a theoretical self-consistent field study of mixed interfacial biopolymer films

7/29/2019 A Theoretical Self-Consistent Field Study of Mixed Interfacial Biopolymer Films

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Chapter 3A Theoretical Self-Consistent Field Studyof Mixed

Interfacial Biopolymer FilmsRammleEttelaie,Anna Akinshina, and EricDickinson

Procter Departmentof Food Science,Universityof Leeds,LeedsLS2 9J T,United Kingdom

The adsorption of a linear polyelectrolyte onto an existinglayer of protein at an interface has been investigated.Calculations using a simple model, involving only the shortrange interactions, show that the polyelectrolyte forms a moreextended distinct secondary layer ifonly ceratin sections of themolecule interact with the protein layer. These results are alsoconfirmed for a more sophisticated model thataccounts for theelectrostatic interactions between the two biopolymers. It isfound that there is a maximum level of adsorption ofpolyelectrolyte as the number of charged segments of thechains is varied. The peak occurs at higher levels of chargingas the background salt concentration is increased. We alsoconsider the effects of pH on the adsorption. The influence ofthe structure of the mixed layers on colloidal forces, mediatedbetween two surfaces covered by such films, is also discussed.

46 2009American Chemical Society

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In Micro/Nanoencapsulation of Active Food Ingredients; Huang, Q., el al.;ACS Symposium Series; American Chemical Society: Washington, DC, 2009.


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Introduction47

Proteins and polysaccharides constitute two of the most important functionalingredients in foods. Polysaccharides are by and large high-molecular-weighthydrophilicmolecules and as such do not show any tendency for adsorption ontohydrophobic interfaces. They are commonly added to food products as rheologymodifiers, thickeners and for their excellent waterholding properties (/). Incontrast, the simultaneous presenceof both hydrophobic and hydrophilic aminoacid groups in the primary structureof proteins makes thesemolecules surface-active. Most of the important functional properties of proteins in food colloidsare the direct consequence of the amphiphilicnatureof thesemolecules. Thus,proteins are widely used as foaming agents, emulsifiers and in particular ascolloidal stabilisers in foods (2,3).The presence of both polysaccharide and protein, in many food colloidformulations, gives rise to the inevitable possibility of these different speciesinteracting with each other. In most cases, where the polysaccharides containcharged groups, these interactions are electrostatic inorigin. However, shorterrange interactions such as bridging by specific ions and hydrogen bonding canalso bepresent (4). When strong enough, and at sufficient concentrations of thetwo biopolymers, these interactions can lead to the phase separation of thesystem intoseparateregions. The type of phaseseparation occurringdependsonwhether the interactions are synergistic or repulsive (4-7). In the latter case thesystem breaks up into two separatesolutions, one rich in protein and the other inpolysaccharide. Associative interactions on the other hand result in thephenomenon of coacervation, where one of the resulting phases has a highconcentration of both biopolymers and the other is depleted in both.

Despite receiving a great deal of interest (5,6) in the past, the interactionsbetween proteins and polysaccharides in food system were often seen as anunwanted complication. However, in recent years it has been recognised thatsuch interactions provide interesting routes to design of a variety of foods withnovel textures and structures (4,8-10). It is predicted that such structures wil llead to significant possibilities for better control of the releaseprofile of flavoursduring consumption of foods, for improving the mouthfeel of the products, formicroencapsulation of nutrients and flavours, and for inhibiting the digestion oflipids in the design of healthier food emulsion systems, to name but a fewapplications.

In choosing the most appropriate structurefor each type of application it isuseful to broadly distinguish between two different typesof structuresthatoccurin the context of such mixed biopolymer systems. The first type involves theentire bulk of the system. An example is the kinetic trapping of a desiredstructure, at an intermediate stage in the phase separation of the protein andpolysaccharide, as a result of gelation of one or both sets of the biopolymers


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48(9,11). The second type of mixed structures comprises those that are formedspecificaly at surfaces, and in particular hydrophobic-hydrophilic interfaces (12-15). Such surface structures rely on the amphiphilicnatureof proteins to form aprimary layer at the interface, with a secondary layer of polysaccharide thenbeing adsorbed on top of the primary one. By suitable adjustment of pH , andaternating the solution, with which the interface is in contact, between onecontaining anionic to one with cationic polyelectrolytes or visa versa, severabiopolymer layers can be deposited on the surface to form a multi-layered fi lm atthe interface. A number of recent studies on oil-in-water emulsions suggestthat,compared to simple protein stabilised emulsions, the coverage of the droplets bythese multi-layer films can provide significantly superior stability aganst pHchanges, therma cycles and increase in background electrolyte concentrations(12,16).

