Effect of protein aggregates on the complex coacervation between [beta]-lactoglobulin and acacia gum...

Effect of protein aggregates on the complex coacervationbetweenb-lactoglobulin and acacia gum at pH 4.2

C. Schmitta,* , C. Sancheza, S. Desponda, D. Renardb, F. Thomasc, J. Hardya

aLaboratoire de Physico-Chimie et Ge´nie Alimentaires, INPL-ENSAIA, 2 Avenue de la Foreˆt de Haye, BP 172, F-54505 Vandoeuvre le`s Nancy Cedex, FrancebLaboratoire de Physico-Chimie des Macromole´cules, INRA, Rue de la Ge´raudiere, BP 71627, F-44316 Nantes Cedex 3, France

cLaboratoire Environnement et Mine´ralurgie, UMR 7569, CNRS-INPL, 15 Avenue du Charmois, BP 40, F-54501 Vandoeuvre le`s Nancy Cedex, France

Received 11 October 1999; accepted 10 March 2000

Abstract

The influence of protein aggregates on complex coacervation betweenb-lactoglobulin (b-lg) and acacia gum (AG) has been studied at pH4.2 using phase diagrams, electrophoretic mobility measurements, laser light scattering and phase contrast optical microscopy. Removal ofprotein aggregates by centrifugation (pH 4.75, 1 h, 10,000g) led to an aggregate-freeb-lg dispersion (AF-BLG) exhibiting a narrowpolydispersity in molecular masses�Mw , 18;500 and 64,500) and hydrodynamic radii�Rh , 2:51 nm� as determined by GPC andQELS measurements, respectively. Phase diagram determination for the AF-BLG system revealed a two-phase region located in thewater-reach corner. In addition, the highest total biopolymer concentration leading to phase separation (4.5 wt%) and the tie-lines shapetypically accounted for complex coacervation. The position and extend of the two-phase region differed from that obtained using BLGdispersions (containing protein aggregates). This was partly due to the lowestMw andRh polydispersity of the AF-BLG, but also to thedecrease of the electrophoretic mobility (mE) of the AF-BLG induced by the protein aggregates elimination (loss of cations in the dispersion).The variation of themE of the AF-BLG/AG complexes with pH was similar to that of the BLG/AG ones, but of lower intensity due to thedecrease of cations in the system. The size distribution of the BLG/AG complexes obtained at pH 4.2 was mainly governed by the presence oflarge-sized aggregates (10–50mm). By contrast, AF-BLG/AG coacervates exhibited a volume average diameter (d43) that reached 220mmfor the 2:1 Pr:Ps ratio at pH 4.2, revealing less stability of particles in the system. These results were in agreement with phase contrast opticalmicroscopy that revealed marked differences in the structure of BLG/AG complexes and microparticles as compared to that of AF-BLG/AGcoacervates.q 2000 Elsevier Science Ltd. All rights reserved.

Keywords: Polyelectrolyte complexes; Coacervate; Phase separation; Aggregation; Microparticles; Phase diagrams; Light scattering; Microscopy

1. Introduction

The control of interactions between the differentingredients involved in modern formulated foods is akey problem regarding their structure and texture(Samant, Singhal, Kulkarni & Rege, 1993). Two parti-cularly important food ingredients are proteins andpolysaccharides, widely used for their high nutritionaland functional properties (Dickinson, 1998; Sanchez &Paquin, 1997). For example, milk, fish, soy or eggproteins exhibit useful interfacial and thermal gelationproperties needed for building structure and stabilise foods(Tuley, 1999). Polysaccharides are rather used for theirthickening and water-binding properties, but sometimesfor their surface properties as for example acacia gum

(McNamee, O’Riordan & O’Sullivan, 1998). When thesetwo macromolecules are mixed together in water, phaseseparation generally occurs, leading either to the segrega-tion of each macromolecule in separated phases or theconcentration of both macromolecules in one phase withthe appearance of an equilibrium dilute phase (Albertsson,1995). This biopolymeric concentration, the so-calledcomplex coacervation, is generally driven by attractiveinteractions between the macromolecules that result in theprimary formation of electrostatic protein–polysaccharidesoluble complexes. The rearrangement of such complexesleads to the formation of soluble and insoluble liquiddroplets, also called coacervates, that ultimately coalesceand sediment forming the coacervated phase. The mostinteresting feature revealed by the formation of complexesand various kinds of particles is that their functional proper-ties are potentially better than those of the protein and thepolysaccharide alone. Even if, much work remains to bedone in this field, these observations allow us to predict

Food Hydrocolloids 14 (2000) 403–413

0268-005X/00/$ - see front matterq 2000 Elsevier Science Ltd. All rights reserved.PII: S0268-005X(00)00022-9

www.elsevier.com/locate/foodhyd

* Corresponding author. Tel.:1 33-3-83-59-58-61; fax:1 33-3-83-59-58-04.

E-mail address:[email protected] (C. Schmitt).

their wide use as new functional food ingredients (Schmitt,Sanchez, Desobry-Banon & Hardy, 1998).

Nevertheless, it should be emphasised that few informa-tion is actually available on either the mechanism of forma-tion (kinetic, influence of processes such as thermal, shearor high-pressure treatments) or the structure of the protein–polysaccharide complexes and coacervates. This lack ofknowledge could be damageable in the prediction of theirtechnofunctional properties since it is well known that thestructure and the function of the macromolecular complexesare tightly related (Nakai & Li-Chan, 1993). Consequently,a global characterisation of the formation, the stability, thestructure and the technofunctional properties of such a kindof biopolymer mixtures, is required.

