Food Hydrocolloids 23 (2009) 1420–1426

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

Influence of molecular weight and degree of substitution of

carboxymethylcellulose on the stability of acidified milk drinks
Baiqiao Du a, Jing Li a, Hongbin Zhang a, *, Long Huang b, Ping Chen b, Jianjun Zhou b
a
Advanced Rheology Institute, Department of Polymer Science and Engineering, School of Chemistry and Chemical Technology, Shanghai Jiao Tong University,
Shanghai 200240, China
b
Danisco-China Co., Ltd., Kunshan 215300, China

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

Article history: The influence of molecular weight (Mw, 250,000, 700,000) and degree of substitution (DS, 0.7, 0.9 and
Received 17 December 2007 1.2) of carboxymethylcellulose (CMC) on the diameter and z-potential of casein micelles during acidi-
Accepted 13 October 2008 fication in diluted dispersions and on the stability of acidified milk drinks was investigated. The
experimental results suggested that CMC with high Mw or low DS would result in thick adsorbed layer
Keywords: onto casein micelles. The z-potential of CMC-coated casein micelle increased with increasing the Mw of
Acidified milk drinks
CMC with the same DS while at a fixed Mw the z-potential for CMC with high DS (1.2) increased in
Carboxymethylcellulose
comparison with those for CMC with low DS (0.7 and 0.9). Both Mw and DS of CMC influenced the
Molecular parameter
Casein micelle stability of acidified milk drinks. CMC with high Mw increased the viscosity of acidified milk drinks
Stability significantly and therefore contributed to the stability. CMC with high DS resulted in high z-potential of
CMC-coated casein micelles, increasing the electrostatic repulsion between casein particles, which
prevented the phase separation in acidified milk drinks. It was also found that the amount of CMC
needed for efficient coverage of casein micelles increased with increasing the Mw of CMC. Above the
efficient coverage concentration, the long-term stability of acidified milk drinks with high Mw CMC was
better than that with low Mw CMC.
Ó 2008 Elsevier Ltd. All rights reserved.

1. Introduction
Acidified milk drinks can be described as an acidified protein
liquid system with stability and viscosity similar to natural milk.
Such drinks usually comprise a large range of products, from those
usually prepared from fermented milk with stabilizers and sugar to
those prepared by direct acidification with fruit juices and/or acids.
The pH of these products ranges from 3.6 to 4.6 (Nakamura,
Yoshida, Maeda, & Corredig, 2006). At neutral pH, caseins exist in
the form of micelles, which are stabilized by steric repulsion due to
the extended conformation of k-casein present mainly on the
surface of micelles (de Kruif, 1998; Tuinier & de Kruif, 2002). During
acidification, at a pH close to the isoelectric point (pH 4.6) the
casein micelles aggregate mainly because of the collapse of the
extended conformation of k-caseins (Holt, 1982). On account of
the instability of casein in the abovementioned pH range, stabilizer
needs to be added to avoid the flocculation of milk proteins and
subsequent macroscopic whey separation. High methoxyl pectin
(Boulenguer & Laurent, 2003; Liu, Nakamura, & Corredig, 2006;
Parker, Boulenguer, & Kravtchenko, 1997) and soybean soluble
polysaccharides (SSPS) (Asai et al., 1994; Nakamura, Furuta, Kato,
* Corresponding author. Fax: þ86 21 54741297.
E-mail address: hbzhang@sjtu.edu.cn (H. Zhang).
Maeda, & Nagamatsu, 2003; Nakamura et al., 2006) are often used
to achieve this, and much attention has been paid to pectin. In
addition, propylene glycol alginate (PGA) and carboxymethylcel-
lulose (CMC) are also mentioned to be able to use as stabilizers
(Keiichi, 2006; Koji, Takeshi, Norihiro, & Hideaki, 2004; Mann,
2004; Masaki, Yoshitaka, Yuka, Yasuyuki, & Tatsuyuki, 2004; Mur-
ray, 2000; Nishiyama, 1978; Ogasawara, Akahoshi, Hashimoto,
Yamashita, & Yamamota, 2003; Syrbe, Bauer, & Klostermeyer, 1998;
Young & Bluestein, 2002).
As one of the most important derivatives of cellulose, CMC is
a typical anionic polysaccharide and has been widely used as
a stabilizer in food. CMC chains are linear b(1 / 4)-linked gluco-
pyranose residues. The average degree of substitution (DS) of CMC
is defined as the average number of carboxymethyl groups per
repeating unit and is usually in the range 0.4–1.5. CMC is generally
found under sodium salt form, a water-soluble product for DS > 0.5.
A maximum degree of substitution of 1.5 is permitted, but more
typically DS is in the range 0.6–0.95 for food applications (Coffey,
Bell, & Henderson, 2006; Murray, 2000).
CMC is commonly chosen as a stabilizing agent for its low cost in
acidified milk drinks instead of pectin in Asia, especially in China
(Chen, Zheng, Chen, & Rao, 1996). The application and the stabili-
zation mechanism of pectin and SSPS in acidified milk drinks have
been extensively studied in recent years (Liu et al., 2006, Nakamura

