Journal of Colloid and Interface Science 378 (2012) 184–190

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Journal of Colloid and Interface Science

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Formation of interfacial milk protein complexation to stabilize oil-in-water

emulsions against calcium
Aiqian Ye ⇑, Jerilyn Lo, Harjinder Singh
Riddet Institute, Massey University, Palmerston North 4442, New Zealand

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

Article history: Oil-in-water emulsions (30% soya oil) stabilized by sodium caseinate or whey protein were destabilized
Received 8 February 2012 in the presence of calcium, whereas lactoferrin-stabilized emulsion was stable under same conditions. At
Accepted 16 April 2012 pH 7, addition of lactoferrin to the caseinate or whey protein-stabilized emulsions led to association of
Available online 26 April 2012
lactoferrin with adsorbed proteins by electrostatic interactions, hence resulted in the formation of lacto-
ferrin-caseins/whey protein interfacial complexes at the interface of the emulsion droplets. The stability
Keywords: of the interfacial complexes-coated emulsion droplets in the presence of Ca2+ was examined by determi-
Milk proteins
nation of particle size, zeta-potential and microstructure as a function of Ca2+ concentration and added
Lactoferrin
Oil-in-water emulsions
lactoferrin concentration. For emulsions made with 1% w/w caseinate or WPI, the stability of emulsions
Interfacial complexes in the presence of 20 mM CaCl2 has been improved markedly after addition of 0.2% w/w lactoferrin, sug-
Calcium stability gesting the small amount of lactoferrin on the interface can largely enhance the calcium stability of milk
Steric repulsion proteins-coated emulsion droplets. Steric repulsion between the interfaces of droplets produced by the
large lactoferrin molecules on the interface was considered to contribute the preventing the flocculation
caused by Ca2+ binding and reduction in the electrostatic repulsion between the emulsion droplets.
Ó 2012 Elsevier Inc. All rights reserved.

1. Introduction
Recently, the formation of multilayers on the interface of food
colloids has been extensively studied [1–8]. The multilayers on
the emulsion droplets are formed through the complexation via
electrostatic interactions between protein and oppositely charged
polysaccharides [4,9,10]; some are formed by two proteins with
opposite charges [2,11,12]. The multilayers formed by inactive
polyelectrolyte are expected to protect the sensitive inner protein
layers against environmental conditions, such as changes in pH
and heat treatment [1,13]. However, studies on the properties of
the multilayers to improve the stability properties of emulsion
droplets under various environmental stresses have so far been
limited [1]. Due to the strict conditions for formation of multilayer
interface and the lack of knowledge on the structure of multilayers,
the applications of the emulsion droplets coated with multilayers
have not been developed extensively [14].
Previous studies in our laboratory have shown that interfacial
electrostatic complexation between lactoferrin and b-lactoglobulin
can be utilized to form multilayered food-grade emulsions [2,11].
Most recently, it has been reported that these multilayered emul-
sions may be more stable against various environmental condition
⇑ Corresponding author. Address: Riddet Institute, Massey University, Private Bag
11 222, Palmerston North 4442, New Zealand. Fax: +64 6 350 5655.
E-mail address: a.m.ye@massey.ac.nz (A. Ye).
such as Ca2+, high ionic strength and heat treatment [15]. Further
studies on the properties of multilayered emulsion should be car-
ried out so that this novel technology could be applied in the
industry.
Whey proteins, caseins and lactoferrin are known to be excel-
lent emulsifying agents, and are often used to stabilize oil-in-water
emulsions. The stability of the emulsions depends largely on the
conditions of their surroundings [16]. The interactions between
Ca2+ and milk proteins have been studied extensively. The forma-
tion of aggregates induced by Ca2+ would affect the stability of
the protein-coated oil-in-water emulsions and their adsorption
characteristics [17–21]. When Ca2+ binds with whey proteins
stabilized emulsion droplets, droplet destabilization and aggrega-
tion occurs. This is due to the binding of calcium ions to the
b-lactoglobulin on the droplet surface that reduces electrostatic
repulsions between droplets. Also, the hydrophobic regions of
b-lactoglobulin are also involved in the aggregation of the oil
droplets [17,18]. Sodium caseinate is composed of as1-, as2-, b- and
j-caseins. The individual caseins are able to protect droplets
against aggregation and flocculation through steric and electro-
static repulsions [19,20,22]. In particular, the as1- and b-caseins
have stronger tendencies to bind to Ca2+. The binding of caseins
to the divalent Ca2+ results in droplet aggregation and flocculation.
This occurs due to the reduction of electrostatic repulsion when
Ca2+ binds to the negatively charged phosphoseryl residues on
the as1- and b-caseins. Lactoferrin (LF) is an 80 kDa glycoprotein

