Interfacial characteristics of food emulsifiers (proteins and lipids) at the air-water interface

Colloids and Surfaces B: Biointerfaces 15 (1999) 235 – 252 www.elsevier.nl/locate/colsurfb

Interfacial characteristics of food emulsifiers (proteins and lipids) at the air-water interface Juan M. Rodrı´guez Patino *, M. Rosario Rodrı´guez Nin˜o Departamento de Ingenierı´a Quı´mica, Facultad de Quı´mica, Uni6ersidad de Se6illa, C/. Professor Garcı´a Gonza´lez s/n, 41012 Se6ille, Spain

Abstract Factors affecting the interfacial characteristics (structure, stability, interfacial rheology, molecular diffusion, and rate of film formation) of food emulsifiers (polar lipids and proteins) at the air-aqueous phase interface are reviewed. The effect of interfacial and aqueous phase (water, and aqueous solutions of ethanol, glycerol, sugars, electrolytes, and pH) compositions have been analyzed as variables. Many measurement methods — such as tensiometry (Wilhelmy plate and pendant drop methods), and Langmuir- and Wilhelmy-type film balances — have been used in the experiments. The effect of the interfacial, aqueous phase composition, and operational conditions (surface density, surface pressure, and temperature) of food emulsifiers (lipids and proteins) at the air-aqueous phase interface are discussed. © 1999 Elsevier Science B.V. All rights reserved. Keywords: Protein; Polar lipid; Monolayer; Interfacial properties; Air-water interface

Abbre6iations: A, molecular area at time u (nm2 molecule − 1); A0, molecular area at the initial moment (nm2 molecule − 1); AE, molecular area at the beginning of the LE to LC transition (nm2 molecule − 1); AC, molecular area at the end of then LE to LC transition (nm2 molecule − 1); A1, coefficient for the rate of dissolution in Eq. 6 (min − 0.5); A2, coefficient for the rate of diffusion in Eq. 7 (min − 1); DA, molecular area require for the molecule to adsorb at the interface (nm2); B1, coefficient for the rate of nuclei formation in Eq. 8 (min − 1); B2, coefficient for the rate of nuclei growth in Eq. 8 (min − 2); Ca, emulsifier concentration in the subsurface (mol l − 1); C0, Cs, concentration in the bulk phase (mol l − 1); C1, C2, coefficients for Eqs. 9 – 11; D, diffusion coefficient (cm − 2 s − 1); DHt, latent heat of a two-dimensional first-order phase transition (J mol − 1K − 1); K, Boltzmann constant; ki, first order rate constant in Eq. 4 (min − 1); k1, first-order rate constant for unfolding (min − 1); k2, first-order rate constant for rearrangement (min − 1); k%, rate constant for adsorption in Eq. 5; N, number of monoglyceride molecules in the monolayer at time u (molecule); N0, number of monoglyceride molecules in the monolayer at the initial moment (molecule); DSt, entropy of a two-dimensional first-order phase transition (kJ mol − 1); T, temperature (°C, K); G, surface concentration (mg m − 2); u*, characteristic time corresponding to mechanism change in Fig. 8 (min); u, time (min, s); n, number of adsorbing groups per protein molecule; p , surface pressure at steady-state (mN/m); p, surface pressure (mN/m); pe, equilibrium surface pressure (mN/m); p0, surface pressure at the initial moment (mN/m); pu, surface pressure at time u (mN/m); pt, surface pressure at the transition between LE and LC structures (mN/m); s, surface tension of the film covered interface (mN/m); s0, subphase surface tension (mN/m); t1, t2, relaxation time in Eqs. 10 and 11 (min − 1). * Corresponding author. Tel.: +34-5-4557183; fax: + 34-5-4557134. E-mail address: [email protected] (J.M. Rodrı´guez Patino) 0927-7765/99/$ - see front matter © 1999 Elsevier Science B.V. All rights reserved. PII: S 0 9 2 7 - 7 7 5 7 ( 9 9 ) 0 0 0 1 2 - 6

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1. Introduction Many real food formulations are complex dispersions such as foams and emulsions. These dispersions are inherently unstable systems because of their large interfacial area. That is, in addition to formation, stability is one of the most important properties of such dispersions. For this reason, food dispersions contain many emulsifiers. Emulsifier-based foods such as margarine, spreads, ice cream, imitative dairy products, and cereal-based products, i.e. bread, cakes, and other bakery products, either as traditional foods or low-fat products and instant foods are some examples of food systems in which proteins and polar lipids are added as emulsifiers. For recent reviews readers are directed to Refs. [1 – 6]. Food dispersions are complicated multicomponent systems containing many emulsifiers which may show surface activity by themselves (proteins and low-molecular-weight surfactants) or by association with other components (proteins, lowmolecular-weight surfactants, polysaccharides, to name a few examples). In addition, food dispersions contain many other organic (ethanol, sugars, etc.) and inorganic (salts) reagents which may interact with emulsifiers in different complicated manners depending on pH, temperature, etc. Interfacial interactions become significant when considering the fouling of food processing equipment [7], or during the production of food dispersions (emulsions and foams), when emulsifiers are used as stabilizers [8]. The analysis of the aqueous phase composition is important not only from a practical point of view, but also from a theoretical point of view due to the fact that ethanol [9–26], sugars [10– 17,19,22,24 – 29], glycerol [10,30], electrolytes [31,32], and pH [33 – 35] can affect the interactions in the layer region between pure polar lipids [9– 13,27,30,31,33,35], pure proteins [14,15,17,23,29,34], mixed lipids [18 – 22,28,36], and protein-lipid mixed films [14 – 16,24–26,32], with important repercussions for the structure [9,10,12,27,30,31,33,34], stability [11 – 13,20,21, 33,34,36], rheological characteristics [22– 25,29,35], molecular diffusion [26], rate of film formation [14,15,17,23 – 25,29], and compatibility