In a recent study, we reported the phase behaviour of aternary system consisting in a whey protein, theb-lactoglo-bulin (b-lg), a plant polysaccharide, acacia gum (AG) andwater (Schmitt, Sanchez, Thomas & Hardy, 1999). Themain results lightened the formation of electrostaticcomplexes between the two biopolymers, leading to phaseseparation of the system through complex coacervation(Bungenberg de Jong, 1936). The extent of phase separationdepended on the pH (3.6–5.0), the protein to polysaccharideweight ratio (Pr:Ps, 50:1 to 1:20) and to a lesser extent onthe total biopolymer concentration (0.1–5 wt%). Thus,formation of coacervates has been suggested as it is gener-ally the case in systems containing oppositely charged poly-electrolytes (Menger & Sykes, 1998). However, parts of theobtained results led us to the conclusion that a morecomplex mechanism could be involved in the system. Forexample, (i) the high total biopolymer concentration of20 wt% that still led to phase separation and disagreed thecritical limiting value of,4 wt% proposed by Tolstoguzov(1986), (ii) the electrophoretic mobility (mE) measurementsperformed on the complexes at pH close to the IEP of theb-lg (5.2), exhibitingmE values close to those of the AG alone,or (iii) the limited effect of the protein to polysaccharideratio (Pr:Ps) at pH 4.2 and 5.0 claimed for a striking influ-ence ofb-lg aggregates on the phase behaviour of blends.Indeed, it is well known thatb-lg molecule forms aggre-gates near its IEP, where repulsive interactions betweenprotein molecules are of very low intensity (Townend,Winterbottom & Timasheff, 1960).

The evaluation of the influence ofb-lg aggregates on theb-lg/AG complex coacervation at pH 4.2 is the purpose ofthe present study. Thus, before presenting the experimentalpart of this work, it appears necessary to define properly theterms referring to theb-lg dispersions. In this paper, theBLG term will be used forb-lg dispersions containingprotein aggregates (arising fromb-lg denaturation duringits purification treatment). At pH 4.2, such a kind of BLGdispersion mainly contained aggregates exhibiting sizesranging from around 200 nm to 8–10mm and molecularmasses from 18,300 to 150,000 (Schmitt et al., 1999). Incontrast, the AF-BLG term will be used forb-lg dispersionsafter elimination of the protein aggregates, i.e. dispersions

containing mainly monomers and dimers ofb-lg. Thefollowing experimental section encloses the building ofternary phase diagrams and the comparison between theBLG/AG and AF-BLG/AG systems by means of laserlight scattering, electrophoretic mobility measurementsand phase contrast optical microscopy.

2. Experimental

2.1. Material

Acid-processed bovineb-lg powder (lot no. 838) waskindly provided by Lactalis (Retiers, France). The composi-tion of the powder was (g/100 g): 4.75% moisture, 91%protein �N × 6:38�; 3.85% lactose and 0.40% ash (mineralcontent: 0.027% Na1, 0.008% K1, 0.0008% Mg21, 0.012%Ca21). Powdered acacia gum (lot no. 97 J 716) was a giftfrom the CNI Company (Rouen, France). The compositionof the powder was (g/100 g): 6.65% moisture, 89.95% poly-saccharide, 0.30% nitrogen and 3.10% ash (mineral content:0.032% Na1, 0.9% K1, 0.2% Mg21, 0.61% Ca21). All otherreagents were of analytical grade (Labosi, France).

2.2. Preparation of BLG and AG stock dispersions

Dispersions of BLG and AG were prepared in percent byweight (wt%) by dispersing known amounts of biopolymerpowder in distilled water under gentle stirring at 2018Cduring at least 2 h. Dispersions were then left overnight at4^ 18C to allow complete hydration of macromolecules.Stock dispersions were then centrifuged at 3100g for30 min to discard insoluble matter and air bubbles. ThepH of the dispersions was then adjusted (^0.01 pH unit)by careful addition of 0.01, 0.1 and 1 N HCl or NaOH.

2.3. Solubility ofb -lg and elimination ofb -lg aggregates

Solubility of the b-lg in a 1 wt% BLG dispersion wasbased on the determination of the nitrogen soluble index(NSI) that gives an estimation of the solubility of the nitro-gen compounds in milk protein powders (FIL-IDF, 1995).The initial pH of BLG dispersions (,2.96–3.05) wasadjusted in the 3.0–6.5 range. The solubility ofb-lg wasdefined as the ratio between the protein content in the super-natant, determined by the Kjeldhal method�N × 6:38� aftercentrifugation at 3100g during 30 min, to the initial totalprotein content. The experiment was duplicated (3 assaysper experiment). The pH corresponding to the lowest valueof b-lg solubility was chosen to eliminateb-lg aggregates(de Wit & van Kessel, 1996). After pH adjustment, the BLGdispersion was centrifuged during 1 h at 10,000g to discardb-lg aggregates. The pH of the resulting aggregate freeb-lgdispersion (AF-BLG) was adjusted to 4.2.

2.4. Gel permeation chromatography

Weight average molecular masses (Mw) of the AF-BLG

C. Schmitt et al. / Food Hydrocolloids 14 (2000) 403–413404

and AG were determined using gel permeation chromato-graphy (GPC). The analysis system has been alreadydescribed (Schmitt et al., 1999). The column was calibratedwith eight protein standards ranging from 1450 to 480,000(ICN Biomedical Inc., Aurora, OH, USA), the latter beingused to determine the void volume of the column. Thesample volume (100ml, pH 4.2, 0.5 wt%) was injectedinto the column after filtration through 0.2mm filters(Lida Manufacturing Corp., WI, USA). The eluent was a30 mM NaCl solution at a flow rate of 1 ml min21. Thetemperature was kept at 200:58C:

