Rheological properties of sodium alginate in an aqueous system during gelation in relation to supermolecular structures and Ca2+ binding
Introduction
Alginate is a collective term for a family of exopolysaccharides produced mainly from brown seaweeds. It has been widely used in food, biomedical, pharmaceutical, and sewage-treating industries, preferentially in sodium form due to the solubility in cold water. In molecular terms, alginate is unbranched binary copolymers of (1-4)-linked β-d-mannuronic acid (M) and α-l-guluronic acid (G) residues as monomers, constituting M-, G-, and MG- sequential block structures (Moe, Draget, Skjåk-Bræk, & Smidsrød, 1995, chap. 9). Since the M and G residues are in the 4C1 and 1C4 conformation, respectively, three types of glycosidic linkages are found generally in the block structures, including diequatorial (MM), diaxial (GG), and equatorial-axial (MG). The G-block is stiffer and more extended in chain configuration than the M-block due to a higher degree of hindered rotation around the glycosidic linkages (Braccini, Grasso, & Perez, 1999). Most applications of alginate is based on its gel-forming ability through cation binding; the transition from water-soluble sodium alginate to water-insoluble calcium alginate, for example. Divalent cations preferentially bind toward the G-block rather than the M-block (Braccini et al., 1999, Braccini and Perez, 2001). The composition of monomers and their sequential character (i.e., blockiness) affect the gelation behavior of alginate. In the presence of Ca2+, G-rich samples generally form hard and brittle gels, while M-rich samples form soft and elastic gels (Moe et al., 1995), and the “egg-box” has been accepted as a general model to describe the gel formation (Morris et al., 1978, Thom et al., 1982). As for the stoichiometry describing Ca2+-induced gelation, three distinct molecular events; monocomplexation, dimerization, and lateral associations among the dimers occur in a step-wise manner upon addition of the cation elucidated by isothermal titration calorimetry (Fang et al., 2007). These three steps occur sequentially rather than simultaneously with critical boundaries at R[Ca]/[G] (the feeding ratio of Ca2+ to unit G residue) of 0.25 and 0.55, confirming 2/1 helical structure as the most probable conformation for the egg-box junctions. At R[Ca]/[G] < 0.25, monocomplexes form between Ca2+ and the G units. The formation of the monocomplexes decreases electrostatic repulsion to make molecular conformation more compact by reducing the charge density on alginate chains and also by giving rise to local charge reversal to form some positively charged patches (Siew, Williams, & Young, 2005). This step can be regarded as a nucleation, prerequisite to the formation of the egg-box dimers. At 0.25 < R[Ca]/[G] < 0.55, the egg-box dimers form through the pair-wise coupling between the monocomplexes. The formation of the dimers increases hydrodynamic molecular size and the degree of structural order. At R[Ca]/[G] > 0.55, egg-box multimers form via lateral associations among the dimers. The formation of the multimers undergoes within individual cluster (i.e., intra-cluster associated multimers) when the chain length is long enough to provide some flexibility with molecules. For industrial usage of sodium alginate, there are essentially two methods to prepare homogeneous gels; so-called dialysis and internal gelation methods (Moe et al., 1995). The polysaccharide instantaneously forms gels or precipitates as a result of rapid and irreversible cation-conversion when water-soluble calcium salts are introduced into sodium alginate solutions. The internal gelation method uses an inactive form of Ca2+ as a cross-linker, either in bound form to a chelating agent or as an insoluble salt (Skjåk-Bræk, Grasdalen, & Smidsrød, 1989). Solutions of a slowly hydrolyzing lactone, generally glucono-δ-lactone (GDL), are then added to the composite of alginate and the inactive cross-linker. The slow hydrolysis releases protons and liberates Ca2+ from the inactive form, leading to the formation of gel matrixes through pH decrease. When water-insoluble calcium salts are used as the cross-linker, the particle size is a parameter for gelation by affecting liberating and sedimentation rates prior to gel setting (Draget, Østgaard, & Smidsrød, 1991). The pH of the system also affects the gelation behavior. Thus, the gelation properties of alginate are controlled by selecting source and proportion of calcium salt, acidic material, and chelating agent. Alginate also undergoes the sol-to-gel transition upon lowering pH even in the absence of cations (Draget et al., 1996, Draget et al., 1994, Draget et al., 2003). For the acidic gels, increased fraction of homopolymeric G-block yields higher shear modulus, similar to the influence of this residual sequence on the ionitropic gels. Homopolymeric M-block also forms junctions under acidic conditions though their contributions are smaller than those of the G-block. Alternating MG-block, the degree of polymerization for which being reportedly 15 (Haug, Larsen, & Smidsrød, 1967), does not contribute to form junctions and rather perturb gel formation.
