Ind. Eng. Chem. Res. 2004, 43, 1681-1689
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Adsorption of Soy Globulin Films at the Air-Water Interface Juan M. Rodrı´guez Patino,*,† Cecilio Carrera Sa´ nchez,† Sara E. Molina Ortiz,‡ Ma. Rosario Rodrı´guez Nin ˜ o,† and Ma. Cristina An ˜ o´ n‡ Departamento de Ingenierı´a Quı´mica, Facultad de Quı´mica, Universidad de Sevilla, c/. Prof. Garcı´a Gonza´ lez, s/nu´ m. 41012-Sevilla, Spain, and Centro de Investigacio´ n y Desarrollo en Criotecnologı´a de Alimentos (CIDCA), Facultad de Ciencias Exactas, Universidad Nacional de la Plata, Calle 47 y 116, 1900-La Plata, Argentina
The adsorption kinetics at the air-water phase interface of soy globulins (β-conglycinin, glycinin, and reduced glycinin) has been studied. The adsorption kinetics was determined by surface tension measurements coupled with Brewster angle microscopy (BAM) as a function of time, protein concentration in the solution (within the range of 1-0.001%, wt/wt), and pH (2.0, 5.0, and 8.0). The ionic strength (0.05 M) and the temperature (20 °C) were maintained constant. The adsorption of soy globulins to the interface increases with the protein concentration in the solution, depending on the protein and, especially, on the pH. The adsorption decreases dramatically at pH 5.0, close to the isoelectric point of the protein. A lag period was observed at lower protein concentrations. The adsorption kinetics at the beginning of the adsorption is diffusion-controlled. However, the mechanism that controls the long-term adsorption is the penetration and unfolding of the protein. The molecular conformation of soy globulins, which depends on the pH, has an effect on the adsorption kinetics. Introduction The rate of adsorption at fluid-fluid interfaces of protein solutions is considered1-3 to play an important role in the formation and stabilization of food dispersions (emulsions and foams). In fact, during the formation of a dispersed system, the protein must be adsorbed at the interface to prevent the recoalescence of the initially formed bubbles or droplets. In addition, during protein adsorption, the surface or interfacial tension of the fluid interfaces decreases, which is an important factor both in optimizing the input of energy involved in the emulsification or foaming process4 and, finally, in achieving a smaller droplet and bubble size, which is an important factor for the stability of the dispersed system.1 The aim of this work is to systematically study the effect of the protein concentration in solution and pH on the adsorption kinetics of soy globulins (β-conglycinin, glycinin), including the reduction of glycinin by dithiothreitol (DTT), at the air-water interface. Although systematic studies dealing with protein adsorption at the water-fluid interface have been published recently,5,6 the kinetics of soy globulin adsorption in solution has not yet been systematically studied.7,8 The strong pH dependence of the molecular conformation and the associated functional properties9,10 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. In this work, we describe the first use of two complementary interfacial techniques (tensiometry and Brewster angle microscopy) to monitor the adsorption of the protein at the interface. The effects of protein * To whom correspondence should be addressed. Fax: +34 95 4557134. Phone: +34 95 4556446. E-mail:
[email protected]. † Universidad de Sevilla. ‡ Universidad Nacional de la Plata.
