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 Sánchez, Mª. Rosario Rodrı́guez Niño, Ana Lucero Caro,
Juan M. Rodrı́guez Patino *
Departamento de Ingeniera Quı́mica, Facultad de Quı́mica, Universidad de Sevilla, C/. Prof. Garcı́a González, 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-phosphat-
idyl-choline, DOPC) at the air–water interface. Combined surface chemistry (surface film balance and static and dynamic tensiom-
etry) 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 inter-
facial 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 pres-
sure (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 stability, shelf-life, or product texture. For up-to-date

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 associa-
tion with other components (polysaccharides). In addi-
tion 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, tempera-
ture, 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: jmrodri@us.es (J.M. Rodrı́guez Patino).
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 mono-
and diglycerides, phospholipids, etc.) and macro-
molecules (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 tex-
tural properties so desired by manufacturers and appre-
ciated by consumers.
Proteins and LMWE have an important physical prop-
erty in common, their amphiphilic nature (Horne &

0260-8774/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.jfoodeng.2004.05.065
226 C.C. Sánchez et al. / Journal of Food Engineering 67 (2005) 225–234
Rodrı́guez Patino, 2003; Rodriguez Niño, Rodrı́guez
Patino, Carrera, Cejudo, & Navarro, 2003). This prop-
erty provides the potential for association, adsorption,
and reorientation at fluid interfaces, depending on the
properties of the components and the protein–LMWE ra-
tio (Rodrı́guez Niño & Rodrı́guez Patino, 1998a, 1998b;
Rodrı́guez Niño, Carrera, Cejudo, & Rodrı́guez Patino,
2001; Rodrı́guez Patino, Rodrı́guez Niño, & Carrera,
2003a). LMWE stabilize the dispersed droplets or bub-
bles 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 ten-
sions (Rodrı́guez Niño & Rodrı́guez Patino, 1998a,
1998b). In contrast, proteins act as polymeric emulsifiers
with multiple anchoring sites at the interface that, to-
gether with the unfolding process of the adsorbing pro-
tein molecule, stabilize the interfacial layer kinetically.
This behavior contributes significantly to the interfacial
rheological properties and immobilizes proteins in the ad-
sorbed 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 (typi-
cally, mono- and diglycerides) are added to break the ad-
sorbed 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; Rodrı́-
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 behav-
ior of milk proteins and LMWE (monoglycerides and
phospholipids). Emphasis will be on the air–water inter-
face as a three-dimensional dynamic entity. We will con-
sider emulsifier (protein, LMWE and their mixtures)
adsorption, structure and topography at the interface,
relaxation phenomena, and interfacial rheology, as re-
lated to the formation and stability of food dispersions
(emulsions and foams).
2. Experimental
2.1. Chemicals
Synthetic 1-monohexadecanoyl-rac-glycerol (mono-
palmitin, DIMODANR PA 90), 1-mono(cis-9-octa-
decanoyl)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 -a-
dipalmitoyl-phosphatidyl-choline (DPPC, Sigma (St.
Louis, MO, USA), 99%) and L -a-dioleoyl-phosphat-
idyl-choline (DOPC, Sigma, 99%) were used as supplied.
Ninety-nine percent pure b-casein was supplied and puri-
fied 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 b-
lactoglobulin (protein 92 ± 2%, b-lactoglobulin >95%,
a-lactalbumin <5%) obtained by fractionation, was sup-
plied by Danisco Ingredients (Brabran, Denmark). To
form the surface film, monoglyceride and phospholipid
were spread in the form of a solution, using hexane:eth-
anol (9:1, v:v) and chloroform:ethanol (4:1, v:v), respec-
tively, 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 relaxa-
tion phenomena in spread monolayers at the air–water
interface (Gaines, 1966). The equilibrium spreading pres-
sure 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, Rodrı́guez Pati-
no, & Rodrı́guez Niño, 1999). The adsorption isotherm
of proteins and LMWE–protein films was studied by tensi-
ometry as described elsewhere (Rodrı́guez Niño & Rodrı́-
guez Patino, 1998a; Rodrı́guez Niño et al., 2001).
2.2.2. Surface film balance
Measurements of the surface pressure (p) versus aver-
age area per molecule (A), the so-called p–A isotherm,
were performed on a fully automated Langmuir- or
 C.C. Sánchez et al. / Journal of Food Engineering 67 (2005) 225–234 227

Wilhelmy-type film balance as described elsewhere

(Rodrı́guez Niño, Carrera, & Rodrı́guez Patino, 1999).