0268-005X/$ – see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.foodhyd.2008.10.004
 B. Du et al. / Food Hydrocolloids 23 (2009) 1420–1426
et al., 2006). However, the stabilizing effects of CMC on this kind of
drinks are less reported. The stability of casein micelles at low pH
could be improved by CMC. In a previous work (Du et al., 2007), we
found that electrosorption of CMC onto casein micelles took place
below pH 5.2 and the adsorbed CMC layer on the surface of casein
could prevent flocculation of casein micelles by steric repulsion. In
addition, the non-adsorbed CMC increased the viscosity of serum
and slowed down the sedimentation of casein particles. The
adsorbed CMC layer caused a repulsive interaction between the
casein micelles at low pH in the same way as k-caseins do at neutral
pH. This phenomenon is related to the interaction between protein
(mainly casein micelles) and CMC.
The stability of acidified milk drinks depends largely on the inter-
actions between casein and polysaccharides, which can be influenced
by the concentrations of protein and polysaccharides (Tromp, de Kruif,
van Eijk, & Rolin, 2004; Tuinier, Rolin, & de Kruif, 2002), pH (Nakamura
et al., 2003), molecular properties of polysaccharides (Laurent & Bou-
lenguer, 2003; Maroziene & de Kruif, 2000; Pereyra, Schmidt, & Wicker,
1997), ionic environment (Ambjerg Pedersen & Jorgensen, 1991), milk
protein composition and processing (Boulenguer & Laurent, 2003;
Glahn, 1982; Sedlmeyer, Brack, Rademacher, & Kulozik, 2004;), and
thermal history of the sample (Horne, 1998; Lucey, Tamehana, Singh, &
Munro, 1999; Zaleska, Ring, & Tomasik, 2000) etc.
Although the interactions between casein micelles and CMC and
the stability of the acidified milk drinks might be primarily
dependent on pH and concentration of CMC as previously reported
(Du et al., 2007), the molecular weight and substitution pattern of
carboxymethyl groups on CMC should be emphasized because in
the practical processing of acidified milk drinks the properties
including stability of the drinks can be obtained by the adjustment
on the molecular parameters of CMC. In the present work, we aim
to investigate the influence of Mw and DS of CMC on the interaction
between CMC and casein micelles and thus on the stability of
acidified milk drinks.
2. Materials and methods
2.1. Materials
A series of CMC with different Mw (250,000 Da and 700,000 Da)
and different DS (0.7, 0.9 and 1.2) were purchased from the Acros
organics (Morris Plains, New Jersey). Skim milk powders were
obtained from Fonterra Co-operative Group (Wellington, New
Zealand). Citric acid monohydrate was obtained from Shanghai
Chemical Reagent Co., Ltd. (Shanghai, China).
2.2. Preparation of samples for dynamic light scattering (DLS) and
z-potential experiments
The sample was made by dispersing 80 g/kg reconstituted skim
milk in simulated milk ultra filtrate (SMUF) (Jenness & Koops, 1962)
(1:100). SMUF contains Na, K, Ca, Mg, phosphate and citrate and
was used to dilute mixtures in an environment which would
simulate the salt system in milk. Then 5 g/kg CMC was added to the
diluted reconstituted skim milk at about neutral pH (6.6–6.7) to
obtain 800 mg/kg skim milk powders containing 400 mg/kg CMC.
All solutions in this measurement were prepared with ultra-pure
water with 18.2 MU/cm (Millipore, Bedford, MA. USA), and filtered
through 0.22 mm membrane filters prior to use. The apparent
diameter and z-potential of casein micelles were monitored during
acidifying the diluted reconstituted skim milk with citric acid.
2.3. Dynamic light scattering measurement (DLS)
Dynamic light scattering measurements were carried out with
a Malvern Zetasizer 3000HSA (Malvern Instruments, Worcestershire,
1421
UK) equipped with a 10 W max output He–Ne laser and at a l of
633 nm. Measurement occurred at 90 from the incident beam and
gave an estimation of the particle mean diameter distribution in
intensity. The temperature of the samples was controlled by a Joule–
Peltier thermostat at 20  C. The stated results of each measurement
were the average of 10 measurements. And all measurements were
performed three times. The biggest variance of the measurement was
below 10%.
2.4. z-Potential measurement
The z-potential of casein micelles as a function of pH was
determined using a particle electrophoresis instrument (Zetasizer
Nano ZS, Malvern Instruments, Worcestershire, UK) which
measures the direction and velocity of droplet movement in
applied electric field at 20  C. The z-potential provides an estimate
of the net charge of a particle measured at the ‘shear plane’, which
