Behavior of Soy Globulin Films at the Airâ Water Interface. Structural

Ind. Eng. Chem. Res. 2003, 42, 5011-5017

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Behavior of Soy Globulin Films at the Air-Water Interface. Structural and Dilatational Properties of Spread Films Juan M. Rodrı´guez Patino,*,† Sara E. Molina Ortiz,‡ Cecilio Carrera Sa´ nchez,† Ma. Rosario Rodrı´guez Nin ˜ o,† and Ma. Cristina An ˜ on‡ Departamento de Ingenierıá Quı´mica, Facultad de Quı´mica, Universidad de Sevilla, c/. Prof. Garcıá Gonza´ lez, s/nu´ m. 41012 Sevilla, Spain, and Centro de Investigacio´ n y Desarrollo en Criotecnologıá de Alimentos (CIDCA), Facultad de Ciencias Exactas, Universidad Nacional de la Plata, Calle 47 y 116, 1900 La Plata, Argentina

We have studied the structural and surface dilatational characteristics (surface dilatational modulus and its elastic and viscous components) of two major fractions of soy globulin from a soy protein isolate, β-conglycinin (a 7S globulin) and glycinin (a 11S globulin)sincluding the effect of chemical reduction of glycinin with dithiothreitol (DTT)sspread at the air-water interface at 20 °C and at pH 2.0, 5.0, and 8.0. The stress response to compression-expansion sinusoidal deformation of the interface in a modified Wilhelmy-type trough with two oscillating barriers was measured at a constant amplitude (5% of the initial area) and as a function of frequency (within the range of 1-100 mHz), and superficial pressure. The same experimental device coupled with Brewster angle microscopy makes it possible to determine the structure, morphology, and relative reflectivity of the monolayer. The monolayer structure was more expanded on an aqueous subphase at pH 2.0 and the opposite was observed at pH 5.0. The chemical reduction of glycinin with DTT produced a significant expansion of the monolayer structure. The monolayer structure determines the surface dilatational characteristics of soy protein films. It was found that not only is the dilatational modulus determined by the interactions between spread molecules (which depend on the surface pressure) but also the structure of the proteins spread on the monolayer plays an important role. Introduction The functional properties of proteins in foods are related to their structural and dynamic characteristics at fluid interfaces. A fundamental understanding of the physical, chemical, and functional properties of proteins and the changes these properties undergo during processing is essential if the performance of proteins in foods is to be improved and if under-utilized proteins, such as plant proteins (legume and cereal), are to be increasingly used in traditional and new processed food products.1-6 The physical principles governing the formation and stability of food colloids (foams and emulsions) are complex, especially if protein macromolecules are involved as emulsifiers.7,8 The protein film structure is important from a practical point of view because it defines its emulsifying and foaming properties. On the other hand, the dynamic behavior of protein films is recognized as being of importance in the formation and stability of food colloids in which proteins are added as emulsifiers (e.g., filled gel).9-13 The study of such dynamic behavior can be described by interfacial rheology. Interfacial rheology can be defined for both compressional deformation (dilatational rheology) and shearing motion of the interface (shear rheology). While shear viscosity may contribute appreciably to the long-term stability of dispersions, dilatational rheology plays an important role in short-term stability. In fact, during formation of * To whom correspondence should be addressed. Phone: +34 95 4556446. Fax. +34 95 4557134. E-mail: [email protected]. † Universidad de Sevilla. ‡ Universidad Nacional de la Plata.

