Journal of Colloid and Interface Science 296 (2006) 332–341www.elsevier.com/locate/jcis

Microstructure of β-lactoglobulin-stabilized emulsions containing non-ionicsurfactant and excess free protein: Influence of heating

Sven Kerstens, Brent S. Murray, Eric Dickinson ∗

Procter Department of Food Science, University of Leeds, Leeds LS2 9JT, UK

Received 7 June 2005; accepted 21 August 2005

Available online 15 September 2005

Abstract

The influence of the non-ionic surfactant Tween 20 on the microstructure of β-lactoglobulin-stabilized emulsions with substantial excess freeprotein present was investigated via confocal microscopy. The separate distributions of oil droplets and protein were determined using two differentfluorescent dyes. In the emulsion at ambient temperature the excess protein and protein-coated oil droplets were associated together in a reversiblyflocculated state. The pore-size distribution of the initial flocculated emulsion was found to depend on the surfactant/protein ratio R, and at highervalues of R the system became more inhomogeneous due to areas of local phase separation. Evidence for competitive displacement of proteinfrom the oil–water interface by surfactant was obtained only on heating (from 25 to 85 ◦C) during the process of formation of a heat-set emulsiongel. By measuring fluorescence intensities of the protein dye inside and outside of the oil-droplet-rich areas, we have been able to quantify theevolving protein distribution during the thermal processing. The results are discussed in relation to previous work on the competitive adsorptionof proteins and surfactants in emulsions and the effect of emulsion droplets on the rheology of heat-set protein gels.© 2005 Elsevier Inc. All rights reserved.

Keywords: Confocal laser scanning microscopy; Competitive displacement; Emulsion gel; Phase separation; β-Lactoglobulin; Non-ionic surfactant; Whey protein;Tween 20

1. Introduction

Obtaining stability in complex food systems remains a ma-jor challenge for the food industry. Whereas two ingredientsmay individually enhance the desired properties of a product,the interplay of the mixed components is sometimes a desta-bilizing factor. Whey proteins are widely used as structuralbuilding blocks in food products [1–3]; their ability to denatureand aggregate into a three-dimensional network makes them es-pecially suitable as gelling agents and texture modifiers. Mostfrequently the gelation of whey proteins is induced by heating,although cold-set gels can also be obtained by the addition ofsalts or by using high pressure or enzymatic treatments [4–7].The main molecular interactions involved in the associationand cross-linking of denatured whey proteins are hydrophobicforces and covalent bonding via intermolecular disulfide link-ages [8–12]. The final gel microstructure is determined by a

* Corresponding author. Fax: +44 (0) 113 343 2982.E-mail address: e.dickinson@leeds.ac.uk (E. Dickinson).

0021-9797/$ – see front matter © 2005 Elsevier Inc. All rights reserved.doi:10.1016/j.jcis.2005.08.046

balance of local attractive and repulsive forces involving bothwhey protein molecules and their aggregates, and it is stronglyinfluenced by pH and ionic strength [13–16].

The presence of small-molecule surfactants (emulsifiers)can affect the functional properties of proteins. For instance,the addition of the water-soluble non-ionic surfactant, poly-oxyethylene sorbitan monolaurate (Tween 20), influences thethermal gelation behavior of the major whey protein, β-lacto-globulin (β-lg), both in solution and in β-lg-stabilized emul-sions [17,18]. Recently, we reported [19] on the use of confocalmicroscopy to follow the microstructure of heated β-lg solu-tions at pH 6.8 with and without added surfactant. While thestrands making up the final heat-set gel were too small to be re-solved by the technique, and the homogeneous final microstruc-ture was visibly unaffected by the presence of Tween 20, weobserved that during the process of heating from 25 to 85 ◦Cthere appeared micrometer-sized aggregates which disappearedagain upon further heating towards the gelation point. More-over, we found that the temperature-dependent evolution of theappearance and sizes of these aggregates was dependent on the

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surfactant/protein molar ratio R. While the exact nature of theaggregates is yet has to be established, we do know from theliterature [20–22] that Tween 20 binds specifically to β-lg in a1:1 fashion; so it is possible that some kind of reversible self-assembly involving β-lg/Tween 20 complexes lies at the originof these pseudo-micellar aggregates.

Due to their much higher adsorption energy per molecule,milk proteins saturate fluid interfaces at much lower concen-trations than do small-molecule surfactants [23]. However, athigher concentrations, due to their more effective interfacialpacking, the interfacial tension is lowered more by small-molecule surfactants than by proteins. Hence, milk proteins arecompetitively displaced by surfactants from oil–water and air–water interfaces, as has been extensively reported [20–28] forsystems containing a mixture of Tween 20 + whey proteins (in-cluding pure β-lg). Typically, in oil-in-water emulsions, thereis complete displacement of β-lg from the oil–water interfaceby non-ionic surfactants at high surfactant/protein ratios (i.e.,R � 15) [29].

