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Journal of Colloid and Interface Science 296 (2006) 332–341

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Microstructure of β-lactoglobulin-stabilized emulsions containing non-ionic
surfactant 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 free
protein present was investigated via confocal microscopy. The separate distributions of oil droplets and protein were determined using two different
fluorescent dyes. In the emulsion at ambient temperature the excess protein and protein-coated oil droplets were associated together in a reversibly
flocculated state. The pore-size distribution of the initial flocculated emulsion was found to depend on the surfactant/protein ratio R, and at higher
values of R the system became more inhomogeneous due to areas of local phase separation. Evidence for competitive displacement of protein
from 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 emulsion
gel. By measuring fluorescence intensities of the protein dye inside and outside of the oil-droplet-rich areas, we have been able to quantify the
evolving protein distribution during the thermal processing. The results are discussed in relation to previous work on the competitive adsorption
of 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 major challenge for the food industry. Whereas two ingredients
may individually enhance the desired properties of a product,
the interplay of the mixed components is sometimes a destabilizing factor. Whey proteins are widely used as structural
building blocks in food products [1–3]; their ability to denature
and aggregate into a three-dimensional network makes them especially suitable as gelling agents and texture modifiers. Most
frequently the gelation of whey proteins is induced by heating,
although cold-set gels can also be obtained by the addition of
salts or by using high pressure or enzymatic treatments [4–7].
The main molecular interactions involved in the association
and cross-linking of denatured whey proteins are hydrophobic
forces and covalent bonding via intermolecular disulfide linkages [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 both
whey protein molecules and their aggregates, and it is strongly
influenced 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, polyoxyethylene sorbitan monolaurate (Tween 20), influences the
thermal gelation behavior of the major whey protein, β-lactoglobulin (β-lg), both in solution and in β-lg-stabilized emulsions [17,18]. Recently, we reported [19] on the use of confocal
microscopy to follow the microstructure of heated β-lg solutions at pH 6.8 with and without added surfactant. While the
strands making up the final heat-set gel were too small to be resolved by the technique, and the homogeneous final microstructure was visibly unaffected by the presence of Tween 20, we
observed that during the process of heating from 25 to 85 ◦ C
there appeared micrometer-sized aggregates which disappeared
again upon further heating towards the gelation point. Moreover, we found that the temperature-dependent evolution of the
appearance and sizes of these aggregates was dependent on the

influences the overall mechanical behavior of the filled gel. The solutions were adjusted to pH 6. using an oil-soluble dye to identify the oil phase.34]. we do know from the literature [20–22] that Tween 20 binds specifically to β-lg in a 1:1 fashion. Chen and coworkers [35] revealed substantial qualitative effects of added emulsifier on the final microstructure of a heat-set emulsion gel.3. in oil-in-water emulsions. during and after thermal processing. due to their more effective interfacial packing. 4 ml of emulsion were placed in a water bath at 85 ◦ C.4. Nile Blue and Nile Red were purchased from Sigma Chemicals (Poole.2% (w/v) and values of the surfactant/protein ratio R varying between 0 and 2. In order to calculate the surfactant/protein molar ratio R.32] and confirmed by rheological measurements on heat-set whey protein emulsion gels [33. The pH was adjusted dropwise with 1 M HCl to pH 6.34–39] that the elastic shear modulus is indeed greatly enhanced by the presence of the active filler particles. Hence. From a material science point of view. Acros Organics. 2.33–37.8. the interfacial tension is lowered more by smallmolecule surfactants than by proteins. we report on the use of confocal microscopy to follow time-dependent changes in microstructure during the formation of a heat-set emulsion gel. Most of this existing knowledge about the effect of surfactant/protein competitive adsorption on emulsion properties has come from a combination of protein surface coverage measurements made at ambient temperature [29. Gelation time test Test tubes containing ca. we assumed average molecular weights of 1228 Da and 18. at higher concentrations. we examine the effect of varying amounts of Tween 20 on the initial and evolving microstructure of heated β-lg-stabilized emulsions containing a high content of excess unadsorbed protein. 