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Biopolymers and emulsifiers at the air–water interface. Implications in food colloid formulations
Journal of Food Engineering 67 (2005) 225–234 www.elsevier.com/locate/jfoodeng
Biopolymers and emulsifiers at the air–water interface. Implications in food colloid formulations Cecilio Carrera Sa´nchez, Mª. Rosario Rodrı´guez Nin˜o, Ana Lucero Caro, Juan M. Rodrı´guez Patino * Departamento de Ingeniera Quı´mica, Facultad de Quı´mica, Universidad de Sevilla, C/. Prof. Garcı´a Gonza´lez, 1, 41012 Sevilla, Spain Received 10 October 2003; accepted 1 May 2004
Abstract In this paper we are concerned with adsorption, structure, topography, and dynamic properties (relaxation phenomena and surface dilatational rheology) of food dairy proteins (b-casein, caseinate, and whey protein isolate, WPI), water-insoluble lipids (monopalmitin, monoolein, and monolaurin) and phospholipids (dipalmitoyl-phosphatidyl-choline, DPPC, and dioleoyl-phosphatidyl-choline, DOPC) at the air–water interface. Combined surface chemistry (surface film balance and static and dynamic tensiometry) and microscopy (Brewster angle microscopy, BAM) techniques have been used to determine the static and dynamic characteristics of these emulsifiers and their mixtures at the air–water interface. The derived information shows that biopolymer (proteins) and low-molecular-weight-emulsifier (LMWE, monoglycerides and phospholipids) type and their mixtures affect the interfacial characteristics of adsorbed and spread films. Important functional differences have been established between proteins, lipids and phospholipids. The static and dynamic characteristics of mixed films depend on the interfacial composition and the surface pressure (p). At higher surface pressures, collapsed protein residues may be displaced from the interface by LMWE molecules with important repercussions on the interfacial characteristics of the mixed films. Ó 2004 Elsevier Ltd. All rights reserved. Keywords: Fluid interfaces; Adsorption; Interfacial rheology; Monolayer; Food emulsifier; Biopolymer; Low-molecular weight emulsifier; Milk protein; Monoglyceride; Phospholipid
1. Introduction Food dispersions (emulsions and foams) are complex multicomponent systems containing many biopolymers and low-molecular weight emulsifiers (LMWE) which may show surface activity by themselves or by association with other components (polysaccharides). In addition food dispersions contain many other organic (ethanol, sugars, etc.) and inorganic (salts) components which may interact with biopolymers and LMWE in different complex fashion depending on pH, temperature, processing history, etc., all of which intensify the problems of the manufacturer attempting to control *
Corresponding author. Tel.: +34 954 556446; fax: +34 954 557134. E-mail address: [email protected] (J.M. Rodrı´guez Patino).
0260-8774/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.jfoodeng.2004.05.065
stability, shelf-life, or product texture. For up-to-date reviews, readers are directed to recent references (Damodaran & Paraf, 1997; Friberg & Larsson, 1997; Hartel & Hasenhuette, 1997). Manufacturers employ two types of emulsifiers or foaming agents in food (Dickinson, 1992), namely: LMWE (mainly monoand diglycerides, phospholipids, etc.) and macromolecules (proteins and certain hydrocolloids). The emulsifier film adsorbed at the oil–water or air–water interface is the source of many of the unique properties of food dispersions, particularly their stability and interactions, which translate into the shelf-life and textural properties so desired by manufacturers and appreciated by consumers. Proteins and LMWE have an important physical property in common, their amphiphilic nature (Horne &
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Rodrı´guez Patino, 2003; Rodriguez Nin˜o, Rodrı´guez Patino, Carrera, Cejudo, & Navarro, 2003). This property provides the potential for association, adsorption, and reorientation at fluid interfaces, depending on the properties of the components and the protein–LMWE ratio (Rodrı´guez Nin˜o & Rodrı´guez Patino, 1998a, 1998b; Rodrı´guez Nin˜o, Carrera, Cejudo, & Rodrı´guez Patino, 2001; Rodrı´guez Patino, Rodrı´guez Nin˜o, & Carrera, 2003a). LMWE stabilize the dispersed droplets or bubbles by formation of a densely packed but much less rigid monomolecular layer, which is stabilized by dynamic processes (i.e. Gibbs–Marangoni effect). LMWE adsorb strongly to fluid interfaces giving close molecular packing at the interface to produce low surface and interfacial tensions (Rodrı´guez Nin˜o & Rodrı´guez Patino, 1998a, 1998b). In contrast, proteins act as polymeric emulsifiers with multiple anchoring sites at the interface that, together with the unfolding process of the adsorbing protein molecule, stabilize the interfacial layer kinetically. This behavior contributes significantly to the interfacial rheological properties and immobilizes proteins in the adsorbed layer (Bos, Nylander, Arnebrant, & Clark, 1997). However more important in some products is the effect of the LMWE in destabilizing the emulsion (Goff & Jordan, 1989). In the formulation of ice cream the LMWE (typically, mono- and diglycerides) are added to break the adsorbed layer of protein and allow the adsorption of fat to the surface of the air bubble. Thus, an important action of LMWE is to promote the displacement of proteins (mainly caseins) from the interface. The competitive adsorption and/or displacement between LMWE and proteins at fluid–fluid interfaces have been studied in detail in several investigations (Bos et al., 1997; Nylander, 1998; Wilde, 2000; Dickinson, 2001; Rodrı´guez Patino et al., 2003a). However, so far, little is known about the structure that biopolymers and LMWE adopt at fluid interfaces. This paper will concentrate on the interfacial behavior of milk proteins and LMWE (monoglycerides and phospholipids). Emphasis will be on the air–water interface as a three-dimensional dynamic entity. We will consider emulsifier (protein, LMWE and their mixtures) adsorption, structure and topography at the interface, relaxation phenomena, and interfacial rheology, as related to the formation and stability of food dispersions (emulsions and foams).
2. Experimental 2.1. Chemicals Synthetic 1-monohexadecanoyl-rac-glycerol (monopalmitin, DIMODANR PA 90), 1-mono(cis-9-octadecanoyl)glycerol (monoolein, RYLOTMMG 19), and 1-monododecanoyl-rac-glycerol (monolaurin, DIMO-
DANR ML 90) were supplied by Danisco Ingredients (Braban, Denmark) with over 95–98% of purity. DL -adipalmitoyl-phosphatidyl-choline (DPPC, Sigma (St. Louis, MO, USA), 99%) and L -a-dioleoyl-phosphatidyl-choline (DOPC, Sigma, 99%) were used as supplied. Ninety-nine percent pure b-casein was supplied and purified from bulk milk from the Hannah Research Institute (Ayr, Scotland). Caseinate (a mixture of 38% b-casein, 39% as1-casein, 12% j-casein, and 11% as2-casein) was supplied and purified from bulk milk from Unilever Research Laboratories (Colworth, UK). Whey protein isolate (WPI), a native protein with high content of blactoglobulin (protein 92 ± 2%, b-lactoglobulin >95%, a-lactalbumin <5%) obtained by fractionation, was supplied by Danisco Ingredients (Brabran, Denmark). To form the surface film, monoglyceride and phospholipid were spread in the form of a solution, using hexane:ethanol (9:1, v:v) and chloroform:ethanol (4:1, v:v), respectively, as a spreading solvent. Analytical grade hexane (Merck, 99%), ethanol (Merck, >99.8%), and chloroform (Sigma, 99%), were used without further purification. Samples for interfacial characteristics of protein films were prepared using Milli-Q ultrapure water at pH 7. The water used as subphase was purified by means of a Millipore filtration device, Milli-Q (Milford, MA, USA). To adjust subphase pH, buffer solutions were used. Acetic acid/sodium acetate aqueous solution (CH3COOH/CH3COONa) was used to achieve pH 5, and a commercial buffer solution called trizma ((CH2OH)3CNH2/(CH2OH)3CNH3Cl) for pH 7. All these products were supplied by Sigma (>99.5%). Ionic strength was 0.05 M in all the experiments. 2.2. Methods 2.2.1. Equilibrium surface pressure and adsorption isotherm The equilibrium surface pressure (pe) is a key parameter for the analysis of the mechanisms that trigger the relaxation phenomena in spread monolayers at the air–water interface (Gaines, 1966). The equilibrium spreading pressure is the maximum surface pressure to which a spread monolayer may be compressed before monolayer collapse. Equilibrium surface pressure of protein and LMWE at the air–water interface was measured by the Wilhelmy plate method as described elsewhere (Carrera, Rodrı´guez Patino, & Rodrı´guez Nin˜o, 1999). The adsorption isotherm of proteins and LMWE–protein films was studied by tensiometry as described elsewhere (Rodrı´guez Nin˜o & Rodrı´guez Patino, 1998a; Rodrı´guez Nin˜o et al., 2001). 2.2.2. Surface film balance Measurements of the surface pressure (p) versus average area per molecule (A), the so-called p–A isotherm, were performed on a fully automated Langmuir- or
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Wilhelmy-type film balance as described elsewhere (Rodrı´guez Nin˜o, Carrera, & Rodrı´guez Patino, 1999). 2.2.3. Brewster angle microscopy For microscopic observation of the monolayer structure, the Brewster angle microscope, BAM 2 plus (NFT, Germany) was used as described elsewhere (Rodrı´guez Patino, Carrera, & Rodrı´guez Nin˜o, 1999a, 1999b). To measure the relative thickness of the film a previous camera calibration is necessary in order to determine the relationship between the gray level (GL) and the relative reflectivity (I), according to a procedure described previously (Rodrı´guez Patino et al., 1999a, Rodrı´guez Patino, Carrera, & Rodrı´guez Nin˜o, 1999b). 2.2.4. Surface relaxation measurements Measurements of surface relaxation in protein or LMWE films at the air–water interface were performed on a fully automated Langmuir-type film balance. The method has been described previously (Carrera et al., 1999). 2.2.5. Surface dilatational rheology To obtain surface rheological parameters at the air– water interface––such as surface dilatational modulus (E), elastic (Ed) and viscous (Ev) components, and loss angle tangent (tan h)––a modified Wilhelmy-type film balance (KSV 3000) was used as described elsewhere (Rodrı´guez Patino, Carrera, Rodrı´guez Nin˜o, & Cejudo, 2001c, 2001d).
