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.     

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.     

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.     

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.     

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.     

Influence of alginate properties and gel reinforcement on fermentation characteristics of immobilized yeast cells

The effect of alginate composition, gel concentration, gelation method, cell loading and surface area on fermentation characteristics of immobilized yeast cells have been investigated. Molecular weight and G/M ratio had only little effect on fermentation velocity and gel strength, while increasing the alginate concentration caused a sizeable decrease in fermentation velocity and an increase in gel strength. The internally gelled immobilizates generally showed a higher fermentation velocity for the same gel strength and no decrease in gel strength was seen during fermentation. With high initial cell loadings, the fermentation velocity per g of immobilizate was higher, but the productivity per cell was lower than with low initial cell loadings. The difference decreased with time. Specific surface area (surface/volume) was shown to be an important factor for the observed productivity per gram of immobilizate, with high S/V ratios giving the highest productivity. Gel shape had no influence on fermentation velocity for a given S/V ratio. Gelation behaviour of externally gelled beads was determined by estimating the amount of cells liberated during gel formation through measurement of invertase activity (yeast-bound) in the gelling solution. A method for reinforcement of internally gelled alginate slabs with a nylon mesh was developed and utilized for production of a continuous fermentation reactor with reinforced gels.     

Tuning structural durability of yeast-encapsulating alginate gel beads with interpenetrating networks for sustained bioethanol production

Microorganisms have become key components in many biotechnological processes to produce various chemicals and biofuels. The encapsulation of microbial cells in calcium cross-linked alginate gel beads has been extensively studied due to several advantages over using free cells. However, industrial use of alginate gel beads has been hampered by the low structural stability of the beads. In this study, we demonstrate that the incorporation of interpenetrating covalent cross-links in an ionically cross-linked alginate gel bead significantly enhances the bead's structural durability. The interpenetrating network (IPN) was prepared by first cross-linking alginate chemically modified with methacrylic groups, termed methacrylic alginate (MA), with calcium ions and subsequently conducting a photo cross-linking reaction. The resulting methacrylic alginate gel beads (IPN-MA) exhibited higher stiffness, ultimate strength and ultimate strain and also remained more stable in media either subjected to high shear or supplemented with chelating agents than calcium cross-linked alginate gel beads. Furthermore, yeast cells encapsulated in IPN-MA gel beads remained more metabolically active in ethanol production than those in calcium cross-linked alginate gel beads. Overall, the results of this study will be highly useful in designing encapsulation devices with improved structural durability for a broad array of prokaryotic and eukaryotic cells used in biochemical and industrial processes.     

Spherical alginate granules formulated for quick-release active subtilisin

Novel attrition-resistant and spherical enzyme granules encapsulating active subtilisin were formed by emulsification of 2% alginate sol loaded with active enzyme, instantaneous gelation triggered through in situ release of Ca(2+) (internal gelation), particle separation, and finally acetone extractive drying. Granular subtilisin was highly active, readily dispersible, and mechanically robust. This technique serves as a new and attractive alternative to established enzyme granulation processes, such as fluid bed coating, extrusion followed by marumerization, drum granulation, or prilling, for use in industrial enzyme applications such as detergents, textile manufacturing, and food processing. The formulation and encapsulation conditions were optimized to maximize the resistance of the granule to compression and impact forces, consistent with enzyme release and particle dispersion in detergent solutions. Well characterized alginates, with specified guluronic/mannuronic acid (G/M) content and molecular weight, were used in the formulation. The characteristics of the resulting microspheres, including their size and distribution, morphology, shrinkage, compression resistance, impact strength, solubility and encapsulation yield, were examined. Spherical dry granules were formulated with a mean diameter of 500 microm with particle sizes ranging from 300 to 800 microm. Dry alginate granules were discrete, spherical, and glossy white and exhibited impact strength, compression resistance, and solubility difference dependent on composition. Reduced starch levels, high alginate concentration, low alginate molecular weight, and use of high guluronate alginates resulted in the lowest dust level and highest compression resistance. Subtilisin mass yields were approximately 50%, and specific activity yields ranged from 60% to 100%. A formulation consisting of 3% SG150 alginate, 10% starch, 10% TiO(2), and 1% CaCO(3) provided granules appropriate for use in detergent application.     

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.     

DECREASE IN DYNAMIC VISCOSITY AND AVERAGE MOLECULAR WEIGHT OF ALGINATE FROM LAMINARIA DIGITATA DURING ALKALINE EXTRACTION1

Alginates are natural polysaccharides that are extracted from brown seaweeds and widely used for their rheological properties. The central step in the extraction protocol used in the alginate industry is the alkaline extraction, which requires several hours. In this study, a significant decrease in alginate dynamic viscosity was observed after 2 h of alkaline treatment. Intrinsic viscosity and average molecular weight of alginates from alkaline extractions 1–4 h in duration were determined, indicating depolymerization of alginates: average molecular weight decreased significantly during the extraction, falling by a factor of 5 between 1 and 4 h of extraction. These results suggested that reducing extraction time could enable preserving the rheological properties of the extracted alginates.     

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.     

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.     