2.2.3. Brewster angle microscopy

For microscopic observation of the monolayer struc-
ture, the Brewster angle microscope, BAM 2 plus (NFT,
Germany) was used as described elsewhere (Rodrı́guez
Patino, Carrera, & Rodrı́guez Niño, 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 rel-
ative reflectivity (I), according to a procedure described
previously (Rodrı́guez Patino et al., 1999a, Rodrı́guez
Patino, Carrera, & Rodrı́guez Niño, 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
(Rodrı́guez Patino, Carrera, Rodrı́guez Niño, & Cejudo,
2001c, 2001d).
3. Results and discussion
3.1. Protein and LMWE films at equilibrium
Fig. 1. (A) Equilibrium surface pressure of monopalmitin (MP),
monoolein (MO), dipalmitoyl-phosphatidyl-choline (DPPC), dioleoyl-
phosphatidyl-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.

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 monoglycer-
ides (Rodriguez Niño et al., 2003) and phospholipids
(unpublished data) and lower for proteins (Rodrı́guez
Niño et al., 2001). The effect of temperature (data not
shown) was also different for LMWE and proteins, espe-
cially 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 interac-
tions within the film forming components thus, with a
more condensed film structure at equilibrium.
Protein–LMWE interactions at the air–water inter-
face can be studied by tensiometry. From these experi-
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 mono-
glyceride (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 (Rodrı́guez Niño & Rodrı́-
guez Patino, 1998a, 2001). However, the protein deter-
228 C.C. Sánchez et al. / Journal of Food Engineering 67 (2005) 225–234
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 pro-
teins and LMWE spread films at the air–water interface
can be deduced from p–A isotherms (Rodrı́guez Patino
& Rodrı́guez Niño, 1999) coupled to information from
BAM (Rodrı́guez Patino et al., 1999a, 1999b). From
the p–A isotherm, different structures can be deduced
for LMWE monolayers as a function of LMWE, temper-
ature, and surface density or surface pressure. For
instance, monopalmitin (and most saturated phospholi-
pids) 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 mono-
palmitin, 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 cir-
cular 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 van-
ishes suddenly, and the presence of monolayer fractures
can be observed in different zones (Rodrı́guez Patino et
al., 1999a). BAM images corroborate that only the homo-
geneous 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 (Rodrı́guez Patino et al.,
1999a). The film thickness increases as the monolayer is
compressed, passes through a maximum and then de-
creases 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 (Rodrı́guez 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 de-
duced 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 isot-
ropy. 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 col-
lapse (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
(Rodrı́guez Patino et al., 1999b). The differences ob-
served between lipids (Rodrı́guez Patino et al., 1999a)
and proteins (Rodrı́guez Patino et al., 1999b) in p–A iso-
therms and BAM images is of great utility for the appli-
cation of BAM to the analysis of more complicated
systems in which proteins and lipids are spread at the
interface (Rodrı́guez Patino, Carrera, & Rodrı́guez
Niño, 1999c, 1999d; Rodrı́guez Patino, Rodrı́guez Niño,
Carrera, & Cejudo, 2001a, Rodrı́guez Patino, Rodrı́guez
Niño, Carrera, & Cejudo, 2001b).
From a systematic study centered on the p–A iso-
therm of protein–monoglyceride mixed monolayers––
including the application of the additivity rule on misci-
bility and the quantification of interactions between
monolayer components by excess free energy––it has
been concluded that, at a macroscopic level, these com-
pounds form a practically immiscible monolayer at the
air–water interface, at p lower than that for the protein
collapse (Rodrı́guez 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 casei-
nate) facilitates the protein displacement by LMWE
from the air–water interface. On the other hand, the low-
er 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, miscibil-
ity and topography, confirming the importance of pro-
tein and LMWE structure in determining the
mechanism of interfacial interactions (Rodrı́guez 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 dis-
placement surface pressure (pd) value for monopalm-
itin–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 elastic-
ity 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 mono-
palmitin 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). Phosp-
holipids and b-casein form a practically immiscible
monolayer at the air–water interface on neutral or acidic
aqueous subphases. However, some degree of interac-
tion exists between phospholipids and b-casein in the
 C.C. Sánchez et al. / Journal of Food Engineering 67 (2005) 225–234 229