concentration, pH, temperature, processing history, and chemical or enzymatic treatment on soy protein adsorption at the air-water interface is of practical importance in the manufacture of model and real food dispersions.7,8,11,12 Materials and Methods Materials. Samples for the adsorption of soy protein at the air-water interface 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 (analyticalgrade, Merck) were used to attain pH 2.0 and to adjust the ionic strength of the solutions, respectively. The ionic strength was 0.05 M in all the experiments. β-Conglycinin (fraction 7S) and glycinin (fraction 11S) were isolated from defatted low-heat soybean meal as described by Molina et al.13 Glycinin was reduced using 10 mM of dithiothreitol (11S + 10 mM DTT) as described by German et al.14 The samples were stored at 4 °C and all work was done without further purification. Surface Pressure Measurements. For time-dependent surface pressure measurements of adsorbed soy globulin films at the air-water interface, an automatic drop tensiometer was used as described elsewhere.15 Briefly, the drop profile was processed according to the fundamental Laplace equation (eq 1) to obtain the surface tension
1 d 2 (x sin Θ) ) - Cz x dx b
(1)
where x and z are the Cartesian coordinates at any point of the drop profile; b is the radius of curvature of the drop apex; Θ is the angle of the tangent to the drop
10.1021/ie0302443 CCC: $27.50 © 2004 American Chemical Society Published on Web 03/09/2004
1682 Ind. Eng. Chem. Res., Vol. 43, No. 7, 2004
profile; and C is the capillary constant, C ) gF/σ, where σ is the surface tension, F is the difference between the densities of the two liquids, and g is the acceleration of gravity. The surface pressure is π ) σ0 - σ, where σ0 is the surface tension of pure water in the absence of protein. Protein solution was prepared freshly by weighing proper amounts of protein and buffer solution to attain the desire protein concentration in solution (within the range of between 1 and 0.001%, wt/wt) and then stirring for 30 min. The protein solution was placed in a 15-µL 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. The solution was allowed to stand for 30 min to reach constant temperature. Then, a drop of protein solution (5-8 µL) was delivered and allowed to stand at the tip of the needle for about 180 min to achieve protein adsorption at the air-water interface. The image of the drop was continuously recorded by a CDD camera and digitized. The surface tension was calculated through analysis of the profile of the drop according to eq 1. Topography and Reflectivity of Adsorbed Soy Globulin Films. Visualization (topography) of the interfacial film with a reasonable spatial resolution (∼2 µm) and analysis of the evolution of the relative reflectivity (or relative film thickness) with time provide useful information, at a microscopic level, about the structural characteristics and interactions of soy globulins during adsorption at the air-water interface. Surface tension-time and BAM-time (topography and reflectivity) measurements on the same film provide complementary information on soy globulin during its adsorption at the air-water interface and thus enable a global consistency check. A commercial Brewster angle microscope (BAM) was used for the first time to study the topography and reflectivity of the film during the adsorption process. Further characteristics of BAM and operating conditions are described elsewhere.16,17 The light reflected from the surface is collected by two achromatic lenses and detected with a CCD camera. The CCD camera converts the reflectivity signal from the sample (measure by the gray level) into a video image. To measure the relative reflectivity (I) of the adsorbed film, a previous camera calibration is necessary to determine the relationship between the gray level (GL) measured directly from the camera and the relative reflectivity (I).16,17 The imaging conditions were adjusted to optimize both image quality and quantitative measurement of reflectivity. Thus, generally, as the surface pressure increased, the shutter speed was also increased. The reflectivity at each point in the BAM image depends on the local thickness and optical properties of the film. These parameters can be measured by determining the light intensity at the camera and analyzing the polarization state of the reflected light. At the Brewster angle, I ) Cδ2, where I is the relative reflectivity, C is a constant, and δ is the relative film thickness. The BAM instrument was positioned on the dish of a Sigma 701 digital tensiometer (KSV Instruments Ltd., Helsinki, Finland), based on the Wilhelmy method, using a roughened platinum plate for the measurement of the surface tension, as described elsewhere.18 Surface tension measurements were reproducible within (0.2 mN/m. The surface tension and relative
Figure 1. Rate of soy globulin adsorption at the air-water interface: (A) β-conglycinin (7S), (B) glycinin (11S), and (C) reduced glycinin (11S + 10 mM DTT). Protein concentration (%, wt/wt): (4) 0.001, (O) 0.1, and (0) 1. Temperature ) 20 °C, pH ) 2.0, and I ) 0.05 M.
