Milk and soy protein films at the air–water interface

Food Hydrocolloids 19 (2005) 417–428 www.elsevier.com/locate/foodhyd

Milk and soy protein films at the air–water interface M. Rosario Rodrı´guez Nin˜o, Cecilio Carrera Sa´nchez, Victor Pizones Ruı´z-Henestrosa, 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 In this contribution we present a systematic comparison of the two major fractions of globulin from a soy protein isolate (b-conglycinin and glycinin, including the chemical reduction of glycinin with dithiothreitol (DTT)) and typical milk proteins (b-casein, caseinate, and WPI). The comparison is centred on the most important functional properties of spread and adsorbed protein films at the air–water interface (such as adsorption, structural, topographical, and dynamic characteristics), as a function of the aqueous phase pH and the protein concentration in the bulk phase and at interface. A combination of surface techniques (tensiometry, surface film balance, Brewster angle microscopy and surface dilatational rheology) was used in this study. A notable feature of soy proteins is the strong pH dependence of the molecular conformation and the associated functional properties, such as surface activity, film structure, surface dilatational viscoelasticity, and especially, the rate of adsorption at a fluid interface. Optimum functionality occurs at pH!5, which limits the application of soy globulins as food ingredients. In this respect the behaviour of milk and soy proteins is quite different. q 2005 Elsevier Ltd. All rights reserved. Keywords: Soy proteins; Milk proteins; Air–water interface; Adsorption; Spreading; Interfacial rheology

1. Introduction A majority of foods are emulsions and foams, which are two-phase systems in which one of the phases (oil or gas or both) is dispersed in an aqueous continuous phase (Dickinson, 1992; McClements, 1999). Because oil/water and air/water interfaces are high-energy interfaces, emulsions and foams collapse as soon as they are created unless an emulsifier or a foaming agent is added to the system. The emulsifier or foaming agent (i.e. surfactant), because of its amphiphilic chemical nature (i.e. its affinity to both water and non-polar phases), adsorbs and orients itself with the lipophilic group towards the non-polar phase and the hydrophilic group towards the aqueous phase. This molecular ordering and film formation by a surfactant decreases the interfacial tension and contributes to the formation and stability of the dispersion (emulsion or foam). Two types of emulsifier or foaming agents are used in foods: low molecular weight surfactants (mono- and diglycerides, phospholipids, etc.) and macromolecules, such as proteins * Corresponding author. Tel.: C34 95 4556446; fax: C34 95 4557134. E-mail address: [email protected] (J.M.R. Patino). 0268-005X/$ - see front matter q 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodhyd.2004.10.008

and some hydrocolloids. Although low molecular weight surfactants are more effective than proteins in reducing the interfacial tension, foams and emulsions formed by such surfactants are mostly unstable. This is because proteins, in addition to lowering interfacial tension, can form continuous viscoelastic gel-like films around oil droplets or air cells via non-covalent intermolecular interactions and via covalent disulfide cross-linking, whereas the low molecular weight surfactant cannot form such a viscoelastic film. Thus, in foods that contain both low molecular and macromolecular surfactants, the stability of colloidal dispersed phases is primarily dependent on protein films adsorbed at the interfaces (Damodaran & Paraf, 1997; Friberg & Larsson, 1997; Hartel & Hasenhuette, 1997; Sjo¨mblom, 1996; Rodrı´quez Patino, Rodrı´quez Nin˜o & Carrera, 2003). Although the study of food-dispersed systems generated from proteins is dominated by research into milk proteins, there exists an increasing interest in the use of vegetable proteins from cereals and legumes for the formation and stabilization of food emulsions and foams. Practical observations indicate that all proteins are not equally surface active, even though all are amphiphilic and a majority of them contain similar percentages of polar and no

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polar amino acid residues (Damodaran & Paraf, 1997). The wide differences in the surface activities of various proteins therefore must be related to their physical, chemical, and conformational properties, which include size, shape, amino acid composition and sequence, charge, and charge distribution. Protein stability is of particular importance in determining their functionality in food systems. This is because a particular functional property is often governed by a specific conformational state of a protein and any alteration of that state affects its functionality. Apart from the above mentioned intrinsic molecular factors, the surface activity of a protein in a complex food systems will be dictated by several other extrinsic factors, such as pH, ionic strength, temperature, and interactions with other food components (Damodaran, 1997; Rodrı´quez Patino, Rodrı´quez Nin˜o et al., 2003). The aim of this paper was to obtain systematic information on the interfacial characteristics of the two major fractions of globulin from a soy protein isolate (b-conglycinin and glycinin, including the chemical reduction of glycinin with dithiothreitol (DTT)) and typical milk proteins (b-casein, caseinate, and WPI). Soy proteins exhibit high functional properties compared to other plant proteins (Utsumi, Matsumura, & Mori, 1997; Martı´n, 2003). In addition, the demand for safe, high-quality, health foods with good nutritional value has increased the use of soy proteins. On the other hand, milk proteins have been utilized traditionally in food dispersions (Dalgleish, 1997). For further information concerning the interfacial characteristics of proteins the readers are referred to recent reviews (Benjamins, 2000; Martı´n, 2003; Horne & Rodrı´guez Patino, 2003).