1-Bromohexadecane (99%.S. The composition of the adsorbed monolayer around the droplets also has important implications for the bulk rheology of the system because it influences the nature of the interaction between the filler particles and the protein gel network [30]. R  15) [29]. and so they behave as ‘inactive’ fillers. Here. for the first time. While the exact nature of the aggregates is yet has to be established. There is very little information available on the influence of added surfactants on the emulsion microstructure. Droplets coated with protein are incorporated into the protein gel network via hydrophobic and covalent bonding. 18. UK).18. there is complete displacement of β-lg from the oil–water interface by non-ionic surfactants at high surfactant/protein ratios (i. 333 But this earlier study [35] provided no quantitative information about the distribution of the protein in the system. Spatial distributions of oil droplets and protein are separately monitored using two different fluorescent dyes. Tween 20 (polyoxyethylene sorbitan monolaurate). the presence of emulsion droplets stabilized by a low-molecular-weight surfactant (such as Tween 20) leads to a lower elastic modulus. as well the size. shape and deformability of the inclusions. Visualizing the microstructure in situ seems essential. Preparation of protein solutions Concentrated protein solutions were used to investigate the effect of temperature and environment on the fluorescence behavior of the protein dye Nile Blue. Typically. In a preliminary confocal microscopy study of this type. 2. 0. This stock emulsion was divided into several aliquots. and hence they act as ‘active’ fillers. milk proteins saturate fluid interfaces at much lower concentrations than do small-molecule surfactants [23]. The tubes were taken out for observation at . Previous work in our laboratory on heat-set gels made from whey protein-stabilized (or β-lg-stabilized) emulsions has shown [17. Kerstens et al. 2. Solutions were prepared containing 13. 2.44–49]. On the other hand.e. nor was the microstructure followed in time during heating.1 M HCl. as has been extensively reported [20–28] for systems containing a mixture of Tween 20 + whey proteins (including pure β-lg).5 and 2. polyethylene glycol. The particle-size distribution and mean diameter d32 of the emulsions were determined by static light scattering using a Malvern Mastersizer 2000 with an assumed value of 1.38–43] and bulk rheological measurements [17. and no further purification was applied. lot number 024K7051). / Journal of Colloid and Interface Science 296 (2006) 332–341 surfactant/protein molar ratio R. Belgium) was used as the emulsion oil phase and distilled water was used for the preparation of the solutions. the rigidity of a heatset protein gel can be strongly affected by the presence of socalled ‘filler particles’ like emulsion droplets.0005 for the particle absorption.3 kDa respectively for Tween 20 and β-lg [50].8 with 0. Materials Bovine β-lactoglobulin (L-3908. so it is possible that some kind of reversible selfassembly involving β-lg/Tween 20 complexes lies at the origin of these pseudo-micellar aggregates. however. During gentle stirring each aliquot was topped up with an aqueous solution containing known concentrations of β-lg and Tween 20 to obtain a set of final oil-inwater emulsions (20 vol% oil) having a total protein content of 13.2 wt% β-lg with Tween 20 present at surfactant/protein ratios of R = 0. Materials and methods 2. 2. but droplets stabilized by small-molecule surfactants have little affinity for the surrounding protein matrix. Due to their much higher adsorption energy per molecule.. However. for a proper understanding of the factors affecting the bulk mechanical behavior and stability of emulsions before.1. Preparation of emulsions A starting oil-in-water emulsion (45 vol% oil.7 wt% β-lg in the aqueous phase) was made using a jet homogenizer operating at a constant pressure of 300 bar. The protein sample was a mixture of β-lactoglobulin variants A and B. This role of the filler–matrix interaction has been demonstrated using scanning electron microscopy [31. Specifically.1 for the relative refractive index and 0. The oil volume fraction.2. milk proteins are competitively displaced by surfactants from oil–water and air– water interfaces.