food colloids a new interface is continuously formed during the dispersion process, over a time scale typical of dilatational rheology. In addition, the type of deformation that interfaces undergo during emulsification and foaming is expansion and, to a lesser extent, compression.11-14 Moreover, interfacial rheology is a very sensitive technique for assessing structure and interactions between film-forming components.15,16 In this work we complement previous studies17-19 by investigating the structural and dilatational characteristics of spread films of two major fractions of soy globulin from a soy protein isolatesβ-conglycinin (a 7S globulin) and glycinin (a 11S globulin), including the chemical reduction of glycinin with 10 mM dithiothreitol (DTT)sat the air-water interface as a function of pH and at 20 °C. With the exception of preliminary studies on adsorbed films,18-22 the structural characteristics of spread soy proteins at the air-water interface have not been systematically analyzed so far. The demand for safe, high-quality health foods with good nutritional value has increased the use of soy proteins.23 Globulins account for about 50-90% of seed proteins. Globulins 7S and 11S are two major storage proteins in soybeans. The 11S globulin has a quaternary structure composed of 12 subunits with a molecular weight of about 360 kDa. Native soy glycinin, because of its compact tertiary structure which is stabilized by disulfide cross-linking, has limited foaming4,5,20,24 and emulsifying4,25,26 properties. However, reduction of some disulfide bonds may improve their foaming and emulsifying ability by allowing greater conformational flexibility.27,28 The 7S globulin is a glycoprotein with a molecular weight of about 180 kDa that does not contain

10.1021/ie030140s CCC: $25.00 © 2003 American Chemical Society Published on Web 09/17/2003

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disulfide bonds. The strong pH dependence of the molecular conformation and the associated functional properties4,5,29,30 mean that the optimum functionality of soy proteins occurs at pH < 5, which limits their application as food ingredients. Thus, more research is required to resolve this and other issues related to the use of soy proteins in food formulations. Materials and Methods Materials. To form the spread surface film, protein was spread in the form of a solution using water at pH 8.0 as a spreading solvent. The sample was stored at 4 °C and all work was done without further purification. Samples for interfacial characteristics of soy protein films were prepared using Milli-Q ultrapure water and were buffered at pH 2.0, 5.0, and 8.0. Analytical-grade acetic acid and sodium acetate, and Trizma [(CH2OH)3CNH2/(CH2OH)3CNH3Cl] for buffered solutions at pH 5.0 and 8.0, respectively, were used as supplied by Sigma (>95%) without further purification. HCl (analytical grade, Panreac) and KCl (analytical grade, Merck) were used for adjusting the pH 2.0 and the ionic strength of the aqueous solutions, respectively. Ionic strength was 0.05 M in all the experiments. The isolation of β-conglycinin (fraction 7S) and glycinin (fraction 11S) soy globulins, solubility, and structural characteristics (including scanning differential calorimetric analysis, polyacrylamide gel electrophoresis under native conditions, surface hydrophobicity, and fluorescence spectroscopy) of 7S and 11S globulins have been described elsewhere.17 Glycinin was reduced using 10 mM DTT (11S + 10 mM DTT) as described elsewhere.27,31 Surface Film Balance. Measurements of the surface pressure (π) versus average area per molecule (A) were performed on a fully automated Wilhelmy-type film balance (KSV 3000, Finland) as described elsewhere.15,16 The maximum area of the trough between the two oscillating barriers is 51.5 × 15 cm2. Aliquots of aqueous solutions of soy protein fractions (1.3 × 10-4-1.5 × 10-4 mg/µL) at pH 8.0 were spread on the interface. To allow for spreading, adsorption, and rearrangements of the protein, 30 min was allowed to elapse before measurements were taken. The spreading method adopted in these experiments allows quantitative spreading of the proteins, as discussed elsewhere.18 The mean deviation was within (0.1 mN/m for surface pressure and (0.125 × 10-3 m2/mg for the area. The subphase temperature was controlled by water circulation from a thermostat, within an error range of (0.5 °C. The experiments were carried out at 20 °C. The compression rate was 3.3 cm‚min-1. Each isotherm was measured at least three times. The reproducibility of the surface pressure results was better than (0.4 mN/m. Brewster Angle Microscope (BAM). A commercial Brewster angle microscope (BAM), BAM2, manufactured by NFT (Go¨ttingen, Germany), was used to study the topography of the film. The BAM was positioned over the film balance. Further characteristics of the device and operational conditions have been described elsewhere.32,33 The surface pressure measurements, area, and gray level as a function of time were carried out simultaneously by means of a device connected between the film balance and BAM. These measurements were performed during continuous compression and expansion of the monolayer. To measure the relative thickness (δ) of the film, a previous camera calibra-

