Inuence of homogenisation and the degradation of stabilizer on the

stability of acidied milk drinks stabilized by carboxymethylcellulose

Juan Wu
a
, Baiqiao Du
b
, Jing Li
c
, Hongbin Zhang
a,
*
a
Advanced Rheology Institute, Department of Polymer Science and Engineering, School of Chemistry and Chemical Technology, Shanghai Jiao Tong
University, Shanghai 200240, China
b
SGS-CSTC Standards Technical Services Co., Ltd., Shanghai 200233, China
c
Bright Dairy & Food Co., Ltd., Shanghai 201103, China
a r t i c l e i n f o
Article history:
Received 4 June 2013
Received in revised form
10 December 2013
Accepted 17 December 2013
Keywords:
Carboxymethylcellulose (CMC)
Homogenization
CMC degradation
Acidied milk drinks
Stability
a b s t r a c t
The present work deals with the inuences of both homogenisation and the degradation of carboxy-
methylcellulose (CMC) on the stability of two kinds of acidied milk drinks (AMDs), directly acidied
milk drinks and yoghurt drinks. The effect of homogenisation pressure for direct acidication process
was investigated and evaluated. The experimental results showed that homogenisation was required to
achieve a signicantly small particle size (0.7 mm in the present work) and to prevent sedimentation and
serum separation. However, homogenisation at too high pressures was not benecial for the stability of
the colloidal systems. The occurrence of degradation of CMC during homogenisation weakened the
stabilisation effect of CMC. A qualied homogenisation pressure of 20 MPa should be chosen to achieve a
good stability when a usually practical pressure range of 0e30 MPa was applied. In addition, the stability
of directly acidied milk and yoghurt drinks prepared under the same homogenisation pressure was also
investigated. While their stability increased with increasing CMC concentration, the degradation of CMC
at low pH during storage gave rise to instability of the nal products.
2013 Elsevier Ltd. All rights reserved.
1. Introduction
Acidied milk drinks (AMDs) are popular food products found
worldwide. Many types of AMDs are available, including those pre-
pared from fermented milk with stabilisers and sugar to those pre-
paredbydirect acidicationwithfruit juices and/or acids, suchas fruit
milkdrinks, yoghurt drinks, soymilk, wheydrinks, andsoon(Laurent
& Boulenguer, 2003; Nakamura, Yoshida, Maeda, & Corredig, 2006).
The nal pHof these products generally ranges from3.6 to 4.6. Inraw
milk, at neutral pH, caseins exist in the form of micelles, which are
stabilised by steric repulsion due to the extended conformation of k-
casein present mainly on their surface (Tuinier & de Kruif, 2002).
During acidication of milk to a pH close to the isoelectric point (pH
4.6) of caseins, casein micelles aggregate mainly because of the
collapse of the extended k-casein (de Kruif, 1998; Nakamura et al.,
2006) such that a stabiliser must to be added to avoid protein ag-
gregation and subsequent macroscopic whey separation due to the
instabilityof caseins at their isoelectric points. Highmethoxyl pectins
(Laurent & Boulenguer, 2003; Liu, Nakamura, & Corredig, 2006;
Parker, Boulenguer, & Kravtchenko, 1994; Tromp, de Kruif, van Eijk,
& Rolin, 2004; Tuinier, Rolin, & de Kruif, 2002), soybean soluble
polysaccharides (Asai et al., 1994; Nakamura, Furuta, Kato, Maeda, &
Nagamatsu, 2003; Nakamura et al., 2006), and carboxymethylcellu-
lose (CMC) (Du et al., 2007, 2009; Wu, Liu, Dai, & Zhang, 2012) are
often used to achieve this purpose.
CMC can improve the stability of casein micelles at low pH. The
concentration, molecular weight (M
w
), and the degree of substi-
tution (DS) of CMC have different inuences on the stability of
AMDs. The system containing 40 g$kg
1
milk solid at pH 4.0 can be
stabilised by 4 g$kg
1
CMC, in which particle size of proteins was
about 0.68 mm, at the moment both the serum and the sedimen-
tation fraction were low. Below4 g$kg
1
CMC, bridging occulation
occurs in the system. While CMC with a high M
w
(700,000) in-
creases the viscosity of AMDs signicantly thereby contributing to
the stability, CMC with a high DS (1.2) results in a high z-potential of
CMC-coated casein micelles increasing the electrostatic repulsion
between casein micelles, also beneting the stability of the
colloidal systems (Du et al., 2007, 2009). Typical electrostatic in-
teractions occur between negatively charged CMC and positively
charged milk proteins at and below pH 5.2 (Du et al., 2007; Wu
et al., 2012). The adsorbed CMC layer causes repulsive in-
teractions between casein micelles at low pH via a manner similar
* Corresponding author. Tel.: 86 21 54745005.
E-mail address: hbzhang@sjtu.edu.cn (H. Zhang).
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http://dx.doi.org/10.1016/j.lwt.2013.12.029
LWT - Food Science and Technology 56 (2014) 370e376
to how k-caseins stabilise casein micelles at neutral pH. In addition,
non-adsorbed CMC increases the viscosity of AMDs, which can slow
down the sedimentation rate, thus also contributing to the stability
of the colloidal systems (Du et al., 2007).
The stability of AMDs has also been found to depend on the size
of the protein particles. Generally, larger particle sizes correspond
to more unstable dispersions, which are prone to syneresis and
wheying off. Usually, homogenisation has to be used in the dairy
industry to reduce the creaming and sedimentation of milk. Sta-
bilisation can be achieved by the size reduction of both protein
particles and fat globules (McCrae, Hirst, Law, & Muir, 1994; Sandra
& Dalgleish, 2005; Sedlmeyer, Brack, Rademacher, & Kulozik, 2004).
Parker et al. (1994) reported that homogenisation is required to
achieve the signicantly improved stabilisation of casein micelles
in acidied skim milk systems by pectin. As an important standard
operation during the AMD process, homogenisation has been
shown to enhance the adsorption of the pectin chains onto casein
particles (Tromp et al., 2004).
When producing directly acidied milk drinks (DAMDs), a
neutral milk dispersion is directly acidied to a certain pH using
acid or fruit juice. During this acidication process, there are many
changes in structure and component of casein micelles. Proteins in
casein micelles undergo extensive dissociation, reassociation and
rearrangement during acidication (Lucey, Tamehana, Singh, &
Munro, 1998a; McMahon, Du, McManus, & Larsen, 2009). Gastald
et al. observed the pH-induced changes in casein micelles during
direct acidication of reconstituted skim milk (low-heat type) at
20

