Structural characteristics of adsorbed protein and monoglyceride mixed monolayers at the air–water interface

ARTICLE IN PRESS

FOOD

HYDROCOLLOIDS Food Hydrocolloids 21 (2007) 906–919 www.elsevier.com/locate/foodhyd

Structural characteristics of adsorbed protein and monoglyceride mixed monolayers at the air–water interface Marta Cejudo Ferna´ndez, 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 Received 16 May 2006; accepted 14 August 2006

Abstract Monolayer technique has been used successfully to study the properties of mixed emulsifiers spread at the air–water interface. In this contribution we are concerned with the analysis of structural characteristics of mixed monolayers formed by milk proteins (b-casein and b-lactoglobulin) and monoglycerides (monopalmitin and monoolein), and how these differ depending on the method used for the formation of the mixed film at the air–water interface (spreading, penetration or by spreading of a lipid into a previously adsorbed protein film). Measurements of the p-A isotherm were obtained in Langmuir- and Wilhelmy-type film balances coupled with Brewster angle microscopy (BAM). The p-A isotherm deduced for adsorbed b-lactoglobulin monolayer in this work is practically the same as that obtained directly by spreading. However, for b-casein the adsorbed monolayer is more condensed than the spread one. For protein– monoglyceride mixed films, the p-A isotherms for adsorbed and spread monolayers at surface pressure (p) higher than the equilibrium spreading pressure of protein (pprotein ) are practically coincident, a phenomenon which may be attributed to protein displacement by the e monoglyceride from the interface. At pope protein and monoglyceride coexist at the interface and the adsorbed and spread p-A isotherms (monolayer structure) are different. Monopalmitin has a higher capacity than monoolein for the displacement of protein from the air–water interface. However, some degree of interaction exists between proteins and monoglycerides and these interactions are higher for adsorbed than for spread films. The topography of the monolayer corroborates these conclusions. r 2006 Elsevier Ltd. All rights reserved. Keywords: Monolayer structure; Monolayer topography; Adsorbed films; Spread monolayers; Monolayer penetration; Air–water interface; Food emulsifiers; Protein–monoglyceride mixed films; Film balance; Brewster angle microscopy

1. Introduction The stability and mechanical properties of dispersed food systems (emulsions and foams) depend on the way in which the constituent emulsifiers (low-molecular weight emulsifiers, LMWE, and biopolymers) adsorb and interact at fluid interfaces. The chemical and physical properties of surface-active molecules are of great interest because they determine the colloidal stability of dispersed systems (Dickinson, 1992). The optimum use of emulsifiers depends on knowledge of their interfacial physico-chemical characteristics—such as surface activity, structure, miscibility, superficial viscosity, etc.—and the kinetics of film formaCorresponding author. Tel.: +34 95 4556446; fax: +34 95 4556447.

E-mail address: [email protected] (J.M.R. Patino). 0268-005X/$ - see front matter r 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodhyd.2006.08.016

tion at fluid interfaces (Damodaran & Paraf, 1997; Dickinson, 1992; McClements, 2005). The distribution of biopolymers and LMWE in food dispersions is determined by competitive and co-operative adsorption between the two types of emulsifiers at the fluid–fluid interfaces, and by the nature of protein–lipid interactions, both at the interface and in bulk phase (Bos, Nylander, Arnebrant, & Clark, 1997). Interactions between molecules of emulsifiers could affect the structure, topography, and dynamic characteristics (relaxation phenomena and viscoelasticity) in mixed films (Bos & van Vliet, 2001; Pugnaloni, Dickinson, Ettelaie, Mackie, & Wilde, 2004; Rodrı´ guez Patino, Rodrı´ guez Nin˜o, & Carrera, 2003; Wilde, Mackie, Husband, Gunning, & Morris, 2004). However, the presence of proteins and lipids in the same system can result in instability as both types of surface-active molecules compete

ARTICLE IN PRESS M. Cejudo Ferna´ndez et al. / Food Hydrocolloids 21 (2007) 906–919

to form different types of adsorbed layers (Bos, Nylander et al., 1997). Thus, information about these phenomena would be very helpful in the prediction of optimised formulations for food foams and emulsions. The main challenge, which we address in this study, is to understand the interfacial structural characteristics of proteins and monoglycerides depending on the method used for film formation at the air–water interface. We have chosen three different methods, which mimic the formation of food emulsions: (i) spreading of film forming components (Rodrı´ guez Patino, Carrera, & Rodrı´ guez Nin˜o, 1999a, 1999b; Rodrı´ guez Patino, Carrera, Rodrı´ guez Nin˜o, & Cejudo, 2001; Rodrı´ guez Patino, Rodrı´ guez Nin˜o, Carrera, & Cejudo, 2001), (ii) penetration of a protein into a lipid monolayer (Carrera, Cejudo, Rodrı´ guez Nin˜o, & Rodrı´ guez Patino, 2006), and (iii) displacement or competition between film forming components by spreading of a lipid into a protein film previously adsorbed at the air– water interface (Rodrı´ guez Patino & Cejudo, 2004; Cejudo, Carrera, Rodrı´ guez Nin˜o, & Rodrı´ guez Patino, 2006; Rodrı´ guez Patino, Cejudo, Carrera, & Rodrı´ guez Nin˜o, 2006a, 2006b). Up to now, most of the fundamental information has been derived from structural measurements on mixed monolayers by spreading the monolayer forming components sequentially using surface film balance, Brewster angle microscopy, atomic force microscopy, etc. (Rodrı´ guez Patino et al., 2003; Wilde, Mackie, et al. 2004). But, adsorbed or penetrated monolayers would be more interesting from a practical point of view. However, there is a gap in our knowledge concerning how the structural characteristics of mixed emulsifiers differ depending on the formation of the interface. In order to gain further insight into mixed emulsifiers at fluid interfaces we have also analysed the effect of the protein (using b-casein and b-lactoglobulin as model disordered and globular proteins, respectively) and monoglycerides (monopalmitin and monoolein as saturated and unsaturated polar lipids, respectively). These emulsifiers are successfully used in many food colloid formulations. Well-known examples of these food dispersions are icecream, whipped toppings, salad dressing, coffee creamers, confectionary and bakery products, etc. (Damodaran & Paraf, 1997; Friberg, Larsson, & Sjo¨blom, 2004; Hartel & Hasenhuette, 1997). Understanding the structural characteristics of emulsifiers at fluid interfaces at different length scales (from microscopic to nanoscopic) is potentially important in order to improve structure, stability, texture, taste, and other organoleptic properties of food colloid formulations (Leser, Michael, & Watzke, 2003; Rodrı´ guez Patino et al., 2006). 2. Experimental 2.1. Chemicals Synthetic 1-monohexadecanoyl-rac-glycerol (monopalmitin, DIMODANR PA 90) and 1-mono (cis-9-octadeca-

