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Dynamic phenomena in caseinate–monoglyceride mixed films at the air–water interface
Food Hydrocolloids 19 (2005) 395–405 www.elsevier.com/locate/foodhyd
Dynamic phenomena in caseinate–monoglyceride mixed films at the air–water interface Cecilio Carrera Sa´nchez, Ma Rosario Rodrı´guez Nin˜o, Juan M. Rodrı´guez Patino* Departamento de Ingenierı´a Quı´mica, Facultad de Quı´mica, Universidad de Sevilla, C/. Prof. Garcı´a Gonza´lez, 1, 41012 Seville, Spain
Abstract The aim of this work is to perform a comparative study of the long-term relaxation phenomena and surface dilatational characteristics in protein and monoglyceride (monopalmitin, monoolein and monolaurin) mixed monolayers spread at the air–water interface as a function of processing variables (surface composition, surface pressure and temperature) and aqueous phase pH. The study has been centred on a real protein (caseinate), to see if the relaxation phenomena observed in protein–monoglyceride mixed films with a model protein (b-casein) are generic. Different and complementary interfacial techniques (surface film balance, Brewster angle microscopy, and interfacial dilatational rheology) have been used to analyse the static (structure, reflectivity, miscibility, and interactions) and dynamic characteristics (long-term relaxation phenomena and surface dilatational properties) of protein–monoglyceride mixed films spread on the air–water interface. The static and dynamic characteristics of the mixed films depend on the protein–monoglyceride ratio and the surface pressure. At higher surface pressures, collapsed protein residues may be displaced from the interface by monoglyceride molecules with important repercussions on the interfacial characteristics of the mixed films. From the frequency dependence of the surface dilatational modulus we have deduced the relationships between interfacial dilatational rheology and changes in molecular structure, interactions, miscibility, and relaxation phenomena. q 2005 Elsevier Ltd. All rights reserved. Keywords: Air–water interface; Mixed film; Proteins
1. Introduction Among food proteins, caseinate is distinguished by its good foaming and emulsifying properties, and for these reasons it is widely used in food formulations. The emulsifying properties of caseinate arise from the structures of the four proteins found in bovine milk (b-, as1-, k-, and as2-casein). All of the individual caseins, except k-casein, show a strong tendency to adsorb to fluid interfaces (air– water and oil–water), and thus they find an important use in the manufacture of stable emulsions (i.e. ice cream, cream liqueurs, whipped toppings, coffee whiteners, products for infant nutrition, etc.), where long-term emulsion stability is essential. In emulsions incorporating caseinate the individual caseins seem to be adsorbed at the oil–water interface in proportion to their incorporation in solution (Hunt & Dalgleish, 1994). However, there seems to be a distinction * Corresponding author. Tel.: C34 5 4556446; fax: C34 5 4557134. E-mail address: [email protected] (J.M.R. Patino). 0268-005X/$ - see front matter q 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodhyd.2004.10.006
between caseinate and mixtures of purified individual caseins, since the latter show competitive adsorption between b-, and as1-casein (Dickinson, 1994; Dickinson, Rolfe, & Dalgleish, 1988; Fan & Dalgleish, 1993). Caseinate gives adsorbed layers similar to those measured for b-casein, the individual casein that shows the highest surface activity of the four individual proteins in caseinate (Fan & Dalgleish, 1993). On the other hand, in many food formulations the caseins are not the only emulsifier present, because small molecule emulsifiers (monoglycerides, phospholipids, Tweens, etc.) are also incorporated into the formulation. The small molecule emulsifiers can faster cover the interface than caseins do, resulting in an emulsion with smaller particles, leading to greater stability. However, more important in some products is the effect of the small molecules in destabilising the emulsion (Goff & Jordan, 1989). In the formulation of ice cream the small molecule emulsifier is added to break the adsorbed layer of protein and allow the adsorption of the fat to the surface of the air bubble.
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Thus, an important action of the small molecule emulsifiers is to promote the displacement of caseins from the interface. The aim of this work is to perform a comparative study of the long-term relaxation phenomena (desorption, collapse, conformation and/or reorganization changes, etc.) and surface dilatational characteristics in caseinate and monoglyceride (monopalmitin, monoolein and monolaurin) mixed monolayers spread at the air–water interface as a function of processing variables (surface composition and surface pressure) and aqueous phase pH. Non-equilibrium processes occurring in systems containing fluid interfaces with a surfactant present are of great practical significance for emulsification and foaming, both in technology and in natural phenomena (Rodrı´guez Patino, Rodrı´guez Nin˜o, & Carrera, 2003a). This work is an extension of previous studies on relaxation phenomena (Carrera, Rodrı´guez Nin˜o, & Rodrı´guez Patino, 1999; Rodrı´guez Nin˜o, Carrera, & Rodrı´guez Patino, 1999) and surface dilatational characteristics (Rodrı´guez Patino, Carrera, Rodrı´guez Nin˜o, & Cejudo, 2001a,b; Rodrı´guez Patino, Rodrı´guez Nin˜o, & Carrera, 2002, 2003b) of monoglyceride and milk proteins. The study has been centred on a real protein (caseinate), to see if the relaxation phenomena observed in protein– monoglyceride mixed films with a model protein (b-casein) are generic.
2. Experimental 2.1. Chemicals Synthetic 1-monohexadecanoyl-rac-glycerol (monopalmitin, DIMODANR PA 90), 1-mono (cis-9-octadecanoyl) glycerol (monoolein, RYLOTM MG 19), and 1-monododecanoyl-rac-glycerol (monolaurin, DIMODANR ML 90) were supplied by Danisco Ingredients with over 95–98% of purity. Caseinate (a mixture of z38% b-casein, z39% as1-casein, z12% k-casein, and z11% as2-casein) was supplied and purified from bulk milk from Unilever Research (Colworth, UK). The sample was stored below 0 8C and all work was done without further purification. Samples for interfacial characteristics of caseinate films were prepared using Milli-Q ultrapure water and were buffered at pH 5 and 7. Analytical-grade acetic acid, sodium acetate, and Trizma [Tris(hydroxymethyl)aminomethane] for buffered solutions were used as supplied by Sigma (O95%) without further purification. To form the surface film, monoglyceride was spread in the form of a solution, using hexane:ethanol (9:1, v:v) as a spreading solvent. Analytical grade hexane (Merck, 99%) and ethanol (Merck, O99.8%) were used without further purification. The water used as subphase was purified by means of a Millipore filtration device (Milli-Q). All these products were supplied by Sigma (O99.5%). Ionic strength was 0.05 M in all the experiments.
2.2. Surface film balance Measurements of surface pressure (p)-area (A) isotherms and surface relaxation in caseinate–monoglyceride mixed films at the air–water interface were performed on a commercial fully automated Langmuir-type film balance (Lauda, Germany). The method has been described previously for mixed components (Rodrı´guez Patino, Carrera, & Rodrı´guez Nin˜o, 1999a,b). Before each measurement, the film balance was calibrated at 20 8C. Mixtures of particular mass ratios—ranging between 0 and 1, expressed as the mass fraction of monoglyceride in the mixture, X—were studied. To allow the quantitative adsorption of the protein on the interface the monolayer was not under any surface pressure during the spreading process. Thus, the caseinate necessary to form the mixed film should be spread before the monoglyceride. Aliquots of aqueous solutions of caseinate (1.553!10K4 mg/ml) at pH 7 were spread on the interface by means of a micrometric syringe. To allow for spreading, adsorption and rearrangements of the protein, 30 min were allowed to elapse before measurements were taken. The spreading method adopted in these experiments ensured the quantitative spreading of the protein on the interface as was discussed in a previous paper (Rodrı´guez Patino, Rodrı´guez Nin˜o, Carrera, & Cejudo, 2001c). Afterwards, a monoglyceride solution in hexane:ethanol mixture was spread at different points on the caseinate film. The monoglyceride solutions were spread on the subphase by means of a micrometric syringe at 20 8C. To allow for spreading and caseinate–monoglyceride interactions, 30 min were allowed to elapse before compression was performed. To ensure interactions and homogeneity, the mixed film was compressed near the collapse point of the mixture and then expanded immediately to avoid the collapse. After 30 min at the maximum area, a new p–A isotherm was performed. The compression rate was 3.3 cm minK1, which is the highest value for which isotherms were found to be reproducible in preliminary experiments. The experiments (p–A isotherm and relaxation phenomena in mixed films) were measured at least three times. The reproducibility of the results was better than G0.5 mN/m for surface pressure and G0.005 nm 2/molecule for area. All isotherms were recorded continuously by a device connected to the film balance and then analyzed off-line. Two kinds of experiment were used for the analysis of relaxation in caseinate–monoglyceride mixed monolayers. First, the surface pressure (p) is kept constant, and the area A is measured as a function of time. In the second type of experiment, the area is kept constant (at the collapse) and the surface pressure decreases. This decrease is measured as a function of time. Various relaxation mechanisms can be fitted to the results derived from the above-mentioned experiments. Desorption of a spread monolayer at any constant surface pressure involves two stages (Ter Minassian-Saraga, 1956). The first is dissolution into the bulk aqueous phase to
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form a saturated aqueous layer. During the initial non-steadystate period of desorption, the rate of monolayer molecular loss can be expressed by Eq. (1). The second stage occurs when, 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. (2). KLog ðA=A0 Þ Z A1 q1=2
(1)
KLog ðA=A0 Þ Z A2 q
(2)
where A and A0 are the molecular area at time q and at the initial moment, and coefficients A1 and A2 account for the rate of dissolution and diffusion, 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 due to a macroscopic film fracture, or by a process of nucleation and the growth of bulk surfactant fragments, or formation of lenses, whenever a characteristic pe surface pressure is exceeded (Carrera et al., 1999; Smith & Berg, 1980). The modelling of monolayer collapse by homogeneous nucleation and the growth of bulk surfactant nuclei can be analysed by applying the Prout-Tompkins (1944) equation to the data derived from relaxation experiments at constant surface collapse area Log
p0 K p Z C1 Log q C C2 p
(3)
where p and p0 are the surface pressures at time q and at the initial moment, respectively, and C1 and C2 are coefficients which depend on the experimental conditions. For proteins, various relaxation mechanisms ascribed to desorption or collapse of spread monolayer were applied to the results derived from relaxation data (Rodrı´guez Nin˜o, Carrera, & Rodrı´guez Patino, 1999). However, the best fit of the results was obtained by means of two exponential equations, Eqs. (4) and (5), for relaxation kinetics at constant surface pressure and at constant surface area, respectively. These relaxation phenomena were associated with conformation–organization change and hydrophilic group hydration (Rodrı´guez Nin˜o et al., 1999). ðA=A0 Þ Z ðA=A0 Þ0 C P1 eðKq=t1 Þ C P2 eðKq=t2 Þ
397
2.3. Brewster angle microscopy A commercial Brewster angle microscope (BAM), BAM2, manufactured by NFT (Go¨ttingen, Germany) was used to study the topography and reflectivity of the monolayer. BAM, coupled with relaxation in surface pressure (p), at constant area, or relaxation in area (A), at constant surface pressure, was used in this work to visualize and determine structural changes during relaxation phenomena of mixed emulsifier monolayers at the air–water interface. Further characteristics of the device and operational conditions have been described elsewhere (Rodrı´guez Patino, Carrera, & Rodrı´guez Nin˜o, 1999c,d). The relative film reflectivity can be measured by determining the light intensity at the camera and analysing the polarization state of the reflected light through the method described elsewhere. In the experiments presented here, the reflectivity for the protein film was relatively high and that for monoglyceride films relatively low. 2.4. Surface dilatational rheology To obtain surface rheological parameters—such as surface dilatational modulus, elastic and viscous components, and loss angle tangent—a modified Wilhelmy-type film balance (KSV 3000) was used as described elsewhere (Rodrı´guez Patino et al., 2001a,b). In this method the surface is subjected to small periodic sinusoidal compressions and expansions by means of two oscillating barriers at a given frequency (u) and amplitude (DA/A) and the response of the surface pressure is monitored. Surface pressure was directly measured by means of two roughened platinum plates situated on the surface between the two barriers. The surface dilatational modulus derived from the change in surface pressure resulting from a small change in surface area may be described by the equation (Lucassen & Van den Tempel, 1972): E ZK
dp Z Ed C iEv d ln A
(6)
The dilatational modulus is a complex quantity and is composed of real and imaginary parts. The real part of the dilatational modulus or storage component is the dilatational elasticity, EdZjEjcos q. The imaginary part of the dilatational modulus or loss component is the surface dilatational viscous modulus, EvZjEjsin q. The loss angle tangent can be defined by Tan qZEv/Ed. Measurements were performed at least three times. The reproducibility of these results was better than 5%.
