Viscoelastic Properties of Diglycerol Ester and Protein Adsorbed Films

Mar 27, 2007 - The values of E reflect not only the amount of emulsifier adsorbed at the interface but also the degree of interaction between adsorbed...
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Ind. Eng. Chem. Res. 2007, 46, 2693-2701

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Viscoelastic Properties of Diglycerol Ester and Protein Adsorbed Films at the Air-Water Interface Jose´ M. A Ä lvarez Go´ mez and 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-SeVilla, Spain

The dynamic surface pressure (π) and surface viscoelastic properties (surface dilatational modulus, E, its elastic, Ed, and viscous, Ev, components, and loss angle tangent, tan φ) of lipids (diglycerol-monostearate and diglycerol-monolaurate) and proteins (sodium caseinate and β-lactoglobulin) at different concentrations in the aqueous phase were measured using a dynamic drop tensiometer. Temperature, pH, and, ionic strength were maintained constant at 20 °C, 7, and 0.05 M, respectively. The surface dilatational properties depend on the chemistry of the molecule (length of the hydrocarbon chain in lipids and disordered or globular characteristics of the protein). Lipid films are essentially elastic, but protein films are viscoelastic. The values of Ed increase with the emulsifier (lipid or protein) concentration in solution and were higher at the critical micellar concentration (CMC) for lipids or at the adsorption efficiency (AE) for proteins. The values of E reflect not only the amount of emulsifier adsorbed at the interface but also the degree of interaction between adsorbed emulsifier molecules. E increases with the van der Waals interactions between lipid hydrocarbon chains, which are higher for diglycerol-monostearate than for diglycerol-monolaurate. The values of E for caseinate adsorbed films were lower than those for β-lactoglobulin adsorbed films. These differences are due mainly to differences in the looping of amino acid residues for adsorbed films of random coil (caseinate) and globular (β-lactoglobulin) proteins at the air-water interface. The values of E are lower for protein compare to lipid adsorbed films. The role of surface dynamic properties of lipid and protein adsorbed films on foam formation and stabilization is discussed. Introduction The interfacial structure of adsorbed emulsifiers (biopolymers and low molecular weight emulsifiers) and the dynamic properties derived from this structure at fluid interfaces (i.e., the surface dynamic properties and surface dilatational characteristics) will play an important role in food dispersion formulations (emulsions and foams). In these systems the desired product property can be obtained by the choice of suitable process functions, such as surface pressure or surface density, surface composition, kinetics of film formation, and so forth. We have observed recently that the thermodynamic and dynamic characteristics of adsorbed protein and lipid films have a significant effect on the formation and stability of food model foams and emulsions formulated with these emulsifiers.1,2 Among the functional effects of emulsifiers, foaming is of particular interest because it provides desirable textures to many aerated foods. Foams are present in many aerated foods (ice cream, whipped topping, breads, cakes, meringues, beers, champagne, etc.) either in the finished product or incorporated during the production as a preliminary processing stage, which can be subjected to further processing before the product is complete. Thus, knowledge of the mechanism of foam formation and stabilization is essential if foam of the required characteristics is to be produced. Stability is an important property of food foams, because consumer perception of quality is influenced by appearance. Foams are thermodynamically unstable, and their relative stability is affected by factors such as (i) drainage of liquid previously present in the foam, (ii) disproportionation (the diffusion of gas from small bubbles into big bubbles), and (iii) coalescence (the breakdown of the bubbles by lamellae rupture), resulting in final * To whom correspondence should be addressed. Tel.: +34 95 4556446. Fax: +34 95 4556447. E-mail: [email protected].

foam breakdown. Foam capacity and the stability of the resulting foam depend on the properties of the surface-active components in the system.3 Foam formation is influenced by the adsorption of the foaming agent at the air-water interface and its ability to reduce surface tension. However, foam stabilization against drainage, disproportionation, and coalescence depends on different surface properties, such as the surface tension at longterm adsorption and the structural, topographical, and mechanical characteristics of the adsorbed film, in a complicated manner. Bubbles in foam are stabilized by a bilayer of emulsifier molecules separated by the continuous aqueous phase. Thus, the characteristics of this thin film determine the stability of the foam. The analysis of the interfacial characteristics of emulsifiers at fluid interfaces is of practical importance, because these monolayers constitute well-defined systems for the analysis of food colloids at the micro- and nanoscale level, with advantages for fundamental studies. It is the properties of the two air/water interfaces of the thin films which make or break a foam.4-8 There are two classes of surface-active molecules (emulsifiers) that can be used in food foam production by different and somewhat incompatible mechanisms:9-11 biopolymers (mainly proteins and some polysaccharides) and low molecular weight emulsifiers (mainly lipids, phospholipids, and surfactants). In this contribution we are concerned with the analysis of dynamic interfacial characteristics (dynamic surface pressure and surface dilatational characteristics) of two typical milk protein (β-lactoglobulin and caseinate) and diglycerol ester (diglycerol-monostearate and diglycerol-monolaurate) adsorbed films at the air-water interface, as a function of the emulsifier concentration in aqueous solution. β-Lactoglobulin is distinguished by its good foaming and emulsifying properties, and for these reasons it is widely used in food formulations.12 As a

