Interfacial, foaming and emulsifying characteristics of sodium caseinate as influenced by protein concentration in solution

Food Hydrocolloids 19 (2005) 407–416 www.elsevier.com/locate/foodhyd

Interfacial, foaming and emulsifying characteristics of sodium caseinate as influenced by protein concentration in solution Cecilio Carrera Sa´nchez, Juan M. Rodrı´guez Patino* Departamento de Ingenierı´a Quı´mica, Facultad de Quı´mica, Universidad de Sevilla, C/, Prof. Garcı´a Gonza´lez 1, 41012 Seville, Spain

Abstract The foaming and emulsifying characteristics of proteins are important attributes during the production stage, storage, transport, and consumer perception of quality (appearance) of food dispersions (emulsions and foams). In this contribution, we are concerned with the analysis of interfacial, foaming, and emulsifying characteristics of a typical milk protein (sodium caseinate) as a function of protein concentration in aqueous solution (C, % w/w). We have observed that there exists close relationships between foaming (power of foaming, foam capacity, foam density, and foam conductivity) and the rate of diffusion (slope of p vs. t1/2) of caseinate to the air–water interface. The foam stability (quantified by the relaxation time (t) due to drainage and disproportionation/collapse) correlates linearly with the equilibrium surface pressure (pe) of aqueous solutions of caseinate. At surface pressures lower than that of monolayer saturation (at C!1!10K3%, w/w) the foaming is zero. The emulsifying capacity (quantified by the droplet size and the specific surface area) was correlated with the protein concentration in solution (surface pressure at equilibrium). Coalescence was observed only at the lower caseinate concentration in solution (C!0.1%, w/w). As a coherent protein layer (multilayer) saturates the interface, at higher protein concentrations, the emulsion instability is due to flocculation and/or creaming. The coalescence and creaming rates correlate well with the protein concentration in solution (thus with the surface pressure and/or surface dilatational modulus). q 2004 Elsevier Ltd. All rights reserved. Keywords: Foam; Emulsion; Food dispersion; Protein; Caseinate; Food colloids; Adsorption; Air–water interface

1. Introduction Food dispersions often take the form of emulsions and foams. The foaming and emulsifying characteristics are important attributes during the production stage of such dispersions. In addition, stability is an important property of food dispersions, since consumer perception of quality is influenced by appearance. These dispersions are thermodynamically unstable, and their relative stability is affected by factors such as flocculation of aggregated dispersed particles, usually followed by creaming (sedimentation), partial or total coalescence of dispersed particles and/or Ostwald ripening (disproportionation), resulting in final dispersion breakdown (Dickinson, 1992; McClements, 1999). Foaming and emulsifying characteristics and the stability of the resulting dispersion depend on the properties

* Corresponding author. Tel.: C34 95 455 6446; fax: C34 95 455 7134. E-mail address: [email protected] (J.M.R. Patino). 0268-005X/$ - see front matter q 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodhyd.2004.10.007

of the surface-active components in the system. The most important surface-active components in foods are proteins and low-molecular weight emulsifiers (lipids, phospholipids, surfactants, etc.). Among food proteins, caseins are distinguished by their good foaming and emulsifying properties, and for these reasons they are widely used in food formulations (Dalgleish, 1997; Damodaran, 1997). All of the individual caseins, except k-casein, show a strong tendency to adsorb the fluid interfaces (air–water and oil–water), and thus they find an important use in the manufacture of stable emulsions (i.e. ice cream, cream liqueurs, whipped toppings, coffee whiteners, products for infant nutrition, etc.), where longterm emulsion stability is essential. The foaming and emulsifying properties of caseinate arise from the structures of the four proteins found in bovine milk (b-casein, as1-casein, k-casein, and as2-casein). In foams and emulsions made of incorporating caseinate the individual caseins seem to be adsorbed at fluid interfaces in proportion to their incorporation in solution (Hunt & Dalgleish, 1994).

