström et al. (1996), Int. J. Biol. Macromol. 18, 223) have shown that kappa-carrageenan helices associate into superhelical rigid rods in mixed 0.1 M aqueous solutions of NaI and CsI above a critical mole fraction (x_Cs = 0.4) of Cs. This work concerns the temperature-dependent rheology of more concentrated systems in mixed and pure solutions of the same salts. Gels with low moduli were even found in NaI alone, although this salt is known to impede the gelation of kappa-carrageenan, but only above 0.9% (w/w) of carrageenan. These gels were reminiscent of iota-carrageenan gels in two respects: the (low) magnitude of the shear storage modulus (G′), and the absence of hysteresis in the sol-gel transition. On the other hand, both the threshold concentration for gelation and the ratio between the loss and storage moduli were substantially higher for the kappa-carrageenan gels in NaI. In mixed solutions of CsI and NaI, two types of kappa-carrageenan gels could be distinguished, depending on the cesium content. The transition occurred at x_Cs = 0.4, as in the previous studies on dilute solutions. Below x_Cs = 0.4, the gels were similar to those in NaI alone. Above x_Cs = 0.4, the gels were similar to ‘conventional’ kappa-carrageenan gels, formed in salts such as KC1: a pronounced thermal hysteresis appeared in the sol-gel transition, the gels showed tendencies for syneresis, and G′ increased dramatically with increasing cesium content.     

Mixtures of pregelatinised maize starch and κ-carrageenan: Compatibility, rheology and gelation

Compatibility, flow and visco-elastic properties of a pregelatinised maize starch mixed with κ-carrageenan were investigated. After cooking of the pregelatinised starch, some undissolved granules remained in solution. Aqueous mixtures of κ-carrageenan and starch were studied at 60 °C and 20 °C by combining rheological measurements and microscopic observations under conditions allowing gelation of carrageenan and non-gelation of starch. The viscometric study of mixed dilute solutions of amylose from pregelatinised starch and carrageenan showed that the components are slightly incompatible. Mixture viscosity and elastic modulus were studied at 60 °C in details as a function of mixture composition for a total polymer concentration of 3%; both were found to be significantly higher than the corresponding theoretical additive values. This finding was interpreted by starch granules excluded volume effect. At 20 °C, no noticeable increase of mixture elastic modulus was found as compared with the additive value. The absence of the synergistic effect is supposed to be due to the formation of highly inhomogeneous gels with agglomerates of undissolved granules.     

Characterization of kappa-carrageenan gels used for immobilization of Bacillus firmus.

In this study, aimed at a biochemical and physical characterization of kappa-carrageenan gels used for entrapment of Bacillus firmus NRS 783 (a superior producer of an alkaline protease), effects of carrageenan concentration, gelation temperature, initial cell loading, and strength of the curing agent (KCl) on the properties of cell-free and cell-laden gels were examined. The physical properties of the differently prepared gels that were examined included density, free volume fraction, mechanical strength, and change in gel volume during gel curing. The biochemical characteristics studied included viability of gel-entrapped cells, cell leakage from cell-laden gels, and cell penetration into cell-free gels. For the range of carrageenan contents investigated [between 2% and 5% (w/v)], the mechanical strength of the gels with/without KCl curing was observed to increase with an increase in carrageenan content of gels. The mechanical strength of each gel increased substantially upon extensive curing. Free volume fractions in excess of 0.8 were observed for all gels. Of cells that were viable prior to immobilization, 90-92% remained viable after formation and extensive curing of gels for cell-gel mixtures prepared at 45 degrees C. Attempts at prolonged storage of cell-laden gel beads at 0 degrees C as stock cultures of immobilized B. firmus were unsuccessful due to a significant decline in cell viability during such storage. On the basis of the cell leakage studies, the average pore sizes of 2%, 3%, 4%, and 5% gels were deduced to increase in the following order of carrageenan content (w/v): 4%, 3%, 2%, and 5%. Commensurate with the decrease in the average pore size (or the increased tightness of the gels) with increasing carrageenan content, both the extent of cell leakage and the extent of net cell penetration decreased with increasing carrageenan content for the first three gels. Owing to non-uniform distribution of free space and much larger pores, the extent of net cell penetration in 5% carrageenan gels was considerably low, while the extent of cell leakage in 5% carrageenan gels was an order of magnitude greater than the extents of cell leakage in the other three gels.     