907

noyl) glycerol (monoolein, RYLOTM MG 19) were kindly supplied by Danisco Ingredients (Brabran, Denmark) with over 95–98% of purity. b-Casein (4 99%) was supplied and purified from bulk milk from the Hannah Research Institute (Ayr, Scotland). Whey protein isolate (WPI), a native protein with very high content of b-lactoglobulin (protein 9272%, b-lactoglobulin495%, a-lactalbumino 5%) obtained by fractionation, was supplied by Danisco Ingredients. Samples for interfacial characteristics of protein-adsorbed films were prepared using Milli-Q ultrapure water and were buffered at pH 7. Monoglycerides were 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, 499.8%) were used. The water used as subphase was purified by means of a Millipore filtration device (Milli-Q). A commercial buffer solution called trizma ((CH2OH)3CNH2/(CH2OH)3CNH3Cl, Sigma, 499.5%) was used to achieve pH 7. Ionic strength was 0.05 M in all the experiments. 2.2. Surface film balance Measurements of surface pressure (p)-area (A) isotherms of protein–monoglyceride mixed films at the air–water interface were performed on a fully automated Langmuirand Wilhelmy-type film balance. Mixtures of particular mass fractions—expressed as the mass fraction of monoglyceride in the mixture, X—were studied. The compression rate was 0.062 nm2  mol1 min1, which is the highest value for which isotherms were found to be reproducible in preliminary experiments. The reproducibility of the results was better than 70.5 mN/m for surface pressure and70.05 m2/mg. Different experimental protocols were used in this study (Fig. 1): (i) Spreading of protein and lipid monolayers at the air– water interface (Fig. 1B). Spread monolayers of mixed emulsifiers have demonstrated to be very important for fundamental studies because the concentrations of the film forming components are well known (Rodrı´ guez Patino et al., 1999a, 1999b, 2001). In addition, spread mixed monolayers can be used as a reference for the analysis of adsorbed or penetrated mixed films because in the latter the concentrations of film forming components are unknown in experiments involving film balances. To allow the quantitative adsorption of the protein onto the interface the monolayer was not under any surface pressure during the spreading process. Thus, the protein necessary to form the mixed film should be spread before the lipid. Aliquots of aqueous solutions of protein 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 methods adopted in these experiments ensured the quantitative spreading of the protein on the interface as was discussed in previous papers (Rodrı´ guez Nin˜o, Carrera, & Rodrı´ guez Patino, 1998; Rodrı´ guez Patino et al., 2001). Afterwards, monoglyceride solutions in

ARTICLE IN PRESS 908

M. Cejudo Ferna´ndez et al. / Food Hydrocolloids 21 (2007) 906–919

Fig. 1. Experimental protocols. (A) Penetration of a protein into a lipid monolayer previously spread at the air–water interface: (1) Lipid spreading at p ¼ 0, (2) compression at pi, (3) protein injection into the aqueous bulk phase at pi, (4) Protein adsorption/penetration, and (5) compression of the mixed film. (B) Spreading of protein and lipid monolayers at the air–water interface: (1) protein spreading at p ¼ 0, (2) lipid spreading at p ¼ 0, and (3) compression of the mixed monolayer. (C) Spreading of a lipid into a previously adsorbed protein monolayer: (1) protein injection into the aqueous bulk phase at p ¼ 0, (2) protein adsorption at p ¼ 0, (3) compression of the protein adsorbed film, (4) lipid spreading at p ¼ 0, and (5) compression of the mixed film.

a hexane:ethanol mixture were spread at different points on the protein film. To allow for spreading and protein– 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 collapse. After 30 min at the maximum area, measurements of compression–expansion cycles were performed with 30 min of waiting time between each expansion–compression cycle. (ii) Spreading of a lipid into a previously adsorbed protein monolayer (Fig. 1C). These experiments mimic the behaviour of emulsifiers in food emulsions in which an oilsoluble lipid (monopalmitin or monoolein) interacts at the interface with a protein film previously adsorbed from the aqueous bulk phase (Cejudo et al. 2006; Rodrı´ guez Patino & Cejudo, 2004; Rodrı´ guez Patino, Cejudo et al. 2006a, 2006b). In fact, during the production of some food emulsions the lipid is added in a second step after the formation of a previously formulated emulsion including the protein as emulsifier (Dickinson, 1992; McClements, 2005). For pure adsorbed protein films the maximum protein concentration in the bulk phase should be selected in order to obtain a reasonable rate of adsorption at the interface, but maintaining the equilibrium surface pressure

as zero (Rodrı´ guez Nin˜o & Rodrı´ guez Patino, 2002). For protein adsorbed films from water a protein solution at 1  106–5  106% (wt/wt) was left in the trough and time allowed for protein adsorption at the interface. These protein concentrations were selected from previous data of the adsorption isotherm (Rodrı´ guez Nin˜o, Carrera, Cejudo & Rodrı´ guez Patino, 2001). At this protein concentration in solution the surface pressure after 24 h of adsorption is zero and then the monoglyceride was spread at different points on the protein film. (iii) Penetration of a protein into a monoglyceride monolayer (Fig. 1A). These experiments mimic the behaviour of mixed emulsifiers in most food emulsions in which an oil-soluble lipid (monopalmitin or monoolein) is adsorbed faster than a protein at the interface (Carrera et al., 2006). Then, after the formation of a lipid monolayer the protein can be penetrated into the previously created lipid monolayer, and this is followed by lipid–protein interactions (Dickinson, 1992; McClements, 2005). The pA isotherms for monoglyceride monolayers penetrated by proteins were measured after the surface pressure relaxed to a steady state value during previous penetration experiments. The monoglyceride solutions in hexane:ethanol (9:1, v:v) were spread on the subphase by means of a micrometric syringe at 20 1C. The same precautions as in

ARTICLE IN PRESS M. Cejudo Ferna´ndez et al. / Food Hydrocolloids 21 (2007) 906–919

previous works were taken to allow for the evaporation of the spreading solvent and for the choice of compression rate (Rodrı´ guez Patino et al., 1999a, 199b). Afterwards, a p-A isotherm for the monoglyceride was recorded and used as a control. Then, the monoglyceride monolayer was compressed up to the desired surface pressure (at 10 mN/m, 20 mN/m, or at the collapse point) before experiments for protein penetration were carried out. For protein-adsorbed films from water protein solution at 1  106–5  106 wt% was left in the trough and time was allowed for protein penetration at the interface. 2.3. Brewster angle microscopy (BAM) A commercial BAM, BAM2, manufactured by NFT (Go¨ttingen, Germany) was used to study the topography of the monolayer. The BAM was positioned over the film balance. Further characteristics of the device and operational conditions have been described elsewhere (Rodrı´ guez Patino, Carrera, & Rodrı´ guez Nin˜o, 1999c, 1999d). The surface pressure measurements, area, and grey level as a function of time were carried out simultaneously by means of a device connected between the film balance and BAM, during surface film balance measurements according to experimental protocols used in this study (Fig. 1). These measurements were performed during the monolayer compression. The imaging conditions were adjusted to optimise image quality. Thus, generally as the surface pressure or the protein content increased the shutter speed was also increased. 3. Results 3.1. Spread and adsorbed protein monolayers Fig. 2 shows the p-A isotherm for adsorbed and spread b-lactoglobulin (Fig. 2A) and b-casein (Fig. 2B) monolayers at the air–water interface (Cejudo et al., 2006; Rodrı´ guez Patino, Cejudo et al., 2006a). Since the surface concentration is actually unknown for the adsorbed film, the values were derived from plots in Fig. 2 by assuming that the A value for adsorbed and spread films was equal at the collapse point of the mixed film (Cejudo et al., 2006; Murray, Faergemand, Trotereau, & Ventura, 1999; Rodrı´ guez Patino & Cejudo, 2004). This assumption can be supported by the fact that for protein films, the equilibrium spreading pressure (pblactoglobulin or pebcasein ) e and the surface pressure at the plateau for a saturated protein adsorbed film (Rodrı´ guez Nin˜o et al., 2001) are the same. The results of the p-A isotherms (Fig. 2A) with the help of the compressional coefficient (data not shown) deduced from the slope of the p-A isotherm (k ¼ dp/dA), confirm that adsorbed b-lactoglobulin films at the air–water interface adopt a liquid expanded-like structure and the collapse phase. However, according to Graham & Phillips (1979), b-lactoglobulin retains elements of the native structure, not

