ARTICLE IN PRESS

International Dairy Journal 13 (2003) 773–782

Preliminary observations on the effects of milk fortification and

heating on microstructure and physical properties of stirred yogurt
F. Remeufa,*, S. Mohammed, I. Sodini, J.P. Tissierb
a
Unit!e Mixte de Recherche de G!enie et Microbiologie des Proc!ed!es Alimentaires, Institut National Agronomique Paris Grignon/Institut National de la
Recherche Agronomique, 78 850 Thiverval Grignon, France
b
Unit!e de Recherche de G!enie des Proc!ed!es et Technologies Alimentaires, Institut National de la Recherche Agronomique, 369, rue J. Guesdes,
B.P. 38, 59 650 Villeneuve d’Ascq, France
Received 26 March 2002; accepted 7 April 2003

Abstract

The aim of this work was to study how milk fortification and heating affect yogurt microstructure (micellar characteristics,
protein network) and physical properties (viscosity, water-holding capacity (WHC), and graininess). Milk was fortified with skim
milk powder (control), whey protein concentrate (WPC), caseinate, or a mixture of caseinate and whey protein. Two heat treatments
were applied, giving average whey protein denaturation levels of 58% and 77%. For caseinate-enriched yogurts, the heating effect
was negligible. When milk was enriched with WPC, heating led to a high level of cross-linking within the gel network. Heating
increased yogurt viscosity and WHC, but also graininess. When milk was fortified with a blend of WPC and caseinate giving a whey
protein-to-casein ratio of 0.20, the yogurt viscosity was greatly improved, while graininess was kept low. The results show a
relationship between micelle solvation and yogurt microstructure, as well as micelle size in milk base and yogurt graininess.
r 2003 Elsevier Science Ltd. All rights reserved.

Keywords: Yogurt; Milk proteins; Microstructure; Texture

1. Introduction isoelectric points of denatured whey proteins (pH 5.2),

Yogurt and fermented milks are among the most
common dairy products eaten in the world. Their
production was estimated at 8.3 million tonnes in 1999
(IDF, 2000). The yogurt manufacturing process changes
the properties of milk proteins in an irreversible way.
First, the milk is fortified with dairy ingredients to
achieve a final protein content of 40–50 g kg
1. Then,
the milk base is submitted to a drastic heat treatment,
which results in a high level of thermal denaturation of
the whey proteins, and their partial fixation on the
casein micelles (Mottar, Bassier, Joniau, & Baert, 1989;
Lucey, Tet Teo, Munro, & Singh, 1997; Corredig &
Dalgleish, 1999). Finally, the lactic acid production
during the fermentation step leads to the destabilization
of the micellar system which results in the gelation of the
proteins (Heertje, Visser, & Smits, 1985). As the
*Corresponding author. Tel.: +33-1-30815383;
fax: +33-1-30815597.
E-mail address: florent.remeuf@grignon.inra.fr (F. Remeuf).
and caseins (pH 4.6) are reached, low-energy bonds,
mainly hydrophobic, are progressively established be-
tween the proteins (Lucey & Singh, 1998).
Skim milk powder (SMP) is used widely to fortify
yogurt milk. However, new technologies for milk and
whey fractionation produce a large diversity of dairy
ingredients, as caseinates (Na or Ca-caseinates) and
whey protein concentrates (WPC) (34% w/w–80% w/w
protein). They have different properties and can be used
separately or blended to replace SMP in yogurt
manufacture. Previous authors studied the effect of
caseinates and WPCs used separately (Tamime, Kalab,
& Davies, 1984; Guinee, Mullins, Reville, & Cotter,
1995; Kailasapathy & Supriadi, 1998; Trachoo &
Mistry, 1998). On the other hand, few have studied the
effect of blends of caseinates and WPCs on yogurt
physical properties (GuzmanGonzalez, Morais, Ramos,
& Amigo, 1999; GuzmanGonzalez, Morais, & Amigo,
2000). Furthermore, the impact of milk heating on
yogurt structure when SMP is replaced by other dairy
powders, has not been evaluated.

0958-6946/03/$ - see front matter r 2003 Elsevier Science Ltd. All rights reserved.
doi:10.1016/S0958-6946(03)00092-X
 ARTICLE IN PRESS
774 F. Remeuf et al. / International Dairy Journal 13 (2003) 773–782

The objective of this research was to study the effect Table 1

of caseinates and WPCs, when used separately and Composition of milk and ingredientsa used in this study, expressed in
blended to fortify milk, on the physical properties of g kg
1
yogurt. Different heat treatments were applied to the Milk SMP NaCn CaCn WPC
fortified milk. In order to characterize its organization,
Dry matter 110.7 960 940 940 950
the acid gel was examined at both microscopic level Fat 25.1 12.5 7 5 6
(protein denaturation, micelle size and hydration, Lactose 47.4 510 2 2 10
microstructure of the protein system) and macroscopic Calcium 1.2 12.75 0.8 13 3.5
level (physical properties such as rheological behavior, Protein 30.9 340 880 880 840
ability to retain water, and graininess). The aim was to a
Ingredients used: SMP—skim milk powder; NaCn—Na caseinate;
establish some links between these two levels of CaCn—Ca caseinate; and WPC—whey protein concentrate.
observations so as to better understand the mechanisms
that control the texture development of yogurt during
processing. Table 2
Level of ingredientsa added to milk, expressed in g kg
1, for the six
milk base used for yogurt manufactureb
2. Material and methods Ingredient Milk base

