ELS EVI E R

Current Opinion in Colloid & Interface Science 5 (2000) 202-214

www.elsevier.nl/locate/cocis

Protein-polysaccharide interactions
J.-L. Doublier"?",C. Garnier a, D. Renarda, C. Sanchezb?'
aUnitd de Physico-Chimiedes Macromoldcules,INRA, Rue de la Gdraudikre, BP 71627, 44316 Nantes Cedex 3, France bLaboratoirede Physico-Chimie et GdnieAlirnentaires,ENSALA / INPL, 2 Avenue de la For&-de-Haye, 54500 Vandoeuvre-les-Nancy, France

Abstract

Numerous investigations on protein-polysaccharide systems have recently been undertaken and are leading to a better understanding of the key parameters implied in protein-polysaccharide interactions. Microscopic methods are being developed to describe the structure formation in the mixed systems in combination with rheological characterisation. Progress is also being made in the description of the mechanisms underlying the phase separation processes by the use of scattering techniques. 0 2000 Elsevier Science Ltd. All rights reserved.
Keywords: Associative phase separation; Segregative phase separation; Demixing; Complex coacervation; Proteins; Polysaccharides

1. Introduction

Proteins and polysaccharides are present together in many kinds of food systems, and both types of food macromolecules contribute to the structure, texture and stability of food through their thickening or gelling behaviour and surface properties. Much is now known at the molecular level about the functional properties of individual biopolymers, except the fact that molecular weight polydispersity is rarely taken into account. Nevertheless, our knowledge of the role of protein-polysaccharide interactions, in relation to their functionality in complex multiphasic systems, such as food mixed solutions, emulsions or gels, is still rather limited.

Abbreviations: NRTL, Non-random two-liquid; S A N S , Small angle neutron scattering; CLSM, Confocal laser scanning microscopy; PCM, Phase contrast microscopy; SLS,Static light scattering; DLS, Dynamic light scattering * Corresponding author. Tel.: + 33-2-40-67-50-55;fax: + 33-2-4067-50-43. E-mail address: doublier@nantes.inra.fr (J.-L. Doublier). 'E-mail: christian.sanchez@ensaia.inpl-nancy.fr

In many biopolymer mixtures, the entropic contribution is often greater than the enthalpic one, so that phase separation of biopolymers is generally the rule. In this case, two phase separation phenomena can be observed, depending on the affinity between the different biopolymers and the solvent. The first one, called thermodynamic incompatibility or segregative phase separation, is generally observed (Fig. 1). It appears when the Flory-Huggins interaction parameter xZ3(accounting for the biopolymerl-biopolymer2 interactions) is positive, indicating a net repulsion between the biopolymers. Clearly, solvent-biopolymerl (biopolymer2) interactions are favoured to the detriment of biopolymerl-biopolymer2 and solvent-solvent interactions, so that the system finally demixes into two phases, each being enriched with one of the two biopolymers [l']. The second phase separation phenomenon is the associative phase separation. It occurs when the interactions between the two biopolymers are favoured (x23< 0). This occurs when both polymers carry an opposite charge, for instance at a pH slightly lower than the isoelectric point of the protein, while the polysaccharide still

1359-0294/00/$ - see front matter 0 2000 Elsevier Science Ltd. All rights reserved. PII: S 13 5 9 - 0 2 9 4 ( 0 0 ) 0 0 0 5 4 - 6

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carries a negative charge. Complexation takes place, which can yield either the formation of soluble complexes or an aggregative phase separation. In an associative phase separation, the two coexisting phases have the following composition: a rich solvent phase with very small amounts of biopolymeds) and a rich biopolymeds) phase forming the so-called coacervate. Both types of phase diagrams may be calculated and analysed in terms of the Flory-Huggins theory using the chemical potentials equality in each phase (Fig. 1)

mena with an emphasis on the mechanisms involved and the final structure of the ternary systems. Theoretical considerations on colloid-polymer mixtures and their phase behaviour have also been given in order to highlight experimental data obtained on globular protein (or colloidal protein particle)-polysaccharide mixtures. Note that protein-polysaccharide covalent interactions leading to the formation of conjugates have not been considered in this review.

D. . 1
Note that polymer pairs can form single-phase mixed solutions only when their mixing process is exothermic. This type of behaviour is rarely encountered, and is scarcely described in the literature for biopolymer mixtures [3]. Exceptions of increase of compatibility by mixing polymers are found in the literature for synthetic weakly charged polyelectrolyte mixtures. This is caused by the enhancement of the co-solubility of polyelectrolytes reflecting an increase in mixing entropy due to contributions of low molecular weight counterions under electrically neutral conditions [4,5]. In this review, we have attempted to give current views of protein-polysaccharide interactions based on segregative and associative phase separation pheno2. Segregative phase separation and theory

The Flory-Huggins approach described in Section 1 (or the second virial approximation) may be valid to analyse protein-polysaccharide phase behaviour only when protein displays polymer characteristics (e.g. gelatin). This approach, originally applied to polymer-organic solvent or polymer melts, may fail in the case of protein-polysaccharide mixtures in water. An alternative model to predict the phase behaviour of aqueous two-phase systems was proposed recently (modified NRTL model), taking into account the polydispersity of the polymer [6]. In the particular case of globular proteins or colloidal protein particles, the

Enthalpic terms

Entropic terms

6 0 A 40

90

80

70

60

50

40
I one-phase region I1 two-phases separated region

ai

a10

% Solvent

% Biopolymer 1

Associative phase separation

Segregative phase separation

Fig. 1. Schematic illustrations of associative and segregative phase separation in biopolymer-biopolymer-solvent mixtures. The chemical potentials equality in each phases based on the Flory-Huggins lattice model is given as a tool to predict experimental tie lines.

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depletion interaction theories classically used to treat the colloid-polymer phase behaviour were seldom applied in the field of protein-polysaccharide mixtures. Note that colloid-polymer mixtures generally phase separate at sufficiently high concentrations of colloids and polymers leading into a colloid-rich (colloidal liquid) and a polymer-rich (colloidal gas) phase. Depletion interaction theories [7,8] apply well in the case of values of polymer to colloid size ratios 5 < 1 (5 = 2rg/0) where 5 is a measure of the relative range of the attractive part of the potential. Tuinier et al. [9"] measured a depletion interaction potential between the casein micelles (a = 200 nm) induced by the presence of a non-adsorbing polysaccharide (rg= 86 nm) by means of S A N S and turbidity. The authors compared data with theoretical predictions based on the depletion interaction theory and the adhesive hard sphere model. In a second paper [lo'], the authors compared the phase diagram of the same system with those calculated from the depletion interaction theories. Discrepancies between theory and experience were explained in terms of a lack of consideration of polydispersity in the different theoretical approaches (colloidal particle polydispersity is known to weaken depletion interaction effects). The application of the adhesive hard sphere model was justified in view of the long range of the depletion interaction potential. The authors did not mention, however, the low purity of their polysaccharide sample 72%) and the quite high polydispersity of casein micelles. In the case of E > 1, little attention has been paid to either the theoretical or experimental points of view. Theoretical work was presented by Schaink and Smit [ll'] to calculate the depletion-induced demixing of a suspension of relatively small spherical colloids (e.g. small globular proteins) and long flexible polymers (neutral and charged polysaccharides which could belong to this category might be pullulans and carrageenans, respectively). Tuinier et al. [12"] applied this theory to a system made of aggregated whey protein colloids mixed with a polysaccharide (5 = 3.2) in the one-phase region and found an effective depletion layer thickness 6 rg/lO. This was much smaller than rg, a value classically taken as the depletion layer thickness for large colloids and small polymers (when 5 < 1). The authors also found that the mechanism of phase separation would proceed via a spinodal decomposition mechanism. Depletion forces and electrostatic repulsions were proposed in earlier works done by Renard et al. [13',14,15'] to explain S A N S results obtained on BSA-cellulose derivatives mixtures located in the one-phase region. Complicated features for the treatment of the intensities in these systems came from the attraction between the protein and segments of the polysaccharide coils (at the IEP of BSA) and the existence of correlation peaks in the
( w w

scattering functions. In the field of polymer-protein interactions, Abbott et al. [16], working on PEO-BSA mixtures, found an attractive interaction energy of approximately 0.05 kT (per polymer segment interacting with the protein). This attractive force coupled with repulsive steric interactions was consistent to explain protein partitioning in two-phase aqueous polymer systems. Concluding and intriguing remarks on particle-polymer mixtures came from the recent theoretical papers of Chatterjee and Schweizer [17',18]. The authors wondered, on the basis of the new theory basis developed, whether reduced solvent quality and non-additive packing effects associated with the interaction of free polymers with particle surfaces, which are rough or coated with grafted or adsorbed polymers, could not be other potential sources of strong polymer-induced particle-particle attractions.
3. Segregative phase separation in protein-polysaccharide systems