3. Results and discussion 3.1. Protein and LMWE films at equilibrium Equilibrium spreading pressure of monoglycerides (monopalmitin, and monoolein), phospholipids (DPPC and DOPC) and proteins (b-casein and WPI) at the air–water interface, at pH 7 and at 20 °C is shown in Fig. 1A. The magnitude of pe was dependent on the LMWE and the protein. pe was higher for monoglycerides (Rodriguez Nin˜o et al., 2003) and phospholipids (unpublished data) and lower for proteins (Rodrı´guez Nin˜o et al., 2001). The effect of temperature (data not shown) was also different for LMWE and proteins, especially for an ordered protein (WPI). pe for LMWE and b-casein was not affected by temperature within the range of 5 and 40 °C. However, pe for WPI increased with temperature, especially at temperatures higher than 25 °C. Higher pe values correlate with higher interactions within the film forming components thus, with a more condensed film structure at equilibrium. Protein–LMWE interactions at the air–water interface can be studied by tensiometry. From these experi-
Fig. 1. (A) Equilibrium surface pressure of monopalmitin (MP), monoolein (MO), dipalmitoyl-phosphatidyl-choline (DPPC), dioleoylphosphatidyl-choline (DOPC), b-casein (BC) and WPI at pH 7 and at 20 °C. (B) The effect of spreading of (s) MP, (n) MO and (j) monolaurin (ML) on a film of WPI previously adsorbed on the air– water interface. The arrows indicate the equilibrium surface pressures (pe) for MP, MO and ML. C is the concentration of WPI in the bulk phase. The amount of monoglyceride spread on the WPI film is enough to saturate the monolayer by itself. Temperature 20 °C. pH 5.
ments it has been observed that the interfacial characteristics of mixed protein and LMWE films at air–water interfaces depend at least on the interfacial composition and on the protein/LMWE ratio (Fig. 1B). At higher concentrations of WPI in the bulk phase the surface activity of the mixed film is similar to that for pure WPI while at lower concentrations the surface activity of the mixed film is similar to pe of the monoglyceride (MP or MO). The solubility of monolaurin proves that the mixed film is dominated by the protein within the overall range, because for the monolaurin– protein mixed film the p–log C plot is practically the same as that for pure protein (Fig. 1B). In general, the surface activity of the protein + LMWE mixed films is determined by the LMWE as p of the mixed film is the same as the pe of LMWE and the monolayer is not saturated by the protein (Rodrı´guez Nin˜o & Rodrı´guez Patino, 1998a, 2001). However, the protein deter-
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mines the surface activity of mixed films as the protein saturates the monolayer. 3.2. Structure and topography of protein and LMWE films Structural and topographical characteristics of proteins and LMWE spread films at the air–water interface can be deduced from p–A isotherms (Rodrı´guez Patino & Rodrı´guez Nin˜o, 1999) coupled to information from BAM (Rodrı´guez Patino et al., 1999a, 1999b). From the p–A isotherm, different structures can be deduced for LMWE monolayers as a function of LMWE, temperature, and surface density or surface pressure. For instance, monopalmitin (and most saturated phospholipids) monolayers (Fig. 2) show that liquid expanded (LE), liquid-condensed (LC) and solid (S) structures, and, finally, the collapse at a p higher than pe, take place as a function of surface pressure. In contrast with monopalmitin, monoolein and DOPC monolayers (data not shown) presents only the liquid expanded structure and the collapse at pe. BAM allows direct visualization of changes in morphology and collapse of monopalmitin monolayer (as an example) at the air–water interface (Fig. 2). Monopalmitin monolayer at 10 mN/m shows circular LC domains from the homogeneous ambient phase with a LE structure. The LC domains grow in size and the monolayer is covered with LC domains as p is increased. At the highest p, the LC domains are so closely packed that they occupy the entire field of view, the contrast vanishes suddenly, and the presence of monolayer fractures can be observed in different zones (Rodrı´guez Patino et al., 1999a). BAM images corroborate that only the homogeneous LE phase is present during the compression of a monoolein and DOPC monolayers (data not shown). The evolution with the monolayer compression of the film thickness (d) gives complementary information about the structural characteristics of LMWE during monolayer compression (Rodrı´guez Patino et al., 1999a). The film thickness increases as the monolayer is compressed, passes through a maximum and then decreases at the monolayer collapse point. The evolution of d for saturated LMWE monolayer with monolayer compression also shows important differences with unsaturated LMWE monolayers and their d is lower than for saturated LMWE (Rodrı´guez Patino et al., 1999a). Results of BAM (specially the relative reflectivity) as a function of p obtained with protein monolayers clearly show the same structural characteristics as those deduced from the p–A isotherm (Fig. 2). The domains that residues of protein molecules adopt at the air–water interface appeared to be of uniform reflectivity (Fig. 2), suggesting homogeneity in thickness and film isotropy. The results of p–A isotherms (Fig. 2) confirm that protein monolayers at the air–water interface adopt two
different structures and the collapse phase. As for LMWE, d increases with p and is maximum at the collapse (at the highest p). At p lower than pe, the relative film thickness is independent of the protein but, at the collapse point, d for b-casein is higher than for WPI (Rodrı´guez Patino et al., 1999b). The differences observed between lipids (Rodrı´guez Patino et al., 1999a) and proteins (Rodrı´guez Patino et al., 1999b) in p–A isotherms and BAM images is of great utility for the application of BAM to the analysis of more complicated systems in which proteins and lipids are spread at the interface (Rodrı´guez Patino, Carrera, & Rodrı´guez Nin˜o, 1999c, 1999d; Rodrı´guez Patino, Rodrı´guez Nin˜o, Carrera, & Cejudo, 2001a, Rodrı´guez Patino, Rodrı´guez Nin˜o, Carrera, & Cejudo, 2001b). From a systematic study centered on the p–A isotherm of protein–monoglyceride mixed monolayers–– including the application of the additivity rule on miscibility and the quantification of interactions between monolayer components by excess free energy––it has been concluded that, at a macroscopic level, these compounds form a practically immiscible monolayer at the air–water interface, at p lower than that for the protein collapse (Rodrı´guez Patino et al., 2001a, 2001b) (Fig. 2). At higher p the collapsed protein is displaced from the interface by LMWE. The existence of low protein interactions in disordered proteins (b-casein and caseinate) facilitates the protein displacement by LMWE from the air–water interface. On the other hand, the lower surface activity of unsaturated LMWE explains the fact that this LMWE has a lower capacity than saturated LMWE for protein displacement. Different proteins and LMWE show different interfacial interactions, miscibility and topography, confirming the importance of protein and LMWE structure in determining the mechanism of interfacial interactions (Rodrı´guez Patino et al., 2001a, 2001b). Thus, displacement of proteins by LMWE from the air–water interface depend on the particular protein–LMWE system (Fig. 3). The displacement surface pressure (pd) value for monopalmitin–b-casein mixed films is lower than those for monopalmitin–caseinate and monopalmitin–WPI mixed films. Thus, protein displacement by monopalmitin is easier for b-casein than it is for caseinate and WPI, in this order. In the same order increases the surface elasticity of the protein (see last section). Thus, the more elastic WPI film is more resistant than the less elastic b-casein films. Monoolein has a lower capacity than monopalmitin for protein displacement due to the fact that monoolein requires higher pd for protein displacement. Phospholipids and proteins mixed films behave in a more complicated fashion (unpublished data). Phospholipids and b-casein form a practically immiscible monolayer at the air–water interface on neutral or acidic aqueous subphases. However, some degree of interaction exists between phospholipids and b-casein in the
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Fig. 2. p–A isotherm and visualization by BAM of (n) b-casein, () monopalmitin, and (––) monopalmitin–b-casein mixed film at XMP = 0.5. BAM images of monopalmitin––(a) LE phase at p < 5 mN/m, (b) coexistence of LE and LC domains at 5 mN/m < p < 30 mN/m, (c) LC domains at p > 35 mN/m, and (d) fracture of a collapse monolayer at p ffi pe (monopalmitin)––and saturated LMWE, b-casein––homogeneous topography at (a) p < pe and (b) at p ffi pe––and unsaturated LMWE, and monopalmitin–b-casein mixed monolayer at XMP = 0.5 and at p < pe––(a) segregated LE–LC monopalmitin and b-casein domains, (b) homogeneous LE monopalmitin–b-casein domains, and (c) segregated LC monopalmitin and b-casein domains––at p > pe––(d) region of monopalmitin LC domains, (e) coexistence of monopalmitin and collapse b-casein, and (f) squeezing out of bcasein by monopalmitin––and at the collapse point––(g) a region of collapsed monopalmitin dominates the topography of the interface, (h) fracture of collapsed monopalmitin, and (i) coexistence of collapse monopalmitin and islands of collapsed b-casein.