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However, there seems to be a distinction between caseinate and mixtures of purified individual caseins, since the latter show competitive adsorption between b-casein, and as1-casein (Dickinson, 1994; Dickinson, Rolfe, & Dalgleish, 1988; Fang & Dalgleish, 1993). Caseinate gives adsorbed layers similar to those measured for b-casein, the individual casein that shows the highest surface activity of the four individual proteins in caseinate (Fang & Dalgleish, 1993). In this contribution, we are concerned with the analysis of interfacial, foaming, and emulsifying characteristics of a typical milk protein (sodium caseinate) as a function of protein concentration in aqueous solution (C, % w/w). Sodium caseinate is commonly used as an ingredient in a wide range of formulated food dispersions (emulsions and foams). Milk-based emulsions can be divided into two categories. First, emulsions which are consumed in liquid form (e.g. milk, coffee cream, non-dairy coffee whiteners, etc.). Stability towards creaming, flocculation and coalescence during storage and transport is very important with such products. Secondly, emulsions which are aerated into a foam before consumption (e.g. whipped cream toppings, ice cream, etc.). In these emulsions both stability towards coalescence and a controlled de-stabilisation is needed (Robins, 2000).

2. Experimental 2.1. Chemicals Caseinate (a mixture of z38% b-casein, z39% as1-casein, z12% k-casein, and z11% as2-casein) was supplied and purified by bulk milk from Unilever Research (Colworth, UK). The sample was stored below 0 8C and all work were done without further purification. Caseinate solutions were prepared using Milli-Q ultrapure water and were buffered at pH 7. Trizma–HCl ((CH2OH)3CNH2/(CH2OH)3CNH3Cl) buffered solution was used as supplied by Sigma (O95%) without further purification. Ionic strength was 0.05 M in all the experiments. Trisun oil (fatty acid composition, C16, 4%; C18, 4%; C18, 1; 80%, C18, 2; 9%; C18, 3: traces, C20, 0.5%, and C22, 1%) was supplied by Danisco Ingredients. The interfacial tension of the Trisun oil–water interface was 29.5G0.5 mN/m. Sodium dodecyl sulfate (SDS) as a dissociating medium for freshly prepared emulsions was acquired from Sigma (O95%). Sodium azide (Sigma) was added to the aqueous protein solution (0.02% w/w) in order to avoid microbial contamination in the sample. The absence of active surface contaminants in the aqueous buffered solutions was checked by interfacial tension measurements before sample preparation. No aqueous solutions with a surface tension other than that accepted in the literature (72–73mN/m at 20 8C) were used.

2.2. Methods 2.2.1. Foaming properties The foaming properties of caseinate solutions were characterised through their foam formation and stability measured in a commercial instrument (Foamscan IT Concept, Longessaigne, France), based on the ideas by Popineau et al. (Guillerme, Loisel, Bertrand, & Popineau, 1993; Loisel, Gue´gan, & Popineau, 1993). With this instrument the foam formation, the foam stability and the drainage of liquid from the foam can be determined by conductimetric and optical measurements. The foam is generated by blowing gas (nitrogen) at a flow of 45 ml/min through a porous glass filter (pore diameter 0.2 mm) at the bottom of a glass tube where 20 ml of caseinate solution under investigation is placed. The foam volume is determined by use of a CCD camera. The drainage of water from the foam is followed via conductivity measurements at different heights of the foam column. A pair of electrodes at the bottom of the column was used for measuring the quantity of liquid that was not in the foam, while the volume of liquid in the foam was measured by conductimetry in three pairs of electrodes located along the glass column. In all experiments, the foam was allowed to reach a volume of 120 ml. The bubbling was then stopped and the evolution of the foam was analysed. Fig. 1 shows data of liquid volume, foam volume, and foam conductivity during foam formation and rupture, for a foam generated from a caseinate solution. Foaming properties were measured at 20 8C from protein aqueous solutions at pH 7 and at an ionic strength of 0.05 M. Four parameters were determined as a measure of foaming capacity. The overall foaming capacity (OFC, ml/s) was determined from the slope of foam volume curve till the end of the bubbling. The foam capacity (FC), a measure of gas retention in the foam, was determined by Eq. (1). The foam maximum density (MD), a measure of