(4) 3. Results and discussion
ðp=p0 Þ Z ðp=p0 Þ0 C P3 e
ðKq=t3 Þ
C P4 e
ðKq=t4 Þ
(5)
where (A/A0)0 and (p/p0)0 are the amplitude of the relative area and relative surface pressure at the initial moment, respectively, t1 to t4 are the relaxation times and P1 to P4 are constants.
3.1. Relaxation phenomena during the compression– expansion of the monolayer The relaxation phenomena during the compression– expansion cycle of caseinate–monopalmitin mixed films
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Fig. 1. Compression–expansion cycles for caseinate–monopalmitin mixed monolayers on water at pH 7. Temperature 20 8C. Monolayer composition (mass fraction of monopalmitin in the mixture): (A) 0.2, (B) 0.4, (C) 0.6, and (D) 0.8. (,) Compression and (B) expansion for a first cycle at 30 min after spreading, at the maximum area. (—) Compression and ($$$$) expansion for a second cycle at 30 min after the first one, at the maximum area. (6) Compression for a third cycle at 30 min after the relaxation experiment at constant surface pressure (pZ20 mN/m), at the maximum area.
(as an example) are presented in Fig. 1. It can be seen that there exists a hysteresis in p–A isotherms during the compression–expansion cycle, which is more evident at the higher caseinate concentration in the mixed film. This is an indication of the importance of the caseinate content in the mixture in the dynamic relaxation phenomena. The higher the caseinate/monopalmitin ratio in the mixture the higher the hysteresis becomes. At the higher content of monopalmitin in the mixture (Fig. 1D) the hysteresis in the p–A isotherm was the same as for pure monopalmitin monolayer. Caseinate–monoolein mixed monolayers behaved in a similar way (data not shown), but the magnitude of the hysteresis is lower than for caseinate–monopalmitin mixed monolayers. Further information about the mechanism or mechanisms that control the relaxation phenomena in caseinate–monoglyceride mixed films can be deduced from the time evolution of relative reflectivity during a compression– expansion cycle for a caseinate–monopalmitin (Fig. 2) or a caseinate–monoolein (Fig. 3) mixed monolayer at two representative mass fractions of monoglyceride in the mixture of 0.2 and 0.8 (as two examples). At surface pressures lower than that for the caseinate collapse
(py29 mN/m), monopalmitin in the mixture has a liquidcondensed structure (LC)—which can be deduced from the existence of the characteristic main transition between the liquid-expanded (LE) and LC phases (Fig. 1) and from the reflectivity peaks (Fig. 2). This main transition is hindered as the content of caseinate in the mixture increases. Thus, at XZ0.2 the main transition between the LE and LC phases was not observed in the p–A isotherm (Fig. 1A). On the other hand, the presence of caseinate in the mixture can also be deduced because some peaks have a reflectivity similar to that for pure caseinate. From these results some differences can be deduced as compared to those recorded for caseinate–monoolein mixed monolayers (Fig. 3). Firstly, monoolein in the mixture (and caseinate as well) has a LE structure at every surface pressure. Secondly, over the overall range of surface pressures the I–p plots for caseinate–monoolein mixed films were practically the same as those for pure monoolein (Rodrı´guez Patino et al., 1999c), which strengthens the conclusion that monoolein, with its lower relative thickness, predominates at the interface. However, at higher surface pressures, near to and after the caseinate collapse, the relative intensity of some spots was the same as for pure caseinate.
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Fig. 2. The time evolution of (6 and B) relative reflectivity (at a shutter speed of 1/250 s) during a compression–expansion cycle for caseinate– monopalmitin mixed monolayers on water at pH 7 and at 20 8C. Monolayer composition (mass fraction of monoglyceride in the mixture): (a, 6) 0.2 and (b, B) 0.8. The vertical arrows indicate the time at which the measure surface pressure is equal to the equilibrium surface pressures of caseinate (pe, CS) and monopalmitin (pe, MP). The coexistence of caseinate and monopalmitin (A), the squeezing out phenomena of caseinate by monopalmitin (B), the prevalence of monopalmitin at the interface (C), and the re-adsorption of caseinate at the interface (D) are also indicated. The vertical lines in plots (a) and (b) indicate the point at which the monolayer collapses.
The higher hysteresis in p–A isotherm during the compression–expansion cycle, at the higher concentrations of caseinate in the mixture, may be associated with the readsorption of the protein at the interface during the expansion, after the squeezing out phenomena observed during the compression of the monolayer, a phenomenon which involves some time. This hypothesis is corroborated by the fact that the I–p plots during the compression– expansion cycle are not symmetrical (Figs. 2 and 3). The presence of numerous and more intense reflectivity peaks of collapsed caseinate are visible at the higher content of caseinate in the mixture (Figs. 2A and 3A). These data also support the hypothesis that the hysteresis is due to molecular
399
Fig. 3. The time evolution of (6 and B) relative reflectivity (at a shutter speed of 1/250 s) during a compression–expansion cycle for caseinate– monoolein mixed monolayers on water at pH 7 and at 20 8C. Monolayer composition (mass fraction of monoglyceride in the mixture): (A, 6) 0.2 and (B, B) 0.8. The vertical arrows indicate the time at which the measure surface pressure is equal to the equilibrium surface pressures of caseinate (pe, CS) and monoolein (pe, MO). The coexistence of caseinate and monoolein (A), the squeezing out phenomena of caseinate by monoolein (B), the prevalence of monoolein at the interface (C), and the re-adsorption of caseinate at the interface (D) are also indicated. The vertical lines in plots (a) and (b) indicate the point at which the monolayer collapses.
reorganisation. But, these changes are more drastic for caseinate–monopalmitin than for caseinate–monoolein mixed films because monopalmitin in the mixture presents more changes in the monolayer structure, a phenomenon that explains the higher degree of hysteresis in monopalmitin–caseinate mixed films. The relaxation phenomena in caseinate–monoglyceride (monopalmitin or monoolein) mixed films are reversible processes, as can be deduced from data presented in Fig. 1 for caseinate–monopalmitin mixed films (as an example). It can be seen that the p–A isotherms are repetitive after different compression–expansion cycles. Monolaurin–caseinate mixed films behaved in a different way (Fig. 4). The hysteresis in the p–A isotherm during a compression–expansion cycle is higher (data not shown)
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films. These results support evidence of the importance of the monolaurin molecular loss, as compared with the structural changes that take place in monolaurin and caseinate molecules present in the mixed films during the relaxation process. The instability of mixed films can be also deduced from the displacement towards the p-axis of the p–A isotherm after continuous compression–expansion cycles (Fig. 4A). The displacement of the p–A isotherms after continuous compression–expansion cycles is irreversible because the isotherm does not return to its original state on recompression after a waiting period of 24 h at the maximum area at pZ0 (data not shown). 3.2. Long-term relaxation phenomena at constant surface pressure 3.2.1. Relaxation phenomena in monopalmitin– and monoolein–caseinate mixed films Fig. 5 shows the relaxation in relative molecular area at 20 8C and at 20 mN/m for caseinate–monopalmitin (Fig. 5A) and caseinate–monoolein (Fig. 5B) mixed films, at pH 7 as an example (caseinate–monoglyceride mixed
Fig. 4. (A) p–A isotherms (compression curves) for (B) caseinate and a caseinate–monolaurin mixed at a mass fraction of monolaurin in the mixture of 0.5: (—) first compression at 30 min after spreading, at the maximum area, (–-–-–) second compression after 30 min after the first one, and ($$$$) third compression after the relaxation experiment at constant surface pressure (pZ20 mN/m). (B) Area relaxation at constant surface pressure (pZ20 mN/m) for (B) caseinate, ($) monolaurin, and (—) caseinate–monolaurin mixed film at a mass fraction of monolaurin in the mixture of 0.5. (C) Relaxation of surface pressure (B) and relative surface pressure (—) at constant surface area (at the collapse point) for caseinate– monolaurin mixed monolayers at a mass fraction of monolaurin in the mixture of 0.5. pH 7. Temperature 20 8C. The equilibrium surface pressure for monolaurin (pe ML) is indicated by means of an arrow.
than for monopalmitin– and monoolein–caseinate mixed films. In addition, the hysteresis in the p–A isotherm during a compression–expansion cycle is higher as the content of monolaurin in the mixture increases (data not shown), a phenomenon opposite to that observed for monopalmitin— (Fig. 1) and caseinate–monoolein (data not shown) mixed
Fig. 5. Relaxation at constant surface pressure (pZ20 mN/m) of (A) caseinate–monopalmitin and (B) caseinate–monoolein mixed monolayers on water at pH 7. Temperature 20 8C. Monolayer composition (mass fraction of monopalmitin or monoolein in the mixture): (B) 0, (—) 0.2, (– – –) 0.4, (..) 0.6, (–$–) 0.8, and (6) 1.