mixed film and these interactions, of an electrostatic character, are more pronounced at pH 9 as the phospholipid molecules become completely ionized.
Competitive adsorption of proteins and LMWE at fluid interfaces can affect the stability of food dispersions (Bos et al., 1997; Wilde, 2000). Thus, knowledge
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Fig. 3. The displacement surface pressure (pd) of (h) b-casein, (s) caseinate, and (n) WPI by (A) monopalmitin and (B) monoolein from spread mixed monolayers at the air–water interface as a function of the mass fraction of monoglyceride in the mixture. The horizontal lines represent the equilibrium surface pressures of (––, pe MP) monopalmitin, ( , pe MO) monoolein, (- - -, pe BC) b-casein, (- Æ -, pe CS) caseinate, and (- -, pe WPI) WPI pure monolayers at 20 °C and at pH 7.
of the proteins, LMWE, and their mixtures at fluid–fluid interfaces is a key factor for the formation and stability of food dispersions (emulsions and foams). Recent works have allowed the indirect study of competitive adsorption at a molecular level (Gunning, Mackie, Wilde, & Morris, 1999; Mackie, Gunning, Wilde, & Morris, 1999). The Norwich group (Gunning et al., 1999; Mackie et al., 1999) has demonstrated how surfactants disrupt and displace proteins from an interface by a three-stage ‘‘orogenic’’ mechanism. Briefly, emulsifier penetrates into the protein film, forming separate adsorbed domains that exert a lateral p which mechanically compresses the protein network until it fails and is displaced from the interface. The mechanism also reveals that protein adsorbed layers with a high surface elasticity are more resistant to displacement. The BAM images (Fig. 2) appear to support the idea that displacement takes place via an orogenic mechanism, but there exist differences between adsorbable watersoluble emulsifiers (Gunning et al., 1999; Mackie et al., 1999) and spread water-insoluble emulsifiers (Mackie, Gunning, Ridout, Wilde, & Rodrı´guez Patino, 2001; Rodrı´guez Patino et al., 2001a, 2001b). The results suggest that for spread water-insoluble emulsifiers (Fig. 4) the first stage of the orogenic mechanism, which occurs at p lower than pe of the protein, involves a displacement front of emulsifier domains (Fig. 4A) instead of the adsorption of water-soluble surfactant molecules at defects in the protein network. The second stage (Fig.
4B), which occurs at p near to and above pe of the protein, involves a buckling of the monolayer and reordering of the molecules as the protein film gets thicker in response to the decreasing surface coverage. Finally, at sufficiently high p the protein network begins to fail (Fig. 4C), freeing proteins, which then desorb from the interface (Rodrı´guez Patino et al., 2001a, 2001b). But, for spread water-insoluble emulsifier monolayers, the protein displacement is not total even at the highest surface pressure, at the collapse point of the mixed film. The orogenic displacement mechanism is a consequence of the low level of interaction between proteins and LMWE at the air–water interface (Rodrı´guez Patino et al., 2001a, 2001b). This model has been shown to work for a range of proteins with different secondary and tertiary structures and different types of LMWE (non-ionic, cationic, anionic and zwitterionic). This mechanism also explains the destabilization of beer foams by lipids with a range of chain lengths (Wilde et al., 2003). 3.3. Relaxation phenomena in protein and LMWE films Non-equilibrium processes occurring in systems containing fluid interfaces with a surfactant present are of
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Fig. 4. Displacement of proteins by LMWE spread at the air–water interface according to the orogenic mechanism. (A) At p < pprotein a e displacement front of emulsifier domains is produced. (B) At p ffi pprotein a buckling of the protein monolayer and reordering of e the molecules is produced and the protein film gets thicker in response to the decreasing surface coverage. (C) At p > pprotein the protein e network begins to fail and desorbs from the interface, forming collapse protein multilayers in the aqueous bulk phase near to the interface.
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From a practical point of view it must be emphasized that under these conditions the mixed film is more stable in relation to monolayer molecular loss than that of the pure components. At the collapse point of the mixed film, the relaxation phenomena may be due either to nucleation and growth of critical nuclei of monoglyceride or to a complex mechanism including competition between desorption and monolayer collapse. The reasons for these behaviors may be associated again 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 protein collapse. 3.4. Interfacial rheological characteristics of protein and LMWE films The breaking of drops and bubbles during emulsification and foaming requires rapid and substantial stretching of the drops or bubbles, and consequently, the surface tension may be far from equilibrium. Thus, dil-
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great practical significance and include important technological operations such as emulsification and foaming. Two experimental approaches can be used for the analysis of long-term relaxation phenomena in emulsifier monolayers (Gaines, 1966). In a first approach, the surface pressure (p) is kept constant, and the area A is measured as a function of time. In the second approach, area is kept constant (at the monolayer collapse) and the decrease in p is monitored as a function of time. Information on various relaxation paths (Marangoni effect, chemical reaction, polar group hydration, conformation/organization changes, film dissolution by desorption and/or diffusion, collapse, etc.) can be derived from these data (Rodriguez Nin˜o et al., 2003; Rodrı´guez Patino et al., 2003a). Desorption of spread LMWE monolayers at any constant surface pressure, at p < pe, involves two stages (Rodriguez Nin˜o et al., 2003; Rodrı´guez Patino et al., 2003a). The first is dissolution into the bulk aqueous phase to form a saturated aqueous layer. The second stage occurs when, after a time, the concentration gradient within the diffusion layer becomes constant and desorption reaches a steady state. The monolayer molecular loss was lower for saturated than for unsaturated LMWE. At p > pe the relaxation phenomena in LMWE films are due to the transformation of a homogeneous monolayer phase into a heterogeneous monolayer-collapse phase system. However, some differences exist between saturated (monopalmitin or DPPC) and unsaturated (monoolein or DOPC) LMWE monolayers. Relaxation phenomena in saturated LMWE monolayer are controlled predominantly by the collapse mechanism because of the p values relaxed to pe value. For unsaturated LMWE monolayer p relaxed from the collapse value, which is close to pe, towards lower p values at longer times. This phenomenon should be ascribed to the concurrence of different phenomena, such as desorption and collapse. Protein monolayer behaves in a different way to LMWE under the same experimental conditions. At p < pe the relaxation is a reversible process, which include monolayer organization/reorientation. The relaxation in relative molecular area and in surface pressure at p > pe should be attributed to processes related to monolayer organization/reorientation and collapse, respectively. The strength of interactions between protein and LMWE can also be studied by relaxation experiments. Long-term relaxation phenomena in protein–monoglyceride mixed films (Fig. 5) at the air–water interface have been analyzed according to models for desorption, collapse and/or organization/reorganization changes (Rodrı´guez Patino, Rodrı´guez Nin˜o, & Carrera, 2002a, 2002b). At lower surface pressures (i.e., p lower than protein collapse pressure), the organization/reorganization change of protein molecules in the mixed film is the mechanism that controls the relaxation process.