interfacial characteristics of protein films were prepared using Milli-Q ultrapure water and were buffered at pH 2, 5, 7, and 8. Analytical-grade acetic acid and sodium acetate for buffered solutions at pH 5 and Trizma-HCl [(CH2OH)3 CNH2/(CH2OH)3CNH3Cl] for buffered solutions at pH 7 and 8 were used as supplied by Sigma (O95%) without further purification. Aqueous solutions at 0.1 M of HCl (analytical-grade, Panreac) and KCl (analytical-grade, Merck) were used for adjusting the pH 2 and the ionic strength of the aqueous solutions, respectively. Ionic strength was 0.05 M in all the experiments. 2.2. Surface tension measurements Surface tension measurements were used to determine protein spreading and protein adsorption. Surface activity was expressed by the surface pressure, pZsoKs, where so and s are the aqueous subphase surface tension and the surface tension of the aqueous solutions of protein, respectively. Measurements were performed with a Sigma 701 digital tensiometer (KSV, Finland), based on the Wilhelmy method, with a roughened platinum plate, as described elsewhere for measurements of equilibrium spreading pressure (pe) and surface pressure isotherm (Rodrı´guez Nin˜o, Carrera, Cejudo, & Rodrı´guez Patino, 2001). True equilibrium adsorption does not seem to be possible with proteins, even after 2 or 3 days (Benjamins, 2000; Rodrı´guez Nin˜o & Rodrı´guez Patino, 1998a,b). Therefore, we considered the surface pressure measured after 24 h as the pseudo-equilibrium value. Surface tension measurements were reproducible within G0.2 mN/m. 2.3. Protein adsorption

2. Materials and methods 2.1. Materials b-Casein (O99%) was supplied and purified from bulk milk from the Hannah Research Institute (Ayr, Scotland). Caseinate (a mixture of z38% b-casein, z39% as1-casein, z12% k-casein, and z11% as2-casein) was supplied and purified from bulk milk from Unilever Research (Colworth, UK). Whey protein isolate (WPI), a native protein with high content of b-lactoglobulin (protein 92G2%, b-lactoglobulin O95%, a-lactalbumin !5%) obtained by fractionation, was supplied by Danisco Ingredients (Brabran, Denmark). The isolation, solubility, and structural characteristics of b-conglycinin (fraction 7S) and glycinin (fraction 11S) soy globulins have been described elsewhere (Molina, Carrera, Rodrı´guez Nin˜o, An˜o´n, & Rodrı´guez Patino, 2003). Glycinin was reduced using 10 mM DTT (11SC10 mM DTT) as described elsewhere (German, O’Neill, & Kinsella, 1985; Kim & Kinsella, 1986). To form the spread surface film, protein was spread in the form of a solution using water at pH 7 for milk proteins and at pH 8 for soy globulins as a spreading solvent. Samples for

The experiments for protein adsorption on water were carried out on an automated drop tensiometer as described elsewhere (Rodrı´guez Nin˜o & Rodrı´guez Patino, 2002). Surface pressure measurements were reproducible within G0.2 mN/m. 2.4. Surface film balance Measurements of the surface pressure (p) vs average area per molecule (A) were performed on fully automated Wilhelmy- and Langmuir-type film balances as described elsewhere (Rodrı´guez Patino, Carrera, & Rodrı´guez Nin˜o, 1999; Rodrı´guez Patino, Carrera, Rodrı´guez Nin˜o, & Cejudo, 2001). The reproducibility was better than G0.5 mN/m for surface pressure and G0.25!10K3 m2 per mg for area. Each isotherm was measured at least five times using four new aliquots. 2.5. Brewster angle microscope (BAM) A commercial Brewster angle microscope (BAM), BAM2, manufactured by NFT (Go¨ttingen, Germany) was

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used to study the topography of the monolayer. Further characteristics of the device and operational conditions were described elsewhere (Rodrı´guez Patino et al., 1999). The surface pressure measurements, area, and relative reflectivity (I) as a function of time were carried out simultaneously during continuous compression and expansion of the monolayer. 2.6. Surface dilatational rheology To obtain surface rheological parameters at the air–water interface—such as surface dilatational modulus (E), elastic (Ed) and viscous (Ev) components, and loss angle tangent (tan q)—of spread monolayers a modified Wilhelmy-type film balance (KSV 3000) was used as described elsewhere (Rodrı´guez Patino, Carrera, Rodrı´guez Nin˜o, & Cejudo, 2001; Rodrı´guez Patino, Carrera, Rodrı´guez Nin˜o, & Cejudo M., 2001). The surface rheological characteristics of adsorbed films at the air–water interface were measured by an automated drop tensiometer as described elsewhere (Rodrı´guez Patino, Carrera, Molina, Rodrı´guez Nin˜o, & An˜o´n, 2003).

3. Results and discussion 3.1. Spreading of soy globulins at the air–water interface at equilibrium Equilibrium spreading pressures of milk (b-casein and WPI) (Rodrı´guez Nin˜o et al., 2001) and soy (fractions 7S, 11S, and 11SC10 mM DTT) (Molina et al., 2003) proteins at the air–water interface as a function of pH and at 20 8C, are shown in Fig. 1. The magnitude of pe was dependent on the protein, and especially, on the aqueous phase pH. For 7S and 11S, pe showed a minimum at pH 5. However, for 11SC10 mM DTT pe decreased with increasing pH. At pH

Fig. 1. Equilibrium surface pressure for milk and soy protein spread monolayers on the air–water interface. Temperature 20 8C. Ionic strength 0.05 M.

