ARTICLE IN PRESS

FOOD
HYDROCOLLOIDS
Food Hydrocolloids 22 (2008) 56–64
www.elsevier.com/locate/foodhyd

Aggregation of casein micelles and k-carrageenan in reconstituted

skim milk
S. Ji, M. Corredig, H.D. Goff
Department of Food Science, University of Guelph, Guelph, Ont., Canada N1G 2W1
Received 9 June 2006; accepted 2 April 2007

Abstract

A novel method to form casein/k-carrageenan aggregates in skim milk, by mixing solutions of each at 60 1C and cooling to 25 1C while
shearing, was investigated. The average particle size of casein/k-carrageenan aggregates decreased with increasing shear rate (200, 400
and 800 s1) but increased with k-carrageenan concentration (0.025% 0.05% and 0.075%). Oscillatory rheology measurements
demonstrated that skim milk containing 0.025% k-carrageenan behaved as a dilute solution, while skim milk with 0.05% or 0.075% k-
carrageenan formed weak gels, although solutions remained fluid and were at a much lower modulus compared to similar solutions
cooled with no shearing. The aggregates were quite stable to shear once formed. The initial mixing temperature of reconstituted skim
milk and k-carrageenan dispersions greatly affected the particle size distribution of the aggregates. Microstructural observations as well
as studies in the presence of NaI demonstrated that at particular protein/polysaccharide ratios, casein/k-carrageenan aggregates formed
in reconstituted skim milk via adsorption of k-carrageenan with casein micelles.
r 2007 Elsevier Ltd. All rights reserved.

Keywords: Casein micelles; k-Carrageenan; Rheology; Microstructure; Casein–k-carrageenan interactions

1. Introduction (de Vries, 2002). No interactions have been measured

Phase separation is quite common in dairy products
containing polysaccharides, since most polysaccharides
used in commercial formulations are thermodynamically
incompatible with milk proteins. ‘‘Wheying off’’ and other
instability effects result in significant changes in product
quality. k-Carrageenan is often added to dairy products
together with other polysaccharides to prevent phase
separation. The mechanism of stabilization of k-carragee-
nan in complex milk systems is still not fully understood,
although much research has been focused in this area in the
recent years. de Vries (2002) demonstrated that the strength
of carrageenan–caseins gel in milk is much higher
compared to mixes containing caseinate and whey protein.
It was suggested that casein micelles play a major role in
the stabilizing and gelling functionality of k-carrageenan
Corresponding author. Tel.: +1 519 824 4120x53878;
fax: +1 519 824 6631.
E-mail address: dgoff@uoguelph.ca (H.D. Goff).
between whey proteins and k-carrageenan (Xu, Stanley,
Goff, Davidson, & Le Maguer, 1992). In addition,
Tziboula and Horne (1999) reported that whey proteins
in either native or denatured form do not play an
important role in k-carrageenan gelation. Phase separation
and independent gelation of whey proteins and k-carra-
geenan occurred, when gelling concentrations of whey
proteins and k-carrageenan were present in the dispersions
(Ould Eleya & Turgeon, 2000; Turgeon & Beaulieu, 2001).
Vega, Dalgleish, and Goff (2005) found that k-carrageenan
was not as efficient at inhibiting phase separation in
sodium caseinate as at inhibiting phase separation of skim
milk powder (SMP) systems, demonstrating that the
micellar structure of casein micelles plays an important
role in the association of k-carrageenan with proteins in
milk.
It is generally accepted that the interaction between
k-carrageenan and casein micelles is dependent on the
concentration of k-carrageenan, that is, at high concentra-
tions of carrageenan, interactions between k-carrageenan