depends on the charge on the actual particle (in this case casein
micelles and polysaccharides) plus the charge associated with any
ions that move along with the particle in the electric field. The
sample was similar to those for DLS measurements. An individual z-
potential was determined from the average of three readings taken
on the same sample. All measurements were performed three
times.
2.5. Preparation of acidified milk drinks
The 80 g/kg reconstituted skim milk was prepared by mixing
skim milk powders and distilled water at 45  C for 30 min. Mean-
while, CMC and sucrose were dry mixed together and then the
mixture was dissolved in distilled water at 75  C by stirring for
20 min. Stabilizer and reconstituted skim milk were mixed at a 1:1
ratio to obtain 40 g/kg skim milk powders containing 4 g/kg CMC
and 80 g/kg sucrose. The pH of this mixture was directly acidified to
4.0 with 500 g/kg citric acid at 20  C. The acidified milk drinks were
preheated to 65  C and then homogenized at 200 bar with a two-
stage value homogeniser Rannie TYPE 8.30 H (APV Rannie A/S,
Denmark). All samples were stored in sealed glass bottles. The
bottle of each sample (500 ml) was heated in a water bath at 90  C
for 30 min. Sodium azide (0.02%) was added to prevent bacterial
growth. Measurements were performed after the samples were
stored at room temperature overnight. All measurements were
performed three times.
2.6. Measurement of apparent viscosity for the acidified milk drinks
The apparent viscosity of acidified milk drinks was measured by
a Brookfield Viscometer LVDV-I, Model NDJ-79 with No. 1 rotor, at
100 rpm/min, and 25 8C.
2.7. Measurement of particle size for the acidified milk drinks
The acidified milk drinks were diluted to a measurable
concentration (100–200 times) with distilled water. Then the
particle size was measured with a Laser Diffraction Particle Size
Analyser, Malvern MasterSizer 2000 (Malvern Instruments Ltd.,
Worcestershire, UK).
2.8. Measurement of storage stability for the acidified milk drinks
Sedimentation and serum phase occurred during storage were
observed with the optical analyser TURBISCAN MA 2000 (Ramon-
ville-St-Agne, France). The cylindrical glass tubes containing 5 ml of
sample were stored at 25  C. Sedimentation and serum phase
fraction as a function of time were followed after the sample was
prepared for 30 days.
1422 B. Du et al. / Food Hydrocolloids 23 (2009) 1420–1426
2.9. Phase separation
The mixtures of 40 g/kg skim milk and CMC with different
concentrations (0–5 g/kg) were acidified to pH values 4.7  0.1 and
3.8  0.1, respectively, while without acidification the pH of the
mixture was ca. 6.7  0.1. 10 ml sample was placed in a graduated
glass tube with lid at room temperature of 25  C. The stability of the
mixture was obtained from visual observation 3 days after prepa-
ration. The sample was regarded as stable if the system was
homogeneous whereas the sample was unstable if obvious phase
separation took place.
3. Results and discussion
3.1. Influence of Mw and DS of CMC on the diameter of casein
micelles during acidification
The particle size evolution of casein micelles in the system
prepared by diluting reconstituted skim milk in SMUF (1:100) was
followed by DLS. The ratio of 800 mg/kg reconstituted skim milk
and 400 mg/kg CMC was selected based on our previous work (Du
et al., 2007), at which CMC could sufficiently form thick adsorbed
layer on casein micelles and maintained their stability. Fig. 1 illus-
trated the effect of Mw of CMC (DS ¼ 0.9) on the particle diameter of
casein micelles during acidification with citric acid. For recon-
stituted skim milk sample without CMC, the diameter was close to
230  10 nm, which was consistent with our previous result
(Du et al., 2007) and was also in agreement with earlier DLS results
regarding the size of casein micelles (de Kruif, 1992, Tuinier et al.,
2002). Upon decreasing the pH from 5.8 to 5.0 the casein micelle
diameter decreased due to a fraction of a- and b-casein coming out
of casein micelles (Tuinier et al., 2002). Around pH 5.0, the apparent
casein micelles size started to increase and exceeded 1 mm at pH 4.7
because of the formation of aggregates. The suspension became
thus unstable and macroscopic flocs were observed in a few
minutes. This observation was the same as those of Du et al. (2007)
and Tuinier et al. (2002).
When CMC was added in diluted reconstituted skim milk,
a small increase in the particle diameter was found, as compared
with the bare casein micelle size at neutral pH. A similar
phenomenon was also observed in the size evolution during acid-