2.5. Quasi-elastic light scattering

Hydrodynamic radii (Rh) of AF-BLG and AG were deter-mined by quasi-elastic light scattering (QELS) at pH 4.2.Dispersions (1 wt%) were prepared in distilled water (forAF-BLG and AG) or 0.1 M NaCl (for AG). The dispersionswere filtered on 0.02 or 0.1mm filters (Anotop, Alltech,France) for AF-BLG and AG, respectively, and put intoglass measuring vials. The light scattering measurementswere performed in duplicate using an ALV-5000 multi-bit, multi-tau correlator in combination with a 4700 Malverngoniometer (Malvern Instruments, Orsay, France) and aSpectra-Physics laser emitting vertically polarised light at514.5 nm. The temperature was kept constant at 25^ 0:18C:The correlation functions,G2(t), were recorded at threeobservation anglesu � 30; 90 and 1508. These functionswere then analysed using the inverse Laplace transformroutine REPES in order to obtain the corresponding relaxa-tion time distribution and the diffusion coefficients of parti-cles (Stepanek, 1993). Using the Stokes–Einstein relation(Eq. (1)), the hydrodynamic radii of macromolecules (Rh,nm) were calculated as:

Rh � kT=6phD �1�whereD is the diffusion coefficient of the particle, k theBoltzman constant,T the absolute temperature andh thesolvent viscosity.

2.6. Phase diagram

The ternary phase diagram of the AF-BLG/AG/Watersystem was obtained by chemical analysis of the two sepa-rated phases obtained after two days. The binodal line of thesystem corresponding to the macroscopic limit between theapparent one-phase and the two-phase region was deter-mined by the cloudy-point method (Reisman, 1970). AnAF-BLG dispersion at pH 4.2 was used to titrate an AGdispersion at both the same pH value and biopolymerconcentration. The turbidity of the mixture was followedat 650 nm with an Ultrospec III UV–visible spectrophot-ometer (Pharmacia LKB, UK). Phase separation wasconsidered to be macroscopically achieved after a 10%increase of the initial turbidity value of the AG dispersion.This experiment was repeated twice and, similarly, an AGdispersion was used to titrate an AF-BLG one. Tie-lines of

the system, corresponding to the compositions of eachcomponent in the two phases at equilibrium, were deter-mined by mixing AF-BLG with AG at a 1:1 weight ratiointo hermetically sealed propylene tubes. The tubes werethen left at 20 18C during 2 days to allow phase separa-tion to occur. The two phases, if any, were then recoveredafter centrifugation at 10,000g during 1 h. Composition inwater in the upper phase was determined by drying samplesto constant weight at 1058C. In the same phase,b-lg and AGcontents were determined using, respectively, the Bradfordcolorimetric method and the phenol–sulfuric method asdescribed previously (Schmitt et al., 1999). The composi-tion of the lower phase was calculated by difference with thetotal initial composition of the blend. The results were thenexpressed in percent and the ternary phase diagram wasdrawn by means of the TContour 4.1 shareware (JohnPilling, Michigan Technology University, USA).

2.7. Electrophoretic mobility

The electrophoretic mobility (mE) of the 0.1 wt% BLG,AF-BLG, b-lg aggregates, AG and AF-BLG/AG disper-sions was determined at 2018C in distilled water. ThepH range from 3.6 to 5.0 was investigated. The velocityof particles was determined by means of image analysisusing a Zetaphoremetre II apparatus (Sephy Technology,France) and converted into electrophoretic mobility accord-ing to the Smoluchowski–Henry equation (Hunter, 1986).Experimental details have been already described (Schmittet al., 1999). Experiments were duplicated (3 assays perexperiment). For reasons of convenience, electrophoreticmobility units (mm cm s21 V21) will be referred as e.m.u.in the following.

2.8. Mineral composition

The effect ofb-lg aggregates discarding on themE of theresulting AF-BLG dispersions was checked by analysis ofthe mineral composition in Ca21, Mg21, K1, Na1 and Cl-

ions before and after centrifugation of the BLG dispersion(10,000g, 1 h). The content in cations was determined induplicate on 0.5, 1 and 5 wt% BLG dispersions using atomicabsorption/emission spectroscopy. The apparatus was aPerkin–Elmer 1100 AAS spectrometer (Perkin–Elmer,France) calibrated with 5 standards solutions containingthe four different cations at rising concentrations (4–12 mg l21). The chloride content was determined by poten-tiometry using a specific chloride electrode (Subra S100,Grosseron, France) calibrated with a 1 g l21 NaCl solution.

2.9. Laser light scattering

The size distributions of BLG/AG and AF-BLG/AGparticles were determined by laser light scattering using aMastersizer S granulometer equipped with a 5 mW He/Nelaser emitting at 632.8 nm (Malvern Instruments, Orsay,France). The optical system was composed by a 300

C. Schmitt et al. / Food Hydrocolloids 14 (2000) 403–413 405

Reverse Fourier lens covering a particle size range from0.05 to 880mm. We selected 8:1, 2:1 and 1:1 Pr:Ps mixingweight ratios at total biopolymer concentrations of 0.1, 0.5,1.0, 3.0 and 5.0 wt% at pH 4.2. The concentrated aliquotswere introduced in the measuring sample unit (120 ml)containing distilled water at pH 4.2 in order to obtain anobscuration value of 15% at a stirring rate of 1000 rpm.Similar experiments were performed on the BLG samplealone. Unfortunately, the AF-BLG dispersions could notbe investigated by this method, obscuration values beingtoo low. All determinations were triplicated (5 assays perexperiment). The size distributions of the particles werecalculated on a volume basis after treatment of the scatteredintensity data with a polydispersed model provided by theMalvern Mastersizer-S v2.17 software. The volume averagediameter defined by,

d43 �

Xni

nid4i

Xni

nid3i

;

whereni is the number of particles exhibiting a diameterdi,was then calculated for each total biopolymer concentrationtested.