In a recent review, almost 100 mixed 'ternary' systems containing proteins and polysaccharides in aqueous medium were described as thermodynamically incompatible [ 191. This gave an illustration that such a mechanism is a general phenomenon when the biopolymers are in solution. Thermodynamic incompatibility generally arises in conditions when the protein is in the presence of a neutral polysaccharide or of an anionic polysaccharide bearing a charge of the same sign as the protein (close to neutrality); obviously, the main parameters involved in the mechanism are pH and ionic strength. A description of the phenomena in the case of thermodynamic incompatibility can be attempted on the basis of the second virial coefficients as obtained from static light scattering or from osmometry. A comparison of the single second virial coefficients (A1, and A 1 3 1 arising from polymer-solvent ) interactions to the cross second virial coefficient ( A, corresponding to the polymer2-polymer3 interactions allows one to estimate the compatibility of the polymers in aqueous solvent. A 2 3 should be sufficiently large while A,, and A , , have relatively low values. The inequality: ( A 2 , ) ,>A12A13 is a general rule employed for the prediction of phase separation. A theoretical description of the phase diagrams can be attempted on this basis by means of the Edmond and Ogston [20] procedure [21-241. However, a major difficulty arises as to the performance of reliable measurements of the second virial coefficients. A theoretical description on the basis of the depletion flocculation theory allowed only a qualitative description of the phase diagram [25,26].

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Segregative phase separation through depletion-flocculation mechanisms is experienced when the proteins are particle-like, such as micellar casein or large aggregates of heat-denaturated proteins. Although apparently similar to limited compatibility in the final results (separation of two phases), this mechanism differs basically from the former one, since it implies a colloidal suspension which is thermodynamically unstable in nature [27']. The result is that the phase separation of a mixed protein-polysaccharide solution requires a total polymer concentration usually higher than 4% while depletion-flocculation takes place at a total concentration of less than 1%. In recent literature, the phase behaviour of dextran (or hydroxyethylcellulose) with different proteins differing in their isoelectric points (bovine serum albumin, y-globulin, lysozyme) was described. Phase separation was experienced which was highly dependent upon the pH with respect to the isoelectric point of the protein and the ionic strength [25,26]. Syrbe et al. [28] also mentioned segregative phase separation between neutral polysaccharides (dextran, maltodextrins, methylcellulose) and native whey proteins within a quite narrow pH range (5-7). When dealing with anionic polysaccharides (high methoxyl pectins, sodium alginate, sodium carboxymethyl cellulose), these authors reported similar phenomena only with denaturated whey proteins. There is, however, evidence of such phenomena taking place in the case of native BSA/CMC mixtures at pH 5.3 [15'1. Gelatin/polysaccharide mixtures obey similar rules. Segregative phase separation occurs only at high ionic strength and under specific pH conditions with respect to the isoelectric point. Under other conditions, compatibility or complex formation would be expected. Isothermal phase diagrams have been described for many systems in conditions when gelatin is not gelled (above the gelling temperature) [29',30',31]. A difficulty arising when dealing with the description of phase-separated systems is related to the fact that phase separation very often competes with the gelation of one or both of the components [32]. This is classically experienced in the case of globular proteins mixed with a polysaccharide, when the gelation of globular protein is triggered by thermal treatment, or with gelatin/polysaccharide mixed systems, the polysaccharide being gelling or non-gelling. In all cases, the resulting system is a gel which may appear homogeneous at the macroscopic level, although heterogeneous at the microscopic one. This explains difficulties often encountered in the description of systems to evidence that segregative phase separation takes place. Besides rheological techniques, which are almost systematically used to describe the properties of the systems, some attempts have been made to

describe the kinetics of the process as well as the structure of the system. When seeking a description of the protein in the medium, scattering techniques appear suitable. Static light scattering has been applied to p-lactoglobulin/K-carrageenan mixtures [33,34'1. In fact, the authors described how p-lactoglobulin denaturation was modified by the presence of K-carrageenan. Different scattering techniques can be combined: small angle neutron scattering ( S A N S ) , dynamic light scattering (DLS) and static light scattering (SLS) to describe the phase separation mechanisms and the kinetics of the process [12",351. Microscopy is another tool that can be useful to describe the microstructure of the system at the end of the gelation process. Numerous examples can be found based on scanning electron microscopy (SEMI, transmission electron microscopy (TEM), or phase contrast microscopy (PCM). Unfortunately, the localisation of the macromolecular components in the medium, which could allow a description of the phase diagram in the gel state, cannot be performed. The use of more appropriate methods like FTIR-microscopy [36], confocal laser microscopy or confocal Raman microspectrometry could be useful in this respect. The consequences of segregative phase separation phenomena have been investigated in the case of a dextran/gelatin system [37"] by means of rheological measurements combined with phase contrast microscopy. When dealing with a gelling polysaccharide, the biopolymer gelling first mainly determines the structure of the final gel, since it develops its own network before gelation of the second component. This has been illustrated in the case of maltodextrin/gelatin mixtures [38], CLSM being used as a tool to detect phase separation as well as to describe the final morphology of the gels. Gelatin concentration was chosen in order that the continuous phase was a gelatin-enriched one. It was confirmed that the time of residence in the region where phase separation competed with gel formation was of critical importance in the morphology of the resulting system. Mixed globular proteins (either native or denaturated)/carrageenan systems have been the object of recent investigations. Capron et al. [33,34'] showed that the first step of the aggregation process of plactoglobulin was not changed by the presence of K-carrageenan, while an acceleration of the gelation process of the protein was experienced in the second step. This was ascribed to a microphase separation. Then, as soon as the protein network was formed, the separation of the phases was 'frustrated' and the system was 'frozen in'. Other rheological studies confirmed the synergistic effects in K-carrageenan/denaturated protein systems [39-421. In detailed rheological studies [40,41] on K'-K-Carrageenan in the

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presence of p-lactoglobulin, these synergistic effects were exhibited in conditions where the two biopolymers gelled at near neutrality. Synergistic interaction was taken as the consequence of two co-continuous networks as a result of a segregative phase separation before gelation takes place. On a similar basis, and in combination with TEM observations, Neiser et al. [42] reached the same conclusions in the case of BSA/K'-K-carrageenan systems. Systems obtained by mixing whey proteins with xanthan at pH 7 or higher also appeared to be governed by segregative phase separation phenomena [43-451. As a result of this process, the limiting concentration for the gelation of proteins was decreased. Synergistic effects were experienced at low xanthan concentrations while antagonistic effects were reported at a high xanthan content. This was consistent with some SEM observations [44] showing that the size of the protein aggregates was increased in the presence of xanthan. Bryant and McClements [45] reached similar conclusions by mixing heat-denatured whey proteins and xanthan. BSA/Na-alginate systems have been described by rheological means [461. Similar trends as those reported in case of whey protein/xanthan systems were evidenced with a dramatic effect of pH and ionic strength on the gel properties; a depressing effect was found upon increasing the ionic strength at a given pH. The lower the pH, the higher the ionic strength to get this effect. These results were interpreted on the basis of a segregative phase separation, despite no evidence of such a mechanism being given. Several recent investigations provided new insights on the important issue of polysaccharide/casein interactions. The phase behaviour of different micellar casein/polysaccharide mixed systems has been studied with either galactomannans [47,48',49,50] or an exocellular high molar weight anionic polysaccharide produced by a lactic bacteria [9",10']. A segregative phase separation phenomenon between the micellar casein and the polysaccharide was reported. This was ascribed to a depletion-flocculation mechanism [9~0,10",47,48',50] or to thermodynamic incompatibility between the components [49]. The large size of the casein micelle and its colloidal (non-polymeric) nature made the former assumption more likely. Indeed, phase diagrams calculated from depletion theories were consistent with the experimental ones [9",50']. Furthermore, the use of different scattering techniques showed that casein micelles became more attractive upon increasing the polysaccharide concentration [lO"]. Decreasing the molecular weight of the polysaccharide resulted in an increase of its concentration to obtain demixing of the system [48',50].