C.C. Sa´nchez et al. / Food Hydrocolloids 19 (2005) 395–405 Table 1 Characteristic parameters for desorption (Eqs. (1) and (2)) of monoglycerides and a caseinate–monolaurin mixed film at XMLZ0.5, at constant surface pressure (pZ20 mN/m) and at 20 8C and at pH 7
Monopalmitin Monoolein Monolaurin Caseinate–monolaurin XMLZ0.5
A1!103 (LR) (minK0.5)
A2!105 (LR) (minK1)
7.0 (0.996) 7.0 (0.997) 90 (0.999) 20.2 (0.996)
16 (0.983) 28 (0.989) 243 (0.938) 3.2 (0.915)
films at pH 5 behaved in a similar way). For pure monopalmitin and monoolein monolayers the relaxation phenomena can be quantified (Table 1) by a desorption mechanism in two steps (Carrera et al., 1999): dissolution (Eq. (1)) and diffusion into the aqueous phase (Eq. (2)). For caseinate, caseinate–monopalmitin, and caseinate– monoolein mixed films the (A/A0)-relaxation has an exponential time-dependence. The best fits of the experimental data were obtained by means of Eq. (4) with two relaxation times. In Table 2 the relaxation times and the value of relative area at 60 min of relaxation time, (A/A0)60, are shown. These phenomena may be due, among other things, to reorganization changes between caseinate native molecules and caseinate molecules in various degrees of unfolding after the compression up to 20 mN/m. The relaxation phenomena at 20 mN/m for caseinate– monoglyceride mixed films (Fig. 5) are reversible processes, as can be deduced from data presented in Fig. 1 for caseinate–monopalmitin mixed films (as an example). It can be seen that the p–A isotherms are repetitive after different compression–expansion cycles, the last one being registered after the relaxation experiment at 20 mN/m. These data confirm the hypothesis that the relaxation phenomena observed with caseinate–monoglyceride mixed monolayers at constant surface pressures lower than pe could be related to the kinetics of the molecular reorganisation.
Table 2 Characteristic parameters (Eq. (4)) for relaxation of caseinate–monopalmitin and caseinate–monoolein mixed films at constant surface pressure (pZ 20 mN/m) and at 20 8C and at pH 7
Caseinate–monopalmitin
Caseinate–monoolein
X
t1 (min)
t2 (min)
(A/A0)60
0 0.2 0.4 0.6 0.8 1.0 0 0.2 0.4 0.6 0.8 1.0
9.0 9.6 7.1 5.0 5.4 – 9.0 3.8 7.1 7.1 9.0 –
66 234 63 52 60 – 66 67 64 160 2206 –
0.92 0.93 0.94 0.93 0.90 – 0.92 0.91 0.93 0.93 0.96 –
401
3.2.2. Relaxation phenomena in caseinate–monolaurin mixed films Fig. 4B shows the relaxation in relative molecular area at 20 8C and at 20 mN/m for caseinate–monolaurin mixed films at a mass fraction of monolaurin in the mixture of 0.5 (as an example). The relaxation phenomena are more pronounced for these systems than for caseinate–monopalmitin (Fig. 5A) and for caseinate–monoolein (Fig. 5B) mixed films. Unlike monopalmitin– and monoolein–caseinate mixed films, a mechanism with two steps in accord with Eqs. (1) and (2) fits the data better. Thus the phenomena of monolayer relaxation may be due to a desorption mechanism by dissolution and diffusion. However, we do not reject the concurrence of this phenomenon with the organization/rearrangement of the aminoacids segments of caseins forming caseinate into the underlying aqueous phase, as discussed in previous sections. The characteristic parameters for destabilisation of caseinate– monolaurin monolayer are included in Table 1. It can be seen that the magnitude of the rates of dissolution (coefficient A1) and diffusion (coefficient A2) is higher for pure monolaurin than for the mixed film. That is, the presence of caseinate in the mixture produces a reduction of the magnitude of the relaxation phenomena in the mixed film. 3.3. Long-term relaxation phenomena at constant surface area (at the collapse point) 3.3.1. Relaxation phenomena in monopalmitin– and monoolein–caseinate mixed films Monopalmitin– and monoolein–caseinate mixed films were also tested under the most adverse conditions, at the maximum superficial density at constant collapse area. Fig. 6A shows the relaxation in surface pressure at constant surface area (at the collapse point) for caseinate–monopalmitin mixed films. It can be seen that monopalmitin and caseinate–monopalmitin mixed films relaxed towards the equilibrium surface pressure (pe) of pure monopalmitin (Carrera et al., 1999), which is indicated in Fig. 6A by means of an arrow. The (p/p0)-relaxation has a logarithmic time-dependence instead of an exponential dependency. In fact, processing the experimental data according to the Prout-Tompkins equation (Eq. (3)) showed that it was possible to obtain a linear fit (Table 3). This indicates that the relaxation phenomena under these conditions may be due to nucleation and growth of critical nuclei of monopalmitin. On the other hand, the magnitude of relaxation in surface pressure is practically the same for all mixed films. Fig. 6B shows the relaxation in surface pressure at constant surface area (at the collapse point) for caseinate– monoolein mixed films. Caseinate–monoolein monolayers behave differently to caseinate–monopalmitin monolayers. It can be seen that in experiments at constant collapse area, the surface pressure relaxes from the collapse value for pure
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402
Table 4 Characteristic parameters (Eq. (5)) for relaxation of caseinate–monoolein mixed films at constant area (at the collapse point) at 20 8C and at pH 7 XMO
t3 (min)
t4 (min)
(p/p0)60
0 0.2 0.4 0.6 0.8 1.0
0.82 21 72 10 59 –
24 102 217 326 228 –
0.92 0.85 0.89 0.81 0.89 0.62
On the other hand, the magnitude of relaxation in surface pressures depends on the monolayer composition. The reason for these behaviours must be associated with the immiscibility between both components (monoglycerides and caseinate) at the air–water interface, as was deduced for these systems at the collapse point (Rodrı´guez Patino, Carrera, & Rodrı´guez Nin˜o, 2001d). In fact, at surface pressures higher than that for the caseinate collapse a squeezing out of caseinate by monoglyceride is produced (the results in Figs. 2 and 3 are an example of this behaviour). The displacement of caseinate by monoglyceride progresses with surface pressure up to the collapse point. Thus, at the collapse point the mixed film is dominated by the presence of monoglyceride.
Fig. 6. Relaxation at constant area (at the collapse point) for (A) caseinate–monopalmitin and (B) caseinate–monoolein mixed monolayers on water at pH 7. Temperature 20 8C. Monolayer composition (mass fraction of monoglyceride in the mixture): (6) 0, (—) 0.2, (– – –) 0.4, (..) 0.6, (–$–) 0.8, and (6) 1. The equilibrium surface pressures for monopalmitin (pe, MP), monoolein (pe, MO), and caseinate (pe, CS) are indicated by means of arrows.
monoolein, which is close to pe (see arrow in Fig. 6B), towards lower p values at longer times. This behaviour could be assigned to different phenomena, such as desorption and collapse occurring concurrently, as observed for a pure monoolein monolayer (Carrera et al., 1999). These hypotheses can be supported by the quantification of the (p/p0)-relaxation by means of an exponential dependency (Eq. (5)), with two relaxation times (Table 4). Table 3 Characteristic parameters (Eq. (3)) for relaxation of caseinate–monopalmitin mixed films at constant area (at the collapse point) and at 20 8C and at pH 7 XMP
C1 (LR)
C2 (LR)
(p/p0)60
0 0.2 0.4 0.6 0.8 1.0
– 0.10 (0.994) 0.13 (0.995) 0.18 (0.972) 0.09 (0.996) 0.38 (0.999)
– – – – – 0.19 (0.996)
– 0.94 0.92 0.88 0.93 0.85
3.3.2. Relaxation phenomena in caseinate–monolaurin mixed films Fig. 4C shows the relaxation in surface pressure at constant surface area (at the collapse point) for caseinate– monolaurin mixed films at a mass fraction of monolaurin in the mixture of 0.5, as an example. It can be seen that in experiments at constant collapse area, the surface pressure relaxes from the collapse value to very low surface pressures. However, important differences with monopalmitin– and monoolein–caseinate mixed films can be observed. Firstly, the magnitude of the relaxation in surface pressure is higher for monolaurin—than for monopalmitin– and monoolein–caseinate mixed films. Secondly, the (p/p0)-relaxation has a logarithmic time-dependence as for caseinate–monopalmitin mixed films, but this dependence cannot be associated with a mechanism of collapse by nucleation and growth of nuclei of monolaurin because the surface pressure decreases to values lower than pe for monolaurin. Clearly, the desorption of monolaurin, which is practically instantaneous for pure films at the collapse point (Carrera et al., 1999), has an important role in the relaxation phenomena observed in the mixed films (Fig. 4C). 3.4. Surface dilatational characteristics of caseinate– monoglyceride mixed films at the air–water interface 3.4.1. The effect of surface pressure The surface dilatational moduli of monopalmitin– and monoolein–caseinate mixed films spread on the air– water interface at pH 7 (as an example) are shown in
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403
Fig. 7. Surface pressure dependence of (A) surface dilatational modulus (E, mN/m) and (B) loss angel tangent (Tan q) for caseinate–monopalmitin mixed films at 20 8C and at pH 7. Amplitude of the area deformation 5%. Frequency 50 mHz. Mass fraction of monopalmitin in the mixture: (!) 0, (6) 0.2, (7) 0.4, ($) 0.6, (B) 0.8, and (,) 1.0.
Figs. 7A and 8A, respectively. For caseinate monolayer the E–p plot showed a typical irregular shape (Rodrı´guez Patino et al., 2001a). It can be seen that E increased with increasing p to a maximum value at py10 mN/m. Upon further increase of the surface pressure E decreased to a minimum at py20 mN/m. Afterwards, E increased again with surface pressure. The pH effect on the surface dilatational modulus is lower, and practically insignificant (data not shown), as compared with the surface pressure effect. For pure monoglyceride monolayer the surface dilatational modulus also depends on the monolayer structure. The more condensed the structure is (at higher p), the higher the surface dilatational modulus of the monolayer becomes until the collapse is reached, which is consistent with an increase in the forces of interaction between molecules at the interface. However, the surface dilatational modulus of monoolein spread monolayer (Fig. 8A) is lower than that for monopalmitin (Fig. 7A). That is, for the more condensed monopalmitin monolayer E is higher than for the more expanded monoolein monolayer, at every surface pressure.