In conjunction with the experimenta work therehas aso been agreat deaof interest in theoretica modelling of the process of polyelectrolyte-proteincomplexation inrecentyears (7). Most of the work has focused on formation ofcomplexes in bulk systems (17-21) where only one polysaccharide chaininteracts with one or a smal number of protein molecules. In contrast to thesestudies, theoretica investigation of the structureof mixed polysaccharide-proteinlayers has received relatively less attention in the literature (23). Within theinterfacia region, the concentration of the biopolymers can be far higher thanthat in bulk, even for very dilute systems (3,24). Given the additionaconfigurationa restrictions that the presence of a surface imposes on themacromolecules, a polyelectrolyte adsorbed at the interface wi l l interact withmany more protein molecules. Additionally therewi l l be strong interactions, atvery least through the excluded volume and the electrostatic repulsion forces,with the neighbouring polyelectrolyte chans. Thus, under the influence of theseforces, even complexes formed in the bulk in the first instance, may have a verydifferent structure once adsorbed on the surface. However, one simplifyingconsequence of the high concentration of macromolecules at the interface isthatthe surface becomes increasing more homogenous. This is particularly the casefor more flexible or highly denatured proteins, or thosewith disordered coil-likestructures. An archetypa example of the latter is the heterogeneous milkprotein casein. In particular, al-casein and -casein are known to have notertiary and very little secondary structure (25).

The current study focuses on the formation of mixed interfacia layers offlexible disordered protein and polyelectrolyte using the method of SelfConsistent Field (SCF) caculations (26,27). L ike Monte Carlo simulations, themethod only deas with the equilibrium properties of the interfacia film.However, being a numerica calculation, it has theadvantagethat thepresenceofa large number of chans can be accounted for. We begin by first giving a briefoutline of the SCF methodology in the next section. We then consider a simplemodel consisting of mixtures of amphiphilic and hydrophilic chain species.


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49Initialy we only introduce short range interactions between the chans. Even atthis level of approximation certain interesting features of the mixed interfaciafilm become evident. We then extend our caculations to surface layers formedby protein-like and polysaccharide-like chans in which the electrostaticinteraction play the dominant role. Our model for the protein is roughly basedon the primary structure of otsl-casein. We investigate the influence of pH , satconcentration and distribution of charge groups of the polyelectrolyte on thedegreeof adsorption of thesechans and its consequence for the structure of theresulting interfacia layers.

Another possible route for achieving protein-polysaccharide mixed films isto have covaently bonded complexes of these molecules adsorb at the surface(28,29). In the present work we shall not study such systems, deferring thediscussion to future publications.

Self ConsistentField Calculationsand theMethodologyThe theoretica basis of the SCF theory and the detals of its implementation

have been extensively described in the literature (26, 27, 30). Therefore, in thissection we shall confine ourselves to presenting an outline of the key aspectsofthe method relevant to the present study. In SCF theories all molecular species,including biopolymers, sat ions and solvents are considered to be made frominterconnection of a set of equa sized segments. While the sat ions and thesolvent molecules only consist of one segment, the biopolymer chans have anappropriately large number of connected units, reflecting their polymeric nature.The monomeric segments on a chain may all be identica, representing ahomopolymer, or they may be chosen to have different properties, as would bethe case for protein chans consisting of many different types of amino acids.The centra quantities of interest to be obtaned from SCF caculations are thespatia variation of a set of density profiles between two approaching interfaces.These density profiles, (), are determined as functions of perpendiculardistance, z, away from one of the interfaces, for each type of segment a,belonging to each kind of biopolymer species / that is present in the solution.The interfaces are taken to be sharply defined, flat surfaces. Units interact withthe solvent molecules, the surface and other neighbouring segments throughshort range interactions. The strength of such interactions between any twodifferent segment types and , as well as those with the surface and thesolvent, are specified by a set of Flory-Huggins parameters As usua, anegative vaue of the Flory-Huggins parameter between two segment typesindicates an affinity between the two, while positive vaues represent anunfavourable interaction. Similarly, we have a good solvent for any given kindof segment, if the solvent-segment parameter between the two is less than 0.5,


