Food Hydrocolloids 25 (2011) 1833e1841

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Food Hydrocolloids
journal homepage: www.elsevier.com/locate/foodhyd

Polysaccharideeprotein interactions in dairy matrices, control

and design of structures
Milena Corredig*, Negin Sharafbafi, Eleana Kristo
Food Science Department, University of Guelph, Guelph, Ontario N1G 2W1, Canada

a r t i c l e i n f o a b s t r a c t

Article history: Proteinepolysaccharide interactions are of great importance in the design of dairy formulations, as they
Received 22 December 2010 play a key role in the formation of structure and texture in dairy products. With a detailed understanding
Accepted 27 May 2011 of the factors affecting the interactions, the ability of charged polysaccharides to associate with the milk
proteins is continuously exploited to create functional complexes, novel ingredients and delivery
Keywords: systems. In addition, formulations containing non-interacting polysaccharides also need to be carefully
Milk proteins
controlled, as these biopolymers may give rise to segregative phase separation, with important conse-
Casein micelles
quences to the stability and quality of the final matrix. As casein micelles play a major role in imparting
Gelation
Proteinepolysaccharide interactions
structure to dairy products, emphasis in this review will be given to the molecular details of the
interactions between polysaccharides with these protein particles. Some of the most researched poly-
saccharides will be highlighted in this context, and the progress made in understanding their role during
structure formation of dairy matrices will be discussed. The opportunity of creating novel microstruc-
tures provided by association or/and incompatibility of milk proteins and different polysaccharides will
be assessed.
Ó 2011 Elsevier Ltd. All rights reserved.

1. Introduction described how polysaccharide molecules may interact with

As main components of foods, polysaccharides and proteins play
a key role in the formation of the building blocks of structure and
texture. Polysaccharides are widely used as stabilizers, thickening
or gelling agents in dairy products, but their concentration levels
have to be carefully controlled, since the properties of the final food
products will depend on their interactions with the other macro-
molecules present in the system.
When mixing proteins with polysaccharides, the interactions
can be segregative or associative in nature. A homogeneous
mixture of co-soluble polymers can be obtained only under very
specific conditions of concentration and polymer ratios (and
usually at very low volume fractions). Depending on the type of the
polymer, concentration of the polysaccharide present, and the
environmental conditions of the solution (i.e. temperature, pH,
ionic strength) the proteinepolysaccharide interactions can
improve the stability or lead to macroscopic destabilization. These
effects can be welcome, for example when the complex formed
improves colloidal stability, or must be avoided, when the result
is a bulk of two separated phases. A number of reviews have
* Corresponding author.
E-mail address: milena.corredig@uoguelph.ca (M. Corredig).
proteins and adsorb to more than one colloidal particle forming
bridges or aggregated structures, or may show incompatibility,
forming regions depleted of polymer and drawing protein particles
closer to one another (Doublier, Garnier, Renard, & Sanchez, 2000;
Grinberg & Tolstoguzov, 1997).
A large number of polysaccharides have been described to be
thermodynamically incompatible with food proteins (see for
example, Grinberg & Tolstoguzov, 1997). This is also the case when
non-interacting polysaccharides are added to milk proteins. Ther-
modynamic incompatibility is observed when the solvente
biopolymer interactions are favored compared to biopolymere
biopolymer interactions. This is also called segregative phase sepa-
ration, as each biopolymer concentrates in a separate phase
(Tolstoguzov, 1988). In the case of casein micelles, the phase sepa-
ration is drawn by a depletion mechanism (Doublier et al., 2000;
Tuinier, Rieger, & de Kruif, 2003), and it has been described for
caseinehigh methoxyl pectin mixtures at pH 6.7 (Acero-Lopez,
Alexander, & Corredig, 2010), casein-exopolysaccharides from lactic
acid bacteria at pH near neutral (Tuinier & de Kruif, 1999; Tuinier,
ten Grotenhuis, Holt, Timmins, & de Kruif, 1999), casein and
galactomannans (Bourriot, Garnier, & Doublier, 1999a), amongst
others.
On the other hand, associative interactions usually occur because
of electrostatic attraction between oppositely charged portions of

0268-005X/$ e see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.foodhyd.2011.05.014
1834 M. Corredig et al. / Food Hydrocolloids 25 (2011) 1833e1841
proteins and polysaccharides. Hydrogen bridging and hydrophobic
interactions also play a part in the stabilization of the complexes
(coacervates) formed (Doublier et al., 2000; Turgeon, Schmitt, &
Sanchez, 2007). Soluble complexes can form at specific pH values,
when the electrostatic repulsion forces are minimized (Turgeon et al.,
2007) and interactions occur between charged patches of the
molecules (de Kruif, Weinbreck, & de Vries, 2004; de Vries, 2004).