[18–21,28,36], etc. of emulsifiers at the air-water interface. The highly complex mechanisms involved in the formation and stabilization/destabilization of food dispersions make fundamental studies in applied systems difficult. Thus, the study of characteristics of food emulsifier monolayers at the air-water interface as a food model presents several advantages. In fact, relationships between structural characteristics of emulsifier monolayers on the air-water interface and dispersion stability have been established, as well as between condensed film formation and emulsifier association in the bulk phase. By incorporating typical food reagents in the aqueous phase in addition to emulsifiers at the interface, the behaviour of food model systems can be approximated to that of real food systems. In this article we are only concerned with the analysis and discussion of the effect of interfacial and aqueous phase compositions on interfacial characteristics (both under equilibrium, steady-state, and dynamic conditions) of typical food emulsifiers (proteins and lipids) at the air-water interface.

2. Structural characteristics of food emulsifier monolayers For a fundamental understanding of the role of emulsifiers in the stabilization of food dispersions, it is essential to obtain information on their packing at the interface. Such information can be obtained through p-A isotherms from investigation of spread monolayers at the air-water interface. The surface pressure is defined as p=s0 −s, where s0 is the subphase surface tension of the bulk aqueous solution and s is the surface tension of the film covered interface.

2.1. Structural characteristics of polar lipids Monoglycerides are the most important group of low-molecular-weight surfactants in the food industry. Mono- and diglycerides spread on water [9,19–21,36] or aqueous solutions of ethanol [9,18–21], sugars [19,20,27,28], salts [31,35], etc. can exist in all classical monolayer states [37,38],

J.M. Rodrı´guez Patino, M.R. Rodrı´guez Nin˜o / Colloids and Surfaces B: Biointerfaces 15 (1999) 235–252

such as liquid-expanded (LE), liquid-condensed (LC), solid (S), and collapse phase, except the gaseous phase (Fig. 1). A decrease in surface density and temperature, and/or an increase in surface pressure produces transitions towards more condensed structures or monolayer collapse. Moreover, the addition of ethanol [9,18– 21] in the aqueous phase produces a condensation in the monolayer structure, but the opposite is observed with sugars [19,20,27,28], salts [31,35], or at pH lower or higher than 7 [33,35].

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2.2. Structural characteristics of proteins In contrast to the rich structural polymorphism observed with polar lipids, protein monolayers have a liquid-expanded-like structure at all the temperatures studied [34]. In addition, the p-A isotherms for protein spread monolayers are very sensitive to the aqueous phase pH. The p-A isotherms were displaced towards the surface pressure axis and the monolayer structure was more condensed in acidic subphases, as the pH approaches the isoelectric point.

Fig. 1. Structures, transitions, and characteristics structural parameters in a standard p-A isotherm. Alim is a measure of the cross-sectional area, and is the limit area obtained by extrapolation of the linear part of the p-A isotherm. Other symbols are explained in the text.

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2.3. Phase diagram The influence of temperature and the interfacial or aqueous phase composition on monolayer structure can be analyzed in a phase diagram obtained directly from the p-A isotherm. Fig. 2 shows phase diagrams for some monoglycerides

spread on water and aqueous solutions of ethanol and sucrose, as an example. In these diagrams the drawn lines represent transitions between structures of the monolayer or transition towards collapse. The area between the transition lines represents the respective zone for each structure. The results show reduced solid structure and

Fig. 2. Phase diagrams for (A) monostearin on water, (B) monopalmitin on water, (C) monoolein on water, (D) monostearin on ethanol 1.0 M, (E) monostearin on glucose 0.5 M, and (F) monostearin on sucrose 0.5 M.

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solutions [35]. However, at pH\7 the S structure was reduced, while those for LC and LE phases did not change [35]. Electrolytes promoted a LC structure in the monolayer, the S structure disappeared, and an LE structure was observed only at higher temperatures [31]. Fig. 3 shows a phase diagram for b-casein, as an example of the behaviour of protein monolayers [34]. b-casein spread films present two structures and the collapse phase. That is, there is a critical surface pressure and a surface concentration at which the film properties change significantly. This transition depends on the temperature and the aqueous phase pH. The film structure was observed to be more condensed and the b-casein interfacial density (G) was higher in acidic subphases. This behaviour must be attributed to a reduction in the repulsive interaction between negative amino-acid residues due to the fact that at pH 5 the overall charge of b-casein molecules approaches zero.

