Edwin R. Morris

Conformations and interactions of pectins. I. Polymorphism between gel and solid states of calcium polygalacturonate

Ca2+ binding to poly-d-galacturonate (the main backbone sequence of the polysaccharide pectin from plant cell walls) has been investigated by equilibrium dialysis and circular dichroism to elucidate the nature of conformational ordering and chain association in the sol-gel transition. Of the total stoichiometric requirement of bound calcium, only (50 ± 5)% is resistant to displacement by swamping concentrations of univalent counterions. Closely similar behaviour has been reported for poly-l-guluronate (derived from the polysaccharide alginate) and was attributed to site-binding of Ca2+ within dimers of chains of 21 helical symmetry. The circular dichroism changes that accompany Ca2+ binding are also closely similar for the two polymers when allowance is made for their near mirror-image stereochemistry by inversion of sign. When polygalacturonate gels, with calcium as sole or principal counterion, are dried to solid films, very profound circular dichroism changes occur, suggesting that the chain conformation and/or packing undergoes some modification during interconversion between gel and solid states. No such circular dichroism changes are seen for polyguluronate, consistent with the persistence of similar (21) chain conformations and packing for this polysaccharide in both the gel and solid state. On the basis of all this evidence, we propose: 1. (1) The co-operative binding of Ca2+ in polyguluronate and polygalacturonate gels, is through “egg-box” complexes with the polysaccharide chains in analogous 21 conformations. 2. (2) Drying of calcium polygalacturonate gels, but not polyguluronate gels, is associated with a polymorphic phase transition, and it is for this reason that diffraction studies on dried films show the 31 helix.

Conformations and interactions of pectins: II. Influence of residue sequence on chain association in calcium pectate gels

Abstract In the accompanying paper (Morris et al. , 1982) we present evidence of Ca 2+ -induced association of poly- d -galacturonate sequences from pectin into dimers of 2 1 chain symmetry, with co-operative (“egg-box”) binding of Ca 2+ on specific sites along the interior faces of each chain. We now investigate the role in calcium pectate gel networks of other structural features, in particular methyl esterification and 1,2-linked l -rhamnosyl residues in the polymer backbone. Acid hydrolysis of citrus, apple and sunflower pectins gave polygalacturonate blocks with a relatively narrow molecular weight distribution, and average chainlength of ~25 residues in each case. Since the known relative stabilities of glycosidic linkages would lead to chain cleavage predominantly at l -rhamnose, this result indicates that the length of polygalacturonate sequences between rhamnose interruptions is approximately constant within and between the pectins studied. Calcium pectate gel strength is reduced dramatically by the incorporation of these chain segments when they are de-esterified, but not when they are esterified. This interference with the development of a network structure that resists applied stress, provides further support for our model of junction zone formation from sequences of contiguous deesterified residues, with Ca 2+ -mediated chain dimers providing the primary associations that can offer resistance to deformation. Samples with different levels and patterns of esterification were prepared by enzymic (blockwise) and chemical (random) de-esterification of almost fully methyl esterified pectin. In the former series, the extent of Ca 2+ binding (as monitored by circular dichroism) increased almost linearly with the fraction of free carboxyl groups, whereas the latter showed a non-linear relationship of a form consistent with the requirement of this binding for blocks of contiguous non-esterified residues and, in the presence of excess univalent cations, binding was negligible when more than ~40% of the carboxyl groups were esterified. Statistical calculations of sequence length distribution at different degrees of random de-esterification show the best fit with experimental data when binding is assumed to require sequences with seven or more consecutive free carboxyl groups along the participating face of the chain. For 2 1 chain symmetry, this corresponds to a sequence length of 14 residues, in excellent agreement with previous independent studies of Ca 2+ binding to oligogalacturonates. In the absence of competing univalent counterions, circular dichroism changes are similar in form but so large in magnitude that site-binding of Ca 2+ must now go beyond the half-stoichiometry at which it is arrested in their presence. Ca 2+ binding monitored by circular dichroism, and gel strength (yield stress) measured mechanically, both show a similar dependence on the pattern as well as the level of esterification, as expected for network formation by co-operative binding of Ca 2+ within interchain junction zones. To fit this binding data quantitatively, it is necessary to postulate a two-stage process. (1) Initial dimerization, probably corresponding to the “strong associations” indicated by evidence from competitive inhibition (see above), for which a critical minimum sequence of seven residues is again required but esterified residues can now be accommodated within individual sites provided that they are paired with a free carboxylate on the complementary chain. (2) Subsequent Ca 2+ -induced aggregation of these preformed dimers, which can occur irrespective of the pattern of esterification on the external faces; the evidence from mechanical measurements shows that these contribute little to gel strength at high stress.