2.10. Phase contrast optical microscopy

BLG and AG initial dispersions were microscopicallycharacterised at 500× magnification using a phase contrastoptical microscope (Olympus BH-2, Olympus Optical,Tokyo, Japan) equipped with a SC-35 Olympus cameracontaining 320 Tungsten Kodak Ektachromew films. 20mlof the considered dispersion was put between glass slidesand examined for particle size determination. At least ninemicrographs were taken in three different samples to ensurea valuable reliability with the particle size obtained viagranulometry measurements. Similarly, BLG/AG and AF-BLG/AG mixtures obtained at 8:1, 2:1 and 1:1 Pr:Ps ratios

were characterised just after mixing (10–15 s) and after twodays at 208C (upper and lower phases).

2.11. Statistical analysis

The obtained results were analysed by Anova using theStatView IIe software (Abacus Concepts Inc., Berkeley,CA, USA). Comparison of means was carried out usingthe Fisher PLSD test at a confidence level of 0.05.

3. Results and discussion

3.1. Solubility ofb -lg

Determination of theb-lg solubility is needed to estimatethe extent of denaturation and aggregation of the proteinduring its extraction and purification from whey. Bothphenomena are expected to affect the complex coacervationwith the AG. The solubility curve of the 1 wt% BLG disper-sion exhibited a minimum at pH 4.75, corresponding to 43%protein solubility (Fig. 1). At pH values below 3.6 andabove 5.5,b-lg solubility is $80%. Similar results havebeen reported by de Wit & van Kessel (1996) who foundsolubility values below 50% between pH 4.5 and 5.0 for asalt-free WPC (whey protein concentrate) dispersioncontaining 90%b-lg. The low solubility obtained at pH4.75, which is not far away from its IEP (5.2), can beexplained by favourable protein–protein interactions arisingfrom the lowest charge carried by theb-lg molecules. Suchinteractions led to the formation ofb-lg oligomers (trimers,tetramers) that could interact to form aggregates by non-covalent interactions (Stading & Hermansson, 1990). Thistrend has been recently reported through an increase of theratio between theMw of b-lg oligomers andMw of theb-lgmonomer from 1 to.4 as determined by SANS measure-ments for ab-lg solution at pH 4.7 in 0.1 M NaCl (Verheul,Pedersen, Roefs & de Kruif, 1999). As reported in theexperimental section, the pH 4.75 value at which the lowestsolubility for b-lg occurred was selected to prepare the AF-BLG dispersions. Thus,b-lg aggregates were removed after1 h centrifugation at 10,000g and 258C.

3.2. Molecular masses and hydrodynamic radii of AF-BLGand AG in the initial dispersions

GPC measurements have been performed at pH 4.2 on0.5 wt% AF-BLG and AG dispersions. The elution patterncorresponding to the AF-BLG displayed two major elutionpeaks at 280 nm (Fig. 2). The first peak (relative intensity:,98%) was characterised by a retention time of 8.6 min,corresponding to aMw of 64,500. A minor second peak wasalso detected at a retention time of 10.75 min. It corre-sponded to aMw of 18,500 (relative intensity:,2%).These two peaks corresponded tob-lg trimers/tetramersand monomers, respectively. If we compare these resultswith those obtained at pH 4.2 in the presence ofb-lg


Fig. 1. Solubility curve of a 1 wt% BLG dispersion at 208C. Bars representstandard deviation.

aggregates, the main difference was the disappearance of theelution peaks corresponding to theb-lg dimers, pentamersand octamers (Schmitt et al., 1999). The QELS measure-ments performed on the 1 wt% AF-BLG dispersion revealeda Rh of 2.51 nm at the three scattering observation anglesaccounting for a monomer/dimer equilibrium state for AF-BLG (Fig. 3).b-lg oligomers exhibiting higherRh were notfound, probably due to the small size exclusion limits of thefilters that were used to prepare samples (0.02mm). Rh

values of the same magnitude have been obtained byAymard, Durand & Nicolai (1996) who found 2.14 nm forthe monomer and 3.19 nm for the dimer at pH 2.0. Inanother study,Rh values ranging from 1.73 to 2.81 nmwere found by Renard (1994) forb-lg solution in 0.03 MNaCl using QELS. The increase ofRh was attributed to theassociation behaviour ofb-lg when the pH increased from 2to 7. These results suggested that our AF-BLG samplecontained mainlyb-lg monomers and dimers, emphasisingthe good efficiency of our purification method. It is worthnoting that traces of BLG aggregates�Rh � 65 nm; relativeproportion� 2%) have been detected in one of our QELSassay. These results could be related to the trimer/tetramer

peak detected by GPC, as some of them could be passedthrough the filter. This reflected the difficulty to obtainexactly the same system in separate experiments. However,the trimer/tetramer peak could also be attributed to someb-lg aggregation favoured by the use of a 30 mM NaCl solu-tion as eluent.

The gel permeation profiles obtained for the AG at pH 4.2exhibited only one peak corresponding to aMw of around180,500 (not shown). This result is close to that previouslydetermined and confirmed the high molecular mass mono-dispersity of the AG sample (Schmitt et al., 1999). Dynamiclight scattering measurements performed on AG (0.1 wt%)in distilled water revealed a meanRh of 13 nm for fourobservation angles. This result was in accordance with the10 nm value obtained by Swenson, Kaustinen & Thompson(1968) using light scattering measurements or more recentlywith the 10–21 nm values reported by Idris, Williams &Phillips (1998) for eight different AG samples. In addition,the polyelectrolyte nature of the AG was analysed measur-ing theRh of the biopolymer in the presence of salt (0.1 MNaCl). TheRh value was found to be in the same order thanin water (11.9 nm atu � 90 and 1508, 15.9 nm atu � 308;Fig. 4). This indicates that the polyelectrolyte effect of AGmolecules could not be so pronounced as the chain expan-sion due to intramolecular electrostatic repulsions was notlargely decreased in screening conditions.