To understand gelation behavior of polysaccharides, information about molecular assemblies or network structures is indispensable because polysaccharides do not function as individual molecules in real systems. These supermolecular structures of polysaccharides, particularly natural ones, are originally heterogeneous due to their polydispersities. To observe such heterogeneity, microscopy is advantageous over other physical methods, and atomic force microscopy (AFM) has been one of the most versatile technique in this research field because its operating range spans nano-scale, realizing molecular resolution under natural conditions that hardly damage the original architectures and enabling us to visualize directly not only individual molecules of polysaccharides but also their assemblies (e.g., aggregation, network, gels, gel precursors) (Morris, 2004). There is still room left to understand the gelation behavior of polysaccharides on a molecular level with growing interest in gelled systems from both fundamental and application aspects to improve overall palatability of food products, including texture, stability, appearance etc.
In the present study, we focused on gelation behavior of sodium alginate in the presence of Ca2+. It is reasonable to think that macroscopic rheological properties of the polysaccharide during gelation are governed by microscopic molecular structures and related Ca2+ binding. Well-characterized samples of different M/G ratios but comparable molecular masses were used to obtain cleat-cut conclusions. The aim of the present study is to increase the knowledge on the gelation mechanism of sodium alginate for better use of the polysaccharide in the industries.
Section snippets
Materials
Two samples of sodium alginate were obtained from San-Ei Gen F.F.I., Inc. (Osaka, Japan). The polysaccharide samples were washed in excess amount of 95 v/v% ethanol twice and were freeze-dried prior to use. Reagent grade of CaCO3 (Wako Pure Chemicals, Osaka, Japan) with an average particle size of 12.0 μm and GDL (Wako Pure Chemicals) were used without further purification.
Characterizations of sodium alginate
Weight-average molar mass (Mw), radius of gyration (Rg), and polydispersity index were determined by size exclusion
Characterizations of alginate samples
Macromolecular characteristics determined were comparable between two alginate samples; Mw, Rg, and polydispersity index were 213 kg/mol, 89.5 nm, and 2.1, respectively, for Sample M, whereas 221 kg/mol, 92.6 nm, and 2.1, respectively, for Sample G (Table 1). More than 95% of counterions was detected as sodium for each sample. As sequential parameters, the M/G ratio for Sample M was 44/56 with calculated G- and MG-blockiness of 5 and 2.5, respectively, whereas the M/G ratio for Sample G was 29/71
Rheological behavior in relation to supermolecular structures during gelation
Alginate samples are essentially the same in macromolecular characteristics and are almost completely in sodium form. Differences in macroscopic and microscopic behaviors during gelation are thus attributed exclusively to sequential parameters between the samples. The effects of pH are almost negligible on gelation because acidic gels form only when pH of the system is below ca. 2.4 from isothermal titration calorimetry (data not shown). Actually, no rheological changes occurred macroscopically
Conclusions
Macroscopic gelation behavior of sodium alginate is governed by the structural consistency and/or the mechanical strength of molecular assemblies in the presence of Ca2+. Although the important role of the G-block structures has been reported (Smidsrød & Haug, 1972) for the ionitropic gelation of the polysaccharide as a predominant binding site, larger amount of bound Ca2+ does not necessarily lead to higher elasticity of the system, indicating that the egg-box dimer among G-blocks is not a
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