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atational properties of adsorbed emulsifier layers are also important. The viscoelastic properties of the surface have often been correlated with functionality (Bos & van Vliet, 2001; Dickinson, 1999, 2001; Murray, 2002). The ability of the protein to resist displacement by emulsifiers is closely linked to the surface dilatational rheology, whereas the precise form of the displacement is considered to be more closely related to the surface shear behavior (Mackie et al., 2003; Murray, 2002; Roth, Murray, & Dickinson, 2000). Different and complementary interfacial techniques (surface film balance, BAM, and interfacial dilatational rheology) are useful in the analysis of the structural and dynamic characteristics of protein, LMWE, and their mixtures at the air–water interface (Rodrı´guez Patino, Rodrı´guez Nin˜o, & Carrera, 2002b; Rodrı´guez Patino, Rodrı´guez Nin˜o, & Carrera, 2003b). A common trend of the p dependence of dilatational modulus (E) for monopalmitin and monoolein monolayers (Fig. 6) is that E increased with increasing p up to the collapse point. This increase is a result of an increase in the interactions between the monolayer molecules (that is, of its structure), as deduced from p–A isotherms and BAM images (Fig. 2). However, for the more condensed monolayer (monopalmitin) this in-
crease is higher than for the more expanded monoolein monolayer. This indicates that E is not only determined by the interactions between spread monoglyceride (or phospholipid) molecules (which depend on p), but that the structure of the spread molecule also plays an important role. In fact, for the more aggregated monopalmitin molecules in LC domains (see Fig. 2) E is higher than that of monoolein molecules with LE structure, at the same p. As for LMWE, for WPI monolayers (Fig. 6) E increased with increasing p up to the collapse point. This increase is the result of an increase in the interactions between the monolayer molecules, as deduced from p–A isotherms and BAM image. However, for the more disordered proteins (b-casein and caseinate) the E–p dependence is more complex. E increases to a maximum with p, but decreases with p and passes to a minimum. Finally, E increases up to the collapse point (Fig. 6). This inflection in the E–p curve may be attributed to the transition from an ‘‘all-train’’ configuration to a ‘‘train-and-loop’’ conformation of the b-casein molecule (Lucassen-Reynders & Benjamins, 1999). The results with protein monolayers indicate that E is not only determined by the structure of protein molecules, but the internal nature of the protein
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Fig. 6. Surface pressure dependence of surface dilatational modulus for protein + monoglyceride mixed films at the air–water interface at pH 7. (A) b-casein + monopalmitin mixed films. (B) b-casein + monoolein mixed films. (C) WPI + monopalmitin mixed films. (D) WPI + monoolein mixed films. Temperature: 20 °C; frequency: 50 mHz; amplitude: 5%. Monolayer composition (mass fraction of monoglyceride): (s) 0, (n) 0.2, (,) 0.4, (}) 0.6, (+) 0.8, and ( ) 1.0.
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spread molecule also plays an important role. In fact, for the more ordered b-lactoglobulin molecules in WPI E is higher than that for b-casein or caseinate molecules (Fig. 6) with disordered structure, at the same surface pressures. Surface dilatational rheology is a very sensitive technique to analyze the competitive adsorption/displacement of protein and LMWE emulsifier at the air–water interface. At higher p, the collapsed protein residues displaced from the interface by LMWE molecules have important repercussions on the dilatational characteristics of the mixed films (Rodrı´guez Patino et al., 2002b, 2003b). However, the mechanical properties of the mixed films also demonstrate that, even at the highest p, the monoglyceride is unable to displace completely protein molecules from the air–water interface (Fig. 6). The surface dilatational properties of mixed protein–emulsifier films also depend on the presence of some food components (ethanol and sucrose) in the aqueous phase. In general, a decrease in the dilatational rheological properties on the addition of ethanol was found for protein–waterinsoluble emulsifiers, whereas the opposite was observed for protein–water-soluble emulsifiers (Rodrı´guez Nin˜o, Wilde, Clark, & Rodrı´guez Patino, 1998c).
3.5. Final considerations In this paper, we have analyzed the structure, topography, adsorption, interactions, miscibility, and dynamic properties of food dairy proteins (b-casein, caseinate, and WPI) and LMWE (monoglycerides and phospholipids) at the air–water interface. The summary includes an assessment of information derived from a variety of chemical and physical techniques. The results demonstrate that protein and LMWE type affect the interfacial characteristics. The nature of biopolymer and LMWE interactions at the interface has an important role on their physicochemical characteristics, including their role in conferring stability on emulsions and foams. Important functional differences have been demonstrated between globular (WPI) and disordered (b-casein and caseinate) proteins, between proteins and LMWE, and between saturated and unsaturated LMWE.
Acknowledgment This research was supported by CICYT through Grant AGL2001-3843-C02-01. References Bos, M., Nylander, T., Arnebrant, T., & Clark, D. C. (1997). Protein/ emulsifier interactions. In G. L. Hasenhuette & R. W. Hartel
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(Eds.), Food emulsifiers and their applications (pp. 95–146). New York: Chapman and Hall. Bos, M. A., & van Vliet, T. (2001). Interfacial rheological properties of adsorbed protein layers and surfactants: A review. Advances in Colloid and Interface Science, 91, 437–471. Carrera, C., Rodrı´guez Patino, J. M., & Rodrı´guez Nin˜o, Mª. R. (1999). Relaxation phenomena in monoglyceride films at the air– water interface. Colloids Surfaces B, 42, 175–192. Damodaran, S., & Paraf, A. (1997). Food proteins and their applications. New York: Marcel Dekker. Dickinson, E. (1992). An introduction to food colloids. Oxford: Oxford University Press. Dickinson, E. (1999). Adsorbed protein layers at fluid interfaces: Interactions structure and surface rheology. Colloids and Surfaces B: Biointerfaces, 15, 161–176. Dickinson, E. (2001). Milk protein interfacial layers and the relationship to emulsion stability and rheology. Colloids and Surfaces B: Biointerfaces, 20, 197–210. Friberg, S. E., & Larsson, K. (1997). Food emulsions (3rd ed.). New York: Marcel Dekker. Gaines, G. L. (1966). Insoluble monolayers at liquid–gas interface. New York: Wiley. 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. Gunning, A. P., Mackie, A. R., Wilde, P. J., & Morris, V. J. (1999). Insitu observation of surfactant-induced displacement of protein by atomic force microscopy. Langmuir, 15, 4636–4640. Hartel, R., & Hasenhuette, G. R. (1997). Food emulsifiers and their applications. New York: Chapman and Hall. Horne, D. S., & Rodrı´guez Patino, J. M. (2003). Adsorbed biopolymers: Behavior in food applications. In M. Malmsten (Ed.), Biopolymers at interfaces (pp. 857–900). New York: Marcel Dekker. Lucassen-Reynders, E. H., & Benjamins, J. (1999). Dilatational rheology of proteins adsorbed at fluid interfaces. In E. Dickinson & J. M. Rodrı´guez Patino (Eds.), Food emulsions and foams: Interfaces, interactions and stability (pp. 195–206). Cambridge: Royal Society of Chemistry. Mackie, A. R., Gunning, P. A., Pugnaloni, L. A., Dickinson, E., Wilde, P. J., & Morris, V. J. (2003). The growth of surfactant domains in protein films. Langmuir, 19, 6032–6038. Mackie, A. R., Gunning, A. P., Ridout, M. J., Wilde, P. J., & Rodrı´guez Patino, J. M. (2001). In situ measurement of the displacement of protein films from the air/water by surfactant. Biomacromolecules, 2, 1001–1006. Mackie, A. R., Gunning, A. P., Wilde, P. J., & Morris, V. J. (1999). The orogenic displacement of proteins from the air/water interface by surfactant. Journal of Colloid and Interface Science, 210, 157–166. Murray, B. S. (2002). Interfacial rheology of food emulsifiers and proteins. Current Opinion in Colloid and Interface Science, 7, 426–431. Nylander, T. (1998). Protein–lipid interactions. In D. Mo¨bius & R. Miller (Eds.), Proteins at liquid interfaces (pp. 365–431). Amsterdam: Elsevier. Rodrı´guez Nin˜o, Mª. R., Carrera, C., Cejudo, M., & Rodrı´guez Patino, J. M. (2001). Protein and lipid films at equilibrium at air–water interface. Journal of the American Oil Chemists Society, 78, 873–879. Rodrı´guez Nin˜o, Mª. R., Carrera, C., & Rodrı´guez Patino, J. M. (1999). Interfacial characteristics of b-casein spread films at the air– water interface. Colloids Surfaces B, 12, 161–173. Rodriguez Nin˜o, Mª. R., Rodrı´guez Patino, J. M., Carrera, C., Cejudo, M., & Navarro, J. M. (2003). Physicochemical characteristics of food lipids and proteins at fluid-fluid interfaces. Chemical Engineering Communications, 190, 15–47.