C. They found that a
s
-, b- and k-casein were dissociated from
the micelles as pH lowered. For these three kinds of caseins, the
dissociation was relatively slow until about pH 6.0, then became
faster, especially with regard to b-casein, and reached a maximum
around pH 5.4. At pH 5.1 virtually all colloidal calcium phosphate
(CCP) was also solubilised and a maximum was also observed with
regard to micellar casein dissociation and solvation at about pH
5.4e5.3. The changes in compositions of casein micelles resulted in
the dissociation and aggregation of proteins observed by SEM
during acidication (Gastaldi, Lagaude, & Tarodo De La Fuente,
1996). In contrast, when producing yoghurt drinks using a fer-
mented base, both whey proteins denatured by pasteurisation and
those denatured whey proteins associated with casein micelles
become more susceptible to aggregate during acidication. Heat
treatments of milk at 90

C cause denaturation of whey proteins,
which will complex with casein micelles, involving k-casein, via
hydrophobic interactions and the formation of intermolecular
disulphide bonds. The presence of b-lactoglobulin was necessary
for any association of whey protein with casein micelles to occur
(Corredig & Dalgleish, 2001). Cross-linking or bridging by dena-
tured whey proteins produces a rigid gel network by casein mi-
celles (Lucey, 2004; Lucey, Tamehana, Singh, & Munro, 1998b;
Lucey, Tet Teo, Munro, & Singh, 1997). Yoghurt drinks are usually
manufactured by homogenisation of acid casein gels (i.e., fer-
mented bases). This means that they can be considered as sus-
pensions of colloidal casein gel particles.
The present work evaluates the inuence of homogenisation
and the degradation of CMC during homogenization on the stability
of DAMDs. The stabilities of DAMDs and yoghurt drinks induced by
CMC via different acidication processes of direct acidication by
an acid and fermentation by a starter culture are also discussed.
2. Materials and methods
2.1. Materials
CMC was provided by DuPont-Danisco Co., Ltd. (Shanghai,
China). The molecular weight and DS of CMC was 5 10
5
and 0.9,
respectively, and the Brookeld viscosity of 10 mg/ml aqueous so-
lution at its neutral pH (7.6) was 352 mPa$s. The Brookeld vis-
cosity was measured by a Brookeld DV-_instrument (Brookeld,
USA) with a No. 3 rotor at 100 rpm and 25