From a material science point of view, the rigidity of a heat-set protein gel can be strongly affected by the presence of so-called ‘filler particles’ like emulsion droplets. The oil volumefraction, as well the size, shape and deformability of the inclu-sions, influences the overall mechanical behavior of the filledgel. The composition of the adsorbed monolayer around thedroplets also has important implications for the bulk rheologyof the system because it influences the nature of the interactionbetween the filler particles and the protein gel network [30].This role of the filler–matrix interaction has been demonstratedusing scanning electron microscopy [31,32] and confirmed byrheological measurements on heat-set whey protein emulsiongels [33,34]. Droplets coated with protein are incorporated intothe protein gel network via hydrophobic and covalent bonding,and hence they act as ‘active’ fillers; but droplets stabilized bysmall-molecule surfactants have little affinity for the surround-ing protein matrix, and so they behave as ‘inactive’ fillers. Pre-vious work in our laboratory on heat-set gels made from wheyprotein-stabilized (or β-lg-stabilized) emulsions has shown [17,18,34–39] that the elastic shear modulus is indeed greatly en-hanced by the presence of the active filler particles. On theother hand, the presence of emulsion droplets stabilized by alow-molecular-weight surfactant (such as Tween 20) leads to alower elastic modulus.

Most of this existing knowledge about the effect of surfac-tant/protein competitive adsorption on emulsion properties hascome from a combination of protein surface coverage mea-surements made at ambient temperature [29,38–43] and bulkrheological measurements [17,18,33–37,44–49]. There is verylittle information available on the influence of added surfactantson the emulsion microstructure. Visualizing the microstructurein situ seems essential, however, for a proper understanding ofthe factors affecting the bulk mechanical behavior and stabil-ity of emulsions before, during and after thermal processing.In a preliminary confocal microscopy study of this type, us-ing an oil-soluble dye to identify the oil phase, Chen and co-workers [35] revealed substantial qualitative effects of addedemulsifier on the final microstructure of a heat-set emulsion gel.

But this earlier study [35] provided no quantitative informationabout the distribution of the protein in the system; nor was themicrostructure followed in time during heating.

Here, for the first time, we report on the use of confocal mi-croscopy to follow time-dependent changes in microstructureduring the formation of a heat-set emulsion gel. Spatial distrib-utions of oil droplets and protein are separately monitored usingtwo different fluorescent dyes. Specifically, we examine the ef-fect of varying amounts of Tween 20 on the initial and evolvingmicrostructure of heated β-lg-stabilized emulsions containinga high content of excess unadsorbed protein.

2. Materials and methods

2.1. Materials

Bovine β-lactoglobulin (L-3908, lot number 024K7051),Tween 20 (polyoxyethylene sorbitan monolaurate), polyethyl-ene glycol, Nile Blue and Nile Red were purchased from SigmaChemicals (Poole, UK). The protein sample was a mixture ofβ-lactoglobulin variants A and B, and no further purificationwas applied. In order to calculate the surfactant/protein molarratio R, we assumed average molecular weights of 1228 Da and18.3 kDa respectively for Tween 20 and β-lg [50]. 1-Bromo-hexadecane (99%, Acros Organics, Belgium) was used as theemulsion oil phase and distilled water was used for the prepa-ration of the solutions.

2.2. Preparation of protein solutions

Concentrated protein solutions were used to investigate theeffect of temperature and environment on the fluorescence be-havior of the protein dye Nile Blue. Solutions were preparedcontaining 13.2 wt% β-lg with Tween 20 present at surfac-tant/protein ratios of R = 0, 0.5 and 2. The solutions wereadjusted to pH 6.8 with 0.1 M HCl.

2.3. Preparation of emulsions

A starting oil-in-water emulsion (45 vol% oil, 2.7 wt% β-lgin the aqueous phase) was made using a jet homogenizer oper-ating at a constant pressure of 300 bar. This stock emulsion wasdivided into several aliquots. During gentle stirring each aliquotwas topped up with an aqueous solution containing known con-centrations of β-lg and Tween 20 to obtain a set of final oil-in-water emulsions (20 vol% oil) having a total protein content of13.2% (w/v) and values of the surfactant/protein ratio R vary-ing between 0 and 2. The pH was adjusted dropwise with 1 MHCl to pH 6.8.

The particle-size distribution and mean diameter d32 of theemulsions were determined by static light scattering using aMalvern Mastersizer 2000 with an assumed value of 1.1 for therelative refractive index and 0.0005 for the particle absorption.

2.4. Gelation time test

Test tubes containing ca. 4 ml of emulsion were placed in awater bath at 85 ◦C. The tubes were taken out for observation at

334 S. Kerstens et al. / Journal of Colloid and Interface Science 296 (2006) 332–341

regular intervals, and the gel point was defined empirically asthe time at which a tube could be turned upside-down withoutobservable downwards flow of its contents, i.e., indicating theformation of a self-supporting gel.