909

fully unfolded at the interface. Thus, most amino-acid residues in b-lactoglobulin adopt loop conformation at the air–water interface. But the loop conformation is more condensed at higher surface pressures and is displaced towards the bulk phase at the collapse point (at ). The film collapses at a surface pressure pXpblactoglobulin e of about 30 mN/m (Fig. 2A), a value that is a little higher than the surface pressure at the plateau for a saturated adsorbed film and the equilibrium spreading pressure (Rodrı´ guez Nin˜o et al., 2001). However, the results of the p-A isotherms indicate that adsorbed b-casein films at the air–water interface adopt two different structures or condensation states and the collapse phase (Fig. 2B). At low surface pressures (at po12 mN/m) adsorbed b-casein films exist (Graham & Phillips, 1979) as trains with amino acid segments located at the interface (structure I). At higher surface pressures (at p412 mN/m), and up to the equilibrium surface pressure, amino-acid segments are extended into the underlying aqueous solution and adopt the form of loops and tails (structure II). The film collapses at a surface pressure of about 21 mN/m (Fig. 2B), a value close to (Rodrı´ guez Nin˜o et al., 2001) the surface pressure at the plateau for a saturated adsorbed film and to the equilibrium spreading pressure (pb-casein ). e BAM images prove that the morphology of adsorbed blactoglobulin and b-casein monolayers during the first compression is uniform (Fig. 2C-a), suggesting homogeneity in thickness and film isotropy. However, interfacial regions with folds or aggregations of collapsed b-casein, which were formed at the higher surface pressures (Fig. 2Cb), were observed at the interface during the monolayer expansion, even at the lowest surface pressure, at p E 0 mN/m. These heterogeneities at a microscopic level were also observed at the interface after repeated compressions of the monolayer. The reflectivity as a function of surface pressure obtained with b-lactoglobulin and b-casein adsorbed films show that film thickness increases with the surface pressure and is a maximum at the collapse, at the highest surface pressure (Rodrı´ guez Patino et al., 1999d, 2001). 3.2. Protein and monoglyceride spread monolayers at the air–water interface Monopalmitin spread monolayers show a rich structural polymorphism as a function of surface pressure (Rodrı´ guez Patino et al., 1999c). The liquid-expanded (LE) phase (at po5 mN/m), an intermediate region at the broad plateau due to a degenerated first-order phase transition between liquidcondensed (LC) and LE structures (at 5opo30 mN/m), the LC structure (at p430 mN/m), and, finally, the solid (S) structure near to the monolayer collapse at a surface pressure of about 53.1 mN/m were observed. The structural characteristics of monoolein monolayers were different from those of monopalmitin as deduced from p-A isotherms. Monoolein monolayers had a LE structure under all experimental

ARTICLE IN PRESS M. Cejudo Ferna´ndez et al. / Food Hydrocolloids 21 (2007) 906–919

910

πeβ-lactoglobulin

Surface pressure (mN/m)

30 25 20 15 10 5 0 0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6 (a)

2

Area (m /mg)

(A)

30

Surface pressure (mN/m)

25 πeβ-casein

20 15 10

(b) (C)

5 0 0.0

(B)

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

2

Area (m /mg)

Fig. 2. p-A isotherms (compression curves) for (- - -) adsorbed and (—) spread monolayers of (A) b-lactoglobulin and (B) b-casein at the air–water interface. (C) Visualisation of protein adsorbed films by Brewster angle microscopy: (a) this picture was observed for b-casein during the first compression. The same image was observed for b-lactoglobulin during the first and after successive compressions. (b) b-casein aggregates in adsorbed films at the end of the first compression (at p421 mN/m) and during successive compressions. The horizontal direction of the image corresponds to 630 mm and the vertical direction to 470 mm. Aqueous subphase at pH 7. Temperature 20 1C. The equilibrium surface pressure for b-lactoglobulin (pb-lactoglobulin ) and b-casein e (pbcasein ) are indicated by arrows. e

conditions, as did protein monolayers. However, the collapse pressure for monoolein monolayers at 20 1C is lower (pc ¼ 44.3 mN/m) than that for monopalmitin, which indicates that hydrophobic interactions between hydrocarbon chains for monoolein are lower than those for monopalmitin monolayers. The monolayer structure in protein+monopalmitin mixed films (Figs. 3 and 4) at surface pressures lower than that for protein collapse (at pprotein ) shows a structural e polymorphism, as for monopalmitin. Moreover, there was a monolayer expansion as the monopalmitin content in the mixture was increased. At surface pressures higher than that for protein collapse, the p-A isotherms for mixed monolayers were parallel to those of monopalmitin. The images for protein+monopalmitin mixed monolayers (Fig. 5) clearly indicate that at low surface pressures (at po5 mN/m) there exists a homogeneous phase due to the coexistence of LE monopalmitin domains and protein

(Fig. 5a). As the surface pressure increases the coexistence between small circular LC monopalmitin domains and homogeneous LE domains of monopalmitin and probably of protein was observed (Fig. 5b). At higher surface pressures, near and after the protein collapse, the squeezing out of protein by monopalmitin can be distinguished (Fig. 5c), in a region with LC domains of monopalmitin (dark area) over a sublayer of collapsed protein (bright area). At the monolayer collapse, the monopalmitin domains were so closely packed that the monolayer morphology acquired a high homogeneity. However, in this region of highest surface pressure different regions of collapsed protein were observed on the interface (Fig. 5d). The p-A isotherms (Figs. 6 and 7) for protein+monoolein mixed monolayers show that the mixed films at surface pressures lower than that for protein collapse adopt a LElike structure, as for pure components. There was a

ARTICLE IN PRESS 60

60

50

50 Surface pressure (mN/m)

Surface pressure (mN/m)

M. Cejudo Ferna´ndez et al. / Food Hydrocolloids 21 (2007) 906–919

40 πeβ-lactoglobulin

30 20 10

0.0

0.2

0.4

0.6

0.8

1.0

1.2

30 πeβ-casein

20

0

1.4

Area (m2/mg)

(A)

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.2

1.4

2

Area (m /mg)

(A)

60

60

50

50 Surface pressure (mN/m)

Surface pressure (mN/m)

40

10

0

40 πeβ-lactoglobulin

30 20 10

40 30 πeβ-casein

20 10

0 0.0

(B)

911

0.2

0.4

0.6

0.8

1.0

1.2

0

1.4

0.0

Area (m2/mg)

(B) Fig. 3. p-A isotherms (compression curves) for mixed monolayers at the air–water interface of b-lactoglobulin and monopalmitin formed by the spreading of monopalmitin on (- - -) adsorbed and (—) spread monolayers of b-lactoglobulin at mass fraction of monopalmitin in the mixture of (A) 20 wt% and (B) 40 wt%. Aqueous subphase at pH 7. Temperature 20 1C. The equilibrium surface pressure for b-lactoglobulin (pblactoglobulin ) is e indicated by an arrow.