Fig. 1. Time evolution of (D) liquid volume, (B) foam volume, and (P) foam conductivity in a foam generated from a caseinate solution at 1% w/w. Temperature 20 8C. Aqueous phase pH 7. Ionic strength 0.05 M. The dashed arrow indicated the end of foaming (tf).

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the liquid retention in the foam, was determined by Eq. (2). The relative foam conductivity (Cf, %) is a measure of the foam density and was determined by Eq. (3). FC Z

Vfoam ðfÞ Vgas ðfÞ

MD Z

Cf Z

Vliq ðiÞ K Vliq ðfÞ Vfoam ðfÞ

Cfoam ðfÞ !100 Cliq ðfÞ

(1)

(2)

(3)

where Vfoam (f) is the final foam volume, Vgas (f) is the final gas volume injected, Vliq (i) and Vliq (f) are the initial and final liquid volumes, and Cfoam (f) and Cliq (f) are the final foam and liquid conductivity values, respectively. The static foam stability was determined from the volume of liquid drained from the foam over time (Rodrı´guez Patino, Naranjo, & Linares, 1995; Rodrı´guez ´ lvarez, 1997). Two integrated Patino, Rodrı´guez Nin˜o, & A empirical equations were used to correlate the experimental data: first order equation (Eq. (4)) and second order equation (Eq. (5)). The kinetic constants k1 and k2 for foam drainage were evaluated from linear representations of Eqs. (4) and (5), respectively. In all of the experiments performed in this study, the second-order empirical equation fits the data of foam drainage better. Moreover, the half-life time (t1/2), referring to the time needed to drain V/2 can be expressed by Eq. (6) corresponding to the empirical second-order

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equation. lnðV=Vo Þ Z Kk1 t

(4)

Vo =V Z 1 C k2 Vo t

(5)

t1=2 Z ðk2 Vo ÞK1

(6)

where V and Vo are the liquid volumes in the foam at time tZt and at tZ0, respectively. The foam stability was also determined by the time evolution of the foam conductivity (Kato, Takahashi, Matsudomi, & Kobayashi, 1983; Wright & Hemmant, 1987). The relative conductivity of the foam (Ct/Ci, where Ct and Ci are the foam conductivity values at time tZt and at tZ0, respectively, of the foam rupture) as a function of time was fitted using a second-order exponential equation Ct =Ci Z A1 expðKt=t1 Þ C A2 expðKt=t2 Þ

(7)

where A1 and A2 are adjustable parameters and t1 and t2 are the relaxation times, which can be related to the kinetics of liquid drainage from the foam (including the gravitational drainage and marginal regeneration) and disproportionation and foam collapse, respectively. 2.2.2. Emulsifying properties Oil-in-water emulsions (o/w) were prepared (Fig. 2) using a valve homogeniser (Emulsiflex-2000-B3, Avestin, Ottawa, Canada), using a single pass at 50 MPa after premixing with a Ultraturrax (Ika Lab., T25 basic, Germany) at 8000 rpm for 10 min. The oil phase (30% by weight of the total emulsion)

Fig. 2. Protocol for the formation and determination of the stability of oil-in-water emulsions formed from caseinate solutions.