For caseinate–monoglyceride mixed films the surface pressure dependence of E (Figs. 7A and 8A) depends on the surface pressure and the interfacial composition. At surface pressures lower than that for caseinate collapse and at XMP or XMO!0.8, the same irregular shape in the p dependence of E, as for pure caseinate, was observed for the mixed films. At surface pressures higher than that for caseinate collapse the E–p and for mixed films were parallel to that of monoglyceride, which demonstrated again that at higher surface pressures the mixed films were practically dominated by monoglyceride molecules. However, the data in Figs. 7A and 8A also demonstrate that the small amounts of caseinate collapsed residues at the interface—as deduced at a microscopic level from I–p plots (Figs. 2 and 3)—have an effect on the surface dilatational properties of the mixed films. In fact, the values of E for mixed films are lower than that for a pure monoglyceride monolayer, even at the collapse point of the mixed films. Thus, the mechanical properties of the mixed films also demonstrated that, even at the highest surface pressure, a monopalmitin monolayer is unable to displace caseinate molecules completely from
Fig. 8. Surface pressure dependence of (A) surface dilatational modulus (E, mN/m) and (B) loss angel tangent (Tan q) for caseinate–monoolein mixed films at 20 8C and at pH 7. Amplitude of the area deformation 5%. Frequency 50 mHz. Mass fraction of monoolein in the mixture: (!) 0, (6) 0.2, (7) 0.4, ($) 0.6, (B) 0.8, and (,) 1.0.
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the air–water interface. From the values of the tangent of the loss angle (Figs. 7B and 8B) it can be concluded that caseinate–monoglyceride mixed films behaved as viscoelastic at every surface pressure. 3.4.2. Effect of frequency Changes in surface dilatational modulus for caseinate– monoglyceride mixed monolayers at pH 7 (as a example) and at the collapse point, as a function of frequency of oscillation over a range of 1–200 mHz, are illustrated in Fig. 9. We did not observe any difference in the frequency dependence of surface rheological parameters at pH 5 (data not shown). It can be seen that in the range 1 mHz!u!50 mHz, the dilatational modulus increased with the frequency, but at higher frequencies E is practically constant. The frequency dependence of E in the frequency range of 1–50 mHz, should be associated with the monoglyceride monolayer collapse. This long-term relaxation process requires (Fig. 6) a time of the same order of magnitude as the scale of the oscillation— in our experiments between 2 and 25 min. Again, the reason
for this behaviour must be associated with the immiscibility between both components at the air–water interface. At higher surface frequencies (50!u!200 mHz), the viscoelastic behaviour of the mixed monolayer is more complex and should be associated with formation/destruction of phase transitions and the formation/destruction of 3D collapse structures in the monoglyceride monolayer (Figs. 1–3). We do not reject the possibility that islands of collapsed caseinate could be displaced and reabsorbed within the interface during the sinusoidal compression– expansion cycles. In fact, the time required for organization/ reorganization changes in monoglyceride monolayer structure is of the same order of magnitude as the time scale of the deformation (Fig. 6). However, the surface dilatational properties at the higher frequencies (at uO100 mHz) may have been subjected to errors due to artefacts of the technique, resulting in an artificially high viscous effect as a consequence of the resonance in the flow of the liquid between the two barriers. In addition, shear effects between monolayer molecules and the trough wall or the Wilhelmy plate should also have a role (Petkov, Gurkov, Cambell, & Borwankar, 2000; Wijmans & Dickinson, 1998).
4. Conclusions
Fig. 9. Frequency dependence of surface dilatational modulus for (A) caseinate–monopalmitin and (B) caseinate–monoolein mixed films at the collapse point. Temperature 20 8C. pH 7. Amplitude of the area deformation 5%. Mass fraction of monoglyceride in the mixture: (!) 0, (6) 0.2, (7) 0.4, ($) 0.6, (B) 0.8, and (,) 1.0.
This article presents experimental studies of relaxation phenomena in caseinate–monoglyceride mixed films at the air–water interface. Data have been analysed according to models for desorption, collapse and/or organization/reorganization changes, during a compression–expansion cycle, after the monolayer compression to a surface pressure of 20 mN/m or at the collapse area of the mixed film. During the compression–expansion cycle and at a constant surface pressure (at 20 mN/m), below the caseinate collapse, the organization/reorganization changes of caseinate molecules in monopalmitin– and monoolein–caseinate mixed films are the mechanisms that control the relaxation process. At constant surface area (at the collapse point of the mixed film) the relaxation phenomena in mixed films may be due either to nucleation and growth of critical nuclei of monoglyceride—such as was deduced for caseinate–monopalmitin mixed films—or to a complex mechanism including competition between collapse and monolayer desorption—such as was deduced for caseinate–monoolein mixed films. For caseinate–monolaurin mixed films at every surface pressure the relaxation phenomena are mainly due to the irreversible loss of monolaurin molecules by desorption into the bulk aqueous phase. Comparing the data presented in this paper to those for b-casein–monoglyceride mixed films (Rodrı´guez Patino et al., 2003b) at pH 5 and 7 we conclude that the relaxation phenomena observed with protein (either a model b-casein or a real caseinate protein) and monoglyceride mixed films appear to be generic. These relaxation phenomena were also observed for protein–monoglyceride mixed films with
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disordered b-casein (Rodrı´guez Patino et al., 2003b) and caseinate (this work) and globular (Rodrı´guez Patino et al., 2002) proteins. The reasons for these behaviours must be associated with the immiscibility between protein and monoglyceride at the air–water interface and to the protein displacement by the monoglyceride at surface pressures higher than that for the protein collapse. These relaxation phenomena have significant repercussion on surface dilatational properties. That is, the surface dilatational characteristics reflect the complex relaxation phenomena that take place in protein–monoglyceride mixed films.
Acknowledgements This research was supported by CICYT through grant AGL2001-3843-C02-01.
References Carrera, C., Rodrı´guez Nin˜o, Ma. R., & Rodrı´guez Patino, J. M. (1999). Relaxation phenomena in monoglyceride films at the air–water interface. Colloids and Surfaces B: Biointerfaces, 12, 175–192. Dickinson, E. (1994). Protein-stabilized emulsions. Journal of Food Engineering., 22, 59–74. Dickinson, E., Rolfe, S. E., & Dalgleish, D. G. (1988). Competitive adsorption of as1-casein and b-casein in oil-in-water emulsions. Food Hydrocolloids, 2, 397–405. Fan, Y., & Dalgleish, D. G. (1993). Dimensions of the adsorbed layers in oil-in water emulsions stabilized by caseins. Journal of Colloid Interface Science, 156, 329–334. Goff, H. D., & Jordan, W. K. (1989). Action of emulsifiers in promoting fat destabilization during the manufacture of ice cream. Journal of Dairy Science, 72, 18–29. Hunt, J. A., & Dalgleish, D. G. (1994). Adsorption behaviour of whey protein isolate and caseinate in soya oil-in-water emulsions. Food Hydrocolloids, 8, 175–187. Lucassen, J., & Van den Tempel (1972). Dynamic measurements of dilational properties of a liquid interface. Chemical Engineering Science, 27, 1283–1291. Petkov, J. T., Gurkov, T. D., Cambell, B. E., & Borwankar, R. P. (2000). Dilatational and shear elasticity of gel-like protein layers on air/water interface. Langmuir, 16, 3703–3711. Prout, E. G., & Tompkins, F. C. (1944). The thermal decomposition of potassium permanganate. Transaction of the Faraday Society, 40, 488–498.
405
Rodrı´guez Nin˜o, Ma. R., Carrera, C., & Rodrı´guez Patino, J. M. (1999). Interfacial characteristics of b-casein films at the air–water interface. Colloids and Surfaces B: Biointerfaces, 12, 161–173. Rodrı´guez Patino, J. M., Carrera, C., & Rodrı´guez Nin˜o, Ma. R. (1999a). Analysis of b-casein–monopalmitin mixed films at the air–water interface. Journal of Agricultural Food Chemistry, 47, 4998–5008. Rodrı´guez Patino, J. M., Carrera, C., & Rodrı´guez Nin˜o, Ma. R. (1999b). Is Brewster angle microscopy a useful technique to distinguish between isotropic domains in b-casein–monoolein mixed monolayers at the air– water interface? Langmuir, 15, 4777–4788. Rodrı´guez Patino, J. M., Carrera, C., & Rodrı´guez Nin˜o, Ma. R. (1999c). Morphological and structural characteristics of monoglyceride monolayers at the air–water interface observed by Brewster angle microscopy. Langmuir, 15, 2484–2492. Rodrı´guez Patino, J. M., Carrera, C., & Rodrı´guez Nin˜o, Ma. R. (1999d). Structural and morphological characteristics of b-casein monolayers at the air–water interface. Food Hydrocolloids, 13, 401–408. Rodrı´guez Patino, J. M., Carrera, C., Rodrı´guez Nin˜o, Ma. R., & Cejudo, M. (2001a). Structural and dynamic properties of milk proteins spread at the air–water interface. Journal of Colloid Interface Science, 242, 141–151. Rodrı´guez Patino, J. M., Carrera, C., Rodrı´guez Nin˜o, Ma. R., & Cejudo, M. (2001b). Structural–dilatational characteristics relationships of monoglyceride monolayers at the air–water interface. Langmuir, 17, 4003–4013. Rodrı´guez Patino, J. M., Rodrı´guez Nin˜o, Ma. R., & Carrera, C. (2002). Static and dynamic properties of a whey protein isolate and monoglyceride mixed films at the air–water interface. Industrial and Engineering Chemistry Research, 41, 2652–2661. Rodrı´guez Patino, J. M., Rodrı´guez Nin˜o, Ma. R., & Carrera, C. (2003a). Protein–emulsifier interactions at the air–water interface. Current Opinion in Colloid and Interface Science, 8, 387–395. Rodrı´guez Patino, J. M., Rodrı´guez Nin˜o, Ma. R., & Carrera, C. (2003b). Structure, miscibility, and rheological characteristics of b-casein– monoglyceride mixed films at the air–water interface. Journal of Agricultural and Food Chemistry, 51, 112–119. Rodrı´guez Patino, J. M., Rodrı´guez Nin˜o, Ma. R., Carrera, C., & Cejudo, M. (2001d). The effect of pH on monoglyceride–caseinate mixed monolayers at the air–water interface. Journal of Colloid Interface Science, 240, 113–126. Rodrı´guez Patino, J. M., Rodrı´guez Nin˜o, Ma. R., Carrera, C., & Cejudo, M. (2001c). Whey protein isolate–monoglyceride mixed monolayers at the air–water interface. Morphology, structure, and interactions. Langmuir, 17, 7545–7553. Smith, R. D., & Berg, J. C. (1980). The collapse of surfactant monolayers at the air–water interface. Journal of Colloid Interface Science, 74, 273–273. Ter Minassian-Saraga, L. (1956). Recent work on spread monolayers. Adsorption and desorption. Journal of Colloid Science, 11, 398–418. Wijmans, C. M., & Dickinson, E. (1998). Simulation of interfacial shear and dilatational rheology of an adsorbed protein monolayer modeled as a network of spherical particles. Langmuir, 14, 7278–7286.