When dealing with gelling carrageenans (K or L) at a temperature above the coil-to-helix transition of the polysaccharide, it has been shown that the major event taking place was a demixing process due to depletion-flocculation of the micelles promoted by the disordered carrageenan chains [51',52',531. Demixing phenomena were noticed with X-carrageenan, which never adopts an ordered conformation [52',54]. In this latter case, however, there was some evidence of an associative demixing process. By using microscopy techniques (CLSM and PCM) on micellar casein/K-carrageenan mixtures [51*,531, it was clearly evidenced that the gelled system was biphasic with carrageenan-rich and casein-rich zones. This meant that, as long as the mixture stayed at a temperature above the coil-to-helix transition temperature of the carrageenan, demixing took place, the extent of which determined the structure of the gelled system. Recent results obtained at a very low cooling rate (loC/l5 min) [55] might be interpreted on this basis. The question arises as to whether carrageenans molecules (K or L) adsorb onto the surface of casein micelles and, hence, if adsorption plays a role in the mechanism, as initially postulated by Snoeren et al. [56]. From variations as a function of temperature of the apparent hydrodynamic diameter of micellar casein in dilute conditions upon the addition of carrageenan (K or L), it was concluded upon the occurrence of carrageenan adsorption onto the casein micelle at a temperature close to the onset of the coil-to-helix transition [52']. It was suggested that adsorption takes place for L-carrageenan (or K ) in the ordered conformation of the carrageenan and that cross-links between carrageenan helices and casein micelles are involved in the gel. There is some evidence, however, that carrageenan-carrageenan interactions and counterions (K', Ca") can be involved in the gel formation [57-591. Although high-methoxyl pectins are widely used in the field of low pH milk beverages, their interaction with casein has been much less investigated. The mechanism of interactions of pectins with casein at low pH ( < 5.0) arises from electrostatic interactions [28',60]; such a mechanism is discussed below. A recent study of interactions of pectin and casein has been performed at neutral pH [61]. In these conditions, depletion-flocculation occurred whatever the type of pectin used. The phase separation boundary was obtained at lower polysaccharide concentrations with low-methoxyl than for high-methoxyl pectins. Upon lowering the pH down to 5.3, pectins adsorbed onto casein micelles and bridging flocculation occurred. Desorption was observed on further pH increase.

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4. Associative phase separation Associative phase separation between proteins and polysaccharides refers to a demixing phenomena induced either by direct interactions between biopolymers, e.g. electrostatic interactions (the famous complex coacervation phenomenon) or hydrogen bonding, or by bad solvent conditions without requiring the involvement of interactions between molecules. In the following, only interacting molecular species in good solvent conditions will be considered. Basically, associative phase separation implies the formation of primary soluble macromolecular complexes that interact to form electrically neutralised aggregates, then unstable liquid droplets and/or precipitates that ultimately sediment to form the coacervated phase containing both biopolymers [1',27']. Electrostatic interactions between oppositely charged proteins and polysaccharides are, in most cases, the prevalent primary interactions in associative mixed biopolymer systems. However, in some examples, primary macromolecule interactions can also be induced by hydrogen bonding or hydrophobic interactions [29',63-681. Now, it is understandable that attractive protein-polysaccharide interactions during associative phase separation actually result from a subtle balance between attractive/repulsive forces [27',601. This topic is rarely considered in the protein-polysaccharide interaction literature and deserves future investigation. One obvious difficulty is to resolve one effect from the others. Also, changes in environmental conditions so as to maximise one kind of interaction may induce biopolymer conformational changes and modifications in biopolymer-solvent interactions, two fundamental parameters in the establishment of protein-polysaccharide interactions. Some more fundamental results have been found in the study of protein-polymer or polymer-micelle interactions. For example, it has been demonstrated that hydrophobic interactions may overcome electrostatic interactions when hydrophobic groups have been anchored along the polymer backbone [69-711. In this case, electrostatic interactions may stabilise the formed macromolecular complex [69]. Conversely, the stabilisation of electrostatically-induced proteinpolysaccharide complexes is supposed to be achieved through secondary hydrogen bonding or hydrophobic interactions [63,72,73]. A surface adsorption mechanism has also been suggested in systems consisting of surface-active anionic polysaccharides and hydrophobic protein aggregates [74]. The existence of weak coulombic interactions at pHs where the two macromolecules have the same net surface charge has been reported. The interaction is possible either at the protein isoelectric point (IEP)

or not too far from the IEP so as to minimise electrostatic repulsion forces between similarly charged groups. For instance this has been demonstrated or hypothesised in mixed systems containing milk or whey proteins and pectin [75,76',77'], (A, L, K ) carrageenan [42,53',75,78,791 sodium alginate [461, carboxymethylcellulose [13'], xanthan [80], ovalbumin-carrageenan [81], ovalbumin-dextran sulfate [82] and gelatin-(L or dcarrageenan [30',83]. Thus, the numerous and different mixed systems studied suggest a widespread phenomenon. In a number of the previously mentioned papers, such interactions have been attributed to the presence of localised positive surface charges onto the proteins (also called 'patches') [84]. Since the effective charge density in these areas is higher than the net protein surface charge density, but, however, linearly dependent on the latter [85"1, a weak electrostatic attraction is expected to occur. As well, the possible protein pKa shift induced by the interaction with the polysaccharide may result in proton migration from NH; to C 0 2 groups, providing an additional mechanism for favourable binding [62,85",86]. Since the hypothesis has been proposed to explain the electrostatic interactions between globular whey proteins such as bovine serum albumin (BSA) or P-lactoglobulin (P-LG) and polymers, one can question whether such a hypothesis is also valid in the case of proteins with a random coil configuration, such as gelatin. The polarisation of the protein by the anionic polysaccharide electric field could possibly explain the measured weak attraction [87"]. The strength of attractive coulombic interactions between proteins and polysaccharides depends to a great extent on the macromolecular charge densities [27'1. This is clearly demonstrated using milk proteins and low methoxyl pectin vs. high methoxyl pectin [61,88], and A-carrageenan vs. L- or K-carrageenans [53',78,79,81]. Sulphated polysaccharides such as carrageenan also interact more strongly with proteins than carboxylated polysaccharides, such as alginates and pectins [60,891. Up to now, the effect of biopolymer charge density, including the effect of ionic strength ( I ) , on the formation of macromolecular complexes and associative phase separation has not been sufficiently considered on a fundamental basis. The difficulty in obtaining biological macromolecules with different charge densities, all other macromolecular parameters being kept constant, may at least partially explain the lack of reliable results. The effect of surface or linear charge density also cannot be dissociated from the biopolymers structure, especially their flexibility and size [90']. For instance, it has been reported that flexible proteins, e.g. caseins or gelatin, bind polysaccharides more strongly than globular proteins, e.g. BSA or P-LG, and that the