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Rodrı´guez Patino, J. M., Carrera, C., & Rodrı´guez Nin˜o, Mª. R. (1999a). Morphological and structural characteristics of monoglyceride monolayers at the air–water interface by Brewster angle microscopy. Langmuir, 15, 2484–2492. Rodrı´guez Patino, J. M., Carrera, C., & Rodrı´guez Nin˜o, J. M. (1999b). Structural and morphological characteristics of b-casein monolayers at the air–water interface by Brewster angle microscopy. Food Hydrocolloids, 13, 401–408. Rodrı´guez Patino, J. M., Carrera, C., & Rodrı´guez Nin˜o, Mª. R. (1999c). Analysis of b-casein–monopalmitin mixed films at the air– water interface. Journal of Agriculture and Food Chemistry, 47, 4998–5008. Rodrı´guez Patino, J. M., Carrera, C., & Rodrı´guez Nin˜o, Mª. R. (1999d). 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., & Cejudo, C. (2001c). Structural-dilatational characteristics relationships of monoglyceride monolayers at the air–water interface. Langmuir, 17, 4003–4013. Rodrı´guez Patino, J. M., Carrera, C., Rodrı´guez Nin˜o, Mª. R., & Cejudo, M. (2001d). Structural and dynamic properties of milk proteins at the air–water interface. Journal of Colloid and Interface Science, 242, 141–151. Rodrı´guez Nin˜o, Mª. R., & Rodrı´guez Patino, J. M. (1998a). Surface tension of protein and insoluble lipids at the air–aqueous phase interface. Journal of the American Oil Chemists Society, 75, 1233–1239. Rodrı´guez Nin˜o, Mª. R., & Rodrı´guez Patino, J. M. (1998b). Surface tension of bovine serum albumin and Tween 20 at the air–aqueous phase interface. Journal of the American Oil Chemists Society, 75, 1241–1248. Rodrı´guez Patino, J. M., & Rodrı´guez Nin˜o, Mª. R. (1999). Interfacial characteristics of food emulsifiers (proteins and lipids) at the air– water interface. Colloids Surfaces A, 15, 235–252. Rodrı´guez Patino, J. M., Rodrı´guez Nin˜o, Mª. R., & Carrera, C. (2002a). Relaxation phenomena in whey protein isolated and
monoglyceride mixed films at the air–water interface. Industrial and Engineering Chemistry Research, 41, 3169–3178. Rodrı´guez Patino, J. M., Rodrı´guez Nin˜o, M. R., & Carrera, C. (2002b). 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 bcasein–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. (2001a). The effect of pH on monoglyceride–caseinate mixed monolayers at the air–water interface. Journal of Colloid and Interface Science, 240, 113–126. Rodrı´guez Patino, J. M., Rodrı´guez Nin˜o, Mª. R., Carrera, C., & Cejudo, M. (2001b). Whey protein isolate-monoglyceride mixed monolayers at the air–water interface. Structure morphology and interactions. Langmuir, 17, 7545–7553. Rodrı´guez Nin˜o, Mª. R., Wilde, P. J., Clark, D. C., & Rodrı´guez Patino, J. M. (1998c). Surface dilational properties of protein and lipid films at the air–water interface. Langmuir, 14, 2160–2166. Roth, S., Murray, B. S., & Dickinson, E. (2000). Interfacial shear rheology of aged and heat-treated b-lactoglobulin films: Displacement by non-ionic surfactant. Journal of Agricultural and Food Chemistry, 48, 1491–1497. Wilde, P. J. (2000). Interfaces: Their role in foam and emulsion behaviour. Current Opinion in Colloid and Interface Science, 5, 176–181. Wilde, P. J., Husband, F. A., Cooper, D., Ridout, M. J., Mackie, A. R., Gunning, A. P., Morris, V. J., Woodward, N., & Clare Mills, E. N. (2003). Interfacial mechanisms underlying lipid damage of beer foams. In E. Dickinson & T. Van Vliet (Eds.), Food colloids biopolymers and materials (pp. 200–206). Cambridge: Royal Society of Chemistry.
Biopolymers and emulsifiers at the air–water interface. Implications in food colloid formulations Cecilio Carrera Sa´nchez, Mª. Rosario Rodrı´guez Nin˜o, Ana Lucero Caro, Juan M. Rodrı´guez Patino * Departamento de Ingeniera Quı´mica, Facultad de Quı´mica, Universidad de Sevilla, C/. Prof. Garcı´a Gonza´lez, 1, 41012 Sevilla, Spain Received 10 October 2003; accepted 1 May 2004
Abstract In this paper we are concerned with adsorption, structure, topography, and dynamic properties (relaxation phenomena and surface dilatational rheology) of food dairy proteins (b-casein, caseinate, and whey protein isolate, WPI), water-insoluble lipids (monopalmitin, monoolein, and monolaurin) and phospholipids (dipalmitoyl-phosphatidyl-choline, DPPC, and dioleoyl-phosphatidyl-choline, DOPC) at the air–water interface. Combined surface chemistry (surface film balance and static and dynamic tensiometry) and microscopy (Brewster angle microscopy, BAM) techniques have been used to determine the static and dynamic characteristics of these emulsifiers and their mixtures at the air–water interface. The derived information shows that biopolymer (proteins) and low-molecular-weight-emulsifier (LMWE, monoglycerides and phospholipids) type and their mixtures affect the interfacial characteristics of adsorbed and spread films. Important functional differences have been established between proteins, lipids and phospholipids. The static and dynamic characteristics of mixed films depend on the interfacial composition and the surface pressure (p). At higher surface pressures, collapsed protein residues may be displaced from the interface by LMWE molecules with important repercussions on the interfacial characteristics of the mixed films. Ó 2004 Elsevier Ltd. All rights reserved. Keywords: Fluid interfaces; Adsorption; Interfacial rheology; Monolayer; Food emulsifier; Biopolymer; Low-molecular weight emulsifier; Milk protein; Monoglyceride; Phospholipid
1. Introduction Food dispersions (emulsions and foams) are complex multicomponent systems containing many biopolymers and low-molecular weight emulsifiers (LMWE) which may show surface activity by themselves or by association with other components (polysaccharides). In addition food dispersions contain many other organic (ethanol, sugars, etc.) and inorganic (salts) components which may interact with biopolymers and LMWE in different complex fashion depending on pH, temperature, processing history, etc., all of which intensify the problems of the manufacturer attempting to control *
Corresponding author. Tel.: +34 954 556446; fax: +34 954 557134. E-mail address: [email protected] (J.M. Rodrı´guez Patino).
0260-8774/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.jfoodeng.2004.05.065
stability, shelf-life, or product texture. For up-to-date reviews, readers are directed to recent references (Damodaran & Paraf, 1997; Friberg & Larsson, 1997; Hartel & Hasenhuette, 1997). Manufacturers employ two types of emulsifiers or foaming agents in food (Dickinson, 1992), namely: LMWE (mainly monoand diglycerides, phospholipids, etc.) and macromolecules (proteins and certain hydrocolloids). The emulsifier film adsorbed at the oil–water or air–water interface is the source of many of the unique properties of food dispersions, particularly their stability and interactions, which translate into the shelf-life and textural properties so desired by manufacturers and appreciated by consumers. Proteins and LMWE have an important physical property in common, their amphiphilic nature (Horne &
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Rodrı´guez Patino, 2003; Rodriguez Nin˜o, Rodrı´guez Patino, Carrera, Cejudo, & Navarro, 2003). This property provides the potential for association, adsorption, and reorientation at fluid interfaces, depending on the properties of the components and the protein–LMWE ratio (Rodrı´guez Nin˜o & Rodrı´guez Patino, 1998a, 1998b; Rodrı´guez Nin˜o, Carrera, Cejudo, & Rodrı´guez Patino, 2001; Rodrı´guez Patino, Rodrı´guez Nin˜o, & Carrera, 2003a). LMWE stabilize the dispersed droplets or bubbles by formation of a densely packed but much less rigid monomolecular layer, which is stabilized by dynamic processes (i.e. Gibbs–Marangoni effect). LMWE adsorb strongly to fluid interfaces giving close molecular packing at the interface to produce low surface and interfacial tensions (Rodrı´guez Nin˜o & Rodrı´guez Patino, 1998a, 1998b). In contrast, proteins act as polymeric emulsifiers with multiple anchoring sites at the interface that, together with the unfolding process of the adsorbing protein molecule, stabilize the interfacial layer kinetically. This behavior contributes significantly to the interfacial rheological properties and immobilizes proteins in the adsorbed layer (Bos, Nylander, Arnebrant, & Clark, 1997). However more important in some products is the effect of the LMWE in destabilizing the emulsion (Goff & Jordan, 1989). In the formulation of ice cream the LMWE (typically, mono- and diglycerides) are added to break the adsorbed layer of protein and allow the adsorption of fat to the surface of the air bubble. Thus, an important action of LMWE is to promote the displacement of proteins (mainly caseins) from the interface. The competitive adsorption and/or displacement between LMWE and proteins at fluid–fluid interfaces have been studied in detail in several investigations (Bos et al., 1997; Nylander, 1998; Wilde, 2000; Dickinson, 2001; Rodrı´guez Patino et al., 2003a). However, so far, little is known about the structure that biopolymers and LMWE adopt at fluid interfaces. This paper will concentrate on the interfacial behavior of milk proteins and LMWE (monoglycerides and phospholipids). Emphasis will be on the air–water interface as a three-dimensional dynamic entity. We will consider emulsifier (protein, LMWE and their mixtures) adsorption, structure and topography at the interface, relaxation phenomena, and interfacial rheology, as related to the formation and stability of food dispersions (emulsions and foams).