Dynamic phenomena in caseinate–monoglyceride mixed films at the air–water interface Cecilio Carrera Sa´nchez, Ma Rosario Rodrı´guez Nin˜o, Juan M. Rodrı´guez Patino* Departamento de Ingenierı´a Quı´mica, Facultad de Quı´mica, Universidad de Sevilla, C/. Prof. Garcı´a Gonza´lez, 1, 41012 Seville, Spain
Abstract The aim of this work is to perform a comparative study of the long-term relaxation phenomena and surface dilatational characteristics in protein and monoglyceride (monopalmitin, monoolein and monolaurin) mixed monolayers spread at the air–water interface as a function of processing variables (surface composition, surface pressure and temperature) and aqueous phase pH. The study has been centred on a real protein (caseinate), to see if the relaxation phenomena observed in protein–monoglyceride mixed films with a model protein (b-casein) are generic. Different and complementary interfacial techniques (surface film balance, Brewster angle microscopy, and interfacial dilatational rheology) have been used to analyse the static (structure, reflectivity, miscibility, and interactions) and dynamic characteristics (long-term relaxation phenomena and surface dilatational properties) of protein–monoglyceride mixed films spread on the air–water interface. The static and dynamic characteristics of the mixed films depend on the protein–monoglyceride ratio and the surface pressure. At higher surface pressures, collapsed protein residues may be displaced from the interface by monoglyceride molecules with important repercussions on the interfacial characteristics of the mixed films. From the frequency dependence of the surface dilatational modulus we have deduced the relationships between interfacial dilatational rheology and changes in molecular structure, interactions, miscibility, and relaxation phenomena. q 2005 Elsevier Ltd. All rights reserved. Keywords: Air–water interface; Mixed film; Proteins
1. Introduction Among food proteins, caseinate is distinguished by its good foaming and emulsifying properties, and for these reasons it is widely used in food formulations. The emulsifying properties of caseinate arise from the structures of the four proteins found in bovine milk (b-, as1-, k-, and as2-casein). All of the individual caseins, except k-casein, show a strong tendency to adsorb to fluid interfaces (air– water and oil–water), and thus they find an important use in the manufacture of stable emulsions (i.e. ice cream, cream liqueurs, whipped toppings, coffee whiteners, products for infant nutrition, etc.), where long-term emulsion stability is essential. In emulsions incorporating caseinate the individual caseins seem to be adsorbed at the oil–water interface in proportion to their incorporation in solution (Hunt & Dalgleish, 1994). However, there seems to be a distinction * Corresponding author. Tel.: C34 5 4556446; fax: C34 5 4557134. E-mail address: [email protected] (J.M.R. Patino). 0268-005X/$ - see front matter q 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodhyd.2004.10.006
between caseinate and mixtures of purified individual caseins, since the latter show competitive adsorption between b-, and as1-casein (Dickinson, 1994; Dickinson, Rolfe, & Dalgleish, 1988; Fan & Dalgleish, 1993). Caseinate gives adsorbed layers similar to those measured for b-casein, the individual casein that shows the highest surface activity of the four individual proteins in caseinate (Fan & Dalgleish, 1993). On the other hand, in many food formulations the caseins are not the only emulsifier present, because small molecule emulsifiers (monoglycerides, phospholipids, Tweens, etc.) are also incorporated into the formulation. The small molecule emulsifiers can faster cover the interface than caseins do, resulting in an emulsion with smaller particles, leading to greater stability. However, more important in some products is the effect of the small molecules in destabilising the emulsion (Goff & Jordan, 1989). In the formulation of ice cream the small molecule emulsifier is added to break the adsorbed layer of protein and allow the adsorption of the fat to the surface of the air bubble.
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Thus, an important action of the small molecule emulsifiers is to promote the displacement of caseins from the interface. The aim of this work is to perform a comparative study of the long-term relaxation phenomena (desorption, collapse, conformation and/or reorganization changes, etc.) and surface dilatational characteristics in caseinate and monoglyceride (monopalmitin, monoolein and monolaurin) mixed monolayers spread at the air–water interface as a function of processing variables (surface composition and surface pressure) and aqueous phase pH. Non-equilibrium processes occurring in systems containing fluid interfaces with a surfactant present are of great practical significance for emulsification and foaming, both in technology and in natural phenomena (Rodrı´guez Patino, Rodrı´guez Nin˜o, & Carrera, 2003a). This work is an extension of previous studies on relaxation phenomena (Carrera, Rodrı´guez Nin˜o, & Rodrı´guez Patino, 1999; Rodrı´guez Nin˜o, Carrera, & Rodrı´guez Patino, 1999) and surface dilatational characteristics (Rodrı´guez Patino, Carrera, Rodrı´guez Nin˜o, & Cejudo, 2001a,b; Rodrı´guez Patino, Rodrı´guez Nin˜o, & Carrera, 2002, 2003b) of monoglyceride and milk proteins. The study has been centred on a real protein (caseinate), to see if the relaxation phenomena observed in protein– monoglyceride mixed films with a model protein (b-casein) are generic.
2. Experimental 2.1. Chemicals Synthetic 1-monohexadecanoyl-rac-glycerol (monopalmitin, DIMODANR PA 90), 1-mono (cis-9-octadecanoyl) glycerol (monoolein, RYLOTM MG 19), and 1-monododecanoyl-rac-glycerol (monolaurin, DIMODANR ML 90) were supplied by Danisco Ingredients with over 95–98% of purity. Caseinate (a mixture of z38% b-casein, z39% as1-casein, z12% k-casein, and z11% as2-casein) was supplied and purified from bulk milk from Unilever Research (Colworth, UK). The sample was stored below 0 8C and all work was done without further purification. Samples for interfacial characteristics of caseinate films were prepared using Milli-Q ultrapure water and were buffered at pH 5 and 7. Analytical-grade acetic acid, sodium acetate, and Trizma [Tris(hydroxymethyl)aminomethane] for buffered solutions were used as supplied by Sigma (O95%) without further purification. To form the surface film, monoglyceride was spread in the form of a solution, using hexane:ethanol (9:1, v:v) as a spreading solvent. Analytical grade hexane (Merck, 99%) and ethanol (Merck, O99.8%) were used without further purification. The water used as subphase was purified by means of a Millipore filtration device (Milli-Q). All these products were supplied by Sigma (O99.5%). Ionic strength was 0.05 M in all the experiments.
2.2. Surface film balance Measurements of surface pressure (p)-area (A) isotherms and surface relaxation in caseinate–monoglyceride mixed films at the air–water interface were performed on a commercial fully automated Langmuir-type film balance (Lauda, Germany). The method has been described previously for mixed components (Rodrı´guez Patino, Carrera, & Rodrı´guez Nin˜o, 1999a,b). Before each measurement, the film balance was calibrated at 20 8C. Mixtures of particular mass ratios—ranging between 0 and 1, expressed as the mass fraction of monoglyceride in the mixture, X—were studied. To allow the quantitative adsorption of the protein on the interface the monolayer was not under any surface pressure during the spreading process. Thus, the caseinate necessary to form the mixed film should be spread before the monoglyceride. Aliquots of aqueous solutions of caseinate (1.553!10K4 mg/ml) at pH 7 were spread on the interface by means of a micrometric syringe. To allow for spreading, adsorption and rearrangements of the protein, 30 min were allowed to elapse before measurements were taken. The spreading method adopted in these experiments ensured the quantitative spreading of the protein on the interface as was discussed in a previous paper (Rodrı´guez Patino, Rodrı´guez Nin˜o, Carrera, & Cejudo, 2001c). Afterwards, a monoglyceride solution in hexane:ethanol mixture was spread at different points on the caseinate film. The monoglyceride solutions were spread on the subphase by means of a micrometric syringe at 20 8C. To allow for spreading and caseinate–monoglyceride interactions, 30 min were allowed to elapse before compression was performed. To ensure interactions and homogeneity, the mixed film was compressed near the collapse point of the mixture and then expanded immediately to avoid the collapse. After 30 min at the maximum area, a new p–A isotherm was performed. The compression rate was 3.3 cm minK1, which is the highest value for which isotherms were found to be reproducible in preliminary experiments. The experiments (p–A isotherm and relaxation phenomena in mixed films) were measured at least three times. The reproducibility of the results was better than G0.5 mN/m for surface pressure and G0.005 nm 2/molecule for area. All isotherms were recorded continuously by a device connected to the film balance and then analyzed off-line. Two kinds of experiment were used for the analysis of relaxation in caseinate–monoglyceride mixed monolayers. First, the surface pressure (p) is kept constant, and the area A is measured as a function of time. In the second type of experiment, the area is kept constant (at the collapse) and the surface pressure decreases. This decrease is measured as a function of time. Various relaxation mechanisms can be fitted to the results derived from the above-mentioned experiments. Desorption of a spread monolayer at any constant surface pressure involves two stages (Ter Minassian-Saraga, 1956). The first is dissolution into the bulk aqueous phase to
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form a saturated aqueous layer. During the initial non-steadystate period of desorption, the rate of monolayer molecular loss can be expressed by Eq. (1). The second stage occurs when, 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. (2). KLog ðA=A0 Þ Z A1 q1=2
(1)
KLog ðA=A0 Þ Z A2 q
(2)
where A and A0 are the molecular area at time q and at the initial moment, and coefficients A1 and A2 account for the rate of dissolution and diffusion, 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 due to a macroscopic film fracture, or by a process of nucleation and the growth of bulk surfactant fragments, or formation of lenses, whenever a characteristic pe surface pressure is exceeded (Carrera et al., 1999; Smith & Berg, 1980). The modelling of monolayer collapse by homogeneous nucleation and the growth of bulk surfactant nuclei can be analysed by applying the Prout-Tompkins (1944) equation to the data derived from relaxation experiments at constant surface collapse area Log
p0 K p Z C1 Log q C C2 p
(3)
where p and p0 are the surface pressures at time q and at the initial moment, respectively, and C1 and C2 are coefficients which depend on the experimental conditions. For proteins, various relaxation mechanisms ascribed to desorption or collapse of spread monolayer were applied to the results derived from relaxation data (Rodrı´guez Nin˜o, Carrera, & Rodrı´guez Patino, 1999). However, the best fit of the results was obtained by means of two exponential equations, Eqs. (4) and (5), for relaxation kinetics at constant surface pressure and at constant surface area, respectively. These relaxation phenomena were associated with conformation–organization change and hydrophilic group hydration (Rodrı´guez Nin˜o et al., 1999). ðA=A0 Þ Z ðA=A0 Þ0 C P1 eðKq=t1 Þ C P2 eðKq=t2 Þ
397
2.3. Brewster angle microscopy A commercial Brewster angle microscope (BAM), BAM2, manufactured by NFT (Go¨ttingen, Germany) was used to study the topography and reflectivity of the monolayer. BAM, coupled with relaxation in surface pressure (p), at constant area, or relaxation in area (A), at constant surface pressure, was used in this work to visualize and determine structural changes during relaxation phenomena of mixed emulsifier monolayers at the air–water interface. Further characteristics of the device and operational conditions have been described elsewhere (Rodrı´guez Patino, Carrera, & Rodrı´guez Nin˜o, 1999c,d). The relative film reflectivity can be measured by determining the light intensity at the camera and analysing the polarization state of the reflected light through the method described elsewhere. In the experiments presented here, the reflectivity for the protein film was relatively high and that for monoglyceride films relatively low. 2.4. Surface dilatational rheology To obtain surface rheological parameters—such as surface dilatational modulus, elastic and viscous components, and loss angle tangent—a modified Wilhelmy-type film balance (KSV 3000) was used as described elsewhere (Rodrı´guez Patino et al., 2001a,b). In this method the surface is subjected to small periodic sinusoidal compressions and expansions by means of two oscillating barriers at a given frequency (u) and amplitude (DA/A) and the response of the surface pressure is monitored. Surface pressure was directly measured by means of two roughened platinum plates situated on the surface between the two barriers. The surface dilatational modulus derived from the change in surface pressure resulting from a small change in surface area may be described by the equation (Lucassen & Van den Tempel, 1972): E ZK
dp Z Ed C iEv d ln A
(6)
The dilatational modulus is a complex quantity and is composed of real and imaginary parts. The real part of the dilatational modulus or storage component is the dilatational elasticity, EdZjEjcos q. The imaginary part of the dilatational modulus or loss component is the surface dilatational viscous modulus, EvZjEjsin q. The loss angle tangent can be defined by Tan qZEv/Ed. Measurements were performed at least three times. The reproducibility of these results was better than 5%.