Since the main forces involved in associative complexation of
proteins and polysaccharides are electrostatic in nature, the extent of
the interactions depends on environmental conditions such as pH
and ionic strength. Furthermore, stiffness and charge density of
polysaccharide may influence the type of aggregates formed (see for
example, Weinbreck, Nieuwenhuijse, Robijn, & de Kruif, 2003). While
a number of studies have focused on the associative interactions of
various proteinepolysaccharide binary systems taken in isolation,
less is known on the changes of the complexes in mixed systems, or
during processing, when the conditions critical to the formation and
stability of the aggregates can change over time. Only recently the
study of the potential for creating different functional structures and
of their stability during processing (Flett, Corredig, & Goff, 2010),
storage (Dickinson, 2008; Nigen, Croguennec, Renard, & Bouhallab,
2007) or digestion (Vandenberg, Drolet, Scott, & de la Noue, 2001)
has become a focus of research.
Considering the complexity of proteinepolysaccharide interac-
tions, gathering complementary information by using several
techniques is becoming critical to describe the molecular details
occurring during changes in processing, storage or digestion. The
nature of the biopolymers (molecular weight, charge, distribution
of charges), the ratio between the polymers and their concentra-
tion, as well as the environmental conditions are all important
parameters affecting the formation of the structure of a dairy
matrix. A number of techniques have been proven useful in
providing information on the overall mechanisms involved during
structuring of dairy products containing polysaccharides. Besides
rheological techniques, spectroscopic, light scattering, and micro-
scopic methods have been employed to better describe the nature
of the interactions and the dynamics of structuring during pro-
cessing of dairy products. Other powerful techniques such as
nuclear magnetic resonance (NMR and diffusivity NMR), fluores-
cence recovery after photo-bleaching and diffusing wave spec-
troscopy are also used to obtain information on the diffusivity of
macromolecules in these mixed systems.
An example of the complexity of dairy matrices and the
dynamics of change during processing is the structuring occurring
to a fermented milk gel in the presence of lactic acid bacteria
producing exopolysaccharides (EPS). At the beginning of fermen-
tation, the pH is near neutral, the proteins are negatively charged,
and little amount of polysaccharide is produced. However,
depending on the charge, molecular dimensions and concentration
of EPS, thermodynamic incompatibility between the proteins and
EPS may occur, even at very low concentrations. During fermenta-
tion, the gradual acidification causes the molecules to decrease their
overall charges and casein micelles to release their colloidal calcium
into the serum phase, with important consequences to the ionic
conditions. These changes will affect the mechanisms involved in
the interactions between the polysaccharide and the casein
micelles. Finally, the casein micelles will form a network at the pH of
gelation and the bacteria, still producing EPS, may now be entrap-
ped in the pores formed by the proteins, creating pockets rich in
polysaccharides. This example of the complex dynamics occurring
in a fermented milk system containing EPS illustrates the impor-
tance of studying the system under relevant conditions, with
minimal sample disruption and with a multi-technique approach.
The fundamentals of polysaccharideeprotein interactions
have been presented before (see for example, Syrbe, Bauer, &
Klostermeyer, 1998; Ye, 2008) and the present review summa-
rizes the recent advances in the study of polysaccharide interac-
tions with casein micelles, focusing mainly on how segregative and
associative interactions can modify the structure of dairy products.
2. Milk proteins
Caseins comprise 80% of the total protein in bovine milk, the rest
being whey proteins (mainly, b-lactoglobulin, a-lactalbumin and
bovine serum albumin). In bovine milk the four main caseins, as1-,
as2-, b- and k-casein, are assembled together in an aggregated
structure, called the casein micelle. These proteins do not possess
well defined tertiary structures, and they, with the exception of k-
casein, have phosphoseryl residues that bind to calcium phosphate
nanoclusters. The casein micelles are highly hydrated (about 4:1
ratio of water to protein), polydisperse in size, with an average
diameter of 200 nm, and the proteins are held together by calcium
phosphate nanoclusters, hydrophobic interactions, hydrogen
bonding and van der Walls interactions (Dalgleish, 2011; Holt,
de Kruif, Tuinier & Timmins, 2003; Horne, 2002).
A large proportion of k-casein is located on the surface of the
casein micelle, where this protein imparts steric stability, by
protruding into the serum phase with its hydrophilic glycosylated
portion. The uneven, rough surface of the casein micelles
(Dalgleish, Spagnuolo, & Goff, 2004) is of particular relevance in the
study of the interactions with other biomolecules, such as poly-
saccharides. To large molecules the interior of the micelles will not
be accessible, and the polyelectrolyte layer of k-casein is what
drives the polysaccharide molecules to attraction or repulsion at
close distance. On the other hand, for relatively small molecules
(such rennet or transglutaminase enzymes, polyphenols, b-casein,
b-lactoglobulin), the casein micelle structure is porous enough to
provide some access to the internal structure of the micelles.
While casein micelles are mostly unaffected by heating at
temperatures < 100
C, whey proteins unfold and denature at
temperatures > 60
C, leading to the formation of aggregates of
whey proteins with themselves or with casein micelles. With
heating, k-casein is also released in the serum phase, where it
associates with the whey proteins. These whey protein aggregates,
formed via hydrophobic interactions or disulphide exchanges
between the proteins, are either dispersed in the soluble phase or
adsorbed on the surface of the casein micelles. The size of the
soluble aggregates is reported to be between 30 and 100 nm in
diameter, and their ratio of soluble/bound aggregates changes
depending on the pH of milk (Donato & Guyomarc’h, 2009).