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 b-
casein 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 Competitive adsorption of proteins and LMWE at
character, are more pronounced at pH 9 as the phospho- fluid interfaces can affect the stability of food disper-
lipid molecules become completely ionized. sions (Bos et al., 1997; Wilde, 2000). Thus, knowledge
230 C.C. Sánchez et al. / Journal of Food Engineering 67 (2005) 225–234
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) monopalm-
itin, (  , 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 surfac-
tants disrupt and displace proteins from an interface by
a three-stage ‘‘orogenic’’ mechanism. Briefly, emulsifier
penetrates into the protein film, forming separate ad-
sorbed domains that exert a lateral p which mechani-
cally compresses the protein network until it fails and
is displaced from the interface. The mechanism also re-
veals 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 water-
soluble emulsifiers (Gunning et al., 1999; Mackie et al.,
1999) and spread water-insoluble emulsifiers (Mackie,
Gunning, Ridout, Wilde, & Rodrı́guez Patino, 2001;
Rodrı́guez Patino et al., 2001a, 2001b). The results sug-
gest 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 displace-
ment 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 pro-
tein, involves a buckling of the monolayer and reorder-
ing 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 (Rodrı́guez Patino et al., 2001a, 2001b). But,
for spread water-insoluble emulsifier monolayers, the
protein displacement is not total even at the highest sur-
face pressure, at the collapse point of the mixed film.
The orogenic displacement mechanism is a conse-
quence of the low level of interaction between pro-
teins and LMWE at the air–water interface (Rodrı́guez
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 con-
taining 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
e a
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.
 C.C. Sánchez et al. / Journal of Food Engineering 67 (2005) 225–234 231

great practical significance and include important tech-
nological operations such as emulsification and foam-
ing. Two experimental approaches can be used for the
analysis of long-term relaxation phenomena in emulsi-
fier 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. Infor-
mation on various relaxation paths (Marangoni effect,
chemical reaction, polar group hydration, conforma-
tion/organization changes, film dissolution by desorp-
tion and/or diffusion, collapse, etc.) can be derived
from these data (Rodriguez Niño et al., 2003; Rodrı́guez
Patino et al., 2003a).
Desorption of spread LMWE monolayers at any con-
stant surface pressure, at p < pe, involves two stages
(Rodriguez Niño et al., 2003; Rodrı́guez 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 gradi-
ent within the diffusion layer becomes constant and
desorption reaches a steady state. The monolayer molec-
ular 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-col-
lapse phase system. However, some differences exist be-
tween saturated (monopalmitin or DPPC) and
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 monoglycer-
ide or to a complex mechanism including competition
between desorption and monolayer collapse. The rea-
sons for these behaviors may be associated again with
the immiscibility between protein and monoglyceride
at the air–water interface and to the protein displace-
ment 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 emulsifica-
tion and foaming requires rapid and substantial stretch-
ing of the drops or bubbles, and consequently, the
surface tension may be far from equilibrium. Thus, dil-
1.0
0.9
0.8
A/A o

β-casein
unsaturated (monoolein or DOPC) LMWE monolayers.
monopalmitin
Relaxation phenomena in saturated LMWE monolayer 0.7
0.2
are controlled predominantly by the collapse mechanism
because of the p values relaxed to pe value. For unsatu- 0.4.
0.6
rated LMWE monolayer p relaxed from the collapse 0.6.
(A)
value, which is close to pe, towards lower p values at 0.8.