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8 the highest pe values were observed for 7S and 11S. At neutral pH the pe values for soy protein fractions are higher than those for milk proteins, with the minimum value for b-casein. However, at pH 5 the pe values for soy protein fractions are between those for milk proteins. The pH has no significant effect on the value of pe for WPI. The minimum pe for 7S and 11S at pH 5 as compared to pe on aqueous solutions at pH different to the isoelectric point (pI) can be explained by the fact that the protein is more difficult to convert into a monolayer at its isoelectric point. In fact, at pH 5 the fractions 7S and 11S are close to the isoelectric point (pIz4.9–5.5) showing the maximum aggregation and the minimum solubility (Molina et al., 2003). This behaviour is not observed for globular milk proteins (like WPI), which have a pI similar to that for soy globulins (pIz4.9–5.2). The reduction of 11S with 10 mM DTT resulted in molecular conformational changes, which enhance surface hydrophobicity (Kim & Kinsella, 1986; Wolf, 1993) and the flexibility of the molecule; these changes increased pe at pH 5 compared to pe for 11S (Fig. 1). Surprisingly, a typical disordered protein, like b-casein, does not follow this trend and shows the lower values of pe at pHypI. 3.2. Adsorption of soy globulins at the air–water interface at equilibrium The protein concentration dependence on surface pressure (surface pressure isotherm) for milk (Rodrı´guez Nin˜o et al., 2001) and soy (Molina et al., 2003) proteins on water at 20 8C and at pH 5 and 7 showed a sigmoidal behaviour (Fig. 2). At low protein concentrations, the initial solutions caused only a small increase in the surface pressure. The surface pressure increased with protein concentration and tended to a plateau. This plateau commenced at the point where surface pressure reached its maximum value over the range of protein concentrations from 1!10K6 to 5%, wt/wt. The behaviour of adsorbed protein films (Fig. 2) can be interpreted in terms of monolayer coverage. At the lower protein concentrations, as the surface pressure is close to zero, the adsorbed protein residues may be considered as a two-dimensional ideal gas. Proteins at higher concentrations, but lower than that of the plateau, form a monolayer of irreversibly adsorbed molecules. As the plateau is attained, the monolayer is saturated by protein that is irreversibly adsorbed. The protein concentration at which the plateau is attained is the adsorption efficiency (AE). At higher protein concentrations, the protein molecules may form multilayers beneath the primary monolayer, but these structures do not contribute significantly to surface pressure (Graham & Phillips, 1979a). The maximum surface pressure at the plateau is the superficial activity (SA). However, some differences exist between milk and soy proteins depending on the pH. The superficial activity (SA) is almost identical for WPI, 7S and 11SC10 mM DTT,

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the pH has a lesser effect on the superficial activity of milk proteins. The lower surface activity at pH 5 coincides with an aggregation of soy proteins at interface and in the bulk phase (Molina et al., 2003). The adsorption efficiency (AE) is also lower at pH 5, especially for soy globulins. 3.3. Structural and topographical characteristics of protein monolayers The structural and topographical characteristics of milk and soy proteins spread at the air–water interface at pH 5 (Fig. 3) and 7 (Fig. 4) were determined from p–A isotherms coupled with BAM. The structural characteristics of soy globulin spread monolayers depend on film ageing (Carrera, Molina, Rodrı´guez Nin˜o, An˜o´n, & Rodrı´guez Patino, 2003a, b). A significant shift of the p–A isotherms towards higher molecular areas over time was observed. That is, the soy globulin film changed significantly over time. For soy globulins the ageing time was higher than for b-lactoglobulin (Garofalakis & Murray, 1999), a phenomenon that may be due to unfolding of globular protein at the interface (Tronin, Dubrovsky, Dubrovskaya, Radicchy, & Nicolini, 1996).

Fig. 2. Concentration dependence on surface pressure (surface pressure isotherm) for milk and soy protein adsorbed films on the air–water interface. Aqueous phase pH (A) 5 and (B) 7.Temperature 20 8C. Ionic strength 0.05 M. Symbols: (:) b-casein, (C) WPI, (6) 7S, (7) 11S, and ($) 11SCDTT.

but lower for b-casein and 11S soy globulin, at neutral pH (Table 1). For pH 5 the superficial activity of soy proteins is lower than at neutral pH. Moreover, for pH 5 the superficial activity for soy proteins is lower than for milk proteins and the range of surface activity is reduced at soy globulin concentrations higher than 1!10K3% wt/wt. However, Table 1 Adsorption efficiency (AE) and superficial activity (SA) of milk and soy globulins as a function of the aqueous phase pH pH 5

b-casein WPI 7S 11S 11SCDTT

pH 7

AE (% wt/wt)

SA (mN/m)

AE (% wt/wt)

SA (mN/m)

0.01 0.1 0.05 0.05 0.05

25.4 29.5 18.5 18.5 18.5

0.15 2.5 0.1 0.1 0.1

23.6 26.6 26.4 21.0 27.3

Temperature 20 8C. IZ0.05 M.

