Stability and rheology of egg-yolk-stabilized concentrated emulsions containing cereal β-glucans of varying molecular size

Food Hydrocolloids 18 (2004) 987–998 www.elsevier.com/locate/foodhyd

Stability and rheology of egg-yolk-stabilized concentrated emulsions containing cereal b-glucans of varying molecular size V. Kontogiorgosa, C.G. Biliaderisa,*, V. Kiosseogloub, G. Doxastakisb a

Laboratory of Food Chemistry and Biochemistry, Department of Food Science and Technology, School of Agriculture, Aristotle University, GR-541 24 Thessaloniki, Greece b Laboratory of Food Chemistry and Technology, School of Chemistry, Aristotle University, GR-541 24 Thessaloniki, Greece Received 11 December 2003; revised 26 January 2004; accepted 2 April 2004

Abstract The effects of barley and oat b-glucans on rheological and creaming behaviour of concentrated egg-yolk-stabilized model emulsions were investigated. Four polysaccharide preparations were used, two from each cereal; one sample with high and one with low molecular weight, i.e. the molecular weights were alike in pairs (, 110 £ 103 and , 40 £ 103, respectively). In order to elucidate the mechanism of action of b-glucans in emulsions, Tween 20-stabilized emulsions were also examined. Tween 20 enhances neither the continuous phase viscosity nor the interactions between the droplets, so the changes could be easily attributed to b-glucans. It appeared that the low Mw b-glucan samples stabilize emulsions against creaming by means of network formation in the continuous phase while their high molecular weight counterparts enhance the viscosity of the continuous phase. Comparison of dynamic rheological tests between a reference emulsion without b-glucans and emulsions containing b-glucans showed that the polysaccharides largely affects the viscoelastic behaviour of the emulsion. Ageing of b-glucan-containing emulsions did not affect significantly the viscoelastic properties except for the emulsions containing low Mw b-glucans extracted from oat. Interestingly, all emulsions containing b-glucans creamed approximately the same after 30 days of storage regardless which preparation was used. The egg yolk constituents seemed to play a dominant role on the viscoelastic and the creaming behaviour of the emulsions, i.e. the viscoelastic behaviour was further enhanced and this could not only be attributed to the presence of the b-glucans but also to the stronger interactions between the oil droplets. Ageing did not affect the viscoelastic properties of b-glucan-containing emulsions while the reference emulsion, prepared only with egg yolk, showed a decrease in the value of storage modulus. The former could be interpreted as a steady consistency of the product during storage independent of the creaming behaviour. The creaming behaviour varied among the samples with the high molecular weight b-glucans from oat showing the highest stability. q 2004 Elsevier Ltd. All rights reserved. Keywords: b-Glucan; Egg yolk; Emulsion stability and rheology; Tween 20

1. Introduction Emulsions are dispersions of one liquid phase in the form of fine droplets in another, immiscible liquid phase. The two immiscible phases are usually oil and water. The change in the free energy during emulsion formation is usually positive and therefore emulsions are thermodynamically unstable. Mainly proteins but also low molecular weight surfactants are employed to confer stability against coalescence in food emulsions in order to provide long shelf-life (kinetic stability). Shelf-life can also be related to * Corresponding author. Tel.: þ 30-2310-998785; fax: þ 30-2310471257. E-mail address: [email protected] (C.G. Biliaderis). 0268-005X/$ - see front matter q 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodhyd.2004.04.003

creaming which is another important form of instability. Polysaccharides are added as thickening agents so as to modify the rheological behaviour of the continuous phase, thereby retarding or inhibiting the process of creaming. In a protein stabilized oil-in-water emulsion containing polysaccharides, interactions between protein and polysaccharide can significantly affect the stability of the system (Dickinson, 2003; Dickinson & Pawlowsky, 1997; Dickinson & Pawlowsky, 1998). It has been found that the addition of non-adsorbing polysaccharides at low concentrations can speed up the process of creaming through the induction of flocculation by a depletion mechanism (Cao, Dickinson, & Wedlock, 1990, 1991; Dickinson, Goller, & Wedlock, 1995). This flocculation results in enhanced emulsion creaming under quiescent storage conditions, as