reflectivity measurements as a function of the adsorption time were carried out simultaneously. Results Soy Globulin Adsorption in Water Solutions. Time-Dependent Surface Pressure of Soy Globulins. Time-dependent surface pressure (π) measurements for soy globulin (β-conglycinin, glycinin, and reduced glycinin) adsorption at the air-water interface from protein solutions at pH 2.0, 5.0, and 8.0 are plotted in Figures 1-3, respectively, as a function of the protein concentration 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 soy globulins is high, which agrees with previous data in the literature for proteins.2,3,5,11 Moreover, the rate of increase of the surface pressure (dπ/ dθ) of soy globulin solutions also depends on the protein and the pH, as will be analyzed and discussed later in this work from a kinetic point of view. Lag Period. Another interesting result was the lag period observed at low protein concentrations in solution, which disappears at higher protein concentrations (Figures 1-3, Tables 1-3). Interestingly, for a native conformation of the protein at pH 8.0,13 the lag period (Figure 3, Table 3) appeared only at the lowest protein concentration studied (0.001%, wt/wt). The lag period is higher for glycinin than for β-conglycinin, because of the more compact structure of glycinin, which is stabilized by SS/SH bonds in the native state.10 The lag
Ind. Eng. Chem. Res., Vol. 43, No. 7, 2004 1683 Table 2. Characteristic Parameters for the Adsorption of Soy Globulins at the Air-Water Interface at 20 °C and at pH 5.0 concentration π vs θ1/2 slope (%, wt/wt) (mN‚m-1‚s-0.5)
θDa (s)
k1 × 104 induction ( s-1) (LRb) time (s)
0.1 0.01 0.001
β-Conglycinin 0.215 (0.995) 3365 1.5 (0.986) 0.24 (0.997) ∼12 100 - -
441 3481 >12 100
0.1 0.01 0.001
Glycinin 0.26 (0.996) 1300 2.2 (0.996) 0.025 (0.987) >12 100 - -
144 5900 >12 100
0.1 0.01 0.001
Glycinin + 10 mM DTT 0.63 (0.997) 360 3.0 (0.999) 0.16 (0.969) >12 100 - -
56 6250 >12 100
a Period during which diffusion controls the kinetics of adsorption of soy globulins at the air-water interface. b LR ) linear regression coefficient.
Figure 2. Rate of soy globulin adsorption at the air-water interface: (A) β-conglycinin (7S), (B) glycinin (11S), and (C) reduced glycinin (11S + 10 mM DTT). Protein concentration (%, wt/wt): (4) 0.001, (O) 0.01, and (0) 0,1. Temperature ) 20 °C, pH ) 5.0, and I ) 0.05 M. Table 1. Characteristic Parameters for the Adsorption of Soy Globulins at the Air-Water Interface at 20 °C and at pH 2.0 concentration (%, wt/wt)
π vs θ1/2 slope (mN‚m-1‚s-0.5)
θDa (s)
k1 × 104 ( s-1) (LRb)
induction time (s)
1 0.1 0.001
β-Conglycinin >15 15 7.5 1.7 (0.997) 9.5 0.68 (0.998) 575
3.1 (0.989) 2.3 (0.997) 1.7 (0.996)
0 0 130
1 0.1 0.001
Glycinin + 10 mM DTT >15 18.5 3.4 (0.993) 1.7 (0.997) 9 1.9 (0.992) 0.49 (0.998) 840 2.7 (0.972)
0 0 196
a Period during which diffusion controls the kinetics of adsorption of soy globulins at the air-water interface. b LR ) linear regression coefficient.
Figure 3. Rate of soy globulin adsorption at the air-water interface: (A) β-conglycinin (7S), (B) glycinin (11S), and (C) reduced glycinin (11S + 10 mM DTT). Protein concentration (%, wt/wt): (4) 0.001, (O) 0.1, and (0) 1. Temperature ) 20 °C, pH ) 8.0, and I ) 0.05 M.
period is higher at pH 5.0 (Figure 2, Table 2), especially for glycinin, due to the fact that, at pH’s close to the isoelectric point, these proteins are more aggregated. At pH 5.0 (Table 2), soy globulins adsorbed at the interface only at the highest concentration studied (0.1%, wt/wt). We cannot perform adsorption experiments at the concentration of 1% (wt/wt) because of the insolubility of these proteins at pH 5.0. At pH 2.0, the lag period appeared only at the lowest protein concentration studied (0.001%, wt/wt) and was higher for β-conglycinin than for glycinin (Table 1).
The reduction of SS/SH bonds in native glycinin by DTT has an effect on the lag period (Figures 1-3). In fact, at pH 8.0, as glycinin was in a native state, the reduction of disulfide bridges by DTT decreased the lag period by more than a factor of 7 (Table 3). However, the reduction of glycinin with DTT at pH 5.0 had a small effect on the lag period (Table 2). At the most acidic pH (pH 2.0), glycinin molecules would be completely denatured,13 and hence, the effect of DTT on the glycinin lag period is null (Figure 1, Table 1). Topography and Reflectivity of Adsorbed Films. In Figure 4, the evolution of the relative reflectivity with
1684 Ind. Eng. Chem. Res., Vol. 43, No. 7, 2004 Table 3. Characteristic Parameters for the Adsorption of Soy Globulins at the Air-Water Interface at 20 °C and at pH 8.0 concentration (%, wt/wt)
π vs θ1/2 slope (mN‚m-1‚s-0.5)
θDa (s)
k1 × 104 ( s-1) (LRb)
induction time (s)
the change in reflectivity corresponds to an increase in the monolayer thickness by
δ60 s δ12500 s
1 0.1 0.001
β-Conglycinin >12.5 15 15 15,000 s) and at pH * pl. (E) BAM image for β-conglycinin or glycinin at 24 hours of adsorption time and at pH * pl, and (F) BAM image for glycinin + 10 mM DTT at long adsorption time. Protein concentration: 0.1% wt/wt. Temperature 20 °C.