2.4. Thermodynamic parameters Fig. 3. (A) Phase diagram and (B) surface coverage at the transition between structures 1 and 2 for b-casein spread monolayers at the air-water interface at ( ) pH 5 and () pH 7, as a function of temperature. Closed symbols: equilibrium surface pressure for b-casein spread monolayers at pH 5 ( ) and pH 7 ().

increased LC structure with sugars [27] and glycerol [30] in the subphase, and in acidic aqueous

The effect of temperature and aqueous phase composition on the structural characteristics of emulsifier spread films can also be analyzed by thermodynamic parameters of the liquid-condensed to liquid-expanded transition. The study of this transition is interesting for mono- and diglyceride monolayers in which this first-order

Fig. 4. Entropies (DSt) and enthalpies (DHt) versus temperature for liquid-condensed to liquid-expanded transition in monostearin monolayers spread on aqueous solutions containing sugars.

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Fig. 5. Rate of BSA adsorption at the air-aqueous phase interface at 20°C. Protein concentration: 0.1 wt/wt%. Aqueous phase composition: (A) water, (B) ethanol 1.0 M, and (C): sucrose 0.5 M.

transition is present depending on the temperature, and aqueous and interfacial compositions. The latent heat, DHt, and the entropy, DSt, of a two-dimensional first-order phase transition can be evaluated from a suitable form of the ClausiusClapeyron equation applied to the isotherm: (pt DHt = (T T(AE −AC)

(1)

DHt T

(2)

DS =

where the molecular areas at the beginning and the end of the transition at temperature T, AE, and AC, are associated with the molecular areas in the LE and LC phases, respectively, and pt is the pressure at which the two-dimensional condensation occurs (Fig. 1). The LC to LE transition requires energy (DHt \ 0) and involves an increase in the system disorder

(DSt \ 0). For most polar lipids (Fig. 4), the numerical values are not very high; therefore, from a thermodynamic point of view, it is not difficult for the phase change to take place. An increase in temperature leads to a lower increment in enthalpy and entropy, which is in agreement with a process that is endothermic and increasingly disordered. The values of DHt and DSt also depend on the addition of solutes in the aqueous phase. In fact, the presence of glycerol [30], sugars [27], electrolytes [31], or a buffer solution (especially in the range of acidic subphases [35]) produces higher values of entropy and enthalpy. DHt and DSt also increase when the concentration of the these reagents in the subphase increases. Monoglyceride monolayers with an LE structure spread on these reagents are favourable to the existence of a large accumulation of water molecules at the interface. Thus, compression involves either the removal of intervening water molecules or a rearrangement of the polar group as the film condenses. In addition, monoglyceride monolayers spread on polyhydroxy compounds (glycerol and sugars) may possess greater configurational freedom than when they are spread on water due to the location of these reagents at the aqueous interface between the monolayer head groups, introducing spatial separation and disorder into the lipid head region with a consequent increase in entropy. This location can be helped by formation of intermolecular hydrogen-bonded complexes between the monoglyceride head group

Fig. 6. Rate of BSA adsorption at the air-water interface at 20°C. Protein concentration: 10 − 2 wt/wt%. The subphase was phosphate buffer (pH 7, I =0.05 M).

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and sugars or glycerol molecules. It is obvious that the overall effect of the reagent or reagent concentration on thermodynamic parameters is very dependent on the monolayer-solute system.

2.5. Structural characteristics at equilibrium The equilibrium spreading pressure (pe) is the maximum surface pressure to which a spread monolayer may be compressed without the possibility of monolayer collapse. Thus the pe value is of primary importance for the stability of a monolayer (Section 4), since at higher surface pressures the monolayer is not in a state of thermodynamic equilibrium. From these data, together with the phase diagram, the structure at equilibrium of a emulsifier (protein or lipid) monolayer can be established as a function of temperature and aqueous phase composition. The LC is the structure at equilibrium for monolayers of saturated monoglycerides, while the LE is the structure at equilibrium for monoolein and most diglycerides [10]. Proteins behave in a different way. For most proteins studied, such as b-casein [34], BSA [14], b-lactoglobulin, and caseinate (data not yet published), the monolayer collapses at a surface pressure which is practically the same as pe. This is an indication that proteins tend to be adsorbed in the most compact structure after rearrangement of hydrophobic residues at the interface.

3. Protein adsorption The adsorption of protein at fluid-fluid interfaces is considered to play an important role in the formation and stabilization of food dispersions [39–41]. 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 the protein adsorption the surface or interfacial tension of fluid-fluid interfaces decreases, which is an important factor both to optimise the input of energy involved in the emulsification or foaming process [42] and,

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finally, to achieve smaller droplet and bubble size, which is an important factor for the stability of the dispersed system [39]. The decrease in surface tension by proteins is caused by different processes [43–45]: (i) the protein has to diffuse from the bulk phase 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.

3.1. Rate of protein adsorption The kinetics of protein adsorption at the airwater interface can be monitored by measuring changes in surface pressure with time (u) [38,46]. The rate of change of surface concentration (G) can be expressed as [44]: dG/du = (dG/dp).(dp/ du). If (dG/dp) is constant (as is the case for BSA adsorbed [47] or spread [48] monolayers at surface pressures lower than 17 mN/m, dp/du can be used to evaluate the rate of protein adsorption. During the first step, at relatively low pressures when diffusion is the rate-determining step, a modified form of the Ward and Torday equation [49] can be used to correlate the change in the surface pressure with time (Eq. (3)): p= 2C0·K·T(D·u/3.14)1/2