C. Low-heat skim milk
powder was obtained from Fonterra Co., Ltd. (Auckland, New Zea-
land). The skim milk powder contains 33.4% protein, 54.1% lactose,
7.9% mineral, 3.8% moisture and 0.8% fat. The citric acid and other
analytical grade chemicals used were purchased from Sinopharm
Chemical Reagent Co., Ltd. (Shanghai, China).
2.2. Preparation of DAMD
A solution of 80 g$kg
1
reconstituted skimmilk was prepared by
mixing the low-heat skimmilk powder with distilled water at 45

C
for 30 min. CMC and sucrose were dry mixed and then dissolved in
distilled water at 75

C by stirring for 20 min. The stabiliser and
skim milk were mixed at a 1:1 ratio to obtain 40 g$kg
1
skim milk
powder containing different CMC concentrations and 80 g$kg
1
sucrose. The pH of this mixture was adjusted to 4.0 using
500 g$kg
1
citrate acid at 20

C under continuous stirring at
1000 rpm. The nal volume was controlled in 5L. The sample was
homogenised at 25

C with a heat exchanger (TG-UHT-0.2 MJ,
Shanghai Nanhua Transducer Manufacture Co., Ltd., Shanghai,
China), followed by pasteurisation at 110

C for 30 s with a pas-
teuriser (HZ-SJJ, Shanghai Huizhan Technology Co., Ltd., Shanghai,
China). The nal product was then stored at 4

C for 24 h.
2.3. Preparation of yoghurt drinks
A solution of 120 g$kg
1
reconstituted skim milk was pas-
teurised at 90

C for 5 min and then cooled to 35

C in a water bath
(W201-B, Shanghai SENCO Technology Co., Ltd., China). About
0.053 g/L of a starter culture (MY-105, Danisco) was added.
Fermentation at 42

C was performed for 6 h to achieve a pH of 4.2.
To cease fermentation, the yoghurt was rapidly cooled to 4

C. CMC
and sucrose were dry mixed, dissolved in distilled water at 75

C by
stirring for 20 min, and then cooled to 15

C. Thereafter, the fer-
mented milk was added to the CMC/sugar solution with stirring at
1000 rpm. The milk solid non-fat (MSNF) concentration was
adjusted to 40 g$kg
1
, and the sucrose concentration was adjusted
to 80 g$kg
1
by addition of water. This mixture was acidied to pH
4.0 by addition of 500 g$kg
1
citrate acid at 20

C with continuous
stirring at 1000 rpm. The nal volume was controlled in 5L. The
sample was homogenised at 25

C, followed by pasteurisation at
110

C for 30 s with a pasteuriser (HZ-SJJ, Shanghai Huizhan
Technology Co., Ltd., Shanghai, China). The nal product was then
stored at 4

C for 24 h.
2.4. Effect of homogenisation
To study the inuence of homogenisation pressure on the sta-
bility of the drinks, a series of DAMDs containing 40 g$kg
1
MSNF,
4 g$kg
1
CMC, and 80 g$kg
1
sucrose with a nal pH value of 4.0
were prepared at different homogenisation pressures in the range
from0 to 30 MPa. Based on our previous study (Du et al., 2007), the
AMDs containing 40 g$kg
1
MSNF can be stabilised by 4 g$kg
1
CMC, so here this concentration of CMC, 4 g$kg
1
, was chosen in the
present work. The batch was pumped at different pressures
through a single-step homogenizer (GYB30-6S, Shanghai Donghua
High Pressure Homogenizer Company, China) with two passes. The
homogenisation pressures at the two passes were the same. The
particle size distribution, Brookeld viscosity and stability of each
DAMD were then measured.
J. Wu et al. / LWT - Food Science and Technology 56 (2014) 370e376 371
2.5. Different acidication processes
To determine the stability of AMDs induced by CMC via different
acidication processes, the particle size, Brookeld viscosity and
sedimentation of mixtures containing 40 g$kg
1
MSNF and
different CMC concentrations at different pH were measured. For
the DAMDs, during acidication, samples at pH 4.6, 4.4, 4.2, 4.0, 3.8
and 3.6 were collected. For the yoghurt drinks, the pH of the fer-
mented base was adjusted to range from4.2 to 3.6 using citrate acid
under continuous stirring. Samples were also collected at 0.2 pH
increments in a manner similar to that for the DAMDs. The samples
at various pH were then homogenised using a single-step homog-
eniser (GYB30-6S, Shanghai Donghua High Pressure Homogenizer
Company, China) at 20 MPa and pasteurised at 110