0021-9797/$ - see front matter Ó 2012 Elsevier Inc. All rights reserved.
http://dx.doi.org/10.1016/j.jcis.2012.04.042
 A. Ye et al. / Journal of Colloid and Interface Science 378 (2012) 184–190 185

with an estimated 700 amino acid residues. In particular, LF is un- of NaOH to pH 6.8. Appropriate quantities of sodium azide were
ique and distinguishable from other milk proteins due to its high then added to be 0.02% of the total emulsions and stored at 4 °C.
isoelectric point (pI  9.0) [23]. LF is positively charged at a neutral All emulsions were prepared at least in duplicate.
pH while the other milk proteins are negatively charged in the
same pH range. Thus, LF has been shown to interact with other
negatively charged milk proteins, such as b-lactoglobulin and case-
ins [15,24,25].
The objective of this study was to investigate the influence of A
the formation of LF-whey proteins and LF-caseins multi-layered
emulsion droplets on the stability of emulsions towards the addi-
tion of calcium. The possible mechanisms of how these multilayers
influence calcium sensitivity of emulsion droplets to calcium are
discussed.

2. Materials and methods

2.1. Materials

Lactoferrin was a gift from Tatua Co-operative Dairy Company

Limited, New Zealand. The powder contained 1.1% moisture and
99.7% protein, of which 92% was lactoferrin. Whey protein isolate
895 (WPI) and sodium caseinate were purchased from Fonterra
Ltd, Auckland, New Zealand. Soya oil was purchased from Davis
Trading Company, Palmerston North, New Zealand. All the chemi-
cals used were of analytical grade and were obtained from either
BDH Chemicals (BDH Ltd., Poole, England) or Sigma Chemical Co.
(St. Louis, MO, USA) unless specified otherwise. B
2.2. Emulsion preparation

Protein solutions (0.2–2.0%) were prepared by dispersing lacto-

ferrin, WPI or sodium caseinate powders into Milli-Q water (water
purified by treatment with a Milli-Q apparatus, Millipore Corp.,
Bedford, MA, USA), and then stirring for 2 h at room temperature
to ensure complete dispersion. Appropriate quantities of soya oil
were then mixed with the protein solution to form the final emul-
sions containing 30 wt.% soya oil and 70 wt.% aqueous phase. The
mixture of protein solution was stirred for another 1 h and then
homogenized in a two-stage homogenizer (APV 2000, Denmark)
at a first-stage pressure of 25 MPa and a second-stage pressure of
5 MPa. The emulsions were homogenized twice for more effective
mixing of the oil phase. Different amounts of CaCl2 were added to
the emulsions after they were made in the absence of CaCl2. The pH
values of the emulsions were adjusted with 0.1 M of HCl or 0.1 M

Fig. 1. Changes in the average droplet size (d43) of emulsions made with 30 wt.% Fig. 2. Particle size distributions of emulsions made with 30 wt.% soya oil, pH 7 and
soya oil, pH 7 and 1% w/w sodium caseinate (d), 1% w/w WPI (j) or 1% w/w LF (N) 1% w/w WPI (A), 1% w/w sodium caseinate (B) and 1% w/w LF (C) at different CaCl2
as a function of added CaCl2 concentration. Each data point is the average of concentrations. 0 mM (); 5 mM (j); 10 mM (N); 20 mM (d); or 50 mM () CaCl2
determinations on two separate emulsions. was added after emulsion formation.
186 A. Ye et al. / Journal of Colloid and Interface Science 378 (2012) 184–190