0268-005X/$ - see front matter r 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.foodhyd.2007.04.005
 ARTICLE IN PRESS
S. Ji et al. / Food Hydrocolloids 22 (2008) 56–64
molecules are favored and casein micelles are trapped
within a continuous three-dimensional network of
k-carrageenan. This has been reported for k-carrageenan
concentrations well above the minimum gelling concentra-
tions in milk (0.03%) (Schorsch, Jones, & Norton, 2000).
At concentrations below gelling, k-carrageenan interac-
tions are fundamental in stabilizing milk protein-based
dispersions; however, the exact mechanism of the associa-
tion of k-carrageenan with caseins is still under investiga-
tion. It has been hypothesized that k-carrageenan interacts
with casein micelles through electrostatic interaction
(Langendorff et al., 1999). Snoeren, Payens, Jeunink, and
Both (1975) showed that an electrostatic attraction occurs
between k-carrageenan and k-casein, but not as or b-casein.
It was suggested that the association of k-carrageenan with
casein micelles at neutral pH is driven by the attraction of
negatively charged carrageenan to the positive patch
between amino acid residues 97 and 112 of k-casein,
although at this pH, the overall charge of the casein micelle
is negative. Dalgleish and Morris (1988) measured the
z-potential of casein micelles with k-carrageenan and showed
that below the coil–helix transition temperature, the charge
density of casein micelles becomes more negative with
increasing concentration of k-carrageenan, indicating the
adsorption of negatively charged k-carrageenan to casein
micelles.
It has been shown that at temperatures below the coil to
helix transition, the apparent diameter of casein micelles
measured by DLS increases in the presence of
k-carrageenan (Langendorff et al., 2000). It was suggested
that casein micelles are bridged via the helical parts of
k-carrageenan and form a network. Recent research
showed that k-carrageenan adsorbs on the surface of
casein micelles (Spagnuolo, Dalgleish, Goff, & Morris,
2005). Below the gelling concentration of k-carrageenan
(0–0.03%) at 25 1C, the increase in size of the casein
micelles is linearly related to the concentration of
k-carrageenan. Microstructural observations showed an
open, porous surface of casein micelles, which could
suggest that a large polymer like carrageenan could
penetrate the hairy layer and adsorb to the surface of
casein micelles. Indeed, Thomsen, Jakobsen, Nielsen,
Petersen, and Rasmussen (1995) showed that the con-
formation of the C-terminal part of k-casein is quite
flexible.
Most studies on casein–k-carrageenan systems have been
carried out at concentrations of k-carrageenan much
higher than its critical gelling concentration. The objectives
of this study are to investigate if k-carrageenan could form
aggregates with casein micelles in skim milk at low
concentration by mixing solutions of each at 460 1C
and cooling through the coil–helix transition region of
k-carrageenan while shearing the mixture and to char-
acterize the aggregates formed. With this aim, the effect of
shear during cooling and of concentration of k-carrageenan
on the particle size distribution of the reconstituted milk were
studied. Rheological properties of casein/k-carrageenan
57
aggregates were determined and the stability of these
aggregates was studied. Microstructures of casein/k-carra-
geenan aggregates were studied with cryo-scanning elec-
tron microscopy, field emission scanning electron
microscopy and transmission electron microscopy. Micro-
particulated complexes of casein micelles and k-carragee-
nan could provide unique functional properties to skim
milk, which may lead to the development of a novel food
ingredient.
2. Materials and methods
Low heat SMP was purchased from Parmalat Canada.
k-Carrageenan was supplied by Danisco Canada without
further purification. Stock SMP solutions (21%) were
prepared by dissolving powder in ultrapure water at 20 1C
and storing at 4 1C overnight. Stock k-carrageenan solution
(0.05%, 0.1% and 0.15%) was prepared by adding
k-carrageenan powder to ultrapure water at 95 1C and
stirring until completely dissolved. The solution was then
kept in a water bath at 60 1C. k-Carrageenan solutions and
SMP solutions were mixed at a 1:1 (w/w) ratio at 60 1C,
transferred to the rheometer and cooled to 25 1C under
continuous shear. The shear applied during cooling (200,
400 or 800 s1) was controlled with a controlled stress
rheometer (AR2000, TA Instruments, New Castle, USA)
using a parallel plate geometry (diameter 40 mm). The
temperature was controlled with a peltier system. A cooling
rate of 10 1C/min was used. A frequency sweep from 0.01
to 10 Hz was then performed within the linear viscoelastic
region of casein/k-carrageenan mixed system at 25 1C. An
oscillatory stress of 0.1 Pa was applied.
The effects of initial mixing temperature of SMP
solution and k-carrageenan solution were studied by
mixing these two solutions at 25 1C and shearing with a
magnetic bar.
The method of MacArtain, Jacquier, and Dawson (2003)
was followed for cryo-scanning electron microscopy (cryo-
SEM). Hitachi S570 SEM (Tokyo, Japan) and EMScope
Cryo-preparation Unit (Kent, UK) were used to capture
the images. The samples were loaded to a sample tube and
then immersed in liquid nitrogen slush, fractured under
vacuum and sublimated at 80 1C for up to 30 min. The
samples were then sputter coated with gold and examined.
The method of Spagnuolo et al. (2005) was followed for
field emission scanning electron microscopy (FE-SEM,
critical point drying). Carbon planchets (Canemco Inc., St.
Laurent, Que., Canada) were polished with fine polishing
paper to obtain a glassy surface as the substrate. The
samples were placed on the polished carbon planchets for
5 min to allow the samples to adhere to the substrate.
Excess sample was then carefully removed and 2%
glutaraldehyde prepared in Sorensen’s phosphate buffer
was applied on the planchets as a primary fixative for 1 h.
Then 1% osmium tetroxide in phosphate buffer was
deposited on the planchets as a secondary fixative for 1 h.
The secondary fixatives were then removed and the
 ARTICLE IN PRESS
58 S. Ji et al. / Food Hydrocolloids 22 (2008) 56–64

planchets were rinsed three times with phosphate buffer at casein/k-carrageenan aggregates appeared as a homoge-
pH 7. The samples were dehydrated with a series of ethanol nous liquid. The particle size measured by integrated light
(70%, 80%, 90%, 95%, 100%, w/w) by immersing for scattering showed a distribution ranging between 1 and
5 min in each. The 95% ethanol step was repeated twice
and 100% ethanol step was repeated three times. The
samples then were critical point dried using carbon dioxide 40
200 s-1
and mounted onto SEM stubs. The images were captured 400 s-1
with a Hitachi S4800 FE-SEM (Tokyo, Japan). 800 s-1
SMP only
The method of Thaiudom and Goff (2003) was followed 30
for transmission electron microscopy (TEM). Samples were

