Ionic and acid gel formation of epimerised alginates; the effect of AlgE4

AlgE4 is a mannuronan C5 epimerase converting homopolymeric sequences of mannuronate residues in alginates into mannuronate/guluronate alternating sequences. Treating alginates of different biological origin with AlgE4 resulted in different amounts of alternating sequences. Both ionically cross-linked alginate gels as well as alginic acid gels were prepared from the epimerised alginates. Gelling kinetics and gel equilibrium properties were recorded and compared to results obtained with the original non-epimerised alginates. An observed reduced elasticity of the alginic acid gels following epimerisation by AlgE4 seems to be explained by the generally increased acid solubility of the alternating sequences. Ionically (Ca(2+)) cross-linked gels made from epimerised alginates expressed a higher degree of syneresis compared to the native samples. An increase in the modulus of elasticity was observed in calcium saturated (diffusion set) gels whereas calcium limited, internally set alginate gels showed no change in elasticity. An increase in the sol-gel transitional rate of gels made from epimerised alginates was also observed. These results suggest an increased possibility of creating new junction zones in the epimerised alginate gel due to the increased mobility in the alginate chain segments caused by the less extended alternating sequences.     

Temperature dependence of the elastic properties of alginate gels

The moduli of elasticity of calcium and lead alginate gels increase with time after preparation, and the temperature dependence of the rate of syneresis suggests an activation energy of 8?12 x 10⁴ J.mol?¹ for the formation of new junctions. At zero time, a negative temperature-dependence was found for the elastic force measured at a low degree of deformation (4%). Deformation of the gels was associated with an increase in entropy and internal energy. When the calcium ions in a preformed calcium alginate gel were exchanged for lead ions, which have a higher affinity for alginate, the modulus increased due to an enhanced increase in internal energy with deformation. Reversal of the sequence of introducing the two types of ions gave the opposite effect. The data suggest that the junctions are “weak points” in the gels, and that even small deformations can cause partial rupture.     

Dose-dependent suppression of hunger by a specific alginate in a low-viscosity drink formulation

Addition of specific types of alginates to drinks can enhance postmeal suppression of hunger, by forming strong gastric gels in the presence of calcium. However, some recent studies have not demonstrated an effect of alginate/calcium on appetite, perhaps because the selected alginates do not produce sufficiently strong gels or because the alginates were not sufficiently hydrated when consumed. Therefore, the objective of the study was to test effects on appetite of a strongly gelling and fully hydrated alginate in an acceptable, low-viscosity drink formulation. In a balanced order crossover design, 23 volunteers consumed a meal replacement drink containing protein and calcium and either 0 (control), 0.6, or 0.8% of a specific high-guluronate alginate. Appetite (six self-report scales) was measured for 5 h postconsumption. Relevant physicochemical properties of the drinks were measured, i.e., product viscosity and strength of gel formed under simulated gastric conditions. Hunger was robustly reduced (20-30% lower area under the curve) with 0.8% alginate (P < 0.001, analysis of covariance), an effect consistent across all appetite scales. Most effects were also significant with 0.6% alginate, and a clear dose-response observed. Gastric gel strength was 1.8 and 3.8 N for the 0.6 and 0.8% alginate drinks, respectively, while product viscosity was acceptable (<0.5 Pa.s at 10 s(-1)). We conclude that strongly gastric-gelling alginates at relatively low concentrations in a low-viscosity drink formulation produced a robust reduction in hunger responses. This and other related studies indicate that the specific alginate source and product matrix critically impacts upon apparent efficacy.     

Alginate as immobilization material: I. Correlation between chemical and physical properties of alginate gel beads

Calcium alginate gel beads were prepared from a range of well characterized alginates. The physical properties of beads depended strongly on the composition, sequential structure, and molecular size of the polymers. Beads with the highest mechanical strength, lowest shrinkage, best stability towards monovalent cations, and highest porosity were made from alginate with a content of L-guluronic acid higher than 70% and an average length of the G-blocks higher than 15. For these "high G" alginates the critical overlap intrinsic viscosities have been determined, and for molecular weight higher than 2.4 x 10(5), the gel strength was independent of the molecular weight.     

