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Thermodynamic and functional properties of legumin (11S globulin from Vicia faba) in the presence of small-molecule surfactants: effect of temperature and pH
Journal of Colloid and Interface Science 278 (2004) 71–80 www.elsevier.com/locate/jcis
Thermodynamic and functional properties of legumin (11S globulin from Vicia faba) in the presence of small-molecule surfactants: effect of temperature and pH Michael M. Il’in, Maria G. Semenova ∗ , Larisa E. Belyakova, Anna S. Antipova, Yurii N. Polikarpov Institute of Biochemical Physics of Russian Academy of Sciences, Vavilov str. 28, Moscow 119991, Russia Received 10 November 2003; accepted 24 May 2004 Available online 3 July 2004
Abstract We report on the effect of a set of water-dispersible small-molecule surfactants (the main and the longest-hydrocarbon components of which are a citric acid ester of monostearate, a sodium salt of stearol–lactoyl lactic acid, and a polyglycerol ester of stearic acid) on molecular, thermodynamic, and functional properties of the major storage protein of broad beans (Vicia faba) legumin in different molecular states (native, heated, and acid-denatured). The interaction between legumin and the surfactants has been characterized by a combination of thermodynamic methods, namely, mixing calorimetry and multiangle laser static and dynamic light scattering. It was found that hydrogen bonds, electrostatic interactions, and hydrophobic contacts provided a basis for the interactions between the surfactants and both the native and the denatured protein in aqueous medium. Intensive association of the protein molecules in a bulk aqueous medium in the presence of the surfactants was revealed by static and dynamic laser light scattering. In consequence of this, both the surface activity and the gel-forming ability of legumin increased markedly, which has been shown by tensiometry, estimation of protein foaming capacity, and steady-state viscometry. A likely molecular mechanism underlying the effects of small-molecule surfactants on legumin structure-forming properties at the interface and in a bulk aqueous medium is discussed. 2004 Elsevier Inc. All rights reserved. Keywords: Protein legumin; 11S globulin; Small-molecule surfactants; Molecular parameters; Thermodynamic parameters; Thermodynamics of interactions; Surface activity; Foaming capacity; Gelation
1. Introduction By now it is well known that the interactions between proteins and small-molecule surfactants (SMS) are of great practical consequence for stabilization of a wide variety of food colloids, because they provide a basis for structure formation at all structural levels of such systems, from molecules through the interfaces to the bulk complex aggregation of colloidal particles. For example, these interactions, which could be specific or nonspecific [1], contribute essentially, first, to morphology, composition, surface tension, and surface rheology of mixed adsorbed layers formed at * Corresponding author. Fax: +7-95-1355085.
E-mail address: [email protected] (M.G. Semenova). 0021-9797/$ – see front matter 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.jcis.2004.05.039
the interfaces of food colloids through crosslinking or protein displacement as a result of protein solubilization [2–13]; second, to the viscosity and structure of both protein and protein-stabilized emulsion gels by enhancing or weakening protein–protein crosslinks [2,14–16]; third, to long-term stability of both emulsions [17–20] and foams [21,22], caused mainly by strengthening or, on the contrary, by weakening of both the structure of the protein adsorbed layers and the interactions between colloidal particles. By this means, though the importance of the interactions between proteins and small-molecule surfactants for properties of food colloids is now accepted and well demonstrated, the molecular and thermodynamic basis underlying these properties is still incompletely understood. Because of this, in this study we have attempted to correlate the effects of SMS on molecular and thermodynamic
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Fig. 1. Chemical structure of the major components of the surfactants PGE, SSL, and CITREM [23].
properties of protein in the bulk with their effects on protein functional properties such as gel-forming ability, surface activity at the air–water interface, and foaming capacity. In addition, the roles of the structure of both protein and SMS in the effects studied has also been the focus of our attention. In pursuing these aims, we have studied the effect of a set of commercially important water-dispersible smallmolecule surfactants (the main and the longest-hydrocarbon components of which are esters, composed of the same hydrophobic part, stearic acid (C18), and different hydrophilic parts (Fig. 1) [23]: CITREM, a citric acid ester of monostearate, SSL, a sodium salt of stearol–lactoyl lactic acid, and PGE(080), polyglycerol ester of stearic acid) on molecular and functional properties of the major storage protein of broad beans (Vicia faba), legumin, in different molecular states (native, heated, and acid-denatured). We have chosen 11S globulin for our study, because the basic molecular and thermodynamic properties of this protein are well known [24–27] and reproducible and, in addition, this protein is homologous in physicochemical properties and biological functions to 11S globulins of the other leguminous plants (soy, pea, etc.) and could be a useful model to predict their behavior in the presence of SMS. Moreover, from the practical point of view both legumin [28–31] and SMS [23] are promising additives for a new food formulation.
2. Experimental 2.1. Materials Legumin (11S globulin) was isolated from broad beans (var. “Agat”) by a method that was described previously [32]. Homogeneity of the isolated 11S globulin was assessed by sedimentation velocity analysis in phosphate buffer at pH 8.0, ionic strength 0.05 mol dm−3 . It was found to be a single peak of 11S globulin with a sedimentation coefficient of about 12S (Fig. 2). Small-molecule surfactants, CITREM, SSL, and PGE (080) [23], were supplied by
Fig. 2. Result of sedimentation velocity analysis of legumin (1% wt/v) in phosphate buffer at pH 8.0 and ionic strength 0.05 mol dm−3 .
Danisco Cultor (Denmark). Phosphate buffer (pH 7.2, ionic strength 0.05 mol dm−3 ) and citrate buffer (pH 3.0, ionic strength 0.01 mol dm−3 ) were prepared using analytical grade reagents (99.9% pure): Na2 HPO4 , NaH2 PO4 , sodium salt of citric acid, and citric acid, respectively. Sodium azide (0.01 wt%) was added to the buffers as an antimicrobial agent. All solutions were prepared using double-distilled water. 2.2. Methods 2.2.1. Protein solution preparation Protein solutions with required concentrations were made in the appropriate buffer. Centrifugation (4000 rpm (3734g), 30 min, 20 ◦ C) of the protein solutions was common for removing of a small part of insoluble protein. Concentration
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Table 1 Thermodynamic parameters of legumin denaturation process: the specific denaturation enthalpy H d , the difference between the specific heat capacities of the native and denatured forms of the protein d C p , and the denaturation temperature T d System Native legumin Acid-denatured legumin Heat-denatured legumin
H d d C p Td (K) (J g−1 ) (J g−1 grad−1 ) 31 0.4 357 5 0.2 329 Absence of any heat effects on the thermogram
of the protein in solutions was checked after centrifugation using a Shimadzu (Japan) refractometer, on the known values of protein refractive index increment ground (see Section 2.2.7). 2.2.2. Preparation of thermo-denatured legumin The protein solutions with required concentrations were made in a 0.05 mol dm−3 phosphate buffer (pH 7.2) as described above (Section 2.2.1). Aliquots (25 ml) of the protein solutions were placed in glass vials and soldered. The vials were heat-treated at 90 ◦ C for 30 min in a water bath and after cooling were allowed to equilibrate for 20 h at room temperature (22 ± 2). The level of protein denaturation as a result of the heat treatment was checked by differential scanning calorimetry and was indicative of a partial loss of the native conformation of the protein; i.e., no change of the heat capacity of heat-treated protein solutions with increasing temperature was found in the thermogram (Table 1). 2.2.3. Preparation of acid-denatured legumin The acid-denatured protein solutions with required concentrations were made in a citric buffer (pH 3.0, ionic strength 0.01 mol dm−3 ) as described above (Section 2.2.1). The level of protein denaturation at pH 3.0 was estimated by differential scanning calorimetry and was suggestive of practical loss of the native protein conformation (Table 1). 2.2.4. Determination of the critical micelle concentration for the small-molecule surfactants The thresholds of concentrations (cmc) of the smallmolecule surfactants at which their micellization begins in aqueous medium were determined by a combination of several thermodynamic methods: (i) by tensiometry using a Krüss GmbH K-10 digital tensiometer (Hamburg, Germany)—indicated by the sharp break at the cmc in the plot of surface tension against the logarithm of concentration of the small-molecule surfactants that is shown in Fig. 3a, as an example; (ii) by static light scattering using an LS-01 apparatus (VA Instruments, St. Petersburg, Russia)—indicated by the sharp increase in the intensity of light scattering from a surfactant solution with increasing surfactant concentration that is illustrated in Fig. 3b, as an example; (iii) by mixing calorimetry using an LKB 2277 flow calorimeter— indicated both by the inflection point of the concentration dependence of the enthalpy of the dilution of a surfactant
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solution by buffer normalized to the surfactant concentration as shown in Fig. 3c, as an example, and by the minimum of the first derivative of this curve with respect to surfactant concentration (Fig. 3c ). Relying on such experiments, it was found that the cmc practically does not vary with temperature or pH. The pH- and temperature-averaged cmc interval is the following for each surfactant: PGE(080) 0.75–1.0 mg dm−3 ; SSL 2.5–3.5 mg dm−3 ; CITREM 12.5– 20 mg dm−3 . As this takes place, the cmc intervals found are in the series in conformity with a change in the ionic nature of the surfactants: PGE < SSL < CITREM (see Fig. 1, where the chemical structure of the SMS is shown more precisely). 2.2.5. Addition of the small-molecule surfactants into the protein solutions Stock solutions of the small-molecule surfactants (102 mg dm−3 ) were prepared by ultrasound sonication over 1 h at a frequency of 4.5 kHz while the solutions were shaken at 65 ◦ C (CPLAN water bath shaker, Type 357, Poland). As a result fine homogeneous dispersions of the small-molecule surfactants in aqueous medium were formed. Thereafter the stock solutions, cooled to room temperature, were used to prepare mixed solutions with the proteins at the required concentrations, whereupon the mixed solutions were shaken at 40 ◦ C for 1 h (CPLAN water bath shaker, Type 357, Poland) and then were allowed to cool to room temperature. 2.2.6. Determination of the surface tension, γ , at the planar air–water interface Values of the surface tension, γ , of the solutions of the small-molecule surfactants, the proteins, and their mixtures were monitored with an accuracy of 1 mN m−1 with the Wilhelmy plate technique [33]. All measurements were made in the thermostatic cell at 25 ± 0.5 ◦ C. The value of the surface tension between double-distilled water and air did not differ from the standard value within the range of experimental error and was 72 mN m−1 at 25 ◦ C independent of time. A platinum plate was used in measuring the surface tension. The surface tension was monitored continuously as a function of time. The values of γ presented in this work are averaged data for at least two repetitions of each of the experiments. 2.2.7. Estimation of protein molecular and interaction parameters in bulk aqueous medium The weight-average molecular weight, M w , radius of gyration, R G , and second virial coefficient, Apr−pr , of legumin alone and in the presence of small-molecule surfactants were determined by static light scattering in dilute aqueous solution (1 × 10−2 –1 × 10−3 g ml−1 , with 5–8 protein concentration points at most). The Rayleigh ratio Rθ was measured using vertically polarized light (633 nm) at angles in the range 40◦ θ 140◦ (13 angles) using a VA Instruments LS-01 apparatus (St. Petersburg, Russia) cali-
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(a)
(b)
(c)
(c )
Fig. 3. Data of cmc determination at pH 7.2, ionic strength 0.05 M for surfactants studied from (a) tensiometry; (b) light scattering: I90 /I0 is the intensity of light scattering normalized to the intensity of the incident light, (") 283, (2) 293, (P) 303, (Q) 313, (!) 323 K; (c, c ) mixing calorimetry.
