Author: Dr Jennifer Harrington
Food products are complex multi-component systems that may contain mixtures of proteins and polysaccharides, either as inherent components of foods such as meat, fish, fruit and vegetables, or added by manufacturers as thickeners, stabilisers, emulsifiers or gelling agents. In consequence, products often contain at least two different structural biopolymers. As these biopolymers are largely responsible for the physical structure (texture) of foods, understanding the mechanisms of interaction between these components is important as the overall textural properties of the product will be greatly affected by the nature and strength of these interactions.
|Micrographs from CSLM of 10% wt WPI gels in 24 mM CaCl2 (top) alone and (bottom) in the presence of 1% wt κ-carrageenan. White areas correspond to fluorescently-labelled protein. (top) shows a comparatively homogeneous network of agglomerated protein. The presence of κ-carrageenan (bottom) causes the structure to become grossly heterogeneous and is attributed to segregative interactions.|
Aqueous mixtures of two different biopolymers have been the subject of increasing research over the last 20 to 30 years, and some general principles are now well established. Interactions between the two polymeric constituents can be classified as associative or segregative, depending on whether they are enthalpically more favourable (associative) or less favourable (segregative) than interactions between individual polymers of the same type (Tolstoguzov, 1986; Piculell et al., 1994; Picullel, Bergfeldt and Nilsson, 1995). Associative interactions occur normally, but not exclusively, by electrostatic attraction e.g. between negatively-charged polysaccharides and positively-charged proteins. Complexes may be soluble or insoluble depending on factors such as ionic environment and the relative and absolute concentrations of the two polymers. Formation of insoluble complexes (complex coacervation) is the more usual outcome.
Segregative interactions (also known as "thermodynamic incompatibility") are more common, and sufficiently high concentrations of the two polymers can lead to resolution of the system into two liquid layers, due to density differences between the constituent phases. At lower concentrations, the polymers, although incompatible, may remain in a single phase. Phase separation can lead to substantial enhancements in gel strength of gelling polymers (since segregation increases the effective concentration of both polymers, by confining them to only part of the total volume). Large enhancements can also be obtained by mixing a gelling polymer and a non-gelling polymer at concentrations where the pre-gel solution remains in a single phase. Restriction of molecules to only part of the total volume causes a large reduction in entropy, and segregation will occur only if the loss of entropy is outweighed by the enthalpic advantage of individual molecules being surrounded by others of the same type.
Several studies have shown that conformationally-disordered soluble polysaccharides cause large enhancements in self-association of other gelling biopolymers in systems which remain single phase on mixing. Further increases in concentration of the soluble polysaccharides, however, often result in weakening of the gel networks.
Picout, Richardson, Rolin, Abeysekera and Morris (2000) found that the networks formed when pectin is cooled in the presence of Ca2+ can be strengthened, weakened, or remain unaffected by progressive incorporation of oxidised starch, depending on the degree of esterification (DE) of the pectin and on the concentration of Ca2+. At each DE there was a critical Ca2+ concentration above which gel strength was decreased, rather than increased, by the presence of oxidised starch. The transition from enhancement to depletion was accompanied by a change in gel structure from homogeneous to grossly heterogeneous. The reduction in final gel strength increased with increasing concentration of starch, and was found to arise from a sharp drop in modulus during cooling, indicating abrupt collapse of the developing network due to excessive aggregation in response to segregative interactions with the oxidised starch. In a continuation of this work, Giannouli, Richardson and Morris (2004a; 2004b; 2004c) explored the effect of a range of different polymeric co-solutes on the Ca2+-induced gelation of pectin, and observed massive (up to ~1000-fold) enhancements in G' at high temperatures, prior to network collapse on cooling.
Recent work by Fitzsimons, Mulvihill and Morris (2008) has shown that low concentrations of the non-gelling polysaccharide guar gum can substantially increase the gel strength of networks formed by thermal denaturation of whey protein isolate (WPI). DSC heating scans demonstrated that the addition of small amounts of guar gum (0.0-0.5 wt %) moved the aggregation exotherm of WPI to progressively lower temperature. This is attributed to segregative interactions with guar gum promoting aggregation of thermally denatured whey protein. At concentrations of added guar gum as low as 0.1 wt % a 12-fold enhancement in gel strength was evident in comparison to WPI alone. The gel strength of these mixtures rose to a maximum and then decreased at higher concentrations, until at 0.5 wt % guar gum the mixture was liquid-like. The increase and subsequent decrease in gel strength on addition of increasing amounts of guar gum is attributed to segregative interactions. At low concentration, the guar gum promoted aggregation of the WPI resulting in stronger networks, but higher concentrations resulted in excessive aggregation of WPI with eventual collapse of the gel network.
Harrington, Foegeding, Mulvihill and Morris (2008) studied the behaviour of mixtures of WPI and κ-carrageenan in the presence of calcium. Mixtures of WPI (10 % wt) with κ-carrageenan (up to 3 % wt) in the solution state at 45°C remained homogeneous, with no indication of either complex formation or phase separation. On gelation of κ-carrageenan in the presence of native WPI or of WPI in the presence of disordered carrageenan, self-association of the gelling component was promoted by segregative interactions with the soluble component. The initial effect of enhanced self-association was to give stronger gels, but further association caused a reduction in gel strength due to excessive aggregation.