Small-angle X-ray scattering and rheological characterization of alginate gels. 3. Alginic acid gels

Alginic acid gels were studied by small-angle X-ray scattering and rheology to elucidate the influence of alginate chemical composition and molecular weight on the gel elasticity and molecular structure. The alginic acid gels were prepared by homogeneous pH reduction throughout the sample. Three alginates with different chemical composition and sequence, and two to three different molecular weights of each sample were examined. Three alginate samples with fractions of guluronic acid residues of 0.39 (LoG), 0.50 (InG), and 0.68 (HiG), covering the range of commercially available alginates, were employed. The excess scattering intensity I of the alginic acid gels was about 1 order of magnitude larger and exhibited a stronger curvature toward low q compared to ionically cross-linked alginate. The I(q) were decomposed into two components by assuming that the alginic acid gel is composed of aggregated multiple junctions and single chains. Time-resolved experiments showed a large increase in the average size of aggregates and their weight fraction within the first 2 h after onset of gelling, which also coincides with the most pronounced rheological changes. At equilibrium, little or no effect of molecular weight was observed, whereas at comparable molecular weights, an increased scattering intensity with increasing content of guluronic acid residues was recorded, probably because of a larger apparent molecular mass of domains. The results suggest a quasi-ordered junction zone is formed in the initial stage, followed by subsequent assembling of such zones, forming domains in the order of 50 A. The average length of the initial junction zones, being governed by the relative fraction of stabilizing G-blocks and destabilizing alternating (MG) blocks, determines the density of the final random aggregates. Hence, high-G alginates give alginic acid gels of a higher aggregate density compared to domains composed of loosely packed shorter junction zones in InG or LoG system.     

Controlling rigidity and degradation of alginate hydrogels via molecular weight distribution

The mechanical rigidity and degradation rate of hydrogels utilized as cell transplantation vehicles have been regarded as critical factors in new tissue formation. However, conventional approaches to accelerate the degradation rate of gels deteriorate their function as a mechanical support in parallel. We hypothesized that adjusting the molecular weight distribution of polymers that are hydrolytically labile but capable of forming gels would allow one to alter the degradation rate of the gels over a broad range, while limiting the range of their elastic moduli (E). We investigated this hypothesis with binary alginate hydrogels formed from both ionically and covalently cross-linked partially oxidized (1% uronic acid residues), low [molecular weight (MW) approximately 60,000 g/mol] and high MW alginates (MW approximately 120,000 g/mol) in order to examine the utility of this approach with various cross-linking strategies. Increasing the fraction of low MW alginates to 0.50 maintained a value of E similar to that for the high MW alginate gels but led to faster degradation, irrespective of the cross-linking mode. This result was attributed to a faster separation between cross-linked domains upon chain breakages for the low MW alginates, coupled with their faster chain scission than the high MW alginates. The more rapidly degrading oxidized binary hydrogels facilitated the formation of new bone tissues from transplanted bone marrow stromal cells, as compared with the nonoxidized high MW hydrogels. The results of these studies will be useful for controlling the physical properties of a broad array of hydrogel-forming polymers.     

Influence of oligoguluronates on alginate gelation, kinetics, and polymer organization

Structural polysaccharides of the alginate family form gels in aqueous Ca2+-containing solutions by lateral association of chain segments. The effect of adding oligomers of alpha-l-guluronic acid (G blocks) to gelling solutions of alginate was investigated using rheology and atomic force microscopy (AFM). Ca-alginate gels were prepared by in situ release of Ca2+. The gel strength increased with increasing level of calcium saturation of the alginate and decreased with increasing amount of free G blocks. The presence of free G blocks also led to an increased gelation time. The gel point and fractal dimensionalities of the gels were determined based on the rheological characterization. Without added free G blocks the fractal dimension of the gels increased from df = 2.14 to df = 2.46 when increasing [Ca2+] from 10 to 20 mM. This increase was suggested to arise from an increased junction zone multiplicity induced by the increased concentration of calcium ions. In the presence of free G blocks (G block/alginate = 1/1) the fractal dimension increased from 2.14 to 2.29 at 10 mM Ca2+, whereas there was no significant change associated with addition of G blocks at 20 mM Ca2+. These observations indicate that free G blocks are involved in calcium-mediated bonds formed between guluronic acid sequences within the polymeric alginates. Thus, the added oligoguluronate competes with the alginate chains for the calcium ions. The gels and pregel situations close to the gel point were also studied using AFM. The AFM topographs indicated that in situations of low calcium saturation microgels a few hundred nanometers in diameter develop in solution. In situations of higher calcium saturation lateral association of a number of alginate chains are occurring, giving ordered fiber-like structures. These results show that G blocks can be used as modulators of gelation kinetics as well as local network structure formation and equilibrium properties in alginate gels.     