10.1021/ie061451g CCC: $37.00 © 2007 American Chemical Society Published on Web 03/27/2007

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Table 1. Analytical Specifications of Diglycerol Esters of Fatty Acids

lipid diglycerolmonolaurate diglycerolmonostearate

diglycerol monoester

diglycerol diesters

free fatty acids

free diglycerol

83.2%

11.8%

0.1%

1.5%

90.1%

5.2%

0.1%

4.6%

fatty acid profile 98% C12 2% C14 48% C16 48% C18 4% others

result of its secondary and tertiary structure, β-lactoglobulin, in common with other globular proteins, has the ability to form viscoelastic gel-like structures at fluid interfaces,12-14 which are important for dispersion stability.7,15,16 The foaming and emulsifying properties of caseinate arise from the structures of the four proteins found in bovine milk (β-casein, Rs1-casein, κ-casein, and Rs2-casein). Although the major caseins (Rs1- and β-casein) do not contain any cysteine residue, both κ- and Rs2casein contain two cysteine residues per molecule, which are able to form disulfide bonds. That is, minor caseins (κ- and Rs2-casein) have the ability to form interfacial protein gel stabilized by covalent disulfide cross-linked networks. The formation of this interfacial gel may be the cause of the higher values of surface shear properties of κ-casein and caseinate compared to those for β-casein adsorbed17-19 and spread20 films, although monolayer structure, topography, dilatational characteristics, and long-term relaxation phenomena of β-casein and caseinate spread films are somewhat similar.21,22 Polyglycerol esters of fatty acids (PGE) have been used to control the properties of aqueous dispersions in cosmetic, pharmaceutical, and food applications.23-25 The physical properties of fatty acid esters of polyglycerols have been analyzed in the literature.26-29 However, the surface rheological characteristics of adsorbed films have not been analyzed so far. Commercial PGE products are mixtures of many different components with variations in the degree of polymerization of the polyols. More specifically defined diglycerol esters can be made by the esterification of purified polyols (e.g., diglycerol) with single fatty acids, followed by a concentration of the monoesters by a distillation process. Experimental Section Chemicals. Linear diglycerol esters were esterified with fatty acids of different chain lengths. The diglycerol monoesters were separated from the equilibrium mixture of mono-, di-, and triesters by a laboratory-scale distillation process, yielding concentrated diglycerol monoesters.26 Analytical specifications of diglycerol-monostearate and diglycerol-monolaurate used in this work as given by suppliers are included in Table 1. Whey protein isolate (WPI), a native protein with very high content of β-lactoglobulin (protein 92 ( 2%, β-lactoglobulin > 95%, R-lactalbumin < 5%) was obtained by fractionation. These products were kindly provided by Danisco Ingredients (Brabrand, Denmark). Caseinate (a mixture of ≈38% β-casein, ≈39% Rs1casein, ≈12% κ-casein, and ≈11% Rs2-casein) was kindly supplied and purified from bulk milk from Unilever Research (Colworth, U.K.). These products were used without further purification. Samples for adsorption and foam properties of foaming agent (protein and lipid) solutions were prepared using Milli-Q ultrapure water and were buffered at pH 7. TrizmaHCl ((CH2OH)3CNH2/(CH2OH)3CNH3Cl) for buffered solution was used as supplied by Sigma (>95%) without further purification. The ionic strength was 0.05 M in all the experiments. 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 (72-73 mN/m at 20 °C) were used. Sodium azide (Sigma) was added to aqueous protein solutions (0.02%, w/w) to avoid microbial contamination in the sample. Dynamic Surface Measurements. Measurements of timedependent surface pressure and surface dilatational properties of adsorbed films at the air-water interface were performed simultaneously by an automatic drop tensiometer as described elsewhere.30 Briefly, an image of the drop was continuously recorded by a CCD camera and digitalized. The surface tension (σ) was calculated by analyzing the profile of the drop according to the fundamental Laplace equation.31 The average standard accuracy of the surface tension for at least two measurements with different drops is roughly (0.5 mN/m. For surface dilatational property measurements of adsorbed emulsifier (diglycerol ester or protein) films at the air-water interface the same automatic drop tensiometer was utilized, as described elsewhere.32 Briefly, the method involved a periodic automatically controlled, sinusoidal interfacial compression and expansion performed by decreasing and increasing the drop volume, at the desired amplitude (∆A/A) and angular frequency (ω). The surface dilatational modulus (E ) Ed + iEv; eq 1), its elastic (Ed ) E cos φ) and viscous (Ev ) E sin φ) components, and the phase angle (φ) were derived from the change in surface pressure resulting from a small change in surface area (A). The surface dilatational properties were measured as a function of time, θ.

