Effects of molecular weight and elastic segment flexibility on syneresis in Ca-alginate gels
Introduction
Alginates are binary heteropolymers consisting of α-l-guluronic acid (G) and β-d-mannuronic acid (M) residues in various proportions and sequences (Smidsrød & Draget, 1996a). The two different monomers are not randomly distributed, but rather in a block-wise pattern of oligo-mannuronate (M-blocks), oligo-guluronate (G-blocks) and sequences of a predominately alternating (MGM) structure (Smidsrød & Draget, 1996a). These different structural elements can be isolated employing various acid hydrolysis procedures and purified by exploiting their inherent acid solubility properties (Draget et al., 1998, Haug et al., 1967, Haug and Smidsrød, 1966). Guluronate blocks are the sequences within the alginate molecule which are responsible for the selective binding of multivalent cations and hence also for the sol-gel transition (Smidsrød, 1974). Once isolated and purified, these structures will not be able to give gel-like structures, but will rather form a precipitate in the presence of e.g. Ca2+. This is due to their low molecular weight (Mn typically 5000 Da) and a complete lack of elastically active segments. It is known, however, that in a blend with a gelling alginate these oligo-guluronates are able to alter both the gelling kinetics and the modulus (Draget, Simensen, Onsøyen, & Smidsrød, 1997).
AlgE4 is a mannuronan C5 epimerase converting homo-polymeric sequences of mannuronate residues in alginates into mannuronate/guluronate alternating sequences. Treating alginates of different biological origin with AlgE4 results in different amounts of alternating sequences (Ertesvåg, Valla, & Skjåk-Bræk, 1996). Conformational energy calculations (Smidsrød et al., 1973, Stokke et al., 1993) have indicated that the relative extension (‘stiffness’) of the three types of blocks increases in the order: MG-blocks<MM-blocks<GG-blocks. Hence, the net result of treating an alginate with AlgE4, i.e. replacing M-blocks with MG-blocks, is that the elastic segments, comprising both these types of blocks, become more flexible. An elastic segment is, in the present context, the part of the alginate molecule spanning two oligo-guluronate sequences involved in different junction zones.
One important property of the alginate gel is its non-equilibrium feature (Smidsrød, 1973). This implies that the obtained mechanical properties depend upon the history of gel formation, and, furthermore, that the apparent equilibrium modulus is altered by e.g. exposing the gel to a heating/cooling cycle (Andresen and Smidsrød, 1977, Moe et al., 1991). This property is, at least partly, due to a certain degree of sub-optimal cross-linking in alginate gels made from isolated and purified alginate as opposed to the more regular structure found in the seaweed (Andresen, Skipnes, Smidsrød, Østgaard, & Hemmer, 1977).
Syneresis is macroscopically characterised by a slow, time-dependent de-swelling of a gel resulting in an exudation of liquid. It is a phenomenon commonly observed over time in various systems undergoing a sol/gel transition and often represents a challenge in the manufacturing of food gels. A number of papers describe the driving force behind this type of behaviour in various biopolymer gel systems (see e.g. Dunstan et al., 2000, Lucey et al., 1997, van Vliet et al., 1991), but the molecular mechanisms leading to syneresis in alginate gels are less clear. One established fact is, however, that in the case of internal gelling of alginate, the degree of syneresis is strongly related to the amount of calcium present (Draget, Østgaard, & Smidsrød, 1991). The scope of the present paper is therefore to summarise the changes taking place in the gel network during syneresis, and to investigate which alginate molecular properties that govern these changes.
Section snippets
Materials and methods
The alginates used in the molecular weight dependence study were commercial samples isolated from Laminaria hyperborea kindly provided by FMC Biopolymer A/S, Drammen, Norway. These samples have a high content of guluronic acid residues (65–68%) as determined by NMR (Grasdalen, 1983). The samples selected for this study had a weight average molecular weight (Mw) from 50 to 320 kDa as estimated from their intrinsic viscosities and SEC-LALLS experiments (Christensen, 2000). Oligo-guluronates
Gel network alterations during syneresis
When the Ca2+ concentration is increased from 10 to 30 mM in a 10 mg/ml high-G alginate solution by internal gelling, the final gel exhibits an increased syneresis going from zero to 20%, respectively (Stokke et al., 2000). Fig. 1 shows the excess small angle X-ray scattering from gels made at these two concentrations of cross-linking ion. At the smallest accessible scattering vector q, it is found that the syneretic gel (30 mM Ca2+) shows a scattering intensity being two to three times higher
Conclusion
Both addition of low molecular weight alginate and an introduction of more flexible elastic segments seem to bring about gels with more equilibrium-like properties, but by highly different molecular mechanisms. Low Mw alginate seems to introduce an equilibrium state by restricting the primary network structure from further contraction (low degree of syneresis), whereas more flexible elastic segments give an equilibrium state by allowing more rapid relaxation (high degree of syneresis).
Acknowledgements
The authors highly appreciate the financial support of FMC Biopolymer A/S, and the Norwegian Research Council.
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