Fig. 3. (A) p–A isotherm and (B) relative reflectivity as a function of surface pressure for milk and soy protein spread monolayers on the air–water interface. Temperature 20 8C. Ionic strength 0.05 M. Aqueous phase pH 5. Symbols: (—) b-casein, (C) WPI, (6) 7S, (7) 11S, and ($) 11SCDTT.

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Fig. 4. (A) p–A isotherm and (B) relative reflectivity as a function of surface pressure for milk and soy protein spread monolayers on the air–water interface. Temperature 20 8C. Ionic strength 0.05 M. Aqueous phase pH 7. Symbols: (—) b-casein, (C) WPI, (6) 7S, (7) 11S, and ($) 11SCDTT.

The results of p–A isotherms (Figs. 3A and 4A), with the help of the compressional coefficient deduced from the slope of the p–A isotherm (kZKdp/dA), indicate that milk and soy protein monolayers at the air–water interface adopt two different structures or condensation states and the collapse phase. The surface pressure at the transition or at the change in the condensation state of the monolayer (pt) is included in Table 2. Disordered proteins such as b-casein at Table 2 Surface pressure at which protein monolayers adopt two different structures or condensation states (pt) and relative intensity at the equilibrium surface pressure (Ipe) as a function of the aqueous phase pH pH 5

b-casein WPI 7S 11S 11SCDTT

pH 7

pt (mN/m)

Ipe (a.u.)

15 – 15.2 14.5 16.5

1.64!10 1.7!10K6 2.5!10K6 4.0!10K6 3.9!10K6 K6

pt (mN/m)

Ipe (a.u.)

10 – 19 20 20

3.1!10K6 1.9!10K6 2.0!10K6 4.4!10K6 2.3!10K6

The value of pt was deduced from the presence of a discontinuity in the p–A isotherm, which corresponds with the presence of a maximum in the plot of compressional coefficient vs surface pressure. Temperature 20 8C. IZ0.05 M.

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low surface pressures (p!pt) exist as trains with all aminoacid segments located at the interface (structure 1). At higher surface pressures (pOpt), and up to the equilibrium surface pressure, amino-acid segments are extended into the underlying aqueous solution and adopt the form of loops and tails (structure 2). As for most globular proteins, at low surface pressures most amino-acid residues in WPI and soy globulin molecules adopt loop conformation at the air– water interface. But the loop conformation is more condensed at higher surface pressures and is displaced towards the bulk phase at the collapse point (Graham & Phillips, 1979b). The collapse was produced at a surface pressure higher than the equilibrium surface pressure for soy globulins, a phenomenon which indicates that the monolayer was in a metastable state at pOpe. The surface pressure at which soy protein monolayers adopt two different condensation states (pt) is lower at pH 5 (14.5– 16.5 mN/m) than at pH 7 (19–20 mN/m), but the opposite was observed for b-casein monolayers (Table 2). The pt values for b-casein and soy globulins are practically the same at pH 5, but the values of pt are higher for soy globulins than for b-casein at neutral pH. For WPI monolayers the transition between different condensation states was not clearly observed. The monolayer topography (BAM image) clearly shows significant differences between milk and soy protein monolayers as a function of pH (Fig. 5) and corroborates the conclusions deduced from the p–A isotherms. The topography of b-casein and WPI monolayers shows that the domains that residues of protein molecules adopt at the air– water interface appear to be of uniform reflectivity, suggesting homogeneity in thickness and film isotropy (Fig. 5A) up to the monolayer collapse. At this point some folds of collapsed protein residues were observed at the interface (Fig. 5B). By BAM we are unable to distinguish between different structures or states of condensation in milk protein monolayer at every pH. However, at a higher level of magnification, using atomic force microscopy (AFM), some in-homogeneities in milk protein layers and significant differences in the topography of b-casein and b-lactoglobulin monolayers were observed (Mackie, Gunning, Wilde, & Morris, 1999). BAM images of b-conglycinin at pH 5 corroborated the fact that a heterogeneous phase was present during the compression (Fig. 5C) up to the monolayer collapse, with numerous flickering microdomains that increased in brightness as the monolayer was compressed up to the collapse (Fig. 5D). Glycinin at pH 5 presented the same features during the compression up to the monolayer collapse (Fig. 5C and D). However, at pOpe glycinin collapsed via a fracturing mechanism in which the monolayer cracks cooperatively over large length scales giving some regions of collapsed proteins separated by other regions with a lower condensation state, observed over the interface from differences in illumination (Fig. 5E and F).

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Fig. 5. Visualization by BAM of milk and soy protein monolayers. (A) BAM image of milk proteins at every pH and surface pressure. (B) BAM image of milk proteins at every pH and at the collapse point. (C) BAM image of b-conglycinin at pH 5 and at pZ0 mN/m. (D) BAM image of b-conglycinin at pH 5 and at pZ32.0 mN/m. (E) BAM image of glycinin at pH 5 and at pZ36.0 mN/m. (F) BAM image of glycinin at pH 5 and at pZ45.0 mN/m. (G) BAM image of bconglycinin at pH 8 and at pZ9.5 mN/m. (H) BAM image of b-conglycinin at pH 8 and at pZ22 mN/m. (I) BAM image of glycininC10 mM DTT at pH 5 and at pZ34 mN/m The horizontal direction of the image corresponds to 630 mm, and the vertical direction corresponds to 470 mm.