tion is necessary to determine the relationship between the gray level (GL) and the relative reflectivity (I), according to a procedure described previously.32,33 At Brewster angle, I ) |Rp|2 ) Cδ2, where C is a constant and Rp is the p-component of the light. Surface Dilatational Rheology. To obtain surface rheological parametersssuch as surface dilatational modulus, elastic and viscous components, and loss angle tangentsthe same modified Wilhelmy-type film balance (KSV 3000) was used as described elsewhere.15,16 In this method the surface is subjected to small periodic sinusoidal compressions and expansions by means of two oscillating barriers at a given frequency (ω) and amplitude (∆A/A) and the response of the surface pressure is monitored (π). Surface pressure was directly measured by means of two roughened platinum plates situated on the surface between the two barriers. The surface dilatational modulus derived from the change in surface tension (dilatational stress), σ (eq 1), resulting from a small change in surface area (dilatational strain), A (eq 2), may be described by eq 3.34

σ ) σo sin(ωt + θ)

(1)

A) Ao sin(ωt)

(2)

E)

dσ dπ )dA/A d ln A

(3)

where σo and Ao are the stress and strain amplitudes, respectively, t is the time, θ is the phase angle between stress and strain, π ) σο - σ is the surface pressure, and σο is the surface tension in the absence of protein. The dilatational modulus is a complex quantity and is composed of real and imaginary parts (eq 4). The real part of the dilatational modulus or storage component is the dilatational elasticity, Ed ) |E| cos θ. The imaginary part of the dilatational modulus or loss component is the surface dilatational viscosity, Ev ) |E| sin θ. The ratio (σo/Ao) is the absolute modulus, |E|, a measure of the total unit material dilatational resistance to deformation (elastic + viscous). For a perfectly elastic surface the stress and strain are in phase (θ ) 0) and the imaginary term is zero. In the case of a perfectly viscous material θ ) 90° and the real part is zero. The loss angle tangent can be defined by eq 5. If the film is purely elastic, the loss angle tangent is zero. The reproducibility of these results was better than 5%.

E ) (σo/Ao)(cos θ + i sin θ) ) Ed + i Ev

(4)

tan θ ) Ev/Ed

(5)

Results and Discussion Structural Characteristics of Spread Soy Proteins at the Air-Water Interface. Results derived from π-A isotherms in the Wilhelmy-type trough are in good agreement with those obtained in the Langmuirtype trough with the same proteins spread on similar subphases.18,19 The results of π-A isotherms for β-conglycinin and glycinin at pH 5.0 and 8.0 indicate (data not shown) that the structural characteristics of spread monolayers depend on a continuous surface denaturation process (film aging). That is, the protein film changed significantly over time. In fact, for these soy globulins we have observed a significant shift of the π-A isotherms toward higher molecular areas as the aging

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Figure 1. Surface pressure-area isotherms for (0) β-conglycinin, (O) glycinin, and (4) glycinin + 10 mM DTT monolayers spread on aqueous solutions at (A) pH 2.0, (B) pH 5.0, and (C) pH 8.0. Temperature: 20 °C.

time increased. In addition, we have also observed that the maximum surface pressure increased with the aging time. This behavior is opposite to that observed for nonquantitative protein spreading.35,36 These results demonstrate that the aging effect observed in soy globulin monolayers is due to unfolding of the protein at the interface, including the possibility that a diffusional loss of aggregated protein takes place during the spreading process, which afterward is incorporated at the interface.18 This aging effect is characteristic of some globular protein (i.e., β-lactoglobulin) monolayers.36-38 However, for glycinin and glycinin + 10 mM DTT (data not shown) spread monolayers at pH 2.0 the π-A isotherms are practically independent of the film aging. These results were explained19 by the conformational change that takes place in glycinin at the highest acidic pH or as a result of the reduction of glycinin by DTT.17 The results for β-conglycinin, glycinin, and glycinin + 10 mM DTT at pH 2.0, 5.0, and 8.0 for an aging time of 330 min indicate that a change in the slope of the π-A isotherms was produced (Figure 1) in the range of 13.5-16.6 mN/m, depending on the protein and pHs with the maximum values of the surface pressure at the transition point for soy globulins at pH 8.0 and for glycinin + DTT at pH 5.0. This behavior was due to a change in the degree of condensation of protein molecules at the interface.18 The change in the state of condensation of the monolayer was confirmed by the evolution of the reflectivity of the monolayer with the surface pressure (Figure 2). In fact, we have observed a step increase in the value of the reflectivity in the regime of surface pressures corresponding to the transition between the two condensation states of the mono-