5) of longer working distance (1. some deconvolution technique has to be used to define the threshold between the background and the objects of interest [51.5 ml emulsion) and the sample was excited at 488 nm. but instead the ‘sequential linescanning mode’ was used. in which case small aggregates appeared and then disappeared again during heating. and the dark enclosed regions were assigned to pores and then quantified.1.5 and 2). the average fluorescence intensity returns to an even higher value than before the sample was . Off-set values (signal cut-off values) were all set to zero. indicating the formation of a self-supporting gel. Nile Red was used for staining of the emulsion oil phase (25 µl of 0. We ignored the small protein aggregates that appeared and disappeared in the sample. The aqueous protein solutions gave a uniform picture with no structure visible. 2. The typical steps involved Gaussian smoothing to reduce noise.e. 2. The temperature was increased from 25 to 85 ◦ C at 2 ◦ C min−1 . These settings were not changed during the course of each heating experiment. Results and discussion 3.52].2) were used to study the microstructure of the sample. the fluorescence intensity declines as temperature increases. Fluorescence intensities measured for the protein solutions during heating are given in Fig.5). whereby the two sub-populations of pixels clearly belong to the background and the foreground. Image analysis Fluorescence intensity distributions in the micrographs were analyzed using the confocal microscope operating software. To determine the change in fluorescence intensity distribution during heating. Fluorescence intensity data for Nile Blue and Nile Red were collected in two separate channels corresponding to 503–613 and 653–750 nm. while a dry 20× lens (NA 0. On holding the temperature at 85 ◦ C for 30 min. So. The containers were placed on a Peltier microscope heating stage.5 ml emulsion to obtain a dye/protein ratio 1:100) and excited at 633 nm. after which time the sample was allowed to cool down to 25 ◦ C.. covered with a cover glass (size 1. the pore size was expressed in terms of area (µm2 ).01% (w/v) dye in polyethylene glycol added to 2. At set time intervals we measured the average grey value intensity of the image. and the gel point was defined empirically as the time at which a tube could be turned upside-down without observable downwards flow of its contents. The protein distribution. and sealed with silicone oil to prevent evaporation. Here we implemented thresholding in a series of consecutive steps using the Leica image analysis software Qwin. where Nile Blue was detected. A bleed-through of the signal from the first channel (Nile Red) could be measured in the second channel. Only one collection channel was required for the protein solutions. Confocal microscopy A Leica TCS SP2 confocal system mounted on a Leica DMRXE upright microscope was used to examine the microstructure of the samples. thereby completely avoiding interference due to crossfluorescence. respectively. / Journal of Colloid and Interface Science 296 (2006) 332–341 regular intervals. Every line of pixels in the image was sequentially scanned for the Nile Red and Nile Blue fluorescence. A crucial step in image analysis is thresholding—in order to differentiate the background from the objects of interest within the image. on the basis that they would not affect the average fluorescence intensity. The type of images we obtained here did not have a clear bimodal distribution of grey levels. corresponding to the fluorescence intensity of the dye. i. On cooling back down to 25 ◦ C.5). After this step the image was thresholded. in images taken with the 63× lens. The general tendency shown in Fig.7) in order to have a bigger field of view than with the 63× lens. Pore sizes were measured on the micrographs showing Nile Red fluorescence (indicating the oil phase). Before and after heating both a dry 20× lens (NA 0.5. Therefore it was necessary first to determine the sensitivity of the dye fluorescence to the different conditions.334 S.7) and a 63× water-immersion lens (NA 1. since this parameter will be used to quantify the distribution of the protein component in the emulsions. taken with the lower magnification 20× lens (NA 0. Effect of environmental conditions on fluorescence behavior We aim to quantify the evolving protein distribution in regions of the emulsion sample images by analyzing the fluorescence intensity of Nile Blue. The proteins were stained with Nile Blue (12. Therefore.6. 1 for three surfactant/protein ratios (R = 0. in principle. the temperature was kept constant at 85 ◦ C for 30 min. Emulsions and protein solutions were put in small cylindrical containers (80 µl). Kerstens et al. the fluorescence intensity increases slightly again.15 mm) was used during the heating process to prevent thermal damage to the other lenses. 3. in the presence or absence of Tween 20. as reported elsewhere [19]. The electronic gain setting was adjusted so that each sample had the same initial fluorescence intensity value (170 ± 10 units) and that the changing fluorescence intensity values remained throughout well within the full 0–255 grey scale.5 µl of dye solution added to 2. after which the bright detail was enhanced by adding a ‘whitetop-hat’ filtered image to the original. 0. the analysis was based on micrographs taken with the 20× lens (NA 0. Following this heating ramp. was determined by measuring the fluorescence intensity of Nile Blue inside and outside the oil-droplet-rich areas (using the Nile Red fluorescence of the oil phase as a mask to define the different regions). 1 is that. Here we are solely interested in the behavior of the Nile Blue fluorescence. except when Tween 20 was present and the system was heated. As these dark regions were generally non-circular (non-spherical in 3-D). Micrographs of emulsion samples were analyzed using the Leica image analysis software QWin. fluorescence data for the two dyes were not collected simultaneously. This enhanced image was transformed using a point function (LUT-transform) whereby lower intensities were suppressed.

