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.     

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.     

Effect of molecular weight on the antioxidant property of low molecular weight alginate from Laminaria japonica

In this study, three alginate fractions with different molecular weights and ratios of mannuronic acid (M) to guluronic acid (G) were prepared by enzymatic hydrolysis and ultrafiltration to assess the antioxidant property of alginates from Laminaria japonica with molecular weight below 10 kDa. The antioxidant properties of different molecular weight alginates were evaluated by determining the scavenging abilities on superoxide, hydroxyl, and hypochlorous acid and inhibitory effect on Fe²⁺-induced lipid peroxidation in yolk homogenate. The results showed that low molecular weight alginates exhibited high scavenging capacities on superoxide, hydroxyl, and hypochlorous acid radicals and good inhibition of Fe²⁺-induced lipid peroxidation in yolk. By comparison, alginate A1 with molecular weight below 1 kDa and M/G of 1.84 had better scavenging activity on superoxide, hydroxyl, and hypochlorous acid radicals in vitro than A2 (1–6 kDa), A3 (6–10 kDa), ascorbic acid, and carnosine. With similar M/G ratio, A2 exhibited better antioxidant activity on superoxide and hypochlorous acid radicals than A3. However, fraction A3 with molecular weight of 6–10 kDa exhibited higher inhibitory ability on lipid peroxidation in yolk in vitro than A1 and A2. The results indicated that molecular weight played a more important role than M/G ratio on alginate to determine the antioxidant ability. By comparison, low molecular weight alginates composed of guluronic acid and mannuronic acid exhibited better antioxidant ability on oxygen free radicals than sulfated polysaccharides from L. japonica in our previous study and represent a good source of marine polysaccharide with potential application as natural antioxidant.     

Structural regime identification in ionotropic alginate gels: influence of the cation nature and alginate structure

The morphologies of several ionotropic alginate hydrogels and aerogels were investigated by SAXS according to the nature of the divalent metal cation (Mn(2+), Co(2+), Zn(2+), Cu(2+)) and the guluronic fraction of the alginate. All alginate hydrogel and aerogel samples show isotropic small-angle X-ray scattering. Gelation results from cooperative associations of cations and chain segments and yields different nanostructures, that is, nanofibrillar morphology or multiple junction morphology, according to cation type and eventually mannuronic/guluronic ratio. Therefore, Mn and Cu gels present the same morphology whatever the guluronic ratio, whereas Co and Zn gels yield different nanostructures. In the size range investigated by SAXS (~10-200 ?), the structure of aerogels obtained by CO(2) supercritical drying is found to be inherited from the morphology of the parent hydrogel whatever the initial structural regime.     

Mechanical properties of C-5 epimerized alginates

There is an increased need for alginate materials with both enhanced and controllable mechanical properties in the fields of food, pharmaceutical and specialty applications. In the present work, well-characterized algal polymers and mannuronan were enzymatically modified using C-5 epimerases converting mannuronic acid residues to guluronic acid in the polymer chain. Composition and sequential structure of controls and epimerized alginates were analyzed by (1)H NMR spectroscopy. Mechanical properties of Ca-alginate gels were further examined giving Young's modulus, syneresis, rupture strength, and elasticity of the gels. Both mechanical strength and elasticity of hydrogels could be improved and manipulated by epimerization. In particular, alternating sequences were found to play an important role for the final mechanical properties of alginate gels, and interestingly, a pure polyalternating sample resulted in gels with extremely high syneresis and rupture strength. In conclusion, enzymatic modification was shown to be a valuable tool in modifying the mechanical properties of alginates in a highly specific manner.     

Alginate Production by Plant-Pathogenic Pseudomonads

Eighteen plant-pathogenic and three non-plant-pathogenic pseudomonads were tested for the ability to produce alginic acid as an exopolysaccharide in vitro. Alginate production was demonstrated for 10 of 13 fluorescent plant-pathogenic pseudomonads tested with glucose or gluconate as the carbon source, but not for all 5 nonfluorescent plant pathogens and all 3 non-plant pathogens tested. With sucrose as the carbon source, some strains produced alginate while others produced both polyfructan (levan) and alginate. Alginates ranged from <1 to 28% guluronic acid, were acetylated, and had number-average molecular weights of 11.3 × 10³ to 47.1 × 10³. Polyfructans and alginates were not elicitors of the soybean phytoalexin glyceollin when applied to wounded cotyledon surfaces and did not induce prolonged water soaking of soybean leaf tissues. All or most pseudomonads in rRNA-DNA homology group I may be capable of synthesizing alginate as an exopolysaccharide.     

Formation of effective channels in alginate gel for immobilization of anchorage-dependent animal cells

Summary The conditions for formation of effective channels in alginate gels for growth of anchorage-dependent animal cells were examined. Many channels were formed in the gels by adding a low concentration solution of a high molecular weight polymer of alginate to a high concentration solution of divalent cations. It is recommended that an alginate with a high molecular weight and a low mannuronic acid/guluronic acid ratio be gelled by contact with strontium ions for the cultivation of immobilized anchorage-dependent cells because the gels produced have many channels and are mechanically strong.     

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.     

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.     

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.     

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.     