E)

dσ dπ )d ln A d(A/A)

(1)

Emulsifier (protein or lipid) solutions were prepared freshly by weighing the correct amounts and the buffer solution to attain the desired concentration in solution, which was then stirred for 30 min. The emulsifier solution was placed in a 0.25 mL glass Hamilton syringe equipped with a stainless steel needle and then in a rectangular glass cuvette (5 mL) covered by a compartment which was maintained at constant temperature (20 ( 0.2 °C) by circulating water from a thermostat. It was then allowed to stand for 30 min to achieve constant temperature and humidity in the compartment. Then a drop of the emulsifier solution (5-8 µL) was delivered and allowed to stand at the tip of the needle for about 15 min to achieve adsorption at the air-water interface. The sinusoidal oscillation for surface dilatational measurements starts after 15 min of adsorption time, to allow initial emulsifier adsorption. Afterward, the drop was subjected to repeated measurements with five oscillation cycles followed by a time corresponding to 50 cycles without any oscillation up to 200 min for emulsifier adsorption. The deformation amplitude (∆A/A) and deformation frequency (ω) were maintained constant at 15% and 100 mHz, respectively. The percentage area change was determined in preliminary experiments to be in the linear region (data not shown). The average standard accuracy of the surface tension is roughly 0.1 mN/m. However, the reproducibility of the viscoelastic properties (for at least two measurements) was better than 5%. All experiments were carried out at 20 °C. The temperature of the system was maintained constant within (0.2 °C by circulating water from a thermostat. Emulsifier solutions were prepared freshly and stirred for 30 min. The ionic strength was maintained constant at 0.05 M. The materials in contact with the emulsifier solution must be clean to prevent any contamination by surface-active compounds.

Ind. Eng. Chem. Res., Vol. 46, No. 9, 2007 2695 Table 2. CMC and Surface Pressure at the CMC (πCMC) for Aqueous Solutions of Lipids and AE and SA for Aqueous Solutions of Proteinsa

diglycerol-monolaurate diglycerol-monostearate caseinate β-lactoglobulin a

CMC (wt %)

πCMC (mN/m)

1 × 10-3 8.83 × 10-5

45.5 30.0

AE (wt %)

SA (mN/m)

1.0 1.0

32 26

Temperature 20 °C, pH ) 7, and ionic strength 0.05 M.

Figure 2. Effect of surface pressure on surface dilatational modulus for diglycerol-monolaurate adsorbed films at the air-water interface. Concentration of diglycerol-monolaurate in the aqueous phase (wt %): (0) 3.5 × 10-4, (O) 3.5 × 10-5, and (4) 3.5 × 10-6. Frequency of oscillation 100 mHz. Amplitude of deformation 15%. Temperature 20 °C, pH 7, and I 0.05 M. The dotted line with slope ) 1 indicates the behavior of an ideal gas.33

Figure 1. Time evolution of (O) surface dilatational modulus (E), its (]) elastic (Ed) and (3) viscous (Ev) components, and (4) loss angle tangent (tan φ) for diglycerol-monolaurate adsorbed films at the air-water interface. Concentration of diglycerol-monolaurate in the aqueous phase (wt %): (A) 3.5 × 10-4, (B) 3.5 × 10-5, and (C) 3.5 × 10-6. Frequency of oscillation 100 mHz. Amplitude of deformation 15%. Temperature 20 °C, pH 7, and I 0.05 M.

Results Viscoelastic Properties of Adsorbed Diglycerol Esters at the Air-Water Interface. The viscoelastic properties of diglycerol-monolaurate and diglycerol-monostearate were measured at lipid concentrations in solution similar to or lower than those for the critical micelle concentration (CMC). The CMC and the surface pressure at the CMC, πCMC, for diglycerol esters are included in Table 2.2 The value of CMC is higher for diglycerol-monolaurate than for diglycerol-monostearate. Thus, the lipid concentration required to saturate the air-water interface decreases as the hydrophobic character of the lipid increases. That is, CMC decreases as the hydrocarbon chain increases because the hydrophobic character of the lipid also increases. However, the value of πCMC (the surface activity, SA) is lower for diglycerol-monostearate than for diglycerolmonolaurate. Thus, as the interface is saturated, the lipid with a shorter hydrocarbon chain can be oriented at the air-water interface in a more favorable conformation than lipids with longer hydrocarbon chains. Viscoelastic Properties of Adsorbed Films of Diglycerolmonolaurate. The transient surface viscoelastic properties (surface dilatational modulus, E, its elastic, Ed, and viscous, Ev, components, and the loss angle tangent, tan φ, for diglycerolmonolaurate are shown in Figure 1. It was observed that at a lipid concentration in the aqueous phase of 3.5 × 10-4 wt % as an example (Figure 1A), the values for the surface dilatational modulus were very similar to those for the dilatational elasticity and the dilatational viscosity values were low. Thus, over the adsorption period studied here the film behaved, from a rheological point of view, as viscoelastic (practically elastic)