2.3. Determination of average droplet size and droplet size distribution
A Malvern MasterSizer 2000 (Malvern Instruments Ltd, Mal-
vern, Worcestershire, UK) was used to determine the average
diameter (d43) of the emulsion droplets [26]. The relative refractive
index (N), i.e. the ratio of the refractive index of the emulsion drop-
lets (1.456) to that of the dispersion medium (1.33), was 1.095. The
absorbance value of the emulsion droplets was 0.001. Droplet size
measurements are reported as average diameters, d43, with d43
4 3
being defined as Rni di =Rni di , where ni is the number of particles
with diameter di. Mean particle diameters were calculated as the
average of duplicate measurements.
2.4. Electrophoretic mobility and f-potential determination
The f-potential values of the emulsion samples were measured
using a laser doppler velocimetry and phase analysis light scatter-
ing (M3-PALS) technique and a Malvern Zetasizer Nano ZS instru-
ment (Malvern Instruments Ltd.). Samples were diluted to 100
times with Milli-Q water. The emulsion samples were placed in
the electrophoretic mobility cell and analyzed at a scattering angle
of 173° using a Malvern Zetasizer Nano ZS instrument (Malvern
Instruments Ltd.). The effective electrical field, E, applied in the
measurement cell was between 50 and 150 V depending on the
ionic strength of the samples. The overall electrophoretic mobility,
l, was calculated at 20 °C according to Eq. (1) (for a spherical
particle).
Similarly, for the 1% sodium caseinate emulsions, d43 values were
relatively unchanged until CaCl2 P 20 mM, where d43 increased
from 1.1 lm to 2.7 lm (Fig. 1).
Fig 2 shows the particle size distributions of emulsions made
with either 1% LF, 1% WPI or 1% sodium caseinate with varying con-
centrations (0–50 mM) of CaCl2 added after homogenization. The
droplet size distributions for the 1% LF emulsion were monomodal
with increasing concentrations of CaCl2 added, implying that the
emulsion droplets were stable and no droplet aggregation took
place (Fig. 2A). In Fig. 2B, a monomodal particle size distribution
was seen for the 1% WPI emulsions until a critical CaCl2 concentra-
tion of P10 mM, where a bimodal size distribution was seen, with
a greater proportion of droplets in the larger size range. For the 1%
sodium caseinate emulsion, the droplet size distributions were
monomodal until a critical CaCl2 concentration of P20 mM, where
bimodal size distributions with a greater proportion of particles in
the larger size range were seen (Fig. 2C). Similarly, the monomodal
particle size distribution observed for the 1% LF emulsion implies
that the emulsion droplets were stable to CaCl2 addition. The
results indicate that Ca2+ induced droplet aggregation did not occur
for LF-coated emulsion droplets, but took place for WPI and
sodium caseinate-coated emulsion droplets.

U ¼ lx=E ð1Þ A
where U is the particle velocity (m s1) and x the frequency (s1) of
the applied electrical field. The f-potential (mV) was then calculated
using the Smoluchoski equation

l ¼ f=g ð2Þ
1
where e is the electric permittivity of the solvent (F m ) and g the
solvent viscosity (Pa s). An individual f-potential measurement was
calculated from the mean of two runs and the standard deviation of
at least 10 readings from an individual sample.

2.5. Confocal laser microscopy

Samples (1 mL) were stained with 1.0% (w/v) Nile Blue and 1.0%
(w/v) Fast Green (fluorescent dye), placed on a concave confocal
microscope slide (Sail; Sailing Medical-Lab Industries Co. Ltd., Suz-
hou, China), covered with a cover slip and finally examined with a
63 magnification lens using a confocal laser scanning microscope B
(Leica DM6000 B, Heidelberg, Germany) with a chosen objective
lens and an Ar/Kr laser with an excitation line of 488 nm for Nile
blue and collect emission in the range of 545–613 nm, an excita-
tion line of 633 nm for Fast Green and collect emission between
643 and 760 nm. The images were recorded to observe the change
in the microstructure of the samples.