brated with dust-free benzene (R90 = 11.84 × 10−6 cm−1 ). Solutions were filtered directly into the light-scattering cell through a Millipore membrane with pore size 0.65 µm. The raw data were used to plot the angle and concentration dependencies of the ratio H C/Rθ according to the Zimm method [34]. Here, C is the protein concentration (g ml−1 ), Rθ is the excess light scattering over that of the solvent at angle θ , and H is an instrumental optical constant equal to 4π 2 n2 ν 2 /N A λ4 , where N A is Avogadro’s number, λ is the wavelength of incident light in vacuo, n is the refractive index of the solvent, and ν is the refractive index increment of the protein. Values of the weight-average molecular weight, M w , were estimated as averages from the intercepts of both the concentration dependence of H C/Rθ as θ → 0 (the extrapolation was performed on 13 angles) and the angular dependence of H C/Rθ as C → 0 (the extrapolation was performed on 5–8 concentrations). Values of the radius of gyration, R G , were estimated from the slope of the angular dependence of H C/Rθ as C → 0. Values of the second virial coefficient, Apr−pr , were estimated from the slope of the concentration dependence of H C/Rθ as θ → 0. The
second virial coefficient characterizes primarily the thermodynamic affinity of protein molecules for solvent (aqueous medium in our case) (it is poor if Apr−pr < 0, or, by contrast, it is good if Apr−pr > 0 and it is ideal if Apr−pr = 0) [35], i.e., providing circumstantial evidence for protein surface hydrophobicity/hydrophilicity. The values of M w , Apr−pr , and R G presented in this work are averaged data for at least two repetitions of each experiment. The experimental error in the determinations of M w and Apr−pr was estimated as ±10%. The error in the R G determination was ±5%. Values of the refractive index increment for legumin at different concentrations of SMS were unchanged within experimental error (±10%) for native, heat-denatured, and acid-denatured legumin: ν = 0.2 × 10−3 , 0.18 × 10−3 , 0.18 × 10−3 m3 kg−1 , respectively. Values of the hydrodynamic radius R h of legumin alone and in the presence of small-molecule surfactants were estimated in aqueous solution (5 × 10−3 g ml−1 ) by dynamic light scattering [36,37]. The time correlation function of the scattering intensity was measured at 90◦ with vertically po-
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larized light (633 nm) using a VA Instruments LS-01 apparatus (St. Petersburg, Russia). Solutions were filtered directly into the light-scattering cell through a Millipore membrane as described above. To determine the hydrodynamic radius from the time correlation function, a special program was used (DYNALS Release 1.5, rights reserved by A. Golding and N. Sidorenko). 2.2.8. Differential scanning calorimetry Calorimetric measurements were made using a DASM4M differential adiabatic scanning microcalorimeter (Special Design Office of Biological Instrument Making, Russian Academy of Sciences) in the temperature range from 20 to 110 ◦ C at a scanning rate of 2 ◦ C min−1 and an excess pressure of 2.5 atm. The concentration of protein samples was equal to 0.005 g ml−1 . The accuracy of the measurements is about 10% of the values of the heat capacities. The sensitivity of the calorimetric measurement is no less than 5 × 10−6 J s−1 . The thermodynamic parameters of protein denaturation were calculated as proposed before [38]. The values of the thermodynamic parameters of protein denaturation presented in this work are averaged data for at least two repetitions of each of the experiments. 2.2.9. Mixing calorimetry Calorimetric measurements were made using an LKB 2277 flow calorimeter set at 22 ◦ C. A peristaltic pump pumped the reactants into the instrument. The pump was calibrated by measuring the time required to pump a known volume of solution into the calorimeter. The flow rate was equal to 9 × 10−6 l s−1 . The ratio of the flow rates in the two channels was close to one; i.e., the solutions of proteins or small-molecule surfactants in all cases were diluted by a factor of 2 under mixing. Both solutions were thermally equilibrated before entering the reaction vessel. The calibration of the calorimeter itself was done electrically at the temperature of measurement. The sensitivity of the calorimetric measurement is no less than 3 × 10−6 J s−1 . Thermal effects were observed during dilution of (i) the protein solution by the pure buffer, Qprotein–buffer; (ii) the solution of small-molecule surfactant by the pure buffer, Qsmall-molecule surfactant–buffer; (iii) the protein solution by the solution of small-molecule surfactant, Q . These thermal effects Q were measured in thermal power units (J s−1 ). The specific enthalpy of the interaction between protein and small-molecule surfactant was obtained from the relationship H protein–small-molecule surfactant = −(Q − Qprotein–buffer − Qsmall-molecule surfactant–buffer)/n,
(1)
where n is the number of grams of protein mixed with small-molecule surfactant per second (g s−1 ).
75
2.2.10. Rheological measurements Steady state shear viscosities were measured at 22 ◦ C as a function of shear stress using Rheotest 2 (Type RV2, Germany). The sample was contained in a concentric cylindrical cell (the ratio of inner diameter, r, to outer diameter is equal to 0.98, sample volume 10 ml). The data are presented as the ratio, µ, of the limiting-zero-shear-rate viscosity of the mixed (protein + surfactant) solution to that of the pure protein solution. The values of µ shown are averaged data for at least two repetitions of each of the experiments. 2.2.11. Estimation of protein foam ability Foam (25 ml) had been generated in an aeration column using a bubbling method. The velocity of air supply through a glass membrane (1 µm) was equal to 1.8 ml s−1 . In these experiments, both the volume of the foaming solutions and the protein concentration were maintained constant: 5 ml and 7 wt/vol%, respectively. (For details of preparation of protein solutions see Sections 2.2.1, 2.2.2, and 2.2.3.) The static stability of the foam (25 ml) was determined from the volume of liquid drained from the foam over time, Vd . The values of Vd presented in this work are averaged data for at least two repetitions of each of the experiments.
3. Results and discussion 3.1. Enthalpy of the interactions between legumin and small-molecule surfactants from mixing calorimetry Data from mixing calorimetry (Figs. 4a, 4b, 4c) show the distinct heat effects of the interaction between legumin and surfactants (corrected for surfactant and protein dilution by buffer in accordance with Eq. (1)), which could be dictated by a great many different physicochemical processes [22,38–45]: (i) the exothermic ones: — direct hydrogen bonding between polar groups of the protein and surfactant [39]; — direct electrostatic interactions between ionic head groups of surfactants and oppositely charged groups on the protein surface through electrostatic attraction [39]; — partial protein folding due to new hydrogen or electrostatic bonding in the protein interior; (ii) the endothermic ones: — direct hydrophobic interactions between nonpolar parts of the protein and surfactant hydrocarbon tail [39]; — partial protein unfolding [38]; — hydrophobically driven protein association in aqueous medium; (iii) either endothermic or exothermic: — changes in interactions with water;
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(a)
(b)
(c)
Fig. 4. Specific enthalpy, H , of the interaction between legumin (0.5% wt/v) and SMS in aqueous medium versus the small-molecule surfactant concentration in the mixed solutions: (a) CITREM, (b) PGE (080) and (c) SSL. (2) Acid-denatured protein (0.01 mol dm−3 , citrate buffer, pH 3.0); (P) native protein (0.05 mol dm−3 , phosphate buffer, pH 7.2); (F) heat-denatured protein (0.05 mol dm−3 , phosphate buffer, pH 7.2) at 298 K.