2.5. Confocal microscopy

A Leica TCS SP2 confocal system mounted on a Leica DM-RXE upright microscope was used to examine the microstruc-ture of the samples. Emulsions and protein solutions were putin small cylindrical containers (80 µl), covered with a coverglass (size 1.5), and sealed with silicone oil to prevent evap-oration. The containers were placed on a Peltier microscopeheating stage. The temperature was increased from 25 to 85 ◦Cat 2 ◦C min−1. Following this heating ramp, the temperaturewas kept constant at 85 ◦C for 30 min, after which time thesample was allowed to cool down to 25 ◦C.

The proteins were stained with Nile Blue (12.5 µl of dye so-lution added to 2.5 ml emulsion to obtain a dye/protein ratio1:100) and excited at 633 nm. Nile Red was used for stain-ing of the emulsion oil phase (25 µl of 0.01% (w/v) dye inpolyethylene glycol added to 2.5 ml emulsion) and the sam-ple was excited at 488 nm. Fluorescence intensity data for NileBlue and Nile Red were collected in two separate channels cor-responding to 503–613 and 653–750 nm, respectively. Onlyone collection channel was required for the protein solutions.A bleed-through of the signal from the first channel (Nile Red)could be measured in the second channel, where Nile Blue wasdetected. Therefore, fluorescence data for the two dyes werenot collected simultaneously, but instead the ‘sequential line-scanning mode’ was used. Every line of pixels in the image wassequentially scanned for the Nile Red and Nile Blue fluores-cence, thereby completely avoiding interference due to cross-fluorescence. Before and after heating both a dry 20× lens (NA0.7) and a 63× water-immersion lens (NA 1.2) were used tostudy the microstructure of the sample, while a dry 20× lens(NA 0.5) of longer working distance (1.15 mm) was used dur-ing the heating process to prevent thermal damage to the otherlenses.

2.6. Image analysis

Fluorescence intensity distributions in the micrographs wereanalyzed using the confocal microscope operating software.The aqueous protein solutions gave a uniform picture withno structure visible, except when Tween 20 was present andthe system was heated, in which case small aggregates ap-peared and then disappeared again during heating, as reportedelsewhere [19]. Here we are solely interested in the behav-ior of the Nile Blue fluorescence, since this parameter will beused to quantify the distribution of the protein component inthe emulsions. At set time intervals we measured the averagegrey value intensity of the image, corresponding to the flu-orescence intensity of the dye. We ignored the small proteinaggregates that appeared and disappeared in the sample, on thebasis that they would not affect the average fluorescence inten-sity.

Micrographs of emulsion samples were analyzed using theLeica image analysis software QWin. The protein distribution,in images taken with the 63× lens, was determined by measur-ing the fluorescence intensity of Nile Blue inside and outsidethe oil-droplet-rich areas (using the Nile Red fluorescence ofthe oil phase as a mask to define the different regions). To de-termine the change in fluorescence intensity distribution duringheating, the analysis was based on micrographs taken with the20× lens (NA 0.5).

Pore sizes were measured on the micrographs showing NileRed fluorescence (indicating the oil phase), taken with the lowermagnification 20× lens (NA 0.7) in order to have a bigger fieldof view than with the 63× lens. A crucial step in image analysisis thresholding—in order to differentiate the background fromthe objects of interest within the image. The type of imageswe obtained here did not have a clear bimodal distribution ofgrey levels, whereby the two sub-populations of pixels clearlybelong to the background and the foreground. So, in princi-ple, some deconvolution technique has to be used to define thethreshold between the background and the objects of interest[51,52]. Here we implemented thresholding in a series of con-secutive steps using the Leica image analysis software Qwin.The typical steps involved Gaussian smoothing to reduce noise,after which the bright detail was enhanced by adding a ‘white-top-hat’ filtered image to the original. This enhanced image wastransformed using a point function (LUT-transform) wherebylower intensities were suppressed. After this step the image wasthresholded, and the dark enclosed regions were assigned topores and then quantified. As these dark regions were generallynon-circular (non-spherical in 3-D), the pore size was expressedin terms of area (µm2).

3. Results and discussion

3.1. Effect of environmental conditions on fluorescencebehavior

We aim to quantify the evolving protein distribution in re-gions of the emulsion sample images by analyzing the fluores-cence intensity of Nile Blue. Therefore it was necessary first todetermine the sensitivity of the dye fluorescence to the differentconditions.

Fluorescence intensities measured for the protein solutionsduring heating are given in Fig. 1 for three surfactant/protein ra-tios (R = 0, 0.5 and 2). The electronic gain setting was adjustedso that each sample had the same initial fluorescence intensityvalue (170 ± 10 units) and that the changing fluorescence in-tensity values remained throughout well within the full 0–255grey scale. Off-set values (signal cut-off values) were all set tozero. These settings were not changed during the course of eachheating experiment.