monolayer expansion as the monoolein content in the mixture was increased. At surface pressures higher than that for protein collapse, the p-A isotherms for mixed monolayers were parallel to that of monoolein. The collapse pressure for mixed monolayers is similar to that for monoolein and did not depend on the monolayer composition. BAM images for protein+monoolein mixed monolayers (Fig. 8) were different to those described above for monopalmitin+protein mixed monolayers. In fact, at surface pressures lower than that for protein collapse (at popprotein ) both components and the mixed monolayer all e form an isotropic (homogeneous) monolayer without any difference in the domain morphology (Fig. 8a). At surface pressures near to and after protein collapse BAM images demonstrated that monoolein and protein molecules adopted an isotropic structure in the mixed monolayer

0.2

0.4

0.6

0.8

1.0

Area (m2/mg)

Fig. 4. p-A isotherms (compression curves) for mixed monolayers at the air–water interface of b-casein and monopalmitin formed by the spreading of monopalmitin on (- - -) adsorbed and (—) spread monolayers of bcasein at mass fraction of monopalmitin in the mixture of (A) 20 wt% and (B) 40 wt%. Aqueous subphase at pH 7. Temperature 20 1C. The equilibrium surface pressure for b-casein (pb-casein ) is indicated by an e arrow.

with some holes associated with the presence of monoolein at the interface, and some bright regions (Fig. 8b), which correspond to the presence of a thicker protein collapsedmonolayer. At the higher surface pressures, and especially at the collapse point of pure monoolein monolayer, the mixed monolayer was dominated by the presence of monoolein at the interface (Fig. 8c). From the p-A isotherm of mixed monolayers (Figs. 3,4,6 and 7)—including the application of the additivity rule on miscibility and the quantification of interactions between monolayer components by the excess free energy (data not shown)—and monolayer topography (Figs. 5 and 8), it has been shown that in protein+monoglyceride mixed films, islands of protein and monoglyceride do exist at the air– water interface, but with few interactions between them, depending on the surface pressure (Rodrı´ guez Patino et al.

ARTICLE IN PRESS 912

M. Cejudo Ferna´ndez et al. / Food Hydrocolloids 21 (2007) 906–919

Fig. 5. Visualisation of protein+monopalmitin mixed monolayers by Brewster angle microscopy at 20 1C. (a) Homogeneous image after the spreading of monopalmitin on a spread or adsorbed protein film at po5 mN/m. (b) Mixed films after spreading of monopalmitin on a spread or adsorbed protein film at popprotein . (c) Mixed films after spreading of monopalmitin on a spread or adsorbed protein film at pXpprotein . (d) Mixed films after spreading of e e monopalmitin on a spread or adsorbed protein film at the collapse point. (e) Mixed films after spreading of monopalmitin on an adsorbed protein film at the collapse point of the mixed film. (f) Mixed films after spreading of monopalmitin on an adsorbed b-casein film. The horizontal direction of the image corresponds to 630 mm and the vertical direction to 470 mm.

1999a, 1999b, 2001). At surface pressures lower than that for protein collapse a mixed monolayer of monoglyceride and protein may exist. However, at surface pressures higher than that for protein collapse, the mixed monolayers were practically dominated by monoglyceride molecules. That is, at higher surface pressures, collapsed protein residues may be displaced from the interface by monoglyceride molecules. Over the overall range of existence of the mixed film the monolayer presents some heterogeneity due to the fact that domains of monoglyceride and protein residues are present during the monolayer compression. Interactions, miscibility, and displacement of proteins by monoglycerides from the air–water interface depend on the particular protein–monoglyceride system. Different proteins show different interfacial topography, confirming the importance of protein secondary structure in determining the mechanism of interfacial interactions. For the more disordered milk proteins (b-casein), large well-defined regions of monoglyceride with low reflectivity can be defined over the isotropic protein-rich region forming a thicker film with high reflectivity, and the frontier between these regions where monoglyceride– protein interactions take place. However, for the more ordered protein (b-lactoglobulin) the topography of the interface is characterised by numerous-smaller segregated regions of proteins and monoglyceride domains distributed homogeneously over the interface. These results suggests the idea that interactions between proteins and monoglycerides are higher for globular than for disordered proteins.

3.3. Spreading of a monoglyceride into an adsorbed protein film As for pure protein adsorbed films, the actual p-A isotherms for protein+monoglyceride adsorbed mixed films were derived by assuming (Rodrı´ guez Patino & Cejudo, 2004; Cejudo et al., 2006; Rodrı´ guez Patino, Cejudo, Rodrı´ guez Nin˜o, & Carrera, 2006a, 2006b) that the A value for adsorbed and spread films was equal at the collapse point. This assumption can be supported by the fact that the surface pressure at the collapse point for adsorbed and spread mixed films is practically equal to that for the pure monoglyceride in the Langmuir- and Wilhelmy-type film balances (Figs. 3, 4, 6, and 7). From these results it can been seen that there was a film expansion as the monoglyceride concentration in the mixture was increased, especially at higher surface pressures. That is, the p-A isotherm is displaced towards higher A as the concentration of monoglyceride in the mixture increases. At surface pressures higher than that for protein collapse (at p4pprotein ), the p-A isotherm for mixed films e was parallel to that of monoglyceride. These results suggest that at p4pprotein protein displacement by the monoglye ceride from the air–water interface takes place. At popprotein both protein and monoglyceride coexist at the e interface and the p-A isotherms of adsorbed and spread mixed films (i.e., the monolayer structures) are different (Figs. 3, 4, 6, and 7). At the highest surface pressures, at the collapse point of the mixed film, immiscibility between film forming components is deduced due to the fact that the

ARTICLE IN PRESS 50

50

40

40

Surface pressure (mN/m)

Surface pressure (mN/m)

M. Cejudo Ferna´ndez et al. / Food Hydrocolloids 21 (2007) 906–919

πeβ-lactoglobulin

30

20

10

0 0.2

0.4

0.6

0.8

1.0

1.2

1.4

πeβ-casein

20

10

1.6

2

Area (m /mg)

(A)

0.0

0.4

40

40

20

10

0

0.8

1.0

1.2

1.4

1.6

Area (m /mg) 50

πeβ-lactoglobulin

0.6

2

50

30

0.2

(A)

Surface pressure (mN/m)

Surface pressure (mN/m)

30

0 0.0

30 πeβ-casein

20

10

0 0.0

(B)

913

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

Area (m2/mg)

0.0

(B)

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

Area (m2/mg)

Fig. 6. p-A isotherms (compression curves) for mixed monolayers at the air–water interface of b-lactoglobulin and monoolein formed by the spreading of monoolein on (- - -) adsorbed and (—) spread monolayers of b-lactoglobulin at mass fraction of monoolein in the mixture of (A) 20 wt% and (B) 40 wt%. Aqueous subphase at pH 7. Temperature 20 1C. The equilibrium surface pressure for b-lactoglobulin (pb-lactoglobulin ) is e indicated by an arrow.