Voulme (%)
prepared using the agar rod sleeve technique (Allan-Wojtas
& Kalab, 1984). Samples were put into the agar sleeve and 20
fixed by 2% glutaraldehyde for 2 h, then immersed in fresh
phosphate buffer three times at 10 min interval and kept in
buffer overnight. The sleeved samples were dehydrated 10
with a series of ethanol (50%, 60%, 70%, 80%, 90%, 95%,
100%, w/w) by immersing for 10 min in each. The samples
were then immersed in 100% ethanol for 15 min twice and 0
in 100% propylene oxide for 15 min twice in order to be 0.1 1 10 100
completely dehydrated. The samples were then immersed in Particle size (µm)
a 1:1 solution of propylene oxide:embedding medium for
1 h at room temperature. The embedding medium was 20
made of 49.76% (w/w) Jembed 812 Resin, 24.49% (w/w) 200 s-1
18 400 s-1
dodecenyl succinic anhydride (DDSA), 24.02% (w/w)
800 s-1
Nadic methyl anhydride (NMA) and 1.73% (w/w) tri- 16
(dimethylaminomethyl) phenol (DMP). The samples were 14
then transferred to a 1:2 solution of propylene oxide:em-
Volume (%)

12
bedding medium for 1 h twice at room temperature,
10
followed by 100% embedding medium for 1 h at room
temperature. The samples were embedded and cured in an 8
oven at 60 1C for 24 h. Blocks were sectioned at a thickness 6
of 90 nm using a LKB ultramicrotome (Leica Reichert 4
Ultracut S., Vienna, Austria) and sections were mounted
2
on gold grids (Marivac Ltd., Halifax, NS). The images
were taken by using Hitachi 7700 TEM (Tokyo, Japan). 0
0.1 1 10 100
Particle size distribution was determined using laser
Particle size (µm)
diffraction with a Malvern Mastersizer X (Worcestershire,
UK). Lenses with a focal length of 45 and 300 mm were
30
used, and a refractive index contrast of 1.06 was chosen for
200 s-1
the particle size distribution calculations. 400 s-1
Effects of EDTA on the stability of aggregates were 25 800 s-1

studied by adding 20 mM EDTA into the solution of

aggregates at 25 1C. SMP solution was used as control. 20
Volume (%)

Effects of NaI on the formation of casein/k-carrageenan

were studied by adding 11.6 mM NaI into 0.025% 15
carrageenan solution at 60 1C. 10.5% SMP solution and
0.025% k-carrageenan solution containing NaI were mixed 10
at 60 1C and cooled to 25 1C with shear.
All experiments were performed in triplicate. 5

3. Results 0
0.1 1 10 100
3.1. Effects of shear on particle size distribution and Particle size (µm)
rheological properties of casein/k-carrageenan aggregates
Fig. 1. The effects of shear rate on particle size distribution of casein/k-
carrageenan aggregates: (a) 0.025% k-carrageenan and 10.5% SMP, (b)
At the lowest concentration of polysaccharide tested 0.05% k-carrageenan and 10.5% SMP, (c) 0.075% k-carrageenan, 10.5%
(0.025% k-carrageenan), reconstituted SMP containing the SMP.
 ARTICLE IN PRESS
S. Ji et al. / Food Hydrocolloids 22 (2008) 56–64 59

100 mm, with most particles from 10 to 100 mm (Fig. 1a).

1
The particle size distribution of casein/k-carrageenan
200 s-1, G'
aggregates decreased with increasing shear rates, showing 200 s-1, G"
a population of smaller particles (o1 mm) at higher shear 400 s-1, G'
0.1 400 s-1, G"
rate (Fig. 1a). When casein/k-carrageenan mixture was 800 s-1, G'
sheared at 800 s1, a bimodal distribution of particles was 800 s-1, G"