the temperature. the measured particle-size distribution was unaffected by the subsequent operations of diluting the aliquots or adjusting the pH. With addition of Tween 20. gain setting = 425 V.2 wt% β-lg. One plausible explanation [54] is that the presence of surfactant micelles prevents radiationless deactivation of the excited dye to its ground state. 2.2% (see below). Together with the fact that complexation of Nile Blue with the protein hinders rotational relaxation processes and increases the quantum yield of the dye [54]. So in our systems the fluorescence intensity is predominantly dependent on the surfactant (micelle) concentration. The original stock emulsion (45 vol% oil. when the temperature reaches 85 ◦ C.75. / Journal of Colloid and Interface Science 296 (2006) 332–341 335 3. Heating of fluorochromes can often induce reduction in quantum efficiency due to the increased vibration and torsion of their flexible side-arms. No surfactant micelles would be expected to exist. resulting in a higher overall fluorescence intensity. therefore. 3 shows the effect of the added surfactant on the microstructure of the emulsion at 25 ◦ C. Others factors that could also potentially affect the measured fluorescence intensity are the relative concentrations of dye and protein and the pH. except for some exceptionally large ones. arising from some immobilization of the dye within the aggregated protein network. For large surfactant additions (R > 1. the protein added after homogenization has greater affinity for the weakly aggregated protein-coated oil droplets than for the aqueous serum. In the low-protein-content stock emulsion. Emulsion systems Fig. Kerstens et al. 3d that. the heterogeneity apparent in Fig. it is clear that the protein-coated oil droplets and the excess protein present in the aqueous continuous phase are in a closely associated state— hence the predominantly yellow appearance of the overlay image (green + red → yellow). depending on the amount of Tween 20 present in the system. the error in mean pore size diverges as the structure becomes quite inhomogeneous.53]. left axis) and the maximum pore size (dashed bars. in Fig. But each of these is kept constant. The flocculation evident in Fig. despite some large open (dark) areas in the image. as reported previously for Rhodamine B [19. For the system represented in Fig. gain setting = 400 V. and the aggregation state of the protein (or protein–dye complexes).38 ± 0. the pore-size distribution varies substantially. we can see how one big oil droplet towards the left side of the picture gives a dark round hole (no discernible intensity) in the image recording the Nile Blue/protein fluorescence. the mean pore size decreases initially. A lower gain setting has to be used when increasing the surfactant concentration in order to obtain similar average grey scale values in the image at room temperature. A further rigidity-enhancing effect in heat-set globular protein gels takes place during the cooling down stage. 4 for the maximum pore size. Fig. Effect of thermal processing on Nile Blue fluorescence of protein solutions (13.5) all of the Tween 20 is expected to be bound to β-lg [19–22]. In the solution of low surfactant content (R = 0. and image (b) represents the fluorescence from the protein stained with Nile Blue. 1. 2 had therefore been induced by ‘topping up’ the high-volume-fraction stock emulsion with the concentrated β-lg solution as described in Section 2. dye/protein ratio 1:100) in the presence and absence of Tween 20. (Q) R = 2. For instance. The most probable droplet diameter was around 0. It would therefore seem that. Fig. 2. While flocculation was induced on subsequently adjusting the protein concentration to 13. there are still some areas with much smaller pores. reversible flocculation. right axis) as derived from the image analysis. there was no evidence of flocculation. Individual oil droplets could not normally be distinguished. at the low ionic strength of this system. until the protein denatures on heating and releases the bound Tween 20 back into the solution. For instance. Fig. and that this manifests itself in the micrographs as local phase separation. 4 shows the effect of surfactant content on the mean pore size (white bars. Image (a) represents the fluorescence collected from the oil phase stained with Nile Red.5 µm and the d32 value given by the Mastersizer was 0. straight after homogenization. we can see in Fig.5.S. gelation takes place [19]. The second main feature of the protein solution data is the increase in fluorescence intensity of Nile Blue on addition of Tween 20. including fluorescence from both the protein (Nile Blue) and the oil (Nile Red) are given for R = 0. (") R = 0. The solid line represents the heating profile. resulting in an internal radiationless transition.02 µm. By eye we can see that. gain setting = 385 V. As no discernible change in mean droplet size d32 was detectable on dilution in the Mastersizer. 2. This latter behavior suggests to us that no substantial photo-bleaching is involved. gelation might explain the increase in fluorescence intensity on holding at 85 ◦ C.2.3. 2 shows a set of images for the 20 vol% oil-in-water emulsion with 13.7 wt% protein) gave a set of images (not shown) with a homogeneous distribution of the oil droplets.25 and 2. For this reason we also include data in Fig. The fluorescence intensity is plotted against time: (!) R = 0. heated. and then it increases strongly. due to strengthening of hydrogen bonding and electrostatic forces [17. 0. The quoted error bars refer to the standard error.2% β-lg in the aqueous phase (no surfactant present). 1.18]. Overlay micrographs. The overlay image (c) permits location of the two dyes with respect to each other. further complicating the interpretation of the fluorescence intensity. In our system. 2 must be the result of a process of weak. Whereas the .5). This could explain the increase in the fluorescence intensity after cooling to a value even higher than for the original unheated sample.