3. Results

3.1. Influence of calcium chloride addition on droplet characteristics of

the single protein-coated emulsions

The primary emulsions (LF, WPI, sodium caseinate) were

prepared at pH 6.8. The average droplet diameters (d43) of the
emulsions were measured as a function of increasing CaCl2 concen-
tration (0–50 mM). As the CaCl2 concentration increased from 0 to
50 mM, the d43 values for emulsions made with 1% LF were rela- Fig. 3. Zeta-Potential of emulsion droplets (30 wt.% soya oil, pH 7) stabilized with
tively unchanged (Fig. 1). However, the d43 values for emulsions different concentrations of WPI (A) and sodium caseinate (B) as a function of added
made with 1% WPI increased sharply for CaCl2 P 10 mM (Fig. 1). lactoferrin concentration.
 A. Ye et al. / Journal of Colloid and Interface Science 378 (2012) 184–190
A
B B
Fig. 4. Changes in the average droplet size (d43) of emulsions made with 30% w/w
soya oil, pH 7 and 1% w/w WPI (A) and 1% w/w sodium caseinate (B) as a function of
added CaCl2 concentration, after 0.2% w/w (d), 0.5% w/w (j) and 1% w/w (N)
lactoferrin were added to the emulsions. Each data point is the average of
determinations on two separate emulsions.
3.2. Formation of protein complex-coated emulsions
Various concentrations of WPI or sodium caseinate were used in
the preparation of primary emulsions in which pH were adjusted
to pH 6.8. Different LF concentrations were added to the primary
emulsions. The f-potential values of emulsions containing a range
of LF concentrations are shown in Fig. 3. In the absence of LF, the
f-potential of the WPI- or sodium caseinate-stabilized emulsions
(pH 6.8) was highly negative at range of 45 to 50 mV. The
f-potential of the emulsion droplets made with both of WPI and
sodium caseinate became increasingly less negative as LF concen-
tration increased. This decrease in the negative f-potential was
more abrupt in the emulsions formed with lower protein concen-
trations. For examples, the f-potential of the 0.3% WPI or sodium
caseinate-coated oil droplets was positive at 2% LF added. This de-
crease in the negative f-potential of the emulsion droplets with
addition of LF indicates the LF (positive charge) associated with
the surface protein (negative charge) of the emulsion droplet to
form a protein complex surface layer on the emulsion droplets. It
has been reported that the LF binds to the surface b-lg by electro-
static interactions and forms complex multilayer on the emulsion
droplets at neutral pH [2,15]. The proportion of LF in the surface
complex layer increased when the concentration of added LF
187
A
Fig. 5. Average droplet size (d43) of emulsions made with (30% w/w soya oil, pH 7)
different concentrations of WPI (A) or sodium caseinate (B) containing 20 mM CaCl2
as a function of added lactoferrin concentration.
increased. Meanwhile, at the same added LF concentration, the
proportion of LF at the droplet surface was higher in emulsions
droplets formed with lower concentration of WPI or sodium case-
inate; in these systems the surface protein concentration in the
primary emulsions is lower [18,20].
In both the emulsions formed with WPI and sodium caseinate,
there was a similar trend in the reversal of charge in f-potential
values with increasing LF concentration (Fig. 3). It appears that
the association of LF with surface was mainly dependent on the
charge of adsorbed surface proteins, not on the type of protein.
The f-potential of the WPI- or sodium caseinate-coated emulsion
droplets (pH 6.8) was similar (at range of 45 to 50 mV) in the
primary emulsions. These studies confirm that stable multilayered
emulsions can be formed through the adsorption of LF onto the
whey protein- or caseinate-coated emulsion droplets [24]; through
electrostatics interaction.
3.3. Influence of calcium addition on droplet characteristics of the
protein complex-coated emulsions
Previous results have shown that droplet aggregation and
destabilization occur when calcium is added to single protein-
coated emulsions prepared with whey proteins or caseinates, but
no destabilization was observed in the emulsion formed with LF
(Fig. 1). Fig. 4 shows that the average droplet size (d43) of the
188 A. Ye et al. / Journal of Colloid and Interface Science 378 (2012) 184–190
Fig. 6. Confocal micrographs of 0.3% w/w WPI-stabilized emulsions (A), containing 1% w/w lactoferrin (B), containing 20 mM CaCl2 (C) or containing 1% w/w lactoferrin and
20 mM CaCl2 (D).
WPI-LF and sodium caseinate-LF complex-coated emulsions as a
function of added CaCl2 concentration. For primary emulsions pre-
pared with 1% w/w WPI, the d43 value began to increase at 5 mM
CaCl2 and at 10 mM CaCl2 for the addition of 0.2% LF and 0.5% LF,
respectively. Addition of LF > 0.5% resulted in d43 values that were