G'& G" (Pa)

shown with one population of particles from 0.1 to 1 mm
and the other from 5 to 40 mm. Viscoelasticity measure- 0.01
ments were performed to determine the rheological proper-
ties of casein/k-carrageenan aggregates, as shown in Fig. 2.
With 0.025% k-carrageenan, the mixture of aggregated and 0.001
non-aggregated casein micelles and k-carrageenan exhib-
ited the characteristics of dilute solution and there was no
gel structure left after casein micelles/k-carrageenan 0.0001
0.01 0.1 1
mixture was sheared at 200, 400 or 800 s1 (Fig. 2a).
Frequency (Hz)
When 0.05% k-carrageenan was added to reconstituted
SMP, the particle size distribution of the dispersions
100
containing casein/k-carrageenan aggregates decreased with
increasing shear rate with a population of large particles
between 10 and 100 mm. However, the population of small 10
particles between 0.1 and 1 mm was not observed after
shearing at rates as high as 800 s1 (Fig. 1b). Frequency G' & G" (Pa)
1
sweep testing of reconstituted skim milk containing 0.05%
k-carrageenan showed that the casein/k-carrageenan ag-
gregates imparted some characteristics of a weak gel to the 0.1
200s-1, G'
dispersion, although solutions remained fluid and were at a 200s-1, G"
400s-1, G'
much lower modulus compared to similar solutions cooled 0.01 400s-1, G"
with no shearing. Sample sheared at low shear rate showed 800s-1, G'
800s-1, G"
a high-frequency dependence of G0 and G00 while samples 0.001
sheared at high shear rates showed a low-frequency 0.01 0.1 1
dependence of G0 and G00 . The values of G0 of samples Frequency (Hz)
increased significantly with shear rate. This indicated that
a stronger gel structure formed with higher shear rate 100
200 s-1, G'
(Fig. 2b). 200 s-1, G"
When 0.075% k-carrageenan was added to reconstituted 400 s-1, G'
400 s-1, G"
SMP, most particles measured showed a diameter 410 mm 800 s-1, G'
(Fig. 1c). While at low shear rate (200 s1) some small gel 10 800 s-1, G"
G'& G" (Pa)

fragments were observed, at higher shear rates the mixture

appeared as a homogenous liquid. Frequency sweep
measurements showed that with 0.075% k-carrageenan,
reconstituted skim milk showed gel-like characteristics 1
caused by the presence of casein/k-carrageenan and
k-carrageenan–k-carrageenan aggregates (Fig. 2c). All
samples showed a low frequency dependence of G0 and
G00 . Shearing the mixtures at 800 s1 showed a significant 0.1
decrease in the value of G0 compared to that of the mixtures 0.01 0.1 1
sheared at lower rates. This may indicate that although Frequency (Hz)
all complexes showed a similar particle size distribution
Fig. 2. Frequency sweep of casein/k-carrageenan aggregates in solution at
(Fig. 1c) the network formed by the k-carrageenan chains
25 1C: (a) 0.025% k-carrageenan and 10.5% SMP, (b) 0.05% k-
was somewhat modified. carrageenan and 10.5% SMP, (c) 0.075% k-carrageenan and 10.5% SMP.

3.2. Microstructure of casein/k-carrageenan aggregates

formed under shear
Cryo-SEM showed that casein/k-carrageenan aggregates
were composed of many spherical subunits. These subunits
were tightly connected to each other. No individual caseins
or k-carrageenans were distinguishable (Fig. 3a). FE-SEM
after critical point drying showed the presence of individual
casein micelles and k-carrageenan strands. Casein micelles
appeared to be spherical with some protuberances on the
surface (Fig. 3b). Spagnuolo et al. (2005) showed some
 ARTICLE IN PRESS
60 S. Ji et al. / Food Hydrocolloids 22 (2008) 56–64

detailed structure of the surface of casein micelles with

some cylindrical or tubular structures protruding out of the
surface. In our studies, although the detailed surface
structures of casein micelles could not be distinguished, it
can be seen that rather than a smooth surface, aggregated
casein micelles possessed an unevenly distributed surface
with some tubular structures sticking out from the surface.
Aggregated casein micelles were bound together with
k-carrageenan, forming a three-dimensional network.
k-Carrageenan appeared as long or short twisted polymer
strands (as indicated by the arrow in Fig. 3b). Higher
resolution images were collected using TEM, and the
detailed structure of casein/k-carrageenan aggregates is
depicted in Fig. 3c. The casein micelles seemed to be
randomly distributed within the aggregates. k-Carrageenan
appeared as long thin stranded polymers (as indicated by
arrow in Fig. 3c) and casein micelles were connected
to each other by k-carrageenan strands. The results from
FE-SEM and TEM show strong evidence that the complex
is indeed comprised of both casein and k-carrageenan.

3.3. Effect of shear on the stability of aggregates

The aggregates formed in reconstituted SMP with

0.025% and 0.05% k-carrageenan added were quite stable
under shear. There was no significant change in the particle
size distribution if the shear was applied again after the
casein/k-carrageenan aggregates were formed. In order to
test the stability of these aggregates under shear, shear rates
equal to or higher than the ones used to produce aggregates
during cooling were chosen. It was shown that with both
0.025% and 0.05% k-carrageenan, no matter at what shear
rate the aggregates were produced (200, 400 or 800 s1) or
at what shear rate the aggregates were treated afterwards
(200, 400, 800 or 1200 s1), there was no significant change
in the particle size distribution. These results indicated that
at a given concentration of k-carrageenan, the size of
aggregates was determined by the shear rate used during
cooling and once the aggregates formed, they were quite
resistant to shear up to 1200 s1 (Figs. 4 and 5).