C for 30 s with a
pasteuriser (HZ-SJJ, Shanghai Huizhan Technology Co., Ltd.,
Shanghai, China).
2.6. Measurement of DAMD stability
Sedimentation and serum phases that appeared during storage
were observed using an optical analyser (Turbiscan MA 2000,
Formulaction, Ramonville-St-Agne, France). Cylindrical glass tubes
containing 5 mL of the samples were stored at 25

C. During
observation, three phases separated out in the tested tubes: sedi-
ments at the bottom, an opaque liquid in the middle, and a clear
liquid at the top. Changes in the sedimentation and serum fractions
as a function of time were monitored for 15 d after the sample was
prepared and calculated by the ratio of the distance of the corre-
sponding transmittance range to the tested tube length.
2.7. Measurement of Brookeld viscosity
The apparent viscosity of the AMDs was measured by a Brook-
eld viscometer Model NDJ-79 Brookeld LVDV-I (Brookeld, USA)
with a No. 1 rotor and 100 rpmat 25

C. The Brookeld viscosities of

the unstable AMDs in which phases separation occurs with obvious
sedimentation or creaming phenomenon, were not measured.
2.8. Measurement of particle size
The samples were diluted at a ratio of 1:100 with simulated milk
ultraltrate containing Na, K, Ca, Mg, phosphate and citrate
(Jenness & Koops, 1962). The particle size of these samples was
measured using a laser diffraction particle size analyser (Malvern
MasterSizer 2000, Malvern Instruments, UK).
2.9. Measurement of sedimentation
About 50 g of the AMDs was centrifuged at 3000 g and room
temperature for 20 min. The supernatant was carefully removed
and the centrifugation tube was inverted for 5 min to drain the
remaining supernatant. Sedimentation was calculated from the
ratio of the weight of the sediment to the weight of the sample.
2.10. Determination of serum CMC concentration
The concentration of the serum CMC was determined using the
method of Tromp et al. (2004). CMC is considered adsorbed when
it follows the protein phase into the pellet during centrifugation
(Beckman Coulter Avanti J-25 centrifuger, Fullerton, USA) at
25,000 g for 2 h. The supernatant or serum contains the non-
adsorbed CMC, hereafter referred to as serum CMC. The viscosity
of the supernatant was measured to determine the serum CMC
concentration. Viscosity measurements of the serum CMC were
performed on a controlled stress rheometer (Bohlin Instruments,
Gemini 200 HR, UK) tted with coaxial cylinders (25 mm and
27.5 mmrespectively in diameter of the inner and outer cylinder) at
a shear stress of 0.4 Pa.
To obtain the CMC concentrations in the serum of the DAMDs
and yoghurt drinks from viscosity measurements, two calibration
curves were made. The CMC was dissolved in solutions centrifuged
at 25,000 g for 2 h from the DAMDs and yoghurt drinks without
CMC addition. The viscosities of these CMC solutions as a function
of CMC concentration were used for the calibration curves, the
results of which are shown in Fig. 1. These curves were used to
determine the serum CMC concentrations in the DAMDs and
yoghurt drinks fromtheir viscosity by interpolation. The fraction of
adsorbed CMC was calculated by 1eC
serum
/C
overall
, where C
serum
is
the concentration of serum CMC, C
overall
is the concentration of
overall CMC.
All measurements were done in triplicate. Error bars shown in
the gures indicate the standard deviation.
3. Results and discussion
3.1. Inuence of homogenisation on the stability of DAMD
Fig. 2 shows the amounts of sedimentation and serum fractions
measured by Turbiscan, as well as the average particle size D[4,3] of
DAMDs produced at different homogenization pressures, ranging
from 0 to 30 MPa. Without homogenisation, the average particle
size of the DAMD was approximately 312 mm, and both sedimen-
tation fraction (ca. 40%) and serum fractions were large (ca. 60%),
0.0
0.1
0.2
0.3
0.4
0.5
0.6
C
M
C

c
o
n
c
e
n
t
r
a
t
i
o
n
/
%
Viscosity/mPa.s
(a)
0 40 80 120 160 200 240 0 40 80 120 160 200
0.0
0.1
0.2
0.3
0.4
0.5
0.6
C
M
C