pletely denatured). The protein concentration at which this induction period appears is some orders of magnitude lower for milk proteins5 than for soy globulins (Tables 1-3). This difference correlates with the fact that the flexibility and susceptibility to conformation changes is lower for globular soy globulins13,20 than for milk random coil and globular proteins.6 If the induction period correlates with the time required to attain a critical, small monolayer coverage, in the range of 0.10.2 mg/m2,5 a soy globulin concentration of 0.001% (wt/ wt) would represent a critical concentration for soy protein foaming. This phenomenon can explain the lower foaming properties of soy proteins8,23 in comparison with milk proteins.24,25
From a kinetic point of view, the rate of surface pressure development by proteins after the lag period is caused by different processes:26 (i) the protein has to diffuse from the solution to the subsurface (a layer immediately adjacent to the fluid interface) by diffusion 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. That is, adsorption of proteins is generally a complex process, involving several types of conformational changes that can be either reversible or irreversible, as well as time-dependent.27
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Figure 6. Diffusion of soy globulin adsorption at the air-water interface: (A) β-conglycinin (7S), (B) glycinin (11S), and (C) reduced glycinin (11S + 10 mM DTT). Protein concentration (%, wt/wt): (4) 0.001, (O) 0.1, and (0) 1. Temperature ) 20 °C, pH ) 2.0, and I ) 0.05 M.
Figure 7. Diffusion of adsorbed soy globulin at the air-water interface: (A) β-conglycinin (7S), (B) glycinin (11S), and (C) reduced glycinin (11S + 10 mM DTT). Protein concentration (%, wt/wt): (4) 0.001, (O) 0.01, and (0) 0,1. Temperature ) 20 °C, pH )5.0, and I ) 0.05 M.
Diffusion of Soy Globulins in Water Solutions. The most important step in the formation of a foam or emulsion is the initial adsorption of the protein at the interface. At low surface concentrations, the surface pressure is low, and molecules adsorb irreversibly by diffusion. In the case of diffusion-controlled adsorption, the diffusion is driven by the concentration gradient.26 Thus, the first step of the adsorption process can be obtained28 from integration of Fick’s second law. The usual, simple Ward and Tordai28 diffusion model accounts for the changes in surface pressure, π, with time (eq 2). This equation indicates that a plot of surface pressure versus the square root of time will be linear.
diffusion, as was observed for milk proteins.15 To analyze the effect of soy globulin, its concentration in the solution, and the solution pH on the kinetics of soy globulin diffusion, the slope derived from the π vs θ1/2 line is reported in Tables 1-3. Effect of Concentration on the Diffusion of Soy Globulin at the Interface. It can be seen that the slope of the π vs θ1/2 line increases with the protein concentration in solution (Tables 1-3). Thus, it can be concluded that the diffusion of the protein is driven by the concentration gradient. The period during which diffusion controls the kinetics of adsorption of soy globulins at the air-water interface increased as the protein concentration decreased (Tables 1-3). At the highest protein concentration studied (1%, wt/wt), 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 Tables 1 and 3 as a qualitative measure of the diffusion of the protein to the interface. That is, at a protein concentration of 1% (wt/wt), the diffusion step does not control the kinetics of the adsorption of soy globulins to the interface. Effect of pH on the Diffusion of Soy Globulin at the Interface. The data presented in Tables 1-3 strongly suggest that the kinetics of adsorption of soy globulins during the first stage is governed not only by diffusion at the interface, but also by the conformational state of the protein in solution. The following observations can be made: (i) At pH 2.0, the rate of diffusions quantified by the slope of the π vs θ1/2 line (Table 1)sof β-conglycinin is practically identical to that of native
Dθ (3.14 )
π(θ) ) 2CokT
1/2
(2)
The application of eq 2 to monitor the kinetics of diffusion of soy globulin (β-conglycinin, glycinin, and reduced glycinin) molecules at the air-water interface is shown in Figures 6-8 for pH’s 2.0, 5.0, and 8.0, respectively. For all protein concentrations in solution, we find a good fit of the experimental data to the Ward and Tordai equation at low surface pressures (Tables 1-3). Thus, it can be concluded that, during the initial period, the kinetics of soy globulin adsorption at the air-water interface is controlled by a diffusion mechanism. The discrepancies observed at longer adsorption times, as the surface pressure is higher than about 10 mN/m, could be due 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