Growth and viability of mycelial fragments of white-rot fungi on some hydrogels

The viability of mycelial fragments of Trametes versicolor and Irpex lacteus and their growth on selected hydrogels are described. The size of mycelial fragments of the fungi did not significantly influence their viability. Alginate hydrogel films supported fungal growth better than agarose, carrageenan, chitosan and gelatin films, and had the highest mechanical strength but were less hydrophilic than the other hydrogels. All commercial alginates that were tested supported aseptic growth of fungal fragments without prior sterilization of the hydrogel solution. The viability of mycelial fragments in the hydrogel solutions was higher for some commercial alginates than that in laboratory grade alginate. The mechanical strength and hydrophilicity of hydrogels from alginate type Sobalg FD 155 and Meer HV were comparable to that of laboratory grade alginate. Sterilization and pH of the alginate hydrogel did not significantly influence the growth of T. versicolor mycelial fragments but affected the growth of I. lacteus. Concentrations of alginate in the range of 1–2% in the hydrogel did not affect the growth of entrapped mycelial fragments of these fungi. Received 25 June 1997/ Accepted in revised form 07 March 1998     

DNA protection from extracapsular nucleases, within chitosan- or poly-L-lysine-coated alginate beads

DNA was immobilized within alginate matrix using an external or an internal calcium source, and then membrane coated with chitosan or poly-L-lysine. Membrane thickness increased with decreasing polymer molecular weight and increasing degree of deacetylation (chitosan). Beads were exposed to a 31,000 molecular weight nuclease to determine the levels of DNA protection offered by different membrane and matrix combinations. Almost total hydrolysis of DNA was observed in alginate beads following nuclease exposure. Less than 1% of total double-stranded DNA remained unhydrolyzed within chitosan- or poly-L-lysine-coated beads, corresponding with an increase in DNA residuals (i.e. double- and single-stranded DNA, polynucleotides, bases). Chitosan membranes did not offer sufficient DNA protection from DNase diffusion since all of the double-stranded DNA was hydrolyzed after 40 min of exposure. Both chitosan and poly-L-lysine membranes reduced the permeability of alginate beads, shown by enhanced retention of DNA residuals after DNase exposure. The highest level of DNA protection within freshly prepared beads was obtained with high molecular weight (197,100) poly-L-lysine membranes coated on beads formed using an external calcium source, where over 80% of the double-stranded DNA remained after 40 min of DNase exposure. Lyophilization and rehydration of DNA beads also reduced permeability to nucleases, resulted in DS-DNA recoveries of 60% for chitosan-coated, 90% for poly-L-lysine-coated, and 95% for uncoated alginate beads.     

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.     

Components in the inoculum determine the kinetics of Azotobacter vinelandii cultures and the molecular weight of its alginate

In cultures of Azotobacter vinelandii inoculated using washed cells (avoiding exhausted broth components) alginates of a higher molecular weight (1200 kDa) than those obtained in cultures conventionally inoculated (350 kDa), were produced. Also, when comparing conventionally inoculated cultures with those inoculated with washed-cells, the alginate lyase activity was delayed and the final polymer concentration decreased from 4.8 to 3.5 g l^–1. This suggests that components in the exhausted inoculum broth play important regulatory roles in alginate biosynthesis and needs to be taken into account when describing polymer biosynthesis.     

Diffusion characteristics of substrates in Ca-alginate gel beads

The diffusion characteristics of several substrates of varying molecular sizes into and from Ca-alginate gel beads in well-stirred solutions were investigated. The values of the diffusion coefficient (D) of substrates such as glucose, L-tryptophan, and alpha-lactoalbumin [with molecular weight (MW) less than 2 x 10(4)] into and from the gel beads agreed with those in the water system. Their substrates could diffuse freely into and from the gel beads without disturbance by the pores in the gel beads. The diffusion of their substrates into and from the gel beads was also not disturbed by increasing the Ca-alginate concentration in the beads and the CaCl(2) concentration used in the gel preparation. In the case of higher molecular weight substances such as albumin (MW = 6.9 x 10(4)), gamma-globulin (MW = 1.54 x 10(5)) and fibrinogen (MW = 3.41 x 10(5)), the diffusion behaviors of the substrates into and from the gel beads were very different. No diffusion of their substrates into the gel beads from solutions was observed, and only albumin was partly absorbed on the surface of the gel beads. The values of D of their substrates from the gel beads into their solutions were smaller than their values in the water system, but all their substrates could diffuse from the gel beads. The diffusion of high molecular weight substrates was limited more strongly by the increase of Ca-alginate concentration in the gel beads than by the increase of the CaCl(2) concentration used in the gel preparation. Using these results, the capacity of Ca-alginate gel as a matrix of immobilization was discussed.     

Alginate production by an <Emphasis Type="Italic">Azotobacter vinelandii</Emphasis> mutant unable to produce alginate lyase

Alginate is an industrially relevant linear copolymer composed of beta-1,4-linked D-mannuronic acid and its C-5 epimer L-guluronic acid. The rheological and gel-forming properties of alginates depend on the molecular weight and the relative content of the two monomers. Alginate produced by Azotobacter vinelandii was shown to be degraded towards the end of the culture, an undesirable situation in terms of potential alginate applications. A gene ( algL) encoding the alginate lyase activity AlgL is present within the alginate biosynthetic gene cluster of A. vinelandii. We constructed strain SML2, an A. vinelandii strain carrying a non-polar mutation within algL. No alginate lyase activity was detected in SML2. Under 3% dissolved oxygen tension, higher values of maximum mean molecular weight alginate were obtained (1240 kDa) with strain SML2, compared to those from the parental strain ATCC 9046 (680 kDa). These data indicate that AlgL activity causes the drop in the molecular weight of alginate produced by A. vinelandii.