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thermal denaturation of the latter enhances their binding affinity [27,29',63]. The explanation proposed is that flexible molecules are able to form a maximum number of contacts with the other oppositely charged molecules, i.e. an increase in local concentration of interacting groups is favoured. On the other hand, it seems that low charge density polysaccharides only interact with proteins if they adopt a more charged ordered (helix) conformation as opposed to a less charged disordered (random coil) conformation [53']. Basically, one may surmise that the optimum interaction between proteins and polysaccharides would occur at a critical balance between the biopolymer charge density and rigidity. An interesting relationship has been obtained on the interaction between proteins and polymers, u, [I~6/<o.2h]K1'2where u, and A are the critical protein surface charge density (to initiate the interaction with the polymer) and the radius of the protein charge patch, respectively. K is the Debye-Huckel parameter, and I , and are the polymer bare persistence length and linear charge density, respectively [91"]. The above equation predicts that the intrinsic stiffness of the polymer chain (I,) will have a greater influence than the polymer linear charge density. The balance between the polymer linear charge density and stiffness is accounted for in recent theoretical developments on polyelectrolyte-sphere electrostatic interactions [92]. The determination of the structure, at the molecular, meso- and macroscopic levels, of protein-polysaccharide associative phase separation represents one of the more challenging and exciting facet of such demixing phenomena. In the future, utilisation of a number of complementary methods such as light and neutron scattering, light and electron microscopy, and spectroscopic methods (e.g. FTIR, circular dichroism, high resolution NMR) will be needed in order to gain an expanded view of the structure. Some scarce results have also been found in the recent years regarding the changes in molecular structure of proteins [65',77',93-96',97,98] and, to a lesser extent, of polysaccharides [29',68'] as induced by their mutual interaction. The structure of primary soluble protein-polysaccharide complexes or higher order aggregated complexes is actually badly known or unknown, and it may be anticipated that an increasing number of papers will be published in this area over the next few years. The knowledge of the structure of the different entities found in demixed systems, due to the often wide molecular polydispersity of natural macromolecules, also deserves much more consideration than it has actually received [76',99',100'1. The use of confocal laser scanning microscopy (C SLM) would allow significant advances in this respect

<

The mechanism of associative phase separation between proteins or surfactant-based micelles and polymers, i.e. the different structural events occurring from the initial interaction towards the formation of coacervates or precipitates, is being intensively studied [101",103',104,105]. Of particular interest are specific pH-induced transitions corresponding to critical structural changes of mixed systems, which have been revealed unambiguously, along with the effects of components charge density and molecular weights. Irrespective of the systems under study, the coarsening processes leading to the coacervated phase from the formation of coacervates, and especially their kinetics, are poorly known. Some recent results on the P-lactoglobulin-acacia gum system suggested a complicated interplay between growth, partial coalescence/flocculation and sedimentation of coacervates [101",1051. Time-resolved small angle light scattering, diffusing wave spectroscopy (DWS) and CLSM are actually used to determine whether a protein-polysaccharide associative phase separation could be described by a spinodal decomposition or nucleation and growth mechanism, or both in sequence [101"] (Schmitt et al., 2000, unpublished results).

5. Conclusions

[101~0,102'].

Active research in the recent past in the field of protein-polysaccharide interactions has provided many new insights into the phase behaviour, rheology and microstructure of mixed systems. Many challenges still remain. Although most parameters affecting protein-polysaccharide interactions are known, their effects on the demixing phenomenon have to be detailed. Future efforts should be focused on the study of the relationships between the structure and the molecular interactions, as well as on the effect of the interaction on the molecular structure. Phase ordering kinetics in biopolymer mixtures should be thoroughly investigated, particularly in the field of gelation-phase separation competing processes. Dynamics in these systems have not been considered in detail, regarding, for instance, relaxation times over a wide frequency range. Scattering and spectroscopic techniques combined with appropriate microscopy techniques and rheological characterisation should provide information on the overall mechanisms involved. Also, it would be important to describe the role of the interface in the liquid-liquid phase separation, the structures at the mesoscopic scale particularly by means of (small angle) light scattering in turbid media and how polydispersity of the biopolymers affects the phase behaviour.

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References and recommended reading

of special interest of outstanding interest

oo

[l] Schmitt C, Sanchez C, Desobry-Banon S , Hardy J. Structure and technofunctional properties of protein-polysaccharide complexes: a review. Crit Rev Food Sci Nut 1998;38:689-753. This paper reviews the main features of protein-polysaccharide associative phase separation, with particular emphasis on complex coacervation, including a detailed description of the different kinds of interactions between macromolecules, the effect of physical and chemical parameters, and the structure of complexes and coacervates. Practical applications and processes are also given in the areas of foods, cosmetics, biomaterials and biotechnology. [2] Clark AH. Direct analysis of experimental tie line data (two 0polymer-one solvent systems) using Flory-Huggins theory. Carbohydr Polym 2000, 42337-351. Theoretical tie-lines of several uncharged ternary systems were predicted according to the Flory-Huggins theory and compared with experimental data. The theoretical procedure is useful to give a qualitative analysis of some (bio)polymer couples but fails in the case where molecular weight polydispersity is relevant. [3] Tolstoguzov V. Compositions and phase diagrams for aqueous systems based on proteins and polysaccharides. In: Walter H, Brooks DE, Srere PA, editors. International review of cytology. A survey of cell biology. Microcompartmentation and phase separation in cytoplasm, vol. 192. San Diego: Academic Press, 20003-31. [4] Khoklov AR, Nyrkova IA. Compatibility enhancement and microdomain structuring in weakly charged polyelectrolyte mixtures. Macromolecules 1992;25:1493-1502. [5] Piculell L, Bergfeldt K, Nilsson S . Factors determining phase behaviour of multicomponent polymer systems. In: Harding SE, Hill SE, Mitchell JR, editors. Biopolymer mixtures. Nottingham: Nottingham University Press, 1995:13-35. [6] Wu Y-T, Lin D-Q, Zhu Z-Q. Thermodynamics of aqueous two-phase systems - the effect of polymer molecular weight on liquid-liquid equilibrium phase diagrams by the modified NRTL model. Fluid Phase Equilibria 1998;147:25-43. [7] Vrij A. Polymers at interfaces and the interactions in colloidal dispersions. Pure Appl Chem 1976;48:471-483. [8] Lekkerkerker HNW, Poon WCK, Pusey PN, Stroobants A, Warren PB. Phase behaviour of colloid polymer mixtures. Europhys Lett 1992;20559-564. [9] Tuinier R, ten Grotenhuis E, Holt C, Timmins PA, de Kruif oo CG. Depletion interaction of casein micelles and an exocellular polysaccharide. Phys Rev E 1999;60:848-856. An interesting approach to quantify the depletion interaction in a biopolymer mixture (casein micelles-exopolysaccharide) using SANS and turbidity measurements was carried out. The authors assumed that the form factor of the casein micelles was unchanged when adding polysaccharide, and neglected the cross term in the expression of the protein-polysaccharide structure factors. All experiments were conducted a priori in the dilute regime ( c p < c : ) and the depletion attraction potential was calculated as a function of c p . [lo] Tuinier R, de Kruif CG. Phase behavior of casein micelles/exocellular polysaccharide mixtures: experiment and theory. J Chem Phys 1999;110:9296-9304. Experimental phase diagrams of casein micelles-exopolysaccharide mixtures are compared with theoretical ones calculated according to depletion theories on the basis of the Vrij and Lekkerkerker approaches. The phase diagram calculated from self-diffusion coefficient measurements is described with special considerations on the effect of the viscosity of the solution on the measured self-diffusion coefficient.