c
o
n
c
e
n
t
r
a
t
i
o
n
/
%
Viscosity/mPa.s
(b)
Fig. 1. Calibration curves used for the determination of serum CMC concentrations from viscosity. Curve equation: Y a b*e
(x/c)
(Y serum CMC concentration and
X viscosity). (a) a 0.505, b 0.585, and c 49.345 in DAMDs. (b) a 0.504, b 0.620, and c 41.237 in yoghurt drinks.
J. Wu et al. / LWT - Food Science and Technology 56 (2014) 370e376 372
indicating the instability of the DAMDs. When the samples were
homogenised at 5 MPa, both the average particle size and the
sedimentation fraction of the DAMDs decreased signicantly, sug-
gesting that the stability of the sample was strongly improved
compared to the unhomogenized dispersions. This result also im-
plies that homogenisation is required to achieve a signicantly
small particle size and prevent sedimentation and serum separa-
tion. With increasing homogenisation pressures from 5 MPa to
20 MPa, the average particle size further decreased. Above 20 MPa,
no signicant changes in average particle sizes were detected
(0.7 mmat 20 MPa, 0.68 mmat 25 MPa, 0.6 mmat 30 MPa), indicating
the limited effect of homogenisation pressure on reducing the
average particle size. However, at high homogenization pressures
both the sedimentation and serum fractions increased slightly.
These ndings suggest that intense homogenisation is not bene-
cial for the stability of the colloidal systems.
The decrease in the Brookeld viscosity of the DAMDs with
increasing the homogenisation pressure shown in Fig. 3(a) is sup-
posed to explain this decrease of stability under intense homoge-
nisation. We previously reported that the presence of non-
adsorbed, excess CMC in AMDs increased the viscosity of drinks
and slowed down the sedimentation of protein particles (Du et al.,
2007). Thus, the stability of our colloidal system is susceptible to
the change in the viscosity of the medium.
Fig. 3(b) shows that the Brookeld viscosity of the pure CMC
solution decreased with increasing homogenisation pressure,
strongly indicating a remarkable decrease in the M
w
of CMC under
homogenisation. Corredig and Wicker (2001) investigated the ef-
fect of homogenisation on the M
w
of pectin and found that the
apparent viscosity and M
w
of pectin decreased at high homogeni-
sation pressure. The degradation of CMC shown in Fig. 3(b) is one of
the reasons for the decrease in viscosity of the DAMDs, leading to
the acceleration in sedimentation of the protein particles and
decrease in DAMD stability. More intense homogenisation may also
result in an increased number of small protein particles and more
bare protein particles, such that relatively more CMC is required to
cover all of these particles sufciently and maintain a stable system.
However, because the M
w
of CMC decreases with increasing ho-
mogenisation pressure, the stabilisation effect of CMC is weakened.
At a given CMC concentration, the fraction of non-absorbed CMC
becomes relatively lower and is unable to effectively increase the
viscosity of the solution. By combining changes in the particle size,
Brookeld viscosity, sedimentation, and serum fractions at
different homogenisation pressures, a qualied homogenisation
pressure of 20 MPa was selected and used in the following
measurements.
3.2. The stability of DAMD and yoghurt drinks induced by CMC
The evolution of particle size D[4,3] of the DAMDs and yoghurt
drinks as a function of pH are shown in Fig. 4. In the DAMD with
over 3 g$kg
1
CMC stabiliser, the particle size was approximately
0.4 mm in the pH range from 3.6 to 4.6; the particle size in the
yoghurt drinks increased from 4 mm to 6 mm when the pH was
reduced from 4.2 to 3.6. CMC adsorption onto the casein micelles
occurs at and below pH 5.2, just before the aggregation of casein
micelles (Du et al., 2007). Thus, the protein particles in the DAMDs
are small and similar to those in natural casein micelles. The ag-
gregation state of caseins is signicantly different in the yoghurt
drinks. The base of yoghurt drinks is an acid milk gel formed during
fermentation (Alting, Hamer, de Kruif, & Visschers, 2000; Lucey
et al., 1997, 1998b; Lucey, 2004; Vasbinder, Alting, Visschers, & de
Kruif, 2003). The milk used in manufacturing of yoghurt is sub-
jected to an extensive heating (90

C for 5 min), which does lead to
the denaturation of whey proteins and their association with casein
micelles via hydrophobic interactions and the formation of inter-
molecular disulphide bonds. This system could aggregate during
acidication, wherein denatured whey proteins associated with
casein micelles act as bridging materials by interacting with other
denatured whey proteins. The cross-linking or bridging by dena-
tured whey proteins could result in a rigid gel network by casein
micelles (Lucey et al., 1997, 1998b; Lucey, 2004). Thus, particles
present in the yoghurt drink after homogenisation are large-sized
gel particles (Tromp et al., 2004).
0 5 10 15 20 25 30
0
10
20
30
40
50
60
70
Pressure / MPa
P
a
r
t
i
c
l
e

s
i
z
e

D
[
4
,
3
]