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was Trisun oil. Caseinate concentration in the aqueous phase at pH 7 was between and 0.1 and 5% w/w. The samples were stored at 5 8C. All experiments were performed in duplicate at room temperature (20–23 8C). The emulsifying capacity was determined from the measurement of the droplet size. The mean droplet size, d32, of freshly made emulsions was measured by light scattering using a Malvern Mastersizer. The measurement was repeated using 0.2% w/w of SDS which dispersed the aggregates formed during emulsification. It was observed that SDS did not affect the droplet size of un-flocculated emulsions. Thus, the difference between the apparent (measurement in water) and real (measurement in SDS aqueous solution) gives a measurement of the existence of flocculation during emulsification (Tomas, Courthaudon, Paquet, & Lorient, 1994). The specific surface area (SSA, or surface area per unit mass of dispersed phase) of the freshly made emulsion was calculated by the Mastersizer using the mean droplet size (d32) and protein concentration adsorbed at the oil–water interface (Cornec et al., 1998). Emulsion stability to coalescence was assessed from the change in droplet-size distribution upon accelerated creaming by centrifugation of the sample at 13,000g for 15 min (Wilde, Cornec, Husband, & Clark, 1999). The cream layer was redispersed in the aqueous phase by agitation and the SSA of the reformed emulsion was measured again by the Mastersizer. The extent of the coalescence was determined from the ratio of the specific surface area of the emulsion after centrifugation (SSAac) and before centrifugation (SSAbc). The creaming rate was monitored by measuring in a commercial instrument (TurbiScan MA2000, Formulation, Toulouse, France) the backscattering of a pulsed near infrared light source (lZ850 nm) from an emulsion as a function of its height (Mengual, Meunier, Cayre´, Puech, & Snabre, 1999). Emulsions were placed into cylindrical glass tubes and stored at 5 8C. The backscattering of light from emulsions was then measured at room temperature (20–23 8C) with height as a function of time. The results are presented as the backscattering profile (DBS/100%) vs. time or as the creaming rate. The creaming rate was determined from the slope of a plot of the height of the lower layer vs. time in the initial stages of creaming (Chanamai & McClements, 2000). However, due to problems in visualisation of the height of the serum layer, we have defined a position where the backscattering of light was half of the peak thickness.

and after bubbling of a caseinate solution at 1% w/w (as an example). Different caseinate concentrations in solution behaved in a similar way. These plots shown the typical increase of liquid and gas (data not shown) incorporated to the foam, the increase in the foam volume, and the foam conductivity during the bubbling. However, during the foam destruction, the foam volume and foam conductivity decreased, and the drainage of liquid increased with time. Foam was not produced with caseinate aqueous solutions at lower concentration than 1!10K3% w/w. From these plots, the foaming capacity and stability were deduced. 3.1.1. Foaming capacity The overall foaming capacity (OFC, ml/s), the foam capacity (FC), the foam maximum density (MD), and the relative foam conductivity (Cf, %) as a function of caseinate concentration in solution are shown in Fig. 3. It can be

3. Results and discussion 3.1. Foaming characteristics of aqueous solutions of caseinate Fig. 1 shows the evolution of the liquid volume in solution, foam volume, and foam conductivity during

Fig. 3. The effect of caseinate concentration (% w/w) on foaming capacity. (,) OFC (ml/s): overall foaming capacity. (O) FC: foam capacity. (D) MD: foam maximum density. (P) Cf (%): relative foam conductivity. Temperature 20 8C. Aqueous phase pH 7. Ionic strength 0.05 M.

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deduced that the overall foaming capacity (OFC) the gas and liquid retentions (FC and MD, respectively) in the foam and the foam density (Cf) increase with caseinate concentration in solution. If the foam capacity depends on the rate of caseinate adsorption at the interface (Kitabatake & Doi, 1988; Martin, Grolle, Bos, Cohen Stuart, & van Vliet, 2002) all these foaming parameters must have a relation with the first step of adsorption of the protein to the interface. In addition, during the adsorption of the protein at the interface the surface dilatational modulus also increases (Horne & Rodrı´guez Patino, 2003). The kinetics of the protein adsorption at the air–water interface can be monitored by measuring changes in surface pressure (p) or surface dilatational modulus with time. During the first step, when diffusion is the rate-determining step, a modified form of the Ward and Tordai equation (Ward & Tordai, 1946) can be used to correlate the change in the surface pressure with time p Z 2C0 KTðDt=3:14Þ1=2