— changes in interactions with counterions; association/dissociation of counterions; — micelle-like cluster formation on the protein surface at 293 K, especially at surfactant concentrations above their cmc [40,41]. It is not possible discriminate the contributions from each of these processes to the heat effects found owing to their compensatory nature. Nevertheless, it seems possible to give some qualitative description of the principal distinctions in the molecular origin of the observed enthalpy changes based on the major structural features of both SMS (Fig. 1) and molecular state of the protein in aqueous medium (Table 1). Thus, for example, under the experimental conditions (pH 7.2 and 3.0, ionic strength 0.05 M) the surfactants that are susceptible to hydrogen bonding with protein, owing to their specific molecular structure (Fig. 1), such as anionic CITREM or nonionic PGE(080), can show an exothermic character in their interaction with protein (Fig. 4a, b). That is more typical of PGE (080), i.e., for the nonionic surfactant with a rather high number of hydroxyl groups in the molecule (Fig. 1). In contrast, SSL, having the smallest number of polar groups among the surfactants studied (Fig. 1), shows a predominantly endothermic character of the interaction with the protein (Fig. 4c) that suggests its hydrophobic nature [39], which is most likely determined by the interaction between the hydrocarbon tail in the SSL molecule and the hydrophobic parts of the protein molecules. The pronounced enhancement of the latter thermal effect revealed above the critical micelle concentration for SSL is likely attributable to a marked increase in the number of hydrophobic contacts, as a
Fig. 5. Specific enthalpy, H , of the interaction between legumin (0.5% wt/v) and SMS in aqueous medium at a concentration of small-molecule surfactants equal to 6 mg dm−3 at 298 K.
result of the addition to the protein of micelles of SSL, combining within a great many of the surfactant molecules. In turn, the role of the different molecular state of the protein in the interaction with SMS is more pronounced in the distinct heat effects observed for the same SMS at the same concentration (Fig. 5). For example, when heat-denatured protein compared to the native protein, there is obviously more accessibility of the reacting groups or parts of the protein molecules as a result of heat denaturation followed by partial protein un-
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Table 2 Effect of small-molecule surfactants (SMS) on the molecular parameter (M w , weight-average molecular weight) and thermodynamics of pair interactions (second virial coefficient Apr–pr ) of legumin in aqueous medium System
M w (kDa) Native legumin Heat-denatured legumin Acid-denatured legumin
With 6 mg dm−3 CITREM
Without SMS
330 6300 43000
104
104
With 1 mg dm−3 SSL
Apr–pr (m3 mol kg−2 ) 0
M w (kDa)
−2.1
19200
0.14
55000
18250
0.67
12000
1.27
375
Apr–pr (m3 mol kg−2 ) −2.45
folding. That is bound to be favorable both to hydrogen bonding, along with electrostatic interactions between opposite charges of the interacting molecules, as is seemingly found in the cases of CITREM and PGE(080), and to hydrophobic interaction, as for SSL. In contrast, protonation of the protein molecules at pH 3.0 (the acid-denatured state of legumin), reducing hydrogen bonding, leads to predominantly hydrophobic interactions between legumin and all studied SMS (Fig. 5). In addition, it is interesting to note that the expected highest total hydrophobicity of the soluble aggregates of heat-denatured legumin, composed of the most hydrophobic basic constituent chains [24], agrees closely with the most endothermic effect of the interaction between legumin and SMS, measured just in this case (Fig. 5). As this takes place, the highest hydrophobicity of the heat-denatured state of the protein is substantiated by both the a greatest extent of the partial protein unfolding observed from differential scanning calorimetry (Table 1) and the negative value of the second virial coefficient (Table 2), suggestive that water is a poor solvent [35] for these protein aggregates. Overall, the mixing calorimetry data indicate the occurrence of different types of interactions between smallmolecule surfactants and legumin in aqueous medium, the dominant character of which seems determined, on the one hand, by the total hydrophobicity and the accessibility of the protein functional groups for SMS, and on the other, by the total hydrophobicity and the availability of specific functional groups in surfactant molecules, themselves. Also, an additional contribution to the measured enthalpies from both protein association and protein unfolding as a result of the interaction of protein with SMS is also expected from our previous work [5,6]. 3.2. Molecular and interaction parameters of the legumin–surfactant complexes First and foremost, Tables 2 and 3 show a marked increase both in the weight-average molecular weight (M w ) and in the size (R g , R h ) of legumin in the presence of SMS, except when CITREM or SSL is added to the acid-denatured protein at studied concentrations. This result suggests an association of the protein in the aqueous medium, as if the
M w (kDa) 690
With 0.5 mg dm−3 PGE(080)
104
2600
Apr–pr 104 (m3 mol kg−2 ) 2.2
3.28
8800
−0.85
0.62
560000
1.42
Apr–pr (m3 mol kg−2 ) −0.07
M w (kDa)
Table 3 Effect of small-molecule surfactants (SMS) on the size and conformational parameter ρ of legumin in aqueous medium System
Without SMS With 6 mg dm−3 CITREM With 1 mg dm−3 SSL With 0.5 mg dm−3 PGE(080)
Heat-denatured legumin
Acid-denatured legumin
RG (nm)
Rh (nm)
ρ= R G /R h
RG (nm)
Rh (nm)
ρ= R G /R h
40 108
58 65
0.7 ≈ 1 1.7
511 325
115 133
4.4 2.4
273
87
3.1
196
132
1.5
119
71
1.7
2100
133
15.8
SMS molecules play the role of components bridging molecules of the protein together. It is interesting to note that the maximal extent of the protein association in the complexes with SMS was found to occur for the nonionic surfactant PGE(080) (Tables 2 and 3). The calculation of the molecular weight of hypothetical protein–surfactant complexes, which rests on the assumption that all amounts of added surfactants were bound equally by the protein in solution, provides evidence for this suggestion by virtue of the fact that the calculated values are several times or even orders of magnitude smaller than those found by light scattering. But it is clear that further determination of the exact composition of protein–surfactant complexes is required. In turn, the surface of the formed protein—SMS associates could become more hydrophobic or alternatively more hydrophilic (Table 2). This is mirrored in the change in the sign of the second virial coefficient [35]. Thus, the negative value of the second virial coefficient shows a decrease in the thermodynamic affinity of the associates for aqueous medium, which could be caused by some rise in their surface hydrophobicity and mutual attraction. The opposite is true for the positive value of the second virial coefficient. The changes found in the properties of the protein surface in the presence of SMS (Table 2) are most likely dictated by the ultimate spatial arrangement of the hydrophobic and hydrophilic parts of the protein and SMS molecules in response to their interaction. What this means is either that most of the hydrophobic parts of both interacting molecules are exposed to aqueous medium (Apr–pr < 0) or, on the contrary, that they
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(a)
(b)
(c)
Fig. 6. Effect of small-molecule surfactants on the surface behavior of native (2) and heat-denatured (") legumin (10 mg dm−3 ) at the planar air–water interface (0.05 M phosphate buffer, pH 7.2, 298 K). Interfacial tension, γ , is plotted as a function of concentration of SMS: (a) CITREM, (b) SSL, (c) PGE (080). The solid lines describe surface behavior of the SMS alone; the dashed lines indicate the critical concentration of micelle formation (cmc) for each case.
are hidden in the interior of the primary protein associations, whereas the hydrophilic ones are directed into the aqueous medium (Apr–pr > 0). Also an additional structural analysis of static and dynamic light-scattering data shows that the ρ parameter, which is highly structure-sensitive [37], changes its value in the presence of SMS (Table 3), suggesting different shapes of the formed associates: (i) spherelike (0.9 < ρ < 1); (ii) randomly coiled/randomly branched (1.5 < ρ < 2); (iii) with more rigid and open architecture (as encountered with rigid polydisperse rods when ρ 2). We would like to note here that the information on the effect of SMS on protein self-association in aqueous medium is very scarce in the literature [40,45], which in our opinion, hinders deeper insight into the molecular mechanisms of the effect of SMS on well-studied structural–functional properties of proteins (mentioned above in the Introduction). Most likely, more systematic studies on this subject are required in order to get this information. 3.3. Functional properties of the legumin–surfactant complexes: surface activity, foaming, and gel-forming abilities The modification of the molecular and thermodynamic properties of legumin in bulk aqueous medium, as a result of interaction with SMS, leads to a marked change in the functional properties of legumin.
A pronounced increase in the legumin surface activity was found for both denatured and native legumin in the presence of SMS (Figs. 6a, 6b, 6c, and 7). As this takes place, the marked distinctions between values of the surface tension of the protein–surfactant mixture and pure components suggest the formation of a mixed protein–surfactant adsorbed layer at the air–water interface. In this case, the determining role of complex formation between protein and surfactants on the surface behavior of their mixed solutions appears as the following: (i) In the values of the surface tension that are lower than those for the more surface-active component. That is more pronounced in the case of the CITREM + legumin mixture practically over the entire range of the concentrations of CITREM studied. (It should be noted here that the concentration of legumin was constant and equal to 10 mg dm−3 for all the mixed systems studied.) (ii) In the much lower values of the surface tension for the mixed systems in comparison with the values that are characteristic of the protein (10 mg dm−3 ) when it is in weight excess in relation to the surfactants. This result was found for the cases of the mixtures of both SSL and PGE with legumin. Based upon light-scattering data (Tables 2 and 3), it may be inferred that this result is primarily governed either by the higher extent of protein association in the presence of SMS, which may be beneficial for exposing more surfaceactive molecules at the interface, or by the deterioration of the thermodynamic affinity of the aqueous medium for the
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Table 4 Effect of small-molecule surfactants on the rate V d (g−1 min) of liquid drainage from foams stabilized by legumin at 7 wt/v% (25 ◦ C; duration of measurement is 5 min just after foam preparation) V d (g−1 min )
System Native legumin Heat-denatured legumin Acid-denatured legumin
Without SMS
With 28 mg dm−3 CITREM
With 28 mg dm−3 SSL
With 28 mg dm−3 PGE(080)
0.51 1.0 0.62
0.1 0.56 0.57
0.63 0.71 0.25
0.64 0 0.45
Table 5 Effect of small-molecule surfactants on the fractional increase µ in the limiting-zero-shear-rate viscosity of solutions of heat-denatured legumin at 7 wt/v% (0.05 M phosphate buffer, pH 7.2, 25 ◦ C) With CITREM
With SSL
With PGE(080)
C sms (mg dm−3 )
µ = ηmixture /ηprotein
C sms (mg dm−3 )
µ = ηmixture /ηprotein
C sms (mg dm−3 )
µ = ηmixture /ηprotein
14 28 42
1.3 2.5 1.1
0.88 3.5 7 9.7 14
3.8 2.4 2.5 2 2.1
3.5 5.3 7 9 21
122 107 24 42 37
Fig. 7. Effect of small-molecule surfactants on the surface behavior of legumin (10 mg dm−3 ) in different molecular states at the planar air–water interface in the presence of SMS at 298 K: CITREM (6 mg dm−3 ); PGE (080) (0.5 mg dm−3 ); SSL (2 mg dm−3 ).
