Enzyme-entrapping behaviors in alginate fibers and their papers

Enzyme immobilization in the form of fiber and paper was easily achieved by wet spinning of aqueous admixture of sodium alginate and enzymes into divalent metallic ion solution as a coagulating bath, followed by paper making of resultant shortly cut fibers. Entrapment yields of enzymes used, e.g., glucoamylase, cyclodextrin glucanotransferase, endo-polygalacturonase, and protease, were always higher in calcium alginate fibers and their papers than those in corresponding beads. It was found that the yields increased with an increase of the discharge rate through the spinning nozzle because the higher discharge rate could provide more highly oriented metal-chelate linear polymer molecules along the fiber axis for preventing leakage of entrapped enzymes. Divalent metallic ions affected greatly the entrapment of glucoamylase in alginate fibers, the order of which followed roughly the ionotropic series of Thiele. Entrapment of glucoamylase in bicomponent systems comprising alginate and other water-soluble polymers was also investigated.     

The interaction of sodium alginate with univalent cations

The M/G ratio, dyad and triad frequencies in the sodium alginate chain, were determined from ¹³C-nmr spectra. The interactions of sodium alginate in solution with the univalent cations K⁺ ion and Na⁺ ion have been investigated by viscometry and membrane osmometry. The dependencies of intrinsic viscosity, Huggins constant, and second virial coefficient on ionic strength were observed, and the maximums in reduced viscosity were obtained in low KCl and NaCl concentrations, respectively. These show that the electroviscous effects play an important role in polyelectrolyte solution, and the effect of the Na⁺ ion on aqueous solution of sodium alginate is greater than the K⁺ ion. The experimental observations are interpreted in terms of ion-pair formation with carboxyl groups of mannuronate and isolated guluronate residues and cooperation “egg-box” binding between polyguluronate chain sequence. The difference of interaction between univalent cations and alginate chains in solution is attributed to the ability of their binding with the polyion, which depends on the properties of ions itself. © 1998 John Wiley & Sons, Inc. Biopoly 46: 395–402, 1998     

Preparation and properties of alginate/carboxymethyl chitosan blend fibers

Alginate/carboxymethyl chitosan blend fibers, prepared by spinning their mixture solution through a viscose-type spinneret into a coagulating bath containing aqueous CaCl₂, were studied for structure and properties with the aid of infrared spectroscopy (IR), X-ray diffraction (XRD) and scanning electron micrography (SEM). The analyses indicated a good miscibility between alginate and carboxymethyl chitosan, because of the strong interaction from the intermolecular hydrogen bonds. The best values of the dry tensile strength and breaking elongation were obtained when carboxymethyl chitosan content was 30 and 10 wt%, respectively. The wet tensile strength and breaking elongation decreased with the increase of carboxymethyl chitosan content. Introduction of CM-chitosan in the blend fiber improved water-retention properties of blend fiber compared to pure alginate fiber. Antibacterial fibers, obtained by treating the fibres with aqueous solution of N-(2-hydroxy)-propyl-3-trimethylammonium chitosan chloride and silver nitrate, respectively, exhibited good antibacterial activity to Staphylococcus aureus.     

Effects of sterilization treatments on some properties of alginate solutions and gels.

Aqueous sodium alginate solutions were subjected to various heat sterilization treatments. Sodium alginate powder was also treated by both gamma-irradiation and ethylene oxide sterilization. The effects of these treatments on the viscosities of sodium alginate solutions and both the diameter and strength of the beads formed in 0.1 M CaCl2 solutions were determined quantitatively. The viscosity of sodium alginate solutions and the gel strength of the calcium alginate beads decreased with increasing sterilization temperature while the bead diameters were found to increase. All these effects can be attributable to a reduction in the degree of polymerization of the alginate molecules as a result of the heat treatments. Ethylene oxide and gamma-irradiation treatments caused similar effects. Standard conditions for sterilization are necessary for comparative studies with alginate beads.     

Photolytic depolymerization of alginate

A photochemical reaction has been developed for the partial de-polymerization of sodium alginate, a polysaccharide utilized in medicine, pharmacy, basic sciences and foods. An aqueous solution of sodium alginate was photochemically depolymerized to ∼40% of its average molecular weight using ultraviolet light in the presence of titanium dioxide catalyst at pH 7 over a period of 3 h. The products were separated giving four fractions all having an average molecular weight that was smaller than that of the starting material. Characterization of the guluronate (G) and mannuronate (M) contents, and determination of the M/G ratio of photochemically depolymerized alginate, were accomplished using ¹H NMR spectroscopy. The resulting M/G ratio was compared to that obtained for alginate fractions produced by acid hydrolysis. The M and G content, of each alginate fraction, was also assigned with regards to their occurrence in G-rich, M-rich or M/G heteropolymeric domains. This new depolymerization method might also be applicable in the preparation of alginate oligosaccharides for use in the food and pharmaceutical industries.     