with a loss angle tangent higher than zero. Different lipid concentrations in solutions behaved in a similar way (data not shown). As a general trend the surface elastic modulus (Ed) increased and the loss angle tangent (tan φ) decreased with the adsorption time for diglycerol-monolaurate (Figure 1). At a concentration of the lipid similar to CMC the values of Ed are practically independent of the adsorption time. That is, after 15 min of adsorption, as the surface rheological measurements start, the fast adsorption of the lipid2 saturated the interface and the Ed values reached the maximum plateau value (Figure 1). At the lower lipid concentration in solution (at C < CMC) the time required to attain a plateau in Ed increased as the lipid concentration in solution decreased, or was even not attained within the experimental time for a lipid concentration in solution of 3.5 × 10-6 wt % (Figure 1C). Diglycerol-monolaurate adsorbed films showed high values of Ed, which reveals the ability of the lipid to form a structured film with solid-like characteristics, even at relatively low concentrations in the bulk phase. This hypothesis is confirmed by the low values of tan φ for this lipid (Figure 1). The increase in surface dilatational modulus and surface dilatational elasticity or the decrease in the phase angle with adsorption time should be associated with both the adsorption of the lipid at the interface and the formation of structured condensed film (solid-like) at the air-water interface. The fact that the time dependence of the surface dilatational elasticity (Ed) follows the same trend as the surface pressure2 indicates that Ed depends on the surface coverage, which increases with time. If the E time dependence of diglycerol-monolaurate is associated with the rate of the lipid adsorption at the interface, these results are consistent with the existence of higher lipidlipid interactions which is expected to be due to a higher lipid concentration at the interface via adsorption and penetration as both the adsorption time and the lipid concentration in solution increase. However, if the surface dilatational modulus is only due to the amount of lipid adsorbed at the air-water interface, all E data should be normalized in a single master curve of E versus π. Figure 2 shows that this normalization was not possible. These results strengthen the hypothesis that diglycerol-monolaurate is adsorbed at the air-water interface with different degrees of association depending on the lipid concentration in solution. As for other emulsifiers, E increased with surface pressure and this dependence reflects the existence of interactions within the adsorbed diglycerol-monolaurate molecules. In

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Figure 4. Effect of surface pressure on surface dilatational modulus for diglycerol-monostearate adsorbed films at the air-water interface. Concentration of diglycerol-monostearate in the aqueous phase (wt %): (3) 4 × 10-3, (4) 4 × 10-4, (O) 4 × 10-5, and (0) 4 × 10-6. Frequency of oscillation 100 mHz. Amplitude of deformation 15%. Temperature 20 °C, pH 7, and I 0.05 M. The dotted line with slope ) 1 indicates the behavior of an ideal gas.33

Figure 3. Time evolution of (O) the elastic component of the surface dilatational modulus (Ed) and (4) loss angle tangent (tan φ) for diglycerolmonostearate adsorbed films at the air-water interface. Concentration of diglycerol-monostearate in the aqueous phase (wt %): (A) 4 × 10-3, (B) 4 × 10-4, (C) 4 × 10-5, and (D) 4 × 10-6. Frequency of oscillation 100 mHz. Amplitude of deformation 15%. Temperature 20 °C, pH 7, and I 0.05 M.

agreement with the theory of Lucassen et al.,33 the plot of Figure 2 suggests that interactions between adsorbed lipids increase with surface pressure. In fact, the slope of the E-π plot was higher than 1 (characteristic of the behavior of an ideal gas), which implies an important nonideal behavior with higher molecular interactions as the amount of lipid at the interface increases. In summary, the E-π plot reflects either the amount of lipid adsorbed at the interface (giving high values of E at the higher surface pressures (Figure 1)) or the degree of lipidlipid interactions, leading to interfacial lipid aggregation at higher lipid concentrations in solution and at higher adsorption time (at high π; Figure 2). Viscoelastic Properties of Adsorbed Films of Diglycerolmonostearate. The transient surface viscoelastic properties (surface dilatational modulus, E, its elastic, Ed, and viscous, Ev, components and the loss angle tangent, tan φ, for diglycerolmonostearate are shown in Figure 3. As for diglycerolmonolaurate, the values for the surface dilatational modulus were very similar to those for the dilatational elasticity, and the dilatational viscosity and loss angle tangent values were low (data not shown). Thus, over the adsorption period studied here, the film behaved, from a rheological point of view, as viscoelastic (practically elastic). As for diglycerol-monolaurate (Figure 1), the surface elastic modulus (Ed) increased and the loss angle tangent (tan φ) decreased with the adsorption time for diglycerol-monostearate (Figure 3). However, a characteristic of diglycerol-monostearate adsorbed films is that the values of Ed did not reach a constant plateau value within the time of the experiment. The same phenomenon was observed for the time evolution of surface pressure.2 That is, although diglycerol-monostearate is able to form a viscoelastic film at short adsorption time (especially at C > CMC), the saturation of the air-water interface by the lipid at each lipid concentration in solution requires high adsorption time. Figure 4 shows a master curve of E versus π for diglycerolmonostearate adsorbed films at the air-water interface, including different lipid concentrations in solution and different