protein + SMS associates (less positive or negative values of the second virial coefficients). Owing to the marked increase in the surface activity of the denatured legumin in the presence of SMS, a clearly defined increase in the stability of the protein foams with time was found, as presented in Table 4. In addition, rheological measurements, shown in Table 5, provide evidence for a large increase in the limiting zeroshear-rate viscosity, η, of solutions of heat-denatured legumin in the presence of the surfactants as compared with the η of pure protein solution, shown by the ratio µ =
ηmixture/ηprotein . This increase is by several times for CITREM and SSL and even by two orders of magnitude in the presence of PGE(080). The last effect agrees well with both the nonionic nature of PGE(080) and the large number of hydroxyl groups in its molecule (Fig. 1), by virtue of the fact that both hydrophobic contacts and hydrogen bonding could contribute to protein network formation in the bulk. On the strength of light-scattering data (Tables 2 and 3), it is believed that both a marked intensification of the protein association in the presence of SMS and a deterioration of the thermodynamic affinity of the aqueous medium for the protein + SMS associates could form the basis of their effect on the rheological properties of the protein solutions. Moreover, it most likely that the observed increase in the apparent viscosity of the solution of heat-denatured legumin in the presence of SMS is the origin, to some extent, of the increase in the time stability of the protein foams revealed. Similar effects of strengthening of protein gels as well as protein emulsion gels were found for a large number of protein–surfactant systems at concentrations of SMS where SMS does not compete for binding sites in protein molecules and does not displace protein from the surface of colloidal particles, but vice versa can enhance protein–protein crosslinks [16,19,46–51].
4. Conclusions The mixing calorimetry data indicate the occurrence of different types of interactions between small-molecule surfactants and legumin in aqueous medium, the dominant character of which seems to be determined, on the one hand, by the total hydrophobicity and the accessibility of the protein functional groups for SMS, and on the other, by the to-
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tal hydrophobicity and the availability of specific functional groups in surfactant molecules themselves. Self-association of the protein molecules in aqueous medium in the presence of surfactants is revealed by static and dynamic laser light scattering, as if the surfactant molecules play the role of the components bridging molecules of the protein together. Both the rather high level of protein association in the protein + surfactant complexes, which is most likely favorable for exposing a large number of surface-active molecules at the interface, and their lower thermodynamic affinity for aqueous medium seem to govern the observed increase in the surface activity of legumin in the presence of surfactants. It is believed that a marked intensification of protein association in the presence of surfactants also forms the basis of a pronounced increase in the apparent viscosity and time stability of the mixed protein + surfactant solutions and foams.
[16] [17] [18] [19] [20] [21] [22]
[23] [24] [25] [26] [27] [28]
Acknowledgment The authors are most grateful to Danisco (Denmark) for free CITREM, SSL, and PGE (080) samples.
[29] [30] [31]
References
[32] [33]
[1] E. Dickinson, J. Chem. Soc. Faraday Trans. 94 (1998) 1657. [2] E. Dickinson, Colloids Surf. B 20 (2001) 197. [3] J.M. Rodriguez Patino, M.R. Rodriguez Nino, in: E. Dickinson, J.M. Rodriguez Patino (Eds.), Food Emulsions and Foams: Interfaces, Interactions and Stability, Royal Society of Chemistry, Cambridge, UK, 1999, p. 246. [4] F.A. Husband, P.J. Wilde, J. Colloid Interface Sci. 205 (1998) 316. [5] L.E. Belyakova, M.G. Semenova, A.S. Antipova, Colloids Surf. B 12 (1999) 271. [6] A.S. Antipova, M.G. Semenova, L.E. Belyakova, Michael M. Il’in, Colloids Surf. B 21 (2001) 217. [7] M.G. Semenova, Curr. Opin. Colloid Interface Sci. 3 (1998) 627. [8] M. Bos, T. Nylander, T. Arnerbrant, D.C. Clark, in: G.L. Hasenheuttl, R.W. Hartel (Eds.), Food Emulsifiers and Their Applications, Chapman & Hall, New York, 1997, p. 95. [9] J.M. Rodriguez Patino, C. Carrera Sanchez, M.R. Rodriguez Nino, J. Agric. Food Chem. 47 (1999) 4998. [10] J. Krägel, R. Wüstneck, F. Husband, P.J. Wilde, A.V. Makievski, D.O. Grigoriev, J.B. Li, Colloids Surf. B 12 (1999) 399. [11] G. Garofalakis, B.S. Murray, Colloids Surf. B 12 (1999) 231. [12] J. Chen, E. Dickinson, J. Agric. Food Chem. 46 (1998) 91. [13] R. Miller, V.B. Fainerman, A.V. Makievski, J. Kragel, D.O. Grigoriev, V.N. Kazakov, O.V. Sinyachenko, Adv. Colloid Interface Sci. 86 (2000) 39. [14] J. Chen, E. Dickinson, in: K. Berger, R.J. Hamilton (Eds.), Emulsifiers: Functionality and Applications, Society of Chemical Industry, London, 1998, p. 19. [15] J. Chen, E. Dickinson, J. Dispers. Sci. Technol. 20 (1999) 197.
[34] [35] [36] [37] [38] [39] [40] [41] [42] [43] [44] [45] [46] [47] [48] [49] [50] [51]
E. Dickinson, C. Ritzoulis, J. Colloid Interface Sci. 224 (2000) 148. E. Dickinson, J. Dairy Sci. 80 (1997) 2607. C.H. McCrae, Int. Dairy J. 9 (1999) 227. S.R. Euston, S.R. Finnigan, R.L. Hirst, Food Hydrocolloids 15 (2001) 253. J.M. Rodriguez Patino, R. Rodriguez Nino, J.M. Alvarez Gomez, Food Hydrocolloids 11 (1997) 49. D.C. Clark, P.J. Wilde, D.R. Wilson, Colloids Surf. 59 (1991) 209. E. Dickinson, in: E.D. Goddard, K.P. Ananthapadmanabhan (Eds.), Interactions of Surfactants with Polymers and Proteins, CRC Press, Boca Raton, FL/Ann Arbor, MI/London/Tokyo, 1993, p. 295. N.J. Krog, in: S.E. Friberg, K. Larsson (Eds.), Food Emulsions, Dekker, New York, 1997, chap. 4, p. 141. E. Derbyshire, D.J. Wright, D. Boulter, Phytochemistry 15 (1976) 3. I.A. Vaintraub, in: V.L. Kretovich (Ed.), Vegetable Proteins and Their Biosynthesis, Nauka, Moscow, 1975, p. 142 (in Russian). P. Plietz, G. Damaschun, D. Zirwer, K. Gast, B. Schlesier, K.D. Schwenke, Kulturpflanzen 32 (1984) 159. M.C. Lawrence, T. Izard, M. Beuchat, R.J. Blagrove, P.M. Colman, J. Mol. Biol. 238 (1994) 748. G. Fauconneau, in: C.E. Bodwell, L. Petit (Eds.), Plant Proteins for Human Food, Martinus Mijhoff, The Hague, 1983, p. 1. H.D. Belitz, W. Grosch, Food Chemistry, Springer-Verlag, Berlin, 1987, chap. 16, p. 536. M.C. Garcia, M. Torre, M.L. Marina, F. Laborda, Crit. Rev. Food Sci. Nutr. 37 (4) (1997) 361. G. Muschiolok, H. Schmandke, Functional Properties of Fava Bean Products (Vicia faba): Nutrition, Biochemistry, Processing, ShakerVerlag, Aachen, 2000 (in German). V.B. Tolstoguzov, Food Hydrocolloids 4 (1991) 429. G.L. Gaines, Insoluble Monolayers at Liquid–Gas Interfaces, Interscience, New York, 1996. J.M. Evans, in: M.B. Huglin (Ed.), Light Scattering from Polymer Solutions, Academic Press, London, 1972, p. 89. C. Tanford, Physical Chemistry of Macromolecules, Wiley, New York, 1961. W. Burchard, Chimia 39 (1985) 10. W. Burchard, in: S.B. Ross-Murphy (Ed.), Physical Techniques for the Study of Food Biopolymers, Blackie, Glasgow, 1994, p. 151. P.L. Privalov, N.N. Khechinashvili, J. Mol. Biol. 86 (1974) 665. W.P. Jenks, Catalysis and Chemistry in Enzymology, McGraw–Hill, New York, 1969. D. Kelly, D.J. McClements, Food Hydrocolloids 17 (2003) 73. S. Paula, W. Süs, J. Tuchtenhagen, A. Blume, J. Phys. Chem. 99 (1995) 11742. M.N. Jones, A. Brass, in: E. Dickinson (Ed.), Food Polymers, Gels and Colloids, Royal Society of Chemistry, Cambridge, UK, 1991, p. 65. M.N. Jones, P. Manley, J. Chem. Soc. Faraday Trans. 76 (1980) 654. W.W. Sukow, H.E. Sandberg, E.A. Lewis, D.J. Eatough, L.D. Hansen, Biochemistry 19 (1980) 912. S. Magdassi, Ye. Vinetsky, P. Relkin, Colloids Surf. B Biointerfaces 6 (1996) 353. E. Dickinson, S.-T. Hong, J. Agric. Food Chem. 43 (1995) 2560. E. Dickinson, Y. Yamamoto, Food Hydrocolloids 10 (1996) 301. E. Dickinson, Y. Yamamoto, J. Food Sci. 61 (1996) 811. E. Dickinson, S.-T. Hong, Y. Yamamoto, Neth. Milk Dairy J. 50 (1996) 199. S. Ikeda, E.A. Foegeding, Food Hydrocolloids 13 (1999) 239. S. Ikeda, E.A. Foegeding, Food Hydrocolloids 13 (1999) 245.