The general tendency shown in Fig. 1 is that, in the presenceor absence of Tween 20, the fluorescence intensity declines astemperature increases. On holding the temperature at 85 ◦C for30 min, the fluorescence intensity increases slightly again. Oncooling back down to 25 ◦C, the average fluorescence inten-sity returns to an even higher value than before the sample was

S. Kerstens et al. / Journal of Colloid and Interface Science 296 (2006) 332–341 335

Fig. 1. Effect of thermal processing on Nile Blue fluorescence of protein so-lutions (13.2 wt% β-lg, dye/protein ratio 1:100) in the presence and absenceof Tween 20. The fluorescence intensity is plotted against time: (!) R = 0,gain setting = 425 V; (") R = 0.5, gain setting = 400 V; (Q) R = 2, gainsetting = 385 V. The solid line represents the heating profile.

heated. This latter behavior suggests to us that no substantialphoto-bleaching is involved. Heating of fluorochromes can of-ten induce reduction in quantum efficiency due to the increasedvibration and torsion of their flexible side-arms, resulting inan internal radiationless transition, as reported previously forRhodamine B [19,53]. In our system, when the temperaturereaches 85 ◦C, gelation takes place [19]. Together with the factthat complexation of Nile Blue with the protein hinders rota-tional relaxation processes and increases the quantum yield ofthe dye [54], gelation might explain the increase in fluorescenceintensity on holding at 85 ◦C, arising from some immobiliza-tion of the dye within the aggregated protein network. A fur-ther rigidity-enhancing effect in heat-set globular protein gelstakes place during the cooling down stage, due to strengthen-ing of hydrogen bonding and electrostatic forces [17,18]. Thiscould explain the increase in the fluorescence intensity aftercooling to a value even higher than for the original unheatedsample.

The second main feature of the protein solution data is theincrease in fluorescence intensity of Nile Blue on addition ofTween 20. A lower gain setting has to be used when increas-ing the surfactant concentration in order to obtain similar av-erage grey scale values in the image at room temperature. Oneplausible explanation [54] is that the presence of surfactant mi-celles prevents radiationless deactivation of the excited dye toits ground state, resulting in a higher overall fluorescence inten-sity. In the solution of low surfactant content (R = 0.5) all ofthe Tween 20 is expected to be bound to β-lg [19–22]. No sur-factant micelles would be expected to exist, therefore, until theprotein denatures on heating and releases the bound Tween 20back into the solution, further complicating the interpretation ofthe fluorescence intensity.

Others factors that could also potentially affect the measuredfluorescence intensity are the relative concentrations of dye andprotein and the pH. But each of these is kept constant. So in oursystems the fluorescence intensity is predominantly dependenton the surfactant (micelle) concentration, the temperature, andthe aggregation state of the protein (or protein–dye complexes).

3.2. Emulsion systems

In the low-protein-content stock emulsion, straight after ho-mogenization, there was no evidence of flocculation. The mostprobable droplet diameter was around 0.5 µm and the d32 valuegiven by the Mastersizer was 0.38 ± 0.02 µm. While floccula-tion was induced on subsequently adjusting the protein concen-tration to 13.2% (see below), the measured particle-size distri-bution was unaffected by the subsequent operations of dilutingthe aliquots or adjusting the pH.

Fig. 2 shows a set of images for the 20 vol% oil-in-wateremulsion with 13.2% β-lg in the aqueous phase (no surfactantpresent). Image (a) represents the fluorescence collected fromthe oil phase stained with Nile Red, and image (b) representsthe fluorescence from the protein stained with Nile Blue. Theoverlay image (c) permits location of the two dyes with re-spect to each other. Individual oil droplets could not normallybe distinguished, except for some exceptionally large ones. Forinstance, in Fig. 2, we can see how one big oil droplet towardsthe left side of the picture gives a dark round hole (no dis-cernible intensity) in the image recording the Nile Blue/proteinfluorescence.

For the system represented in Fig. 2, it is clear that theprotein-coated oil droplets and the excess protein present in theaqueous continuous phase are in a closely associated state—hence the predominantly yellow appearance of the overlayimage (green + red → yellow). The original stock emulsion(45 vol% oil, 2.7 wt% protein) gave a set of images (not shown)with a homogeneous distribution of the oil droplets. The floccu-lation evident in Fig. 2 had therefore been induced by ‘toppingup’ the high-volume-fraction stock emulsion with the concen-trated β-lg solution as described in Section 2.3. As no dis-cernible change in mean droplet size d32 was detectable ondilution in the Mastersizer, the heterogeneity apparent in Fig. 2must be the result of a process of weak, reversible floccula-tion. It would therefore seem that, at the low ionic strength ofthis system, the protein added after homogenization has greateraffinity for the weakly aggregated protein-coated oil dropletsthan for the aqueous serum, and that this manifests itself in themicrographs as local phase separation.