Figure 2. Relative reflectivity as a function of surface pressure for (0) β-conglycinin, (O) glycinin, and (4) glycinin + 10 mM DTT monolayers spread on aqueous solutions at (A) pH 2.0, (B) pH 5.0, and (C) pH 8.0. Temperature: 20 °C. Shutter speed: 1/250 s.

layer. As for most globular proteins,15,39-41 at low surface pressures most amino acid residues in soy globulin molecules adopt loop conformation at the airwater interface. However, the loop conformation is more condensed at higher surface pressures and is displaced toward the bulk phase by the formation of multilayers at the collapse point. The progressive unfolding of β-conglycinin and glycinin as the pH decreases and the associated conformational changes in the molecule5,17,29,30,42 have a significant repercussion on the structural characteristics of the monolayer (Figures 1 and 2). The most condensed monolayer structure (as deduced by the translation of the π-A isotherm toward lower molecular areas) was observed at pH 5.0 as both proteins would be partially denatured and aggregated, close to the isoelectric point. The aggregation of these proteins at the air-water interface at pH 5.0 was confirmed at a microscopic level by BAM images.19 Conversely, the most expanded monolayer structure was observed at pH 2.0, as the proteins would be completely denatured and dissociated.17 In addition, for glycinin + 10 mM DTT the π-A isotherms at pH 2.0 and 8.0 (Figure 1) are practically coincident. Therefore, the conformational changes due to denaturation of glycinin by DTT may be similar to that produced by pH. On the other hand, the monolayer structure at constant pH also depends on the protein: (i) at pH 2.0 the π-A isotherm was the same no matter what the protein (β-conglycinin, glycinin, or glycinin + 10 mM DTT). That is, at the higher acidic pH β-conglycinin and glycinin molecules would be completely denatured and, thus, the effect of DTT on the glycinin monolayer


Figure 3. Effect of surface pressure on surface dilatational modulus (open symbols) and loss angle tangent (close symbols) for (0) β-conglycinin, (O) glycinin, and (4) glycinin + 10 mM DTT monolayers spread on aqueous solutions at pH 2.0. Temperature: 20 °C. Amplitude: 5%. Frequency: 50 mHz.

structure is null. (ii) At pH 5.0 the π-A isotherms were displaced toward higher molecular areas from β-conglycinin to glycinin to glycinin + 10 mM DTT. That is, the cleavage of SS bonds in native glycinin by DTT produces a significant expansion of the monolayer structure. Upon cleaving the SS bridge using DTT31,43 the unfolding of glycinin at the air-water interface is facilitated, which explains with the monolayer expansion. (iii) At pH 8.0, as β-conglycinin and glycinin are in a native state, the monolayer structure was more condensed for glycinin than for β-conglycinin. However, the treatment of glycinin with 10 mM DTT produced an expansion of the monolayer structure, even toward molecular areas higher than that for β-conglycinin. Thus, the same reasoning as above can be applied here. The more condensed structure of glycinin under native conditions should be associated with differences in ternary structuresthat for glycinin is stabilized by hydrophobic and electrostatic interactions besides the disulfide bond between the acidic and basic polypeptides44samong the differences in molecular mass. The SH/SS groups in glycinin have an important role in the formation of intermolecular disulfide bonds, giving a more condensed film at the air-water interface, and preventing the aggregation of the protein at interface, in relation to β-conglycinin. (iv) At every pH the surface pressure (Figure 1) and the reflectivity or thickness (Figure 2) of the monolayer were higher for glycinin than for β-conglycinin, in agreement with the more condensed glycinin film. The reflectivity of glycinin + 10 mM DTT is halfway between those for untreated soy globulins. Dilatational Characteristics of Soy Protein Films Spread on the Air-Water Interface. (1) Effect of Surface Pressure. The surface viscoelastic properties of β-conglycinin, glycinin, and glycinin + 10 mM DTT at pH 2.0 (Figure 3), 5.0 (Figure 4), and 8.0 (Figure 5) monolayers spread on the air-water interface were studied as a function of surface pressure and at 20 °C, at an amplitude of the sinusoidal deformation of 5%, and at a frequency of 50 mHz. In these experiments we have determined the surface dilatational modulus (E) and its elastic (Ed) and viscous components (Ev), including the loss angle tangent (Tan θ). However, in Figures 3-5 only E and Tan θ were included for simplicity, as a function of surface pressure. Similar dependency between E or Tan θ and π was obtained at 20 or 100 mHz (data not shown). Interestingly, for soy globulins at every pH the E-π plot did not depend on the aging of the monolayer, but only depended on the surface pressure (Figures 3-5). These results cor-