Fig. 7. p-A isotherms (compression curves) for mixed monolayers at the air–water interface of b-casein and monoolein formed by the spreading of monoolein on (- - -) adsorbed and (—) spread monolayers of b-casein at mass fraction of monoolein in the mixture of (A) 20 wt% and (B) 40 wt%. Aqueous subphase at pH 7. Temperature 20 1C. The equilibrium surface pressure for b-casein (pb-casein ) is indicated by an arrow. e

collapse pressure of mixed films is similar to that of a pure monoglyceride monolayer (Figs. 3, 4, 6, and 7). The evolution with the surface pressure of BAM images (Fig. 5) gives complementary information, at a microscopic level, on the structural characteristics and interactions of adsorbed protein–monopalmitin mixed films, as deduced from p-A isotherms. At popprotein a mixed film of e monopalmitin and protein may exist with small circular LC domains of monopalmitin uniformly distributed on the protein layer (Fig. 5b). The circular LC domains of monopalmitin in the mixed film were more numerous as the surface pressure increased, as for a pure monopalmitin monolayer. The homogeneous phase may be due to the coexistence of LE monopalmitin domains and protein (image similar to Fig. 5a). During repeated compressions of the mixed film some spots with folds or aggregations of collapsed b-casein, which were formed during the first compression of the film, were also observed (Fig. 5f), a

phenomenon that was not observed for b-lactoglobulin– monopalmitin adsorbed mixed films. At p4pprotein a e characteristic squeezing out phenomena of the protein by monopalmitin was observed (Fig. 5c) and the mixed films were practically dominated by monopalmitin molecules. That is, at higher surface pressures, collapsed protein residues (bright region) may be displaced from the interface by monopalmitin molecules (circular dark regions). A topographical characteristic of the adsorbed film was the presence of short fractures in the film at the higher surface pressures, near to the collapse point of the mixed film (Fig. 5e), which are characteristic of protein–monoglyceride adsorbed films (Rodrı´ guez Patino & Cejudo, 2004). BAM images for adsorbed protein–monoolein mixed films were also different to those described above for adsorbed protein+monopalmitin mixed films (Fig. 5). At popprotein the topography of pure components and the e mixed film is practically identical because in this region

ARTICLE IN PRESS 914

M. Cejudo Ferna´ndez et al. / Food Hydrocolloids 21 (2007) 906–919

Fig. 8. Visualisation of protein+monoolein mixed monolayers by Brewster angle microscopy at 20 1C. (a) Homogeneous image for pure components and after the spreading of monoolein on a spread or adsorbed protein film at popprotein . (b) Mixed films after spreading of monoolein on a spread or adsorbed e protein film at p4pprotein . (c) Mixed films after spreading of monoolein on an adsorbed protein film at the collapse point of the mixed film. The horizontal e direction of the image corresponds to 630 mm and the vertical direction to 470 mm. 60 50 40 π (mN/m)

both components and the mixed film form an isotropic (homogeneous) film without any difference in the domain topography (Fig. 8a). However, during repetitive compressions of the mixed film some spots with folds or aggregations of collapsed b-casein, which were formed during the first compression of the film, were also observed (image similar to Fig. 5f). At surface pressures near to and after protein collapse BAM images (Fig. 8b) demonstrated that monoolein and protein molecules adopted an isotropic structure in the mixed film with some white regions, which correspond to the presence of a thicker protein collapsedfilm. At the higher surface pressures, and especially at the collapse point, the topography of the mixed film was dominated by the presence of small domains of collapsed protein and monoolein at the interface (Fig. 8c).

30

πeβ-lactoglobulin

20 10 0 0.2

0.4

0.6

(A)

3.4. Penetration of a protein into a spread monoglyceride monolayer

0.8

1.0

1.2

1.4

1.6

A (m2/mgMONOPALMITIN) 60 50 40

π (mN/m)

These experiments were performed after the study of the dynamics of penetration of the protein into a monoglyceride monolayer spread at the air–water interface, by following the time evolution of the surface pressure after the injection of a protein solution underneath the monoglyceride monolayer, and when the surface pressure relaxed to a steady state value. The surface pressure of the monoglyceride monolayer at the beginning of the penetration process (at pMP and pMO ) was the variable studied i i (Carrera et al., 2006). The p-A isotherms for monopalmitin monolayers penetrated by proteins on the basis that only monopalmitin was present at the interface (A, m2/mgMONOPALMITIN) at different pMP and at 20 1C are shown in Fig. 9. In the same i figure we have included the p-A isotherm for a spread monopalmitin monolayer registered at the beginning of each experiment, which can be used as a control and for comparison. For monopalmitin spread monolayers penetrated by proteins (b-lactoglobulin and b-casein) at different pMP it can be seen (Fig. 9) that, on the basis i that only monopalmitin was present at the interface, the pA isotherms for monopalmitin and for protein–monopalmitin mixed monolayers p4pprotein are practically coine cident. That is, the structures of the mixed monolayers are

30 πeβ-casein

20 10 0 0.0

(B)

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

2

A (m /mgMONOPALMITIN)

Fig. 9. p-A isotherms (compression curves) for mixed monolayers at the air–water interface of monopalmitin penetrated by (A) b-lactoglobulin at 1  105 wt% and (B) b-casein at 5  106 wt%. Initial surface pressure of a monopalmitin monolayer before the injection of the protein into the aqueous phase: (—) 10 mN/m, (- - -) 20 mN/m, and (?) at the collapse point. The p-A isotherm of a pure monopalmitin spread monolayers is included as a reference (J). Aqueous subphase at pH 7. Temperature 20 1C. The equilibrium surface pressures for b-lactoglobulin (pb-lactoglobulin ) e ) are indicated by arrows. and b-casein (pb-casein e

practically dominated by the presence of monopalmitin. In fact, the first-order transition between LC and LE structures can be distinguished, which is typical of a pure

ARTICLE IN PRESS M. Cejudo Ferna´ndez et al. / Food Hydrocolloids 21 (2007) 906–919 50

π (mN/m)

40 πeβ-lactoglobulin

30

20

10

0 0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

A (m2/mgMONOOLEIN)

(A) 50

40

π (mN/m)

monopalmitin monolayer. In addition, the mixed film collapsed at the collapse pressure of a pure monopalmitin monolayer. These results suggest that at p4pprotein a e protein displacement by the monoglyceride from the air– water interface takes place. At popprotein both protein and e monopalmitin may coexist at the interface, but the structural characteristics of the mixed monolayers are dominated by the presence of monopalmitin. b-Casein– monopalmitin mixed monolayers at pMP ¼ 10 mN/m are i an exception. For this system and at popb-casein the p-A e isotherms for b-casein–monopalmitin mixed monolayers are different from those of pure components. BAM images and the evolution of the reflectivity of the interface with the surface pressure (data not shown) corroborate at a microscopic level these conclusions. In summary, although protein molecules have the capacity to penetrate a spread monopalmitin monolayer, reaching a steady state surface pressure close to pprotein , e monopalmitin molecules have the capacity to re-enter the monolayer after the expansion and recompression of the mixed monolayer. As monopalmitin re-enters the air–water interface the structure of protein–monopalmitin mixed monolayers is practically dominated by the presence of monopalmitin. These results also suggest that the monopalmitin molecular loss by collapse and/or desorption deduced from dynamic surface pressure measurements is reversible (Carrera et al., 2006). We speculate that during the protein penetration into a spread monopalmitin monolayer at the collapse point, monopalmitin molecules are displaced from the interface towards the subphase region and that an irreversible molecular loss into the bulk phase does not take place. The p-A isotherms for monoolein monolayers penetrated by proteins (b-lactoglobulin and b-casein) on the basis that only monoolein was present at the interface (A, m2/ mgMONOOLEIN) at different pMO and at 20 1C are shown i in Fig. 10. In the same figure we have included the p-A isotherms for a spread monoolein monolayer registered at the beginning of each experiment. For monoolein spread monolayers penetrated by protein at different pMO i (Fig. 10) it can be seen that, on the basis that only monoolein was present at the interface, the p-A isotherms for pure monoolein monolayers are different from those for protein–monoolein mixed monolayers. That is, although pure and mixed monolayers present the same LE-like structure, the p-A isotherms for protein–monoolein mixed monolayers are displaced in relation to pure monolayer components. Interestingly, at p4pprotein the e p-A isotherms for protein–monoolein mixed monolayers are parallel, but displaced towards lower monolayer mass areas. These results suggest that the monoolein monolayer molecular loss due to collapse and/or desorption that takes place during the preceding penetration experiments is irreversible (Carrera et al., 2006). In fact, the displacement of p-A isotherms for protein–monoolein mixed monolayers towards lower monolayer mass areas is more intense as pMO increases, especially at the collapse point, in agreei