The initial characterisation of the biopolymers indicatedthat the monodispersity (Mw and Rh) of our samples wasreasonably good. Consequently, we expect that the effectof b-lg aggregates on theb-lg/AG complex coacervationwill not be hindered by theb-lg sample polydispersity.

3.3. Phase behaviour of the AF-BLG/AG/water ternarysystem

The building of the AF-BLG/AG/Water ternary phasediagram revealed the presence of a two-phase regionenclosed into a one-phase region (Fig. 5). The two-phaseregion was anchored in the solvent rich corner (region II on


Fig. 3. Hydrodynamic radii distribution of AF-BLG in water at pH 4.2 asdetermined by QELS (REPES method analysis). Scattering angles: (—)308; (· ··) 908; (– – –) 1508.

Fig. 4. Hydrodynamic radii distribution of AG in 0.1 M NaCl at pH 4.2 asdetermined by QELS (REPES method analysis). Scattering angles: (—)308; (· ··) 908; (– – –) 1508.

Fig. 2. GPC profile of a 0.5 wt% AF-BLG dispersion at pH 4.2 and 208C.Arrow represents the void volume of the column.

Fig. 5). Moreover, the direction of the tie-lines obtainedafter two days at 208C traduced the formation of asolvent-rich phase (left-handed points of the tie-lines onFig. 5) in equilibrium with a polymer rich phase (right-handed points of the tie-lines on Fig. 5). In addition, thehighest total biopolymer concentration giving rise to phaseseparation was 4.5 wt%. These findings both indicated thatphase separation was achieved through complex coacerva-tion rather than thermodynamic incompatibility between thetwo biopolymers (Tolstoguzov, 1986). The comparison ofthe phase diagram obtained for the ternary system contain-ing AF-BLG with the BLG one (dashed-line in Fig. 5),revealed important discrepancies. Firstly, a decrease of thetwo-phase region area was pointed out after elimination ofb-lg aggregates. Secondly, the position of the two-phaseregion changed. On the one hand, it was located betweenthe water corner bisector and theb-lg axis for the BLG/AG/Water system (dashed-line on Fig. 5). On the other, the two-phase region moved between the same water corner bisectorbut along the water axis for the AF-BLG/AG/Water system(continuous-line in Fig. 5). At low biopolymer concentra-tions (below 2 wt%), it should be noted that the two-phaseregion obtained for both systems exhibited a common area.Differences observed in the phase behaviour of our systemafter discardingb-lg aggregates could be explained byconsidering entropic and enthalpic effects induced by thepresence of aggregates. The main entropic effect related tothe presence ofb-lg aggregates in the system could be dueto polydispersity (in size and molecular mass). Such a para-meter is known to sharply influence the complex coacerva-tion phenomenon. It generally led to the formation of a thirdregion on the phase diagram, the so-called mesostableregion, located between the binodal and the spinodal linesof the system (Albertsson, 1971; Frugier & Audebert, 1994).Considering the effect of the elimination ofb-lg aggregates,

we observed by GPC and QELS measurements a decrease oftheb-lg Mw andRh polydispersity. In this case, the differ-ence of molecular mass and hydrodynamic radius betweenthe two macromolecules decreases, lowering the excludedvolume effect arising from the mixing of the macromole-cules (Patterson, 1982; Tolstoguzov, 1998, 1999). Thus, thehighest total biopolymer concentration leading to self-suppression of coacervation for the AF-BLG system(4.5 wt%) was lower as compared to the BLG system(20 wt%). However, assuming that the size distribution ofb-lg aggregates is the main parameter influencing the phasebehaviour of the system, the two-phase region arising in theAF-BLG/AG/Water system should have been enclosed inthe two-phase region obtained for the BLG/AG/Watersystem, and not at the observed location. Therefore, toexplain these results, we also consider enthalpic effectsinduced by the presence ofb-lg aggregates, especiallytheir surface properties that could be estimated throughmeasurements of the electrophoretic mobility (mE). Forthis reason, we investigated the variation of themE of theBLG and AF-BLG dispersions as a function of pH.

3.4. Effect ofb -lg aggregates on the electrophoreticmobility (mE) of BLG dispersions

Measurement of themE of protein dispersion is a mean tostudy the surface properties of proteins as for example theestimation of the solvent accessibility or the interaction withmicroions (Douglas, Humffray, Pratt & Stevens, 1995). Fig.6 depicts the pH-induced variation of themE of BLG disper-sions. It could be seen that themE of BLG decreasedcontinuously from 1 1.5 e.m.u. at pH 3.6 to20.5 e.m.u.at pH 5.0. The zero mobility, and therefore the IEP of theb-lg, was reached at pH 4.8 and corresponded to the lowestBLG solubility (Fig. 1). The variation ofmE depended onthe charge balance between the amino and carboxylicgroups carried by the BLG. Considering the AG,mE isnegative in the pH range 3.6–5.0, due to the presence ofglucuronic acid residues on the gum�pKa , 3:6; Fig. 6). For


Fig. 5. Ternary phase diagram obtained for the AF-BLG/AG/Water systemat pH 4.2 and 208C. I: one-phase region; II: two-phase region; (B) binodalpoints; (A) tie-line points. (—) binodal line of the AF-BLG/AG/Watersystem. (– – –) binodal line of the BLG/AG/Water system.