adsorption times. These results indicate that diglycerolmonostearate is adsorbed at the air-water interface with different degrees of association depending on the lipid concentration in solution. The increase of E with π indicates that the interactions between lipid molecules at the air-water interface increased with the adsorption time as the number of adsorbed molecules increased, giving high π values.2 Viscoelastic Properties of Adsorbed Proteins at the AirWater Interface. The viscoelastic properties of caseinate and β-lactoglobulin were measured as a function of the protein concentration in solution, at concentrations similar to and lower than that for the adsorption efficiency (AE). This is the highest protein concentration at which the plateau in the surface pressure isotherm is attained.2 At higher protein concentrations, the protein molecules may form multilayers beneath the primary monolayer, but these structures do not contribute significantly to surface pressure.34 The maximum surface pressure at the plateau is the SA. The values of AE and SA for caseinate and β-lactoglobulin adsorbed films on aqueous solutions are included in Table 2.2,35 The results confirm3 that biopolymers (such as caseinate and β-lactoglobulin) present higher AE but lower SA than lipids (as diglycerol esters) at the air-water interface. Moreover, the viscoelastic properties of adsorbed protein films depend on the chemistry of the molecule. Viscoelastic Properties of Caseinate Adsorbed Films. For caseinate adsorbed films at the air-water interface the values of the surface dilatational modulus and its elastic component are similar (data not shown) but not the same, as for diglycerol esters (Figure 1A). Thus, the values of the viscous component (Ev) are not zero, and the film presents a viscoelastic behavior with values of tan φ higher than zero (Figure 5). Time-dependent surface dilatational properties for adsorbed films of caseinate are shown in Figure 5. The increase in surface dilatational elasticity, or the decrease in the phase angle tangent, with time should be associated with adsorption of proteins at the air-water interface,36 as was observed for proteins in general at the air-water13,14,22 and oil-water13 interfaces. In fact, the results of time-dependent surface dilatational property measurements are consistent with the existence of protein-protein interactions which are thought to be due to 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.2 The time evolution of Ed showed an anomalous behavior as a function of the caseinate concentration in solution. It can be seen that during adsorption the values of Ed are lower at caseinate concentrations in solution of 1 and 1 × 10-4 wt %,

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Figure 5. Time evolution of (O) the elastic component of the surface dilatational modulus (Ed) and (4) loss angle tangent (tan φ) for caseinate adsorbed films at the air-water interface. Concentration of caseinate in the aqueous phase (wt %): (A) 1, (B) 1 × 10-2, and (C) 1 × 10-4. Frequency of oscillation 100 mHz. Amplitude of deformation 15%. Temperature 20 °C, pH 7, and I 0.05 M.

Figure 6. Effect of surface pressure on surface dilatational modulus for caseinate adsorbed films at the air-water interface. Concentration of caseinate in the aqueous phase (wt %): (4) 1 × 10-4, (O) 1 × 10-2, and (0) 1. Frequency of oscillation 100 mHz. Amplitude of deformation 15%. Temperature 20 °C, pH 7, and I 0.05 M. The dotted line with slope ) 1 indicates the behavior of an ideal gas.33

compared to those at an intermediate concentration (at 1 × 10-2 wt %). The same behavior was observed for disordered proteins (caseinate and β-casein) spread at the air-water interface,37,38 as will be analyzed later. It is possible that the values of Ed decrease at higher adsorption time even at a value close to zero, if a reversible diffusion of collapsed protein takes place between the interface and the bulk phase during the sinusoidal compression-expansion of the interface.39,40 The evolution of surface dilatational modulus (E) with surface pressure (π) for the adsorption of caseinate at the air-water interface at every protein concentration in solution and adsorption time is shown in Figure 6. These results indicate that caseinate is adsorbed at the air-water interface with different degrees of association depending on the protein concentration in solution. The increases of E with π at a concentration of caseinate in solution of 1 × 10-2 wt % reflects a nonideal behavior, with interactions between film forming amino acid caseinate residues, which are higher at higher π. However, at lower (at 1 × 10-4 wt %) and higher (at 1 wt %) caseinate concentrations the E values are lower from those corresponding to an ideal behavior. These results, which were unexpected, can be explained by the hypothesis that adsorption of caseinate aggregate at the highest concentration in solution takes place (at 1 wt %), a phenomenon that can reduce the interactions

Figure 7. Time evolution of (O) the elastic component of the surface dilatational modulus (Ed) and (4) loss angle tangent (tan φ) for β-lactoglobulin adsorbed films at the air-water interface. Concentration of β-lactoglobulin in the aqueous phase (wt %): (A) 1, (B) 1 × 10-2, and (C) 1 × 10-4. Frequency of oscillation 100 mHz. Amplitude of deformation 15%. Temperature 20 °C, pH 7, and I 0.05 M.