ification of pectin/casein mixtures (Tuinier et al., 2002). The
increase in size was interpreted based on the Stokes–Einstein
relation as follows.
1000
skim milk
950
Mw 250000
900
Mw 700000
Particle diameter/nm
500
400
300
200
100
0 420  15 nm and 380  15 nm for DS 0.7, 0.9, and 1.2, respectively).
7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5
pH
Fig. 1. Influence of Mw of CMC (DS ¼ 0.9) on the diameter of casein micelles during
acidification with citric acid. Skim milk concentration is 800 mg/kg and CMC
concentration is 400 mg/kg.
D ¼ kT=3phm deff (1)
eff
where hm was the medium viscosity, d was the effective hydro-
dynamic diameter. In diluted dispersions, the self-diffusion coeffi-
cient D was related to the effective hydrodynamic diameter of the
sphere, deff according to equation (1). The viscosity of SMUF was
taken as the medium viscosity hm. However, upon the addition of
CMC, the actual medium viscosity will increase. It was thus
supposed that the increase in the particle diameter was traceable
from the calculation method of DLS (Tuinier et al., 2002). Alterna-
tively, Nakamura et al. (2006) also found that the initial diameter of
skim milk increased with increasing pectin concentration at neutral
pH. Taking into account that the DLS measurements were carried
out after the samples were extensively diluted in the corresponding
ultra filtration permeate and assuming that the measurement of
particle diameter was not affected by the viscosity of the pectin,
they attributed this increase in size to an association of pectin to
casein micelles at neutral pH, possibly because of the existence of
calcium ions. In our case, the increase in diameter of casein micelles
upon addition of CMC at neutral pH needed further clarification.
The addition of CMC had no effect on the decrease of the casein
micelles size between pH 5.8 and 5.2, which indicated that the
addition of CMC did not influence the coming out of a fraction of a-
and b-casein from casein micelles. Upon decreasing the pH close to
5.2, it was found that the diameter of casein micelle in the mixture
with CMC increased in comparison with that in the absence of CMC.
This was caused by the adsorption of CMC onto casein micelles,
which led to effectively larger casein micelles. The negative charge
might be statistically distributed along the CMC chains, yielding
a conformation with many loops like CMC chains on amino-
terminated surface (Fujimoto & Petri, 2001) and pectin on casein
surface (Tromp et al., 2004). For adsorbed CMC, these loops
extended into the solution and caused a repulsive interaction
between the casein micelles at low pH in the same way as k-casein
chains did at neutral pH. The adsorption of CMC onto casein
micelles took place just before casein micelles aggregation started
in the absence of CMC.
In the present work, the influences of Mw and DS of CMC on the
diameter of casein micelles were investigated upon addition of
enough CMC (400 mg/kg) and when the diameter leveled off in the
low pH range 4.3–3.0. The casein micelles diameter (500  25 nm)
for high Mw CMC (Mw ¼ 700,000) was larger than that
(420  25 nm) for low Mw CMC (Mw ¼ 250,000). These results
suggested that the thickness of adsorbed layer should be related to
the Mw of CMC. CMC with high Mw would result in thick adsorbed
layer onto casein micelles. When CMC was adsorbed to casein
micelles, the segment of the chain between two charged points
protruding from the surface of the casein micelle would be longer
for high Mw CMC than that for low Mw CMC, resulting in a bigger
loop conformation. Similar phenomenon was observed in other
system such as the emulsion stabilized by SSPS (Nakamura, Taka-
hashi, Yoshida, Maeda, & Corredig, 2004), in which the layer
thickness of SSPS on the surface of the oil droplets increased with
increasing the molecular mass of SPSS containing high protein
content.
Fig. 2 showed the influence of DS (0.7, 0.9 and 1.2) of CMC on the
particle diameter of casein micelles for the same concentration
(400 mg/kg) and Mw of CMC (250,000) during acidification with
citric acid. The apparent diameter of the particles also leveled off to
a maximum in low pH range (pH 4.3–3.0) (450  15 nm,
3.0
The results indicated that the higher the charge of CMC, the smaller
the particle size in low pH range. This phenomenon was probably
attributed to two aspects: firstly, CMC with high DS afforded more
negative charges, which could adsorb strongly onto casein micelles,
while the uncharged stretches in charged blocks formed small
 B. Du et al. / Food Hydrocolloids 23 (2009) 1420–1426 1423