2. Experimental 2.1. Chemicals Synthetic 1-monohexadecanoyl-rac-glycerol (monopalmitin, DIMODANR PA 90), 1-mono(cis-9-octadecanoyl)glycerol (monoolein, RYLOTMMG 19), and 1-monododecanoyl-rac-glycerol (monolaurin, DIMO-
DANR ML 90) were supplied by Danisco Ingredients (Braban, Denmark) with over 95–98% of purity. DL -adipalmitoyl-phosphatidyl-choline (DPPC, Sigma (St. Louis, MO, USA), 99%) and L -a-dioleoyl-phosphatidyl-choline (DOPC, Sigma, 99%) were used as supplied. Ninety-nine percent pure b-casein was supplied and purified from bulk milk from the Hannah Research Institute (Ayr, Scotland). Caseinate (a mixture of 38% b-casein, 39% as1-casein, 12% j-casein, and 11% as2-casein) was supplied and purified from bulk milk from Unilever Research Laboratories (Colworth, UK). Whey protein isolate (WPI), a native protein with high content of blactoglobulin (protein 92 ± 2%, b-lactoglobulin >95%, a-lactalbumin <5%) obtained by fractionation, was supplied by Danisco Ingredients (Brabran, Denmark). To form the surface film, monoglyceride and phospholipid were spread in the form of a solution, using hexane:ethanol (9:1, v:v) and chloroform:ethanol (4:1, v:v), respectively, as a spreading solvent. Analytical grade hexane (Merck, 99%), ethanol (Merck, >99.8%), and chloroform (Sigma, 99%), were used without further purification. Samples for interfacial characteristics of protein films were prepared using Milli-Q ultrapure water at pH 7. The water used as subphase was purified by means of a Millipore filtration device, Milli-Q (Milford, MA, USA). To adjust subphase pH, buffer solutions were used. Acetic acid/sodium acetate aqueous solution (CH3COOH/CH3COONa) was used to achieve pH 5, and a commercial buffer solution called trizma ((CH2OH)3CNH2/(CH2OH)3CNH3Cl) for pH 7. All these products were supplied by Sigma (>99.5%). Ionic strength was 0.05 M in all the experiments. 2.2. Methods 2.2.1. Equilibrium surface pressure and adsorption isotherm The equilibrium surface pressure (pe) is a key parameter for the analysis of the mechanisms that trigger the relaxation phenomena in spread monolayers at the air–water interface (Gaines, 1966). The equilibrium spreading pressure is the maximum surface pressure to which a spread monolayer may be compressed before monolayer collapse. Equilibrium surface pressure of protein and LMWE at the air–water interface was measured by the Wilhelmy plate method as described elsewhere (Carrera, Rodrı´guez Patino, & Rodrı´guez Nin˜o, 1999). The adsorption isotherm of proteins and LMWE–protein films was studied by tensiometry as described elsewhere (Rodrı´guez Nin˜o & Rodrı´guez Patino, 1998a; Rodrı´guez Nin˜o et al., 2001). 2.2.2. Surface film balance Measurements of the surface pressure (p) versus average area per molecule (A), the so-called p–A isotherm, were performed on a fully automated Langmuir- or
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Wilhelmy-type film balance as described elsewhere (Rodrı´guez Nin˜o, Carrera, & Rodrı´guez Patino, 1999). 2.2.3. Brewster angle microscopy For microscopic observation of the monolayer structure, the Brewster angle microscope, BAM 2 plus (NFT, Germany) was used as described elsewhere (Rodrı´guez Patino, Carrera, & Rodrı´guez Nin˜o, 1999a, 1999b). To measure the relative thickness of the film a previous camera calibration is necessary in order to determine the relationship between the gray level (GL) and the relative reflectivity (I), according to a procedure described previously (Rodrı´guez Patino et al., 1999a, Rodrı´guez Patino, Carrera, & Rodrı´guez Nin˜o, 1999b). 2.2.4. Surface relaxation measurements Measurements of surface relaxation in protein or LMWE films at the air–water interface were performed on a fully automated Langmuir-type film balance. The method has been described previously (Carrera et al., 1999). 2.2.5. Surface dilatational rheology To obtain surface rheological parameters at the air– water interface––such as surface dilatational modulus (E), elastic (Ed) and viscous (Ev) components, and loss angle tangent (tan h)––a modified Wilhelmy-type film balance (KSV 3000) was used as described elsewhere (Rodrı´guez Patino, Carrera, Rodrı´guez Nin˜o, & Cejudo, 2001c, 2001d).
3. Results and discussion 3.1. Protein and LMWE films at equilibrium Equilibrium spreading pressure of monoglycerides (monopalmitin, and monoolein), phospholipids (DPPC and DOPC) and proteins (b-casein and WPI) at the air–water interface, at pH 7 and at 20 °C is shown in Fig. 1A. The magnitude of pe was dependent on the LMWE and the protein. pe was higher for monoglycerides (Rodriguez Nin˜o et al., 2003) and phospholipids (unpublished data) and lower for proteins (Rodrı´guez Nin˜o et al., 2001). The effect of temperature (data not shown) was also different for LMWE and proteins, especially for an ordered protein (WPI). pe for LMWE and b-casein was not affected by temperature within the range of 5 and 40 °C. However, pe for WPI increased with temperature, especially at temperatures higher than 25 °C. Higher pe values correlate with higher interactions within the film forming components thus, with a more condensed film structure at equilibrium. Protein–LMWE interactions at the air–water interface can be studied by tensiometry. From these experi-
Fig. 1. (A) Equilibrium surface pressure of monopalmitin (MP), monoolein (MO), dipalmitoyl-phosphatidyl-choline (DPPC), dioleoylphosphatidyl-choline (DOPC), b-casein (BC) and WPI at pH 7 and at 20 °C. (B) The effect of spreading of (s) MP, (n) MO and (j) monolaurin (ML) on a film of WPI previously adsorbed on the air– water interface. The arrows indicate the equilibrium surface pressures (pe) for MP, MO and ML. C is the concentration of WPI in the bulk phase. The amount of monoglyceride spread on the WPI film is enough to saturate the monolayer by itself. Temperature 20 °C. pH 5.