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50and a poor solvent i f is larger than this vaue (31). Each segment, dependingon its type and its location, experiences a potentia that in parts is due to theabove short range interactions with the neighbouring units. However, there areaso two further terms contributing to this potentia. The first of these is a hardcore potentia, ( ) , thatenforces the incompressibilitycondition

In the above equation the quantity ? refers to the bulk concentration ofsegmentsof kind a, belonging to species of type /. The hard core potentia termensures that the tota concentration of all segments, including ions and solventmolecules, addsup to the samevaue everywhere in the system. It can be shown(26,27), that at any given location, this hard component is the same for allsegments, irrespective of their type. The second additiona contribution to theoveral potentia only arises for those monomeric groups that are charged. Inthat case a charged group aso interacts with ions and other charged segmentsthrough the longer rangeelectrostatic forces. This can easily be accounted for ifthe local electric potentia,(), at the location of the charged unit is known.The overall potentia resulting from addition of all the three differentcomponents mentioned above, for a segment of type a with a net electric chargeof qa, placed at location z, is given by the following expression:

{)={2) +% ()-) + * ) (2)A n important assumption in SCF theory is that chans can adopt all

configurations available to them. Therefore, the properties related to any givenbiopolymer species, such as its segment density profile, can be obtained byappropriate statistica mechanics averaging of the quantities of interest over allpossible configurations of the chans. This requires the calculation of theBoltzmann factor associated with each configuration, which in turn can only bedone if the segment potentias are known. Since thesepotentias are related tothe concentration profiles of the segments in the first place, we have a situationwhere in order to caculate the desired density profiles one requires a priorknowledge of such profiles. The problem is resolved through an iterativescheme. One begins by choosing a rough initial guess of the concentrationprofile for each segment type. With the distribution of all groups, including thecharged ones now specified, the segment potentias can be caculated fromequation 2. For the electric potentia term this is made marginaly more

(1)a a

i


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51complicated by the fact that one first has to solve the Poisson equation- 2 , / =(*)/, where p(z) is the charge density at point and is the dielectricconstant of the solution. Using the segment potentias, a new set of densityprofiles can be caculated by averaging the segment distribution functions overall configurations available to each molecular species present in the solution.Despite the huge number of interna configurations available to polymericchains, this procedure can be carried out very efficiently using the numericascheme originaly proposed by Scheutjens and Fleer (26,27). The newlyobtained density profiles are next used to recaculate the segment potentias andthe whole process is repeated until convergence is obtaned.

The resulting self-consistent concentration profiles can aso be used toobtain the free energy of the system. It can be shown that the set of densityprofiles computed through the SCF procedure are precisely those thatminimisethe free energy of the system (32). In particular, by considering changes in thefree energy of the system, as the separation distance between two surfaces isatered, the colloidal interaction forces mediated by the biopolymers betweentwo particles can be computed.

It must be emphasised that, while the assumption regarding the chansadopting all configurations available to them is broadly true of disorderedproteins (25,33% that is not true of globular proteins. These, in their nativestates, only sample a very small number of states available to them. For thisreason, the current work wi l l primarily focus on coil-like proteins. Applicationof self consistent field theories to adsorbed films of the disorderedmilk protein-casein (25,30,33), has aready been shown to produce results which are ingood agreement with the neutron reflectivity experiments on the same protein.Such studies have aso been successful in providing a clear explanation for theobserved differences in sat-dependent emulsion stability behaviour of dropletsstabilised by as r and -casein (33).

Resultsand DiscussionSimpleModel InvolvingShort RangeInteractions

Before we consider the issue of electrostatic interactions and other featuresarising from the primary structure of protein chains, it is instructive first to studya very simple model. This involves considering a mixture of amphiphilic andhydrophilic polymers. For the amphiphilic molecules we assume a diblockstructure, consisting of 100 hydrophobic and 100 hydrophilic segments. Weshal refer to these as monomer type A and B, respectively. The hydrophilicpolymer wi l l initially be taken as a homopolymer made from = 1500


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52monomers of type C. The large vaue of is to reflect the fact thatpolysaccharide chans are usualy considerably larger than their proteincounterparts. The interaction parameter for the solvent-hydrophobicmonomers is set at 1 (in units ofkT). These groups aso have an affinity for thesurface with an adsorption energy of -1 kT per monomer. Hydrophilicsegments, belonging to both species, have no tendency to adsorb at the interfaceand the solvent is assumed to be atherma ( =0) for these. Thus, only in thepresence of a favourable interaction between the hydrophilic and amphiphilicchans, is the former expected to adsorb at ahydrophilic-hydrophobic interface.