(3)

where C0 is the concentration in the bulk phase, K is the Boltzmann constant, T is the absolute temperature and D is the diffusion coefficient. If the diffusion of proteins at the interface controls the adsorption process, a plot of p against u 1/2 will then be linear [44,50,51]. To monitor unfolding at the interface and configurational rearrangements of adsorbed protein molecules, two different approaches can be used. The rate of these processes can be analyzed by the first-order equation [43,45]: p − pu = − ki ·u (4) ln p − p0

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where p , p0, and pu, are the surface pressures at steady-state conditions, at time u = 0, and at any time, u, respectively, and ki is the first-order rate constant. In practice, a plot of Eq. (4) 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. When an energy barrier exists, the rate of protein penetration into the surface film will be rate-limiting. Ward and Regan [52] have used a modified form of the Ward and Tordai equation to monitor this process (Eq. (5)). ln (dp/du)=ln (k%·n·C0) −pDA/(K·T)

(5)

where k% is the rate constant of adsorption, DA is the molecular area required for the molecule to adsorb at the interface, and n is the number of adsorbing groups per protein molecule. A plot of ln (dp/du) versus p must be linear. A range of techniques has been developed for the measurement of the dynamic surface tension [38,46]. The Wilhelmy plate is an adequate method to monitor the long-term adsorption of protein at the air-water interface, as the adsorption is controlled by the unfolding at the interface and the

configurational rearrangements of adsorbed protein. However, the diffusion step is too fast to be detected with the Wilhelmy plate method [14,15,23,29]. The dynamic surface tension of protein solutions at short-time, as the adsorption is controlled by the protein diffusion towards the interface, can be determined by means of the dynamic drop tensiometer [17].

3.1.1. Diffusion of protein molecules to the airaqueous phase interface The adsorption kinetic of bovine serum albumin (BSA) at short adsorption time [17], up to approximately 2 s, as the surface pressure is lower than about 5 mN/m, is controlled by the diffusion of the protein towards the interface, in agreement with the Ward and Torday model (Fig. 5). The presence of sucrose in the aqueous phase does increase the rate of BSA diffusion towards the interface, but the opposite was observed for aqueous solutions of ethanol, especially at higher concentrations of this reagent in the bulk phase. The presence of ethanol in the bulk phase exerts the function of an energy barrier for the BSA diffusion towards the interface, which could be attributed to the competition for the penetration of the protein into the interface due to the presence of previously adsorbed ethanol molecules. At higher adsorption time, in the period

Table 1 Characteristic parameters for adsorption of BSA at the airaqueous subphase interface in the presence [29] and the absence [14] of convection, at 20°C System

Presence of convection, k1×103 ( min−1)

Absence of convection, k1×103 (min−1)

BSA BSA BSA BSA BSA BSA BSA BSA BSA BSA BSA BSA BSA BSA

– 56.7 – – – 16.8 26.9 29.3 – 22.3 9.55 47.6 33.6 72.0

30.7 31.9 18.8 16.0 18.3 – – 24.0 6.74 – – – 36.7 –

(1%, wt/wt)-water (0.1%, wt/wt)-water (1.10−2%, wt/wt)-water (1.10−3%, wt/wt)-water (1.10−4%, wt/wt)-water (0.1%, wt/wt)-ethanol 0.1 M (0.1%, wt/wt)-ethanol 0.5 M (0.1%, wt/wt)-ethanol 1 M (1.10−2%, wt/wt)-ethanol 1 M (2.10−2%, wt/wt)-ethanol 1 M (3.10−3%, wt/wt)-ethanol 1 M (0.1%, wt/wt)-sucrose 0.25 M (0.1%, wt/wt)-sucrose 0.5 M (0.1%, wt/wt)-sucrose 1 M

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after that affected by the diffusion, an energy barrier for the BSA adsorption exists which could be attributed to the penetration, unfolding, and rearrangements of the protein at the interface [17].

3.1.2. Protein adsorption in absence of con6ection The long-term adsorption of BSA at the air-water interface, as an example, is given in Fig. 6. Different temperatures, protein concentrations in the bulk phase, and aqueous phase composition (aqueous solutions of ethanol and sucrose) give similar results [14]. We find, for all experiments of BSA adsorption, two or more linear regions in the plot of ln [(p −pu )/(p −po)] versus u or in the plot of ln (dp/du) versus p, according to Eq. (4) or Eq. (5), respectively. When ln (dp/du) is independent of p this is taken as an indication that only the rearrangement process is taking place [14]. It must be emphasised that plots of both Eqs. (4) and (5) give complementary results for protein adsorption, that is, the time and surface pressure at which unfolding or molecular rearrangement is the mechanism that controls the adsorption are practically the same no matter which plot is used, either that from Eq. (4) or from Eq. (5). To summarize the effect of temperature, protein concentration and aqueous phase composition on the time dependence of surface pressure during BSA adsorption from the bulk phase, the first-order rate constants derived from Eq. (4) are collected in Table 1. The following conclusions were drawn. The rate of BSA adsorption at the interface increases with both BSA concentration in the aqueous phase and temperature. With ethanol in the subphase the existence of an induction period is observed, which could be associated with the competitive adsorption of BSA on an ethanol film; BSA has a higher affinity than ethanol for the interface due to its higher hydrophobicity. This phenomenon could also reflect the existence of BSA-solute interactions in the aqueous phase and at the interface. However, sucrose has no affinity for the interface but exerts a strong cohesive force on water molecules. This phenomenon could be associated with the fact that protein molecules are preferentially hydrated in the presence of sucrose, limiting the protein unfolding and protein-protein interactions and, as consequence, the rate of BSA

Fig. 7. (A) Adsorption isotherm of BSA on water as a function of temperature (°C): () 5, ( ) 20, and ( ) 30. Adsorption isotherm of BSA on (1) water and aqueous solutions of (B) ethanol at (2) 0.5 M, (3) 1.0 M and (4) 2.0 M, and (C) sucrose at (5) 0.5 M and (6) 1.0 M, at 20°C.

adsorption increases when sucrose is present in the bulk phase, as was observed experimentally (Table 1).