The destabilization of the casein micelle in milk is at the basis of
the formation of structure and texture in dairy products. Gels can
be formed by enzymatic hydrolysis (usually using chymosin) or by
acidification, or a combination of both. Rennet coagulation can be
described as a two stages process. In the primary enzymatic phase,
the hydrophilic portion of k-casein (the C terminal casein-
omacropeptide (CMP)) is released from the surface of the casein
micelles, as a consequence of the specific hydrolysis of the protein
at the Phenylalanine105eMethionine106 bond. The release of CMP
decreases steric repulsion of the casein micelles, enhancing the
interactions and leading to aggregation. When >85% of k-casein is
hydrolyzed (Dalgleish, 1980), the caseins form a gel. Heating
treatment impairs rennet-induced aggregation, because of the
presence of whey protein complexes on the surfaces of the casein
micelles, the complexes of whey proteins and k-casein in the
serum, as well as the changes in composition of the serum phase.
In the case of fermentation or direct acidification, destabiliza-
tion of the micelles occurs near the isoelectric point of the caseins.
The reduction of pH causes shrinking of casein micelle size, which is
attributed to the collapse of the polyelectrolyte layer of k-casein on
 M. Corredig et al. / Food Hydrocolloids 25 (2011) 1833e1841
the surface of the micelle. This decreases the steric repulsion and
increases the interactions between the particles, leading to aggre-
gation. In addition, during acidification, the colloidal calcium
phosphate, bridging between the caseins is gradually released in
the serum phase. This causes a shift in the balance between the
colloidal and the soluble calcium in milk, and this is of particular
relevance when charged polysaccharides are present in acidified
milk systems, as the concentration of calcium in solution is modi-
fied during the process. While the caseins in unheated milk show
a gelation pH of about 4.9, in heated milk, gelation occurs at
a higher pH, because of the presence of whey protein aggregates,
bridging between the casein micelles. The participation of the whey
proteins in the acid gel affects the structure development and the
gel modulus, measured by small oscillatory rheology (Guyomarc’h,
Law, & Dalgleish, 2003).
3. Associative interactions between casein micelles
and polysaccharides
The association of polysaccharides with casein micelles is
mostly electrostatic in nature, and the strength of the attraction
depends on the type and density of the charges present on the
molecules. This is clearly demonstrated by the differences in the
interactions between caseins and different types of carrageenan or
pectin molecules (see below). With the exception of chitosan
(a(1,4) 2-amino-2 deoxy-d-glucose polymer), which is positively
charged at pH close to neutrality (with a pKa of 6.5), most poly-
saccharides employed in dairy systems are negatively charged.
Hence, in general, most of the complexes between caseins and
polysaccharides are formed close or below the isoelectric point of
the proteins.
An exception is the specific association of k-carrageenan with
casein micelles at neutral pH. Carrageenans are polysaccharides
extracted from red seaweed, composed of galactose and 3,6 anhydro-
galactose repeating units. The three main forms of carrageenan differ
for the number of sulfate groups per disaccharide unit (1 for k-, 2 for
i-, 3 for l-carrageenan). This charge difference affects their function,
as k- and i-carrageenan undergo a coilehelix transition with cool-
ing, at a temperature that depends on the ionic environment, while
the most charged of the carrageenans, l-carrageenan does not show
a transition from coil to helix, and does not gel (Nilsson & Piculell,
1991). This molecule, with a distance between the sulfate groups
of 0.3 nm, uniquely associates with the casein micelles at tempe-
ratures > 60
C (Langendorff et al., 2000; Nilsson & Piculell, 1991).
On the other hand, the association of k- and i-carrageenan with the
casein micelles occurs only at temperatures below the coilehelix
transition, when the polysaccharides are in their ordered confor-
mation and the mean distances between the sulfate groups
decreases from 1 and 0.5 nm (coil) to 0.4 and 0.2 nm (helix) for k-
and i-carrageenan, respectively (Nilsson & Piculell, 1991). At
temperatures above the coilehelix transition, k- and i-carrageenan
molecules will cause phase separation because of depletion floc-
culation of casein micelles (Langendorff et al., 2000).
The unique interacting behavior of k-carrageenan with the
casein micelles at neutral pH has been attributed to a specific
charge interaction with the positively charged patch of k-casein
between residues 97 and 112 (Snoeren, Both, & Schmidt, 1976). This
associative interaction is widely exploited in dairy systems, as it
prevents macroscopic phase separation and destabilization of
mixtures containing non-interacting polysaccharides such as guar
gum, locus bean gum or flaxseed gum (Chappellaz, Alexander, &
Corredig, 2010; Thaiudom & Goff, 2003). At concentrations below
gelling (<0.03%) (Schorsch, Jones, & Norton, 2000), k-carrageenan
adsorbs on the surface of the casein micelles, and helixehelix
associations between the chains cause the molecules to bridge with
1835
each other, resulting in the formation of a weak three dimensional
network, which is very important for stabilization of the structures
(Bourriot, Garnier, & Doublier, 1999b). Indeed, it has been demon-
strated that both adsorption to the micelles and helixehelix asso-
ciations are required to prevent macroscopic phase separation.