1000 20
skim milk skim milk
950 15
DS 0.7 DS 0.7
900 DS 0.9 10 DS 0.9
DS 1.2
Particle diameter/nm

5 DS 1.2

-potential/mV
400
0

-5
300
-10
200 -15

-20
100
-25

0 -30
7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0
pH pH

Fig. 2. Influence of DS of CMC (Mw ¼ 250,000) on the diameter of casein micelles Fig. 4. Influence of DS of CMC (Mw ¼ 250,000) on the z-potential of casein micelles
during acidification with citric acid. CMC concentration is 400 mg/kg. Skim milk during acidification with citric acid. CMC concentration is 400 mg/kg. Skim milk
concentration is 800 mg/kg and CMC concentration is 400 mg/kg. concentration is 800 mg/kg and CMC concentration is 400 mg/kg.

loops; and secondly, the already adsorbed chains might hinder the
adsorption of the other arriving chains due to the electrostatic
repulsion. CMC interacted with casein micelles via electrostatic
interactions, and this interaction was pH-dependent. The charge of
CMC molecule was fundamental in driving the adsorption of the
casein micelles at low pH; however, the size of CMC chain affected
the extent of the steric repulsion between particles.
3.2. Influences of Mw and DS of CMC on the z-potential of casein
micelles during acidification
The influences of Mw and DS of CMC on the z-potential of casein
micelles as a function of pH were shown in Figs. 3 and 4, respec-
tively. The relationship between the z-potential of casein micelles
and pH was complex because of the composition of casein micelles
and the SMUF dilution used (Anema & Klostermeyer, 1996; Schmidt
& Poll, 1986; Shaw, 1980). In this SMUF medium, the change from
negative to positive z-potential was observed at pH 4.7. Fox and
McSweeney (1998) reported that the casein in milk would
precipitate out of solution on acidification to pH 4.6 due to a zero
overall charge at this pH. In contrast to casein micelles without
20
skim milk
15
Mw 250,000
10
Mw 700,000
5 work (Du et al., 2007), the evolution of the z-potential of casein
-potential/mV
CMC, those in the presence of CMC were more negative after
acidification, which was due to the adsorption of CMC onto casein
micelles between pH 5.4 and 4.8. Similar phenomenon was previ-
ously observed with pectin (Sejersen et al., 2007; Shaw, 1980; Surh,
Decker, & McClements, 2006). Below pH 4.8, the z-potential
became less negative with lowering pH. This phenomenon might
be explained by the fact that the carboxylate groups on the
adsorbed CMC layer were protonated in lower pH range (Thakur,
Singh, & Handa, 1997).
The addition of CMC to casein micelles caused some increase in
the absolute magnitude of z-potential of casein micelles. And the
high Mw CMC (Mw ¼ 700,000) seemed to result in a bigger increase
in z-potential of CMC-coated casein micelles above pH 3.7 than low
Mw CMC (Mw ¼ 250,000) (Fig. 3). Below pH 3.7, the effect of Mw of
CMC on the z-potential was not perceptible.
As seen in Fig. 4, the z-potential of CMC-coated casein micelles
did not change so much when the DS of CMC increased from 0.7 to
0.9. Further increasing the DS to 1.2 gave rise to an increase of ca.
3 mV in z-potential. As for the effect of high and low degree of
methylesterification (DE) of pectin on the z-potential of sodium
caseinate emulsions, it has been found that the z-potential was
independent of DE of pectin if its charge density is not too low (Surh
et al., 2006). This phenomenon is attributed to the fact that once the
surface charge has reached a certain value there will be a strong
electrostatic repulsion between the surface and similarly charged
polyelectrolyte in the aqueous solution, which limits further
adsorption of the polyelectrolyte (Schonhoff, 2003). In our previous
micelles during acidification without CMC and with different