3.1.3. Protein adsorption in the presence of con6ection The effect of convection due to the sinusoidal movement of a ring in the interface and in the bulk phase [29] can be analyzed from the data included in Table 1. We found that the rate of BSA adsorption is higher in the presence of convection. However, the convection affects the rate of BSA adsorption at the interface, depending on the reagent present in the aqueous phase (ethanol or sucrose) and its concentration. The rate of BSA adsorption decreased with ethanol in the subphase and at sucrose concentrations lower than 1.0 M,

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Fig. 8. Relaxation phenomena in polar lipid monolayers, where Ca and Cs are the emulsifier concentrations in the subsurface and in the bulk aqueous phase, respectively, and u* is the characteristic time corresponding to a change in the desorption mechanism.

but it increased significantly at the highest sucrose concentration studied (1.0 M).

3.2. Protein adsorption at equilibrium The BSA concentration dependence on surfacepressure at equilibrium showed classical sigmoidal behaviour (Fig. 7). At low BSA concentrations the initial solutions caused only a small increment in the surface pressure. The surface pressure increase with BSA concentration and tends to a plateau. This plateau commences at the point where surface

pressure reaches its maximum value over the range of protein concentrations from 10 − 3 to 10 − 1 wt% [53,54]. The general characteristics of BSA adsorption are practically the same at the three temperatures studied. However, the value of surface pressure at the plateau decreased as the temperature increased. The behaviour of adsorbed BSA films can be interpreted in terms of monolayer coverage. Adsorption of BSA at lower concentrations than that of the plateau forms a monolayer of irreversibly adsorbed molecules. As the plateau is attained the monolayer is saturated by protein that is irreversibly

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adsorbed. At higher protein concentrations the BSA molecules form multilayers beneath the primary monolayer but these structures do not contribute significantly to surface pressure [47]. The effect of aqueous phase composition on the surface activity of BSA is also shown in Fig. 7. These results indicate that the adsorption of BSA is very dependent on the solutes in the aqueous phase. In ethanol aqueous solutions the surface activity of BSA decreased and a higher protein concentration was necessary to reach the plateau. The presence of sucrose in the bulk aqueous phase also strongly affects the adsorption behaviour of BSA (Fig. 7). The adsorption characteristics of BSA as a function of the aqueous phase composition could be interpreted in terms of the competitive BSA-ethanol adsorption at the interface and by the effect of sucrose on the structure of BSA in the bulk phase and at the interface [53,54].

4. Relaxation phenomena in food emulsifier monolayers

4.1. Relaxation phenomena in emulsifier monolayers Two kinds of experiments can be performed for the analysis of relaxation in emulsifiers monolayers [11,33]. First, the surface pressure is kept constant and the area measured as a function of time. In the second type of experiment, the area is kept constant and the surface pressure decreases with time. Several relaxation mechanisms can be fitted to the results derived from these experiments, as shown in Fig. 8. Due to the fact that relaxation mechanisms are different for low-molecular-weight surfactants than for proteins it is convenient to analyze these emulsifiers separately.

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of spread monolayers at any constant surface pressure involves two process: (i) dissolution into the bulk aqueous phase forming a saturated aqueous layer (during the initial non-steady-state period of desorption, the rate of monolayer molecular loss can be expressed by Eq. (6); and (ii) after a time the concentration gradient within the diffusion layer becomes constant and desorption reaches a steady state. The rate of monolayer molecular loss is then given by Eq. (7). In Eqs. (6) and (7), coefficients A1 and A2 account for the rate of dissolution and diffusion, respectively, and N and N0 are the number of monoglyceride molecules in the monolayer that remain on the interface at time u and those at the initial moment, respectively. At a surface pressure higher than the equilibrium surface pressure (pe), with insoluble monolayers, the relaxation phenomena are due to the transformation of a homogeneous monolayer phase into a heterogeneous monolayer-collapse phase system. Monolayer collapse may occur either by a macroscopic film fracture, by a process of nucleation and growth of bulk surfactant fragments, or by the formation of lenses whenever a characteristic surface pressure is exceeded [10,11,33,55–60]. The collapse rate should follow the Eq. (8), where B1 and B2 account for the formation of nuclei and the growth rate of nuclei, respectively. log (N/N0)= A1·u 1/2

(6)

log (N/N0)= A2·u

(7)

log (N/N0)= B1·u+ B2·u 2

(8)

4.2. Relaxation mechanisms in low-molecular weight surfactant monolayers Relaxation mechanism other than desorption and collapse are difficult to quantify in typical relaxation experiments performed in a film balance. Thus the results derived from relaxation experiment for polar lipids were tested by the equations derived for desorption or monolayer collapse. The desorption

Fig. 9. Relaxation of monoglyceride monolayers on water at pH 7 and 20°C. Relaxation at constant surface pressure: ( , , ) 20 mN/m, and () 40 mN/m. Monoglyceride: ( ) monopalmitin, (, ) monoolein, and () monolaurin.