It is known that homopolymers wi l l adsorb at a flat well defined solidinterface only if the magnitude of the adsorption energy per monomer exceeds acertan critical vaue (26). This critical adsorption energy is required tocompensate for the configurationa entropy loss suffered by the chans residingin the interfacia region. It is useful to consider whether the same is aso trueofadsorption of one species of polymer on top of a layer of another polymer. Wehave used our SCF caculations to predict the variation in the excess amount ofhydrophilic polymers at the interface. The number of amphiphilic chans on thesurface is kept constant at 0.001 chans per unit monomer area, a02 . This isensured by having the end monomer, on the hydrophobic side for each chain,tethered to the surface. Unless stated otherwise weshal take a0 as being 0.3 nmthroughout this work. The bulk volume fraction of the hydrophilic chans is setat 0.01 %.The results presented in Figure 1 show the excess amount, #x, ofhomopolymer at the surface, plotted as a function of the strength of the shortrange interactions between these chans and the segments of the diblockpolymer. Note that the excess amount is caculated by integrating the volumefraction of the homopolymer, up and beyond its bulk vaue, over the entireinterfacia region. For large polymers, this interfacia region can extend somedistance away from the solid surface. It is seen thatbelow the critical interactionstrength of around15kT,the hydrophobic chans are not adsorbed at the surface.Althoughnot very clearly seen on the scae used in the graph, the vaue of 6x isin fact negative for \\


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53

0 1 J Ixl (kBT)Figure 1. Theexcessamountof hydrophilicpolymers in the interfacial region, , plotted as afunction of thestrengthof interaction, , betweentheseand

the segmentsof the amphiphilic chains.

0.03

0.02

0.01

A mphi p hi l i c polymer

V , Hydrophil ic polymer

10 20 30

Ma0)40 50

Figure 2. Variation of thedensityprofile (p(r)of the hydrophilic (--) andamphiphilic (-) chainsas afunction of distances, awayfroma solid surface.


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54are plotted aganst the distance into the bulk, away from the solid interface.There is a large concentration of the amphiphilic diblock species, nearly allconsisting of the hydrophobic A segments, in direct contact with the wall. Asexpected, the hydrophilic part of the molecules protrudes further into the bulk.What is more interesting is the presence of the homopolymer, drawn to theinterfacia region, through the favourable interactions with the diblock chains.However, it is aso quite evident that the homopolymer molecules do not form aclear distinct secondary layer of their own. Instead, they lie relatively flat at theinterface, with a large degree of overlapwith the hydrophilic segments of theamphiphilicpolymer.

Theabove situation is drastically atered i fonly certain segments or partsofthe homopolymer were allowed to interact with the diblock molecules. In Figure3 we have presented the results of our caculations for such a case. Now only500 out of the tota 1500 monomers of the hydrophilic chans have a favourableinteraction with the segments of the diblock chains, but with aslightly higherstrength of -5kT. For the rest, this interaction is switched off by setting theappropriate vaues to zero. A l l other parameters are kept exactly the same asthose in Figure 2. The graph of the density profile for the hydrophilic chains inFigure 3 now shows the emergence of a distinct secondary layer. Thispredominately consists of the non-interacting groups of the hydrophilicpolymer.The layer is aso seen to extend well into the aqueous phase, far beyond theinitial primary layer. As we shal see in the next section, the differencesexhibited between these two simple models, as presented in Figures 2 and 3,reman true even when the short range interactions are replaced by long rangeelectrostatic ones.