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flocs tend to separate at a faster rate than individual dispersed droplets. However, the presence of a nonadsorbing polymer at higher concentrations may produce restabilization by conferring a very high apparent viscosity to the continuous phase and/or generating a strong emulsion network in the continuous phase (Cao et al., 1990; Dickinson, Goller, & Wedlock, 1993; Dickinson et al., 1995). Dickinson, Golding, and Povey (1997) working with oil-in-water emulsions containing sodium caseinate identified various regions of creaming stability depending on the overall protein content. Moreover, the important destabilizing role of unadsorbed protein has been recognized. The mechanism of this behaviour was attributed also to depletion flocculation where after saturation of the droplets surface, excess unadsorbed proteins act as non-adsorbing polymers. A number of workers have reported the occurrence of a delay period before the onset of creaming (Manoj, Fillery-Travis, Watson, Hibberd, & Robins, 1998; Moates, Watson, & Robins, 2001; Parker, Gunning, Ng, & Robins, 1995; Ve´lez, Fernandez, & Munoz, 2003). This was first discussed by Parker et al. (1995) who investigated the creaming behaviour of model salad dressings at various oil volume fractions and in the presence of various concentrations of xanthan gum. They concluded that xanthan gum at concentrations . 0.01% induced droplets to flocculate. This resulted in the formation of a three-dimensional droplet network, and the time-dependent yield stress of this network is the mechanism responsible for the delay period. Generally, the influence of the polysaccharides or, in general, of the non-adsorbing macromolecules on the creaming stability is not straightforward and depends on the characteristics of the system. The rheology of emulsions is very important from both fundamental and applied point of view. The factors that affect emulsion rheology are mainly (i) the rheological properties of the continuous phase, (ii) the nature of the droplets, size distribution, deformability, internal viscosity and concentration, and (iii) the nature of the particle – particle interaction (Barnes, 1994). Oscillation and steady state rheology have been employed for the characterization of such dispersions having either small molecular weight surfactant-coated (Manoj, Fillery-Travis, Watson, Hibberd, & Robins, 2000; Moates et al., 2001) or protein-coated droplets (Chen & Dickinson, 1998; Moros, Franco & Gallegos, 2002). Moates et al. (2001), working with an alkane emulsion flocculated by hydroxy-ethyl-cellulose found a correlation between the delay time and the storage modulus. Particularly, they stated that for that system to have a significant delay time, it must possess a solid-like character in the sense of G0 . G00 at some frequencies. In general, ‘stronger’ systems, i.e. those having a larger storage modulus, have longer delay times. Oscillation rheology can differentiate between those compositions of oil flocculated with polymer that will exhibit delay and those that will not. Hemar, Tamehana, Munro, and Singh (2001) argued that the creaming stability of emulsions formed with mixtures of

sodium caseinate and xanthan is more related to the structure and rheology of the emulsion itself rather than to the rheology of the xanthan/protein solution which constituted the continuous phase. The (1-3)(1-4) mixed-linkage b-D -glucans are polysaccharides which exhibit health benefits such as lowering of blood cholesterol (Kahlon, Chow, Knuckles, & Chiu, 1993), regulating blood glucose level and insulin responses in diabetics (Wood, 1993), and even antitumor activity (Eastwood, 1987). Oat and barley are the richest commercial natural sources of b-glucans with levels usually in the range of 4 –7%. Some work has been done on the stabilization of emulsions using b-glucans as stabilizers. Burkus and Temelli (2000) working with b-glucans from barley investigated the effect of this polysaccharide on the stability of emulsions and foams. They attributed the mechanism of foam and emulsion stabilization to the increased viscosity of the continuous phase. They also stated that yield stress caused by the formation of a b-glucan network may be an additional stabilizing factor. In recent studies on structure-functional property relations of cereal b-glucans it was shown that these hydrocolloids are capable of gel network formation in an aqueous medium; the rate and extent of gelation was related to the molecular size and fine structure of the polysaccharide (Lazaridou, Biliaderis, & Izydorczyk, 2003; Vaikousi, Biliaderis, & Izydorzcyk, 2004). The aim of the present investigation was to further elucidate the mechanism of emulsion stabilization using bglucans extracted from oat and barley with varying molecular size, as well as to further characterize the rheological and creaming behaviour of model salad dressings prepared with hen’s egg yolk and containing different concentrations of b-glucans as hydrocolloids. These polysaccharides have been isolated from two different sources and varied in molecular structure and size.

2. Materials and methods 2.1. b-Glucan extraction Four selected b-glucan samples, two extracted from barley and two from oat, were used in this work. Sample HB was a b-glucan extracted from the whole flour of a Greek barley cultivar. The protocol of extraction and purification of this sample was described in detail by Skendi, Biliaderis, Lazaridou, and Izydorczyk (2003). Samples LB, HO, LO were isolates from oat or barley concentrates provided by CEBA (Lund, Sweden). The purification procedure of these concentrates is described elsewhere in detail (Lazaridou et al., 2003). The aim of the extraction and purification procedures adopted in the present work was to obtain four samples with the same Mw in pairs, namely HB with HO and LB with LO. ‘H’ stands for high and ‘L’ for low Mw samples, while ‘B’ for barley and ‘O’ for oat.