relatively unchanged with increasing CaCl2 concentration up to
50 mM. Similar trend was found in primary emulsions prepared
with 1% sodium caseinate. Addition of 0.2% LF had delayed the in-
crease in d43 of emulsions formed with 1% sodium caseinate with
added 20 mM CaCl2. Addition of LF P 0.5% did not influence in
d43 values with increasing CaCl2 concentration up to 50 mM.
The effect of varying LF concentration on the average droplet
size of the emulsions containing 20 mM CaCl2 is shown in Fig. 5.
It was observed that the d43 of emulsions made with 1% WPI were
high at 20 mM CaCl2 concentrations when no LF was added. When
0.2% of LF was added to the emulsions formed with 0.5% and 1%
WPI, the d43 values fell sharply to 0.8 to 1.1 lm. Further in-
creases in LF concentration added to the emulsions resulted in
d43 values that were relatively unchanged. For emulsions formed
with 0.3% WPI, higher LF (0.5% w/w) was needed to decrease the
d43 values fell sharply to 1.0 lm. For emulsions prepared with
sodium caseinate, 2% and 0.5% LF were required to reduce the d43
values to 1 lm for the emulsions formed with 0.3% and 0.5%
sodium caseinate, respectively.
3.4. Confocal laser scanning microscopy
For WPI emulsions with no CaCl2 added, droplets were very sta-
ble, evenly distributed and remained homogenous with no signs of
flocculation (Fig. 6A). When 20 mM of CaCl2 was added to 0.3% WPI
emulsion droplets, small droplets that had aggregated together
were observed (Fig. 6C). When LF was added to the 1% WPI emul-
sion, droplets were stable, homogenous and evenly distributed
(Fig. 6B). When LF was added to the 1% WPI emulsion droplets with
20 mM CaCl2 added, there was no sign of droplet aggregation
(Fig. 6D). The images obtained from the confocal microscope
agreed with the results of particle size of the emulsions deter-
mined by light scattering (Fig. 4). Similar to WPI stabilized emul-
sions, flocculated sodium caseinate-stabilized emulsion droplets
were observed at addition of 20 mM CaCl2, but the flocculation dis-
appeared in the presence of LF (Fig. 7). Also stable, homogenous
and evenly distributed emulsion droplets were observed in the
sodium caseinate-LF multilayer coated emulsion (Fig. 7B).
4. Discussion
The present results are in general agreement with previous
studies, where LF-coated oil droplets exhibited good salt stability
 A. Ye et al. / Journal of Colloid and Interface Science 378 (2012) 184–190
Fig. 7. Confocal micrographs of 0.3% w/w sodium caseinate-stabilized emulsions (A), containing 1% w/w lactoferrin (B), containing 20 mM CaCl2 (C) or containing 1% w/w
lactoferrin and 20 mM CaCl2 (D).
whereas b-lactoglobulin along with sodium caseinate coated oil
droplets had aggregated at high CaCl2 concentrations [17–20]. In
these systems, aggregation of emulsion droplets is due to floccula-
tion rather than coalescence as shown by the confocal microscopy
images. Droplet flocculation in whey protein or casein-coated
emulsions can be attributed to Ca2+ bridging between droplets
and a reduction in the electrostatic repulsions between the drop-
lets due to ion binding and screening effects. The extent of Ca2+ in-
duced destabilization of the emulsions was dependent on the
protein concentration. Emulsions made with higher protein con-
centrations (both WPI and caseinate) were less sensitive to Ca2+
(as indicated by smaller particle size) than those made with low
protein concentrations at 20 mM added calcium (Fig. 5); this is in
agreement with the previous work [18,20]. It is likely that excess
protein in the aqueous phase of emulsions with high protein con-
centrations would bind calcium, thus reducing the impact of added
calcium on the surface of emulsion droplets.
The emulsion containing LF-coated lipid droplets was stable to
droplet flocculation, even at relatively high calcium concentration.
The stability against Ca2+ is likely because LF-coated droplets were
positively charged at neutral pH since pI of LF is about pH 8. Ca2+
may not bind with adsorbed LF molecules to form bridging droplet
flocculation. In addition, LF is a glycoprotein with hydrophilic car-
bohydrate groups that protrude into the surrounding aqueous
phase thereby forming thicker hydrophilic layers [2,11,12,24,25].
189
These protein layers formed by LF may have generated a stronger
steric repulsion between the LF-coated droplets, which may have
prevented flocculation.
The results indicate that the presence of LF significantly reduced
the destabilization sensitivity of WPI and sodium caseinate-stabi-
lized emulsion droplet by calcium (Figs. 4–7). It is easy to consider
that the LF associated to the surface of whey protein and caseinate-
coated droplets to form a LF coated multilayer through electrostat-