between film forming residues. Thus, the interactions between aggregates of caseinate at the interface would be lower than those between free amino acid residues and, consequently, the values of E would be lower from those for an ideal behavior. The low E values for a caseinate concentration of 1 × 10-4 wt % must be due to the fact that at low protein concentration in solution the amount of protein adsorbed at the interface is also too low to form a coherent film. Moreover, the low values of E may be the consequence of a change in the caseinate structure at the air-water interface, as was observed for spread film.37,38 In fact, for caseinate (and for β-casein) spread monolayers, the E versus π (or surface density) plots showed an irregular shape.37 The modulus increased with increasing π to a maximum value at π ≈ 10 mN/m. Upon further increase of the surface density E decreased to a minimum at π ≈ 20 mN/m, close to the collapse point. Afterward, E increased again with π. Interestingly, the same irregular shape in the surface pressure dependence of the surface dilatational modulus was observed by other authors for β-casein and caseinate spread37 and adsorbed13,41 films, the values of E being of the same order of magnitude as those in Figure 6. At these points of inflection we have observed the transition between structure 1 and structure 2 and between structure 2 and monolayer collapse, respectively.37 That is, the interactions between amino acid residues in caseinate films with a tail conformation (structure 1) are stronger than those between amino acid residues with tails and loops conformations (structure 2). The same phenomenon can explain the results shown in Figure 6 for caseinate adsorbed films from aqueous solutions at 1 × 10-4 wt %. Viscoelastic Properties of β-Lactoglobulin Adsorbed Films. For β-lactoglobulin the viscoelasticity of adsorbed films at the air-water interface is very dependent on the protein concentration in solution and the adsorption time (Figure 7). As the protein concentration decreased, the values of E and Ed approached each other, the value of Ev decreased (data not shown), and the values of tan φ approached zero. Thus, the film elasticity increased as the β-lactoglobulin concentration in solution decreased. The viscous character also decreased with the adsorption time, especially at the higher β-lactoglobulin concentration in solution (Figure 7A,B). This behavior is characteristic of globular proteins14 because the adsorbed protein increases at long-term adsorption giving the possibility of increasing the interactions

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(that is, of its structure), as deduced from π-A isotherms and monolayer thickness of spread monolayers.37 That is, more condensed monolayer structures may lead to an increase in the forces of interaction between molecules at the interface, which is consistent with the observed increase in E with π (Figure 8). Upon further increase of π, E remains constant, or even decreases, as multilayer formation takes place. In addition, these data support the hypothesis that the compression of β-lactoglobulin monolayers does not produce any change in loop conformation adopted by amino acid residues but that a condensation of this conformation takes place.14,37 Figure 8. Effect of surface pressure on surface dilatational modulus for β-lactoglobulin adsorbed films at the air-water interface. Concentration of β-lactoglobulin in the aqueous phase (wt %): (4) 1 × 10-4, (O) 1 × 10-2, and (0) 1. Frequency of oscillation 100 mHz. Amplitude of deformation 15%. Temperature 20 °C, pH 7, and I 0.05 M. The dotted line with slope ) 1 indicates the behavior of an ideal gas.33

between amino acid residues and the formation of a gel-like interfacial film. The time-dependent surface dilatational modulus for adsorbed films of β-lactoglobulin showed a complex behavior depending on the protein concentration in solution (Figure 7). The same behavior was observed for other globular proteins42-44 and can be explained by competition between the rates of diffusion/ adsorption and reorganization of the protein at the interface. In fact, (i) at the highest protein concentration in solution (at 1 wt %) the rate of protein diffusion is very high2 and the unfolding and further reorganization of amino acid residues with the formation of a coherent film must be reduced. Thus the formation of a coherent film with high Ed values requires high adsorption time (Figure 7A). (ii) As the protein concentration in solution decreases at 1 × 10-2 wt %, the rate of adsorption also decreases;2 thus, the unfolding and further reorganization of β-lactoglobulin amino acid residues must be facilitated, giving a coherent film with high Ed values, even at low adsorption time (Figure 7A). (iii) Finally, at the lower protein concentration in solution (at 1 × 10-4 wt %) the rate of protein diffusion is very low,2 and the unfolding and further reorganization of amino acid residues is facilitated. However, the surface density is lower compared to that for higher protein concentrations in solution, especially at low adsorption time, giving low values of Ed (Figure 7C). In summary, the packing of protein amino acid residues with an unfolded secondary structure is an important factor that determines the elasticity of globular proteins at a fluid interface.45 That is, the results shown in Figure 7 suggest that the secondary and tertiary structures of a globular protein in solution in addition to the rate of adsorption at the interface have an effect on the unfolding of the protein and further reorganization of amino acid residues, with direct repercussions on the film viscoelasticity. Finally, the lower values of E compared to the behavior of an ideal gas in the E-π plot (Figure 8), at the highest β-lactoglobulin concentration in solution and at low adsorption time (at low π), corroborate the idea that this protein is adsorbed at the interface forming aggregated clusters under these experimental conditions. As for globular soy globulin adsorbed films,43,44 these data confirm that β-lactoglobulin is adsorbed at the air-water interface with different degrees of association depending on the protein concentration in solution and the adsorption time. A common trend of the surface pressure dependence of E for β-lactoglobulin adsorbed films is that E increased with increasing π (Figure 8). This increase is a result of an increase in the interactions between the film molecules