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The protein content of all b-glucan preparations was determined by the method of Lowry, Rosebrough, Farr, and Randall (1951). The b-glucan content was determined by the method of McCleary and Glennie-Holmes (1985) using the Megazymew mixed linkage b-glucan assay kit. Analytical grade reagents and distilled water were used in all experiments. 2.2. Molecular and structural characterization of b-glucan The molecular size distributions and the average molecular weight of b-glucans were determined with a high performance size exclusion chromatography system. The molar ratio of cellotriocyl/cellotetraosyl (DP3/DP4) was determined with lichenase hydrolysis and high performance anion exchange chromatography. The purity and some structural features of the b-glucan samples were also assessed with 13C-NMR spectroscopy. All experimental conditions and the instrumentation used in these analyses have been described in detail by Lazaridou et al. (2003). 2.3. Emulsion preparation All polysaccharide and egg yolk concentrations in the emulsions given below are expressed with respect to the continuous phase. Emulsions using Tween 20 (Sigma, Chemical Co., St Louis, MO, USA) as emulsifier were prepared with w ¼ 0:5 and 0.1. Tween 20 was first dissolved in a buffer solution containing 0.02N di-sodium hydrogen citrate, 0.2 M NaCl, and 0.1% NaN3 with the pH adjusted at 3.8 at a concentration of 16% w/v. Corn oil (Elais S.A, Greece) was poured slowly into the Tween 20 solution and left under continuous intense agitation for 10 min with the help of a mechanical stirrer. The resulting master emulsion with w ¼ 0:75 was then homogenized for 1.5 min using an UltraTurrax T25 homogenizer equipped with a S25KG-25F dispersing tool and operated at 22000 rpm. Aliquots of the master emulsion were then diluted with the appropriate volume of a polysaccharide solution giving emulsions with w ¼ 0:5 and 2% w/v polysaccharide. For preparation of emulsions with w ¼ 0:1; a master emulsion was prepared with w ¼ 0:2 and 1.2% w/v Tween 20. Then it was diluted with the appropriate polysaccharide solution yielding emulsions with w ¼ 0:1 and polysaccharide concentrations of 0.1, 0.5, 1, 2, 3% w/v. Emulsions using egg yolk were prepared with w ¼ 0:5 and 0.1. Fresh egg yolk (eggs were purchased from the American School of Agriculture, Greece) was diluted in the buffer solution. Then, the appropriate amounts of polysaccharide solution and oil were added to give emulsions with w ¼ 0:5; 2% w/v polysaccharide and 4% w/v egg yolk dry matter. For the preparation of emulsions with w ¼ 0:1; the appropriate quantity of egg yolk diluted in buffer solution was mixed with the polysaccharide solution and oil,

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yielding emulsions with 4% egg yolk dry matter and polysaccharide concentrations of 0.1, 0.5, 1, 2, 3% w/v. 2.4. Droplet size distribution Droplet size distributions were determined immediately after the emulsions were made using a Malvern Mastersizer 2000 (Malvern Instruments, England). Emulsions prepared with Tween 20 were measured by diluting before the measurement, a few drops of the emulsion in distilled water. Emulsions prepared with egg yolk were previously treated with 1% w/v SDS solution in order to quench the interdroplet interactions and break up the flocs. The action of the SDS solution on the breakage of the flocs was confirmed by optical microscopy. The refractive index of the corn oil and the water were used for calculations and the measurements were performed in triplicate. 2.5. Determination of emulsion stability against creaming Emulsions were placed in cylinders (diameter 15 mm) at 25 8C and the movement of the creaming boundaries were followed with time with visual observation. The delay time was taken as the time for the emulsion to cream 1 mm from the bottom of the cylinder. The creaming was expressed as the percentage of the height of the serum layer over the total height of the emulsion in the cylinder: Creaming ¼

Height of serum layer £ 100 Total height of the emulsion

2.6. Interfacial protein concentration The egg yolk stabilized emulsions after dilution with the same volume of buffer were centrifuged for 15 min at 4400 rpm in order to separate the oil droplets from the aqueous phase. The interfacial protein concentration (G; mg m22) was calculated as

G¼

mg of adsorbed protein S £ ml of oil in the emulsion

where S (m2 ml21) is the specific surface area: S¼

d3:2 6

The quantity of the proteins was estimated according to the method of Markwell, Hass, Bieber, and Tolberd (1978). Calculation of the protein concentration was performed using a calibration curve made with BSA standard solutions and the results are the means of three measurements. Emulsions of two days old were used for these measurements. 2.7. Rheological measurements of b-glucans and emulsions The intrinsic viscosities [h] of aqueous solutions of bglucans were measured with Ubbelodhe capillary

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viscometers and a rotational rheometer at 25 ^ 0.1 8C; calculations were performed according to the Huggins equation, hsp =c ¼ ½h þ kH ½h2 c; where hsp ¼ ðhsolution =hsolvent Þ 2 1; and kH is the Huggins constant. The flow and viscoelastic behaviour of b-glucan solutions, the continuous phase as well as of the emulsions were performed on a rotational Physica MCR 300 rheometer (Physica Messtechnic GmbH, Stuttgard, Germany) using a double-gap cylindrical geometry (internal and external gap 0.42 and 0.47 mm, respectively) or a serrated plate – plate geometry (1 mm gap, 24.9 mm diameter). The temperature was regulated by a Paar Physica circulating bath and a controlled peltier system (TEZ 150P/MCR) with an accuracy of ^ 0.1 8C. The data of the rheological measurements were analyzed with a supporting the rheometer software US200 V 2.21. Buffer solutions of b-glucans were measured immediately after their preparation and after 5, 15 and 25 days, while rheological testing of the continuous phase was carried out immediately after preparation and after 10, 20 and 30 days. All these preparations were kept in sealed vials at room temperature, one for each measurement separately. The b-glucan solutions and the continuous phase were subjected to oscillatory measurements with a strain 0.1% and a range of frequencies between 0.1 and 100 Hz, and to shear rate sweeps between 0.5 and 1000 s21. Emulsions were measured immediately after their preparation and after 10, 20 and 30 days of storage. Preliminary experiments showed that a 10 min rest in the rheometer’s geomerty was adequate to ensure structure recovery following the load of the sample in the rheometer. Emulsions were stored at room temperature in sealed vials, one for each day of measurement and were subjected to oscillatory measurements with a strain of 0.1% and a range of frequencies between 0.1 and 100 Hz, and to controlled stress measurements within the range of 0– 40 Pa. The Casson model was used for the determination of the yield stress values. All experiments were performed at least in triplicate.