Viability of ectomycorrhizal fungus mycelium entrapped in calcium alginate gel

 We studied the viability of fragmented mycelium of Pisolithus tinctorius and Paxillus involutus entrapped in calcium alginate gel to determine the efficacy of this method of producing ectomycorrhizal fungus inoculum. Fungi were grown in MMN solution at 28  °C before being fragmented in a blender and subsequently entrapped in calcium alginate. We tested different ratios of alginate and mycelium suspension to 0.7 M CaCl₂. The ratio 8 : 10 resulted in well-formed beads of the highest viability for Paxillus involutus (99%) and for Pisolithus tinctorius (75%). Paxillus involutus mycelium was more than 90% viable when entrapped mycelium was 10 to 50 days old, and Pisolithus tinctorius attained its highest viability (55%) for 20- to 40-day-old mycelium. Gel entrapped Paxillus involutus mycelium grew well at all temperatures after 30 days of storage, but viability significantly decreased after 60 days storage at 6  °C on dry filter paper. For gel-entrapped Pisolithus tinctorius mycelium, viability was highest when stored at 25  °C in 0.7 M CaCl₂. Entrapment of Paxillus involutus fragmented mycelium in calcium alginate beads under the conditions that we propose can be used successfully to produce inoculum. Accepted: 11 October 1998     

Extraction and characterization of an alginate from the brown seaweed <Emphasis Type="Italic">Sargassum turbinarioides</Emphasis> Grunow

Matrix polysaccharide from the brown algae Sargassum turbinarioides collected in the coastal waters of Nosy Be (Madagascar) in the Indian Ocean was isolated and its structure was studied by ¹H-NMR spectroscopy, FT-IR, SEC-MALLS and HPAEC. An alginate with a molecular weight of 5.528 × 10⁵ g mol⁻¹ was identified as sole polysaccharide. Values of the M/G ratio, F _GG, F _MM and F _GM (or F _GM) blocks were measured at respectively 0.94, 0.39, 0.36 and 0.25 and compared with those of alginates from other Sargassum species. This sodium alginate appeared similar to some of the other Sargassum alginates with M/G < 1, high values of homopolymeric blocks (η < 1) and significant polyguluronic block content.     

Microwave assisted rapid method for hydrolysis of sodium alginate for M/G ratio determination

A rapid one-pot method for hydrolysis of sodium alginate for determining M/G ratio has been developed under mild conditions, using microwave irradiation. Poly-mannuronic acid (PMA) and poly-guluronic acid (PGA) ratio was determined (M/G 0.38), which was similar to that (M/G 0.39) obtained by the conventional method, using sodium alginate of Sigma as reference sample for bench marking. For validation of the method PMA and PGA were characterized by density, porosity, viscosity, optical rotation measurements, ¹³C NMR, FT-IR, thermogravimetric analysis (TGA), X-ray diffraction, circular dichroism (CD), molar mass distribution (GPC) and scanning electron microscopy (SEM).     

Optical anisotropy of calcium-induced alginate gels

Alginate gels formed by diffusion of calcium ions into solutions of sodium alginate were found to exhibit optical anisotropy depending on preparation conditions. When observed under crossed nicols, the anisotropic alginate gels showed a birefringence pattern which is characteristic of radial orientation of polymer chains. Calcium alginate gels were prepared from different concentrations of sodium alginate and calcium ion, and the conditions for formation of the anisotropic gels were determined. The gel-formation process was measured by monitoring the development of the birefringent layer and was compared with the model in which the diffusion of calcium ions dominates gel formation.     

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.     

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.     

Characterisation of the shrinkage of calcium alginate gel membrane with immobilised Lactobacillus rhamnosus

To quantify the shrinkage of calcium alginate gel membrane as a support matrix for immobilising cells during the fermentation of Lactobacillus rhamnosus, factors including time, pH, membrane thickness, and the concentrations of immobilised cells, lactic acid, glucose, and calcium chloride were examined by statistical experimental design. A Plackett-Burman design was used for the first screening experiment to identify the important factors which caused the divergent effects. Uniform Design, a powerful modelling design technique, was thus chosen to design the modelling experiments. Through a non-linear step-wise regression analysis, the predictive mathematical model of the shrinkage in membrane thickness was established and the significant main effects and two-factor interactions were identified. However, no significant model equations could be obtained for the shrinkage in area and volume of gel membranes. The methodology developed can be extrapolated to the quantitative characterisation of shrinkage in other immobilised gel matrices, which will be very useful in mathematical modelling, design, operation and scale-up of gel immobilised cell systems. Received: 8 November 1999 / Revision received: 17 January 2000 / Accepted: 23 January 2000     