Discussion Viscoelastic Characteristics of Lipid and Protein Adsorbed Films. For the lipids analyzed in this study (diglycerolmonolaurate and diglycerol-monostearate) the values of Ed increased with the lipid concentration in solution (Figures 1 and 3), especially for diglycerol-monostearate (Figure 3). In fact, the values of Ed for diglycerol-monostearate were much higher at C > CMC (Figure 3A), compared to the dilatational elasticity of adsorbed films of this lipid at C < CMC (Figure 3). This phenomenon corroborates the opinion that interactions between lipid molecules at the air-water interface are higher as the film is saturated by the lipid, at lipid concentrations in solutions higher than the CMC. At lipid concentrations in solutions lower than the CMC, the level of Ed is similar and does not depend on the lipid concentration in solution, even at long-term adsorption as the adsorbed film develops high values of π (Figures 2 and 4). Interestingly, at low lipid concentration in solution (at C < CMC) the diluted adsorbed film presents the same values of E no matter what the lipid, either diglycerolmonolaurate (Figure 2) or diglycerol-monostearate (Figure 4). In fact, during the adsorption at the interface the values of E are more than two times higher for diglycerol-monostearate at C > CMC than at C < CMC (Figure 4). Thus, the master curves E versus π (Figures 2 and 4) reflect not only the amount of lipid adsorbed at the interface (giving high E values at higher π) but also the degree of interactions between lipid forming films, which are higher (i) as the surface pressure and/or the lipid concentration in solution increases and, thus, on the interface increases and (ii) as the van der Waals interactions between lipid hydrocarbon chains increase, these being higher for diglycerol-monostearate than for diglycerol-monostearate. However, for protein adsorbed films (caseinate and β-lactoglobulin in this work) the consequences for E of the formation of an adsorbed film at the air-water interface are more complex (Figures 6 and 8). (i) The surface dilatational characteristics of disordered (caseinate) and globular (β-lactoglobulin) adsorbed films are viscoelastic (Figures 5 and 7). (ii) The time evolution of Ed is complex and depends on the chemistry of the molecule, the protein concentration in solution, and the rate of protein adsorption at the air-water interface. For a disordered protein (caseinate) the interfacial structure (tail or tail/loops conformation of amino acid residues), which depends on the protein concentration in solution, has an effect on the values of Ed. Lower Ed values are observed as the protein adopts a tail conformation (structure 1) at the lower protein concentrations in solution. The higher values of Ed at higher protein concentrations in solutions correspond to a tail/loop conformation of amino acid residues (structure 2). At the highest values of protein concentration in solution the minimum values of Ed correspond to a collapsed protein film and the formation of multilayers.37 For a globular protein (β-lactoglobulin) the unfolding and reorganization of amino acid residues at the interface determine

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the influence of the protein concentration in solution on the values of Ed. The rate of protein adsorption at the interface, which is higher at the higher protein concentration in solution,37 has an effect on the unfolding and reorganization of amino acid residues and, thus, on the viscoelasticity of the interface. (iii) The evolution of E with π reflects a nonideal behavior of protein (caseinate and β-lactoglobulin) adsorbed films (Figures 6 and 8) because of the interactions between amino acid residues at the air-water interface. However, at the highest protein concentration in solution the values of E are lower than those for an ideal behavior, a phenomenon that can be associated with a reduction in the interactions between aggregates of adsorbed proteins. (iv) Over the range of surface pressures studied the values of E for caseinate adsorbed films (Figure 6) were lower than those for β-lactoglobulin adsorbed films (Figure 8). These differences are due mainly to differences in the looping of amino acid residues for adsorbed films of random coil (caseinate) and globular (β-lactoglobulin) proteins at the air-water interface. Finally, two significant differences between lipids (diglycerol esters) and protein (caseinate and β-lactoglobulin) adsorbed films emerge from this study: (i) the surface dilatational characteristics are essentially elastic for lipids and viscoelastic for proteins, and (ii) the values of Ed are lower for proteins compared to lipids. Role of Viscoelastic Characteristics of Lipid and Protein Adsorbed Films on Foaming. Previous papers1,2 analyzed the effect of interfacial (adsorption isotherm and rate of adsorption) and foaming (foaming power and foam stability) properties of milk proteins (caseinate and β-lactoglobulin) and diglycerol esters (diglycerol-monostearate and diglycerol-monolaurate), as a function of the emulsifier concentration in solution. We have observed that the adsorption (surface pressure and rate of diffusion) and foaming power (foam capacity, gas and liquid retention in the foam, and foam density) increase with the emulsifier concentration in the aqueous phase. A close relationship was observed between foaming (overall foaming capacity, OFC) and the rate of diffusion (kdiff) of the emulsifier to the air-water interface (Figure 9A). That is, at higher emulsifier concentrations in solution, as the rate of diffusion is higher or the lag period (i.e., the time at which the surface pressure starts to increase from zero) is lower or absent, the foaming capacity is also higher because the concentration of emulsifier at the interface is also higher.1,2 As the interface is saturated by the diglycerol esters at the CMC (Table 2) or by proteins at a concentration coinciding with the AE (Table 2), a maximum in the foaming capacity was observed. Thus, the saturation of the interface by the emulsifier (at the CMC or AE for lipids or proteins, respectively)46 in addition to the rate of emulsifier diffusion at the air-water interface (and/or the absence of a lag period)1,2 can be used as two criteria for foaming. However, the results in Figure 9A also indicate that the foam capacity (in terms of emulsifier concentration in solution) also depends on the chemistry of the emulsifier, lipid (especially the length of the chain or head group), or biopolymer (especially on whether it is a disordered or globular protein). The surface dilatational properties of emulsifier films are considered essential for foam formation.7,13,47-51 The effect of dilatational properties (especially the value of Ed) on foam capacity is different for lipids and proteins, as can be deduced from the evolution of the overall foam capacity (OFC) with the value of Ed at 15 min of adsorption time (Ed15), at the beginning of the measurements (Figure 9B). High values of Ed15 can be produced by a high rate of diffusion of the emulsifier to the interface and/or may be due to the existence of high