915

30 πeβ-casein

20 10 0 0.0

0.2

(B)

0.4

0.6

0.8

1.0

1.2

1.4

1.6

2

A (m /mgMONOOLEIN)

Fig. 10. p-A isotherms (compression curves) for mixed monolayers at the air–water interface of monoolein penetrated by (A) b-lactoglobulin at 1  105 wt% and (B) b-casein at 5  106 wt%. Initial surface pressure of a monoolein monolayer before the injection of the protein into the aqueous phase: (—) 10 mN/m, (- - -) 20 mN/m, and (?) at the collapse point. The p-A isotherm of a pure monoolein spread monolayer is included as a reference (J). Aqueous subphase at pH 7. Temperature 20 1C. The equilibrium surface pressures for b-lactoglobulin (pb-lactoglobulin ) e and b-casein (pb-casein ) are indicated by arrows. e

ment with long-term relaxation data for a pure monoolein monolayer (Carrera, Rodrı´ guez Nin˜o & Rodrı´ guez Patino, 1999). a protein displacement by To summarise, at p4pprotein e monoolein from the air–water interface, preceded by a previous monoolein molecular loss, takes place. At popprotein both protein and monoolein may coexist at e the interface. The evolution of the reflectivity of the interface with the surface pressure (data not shown) corroborates at a microscopic level these conclusions. 4. Discussion The p-A isotherms deduced for adsorbed b-lactoglobulin films (Fig. 2A) in the Wilhelmy-type film balance (Rodrı´ guez Patino, Cejudo et al., 2006b) are in good

ARTICLE IN PRESS 916

M. Cejudo Ferna´ndez et al. / Food Hydrocolloids 21 (2007) 906–919

agreement with those observed in a Langmuir-type film balance (Rodrı´ guez Patino & Cejudo, 2004). On the other hand, the p-A isotherm deduced for adsorbed b-lactoglobulin film (Fig. 2A) is similar to that obtained by spreading in the Wilhelmy- (Rodrı´ guez Patino et al., 2001) and Langmuir-type (Rodrı´ guez Nin˜o, Carrera, Pizones, & Rodrı´ guez Patino, 2005) film balances. Thus, the structures of b-lactoglobulin films formed in the two different ways must be identical, at least for adsorption from low bulk protein concentrations. The p–A isotherm deduced for adsorbed b-casein films (Cejudo et al., 2006; Rodrı´ guez Patino, Cejudo et al. 2006a) is more condensed (Fig. 2B) than that obtained directly by spreading (Rodrı´ guez Patino, Carrera et al., 1999d, 2001b). One explanation is that the slow formation of the adsorbed film in this study will allow the protein extended time and space to unfold, whereas in spread films the protein is forced into an interfacial space with little time or area to unfold. Thus, the structures of the films formed in the two different ways must be different, at least for adsorption from low bulk protein concentrations. The surface pressure at the transition between structures I and II of an adsorbed bcasein film (pat E12 mN/m) is higher than that for a spread monolayer (pst E10 mN/m). However, the transition between these structures is not as evident in the p-A isotherm for b-casein adsorbed films, as compared with spread monolayers. In this regard, b-lactoglobulin (a globular protein) and b-casein (a disordered protein) (Rodriguez Patino, Cejudo et al., 2006a) behaved in a different way. The morphology (Fig. 2C-a) and, especially, the reflectivity as a function of surface pressure obtained with b-lactoglobulin adsorbed films reproduce those for spread monolayers (Rodrı´ guez Patino et al., 1999d; Rodrı´ guez Patino, Carrera et al., 2001). This shows that this protein adopts a homogeneous morphology during the film compression and corroborates the idea that a condensation in the monolayer structure takes place during the compression of the monolayer because the film thickness increases with the surface pressure. BAM images also prove that b-casein spread (Rodrı´ guez Patino et al., 1999d) and adsorbed films adopt a homogeneous morphology during the first compression of the film (Fig. 2C-a). However, after successive compressions b-casein adsorbed films present some aggregations forming a network on a dark background (Fig. 2C-b), which were not observed for spread monolayers (Rodrı´ guez Patino et al., 1999d). From a systematic study centred on the p-A isotherm of spread 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 (Rodrı´ guez Patino et al., 1999a, 1999b, 2001) that these compounds form a practically immiscible monolayer at the air–water interface, at popprotein . The topographical e characteristics of the adsorbed and spread mixed films are essentially similar, except in the presence of both short fractures near to the collapse point of the mixed film

(Fig. 5) and folds or aggregations of collapsed b-casein (Figs. 5), which were not observed in spread mixed films. At higher surface pressures the collapsed protein is displaced from the interface by monoglycerides. However, from the results presented in this study the idea also emerges that the displacement of disordered (b-casein) and globular (b-lactoglobulin) proteins by monoglyceride from the air–water interface depends on the protein–monoglyceride system and the formation of the mixed film. From the p-A isotherms shown in Figs. 3, 4, 6, and 7 we define a displacement surface pressure (pd) as the minimum surface pressure above which the p-A isotherms for monoglyceride and monoglyceride–protein mixed monolayers are coincident. Thus, at low pd the protein displacement by the monoglyceride is facilitated. In Figs. 11 and 12 we show the pd as a function of the mixture composition for different monoglyceride–protein mixed films formed by spreading of film forming components (Fig. 11) or by the spreading of monoglyceride on a protein-adsorbed film (Fig. 12). For spread mixed films (Fig. 11) the protein displacement by monopalmitin is easier for b-casein than b-lactoglobulin. In fact, at X40.2 the pd values for b-casein+monopalmitin mixed films are lower than those for b-lactoglobulin+monopalmitin. That is, the existence of low protein interactions in disordered proteins, as deduced from dilatational properties for bcasein (Rodrı´ guez Patino, Carrera et al., 2001), facilitates the protein displacement by monopalmitin from the air– water interface. These results are in agreement with the opinion that the displacement of proteins by surfactants depends on the extent of protein–protein interactions (Dickinson & Hong, 1994). On the other hand, the lower surface activity of monoolein justifies the idea that this lipid has a lower capacity than monopalmitin for protein displacement. In fact, monoolein requires higher surface pressures than monopalmitin for protein displacement from the air–water interface (Fig. 11). The displacement of a protein-adsorbed film by the spreading of a monoglyceride is more complex (Fig. 12). At low monoglyceride concentration (at X ¼ 0.2) protein displacement is easier for monoolein than for monopalmitin. In fact, under these conditions it is necessary to compress the mixed film up to the pe value of the monoglyceride, which is higher for monopalmitin than for monoolein. However, the opposite is observed at higher monoglyceride concentrations. In fact, at XX0.4 the pd value for protein+monopalmitin mixed films is lower than those for protein+monoolein mixed films. That is, under these conditions protein displacement is easier for monopalmitin than for monoolein. However, higher monoglyceride concentrations in the mixture than those included in this study are necessary for a quantitative displacement of protein from the interface, as observed for protein+monoglyceride spread films (Fig. 11). That is, the capacity of a monoglyceride for protein displacement from the air–water interface is easier for spread (Fig. 11) than for adsorbed (Fig. 12) monolayers. Finally, the displacement of a protein