Mixed Layers of asl-Caseinand PolyelectrolyteIn this section weshal consider a more reaistic model for our amphiphilic

molecules based on the known primary structure of themilk protein arcasein.We aso account for the presence of individua sat ions, as well as the chargegroups on both the polyelectrolyte and our as\-casein-like chains. These chargegroups interact with each other via the usua electrostatic forces. Previous SCFcaculations (25,33)have shown that many of the features of the adsorbed layersof aj or -casein can be reproduced using a model inwhich the various type ofamino acid residues making up the proteins can bedivided into a relativelysmalnumber of groups. In the caculations presented here we shal use six types ofdistinct residue units. The first two of these are the non-charged hydrophobicand polar segments. Histidineand phosphoserine are given there ownseparategroups, but otherwise all the other charge-carrying residues are grouped togetherinto either the negative or the positively charged groups. The charge on these


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0.03A mphi p hi l i c polymer

55

0.02

r(ao)Figure 3.Sameresults asthose in Figure 2, but nowwthonly500 out of a

total of1500 segmentsof the hydrophilic chains having an attractive interactionwththe amphphilic polymer molecules.

groups can change according to the vaue of the pKa and the bulk pH. Theprimary structure of


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56In what follows we shal present the results of our self consistent field

caculation for systems involving a polyelectrolyte, at a bulk volume fraction of0.01%, adsorbing from the solution onto an aready existing interfacia proteinfilm. The surface coverage for the protein film is set to 0.001 chans permonomer square area(al). With a molecular weight of around 24 kDa for thea rcasein, and assuming a0 = 0.3 nm, this translates to a protein surfacecoverage of 0.44 mg m~. Once again, we ensure that this degree of coverageremans constant in the caculations by having the C-termina end of the proteinchans grafted onto the surface.

Table I. TheFlory-Hugginsinteraction parameters, (ArB7), betweendifferent monomer typesand pKa valuesfor charged aminoacidsused inthemode.

Type 0 1 2 3 4 5 6 7 8 90 - Solvent 0 1 0 0 0 0 0 0 -1 -11 - Hydrophobic 1 0 2.0 2.5 2.5 2.5 2.5 2.5 2.5 2.52 - Polar 0 2.0 0 0 0 0 0 0 0 03 - Positive residues 0 2.5 0 0 0 0 0 0 0 04 - Histidine 0 2.5 0 0 0 0 0 0 0 05- Negative residues 0 2.5 0 0 0 0 0 0 0 06 - Phosphoserine 0 2.5 0 0 0 0 0 0 0 07 - Polyelectrolyte 0 2.5 0 0 0 0 0 0 0 08 - Positive ions -1 2.5 0 0 0 0 0 0 0 09 - Negative ions -1 2.5 0 0 0 0 0 0 0 0S - Surface 0 -2 0 0 0 0 0 0 0 0P al - - - 10 6.75 4.5 3 - - -pATa2 - - - - - - 7 - - -

In Figures 4a and 4b we present the caculated density profiles of bothbiopolymers, for two different background sat volume fractions of 0.001 and0.01, respectively. The pH of the solutions in both cases was chosen to be 4,thusmaking the net charge of the protein molecules positive. It is seen thatthereis some adsorption of the negatively charged polyelectrolyte onto the proteinlayer in both systems, though this is marginaly less when the sat concentrationis higher. In a similar manner to the homopolymer molecules studied in thesimple model of the previous section, we find agan that there are no distinct


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57

0.003 C a se in (a)

0.002 H

o.ooH

15005 10 15 20

d i s t a n c e f r o m t h e s u r f a c e , r(a0)

0.003 -

0.002

0 . 0 0 1

(b)C a se in

5 10 15d i s t a n c e f r o m t h e s u r f a c e , r (a0)

Figure4. The densityprofilesofthe oc y- casei- like proteinand thepolyelectrolyte, adsorbedat the interface, at two differentbackgroundsaltvolumefractionsofa) 0.001 andb) 0.01. Thepolyelectrolyte(PE) consistof1500negativelychargedsegments.


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58secondary layers formed. Instead, the protein and the polysaccharide chainsstrongly overlap, forming what is best described as a mixed single layer. It isalso interesting to notethat the total amount of polyelectrolyte at the interface isactually rather small. We believe this is due to the very high charge on thepolyelectrolyte chains used inthesecalculations. The picture that emerges thenisone in which a relatively few, highly charged, polyelectrolyte chains lie flat atthe surface, entangling with the protein layer. In doing so they quickly reversethe charge of the layer, to anextentthat any further adsorption of polyelectrolytechains is inhibited.