Effect of Ca2+, Ba2+, and Sr2+ on alginate microbeads

Microcapsules of alginate cross-linked with divalent ions are the most common system for cell immobilization. In this study, we wanted to characterize the effect of different alginates and cross-linking ions on important microcapsule properties. The dimensional stability and gel strength increased for high-G alginate gels when exchanging the traditional Ca2+ ions with Ba2+. The use of Ba2+ decreased the size of alginate beads and reduced the permeability to immunoglobulin G. Strontium gave gels with characteristics lying between calcium and barium. Interestingly, high-M alginate showed an opposite behavior in combination with barium and strontium as these beads were larger than beads of calcium-alginate and tended to swell more, also resulting in increased permeability. Binding studies revealed that different block structures in the alginate bind the ions to a different extent. More specifically, Ca2+ was found to bind to G- and MG-blocks, Ba2+ to G- and M-blocks, and Sr2+ to G-blocks solely.     

Diffusion characteristics of calcium alginate gels.

The diffusivity of a protein solute (bovine serum albumin) within calcium alginate gels made from sodium alginate of different guluronic acid content was determined. It was found that protein diffusion within alginate gels, prepared to be isotropic in structure, was greatest for gels prepared from sodium alginate of low guluronic acid content as opposed to those prepared from sodium alginate of high guluronic acid content. This finding was explained in terms of the difference in flexibility of the polymer backbone of the two alginates. The greater the polymer backbone flexibility, the greater the solute diffusivity within the gel.     

New hypothesis on the role of alternating sequences in calcium-alginate gels

The availability of mannuronan and mannuronan C-5 epimerases allows the production of a strictly alternating mannuronate-guluronate (MG) polymer and the MG-enrichment of natural alginates, providing a powerful tool for the analysis of the role of such sequences in the calcium-alginate gel network. In view of the calcium binding properties of long alternating sequences revealed by circular dichroism studies which leads eventually to the formation of stable hydrogels, their direct involvement in the gel network is here suggested. In particular, 1H NMR results obtained from a mixed alginate sample containing three polymeric species, G blocks, M blocks, and MG blocks, without chemical linkages between the block structures, indicate for the first time the formation of mixed junctions between G and MG blocks. This is supported by the analysis of the Young's modulus of hydrogels from natural and epimerized samples obtained at low calcium concentrations. Furthermore, the "zipping" of long alternating sequences in secondary MG/MG junctions is suggested to account for the shrinking (syneresis) of alginate gels in view of its dependence on the length of the MG blocks. As a consequence, a partial network collapse, macroscopically revealed by a decrease in the Young's modulus, occurred as the calcium concentration in the gel was increased. The effect of such "secondary" junctions on the viscoelastic properties of alginate gels was evaluated measuring their creep compliance under uniaxial compression. The experimental curves, fitted by a model composed of a Maxwell and a Voigt element in series, revealed an increase in the frictional forces between network chains with increasing length of the alternating sequences. This suggests the presence of an ion mediated mechanism preventing the shear of the gel.     

Aerobic digestion of Ca-alginate gels studied as a model system of seaweed tissue degradation

The Ca-crosslinked alginate matrix of brown seaweeds may present a limiting factor when microbes decompose algal tissue. Ca-alginate gels made from Ascophyllum nodosum and Laminaria hyperborea stipe alginates were digested in aerated batch reactors at 35 °C and pH 7 using an alginate decomposing inoculum harvested during aerobic degradation of L. hyperborea stipe. The mineralisation of Ca-alginate gels was independent of the substrate source, with consumption rates of alginate similar to those of algal alginates in L. hyperborea stipe. Despite a high guluronate lyase activity, the fractional content of guluronate in the remaining Ca-alginate gels increased during digestion as observed earlier for algal tissue. Thus, the Ca-crosslinked guluronate residues were the most recalcitrant material in both gels and algal tissue.Since the access for enzymes to the Ca-crosslinked guluronate residues probably is restricted, ionic washout may represent an important factor for the degradation process. In total, the alginate in algal tissue and Ca-alginate gels behaved similarly during biodegradation. This revised version was published online in August 2006 with corrections to the Cover Date.     

Studies on the characteristics of alginate gels in relation to their use in separation and immobilized applications

Studies on diffusion of NAD and hemoglobin from calcium and barium gels are reported where alginate grade, concentration, and gel dimensions were varied. These show that NAD diffusion characteristics are unaffected by alginate and ion concentrations; however, hemoglobin diffusion is affected by alginate concentration. Both hemoglobin and NAD diffusion patterns were shown to be affected by alginate gel dimensions. Studies are reported that show that polymannuronic alginate gels posses good porosity characteristics while polyguluronic alginates from gels with lower porosity, specifically with respect to high-molecular-weight compounds. These findings are discussed with the view to the use of alginate gels for immobilization, solids separation, and diffusion chromatography techniques.     