3.4. Effects of initial mixing temperature of SMP solution

and k-carrageenan solution on particle size distribution

We found that by mixing SMP solution and k-carrageenan

Fig. 3. Microstructure of casein/k-carrageenan aggregates: (a) Cryo-SEM,
bar ¼ 1 mm, (b) FE-SEM, bar ¼ 100 nm, (c) TEM, bar ¼ 200 nm. 0.025%
k-carrageenan, 10.5% SMP.
solution at 25 1C, casein/k-carrageenan aggregates can
also be formed. The particle size distribution was
dependent on shear and on the concentration of
k-carrageenan (data not shown). However, the particle
size distribution of aggregates was quite different if SMP
solution and k-carrageenan solution were mixed at 25 1C
compared to mixing these two components at 60 1C. It was
shown that by mixing reconstituted SMP and k-carragee-
nan solutions at 25 1C, the aggregates were larger in size
and showed a wider size distribution. Those aggregates
made by mixing reconstituted SMP and k-carrageenan at
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S. Ji et al. / Food Hydrocolloids 22 (2008) 56–64 61

12 10
200 s-1 200 s-1
400 s-1 400 s-1
10 800 s-1 800 s-1
8

Volume (%)
Volume (%)

6
6
4
4

2
2

0 0
0.1 1 10 100 1000 0.1 1 10 100 1000
Particle size (µm) Particle size (µm)

12 10
400 s-1 400 s-1
800 s-1 800 s-1
10 1200 s-1 1200 s-1
8

8
Volume (%)
Volume (%)

6
6
4
4

2
2

0 0
0.1 1 10 100 1000 0.1 1 10 100 1000
Particle size (µm) Particle size (µm)

Fig. 4. The stability of casein/k-carrageenan aggregates: (a) the aggregates

12
were made at 200 s1 and sheared at 200, 400, 800 s1 afterwards, 0.025%
800 s-1
k-carrageenan, 10.5% SMP, (b) the aggregates were made at 400 s1 and
1200 s-1
sheared at 400, 800, 1200 s1 afterwards, 0.025% k-carrageenan, 10.5% 10
SMP.
8
Volume (%)

60 1C had a significantly smaller particle size and a

narrower size distribution (Fig. 6). 6

4
3.5. Characterization of casein/k-carrageenan aggregates
formed under shear
2
To determine if complexes were still present after the
addition of calcium chelating agents, 20 mM EDTA was 0
0.1 1 10 100 1000
added to a 10.5% SMP solution and to the samples
Particle size (µm)
containing casein/k-carrageenan aggregates. Visual obser-
vation showed that both systems lost their white, milky Fig. 5. The stability of casein/k-carrageenan aggregates: (a) the aggregates
color and became transparent after the addition of EDTA, were made at 200 s1 and sheared at 200, 400, 800 s1 afterwards, 0.05%
suggesting that casein micelles in both SMP solution and k-carrageenan, 10.5% SMP, (b) the aggregates were made at 400 s1 and
sheared at 400, 800, 1200 s1 afterwards, 0.05% k-carrageenan, 10.5%
casein/k-carrageenan aggregates completely lost their
SMP, (c) the aggregates were made at 800 s1 and sheared at 800, 1200 s1
micellar structure. afterwards, 0.05% k-carrageenan, 10.5% SMP.
Specific anions such as iodide ions are known to bind to
single k-carrageenan helical chains and prevent the
aggregation of the helices, hence prevent the gelation of
 ARTICLE IN PRESS
62 S. Ji et al. / Food Hydrocolloids 22 (2008) 56–64

12 12
mixed at 25°C 200 s-1
10 mixed at 60°C 10 400 s-1
800 s-1

8 8
Volume (%)

Volume (%)
6 6

4 4

2 2

0 0
0.1 1 10 100 1000 0.1 1 10 100 1000
Particle size (µm) Particle size (µm)

Fig. 6. Effects of initial mixing temperature of SMP solution (10.5%) and Fig. 7. Effects of NaI (11.6 mM) on the formation of casein/k-
k-carrageenan solution (0.025%) on the particle size distribution of casein/ carrageenan aggregates in reconstituted SMP cooled at different shear
k-carrageenan aggregates. SMP solution and k-carrageenan solution were rates (10.5% SMP, 0.025% k-carrageenan).
sheared at 300 rpm.