(8)

where C0 is the concentration in the bulk phase, K is the Boltzmann constant, T is the absolute temperature, and D is the diffusion coefficient. For adsorption of caseinate from aqueous solutions we have observed that the diffusion at the interface controls the adsorption process at short adsorption time, typical for foam production (Rodrı´guez Nin˜o, Carrera, Pizones, & Rodrı´guez Patino, 2004). Thus, from the slope of the plot of p against t1/2 we deduce the diffusion rate (kdiff) of caseinate towards the interface.

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From the data shown in Fig. 4 we observe that there exists a linear relationship between the foaming capacity and the rate of diffusion of caseinate towards the air–water interface. That is, at higher protein concentrations in solution, as the rate of diffusion is higher, the foaming capacity is also higher because the protein concentration at the interface and the surface dilatational modulus are also higher. As at the higher caseinate concentration in solution analysed (at 4–5% w/w) the interface is saturated by the protein (Rodrı´guez Nin˜o et al., 2001), this concentration coincides with the maximum foaming capacity of a caseinate solution, within the concentration range investigated. 3.1.2. Foam stability The static foam stability was determined from the volume of liquid drained from the foam over time (Rodrı´guez Patino et al., 1995, 1997). In all of the experiments performed in this study, the half-life time (t1/2), corresponding to the empirical second-order equation (Eq. (6)), fits the data of foam drainage best. Fig. 5 shows the foam stability quantified by the half-life time as a function of caseinate concentration in aqueous solution. The foam stability increased with protein concentration in solution, up to approximately 0.1–1% w/w. At higher protein concentrations in solution the foam consisted of smaller and denser bubbles as indicated by the higher foam density (MD) and higher relative foam conductivity (Cf) in Fig. 3. At the lower caseinate concentrations, the foam consisted of coarser

Fig. 4. Foaming capacity of aqueous solutions of caseinate as a function of the rate of diffusion (slope of p vs. t1/2) of caseinate to the air–water interface. OFC (ml/s): overall foaming capacity. FC, foam capacity; MD, foam maximum density; Cf (%), relative foam conductivity; temperature, 20 8C; aqueous phase, pH 7; ionic strength, 0.05 M; Caseinate concentration (% w/w): (,) 1.0 (O) 0.5, (D) 0.25, (P) 0.1, ($) 1!10K2, (3) 1!10K3, (")1!10K4.

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polyhedral bubbles. For this particular protein a concentration of 0.1% w/w represents the optimum point for foam stability. The kinetics of liquid drainage from the foam (including the gravitational drainage and marginal regeneration) and disproportionation, and foam collapse, were determined by the fitting of the relative conductivity of the foam vs. time using a second-order exponential equation (Eq. (7)). The caseinate concentration dependence on relaxation times t1 and t2 corresponding to gravitational drainage and marginal regeneration, on the one hand, and disproportionation and foam collapse, on the other hand, respectively, is shown in Fig. 6. As for the static foam stability (i.e. for the half-life time of the foam), the relaxation times t1 and t2 increased with protein concentration and tended to a plateau at caseinate concentration in solution higher than 0.1% w/w. That is, the stability of the foam against the drainage/ marginal regeneration and disproportionation/collapse increased at higher protein concentrations in solution.