(4) 3. Results and discussion
ðp=p0 Þ Z ðp=p0 Þ0 C P3 e
ðKq=t3 Þ
C P4 e
ðKq=t4 Þ
(5)
where (A/A0)0 and (p/p0)0 are the amplitude of the relative area and relative surface pressure at the initial moment, respectively, t1 to t4 are the relaxation times and P1 to P4 are constants.
3.1. Relaxation phenomena during the compression– expansion of the monolayer The relaxation phenomena during the compression– expansion cycle of caseinate–monopalmitin mixed films
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Fig. 1. Compression–expansion cycles for caseinate–monopalmitin mixed monolayers on water at pH 7. Temperature 20 8C. Monolayer composition (mass fraction of monopalmitin in the mixture): (A) 0.2, (B) 0.4, (C) 0.6, and (D) 0.8. (,) Compression and (B) expansion for a first cycle at 30 min after spreading, at the maximum area. (—) Compression and ($$$$) expansion for a second cycle at 30 min after the first one, at the maximum area. (6) Compression for a third cycle at 30 min after the relaxation experiment at constant surface pressure (pZ20 mN/m), at the maximum area.
(as an example) are presented in Fig. 1. It can be seen that there exists a hysteresis in p–A isotherms during the compression–expansion cycle, which is more evident at the higher caseinate concentration in the mixed film. This is an indication of the importance of the caseinate content in the mixture in the dynamic relaxation phenomena. The higher the caseinate/monopalmitin ratio in the mixture the higher the hysteresis becomes. At the higher content of monopalmitin in the mixture (Fig. 1D) the hysteresis in the p–A isotherm was the same as for pure monopalmitin monolayer. Caseinate–monoolein mixed monolayers behaved in a similar way (data not shown), but the magnitude of the hysteresis is lower than for caseinate–monopalmitin mixed monolayers. Further information about the mechanism or mechanisms that control the relaxation phenomena in caseinate–monoglyceride mixed films can be deduced from the time evolution of relative reflectivity during a compression– expansion cycle for a caseinate–monopalmitin (Fig. 2) or a caseinate–monoolein (Fig. 3) mixed monolayer at two representative mass fractions of monoglyceride in the mixture of 0.2 and 0.8 (as two examples). At surface pressures lower than that for the caseinate collapse
(py29 mN/m), monopalmitin in the mixture has a liquidcondensed structure (LC)—which can be deduced from the existence of the characteristic main transition between the liquid-expanded (LE) and LC phases (Fig. 1) and from the reflectivity peaks (Fig. 2). This main transition is hindered as the content of caseinate in the mixture increases. Thus, at XZ0.2 the main transition between the LE and LC phases was not observed in the p–A isotherm (Fig. 1A). On the other hand, the presence of caseinate in the mixture can also be deduced because some peaks have a reflectivity similar to that for pure caseinate. From these results some differences can be deduced as compared to those recorded for caseinate–monoolein mixed monolayers (Fig. 3). Firstly, monoolein in the mixture (and caseinate as well) has a LE structure at every surface pressure. Secondly, over the overall range of surface pressures the I–p plots for caseinate–monoolein mixed films were practically the same as those for pure monoolein (Rodrı´guez Patino et al., 1999c), which strengthens the conclusion that monoolein, with its lower relative thickness, predominates at the interface. However, at higher surface pressures, near to and after the caseinate collapse, the relative intensity of some spots was the same as for pure caseinate.
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Fig. 2. The time evolution of (6 and B) relative reflectivity (at a shutter speed of 1/250 s) during a compression–expansion cycle for caseinate– monopalmitin mixed monolayers on water at pH 7 and at 20 8C. Monolayer composition (mass fraction of monoglyceride in the mixture): (a, 6) 0.2 and (b, B) 0.8. The vertical arrows indicate the time at which the measure surface pressure is equal to the equilibrium surface pressures of caseinate (pe, CS) and monopalmitin (pe, MP). The coexistence of caseinate and monopalmitin (A), the squeezing out phenomena of caseinate by monopalmitin (B), the prevalence of monopalmitin at the interface (C), and the re-adsorption of caseinate at the interface (D) are also indicated. The vertical lines in plots (a) and (b) indicate the point at which the monolayer collapses.
The higher hysteresis in p–A isotherm during the compression–expansion cycle, at the higher concentrations of caseinate in the mixture, may be associated with the readsorption of the protein at the interface during the expansion, after the squeezing out phenomena observed during the compression of the monolayer, a phenomenon which involves some time. This hypothesis is corroborated by the fact that the I–p plots during the compression– expansion cycle are not symmetrical (Figs. 2 and 3). The presence of numerous and more intense reflectivity peaks of collapsed caseinate are visible at the higher content of caseinate in the mixture (Figs. 2A and 3A). These data also support the hypothesis that the hysteresis is due to molecular
399
Fig. 3. The time evolution of (6 and B) relative reflectivity (at a shutter speed of 1/250 s) during a compression–expansion cycle for caseinate– monoolein mixed monolayers on water at pH 7 and at 20 8C. Monolayer composition (mass fraction of monoglyceride in the mixture): (A, 6) 0.2 and (B, B) 0.8. The vertical arrows indicate the time at which the measure surface pressure is equal to the equilibrium surface pressures of caseinate (pe, CS) and monoolein (pe, MO). The coexistence of caseinate and monoolein (A), the squeezing out phenomena of caseinate by monoolein (B), the prevalence of monoolein at the interface (C), and the re-adsorption of caseinate at the interface (D) are also indicated. The vertical lines in plots (a) and (b) indicate the point at which the monolayer collapses.
reorganisation. But, these changes are more drastic for caseinate–monopalmitin than for caseinate–monoolein mixed films because monopalmitin in the mixture presents more changes in the monolayer structure, a phenomenon that explains the higher degree of hysteresis in monopalmitin–caseinate mixed films. The relaxation phenomena in caseinate–monoglyceride (monopalmitin or monoolein) mixed films are reversible processes, as can be deduced from data presented in Fig. 1 for caseinate–monopalmitin mixed films (as an example). It can be seen that the p–A isotherms are repetitive after different compression–expansion cycles. Monolaurin–caseinate mixed films behaved in a different way (Fig. 4). The hysteresis in the p–A isotherm during a compression–expansion cycle is higher (data not shown)
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films. These results support evidence of the importance of the monolaurin molecular loss, as compared with the structural changes that take place in monolaurin and caseinate molecules present in the mixed films during the relaxation process. The instability of mixed films can be also deduced from the displacement towards the p-axis of the p–A isotherm after continuous compression–expansion cycles (Fig. 4A). The displacement of the p–A isotherms after continuous compression–expansion cycles is irreversible because the isotherm does not return to its original state on recompression after a waiting period of 24 h at the maximum area at pZ0 (data not shown). 3.2. Long-term relaxation phenomena at constant surface pressure 3.2.1. Relaxation phenomena in monopalmitin– and monoolein–caseinate mixed films Fig. 5 shows the relaxation in relative molecular area at 20 8C and at 20 mN/m for caseinate–monopalmitin (Fig. 5A) and caseinate–monoolein (Fig. 5B) mixed films, at pH 7 as an example (caseinate–monoglyceride mixed
Fig. 4. (A) p–A isotherms (compression curves) for (B) caseinate and a caseinate–monolaurin mixed at a mass fraction of monolaurin in the mixture of 0.5: (—) first compression at 30 min after spreading, at the maximum area, (–-–-–) second compression after 30 min after the first one, and ($$$$) third compression after the relaxation experiment at constant surface pressure (pZ20 mN/m). (B) Area relaxation at constant surface pressure (pZ20 mN/m) for (B) caseinate, ($) monolaurin, and (—) caseinate–monolaurin mixed film at a mass fraction of monolaurin in the mixture of 0.5. (C) Relaxation of surface pressure (B) and relative surface pressure (—) at constant surface area (at the collapse point) for caseinate– monolaurin mixed monolayers at a mass fraction of monolaurin in the mixture of 0.5. pH 7. Temperature 20 8C. The equilibrium surface pressure for monolaurin (pe ML) is indicated by means of an arrow.
than for monopalmitin– and monoolein–caseinate mixed films. In addition, the hysteresis in the p–A isotherm during a compression–expansion cycle is higher as the content of monolaurin in the mixture increases (data not shown), a phenomenon opposite to that observed for monopalmitin— (Fig. 1) and caseinate–monoolein (data not shown) mixed
Fig. 5. Relaxation at constant surface pressure (pZ20 mN/m) of (A) caseinate–monopalmitin and (B) caseinate–monoolein mixed monolayers on water at pH 7. Temperature 20 8C. Monolayer composition (mass fraction of monopalmitin or monoolein in the mixture): (B) 0, (—) 0.2, (– – –) 0.4, (..) 0.6, (–$–) 0.8, and (6) 1.