Table 1 Compositional, molecular and structural features of b-glucan preparations Samples

HO

LO

HB

LB

b-Glucans (%) Protein (%) Mw ( £ 103) c* * (g/dl)a Molar ratio DP3/DP4b

91.8 2.3 109 1.6 2.22

93.1 1.9 36 2.0 2.25

90.3 3.5 107 1.5 2.80

91.2 4.2 42 2.4 2.79

a b

Measurements at 25 8C. DP, degree of polymerisation.

The polysaccharide concentration used for the preparation of the emulsions were 0.5% w/v, which means that emulsions had contaminant proteins ranging from 0.010 to 0.021% w/v, while the concentrations of the yolk constituents and Tween 20 were , 1% w/v, as expressed on emulsion volume basis. Mine and Keeratiurai (2000) demonstrated the significant ability of yolk proteins in displacing other highly surface active proteins (caseins). On the other hand, for emulsions prepared with Tween 20, the displacement of Tween 20 from the interface by the contaminant proteins of the b-glucan preparations can be considered as unrealistic. Therefore, the concentration of contaminant proteins must not be deemed as important for the behaviour of the emulsions prepared with either the egg yolk or Tween 20. The apparent molecular weights of the samples were akin in pairs as is shown in Table 1. Another important structural feature of b-glucans is their cellotriosyl/cellotetraosyl ratio (DP3/DP4), which constitutes a structural fingerprint for cereal b-glucans (Cui & Wood, 2000). As this ratio is increased, the ability of b-glucans to gel increases (Bo¨hm & Kulicke, 1999; Lazaridou et al., 2003; Vaikousi et al., 2004). Moreover, the low molecular weight samples have the ability to gel more rapidly than their high molecular weight counterparts and this has been attributed to the higher mobility of the shorter chains (Bo¨hm & Kulicke, 1999). Such gelation ability of these hydrocolloids could have a significant impact on emulsion behaviour.

3. Results and discussion

3.2. Solution rheology

3.1. Purity and molecular characterization of b-glucans

The intrinsic viscosity ½h values at 25 8C were obtained by extrapolation of viscometric data to zero concentration. Double logarithmic plots of hsp vs. c½h gave estimates of the c* * (Table 1), a critical concentration above which the coil dimensions become independent of polymer concentration (Doublier & Couvelier, 1996; Morris, Culter, Ross-Murphy, Rees, & Price, 1981). Critical concentrations play an important role on the creaming behaviour of emulsions. The theory indicates that the onset of depletion flocculation occurs above some critical polymer concentration ccr ; and that the strength of the depletion interaction is dependent on such factors as the polymer concentration ðc . ccr Þ and the molecular weight (Feigin & Napper, 1980;

The b-glucan and protein contents as well as other structural features of all samples are presented in Table 1. The purity of the isolated b-glucan samples was further confirmed by 13C-NMR spectroscopy (data not shown) which indicated the absence of characteristic C1-resonances from other polymers (starch, arabinoxylan). The isolation/ purification protocol seemed to provide b-glucan samples of relatively high purity. The aquisition of samples with low protein content is important because proteins may affect emulsion stability by the mechanism of competitive adsorption with other proteins (Dickinson, 1992).

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Joanny, Leibler, & de-Gennes, 1979; Vincent, Edwards, Emmet, & Jones, 1986). While the most obvious evidence for depletion flocculation is the greatly increased rate of emulsion creaming at c . ccr ; it is commonly observed that the presence of non-adsorbing polymer at even higher concentrations (c q ccr ; but c still low in absolute terms) may produce restabilization by conferring a very high apparent viscosity to the continuous phase and/or generating a strong emulsion gel network (Cao et al., 1990; Dickinson et al., 1993; Dickinson et al., 1995). This critical concentration ðccr Þ; for the onset of depletion flocculation is often identified with the critical polymer concentration (c* or c* * ), a parameter derived from viscosimetric data. Dynamic and steady shear measurements of b-glucans prepared in the buffer solution were also performed. Fresh solutions at all concentrations studied showed Newtonian flow and liquid-like behaviour with G00 being greater than G0 at all frequencies (Fig. 1). The rheological behaviour of the high molecular weight samples at concentration of 2% w/v, which is the concentration of the polysaccharide in the continuous phase of the emulsions, was not affected by ageing, as the shape of the flow curves as well as of the mechanical spectra did not change significantly upon storage (Fig. 1a,b). On the other hand, ageing had

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a pronounced effect on the low molecular weight samples showing pseudoplastic flow before the fifth day even at concentration of 2% w/v (Fig. 1c). This can be attributed to the ability of the low molecular weight samples to aggregate and form networks more easily than their high molecular weight counterparts (Bo¨hm & Kulicke, 1999; Lazaridou et al., 2003; Vaikousi et al., 2004). The mechanical spectra of low molecular weight samples showed solid-like character before the fifth day with the LB sample forming stronger networks than those of the LO. The HO exhibited a slightly greater zero shear viscosity values for both fresh and aged solutions than its HB counterpart. Moreover, aged solutions of both LB and LO had greater zero shear viscosity values from the high molecular weight samples. Ve´lez et al. (2003), studying the effect of xanthan and guar gums on creaming, concluded that the delay time before creaming is dependent on zero shear viscosity of the continuous phase and is largely independent of polymer type. They also stated that the delay time increases dramatically at zero shear viscosities approaching 1 Pa s. In the present study, LB (2% w/v) before the fifth day approached 1 Pa s (Fig. 1c), while LO (2% w/v) was at , 0.1 Pa s.