[ l l ] Schaink HM, Smit JAM.Mean field calculation of polymer segment depletion and depletion induced demixing in ternary systems of globular proteins and flexible polymers in a common solvent. J Chem Phys 1997;107:1004-1015. The authors developed a new method based on a mean field calculation of the local polymer volume fraction with the use of a spherical cell-model to calculate phase diagrams for mixtures of small spheres in the presence of long polymers. Despite elegant considerations in the calculations, including corrections for sphere-cell and cell-cell correlations, agreement of experimental phase diagrams with the theory is poor in the case of BSA-PEG and BSA-dextran systems. [12] Tuinier R, Dhont JKG, de Kruif CG. Depletion-induced oo phase separation of aggregated whey protein colloids by an exocellular polysaccharide. Langmuir 2000;16:1497-1507. Extensive work with new theoretical interpretations of the phase behaviour of protein-polysaccharide mixtures is proposed. Small angle light scattering measurements allow the mechanism of phase separation to be described. What is nevertheless unclear is whether the depletion-induced colloidal protein particles flocculation proceeds along the all polysaccharide concentration range studied (dilute and semi-dilute regimes) or if bridging flocculation could occur above c*. [13] Renard D, BouC F, Lefebvre J. Protein-polysaccharide mixtures: structure of the systems and the effect of shear studied by SANS. Physica B 1997; 234-236 289-291. This paper brings direct evidence by means of S A N S of a nonspecific interaction between a globular protein (BSA) and a nongelling polysaccharide (CMC). The interaction is revealed by the appearance of a correlation peak in the scattering intensities (i.e. liquid-like short range order). This peak is ascribed to an adsorption of proteins onto the polysaccharide coil segments (in the dilute regime) or an entrapment in the cross-links of the entangled polysaccharide coils network (in the semi-dilute regime). [14] Renard D, BouB F, Lefebvre J. Protein-polysaccharide mixtures: structure and effect of shear studied by small-angle neutron scattering. In: Dickinson E, Bergenstlhl B, editors. Food colloids - proteins, lipids and polysaccharides. Cambridge: Royal Society of Chemistry, 1997:303-316. [15] Renard D, B o d F, Lefebvre J. Solution and gelation properties of protein-polysaccharide mixtures: signature by smallangle neutron scattering and rheology. In: Williams PA, Phillips GO, editors. Gums and stabilisers for the food industry 9. Royal Society of Chemistry, Cambridge, 1998; 189-201. The surprising result of this work is that soluble complexes formed between BSA and CMC ([12,13]) are maintained after thermal treatment leading to a radical change in the aggregate structure compared to those formed with the protein alone. A consequence of the 'directed' aggregation is a considerable increase in the viscoelasticity of the network. [16] Abbott NL, Blankschtein D, Hatton TA. Protein partitioning in two-phase aqueous polymer systems. 3. A neutron scattering investigation of the polymer solution structure and protein-polymer interactions. Macromolecules 1992;25: 3932-3941. [17] Chatterjee A' Schweizer KS: Correlation effects in dilute E, particle-polymer mixtures. J Chem Phys 1998;109: 10477-10488. This paper reviews theories developed in the field of colloid-polymer mixtures and proposes a new one based on the influence of chain connectivity and polymer-excluded volume correlations on macromolecule-induced depletion interactions between spherical particles. Which are the molecular origins of phase transitions is questioned by the authors, reporting an interesting recent example of liquid-liquid phase separation occurring in polymer blends composed of chains of different local stiffness.

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Chatterjee AP, Schweizer KS. Microscopic theory of polymer-mediated interactions between spherical particles. J Chem Phys 1998;109:10464-10476. Grinberg V, Tolstoguzov VB. Thermodynamic incompatibility of proteins and polysaccharides in solutions. Food Hydrocoll 1997;11:145-158. Edmond E, Ogston AG. An approach to the study of phase separation in ternary aqueous systems. Biochem J 1968; 109569-576. Wasserman LA, Semenova MG, Tsapkina EN. Thermodynamic properties of the 1 s globulin of vicia faba-ovalbumin1 aqueous solvent system: phase behaviour and light scattering. Food Hydrocoll 1997;11:327-337. Antipova AS, Semenova MG. Effect of sucrose on the thermodynamic incompatibility of different biopolymers. Carbohydrate Polym 1995;28:359-365. Antipova AS, Semenova MG. Influence of sucrose on the 1 thermodynamic properties of the 1 s globulin of vicia-fabadextran aquous solvent system. Food Hydrocoll 199711: 415-421. Semenova MG, Savilova LB. The role of biopolymer structure in interactions between unlike biopolymers in aqueous medium. Food Hydrocoll 1998;1265-75. Hoskins R, Robb ID, Williams PA, Warren P. Phase separation in mixtures of polysaccharides and proteins. J Chem SOC Faraday Trans 1996;92:4515-4520. Hoskins R, Robb ID, Williams PA. Selective separation of proteins from mixtures using polysaccharides. Biopolymers 1998;45:97-104. Tolstoguzov VB. Protein-polysaccharide interactions. In: Damodaran S, Paraf A, editors. Food proteins and their applications. Marcel Dekker, New York 1997: 171-198. .. A comprehensive review describing the basic mechanisms involved in the formation of protein-polysaccharide complexes, and their possible structures in relation to pH and ionic strength. [28] Syrbe A, Bauer WJ, Klostermeyer H. Polymer science concepts in dairy systems - an overview of milk protein and food hydrocolloid interaction. Int Dairy J 1998;8:179-193. The role of food polysaccharides in dairy products is reviewed using the theoretical basis of the mixing behaviour of polymer solutions and polymer interactions with colloidal particles. The consequences of these phenomena on the structures that can be created are clearly illustrated. The importance of using high molecular weight samples is emphasised. Some examples are given. [29] Antonov YuA, Lashko NP, Glotova YK, Malovikova A, Markovich 0. Effects of the structural features of pectins and alginates on their thermodynamic compatibility with gelatin in aqueous media. Food Hydrocoll 1996;lOl-9. One of the few studies on the effect of polysaccharide structures in protein-polysaccharide interactions. Pectins with various degrees of esterification, block and statistical distribution of free carboxyl and ester groups as well as alginates of various block structures are used in conjunction with gelatin. Segregative phase separation occurred at a high ionic strength (> 0.2 M) while the system was one-phase at a low ionic strength. Phase diagrams indicate that hydrogen bonding greatly contributes to the formation of pectingelatin complexes. An increase in the content of mixed MG-blocks in alginate molecules promotes miscibility with gelatin, showing the importance of polysaccharide flexibility. [30] Antonov YuA, Goncalves MP. Phase separation in aqueous gelatin-K-carrageenan mixture. Food Hydrocoll 1999;13: 517-524. The phase behaviour of gelatin (type B)/K-carrageenan systems is described at pH 5 as a function of ionic strength. Water-insoluble complexes are formed at a low ionic strength (0.002 M). Incompatibility is found only at ionic strength higher than 0.35 M. Electrostatic and possibly non-coulombic interactions are suggested to be at the origin of the formed complexes. The authors discuss the balance between biopolymer-biopolymer and biopolymer-solvent interactions, which rarely appear in recently published papers. [31] Alves MM, Antonov YuA, Goncalves MP. The effect of structural features of gelatin on its thermodynamic compatibility with locust bean gum in aqueous media. Food Hydro~011 1999;13:157-166. [32] Zazypkin DV, Braudo EE, Tolstoguzov VB. Multicomponent biopolymer gels. Food Hydrocoll 1997;11:156-170. [33] Capron I, Nicolai T, Durand D. Heat induced aggregation and gelation of P-lactoglobulin in the presence of K-Carrageenan. Food Hydrocoll 1999; 13: 1-5. Aggregation of proteins during the heat treatment is monitored using dynamic light scattering allowing variations in the size of the protein aggregates. The presence of Na+-carrageenan in 0.1 M NaCl, i.e. in conditions where the polysaccharide does not gel, yields an acceleration of the kinetics of the process. The results are interpreted on the basis of a microphase separation. [34] Capron I, Nicolai T, Smith C. Effect of addition of K-Carrageenan on the mechanical and structural properties of Plactoglobulin gels. Carbohydrate Polym 1999;40233-238. Rheological measurements are combined with a TEM technique. The kinetics of aggregation of the protein is accelerated in the presence of the polysaccharide; this is ascribed to the phase separation process. This cannot be completed owing the formation of the protein network. The structure of the resulting gel and therefore its properties will depend upon the size of aggregates just after the gel point. [35] De Kruif GC, Tuinier R. Whey protein aggregates and their interaction with exopolysaccharides. Int J Food Sci Techno1 1999;34:487-492. [36] Durrani CM, Prystupa DA, Donald AM. Phase diagram of mixtures of polymers in aqueous solution using Fourier transform infrared spectroscopy. Macromolecules 1993;26: 981-987. [37] Owen AJ, Jones RAL. Rheology of simultaneously phaseH separating and gelling biopolymer mixtures. Macromolecules. 1998;31:7336-7339. Rheological measurements are combined with phase contrast microscopy to describe the effect of thermal history on the properties of a dextran/gelatin mixture, in relation to the thermal history with respect to the upper critical solution temperature (38C) and the gelatin gelling temperature (- 25C). Quenching from 45C to below 25C induced phase separation by spinodal decomposition simultaneously with gelation. The result was a bicontinuous morphology. When a first quench was performed at intermediate temperature (30C), a phase separation through spinodal decomposition was yielded with an interconnected structure in the first stages followed by a transition to a droplet morphology with the gelatinenriched phase dispersed in the dextran-rich continuous phase. A further quench to below 25C froze the structure and resulted in much weaker gels. [38] Lor& N, Langton M, Hermansson AM. Confocal laser scanning microscopy and image analysis of kinetically trapped phase-separated gelatin/maltodextrin gels. Food Hydrocoll 1999;13:185-198. [39] Tziboula A, Horne DS. Influence of whey protein denaturation on K-Carrageenan gelation. Colloid Surf B: Biointerf 1999;12:299-308. [40] Ould Eleya MM, Turgeon SL. Rheology of +Carrageenan and P-lactoglobulin mixed gels. Food Hydrocoll 2000; 14:29-40. [41] Ould Eleya MM, Turgeon SL. The effect of pH on the rheology of P-laCtOglObUlin/K-Carrageenan mixed gels. Food Hydrocoll2000; 14245-2s 1.