Fig. 3 shows the effect of the added surfactant on the mi-crostructure of the emulsion at 25 ◦C. Overlay micrographs,including fluorescence from both the protein (Nile Blue) andthe oil (Nile Red) are given for R = 0, 0.75, 1.25 and 2. Byeye we can see that, depending on the amount of Tween 20present in the system, the pore-size distribution varies substan-tially. Fig. 4 shows the effect of surfactant content on the meanpore size (white bars, left axis) and the maximum pore size(dashed bars, right axis) as derived from the image analysis.With addition of Tween 20, the mean pore size decreases ini-tially, and then it increases strongly. The quoted error bars referto the standard error. For large surfactant additions (R > 1.5),the error in mean pore size diverges as the structure becomesquite inhomogeneous. For instance, we can see in Fig. 3d that,despite some large open (dark) areas in the image, there are stillsome areas with much smaller pores. For this reason we alsoinclude data in Fig. 4 for the maximum pore size. Whereas the

336 S. Kerstens et al. / Journal of Colloid and Interface Science 296 (2006) 332–341

(a)

(b)

(c)

Fig. 2. Confocal micrograph of a sample of an oil-in-water emulsion (20 vol%oil, 13.2 wt% β-lg in the aqueous phase, 25 ◦C) with no Tween 20 added:(a) image from Nile Red fluorescence (highlighting the oil phase); (b) imagefrom Nile Blue/protein fluorescence; (c) overlay image combining fluores-cence from both dyes. The arrow identifies a circular dark hole in the NileBlue/protein image corresponding to a single large oil droplet. Scale bar repre-sents 30 µm.

average pore size only changes by a factor of ∼5 over the wholerange of surfactant contents, we can see that there is a nearly30-fold difference between the lowest (R = 0.5) and the high-est (R = 1.75) values of the maximum pore size. So it wouldseem that, while the average pore size is a reasonable indicatorof structural homogeneity, it should properly be supplementedby information on the whole range of pore sizes.

It is noteworthy that, at the lower Tween 20 concentra-tions (R � 1), the pores are smaller than in the absence ofsurfactant. This is possibly attributable to the known 1:1 com-plexation of Tween 20 with β-lg [20–22]. From our previ-ous calculations [19], all the surfactant present is expectedto be bound to the protein for R � 1. The protruding poly-oxyethylene chains might thereby affect the solubility of theaggregated proteins. At higher surfactant/protein ratios, how-ever, free surfactant (monomer + micelles) would be expectedto be present in the system, inducing local phase separation, asseen in Figs. 3c and 3d. Such reversible depletion flocculationof emulsion droplets due to the presence of surfactant micellesis well recognized in the literature [55,56]. The R values quotedabove are, of course, calculated with respect to all the proteinpresent in the system, but it is likely that the protein adsorbedat the oil–water interface interacts differently with the Tween20. Furthermore, small amounts of surfactant probably alreadyabsorb at the interface for R � 1. These combined factors addsome uncertainty as to the precise value of the free surfactantconcentration in the system.

Fig. 3 shows that the surfactant content strongly affectsthe way that the protein is distributed within the microstruc-ture. In micrographs (a), (b) and (d), corresponding to R = 0,0.75 and 2, respectively, we can see that the protein-coated oildroplets and the excess protein are roughly located within thesame local regions. At higher magnification, the images cor-responding to the separately collected fluorescence data havethe same general appearance as images (a) and (b) in Fig. 2for R = 0. In strong contrast, however, Fig. 3c (R = 1.25) hasa red colored background instead of a black one, indicatingthat at this composition the non-adsorbed protein is spread outevenly throughout the aqueous continuous phase, instead of be-ing closely associated with the aggregated protein-coated oildroplets. Corresponding to Fig. 3c, the primary image indicat-ing just the Nile Blue/protein fluorescence has a rather uniformappearance (not shown), in contrast to the clustered regions ofaggregated protein as seen in Fig. 2b.

In order to quantify how the protein is distributed withinthese samples, we have separately estimated the average flu-orescence intensities of Nile Blue/protein complex inside andoutside of the oil-droplet-rich areas. Absolute quantificationwould require a single calibration curve under strictly definedconditions [54]. In our case this is not feasible, because the en-vironment experiences continuously changing conditions thataffect the fluorescence behavior in a complex manner (see Sec-tion 3.1). Furthermore, an increase in the Tween 20 concentra-tion in our emulsion systems was found to lead to a decreasein fluorescence intensity. This is the opposite effect to that ob-served for the pure protein solutions (Section 3.1), illustratingthe further complexity of this notionally relatively simple sys-tem of β-lg + Tween 20. In practice, the electronic gain and‘off-set’ settings on the confocal microscope had to be changedfrom sample to sample, and adjusted during heating in orderto keep the measured fluorescence level within the 0–255 greyscale range. In order to standardize this process, we took theoccasional voids of big oil droplets in the micrographs of theprotein fluorescence (e.g., see Fig. 2b) as points of zero fluores-

S. Kerstens et al. / Journal of Colloid and Interface Science 296 (2006) 332–341 337

(a) (b)

(c) (d)

Fig. 3. Effect of the addition of surfactant on the microstructure of an oil-in-water emulsion (20 vol% oil, 13.2 wt% β-lg in the aqueous phase, 25 ◦C). Each of thefour confocal micrographs is an overlay image incorporating both Nile Red and Nile Blue fluorescence: (a) R = 0; (b) R = 0.75; (c) R = 1.25; (d) R = 2. Scale barrepresents 100 µm.