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The modelling of monolayer collapse by homogeneous nucleation and growth of bulk surfactant nuclei can also be analyzed by applying the ProutTompkins equation (Eq. (9)) to the data derived from relaxation experiments at constant surface collapse area [55]. log

p0 −p =C1·log u + C2 p

(9)

where p and p0 are the surface pressures at time u and at the initial moment, respectively, and C1 and C2 are coefficients which depend on the experimental conditions. It is unlikely that, at the temperatures used in these experiments, evaporation contributed significantly to the relaxation phenomena observed with monoglyceride monolayers [11]. Moreover, as the same relationship fits the relaxation of relative molecular area by evaporation and diffusion (Eq. (7)) in the processing of the experimental data it is convenient to consider evaporation as being included in diffusion.

4.2.1. Relaxation phenomena at constant surface pressure The relaxation of monopalmitin, monoolein, and monolaurin monolayers at 20°C, as some examples, is shown in Fig. 9. It can be seen that the rate of monolayer molecular loss depends on the surface pressure and the lipid spread on the interface, as was observed with other mono- and diglycerides at

Fig. 10. Destabilization of monomyristin monolayers spread on different aqueous solutions at 20°C and 35 mN/m. Subphase: ( ) water, () glucose 0.5 M, () sucrose 0.5 M, and () ethanol 0.5 M.

different temperatures, surface pressures (lower than pe), and aqueous phase compositions [33]. In general, as the surface pressure is lower than the equilibrium surface pressure, the monolayer molecular loss can be quantified by a desorption mechanism. This molecular loss takes place in two steps: dissolution (Eq. (6)) and diffusion into the aqueous phase (Eq. (7)). The monolayer molecular loss by desorption is an irreversible process. In fact, when experiments were repeated after a wait time of 24 h at the maximum surface area, the rate of molecular loss was lower than for the original experiment [33]. From the values of the kinetic coefficients (A1 and A2) it can be concluded that, as the monolayer molecular loss is controlled by a desorption mechanism, the rate of molecular loss increases with temperature and surface pressure no matter wha tthe lipid is [11,33]. That is, the film stability is very dependent on the monolayer structure, as detected by the film elasticity-stability relationships [12,13]. However, the monolayer molecular loss was lower for monostearin [11,12], and increasingly higher for monopalmitin [33], monoolein [11,33], monomyristin [12], and monolaurin [33], respectively. That is, the maximum monolayer molecular loss was observed with monolaurin monolayer, especially at higher surface pressure and temperature. The effect of the subphase composition on the relaxation phenomena at constant surface pressures lower than pe is complex and depends on the lipid

Fig. 11. Relaxation of monoglyceride monolayers on water at constant area (at the collapse point). Aqueous subphase pH 7. Monoglyceride: ( , ) monopalmitin, and () monoolein. Temperature: () 20°C, and ( , ) 40°C.

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can be quantitatively important with a liquid-expanded structure, as with monomyristin, monolaurin or monoolein, and could produce an increase in the monolayer instability.

4.2.2. Relaxation phenomena at constant collapse area The analysis of the relaxation phenomena under the most adverse conditions (at the collapse point and at higher temperatures) is important from a practical point of view. From a mechanistic point of view some precautions have to be adopted, especially when the monolayers present some instability due to desorption, as analyzed in the previous section. In these cases, the mechanisms of monolayer molecular loss by desorption and collapse could occur concurrently, and the analysis of the overall process could be more complex. Fig. 11

Fig. 12. (A) Relaxation of monoolein monolayers on water at constant surface pressure (at the collapse point). Aqueous subphase pH 7. Temperature: 20°C. Symbols: ( ) overall monolayer molecular loss (N/N0)av at the collapse point, () monolayer molecular loss by dissolution, (N/N0)dis, at 40 mN/m, and () monolayer molecular loss attributed to collapse: (N/N0)col = 1−[(N/N0)av − (N/N0)dis]. (B) Fits of experimental data according with a desorption and/or collapse mechanisms: Eqs. (1)–(3). (C) Fits of (N/N0)col data according with Smith and Berg [56], Eq. (8).

and the surface pressure [11 – 13,33]. In general, sugars and especially ethanol, do increase the instability of lipid monolayers (Fig. 10). However, the effect of solutes in the subphase is also dependent on the monolayer structure. The film-sub-phase interactions, together with the effect of film-film interactions, can have an effect on the monolayer structure. The effect of film-subphase interactions

Fig. 13. (A) Relaxation of b-casein monolayers at the air-water interface at pH 7, as a function of surface pressure (mN/m): ( ) 10, and () 20. Temperature: 20°C. Ionic strength: 0.05 M. (B) Relaxation of b-casein monolayers at the air-water interface at constant surface area (at the collapse point), as a function of temperature: ( ) 20°C, and () 40°C. pH 7, I =0.05 M.