/

m
%
/
n
o
i
t
c
a
r
f
m
u
r
e
S
&
%
/
n
o
i
t
c
a
r
f
n
o
i
t
a
t
n
e
m
i
d
e
S
0
20
40
250
300
350
Fig. 2. Effects of homogenisation on the stability and particle size of DAMDs
(40 g$kg
1
MSNF, 4 g$kg
1
CMC, and 80 g$kg
1
sucrose; pH 4.0). Filled square -:
particle size; lled circle C: sedimentation fraction; empty circle B: serum fraction.
20
30
40
50
60
70
B
r
o
o
k
f
i
e
l
d

v
i
s
c
o
s
i
t
y

/

m
P
a
.
s
Pressure / MPa
a
0 5 10 15 20 25 30 35
0 5 10 15 20 25 30
100
105
110
115
120
125
130
135
140
b
Pressure / MPa
B
r
o
o
k
f
i
e
l
d

v
i
s
c
o
s
i
t
y

/

m
P
a
.
s
Fig. 3. Brookeld viscosities of (a) AMDs (40 g$kg
1
MSNF, 4 g$kg
1
CMC and 80 g$kg
1
sucrose; pH 4.0) and (b) CMC solution (c 10 mg/ml) as a function of homogenisation
pressure.
J. Wu et al. / LWT - Food Science and Technology 56 (2014) 370e376 373
The Brookeld viscosities of the DAMDs and yoghurt drinks
stabilised by different CMC concentrations are shown in Fig. 5(a)
and (b), respectively. Large casein aggregates were observed in the
unstable samples at low CMC concentrations. The Brookeld vis-
cosity cannot be measured in these unstable AMDs. For stable
samples, the Brookeld viscosity increased with increasing con-
centration of CMC and decreased with decreasing pH. These results
may be attributed to two aspects. Firstly, the net charge of the
casein micelle is less negative during acidication. Therefore, more
CMC is adsorbed onto the casein micelles or milk gel particles by
electrosorption. The amount of non-adsorbed CMC that contributes
to the AMD viscosity decreases, leading to a decrease in the
Brookeld viscosity. Secondly, the conformation of CMC chains
becomes compact with lowering pH, thus decreasing the viscosity.
The Brookeld viscosity of the yoghurt drinks is experimentally
larger than that of the DAMDs under the same conditions and at the
same amount of milk solids. This is because in the system of
yoghurt drinks, there exists gel-like network of proteins cross-
linked or bridged by denatured whey proteins, while the protein
particles in the DAMDs are small and similar to those in natural
casein micelles.
Sedimentation results obtained by centrifuging the DAMDs and
yoghurt drinks are shown in Fig. 6. In the absence and presence of
1 g$kg
1
CMC, both of the sedimentation fractions of the DAMDs
and yoghurt drinks are larger than 6%, and these systems are un-
stable (Fig. 6(a)). In the DAMDs, addition of 2 g$kg
1
CMC stabilised
the milk proteins in the narrowpHrange from4.6 to 4.4, addition of
3 g$kg
1
CMC stabilised them in the pH range from 3.8 to 4.6, and
addition of over 4 g$kg
1
CMC stabilised them over the entire pH
range tested. The sedimentation values are almost the same (below
1%) when the CMC concentration is above 4 g$kg
1
. For the yoghurt
drinks, stability over the whole pH range can only be achieved with
addition of more than 4 g$kg
1
CMC (Fig. 6(b)). The sedimentation
of yoghurt drinks decreased with increasing CMC concentration.
3.3. Fraction of CMC adsorption in the DAMDs and yoghurt drinks
To investigate the different stabilising effects of CMC in the
DAMDs and yoghurt drinks, the degree of CMC adsorption, i.e., the
total amount of CMC added and adsorbed onto the casein micelles,
was measured. The adsorbed CMC fraction was determined by
measuring the viscosity of the serum after AMD centrifugation.
Using the viscosity calibration shown in Fig. 1, the viscosity was
converted into a CMC concentration.
Fig. 7 shows the degrees of CMC adsorption obtained in this
manner from AMD samples containing 40 g$kg
1
MSNF as a
function of overall CMC concentration. Almost all of the added CMC
was adsorbed onto the protein particles when the CMC concen-
tration was between 1 and 2 g$kg
1
. However, these AMDs were
unstable. Addition of 1 and 2 g$kg
1
CMCs was not enough to fully
cover the protein particles, and the protein particles may aggregate
by bridging occulation. These results are consistent with those
shown in Fig. 6. At a CMC concentration of 3 g$kg
1
, the degrees of
CMC adsorption were identical in the DAMDs and yoghurt drinks
(73%). A considerable amount of the added CMC was adsorbed onto
the casein micelles and milk gel particles. The AMD appeared to be
stable when the CMC concentration was above the full coverage
concentration, and some of the CMC was not adsorbed at 3 g$kg
1
CMC. By increasing the CMC concentration to range from4 g$kg
1
e
6 g$kg
1
, the degree of CMC adsorption decreased in both the
DAMDs and yoghurt drinks. However, the fraction of CMC adsorbed
by the yoghurt drinks was higher than that adsorbed by the
DAMDs. The fraction of CMC adsorbed by the DAMDs decreased
from 52% to 38% when the CMC concentration was increased from
4 g$kg
1
e6 g$kg
1
. However, the amount of adsorbed CMC per
kilogram of DAMD did not change so much, only from ca. 2.1 g for
the sample containing 4 g/kg CMC to 2.2 g for the sample con-
taining 6 g/kg CMC. We thus conclude that, in 4% MSNF, casein
micelles in DAMDs are efciently covered by the addition of
3 g$kg
1
CMC. Addition of excess CMC (from 4 g$kg
1
e6 g$kg
1
)
Fig. 4. Changes in the average particle diameter as a function of pH for DAMDs (lled
symbols) and yoghurt drinks (open symbols) containing different CMC concentrations:
(A, >) 3 g/kg
1
CMC; (:, 6) 4 g/kg
1
CMC; (C, B) 5 g/kg
1
CMC and (-, ,) 6 g/
kg
1
CMC.
0
10
20
30
40
50
60
70
80
B
r
o
o
k
f
i
e
l
d