Effect of Varying the Chargeof the PolyelectrolyteSome recent experimental studies indicate that the effective charge of

polysaccharides such as pectin, can be as low as -14e, far smaller than theexpected value based on theoretical considerations (13). Reducing the netcharge of the polyelectrolyte decreases the strength of the interactions betweenthesechains and the oppositely charged protein layer. On the other hand, ittakesa larger number of adsorbed chains to reversethe charge of the primary layer. Itis therefore interesting to see how these two potentially competing effects alterthe degree of adsorption of the polyelectrolyte chains to the interface. Toaccomplish this, we change our model such that only a fraction of segmentsbelonging to the polyelectrolyte molecules is now charged. We alsoassumethatthesesegmentsare distributed at one end of the chain, giving the polyelectrolytea more heterogeneousstructure. For the units retaining their charge, this is set at-2e per segmentas before.

Figure5 shows the calculated density profile variationof the polyelectrolytein the interfacial region, obtained for a number of systems involving varyingdegrees of charging of the chains. This calculation is done at a salt volumefraction of 0.001 and at pH =4. The labels on the graphs give the number ofcharged segments, followed by the number of uncharged units, in each case. Thedensity profile for the asrcasein-like molecules is also included. This showsvery little variation from one system to another and is therefore only presentedfor one of the studied cases. It is very clear that, as the number of chargedsegments is reduced, the amount of adsorbed polyelectrolyte at the interfaceincreases. Furthermore, with only part of the polyelectrolyte (as opposed to thewhole chain) interacting with the protein, a secondary polyelectrolyte layer,distinct from the primary protein layer, now begins to emerge.

O f course, the increase in the amount of adsorbed polyelectrolyte withreduction in the overall charge of the chains cannot continue indefinitely. FromFigure 1, we have already seen that there has to be a sufficient degree ofattraction between the two biopolymers before any adsorption of the hydrophilic


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590.006

0.005

0.004

0.003

0.002

0.001

00 20 40 60 80 100

d i s t a n c e f r om the s u r f a c e , r(a0)Figure5. AsFigure4, but now involving polyelectrolytes(PE) carryingdifferentnumbersofchargesegments. Thelabel oneachcurve indicatesthenumber ofchargedsegments,followedbyunchargedunits,for each

polyeletrolyte typestudied.chains can take place at all. For electrostatic interactions, when takentogetherwith the above results, this implies that therehas to be an optimum level ofcharge on the polyelectrolyte. The maximum degree of adsorption ofpolyelectrolytechains onto the primary protein layerwouldbe expected to occurat this level ofcharging. This is precisely what has been foundhere. InFigure6 we have plotted the amount of excess polyelectrolyte at the interface againstthe number of charge groups on the chains at pH = 4. For both saltconcentrations considered here, there is a peak in the amount of adsorbedpolyelectrolyte. The peak occurs at a higher level ofchargingas the backgroundelectrolyte concentration is increased.

Colloidal InteractionsMediatedby Mixed Adsorbed FilmsThe structure ofa mxed layer of two biopolymers, adsorbed on the surface

of two particles, strongly influences thenatureof colloidal interactions betweenthe particles. We have calculated such interactions between two flat surfaces,usingSCFtheory, for each of thesystems considered inFigure5 and alsothatof

Case in


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600.5

Figure 6. Variation of the adsorbed amountofpolyelectrolyte, 9x, wththenumberof chargedsegmentsoneachchain, in twodifferentsolutionswth

electrolyte volumefractions of () 0.001{) 0.0L

Figure 4a. The results are summarised in graphs displayed in Figure 7. Theseshow the interaction potentia per unit area (in units of kT/a02) plotted as afunction of distance between the two surfaces. The interaction mediated by thepure arcasein-like protein layers, in the absenceof any polyelectrolyte, is asoincluded for comparison. Both the steric and the electrostatic contributionsarising from the mixed layers are taken into account. However, the van derWalls interactions are not included, since theseare not significantly affected bythe nature of adsorbed biopolymer films. Under the sat concentration and pHconditions studied here, pure a rcasein layers lead to a repulsive interaction, atall separation distances between the plates. The addition of a highly chargedpolyelectrolyte, where all segments of the chans are charged, is seen todrasticaly reduce this repulsion. Indeed, at certan separations, the forcesbecome marginaly attractive. The problem of ensuring the stability of theemulsions during the process of layer-by-layer deposition is arather well knownone, and is attributed to possible bridging of the droplets by the polyelectrolytechans (13). When all segmentsof the polyelectrolyte are charged, such bridgingis more likely. For now, it is more feasible for the polyelectrolyte chans tointeract simultaneouslywith two protein layers on neighbouring surfaces.