k-carrageenan, even though double helices are still formed
in the presence of NaI (Viebke, Borgström, & Piculell,
1995). It was shown that with NaI, casein/k-carrageenan
aggregates still can be formed. There was no significant
difference in the particle size distribution at low shear rates
compared to the system that did not contain NaI.
However, at high shear rate (800 s1), small particles
(0.1–1 mm) were not measured for the reconstituted SMP
containing k-carrageenan in the presence of NaI. These
mixes were quite different when compared to the same
system not containing NaI (Fig. 7).
4. Discussion
Preliminary experiments showed that with concentration
of k-carrageenan 40.025%, reconstituted SMP/k-carra-
geenan systems formed gels during cooling, when no shear
was applied. In addition, the gel strength increased with the
concentration of k-carrageenan. At 0.025% k-carrageenan,
when shear was applied during cooling, the gel structure
was disrupted and the samples had the appearance of a
homogenous liquid.
When the casein/k-carrageenan mixture was sheared at
800 s1, a bimodal distribution of particles was shown with
one population of particles from 0.1 to 1 mm and the other
from 5 to 40 mm. The small population of particles between
0.1 and 1 mm corresponds to the population of particles
measured in a SMP solution. This may suggest that after
shearing at 800 s1, there were more casein particles that
did not interact with k-carrageenan. At lower shear rates,
only large particles were measured, indicating that the
interaction between casein micelles and k-carrageenan may
be shear rate dependent. Even though casein micelles
can form some structures with k-carrageenan under the
above conditions, these structures are weak and can be
disrupted by high shear rate right after they are formed.
Moreover, cooling at high shear rates, casein micelles and
k-carrageenan dispersions were completely mixed and the
system was quite homogenous. As particle size decreased
with increasing shear rate, the number of particles
increased dramatically. With 0.025% k-carrageenan, there
were not enough k-carrageenan molecules to interact with
all of casein particles at high shear rate. However, if the
concentration of k-carrageenan was high enough, the
particle size distribution showed less change with shear,
indicating that the aggregation is concentration dependent.
With 0.05% k-carrageenan, it is possible to hypothesize
that most of the casein particles were involved in the
complex formation after shearing. All of the casein micelles
either interacted with k-carrageenan or were entrapped by
a k-carrageenan network. Our results also demonstrated
that the interactions between k-carrageenan and casein
micelles are not only dependent on the shear rate during
cooling but also on the concentration of k-carrageenan.
The viscosity of the SMP solution increased with increasing
concentration of k-carrageenan. It is possible to hypothe-
size that the molecular diffusion was slower as the
concentration of k-carrageenan increased. From a kinetic
point of view, the chance for casein micelles/k-carrageenan
interactions increased with concentration of polysacchar-
ide. At high concentrations, k-carrageenan itself could
form a strong polysaccharide network with casein micelles
physically entrapped. However, depending on the amount
of k-carrageenan, even though the k-carrageenan network
may form, it could be disrupted by the shear applied during
cooling.
The rheological studies indicate that at higher concen-
tration of k-carrageenan, especially at the concentration
above gelling concentration (0.03%, Schorsch et al., 2000),
some of the k-carrageenan molecules were not involved
in casein–k-carrageenan interactions but in k-carrageenan–
k-carrageenan interactions. A weak but flexible k-carrageenan
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S. Ji et al. / Food Hydrocolloids 22 (2008) 56–64
network was formed in the system and a microgel structure
could be formed upon shear. Therefore, the structure
of aggregates formed with 0.05% k-carrageenan could
be quite different from those formed with 0.025%
k-carrageenan. It can be hypothesized that for those
aggregates formed with 0.025% k-carrageenan, most of
k-carrageenan was associated with the casein micelles.
During rheological analysis these milk aggregates behaved
like a dilute solution. However, for those aggregates
formed with 0.05% k-carrageenan, k-carrageenann-
dash;k-carrageenan interactions may play a more impor-
tant role. Free casein particles and casein/k-carrageenan
aggregates may be entrapped within a three-dimensional
network of k-carrageenan. Therefore, the milk containing
0.05% k-carrageenan showed more gel-like behavior than
samples containing less polysaccharide. The high-frequency
dependence of G0 and G00 at low rate suggested that as the
concentration of k-carrageenan increased, more shear was
required to make the system homogenous and allow casein
micelles and k-carrageenan more effectively interact with
each other.
Rheological testing indicated that reconstituted SMP
containing 0.075% k-carrageenan showed gel-like char-
acteristics and higher G0 values than the other samples
(Fig. 2c). This suggested that at this concentration,
k-carrageenan–k-carrageenan interactions were predomi-
nant and aggregation may have resulted from carrageenan
gelation. The strength of gel (as indicated by the G0 values)