Such reversible depletion flocculation of emulsion droplets due to the presence of surfactant micelles is well recognized in the literature [55. free surfactant (monomer + micelles) would be expected to be present in the system. 2b) as points of zero fluores- . inducing local phase separation. The arrow identifies a circular dark hole in the Nile Blue/protein image corresponding to a single large oil droplet. In our case this is not feasible. the electronic gain and ‘off-set’ settings on the confocal microscope had to be changed from sample to sample. Confocal micrograph of a sample of an oil-in-water emulsion (20 vol% oil. 2 for R = 0. In strong contrast. Fig. and adjusted during heating in order to keep the measured fluorescence level within the 0–255 grey scale range. of course. at the lower Tween 20 concentrations (R  1). Fig. the images corresponding to the separately collected fluorescence data have the same general appearance as images (a) and (b) in Fig.336 S. Corresponding to Fig. in contrast to the clustered regions of aggregated protein as seen in Fig. In order to standardize this process. however. 3c. 3c (R = 1. 2.75 and 2. (b) image from Nile Blue/protein fluorescence.75) values of the maximum pore size.25) has a red colored background instead of a black one. calculated with respect to all the protein present in the system. Furthermore. This is possibly attributable to the known 1:1 complexation of Tween 20 with β-lg [20–22]. we can see that the protein-coated oil droplets and the excess protein are roughly located within the same local regions.56]. 3 shows that the surfactant content strongly affects the way that the protein is distributed within the microstructure. indicating that at this composition the non-adsorbed protein is spread out evenly throughout the aqueous continuous phase. 3c and 3d. The R values quoted above are. small amounts of surfactant probably already absorb at the interface for R  1. Kerstens et al. 2b. (c) overlay image combining fluorescence from both dyes. At higher magnification. 13. we can see that there is a nearly 30-fold difference between the lowest (R = 0.g. (b) and (d). In practice. we have separately estimated the average fluorescence intensities of Nile Blue/protein complex inside and outside of the oil-droplet-rich areas. Absolute quantification would require a single calibration curve under strictly defined conditions [54]. The protruding polyoxyethylene chains might thereby affect the solubility of the aggregated proteins. Furthermore. the primary image indicating just the Nile Blue/protein fluorescence has a rather uniform appearance (not shown). 0. Scale bar represents 30 µm. / Journal of Colloid and Interface Science 296 (2006) 332–341 (a) (b) (c) Fig.. while the average pore size is a reasonable indicator of structural homogeneity. all the surfactant present is expected to be bound to the protein for R  1. corresponding to R = 0. These combined factors add some uncertainty as to the precise value of the free surfactant concentration in the system. see Fig. From our previous calculations [19]. In micrographs (a). At higher surfactant/protein ratios. we took the occasional voids of big oil droplets in the micrographs of the protein fluorescence (e.1).5) and the highest (R = 1. illustrating the further complexity of this notionally relatively simple system of β-lg + Tween 20. In order to quantify how the protein is distributed within these samples. but it is likely that the protein adsorbed at the oil–water interface interacts differently with the Tween 20. the pores are smaller than in the absence of surfactant.1). average pore size only changes by a factor of ∼5 over the whole range of surfactant contents. 25 ◦ C) with no Tween 20 added: (a) image from Nile Red fluorescence (highlighting the oil phase). instead of being closely associated with the aggregated protein-coated oil droplets. respectively. however. as seen in Figs. So it would seem that. It is noteworthy that. it should properly be supplemented by information on the whole range of pore sizes. This is the opposite effect to that observed for the pure protein solutions (Section 3. an increase in the Tween 20 concentration in our emulsion systems was found to lead to a decrease in fluorescence intensity.2 wt% β-lg in the aqueous phase. because the environment experiences continuously changing conditions that affect the fluorescence behavior in a complex manner (see Section 3.

Fig. a PDR substantially higher than unity implies that the excess unadsorbed protein is mainly associated with the oil droplets. (b) R = 0. Effect of the addition of surfactant on the microstructure of an oil-in-water emulsion (20 vol% oil. cence intensity.2 wt% β-lg in the aqueous phase. 13. (d) R = 2.S. a value near . 25 ◦ C). (c) R = 1. Standard errors are given for the mean pore size. Effect of the surfactant/protein ratio R on the mean pore size (white bars.75. Scale bar represents 100 µm. Each of the four confocal micrographs is an overlay image incorporating both Nile Red and Nile Blue fluorescence: (a) R = 0.25. then any other region in the micrograph showing a higher intensity value could be properly regarded as a region with a true positive fluorescence contribution. So. On this basis we introduce a protein distribution ratio (PDR) defined as the protein fluorescence within the oil-droplet-rich areas divided by the protein fluorescence outside the oil-droplet-rich areas. Kerstens et al. as opposed to one with just amplified background noise. Setting this reference level to a zero intensity value. 4. / Journal of Colloid and Interface Science 296 (2006) 332–341 (a) (b) (c) (d) 337 Fig. left scale) and maximum pore size (shaded bars. right scale). 3.