V
i
s
c
o
s
i
t
y

/

m
P
a
.
s
pH
(a)
3.6 3.8 4.0 4.2 4.4 4.6
3.6 3.8 4.0 4.2
0
10
20
30
40
50
60
70
80
(b)
pH
B
r
o
o
k
f
i
e
l
d

V
i
s
c
o
s
i
t
y

/

m
P
a
.
s
Fig. 5. Effects of CMC concentration on the Brookeld viscosity for (a) DAMDs and (b) yoghurt drinks: (B) 2 g/kg
1
CMC; (,) 3 g/kg
1
CMC; (:) 4 g/kg
1
CMC; (C) 5 g/kg
1
CMC
and (-) 6 g/kg
1
CMC.
J. Wu et al. / LWT - Food Science and Technology 56 (2014) 370e376 374
does not increase the content of adsorbed CMC. In contrast to the
nding that the full coverage content of CMC in the DAMD is not
inuenced by further CMC addition, the amount of adsorbed CMC
increased with increasing CMC concentration in the yoghurt drinks.
The addition of excess CMC contributes to CMC adsorption onto the
milk gel particles in the yoghurt drinks. This phenomenon may be
due to the enhanced interaction of CMC with denatured whey
proteins in yoghurt drinks.
3.4. Degradation of CMC in AMDs
Polysaccharides can be degraded by heating, high pressure, and
acidic solutions (Corredig & Wicker, 2001; Gautier & Lecourtier,
1991; Kok, Kill, & Mitchell, 1999). The pH of the DAMDs and
yoghurt drinks is usually adjusted to 4.0; at low pH, CMC could be
degraded (Gautier & Lecourtier, 1991). As previously mentioned,
non-absorbed CMC helps maintaining the stability of AMDs by
increasing their viscosity and slowing down the sedimentation of
protein particles. Changes in the serum CMC viscosity of DAMDs
and yoghurt drinks containing 4 and 6 g$kg
1
CMC as a function of
storage time are shown in Fig. 8. The serumviscosity of AMDs with
6 g$kg
1
CMC was much higher than those with 4 g$kg
1
CMC
because of the larger amount of non-adsorbed CMC in the serum
phase with the addition of 6 g$kg
1
CMC. Furthermore, the serum
CMC viscosity in the DAMDs was higher than that in the yoghurt
drinks because the low adsorbed fraction of CMC in the DAMDs
resulted in higher amounts of non-absorbed CMC and higher serum
CMC viscosity, consistent with the ndings shown in Fig. 7. The
viscosities of serum CMC were measured after 10, 15, and 20 d for
calibration. Fig. 8 shows that the viscosity of the serum CMC
decreased during storage. The decrease in viscosity with time is
related to the degradation of CMC at low pH (4.0) (Glinel, Sauvage,
Oulyadi, & Huguet, 2000). The viscosity of serum CMC was also
measured 10, 15, and 20 d after the AMD samples had been pre-
pared and compared with the calibration curve. The serum
0
2
4
6
8
10
12
S
e
d
i
m
e
n
t
a
t
i
o
n