Figure 9. Evolution of the OFC with (A) the rate of diffusion at the airwater interface (kdiff) and (B) the surface dilatational modulus at 15 min of adsorption time (Ed15) for aqueous solutions of diglycerol esters and proteins. Symbols in part A: (O) diglycerol-monolaurate, (4) diglycerol-monostearate, (]) caseinate, and (stars) β-lactoglobulin. Symbols in part B: (4) diglycerol esters (diglycerol-monolaurate and diglycerol-monostearate) and (O) proteins (caseinate and β-lactoglobulin). Bubbling gas: nitrogen. Gas flow: 60 mL/s. Temperature 20 °C, pH ) 7, and ionic strength 0.05 M.

emulsifier-emulsifier interactions. As sinusoidal oscillation for surface dilatational measurements start after 15 min of adsorption time, the values of Ed15 correspond to a time much longer that the time necessary for adsorption of the emulsifier at the bubble interface, which is shorter than the time required for foam formation (less than 2 min in our experiments). However, the results in Figure 9B corroborate that the mechanism of foam formation must be different for lipids compared to proteins. In fact, foam formation from aqueous solutions of diglycerol esters requires Ed15 values higher than 15 mN/m, while proteins can form foam at lower Ed15 values and the foam capacity is independent of the actual Ed15 value (Figure 9B). That is, independent measurements of Ed15 are not relevant for explaining the foam capacity of proteins. We speculated that the viscoelastic characteristics of the adsorbed film, which is practically elastic for lipids (as analyzed in preceding sections), determine the effect of surface dilatational characteristics on foam capacity. Thus, we conclude that for foam formation it may be important to have a high surface pressure, as for lipids,2 to produce small bubbles and a certain value of Ed15 to stabilize the bubbles during their formation (at short time). Foam stability, quantified by the relaxation time due to drainage (td) and disproportionation/coalescence (tdc), correlates with the long-term surface pressure (the equilibrium surface pressure, πe) of emulsifier film adsorbed from aqueous solution, as the amount of emulsifier adsorbed at the air-water interface is high.1,2 During 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.4,5 The higher shear viscosity of protein films compared to lipid films20

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the formation of a thin gel-like adsorbed layer with high elasticity, which can explain the differences between the stability of protein and the stability of lipid foams (Figure 10B). Acknowledgment The authors acknowledge the support of CICYT through Grant AGL2004-0136/ALI. Literature Cited

Figure 10. Evolution of the relaxation time for foam (A) drainage (td) and (B) disproportionation/collapse (tdc) with the surface dilatational modulus at 180 min of adsorption time (Ed180) for aqueous solutions of (4) diglycerol esters and (O) proteins. Bubbling gas: nitrogen. Gas flow: 60 mL/s. Temperature 20 °C, pH ) 7, and ionic strength 0.05 M.

can explain the higher stability of protein foams against drainage compared to lipid foams (Figure 10A). Moreover, in the case of protein adsorbed films, multilayer formation at high π, with a possible cross-linked network, can contribute to a higher foam stability against drainage compared to lipid foams, although the values of Ed180 are lower for protein than for lipid films (Figure 10A). However, the relationships between surface dilatational properties and foam stability against disproportionation and foam collapse are not entirely clear.48-53 Martin et al.48 showed that only for stability against disproportionation were surface dilatational properties found to play a role. A generalized stability criterion against disproportionation establishes that Ed/σ > 0.5.3 This criterion is fulfilled in our experiments for diglycerol ester and protein foam stability quantified by the relaxation time due to drainage (td) and disproportionation/ coalescence (tdc; Figure 10), with few exceptions. In fact, assuming as restrictive values for σ those derived from CMC for lipids or AE for proteins (Table 2), the foam stability against individual mechanisms of drainage and disproportionation/ collapse is higher as the values of Ed are also higher. The data in Figure 10 also corroborate that the mechanisms of stability of lipid and protein foams are quite different. In fact, the stability of lipid foams requires higher values of Ed compared to those of protein foams. Moreover, at the same Ed value the stability of protein foams is higher than that of lipid foams (Figure 10B). These data support the idea19 that for optimum stability against coalescence, lipid films should be rigid (with high Ed values) and mobile, to allow for a quicker recovery of the film after any deformation. In addition, the presence of a completely elastic interface (as for lipid films) can stop bubble shrinkage during disproportionation. Interestingly, the maximum stability of protein foams was observed at the higher protein concentrations in solutions, as interfacial protein aggregates and multilayer formation at the interface and in the bulk phase takes place, as deduced from viscoelastic properties of protein films in a preceding section. Thus, adsorbed proteins can provide additional foam stability against disproportionation/collapse via

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ReceiVed for reView November 13, 2006 ReVised manuscript receiVed February 14, 2007 Accepted February 27, 2007 IE061451G