ments it has been observed that the interfacial characteristics of mixed protein and LMWE films at air–water interfaces depend at least on the interfacial composition and on the protein/LMWE ratio (Fig. 1B). At higher concentrations of WPI in the bulk phase the surface activity of the mixed film is similar to that for pure WPI while at lower concentrations the surface activity of the mixed film is similar to pe of the monoglyceride (MP or MO). The solubility of monolaurin proves that the mixed film is dominated by the protein within the overall range, because for the monolaurin– protein mixed film the p–log C plot is practically the same as that for pure protein (Fig. 1B). In general, the surface activity of the protein + LMWE mixed films is determined by the LMWE as p of the mixed film is the same as the pe of LMWE and the monolayer is not saturated by the protein (Rodrı´guez Nin˜o & Rodrı´guez Patino, 1998a, 2001). However, the protein deter-
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mines the surface activity of mixed films as the protein saturates the monolayer. 3.2. Structure and topography of protein and LMWE films Structural and topographical characteristics of proteins and LMWE spread films at the air–water interface can be deduced from p–A isotherms (Rodrı´guez Patino & Rodrı´guez Nin˜o, 1999) coupled to information from BAM (Rodrı´guez Patino et al., 1999a, 1999b). From the p–A isotherm, different structures can be deduced for LMWE monolayers as a function of LMWE, temperature, and surface density or surface pressure. For instance, monopalmitin (and most saturated phospholipids) monolayers (Fig. 2) show that liquid expanded (LE), liquid-condensed (LC) and solid (S) structures, and, finally, the collapse at a p higher than pe, take place as a function of surface pressure. In contrast with monopalmitin, monoolein and DOPC monolayers (data not shown) presents only the liquid expanded structure and the collapse at pe. BAM allows direct visualization of changes in morphology and collapse of monopalmitin monolayer (as an example) at the air–water interface (Fig. 2). Monopalmitin monolayer at 10 mN/m shows circular LC domains from the homogeneous ambient phase with a LE structure. The LC domains grow in size and the monolayer is covered with LC domains as p is increased. At the highest p, the LC domains are so closely packed that they occupy the entire field of view, the contrast vanishes suddenly, and the presence of monolayer fractures can be observed in different zones (Rodrı´guez Patino et al., 1999a). BAM images corroborate that only the homogeneous LE phase is present during the compression of a monoolein and DOPC monolayers (data not shown). The evolution with the monolayer compression of the film thickness (d) gives complementary information about the structural characteristics of LMWE during monolayer compression (Rodrı´guez Patino et al., 1999a). The film thickness increases as the monolayer is compressed, passes through a maximum and then decreases at the monolayer collapse point. The evolution of d for saturated LMWE monolayer with monolayer compression also shows important differences with unsaturated LMWE monolayers and their d is lower than for saturated LMWE (Rodrı´guez Patino et al., 1999a). Results of BAM (specially the relative reflectivity) as a function of p obtained with protein monolayers clearly show the same structural characteristics as those deduced from the p–A isotherm (Fig. 2). The domains that residues of protein molecules adopt at the air–water interface appeared to be of uniform reflectivity (Fig. 2), suggesting homogeneity in thickness and film isotropy. The results of p–A isotherms (Fig. 2) confirm that protein monolayers at the air–water interface adopt two
different structures and the collapse phase. As for LMWE, d increases with p and is maximum at the collapse (at the highest p). At p lower than pe, the relative film thickness is independent of the protein but, at the collapse point, d for b-casein is higher than for WPI (Rodrı´guez Patino et al., 1999b). The differences observed between lipids (Rodrı´guez Patino et al., 1999a) and proteins (Rodrı´guez Patino et al., 1999b) in p–A isotherms and BAM images is of great utility for the application of BAM to the analysis of more complicated systems in which proteins and lipids are spread at the interface (Rodrı´guez Patino, Carrera, & Rodrı´guez Nin˜o, 1999c, 1999d; Rodrı´guez Patino, Rodrı´guez Nin˜o, Carrera, & Cejudo, 2001a, Rodrı´guez Patino, Rodrı´guez Nin˜o, Carrera, & Cejudo, 2001b). From a systematic study centered on the p–A isotherm of protein–monoglyceride mixed monolayers–– including the application of the additivity rule on miscibility and the quantification of interactions between monolayer components by excess free energy––it has been concluded that, at a macroscopic level, these compounds form a practically immiscible monolayer at the air–water interface, at p lower than that for the protein collapse (Rodrı´guez Patino et al., 2001a, 2001b) (Fig. 2). At higher p the collapsed protein is displaced from the interface by LMWE. The existence of low protein interactions in disordered proteins (b-casein and caseinate) facilitates the protein displacement by LMWE from the air–water interface. On the other hand, the lower surface activity of unsaturated LMWE explains the fact that this LMWE has a lower capacity than saturated LMWE for protein displacement. Different proteins and LMWE show different interfacial interactions, miscibility and topography, confirming the importance of protein and LMWE structure in determining the mechanism of interfacial interactions (Rodrı´guez Patino et al., 2001a, 2001b). Thus, displacement of proteins by LMWE from the air–water interface depend on the particular protein–LMWE system (Fig. 3). The displacement surface pressure (pd) value for monopalmitin–b-casein mixed films is lower than those for monopalmitin–caseinate and monopalmitin–WPI mixed films. Thus, protein displacement by monopalmitin is easier for b-casein than it is for caseinate and WPI, in this order. In the same order increases the surface elasticity of the protein (see last section). Thus, the more elastic WPI film is more resistant than the less elastic b-casein films. Monoolein has a lower capacity than monopalmitin for protein displacement due to the fact that monoolein requires higher pd for protein displacement. Phospholipids and proteins mixed films behave in a more complicated fashion (unpublished data). Phospholipids and b-casein form a practically immiscible monolayer at the air–water interface on neutral or acidic aqueous subphases. However, some degree of interaction exists between phospholipids and b-casein in the
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Fig. 2. p–A isotherm and visualization by BAM of (n) b-casein, () monopalmitin, and (––) monopalmitin–b-casein mixed film at XMP = 0.5. BAM images of monopalmitin––(a) LE phase at p < 5 mN/m, (b) coexistence of LE and LC domains at 5 mN/m < p < 30 mN/m, (c) LC domains at p > 35 mN/m, and (d) fracture of a collapse monolayer at p ffi pe (monopalmitin)––and saturated LMWE, b-casein––homogeneous topography at (a) p < pe and (b) at p ffi pe––and unsaturated LMWE, and monopalmitin–b-casein mixed monolayer at XMP = 0.5 and at p < pe––(a) segregated LE–LC monopalmitin and b-casein domains, (b) homogeneous LE monopalmitin–b-casein domains, and (c) segregated LC monopalmitin and b-casein domains––at p > pe––(d) region of monopalmitin LC domains, (e) coexistence of monopalmitin and collapse b-casein, and (f) squeezing out of bcasein by monopalmitin––and at the collapse point––(g) a region of collapsed monopalmitin dominates the topography of the interface, (h) fracture of collapsed monopalmitin, and (i) coexistence of collapse monopalmitin and islands of collapsed b-casein.
mixed film and these interactions, of an electrostatic character, are more pronounced at pH 9 as the phospholipid molecules become completely ionized.
Competitive adsorption of proteins and LMWE at fluid interfaces can affect the stability of food dispersions (Bos et al., 1997; Wilde, 2000). Thus, knowledge
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Fig. 3. The displacement surface pressure (pd) of (h) b-casein, (s) caseinate, and (n) WPI by (A) monopalmitin and (B) monoolein from spread mixed monolayers at the air–water interface as a function of the mass fraction of monoglyceride in the mixture. The horizontal lines represent the equilibrium surface pressures of (––, pe MP) monopalmitin, ( , pe MO) monoolein, (- - -, pe BC) b-casein, (- Æ -, pe CS) caseinate, and (- -, pe WPI) WPI pure monolayers at 20 °C and at pH 7.
of the proteins, LMWE, and their mixtures at fluid–fluid interfaces is a key factor for the formation and stability of food dispersions (emulsions and foams). Recent works have allowed the indirect study of competitive adsorption at a molecular level (Gunning, Mackie, Wilde, & Morris, 1999; Mackie, Gunning, Wilde, & Morris, 1999). The Norwich group (Gunning et al., 1999; Mackie et al., 1999) has demonstrated how surfactants disrupt and displace proteins from an interface by a three-stage ‘‘orogenic’’ mechanism. Briefly, emulsifier penetrates into the protein film, forming separate adsorbed domains that exert a lateral p which mechanically compresses the protein network until it fails and is displaced from the interface. The mechanism also reveals that protein adsorbed layers with a high surface elasticity are more resistant to displacement. The BAM images (Fig. 2) appear to support the idea that displacement takes place via an orogenic mechanism, but there exist differences between adsorbable watersoluble emulsifiers (Gunning et al., 1999; Mackie et al., 1999) and spread water-insoluble emulsifiers (Mackie, Gunning, Ridout, Wilde, & Rodrı´guez Patino, 2001; Rodrı´guez Patino et al., 2001a, 2001b). The results suggest that for spread water-insoluble emulsifiers (Fig. 4) the first stage of the orogenic mechanism, which occurs at p lower than pe of the protein, involves a displacement front of emulsifier domains (Fig. 4A) instead of the adsorption of water-soluble surfactant molecules at defects in the protein network. The second stage (Fig.
4B), which occurs at p near to and above pe of the protein, involves a buckling of the monolayer and reordering of the molecules as the protein film gets thicker in response to the decreasing surface coverage. Finally, at sufficiently high p the protein network begins to fail (Fig. 4C), freeing proteins, which then desorb from the interface (Rodrı´guez Patino et al., 2001a, 2001b). But, for spread water-insoluble emulsifier monolayers, the protein displacement is not total even at the highest surface pressure, at the collapse point of the mixed film. The orogenic displacement mechanism is a consequence of the low level of interaction between proteins and LMWE at the air–water interface (Rodrı´guez Patino et al., 2001a, 2001b). This model has been shown to work for a range of proteins with different secondary and tertiary structures and different types of LMWE (non-ionic, cationic, anionic and zwitterionic). This mechanism also explains the destabilization of beer foams by lipids with a range of chain lengths (Wilde et al., 2003). 3.3. Relaxation phenomena in protein and LMWE films Non-equilibrium processes occurring in systems containing fluid interfaces with a surfactant present are of
(A)
(B)
(C)
LMWE
PROTEIN
Fig. 4. Displacement of proteins by LMWE spread at the air–water interface according to the orogenic mechanism. (A) At p < pprotein a e displacement front of emulsifier domains is produced. (B) At p ffi pprotein a buckling of the protein monolayer and reordering of e the molecules is produced and the protein film gets thicker in response to the decreasing surface coverage. (C) At p > pprotein the protein e network begins to fail and desorbs from the interface, forming collapse protein multilayers in the aqueous bulk phase near to the interface.