0 concentration of CMC was discussed. It was found that the evolu-

-5 tion of the z-potential of casein micelles was independent of CMC
concentration above full coverage in low pH range 4.3–3. This result
-10
also confirmed that the adsorption of CMC onto casein micelles was
-15 not only induced but also controlled by the electrostatic force. CMC
with high DS resulted in a high z-potential of CMC-coated casein
-20
micelles, indicating an increase in the electrostatic repulsion
-25 between casein particles.
-30
7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0
3.3. Influence of Mw and DS of CMC on the stability of acidified
pH milk drinks
Fig. 3. Influence of Mw of CMC (DS ¼ 0.9) on the z-potential of casein micelles during
acidification with citric acid. CMC concentration is 400 mg/kg. Skim milk concentration The stability of acidified milk drinks was expressed as the
is 800 mg/kg and CMC concentration is 400 mg/kg. percentages of the sedimentation and serum which were measured
1424 B. Du et al. / Food Hydrocolloids 23 (2009) 1420–1426

by turbimetry after the sample was prepared for 30 days. The
results in Table 1 showed that there was an obvious phase sepa-
ration in reconstituted skim milk without addition of CMC. Proteins
were completely flocculated and settled down at the bottom
(sedimentation fraction was 16.8%). The large fraction (83.2%) was
the clear serum phase. The addition of CMC increased the stability
of reconstituted skim milk at low pH with both a less serum fraction
and a sedimentation fraction (Table 1).
When compared the effect of Mw of CMC on stability of acidified
milk drinks, it was found that CMC with higher Mw resulted in
a more stable acidified milk drink system than CMC with lower Mw.
Previous work indicated that the stability of acidified milk drinks
increased with increasing the concentration of CMC (Du et al.,
2007). The better stability of the sample with high Mw (700,000)
could be explained by that the stability of acidified milk drinks was
related not only to the stability of casein micelles themselves, but
also to the serum viscosity. When CMC concentration was sufficient
to cover the casein micelles efficiently, the redundantly non-
adsorbed CMC increased the viscosity of acidified milk drinks and
thus contributed to the stability of acidified milk drinks. The
particle size and apparent viscosity of casein in acidified milk
drinks stabilized by CMC with different Mw and DS were also
summarized in Table 1. The apparent viscosity (35.9  0.1 cp) of
acidified milk drinks stabilized by CMC with high Mw (700,000)
was much higher than that with low Mw (250,000) (24.8  0.2 cp),
thus consequently contributed to the stability of acidified milk
drinks. There was no population of aggregates in all samples
because the CMC was sufficient to completely cover the casein
micelles. The value of the particle size of casein in acidified milk
drinks containing CMC with Mw of 700,000 and DS of 0.9,
0.68  0.12 mm, was slightly larger than that (0.60  0.22 mm) of the
sample containing CMC with Mw of 250,000 and the same DS;
however, the sample containing high Mw CMC was more stable
3.4. Influence of Mw on phase behavior of reconstituted skim milk/
CMC mixture
Since Mw of CMC played an important role on the stability of
acidified milk drinks, the phase behavior of reconstituted skim
milk/CMC mixtures was further investigated. Fig. 5 represented the
stability diagram for the mixtures, obtained from visual observation
3 days after preparation. The closed circles referred to stable states
and the open circles referred to unstable. At pH 6.7, the mixtures
(250,000 Mw CMC) were stable at low CMC concentration;
however, higher CMC concentration (>1 g/kg) led to the loss of
stability (Fig. 5a). Phase separation of mixtures with higher Mw
CMC (700,000) occurred at lower CMC concentration (>0.6 g/kg,
Fig. 5b). Our previous work (Du et al., 2007) showed that the
interaction between casein micelles and CMC was closely related to
pH. At pH 5.2–6.8, both casein and CMC are negatively charged and
thus repel each other. We have previously shown that CMC started
to adsorb on casein at pH 5.2; therefore, there was no CMC
adsorption above pH 5.2. At these pH values, non-adsorbing CMC
could lead to phase separation by depletion flocculation. The
similar phenomenon was also found in other non-adsorbing poly-
saccharides in the system mixed with casein micelles, such as
pectin above a certain pH (Maroziene & de Kruif, 2000; Tuinier
a 8
7
phase separation
6
pH