The spreading of b-conglycinin at pH 8 is not uniform over the interface. In the region of low surface pressures we have observed (Fig. 5G) the existence of protein domains alternating with an isotropic interface, but with a diffuse frontier between these regions. b-Conglycinin at pH 8 showed interfacial regions with different thickness at lower surface pressures and the presence of large aggregations of protein at surface pressures higher that the equilibrium surface pressure (Fig. 5H). These features were also observed during the monolayer expansion, even at the lower surface pressures. Glycinin monolayers at pH 8 show significant differences from b-conglycinin monolayers. BAM images corroborate that only a homogeneous phase was present during the compression of glycinin up to the monolayer collapse (Fig. 5A). However, at the collapse point some folds were observed along the interface with different illumination (see arrow in Fig. 5B). Differences in the image contrast are an indication that collapsed residues of glycinin monolayers are aligned, on average, parallel to the barrier movement. GlycininC10 mM DTT monolayers at p!pe and at pH 5 and 8 shown similar features to milk proteins (Fig. 5A). However at the collapse point and at pH 5 a typical feature of GlycininC10 mM DTT monolayers was the presence of

numerous short fractures in the monolayer (Fig. 5I) which led to large fractures defining a plane of shear between regions with different thickness during the monolayer expansion (Carrera et al., 2003b). The relative reflectivity (Figs. 3B and 4B) increases as the monolayer is compressed, and passes through a maximum at the monolayer collapse. The increase in reflected light intensity with surface pressure, and especially at the monolayer collapse, suggests that an increase in the monolayer thickness from more expanded to more condensed structure, and a further increase at the monolayer collapse, takes place. The relative intensity at the equilibrium surface pressure (Ipe) is included in Table 2. At pH 5 the values of Ipe were lower for milk than for soy globulin proteins. However at neutral pH higher Ipe values were observed for b-casein and 11S globulin. The progressive unfolding of soy globulins as the pH decreases and the associated conformational changes in the molecule have a significant repercussion on the structural (Figs. 3 and 4) and topographical (Figs. 3–5) characteristics of the monolayer. The most condensed monolayer structure (as deduced by the translation of the p–A isotherm towards lower molecular areas) was observed at pH 5 as both proteins would be partially denatured and aggregated, close

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to the isoelectric point. The aggregation of these proteins at the air–water interface at pH 5 was confirmed at a microscopic level by BAM images (Fig. 5). The same phenomenon at the isoelectric point was observed for b-casein. At neutral pH the p–A isotherms for 7S and b-casein were displaced towards higher molecular areas. That is, the presence of a net negative charge in b-casein and 7S soy globulin produces a significant expansion of the monolayer structure. Surprisingly, the most condensed structure was observed for glycinin at neutral pH (Fig. 4A). As both glycinin and b-conglycinin globulins are at pH 7 under native conditions (Molina et al., 2003), the differences observed in this work in the structural and topographical characteristics of these soy globulins should be associated with differences in ternary structure—that for glycinin is stabilized by hydrophobic and electrostatic interactions besides the disulfide bond between the acidic and basic polypeptides (Peng, Quass, Dayton, & Allen, 1984)—as well as the differences in molecular mass. The SH/SS groups in glycinin have an important role in the formation of intermolecular disulfide bonds, giving a condensed film at the air–water interface, and preventing the aggregation of the protein at interface, which explains the homogeneous glycinin film structure (Fig. 5). The fact that WPI monolayer structure did not depend on the pH (Figs. 3A and 4A) is consistent with the opinion that the protein components of WPI (which is mainly b-lactoglobulin) retain elements of the native structure, not fully unfolded, at the air–water interface. In summary, significant differences were observed in the structural, topographical, and dynamic characteristics between milk protein and soy globulin monolayers at the air–water interface. The rate of change of structural characteristics with ageing time was higher for soy globulin than for milk proteins. Moreover, the pH exerts higher influence on structural characteristics of soy globulin than on those of milk proteins. 3.4. Adsorption of protein films Time-dependent surface pressure (p) for soy globulin (b-conglycinin, and glycinin) (Rodrı´guez Patino, Molina, Carrera, Rodrı´guez Nin˜o, & An˜o´n, 2004) and milk protein (caseinate and WPI) adsorption at the air–water interface from protein solutions at pH 5 and 7 are plotted in Figs. 6 and 7, at two representative protein concentrations in solution. As a general rule it can be seen that the rate of surface pressure change over time increased when the protein concentration in the solution increased. That is, at higher concentrations the surface activity of proteins is high which agrees with previous data in the literature for proteins (Damodaran, 1990, 1997; Halling, 1981). Moreover, the rate of surface pressure increase (dp/dq) of protein solutions also depends on the protein, and the pH. Another interesting result was the lag period observed at pH 5, especially for soy globulins, which disappears at higher protein concentrations

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Fig. 6. Surface pressure as a function of adsorption time for milk and soy proteins on the air–water interface. Temperature 20 8C. Ionic strength 0.05 M. Aqueous phase pH 5. Protein concentration in solution (% wt/wt): (A) 0.01, and (B) 0.1. Symbols: (%) caseinate, (C) WPI, (6) 7S, and (7) 11S.