Fig. 6. Electrophoretic mobility in water of the BLG, AF-BLG,b-lg aggre-gates and AG dispersions at 208C. AF-BLG (W), (X); BLG, (A), (B); b-lgaggregates (K); AG (O). Bars represent standard deviation.

the AF-BLG dispersion, elimination of the BLG aggregatesled to a decrease of themE from 1 e.m.u. as compared to theBLG dispersion. Another marked difference was thedecrease of the IEP of the AF-BLG which went downfrom 4.8 to 4.4 (Fig. 6). However, the global shape of thetwo curves was similar. The contribution of theb-lg aggre-gates in the measurement of themE of BLG dispersions hasbeen achieved by measuring themE of a dispersion ofb-lgaggregates separated at pH 4.75 after centrifugation at10,000g during 1 h (Fig. 6). We found that the obtainedmE values were very close to those of BLG dispersions.This observation led us to the conclusion thatb-lg aggre-gates mainly contributed to the pH-induced variation of thenon-centrifuged dispersionsmE.

To go further into details on how aggregates act on theelectrophoretic properties of BLG dispersions, mineralcontents of dispersions were determined before and afterelimination of the protein aggregates. As presented onFig. 7, the purification of the BLG dispersions resulted ina decrease of the cation content, specifically the divalentCa21 and Mg21 and the less hydrated K1 (concentrationsdecreased by, respectively, 20, 60 and 40% of the initialvalue). These kinds of ions are well known to promoteaggregation of colloids. Surprisingly, the Na1 contentremained unchanged (b-lg is known to specifically interactwith Na1 through specific molecular sites; see Arakawa andThimasheff (1988) and Fanni, Canet, Elbayed & Hardy(1989)) as well as the Cl2 content. This result was howeverin good agreement with the decrease of themE of the BLGdispersion after elimination of the aggregates, since lesspositive charges were present in the dispersion. The effectof b-lg aggregates on themE of BLG dispersions was finallyinvestigated by addition of CaCl2 in the AF-BLG dispersionat pH 4.2 to compensate the loss of cations due to the elim-ination of b-lg aggregates. No variation of themE wasdetected after stabilisation of the system during 18 h, themeasuredmE remaining equal to that of the AF-BLG disper-sion (Fig. 6). The release or uptake of protons, anions orcations by proteins has been already reported during theadsorption of human plasma albumin or bovine pancreas

ribonuclease onto negatively charged polystyrene lattice(Norde & Lyklema, 1978), but to our knowledge, not forb-lg aggregates. However, our latter experiment with CaCl2

proved that cations specifically interact withb-lg aggregatessince simple compensation of the charges did not allow therecovering of themE values obtained before aggregate elim-ination. These results emphasised the marked effect ofb-lgaggregates on the initial enthalpic (electrostatic interactions,surface properties) and entropic (excluded volume effect)properties of the BLG dispersion. In our case, the questionremaining to solve is how and where the cations or anionsinteract with the protein aggregates. Thus, asb-lg aggre-gates might be porous, one could assume that interactionswould arise at the surface or inside the aggregates.

The next part of the paper will compare the electrophore-tic mobility, the size and shape of the complexes, coacer-vates or microparticles obtained after mixing AGdispersions with BLG or AF-BLG dispersions at pH 4.2 inwater.

3.5. Characteristics of AF-BLG/AG and BLG/AG mixtures

3.5.1. Electrophoretic mobilityElectrophoretic mobilities of AF-BLG/AG mixtures were

investigated for Pr:Ps ratio ranging from 8:1 to 1:1 at a totalbiopolymer concentration of 0.1 wt%. As depicted on Fig. 8,themE values corresponding to the extreme Pr:Ps ratios (8:1and 1:1) were close to those of the AF-BLG and AG alone,respectively. Coacervates obtained for 8:1 ratio exhibited anIEP at pH 4.0, themE values decreasing from10.25 e.m.u.at pH 3.6 to22 e.m.u. at pH 5.0. For the 1:1 ratio, theelectrophoretic mobility was close to zero at pH 3.6, beingthereafter negative to reach a value of22 e.m.u. at pH 5.0.This result clearly revealed that the measuredmE was that ofthe AG, which was therefore probably in contact with thesolvent (Schmitt et al., 1999). For the two other Pr:Ps ratios,intermediatemE values were obtained. The IEP of thecoacervates was still obtained at pH 4.0, then themE sharplydropped from22 e.m.u. for the 2:1 ratio. This decrease was


Fig. 7. Mineral composition of the AF-BLG at pH 4.75 after discardment ofb-lg aggregates. Bars represent standard deviation.

Fig. 8. Electrophoretic mobility as a function of pH for 0.1 wt% AF-BLG/AG coacervates at 208C. Pr:Ps ratio: 8:1 (W); 4:1 (X); 2:1 (A); 1:1 (B). Barsrepresent standard deviation.

more progressive for the 4:1 ratio. Concerning the shape ofthe mE curves, these results are similar to those obtainedwithout elimination of b-lg aggregates (Schmitt et al.,1999). The marked effect was the control of themE valueby theb-lg at high Pr:Ps ratio and by the AG at low Pr:Psratio. However, some discrepancies arose, in particular thelowermE intensities obtained for the AF-BLG/AG mixtures,especially at low pH values (,4.4). A likely explanationcould reside in the decrease of the charges able to facilitatethe interaction (due to the elimination ofb-lg aggregates)betweenb-lg and AG. It would result in the formation ofnegatively charged AF-BLG/AG complexes that could stillbe formed due to the presence of positive charge patches onthe protein (Mattison, Dubin & Brittain, 1998).