4 2. This means that five times more fluorescence is measured in the oil-droplet-rich areas than outside.338 S. both before and after heating.9 6.4 191.7 8.8 28.1 36.4 183.2 wt% protein. The systems before thermal treatment were stable towards changes in visible appearance and microstructure for at least 3 days on storage at refrigerator temperature. The PDR values for the different surfactant/protein ratios after heating are plotted in Fig. Comparison of the data in Figs. (!) after heating. there is a local phase separation between a surfactant-rich phase and a phase containing non-adsorbed protein + proteincoated oil droplets. This is not really surprising. for R  1. it appears that the excess Tween 20 makes the protein more soluble (dispersible) within the continuous phase. Indeed. when the surfactant/protein ratio exceeds unity. At even higher surfactant concentrations. but that it was not detectable under the resolution of the confocal microscope.8 13.5 7. 1 wt% protein. we have PDR ≈ 1.4 184.3 152.4 204.7 95. Previous research has demonstrated [20–29] that addition of Tween 20 or some other non-ionic surfactant after emulsion formation leads to complete spontaneous displacement of protein from the oil–water interface at high surfactant/protein ratios (R  15).6 38.25 and R = 1.3 2. In the present study we are only considering relatively low surfactant/protein ratios (up to R = 2).2 9.3 82. for R  1. we have confirmed separately that an emulsion of much higher surfactant/protein ratio (20 vol% oil.0 179.2 19. however. Hence. indicating that the protein is fairly evenly distributed throughout the system. It is clear that.3 176. At R = 1.6 1.3 11. we see that the PDR value is high (5 ± 1). with Tween 20 added.75 is already higher than for R = 15 in the earlier study [29]. which means . incidentally.7 98.75.5 for R  0. as already qualitatively observed in Fig. the protein is evenly distributed within the system (PDR = 1). is that some limited displacement did occur in our unheated emulsions. the PDR value decreases until it reaches the value of PDR ≈ 0.4 64. Fig. Table 1 lists the sets of measured grey level intensities inside and outside the oil-droplet-rich areas. due to the known 1:1 complexation between Tween 20 and β-lg [19–22].25 1.6 128.5 101.8 177..2 9. as the thickness of an adsorbed monolayer at the oil–water interface is no more than a few nanometers.5 8. in our emulsions the large amount of free protein may ‘neutralize’ the competitive adsorption effect of the surfactant present in the system. 4 and 5 shows no obvious direct correlation between the effects of the added surfactant on the pore size and the protein distribution.5 0. 5. possibly by a molecular mechanism involving the shielding of hydrophobic regions of the protein from the solvent [57].0 6.6 81.6 19.2 3. 3c.75 2 Before heating After heating Oil-rich areas Oil-free areas Oil-rich areas Oil-free areas Mean SD Mean SD Mean SD Mean SD 164.0 188.0 51. In our emulsions containing 20 vol% oil and 13.2 9.0 Fig.8 7. Under the confocal microscope there was no obvious evidence of displacement of protein from the oil–water interface by Tween 20. no change in microstructure on heating).7 6.18]. 5.1 157.7 16. with no evidence of any discernible protein depletion from the surface of the largest oil droplets (and.9 9.5 10.2 wt% for the emulsions studied here. unity corresponds to a fairly homogeneous distribution of protein within the system. It has been recognized recently [58] that the stability properties of β-lg-based emulsions at ambient temperature are affected considerably by the presence of free protein.6 5.75 1 1.6 2. the excess (non-adsorbed) protein has a high affinity for the oil-coated droplets.0 135. as compared with a resolution of not less than a few hundred nanometers for the confocal microscope. as compared to 13. Effect of the surfactant/protein ratio R on the protein distribution ratio PDR as calculated from the grey values listed in Table 1: (") before heating.7 178. for the case of no surfactant present or only a small amount. / Journal of Colloid and Interface Science 296 (2006) 332–341 Table 1 Mean values of absolute grey levels and their standard deviations (SD) for Nile Blue/protein fluorescence before and after heating measured inside and outside of oil-droplet-rich areas as a function of the surfactant/protein ratio R R 0 0.8 161. Furthermore. R ≈ 16) possessed a completely uniform microstructure.5 12.5 wt% [29]). most of the Tween 20 may be unavailable for disrupting the protein adsorbed layer or for displacing protein from the interface [17. although substantially more protein is still present outside the oil-droplet-rich areas as compared to the systems of low surfactant content. However. But typically in this earlier work the emulsion contained a low concentration of protein (e.3 174.1 193. 5 shows that the protein again becomes more closely associated with the oil droplets. 1).2 175. This means that.3 166. in the final gel state of the surfactant-free emulsion after heating and cooling down.0 11.7 2. On the other hand. it was only during the heat treatment that confocal microscopy gave evidence of protein becoming displaced from the oil–water interface. and a value considerably below unity means that the protein is mainly distributed outside the oildroplet-rich areas. 0. For higher surfactant/protein ratios.5 8. 5. The calculated PDR values are plotted as a function of R in Fig. What is more likely.9 177.9 2. Before heating. even though in absolute terms the concentration of Tween 20 in our emulsion at R = 0.5 1.1 6.1 150. All the systems exhibited gelation when held at 85 ◦ C for several minutes in tubes in a water bath.5 16.5. The heating profile used for the emulsions was exactly the same as that used for the protein solutions (see Fig.4 8.3 83.1 8. Kerstens et al.g.