Thermogelation of methylcellulose. Part I: molecular structures and processes

Abstract Thermogelation of methylcellulose (A4M from Dow) shows two distinct ‘waves’ of increase in G ′, preceded by an initial reduction at lower temperature. The reduction and first wave of increase are accompanied by a sigmoidal change in optical rotation (indicating a co-operative conformational transition). Light transmission and detectable 1 H-NMR reach their maximum values at the end of the first structuring process, but drop to almost zero over the temperature range of the second. Both structuring processes are reversible on cooling, but offset, to lower temperature, and are accompanied by enthalpy changes in DSC (endothermic on heating; exothermic on cooling). About 40% of the high-resolution NMR signal remains undetectable in the solution state at low temperature, and the shear rate dependence of viscosity is appreciably different from that of disordered polysaccharide coils. The proposed interpretation of these findings is that methylcellulose chains exist in solution as aggregated ‘bundles’, held together by packing of unsubstituted or sparingly substituted regions of cellulosic structure, and by hydrophobic clustering of methyl groups in regions of denser substitution. As the temperature is raised, the ends of the bundles come apart, exposing methyl groups to the aqueous environment and causing a large increase in volume (with consequent increase in G ′). At higher temperature the methyl substituents shed structured water, and form a hydrophobically crosslinked network (giving the second ‘wave’ of increase in G ′). As in other polysaccharide systems, the thermal hysteresis is attributed to aggregation stabilising the cellulosic ‘bundles’ to temperatures higher than those at which they will re-form on cooling.

Flavour/taste perception in thickened systems: the effect of guar gum above and below c*

Abstract The effect of polymer concentration on flavour/taste perception in thickened systems has been investigated using solutions incorporating a fixed concentration of sucrose and flavouring and a wide range of concentrations of three different samples of guar gum, with intrinsic viscosity values of [η] = 16.7, 8.4 and 5.04 dl/g, respectively. For all three samples the perceived intensity of both attributes (assessed by 18 panellists using magnitude estimation against a fixed control solution) was independent of polymer concentration up to c* ( ≈ 2.85/[η]), but decreased steeply at higher degrees of space occupancy by the polymer, with both sweetness and flavour intensity showing essentially the same dependence on polymer concentration. This behaviour is interpreted in terms of restricted replenishment of surface depletion with increasing coil overlap and entanglement.

Conformational and rheological transitions of welan, rhamsan and acylated gellan

Abstract Native (‘high acyl’) gellan adopts double helix geometry at a much higher temperature than the deacylated polymer (commercial gellan gum), but the resulting gels are weaker, more elastic, and show no thermal hysteresis between formation and melting, indicating that acetyl groups, which are located on the periphery of the helix, prevent aggregation. On progressive removal of glyceryl substituents, which are located in the core of the helix and modify its geometry, the disorder-order transition becomes broader (i.e. less co-operative) and moves to a lower temperature. Eventually a second transition appears at the position characteristic of the fully deacylated polymer. Comparison of the relative magnitudes of the two transitions with the proportion of residual glycerate indicates that conversion from ‘high acyl’ to ‘deacetylated’ geometry requires six consecutive repeating units devoid of glyceryl groups. In welan and rhamsan, the double helix is stabilised to temperatures above 100 °C by incorporation of, respectively, monosaccharide and disaccharide sidechains in the ordered structure. Both have ‘weak gel’ properties similar to those of xanthan. However, ‘true’ gels are formed when the helix structure is dissociated and regenerated (by dissolving welan in dimethyl sulphoxide and adding water, or by heating and cooling deacylated rhamsan in aqueous solution). Our interpretation of this behaviour is that the native structures of both polymers are perfect double helices, with exact pairing of strands along the full length of the participating chains. Dissociation of these ‘perfect’ structures allows development of a cross-linked network by individual chains forming shorter helices with more than one partner.

Shear-thinning of ‘random coil’ polysaccharides: Characterisation by two parameters from a simple linear plot

Abstract The shear-thinning behaviour of solutions of conformationally disordered (‘random coil’) polysaccharides, at concentrations above the onset of coil-overlap and entanglement (i.e. above c∗), can be matched, with reasonable precision, by the equation: η=η= 0 ⧸[1 + ( λ λ 1 2 ) 0.76 ] Thus viscosity (η) at any shear rate (γ) can be characterised by two parameters: the maximum ‘zero-shear’ viscosity (η0) and the shear rate ( γ 1 2 ) at which viscosity is reduced to η 0 2 . The values of η0 and γ 1 2 can be derived from, respectively, the intercept and slope of a simple linear plot of η versus η γ 0·76 . The method can also be applied to other ‘random coil’ polymers of reasonably high polydispersity, and to the analysis of the frequency (ω) dependence of dynamic viscosity (η∗) from small-deformation oscillatory measurements.

