THE EFFECT OF COMPETITIVE ADSORPTION BETWEEN PROTEINS OF DRIED EGG YOLK AND TWEEN 65 ON THE STABILITY AND MICROSTRUCTURE OF OIL-IN-WATER EMULSIONS

Grażyna Bortnowska
Department of Food Technology, West Pomeranian University of Technology, Szczecin, Poland

ABSTRACT

Stability of the oil-in-water emulsions composed of 30% (w/w) rapeseed oil and emulsifying mixtures containing dried egg yolk and Tween 65 were investigated. It has been shown that, stability of emulsions was in range from 37.5% to 58.2% and increased in most cases with the addition of emulsifiers, particularly Tween 65. It was found that surface protein concentration in emulsions decreased as concentration of Tween 65 increased, as the result of competitive adsorption. Changes in interfacial protein concentration probably caused instability of emulsions, observed as oiling off effect, especially in samples heated at 80°C and containing 2.0% (w/w) dried egg yolk and 0.8 or 1.0% (w/w) Tween 65. Furthermore, increase of synthetic emulsifier concentration decreased average droplet diameters D [3,2] from 1.42 mm to 0.28 mm, considerable increasing interfacial areas. Composition of the emulsifying mixtures affected modal diameter of oil droplets especially in the emulsions containing: 0.2; 0.6 and 1.0% (w/w) of dried egg yolk, what may be explained by the fact that the mixtures exhibited different emulsifying properties in the studied range of concentrations. Visual inspection of the investigated emulsions in the microscope showed that all emulsions had individual oil droplets with no signs of flocculation.

Key words: oil-in-water emulsion, dried egg yolk, Tween 65, competitive adsorption, surface protein concentration, emulsion stability.

INTRODUCTION

An oil-in-water emulsion is a thermodynamically unstable system and forms the basis of many food products. It is created when oil and water are rapidly mixed in the presence of food emulsifier [7,19].

Hen egg yolk associates appreciated organoleptic characteristics with very good emulsifying properties so that it has become a key ingredient for the preparation of a large variety of food emulsions [2,6,17,18]. Typical examples of emulsions made with egg yolk are mayonnaises and sauces, which are oil-in-water emulsions with a texture that is particularly appreciated by consumers [21,22,24]. The emulsifying ability of egg yolk is related mainly to the lipoproteins whose amphipathic character allows them to interact with the oil-water interface and also lower the surface tension of the aqueous phase [1,3]. However, most formulated food emulsions contain very often not only proteins but also small-molecule surfactants (emulsifiers) which compete for the emulsion interface during and after formation of the emulsion [26]. The ability of small-molecule surfactants to displace macromolecules from interfaces is related to their high adsorption energy compared to individual segments of the macromolecule [14]. The distribution of proteins and surfactants between the droplet surface and the bulk phase is an important factor affecting the stability, texture and ease of formation of an emulsion [12]. It has been reported that addition of Tween 20 to a β-lactoglobulin-stabilized emulsion results in competitive displacement of the adsorbed globular protein film and the formation of less flocculated emulsion [9]. However, the presence of some of the others molecules at the interface may lead to the protein-protein interactions, flock formation in the emulsion continuous phase and very rapid serum separation [11].

Most of the above studies focused mainly on mixtures containing proteins and surfactants, therefore from technological point of view is more relevant to study systems containing more complex emulsifying mixture as egg yolk with added surfactants.

The objective of this study was evaluation, in which range the competitive adsorption between proteins of commercial dried egg yolk and Tween 65 affects stability and microstructure of oil-in-water emulsions.

MATERIALS AND METHODS

Materials
For the preparation of emulsions the following ingredients were used: vegetable rapeseed oil manufactured by the Oil Plant at Kruszwica and purchased in retail outlet; dried egg yolk produced by "BASSO" (Nowy Sącz), containing according to manufacturer specification 58.6% (w/w) fat, purchased from Hortimex (Konin); Tween 65 (polyoxyethylene sorbitan tristearate) with the estimated hydrophile-lipophile balance (HLB) of 11, purchased from Sigma-Aldrich (Poznań).

Emulsion preparation
The emulsion aqueous phase was prepared by dissolving an appropriate amount of surface-active material (dried egg yolk or its mixture with Tween 65) in disodium hydrogen phosphate – citric acid aqueous solution at pH 7.0 [20] with 0.04% (w/w) sodium azide added to protect against microbial contamination. The mixture of oil and aqueous solution was homogenized using an MPW 302 laboratory homogeniser (Mechanika Precyzyjna, Warszawa) for 30 s at 3.500 rpm. Final concentration of emulsifiers in the emulsions were as specified in Table 1 while the rapeseed oil content was constant at 30% (w/w) in all emulsions.

Surface protein concentration
The surface protein concentration (mg protein · m^-2) was determined by the method described by [12,15]. The protein content was assayed by the Kjeldahl method (1026 Distilling Unit and 1007 Digester, Tecator AB, Höganäs, Sweden). A factor 6.68 was used to convert nitrogen to protein content [23].