Microstructure and rheological behavior of pure and mixed pectin gels

The microstructure and the rheological properties of pure HM (high methoxyl) and LM (low methoxyl) pectin gels and of mixed HM/LM pectin gels have been investigated. Gel formation of either the HM or LM pectin, or both, was initiated in the mixed gels by varying the sucrose and Ca(2+) content. The microstructure was characterized by transmission electron microscopy, light microscopy, and confocal laser scanning microscopy. HM and LM pectin gels showed aggregated networks with large pores around 500 nm and network strands of similar character. Small differences could be found, such as a more inhomogeneous LM pectin network with shorter and more branched strands of flexible appearance. LM pectin also formed a weak gel in 60% sucrose in the absence of calcium. A highly inhomogeneous mixed gel structure was formed in the presence of 60% sucrose and Ca(2+) ions, which showed large synergistic effects in rheological properties. Its formation was explained by the behavior of the corresponding pure gels. In the presence of 60% sucrose alone, a homogeneous, fine-stranded mixed network was formed, which showed weak synergistic effects. It is suggested that LM pectin interacts with HM pectin during gel formation, thereby hindering secondary aggregation leading to the aggregated networks observed for the pure gels.     

Conformation and association of κ-carrageenan in the presence of locust bean gum in mixed NaI/CsI solutions from rheology and cryo-TEM

Mixtures of locust bean gum (LBG) with κ-carrageenan (KC) in 0.1 M aqueous solutions of the mixed salts NaI/CsI were investigated by cryo-transmission electron microscopy (cryo-TEM) and dynamic viscoelastic measurements. Previous studies have shown that as the cesium content is increased in such mixed salt solutions, a transition occurs from molecularly dispersed helices to ‘superhelical rods’ of KC. We now found that LBG stabilises the superhelical rods, shifting the transition to a lower content of Cs for the mixtures than for KC alone. The formation of superhelical rods was evidenced both by cryo-TEM images and by an onset of thermal hysteresis in the coil–helix transition of KC. In the mixtures, the transition temperatures on cooling and heating were insensitive to the proportions of LBG and KC present at all cesium contents. Under conditions where no helix aggregation occurred (no hysteresis) the mixtures showed high tan δ values and low storage moduli. Under aggregated conditions, gels formed, and gels with added LBG had enhanced moduli compared to gels with KC alone. On the basis of these results we propose that LBG associates to the super-helical rods of KC.     

Photon transmission study on swelling of kappa-carrageenan gels prepared in various concentrations

κ-Carrageenan gels prepared with various carrageenan concentrations in pure water were completely dried and then swelled in pure water. Photon transmission measurements were performed using a UV-Vis (UVV) spectrometer during the swelling of κ-carrageenan gels. Transmitted photon intensity, I_tr, increased exponentially as swelling time is increased for all gel samples. The behaviour of I_tr was interpreted by Monte-Carlo Simulation. The increase in I_tr was quantified by employing Li-Tanaka equation, from which time constants τ₁ and collective diffusion coefficients, D_o were determined for the gels in various carrageenan concentrations. Gravimetric and volumetric measurements were also carried out during swelling of gels. It is observed that gel with high carrageenan content possess more double helices and more lattice dislocations and swell slower than gels with low carrageenan content which may contain less double helices and less lattice imperfections. Increase in I_tr was interpreted by the homogeneous distribution of double helices in the carrageenan gel system.     

Assessment of UVB-photoprotective and antioxidative activities of carrageenan in keratinocytes

Ultraviolet B (UVB) (290–320 nm) is the foremost cause of photoaging, sunburn, wrinkles and skin cancer. Photoprotection against harmful UVB radiation is essential through various means including the use of skincare products. The seaweed polysaccharide carrageenan is widely used as an excipient in cosmetics and skincare products. However, its effects on normal skin keratinocytes or potential use as a photoprotective agent have yet to be established. The primary aim of this study was to assess the cytotoxic, photoprotective and antioxdative effects of carrageenan in UVB-induced immortalised normal human keratinocytes (HaCaT cells). Results showed that the percentage of cell viability decreased linearly with increasing UVB doses from 10, 50, 100, 222 to 1,000 mJ cm^?2. Four isomers of carrageenan, namely iota 2 [ι (??)], iota 5 [ι (V)], lambda (λ) and kappa (κ) carrageenan were used in this study. Vitamin E was used as a positive control. In terms of cytotoxicity, the CD₅₀ of kappa carrageenan was ~200 μg mL^?1 while for the other isomers, the values ranged from 122 to 162 μg mL^?1. Carrageenan showed significant protection against detrimental effects of UVB-induced cell killing and reactive oxygen species (ROS) release based on 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and 2′,7′-dichlorfluorescein-diacetate (DCFH-DA) assays, respectively. Carrageenan was also able to quench 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radicals. The ability to protect against UVB suggests that carrageenan has potential application as a photoprotective agent in addition to just being used as an excipient.