Mechanical properties of hydrocolloid gels filled with internally produced CO2 gas bubbles.

Agar (2%), alginate (1% algin), and kappa-carrageenan (1.5%) gel specimens were prepared from mother solutions that contained 0-2.5% sodium bicarbonate (agar and carrageenan) or calcium carbonate (alginate). Upon immersion in a citric acid bath (0-2%), the carbonate reacted with the diffusing acid to produce numerous carbon dioxide bubbles. The compressive strength and deformability of the gas-filled gels so produced were determined using a Universal testing machine and compared with those of pure gels and gels containing the carbonate but not subjected to the process after various immersion times. While the agar and alginate gels retained considerable mechanical integrity even after several hours, the carrageenan gels disintegrated after about 2-5 h. Under similar conditions, the number of bubbles produced in the agar gels was about twice that in the alginate gels, an observation that cannot be explained solely by stoichiometric considerations.     

Properties of spherical calcium pectate and alginate gels and their use in diffusion chromatography,solids separations and immobilization of enzymes and cells

Water-soluble acidic polysaccharides—deesterified pectins and carboxy-derivatives of starch—precipitated with calcium ions were tested as precursors of spherical calcium gels. Pectates prepared from apple or citrus pectin, similarly to alginates, are compounds forming spherical calcium gels stable in aqueous medium which have a relatively highly reproducible mass, particle size, water content, shape, mechanical strength and shearing. Both the liquid-solid partition of low- and high-molar-mass solutes and its kinetics proved to be reasonable features. Distribution of pore size in the above materials was estimated. Detailed pictures of surface and of the interior of calcium beads in the scanning electron microscope are presented. The possible use of calcium beads as enzyme carriers, as affinity matrixes and entrapment materials for diffusion chromatography, solids separations and bioindication of a specific water pollution was evaluated. Calcium alginate beads were always used as reference material.     

Some properties of alginate gels derived from algal sodium alginate

Alginic acid in soluble sodium alginate turns to insoluble gel after contact with divalent metal ions, such as calcium ions. The sodium alginate character has an effect on the alginate gel properties. In order to prepare a suitable calcium alginate gel for use in seawater, the effects of sodium alginate viscosity and M/G ratio (the ratio of D-mannuronate to L-guluronate) on the gel strength were investigated. The wet tensile strengths of gel fibers derived from high viscosity sodium alginate were higher than those from low viscosity sodium alginate. The tensile strength increased with diminishing sodium alginate M/G ratio. Among the gel fibers tested, the gel fiber obtained from a sodium alginate I-5G (1% aqueous solution viscosity = 520 mPa·s, M/G ratio = 0.6) had the highest wet tensile strength. After 13 days treatment in seawater, the wet tensile strength of the gel fiber retained 36% of the original untreated gel strength. For sodium alginates with similar viscosities, the seawater tolerance of low M/G ratio alginate was greater than that of the high M/G ratio one. This study enables us to determine a suitable calcium alginate gel for use in seawater.     

FTIR spectroscopy of NH3 on acidic and ionotropic alginate aerogels

The acidity of alginate aerogel films has been investigated by infrared spectroscopy of adsorbed NH(3). Supercritical drying of the alginate provided samples with a surface area of several hundred square meters per gram, in which the probe molecule could reach all acidic sites. Free carboxylic groups were studied on acid-gelled alginates and were found to behave as effective Br?nsted acid sites. Ionotropic alginate gels formed by alkaline earth cations presented only the Lewis acidity of the cations. Ionotropic gels formed by transition metal cations presented both Lewis and Br?nsted sites, because of the presence of a fraction of free carboxylic groups. The incomplete salification was correlated to the pH of the gelling solutions.     

Production of algal gels from the brown alga, <Emphasis Type="Italic">Laminaria japonica</Emphasis> Aresch., and their biotechnological applications

Alginic acid is localised in the cell walls and intercellular spaces of the brown alga, Laminaria japonica Aresch., and the salts of this compound occur mainly as calcium alginates. However, the alginates in this alga do not have the viscosity and the ability to create and stabilise structural products. Hence, the structure and properties of the alginates in Laminaria were changed by chemical modification to produce new products such as sodium alginates and other substances capable of forming gels. The rheological properties of the algal gel from Laminaria depended on the properties of the sodium alginate. Heating the algal product up to 90°C did not change its physical and chemical properties; the viscosity did not differ from that of the initial product. The viscosity and molecular weight of the sodium alginate isolated from the algal gel were stable from 20°C up to 95°C. However, about 30% of the viscosity was lost at 100°C. Recipes and various methods of preparing the gel products as fish sauces, jelly-like fish products, fruit jellies, drinks, cosmetic and pharmaceutical products are presented. The algal gel and the gel products did not lose their integrity with heat processing. Presented at the 6th Meeting of the Asian Pacific Society of Applied Phycology, Manila, Philippines.     