Thermodynamic and functional properties of legumin (11S globulin from Vicia faba) in the presence of small-molecule surfactants: effect of temperature and pH Michael M. Il’in, Maria G. Semenova ∗ , Larisa E. Belyakova, Anna S. Antipova, Yurii N. Polikarpov Institute of Biochemical Physics of Russian Academy of Sciences, Vavilov str. 28, Moscow 119991, Russia Received 10 November 2003; accepted 24 May 2004 Available online 3 July 2004
Abstract We report on the effect of a set of water-dispersible small-molecule surfactants (the main and the longest-hydrocarbon components of which are a citric acid ester of monostearate, a sodium salt of stearol–lactoyl lactic acid, and a polyglycerol ester of stearic acid) on molecular, thermodynamic, and functional properties of the major storage protein of broad beans (Vicia faba) legumin in different molecular states (native, heated, and acid-denatured). The interaction between legumin and the surfactants has been characterized by a combination of thermodynamic methods, namely, mixing calorimetry and multiangle laser static and dynamic light scattering. It was found that hydrogen bonds, electrostatic interactions, and hydrophobic contacts provided a basis for the interactions between the surfactants and both the native and the denatured protein in aqueous medium. Intensive association of the protein molecules in a bulk aqueous medium in the presence of the surfactants was revealed by static and dynamic laser light scattering. In consequence of this, both the surface activity and the gel-forming ability of legumin increased markedly, which has been shown by tensiometry, estimation of protein foaming capacity, and steady-state viscometry. A likely molecular mechanism underlying the effects of small-molecule surfactants on legumin structure-forming properties at the interface and in a bulk aqueous medium is discussed. 2004 Elsevier Inc. All rights reserved. Keywords: Protein legumin; 11S globulin; Small-molecule surfactants; Molecular parameters; Thermodynamic parameters; Thermodynamics of interactions; Surface activity; Foaming capacity; Gelation
1. Introduction By now it is well known that the interactions between proteins and small-molecule surfactants (SMS) are of great practical consequence for stabilization of a wide variety of food colloids, because they provide a basis for structure formation at all structural levels of such systems, from molecules through the interfaces to the bulk complex aggregation of colloidal particles. For example, these interactions, which could be specific or nonspecific [1], contribute essentially, first, to morphology, composition, surface tension, and surface rheology of mixed adsorbed layers formed at * Corresponding author. Fax: +7-95-1355085.
E-mail address: [email protected] (M.G. Semenova). 0021-9797/$ – see front matter 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.jcis.2004.05.039
the interfaces of food colloids through crosslinking or protein displacement as a result of protein solubilization [2–13]; second, to the viscosity and structure of both protein and protein-stabilized emulsion gels by enhancing or weakening protein–protein crosslinks [2,14–16]; third, to long-term stability of both emulsions [17–20] and foams [21,22], caused mainly by strengthening or, on the contrary, by weakening of both the structure of the protein adsorbed layers and the interactions between colloidal particles. By this means, though the importance of the interactions between proteins and small-molecule surfactants for properties of food colloids is now accepted and well demonstrated, the molecular and thermodynamic basis underlying these properties is still incompletely understood. Because of this, in this study we have attempted to correlate the effects of SMS on molecular and thermodynamic
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Fig. 1. Chemical structure of the major components of the surfactants PGE, SSL, and CITREM [23].
properties of protein in the bulk with their effects on protein functional properties such as gel-forming ability, surface activity at the air–water interface, and foaming capacity. In addition, the roles of the structure of both protein and SMS in the effects studied has also been the focus of our attention. In pursuing these aims, we have studied the effect of a set of commercially important water-dispersible smallmolecule surfactants (the main and the longest-hydrocarbon components of which are esters, composed of the same hydrophobic part, stearic acid (C18), and different hydrophilic parts (Fig. 1) [23]: CITREM, a citric acid ester of monostearate, SSL, a sodium salt of stearol–lactoyl lactic acid, and PGE(080), polyglycerol ester of stearic acid) on molecular and functional properties of the major storage protein of broad beans (Vicia faba), legumin, in different molecular states (native, heated, and acid-denatured). We have chosen 11S globulin for our study, because the basic molecular and thermodynamic properties of this protein are well known [24–27] and reproducible and, in addition, this protein is homologous in physicochemical properties and biological functions to 11S globulins of the other leguminous plants (soy, pea, etc.) and could be a useful model to predict their behavior in the presence of SMS. Moreover, from the practical point of view both legumin [28–31] and SMS [23] are promising additives for a new food formulation.
2. Experimental 2.1. Materials Legumin (11S globulin) was isolated from broad beans (var. “Agat”) by a method that was described previously [32]. Homogeneity of the isolated 11S globulin was assessed by sedimentation velocity analysis in phosphate buffer at pH 8.0, ionic strength 0.05 mol dm−3 . It was found to be a single peak of 11S globulin with a sedimentation coefficient of about 12S (Fig. 2). Small-molecule surfactants, CITREM, SSL, and PGE (080) [23], were supplied by
Fig. 2. Result of sedimentation velocity analysis of legumin (1% wt/v) in phosphate buffer at pH 8.0 and ionic strength 0.05 mol dm−3 .
Danisco Cultor (Denmark). Phosphate buffer (pH 7.2, ionic strength 0.05 mol dm−3 ) and citrate buffer (pH 3.0, ionic strength 0.01 mol dm−3 ) were prepared using analytical grade reagents (99.9% pure): Na2 HPO4 , NaH2 PO4 , sodium salt of citric acid, and citric acid, respectively. Sodium azide (0.01 wt%) was added to the buffers as an antimicrobial agent. All solutions were prepared using double-distilled water. 2.2. Methods 2.2.1. Protein solution preparation Protein solutions with required concentrations were made in the appropriate buffer. Centrifugation (4000 rpm (3734g), 30 min, 20 ◦ C) of the protein solutions was common for removing of a small part of insoluble protein. Concentration
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Table 1 Thermodynamic parameters of legumin denaturation process: the specific denaturation enthalpy H d , the difference between the specific heat capacities of the native and denatured forms of the protein d C p , and the denaturation temperature T d System Native legumin Acid-denatured legumin Heat-denatured legumin
H d d C p Td (K) (J g−1 ) (J g−1 grad−1 ) 31 0.4 357 5 0.2 329 Absence of any heat effects on the thermogram
of the protein in solutions was checked after centrifugation using a Shimadzu (Japan) refractometer, on the known values of protein refractive index increment ground (see Section 2.2.7). 2.2.2. Preparation of thermo-denatured legumin The protein solutions with required concentrations were made in a 0.05 mol dm−3 phosphate buffer (pH 7.2) as described above (Section 2.2.1). Aliquots (25 ml) of the protein solutions were placed in glass vials and soldered. The vials were heat-treated at 90 ◦ C for 30 min in a water bath and after cooling were allowed to equilibrate for 20 h at room temperature (22 ± 2). The level of protein denaturation as a result of the heat treatment was checked by differential scanning calorimetry and was indicative of a partial loss of the native conformation of the protein; i.e., no change of the heat capacity of heat-treated protein solutions with increasing temperature was found in the thermogram (Table 1). 2.2.3. Preparation of acid-denatured legumin The acid-denatured protein solutions with required concentrations were made in a citric buffer (pH 3.0, ionic strength 0.01 mol dm−3 ) as described above (Section 2.2.1). The level of protein denaturation at pH 3.0 was estimated by differential scanning calorimetry and was suggestive of practical loss of the native protein conformation (Table 1). 2.2.4. Determination of the critical micelle concentration for the small-molecule surfactants The thresholds of concentrations (cmc) of the smallmolecule surfactants at which their micellization begins in aqueous medium were determined by a combination of several thermodynamic methods: (i) by tensiometry using a Krüss GmbH K-10 digital tensiometer (Hamburg, Germany)—indicated by the sharp break at the cmc in the plot of surface tension against the logarithm of concentration of the small-molecule surfactants that is shown in Fig. 3a, as an example; (ii) by static light scattering using an LS-01 apparatus (VA Instruments, St. Petersburg, Russia)—indicated by the sharp increase in the intensity of light scattering from a surfactant solution with increasing surfactant concentration that is illustrated in Fig. 3b, as an example; (iii) by mixing calorimetry using an LKB 2277 flow calorimeter— indicated both by the inflection point of the concentration dependence of the enthalpy of the dilution of a surfactant
73
solution by buffer normalized to the surfactant concentration as shown in Fig. 3c, as an example, and by the minimum of the first derivative of this curve with respect to surfactant concentration (Fig. 3c ). Relying on such experiments, it was found that the cmc practically does not vary with temperature or pH. The pH- and temperature-averaged cmc interval is the following for each surfactant: PGE(080) 0.75–1.0 mg dm−3 ; SSL 2.5–3.5 mg dm−3 ; CITREM 12.5– 20 mg dm−3 . As this takes place, the cmc intervals found are in the series in conformity with a change in the ionic nature of the surfactants: PGE < SSL < CITREM (see Fig. 1, where the chemical structure of the SMS is shown more precisely). 2.2.5. Addition of the small-molecule surfactants into the protein solutions Stock solutions of the small-molecule surfactants (102 mg dm−3 ) were prepared by ultrasound sonication over 1 h at a frequency of 4.5 kHz while the solutions were shaken at 65 ◦ C (CPLAN water bath shaker, Type 357, Poland). As a result fine homogeneous dispersions of the small-molecule surfactants in aqueous medium were formed. Thereafter the stock solutions, cooled to room temperature, were used to prepare mixed solutions with the proteins at the required concentrations, whereupon the mixed solutions were shaken at 40 ◦ C for 1 h (CPLAN water bath shaker, Type 357, Poland) and then were allowed to cool to room temperature. 2.2.6. Determination of the surface tension, γ , at the planar air–water interface Values of the surface tension, γ , of the solutions of the small-molecule surfactants, the proteins, and their mixtures were monitored with an accuracy of 1 mN m−1 with the Wilhelmy plate technique [33]. All measurements were made in the thermostatic cell at 25 ± 0.5 ◦ C. The value of the surface tension between double-distilled water and air did not differ from the standard value within the range of experimental error and was 72 mN m−1 at 25 ◦ C independent of time. A platinum plate was used in measuring the surface tension. The surface tension was monitored continuously as a function of time. The values of γ presented in this work are averaged data for at least two repetitions of each of the experiments. 2.2.7. Estimation of protein molecular and interaction parameters in bulk aqueous medium The weight-average molecular weight, M w , radius of gyration, R G , and second virial coefficient, Apr−pr , of legumin alone and in the presence of small-molecule surfactants were determined by static light scattering in dilute aqueous solution (1 × 10−2 –1 × 10−3 g ml−1 , with 5–8 protein concentration points at most). The Rayleigh ratio Rθ was measured using vertically polarized light (633 nm) at angles in the range 40◦ θ 140◦ (13 angles) using a VA Instruments LS-01 apparatus (St. Petersburg, Russia) cali-
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(a)
(b)
(c)
(c )
Fig. 3. Data of cmc determination at pH 7.2, ionic strength 0.05 M for surfactants studied from (a) tensiometry; (b) light scattering: I90 /I0 is the intensity of light scattering normalized to the intensity of the incident light, (") 283, (2) 293, (P) 303, (Q) 313, (!) 323 K; (c, c ) mixing calorimetry.