J.-L. Doublier et al. /Current Opinionin Colloid & Inte8ace Science 5 (2000)202-214
Neiser S, Draget KI, Smidsrod 0. Gel formation in heattreated bovine serum albumin-K-carrageenan systems. Food

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Hydrocoll2000;14:95-110.
Sanchez C, Schmitt C, Babak VG, Hardy J. Rheology of whey protein isolate xanthan mixed solutions and gels. Effect of pH and xanthan concentration. Nahrung 1997;41:336-343. Zasypkin DV, Dumay E, Cheftel JC. Pressure- and heat-induced gelation of mixed P-lactoglobulin/xanthan solutions. Food Hydrocoll 1996;10203-211. Bryant CM, McClements DJ. Influence of xanthan gum on physical characteristics of heat-denaturated whey protein solutions and gels. Food Hydrocoll2000;14:383-390. Neiser S, Draget KI, Smidsr~d0. Gel formation in heattreated bovine serum albumin-sodium alginate systems. Food Hydrocoll 1998;12127-132. Bourriot S, Garnier C, Doublier JL. Phase separation, rheology and microstructure of micellar casein-guar gum mixtures. Food Hydrocoll 1999;13:43-49. Bourriot S, Garnier C, Doublier JL. Phase separation, rheology and microstructure of micellar casein-galactomannan mixtures. Int Dairy J 1999;9:353-357. This study of a model system clearly evidences the phase behaviour of neutral polysaccharide/casein mixtures and shows that aggregation of the proteins are sufficient to structure the system. The predominant role of the molar weight of the polysaccharide is shown. [49] Schorsch C, Jones MG, Norton IT. Thermodynamic incompatibility and microstructure of milk protein/locust bean gum/sucrose systems. Food Hydrocoll 1999;13:89-99. [50] Tuinier R, ten Grotenhuis E, de Kruif CG. The effect of depolymerised guar gum on the stability of skim milk. Food Hydrocoll 2000;14:1-7. It is shown that the theory of Vrij allows a qualitative prediction of the phase boundary with respect to the molar weight dependence. [51] Bourriot S, Garnier C, Doublier JL. Micellar casein/kappacarrageenan mixtures. 1. Phase separation and ultrastructure Carbohydrate Polym 1999;40:145-157. This study highlights the fact that the major event taking place in K-carrageenan/micellar casein systems is a segregative phase separation process. The phase diagram of the system is described at 60C. The existence in the gel at 25C of protein-rich and polysaccharide-rich zones at the microscopic level is clearly evidenced by microscopy tools. [52] Langendorff V, Cuvelier G, Michon C, Launay B, Parker A, de Kruif CG. Effects of carrageenan type on the behaviour of carrageenan/milk mixtures. Food Hydrocoll 2000;14: 273-280. Based on photocorrelation spectroscopy using a Malvern Autosizer, the apparent hydrodynamic diameter of casein micelles is estimated as a function of carrageenan concentration at different temperatures. A dramatic increase of this parameter is observed in the vicinity of the coil-to-helix transition temperature of K and &-carrageenans. In the presence of h-carrageenan, the apparent diameter of the casein micelles does not vary with temperature but is much higher than of casein micelles by themselves. These results are taken as a proof that A-carrageenan adsorbs onto casein micelles whatever the temperature while K and L-carrageenans adsorb only on the onset of the transition, that is in the ordered conformation. It is suggested that these differences are related to the higher charge density of h-carrageenan molecules with respect to L and K-carrageenan in the disordered state. [53] Schorsch C, Jones MG, Norton IT. Phase behaviour of pure micellar casein/K-carrageenan systems in milk salt ultrafiltrate. Food Hydrocoll 2000;14:347-358.

Langendorff V, Cuvelier G, Launay B, Parker A. Gelation and flocculation of casein micelle/carrageenan mixtures. Food Hydrocoll 1997;11:35-40. Tziboula A, Horne DS. Influence of milk protein on K-carrageenan gelation. Int Dairy J 1999;9:359-364. Snoeren THM, Payens TAJ, Jevnink J, Both P. Electrostatic interactions between K-carrageenan and K-casein. Milchwissenschaft 1976;30:393-396. Drohan DD, Tziboula A, McNulty D, Horne DS. Milk protein-carrageenan interactions. Food Hydrocoll 1997;ll: 101-107. Augustin MA, Puvanenthiran A, McKinnon IR. The effect of K-carrageenan conformation on its interaction with casein micelles. Int Dairy J 1999;9:413-414. Oakenfull D, Miyoshi E, Nishinari K, Scott A. Rheological and thermal properties of milk gels formed with K-carrageenan. 1. Sodium caseinate. Food Hydrocoll 1999;13: 525-533. Dickinson E. Stability and rheological implications of electrostatic milk protein-polysaccharide interactions. Trends Food Sci Techno1 1998;9:347-354. Maroziene A, de Kruif CG. Interaction of pectin and casein micelles. Food Hydrocoll 2000;14:391-394. Yoshida K, Sokhakian S, Dubin PL. Binding of polycarboxylic acids to cationic mixed micelles: effects of polymer counterion binding and polyion charge distribution. J Colloid Interf Sci 1998;205:257-264. Braudo EE. Functional interactions in multicomponent polysaccharide-containingsystems. In: Williams PA, Phillips GO, editors. Gums and stabilizers for the food industry 9. Cambridge: Royal Society of Chemistry, 1998:169-183. Tribet C, Porcar I, Bonnefont PA, Audebert R. Association between hydrophobically modified polyanions and negatively charged bovine serum albumin. J Phys Chem B 1998; 1021327-1333. Interactions between hydrophobically modified poly(acry1ic acidls and BSA are studied. It is demonstrated by light scattering that mixed aggregates are formed depending on the polymer hydrophobicity and its molecular weight. Interactions between the two molecules are stronger when increasing polymer hydrophobicity. Polymers of a low molecular weight could form a shell around BSA whereas BSA could bind along one long polymer forming a necklace-type structure. [65] Sedl5k E, Antalik M. Coulombic and noncoulombic effect of polyanions on cytochrom c structure. Biopolymers 1998; 46:145-154. High ionic strength (0.5 M NaCl) does not prevent the formation of PSS-ferrycyt c complexes providing indication for non coulombic interactions. The shift of ferrycyt c pK, induced by the interaction with polyanions improves the affinity of cyt c for cardiolipids. Circular dichroism shows that the addition of PSS to ferrycyt c decreases the negative Cotton effect of ferrycyt c in the Soret region indicating a weakening of the heme-protein interaction. [66] Porcar I, Cottet H, Gareil P, Tribet C. Association between protein particles and long amphiphilic polymers: effect of the polymer hydrophobicity on binding isotherms. Macromolecules 1999;323922-3929. [67] Azegami S,Tsuboi A, Izumi T, Hirata M, Dubin PL, Wang B, Kokufuta E. Formation of an intrapolymer complex from human serum albumin and poly(ethy1ene glycol). Langmuir 1999;15:940-947. [68] Gao JY, Dubin PL. Binding of proteins to copolymers of varying hydrophobicity. Biopolymers 1999;49:185-193. Binding isotherms of complexing between P-lactoglobulin or BSA and copolymers of maleic acid and alkyl-vinyl ethers of varying