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explain the higher diffusion of glycinin at 0.1% (wt/wt) in relation to that for β-conglycinin (Table 3). This conclusion is in line with the higher viscosity of β-conglycinin solutions in relation to glycinin solutions.29 However, according to Kim and Kinsella,30 the reduction of glycinin with 10 mM DTT leads to a marked increase in the hydrophobicity, and this might have enhanced the rate of diffusion of the smaller subunits, a phenomenon different from that observed in Table 3. Conversely, the reduction of glycinin with DTT increased the exposure of apolar groups previously buried in the interior of the molecule, which should result in an increase in its size. The reduction of SS bonds in glycinin by DTT caused dissociation of glycinin, and this might have enhanced the rate of diffusion of the smaller subunits, particularly the acidic subunits of glycinin, to the interface. However, this might have been counteracted to some extent by the increased molecular drag as reflected in the increased viscosity of the reduced molecules.30 Clearly, the results shown in Table 3 reflect the concurrent effects of opposite phenomena on the diffusion of proteins to a fluid interface. Penetration and Rearrangements of Soy Globulins at the Air-Water Interface. The stabilization of a foam or emulsion depends to a great extends on the penetration and further rearrangements of previously diffused molecules at the air-water interface. To monitor unfolding at the interface and configurational rearrangements of adsorbed protein molecules, the following first-order equation can be used31 Figure 8. Diffusion of adsorbed soy globulin at the air-water interface: (A) β-conglycinin (7S), (B) glycinin (11S), and (C) reduced glycinin (11S + 10 mM DTT). Protein concentration (%, wt/wt): (4) 0.001, (O) 0.1, and (0) 1. Temperature ) 20 °C, pH 8.0, and I ) 0.05 M.
or reduced glycinin. (ii) At pH 5.0, the diffusion of β-conglycinin is faster than that of glycinin (Table 2), especially at 0.01% (wt/wt). Interestingly, the period during which diffusion controls the kinetics of adsorption of soy globulins at the air-water interface increased dramatically at pH 5.0 (Table 2). At this pH, the diffusion controls the adsorption of the protein at 0.01% (wt/wt) within the time of the experiment. (iii) Surprisingly, at pH 8.0, where soy globulins are in a native state, the rate of diffusion toward the interface was practically the same no matter what the proteinsβconglycinin, glycinin, or reduced glycinin (Table 3). (iv) Whereas DTT positively affected the diffusion of native glycinin at pH 5.0 (Table 2), it had a limited effect on the completely denatured glycinin molecules at pH 2.0 (Table 1) or the native conformation of the protein at pH 8.0 (Table 3). However, the rate of diffusion to the interface of reduced glycinin was higher at pH 8.0 than at pH 2.0. Because the diffusion coefficient is inversely proportional to the cube root of the molecular weight, in agreement with penetration theory,28 the pH can affect the diffusion rate to the interface. In fact, if pH 5.0 causes an aggregation of soy globulins in the solution,13 the protein’s diffusion toward the interface could diminish, as observed in Table 2. However, at pH * pI, the effect of pH on the diffusion of soy globulins is more complex. The diffusion coefficient depends on the molecular size and shape and the chemical nature of the protein surface (such as the hydrophobicity), among other factors. At pH 8.0, glycinin exhibited greater surface hydrophobicity than β-conglycinin,13 which can
ln
π180 - πθ ) -kiθ π180 - π0
(3)
where π180, π0, and πθ are the surface pressures after 180 min of adsorption, at time θ ) 0, and at any time θ, respectively, and ki is the first-order rate constant. In practice, a plot of eq 3 usually yields two or more linear regions. The initial slope is taken to correspond to a first-order rate constant of unfolding (k1), while the second slope is taken to correspond to a first-order rate constant of rearrangement (k2), occurring among a more or less constant number of adsorbed molecules. For all experiments of soy globulin adsorption, we found two linear regions in the plot of log[(π180 - πθ)/(π180 - π0)] vs θ. However, because protein adsorption at fluid interfaces is very time-consuming, no attempt was made to analyze the experimental data for the second rearrangement step of previously adsorbed soy globulin molecules. Effect of Concentration on the Penetration of Soy Globulin at the Interface. When an activation energy barrier to adsorption exists (as deduced previously from the data in Figures 6-8), the ability of the protein molecules to create space in the existing film and penetrate and rearrange at the interface is ratedetermining. As a general trend, it can be established that the value of k1 increases with increasing protein concentration (Tables 1-3). That is, penetration of soy globulins at the interface is facilitated by higher protein concentrations in the solution. Effect of pH on the Penetration of Soy Globulin at the Interface. The pH plays a significant role in the penetration of soy globulins at the air-water interface. In fact, at pH 2.0 (Table 1), the values of k1 at the lowest concentration (0.001%, wt/wt) are practically the same for 7S, 11S, and 11S + 10 mm DTT.