Although bulk phase separation is prevented, the mixtures are
microscopically phase separated with micro-domains rich in casein
micelles (Spagnuolo, Dalgleish, Goff, & Morris, 2005; Thaiudom &
Goff, 2003).
At the stabilizing concentration of k-carrageenan, below gelling,
the casein micelles still show considerable diffusivity (Alexander &
Dalgleish, 2007). In spite of the presence of these loose networks of
k-carrageenan (present at concentrations of 0.015%) in milk there is
no effect on the development or the timing of the solegel transition
during renneting, compared to that of a milk system without
polysaccharide (Acero-Lopez et al., 2010). It is worth noting that the
specific site for the interactions seems to be in proximity of the
same region of k-casein where the enzymatic cleavage would occur.
As the functionality of the casein micelles is then preserved, it is
possible to use this associative behavior of k-carrageenan to control
the formation and development of phase separated micro-domains
within casein networks.
At low pH, charge interactions between pectin and acidified
casein particles are often exploited to stabilize acid milk beverages.
Pectin is an anionic cell-wall polysaccharide (pKa around 3.5) that
is generally extracted from citrus, apple or beet, and composed of
D-galacturonic acid residues, esterified with methoxyl groups. The
extent of esterification influences the charge distribution of the
chain and the overall charge of the polysaccharide chains. When
pectin molecules with different charges and charge distributions
were tested at various concentrations and pH, it was shown that
pectins with a high degree of esterification can stabilize milk at
pH > 4.0, whereas those with low degree of esterification (con-
taining more overall charge) are less effective in stabilizing the
protein particles (Liu, Nakamura, & Corredig, 2006). In experiments
with diluted milk slowly acidified with glucono-d-lactone, the
dynamics of these interactions have been clearly demonstrated.
The high methoxyl pectin adsorbs on the casein micelles by elec-
trosorption (Maroziene & de Kruif, 2000), and the interactions
occur between pH 5 and 4.5, at the point where the casein micelles
show the shrinking of size and collapse of the polyelectrolyte layer.
The mechanism of stabilization of the acid casein particles is
attributed to the adsorption of the pectin onto these particles, but
the formation of a three dimensional network of casein/pectin
complexes is also, at least in part, responsible (Laurent &
Boulenguer, 2003; Tromp, de Kruif, van Eijk, & Rolin, 2004).
However, the non-adsorbed pectin does not seem to be needed to
maintain stability, as stability can be maintained if removed from
the particles after homogenization (Tromp et al., 2004). The pres-
ence of heat-induced aggregates of whey protein and k-casein
present in the milk serum does not seem to affect the interaction
between high methoxyl pectin and casein micelles (Rediguieri, de
Freitas, Lettinga, & Tuinier, 2007).
Another polysaccharide molecule with similar applications in
acid milk gels is soybean soluble polysaccharide (SSPS). SSPS,
extracted from soybean cotyledons, contains about 20% galactur-
onic acid and has a structure less elongated that that of pectin
molecules, with a hydrodynamic radius of about 30 nm (Wang,
Huang, Nakamura, Burchard, & Hallet, 2005). During homogeni-
zation of casein particles at low pH, SSPS adsorbs on the surface of
the casein particles and stabilizes acid drinks at pH values as low as
3.8 (Liu et al., 2006). It is also important to mention that at pH near
neutral, while pectin induces depletion flocculation, SSPS at similar
concentrations does not show macroscopic phase separation and
the mixtures still have low viscosity (Liu, Verespej, Alexander, &
1836 M. Corredig et al. / Food Hydrocolloids 25 (2011) 1833e1841
Corredig, 2007). The differences in the associative behavior of SSPS
and high methoxyl pectin molecules have been recently illustrated
with a model system consisting of a soy oil emulsion droplet
covered with sodium caseinate (Liu et al., 2007). Fig. 1 summarizes
the differences in the stabilizing behavior of SSPS and high
methoxyl pectin when slowly acidifying the mixture using glu-
cono-d-lactone. While the control emulsion, with no poly-
saccharide added, showed destabilization at pH of 5.4, in the
presence of 0.2% high methoxyl pectin, there was no aggregation,
but a small increase in the hydrodynamic radius at the pH of
adsorption. The change in radius (relative to the initial radius)
indicated the adsorption of the pectin molecules onto the interface.
On the other hand, in the case of SSPS, although destabilization was
again inhibited, no change in size was noted. Because of its smaller
structure, it could be suggested that SSPS penetrates better
between the inhomogeneities of the interface, causing charge and
steric stabilization, but with little change in the overall hydrody-
namic radius of the particles. Fig. 1 also shows the changes in the
ultrasonic attenuation for the same samples undergoing acidifica-
tion. Ultrasonic spectroscopy is well suited to look at the interac-
tions occurring in concentration dependent systems, as dilution or
sample disruption is not required.