For a particular protein the overall foam destabilization (the half-life time of the foam) and the individual destabilization processes (drainage, disproportionation and coalescence) must be related to the interfacial characteristics (protein concentration at the interface, interfacial shear and dilatational characteristics) of the protein film adsorbed around the bubbles. At very low caseinate concentrations in solution the amount adsorbed at the interface is also low, as deduced from the surface pressure evolution with the protein concentration in solution (Rodrı´guez Nin˜o et al., 2004). Therefore, the greater stability of foams formed with higher concentrations of caseinate (Figs. 5 and 6) could be related to the amount of caseinate adsorbed at the air–water interface. This hypothesis is confirmed in data presented in Fig. 7. In fact, there exists a close linear relationship between the relaxation times for drainage/marginal regeneration and disproportionation/collapse or the half-life time of the foam (data not shown) and the equilibrium surface pressure (pe) deduced from the adsorption isotherm of caseinate solutions (Rodrı´guez Nin˜o et al., 2004). This close relationship is not unexpected because at the higher protein concentrations adsorbed at the interface (at the higher pe) the surface dilatational (Rodrı´guez Patino, Carrera, Rodrı´guez Nin˜o, & Cejudo, 2001) and shear (Rodrı´guez Patino & Carrera, 2004) characteristics of the adsorbed caseinate films are also higher. During the film drainage the surface shear properties seem to be important because the higher the surface shear viscosity, the slower the drainage, and the more stable the foam (Prins, 1999). However, some controversy exists between surface dilatational properties and foam stability (Baeza, Carrera, Pilosof, & Rodrı´guez Patino, 2004a,b; Langevin, 2000; Martin et al., 2002). Martin et al. (2002) showed that only for stability against disproportionation were surface dilatational properties found to play a role. Clearly, this point requires further investigation regarding the effect of interfacial characteristics on individual mechanisms of foam destabilization.

Fig. 6. The effect of caseinate concentration (% w/w) on relaxation time for foam drainage (t1: O) and disproportionation/collapse (t2: D). Temperature, 20 8C; aqueous phase, pH 7; ionic strength, 0.05 M.

Fig. 7. The equilibrium surface pressure (pe) dependence of relaxation time for foam drainage (t1: O) and disproportionation/collapse (t2: D). Temperature 20 8C. Aqueous phase pH 7. Ionic strength 0.05 M.

Fig. 5. The effect of caseinate concentration (% w/w) on half-life time of foam drainage. Temperature, 20 8C; aqueous phase, pH 7; ionic strength; 0.05 M.

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In summary, a common characteristic of foams obtained with aqueous caseinate solutions is that the amount of protein in solution held in the foam increases with protein concentration. Foams retain a higher amount of liquid, are denser, and the bubbles are smaller when the protein concentration increases. The effect of caseinate in increasing the surface pressure and surface dilatational and shear properties also improves the foam stability. 3.2. Emulsifying characteristics of caseinate 3.2.1. Emulsifying capacity Fig. 8 shows the evolution of mean droplet size (d3,2) with the concentration of caseinate in the aqueous phase in the presence and in the absence of SDS. For emulsions formed in the presence of SDS a sharp drop in the particle size was observed up to a concentration of 1% w/w of caseinate. After this point d3,2 was independent of the amount of caseinate in solution. The evolution of d3,2 in the absence of SDS shows the same trend but, at lower caseinate concentrations, the values of d3,2 were lower. Consequently, the evolution of the specific surface area (SSA) with caseinate concentration in the presence and the absence of SDS shows the opposite trend (data not shown). These results indicate that: (i) there exists a critical concentration of caseinate in the aqueous phase of ca. 1% w/w at which the emulsifying capacity was maximum. At this concentration the mean droplet size was minimum. (ii) At caseinate concentrations in solution above ca. 1% w/w the emulsifying capacity was constant and the size distribution of droplets in emulsion was monomodal. From this critical caseinate concentration the interfacial film around the droplets may be saturated by the protein. Thus, the excess of caseinate would participate in the formation of multilayers and/or would be present in the aqueous phase, with repercussions on the stability of the emulsions as will be analysed latter. (iii) At caseinate concentrations lower

Fig. 8. The effect of caseinate concentration (% w/w) on mean particle diameter of oil-in water emulsions in the absence of SDS (D) or in the presence of SDS (O). Temperature 20 8C. Aqueous phase pH 7. Ionic strength 0.05 M.