Sulfated extracellular polysaccharide production by the halophilic cyanobacterium Aphanocapsa halophytia immobilized on light-diffusing optical fibers

 The cyanobacterium, Aphanocapsa halo-phytia MN-11, was immobilized in calcium alginate gel and coated on light-diffusing optical fibers (LDOF) for sulfated extracellular polysaccharide production. Results indicated that sulfated extracellular polysaccharide production depends on the number of immobilized cells and the light intensity. In addition, the production rate reached 116.0 mg (mg dry cells)^-1 day^-1 when the cells that were immobilized on LDOF were incubated under a light intensity of 1380 cd sr m^-2 at a cell concentration of 1.0×10⁸ cells/cm³ gel. Cells immobilized on LDOF produced about ten times more sulfated extracellular polysaccharide than those immobilized in calcium alginate beads only (11.7 mg(mg dry cells)^-1day^-1). Received: 31 March 1995/Revised last revision 12 June 1995/Accepted 26 July 1995     

External versus internal source of calcium during the gelation of alginate beads for DNA encapsulation

Alginate gels produced by an external or internal gelation technique were studied so as to determine the optimal bead matrix within which DNA can be immobilized for in vivo application. Alginates were characterized for guluronic/mannuronic acid (G/M) content and average molecular weight using 1H-NMR and LALLS analysis, respectively. Nonhomogeneous calcium, alginate, and DNA distributions were found within gels made by the external gelation method because of the external calcium source used. In contrast, the internal gelation method produces more uniform gels. Sodium was determined to exchange for calcium ions at a ratio of 2:1 and the levels of calcium complexation with alginate appears related to bead strength and integrity. The encapsulation yield of double-stranded DNA was over 97% and 80%, respectively, for beads formed using external and internal calcium gelation methods, regardless of the composition of alginate. Homogeneous gels formed by internal gelation absorbed half as much DNAse as compared with heterogeneous gels formed by external gelation. Testing of bead weight changes during formation, storage, and simulated gastrointestinal (GI) conditions (pH 1.2 and 7.0) showed that high alginate concentration, high G content, and homogeneous gels (internal gelation) result in the lowest bead shrinkage and alginate leakage. These characteristics appear best suited for stabilizing DNA during GI transit.     

Biocompatible alginate from freshly collected Laminaria pallida for implantation

A simple procedure is described for the extraction and purification of alginate from the inner stipes of the kelp Laminaria pallida. Alginate yield was about 10–15% of the dry mass, with a 70:30 mannuronic/ guluronic acid ratio. Analysis of the purified alginate revealed a low polyphenol content while proteins were below detection level. The purified alginate was highly viscous, with 10–15 mPa s and 281 mPa s for a 0.1% and 0.5% solution, respectively, indicating a very high molecular mass (larger than 250 kDa). Bead formation occurred in the presence of divalent cations, but also in the presence of artificial serum (FCSIII) without added divalent cations. The biocompatibility of the alginate was tested with the in vitro mice lymphocyte test as well as by implantation of Ba²⁺ cross-linked beads beneath the kidney capsule of BB/OK rats. There was no evidence for significant mitogenic activity or fibrotic reaction. Biocompatibility of the alginate was also demonstrated by the encapsulation of human chondrocytes into Ca²⁺ cross-linked alginate beads. Immobilized chondrocytes grew and remained functional (i.e. they produced collagen). Received: 14 June 1999 / Received revision: 6 September 1999 / Accepted: 10 September 1999     

Biopolymer of alginate nature with a predominance of L-guluronic acid

Highly viscous polysaccharide (250–350 kDa) of an alginate nature with a predominance of α-L-guluronic acid (M/G = 0.22) was obtained from Azotobacter vinelandi. The yield of polysaccharide was 20.5 ± 0.5 g/l when cultured in a medium containing molasses at a viscosity of the cultural liquid of over 30000 cSt. The biopolymer is stable at pH 4.0–9.0 in a wide temperature range and well soluble in highly mineralized water; it retains a high viscosity level and can be used in the petroleum industry for enhanced oil recovery.     