(Table 3). The lag period is due to the fact that at pH close to the isoelectric point these proteins are more aggregated. At pH 5 (Table 3) 11S globulin was only adsorbed at the interface at the higher concentration studied (0.1%, wt/wt). We cannot perform adsorption experiments with soy globulins at 1% wt/wt due to the insolubility of these proteins at pH 5. For a native conformation of the protein at neutral pH (Molina et al., 2003) the lag period appeared only at low protein concentration (at 0.001%, wt/wt). The reduction of glycinin with DTT at pH 5 had a little effect on the lag period (Table 3). The protein concentration at which this induction period appears is some orders of magnitude lower for milk proteins than for soy globulins (Table 3). This correlates with the fact that the flexibility and susceptibility of conformation changes is lower for globular soy globulins (Molina et al., 2003; Carrera et al., 2003a) than for milk random coil and globular proteins (Miller, Aksenenko, Fainerman, & Pison, 2001). From a kinetic point of view, the rate of surface pressure development by proteins after the lag period is caused by different processes (MacRitchie, 1989): (i) the protein has to diffuse from the solution to the subsurface (a layer immediately adjacent to the fluid interface) by diffusion

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and/or convection, (ii) this step is followed by the adsorption and unfolding of the protein at the interface, and (iii) the adsorbed protein segments rearrange at the fluid interface, a slow process caused by reorganization of the amino-acids segments previously adsorbed on the interface. For all protein concentrations in solution, a good fit of the experimental data in agreement with the Ward and Tordai equation (1946), at low surface pressures (p!10 mN/m) was observed (the slope derived from the p vs q1/2 line is included in Table 3). Thus, it can be concluded that during the initial period the kinetics of milk and soy globulins adsorption at the air–water interface is controlled by a diffusion mechanism. The discrepancies observed at longer adsorption time, as the surface pressure is higher than about 10 mN/m, could be attributed to an energy barrier for the adsorption of the protein, related to the penetration and unfolding of the soy globulin at the interface after the diffusion. The slope of the p vs q1/2 line increases with the protein concentration in solution (Table 3). The period at which diffusion controls the kinetics of adsorption of milk and soy globulins at the air–water interface increased as the protein concentration decreased (Table 3). At the higher protein concentration, and especially for milk proteins, the diffusion step is too fast to be detected with the experimental method used in this work. Thus, the jump in the surface pressure at the beginning of the adsorption is included in Table 3 as a qualitative measure of the diffusion of the protein to the interface. The diffusion at the interface depends on the proteins and on their conformation state in the solution. At every pH the diffusion is faster for milk than for soy globulins (Table 3), a phenomenon that could be associated with the molecular

Fig. 7. Surface pressure as a function of adsorption time for milk and soy proteins on the air–water interface. Temperature 20 8C. Ionic strength 0.05 M. Aqueous phase pH 7. Protein concentration in solution (% wt/wt): (A) 0.01, and (B) 1. Symbols: (%) caseinate, (C) WPI, (6) 7S, and (7) 11S.

Table 3 Characteristic parameters for adsorption of milk and soy globulin proteins at the air–water interface at 20 8C Protein

pH 5

pH 7 (8) 1/2

Caseinate 1% (wt/wt) 0.1% (wt/wt) 0.01% (wt/wt) WPI 1% (wt/wt) 0.1% (wt/wt) 0.01% (wt/wt) b-Conglycinin 1% (wt/wt) 0.1% (wt/wt) 0.01% (wt/wt) Glycinin 1% (wt/wt) 0.1% (wt/wt) 0.01% (wt/wt) GlycininC10 mM DTT 1% (wt/wt) 0.1% (wt/wt) 0.01% (wt/wt)

Induction time (s)

p vs q1/2 slope (mN mK1$sK0.5)

qaD (s)

k1!104 (sK1) (LR)

Induction time (s)

1.8 (0.995) 10.9 (0.962) 2.1 (0.996) 2.3 (0.997) 1.5 (0.989) 2.5 (0.995) –

0 0 56.2 0 0 0 –

– – – – – – O12.5

(b) – (b) (b) – (b) 0.5!

1.8 (0.992) – 1.6 (0.983) 3.2 (0.980) – 2.3 (0.997) 3.1 (0998)

0 – 0 0 – 0 0

3365 z12100 – 1300 O12100 –

1.5 (0.986) – – 2.2 (0.996) – –

441 3481 – 144 5900 –

2.35 (0.998) – O15 (0.900) 2.87 (0.998) – O15

15 – 0.5! 6 – 0.5!

2.7 (0.988) – 2.3 (0.989) 2.7 (0.996) – 2.2 (0.989)

0 – 0 0 – 0

360 O12100

3.0 (0.999) –

56 6250

2.53 (0.998) –

9 –

1.8 (0.981) –

0 –

p vs q slope (mN mK1$sK0.5)

qaD

– 4.9 (0.978) 0.83 (0.995) – – 4.24 –

(b) 7.3 412 (b) (b) 2.2 –

0.215 (0.995) 0.24 (0.997) – 0.26 (0.996) 0.025 (0.987) – 0.63 (0.997) 0.16 (0.969)

(s)

4

k1!10 (s (LR)

K1

)

(a) Period at which diffusion controls the kinetic of adsorption of soy globulins at the air–water interface. (b) The period at which diffusion controls the kinetic of adsorption of soy globulins at the air–water interface is lower than 0.5 s. (LR) Linear regression coefficient.