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[81] Galazka VB, Smith D, Ledward DA, Dickinson E. Interactions of ovalbumin with sulphated polysaccharides: effects of pH, ionic strength, heat and high pressure treatment. Food Hydrocoll 1999;13:81-88. [82] Matsudomi N. Characteristics of heat-induced transparent gels from egg white by the addition of dextran sulfate and the protein-polysaccharide interactions (short communication). Nahrung 1998;42238-239. [83] Michon C, Vigouroux F, Boulenguer P, Cuvelier G, Launay B. Gelatin/iota-carrageenan interactions in non-gelling conditions. Food Hydrocoll2000;14:203-208. [84] Park JM, Muhoberac BB, Dubin PL, Xia J. Effects of protein charge heterogeneity in protein-polyelectrolyte complexation. Macromolecules 1992;25:290-295. [85] Mattison KW, Dubin PL, Brittain IJ. Complex formation w between bovine serum albumin and strong polyelectrolytes: effect of polymer charge density. J Phys Chem B 1998; 102:3830-3836. Synthetic polyanions and polycations differing in their linear charge density are checked for their ability to interact with BSA. As shown previously by the same authors, the critical protein charge required to induce protein-polymer interaction is found to vary linearly with the square root of the ionic strength. Interestingly, intrinsic chain flexibility and linear charge density of polymers are intrinsically related regarding interactions with the protein. This suggests that the so-called protein surface charge patch is an array of charges complementary to the distribution of charges on the polymer binding segment. [86] Wen Y-P, Dubin PL. Potentiometric studies of the interaction of bovine serum albumin and poly(dimethyldially1ammonium chloride). Macromolecules 1997;30:7856-7861. [87] Bowman WA, Rubinstein M, Tan JS. Polyelectrolyte-gelatin oo complexes: light scattering study. Macromolecules 1997; 30:3262-3270. A static and dynamic light scattering study of the complex formation between polyanionic polymers and positively charged gelatin in the random coil conformation. Instability of gelatin-polymer complexes occurs at a given stoichiometry dependent on the polymer used. The attractive interaction is assumed to be caused by polarisation of gelatin by the electric field of the strong polyelectrolytes. More compact conformation of polymers induces higher charge density and therefore stronger interaction with the protein. It is not clear whether the weak polyelectrolyte character of polysaccharides would yield a similar polarisation of gelatin. [88] Pereyra EFE, Schmidt KA, Wicker L. Interaction and stabilization of acidified casein dispersions with low and high methoxyl pectins. J Agric Food Chem 1997;45:3448-3451. [89] Dautzenberg H. Polyelectrolyte complex formation in highly aggregating systems. 1. Effect of salt: polyelectrolyte complex formation in the presence of NaCl. Macromolecules 1997;30:7810-7815. [90] McArthur SL, McLean KM, Kingshott P, St John HAW, Chatelier RC, Griesser HJ. Effect of polysaccharide structure on protein adsorption. Colloids Surf B: Biointerf 2000; 17:37-48. The adsorption of four different proteins on surfaces covalentlymodified by polysaccharides is studied using X-ray photoelectron spectroscopy. It is shown that some proteins may adsorb even whether the surfaces are similarly charged. This is interpreted as a consequence of their multidomain structures, with each domain having different properties in terms of charge and stability. More flexible polysaccharide chains are supposed to produce more steric-entropic forces and reduce protein interactions. [91] Mattison KW, Wang Y, Grymonprt K, Dubin PL. Micro-

hydrophobicity are determined using capillary electrophoresis. The binding of globular proteins increases with the increased hydrophobicity of copolymers. Copolymers in the random coil conformation exhibit a stronger hydrophobic interaction with P-lactoglobulin than those in the hypercoil (less flexible) conformation. Copolymer conformational changes are suggested to be induced by the binding of proteins. [69] Mizusaki M, Morishima Y, Dubin PL. Interaction of pyrenelabeled hydrophobically modified polyelectrolytes with oppositely charged mixed micelles studied by fluorescence quenching. J Phys Chem B 1998;1021908-1915. [70] Borrega R, Tribet C, Audebert R. Reversible gelation in hydrophobic polyelectrolyte/protein mixtures: an example of cross-links between soft and hard colloids. Macromolecules 1999;32:7798-7806. [71] Tsianou M, Kj~niksen A-L, Thuresson K, Nystrom B. Light scattering and viscoelasticity in aqueous mixtures of oppositely charged and hydrophobically modified polyelectrolytes. Macromolecules 1999;322974-2982. [72] Sanchez C, Paquin P. Protein and protein-polysaccharide microparticles. In: Damodaran S, Paraf A, editors. Food proteins and their applications. New York Marcel Dekker, 1997:503-528. [73] Chang H-M, Lu T-C, Chen C-C, Tu Y-Y, Hwang J-Y. Isolation of immunoglobulin from egg yolk by anionic polysaccharides. J Agric Food Chem 2000;48:995-999. [74] Schmitt C, Sanchez C, Thomas F, Hardy J. Complex coacervation of P-lactoglobulin and acacia gum in aqueous medium. Food Hydrocoll 1999;13:483-496. [75] Dalgleish DG, Hollocou A-L. Stabilization of protein-based emulsions by means of interacting polysaccharides. In: Dickinson E, Bergenstahl B, editors. Food colloids proteins, lipids and polysaccharides. Cambridge: Royal Society of Chemistry, 1997:236-244. [76] Dickinson E, James JD. Influence of high-pressure treatment on P-lactoglobulin-pectin associations in emulsions and gels. Food Hydrocoll2000; 14: 365-376. Bridging flocculation of P-lactoglobulin-covered oil droplets is induced by pectin following hydrostatic high pressure treatment at pH above the protein IEP. It is supposed that the bridging mechanism is induced by electrostatic interactions between the polysaccharide and the high pressure unfolded protein. Interestingly, mild high pressure treatment also promotes oil droplet flocculation, which is explained by the dissociation of p-LG dimers in monomers revealing new interaction sites. Although the hypothesis is not demonstrated, this implies that the quaternary structure of the protein could influence the extent of interaction [77] Zaleska H, Ring SG, Tomasik P. Apple pectin complexes with whey protein isolate. Food Hydrocoll 2000;14:37-382. It is suggested from thermogravimetric measurements that formation of whey proteins-pectin complexes decreases the thermal stability of pectins and increases the thermal stability of proteins. This paper also indicates that water was sorbed at the surface of whey proteins-pectin complexes rather than in the formed matrix. [78] Dickinson E, Pawlowsky K. Influence of K-carrageenan on the properties of a protein-stabilized emulsion. Food Hydro~0111998;12:417-423. [79] Galazka VB, Smith D, Ledward DA, Dickinson E. Interactions of bovine serum albumin with sulphated polysaccharides: effects of pH, ionic strength and high pressure treatment. Food Chem 1999;64:303-310. [SO] Sanchez C, Zuniga-Lopez R, Schmitt C, Despond S, Hardy J. Microstructure of acid-induced skim milk-locust bean gum-xanthan gum gels. Int Dairy J 2000;10:199-212.