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shows the relaxation phenomena of monopalmitin and monoolein monolayers at 20 and 40°C at pH 7, as an example [33]. The relaxation phenomena in monopalmitin monolayers at 20°C are controlled predominantly by the collapse mechanism, according to the Prout-Tompkins equation (Eq. (9)). Moreover, the surface pressure relaxed to pe, indicated in the figure by arrows. At 40°C, the fulfilment of the Prout-Tompkins law indicates that the relaxation could be due to nucleation and growth of critical nuclei, as for 20°C. However, as the surface pressure at relaxation time higher than 1 min is lower than the pe, the relaxation mechanisms for monolayer collapse competes with a desorption mechanism. Monoolein monolayers behave differently to monopalmitin monolayers. It can be seen that the surface pressure relaxed from the collapse value, which is close to pe, towards lower values at longer times. Constant area relaxation studies at p \pe are difficult to interpret due to the interference of different relaxation processes and to the fact that the monolayer is continuously passing through the various monolayers states during the course of the experiment.

4.2.3. Relaxation phenomena at constant collapse pressure For a more detailed interpretation of the relaxation phenomena observed under collapse conditions, we have studied the collapse at constant surface pressure by observing the relaxation in molecular area at constant temperature [33]. The loss of relative molecular area as a function of time for monoolein monolayers at 20°C and at pH 7, as an example, is shown in Fig. 12. The initial relative molecular loss could be quantified (Fig. 12B) in accordance with the second step for desorption of monolayer molecules, that is for a diffusion mechanism (Eq. (7)), and/or with the first step for the formation of nuclei corresponding to a collapse mechanism (Eq. (8)). At higher relaxation time, N/N0 could be fitted by Eq. (8), which suggests that collapse could control the relaxation process. If the mechanisms of desorption and collapse are two concurrent processes, we can separate the monolayer molecular loss by desorption, (N/N0)des, and collapse, (N/N0)col, from the average monolayer molecular loss (N/N0)av (Fig. 12C) [33]. Processing

of the experimental data of monolayer molecular loss by collapse, (N/N0)col, according to Eq. (8) showed (Fig. 12C) an excellent fit, which suggests that the relaxation phenomena at the collapse point can be quantified separately with a satisfactory result [33].

4.3. Relaxation mechanisms in protein monolayers 4.3.1. Relaxation phenomena at constant surface pressure The relaxation behaviour (loss of monolayer relative area, A/A0) of b-casein monolayers at 20°C at 10 and 20 mN/m is shown in Fig. 13, as an example [34]. Other proteins, such as b-lactoglobulin and caseinate (data not published), behave in a similar way. Because pB pe, an attempt could be made to interpret the relaxation data according to a desorption mechanism. However, no hysteresis was observed in p-A isotherms after continuous compression-expansion cycles or over time, which is an indication that b-casein monolayers were stable against desorption at pB pe (in this respect, b-lactoglobulin films behave in a different way). The best fit of the experimental data was obtained by means of two exponential equations (Eq. (10)). (A/A0)= (A/A0)0 + C1·e(-u/t1) + C2·e(-u/t2)

(10)

where (A/A0)0 is the amplitude of the relative area at the initial moment, t1 and t2 are the relaxation times, and C1 and C2 are constants. The relaxation of b-casein monolayer is not a simple process and shows important differences compared to typical lipids under the same experimental conditions. The relaxation rate (quantified by means of the relaxation time, t) is higher at the highest surface pressure and in acidic subphases. The reversible relaxation phenomena observed with spread protein monolayer can be attributed to the reorganization/organization at the interface due in part to the looping of the amino acid segments on the protein in the underlying aqueous solutions and to the effect of the viscoelastic characteristics of the protein in this process [34].

4.3.2. Relaxation phenomena at constant surface area at the collapse point Proteins were also tested under the most adverse

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conditions, at the maximum superficial density at the collapse area [34]. It was observed (Fig. 13B) that the surface pressure relaxed to the equilibrium surface pressure. The best fit of the results was obtained by means of two exponential equations (Eq. (11)), for relaxation kinetics of protein monolayers at constant surface area. p/p0 =(p/p0)0 +C1·e(-u/t1) +C2·e(-u/t2)

(11)

where (p/p0)0 is the amplitude of the relative surface pressure at the initial moment, t1 and t2 are the relaxation times, and C1 and C2 are constants. These results confirm that the collapse could be the main cause of removal of protein molecules from the monolayer. However, the collapse of b-casein monolayers does not follow the formation of nuclei or the presence of a monolayer fracture, typical of lipids with a condensed monolayer structure, nor is there formation of lenses upon collapse, attributed to lipids with an expanded monolayer structure. According to Graham and Phillips [43,47], the collapse of protein monolayer could take place through the formation of multilayers in the interface and in the underlying region, as can be confirmed by recent experiments with Brewster angle microscopy (data not published).

5. Rheological characteristics of food emulsifier monolayers Rheological interfacial properties can be defined for compressional deformation and shearing motion at interface, and both types of parameters can be measured experimentally [46,61]. While surface shear viscosity may contribute appreciably to the long-term stability of dispersed systems, the surface dilatational viscoelasticity is more relevant for the stability of the emulsion or foam in the production stage. The surface rheology of emulsifiers at the air-water interface is of interest not only due to its importance in relation to dispersion stability, but also because of its extreme sensitivity to the nature of intermolecular interactions at the interface.