The difference in the signature of ultrasonic attenuation
between the samples containing SSPS and high methoxyl pectin is
caused by the segregative behavior of high methoxyl pectin with
the emulsion droplets at high pH, causing micro-phase separation.
Hence, the values of ultrasonic attenuation of the emulsions con-
taining high methoxyl pectin were lower than those of control and
samples with SSPS at the initial pH. Depletion flocculation caused
spatial rearrangements, and droplet rich domains showed an
overlap that reduced ultrasonic wave thermal losses and a decrease
in attenuation. At a pH of about 5.6, the pectin molecules start
decrease their incompatibility, because of the loss in negative
charges on the casein surface, and with the decrease in pH, they
start to associate with the oil droplets and titrate the positive
patches, causing a change in the interparticle distance between the
oil droplets. The changes in ultrasonic attenuation (Fig. 1, empty
symbols) reflect this behavior.
The model system described in Fig. 1 illustrates the complexity
of the polysaccharide interactions with dairy proteins during pro-
cessing, and demonstrates how, understanding the dynamics of the
Change from the initial diameter (nm)
600
400
200
0 28
6.5 6.0 5.5 5.0 4.5
pH
Fig. 1. Effect of acidification (0.3% glucono-d-lactone) on the stability of 10% soybean
oil emulsions stabilized with 1% sodium caseinate. Control emulsions (triangles),
emulsions containing 0.2% high methoxyl pectin (circles) or 0.2% soy soluble poly-
saccharide (squares). Filled symbols show the change in droplet diameter (left hand
scale) as measured by dynamic light scattering, with lines drawn for more clarity.
Empty symbols show the changes in the ultrasonic attenuation (measured at 8 MHz) of
the samples (right hand scale). Samples were resuspended in their own permeate in
light scattering experiments. For details, see Liu et al. (2007).
interactive and segregative processes, may assist in fine-tuning the
formation of different microstructure. It is important to note,
however, that, while mixing of the negatively charged poly-
saccharides with the emulsion droplets could stabilize the particles
during acidification, the same conditions do not apply in milk, as
calcium is released from the micelles, charges are shielded, and the
adsorption of the polysaccharides efficiently stabilizes the particles
only after homogenization at acidic pH (Liu et al., 2006).
A group of polysaccharides of particular interest in dairy appli-
cations are those produced by lactic acid bacteria during fermen-
tation of milk. Their presence in starter cultures can modify the
texture and increase the water retention of fermented products,
acting as natural stabilizers and thickeners. The presence of asso-
ciative interactions between the exopolysaccharide (EPS) and milk
proteins is still under debate. It is important to note, however, that
the production of EPS is strain and media specific therefore different
strains could produce different types of EPS. As previously
mentioned, the study of EPS during structure formation of a fer-
mented product is particularly challenging because of the dynamics
of change of polymer concentration, casein micelles charge, desta-
bilization conditions and ionic equilibrium. Therefore, while at pH
near neutral phase separation may be induced by EPS (Girard &
Schaffer-Lequart, 2007; Tuinier & de Kruif, 1999), at acidic pH
interactions may occur with negatively charged polysaccharides.
Microstructural studies on fermented milk gels (Hassan, Ipsen,
Janzen, & Qvist, 2003) have shown networks with large interstitial
cavities filled with bacterial colonies surrounded by polysaccharide.
The interactions between milk proteins and EPS have been
studied using scanning electron microscopy (Ayala-Hernandez,
Goff, & Corredig, 2008). This technique is employed to observe
interactions between proteins and polysaccharides, as the sample
preparation includes a step where the proteins are covalently
linked to the carbon support. A washing step can then be carried
out and anything that remains after the washing step can be
assumed to be associated to the proteins that are linked to the
carbon support (Ayala-Hernandez et al., 2008; Azim, Alexander,
Koxholt, & Corredig, 2010; Martin, Goff, Smith, & Dalgleish, 2006).
Fig. 2 is a micrograph taken at pH 4.6 for a whey protein suspension
fermented with Lactococcus lactis. The EPS is clearly present with
the proteins and appears as filament strands attached to the protein
42
40
Ultrasound attenuation (m-1)
38
36
34
32
30
26
4.0
Fig. 2. Field emission scanning electron micrograph prepared using a self assembled
monolayer technique to covalently bind the proteins to the gold support, and then
wash any material not attached to the proteins. Image was fixed at pH 4.5, after
fermentation of milk serum permeate containing 6% whey protein isolate. Scale bar is
600 nm. For details, see Ayala-Hernandez et al. (2008).
 M. Corredig et al. / Food Hydrocolloids 25 (2011) 1833e1841
aggregates as well as to the bacteria cells. Therefore, although it is
possible to hypothesize that although once the protein network has
formed around the bacteria, the EPS will reside in separated
pockets within the gel, interactions with the milk proteins may also
play a major role in the structuring behaviour of EPS.