3.5.2. Particle and coacervate size distributionsThe size distributions (on a volume basis) of the mixtures

(on a volume basis) at pH 4.2 exhibited a bimodal tendency,revealing the presence of at least two granulometric classesof particles (Fig. 9). In the presence ofb-lg aggregates (Fig.9A–D, for the lowest biopolymer concentrations (0.5 and

1 wt%), the major intensity peak was generally Pr:Ps ratio-independent with an average particle diameter of 5–15mm.The second peak was always of lesser intensity with a parti-cle diameter of 200–300 nm. Moreover, its intensitydecreased progressively with the increase of the biopolymerconcentration, to the profit of the first peak (Fig. 9A–C).Considering the number average size distribution, BLG/AGmixtures exhibited a slight increase of the mean diameterwhen the biopolymer concentration was increased (90% ofthe particles exhibited a size comprised between 0.31 and2.72mm depending on total biopolymer concentration),independently from the Pr:Ps ratio. This result agreed foran effect of the total biopolymer concentration. For the AF-BLG/AG mixtures (Fig. 9E–H), size distributions differedfrom the previous ones. If the distributions also exhibited abimodal shape, the Pr:Ps ratios markedly affected the parti-cles size distribution, even at low biopolymer concentration.The 8:1 Pr:Ps ratio showed a major peak centred around25mm, whose intensity increased with increasing the totalbiopolymer concentration. The 2:1 and 1:1 Pr:Ps ratiosexhibited a major peak whose intensity (,40%) did notvaried with the total biopolymer concentration. The maxi-mum value for the volume average diameter was howeverobtained for the 1 wt% total biopolymer concentration (Fig.9F). For the AF-BLG/GA mixture, no variation wasobserved in the number average size distribution with theincrease of total biopolymer concentration (90% of theparticles had a diameter comprised between 0.54 and2.74mm). The exception was obtained in the case of the1 wt% concentration that exhibited a drop of the particlediameter whatever the Pr:Ps ratio considered (0.58, 0.78and 1.82mm for the diameter of 90% of the particles atratios 8:1, 2:1 and 1:1, respectively). Differences betweenthe size distributions obtained with or withoutb-lg aggre-gates was highlighted by plotting the variation of thevolume average diameterd43 as a function of the total biopo-lymer concentration (Fig. 10). In the presence ofb-lg aggre-gates, an increase ofd43 when increasing biopolymerconcentration was clearly visible (Fig. 10A). Fig. 10Aalso revealed that the volume average diameter of theBLG/AG particles was dependent on the Pr:Ps ratio fortotal biopolymer concentration above 1 wt%. Thus thed43

value ranged from,10mm (0.5–1 wt%) to 50mm (5 wt%)for the 8:1 ratio only, i.e. when the protein content was inexcess. For the system obtained after elimination of theb-lgaggregates (Fig. 10B), thed43 sharply increased for the 2:1and 1:1 ratios when the total biopolymer concentration wasincreased to 1 wt%. For instance,d43 values varied from 40to 200mm for the 2:1 ratio. This could reveal an instabilityof the system caused by aggregation or coalescence of parti-cles. For total biopolymer concentrations above 1 wt%, thesize of the complexes decreased and stabilised around50mm diameter values.

3.5.3. Optical phase contrast microscopyPhase contrast migrographs obtained at pH 4.2 for the


Fig. 9. Volume size distributions of particles in BLG/AG and AF-BLG/AGmixtures at pH 4.2 and 208C. (—) ratio 8:1; (···) ratio 2:1; (– – –) ratio 1:1.BLG/AG mixture: (A) 0.5; (B) 1; (C) 3; (D) 5 wt%. AF-BLG/AG mixture:(E) 0.5; (F) 1; (G) 3; (H) 5 wt%.

BLG/AG and AF-BLG/AG systems at 1 wt% total biopoly-mer concentration are presented on Fig. 11. For the systemcontaining aggregates, micrographs obtained for the 8:1Pr:Ps ratio exhibited clear particles with a diameter valuearound 20–40mm (Fig. 11A). According to their irregularshape, these structures are expected to beb-lg aggregates orprecipitates. On the same picture, smaller dark particles arealso visible, exhibiting sizes ranging from,1 to 5mm (Fig.11A). As they are generally of spherical shape, these struc-tures must be liquid in nature and could be coacervates(Newton, McMullen & Becker, 1977; Remun˜an-Lopezand Bodmeier, 1996). For the 2:1 and 1:1 Pr:Ps ratios,these precipitates were still visible, but their size decreased(5–20mm). In contrast, the small rounded particles disap-peared when the Pr:Ps ratio decreased (Fig. 11B and C). Forthe 1:1 ratio, onlyb-lg aggregates were present, revealing asmall yield of interaction between protein and gum. Parti-cles formed in the aggregate-free system markedly differedin shape and structure as compared to the previous ones(Fig. 11D–F). In this case, nob-lg aggregates or precipitateswere visible. The particles were spherical at all Pr:Ps ratiosand seemed to be more or less porous (clear or dark core,respectively). The smallest particles were obtained for the8:1 Pr:Ps ratio (,1 to 10mm), bigger particules wereformed for the 2:1 and 1:1 Pr:Ps (5–40mm). We have tonotice that sizes measured on micrographs were not in total

agreement with the sizes determined by granulometry.Thus, one should keep in mind that the particle size deter-mined by granulometry is given on a volume basis that hasthe drawback to overestimate the contribution of the largeparticles to the detriment of the small ones. For this reason,particles exhibiting size as large as 200mm were notobserved by optical microscopy due to their low numberin the dispersion. Moreover, if optical microscopy allowedthe observation of microparticles (.1 mm), the nanoparti-cles detected by light scattering could not be seen.