M.R. Rodrı´guez Nin˜o et al. / Food Hydrocolloids 19 (2005) 417–428

weight of the protein, in agreement with the penetration theory (Ward and Tordai, 1946), and with the degree of aggregation of the protein in solution. Interestingly, the period at which diffusion controls the kinetics of adsorption of soy globulins at the air–water interface increased drastically at pH 5 (Table 3). At this pH the diffusion controls the adsorption of the protein at 0.01% wt/wt within the time of the experiment. If pH 5 causes an aggregation of caseinate and soy globulins in the solution (Molina et al., 2003) their diffusion towards the interface could diminish as observed in Table 3. At neutral pH, as soy globulins are in a native state, the rate of diffusion towards the interface was practically the same no matter what the protein. While DTT positively affected the diffusion of native glycinin at pH 5 it had a limited effect on the native conformation of the protein at neutral pH. To monitor unfolding at the interface and configurational rearrangements of adsorbed protein molecules, a first-order equation can be used. We find, for all experiments of soy globulin adsorption, two linear regions in the plot of log[(p180Kpq)/(p180Kpo)] vsq where p180, po, and pq, are the surface pressures at 180 min of adsorption time, at time qZ0, and at any time, q, respectively. The initial slope is taken to correspond to a first-order rate constant of unfolding (k1)—which is included in Table 3, while the second slope is taken to correspond to a first-order rate constant of rearrangement (k2). As a general trend it can be established that the values of k1 increase with protein concentration (Table 3). That is, penetration of proteins at the interface is facilitated at higher protein concentrations in the solution. At low protein concentrations and at pH 5 soy proteins do not penetrate at the air–water interface. However, minor differences were observed between the rate of penetration of milk and soy globulins (Table 3). 3.5. Surface dilatational properties of protein films 3.5.1. Dilatational characteristics of protein monolayers spread on the air–water interface The surface viscoelastic properties of milk (Rodrı´guez Patino, Carrera, Rodrı´guez Nin˜o, & Cejudo M., 2001) and soy (Rodrı´guez Patino, Carrera, Molina, Rodrı´guez Nin˜o & An˜o´n, 2003) protein monolayers spread on the air–water interface were studied as a function of surface pressure (Fig. 8). For milk proteins and soy globulins at every pH the E–p plot did not depend on the ageing of the monolayer, but only depended on the surface pressure. These results corroborate the idea (Rodrı´guez Patino, Rodrı´guez Nin˜o, & Carrera, 1999) that the E–p curve could reflect the surface equation of state of the spread material at the air–water interface. The E values of soy globulins were half way between those of WPI and b-casein, with the lower E values for the former. The E–p plots for b-casein and soy globulins showed an irregular shape. The modulus increased with increasing surface pressure to a maximum value. Upon

425

Fig. 8. Surface dilatational modulus as a function of surface pressure for milk and soy protein spread monolayers on the air–water interface. Temperature 20 8C. Ionic strength 0.05 M. Aqueous phase pH (A) 5 and (B) 7. Symbols: (&) b-casein, (C) WPI, (6) 7S, (7) 11S, and ($) 11SCDTT.

further increase of the surface pressure E decreased to a minimum at a surface pressure close to the transition between two structures or condensed states in the monolayer, as deduced from the p–A isotherms and reflectivity (Figs. 2 and 3). Afterwards, the surface dilatational modulus increased again with surface pressure. However, the behaviour of milk globular protein (like WPI) spread films is different. For WPI E increased monotonically with surface pressure up to the collapse point. From this point, E did not depend on the surface pressure. Moreover, over the range of surface pressures studied the values of E for WPI spread films were higher than those for soy globulins, especially at pH 5 and at the higher surface pressures (Fig. 8). Differences in the surface dilatational modulus are due mainly to looping of amino-acid residues at higher surface pressures and interactions between collapse residues, including multilayer formation at surface pressures higher than the equilibrium surface pressure (Carrera et al., 2003a,b). On the other hand, the loss angle tangent decreased with surface pressure no matter what the protein (data