Fig. 4. Effect of the surfactant/protein ratio R on the mean pore size (white bars, left scale) and maximum pore size (shaded bars, right scale). Standard errors aregiven for the mean pore size.

cence intensity. Setting this reference level to a zero intensityvalue, then any other region in the micrograph showing a higherintensity value could be properly regarded as a region with atrue positive fluorescence contribution, as opposed to one withjust amplified background noise. On this basis we introduce a

protein distribution ratio (PDR) defined as the protein fluores-cence within the oil-droplet-rich areas divided by the proteinfluorescence outside the oil-droplet-rich areas. So, a PDR sub-stantially higher than unity implies that the excess unadsorbedprotein is mainly associated with the oil droplets; a value near

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Table 1Mean values of absolute grey levels and their standard deviations (SD) for NileBlue/protein fluorescence before and after heating measured inside and outsideof oil-droplet-rich areas as a function of the surfactant/protein ratio R

R Before heating After heating

Oil-rich areas Oil-free areas Oil-rich areas Oil-free areas

Mean SD Mean SD Mean SD Mean SD

0 164.3 2.7 36.8 2.6 188.8 8.0 183.1 8.90.5 176.7 8.0 28.6 1.8 161.3 6.2 193.1 9.30.75 178.4 11.2 38.0 7.5 83.7 3.2 157.4 2.71 191.2 9.5 51.1 8.6 95.7 9.7 184.0 16.61.25 175.8 16.1 150.0 19.5 98.6 6.2 179.4 5.81.5 177.3 8.9 135.5 7.4 128.3 19.1 204.3 13.31.75 166.3 2.5 101.4 2.5 82.6 6.9 174.9 11.22 152.7 10.1 64.6 12.0 81.4 6.4 177.5 9.0

Fig. 5. Effect of the surfactant/protein ratio R on the protein distribution ratioPDR as calculated from the grey values listed in Table 1: (") before heating;(!) after heating.

unity corresponds to a fairly homogeneous distribution of pro-tein within the system; and a value considerably below unitymeans that the protein is mainly distributed outside the oil-droplet-rich areas.

Table 1 lists the sets of measured grey level intensities in-side and outside the oil-droplet-rich areas, both before and afterheating. The calculated PDR values are plotted as a function ofR in Fig. 5. Before heating, for R � 1, we see that the PDRvalue is high (5 ± 1). This means that five times more fluo-rescence is measured in the oil-droplet-rich areas than outside.At R = 1.25 and R = 1.5, we have PDR ≈ 1, indicating thatthe protein is fairly evenly distributed throughout the system,as already qualitatively observed in Fig. 3c. For higher surfac-tant/protein ratios, Fig. 5 shows that the protein again becomesmore closely associated with the oil droplets, although substan-tially more protein is still present outside the oil-droplet-richareas as compared to the systems of low surfactant content.

Hence, for the case of no surfactant present or only a smallamount, the excess (non-adsorbed) protein has a high affinityfor the oil-coated droplets. On the other hand, when the sur-factant/protein ratio exceeds unity, it appears that the excessTween 20 makes the protein more soluble (dispersible) withinthe continuous phase, possibly by a molecular mechanism in-volving the shielding of hydrophobic regions of the protein

from the solvent [57]. At even higher surfactant concentra-tions, there is a local phase separation between a surfactant-richphase and a phase containing non-adsorbed protein + protein-coated oil droplets. Comparison of the data in Figs. 4 and 5shows no obvious direct correlation between the effects ofthe added surfactant on the pore size and the protein distrib-ution.