brated with dust-free benzene (R90 = 11.84 × 10−6 cm−1 ). Solutions were filtered directly into the light-scattering cell through a Millipore membrane with pore size 0.65 µm. The raw data were used to plot the angle and concentration dependencies of the ratio H C/Rθ according to the Zimm method [34]. Here, C is the protein concentration (g ml−1 ), Rθ is the excess light scattering over that of the solvent at angle θ , and H is an instrumental optical constant equal to 4π 2 n2 ν 2 /N A λ4 , where N A is Avogadro’s number, λ is the wavelength of incident light in vacuo, n is the refractive index of the solvent, and ν is the refractive index increment of the protein. Values of the weight-average molecular weight, M w , were estimated as averages from the intercepts of both the concentration dependence of H C/Rθ as θ → 0 (the extrapolation was performed on 13 angles) and the angular dependence of H C/Rθ as C → 0 (the extrapolation was performed on 5–8 concentrations). Values of the radius of gyration, R G , were estimated from the slope of the angular dependence of H C/Rθ as C → 0. Values of the second virial coefficient, Apr−pr , were estimated from the slope of the concentration dependence of H C/Rθ as θ → 0. The
second virial coefficient characterizes primarily the thermodynamic affinity of protein molecules for solvent (aqueous medium in our case) (it is poor if Apr−pr < 0, or, by contrast, it is good if Apr−pr > 0 and it is ideal if Apr−pr = 0) [35], i.e., providing circumstantial evidence for protein surface hydrophobicity/hydrophilicity. The values of M w , Apr−pr , and R G presented in this work are averaged data for at least two repetitions of each experiment. The experimental error in the determinations of M w and Apr−pr was estimated as ±10%. The error in the R G determination was ±5%. Values of the refractive index increment for legumin at different concentrations of SMS were unchanged within experimental error (±10%) for native, heat-denatured, and acid-denatured legumin: ν = 0.2 × 10−3 , 0.18 × 10−3 , 0.18 × 10−3 m3 kg−1 , respectively. Values of the hydrodynamic radius R h of legumin alone and in the presence of small-molecule surfactants were estimated in aqueous solution (5 × 10−3 g ml−1 ) by dynamic light scattering [36,37]. The time correlation function of the scattering intensity was measured at 90◦ with vertically po-
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larized light (633 nm) using a VA Instruments LS-01 apparatus (St. Petersburg, Russia). Solutions were filtered directly into the light-scattering cell through a Millipore membrane as described above. To determine the hydrodynamic radius from the time correlation function, a special program was used (DYNALS Release 1.5, rights reserved by A. Golding and N. Sidorenko). 2.2.8. Differential scanning calorimetry Calorimetric measurements were made using a DASM4M differential adiabatic scanning microcalorimeter (Special Design Office of Biological Instrument Making, Russian Academy of Sciences) in the temperature range from 20 to 110 ◦ C at a scanning rate of 2 ◦ C min−1 and an excess pressure of 2.5 atm. The concentration of protein samples was equal to 0.005 g ml−1 . The accuracy of the measurements is about 10% of the values of the heat capacities. The sensitivity of the calorimetric measurement is no less than 5 × 10−6 J s−1 . The thermodynamic parameters of protein denaturation were calculated as proposed before [38]. The values of the thermodynamic parameters of protein denaturation presented in this work are averaged data for at least two repetitions of each of the experiments. 2.2.9. Mixing calorimetry Calorimetric measurements were made using an LKB 2277 flow calorimeter set at 22 ◦ C. A peristaltic pump pumped the reactants into the instrument. The pump was calibrated by measuring the time required to pump a known volume of solution into the calorimeter. The flow rate was equal to 9 × 10−6 l s−1 . The ratio of the flow rates in the two channels was close to one; i.e., the solutions of proteins or small-molecule surfactants in all cases were diluted by a factor of 2 under mixing. Both solutions were thermally equilibrated before entering the reaction vessel. The calibration of the calorimeter itself was done electrically at the temperature of measurement. The sensitivity of the calorimetric measurement is no less than 3 × 10−6 J s−1 . Thermal effects were observed during dilution of (i) the protein solution by the pure buffer, Qprotein–buffer; (ii) the solution of small-molecule surfactant by the pure buffer, Qsmall-molecule surfactant–buffer; (iii) the protein solution by the solution of small-molecule surfactant, Q . These thermal effects Q were measured in thermal power units (J s−1 ). The specific enthalpy of the interaction between protein and small-molecule surfactant was obtained from the relationship H protein–small-molecule surfactant = −(Q − Qprotein–buffer − Qsmall-molecule surfactant–buffer)/n,
(1)
where n is the number of grams of protein mixed with small-molecule surfactant per second (g s−1 ).
75
2.2.10. Rheological measurements Steady state shear viscosities were measured at 22 ◦ C as a function of shear stress using Rheotest 2 (Type RV2, Germany). The sample was contained in a concentric cylindrical cell (the ratio of inner diameter, r, to outer diameter is equal to 0.98, sample volume 10 ml). The data are presented as the ratio, µ, of the limiting-zero-shear-rate viscosity of the mixed (protein + surfactant) solution to that of the pure protein solution. The values of µ shown are averaged data for at least two repetitions of each of the experiments. 2.2.11. Estimation of protein foam ability Foam (25 ml) had been generated in an aeration column using a bubbling method. The velocity of air supply through a glass membrane (1 µm) was equal to 1.8 ml s−1 . In these experiments, both the volume of the foaming solutions and the protein concentration were maintained constant: 5 ml and 7 wt/vol%, respectively. (For details of preparation of protein solutions see Sections 2.2.1, 2.2.2, and 2.2.3.) The static stability of the foam (25 ml) was determined from the volume of liquid drained from the foam over time, Vd . The values of Vd presented in this work are averaged data for at least two repetitions of each of the experiments.
3. Results and discussion 3.1. Enthalpy of the interactions between legumin and small-molecule surfactants from mixing calorimetry Data from mixing calorimetry (Figs. 4a, 4b, 4c) show the distinct heat effects of the interaction between legumin and surfactants (corrected for surfactant and protein dilution by buffer in accordance with Eq. (1)), which could be dictated by a great many different physicochemical processes [22,38–45]: (i) the exothermic ones: — direct hydrogen bonding between polar groups of the protein and surfactant [39]; — direct electrostatic interactions between ionic head groups of surfactants and oppositely charged groups on the protein surface through electrostatic attraction [39]; — partial protein folding due to new hydrogen or electrostatic bonding in the protein interior; (ii) the endothermic ones: — direct hydrophobic interactions between nonpolar parts of the protein and surfactant hydrocarbon tail [39]; — partial protein unfolding [38]; — hydrophobically driven protein association in aqueous medium; (iii) either endothermic or exothermic: — changes in interactions with water;
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(a)
(b)
(c)
Fig. 4. Specific enthalpy, H , of the interaction between legumin (0.5% wt/v) and SMS in aqueous medium versus the small-molecule surfactant concentration in the mixed solutions: (a) CITREM, (b) PGE (080) and (c) SSL. (2) Acid-denatured protein (0.01 mol dm−3 , citrate buffer, pH 3.0); (P) native protein (0.05 mol dm−3 , phosphate buffer, pH 7.2); (F) heat-denatured protein (0.05 mol dm−3 , phosphate buffer, pH 7.2) at 298 K.