Characterization of the alginates from algae harvested at the Egyptian Red Sea coast

The alginates from five species of brown algae from the Egyptian Red Sea coast, namely: Cystoseira trinode, Cystoseira myrica, Sargassum dentifolium, Sargassum asperifolium, and Sargassum latifolium, were isolated and their compositions and structures studied by 1H NMR spectroscopy. All the alginates studied contain more guluronic acid (G) than mannuronic acid (M) and have a homopolymeric block-type structure (eta<1). The intrinsic viscosity of the alginate samples range from 8.6 to 15.2 and the gel strength ranges from 10.97 to 15.51. The constitutional G- and M-blocks of alginates from two different species (C. trinode and S. latifolium) were separated after partial acid hydrolysis. The 1H NMR spectral data of the blocks GG and MM obtained by chemical fractionation were compared with those of polymeric alginates. The monomeric uronic acids were separated by complete acid hydrolysis of S. asperifolium alginate and the G and M monomers were characterized by 1H, 13C NMR spectroscopy as well as by paper electrophoresis.     

Phase separation in calcium alginate gels

Alginates are polysaccharides consisting of beta-D-mannuronate and alpha-L-guluronate units. In the presence of bivalent cations like calcium the guluronate blocks form physically cross-linked gels. The gelation properties of alginates play an important role in the stability of extracellular polymer substances and in the food industry. When stock solutions of Ca2+ ions and alginate are mixed, the gelation starts before the Ca2+ ions are evenly distributed, which leads to non-uniform gels. In this contribution, Ca alginate gels were prepared by in situ gelation using glucono-delta-lactone and CaCO3. In this way, uniform gels could be prepared directly in the measuring cell. Below a critical concentration, highly viscous solutions were obtained, which were below the critical point of gel formation. In these solutions at low rotational speeds a Schlieren peak arose, which became smaller and steeper with increasing time until a new meniscus could be detected. This behaviour is in contrast to the peak broadening due to diffusion after a synthetic boundary was formed. Evaluation of the data leads to negative diffusion coefficients. It has been shown by others that the mutual diffusion coefficient must be negative in the spinodal region. This phenomena is known as uphill diffusion and leads to phase separation of a binary system. The formation of the gel phase in this case is therefore discussed as uphill diffusion.     

SYNERESIS AND SWELLING OF GELATIN

1. When solid blocks of isoelectric gelatin are placed in cold distilled water or dilute buffer of pH 4.7, only those of a gelatin content of more than 10 per cent swell, while those of a lower gelatin content not only do not swell but actually lose water. 2. The final quantity of water lost by blocks of dilute gelatin is the same whether the block is immersed in a large volume of water or whether syneresis has been initiated in the gel through mechanical forces such as shaking, pressure, etc., even in the absence of any outside liquid, thus showing that syneresis is identical with the process of negative swelling of dilute gels when placed in cold water, and may be used as a convenient term for it. 3. Acid- or alkali-containing gels give rise to greater syneresis than isoelectric gels, after the acid or alkali has been removed by dialysis. 4. Salt-containing gels show greater syneresis than salt-free gels of the same pH, after the salt has been washed away. 5. The acid and alkali and also the salt effect on syneresis of gels disappears at a gelatin concentration above 8 per cent. 6. The striking similarity in the behavior of gels with respect to syneresis and of gelatin solutions with respect to viscosity suggests the probability that both are due to the same mechanism, namely the mechanism of hydration of the micellæ in gelatin by means of osmosis as brought about either by diffusible ions, as in the presence of acid or alkali, or by the soluble gelatin present in the micellæ. The greater the pressures that caused swelling of the micellæ while the gelatin was in the sol state, the greater is the loss of water from the gels when the pressures are removed. 7. A quantitative study of the loss of water by dilute gels of various gelatin content shows that the same laws which have been found by Northrop to hold for the swelling of gels of high concentrations apply also to the process of losing water by dilute gels, i.e. to the process of syneresis. The general behavior is well represented by the equations: See PDF for Equation and See PDF for Equation where P ₁ = osmotic pressure of the soluble gelatin in the gel, P ₂ = stress on the micellæ in the gelatin solution before setting, K_e = bulk modulus of elasticity, V_o = volume of water per gram of dry gelatin at setting and V_e = volume of water per gram of gelatin at equilibrium.