Fig. 1. Influence of ageing on the rheological behaviour of high and low Mw b-glucan solutions (25 8C): (a, c) typical flow curves of the high and low Mw samples, respectively; and (c, d) typical mechanical spectra of the high and low Mw samples, respectively.

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Fig. 2. (a) Mechanical spectra of Tween 20-stabilized emulsion without b-glucans (REF), (b) tan d values (frequency 1 Hz, strain 0.1%) at the day of preparation (open bars) and after 30 days of storage (solid bars) for Tween 20-stabilized emulsions with (concentration 2% w/v) or without b-glucans; halflines indicate the standard deviations of triplicate measurements (25 8C).

3.3. Rheology of emulsions stabilized with Tween 20 Preparation of emulsions using Tween 20 as emulsifier aimed at interpreting the role of b-glucans on emulsion behaviour, since Tween 20 is generally considered as ‘neutral’ emulsifier. This is because at the concentration used, Tween 20 did not enhance neither the continuous phase viscosity nor the interactions between the droplets. Moreover, all emulsions had identical mean droplet size diameter (, 1.8 mm) since were prepared by diluting a master emulsion. Thus, the changes could be easily attributed to b-glucans. Measurements of the d4:3 values for all emulsions during ageing did not show any pronounced change. In order to clarify the action of b-glucans as modifiers of the rheological behaviour of the continuous phase an emulsion without b-glucans was also prepared (only Tween 20), named REFTween. Dynamic rheological tests of the REFTween emulsions, showed liquid-like character at lower frequencies and the moduli crossing each other at high frequencies (Fig. 2a). Controlled stress measurements showed almost Newtonianlike flow. Ageing did not affect the viscoelasticity and the flow behaviour of this emulsion. The addition of b-glucans affected radically the visoelastic behaviour of the emulsion, and the flow became pseudoplastic. Emulsions with HO, HB and LO showed typical behaviour of a viscoelastic fluid but the frequency of cross-over for the two moduli moved at lower values compared to REFTween which is indicative of a ‘stronger’ structure. This stronger structure in the case of the high Mw samples could be attributed to the formation of a highly flocculated droplet network structure that gives this pronounced viscoelastic behaviour whereas in the case of low Mw samples this could be related to the formation of a gel-like structure in the continuous phase by the polysaccharide itself. A comparison of the tan d values for all emulsions (1 Hz) for days 1 and 30 is given in Fig. 2b. Manoj et al. (2000), working with 1-bromohexadecane

emulsions stabilized with Brij 35 with or without hydroxyethylcellulose (HEC), observed that emulsions without polymer had liquid-like behaviour at w ¼ 0:37: With the addition of HEC at various concentrations (0.04 – 0.35% w/w) they observed a gradual change on the relative values of G0 and G00 , with G0 becoming dominant as the polymer concentration was increased. The influence of ageing of emulsions on the storage modulus over a period of 30 days is shown in Fig. 3. The modulus values are normalized with the values of G0 at the day of preparation (G0o ) at 1 Hz. The storage modulus increased slightly for HO and HB samples, significantly for LO while for LB samples

Fig. 3. Storage modulus measurements (at 1 Hz, 0.1% strain, 25 8C) taken over a period of 30 days (25 8C) and normalized with values at the day of preparation for Tween 20-stabilized emulsions containing b-glucans (w ¼ 0:5; polysaccharide concentration 2.0% w/v). The inset shows the storage modulus values of the emulsions at the day of preparation (open bars) and after 30 days of storage (solid bars); half-lines indicate the standard deviations.

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3.4. Creaming of emulsions stabilized with Tween 20

Fig. 4. Evolution of yield stress values for emulsions with b-glucans (2% w/v) stabilized by Tween 20 during 30 days of storage (w ¼ 0:5; 25 8C).