426

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not shown). That is, the more condensed the structure is (at higher surface pressures), the higher the elasticity of the monolayer becomes. Thus, at surface pressures close to the film collapse the viscoelastic behaviour of soy proteins was almost elastic. In this respect, milk proteins and soy globulin films may be considered as thin layers of protein gel as a consequence of the changes in the molecular conformation and further protein–protein interactions as the surface pressure increased. The surface dilatational characteristics of protein spread films at the air–water interface depend on the protein structure. As at pH 5 the soy globulins would be partially denatured and aggregated, the interactions between protein residues at the interface would be reduced, a phenomenon which coincides with the lower values of E for bconglycinin and native glycinin (Fig. 8A). At neutral pH (Fig. 8B) the higher values of E for glycinin films are in line with the more condensed structure and higher film thickness for glycinin monolayers (Fig. 3), especially at higher surface pressures. The reduced interactions between residues of disordered b-casein explain its lower E values at every pH and surface pressure. In contrast, the higher values of E for WPI correlate with the formation of a more coherent monolayer of this globular protein at the air– water interface. 3.5.2. Dilatational characteristics of protein monolayers adsorbed on the air–water interface Time-dependent surface dilatational moduli for adsorbed films of milk proteins and soy globulins (Rodrı´guez Patino, Molina et al., 2003; Rodrı´guez Patino, Rodrı´guez Nin˜o, Carrera, Molina, & An˜o´n, 2004) are essentially the same (data not shown) as the p–time dependence (Fig. 6). (i) The increase in E, or the decrease in the phase angle with time may be associated with adsorption of the protein at the interface (Liu, Lee, & Damodaran, 1999). (ii) At higher protein concentrations the surface dilatational modulus is high which agrees with previous data in the literature for other proteins (Bos & Van Vliet, 2001). (iii) The same lag period, as for the p–time dependence, was observed in the E–time plots for soy globulins at lower concentrations and at pH 5. The results of time dependent surface dilatational properties are consistent with the existence of protein– protein interactions which is thought to be due to the protein adsorption at the interface via diffusion, penetration and rearrangement (looping of the amino acid residues) as both the adsorption time and the protein concentration in the bulk phase increase. As for spread proteins, E increased with interfacial pressure and this dependence reflects the existence of increased interactions within the adsorbed protein residues (Fig. 9). The results of the soy globulin adsorption at different adsorption times are aligned in different E–p lines for different protein concentrations. These results support the hypothesis that these proteins, are adsorbed at the air– water interface with different degrees of association

Fig. 9. Surface dilatational modulus as a function of surface pressure for milk and soy protein adsorbed films on the air–water interface. Temperature 20 8C. Ionic strength 0.05 M. Aqueous phase pH (A) 5 and (B) 7. Symbols: (C) Caseinate, (:) WPI, (6) 7S, and (7) 11S. The discontinuous lines represent the characteristic behaviour of an ideal gas (Lucassen-Reynders et al., 1975).

(aggregation) at different concentrations in the bulk phase (Rodrı´guez Patino, Molina et al., 2003). The plot of Fig. 9 suggests (Lucassen-Reynders, Lucassen, Garrett, Giles, & Hollway, 1975) that interactions between adsorbed protein residues increase with surface pressure or with the amount of protein at the interface. Moreover, over the range of surface pressures studied the values of E for milk and soy globulin protein spread films (Fig. 8) were different from those for adsorbed films (Fig. 9), especially at pH 5. These differences are due mainly to differences in the looping of amino-acid residues for spread and adsorbed films at the air–water interface, including multilayer formation at the higher surface pressures, as observed recently by Brewster angle microscopy of spread soy globulin films (Carrera et al., 2003a,b). The maximum difference between the E–p plots for milk and soy globulin proteins was observed at pH 5, a behaviour similar to that observed with equilibrium (spreading and adsorption), thermodynamic (structure and topography), and dynamic

M.R. Rodrı´guez Nin˜o et al. / Food Hydrocolloids 19 (2005) 417–428

(adsorption) characteristics of spread and adsorbed films at the air–water interface.

427

physicochemical characteristics of proteins films at fluid interfaces and the formation and stability of food dispersions (emulsions and foams) requires more systematic studies with well defined systems.

4. Conclusions In this paper, we have sought to demonstrate that the structural and topographical characteristics of milk (bcasein, caseinate, and WPI) and soy (b-conglycinin, glycinin, including reduced glycinin) protein films spread and adsorbed at the air–water interface depend on the molecular structure of the protein and the pH of the aqueous phase. A notable feature of soy proteins is the strong pH dependence of the molecular conformation and the associated functional properties (surface activity, film structure and surface dilatational viscoelasticity). The adsorption of protein to the interface increases with the protein concentration in the bulk phase, depending on the protein and, especially, on the aqueous phase pH. The adsorption of soy globulins decreases drastically at pH 5, close to the isoelectric point of the protein. A characteristic ageing effect was observed in the monolayer structure and topography for soy globulins due to unfolding of the protein at the interface. The most condensed monolayer structure was observed for glycinin, being respectively more expanded for b-conglycinin, b-casein, and WPI. At a microscopic level, the heterogeneous monolayer structures visualized at every surface pressure, but especially near to the monolayer collapse, proved the existence of large regions of soy protein aggregates, which were not observed for milk proteins. The monolayer thickness is higher for soy proteins than for milk proteins. The dilatational modulus is not only determined by the interactions between spread and adsorbed protein molecules (which depend on the surface pressure), but the structure of the protein also plays an important role. The surface dilatational properties of soy globulins were similar to those of globular milk proteins and higher than those of b-casein and caseinate. The chemical reduction of glycinin with DTT has a significant effect on the main interfacial characteristics of adsorbed and spread films. Upon cleaving the SS bridge using DTT the unfolding of glycinin at the air–water interface is facilitated, which can increase the number of points of contact of the protein with the interface, in agreement with the monolayer expansion at neutral (Fig. 4A) and acidic aqueous subphase (Fig. 3A). The more expanded structure of glycinin treated with DTT was observed at neutral pH. The limited foaming and emulsifying properties of native soy globulins at neutral or acidic aqueous solutions compared to milk proteins may be due to differences in the rate of protein adsorption at short adsorption time, among other factors. Clearly, the connection between

Acknowledgements The authors acknowledge the support of CICYT thought the grant AGL2001-3843-C02-01.

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