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However, at higher concentrations (1%, wt/wt), the values of k1 for 11S and 11S + 10 mM DTT are higher than that for 7S. On the basis of molecular mass, the probability of penetration into the interface of previously diffused molecules should be higher for 7S globulins (with a lower molecular mass than 11S globulin), but this was not observed in this study. Thus, the most likely explanation for these results is that, at highly acidic pH (pH 2.0), 7S is dissociated into 2S and 5S forms and 11S into 7S and 3S forms.13 Moreover, at this pH, the effect of DTT on native 11S might be null. That is, at pH 2.0, dissociated 7S and 11S globulins might penetrate at the interface at the same rate. Close to the isoelectric point of the protein (pH 5.0), the penetration of the protein into the interface was observed (Table 2) only at the highest protein concentration studied (0.1%, wt/wt). At lower protein concentrations in the solution, the diffusion step controlled the protein adsorption within the time of the experiment, as was discussed in previous sections. From a practical point of view, we can state that, at pH 5.0 and at concentrations lower than 0.1%, soy globulins do not adsorb at the air-water interface. At pH 8.0, the values of k1 for β-conglycinin and glycinin are practically the same (Table 3). At this pH, the hydrophobicity of glycinin is higher than that of β-conglycinin,26 and its rate of penetration at the interface should be higher than that of β-conglycinin. However, on the basis of the molecular mass, the penetration of β-conglycinin at the interface should be faster than that of glycinin. Clearly, the concurrence of opposite effects leads to a compensation that results in the same values of k1 for β-conglycinin and glycinin (Table 3). In summary, the adsorption of soy globulins onto the interface increases with increasing protein concentration in the solution, depending on the protein and, especially, on the pH. A lag period was observed at lower protein concentration and especially at pH 5.0. At this pH, soy globulins do not penetrate into the interface within the time of the experiment. At a microscopic level, the interfacial film formed from adsorbed soy globulins can be considered to consist of thin layers of protein gel as a consequence of the changes in molecular conformation and further protein-protein interactions as the adsorption progressed. The topography of the film turns from homogeneous to heterogeneous after longer adsorption times. The maximum heterogeneity was observed for soy globulins at pH 5.0. The molecular conformation of soy globulins, which depends on the pH, has an effect on the adsorption kinetics. The adsorption kinetics at the beginning of the adsorption (π < 10 mN/ m) and at protein concentrations lower than 1% (wt/ wt) is diffusion-controlled. The rates of diffusion of native soy globulins at pH 8.0 or dissociated soy globulins at pH 2.0 are practically the same. The chemical reduction of glycinin with DTT significantly improves its diffusion to the interface. The penetration and unfolding of the protein is the mechanism that controls the long-term adsorption. The penetration of soy globulins at the interface is facilitated at higher protein concentrations. However, at low protein concentrations and at pH 5.0, the protein does not penetrate at the air-water interface. In fact, the adsorption of soy globulins decreases dramatically at pH 5.0. This means that the optimum functionality of soy proteins occurs at pH * 5, which limits their application as food