Average droplet diameter
Sample droplet size and distribution were measured by the method described by [25], using microscope (Matic^®B1 Series, Carlzeiss Jena equipped with a built in camera and software Multiscan v.11.06, Computer Scanning Systems). Droplet size measurements were made using 0.1 and 0.01 scales. Average droplets diameters D [3,2] and D [3,23] µm of the emulsion droplets were determined by the methods described by [8,9].

Emulsion stability
Emulsion stability has been characterized in terms of changes in the average particle size parameter D [4,3] and towards creaming [9]. The stability of the emulsions (ES) towards creaming was assessed after accelerated ageing [16,18,26]. The emulsions (8.00 ±0.03 ml) were poured into the 10 ml centrifugation tubes and were stored for 30 min at ambient temperature (25 ±0.5°C) or heated at 80 ±0.5°C for 30 min in a MT -1 thermostat (Horyzont) and then heated samples were cooled to the temperature 25 ±0.5°C. The tubes with emulsions were then centrifuged at 1983.6 g for 10 min at 25 ±0.5°C. Centrifugation produced an aqueous phase on the bottom, an emulsion phase in the middle and an oil phase on top. The emulsion stability (ES) was calculated as follows:

ES (%) = (remaining emulsion height / initial emulsion height) · 100

Viscosity measurement
Viscosity of emulsions was investigated using a Rheotest (2-50 Hz), RV2 viscometer equipped with S/S1 cylinder. The measurements were carried out at 25 ±0.5°C and constant shear rate 437.4 (s^-1).

Statistical analysis
Three replicates were conducted for all measured parameters and data were statistically treated using One-way ANOVA program Statistica 6.0. Significant differences between means were identified by LSD procedure (Tukey's test, p < 0.05).

RESULTS AND DISCUSSION

Oil-in-water emulsions with the addition of emulsifying mixtures containing dried egg yolk and Tween 65 were studied. Two temperatures have been applied (25 and 80°C) to evaluate their utility for preparation of cold and hot sauces. The emulsions heated at 80°C were found to exhibit very large changes in stability, which occurred as oiling off effect, therefore these samples were evaluated only towards creaming. However, emulsions  heated at 25°C did not indicate those changes and their stability was investigated both towards creaming and average particle size parameter D [3,23].

Fig. 1 shows that stability of all investigated emulsions towards creaming was in range from 37.5% to 58.2% and depended on concentration as well on the weight ratio of emulsifiers applied. The highest instability showed emulsions without Tween 65 addition, containing 0.2% (w/w) dried egg yolk, stored at 25°C and heated at 80°C and the highest stability was observed for the emulsions stabilized with the mixture consisting of 2.0% (w/w) dried egg yolk and 1.0% (w/w) Tween 65 but only stored at 25°C. As stability of the emulsions stored at 25°C is generally considered, increase of Tween 65 concentration caused higher improvement in the emulsified layer than dried egg yolk addition. The emulsions marked with codes: A, C, D, containing: 0.2; 1.0 and 2.0% (w/w) dried egg yolk, respectively heated at 80°C exhibited oiling off effect as the result of the oil droplets coalescence and their migration towards the surface (Table 1). The amount of oil phase present on the surface of emulsions depended on the weight ratio and concentration of the emulsifiers applied. For example, increase of the concentration of Tween 65 in the emulsifying mixture containing 0.2% (w/w) dried egg yolk (samples: A1 – A6) decreased the amount of oil phase observed on the surface of emulsions, while in the samples D1 – D6, containing 2.0% (w/w) dried egg yolk the amount of this phase increased, promoting rapid destabilization (Table 1). The highest percentage of oil in the surface layer of emulsions was observed in the samples D5 and D6 and averaged to 42.2% in relation to the amount of oil used to prepare these samples (Fig. 1 and Table 1). This can be interpreted in terms of close relationship between stability of the emulsions and concentration of the emulsifiers. At very low concentrations, the emulsifying agent can not adequately stabilize the emulsion. In this case, the emulsion may degrade through bridging flocculation and coalescence. It also was found that bulk surface-active material present in excess, can cause destabilization of the emulsions as the result of depletion flocculation process [7,10].  Some of these studies suggest that  the most likely reason of observed changes in the stability of  investigated emulsions could be competitive adsorption between highly surface active ingredients of egg yolk and Tween 65 and resulting displacement of egg lipoproteins from interfacial area. This process could be contributed to the break a stable network at oil-water interface and imply lower stability of the emulsions [21,22,24]. The amount of the protein adsorbed at the droplet surface depended mainly on the concentration of protein in emulsions, but also close correlation was found between surfactant to protein weight ratios. Fig. 2 shows that in emulsions containing for example: 0.2; 0.6; 1.0 and 2.0% (w/w) dried egg yolk (samples: A1; B1; C1 and D1), the appropriate protein surface concentrations were as follows: 0.22; 0.42; 0.55 and 0.86 (mg · m^-2). Taking into account excellent emulsifying properties of the egg yolk proteins [21,22], it possible that  increasing surface protein concentrations could contribute to the stability of the emulsions (Fig. 1). The addition of Tween 65 caused displacement of the proteins from interfacial area. For example, in the emulsions containing the highest concentration of Tween 65 (samples: A6, B6, C6 and D6), the surface protein concentration decreased in comparison to the control samples without synthetic emulsifier (samples: A1, B1, C1 and D1) by: 1,8; 11,2; 38,2 and 48,8%, respectively (Fig. 2). General view of proteins – Tween 65 interactions is given in Fig. 3 which shoves that adsorption of the proteins on the interfacial area depended on the Tween 65 to dried egg yolk weight ratio (R). Similar results were achieved by Euston et al. [12], who studied competitive adsorption between caseins and Tween 60 and noticed  that protein surface concentration depended only on Tween 60-to-protein molar ratio. It is supposed that stability of the emulsions stored at 25°C did not depend on the surface protein concentration. However, the amount of adsorbed proteins could affect stability of the emulsions heated at 80°C [4,13]. The apparent viscosity of the studied emulsions was relative to the concentration of surface-active material. Increase of the concentration of dried egg yolk caused increase of the apparent viscosity and addition of Tween 65 gradually decreased this parameter what was particularly noticeable between samples: B1-B2; C1-C2 and D1-D2 (Table 1). This may be explained as the result of changes in the composition of the interfacial layer [7] (Fig. 3).