Tailor-made alginate bearing galactose moieties on mannuronic residues: selective modification achieved by a chemoenzymatic strategy

1-Amino-1-deoxygalactose (12%, mole) has been chemically introduced on a mannuronan sample via an N-glycosidic bond involving the uronic group of the mannuronic acid (M) residues. The unsubstituted M residues in the modified polymer were converted into guluronic moieties (G) by the use of two C-5 epimerases, resulting in an alginate-like molecule selectively modified on M residues. The molecular details of the newly formed polymer, in terms of both composition and molecular dimensions, were disclosed by use of (1)H NMR, intrinsic viscosity, and high-performance size-exclusion chromatography-multiple-angle laser light scattering (HPSEC-MALLS). Circular dichroism has revealed that the modified alginate-like polymer obtained after epimerization was able to bind calcium due to the introduction of alternating and homopolymeric G sequences. The gel-forming ability of this M-selectively modified material was tested and compared with an alginate sample containing 14% galactose introduced on G residues. Mechanical spectroscopy pointed out that the modified epimerized material was able to form stable gels and that the kinetics of the gel formation was similar to that of the unsubstituted sample. In contrast, the G-modified alginate samples showed a slower gel formation, eventually leading to gel characterized by a reduced storage modulus. The advantage of the selective modification on M residues was confirmed by measuring the Young's modulus of gel cylinders of the different samples. Furthermore, due to the high content in alternating sequences, a marked syneresis was disclosed for the modified-epimerized sample. Finally, calcium beads obtained from selectively M-modified alginate showed a higher stability than those from the G-modified alginate, as evaluated upon treatment with nongelling ions.     

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.     

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.     

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.     

Tailoring of alginates by enzymatic modification in vitro

High molecular weight alginates having a variety of initial composition and sequential structures were modified with a mannuronan-C-5 epimerase from Azotobacter vinelandii to yield polymers with a high content of guluronic acid and, hence, an enhanced ability to form gels with calcium ions. The monad, diad and triad frequencies in the modified polymers were determined by n.m.r. spectroscopy, and the strength of homogeneous gels prepared from them with calcium ions were measured and compared with those prepared from the starting materials and other naturally occurring alginates. Immobilization of the bacterial enzyme on Eupergite beads greatly increased its stability and favoured high degree of conversion.     

Alginate aerogels as adsorbents of polar molecules from liquid hydrocarbons: Hexanol as probe molecule

Ionotropic gels of alginate, a polysaccharide, can be easily converted to aerogels of high surface area. The potential of alginate aerogels as adsorbents for trace polar contaminants in hydrocarbon feedstocks is evaluated, n-hexanol being used as a polar probe molecule. The influence of the nature of the gelling cation has been studied by testing Ca-, Ba-, Ni-, Co-, and Cu-alginate aerogels and a gel of alginic acid, formed by proton exchange of Na-alginate. Adsorption capacity can reach 15% hexanol (w/w) without any swelling of the gel. The amount adsorbed in the monolayer allows to evaluate the surface area of the adsorbent and confirms that the immersion in hydrocarbon does not modify the size and the dispersion of the polysaccharide fibrils. The comparison of the surface density of adsorbate with the structure of the surface indicates that hexanol is adsorbed on alginic acid by the formation of hydrogen bonds between the alcohol heads and two hydroxyls of the polymer surface. In the case of alginates gelled by divalent cations, stronger adsorption sites allows completion of a monolayer at lower concentrations of the polar molecule.     

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.     

Galactose-substituted alginate: preliminary characterization and study of gelling properties

Coupling of alginate with 1-amino-1-deoxygalactose in the presence of 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide results in a substituted polymer containing galactose side linked via an amide bond. To clarify the degree and pattern of substitution, a (1)H NMR study on the anomeric region of modified alginate, polymannuronate, alginate enriched in guluronic acid (G-enriched alginate), and polyalternating MG, was carried out (G, alpha-l-guluronic acid; M, beta-d-mannuronic acid). From the resonance of the proton at position 1 of galactosylamine, it was possible to determine the amount of galactose linked to mannuronic and to guluronic residues, respectively. Furthermore, (1)H NMR spectroscopy revealed a higher reactivity of guluronic residues for low degrees of conversion. Modified alginates with 7% and 19% of substitution are both able to form stable beads in the presence of calcium ions. The effect of galactose substitution on the dimensions, swelling, and stability of the beads has been studied and the cytotoxicity of the modified polymer evaluated in preliminary biological tests.     

Gel strength of Ca-limited alginate gels made in situ

The gel strengths of Ca-alginate gels made in situ with different degree of cross-linking were determined by adapting three different methods: FIRA Jelly Tester, initial deformation (Youngs modulus, E) with a Stevens LFRA Texture Analyzer, and dynamic measurements with a Bohlin VOR Rheometer (dynamic storage modulus, G). The results showed that there were relatively large differences in absolute values, but that the deviations diminished when the results were expressed as relative gel strengths. The deviations of the Youngs modulus (E) from G increased with decreasing gel strength. Only dynamic measurements were suitable for quantifying low gel strengths.The gel strength and the breaking point were also measured as a function of the molecular weight of alginates isolated from stipes of Laminaria hyperborea. In the present Ca limited system, both the gel strength and the breaking point showed a marked increase with increasing molecular weight (Mw) up to 320–340 kD. This is considerably higher than with gels made by dialysis (Ca saturated), where the gel strength becomes independent on molecular weight around 100 kD. These results may have an impact on applications of alginate gels where the source of Ca crosslinking ions is limited.