(less serum fraction and sedimentation fraction). This suggested

that the higher viscosity endued by the margin of the non-adsorbed 5
high Mw CMC should contribute to the higher stability of the
system. bridging
The influence of DS of CMC on the stability of acidified milk 4
drinks could also be seen in Table 1. The addition of CMC with DS
0.7 and DS 0.9 had no obvious difference on the stability of acidified
milk drinks. The serum fraction (ca. 40%) of acidified milk drinks 3
with DS 0.9 was a little higher than that (ca. 36%) with DS 0.7, and 0 1 2 3 4 5
the sedimentations (ca. 14%) were similar for both samples. There CMC concentration g/kg
was no large aggregation of particles in the system, in which the
particle size was below 1 mm with different DS of CMC. The b 8
apparent viscosity of acidified milk drinks containing CMC
(Mw ¼ 250,000) with various DS was similar. The reason was that
the rheological behavior of an aqueous CMC solution was mainly 7
influenced by Mw of CMC and its concentration (Kulicke, Kull, Kull,
& Thielking, 1996). The stability of acidified milk drinks induced by phase separation
CMC with DS 1.2 was better than that with DS 0.7 and 0.9. This 6
improved stability might be related to the increase electrorepulsion
pH

which generated by the high z-potential of casein micelles with

high DS of CMC, as indicated in Fig. 4. 5

bridging
4
Table 1
Influence of Mw and DS of CMC on the apparent viscosity, particle size and stability of
acidified milk drinks (40 g/kg MSNF, 4 g/kg CMC, and 80 g/kg sucrose, pH 4.0).
3
CMC sample added Apparent Particle size Serum Sedimentation
viscosity/cp D[4,3]/mm fraction/% fraction/% 0 1 2 3 4 5