Thermodynamic instability of the emulsions has been characterized in terms of changes in the particle size parameter D [3,23] [5,9]. Statistical results indicated that the addition of Tween 65 had significant effect on the average diameter D [4,3] in nearly all investigated emulsions. The highest differences were observed between emulsions A1 and A6, where addition of Tween 65 decreased D [4,3] from 1.67 to 0.35 µm, respectively. In others emulsions, changes in D [4,3] parameter were smaller and depended on Tween 65 to dried egg yolk weight ratio (Table 1). Analysis of volume-surface average droplet diameters also showed that there was close relation between composition of emulsifying mixture and particle size parameter D [3,2]. Average droplet diameters D [3,2] decreased with increasing concentration of Tween 65  in the range from 1.42 to 0.28 µm and additionally it was found that this trend was nearly the same as relation between adsorbed proteins and synthetic emulsifier concentration (Fig. 2). The average droplet diameters D [3,2] in the emulsions stabilized with addition of dried egg yolk (samples A1, B1, C1 and D1) had the values of: 1.42; 0.65; 0.56 and 0.60 µm, respectively and in emulsions containing mixture consisting of dried egg yolk and 1% (w/w) Tween 65 (samples: A6, B6, C6 and D6) had the values of: 0.29; 0.43; 0.39 and 0.28 µm, respectively (Fig. 2). Changes in the average droplet diameters may  be explained by partial displacement of proteins from interfacial area by Tween 65 which could be stronger surface active agent lowering interfacial tension to a greater extent [25]. Regular decrease of D [4,3] and D [3,2] parameters observed with increasing Tween 65 concentration, may be explained by slightly antagonistic effect of the protein and surfactant, rather than synergistic at those weight ratios [12]. In relation  to the volume-surface average droplet diameters D [3,2] the specific surface area (m² · cm^-3 oil) was calculated. Table 1 shows that the highest changes in interfacial areas were observed in the samples containing 0.2% (w/w) of dried egg yolk, and in emulsions: A1, A 2, A3, A4, A5 and A6 the specific surface areas had the values of: 4.23; 10.90; 11.32; 15.38; 14.63 and 20.69 (m² · cm^-3 oil), respectively. The particle size distribution of the emulsion droplets was studied. Fig. 4 shows that there were significant changes in modal droplet diameters. Studies of particle diameter ranges showed that irrespective of concentration of dried egg yolk, the addition of Tween 65 caused increase of the percentage of small particles in the range of 0.0-0.2 µm (Table 1). The most reasonable explanation of this process is that the mixtures exhibited different emulsifying properties at different dried egg yolk to Tween 65 weight ratios [17]. Visual inspection of the investigated emulsions in the microscope showed that all emulsions had individual oil droplets with no signs of flocculation. The typical micrographs of emulsions stabilized by: 0.2% (w/w) dried egg yolk; 0.2% (w/w) dried egg yolk and 1% (w/w) Tween 65; 2.0% (w/w) dried egg yolk; 2.0% (w/w) dried egg yolk and 1% (w/w) Tween 65 are shown in Figs. 5a–d, respectively.

In summary, it seems that the competitive adsorption between proteins of dried egg yolk and Tween 65 may significantly affect stability and microstructure of o/w emulsions especially those which will be used to prepare hot sauces.

CONCLUSIONS

1. Increase of emulsifiers concentration led in most cases to the improvement of the stability of the emulsions and depended more on Tween 65 than dried egg yolk concentration.

2. The most probable reason of instability of the emulsions heated at 80°C, particularly those containing the highest concentration of emulsifiers, was competitive adsorption between proteins of dried egg yolk and Tween 65.

3. Analysis of the modal diameters of the droplets showed significant differences of emulsifying mixtures properties containing dried egg yolk and Tween 65.

4. Emulsions stabilized by dried egg yolk or its mixture with Tween 65 contained individual oil droplets with no signs of flocculation.

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Accepted for print: 5.06.2008

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