the gradually unfolding protein releases the bound Tween 20 back into the aqueous phase. Confocal micrograph of the final gel state. Much of the protein is concentrated in the gaps between the oil-droplet-rich areas. resulting in the composite yellow color. / Journal of Colloid and Interface Science 296 (2006) 332–341 (a) (b) (c) Fig. The separate timedependent images from Nile Red and Nile Blue fluorescence are displayed in this movie. of a heated emulsion with surfactant/protein ratio R = 2: (a) Nile Red fluorescence (oil phase). (b) Nile Blue/protein fluorescence. 2c. Initially more (excess) protein is located within the oil-dropletrich-areas.75). together with the overlay image. the protein and oil droplets were observed to remain evenly dispersed together during the heating process (not shown).75 in the heated emulsion to completely remove the protein from the oil–water interface. The newly formed surfactant-coated oil droplets are incompatible with the high content of protein in the aqueous phase. we infer.4 at 25 ◦ C to PDR ≈ 0. there is no visible loss of stability due to creaming or oiling off in the final heat-set emulsion system because the coarse aggregated droplets eventually become immobilized in the cross-linked protein gel matrix. During heating. representing the distinct regions of Nile Red and Nile Blue fluorescence. is that the Tween 20 has displaced the β-lg from the oil–water interface. but during heating the protein distribution becomes shifted towards the areas devoid of oil droplets. Evidence for considerable droplet coalescence during emulsion gel formation was also reported previously by Chen and co-workers [35]. and it stays around this latter value until the temperature is lowered again to 25 ◦ C. In the corresponding overlay image (Fig. 5). Probably this implies that there is already enough surfactant present at R = 0.18]. 7 is presented in an accompanying movie. but clearly these areas are now depleted of protein (Fig. 7. The distribution value changes steadily from PDR ≈ 1.) What has happened. 6c) we can see clear separation of the green and red colors. (c) overlay image. Table 2 presents the absolute fluorescence values from the Nile Blue/protein complex recorded during heating within the two different regions of the emulsion sample with R = 2. The PDR value of the final gel state for R = 0. and consequently they exclude themselves from the developing protein network.5 at 85 ◦ C. That is. the system is transformed from a protein-stabilized emulsion into a surfactant-stabilized emulsion. 6. For lower Tween 20 contents (R < 0. 6b).75 is the same as that for R = 2 (see Fig. That is. (Contrast this with Fig. The sequence of micrographs corresponding to the data points in Table 2 and Fig. the oil droplets start to act as ‘inactive’ fillers. and its associated displacement from the oil–water interface. Before heating. 6 shows a set of images for the final gel emulsion system with R = 2 after heating and cooling down. Clearly we can see that there is more protein initially located in the oildroplet-rich areas. 6a). where fluorescence from the protein overlaps directly the fluorescence from the oil phase. Heat treatment in the presence of surfactant leads to conversion of the initial fine emulsion into a coarse emulsion. The corresponding PDR values are plotted in Fig. 6 that there is a considerable fraction of large droplets of several micrometers diameter in the system with R = 2 after heating. the free surfactant and protein–surfactant complex are in equilibrium. after cooling down. Thus adding more surfactant does not necessarily mean that any more protein becomes excluded from the oil-droplet-rich areas. We can observe in the images of Fig. The oil droplets still appear in a flocculated state (Fig. This suggests that the heat denaturation of β-lg. Scale bar represents 30 µm. despite this coalescence. Fig. reducing the overall strength of the final emulsion gel [17. during heating. Gradually protein moves away from these regions .S. that a significant fraction of the protein has moved out of the oil-droplet-rich areas. is accompanied by significant oil droplet coalescence. 339 respectively. Kerstens et al. the relative amounts depending on the value of R. where the increasing concentration of free surfactant starts to displace the protein from the oil–water interface. Nevertheless.