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than 1% w/w the fresh emulsion shows some degree of flocculation, which disappears in a dispersing medium, with the presence of SDS; the size distribution of droplets in emulsion was bimodal. That is, at low caseinate concentrations (C!1% w/w), as the interface is not saturated by the protein and/or in the absence of protein multilayers, droplet flocculation occurred in the fresh emulsion. In emulsions, SDS displaces any caseinate from the interface and thus breaks up flocs formed from bridging flocculation. The fact that there is a discrepancy between the d3,2 values of SDS and non-SDS treated samples below 1% caseinate hints that these samples are apparently bridgeflocculated, and SDS-mediated displacement of interfacial caseinate releases the droplets. SDS has no impact in the emulsions containing 1% caseinate or more, since there is enough protein there to fully saturate the newly formed interfaces after emulsification, so no bridged flocs exist to be broken up by SDS. Furthermore, the sharp drop in droplet size in 1% protein (SDS treated samples) shows that this is the minimum concentration of protein to fully cover the newly formed interface during emulsification. 3.2.2. Emulsion stability Emulsion stability was determined by monitoring the extent of droplet coalescence induced by accelerated creaming. The extent of the coalescence was determined from the ratio between observed SSA after centrifugation (SSAac) and before centrifugation (SSAbc). Fig. 9 shows the effect of caseinate concentration in solution on emulsion stability. At caseinate concentrations higher than 0.1% w/w, emulsions were stable to coalescence. Thus, at lower caseinate concentrations as the interfacial film is not saturated by the protein, both bridging flocculation and coalescence contribute to the emulsion instability.

Fig. 9. The effect of caseinate concentration (% w/w) on stability of oil-in water emulsions, after centrifugation. The stability of oil-in water emulsions after centrifugation is expressed as the ratio of the specific surface area of the emulsion after centrifugation (SSAac) and before centrifugation (SSAbc). Temperature 20 8C; aqueous phase pH, 7; ionic strength, 0.05 M.

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Fig. 10. The emulsion stability by static coalescence expressed as the time dependence of mean droplet size (measured in the presence of SDS) of oil/water emulsions. Caseinate concentration (% w/w): (,) 0.1. (O) 0.25, (D) 0.5, (P) 1, ($) 2, (C) 3, and (!) 4. Temperature 20 8C. Aqueous phase pH 7. Ionic strength 0.05 M.

Emulsion stability by static coalescence was also tested by the evolution of the droplet size with time (Fig. 10). A low emulsion stability was observed for emulsions stabilised by low caseinate concentration in solution. In fact, at the lowest caseinate concentration (at 0.1% w/w) a small increase in the values of d3,2 was observed after the emulsification, a phenomenon which confirms the existence of coalescence. The initial and time-averaged d3,2 values for 0.1–0.5% w/w emulsions are higher than the 1% ones, which means that the 0.1–0.5% w/w emulsions recoalesce immediately after emulsification. In addition, at caseinate concentration higher than 1% w/w the values of d3,2 were constant within the time range investigated and do not depend on the protein concentration, which is an indication of the emulsion stability against coalescence at higher caseinate concentrations. Thus, at this critical concentration of caseinate in solution (at 1% w/w) the emulsions were stable against accelerated and static coalescence. Interestingly, at this concentration we have observed the maximum values in surface pressure (Rodrı´guez Nin˜o et al., 2001, 2004), surface dilatational modulus (Rodrı´guez Patino et al., 2001; Rodrı´guez Nin˜o et al., 2004), and surface shear viscosity (Rodrı´guez Patino et al., 2004) and a more compact interfacial structure of caseinate films (Rodrı´guez Patino et al., 2001). The creaming, that is the process by which buoyant emulsion droplets tend to rise to the top of a container, was also analysed from a kinetic point of view by the backscattering of the emulsion as a function of caseinate concentration in solution. In Fig. 11 we show the time evolution of creaming profiles for an emulsion with a concentration of caseinate in solution of 1% w/w (as an example). The differential profiles show the backscattering variations when a sample destabilises as a function of time. For all the emulsions the backscattering of light was fairly constant along their entire height at the beginning of