ARTICLE IN PRESS M. Cejudo Ferna´ndez et al. / Food Hydrocolloids 21 (2007) 906–919 60

917

60 (A)

πe MP

50

50

40

40

πe MP

π (mN/m)

π (mN/m)

πe MO

30 πe β-lg 20

πe β-lg

30 20

πe β-cs

10

10

0

0 60

0.1

(B) πe MO

50

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

XMONOGLYCERIDE

(A) 60 50

πe MP

30 πe β-lg 20

π (mN/m)

π (mN/m)

40

πe β-cs

10

πe MO

40 30

πe β-cs 20

0 0.2

0.3

0.4

0.5

0.6

0.7

0.8

10

XMONOGLYCERIDE

Fig. 11. The displacement surface pressure of (D) b-lactoglobulin and (p) b-casein 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 pressure of pure b-lactoglobulin (—, pe b-lg), b-casein (—, pe b-cs), monoolein (?, pe MO), and monopalmitin (—, pe MP) pure monolayers at 20 1C and at pH 7.

by a monoglyceride from the air–water interface is easier for b-casein–monoglyceride than for b-lactoglobulin– monoglyceride mixed monolayers. The protein displaced by monoglyceride from the interface during compression remains underneath the monoglyceride film either through hydrophobic interactions between protein and lipid or by local anchoring through the monoglyceride layer (Rodrı´ guez Patino & Cejudo, 2004; Rodrı´ guez Patino, Cejudo et al., 2006a, 2006b; Li et al., 1998) and re-enters the mixed film during the expansion. This statement is supported by the fact that the p-A isotherms were repetitive after continuous compression–expansion cycles (data not shown). However, for adsorbed protein+monoglyceride mixed films a first orderlike phase transition was observed upon film expansion at surface pressures close to pprotein . This result suggests that e the re-adsorption of previously displaced protein has a kinetic character (Rodrı´ guez Patino & Cejudo, 2004; Rodrı´ guez Patino, Cejudo et al., 2006a, 2006b). The coexistence of protein and monoglyceride at popprotein or the protein displacement by monoglyceride e

0 0.1

(B)

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

XMONOGLYCERIDE

Fig. 12. The displacement surface pressure of (A) b-lactoglobulin and (B) b-casein adsorbed film by (n) monopalmitin and (J) monoolein spread at the air–water interface as a function of the mass fraction of monoglyceride in the mixture. The horizontal lines represent the equilibrium surface pressure of pure b-lactoglobulin (—, pe b-lg), b-casein (—, pe b-cs), monoolein (?, pe MO), and monopalmitin (—, pe MP) films at 20 1C and at pH 7.

have repercussions on the surface mechanical at p4pprotein e properties of the mixed films (Rodrı´ guez Patino, Cejudo et al., 2006a, 2006b; Rodrı´ guez Patino, Cejudo, Carrera & Rodrı´ guez Nin˜o, 2006c). For instance, as protein and monoglyceride coexist in the mixed films at popprotein the e surface dilatational properties (Rodrı´ guez Patino, Cejudo et al., 2006a, 2006b) are practically the same for adsorbed and spread films. However, at the higher surface pressures the surface dilatational modulus (E) for adsorbed protein+monoglyceride mixed films are lower than for spread mixed monolayers at the same surface pressures. These results corroborate the idea that protein in adsorbed mixed films presents a higher resistance to its displacement by monoglyceride from the interface as compared with spread mixed monolayers. Recent studies have shown that the displacement of proteins from fluid–fluid interfaces by water- and oilsoluble emulsifiers involves an orogenic mechanism

ARTICLE IN PRESS 918

M. Cejudo Ferna´ndez et al. / Food Hydrocolloids 21 (2007) 906–919

(Mackie, Gunning, Wilde, & Morris, 1999 & 2000; Gunning, Mackie, Wilde, & Morris, 1999). The mechanism reveals that emulsifier penetrates into the protein film, forming separate adsorbed domains that exert a lateral surface pressure, 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 (Mackie, Gunning, Ridout, Wilde, & Rodrı´ guez Patino, 2001). This model has been shown to work for a range of proteins with different secondary and tertiary structures, and at solid/liquid, liquid/liquid and gas/liquid interfaces. It has also been shown to apply to different types of emulsifiers (non-ionic, cationic, anionic and zwitterionic). However, there exist differences between adsorbable water- or oil-soluble emulsifiers and protein–monoglyceride monolayers anchored at the air–water interface by spreading, penetration or displacement/competition (Fig. 1). The results of this study (Figs. 3–10) suggest that during compression of protein–monoglyceride monolayers the first stage of the orogenic mechanism, which occurs at surface pressures lower than the equilibrium surface pressure of the protein (at popprotein ), involves a displacement front of monoe glyceride domains instead the adsorption of water- or oilsoluble emulsifier molecules at defects in the protein network. The second stage, which occurs at surface pressures near to and above the equilibrium surface pressure of the protein (at pX pprotein ), involves a buckling e 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 surface pressures (at p4pprotein ) the protein network begins to e fail, freeing proteins, which then desorb from the interface. But, for protein–monoglyceride anchored 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 applied to protein– monoglyceride monolayers is a consequence of both the existence of few interactions between protein and monoglyceride molecules at the air–water interface and the absence of formation of complexes by ionic and/or hydrophobic interactions, as observed for protein and surfactant or lipid molecules at the interface (Miller et al., 2000; Fainerman, Leser, Michel, Lucassen-Reynders, & Miller, 2005). The level of interactions between adsorbed protein and monoglyceride molecules at the air–water interface and the consequences of these interactions on the structural and topographical characteristics of the mixed monolayers depend on the method used for the formation of the proper monolayer (by spreading, penetration or displacement/competition). 5. Conclusions The phenomena analysed here, using Langmuir- and Wilhelmy-type film balances coupled with BAM, are

essentially the same for spread and adsorbed protein films and for spread, adsorbed or penetrated mixed films. These results demonstrate that it is possible to measure reproducible p-A isotherms for adsorbed protein films from low protein concentration in the bulk phase. This is clear evidence that the p-A isotherms obtained after long adsorption time have a thermodynamic character. Thus, under these conditions the p-A isotherms for spread pure and mixed films, which are more commonly used for fundamental studies (Rodrı´ guez Patino et al., 2003), with a strict control of the concentration of the film forming components at the air–water interface, can be used to analyse adsorbed or penetrated mixed films, which are more interesting from a practical point of view. Some differences between adsorbed and spread mixed films are: (i) the interactions between film forming components are higher for adsorbed than for spread mixed films, (ii) the adsorbed films are more segregated than spread films, (iii) the protein displacement by monoglycerides is easier for spread than for adsorbed films, (iv) the collapsed protein domains in adsorbed films are smaller than for spread films. All these phenomena are a consequence of the increased interactions between components at the interface in the former. Finally, (v) the rate of re-adsorption of previously displaced protein is slower for adsorbed than for spread films. These results may be of direct relevance to product processing (especially for foam formation and stabilisation) and as a model study for extrapolation to more complex real systems (food foams and emulsions). Acknowledgement This research was supported by CICYT through Grant AGL2004-1306/ALI. References 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. Bos, M., Nylander, T., Arnebrant, T., & Clark, D. C. (1997). Protein/ emulsifier interactions. In G. L. Hasenhuette, & R. W. Hartel (Eds.), Food Emulsions and their Applications (pp. 95–146). New York: Chapman & Hall, pp. Carrera, C., Cejudo, M., Rodrı´ guez Nin˜o, Ma. R., & Rodrı´ guez Patino, J. % characteristics of monoM. (2006). Thermodynamic and dynamic glyceride monolayers penetrated by b-casein. Langmuir, 22, 4215– 4224. Carrera, C., Rodrı´ guez Nin˜o, M. 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. Cejudo, M., Carrera, C., Rodrı´ guez Nin˜o, M. R., & Rodrı´ guez Patino, J. M. (2006). The effect of monoglyceride on structural and topographical characteristics of adsorbed b-casein films at the air–water interface. Biomacromolecules, 7, 507–514. 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., & Hong, S. K. (1994). Surface coverage of b-lactoglobulin at the oil–water interface: influence of protein heat treatment and