ics interactions (Fig. 3). This LF coated surface layer is able to
prevent the flocculation induced by Ca2+ bridging and screening the
repulsion charges as shown in the calcium stability of LF-stabilized
emulsions (Fig. 1). Similar results that show LF-b-lactoglobulin
multilayer not only prevent the flocculation of the emulsion drop-
lets induced by Ca2+, but also salt and heat treatment have been re-
ported recently [12,25]. This multi surface layer combined LF and
other whey protein or caseinate surface layer represents most
properties of LF surface layer on LF-stabilized droplets, suggesting
LF may coat the outside of this multi-surface layer.
However, it is interesting to note that the calcium stability of
WPI or caseinate stabilized emulsions was markedly improved at
a considerable low LF concentration (0.2–0.5%) (Figs 3 and 4). At
this low LF concentration, the zeta-potential of whey protein or
caseinate-coated emulsion droplets only slightly increased from
45 mV to 30 mV (Fig. 3). This suggests that LF has not fully
coated or covered the surface of the droplets; the surface of
190 A. Ye et al. / Journal of Colloid and Interface Science 378 (2012) 184–190
Fig. 8. Schematic representation of the effect of lactoferrin on the colloidal structure of a milk protein (WPI or sodium caseinate)- stabilized emulsion in the presence of Ca2+:
(A) emulsion droplets coated with milk protein; (B) flocculated emulsion droplets induced by Ca2+ bridging; (C) Lactoferrin binding on the milk protein surface layer of
emulsion droplets via electrostatic interaction; (D) non-flocculated emulsion sterically stabilized by low density of dangling lactoferrin molecules.
droplets was still dominated by casein or whey protein. It has been
reported that the zeta-potential of LF-coated emulsions range from
+40 to +60 mV at LF concentrations from 0.3% to 3% at pH 7 [2]. The
question is why calcium does not cause flocculation of droplets. It
has been previously mentioned that droplet flocculation in whey
protein or caseins coated emulsions in the presence of Ca2+ is
attributed to Ca2+ bridging between droplets and a reduction in
the electrostatic repulsion between the droplets due to ion binding
and screening effects. LF coating at the surface of caseins or whey
protein-stabilized emulsion droplets can prevent the Ca2+ bridging
the droplets. In the case of LF partially coating at the surface (small
amount of LF on the surface), when caseins or whey protein dom-
inate the surface, the Ca2+ bridging between the droplets could be
prevented by the small amount of LF on the surface through steric
repulsions. The tentative speculation is illustrated schematically in
Fig. 8. Large molecule of LF bound with the casein and whey pro-
tein coated surface by electrostatic interaction makes the Ca2+
bridging (close distance) impossible to occur. This steric repulsion
required to prevent Ca2+ induced flocculation of emulsion droplets
may not need a large amount of LF bound to surface or cover large
area of surface as illustrated in Fig. 8. The amount of LF bound to
surface needed for preventing the flocculation may be dependent
on the primary protein surface (Fig. 5), which is interesting for fur-
ther investigation in future. In the case of heating-induced destabi-
lization, Dickinson and Parkinson [27] demonstrated that lower
than 5% casein in the total surface protein in the whey protein
coated surface of droplets can prevent the heating induced aggre-
gation of whey protein emulsions through enhanced steric stabil-
ization. Meanwhile, this steric stabilization due to LF at the
surface may also inhibit the flocculation of droplets induced by
the screening effect of increased ionic strength by addition of CaCl2
[2,11].
5. Conclusions
Emulsions stabilized by caseins or whey protein were destabi-
lized in the presence of calcium, whereas lactoferrin stabilized
emulsion demonstrated high stability against calcium. At neutral
pH, addition of lactoferrin to the caseinate or whey protein-stabi-
lized emulsions led to association of lactoferrin with adsorbed pro-
teins by electrostatic interaction, hence resulted in the formation
of lactoferrin-caseins/whey protein multi-layer at the surface of
the emulsion droplets. The multi-surface layer consisted of lacto-
ferrin significantly reduced the destabilization of the emulsions in-
duced by calcium ions, even with small amount of lactoferrin
involved in the surface layer. Steric repulsion interaction produced
by the large lactoferrin molecules on the surface is considered to
contribute the preventing the flocculation caused by Ca2+ binding
and reduction in the electrostatic repulsion between the emulsion
droplets.
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