Fig. 14. Superficial density dependence of () surface dilational modulus (E, mN/m) and ( ) surface tension (mN/m) for (A) monostearin and (B) monoolein films spread on water at 20°C. Elapse time after spreading: 30 min. Angular frequency: 0.81 rad s − 1.

5.1. Surface rheological characteristics of polar lipids at the air-aqueous phase interface The surface dilational characteristics of polar lipids show practically the same features [22,35]: (i) the values of the surface dilational modulus, E, are very similar to that of the dilational elasticity, (ii) the values of the surface dilational viscosity and the loss angle tangent are low and practically zero, and (iii) the surface rheological parameters are practically independent of frequency over the range examined. As a consequence of this behaviour, it can be concluded that the surface rheological characteristics of polar lipids spread on water or aqueous solutions of some typical food reagents (ethanol, sugars, salts, electrolytes, etc.) are essentially elastic. The values of E for different film structures under dilational conditions are similar to those deduced directly from the p-A isotherms slope [35]. From these experiments [22,35] it can be concluded that the more

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condensed the structure is, the higher the E of the film becomes until collapse is reached. From this point, there is a stabilization or a decrease in E values with a higher superficial density, depending on the spread emulsifier. Finally, the rheological characteristics of the lipid monolayer are also sensitive to the existence of lipid-lipid and/or lipid-subphase interactions due to the presence of some solutes in the aqueous phase (Fig. 14) [22,35]. Measurements of surface tension as a function of lipid superficial density suggests that there exists a critical concentration at which the interactions between lipid molecules lead to a constant surface activity as the interface is saturated with lipids (Fig. 14). This critical superficial concentration (CSC) is close to surface density at the collapse point. For monostearin, at surface concentration near the CSC, the surface dilational modulus increases markedly, rising to a plateau value at higher lipid concentrations. The occurrence of the maximum in E at monostearin concentration higher than the CSC was attributed to the liquid-condensed monolayer structure at equi-

Fig. 15. Superficial density dependence of surface dilational modulus (E, mN/m) for (A) monostearin and (B) monoolein films spread on aqueous solutions of () ethanol at 1.0 M, and () sucrose at 0.5 M. Temperature: 20°C. Elapse time after spreading: 30 min. Angular frequency: 0.81 rad s − 1.

librium and the formation of crystals of lipids on the interface due to collapse caused by oversaturation of monostearin at the highest superficial density [22,35]. The superficial density dependence of surface tension and surface dilational modulus for monoolein films show important differences compared to the monostearin films. First, the magnitude of the maximum value of E is reduced by a factor of approximately 4. Second, E describes a maximum at monoolein concentrations just below the CSC, as indicated by the minimum value of the surface tension. The differences between superficial rheological characteristics of monostearin and monoolein spread films, at concentrations above CSC, were attributed to the differences in collapse behaviour and film structure at equilibrium [10,22,35]. As previously stated, the structure of monoolein films at equilibrium is liquid-expanded and this structure favours the formation of lenses with reduced lipid-lipid interactions. The effects of solutes in subphase (ethanol and sucrose) on surface rheological characteristics of monostearin and monoolein monolayers are shown in Fig. 15. The presence of solutes in the subphase produces a significant reduction in E, especially for monoolein in the presence of ethanol at the CSC (Fig. 15B). The low E value for monoolein films must be emphasized because of the potential practical applications. The effect of aqueous subphase composition on surface rheological characteristics of monostearin and monoolein can be attributed to the film-subphase interactions. However, these in-

Fig. 16. () Ethanol and () sucrose dependence of surface dilational modulus (E) for BSA adsorbed films at the air-water interface. BSA concentration: 0.1% wt/wt. Temperature: 20°C. Adsorption time: 60 min. Angular frequency: 0.81 rad s − 1.

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teractions must be different for ethanol and sugars, as previously analyzed.

5.2. Surface rheological characteristics of proteins at the air-aqueous phase interface Most milk proteins films adsorbed or spread at the air-water interface show a viscoelastic behaviour which is almost purely elastic over the range of frequencies studied [23,29,34]. The surface rheological characteristics of protein films are very dependent on the aqueous phase composition. For BSA adsorbed films on ethanol aqueous solutions (Fig. 16), E decreases with increased ethanol concentration. These data are of practical importance because the film viscoelasticity decreases to a low value at the highest ethanol concentration. The phenomena reported here were associated with the protein-ethanol interactions, which could reduce the amount of adsorbed protein or disrupt the protein-protein interactions by forming a mixed BSA-ethanol adsorbed film [23]. However, surface rheological characteristics, especially E, are practically independent of sucrose concentration at concentrations lower than 0.5 M, but decrease at the highest sucrose concentration studied (1.0 M). The E values for BSA adsorbed from a 1.0 M aqueous sucrose solution are three times higher than those from 1.0 M ethanol solutions. The decrease in E in aqueous sucrose solutions was associated with decreased protein-protein interactions and/or the effect of sucrose on the molecular structure of the protein [29]. 6. Conclusions What this article clearly establishes is that the interfacial and aqueous phase compositions, and operational conditions (temperature, surface pressure, surface density, etc.) have an effect on the interfacial characteristics (structure, stability, rheology, etc.) of emulsifiers (proteins and lipids) at the air-water interface. Furthermore, this work has highlighted significant differences between interfacial characteristics of emulsifiers at interfaces which could be of use in the approximation of the

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