ARTICLE IN PRESS M. Cejudo Ferna´ndez et al. / Food Hydrocolloids 21 (2007) 906–919 various emulsifiers. Journal of Agricultural and Food Chemistry, 42, 1602–1606. Fainerman, V. B., Leser, M. E., Michel, M., Lucassen-Reynders, E. H., & Miller, R. (2005). Kinetics of the desorption of surfactants and proteins from adsorption layers at the solution/air interface. Journal of Physical Chemistry B, 109, 9672–9677. Friberg, S. E., Larsson, K., & Sjo¨blom, J. (2004). Food Emulsions (4th ed). New York: Marcel Dekker. Graham, D. E., & Phillips, M. C. (1979). Proteins at liquid interfaces. III. Molecular structures of adsorbed films. Journal of Colloid and Interface Science, 70, 427–439. Gunning, A. P., Mackie, A. R., Wilde, P. J., & Morris, V. J. (1999). In-situ 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. Leser, M. E., Michael, M., & Watzke, H. J. (2003). Food goes nano-new horizonts for food structure research. In E. Dickinson, & E. T. van Vliet (Eds.), Food Colloids: Biopolymers and Materials. Cambridge: Royal Society Chemistry, 3–13. Li, J. B., Kra¨gel, J., Makievski, A. V., Fainermann, V. B., Miller, R., & Mo¨hwald, H. (1998). A study of mixed phospholipid/b-casein monolayers at the water/air surface. Colloids and Surfaces A: Physical and Engineering Aspects, 142, 355–360. 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 interface 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. Mackie, A. R., Gunning, A. P., Wilde, P. J., & Morris, V. J. (2000). Competitive displacement of b-lactoglobulin from the air–water interface by sodium dodecyl sulfate. Langmuir, 16, 8176–8181. McClements, D. J. (2005). Food Emulsions: Principles, Practice and Techniques (2nd edition). Boca Raton, FL: CRC Press. Miller, R., Fainerman, V. B., Makievski, A. V., Kragel, J., Grigoriev, D. O., & Kazakov, V. N. (2000). Dynamics of protein and mixed protein surfactant adsorption layers at the water/fluid interface. Advances in Colloid and Interface Science, 86, 39–82. Murray, B. S., Faergemand, M., Trotereau, M., & Ventura, A. (1999). Comparison of the dynamic behaviour of protein films at air–water and oil–water interfaces. In E. Dickinson, & J. M. Rodrı´ guez Patino (Eds.), Food Emulsions and Foams: Interfaces, Interactions and Stability (pp. 223–235). Cambridge: Royal Soc. Chem. Pugnaloni, L. A., Dickinson, E., Ettelaie, R., Mackie, A. R., & Wilde, P. J. (2004). Competitive adsorption of proteins and low-molecular-weigh surfactants: computer simulation and microscopic imaging. Advances in Colloid and Interface Science, 107, 27–49. Rodrı´ guez Nin˜o, M. R., Carrera, C., Pizones, V., & Rodrı´ guez Patino, J. M. (2005). Milk and soy protein films at the air–water interface. Food Hydrocolloids, 19, 417–428. Rodrı´ guez Nin˜o, Ma. R., & Rodrı´ guez Patino, J. M. (2002). Effect of the % aqueous phase composition on the adsorption of bovine serum albumin to the air–water interface. Industrial and Engineering Chemical Research, 41, 1489–1495.

919

Rodrı´ guez Nin˜o, Ma. R., Carrera, C. S., & Rodrı´ guez Patino, J. M. % (1998). Interfacial characteristics of b-casein spread films at the air–water interface. Colloids Surfaces : Biointerfaces, 12, 161–173. Rodrı´ guez Nin˜o, Ma. R., Carrera, C., Cejudo, M., & Rodrı´ guez Patino, J. % M. (2001). Protein and lipid adsorbed and spread films at the air–water interface at the equilibrium. Journal of the American Oil Chemical Society, 78, 873–879. Rodrı´ guez Patino, J. M., & Cejudo, M. (2004). Structural and topographical characteristics of adsorbed WPI and monoglyceride mixed monolayers at the air–water interface. Langmuir, 20, 4515–4522. Rodrı´ guez Patino, J. M., Carrera, C., & Rodrı´ guez Nin˜o, M. R. (1999c). Morphological and structural characteristics of monoglyceride monolayers at the air–water interface. Langmuir, 15, 2484–2492. Rodrı´ guez Patino, J. M., Carrera, C., & Rodrı´ guez Nin˜o, M. 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. (1999a). % air–water Analysis of b-casein–monopalmitin mixed films at the interface. Journal of Agricultural and 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. S., Rodrı´ guez Nin˜o, Ma., & Cejudo, % F. M. (2001). Structural and dynamic properties of milk proteins spread at the air–water interface. Journal of Colloid and Interface Science, 242, 141–151. Rodrı´ guez Patino, J. M., Cejudo, M., Carrera, C., & Rodrı´ guez Nin˜o, M. R. (2006c). Structural and shear characteristics of adsorbed b-casein and monoglyceride mixed monolayers at the air/water interface. Industrial and Engineering Chemical Research, 45, 1886–1895. Rodrı´ guez Patino, J. M., Cejudo, M., Carrera, C., & Rodrı´ guez Nin˜o, M. R. (2006a). Spreading of monoglyceride onto b-casein adsorbed film. Structural and dilatational characteristics. Journal of Agricultural and Food Chemistry, 54, 3723–3732. Rodrı´ guez Patino, J. M., Cejudo, Rodrı´ guez Nin˜o, M. R., & Carrera, C. (2006b). Self-assembly of monoglyceride in b-lactoglobulin adsorbed films at the air–water interface. Structure, topographical and rheological consequences. Biomacromolecules, 7, 2661–2670. Rodrı´ guez Patino, J. M., Lucero, A., Rodrı´ guez Nin˜o, Ma. R., Mackie, A. % R., Gunning, A. P. & Morris, V. J. (2006). Some implications of nanoscience in food dispersion formulations containing phospholipids as emulsifiers. Food Chemistry, in press, doi:10.1016/j.foodchem.2006.06.010. Rodrı´ guez Patino, J. M., Rodrı´ guez Nin˜o, M. R., & Carrera, C. (2003). 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., & Cejudo, % M. (2001). Whey protein isolate–monoglyceride mixed monolayers at the air–water interface. Structure, morphology, and interactions. Langmuir,, 17, 7545–7553. Wilde, P. J., Mackie, A. R., Husband, F., Gunning, P., & Morris, V. (2004). Protein and emulsifiers at liquid interfaces. Advances in Colloid and Interface Science, 108–109, 63–71.