Without CMC – >10 83.2  0.1 16.8  1.0 CMC concentration g/kg
Mw 250,000; DS 0.7 25.7  0.2 0.87  0.30 36.4  2.2 14.2  2.8
Fig. 5. Influence of CMC Mw (DS ¼ 0.9), CMC concentration, and pH on the phase
Mw 250,000; DS 0.9 24.8  0.2 0.60  0.22 40.8  2.9 14.6  2.1
separation of mixtures of reconstituted skim milk (40 g/kg MSNF) and CMC. (a)
Mw 250,000; DS 1.2 25.5  0.1 0.38  0.20 11.9  2.5 10.3  2.4
Mw ¼ 250,000; (b) Mw ¼ 700,000. Open circle indicated unstable system, and closed
Mw 700,000; DS 0.9 35.9  0.1 0.68  0.12 6.8  1.0 5.7  1.5
circle indicated stable system.
 B. Du et al. / Food Hydrocolloids 23 (2009) 1420–1426
et al., 2002) and guar gum at neutral condition (Tuinier, ten Gro-
tenhuis, & de Kruif, 2000). In Fig. 5, CMC concentration at the phase
boundary decreased with increasing CMC Mw. This was consistent
with the Vrij’s theory that could be used to calculate the phase
boundary from the size of the casein micelles and the size and
molar mass of the non-adsorbed polysaccharides (Mao, Cates, &
Lekkerkerker, 1995; Tuinier, ten Grotenhuis, & de Kruif, 2000; Vrij,
1976), i.e., increasing the chain length of the polysaccharides
causing the phase boundary to move to lower polysaccharide
concentrations.
Below pH 5.2, at pH 4.7 and 3.8, CMC could adsorb onto casein
micelles. Here, pH 4.7 and 3.8 were selected because pH 4.7 was
near the pI of casein micelle and pH 3.8 was in the range of acidified
milk drinks. In these cases, the state diagrams were quite different
from that at the pH at which the adsorption of CMC could not take
place. In contrast to the mixtures at pH 6.7, the system at low pH
was phase separated. When CMC adsorbs onto casein micelles it
could cause either bridging flocculation or steric stabilization
depending on its concentration. Bridging flocculation occurred at
a CMC concentration that was not enough for full coverage of the
casein micelles. A single polymer chain may adsorb onto two or
more particles with low polymer concentration, thereby connect-
ing the particles. The system may thus separate into one phase
concentrated in complexes and the other phase in solvent (Mar-
oziene & de Kruif, 2000; Syrbe et al., 1998; Tuinier et al., 2002).
More CMC was required for stabilization at pH 3.8, which was
attributed to the increased positive charges on casein micelles. The
more positively charged casein micelles would adsorb more CMC to
form thick layer to maintain the stability of casein micelles.
Therefore CMC concentration at which the system was stable
shifted to higher CMC concentration at lower pH.
In the case of pH 3.8, for Mw ¼ 700,000 the boundary concen-
tration of CMC, 3.4 g/kg, required to stable the acidified milk drinks,
was higher than that for Mw ¼ 250,000, i.e., 2.6 g/kg. It was
supposed that when the amount of CMC with high and low Mw was
the same, CMC with high Mw would have a smaller chain number
than that with low Mw. Thus, in order to obtain the stability for the
same amount of acidified milk drinks, relatively more molecular
chains of CMC with high Mw (i.e., higher concentration) were
required than that with low Mw, to cover the protein particles
efficiently. However, it should be noted that beyond this boundary
concentration, the long-term stability of the acidified milk drinks
with high Mw CMC was better than that with low Mw as described
in Table 1.
4. Conclusions
Both Mw and DS of CMC influenced the interaction between
CMC and casein micelles and thus the stability of acidified milk
drinks. At pH 6.7, there was no interaction between caseins and
CMC due to charge repulsion and mixtures of casein and CMC were
stable at low CMC concentrations. Above a certain CMC concen-
tration, depletion flocculation occurred leading to phase separa-
tion. Below pH 5.2 CMC adsorbed onto casein micelles. In the case
of low CMC concentrations, CMC/casein micelles mixture was
phase separated via bridging flocculation. With increasing CMC
concentrations, the casein micelles were effectively coated and
consequently sterically stabilized. The amount of CMC needed for
effective coverage onto casein micelles increased with increasing
the Mw of CMC. It was found that the high Mw CMC or low DS CMC
led to a thick adsorbed layer onto casein micelles. However, this
thick layer was not directly related to the long-term stability of
acidified milk drinks because the stability of acidified milk drinks
was also related to the z-potential of casein micelles, the particle
size of casein micelles, and the viscosity of the system. Acidified
milk drinks with high Mw CMC showed good stability due to both
1425
the thick adsorbed layer and the high viscosity of the system. The
absolute value of z-potential of CMC-coated casein micelles
increased when the DS of CMC was high enough to 1.2 compared
with CMC with DS of 0.7 and 0.9. The high z-potential contributed
to the stability of the system by electrostatic repulsion. It was
supposed that both steric repulsion of the absorbed layer and
electrostatic repulsion influenced the stability of acidified milk
drinks.
Acknowledgements
H. Zhang thanks for the support from Danisco-China Co., Ltd.
and Shanghai Leading Academic Discipline Project (No. B202).
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