4. Non-interacting polysaccharides: segregative
phase separation
As already mentioned, when adding a non-interacting
biopolymer to milk, depletion flocculation of the casein micelles
occurs. The incompatibility between the biopolymers causes the
formation of a depleted layer around the casein micelles, with an
osmotic pressure gradient, which drives the casein micelles to
attract one another. These types of interactions result in the sepa-
ration of the mixture in two liquid layers, one rich in protein
(caseins), and the other in polysaccharide. This mechanism has
been described theoretically and compared to experimental data
(see for example, Bhat, Tuinier, & Schurtenberger, 2006; Tuinier
et al., 2003). In a study of guar gums with different degrees of
hydrolysis, it has been demonstrated that phase separation is
affected by the molecular mass of the biopolymer. Thus, the critical
concentration of guar gum to cause phase separation increases with
decreasing the molecular weight of the gum (Tuinier et al., 2003).
The phase behavior of mixtures of polysaccharides and milk
proteins has been described in detail with phase diagrams for
a number of polysaccharides (see for example, Bourriot et al.,
1999a; de Bont, van Kempen, & Vreeker, 2002; Schorsch et al.,
2000; Tuinier & de Kruif, 1999). It is important to note that in
most cases, the volume fraction of casein micelles is obtained by
reconstituting skim milk or protein concentrates. This would result
in differences in the ionic composition as well as lactose concen-
tration of the mixture. Fig. 3 illustrates the phase diagram of
a mixture of milk proteins and high molecular weight b-glucan
(106 Da). In this case, the mixtures were prepared by mixing
concentrated milk (prepared by ultrafiltration) with b-glucan
dispersed in milk serum, preserving in all cases the ionic environ-
ment of the casein micelles. While this may not be critical for other
proteinepolysaccharide mixtures, maintaining the correct ionic
equilibrium is important for maintaining the integrity of the
structure of the casein micelles.
Fig. 3. Phase diagram for a mixture of milk proteins with high molecular weight
b-glucan. Initial mixtures (>); stable initial mixtures (C), upper phase (6), lower
phase (7), binodal curve (e), and tie line (.). Confocal scanning laser microscopy
images of selected mixtures taken using rhodamine B and calcofluor stains. Scale bar is
50 mm.
1837
In the phase separation diagram, the binodal curve shows the
concentration of protein and polysaccharide present in the upper
and lower phases of the mixture after phase separation. The tie line
connects the points related to the composition of protein and
polysaccharide in each phase after demixing, together with the
initial composition of the mixture located in between. Any mixture
composition sitting on the tie line will result in the same concen-
tration of protein and polysaccharide in the upper and lower phases
after demixing. In the case of a high molecular weight b-glucane
milk mixture it is clearly shown that there is very little room below
the binodal curve where the biopolymers would be co-soluble
(Fig. 3). In most cases, the mixture would readily phase separate.
In general, mixtures with polymers concentrations below the
binodal curve will not phase separate, while mixtures with
biopolymers concentrations higher than the binodal will be in
a state of thermodynamic instability or metastability. As a result,
mixtures will not be homogeneous with mixing, and colloidal
structures will form. A few examples of such microstructures are
illustrated in Fig. 3. The type of these microstructures mainly
depends on the initial volume fraction of protein and poly-
saccharide. Droplet-like structures form when the volume fraction
of one phase is higher than the other (similar to those found in oil in
water emulsions), while undefined, interconnected, or bi-
continuous structures are formed when the volume fractions of
both phases are similar to each other. These structures grow fast
over time and lead to formation of two liquid layers.
It may be possible to create kinetically stable systems and
different microstructures in food matrices, with consequences to
texture, mouthfeel or digestion functionality. Research on the
evolution of these structures, the formation and coarsening of the
domains over time or under shear, has become of increasing
interest (Chappellaz et al., 2010; de Bont, Hendriks, van Kempen, &
Vreeker, 2004; Gaaloul, Turgeon, & Corredig, 2009; Hemar,
Tamehana, Munro, & Singh, 2001). It has been shown that, the
structures resulting from the demixing lead to an increase in the
viscosity of the system, which should be considered in formulating
new dairy products with high concentrations of polysaccharides
(Garnier, Bourriot, & Doublier, 1999; Hemar et al., 2001).
Scattering techniques, combined with microscopy and rheo-
logy, provide information on the mechanisms involved in phase
separation (Chappellaz et al., 2010; Corredig & Alexander, 2008),
and how to control the dynamics to kinetically stabilize these
structures (Acero-Lopez et al., 2010; Doublier et al., 2000; Tuinier &
de Kruif, 1999). Although the resulting mix may still show homo-
geneous appearance, microscopically it will be non-homogeneous.
Starch is a polysaccharide with widespread applications in dairy
technology, because of its structure enhancing properties, its
availability and low cost. Despite the industrial significance of
starchemilk protein mixtures, little fundamental research is
available on starch-milk protein interactions. It has been shown
that concentration, botanical origin, composition and processing
history are critical in understanding the changes occurring in the
starchemilk protein systems. As native starches possess low shear
and thermal resistance and high tendency to retrogradation,
chemically modified starches that maintain the granular structure
during processing are preferred in dairy applications.