was also higher than those aggregates formed with 0.05%
k-carrageenan. However, the strength of casein/k-carrageenan
gel decreased with increasing shear rate, indicating that
some gel structures were disrupted at high shear rates. As
the strength of casein/k-carrageenan gel increased with
concentration of k-carrageenan, the gel structure became
more and more resistant to shear. Therefore, under the
same shear rate applied during cooling, larger particles
were measured in milk containing 0.075% k-carrageenan
compared to milk with lower k-carrageenan concentrations
(compare Figs. 1a, b and c). These results are in agreement
with Michon, Chapuis, Langendorff, Boulenguer, and
Cuvelier (2005) who also reported an increase in the size
of the aggregates with decreasing k-carrageenan/milk ratio.
Although microscopy observations of casein/k-carrageenan
aggregates showed that casein micelles were tightly
connected by k-carrageenan strands, this connection was
not strong enough to hold the micellar structure of casein
micelles if calcium ions were removed. The results from
EDTA test also suggested that the aggregates were indeed
formed by casein micelles rather than individual caseins
with k-carrageenan. Once casein micelles lost their micellar
structures, the aggregates were dissociated or were
sufficiently reduced in size to be no longer measured by
light scattering.
The stability test of casein/k-carrageenan aggregates
suggests that interactions between k-carrageenan and
casein micelles are relatively strong. Once these two
biopolymers aggregate together and reach equilibrium on
63
particle size by shear, it is difficult to separate them
mechanically. The size of casein/k-carrageenan aggregates
was primarily determined by the shear during cooling
rather than the shear applied after the aggregates were
formed. The shear applied during cooling is quite
important; it may decide the length of k-carrageenan
strands during its coil to helix transition. As shown in the
micrographs, casein micelles were tightly connected with
k-carrageenan strands. The size of casein/k-carrageenan
aggregates could be determined by the length of these
k-carrageenan strands. The longer the k-carrageenan
strands are, the more casein micelles are wrapped and the
bigger the particles will be. The association of casein
micelles and k-carrageenan become stronger when the
temperature goes below the coil to helix transition tempe-
rature and k-carrageenan chains are aggregated to form
helices. Therefore, the sizes of casein/k-carrageenan aggregates
will not be changed significantly after they are formed.
It was not surprising that casein/k-carrageenan aggre-
gates can be formed at 25 1C, which is below the coil to
helix transition temperature of k-carrageenan. Nilsson and
Piculell (1991) showed that the mean distance between
sulfate groups on k-carrageenan decreased from 1 nm (coil)
to 0.4 nm (helix) as temperature went below the coil to helix
transition temperature, which could cause the increase in
the charge density on k-carrageenan backbone as sulfate
groups come closer. Therefore, the strength of electrostatic
interactions between casein micelles and k-carrageenan
could be enhanced. However, compared to the aggre-
gates that were formed by mixing SMP solution and
k-carrageenan solution at 60 1C, the aggregates formed at
25 1C were larger and distributed in a wider range. We
believe that with the same concentration of SMP and
k-carrageenan, the aggregates formed through the different
processes should be the same type of aggregates, i.e., the
predominant interactions between these two biopolymers
are concentration dependent, particularly k-carrageenan
concentration. However, when these two biopolymers are
mixed at 60 1C, from a thermodynamic point of view, the
mixed system should be more homogenous. Therefore, the
particles formed were more uniform in size. When SMP
solution and k-carrageenan solution were mixed at 25 1C,
the system was more heterogeneous: there might be some
areas rich in carrageenan and some areas rich in casein
micelles. Although there was no visual phase separation,
phase separation still occurs between casein micelles and
k-carrageenan at a microscopic level. Thaiudom and Goff
(2003) showed that even with 0.05% k-carrageenan, phase
separation between casein micelles and k-carrageenan still
can be observed under TEM. Therefore, larger particles
could be formed in the areas that are rich in carrageenan
and smaller particles could be formed in those area rich in
casein micelles.
It was reported that by adding 11.6 mM NaI, aggrega-
tion of k-carrageenan helices was inhibited and there
were no gel-like properties in solution containing 0.1%
k-carrageenan. However, NaI did not block the interaction
 ARTICLE IN PRESS
64 S. Ji et al. / Food Hydrocolloids 22 (2008) 56–64
between casein micelles and k-carrageenan (Spagnuolo
et al., 2005). By adding NaI into k-carrageenan solution
and then mixing with SMP solution, the mechanism of
interactions between casein micelles and k-carrageenan was
further explored at low concentration of k-carrageenan
(0.025%) in this study. The results from our study
suggested that the aggregation of k-carrageenan helices
was not needed to form casein/k-carrageenan aggregates;
therefore, the formation of casein/k-carrageenan aggre-
gates did not rely on the network of k-carrageenan. It
was the direct interaction between casein micelles and