it was decreased slightly. The network of low molecular weight b-glucans from oats (Lazaridou et al., 2003) is formed at a much lower rate than that of b-glucans from barley (Vaikousi et al., 2004) due to the lower DP3/DP4 ratio of the former. Thus, the gradual network formation of LO sample during storage explains this behaviour, in contrast with the LB sample where the network formation proceeded rapidly reaching a plateau value even within the first few hours of emulsion preparation; the latter sample exhibited a decline in the modulus upon ageing, presumably due to further chain aggregation events which could affect the continuity of the network structure. The evolution of yield stress over a period of 30 days is presented in Fig. 4. The REFTween sample had very low yield stress values (, 0.1 Pa) that remained constant after 30 days. The addition of b-glucans increased the yield stress values between 0.1 and 1.1 Pa. Aged emulsions containing HB, HO and LO had higher and LB lower yield stress values than fresh emulsions which is consistent with the increase or decrease, respectively, in the storage modulus of these particular samples. Moreover, the close values of yield stress obtained for all emulsion samples (0.8 – 1.1 Pa), suggest that the yield stress is mainly affected by the concentration of b-glucans and not by the molecular weight. It is clear from these results that the addition of b-glucans changed the rheology of REFTween emulsion. The flow became pseudoplastic, an elastic character developed and the resistance to flow increased. Therefore, the rheology of the continuous phase containing b-glucans dominates the flow behaviour of the emulsions even at w ¼ 0:5: For the high molecular weight samples, regardless of their origin, there was substantial differentiation in the behaviour of the emulsions. In contrast, for the low molecular weight samples, the botanical origin of the b-glucan (structural effects related to the DP3/DP4 ratio, Table 1) seems to play an important role and differentiates the two preparations as far as emulsion rheology.

When a non-adsorbing polymer is introduced into a colloidal system, flocculation by a depletion mechanism is very often observed (Jerkins & Snowden, 1996). In Fig. 5 the creaming data for the series of emulsions are shown. The REFTween sample creamed more than the emulsions containing b-glucans; the latter creamed approximately the same after 30 days of storage. The serum layer of REFTween was visible after two hours, while the b-glucan containing samples demonstrated an extended delay to the manifestation of creaming for one day. The increase of delay time is attributed to the depletion flocculated network which was formed because of the presence of the b-glucans. That is, the droplet network that is strongly held in place by the short range attractive forces between the Tween 20 covered droplets cannot easily rearrange itself in order to quickly expel serum from the particle-gel structure (Dickinson et al., 1995). While high molecular weight samples stabilize emulsions through viscosity enhancement of the continuous phase, the network formation from the low molecular weight b-glucan samples seems to be a more probable mechanism of stabilization. Such a network may immobilize the droplets in place so they cannot move under the effect of gravity. For the extrication of the hydrodynamic hindrance at a high oil volume fraction, the onset of creaming was also studied in emulsions at w ¼ 0:1 and different concentrations of b-glucans. The emulsions had the same initial droplet diameter (d3:2 ¼, 3:5 mm). The effect of concentration on delay time is shown in Fig. 6 where the time needed for a clear serum to appear is plotted against b-glucan concentration. Emulsions containing HO and HB b-glucans had the same delay time at concentrations of 2 and 3% (w/v).

Fig. 5. Percentage of serum layer of the total height of creamed Tween-20 stabilized emulsions containing b-glucans (2% w/v, w ¼ 0:5; 25 8C); inset shows a magnification of the same diagram up to 48 h of observation. Symbols are means of three replicates and half-bars are the respective standard deviations.

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Fig. 6. Delay time of Tween 20-stabilized emulsions containing b-glucans at different concentrations (w ¼ 0:1; 25 8C); the inset shows a magnification of the same diagram up to 50 min.

At lower concentrations no delay time was observed. The increase of the delay time of the high Mw polymers appeared to be related with the critical concentration c* * ; above which there is a sudden increase in zero shear viscosity. This relation was not observed for the low molecular weight b-glucans. The LO did not show any delay in the manifestation of creaming at all concentrations used. In contrast, the delay time for the emulsion containing LB showed to be approximately the same with the high molecular weight samples between 0.1 and 2% w/v, whereas at 3% w/v it was much higher. This behaviour could not be attributed to the viscosity modification of the continuous phase. It appears, in accordance to the rheological data (Fig. 1), that network formation is the dominant mechanism. The LO sample did not show the same behaviour as the LB due to its lower tendency for chain aggregation. It is also important to mention that the creamed emulsions containing LB did not completely cream, being instead immobilized for several days as shown in Fig. 7. Note that the photographs for the emulsions containing LB were obtained after 10 days of storage compared with those of HO, which were taken after one day. All the remaining emulsions behaved similarly as the HO dispersion. This set of experiments confirmed that the stabilization mechanism of high molecular weight b-glucans is mostly by means of viscosity enhancement of the continuous phase as well as by a depletion stabilization mechanism in the case of high oil volume fraction emulsions. For the low Mw bglucans the contribution of network formation by the polysaccharide in the continuous phase, which further enhances the viscosity, becomes important. The latter is more pronounced in the case of LB. In this respect, the rate of polysaccharide network formation seems to play an important role to the stability of the emulsion because if it is

Fig. 7. Photographs of tubes of emulsions ðw ¼ 0:1Þ containing b-glucans at different concentrations stabilized with Tween 20. From left to right: 3, 2, 1, 0.5, 0.1, 0% (w/v) b-glucan. Photographs were taken for top emulsions (with HO) after 1 day, and for bottom emulsions (with LB) after 10 days.