Studies concerning mixtures of milk with starch have been
conflicting, as both associative and segregative interactions with
milk proteins have been reported. Associative interactions are
mostly observed in mixtures of potato starch (which shows anionic
character due to phosphate ester groups of amylopectin) and milk
proteins (Grega et al., 2003; Oh, Anema, Wong, Pinder, & Hemar,
2007). Reports of interactions of modified waxy maize granules
with whey proteins during heating are also available (Vu Dang,
Loisel, Desrumaux, & Doublier, 2009). However, the majority of
1838 M. Corredig et al. / Food Hydrocolloids 25 (2011) 1833e1841
the reports on milk protein mixes with non-ionic starches seem to
be in agreement that the properties of these mixtures are a conse-
quence of thermodynamic incompatibility or segregative interac-
tions between the components.
The starch components, amylose and amylopectin, that leach
from disrupted starch granules during pasting at elevated
temperatures, cause phase separation in skim milk or milk protein
concentrate dispersions (Noisuwan, Hemar, Wilkinson, & Bronlund,
2009). The rheological properties of the milk mixtures containing
native starches depend on the starch source. For example, in
sodium caseinate mixtures, the apparent viscosity of the casein/
tapioca starch mixture decreases and that of casein/wheat starch
and casein/potato starch increases, despite the fact that in all cases
the swelling and solubility of starch decreases in the presence of
the protein (Doublier, Marzin, Visdeloup, & Lefebvre, 1994). On the
other hand, the addition of modified starches (where there is no
leaching of starch components during pasting) to milk, causes
a substantial volume fraction effect of swollen starch granules,
which in turn causes an increase in local concentration of milk
proteins with consequences to processing functionality (Azim et al.,
2010; Matser & Steeneken, 1997).
5. Structuring of polysaccharides in casein gels
Gelation of milk is usually triggered by either the release of the
caseinomacropeptide (renneting) or acidification, or a combination
Fig. 4. Confocal scanning electron microscopy images of milk fermented to pH 4.6. Set (a, c) and stirred (b, d) acid casein gel containing 0% starch (a, b) and 2.5% starch (c, d). The
protein network was stained with rhodamine B. Bar ¼ 50 mm.
of the two mechanisms. Polysaccharides present in the mixture
have a profound effect on the formation of the building blocks of
the gel structure and the dynamics of the system.
Agar-milk protein gels are interesting systems with unique
properties. Agar, an algal polysaccharide comprised of repeating
units of D-galactose and 3-6 anhydro-L-galactose, forms fully
reversible gels with an open structure with pore sizes that depend
on the concentration of polysaccharide. The gels hold a substantial
amount of unbound water. In agar-milk gels the polysaccharide is
homogeneously distributed, and the elastic modulus (G0 ) of the gel
is dominated by the polysaccharide network. In these structures
the casein micelles are still free diffusing in the void spaces
(Alexander & Dalgleish, 2007). So, albeit there is a high G0 in these
gels, the casein micelles could still be destabilized in clusters.
Similar, but reverse dynamics have been shown for non-interacting
polyethylene glycol (PEG) molecules in casein gels (rennet or acid)
where these biopolymers are employed as diffusivity probes, which
have been reported to travel undisturbed through the gel with
a diffusion rate dependent on the size of PEG molecules
(Le Feunteun & Mariette, 2007). These types of gels will not be
discussed further in this review, as they represent bi-continuous
homogeneous systems.
When normal starch pasted at temperatures lower that the
temperature of granule disruption or when modified starch gran-
ules (resistant to disruption) are added in milk, the starch granules
act as inert fillers that absorb water increasing the local
 M. Corredig et al. / Food Hydrocolloids 25 (2011) 1833e1841
concentration of casein micelles. This increases the strength of the
continuous network of caseins formed during acidification or
fermentation (Azim et al., 2010; Zuo, Hemar, Hewitt, & Saunders,
2008). In the case of modified waxy maize starch, although when
added at a 1% concentration it does not affect the gelation pH
during milk acidification, increasing the concentration to 2.5%
results in a higher pH of gelation and a shorter time of gelation,
most probably due to the volume fraction effect of starch granules
distributed in the continuous phase (Azim et al., 2010). The differ-
ence between acidified gels prepared with or without modified
waxy maize starch is illustrated in the confocal images shown in
Fig. 4. Prior to the confocal imaging, the milk was homogenized,
heated at 95
C for 5 min, cooled and fermented at 40
C until pH
4.6. Stirred and set yogurt gels with or without modified waxy
maize are compared in Fig. 4. Set gels show an open and uniform
structure, but in the presence of starch, the proteins form a denser
network with larger dark voids than those in the milk gels without
starch. Swollen starch granules are embedded throughout the
pores of the protein network, and no direct interactions between
the starch granules and the milk proteins are observed. It has been
shown previously (Azim et al., 2010) that the density of the gel
network increases with increasing starch concentration and that
casein and starch domains can be distinguished by microscopy. In
the stirred gels (Fig. 4b and d), the presence of 2.5% modified waxy
maize starch granules seem to cause the aggregates to reorganize
themselves in greater clusters compared to those shown in the
control sample. Even after stirring the starch granules are
Fig. 5. Confocal scanning laser micrographs of rennet-induced gels after 120 min of incubation. Control skim milk (a) and skim milk containing 0.18% pectin (b) and 0.18% pectin,
and 0.015% k-carrageenan (c). Scale bar is 50 mm. For details, see Acero-Lopez et al. (2010).