k-carrageenan that combine these two biopolymers to-
gether and form the aggregates. At high shear rate
(800 s1), since the aggregation of k-carrageenan helices
were blocked by NaI, there were more non-aggregated
k-carrageenan strands available for casein micelles in the
system, therefore, all of casein micelles were interacted with
k-carrageenan and there was no free casein micelles left.
5. Conclusions
Casein micelles and k-carrageenan formed aggregates in
skim milk under shear. The mechanism of how these two
biopolymers form aggregates is different depending on the
concentration of k-carrageenan: the aggregates could be
formed either through direct casein micelle–k-carrageenan
interaction or the gelation of k-carrageenan. Therefore, the
structures of casein/k-carrageenan aggregates will be
different at different concentration of k-carrageenan. The
particle size distribution of casein/k-carrageenan aggre-
gates was significantly affected by both shear rate and
concentration of k-carrageenan. The viscoelastic properties
of the aggregates were significantly affected by the
concentration of k-carrageenan but not shear. The case-
in/k-carrageenan aggregates were quite stable under shear.
The size of these particles was not changed significantly
if shear was applied after the aggregates were formed.
The initial mixing temperature of SMP solution and
k-carrageenan solution had significant effects on particle
size distribution. The particle size of casein/k-carrageenan
aggregates was easier to control when SMP solution and
k-carrageenan solution were mixed at high temperature.
Acknowledgments
The financial contribution from Natural Sciences and
Engineering Research Council of Canada and the assis-
tance of Dr. Smith (University of Guelph) for the
microscopy study are gratefully acknowledged. The
authors also would like to thank Dr. S. Turgeon
(Université Laval) for her collaboration and valuable
suggestions.
References
Allan-Wojtas, P., & Kalab, M. (1984). A simple procedure for the
preparation of stirred yoghurt for scanning electron microscopy. Food
Microstructure, 3, 197–198.
Dalgleish, D. G., & Morris, E. R. (1988). Interactions between
carrageenans and casein micelles: Electrophoretic and hydrodynamic
properties of the particles. Food Hydrocolloids, 2, 311–320.
de Vries, J. (2002). Interaction of carrageenan with other ingredients in
dairy dessert gels. In P. A. Williams, & G. O. Phillips (Eds.), Gums and
stabilizers for the food industry, Vol. 11 (pp. 200–210). London: Royal
Society of Chemistry.
Langendorff, V., Cuvelier, G., Michon, C., Launay, B., Parker, A., & De
Kruif, C. G. (1999). Casein micelles/iota–carrageenan interactions in
milk: Influence of temperature. Food Hydrocolloids, 13, 211–218.
Langendorff, V., Cuvelier, G., Michon, C., Launay, B., Parker, A., & De
Kruif, C. G. (2000). Effects of carrageenan type on the behaviour of
carrageenan/milk mixtures. Food Hydrocolloids, 14, 273–280.
MacArtain, P., Jacquier, J. C., & Dawson, K. A. (2003). Physical
characteristics of calcium induced k-carrageenan networks. Carbohy-
drate Polymers, 53, 395–400.
Michon, C., Chapuis, C., Langendorff, V., Boulenguer, P., & Cuvelier, G.
(2005). Structure evolution of carrageenan/milk gels: Effect of
shearing, carrageenan concentration and nu fraction on rheological
behavior. Food Hydrocolloids, 19, 541–547.
Nilsson, S., & Piculell, P. (1991). Helix-coil transitions of ionic
polysaccharides analysed within the Poisson–Boltzmann cell model.
4—Effects of site specific counterion binding. Macromolecules, 24,
3804–3811.
Ould Eleya, M. M., & Turgeon, S. L. (2000). Rheology of k-carrageenan
and b-lactoglobulin mixed gels. Food Hydrocolloids, 14, 29–40.
Schorsch, C., Jones, M. G., & Norton, I. T. (2000). Phase behavior of pure
micellar casein/k-carrageenan systems in milk salt ultrafiltrate. Food
Hydrocolloids, 14, 347–358.
Snoeren, T. H. M., Payens, T. A. J., Jeunink, J., & Both, P. (1975).
Electrostatic interaction between kappa-carrageenan and kappa-case-
in. Milchwissenschaft, 30, 393–395.
Spagnuolo, P. A., Dalgleish, D. G., Goff, H. D., & Morris, E. R. (2005).
Kappa-carrageenan interactions in systems containing casein micelles
and polysaccharide stabilizers. Food Hydrocolloids, 19, 371–377.
Thaiudom, S., & Goff, H. D. (2003). Effect of k-carrageenan on milk
protein polysaccharide mixtures. International Dairy Journal, 13,
763–771.
Thomsen, J. K., Jakobsen, H. J., Nielsen, N. C., Petersen, T. E., &
Rasmussen, L. K. (1995). Solid state magic-angle spinning 31P-NMR
studies of native casein micelles. European Journal of Biochemistry,
230, 454–459.
Turgeon, S. L., & Beaulieu, M. (2001). Improvement and modification of
whey protein gel texture using polysaccharides. Food Hydrocolloids,
15, 583–591.
Tziboula, A., & Horne, D. S. (1999). Influence of whey protein
denaturation on k-carrageenan gelation. Colloids and Surfaces B:
Biointerfaces, 12, 299–308.
Vega, C., Dalgleish, D. G., & Goff, H. D. (2005). Effect of k-carrageenan
addition to dairy emulsions containing sodium caseinate and locust
bean gum. Food Hydrocolloids, 19, 187–195.
Viebke, C., Borgström, J., & Piculell, L. (1995). Characterization of k- and
t-carrageenan coils and helices by MALLS/GPC. Carbohydrate
Polymers, 27, 145–154.
Xu, S. Y., Stanley, D. W., Goff, H. D., Davidson, V. J., & Le Maguer, M.
(1992). Hydrocolloid/milk gel formation and properties. Journal of
Food Science, 57(1), 96–102.