not established fast enough the emulsion will cream, as for instance do the emulsions containing LO. Therefore, for improved emulsion stability, a network structure in the continuous phase must be quickly developed following the preparation of the emulsion. 3.5. Rheology of emulsions stabilized with egg yolk Egg yolk is used extensively in the food industry as emulsifier of mayonnaises and salad dressings, as well as an ingredient in other products. The influence of egg yolk on creaming and rheology of model salad dressings containing b-glucans was studied and the results compared to those obtained by Tween 20. The rheology of the continuous phase of emulsions stabilized with yolk was investigated first. The viscoelastic data of the continuous phase containing b-glucans and egg yolk suggested that only the b-glucans and not the yolk solids are responsible for the viscoelasticity. Mun˜oz, Hudson, Ve´lez, Alfaro, and Ferguson (2001), studying the rheological behaviour of spray-dried egg yolk/xanthan gum aqueous dispersions, found that the linear viscoelastic properties are governed by the xanthan gum/water solution. Egg yolk slightly increased the viscosity of the fresh b-glucan solutions. For the low molecular weight samples, the contribution of egg yolk to the viscosity diminished

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Fig. 8. Mechanical spectra of egg-yolk-stabilized emulsions ðw ¼ 0:5Þ : (a) without b-glucans (REF); and (b) with b-glucans (2% w/v HO, strain 0.1%, 25 8C) at the first day of preparation.

during storage due to network formation and the viscosity was the same as for the solutions without egg yolk. In order to elucidate the properties of egg-yolk in the presence of b-glucans and to simulate real industrial conditions, emulsions were prepared in the presence of both yolk and b-glucan. This resulted in different initial droplet sizes with LB and LO samples having larger droplet size (d3:2 ¼, 2:7 mm) than the HO and HB samples (d3:2 ¼, 2:1 mm). A reference sample (REFyolk), prepared without b-glucans, had even larger droplets (d3:2 ¼, 3:2 mm). This difference is attributed to the higher viscosity of the freshly prepared high molecular weight samples so the recoalescence rate during emulsion formation is decreased stabilizing thus the droplets at smaller size. The size of the droplets did not change significantly after 30 days of storage. The flow of the REFyolk sample was pseudoplastic at all shear rates in contrast with REFTween where it was Newtonian. It seems that the egg yolk played an important role to the formation of a strong flocculated structure that could not be disrupted under a strong shear field, and additionally this behaviour remained unchanged after 30 days of storage. Aluko and Mine (1998) working with emulsions stabilized with egg yolk supported that at pH 4.0 there is increased attraction between the oil droplets. Emulsions prepared in the present work had a pH of 3.8 that is very close to 4.0. Such attraction explains the strong flocculated structure of the REFyolk emulsions. This resulted in a dramatic change on viscoelasticity compared with the system REFTween, with G0 dominating at all frequencies (Fig. 8a). The effect of ageing of REFyolk emulsion as well as of the rest emulsions are presented in Fig. 9. Ageing seemed to affect the viscoelasticity of REFyolk emulsion where the value of storage modulus decreased. Paraskevopoulou, Kiosseoglou, Alevisopoulos, and Kasapis (1997) observed similar trends of egg-yolk-stabilized emulsions. They attributed this drop of storage modulus to the increase of droplet size. However, data in the present work did not show any increase in droplet size of REFyolk sample, thus the behaviour must be attributed to a modification of the interdroplet interactions due to ageing of the viscoelastic protein film, resulting in weaker interactions.

The viscoelastic behaviour of emulsions after the addition of b-glucans was further enhanced. The flow became more pseudoplastic with increased values of viscosity, and the storage modulus was also increased. A typical mechanical spectrum of the b-glucan containing emulsions is shown in Fig. 8b. It is apparent that the rheological behaviour of the emulsions could not only be attributed to the incorporation of b-glucans, but also to the interactions between the droplets due to the presence of egg yolk proteins. The slight increase of the continuous phase viscosity generated by the addition of yolk could not explain by itself such dramatic rheological changes. In contrast to REFyolk, the viscoelastic behaviour of the samples containing b-glucans was not affected significantly by ageing (Fig. 9). It appears that the droplet network that is formed in these emulsions remains relatively unchanged. This would be reflected by a steady consistency of the product during the storage independent of the creaming behaviour.

Fig. 9. Storage modulus measurements (at 1 Hz, 0.1% strain, 25 8C) taken over a period of 30 days and normalized with values at the day of preparation for egg-yolk-stabilized emulsions containing b-glucans (2% w/v); inset shows the storage modulus values at the day of preparation (open bars) and after 30 days of storage (solid bars).

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Table 2 Total protein adsorption and interfacial protein concentration of egg-yolkstabilized emulsions containing b-glucans Sample

HB

LB

HO

LO

REFyolk

% of adsorbed protein G (mg m22)

78.8a 3.95c

70.1b 4.87b

79.8a 4.13c

75.9a 5.05b

78.0a 6.44a

Values with the same letter (in each row) do not differ significantly (p , 0:05).