The systems before thermal treatment were stable towardschanges in visible appearance and microstructure for at least 3days on storage at refrigerator temperature. Under the confocalmicroscope there was no obvious evidence of displacement ofprotein from the oil–water interface by Tween 20. This is notreally surprising, as the thickness of an adsorbed monolayerat the oil–water interface is no more than a few nanometers,as compared with a resolution of not less than a few hundrednanometers for the confocal microscope. Previous research hasdemonstrated [20–29] that addition of Tween 20 or some othernon-ionic surfactant after emulsion formation leads to completespontaneous displacement of protein from the oil–water inter-face at high surfactant/protein ratios (R � 15). But typically inthis earlier work the emulsion contained a low concentration ofprotein (e.g., 0.5 wt% [29]), as compared to 13.2 wt% for theemulsions studied here. In the present study we are only con-sidering relatively low surfactant/protein ratios (up to R = 2),even though in absolute terms the concentration of Tween 20in our emulsion at R = 0.75 is already higher than for R = 15in the earlier study [29]. It has been recognized recently [58]that the stability properties of β-lg-based emulsions at ambienttemperature are affected considerably by the presence of freeprotein. Furthermore, in our emulsions the large amount of freeprotein may ‘neutralize’ the competitive adsorption effect of thesurfactant present in the system, due to the known 1:1 complex-ation between Tween 20 and β-lg [19–22]. This means that, forR � 1, most of the Tween 20 may be unavailable for disruptingthe protein adsorbed layer or for displacing protein from the in-terface [17,18]. What is more likely, however, is that some lim-ited displacement did occur in our unheated emulsions, but thatit was not detectable under the resolution of the confocal micro-scope. Indeed, we have confirmed separately that an emulsionof much higher surfactant/protein ratio (20 vol% oil, 1 wt% pro-tein, R ≈ 16) possessed a completely uniform microstructure,with no evidence of any discernible protein depletion from thesurface of the largest oil droplets (and, incidentally, no changein microstructure on heating).

In our emulsions containing 20 vol% oil and 13.2 wt% pro-tein, it was only during the heat treatment that confocal mi-croscopy gave evidence of protein becoming displaced fromthe oil–water interface. All the systems exhibited gelation whenheld at 85 ◦C for several minutes in tubes in a water bath. Theheating profile used for the emulsions was exactly the sameas that used for the protein solutions (see Fig. 1). The PDRvalues for the different surfactant/protein ratios after heatingare plotted in Fig. 5. It is clear that, in the final gel state ofthe surfactant-free emulsion after heating and cooling down,the protein is evenly distributed within the system (PDR = 1).However, with Tween 20 added, the PDR value decreases untilit reaches the value of PDR ≈ 0.5 for R � 0.75, which means

S. Kerstens et al. / Journal of Colloid and Interface Science 296 (2006) 332–341 339

(a)

(b)

(c)

Fig. 6. Confocal micrograph of the final gel state, after cooling down, of aheated emulsion with surfactant/protein ratio R = 2: (a) Nile Red fluorescence(oil phase); (b) Nile Blue/protein fluorescence; (c) overlay image. Scale barrepresents 30 µm.

that a significant fraction of the protein has moved out of theoil-droplet-rich areas.

Fig. 6 shows a set of images for the final gel emulsion sys-tem with R = 2 after heating and cooling down. The oil dropletsstill appear in a flocculated state (Fig. 6a), but clearly theseareas are now depleted of protein (Fig. 6b). Much of the pro-tein is concentrated in the gaps between the oil-droplet-richareas. In the corresponding overlay image (Fig. 6c) we cansee clear separation of the green and red colors, representingthe distinct regions of Nile Red and Nile Blue fluorescence,

respectively. (Contrast this with Fig. 2c, where fluorescencefrom the protein overlaps directly the fluorescence from the oilphase, resulting in the composite yellow color.) What has hap-pened, we infer, is that the Tween 20 has displaced the β-lgfrom the oil–water interface. Before heating, the free surfactantand protein–surfactant complex are in equilibrium, the rela-tive amounts depending on the value of R. During heating, thegradually unfolding protein releases the bound Tween 20 backinto the aqueous phase, where the increasing concentration offree surfactant starts to displace the protein from the oil–waterinterface. That is, during heating, the system is transformedfrom a protein-stabilized emulsion into a surfactant-stabilizedemulsion. The newly formed surfactant-coated oil droplets areincompatible with the high content of protein in the aqueousphase, and consequently they exclude themselves from the de-veloping protein network. That is, the oil droplets start to act as‘inactive’ fillers, reducing the overall strength of the final emul-sion gel [17,18].

Heat treatment in the presence of surfactant leads to con-version of the initial fine emulsion into a coarse emulsion. Wecan observe in the images of Fig. 6 that there is a consider-able fraction of large droplets of several micrometers diame-ter in the system with R = 2 after heating. This suggests thatthe heat denaturation of β-lg, and its associated displacementfrom the oil–water interface, is accompanied by significant oildroplet coalescence. Evidence for considerable droplet coales-cence during emulsion gel formation was also reported previ-ously by Chen and co-workers [35]. Nevertheless, despite thiscoalescence, there is no visible loss of stability due to cream-ing or oiling off in the final heat-set emulsion system becausethe coarse aggregated droplets eventually become immobilizedin the cross-linked protein gel matrix.

The PDR value of the final gel state for R = 0.75 is thesame as that for R = 2 (see Fig. 5). Probably this implies thatthere is already enough surfactant present at R = 0.75 in theheated emulsion to completely remove the protein from theoil–water interface. Thus adding more surfactant does not nec-essarily mean that any more protein becomes excluded from theoil-droplet-rich areas. For lower Tween 20 contents (R < 0.75),the protein and oil droplets were observed to remain evenly dis-persed together during the heating process (not shown).