roborate the idea45 that the E-π curve could reflect the surface equation of state of the spread material at the air-water interface. This master curve is characteristic for any material spread at the air-water interface. It was observed that (i) the values for the surface dilatational modulus were very similar to those for the dilatational elasticity at every subphase pH and (ii) the dilatational viscosity and the loss angle tangent values were low and practically zero. As a consequence of this behavior, it can be established that the surface dilatational characteristics of protein monolayers are essentially elastic over the range of surface pressure studied. The E-π plots showed an irregular shape. The modulus increased with increasing surface pressure to a maximum value. Upon further increase of the surface pressure E decreased to a minimum at a surface pressure of about 13-17 mN/m, close to the transition between two condensed states in the monolayer structure, as deduced from the π-A isotherms (Figure 1) and reflectivity (Figure 2). Afterward, the surface dilatational modulus increased again with surface pressure. Interestingly, the same irregular shape in the surface density dependence of the surface dilatational modulus was observed by other authors for β-casein and caseinate (typical disordered proteins) adsorbed11,13,46 and spread16 films, the values of E being lower than those in Figures 3-5. Surprisingly, the behavior of milk globular proteins was different. For WPI and β-lactoglobulin adsorbed11,13 and spread16 films, 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 films16 were

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significantly higher than those for soy globulins (Figures 3-5). In conclusion, 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.18,19 On the other hand, the loss angle tangent decreased with surface pressure no matter what the protein (Figures 3-5). That is, the more condensed the structure (at higher surface pressures), the higher the elasticity of the monolayer. Thus, at surface pressures close to the film collapse the viscoelastic behavior of soy proteins was almost elastic. In this regard, 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. These findings support the hypothesis that the surface dilatational properties are not only determined by the interactions between spread protein molecules (which depend on the surface pressure) but also that the internal structure of the spread protein molecule also plays an important role.16 Benjamins et al.11,13 has pointed out the conclusion that molecular structure is important for understanding the dynamic interfacial behavior of adsorbed protein at fluid interfaces. We have observed the same behavior in this work for soy proteins spread on the air-water interface. That is, the surface dilatational characteristics of soy globulin spread films depends on the proper structure of the protein (βconglycinin, glycinin, or glycinin reduced with DTT). (2) Effect of the Aqueous Phase pH. As the quaternary structure of soy proteins depends to a great extent on the aqueous phase pH,17,42 which leads to differences in surface activity,17 foaming,4,5,20,24 emulsifying,4,25,26 and gelation5,47 properties, the analysis of this variable on surface dilatational characteristics of soy protein spread films may be of practical importance. The surface dilatational characteristics of soy globulins spread films at the air-water interface depend on the pH. At pH 2.0 (Figure 3) the surface dilatational modulus and the loss angle tangent were practically the same for β-conglycinin, glycinin, and glycinin + 10 mM DTT. This behavior is similar to that observed from the π-A isotherms (Figure 1). Thus, the same reasons as above can be applied here. That is, at higher acidic pH β-conglycinin and glycinin molecules would be completely denatured and, thus, the effect of DTT on the glycinin superficial dilatational characteristics is null. At pH 5.0 (Figure 4) the values of E were higher for glycinin + 10 mM DTT than for β-conglycinin and native glycinin. Conversely, the loss angle tangent was lower for reduced glycinin. Clearly, the surface dilatational properties of soy proteins depend on the protein structure. As at pH 5.0 the proteins 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 β-conglycinin and native glycinin. However, the cleavage of SS bonds in native glycinin by DTT at pH 5.0 produces a significant unfolding of the glycinin molecule in the aqueous phase31,43 and also at the airwater interface,19 which facilitates the interactions between proteins residues at the interface, in agreement with the higher values of E in relation to those for unreduced glycinin (Figure 4). Finally, at pH 8.0 (Figure