longer times. This phenomenon should be ascribed to 0.5

0 50 100 15 0 200 25 0
the concurrence of different phenomena, such as desorp-
time (min)
tion and collapse. Protein monolayer behaves in a differ-
ent way to LMWE under the same experimental 60
conditions. At p < pe the relaxation is a reversible proc-
55
ess, which include monolayer organization/reorienta- πe
MP

tion. The relaxation in relative molecular area and in 50

surface pressure at p > pe should be attributed to proc- 45
π(m N/m )

esses related to monolayer organization/reorientation

40
and collapse, respectively.
The strength of interactions between protein and 35
LMWE can also be studied by relaxation experiments. 30
Long-term relaxation phenomena in protein–monoglyc- 25
eride mixed films (Fig. 5) at the air–water interface have
20 (B)
been analyzed according to models for desorption, col-
lapse and/or organization/reorganization changes 15
0 20 40 60 80 100 120 140
(Rodrı́guez Patino, Rodrı́guez Niño, & Carrera, 2002a,
2002b). At lower surface pressures (i.e., p lower than time (min)
protein collapse pressure), the organization/reorganiza- Fig. 5. (A) Relaxation at constant surface pressure (p = 20 mN/m) and
tion change of protein molecules in the mixed film is (B) relaxation at constant area (at the collapse point) for of b-casein–
the mechanism that controls the relaxation process. monopalmitin mixed monolayers on water at pH 7 and at 20 °C.
232 C.C. Sánchez et al. / Journal of Food Engineering 67 (2005) 225–234
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 emulsifi-
ers is closely linked to the surface dilatational rheology,
whereas the precise form of the displacement is consid-
ered to be more closely related to the surface shear
behavior (Mackie et al., 2003; Murray, 2002; Roth,
Murray, & Dickinson, 2000). Different and complemen-
tary 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 (Rodrı́guez Patino, Rodrı́guez Niño, &
Carrera, 2002b; Rodrı́guez Patino, Rodrı́guez Niño, &
Carrera, 2003b).
A common trend of the p dependence of dilatational
modulus (E) for monopalmitin and monoolein mono-
layers (Fig. 6) is that E increased with increasing p up
to the collapse point. This increase is a result of an in-
crease in the interactions between the monolayer mole-
cules (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-
β-casein + LMWE
250 (A)
200
E (mN/m)
150
100
50
0 0
100
(B)
80
E (mN/m)
60
40
20
0 10 20 30
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.
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 impor-
tant 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 in-
creased with increasing p up to the collapse point.
This increase is the result of an increase in the interac-
tions 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’’ con-
figuration 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 pro-
tein molecules, but the internal nature of the protein
WPI + LMWE
250 (C)
200
E (mN/m)
150
100
50
100
(D)
80
E (mN/m)
60
40
20
40 50 0 10 20 30 40 50
 C.C. Sánchez et al. / Journal of Food Engineering 67 (2005) 225–234
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 mole-
cules (Fig. 6) with disordered structure, at the same
surface pressures.
Surface dilatational rheology is a very sensitive tech-
nique to analyze the competitive adsorption/displace-
ment of protein and LMWE emulsifier at the air–water
interface. At higher p, the collapsed protein residues dis-
placed from the interface by LMWE molecules have
important repercussions on the dilatational characteris-
tics of the mixed films (Rodrı́guez 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 sur-
face dilatational properties of mixed protein–emulsifier
films also depend on the presence of some food compo-
nents (ethanol and sucrose) in the aqueous phase. In gen-
eral, a decrease in the dilatational rheological properties
on the addition of ethanol was found for protein–water-
insoluble emulsifiers, whereas the opposite was observed
for protein–water-soluble emulsifiers (Rodrı́guez Niño,
Wilde, Clark, & Rodrı́guez Patino, 1998c).
3.5. Final considerations
In this paper, we have analyzed the structure, topog-
raphy, adsorption, interactions, miscibility, and dynamic
properties of food dairy proteins (b-casein, caseinate,
and WPI) and LMWE (monoglycerides and phospholi-
pids) at the air–water interface. The summary includes
an assessment of information derived from a variety of
chemical and physical techniques. The results demon-
strate 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. Impor-
tant 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.
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