3.6. General discussion

The presence of protein aggregates (arising from the puri-fication treatment of theb-lg) in the initial b-lg dispersionhas been found to have a marked effect on both the extent ofphase separation phenomenon, the size and surface proper-ties of the obtained complexes, coacervates or microparti-cles. This effect was probably due to an entropic variation inthe system due to a decrease of the polydispersity in mole-cular masses and size in the BLG dispersion after elimina-tion of the aggregates. Withoutb-lg aggregates, phase


Fig. 11. Phase contrast optical micrographs of 1 wt% BLG/AG and AF-BLG/AG mixtures. Magnification: 500× . Bar represents 20mm. (A) BLG/AG, ratio 8:1; (B) BLG/AG, ratio 2:1; (C) BLG/AG, ratio 1:1 (D) AF-BLG/AG, ratio 8:1; (E) AF-BLG/AG, ratio 2:1; (F) AF-BLG/AG, ratio 1:1.

Fig. 10. Evolution of the volume average diameter (d43) of particles in1 wt% mixtures at pH 4.2 and 208C. (A) BLG/AG mixture, (B) AF-BLG/AG mixture. Pr:Ps ratio: 8:1 (W); 2:1 (X); 1:1 (A). Bars represent standarddeviation.

separation should be mainly due to complex coacervation.Thus, discarding aggregates should avoid non-electrostaticinteractions betweenb-lg and AG, as it has been describedrecently in the formation of electrostatic complexes betweenlysozyme aggregates and pyrene-labelled polyanions at highpH values (Sato, Mattison, Dubin, Kamachi & Morishima,1998). This assumption is confirmed by the self-suppressionof complex coacervation at a low total biopolymer concen-tration (4.5 wt%) as already suggested by Tolstoguzov(1991). For higher biopolymer concentrations, favourableenthalpic effects could not overcome the increase of free-energy in the system induced by biopolymer interactions sothat coacervation was then self-suppressed (Veis, 1963).However, enthalpic effects were also involved as the elim-ination ofb-lg aggregates led to a decrease of the cationscontent in the resulting AF-BLG dispersion. The size distri-bution and the shape of the complexes, coacervates andmicroparticles markedly differed between BLG/AG andAF-BLG/AG systems. In the AF-BLG/AG system, coacer-vates exhibited various sizes depending on the Pr:Ps ratioand the total biopolymer concentration, revealing the forma-tion of large-sized coacervates by coalescence of smallerones or interactions between soluble Pr:Ps complexes(Mattison et al., 1998; Xia, Dubin, Ahmed & Kakofuta,1994). Moreover, these particles were more unstable thanthose obtained when aggregates were present. This stabili-sation of the particle size by aggregates was not due to adecrease of the soluble protein content. Thus, if wecompared the particle diameters obtained for the 1 wt%BLG/AG dispersion to those obtained for the 0.5 wt% AF-BLG/AG dispersion, to take account for the,50% b-lgsolubility at pH 4.2 (see Fig. 1), values did not coincide,whatever the Pr:Ps ratio. Consequently, other mechanismsshould be involved, in addition to complex coacervation. Inpresence of aggregates, the particles size was limited by theinitial b-lg aggregate size, leading to the hypothesis thatinteraction betweenb-lg and AG was not only electrostaticin nature. In that case,b-lg aggregates should be consideredas peculiar entities reacting differently with AG than mono-mers or dimers due to their largeMw and different surfaceproperties. As depicted by the electrophoretic mobilitymeasurements, large-sized aggregates exhibited a positivecharge value favouring the electrostatic interaction with AG(Buchhammer, Petzolt & Lunkwist, 1993). Moreover, inter-actions could also arise from adsorption phenomenon of AGor b-lg/AG coacervates onto the surface ofb-lg aggregates,which are known to be hydrophobic (Gao & Dubin, 1999).Thus, based on interfacial tension differences between thedifferent constituents in the mixture, one could assume thatb-lg aggregates should exhibit a high thermodynamicallyunfavourable surface tension value, allowing the absorptionof free biopolymers on their surface (Thomasin, Merkle &Gander, 1997; Thomasin, Nan-Traˆn, Merkle & Gander,1998). Another possible mechanism, which is largely usedfor the microencapsulation of proteins, could be the engulf-ment ofb-lg aggregates by theb-lg/AG coacervates due to

an interfacial tension value intermediate between that of thesolvent and theb-lg aggregates (Torza & Mason, 1970).

4. Conclusion

The effect ofb-lg aggregates has been examined on thecomplex coacervation phenomenon betweenb-lg andacacia gum. Regarding the particles size distributions, theternary system containing aggregates seemed to be morestable than the free-aggregate one. Surface properties, sizeand structure of the obtained particles markedly differedbetween the two systems. We could reasonably expectthat it would be the same for their functional properties.Thus, new food ingredients exhibiting large potentialitiescould be obtained by simple control of theb-lg aggregationstate. In this context, several questions arise from theobtained results. It would be necessary to investigate thekinetic of formation of theb-lg/AG complexes, and to eluci-date the influence of the protein aggregates on the kineticparameters. A promising way of investigation could be tostudy the effect of the back-addition of aggregates ofcontrolled size (obtained by controlled heat treatment ofmonomericb-lg dispersions) into the purified system atincreasing concentrations on the phase diagram shape, elec-trophoretic mobility values and size distribution of the parti-cles. Moreover, characterisation of the particles’ structure inboth systems by means of non-destructive techniques suchas confocal scanning laser microscopy (CSLM), coupledwith interfacial tension measurements could be envisagedto check the proposed hypothesis relative to adsorption/coacervation phenomena. Finally, the stability of thecomplexes obtained in both conditions should be exploredto go deeper into the mechanisms involved in the interactionbetween the two macromolecules.

Acknowledgements

We acknowledge the PhD grant from the “Ministe`reFrancais de l’Education Nationale, de la Recherche et dela Technologie”.

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