— changes in interactions with counterions; association/dissociation of counterions; — micelle-like cluster formation on the protein surface at 293 K, especially at surfactant concentrations above their cmc [40,41]. It is not possible discriminate the contributions from each of these processes to the heat effects found owing to their compensatory nature. Nevertheless, it seems possible to give some qualitative description of the principal distinctions in the molecular origin of the observed enthalpy changes based on the major structural features of both SMS (Fig. 1) and molecular state of the protein in aqueous medium (Table 1). Thus, for example, under the experimental conditions (pH 7.2 and 3.0, ionic strength 0.05 M) the surfactants that are susceptible to hydrogen bonding with protein, owing to their specific molecular structure (Fig. 1), such as anionic CITREM or nonionic PGE(080), can show an exothermic character in their interaction with protein (Fig. 4a, b). That is more typical of PGE (080), i.e., for the nonionic surfactant with a rather high number of hydroxyl groups in the molecule (Fig. 1). In contrast, SSL, having the smallest number of polar groups among the surfactants studied (Fig. 1), shows a predominantly endothermic character of the interaction with the protein (Fig. 4c) that suggests its hydrophobic nature [39], which is most likely determined by the interaction between the hydrocarbon tail in the SSL molecule and the hydrophobic parts of the protein molecules. The pronounced enhancement of the latter thermal effect revealed above the critical micelle concentration for SSL is likely attributable to a marked increase in the number of hydrophobic contacts, as a
Fig. 5. Specific enthalpy, H , of the interaction between legumin (0.5% wt/v) and SMS in aqueous medium at a concentration of small-molecule surfactants equal to 6 mg dm−3 at 298 K.
result of the addition to the protein of micelles of SSL, combining within a great many of the surfactant molecules. In turn, the role of the different molecular state of the protein in the interaction with SMS is more pronounced in the distinct heat effects observed for the same SMS at the same concentration (Fig. 5). For example, when heat-denatured protein compared to the native protein, there is obviously more accessibility of the reacting groups or parts of the protein molecules as a result of heat denaturation followed by partial protein un-
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77
Table 2 Effect of small-molecule surfactants (SMS) on the molecular parameter (M w , weight-average molecular weight) and thermodynamics of pair interactions (second virial coefficient Apr–pr ) of legumin in aqueous medium System
M w (kDa) Native legumin Heat-denatured legumin Acid-denatured legumin
With 6 mg dm−3 CITREM
Without SMS
330 6300 43000
104
104
With 1 mg dm−3 SSL
Apr–pr (m3 mol kg−2 ) 0
M w (kDa)
−2.1
19200
0.14
55000
18250
0.67
12000
1.27
375
Apr–pr (m3 mol kg−2 ) −2.45
folding. That is bound to be favorable both to hydrogen bonding, along with electrostatic interactions between opposite charges of the interacting molecules, as is seemingly found in the cases of CITREM and PGE(080), and to hydrophobic interaction, as for SSL. In contrast, protonation of the protein molecules at pH 3.0 (the acid-denatured state of legumin), reducing hydrogen bonding, leads to predominantly hydrophobic interactions between legumin and all studied SMS (Fig. 5). In addition, it is interesting to note that the expected highest total hydrophobicity of the soluble aggregates of heat-denatured legumin, composed of the most hydrophobic basic constituent chains [24], agrees closely with the most endothermic effect of the interaction between legumin and SMS, measured just in this case (Fig. 5). As this takes place, the highest hydrophobicity of the heat-denatured state of the protein is substantiated by both the a greatest extent of the partial protein unfolding observed from differential scanning calorimetry (Table 1) and the negative value of the second virial coefficient (Table 2), suggestive that water is a poor solvent [35] for these protein aggregates. Overall, the mixing calorimetry data indicate the occurrence of different types of interactions between smallmolecule surfactants and legumin in aqueous medium, the dominant character of which seems determined, on the one hand, by the total hydrophobicity and the accessibility of the protein functional groups for SMS, and on the other, by the total hydrophobicity and the availability of specific functional groups in surfactant molecules, themselves. Also, an additional contribution to the measured enthalpies from both protein association and protein unfolding as a result of the interaction of protein with SMS is also expected from our previous work [5,6]. 3.2. Molecular and interaction parameters of the legumin–surfactant complexes First and foremost, Tables 2 and 3 show a marked increase both in the weight-average molecular weight (M w ) and in the size (R g , R h ) of legumin in the presence of SMS, except when CITREM or SSL is added to the acid-denatured protein at studied concentrations. This result suggests an association of the protein in the aqueous medium, as if the
M w (kDa) 690
With 0.5 mg dm−3 PGE(080)
104
2600
Apr–pr 104 (m3 mol kg−2 ) 2.2
3.28
8800
−0.85
0.62
560000
1.42
Apr–pr (m3 mol kg−2 ) −0.07
M w (kDa)
Table 3 Effect of small-molecule surfactants (SMS) on the size and conformational parameter ρ of legumin in aqueous medium System
Without SMS With 6 mg dm−3 CITREM With 1 mg dm−3 SSL With 0.5 mg dm−3 PGE(080)
Heat-denatured legumin
Acid-denatured legumin
RG (nm)
Rh (nm)
ρ= R G /R h
RG (nm)
Rh (nm)
ρ= R G /R h
40 108
58 65
0.7 ≈ 1 1.7
511 325
115 133
4.4 2.4
273
87
3.1
196
132
1.5
119
71
1.7
2100
133
15.8
SMS molecules play the role of components bridging molecules of the protein together. It is interesting to note that the maximal extent of the protein association in the complexes with SMS was found to occur for the nonionic surfactant PGE(080) (Tables 2 and 3). The calculation of the molecular weight of hypothetical protein–surfactant complexes, which rests on the assumption that all amounts of added surfactants were bound equally by the protein in solution, provides evidence for this suggestion by virtue of the fact that the calculated values are several times or even orders of magnitude smaller than those found by light scattering. But it is clear that further determination of the exact composition of protein–surfactant complexes is required. In turn, the surface of the formed protein—SMS associates could become more hydrophobic or alternatively more hydrophilic (Table 2). This is mirrored in the change in the sign of the second virial coefficient [35]. Thus, the negative value of the second virial coefficient shows a decrease in the thermodynamic affinity of the associates for aqueous medium, which could be caused by some rise in their surface hydrophobicity and mutual attraction. The opposite is true for the positive value of the second virial coefficient. The changes found in the properties of the protein surface in the presence of SMS (Table 2) are most likely dictated by the ultimate spatial arrangement of the hydrophobic and hydrophilic parts of the protein and SMS molecules in response to their interaction. What this means is either that most of the hydrophobic parts of both interacting molecules are exposed to aqueous medium (Apr–pr < 0) or, on the contrary, that they
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(a)
(b)
(c)
Fig. 6. Effect of small-molecule surfactants on the surface behavior of native (2) and heat-denatured (") legumin (10 mg dm−3 ) at the planar air–water interface (0.05 M phosphate buffer, pH 7.2, 298 K). Interfacial tension, γ , is plotted as a function of concentration of SMS: (a) CITREM, (b) SSL, (c) PGE (080). The solid lines describe surface behavior of the SMS alone; the dashed lines indicate the critical concentration of micelle formation (cmc) for each case.
are hidden in the interior of the primary protein associations, whereas the hydrophilic ones are directed into the aqueous medium (Apr–pr > 0). Also an additional structural analysis of static and dynamic light-scattering data shows that the ρ parameter, which is highly structure-sensitive [37], changes its value in the presence of SMS (Table 3), suggesting different shapes of the formed associates: (i) spherelike (0.9 < ρ < 1); (ii) randomly coiled/randomly branched (1.5 < ρ < 2); (iii) with more rigid and open architecture (as encountered with rigid polydisperse rods when ρ 2). We would like to note here that the information on the effect of SMS on protein self-association in aqueous medium is very scarce in the literature [40,45], which in our opinion, hinders deeper insight into the molecular mechanisms of the effect of SMS on well-studied structural–functional properties of proteins (mentioned above in the Introduction). Most likely, more systematic studies on this subject are required in order to get this information. 3.3. Functional properties of the legumin–surfactant complexes: surface activity, foaming, and gel-forming abilities The modification of the molecular and thermodynamic properties of legumin in bulk aqueous medium, as a result of interaction with SMS, leads to a marked change in the functional properties of legumin.
A pronounced increase in the legumin surface activity was found for both denatured and native legumin in the presence of SMS (Figs. 6a, 6b, 6c, and 7). As this takes place, the marked distinctions between values of the surface tension of the protein–surfactant mixture and pure components suggest the formation of a mixed protein–surfactant adsorbed layer at the air–water interface. In this case, the determining role of complex formation between protein and surfactants on the surface behavior of their mixed solutions appears as the following: (i) In the values of the surface tension that are lower than those for the more surface-active component. That is more pronounced in the case of the CITREM + legumin mixture practically over the entire range of the concentrations of CITREM studied. (It should be noted here that the concentration of legumin was constant and equal to 10 mg dm−3 for all the mixed systems studied.) (ii) In the much lower values of the surface tension for the mixed systems in comparison with the values that are characteristic of the protein (10 mg dm−3 ) when it is in weight excess in relation to the surfactants. This result was found for the cases of the mixtures of both SSL and PGE with legumin. Based upon light-scattering data (Tables 2 and 3), it may be inferred that this result is primarily governed either by the higher extent of protein association in the presence of SMS, which may be beneficial for exposing more surfaceactive molecules at the interface, or by the deterioration of the thermodynamic affinity of the aqueous medium for the
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Table 4 Effect of small-molecule surfactants on the rate V d (g−1 min) of liquid drainage from foams stabilized by legumin at 7 wt/v% (25 ◦ C; duration of measurement is 5 min just after foam preparation) V d (g−1 min )
System Native legumin Heat-denatured legumin Acid-denatured legumin
Without SMS
With 28 mg dm−3 CITREM
With 28 mg dm−3 SSL
With 28 mg dm−3 PGE(080)
0.51 1.0 0.62
0.1 0.56 0.57
0.63 0.71 0.25
0.64 0 0.45
Table 5 Effect of small-molecule surfactants on the fractional increase µ in the limiting-zero-shear-rate viscosity of solutions of heat-denatured legumin at 7 wt/v% (0.05 M phosphate buffer, pH 7.2, 25 ◦ C) With CITREM
With SSL
With PGE(080)
C sms (mg dm−3 )
µ = ηmixture /ηprotein
C sms (mg dm−3 )
µ = ηmixture /ηprotein
C sms (mg dm−3 )
µ = ηmixture /ηprotein
14 28 42
1.3 2.5 1.1
0.88 3.5 7 9.7 14
3.8 2.4 2.5 2 2.1
3.5 5.3 7 9 21
122 107 24 42 37
Fig. 7. Effect of small-molecule surfactants on the surface behavior of legumin (10 mg dm−3 ) in different molecular states at the planar air–water interface in the presence of SMS at 298 K: CITREM (6 mg dm−3 ); PGE (080) (0.5 mg dm−3 ); SSL (2 mg dm−3 ).