J.-L. Doublier et al. /Current Opinionin Colloid & Inte8ace Science 5 (2000)202-214
and macro-phase behavior in protein-polyelectrolyte complexes. Macromol symp 1999; 140 53-76. A interesting review of the fundamental contribution by Dubin and co-workers in the field of protein-polymer attractive interactions and phase separation. The effect of pH, ionic strength, proteinpolymer stoichiometry and polymer chain stiffness on the formation, stoichiometry and stability of primary soluble complexes is discussed and illustrated. The authors expect that their conclusions could be applicable to biological macromolecules, which remains to be demonstrated. [92] Netz RR, Joanny J-F. Complexation between a semiflexible polyelectrolyte and an oppositely charged sphere. Macromolecules 1999;329026-9040. [93] Yang C-C, Chen C-C, Chang H-M. Separation of egg white lyzozyme by anionic polysaccharides. J Food Sci 2000;63: 962-965. [94] Delben F, Stefancich S. Interaction of food polysaccharides with ovalbumin. Food Hydrocoll 1998;12291-299. [95] Paradossi G, Chiessi E, Malovikova A. Study of the interactions of D- and L-polylysine enantiomers with pectate in aqueous solutions. Biopolymers 1999;50:201-209. The electrostatic interaction between potassium pectate and D- and L-enantiomers of polylysin is determined using circular dichroism, microcalorimetry and osmometry. A coil-helix transition is detected for poly(L-lysin) after interaction with the polysaccharide whereas the poly(D-lysin) remains in the disordered state. It infers that both electrostatic interactions and stereochemical constraints play a role in the energetics of the interaction. [96] Schmitt C, Sanchez C, Despond S, Renard D, Robert P, Hardy J. Structural modifications of P-lactoglobulin as induced by complex coacervation with acacia gum. In: Dickinson E, Miller R, editors. Food Colloids Fundamental of Formulation. Royal Society of Chemistry, Cambridge, 2000 (in press). FTIR spectroscopy, circular dichroism and front face fluorescence spectroscopy are used to detect conformational changes in plactoglobulin upon complexation with acacia gum. Independently of the pH, protein-polysaccharide ratio and protein polydispersity, circular dichroism reveals a decrease in the mean residue ellipticity at 208 and 222 nm, indicative of a loss in the protein (a-helix content. It is supposed that the detected conformational change involves the outer part of the protein calyx, which is positively charged at the pHs selected in the study. [97] Anderson MM, Hatti-Kaul R, Brown W. Dynamic and static light scattering and fluorescence studies of the interactions between lactate dehydrogenase and poly(ethy1eneimine). J Phys Chem B 2000;104:3660-3667. [98] Xia J, Dubin PL, Kokufuta E, Have1 H, Muhoberac BB. Light scattering, CD, and ligand binding studies of ferrihemoglobin-polyelectrolyte complexes. Biopolymers 1999;50: 153-161. [99] Schmitt C, Sanchez C, Despond S, Renard D, Thomas F, -0 Hardy J. Effect of protein aggregates on the complex coacervation between P-lactoglobulin and acacia gum at pH 4.2. Food Hydrocoll2000;14:403-413. The presence of aggregates of P-lactoglobulin enlarges the biphasic area of the phase diagram and modifies its position, revealing both an effect of aggregate size and surface properties. Volume diameter of particles depends on the pr0tein:polysaccharide ratio and the presence or not of aggregates. Precipitates and coacervates are formed in presence of protein aggregates whereas only coacervates are produced in the absence of aggregates. Highly unstable systems are obtained at a given total concentration for mixtures without aggregates, which suggest that insoluble aggregates could stabilise the coacervates.
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[loo] Laneuville SI, Paquin P, Turgeon SL. Effect of preparation conditions on the characteristics of whey protein-xanthan gum complexes. Food Hydrocoll2000;14:305-314. High-pressure microfluidization is applied to xanthan dispersion to reduce its polydispersity. As a consequence, whey protein-xanthan microparticles are obtained rather than large fibrous macromolecular complexes. This study suggests that physical parameters could be used to control the size of protein-polysaccharide supramolecular entities. [loll Schmitt C, Sanchez C, Lamprecht A, Renard D, Lehr C-M, 0. de Kruif CG, Hardy J. Study of P-lactoglobulin-acacia gum complex coacervation by diffusing wave spectroscopy and confocal laser scanning microscopy. Colloids Surf B: Biointerf 2000 (in press). CLSM on FITC-P-lactoglobulin-RITC-acaciagum complex coacervation reveals the presence of both biopolymers in precipitates and coacervates. The formation of protein aggregate-based co-precipitates is suggested to be due to the adsorption of the surface active polysaccharides onto the hydrophobic insoluble particles. Coacervates form vesicles or multi-hollow spheres. Flocculation and partial coalescence of vesicles appear in conditions of maximum interaction in blends not containing protein aggregates. Diffusing wave spectroscopy correlation function shows that small coacervates increase in size as a function of time. Normalised backscattered intensity also shows that the time stability of mixed P-lactoglobulin-acacia gum systems depends on the strength of interaction. Highly interacting systems display a complex turbidity pattern with different particle coarsening phases. [lo21 Lamprecht A, Schafer UF, Lehr C-M. Characterization of microcapsules by confocal laser scanning microscopy: structure, capsule wall composition and encapsulation rate. Europ J Pharm Biopharm 2000;49:1-9. Oil droplets encapsulated by gelain-acacia gum or gelatin acacia gum coacervates are characterised using CLSM. A new method is developed to quantify the amount and location of proteins and polysaccharides in the particle wall. It is demonstrated that gelatin-acacia gum are distributed homogeneously across the wall. On the other hand, casein-acacia gum coacervates present an inhomogenous distribution across the particle wall, with the highest concentration of casein found at the oil-wall interface. [lo31 Wang Y, Kimura K, Jaeger W, Dubin P. PolyelecOD trolyte-micelle coacervation: Effects of micelle surface charge density, polymer molecular weight, and polymer/surfactant ratio. Macromolecules 2000;33:3324-3331. An important paper on the coacervation mechanism between polymers and micelles. An important finding is the existence of a critical molecular weight for coacervation at any fixed polymer concentration, micelle charge density and ionic strength. An increase in the micelle surface charge density can either suppress or enhance coacervation. The size of aggregated polymer-micelles complexes just before coacervation is of the order of 45 nm as determined by dynamic light scattering. However, the path between aggregated complexes and coacervates remains totally mysterious. In addition, it is doubtful that such a supposed well defined borderline exists in the case of the inherently polydisperse biopolymers. [lo41 Kaibara K, Okazaki T, Bohidar HB, Dubin PL. pH-induced coacervation in complexes of bovine serum albumin and cationic polysaccharides. Biomacromolecules 2000;l: 100-107. Turbidimetric titration, light scattering (LS) and phase contrast light microscopy are used to follow the pH-induced coacervation between BSA and a polycationic polymer (PDADMAC). On the basis of LS measurements, six specific pH are identified corresponding to different structural transitions. This includes the

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entities are likely to be found simultaneously or sequentially in biopolymeric systems. [lo51 Sanchez C. Schmitt C, Despond S , Hardy J. Effect of heat and shear on P-lactoglobulin-acacia gum complex coacervation. In: Dickinson E, Miller R, editors. Food Colloids Fundamental of Formulation. Royal Society of Chemistry, Cambridge, 2000 (in press).

beginning of formation of primary soluble BSA-PDADMAC complexes (pH,) that is completed at pH,, aggregation of soluble complexes (pHpre),appearance of coacervates (pH,), changes in coacervates morphology (pHmorph) precipitation of coacervates and (pHprecip). real significance of pHmorph unclear. It is possible The is that this pH corresponds to coacervate coalescence or flocculation, or inner rearrangement as shown in references [99,101]. All these