Figure 6. Effect of frequency on rheological parameters O, E (mN/ m); 4, Ed (mN/m); 3, Ev (mN/m); and ), Tan θ) for β-conglycinin monolayers spread on aqueous solutions at (A) pH 2.0, (B) pH 5.0, and (C) pH 8.0. Temperature: 20 °C. Surface pressure: 10 mN/ m. Amplitude: 5%.

5) the higher values of E (and minimum values of Tan θ) for glycinin films are in line with the more condensed structure (Figure 1C) and higher film thickness for glycinin monolayers (Figure 2C), especially at higher surface pressures. In summary, for β-conglycinin the maximum values for E and the more elastic films were observed at pH 2.0, as the molecular structure of the protein was more denatured. For glycinin the maximum values of E and the more elastic films were observed at pH 8.0, as the monolayer structure was more condensed in a native state. The effect of pH on reduced glycinin with DTT is characteristic for this system. In fact, this is the only system in which the values of E were higher at pH 5.0. Thus, if glycinin is unfolded by the effect of DTT, the negative effect of pH 5.0 on E in native glycinin disappeared in the reduced protein. (3) Effect of Frequency. Changes in surface dilatational properties for β-conglycinin film at 10 mN/m (as an example), as a function of pH and the frequency of oscillation over a range of 1 to 100 mHz, is illustrated in Figure 6. We did not observe any difference in the frequency dependence of surface rheological parameters for native or reduced glycinin, or for these soy proteins at different surface pressures (data not shown). It can be seen that (i) the dilatational modulus increased with the frequency and tends to a plateau at frequencies higher than 50 mHz. (ii) The dilatational modulus and its elastic component were essentially the same at frequencies lower than 50 mHz. However, significant differences between both rheological parameters were observed at frequencies higher than 50 mHz, mainly due


to the decrease of the elastic component at increasing frequencies. (iii) The value of the viscous component also increased with the frequency. These results are in good agreement with those obtained for β-lactoglobulin,9,48 β-casein,49 and BSA49,50 adsorbed films and for β-casein, caseinate, and WPI spread films.16 From the effect of frequency on surface dilatational parameters it can be concluded that protein monolayers present rheological behavior in dilatational conditions that is essentially elastic at low frequencies (ω < 50 mHz) and viscoelastic at higher frequencies (ω > 50 mHz). As a consequence of the viscoelastic behavior, the loss tangent angle increased with frequency (Figure 6). The frequency dependence of surface dilatational properties may be associated with the effect of the rate of deformation on the structure and stability of the protein monolayer. Relaxation of the surface pressure by diffusion is the most common relaxation mechanism in soluble surfactants and proteins.34,49,51-53 However, for spread protein monolayers other relaxation mechanisms should be more operative, as a function of surface pressure and the time scale considered.54,55 Thus, the viscoelastic, practically elastic, behavior observed for protein monolayers in the frequency range of 1-50 mHz may be associated with the slow52,56 organization/ reorganization of loop conformation in soy monolayer structure at 10 mN/m. However, at surface pressures higher than the equilibrium surface pressure the frequency dependence on surface dilatational properties (data not shown) may be associated with organization/ reorganization of soy monolayer structure coupled with the formation/destruction of 3-D collapse structures (including multilayer formation) at the collapse point. At higher frequencies (ω > 50 mHz), as viscoelastic behavior characterized the protein monolayer, unfolding and reorganization may contribute little to the dilatational modulus. Thus, for short time scales the exchange of protein residues in the conformation of loops from the interface may play an important role.10,11,13,48,52 That is, at short time scales the relaxation mechanism is essentially the same no matter what the surface pressure (data not shown) or what the proteinsthe more rigid globular β-conglycinin (Figure 6) or the more unfolded reduced glycinin due to the effect of DTT (data not shown). The fact that the frequency dependence of the surface dilatational properties was the same no matter what the protein and the aqueous phase pH (Figure 6) proves the hypothesis that the looping of the amino acid residues at low frequencies (ω < 50 mHz) and that the diffusion of these residues between the interface and the bulk aqueous phase at high frequencies (ω > 50 mHz) delimit the elastic and viscoelastic behavior of the film, respectively. From the surface pressure response to the sinusoidal change in the area for β-conglycinin films at pH 2.0 and at 10 mN/m (Figure 7) we have observed that the monolayer structure was constant during the sinusoidal compression/expansion of the film. In fact, during the relaxation mechanism at 30 and 100 mHzsat which the dilatational properties are elastic and viscoelastic, respectivelysthe oscillation in the surface pressure was in the range between 7 and 11 mN/m where a constant conformation in the looping of the amino acid residues can be deduced from the π-A isotherm (Figure 1). That is, the elastic and/or viscoelastic behavior of soy protein films is not necessarily