Reducing the number of charged segmentsof the polyelectrolyte from 1500down to 300, and confiningthese to one end of the chains, results in a purelyrepulsive force between the surfaces. However, the net repulsion isstill less than


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61

separation between the surfaces, d (a)Figure 7. Interaction potential, v(d), betweentwo surfaces, mediated by themxedprotein-polyelectrolyte layers,for anumber ofpolyelectrolytes of the

samesizebutwthdifferentdegreeof charge, () 1500; () 300; () dashedline 100; and ( ) 20 chargedsegments. The solid lineshowsthe potentialresultingfromthe pureprotein layer, in theabsenceof the polyelectrolyte.

that mediated by the pure protein layers, even though it comes into operation atfurther surface separations. Only when the number of charged segments isdecreased to 100, is a stronger repulsive interaction, operating over longerseparation distances, predicted for the mixed layers. This can be understood interms of the structure of themixed layers at the interface, as presented in Figure5. These more lightly charged polymers formmore extended secondary layers atthe interface. The secondary layers consist mainly of the uncharged segmentsthat have no affinity for the protein layers. The stronger and longer rangerepulsion results fromthe steric effects, as two suchsecondary layersoverlap.

Influenceof pH and Background ElectrolyteTheaddition of sat to a solution has the effect of screening the electrostatic

interactions between the charged groups. Our caculations indicate that, forpolyelectrolyte chains of any charge, the level of adsorption onto the primaryprotein layer is reduced as the electrolyte volume fraction is increased from0.001 to 0.01. However, as the graphs in Figure 6 show, the decrease in theamount of excess polyelectrolyte at the interface is far more pronounced for the


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621.2

1

0.8

5 0.6 0.4

0.2 -

0 -2

-0.2-

Figure 8. Theamountof polyelectrolyte, adsorbed ontoaprimary protein layer,wththe variation in the pH of the solution. The label oneach curve indicatesthenumber of chargedsegments, followed by unchargedsegments, for eachof

the two polyelectrolyte typesstudied.

lightly charged polymers. Despite their higher level of adsorption at low satconcentrations, each individua chain of a lightly charged polyelectrolyte has aweaker binding to the oppositely charged protein layer. As the backgroundelectrolyte is increased, the screening of electrostatic interactions is expected tohave a stronger effect on the adsorption of these low charged chains. This asoexplains the shift in the maximum vaue of the adsorbed polyelectrolyte, withincreasingsat concentration, towards higher charged chans (see Figure 6).

The greater sensitivity of the adsorption of the lightly chargedpolyelectrolytes, to the weakeningof the electrostatic interactionswith protein, isaso reflected in the manner inwhich the absorbed amount varieswith changes inthe pH of the solution. In Figure 8, we have plotted the excess amount ofpolyelectrolyte at the interface, as a function of pH, for two differentpolyelectrolytes. In one case, out of a tota of 1500 units, the chans carry 300charged segments. In the other system, there are only 20 charged units perchain, giving each molecule a tota charge of -40e. The coverage of the surfaceby the primary protein layer is fixed at 0.001 chans per unit monomer area. Thethe bulk volume fraction of polyelectrolyte and that of the backgroundelectrolyte are 0.01 and 0.001, respectively. As the pH of the solution isreduced, the net charge of the primary protein layerchanges frombeing negativeto positive, increasingly further with the lowering of pH . At pH vaues below


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63the pi value (~ 4.5) of our ctsrcasein-like protein, the amount of adsorbedpolyelectrolyte increases as the pH of the solution is decreased. However, thechange effect is more pronounced for the lighter charged polyelectrolyte, withonly 20 charged segments. At the higher salt volume fraction of 0.01, we foundthat the excess amount of polyelectrolyte at interface is more comparable for thetwo polyelectrolytes. Indeed, for this value of background electrolyteconcentration, the amount of polyelectrolyteat interface only becomes larger forthe lighter charged chain, when pH drops below 3.5.