Effect of xanthan on the small-deformation rheology of crosslinked and uncrosslinked waxy maize starch

Abstract Small-deformation oscillatory measurements have been used to characterise the effect of ordered xanthan (in 0.1 M KCl) on the rheological properties of gelatinised waxy maize starch (crosslinked and uncrosslinked). The uncrosslinked material shows a single DSC endotherm on heating, centred at ~70 °C. The overall enthalpy change on gelatinisation of the crosslinked starch is the same (ΔH = 13.2 J/g), but occurs in two endothermic processes (centred at ~60 and ~74 °C), tentatively attributed to thermal dissociation of, respectively, chemically modified and native granule structure. The thermal transitions for both samples are complete by 80 °C, which was therefore chosen as the maximum temperature in the rheological investigations, to minimise loss of granule integrity and release of starch polysaccharides into the xanthan matrix. Both samples, after gelatinisation in water, gave gel-like mechanical spectra at volume fractions below 20%, suggesting association between the swollen granules, in addition to the steric interactions which become dominant at higher concentrations. The magnitude of the increase in moduli observed on progressive addition of starch (1–5 wt%) to solutions of xanthan (0.25 or 0.50 wt%) is far too great to be explained by increased concentration of the xanthan phase on swelling of the starch granules. Comparison with the concentration-dependence of moduli for starch alone, however, suggests that xanthan acts by promoting association between the gelatinised granules, possibly by a depletion flocculation mechanism.

Characterization of the perceived texture of thickened systems by dynamic viscosity measurements

Abstract Previous attempts to correlate perceived thickness in the mouth with shear viscosity (e.g. from rotational measurements) have worked well for ‘true’ solutions (i.e. fluid materials which do not exhibit a so-called ‘yield stress’) but underestimate the oral thickness of ‘weak gels’ (materials such as ketchup and mayonnaise, which show gel-like properties at rest but flow under sufficient stress). We now report that small deformation measurements of dynamic viscosity (η*) under oscillatory shear at a single frequency (~50 rad/s) correlate directly (r2 = 0.95) with panel scores for the perceived thickness of both true solutions and weak gels. Contrary to previous suggestions that perceived ‘sliminess’ decreases with increased ‘shear thinning’, we show that panel scores for ‘sliminess’ (and for ‘stickiness’) are also directly correlated with objective values of η* at ~50 rad/s, irrespective of the extent of shear thinning. The origin of this apparent conflict of evidence is traced to the experimental design used in the earlier studies.

Interactions between carrageenans and casein micelles: electrophoretic and hydrodynamic properties of the particles

Abstract Complexes of casein micelles with λ-, ι- and κ-carrageenans have been made in solutions containing milk diluted 100-fold and concentrations of up to 0.2 mg/ml of the polysaccharides. The electrophoretic mobilities and diffusion coefficients of the complexes were measured, with some studies of the molecular weights. The results are consistent with binding of carrageenan to the casein micelles until the surfaces of the micelles were saturated, after which some further aggregation of the complexes could occur. Conformationally ordered (helical) ι-carrageenan and conformationally disordered (‘random coil’) λ-carrageenan both showed similar aggregation behaviour. κ-Carra-geenan, by contrast, gave no evidence of micellar aggregation when present as a random coil, but on conversion to the helical form (by addition of K + ) it caused precipitation of casein micelles under all conditions studied.

Phase equilibria and gelation in gelatin/maltodextrin systems — Part II: polymer incompatibility in solution

Abstract The effect of thermodynamic incompatibility in mixed solutions of gelatin and Paselli maltodextrins SA-6 and SA-2 has been studied at a temperature (45°C) where the individual polymers are stable as disordered coils. Concentrated mixtures of SA-6 and gelatin showed classic phase separation into two co-existing liquid layers, with compositions lying along a well-defined binodal. On decreasing SA-6 concentration below the binodal, however, a substantial proportion (up to 60%) of the maltodextrin was precipitated, with normal single-phase solutions occurring only at much lower concentrations of both polymers. SA-2 showed a more extreme version of the same behaviour, with precipitation of up to 100% of the maltodextrin and no evidence of co-existing liquid phases at any accessible concentrations. In both cases, the amount of maltodextrin precipitated was proportional to the square of its initial concentration and to the first power of gelatin concentration, indicating that gelatin drives self-association and aggregation of maltodextrin when both polymers are present in a single liquid phase. 1 H NMR showed the precipitated maltodextrin to be higher in molecular weight and in degree of branching than the material remaining in solution, and particlesize analysis indicated that the volume of the individual maltodextrin particles increased linearly with the total mass precipitated.