The addition of egg yolk increased the yield stress of REFyolk (, 1 Pa) compared with REFTween (, 0.1 Pa), while the addition of b-glucans increased it even more (1.4 – 2.2 Pa). With the exception of the LB sample that showed a slight decrease in yield stress values, all other samples did not show any significant change of this parameter during ageing. 3.6. Interfacial protein concentration Table 2 contains data on the total protein adsorption at the interface and the interfacial protein concentration of egg-yolk-stabilized emulsions. As can be seen from the comparison with the REFyolk sample, the total protein adsorption was not affected by the presence of b-glucans. The emulsion containing LB adsorbed slightly lower amount of protein. The LB b-glucan as stated earlier forms rapidly a network in the continuous phase; this network probably restricts the diffusion of proteins towards the interface and thereby reduces their adsorption. Similar values of total adsorbed protein were found by Mine (1998) for emulsions prepared with egg yolk and with oil volume fractions ranging between 0.025 and 0.400. Large interfacial protein concentration is reasonably correlated to greater stability against coalescence and appeared to be sufficient for its prevention for all the emulsions. The interfacial protein concentration seems to have its greater value for the REF sample. This is reasonable since G is proportional to droplet diameter and the droplet diameter of REF emulsions was larger than the others. Similar G values were reported by Anton, Beaumal, and Gandemer (2000) and Aluko and Mine (1998) using egg yolk as emulsifier. 3.7. Creaming of emulsions stabilized with egg yolk Fig. 10 shows the creaming behaviour of egg-yolkstabilized emulsions containing b-glucans. The creaming behaviour of REFyolk was different from that of the REFTween emulsion. The visible serum layer decreased compared with the REFTween emulsion. It must be noted that while REFTween had smaller droplets, it was more unstable towards creaming compared with the REFyolk. This is indicative that a flocculated network is formed in the presence of egg yolk. With the addition of b-glucans, creaming decreased even more. The droplet size did not

Fig. 10. Percentage of serum layer of the total height of creamed egg-yolkstabilized emulsions containing b-glucans (w ¼ 0:5; 2% w/v, 25 8C); symbols are means of three replicates and half-lines are the respective standard deviations.

play an important role; all Tween 20 emulsions had smaller droplet size but were more unstable towards creaming compared to egg yolk emulsions. A three-dimensional droplet network was observed by other researchers working with BSA stabilized emulsions and i-carrageenan (Dickinson & Pawlowsky, 1997) or with sodium caseinatestabilized emulsions and xanthan (Hemar et al., 2001). The results of the present work confirm the arguments of Hemar et al. (2001) who suggested that the creaming stability of emulsions is more related to the structure and rheology of the emulsion itself rather than to the rheology of the continuous phase alone as: (a) while the continuous phase containing HO b-glucan had practically the same rheological behaviour with the HB sample, the former did not cream at all (Fig. 10), and (b) although the rheology of the continuous phase of emulsions stabilized with Tween 20 varied with the type of b-glucan used (Fig. 1), all the emulsions creamed similarly (Fig. 5). Consequently, the extend as well as the mechanism of flocculation, in the case of Tween 20-stabilized emulsions can be attributed to depletion flocculation. In the case of egg-yolk-stabilized emulsions may be related both to a depletion mechanism and to the contribution from egg yolk proteins through steric effects. Overall, both egg yolk and b-glucans and not bglucans alone seem to stabilize the egg yolk containing emulsions against creaming. The creaming behaviour of emulsions with low oil volume fraction was also studied with an intention to further clarify the effect of egg yolk and identify any possible synergistic action with b-glucans on the formation of a three dimensional droplet network. A low volume fraction was selected in order to dissipate the hydrodynamic hindrance caused by the high volume fractions. The droplets had different d3:2 values ranging from 1 to 2 mm because the emulsions were prepared separately. Fig. 11 shows photographs taken after 20 days of storage. The first interesting observation was that egg yolk increased the delay time from minutes to hours, as compared with

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synergistically with b-glucans offering protection from creaming even at low oil volume fractions.

Acknowledgements This work was supported in part by funds from the Commission of the European Communities; project QLKICT-2000-00535 ‘Design of Foods with Improved Functionality and Superior Health Effects using Cereal b-glucans’. The authors also wish to thank Dr C. Ritzoulis for his valuable comments concerning the experimental design and interpretation of some research findings.

References

Fig. 11. Photographs of emulsions ðw ¼ 0:1Þ taken after 20 days of storage stabilized with egg yolk solids and containing b-glucans at different concentrations. From left to right: 3, 2, 1, 0.5, 0.1, 0% w/v b-glucan. Emulsions at top contain HO, whereas those at bottom have LO.

the corresponding Tween 20-stabilized emulsions. From Fig. 11 is apparent that as the polysaccharide concentration is increased the extent of creaming decreases substantially (Fig. 7 vs. Fig. 11). This indicates a synergistic action between egg yolk and b-glucans, resulting in the formation of a three-dimensional droplet network that is slowly reorganized resulting in more stable emulsions.

4. Conclusions From the results of the present work it can be concluded that the high Mw b-glucans stabilize emulsions mainly by increasing the viscosity of the continuous phase while the low Mw b-glucans can influence stability through network formation in the continuous phase. The source of extraction, for the high Mw b-glucans, did not seem to play an important role to their functional properties and the Mw seems to be the main factor that affects their behaviour. In contrast, for the low Mw b-glucans the botanical origin of the sample is important since structural variation (DP3/DP4 ratio) among the b-glucans has an impact on the rheology of the system. The increased interdroplet attraction at pH of 3.8 leads to the formation of a strongly flocculated droplet network which is slowly reorganized giving a reduction of creaming. With the addition of b-glucans this structure is further enhanced and the creaming is reduced even more. A comparison of egg yolk with the Tween 20stabilized emulsions demonstrated that the egg yolk acts

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