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From a practical point of view it must be emphasized that under these conditions the mixed film is more stable in relation to monolayer molecular loss than that of the pure components. At the collapse point of the mixed film, the relaxation phenomena may be due either to nucleation and growth of critical nuclei of monoglyceride or to a complex mechanism including competition between desorption and monolayer collapse. The reasons for these behaviors may be associated again 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 protein collapse. 3.4. Interfacial rheological characteristics of protein and LMWE films The breaking of drops and bubbles during emulsification and foaming requires rapid and substantial stretching of the drops or bubbles, and consequently, the surface tension may be far from equilibrium. Thus, dil-
1.0
A/A o
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great practical significance and include important technological operations such as emulsification and foaming. Two experimental approaches can be used for the analysis of long-term relaxation phenomena in emulsifier monolayers (Gaines, 1966). In a first approach, the surface pressure (p) is kept constant, and the area A is measured as a function of time. In the second approach, area is kept constant (at the monolayer collapse) and the decrease in p is monitored as a function of time. Information on various relaxation paths (Marangoni effect, chemical reaction, polar group hydration, conformation/organization changes, film dissolution by desorption and/or diffusion, collapse, etc.) can be derived from these data (Rodriguez Nin˜o et al., 2003; Rodrı´guez Patino et al., 2003a). Desorption of spread LMWE monolayers at any constant surface pressure, at p < pe, involves two stages (Rodriguez Nin˜o et al., 2003; Rodrı´guez Patino et al., 2003a). The first is dissolution into the bulk aqueous phase to form a saturated aqueous layer. The second stage occurs when, after a time, the concentration gradient within the diffusion layer becomes constant and desorption reaches a steady state. The monolayer molecular loss was lower for saturated than for unsaturated LMWE. At p > pe the relaxation phenomena in LMWE films are due to the transformation of a homogeneous monolayer phase into a heterogeneous monolayer-collapse phase system. However, some differences exist between saturated (monopalmitin or DPPC) and unsaturated (monoolein or DOPC) LMWE monolayers. Relaxation phenomena in saturated LMWE monolayer are controlled predominantly by the collapse mechanism because of the p values relaxed to pe value. For unsaturated LMWE monolayer p relaxed from the collapse value, which is close to pe, towards lower p values at longer times. This phenomenon should be ascribed to the concurrence of different phenomena, such as desorption and collapse. Protein monolayer behaves in a different way to LMWE under the same experimental conditions. At p < pe the relaxation is a reversible process, which include monolayer organization/reorientation. The relaxation in relative molecular area and in surface pressure at p > pe should be attributed to processes related to monolayer organization/reorientation and collapse, respectively. The strength of interactions between protein and LMWE can also be studied by relaxation experiments. Long-term relaxation phenomena in protein–monoglyceride mixed films (Fig. 5) at the air–water interface have been analyzed according to models for desorption, collapse and/or organization/reorganization changes (Rodrı´guez Patino, Rodrı´guez Nin˜o, & Carrera, 2002a, 2002b). At lower surface pressures (i.e., p lower than protein collapse pressure), the organization/reorganization change of protein molecules in the mixed film is the mechanism that controls the relaxation process.
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45 40 35 30 25 20
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40
60
80
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time (min) Fig. 5. (A) Relaxation at constant surface pressure (p = 20 mN/m) and (B) relaxation at constant area (at the collapse point) for of b-casein– monopalmitin mixed monolayers on water at pH 7 and at 20 °C.
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atational properties of adsorbed emulsifier layers are also important. The viscoelastic properties of the surface have often been correlated with functionality (Bos & van Vliet, 2001; Dickinson, 1999, 2001; Murray, 2002). The ability of the protein to resist displacement by emulsifiers is closely linked to the surface dilatational rheology, whereas the precise form of the displacement is considered to be more closely related to the surface shear behavior (Mackie et al., 2003; Murray, 2002; Roth, Murray, & Dickinson, 2000). Different and complementary interfacial techniques (surface film balance, BAM, and interfacial dilatational rheology) are useful in the analysis of the structural and dynamic characteristics of protein, LMWE, and their mixtures at the air–water interface (Rodrı´guez Patino, Rodrı´guez Nin˜o, & Carrera, 2002b; Rodrı´guez Patino, Rodrı´guez Nin˜o, & Carrera, 2003b). A common trend of the p dependence of dilatational modulus (E) for monopalmitin and monoolein monolayers (Fig. 6) is that E increased with increasing p up to the collapse point. This increase is a result of an increase in the interactions between the monolayer molecules (that is, of its structure), as deduced from p–A isotherms and BAM images (Fig. 2). However, for the more condensed monolayer (monopalmitin) this in-
crease is higher than for the more expanded monoolein monolayer. This indicates that E is not only determined by the interactions between spread monoglyceride (or phospholipid) molecules (which depend on p), but that the structure of the spread molecule also plays an important role. In fact, for the more aggregated monopalmitin molecules in LC domains (see Fig. 2) E is higher than that of monoolein molecules with LE structure, at the same p. As for LMWE, for WPI monolayers (Fig. 6) E increased with increasing p up to the collapse point. This increase is the result of an increase in the interactions between the monolayer molecules, as deduced from p–A isotherms and BAM image. However, for the more disordered proteins (b-casein and caseinate) the E–p dependence is more complex. E increases to a maximum with p, but decreases with p and passes to a minimum. Finally, E increases up to the collapse point (Fig. 6). This inflection in the E–p curve may be attributed to the transition from an ‘‘all-train’’ configuration to a ‘‘train-and-loop’’ conformation of the b-casein molecule (Lucassen-Reynders & Benjamins, 1999). The results with protein monolayers indicate that E is not only determined by the structure of protein molecules, but the internal nature of the protein
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Fig. 6. Surface pressure dependence of surface dilatational modulus for protein + monoglyceride mixed films at the air–water interface at pH 7. (A) b-casein + monopalmitin mixed films. (B) b-casein + monoolein mixed films. (C) WPI + monopalmitin mixed films. (D) WPI + monoolein mixed films. Temperature: 20 °C; frequency: 50 mHz; amplitude: 5%. Monolayer composition (mass fraction of monoglyceride): (s) 0, (n) 0.2, (,) 0.4, (}) 0.6, (+) 0.8, and ( ) 1.0.
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spread molecule also plays an important role. In fact, for the more ordered b-lactoglobulin molecules in WPI E is higher than that for b-casein or caseinate molecules (Fig. 6) with disordered structure, at the same surface pressures. Surface dilatational rheology is a very sensitive technique to analyze the competitive adsorption/displacement of protein and LMWE emulsifier at the air–water interface. At higher p, the collapsed protein residues displaced from the interface by LMWE molecules have important repercussions on the dilatational characteristics of the mixed films (Rodrı´guez Patino et al., 2002b, 2003b). However, the mechanical properties of the mixed films also demonstrate that, even at the highest p, the monoglyceride is unable to displace completely protein molecules from the air–water interface (Fig. 6). The surface dilatational properties of mixed protein–emulsifier films also depend on the presence of some food components (ethanol and sucrose) in the aqueous phase. In general, a decrease in the dilatational rheological properties on the addition of ethanol was found for protein–waterinsoluble emulsifiers, whereas the opposite was observed for protein–water-soluble emulsifiers (Rodrı´guez Nin˜o, Wilde, Clark, & Rodrı´guez Patino, 1998c).
3.5. Final considerations In this paper, we have analyzed the structure, topography, adsorption, interactions, miscibility, and dynamic properties of food dairy proteins (b-casein, caseinate, and WPI) and LMWE (monoglycerides and phospholipids) at the air–water interface. The summary includes an assessment of information derived from a variety of chemical and physical techniques. The results demonstrate that protein and LMWE type affect the interfacial characteristics. The nature of biopolymer and LMWE interactions at the interface has an important role on their physicochemical characteristics, including their role in conferring stability on emulsions and foams. Important functional differences have been demonstrated between globular (WPI) and disordered (b-casein and caseinate) proteins, between proteins and LMWE, and between saturated and unsaturated LMWE.
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