protein + SMS associates (less positive or negative values of the second virial coefficients). Owing to the marked increase in the surface activity of the denatured legumin in the presence of SMS, a clearly defined increase in the stability of the protein foams with time was found, as presented in Table 4. In addition, rheological measurements, shown in Table 5, provide evidence for a large increase in the limiting zeroshear-rate viscosity, η, of solutions of heat-denatured legumin in the presence of the surfactants as compared with the η of pure protein solution, shown by the ratio µ =
ηmixture/ηprotein . This increase is by several times for CITREM and SSL and even by two orders of magnitude in the presence of PGE(080). The last effect agrees well with both the nonionic nature of PGE(080) and the large number of hydroxyl groups in its molecule (Fig. 1), by virtue of the fact that both hydrophobic contacts and hydrogen bonding could contribute to protein network formation in the bulk. On the strength of light-scattering data (Tables 2 and 3), it is believed that both a marked intensification of the protein association in the presence of SMS and a deterioration of the thermodynamic affinity of the aqueous medium for the protein + SMS associates could form the basis of their effect on the rheological properties of the protein solutions. Moreover, it most likely that the observed increase in the apparent viscosity of the solution of heat-denatured legumin in the presence of SMS is the origin, to some extent, of the increase in the time stability of the protein foams revealed. Similar effects of strengthening of protein gels as well as protein emulsion gels were found for a large number of protein–surfactant systems at concentrations of SMS where SMS does not compete for binding sites in protein molecules and does not displace protein from the surface of colloidal particles, but vice versa can enhance protein–protein crosslinks [16,19,46–51].
4. Conclusions The mixing calorimetry data indicate the occurrence of different types of interactions between small-molecule surfactants and legumin in aqueous medium, the dominant character of which seems to be determined, on the one hand, by the total hydrophobicity and the accessibility of the protein functional groups for SMS, and on the other, by the to-
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tal hydrophobicity and the availability of specific functional groups in surfactant molecules themselves. Self-association of the protein molecules in aqueous medium in the presence of surfactants is revealed by static and dynamic laser light scattering, as if the surfactant molecules play the role of the components bridging molecules of the protein together. Both the rather high level of protein association in the protein + surfactant complexes, which is most likely favorable for exposing a large number of surface-active molecules at the interface, and their lower thermodynamic affinity for aqueous medium seem to govern the observed increase in the surface activity of legumin in the presence of surfactants. It is believed that a marked intensification of protein association in the presence of surfactants also forms the basis of a pronounced increase in the apparent viscosity and time stability of the mixed protein + surfactant solutions and foams.
[16] [17] [18] [19] [20] [21] [22]
[23] [24] [25] [26] [27] [28]
Acknowledgment The authors are most grateful to Danisco (Denmark) for free CITREM, SSL, and PGE (080) samples.
[29] [30] [31]
References
[32] [33]
[1] E. Dickinson, J. Chem. Soc. Faraday Trans. 94 (1998) 1657. [2] E. Dickinson, Colloids Surf. B 20 (2001) 197. [3] J.M. Rodriguez Patino, M.R. Rodriguez Nino, in: E. Dickinson, J.M. Rodriguez Patino (Eds.), Food Emulsions and Foams: Interfaces, Interactions and Stability, Royal Society of Chemistry, Cambridge, UK, 1999, p. 246. [4] F.A. Husband, P.J. Wilde, J. Colloid Interface Sci. 205 (1998) 316. [5] L.E. Belyakova, M.G. Semenova, A.S. Antipova, Colloids Surf. B 12 (1999) 271. [6] A.S. Antipova, M.G. Semenova, L.E. Belyakova, Michael M. Il’in, Colloids Surf. B 21 (2001) 217. [7] M.G. Semenova, Curr. Opin. Colloid Interface Sci. 3 (1998) 627. [8] M. Bos, T. Nylander, T. Arnerbrant, D.C. Clark, in: G.L. Hasenheuttl, R.W. Hartel (Eds.), Food Emulsifiers and Their Applications, Chapman & Hall, New York, 1997, p. 95. [9] J.M. Rodriguez Patino, C. Carrera Sanchez, M.R. Rodriguez Nino, J. Agric. Food Chem. 47 (1999) 4998. [10] J. Krägel, R. Wüstneck, F. Husband, P.J. Wilde, A.V. Makievski, D.O. Grigoriev, J.B. Li, Colloids Surf. B 12 (1999) 399. [11] G. Garofalakis, B.S. Murray, Colloids Surf. B 12 (1999) 231. [12] J. Chen, E. Dickinson, J. Agric. Food Chem. 46 (1998) 91. [13] R. Miller, V.B. Fainerman, A.V. Makievski, J. Kragel, D.O. Grigoriev, V.N. Kazakov, O.V. Sinyachenko, Adv. Colloid Interface Sci. 86 (2000) 39. [14] J. Chen, E. Dickinson, in: K. Berger, R.J. Hamilton (Eds.), Emulsifiers: Functionality and Applications, Society of Chemical Industry, London, 1998, p. 19. [15] J. Chen, E. Dickinson, J. Dispers. Sci. Technol. 20 (1999) 197.
[34] [35] [36] [37] [38] [39] [40] [41] [42] [43] [44] [45] [46] [47] [48] [49] [50] [51]
E. Dickinson, C. Ritzoulis, J. Colloid Interface Sci. 224 (2000) 148. E. Dickinson, J. Dairy Sci. 80 (1997) 2607. C.H. McCrae, Int. Dairy J. 9 (1999) 227. S.R. Euston, S.R. Finnigan, R.L. Hirst, Food Hydrocolloids 15 (2001) 253. J.M. Rodriguez Patino, R. Rodriguez Nino, J.M. Alvarez Gomez, Food Hydrocolloids 11 (1997) 49. D.C. Clark, P.J. Wilde, D.R. Wilson, Colloids Surf. 59 (1991) 209. E. Dickinson, in: E.D. Goddard, K.P. Ananthapadmanabhan (Eds.), Interactions of Surfactants with Polymers and Proteins, CRC Press, Boca Raton, FL/Ann Arbor, MI/London/Tokyo, 1993, p. 295. N.J. Krog, in: S.E. Friberg, K. Larsson (Eds.), Food Emulsions, Dekker, New York, 1997, chap. 4, p. 141. E. Derbyshire, D.J. Wright, D. Boulter, Phytochemistry 15 (1976) 3. I.A. Vaintraub, in: V.L. Kretovich (Ed.), Vegetable Proteins and Their Biosynthesis, Nauka, Moscow, 1975, p. 142 (in Russian). P. Plietz, G. Damaschun, D. Zirwer, K. Gast, B. Schlesier, K.D. Schwenke, Kulturpflanzen 32 (1984) 159. M.C. Lawrence, T. Izard, M. Beuchat, R.J. Blagrove, P.M. Colman, J. Mol. Biol. 238 (1994) 748. G. Fauconneau, in: C.E. Bodwell, L. Petit (Eds.), Plant Proteins for Human Food, Martinus Mijhoff, The Hague, 1983, p. 1. H.D. Belitz, W. Grosch, Food Chemistry, Springer-Verlag, Berlin, 1987, chap. 16, p. 536. M.C. Garcia, M. Torre, M.L. Marina, F. Laborda, Crit. Rev. Food Sci. Nutr. 37 (4) (1997) 361. G. Muschiolok, H. Schmandke, Functional Properties of Fava Bean Products (Vicia faba): Nutrition, Biochemistry, Processing, ShakerVerlag, Aachen, 2000 (in German). V.B. Tolstoguzov, Food Hydrocolloids 4 (1991) 429. G.L. Gaines, Insoluble Monolayers at Liquid–Gas Interfaces, Interscience, New York, 1996. J.M. Evans, in: M.B. Huglin (Ed.), Light Scattering from Polymer Solutions, Academic Press, London, 1972, p. 89. C. Tanford, Physical Chemistry of Macromolecules, Wiley, New York, 1961. W. Burchard, Chimia 39 (1985) 10. W. Burchard, in: S.B. Ross-Murphy (Ed.), Physical Techniques for the Study of Food Biopolymers, Blackie, Glasgow, 1994, p. 151. P.L. Privalov, N.N. Khechinashvili, J. Mol. Biol. 86 (1974) 665. W.P. Jenks, Catalysis and Chemistry in Enzymology, McGraw–Hill, New York, 1969. D. Kelly, D.J. McClements, Food Hydrocolloids 17 (2003) 73. S. Paula, W. Süs, J. Tuchtenhagen, A. Blume, J. Phys. Chem. 99 (1995) 11742. M.N. Jones, A. Brass, in: E. Dickinson (Ed.), Food Polymers, Gels and Colloids, Royal Society of Chemistry, Cambridge, UK, 1991, p. 65. M.N. Jones, P. Manley, J. Chem. Soc. Faraday Trans. 76 (1980) 654. W.W. Sukow, H.E. Sandberg, E.A. Lewis, D.J. Eatough, L.D. Hansen, Biochemistry 19 (1980) 912. S. Magdassi, Ye. Vinetsky, P. Relkin, Colloids Surf. B Biointerfaces 6 (1996) 353. E. Dickinson, S.-T. Hong, J. Agric. Food Chem. 43 (1995) 2560. E. Dickinson, Y. Yamamoto, Food Hydrocolloids 10 (1996) 301. E. Dickinson, Y. Yamamoto, J. Food Sci. 61 (1996) 811. E. Dickinson, S.-T. Hong, Y. Yamamoto, Neth. Milk Dairy J. 50 (1996) 199. S. Ikeda, E.A. Foegeding, Food Hydrocolloids 13 (1999) 239. S. Ikeda, E.A. Foegeding, Food Hydrocolloids 13 (1999) 245.