Addition of native WPI promoted earlier gelation of κ-carrageenan during cooling and displaced the disorder/order transition by DSC to higher temperature. Where availability of Ca2+ cations was not a limiting factor in gelation of the κ-carrageenan, the effect of segregative interactions could be seen as an increase in gel strength.
The presence of κ-carrageenan caused earlier gelation and aggregation of WPI. Low concentrations of added carrageenan increased WPI gel strength while higher concentrations resulted in weaker networks. These effects were attributed to segregative interactions between the two polymers. On heating to gel the WPI, the presence of κ-carrageenan led to the earlier appearance of an aggregation exotherm in DSC and to network formation at shorter times, which can be explained by enhanced self-association of denatured WPI in response to segregative interactions with disordered carrageenan. An initial large (~10 fold) increase in gel strength with increasing concentration of carrageenan to 0.25 wt % was followed by a reduction at higher concentrations, attributed to excessive aggregation of the WPI.
A similar increase and subsequent decrease in gel strength with increasing concentration of carrageenan was reported by Neiser, Draget and Smidsrod (2000) for thermally-gelled mixtures with BSA. This effect is not specific to κ-carrageenan. Increasing concentrations of other polysaccharides, including galactomannans (Goncalves, Torres, Andrade, Azero and Lefebvre, 2004) and xanthan (Li, Ould Eleya and Gunasekaren, 2006) have also been shown to cause an initial increase and subsequent decrease in the strength of whey protein gels.
Tara gum has also been found to increase the gel strength of β-lactoglobulin (Sittikijyothin, Sampaio and Goncalves, 2007). Aggregation of micellar casein in mixtures of reconstituted skim milk powder with amylopectin has also been observed (de Bont, Hendriks, van Kempen and Vreeker, 2004), leading, at high concentrations of amylopectin, to a network of protein particles with gel-like properties.
It has been observed by Penrog, Mitchell, Hill and Ganjanagunchorn (2005) that the concentration of konjac glucomannan required to form a gel network (on removal of its stabilizing acetyl substituents by alkaline hydrolysis) was reduced substantially by the presence of disordered κ-carrageenan. The enhancement in self-association of the konjac chains was attributed to segregative interactions with the κ-carrageenan in the single phase mixtures formed at temperatures above the coil/helix transition temperature of the carrageenan.
One possible mechanism of enhanced aggregation is depletion flocculation as mentioned by Croquennoc, Nicolai, Durand and Clark (2001). Evidence for favouring a mechanism based on segregative interactions comes from previous studies of the effect of a wide range of disordered polysaccharides. In one of these studies (Giannouli et al., 2004a) guar gum samples of widely varying molecular weight were used as the soluble component in the mixtures, and it was found that their effectiveness in inducing self-association of calcium pectinate increased as their molecular weight decreased. This is consistent with segregative interactions, since the proportion of chain segments buried in the interior of the coil, where they cannot interact with other species, will decrease as the size of the coils decreases. Depletion flocculation, by contrast, would increase with molecular weight, since the range of inter-particle spaces that the coils cannot penetrate will increase as their size increases.
A common feature of many of the systems described above is that the junction zones of the gel networks consist of large aggregates of individually-rigid subunits e.g. aggregation of double helices, protein aggregates and inflexible β-(1-4) glycans. In systems where the junction zones are made up of more flexible entities the outcome may be very different. Harrington and Morris (2008a; 2008b) studied the conformational ordering and gelation of gelatin in mixtures with several soluble, disordered polysaccharides. The polysaccharides studied were gum arabic, inulin, guar gum, dextran (up to 3%), konjac glucomannan, carboxymethyl cellulose (CMC) and hydroxypropyl cellulose (HPMC). The aim of the investigation was to screen for increases in the rate and extent of triple-helix formation, and associated gelation of gelatin in response to segregative interactions with the polysaccharide component of the mixtures. However, incorporation of the soluble polymers caused no significant changes in gel strength, and the rate of conformational ordering (measured by optical rotation) was within ~10 % of that for gelatin alone. A possible explanation for this is that the enthalpic advantage of segregation is not sufficient to overcome the large reduction in entropy associated with conversion of flexible gelatin coils to conformationally-immobile triple helices.
From the investigations discussed here, it seems that segregative interactions are more important (i.e. large effects on rheological properties are seen) where the gelling biopolymer is in a conformationally-rigid state, e.g. denatured whey protein aggregates or κ-carrageenan double helices. In this case, the reduction of entropy from the association of conformationally rigid species is quite low. When the gelling process involves the transition of flexible coils, like those of disordered gelatin, to a conformationally restricted form (e.g. gelatin triple helices) segregative effects do not appear to promote such large changes in rheological properties. For polymers like gelatin, the transition from flexible coils to triple helices results in a considerable loss of entropy, which cannot be overcome by the enthalpic advantage of segregation.
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