as calculated from the data in Table 2. Kinsella. so that finally the surfactant-coated droplets became phase-separated inactive filler particles within the heat-set protein gel network. Bryant.2005. A best-fit polynomial has been added as a guide to the eye. Kinsella. until the Nile Blue and Nile Red images become completely complementary.V. whereas here we are concerned with study- The authors thank EPSRC (Grant No. despite the differences. indicating that the aggregated oil droplets and the gelling protein are mutually excluded from each other. In the emulsion system before heat treatment. McSwiney. These findings may have potential implications for using the combination of added emulsifiers and thermal processing to control the texture and rheology of protein-based emulsion gels. Res. Condon (Eds. Morr. Conclusions Model whey protein emulsions containing a high concentration of non-adsorbed protein (β-lg) and variable amounts of non-ionic surfactant Tween 20 have been examined by confocal microscopy before. 44 (4) (1990) 100.J. whereas here we describe changes in protein distribution with time. as is the emulsion morphology as measured by the pore-size distribution. both these studies have demonstrated the power of the technique for exploring timedependent changes induced by surfactants in food colloid systems. Campanello. Singh. Fox. 9 (1998) 143. Firstly. with time as the only variable factor. D. Heertje’s experiments were done at a semi-macroscopic oil–water interface.08. we found no evidence for displacement of β-lg from the oil–water interfacial layer (thickness: a few nanometers) based on observations at ambient temperature with the confocal microscope (resolution: hundreds of nanometers).340 S. However. Food Technol. Foegeding.jcis.F. p. Trends Food Sci. 1982. Applied Science. McClements. their experiments were carried out in a more uniform physico-chemical environment. Technol. Food Proteins. Not unexpectedly. GR/S01566/01) and the IMPACT Faraday Partnership (UK) for financial support of this project. H. Food Hydrocoll.E. Please visit DOI: 10. the manifestation of such competitive displacement was very apparent on heating the samples (up to 85 ◦ C).M. However.M. Supplementary material The online version of this article contains additional supplementary material (the movie). 33 (1989) 343. O. J. 4. London.1016/j. References [1] J.60] demonstrating the displacement of protein from the oil–water interface by emulsifier. / Journal of Colloid and Interface Science 296 (2006) 332–341 Table 2 Mean values of absolute grey levels and their standard deviations (SD) for Nile Blue/protein fluorescence measured inside and outside of oil-droplet-rich areas for a system of surfactant/protein ratio R = 2 as a function of temperature/time during thermal processing Temperature (◦ C) (time) Oil-rich areas Mean SD Mean SD 25 30 35 40 45 50 55 60 65 70 75 80 85 85 (5 min) 85 (10 min) 85 (15 min) 85 (20 min) 85 (25 min) 85 (30 min) 25 177 150 154 138 123 118 119 120 128 93 87 63 63 72 72 93 70 73 87 88 30 29 27 29 26 29 31 27 34 33 36 36 35 38 36 40 36 38 42 40 124 115 116 112 102 117 133 134 149 150 161 141 156 170 165 189 163 161 170 177 26 25 23 24 23 26 31 28 38 39 43 52 44 43 41 39 43 47 48 47 Oil-free areas ing the changing microstructure of fine emulsions with droplets of mean size of the order of the resolution of the microscope. But. . Time-dependent change in protein distribution ratio PDR during thermal processing for the emulsion system of surfactant/protein ratio R = 2. Finally. temperature and emulsifier concentration. the protein distribution is substantially affected by the concentration of added surfactant. Adv. Secondly.046.). 7. D. although these two structural characteristics appear not to be directly correlated. Also shown is the temperature/time profile. [4] C. we have observed that the excess protein has more affinity for the protein-coated droplets than for the aqueous medium.A.E. Whitehead. [3] J. E. it seems appropriate to make mention of the pioneering confocal microscopy investigation of Heertje and coworkers [59. 51. Acknowledgments Fig. [2] C. During this process the protein component was seen to move away from the aggregated oil droplets into the aqueous continuous phase regions of the emulsion microstructure.H. during and after heat-induced gelation. Kerstens et al. 8 (1994) 441. [5] M. Food Nutr. in: P.E. There are two main differences between Heertje’s experiments and those reported here.

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