1839
distributed in the void spaces and the rearrangement of the casein
aggregates in bigger, denser structures in the presence of swollen
starch granules is one more indication of the lack of interaction
between starch granules and caseins.
Gels prepared with native starch granules can be quite different
than those described above for crosslinked starch. When milk is
heat-treated and homogenized, the native starch granules are
disrupted and amylase and amylopectin leach into the continuous
phase, and will cause phase separation in milk. It has been reported
that acidified milk gels containing native rice starch show a signi-
ficantly lower complex modulus compared to the gels that con-
tained rice starch pasted at low temperatures (where granules are
not disrupted) (Zuo et al., 2008).
EPS produced by lactic acid bacteria during milk fermentation
present a specific case of milkepolysaccharide interactions in acid
milk gels since EPS are produced continuously during fermentation,
gelation and to some extent during cooling and storage. If EPS
continues to be produced after the onset of aggregation of the
casein micelles, it will remain in proximity of the bacteria cells, and
will be constrained in pockets. Such confinement may minimize
interactions of EPS with caseins and intensify the formation of thick
casein networks. Breaking of the acid-induced gel containing EPS
will result in less dense protein aggregates, with less connectivity
and a large number of void spaces containing EPS (Kristo, Miao, &
Corredig, in press).
In recent years, increasing attention has been devoted to non-
equilibrium systems, where non-interacting polysaccharides
1840 M. Corredig et al. / Food Hydrocolloids 25 (2011) 1833e1841
cause an attractive interaction between the casein micelles. These
metastable systems may show great potential to develop novel
structures and textures in dairy products. Understanding how to
control and modulate different destabilization mechanisms at the
same time will allow the design of novel microstructures. The
phase separation systems can be controlled by the interplay
between gelation and phase separation, as this will have an effect
on the coarsening of the domains, and their stabilization.
Increasing the amount of non-interacting polysaccharides
affects the rheological properties of the gels, as well as their
microstructure (Acero-Lopez et al., 2010; Lazaridou, Vaikousi, &
Biliaderis, 2008). Incorporation of high molecular weight
b-glucan in skim milk results in weakening of the protein network
of acid milk gels, increasing the porosity of the structure (Lazaridou
et al., 2008). This weakening of the gel structure, however, could
be reduced using higher concentrations of b-glucan, as a higher
concentration promotes an increase in viscosity of the continuous
phase, with a consequent decrease in the rate of formation of the
phase separated domains. Recently, it has been clearly demon-
strated that renneting of milk in the presence of pectin causes the
formation of different structures (Acero-Lopez et al., 2010). At
concentrations of pectin leading to macroscopic phase separation,
the phase separated domains may be controlled by addition of k-
carrageenan, so that while renneting occurs, the casein micelles gel
within and between the domains. Fig. 5 illustrates the difference in
the microstructure between a rennet gel containing 0.18% high
methoxyl pectin and the same gel containing also 0.015% k-carra-
geenan. While in the case of the phase separating system (Fig. 5b)
the casein micelles form a gel within the domains and do not show
interconnectivity, in the presence of k-carrageenan an inter-
connected structure different from that of control milk (with no
polysaccharide) can be formed (Fig. 5c). By better understanding
these systems and their phase separation dynamics it will be
possible to design better functional dairy matrices.
6. Conclusions
Polysaccharides have traditionally been incorporated in dairy
products to improve bulk properties and stability. A better under-
standing of the behavior of biopolymers in dairy mixtures during
processing will allow to predict their performance as functional
ingredients and to obtain optimal quality in terms of texture, struc-
ture, mouthfeel, shelf life, and digestion performance. Indeed,
increasing evidence is becoming available on how structure of dairy
matrices affect the delivery of nutrients. For example, a complex
could be employed to encapsulate and deliver molecules for
improved taste or improved biological function. In the case of non-
interacting polysaccharides non-equilibrium phenomena will
continue to drive the curiosity of the food scientist, as predicting and
controlling the formation and development of bioactive nutrients in
the gastrointestinal tract, more attention will need to be focused on
the control of the formation of aggregates and microstructure for
optimal functionality, from fat replacing to increased satiety.
In associative interactions, efforts are increasing on how to
stabilize the complexes formed by polysaccharides and proteins.
How to exploit their functionality in micro-phase separated
domains will become important for improving product quality as
well as for designing novel food matrices. Gelation is one of the
most important functional properties of the milk proteins, and it is
exploited in a variety of dairy processes, but while the behavior of
proteins in various stages of processing is well understood, the
intensifying effect of addition of incompatible polysaccharides on
their aggregation, and formation of self-organized structures and
texture needs to be better understood.
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