Changes in alginate molecular mass distributions, broth viscosity and morphology of Azotobacter vinelandii cultured in shake flasks

The effect of different aeration conditions during the culture of Azotobacter vinelandii on the production and molecular mass of alginate was evaluated in shake flasks. In baffled flasks, the bacteria grew faster and produced less alginate (1.5 g/l) than in conventional (unbaffled) flasks (4.5 g/l). The viscosity of the culture broth was also influenced by the type of flask. Higher final viscosities were attained in unbaffled flasks [520 cP (520 mPa s)] as compared to baffled flasks (30 cP). This latter phenomenon was closely related to the changes in the molecular mass distribution. In either cases, the mean molecular mass increased with culture age; however, at the end of the fermentation, the mean molecular mass of the alginate obtained in unbaffled flasks was fivefold higher than that obtained in baffled flasks. As the culture proceeded, the cells of Azotobacter grown in unbaffled flasks increased in diameter, whereas those cultured in baffled flasks decreased in size. Received: 13 December 1996 / Received revision: 10 April 1997 / Accepted: 27 April 1997     

Effect of calcium on immobilization of rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.) peroxidase for bioassays in sodium alginate and Agarose gel

The direct immobilization of soluble peroxidase isolated and partially purified from shoots of rice seedlings in calcium alginate beads and in calcium agarose gel was carried out. Peroxidase was assayed for guaiacol oxidation products in presence of hydrogen peroxide. The maximum specific activity and immobilization yield of the calcium agarose immobilized peroxidase reached 2,200 U mg⁻¹ protein (540 mU cm⁻³ gel) and 82%, respectively. In calcium alginate the maximum activity of peroxidase upon immobilization was 210 mU g⁻¹ bead with 46% yield. The optimal pH for agarose immobilized peroxidase was 7.0 which differed from the pH 6.0 for soluble peroxidase. The optimum temperature for the agarose immobilized peroxidase however was 30°C, which was similar to that of soluble peroxidase. The thermal stability of calcium agarose immobilized peroxidase significantly enhanced over a temperature range of 30∼60°C upon immobilization. The operational stability of peroxidase was examined with repeated hydrogen peroxide oxidation at varying time intervals. Based on 50% conversion of hydrogen peroxide and four times reuse of immobilized gel, the specific degradation of guaiacol for the agarose immobilized peroxidase increased three folds compared to that of soluble peroxidase. Nearly 165% increase in the enzyme protein binding to agarose in presence of calcium was noted. The results suggest that the presence of calcium, ions help in the immobilization process of peroxidase from rice shoots and mediates the direct binding of the enzyme to the agarose gel and that agarose seems to be a better immobilization matrix for peroxidase compared to sodium alginate.     

Utilization in alginate beads for Cu(II) and Ni(II) adsorption of an exopolysaccharide produced by <Emphasis Type="Italic">Chryseomonas luteola</Emphasis> TEM05

Copper and nickel adsorption onto calcium alginate, sodium alginate with an extracellular polysaccharide (EPS) produced by the activated sludge bacterium Chryseomonas luteola TEM05 and the immobilized C. luteola TEM05 from aqueous solutions were studied. After that, the multi metal ions containing these ions together were prepared and partial competitive adsorptions of these mixtures were also investigated. The metal adsorption of gel beads were carried out at pH 6.0, 25 °C. The maximum adsorption capacities in Langmuir isotherm for calcium alginate, calcium alginate + EPS, calcium alginate + C. luteola TEM05 and calcium alginate + EPS + C. luteola TEM05 were 1.505, 1.989, 1.976, 1.937 mmol/g dry weight for Cu(II) and 0.996, 1.224, 1.078, 1.219 mol/g dry weight for Ni(II), respectively.The competitive biosorption capacities of the carrier for all metal ions were lower than single conditions.     

Culture of chondrocytes in alginate gel: Variations in conditions of Gelation influence the structure of the alginate gel, and the arrangement and morphology of proliferating chondrocytes

Summary Sodium alginate, which gels in the presence of calcium ions, is commonly used for culture of anchorage-independent cells, such as chondrocytes. Normally, the gel appears microscopically homogeneous but, depending on the conditions of gelation, it may contain a varying number of small channels that extend inward from the surface. We have examined the influence of these channels on the morphology of cultured chondrocytes entrapped in alginate beads. Growth-plate or articular chondrocytes cultured in alginate normally proliferate and form rounded cell clusters but, in alginate beads containing numerous channels, many chondrocytes become aligned and form columns similar to those in the growth plate in vivo. As the pattern of cellular growth and morphology in alginate is profoundly influenced by the presence of channels in the gel, further studies were conducted to determine what specific conditions of gelation affect their formation. The channels are especially numerous when both the alginate and the gelling solutions lack sodium ions or other monovalent cations. The channels are cavities in the gel formed by particulate blocking of the rapid diffusion of calcium ions from the gelling solution into the boundary of the calcium alginate solution, and hence they extend inward from cells at the surface of the alginate gel. An understanding of the conditions under which these channels develop makes it possible either to avoid their formation or, alternatively, to enhance the number of channels in order to encourage proliferating cells to grow in radial columns, rather than in a less organized pattern characteristic of most culture systems.