/

%
pH
(a)
3.6 3.8 4.0 4.2 4.4 4.6
3.6 3.8 4.0 4.2
2
4
6
8
10
pH
S
e
d
i
m
e
n
t
a
t
i
o
n

/

%
(b)
Fig. 6. Effects of CMC concentration on the sedimentation of (a) DAMDs and (b) yoghurt drinks: () 0 g/kg
1
CMC; (6) 1 g/kg
1
CMC; (B) 2 g/kg
1
CMC; (,) 3 g/kg
1
CMC; (:)
4 g/kg
1
CMC; (C) 5 g/kg
1
CMC and (-) 6 g/kg
1
CMC. The samples were centrifuged at 3,000 g for 20 min.
0 1 2 3 4 5 6 7
0.0
0.2
0.4
0.6
0.8
1.0
A
d
s
o
r
p
t
i
o
n

f
r
a
c
t
i
o
n
Overall CMC concentration g/kg
Fig. 7. Fractions of CMC adsorbed for DAMDs (B) and yoghurt drinks (6) (40 g$kg
1
MSNF; pH 4.0) containing different CMC concentrations.
20
30
40
50
60
70
V
i
s
c
o
s
i
t
y

/

m
P
a
.
s
t / day
(a)
0 5 10 15 20 0 5 10 15 20
15
20
25
30
35
40
45
50
v
i
s
c
i
o
s
i
t
y

/

m
P
a
.
s
t / day
(b)
Fig. 8. Serum CMC viscosity of (a) DAMDs and (b) yoghurt drinks as a function of time (40 g$kg
1
MSNF and pH 4.0): (7, ) and (6, ) refers to the milk drinks stabilised by 4
and 6 g$kg
1
CMC, respectively. Calibrated (7, 6) refers to viscosity changes in the serum centrifuged after preparation as a function of time. Centrifuged (, ) refers to
viscosity changes in the serum centrifuged at different storage times. Some overlaps in the values are observed between the calibrated and centrifuged samples.
J. Wu et al. / LWT - Food Science and Technology 56 (2014) 370e376 375
viscosities of the AMDs decreased with time, with the rate of
decrease similar to that observed in the calibration curve. The
decrease in viscosity of the serum CMC strongly suggests the
degradation of CMC at acidic condition (Glinel et al., 2000), which is
probably the main reason that leads to the instability of AMD
during storage.
4. Conclusions
The stability of AMD induced by CMC depends on several pa-
rameters, including the molecular properties of CMC and the pro-
cessing conditions. The present work reveals the signicance of
homogenisation and degradation of cellulose gum stabiliser on the
stability of AMDs via either directly acidication by citric acid or
fermentation.
Small protein particles generated by homogenisation are
hypothesised to result in stable DAMDs. However, their stability
deteriorates when high homogenisation pressure is applied
because of the degradation of CMC that results in the decrease in
viscosity of the colloidal system and the new-born surface of pro-
tein particles to which more CMC is needed to adsorb. Thus, a
qualied choice for homogenisation pressure (20 MPa in the pre-
sent work) is required to achieve a good stability when a usually
practical pressure range, such as 0e30 MPa, is applied.
CMC is an effective stabiliser for both DAMDs and yoghurt
drinks, and the stability of these two colloidal systems increases
with increasing CMC concentration. Given the same amount of milk
solids, concentration of CMC and homogenisation pressure, the gel
particle size of yoghurt drinks is larger than that of the casein mi-
celles in DAMDs. The amount of CMC adsorbed onto the milk gel
particles in yoghurt drinks is larger than that in casein micelles in
DAMDs at the same CMC concentration and pH. The nal instability
of the acidied milk products is closely related to the degradation of
CMC during storage at low pH.
Acknowledgements
The authors are thankful for the nancial support for this work
from the Shanghai Leading Academic Discipline Project (No. B202).
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