C.C. Sa´nchez et al. / Food Hydrocolloids 19 (2005) 395–405 Table 1 Characteristic parameters for desorption (Eqs. (1) and (2)) of monoglycerides and a caseinate–monolaurin mixed film at XMLZ0.5, at constant surface pressure (pZ20 mN/m) and at 20 8C and at pH 7
Monopalmitin Monoolein Monolaurin Caseinate–monolaurin XMLZ0.5
A1!103 (LR) (minK0.5)
A2!105 (LR) (minK1)
7.0 (0.996) 7.0 (0.997) 90 (0.999) 20.2 (0.996)
16 (0.983) 28 (0.989) 243 (0.938) 3.2 (0.915)
films at pH 5 behaved in a similar way). For pure monopalmitin and monoolein monolayers the relaxation phenomena can be quantified (Table 1) by a desorption mechanism in two steps (Carrera et al., 1999): dissolution (Eq. (1)) and diffusion into the aqueous phase (Eq. (2)). For caseinate, caseinate–monopalmitin, and caseinate– monoolein mixed films the (A/A0)-relaxation has an exponential time-dependence. The best fits of the experimental data were obtained by means of Eq. (4) with two relaxation times. In Table 2 the relaxation times and the value of relative area at 60 min of relaxation time, (A/A0)60, are shown. These phenomena may be due, among other things, to reorganization changes between caseinate native molecules and caseinate molecules in various degrees of unfolding after the compression up to 20 mN/m. The relaxation phenomena at 20 mN/m for caseinate– monoglyceride mixed films (Fig. 5) are reversible processes, as can be deduced from data presented in Fig. 1 for caseinate–monopalmitin mixed films (as an example). It can be seen that the p–A isotherms are repetitive after different compression–expansion cycles, the last one being registered after the relaxation experiment at 20 mN/m. These data confirm the hypothesis that the relaxation phenomena observed with caseinate–monoglyceride mixed monolayers at constant surface pressures lower than pe could be related to the kinetics of the molecular reorganisation.
Table 2 Characteristic parameters (Eq. (4)) for relaxation of caseinate–monopalmitin and caseinate–monoolein mixed films at constant surface pressure (pZ 20 mN/m) and at 20 8C and at pH 7
Caseinate–monopalmitin
Caseinate–monoolein
X
t1 (min)
t2 (min)
(A/A0)60
0 0.2 0.4 0.6 0.8 1.0 0 0.2 0.4 0.6 0.8 1.0
9.0 9.6 7.1 5.0 5.4 – 9.0 3.8 7.1 7.1 9.0 –
66 234 63 52 60 – 66 67 64 160 2206 –
0.92 0.93 0.94 0.93 0.90 – 0.92 0.91 0.93 0.93 0.96 –
401
3.2.2. Relaxation phenomena in caseinate–monolaurin mixed films Fig. 4B shows the relaxation in relative molecular area at 20 8C and at 20 mN/m for caseinate–monolaurin mixed films at a mass fraction of monolaurin in the mixture of 0.5 (as an example). The relaxation phenomena are more pronounced for these systems than for caseinate–monopalmitin (Fig. 5A) and for caseinate–monoolein (Fig. 5B) mixed films. Unlike monopalmitin– and monoolein–caseinate mixed films, a mechanism with two steps in accord with Eqs. (1) and (2) fits the data better. Thus the phenomena of monolayer relaxation may be due to a desorption mechanism by dissolution and diffusion. However, we do not reject the concurrence of this phenomenon with the organization/rearrangement of the aminoacids segments of caseins forming caseinate into the underlying aqueous phase, as discussed in previous sections. The characteristic parameters for destabilisation of caseinate– monolaurin monolayer are included in Table 1. It can be seen that the magnitude of the rates of dissolution (coefficient A1) and diffusion (coefficient A2) is higher for pure monolaurin than for the mixed film. That is, the presence of caseinate in the mixture produces a reduction of the magnitude of the relaxation phenomena in the mixed film. 3.3. Long-term relaxation phenomena at constant surface area (at the collapse point) 3.3.1. Relaxation phenomena in monopalmitin– and monoolein–caseinate mixed films Monopalmitin– and monoolein–caseinate mixed films were also tested under the most adverse conditions, at the maximum superficial density at constant collapse area. Fig. 6A shows the relaxation in surface pressure at constant surface area (at the collapse point) for caseinate–monopalmitin mixed films. It can be seen that monopalmitin and caseinate–monopalmitin mixed films relaxed towards the equilibrium surface pressure (pe) of pure monopalmitin (Carrera et al., 1999), which is indicated in Fig. 6A by means of an arrow. The (p/p0)-relaxation has a logarithmic time-dependence instead of an exponential dependency. In fact, processing the experimental data according to the Prout-Tompkins equation (Eq. (3)) showed that it was possible to obtain a linear fit (Table 3). This indicates that the relaxation phenomena under these conditions may be due to nucleation and growth of critical nuclei of monopalmitin. On the other hand, the magnitude of relaxation in surface pressure is practically the same for all mixed films. Fig. 6B shows the relaxation in surface pressure at constant surface area (at the collapse point) for caseinate– monoolein mixed films. Caseinate–monoolein monolayers behave differently to caseinate–monopalmitin monolayers. It can be seen that in experiments at constant collapse area, the surface pressure relaxes from the collapse value for pure
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402
Table 4 Characteristic parameters (Eq. (5)) for relaxation of caseinate–monoolein mixed films at constant area (at the collapse point) at 20 8C and at pH 7 XMO
t3 (min)
t4 (min)
(p/p0)60
0 0.2 0.4 0.6 0.8 1.0
0.82 21 72 10 59 –
24 102 217 326 228 –
0.92 0.85 0.89 0.81 0.89 0.62
On the other hand, the magnitude of relaxation in surface pressures depends on the monolayer composition. The reason for these behaviours must be associated with the immiscibility between both components (monoglycerides and caseinate) at the air–water interface, as was deduced for these systems at the collapse point (Rodrı´guez Patino, Carrera, & Rodrı´guez Nin˜o, 2001d). In fact, at surface pressures higher than that for the caseinate collapse a squeezing out of caseinate by monoglyceride is produced (the results in Figs. 2 and 3 are an example of this behaviour). The displacement of caseinate by monoglyceride progresses with surface pressure up to the collapse point. Thus, at the collapse point the mixed film is dominated by the presence of monoglyceride.
Fig. 6. Relaxation at constant area (at the collapse point) for (A) caseinate–monopalmitin and (B) caseinate–monoolein mixed monolayers on water at pH 7. Temperature 20 8C. Monolayer composition (mass fraction of monoglyceride in the mixture): (6) 0, (—) 0.2, (– – –) 0.4, (..) 0.6, (–$–) 0.8, and (6) 1. The equilibrium surface pressures for monopalmitin (pe, MP), monoolein (pe, MO), and caseinate (pe, CS) are indicated by means of arrows.
monoolein, which is close to pe (see arrow in Fig. 6B), towards lower p values at longer times. This behaviour could be assigned to different phenomena, such as desorption and collapse occurring concurrently, as observed for a pure monoolein monolayer (Carrera et al., 1999). These hypotheses can be supported by the quantification of the (p/p0)-relaxation by means of an exponential dependency (Eq. (5)), with two relaxation times (Table 4). Table 3 Characteristic parameters (Eq. (3)) for relaxation of caseinate–monopalmitin mixed films at constant area (at the collapse point) and at 20 8C and at pH 7 XMP
C1 (LR)
C2 (LR)
(p/p0)60
0 0.2 0.4 0.6 0.8 1.0
– 0.10 (0.994) 0.13 (0.995) 0.18 (0.972) 0.09 (0.996) 0.38 (0.999)
– – – – – 0.19 (0.996)
– 0.94 0.92 0.88 0.93 0.85
3.3.2. Relaxation phenomena in caseinate–monolaurin mixed films Fig. 4C shows the relaxation in surface pressure at constant surface area (at the collapse point) for caseinate– monolaurin mixed films at a mass fraction of monolaurin in the mixture of 0.5, as an example. It can be seen that in experiments at constant collapse area, the surface pressure relaxes from the collapse value to very low surface pressures. However, important differences with monopalmitin– and monoolein–caseinate mixed films can be observed. Firstly, the magnitude of the relaxation in surface pressure is higher for monolaurin—than for monopalmitin– and monoolein–caseinate mixed films. Secondly, the (p/p0)-relaxation has a logarithmic time-dependence as for caseinate–monopalmitin mixed films, but this dependence cannot be associated with a mechanism of collapse by nucleation and growth of nuclei of monolaurin because the surface pressure decreases to values lower than pe for monolaurin. Clearly, the desorption of monolaurin, which is practically instantaneous for pure films at the collapse point (Carrera et al., 1999), has an important role in the relaxation phenomena observed in the mixed films (Fig. 4C). 3.4. Surface dilatational characteristics of caseinate– monoglyceride mixed films at the air–water interface 3.4.1. The effect of surface pressure The surface dilatational moduli of monopalmitin– and monoolein–caseinate mixed films spread on the air– water interface at pH 7 (as an example) are shown in
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403
Fig. 7. Surface pressure dependence of (A) surface dilatational modulus (E, mN/m) and (B) loss angel tangent (Tan q) for caseinate–monopalmitin mixed films at 20 8C and at pH 7. Amplitude of the area deformation 5%. Frequency 50 mHz. Mass fraction of monopalmitin in the mixture: (!) 0, (6) 0.2, (7) 0.4, ($) 0.6, (B) 0.8, and (,) 1.0.
Figs. 7A and 8A, respectively. For caseinate monolayer the E–p plot showed a typical irregular shape (Rodrı´guez Patino et al., 2001a). It can be seen that E increased with increasing p to a maximum value at py10 mN/m. Upon further increase of the surface pressure E decreased to a minimum at py20 mN/m. Afterwards, E increased again with surface pressure. The pH effect on the surface dilatational modulus is lower, and practically insignificant (data not shown), as compared with the surface pressure effect. For pure monoglyceride monolayer the surface dilatational modulus also depends on the monolayer structure. The more condensed the structure is (at higher p), the higher the surface dilatational modulus of the monolayer becomes until the collapse is reached, which is consistent with an increase in the forces of interaction between molecules at the interface. However, the surface dilatational modulus of monoolein spread monolayer (Fig. 8A) is lower than that for monopalmitin (Fig. 7A). That is, for the more condensed monopalmitin monolayer E is higher than for the more expanded monoolein monolayer, at every surface pressure.