One noticeable aspect of observed experimental results on such mixedsystems is the possibility of bindingof polyelectrolyte to the protein layers at pHvalues above the iso-electric point for the protein (4,7). In thesecircumstances,the overall charge on the protein has the samesign as the polyelectrolyte. Thisfeature is not reproduced by the SCF calculations here. This might be due tolack of lateral heterogeneity in our model. While such heterogeneitiesundoubtedly exist at the lower coverage of the surface by the protein molecules,they should tend to average out at higher surface concentrations. Thiswould beexpected to be particularly thecasefor more coil-like disordered proteins, as wasargued in the Introduction. There may also exist other kind of short rangeattraction between protein and polysaccharide molecules, neglected in thepresent model. These need to be at least as strong as the electrostaticinteractions i f they are to make any significant difference to the adsorption of thepolyelectrolyte at thesehigher pH values.

Summaryand ConclusionsThe adsorption behaviour of polysaccharides, onto an existing layer of

protein at an interface, is not only determined by protein-polysaccharideinteractions but also by interactions between neighbouring polysaccharidechains. We have used Self Consistent Field calculations to study the structureand properties of mixed films of these biopolymers. In the model systemconsidered, it is only the hydrophobic amino acid residues of the protein thathave an affinity for adsorption onto a hydrophobic solid surface. Theaccumulation of the polyelectrolyte at the interface occurs as a result ofelectrostatic interactions between the positive segments of the protein and thenegatively charged polyelectrolyte chains. For the primary structure of ourmodel protein, we have adopted asequenceof amino acids loosely based on thestructure of the disorderedmilk protein ai-casein.

Our results for the density profile of each biopolymer at the interface showthat the adsorption of polysaccharide molecules onto the primary protein film,where all segments of the polyelectrolyte are equally charged, leads to a rathermixed, entangled layer. We find that for this system the polyelectrolyte chainslie rather flat at the interface. Thus, emulsion layers stabilised by thesetypes of


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64mixed layers are prone to bridging flocculation. Our calculations predict aweakening of colloidal repulsion forces, and even attraction, when compared tothoseproduced by pure protein layers. In contrast, for polyelectrolyte moleculeswith only certain sections of the chains charged, the adsorption process results ina significantly more extended interfacial film. In this case, a distinct secondarylayer is seen to be formed. This predominately consists of hydrophilicuncharged units of the polyelectrolyte. The steric interactions, resulting from theoverlap of thesesecondary layers, greatly enhance the repulsion forces betweenapproaching emulsion droplets stabilised by such interfacial layers. Therepulsion is also seen to become longer range, when compared to that mediatedby protein layers in the absence of polyelectrolyte. The above results are alsofound to be true of a much simpler model, where the attractive interactionsbetween the two biopolymer species is assumed to be short range.

We have found that the extent of adsorption of the polyelectrolyte chainsonto the protein layer varies greatly with the degree of charging of thepolyelectrolyte. Highly charged chains have a stronger affinity for theoppositely charged protein film. However, this also leads to a more effectiveneutralisation and then reversal of the charge of the interfacial layer as thepolyelectrolytemolecules adsorb to the surface. Furthermore, stronger repulsionbetween neighbouring chains limits the amount of adsorption of thepolyelectrolyte. We have shown that these two competing effects result in anoptimum level of charging of the chains for which the adsorption level is amaximum. This optimumdegreeof charging varies with salt concentration andpH of the solution. In particular, for low electrolyte concentrations and low pHvalues, the adsorption favours lightly charged polyelectrolyte chains.Nevertheless, from a practical point of view, a polyelectrolyte with a higherdensity of charge segments might be preferable. We find that the amount ofadsorption of more highly charged chains show less sensitivity to changes in pHand background salt concentration.

Polyelectrolyte chains in our model are assumed to be flexible. However,many polysaccharide molecules have a rather rigid rod-like backbone. Rigidityof the molecules is known to have important implications for the formation ofcomplexes between protein and polysaccharides in bulk solutions (4,18). Thesame is expected to be trueat the interface. Current work is underway to studyadsorption of more rigid polyelectrolyte molecules within the framework of ourpresentmodel.

References1. Dickinson, E.Food Hydrocolloids 2003, 17, 25.2. McClements, D. J . Curr. Opin. Colloid InterfaceSci. 2004, 9, 305.3. Walstra, P.Physical Chemsitry of Foods; Marcel Dekker: NewY ork, 2003.


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a theoretical self-consistent field study of mixed interfacial biopolymer films

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