Figure 7. Dynamic surface pressure response to a sinusoidal change in area for β-conglycinin monolayers spread on aqueous solutions at pH 2.0. Frequency of the oscillation: (A) 30 mHz and (B) 100 mHz. Amplitude: 5%. Surface pressure: 10 mN/m. Temperature: 20 °C.

associated with the change in the film structure, as observed for lipids15 and milk protein.16 Acknowledgment This research was supported by CYTED through Project XI.17, for CICYT (Spain) and ANPCyT (Argentine) through Grants AGL2001-3843-C02-01 and PICT98 09-04265, respectively. Literature Cited (1) Damodaran, S. In Food Proteins and their Applications; Damodaran, S., Paraf, A., Eds.; Marcel Dekker: New York, 1997. (2) MacRitchie, F.; Lafiandra, D. In Food proteins ad their application; Damodaran, S., Paraf. A., Eds.; Dekker: New York, 1997. (3) O ¨ rnebro, J.; Nylander, T.; Eliasson, A.-C. Interfacial behaviour of wheat proteins. J. Cereal Sci. 2000, 31, 195. (4) Kinsella, J. E. Functional properties of soy proteins. J. Am. Oil Chem. Soc. 1979, 56 (March), 242. (5) Utsumi, S.; Matsumura Y.; Mori, T. In Food proteins and their application; Damodaran, S., Paraf. A., Eds.; Dekker: New York, 1997. (6) Hettiarachchy, N. S.; Kalapathy, U. In Functional Properties of Proteins and Lipids; Whitaker, J. R., Fereidoon, S., Munguia, A. L., Yada, R. Y., Fuler, G., Eds.; American Chemical Society: Washington, DC, 1998. (7) Walstra, P. Principles of emulsion formation. Chem. Eng. Sci. 1993, 48, 333. (8) Clark, D. C.; Wilde, P. J. In Proteins at Liquid Interfaces; Mo¨bius, D., Miller, R., Eds.; Elsevier: Amsterdam, 1998. (9) Murray, B. S.; Dickinson, E. Interfacial rheology and the dynamic properties of adsorbed films of food proteins and surfactants. Food Sci. Technol. Inst. 1996, 2, 131. (10) Murray, B. S. In Proteins at Liquid Interfaces; Mo¨bius, D., Miller, R., Eds.; Elsevier: Amsterdam, 1998. (11) Lucassen-Reynders, E. H.; Benjamins, J. In Food Emulsions and Foams: Interfaces, Interactions and Stability; Dickinson, E., Rodrı´guez Patino, J. M., Eds.; Royal Society of Chemistry: Cambridge, 1999. (12) Wasan, D. T. In Emulsions, Foams, and Thin Films; Mittal, K. L., Kumar, P., Eds.; Marcel Dekker: New York, 2000.

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Received for review February 13, 2003 Revised manuscript received July 30, 2003 Accepted August 2, 2003 IE030140S

Behavior of Soy Globulin Films at the Airâ Water Interface. Structural

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