For caseinate–monoglyceride mixed films the surface pressure dependence of E (Figs. 7A and 8A) depends on the surface pressure and the interfacial composition. At surface pressures lower than that for caseinate collapse and at XMP or XMO!0.8, the same irregular shape in the p dependence of E, as for pure caseinate, was observed for the mixed films. At surface pressures higher than that for caseinate collapse the E–p and for mixed films were parallel to that of monoglyceride, which demonstrated again that at higher surface pressures the mixed films were practically dominated by monoglyceride molecules. However, the data in Figs. 7A and 8A also demonstrate that the small amounts of caseinate collapsed residues at the interface—as deduced at a microscopic level from I–p plots (Figs. 2 and 3)—have an effect on the surface dilatational properties of the mixed films. In fact, the values of E for mixed films are lower than that for a pure monoglyceride monolayer, even at the collapse point of the mixed films. Thus, the mechanical properties of the mixed films also demonstrated that, even at the highest surface pressure, a monopalmitin monolayer is unable to displace caseinate molecules completely from
Fig. 8. Surface pressure dependence of (A) surface dilatational modulus (E, mN/m) and (B) loss angel tangent (Tan q) for caseinate–monoolein mixed films at 20 8C and at pH 7. Amplitude of the area deformation 5%. Frequency 50 mHz. Mass fraction of monoolein in the mixture: (!) 0, (6) 0.2, (7) 0.4, ($) 0.6, (B) 0.8, and (,) 1.0.
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the air–water interface. From the values of the tangent of the loss angle (Figs. 7B and 8B) it can be concluded that caseinate–monoglyceride mixed films behaved as viscoelastic at every surface pressure. 3.4.2. Effect of frequency Changes in surface dilatational modulus for caseinate– monoglyceride mixed monolayers at pH 7 (as a example) and at the collapse point, as a function of frequency of oscillation over a range of 1–200 mHz, are illustrated in Fig. 9. We did not observe any difference in the frequency dependence of surface rheological parameters at pH 5 (data not shown). It can be seen that in the range 1 mHz!u!50 mHz, the dilatational modulus increased with the frequency, but at higher frequencies E is practically constant. The frequency dependence of E in the frequency range of 1–50 mHz, should be associated with the monoglyceride monolayer collapse. This long-term relaxation process requires (Fig. 6) a time of the same order of magnitude as the scale of the oscillation— in our experiments between 2 and 25 min. Again, the reason
for this behaviour must be associated with the immiscibility between both components at the air–water interface. At higher surface frequencies (50!u!200 mHz), the viscoelastic behaviour of the mixed monolayer is more complex and should be associated with formation/destruction of phase transitions and the formation/destruction of 3D collapse structures in the monoglyceride monolayer (Figs. 1–3). We do not reject the possibility that islands of collapsed caseinate could be displaced and reabsorbed within the interface during the sinusoidal compression– expansion cycles. In fact, the time required for organization/ reorganization changes in monoglyceride monolayer structure is of the same order of magnitude as the time scale of the deformation (Fig. 6). However, the surface dilatational properties at the higher frequencies (at uO100 mHz) may have been subjected to errors due to artefacts of the technique, resulting in an artificially high viscous effect as a consequence of the resonance in the flow of the liquid between the two barriers. In addition, shear effects between monolayer molecules and the trough wall or the Wilhelmy plate should also have a role (Petkov, Gurkov, Cambell, & Borwankar, 2000; Wijmans & Dickinson, 1998).
4. Conclusions
Fig. 9. Frequency dependence of surface dilatational modulus for (A) caseinate–monopalmitin and (B) caseinate–monoolein mixed films at the collapse point. Temperature 20 8C. pH 7. Amplitude of the area deformation 5%. Mass fraction of monoglyceride in the mixture: (!) 0, (6) 0.2, (7) 0.4, ($) 0.6, (B) 0.8, and (,) 1.0.
This article presents experimental studies of relaxation phenomena in caseinate–monoglyceride mixed films at the air–water interface. Data have been analysed according to models for desorption, collapse and/or organization/reorganization changes, during a compression–expansion cycle, after the monolayer compression to a surface pressure of 20 mN/m or at the collapse area of the mixed film. During the compression–expansion cycle and at a constant surface pressure (at 20 mN/m), below the caseinate collapse, the organization/reorganization changes of caseinate molecules in monopalmitin– and monoolein–caseinate mixed films are the mechanisms that control the relaxation process. At constant surface area (at the collapse point of the mixed film) the relaxation phenomena in mixed films may be due either to nucleation and growth of critical nuclei of monoglyceride—such as was deduced for caseinate–monopalmitin mixed films—or to a complex mechanism including competition between collapse and monolayer desorption—such as was deduced for caseinate–monoolein mixed films. For caseinate–monolaurin mixed films at every surface pressure the relaxation phenomena are mainly due to the irreversible loss of monolaurin molecules by desorption into the bulk aqueous phase. Comparing the data presented in this paper to those for b-casein–monoglyceride mixed films (Rodrı´guez Patino et al., 2003b) at pH 5 and 7 we conclude that the relaxation phenomena observed with protein (either a model b-casein or a real caseinate protein) and monoglyceride mixed films appear to be generic. These relaxation phenomena were also observed for protein–monoglyceride mixed films with
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disordered b-casein (Rodrı´guez Patino et al., 2003b) and caseinate (this work) and globular (Rodrı´guez Patino et al., 2002) proteins. The reasons for these behaviours must be associated with the immiscibility between protein and monoglyceride at the air–water interface and to the protein displacement by the monoglyceride at surface pressures higher than that for the protein collapse. These relaxation phenomena have significant repercussion on surface dilatational properties. That is, the surface dilatational characteristics reflect the complex relaxation phenomena that take place in protein–monoglyceride mixed films.
Acknowledgements This research was supported by CICYT through grant AGL2001-3843-C02-01.
References Carrera, C., Rodrı´guez Nin˜o, Ma. R., & Rodrı´guez Patino, J. M. (1999). Relaxation phenomena in monoglyceride films at the air–water interface. Colloids and Surfaces B: Biointerfaces, 12, 175–192. Dickinson, E. (1994). Protein-stabilized emulsions. Journal of Food Engineering., 22, 59–74. Dickinson, E., Rolfe, S. E., & Dalgleish, D. G. (1988). Competitive adsorption of as1-casein and b-casein in oil-in-water emulsions. Food Hydrocolloids, 2, 397–405. Fan, Y., & Dalgleish, D. G. (1993). Dimensions of the adsorbed layers in oil-in water emulsions stabilized by caseins. Journal of Colloid Interface Science, 156, 329–334. Goff, H. D., & Jordan, W. K. (1989). Action of emulsifiers in promoting fat destabilization during the manufacture of ice cream. Journal of Dairy Science, 72, 18–29. Hunt, J. A., & Dalgleish, D. G. (1994). Adsorption behaviour of whey protein isolate and caseinate in soya oil-in-water emulsions. Food Hydrocolloids, 8, 175–187. Lucassen, J., & Van den Tempel (1972). Dynamic measurements of dilational properties of a liquid interface. Chemical Engineering Science, 27, 1283–1291. Petkov, J. T., Gurkov, T. D., Cambell, B. E., & Borwankar, R. P. (2000). Dilatational and shear elasticity of gel-like protein layers on air/water interface. Langmuir, 16, 3703–3711. Prout, E. G., & Tompkins, F. C. (1944). The thermal decomposition of potassium permanganate. Transaction of the Faraday Society, 40, 488–498.
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Rodrı´guez Nin˜o, Ma. R., Carrera, C., & Rodrı´guez Patino, J. M. (1999). Interfacial characteristics of b-casein films at the air–water interface. Colloids and Surfaces B: Biointerfaces, 12, 161–173. Rodrı´guez Patino, J. M., Carrera, C., & Rodrı´guez Nin˜o, Ma. R. (1999a). Analysis of b-casein–monopalmitin mixed films at the air–water interface. Journal of Agricultural Food Chemistry, 47, 4998–5008. Rodrı´guez Patino, J. M., Carrera, C., & Rodrı´guez Nin˜o, Ma. R. (1999b). Is Brewster angle microscopy a useful technique to distinguish between isotropic domains in b-casein–monoolein mixed monolayers at the air– water interface? Langmuir, 15, 4777–4788. Rodrı´guez Patino, J. M., Carrera, C., & Rodrı´guez Nin˜o, Ma. R. (1999c). Morphological and structural characteristics of monoglyceride monolayers at the air–water interface observed by Brewster angle microscopy. Langmuir, 15, 2484–2492. Rodrı´guez Patino, J. M., Carrera, C., & Rodrı´guez Nin˜o, Ma. R. (1999d). Structural and morphological characteristics of b-casein monolayers at the air–water interface. Food Hydrocolloids, 13, 401–408. Rodrı´guez Patino, J. M., Carrera, C., Rodrı´guez Nin˜o, Ma. R., & Cejudo, M. (2001a). Structural and dynamic properties of milk proteins spread at the air–water interface. Journal of Colloid Interface Science, 242, 141–151. Rodrı´guez Patino, J. M., Carrera, C., Rodrı´guez Nin˜o, Ma. R., & Cejudo, M. (2001b). Structural–dilatational characteristics relationships of monoglyceride monolayers at the air–water interface. Langmuir, 17, 4003–4013. Rodrı´guez Patino, J. M., Rodrı´guez Nin˜o, Ma. R., & Carrera, C. (2002). Static and dynamic properties of a whey protein isolate and monoglyceride mixed films at the air–water interface. Industrial and Engineering Chemistry Research, 41, 2652–2661. Rodrı´guez Patino, J. M., Rodrı´guez Nin˜o, Ma. R., & Carrera, C. (2003a). Protein–emulsifier interactions at the air–water interface. Current Opinion in Colloid and Interface Science, 8, 387–395. Rodrı´guez Patino, J. M., Rodrı´guez Nin˜o, Ma. R., & Carrera, C. (2003b). Structure, miscibility, and rheological characteristics of b-casein– monoglyceride mixed films at the air–water interface. Journal of Agricultural and Food Chemistry, 51, 112–119. Rodrı´guez Patino, J. M., Rodrı´guez Nin˜o, Ma. R., Carrera, C., & Cejudo, M. (2001d). The effect of pH on monoglyceride–caseinate mixed monolayers at the air–water interface. Journal of Colloid Interface Science, 240, 113–126. Rodrı´guez Patino, J. M., Rodrı´guez Nin˜o, Ma. R., Carrera, C., & Cejudo, M. (2001c). Whey protein isolate–monoglyceride mixed monolayers at the air–water interface. Morphology, structure, and interactions. Langmuir, 17, 7545–7553. Smith, R. D., & Berg, J. C. (1980). The collapse of surfactant monolayers at the air–water interface. Journal of Colloid Interface Science, 74, 273–273. Ter Minassian-Saraga, L. (1956). Recent work on spread monolayers. Adsorption and desorption. Journal of Colloid Science, 11, 398–418. Wijmans, C. M., & Dickinson, E. (1998). Simulation of interfacial shear and dilatational rheology of an adsorbed protein monolayer modeled as a network of spherical particles. Langmuir, 14, 7278–7286.