ingredients. These results led us to investigate the possible improvement of the interfacial characteristics of spread and adsorbed soy globulin films by means of chemical or enzymatic treatments,9-11 by the inclusion in the protein solution of typical food reagents with repercussions on the behavior of the protein at fluid interfaces,32,33 or by the inclusion in the adsorbed film of other emulsifiers (lipids, phospholipids, surfactants, etc.) that are present in the majority of manufactured, processed foods.34 Acknowledgment This research was supported by CYTED through Project XI.17, for CICYT (Spain) and ANPCyT (Argentina), through Grants AGL2001-3843-C02-01 and PICT98 09-04265, respectively. Literature Cited (1) Dickinson, E. An Introduction to Food Colloids; Oxford University Press: Oxford, U.k., 1992. (2) Damodaran, S. Interfaces, Protein Films, and Foams. Adv. Food Nutr. Res. 1990, 34, 1. (3) Halling, P. J. Protein-Stabilized Foams and Emulsions. Crit. Rev. Food Sci. Nutr. 1981, 13, 155. (4) Walstra, P. Principles of Emulsion Formation. Chem. Eng. Sci. 1993, 48, 333. (5) Miller, R.; Fainerman, V. B.; Makievski, A. V.; Kra¨gel, J.; Grigoriev, D. O.; Kazakov, V. N.; Sinyachenko, O. V. Dynamic of Protein and Mixed Protein/Surfactant Adsorption Layers at the Water/Fluid Interface. Adv. Colloid Interface Sci. 2000, 86, 39. (6) Miller, R.; Aksenenko, E. V.; Fainerman, V. B.; Pison, U. Kinetics of Adsorption of Globular Proteins at Liquid/Fluid Interfaces. Colloids Surf. A 2001, 183, 381. (7) Wagner, J. R.; Gue´guen, J. Effects of Dissociation, Deamidation, and Reducing Treatment on Structural and Surface Properties of Soy Glycinin. J. Agric. Food Chem. 1995, 43, 1993. (8) Martin, A. H.; Grolle, K.; Bos, M. A.; Cohen Stuart, M. A.; van Vliet, T. Network Forming Properties of Various Protein Adsorbed at the Air/Water Interface in Relation to Foam Stability. J. Colloid Interface Sci. 2002, 254, 175. (9) Kinsella, J. E. Functional Properties of Soy Proteins. J. Am. Oil Chem. Soc. 1979, 56, 242. (10) Utsumi, S.; Matsumura, Y.; Mori, T. Structure Function Relationships of Soy Proteins. In Food Proteins and Their Applications; Damodaran, S., Paraf, A., Eds.; Marcel Dekker: New York, 1997. (11) Damodaran, S.; Paraf, A., Eds. Food Proteins and Their Applications; Marcel Dekker: New York, 1997. (12) Yu, M.-A.; Damodaran, S. Kinetics of Destabilization of Soy Protein Foams. J. Agric. Food Chem. 1991, 39, 1563. (13) Molina, S.; Carrera, C.; Rodrı´guez Nin˜o, Ma. R.; An˜o´n, C.; Rodrı´guez Patino, J. M. Structural Characterization and Surface Activity of Spread and Adsorbed Soy Globulin Films at Equilibrium. Colloids Surf. B 2003, 32, 57. (14) German, J. B.; O’Neill, T. E.; Kinsella, J. E. Film Formation and Foaming Behavior of Food Proteins. J. Am. Oil Chem. Soc. 1985, 62, 1358. (15) Rodrı´guez Nin˜o, Ma. R.; Rodrı´guez Patino, J. M. Effect of the Aqueous Phase Composition on the Adsorption of Bovine Serum Albumin to the Air-Water Interface. Ind. Eng. Chem. Res. 2002, 41, 1489. (16) Rodrı´guez Patino, J. M.; Carrera, C.; Rodrı´guez Nin˜o, Ma. R. Morphological and Structural Characteristics of Monoglyceride Monolayers at the Air-Water Interface Observed by Brewster Angle Microscopy. Langmuir 1999, 15, 2484. (17) Rodrı´guez Patino, J. M.; Carrera, C.; Rodrı´guez Nin˜o, Ma. R. Structural and Morphological Characteristics of β-Casein Monolayers at the Air-Water Interface. Food Hydrocolloids 1999, 13, 401. (18) Rodrı´guez Nin˜o, Ma. R.; Carrera, C.; Rodrı´guez Patino, J. M. Protein and Lipid Films at Equilibrium at Air-Water Interface. J. Am. Oil Chem. Soc. 2001, 78, 873.
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Received for review March 14, 2003 Revised manuscript received January 21, 2004 Accepted January 22, 2004 IE0302443