Table 2 presents the absolute fluorescence values from theNile Blue/protein complex recorded during heating within thetwo different regions of the emulsion sample with R = 2. Thecorresponding PDR values are plotted in Fig. 7. Clearly wecan see that there is more protein initially located in the oil-droplet-rich areas, but during heating the protein distributionbecomes shifted towards the areas devoid of oil droplets. Thedistribution value changes steadily from PDR ≈ 1.4 at 25 ◦C toPDR ≈ 0.5 at 85 ◦C, and it stays around this latter value untilthe temperature is lowered again to 25 ◦C. The sequence of mi-crographs corresponding to the data points in Table 2 and Fig. 7is presented in an accompanying movie. The separate time-dependent images from Nile Red and Nile Blue fluorescenceare displayed in this movie, together with the overlay image.Initially more (excess) protein is located within the oil-droplet-rich-areas. Gradually protein moves away from these regions

340 S. Kerstens et al. / Journal of Colloid and Interface Science 296 (2006) 332–341

Table 2Mean values of absolute grey levels and their standard deviations (SD) for NileBlue/protein fluorescence measured inside and outside of oil-droplet-rich areasfor a system of surfactant/protein ratio R = 2 as a function of temperature/timeduring thermal processing

Temperature (◦C)(time)

Oil-rich areas Oil-free areas

Mean SD Mean SD

25 177 30 124 2630 150 29 115 2535 154 27 116 2340 138 29 112 2445 123 26 102 2350 118 29 117 2655 119 31 133 3160 120 27 134 2865 128 34 149 3870 93 33 150 3975 87 36 161 4380 63 36 141 5285 63 35 156 4485 (5 min) 72 38 170 4385 (10 min) 72 36 165 4185 (15 min) 93 40 189 3985 (20 min) 70 36 163 4385 (25 min) 73 38 161 4785 (30 min) 87 42 170 4825 88 40 177 47

Fig. 7. Time-dependent change in protein distribution ratio PDR during thermalprocessing for the emulsion system of surfactant/protein ratio R = 2, as calcu-lated from the data in Table 2. A best-fit polynomial has been added as a guideto the eye. Also shown is the temperature/time profile.

until the Nile Blue and Nile Red images become completelycomplementary, indicating that the aggregated oil droplets andthe gelling protein are mutually excluded from each other.

Finally, it seems appropriate to make mention of the pio-neering confocal microscopy investigation of Heertje and co-workers [59,60] demonstrating the displacement of proteinfrom the oil–water interface by emulsifier. There are two maindifferences between Heertje’s experiments and those reportedhere. Firstly, their experiments were carried out in a more uni-form physico-chemical environment, with time as the only vari-able factor, whereas here we describe changes in protein distrib-ution with time, temperature and emulsifier concentration. Sec-ondly, Heertje’s experiments were done at a semi-macroscopicoil–water interface, whereas here we are concerned with study-

ing the changing microstructure of fine emulsions with dropletsof mean size of the order of the resolution of the micro-scope. But, despite the differences, both these studies havedemonstrated the power of the technique for exploring time-dependent changes induced by surfactants in food colloid sys-tems.

4. Conclusions

Model whey protein emulsions containing a high concen-tration of non-adsorbed protein (β-lg) and variable amounts ofnon-ionic surfactant Tween 20 have been examined by con-focal microscopy before, during and after heat-induced gela-tion. In the emulsion system before heat treatment, we haveobserved that the excess protein has more affinity for theprotein-coated droplets than for the aqueous medium. How-ever, the protein distribution is substantially affected by theconcentration of added surfactant, as is the emulsion morphol-ogy as measured by the pore-size distribution, although thesetwo structural characteristics appear not to be directly corre-lated.

Not unexpectedly, we found no evidence for displacementof β-lg from the oil–water interfacial layer (thickness: a fewnanometers) based on observations at ambient temperature withthe confocal microscope (resolution: hundreds of nanometers).However, the manifestation of such competitive displacementwas very apparent on heating the samples (up to 85 ◦C). Dur-ing this process the protein component was seen to moveaway from the aggregated oil droplets into the aqueous con-tinuous phase regions of the emulsion microstructure, so thatfinally the surfactant-coated droplets became phase-separatedinactive filler particles within the heat-set protein gel network.These findings may have potential implications for using thecombination of added emulsifiers and thermal processing tocontrol the texture and rheology of protein-based emulsiongels.

Acknowledgments

The authors thank EPSRC (Grant No. GR/S01566/01) andthe IMPACT Faraday Partnership (UK) for financial support ofthis project.

Supplementary material

The online version of this article contains additional supple-mentary material (the movie).

Please visit DOI: 10.1016/j.jcis.2005.08.046.

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