Fig. 11. Creaming profiles of backscattering as a function of time for an oilin-water emulsion formed by a caseinate solution at 1% w/w. Temperature 20 8C. Aqueous phase pH 7. Ionic strength 0.05 M. The creaming profiles were obtained every two days. Thus, the last line corresponds to an emulsion 40 days old.

the experiment because there was an even distribution of droplets throughout the system. Over time the droplets moved upwards due to gravity which caused a decrease in the backscattering at the bottom of the emulsions (because the droplet concentration decreased) and an increase at the top (because the droplet concentration increased). From the time evolution of creaming profiles we have obtained the time evolution of the peak thickness—defined as the thickness of backscattering peak at 50% of the peak maximum length (Fig. 12A)—and the creaming rate— determined by the slope of the time evolution of the peak thickness in Fig. 12A in the initial stages of creaming (Chanamai & McClements, 2000)—as a function of caseinate concentration in solution (Fig. 12B). We have observed that the presence of flocculation has a significant effect on the rate of creaming (Fig. 12B). The creaming rate was higher in flocculated and coarser emulsions (at Ccaseinate! ca. 1% w/w) than in the nonflocculated emulsions, as would be expected because of the increase in the size of the droplets (Chanamai & McClements, 2000; Robins, 2000). At sufficiently high caseinate concentration in solution the creaming rate became extremely low, probably because an emulsion gel with closely packed droplets was formed in the middle and at the top of the sample, which restricted the creaming of the droplets. In addition, increased aqueous phase viscosity and smaller droplet size may also contribute to the low creaming rate of emulsions at high caseinate concentrations in solution. Surprisingly, at caseinate concentrations in solution higher than 2% w/w an increase in the rate of creaming (which is difficult to quantify) was observed (Fig. 12A). This phenomenon may be attributed to the presence of depletion flocculation. In fact, at these concentrations the interface is saturated by the protein and the excess could form micelles of caseinate in solution. Dickinson and Golding (1997) demonstrated that emulsions made with

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the equilibrium surface pressure of aqueous solutions of caseinate. At surface pressures lower than that of monolayer saturation (at C!1!10K3%, w/w) the foaming is zero. The emulsifying capacity was correlated with the protein concentration in solution. Coalescence was observed only at the lower caseinate concentrations in solution. As a protein layer saturates the interface, at higher protein concentrations, the emulsion instability is due to flocculation and/or creaming. The coalescence and creaming rate correlate well with the protein concentration in solution.

Acknowledgements The authors acknowledge the support of CICYT thought the grant AGL2001-3843-C02-01. The comments and suggestions raised by the referees are also acknowledged.

References

Fig. 12. The caseinate concentration dependence of creaming velocities of oil-in-water emulsion formed by caseinate solutions. (A) The time evolution of peak thickness as a function of caseinate concentration (% w/w): (,) 0.1, (O) 0.25, (D) 0.5, (P) 1.0, and ($) 2.0. (B) Creaming rate as a function of caseinate concentration in solution (*). Temperature 20 8C. Aqueous phase pH 7. Ionic strength 0.05 M.

O2% sodium caseinate were more unstable towards creaming than emulsions made with lower caseinate concentrations. This destabilization was attributed to depletion flocculation caused by the presence of high concentrations of non-adsorbed caseinate. The addition of CaCl2 to sodium caseinate solutions (O2%) prior to emulsification enhanced the creaming stability probably due to the fact that the addition of CaCC resulted in association of caseinate into large aggregates which were incapable of inducing depletion flocculation (Dickinson & Golding, 1998; Ye & Singh, 2001).

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