SMP NaCn CaCn Blend 1 Blend 2 WPC

2.1. Milk base preparation
Commercial whole and half skimmed pasteurized
milks (Lactalis, Laval, France), were mixed in order to
standardize the fat level to 25 g kg
1. The milk was then
fortified with dairy ingredients to give a protein content
of 45 g kg
1. Four dairy ingredients were used: one low-
heat SMP (Elle et Vire, Conde! Sur Vire, France), and
three dairy powders provided by Armor Prote! ines (Saint
Brice en Cogle" s, France), sodium and calcium caseinate,
and a WPC. The composition of dairy ingredients is
given in Table 1. Six different milk bases were prepared,
using the dairy ingredients alone or in blend. Four milk
bases contained only one kind of dairy ingredient: SMP,
Na-caseinate (NaCn), Ca-caseinate (CaCn), or WPC.
Two contained a blend of CaCn and WPC, with
caseinate to whey protein ratios of 2:1 (Blend 1) or 1:2
(Blend 2). The addition levels for milk fortification are
given in Table 2.
Each milk base was heated at 90 C for 1 or 5 min, by
circulating in a hot water bath. Then, it was quickly
cooled to 4 C in an iced water bath, poured into 250-mL
Erlenmeyer flasks, and stored 24 h before use.
2.2. Yogurt manufacture
Two commercial yogurt strains were used: Strepto-
coccus thermophilus ST7, and Lactobacillus bulgaricus
LB12 (Chr. Hansen, Arpajon, France). Inocula of the
strains were stored at
70 C in concentrate form. They
were thawed and diluted 10 times in sterile tryptone
diluent (0.1% w/v) just before inoculation.
The flasks were inoculated at the fermentation
temperature (42 C) with each strain at the level of
0.2 g L
1. Fermentation was stopped when the pH
reached 4.5 by rapidly cooling the fermented milk in
an ice bath, manually stirring it with a stainless-steel
bored disk by up and down movements for almost
SMP 66
NaCn 23
CaCn 23 15 8
WPC 8 16 25
a
Ingredients added: SMP—skim milk powder; NaCn—Na case-
inate; CaCn—Ca caseinate; WPC—whey protein concentrate, Blends 1
and 2—blends of Ca caseinate and WPC with caseinate to whey
protein ratios of 2:1 (Blend 1) or 1:2 (Blend 2).
b
The total protein content in all milk bases was fixed at 45 g kg
1.
1 min, setting the stirred product into 100 mL cups, and
storing it at 4 C.
2.3. Physicochemical determinations
The total solid content of the milk bases was
determined by dehydration in a microwave Ultra X
Analysator 3503 oven (LABO STANDA, Caen,
France).
The concentrations of total (TN), N soluble at pH 4.6
(SN) and non-protein nitrogen (NPN) were quantified in
milk bases before and after heating, by the Kjeldahl
method (Rowland, 1938). The protein content was
calculated as: (TN
NPN)  6.38; the undenatured whey
protein content was calculated as (SN
NPN)  6.38.
The casein content was estimated for each milk base,
considering that casein represented 780 g kg
1 of total N
in pasteurized milk and SMP (low heat), 1000 g kg
1 of
total N in caseinates, and 150 g kg
1 of total N in WPC
(technical specifications given by ‘‘Armor Prote! ines’’). It
was assumed that the difference between the non-soluble
protein fraction, (TN
SN)  6.38, and the casein
content was due to denatured whey protein that
precipitated with casein at pH 4.6. This allowed us to
calculate the content of denatured whey proteins. The
total whey protein content was obtained by adding
undenatured and denatured whey protein.
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F. Remeuf et al. / International Dairy Journal 13 (2003) 773–782
The mean micelle diameter in the milk bases was
determined by photon correlation spectroscopy with an
N4 Coultronics Particle Analyzer (Coulter Electronics,
Miami, USA). The measurements were made, according
to the method described by Remeuf, Lenoir, and Duby
(1989), on each sample of milk base, after a 1:100
dilution in milk ultra-filtrate and filtration through a
0.45 mm syringe membrane.
The casein micelle solvation in milk bases and yogurts
was estimated as the quantity of water in the sediment
pellets produced on ultra-centrifugation at 80,000  g
for 1 h (Remeuf et al., 1989).
Each physicochemical measurement was made in
triplicate.
2.4. Yogurt microstructure
After 1 week of storage at 4 C, three samples of
yogurts were taken with a 0.2 mL positive displacement
microdispenser and prepared for scanning electron
microscopy (SEM), according to the method adapted
from Kalab (1981). The yogurt samples were fixed for at
least 4 h in a glutaraldehyde solution (12.5 mL L
1).
Samples were then rinsed five times for 15 min in ultra-
filtered water. The samples underwent further dehydra-
tion in a graded acetone series (500, 750 and
950 mL L
1) followed by rinsing in anhydrous acetone.
Dehydrated samples were dried by the critical point
method under CO2 with an Emscope CPD 750
apparatus (Ashford, Great Britain). The dehydrated
cylinders of samples were stuck on stubs with an epoxy
resin (Araldites), and then they were broken with a
scalpel in order to exhibit sample microstructure. A final
coating of about 15 nm gold–palladium thick was
performed by cathodic spreading of the samples in a
Polaron E 5100 coater (West Sussex, Great Britain).
Sample observation and photomicrography were per-
formed in a Hitachi S-3000N SEM (Tokyo, Japan)
operating at a voltage of 4 kV. Six fields were observed
for each sample.
2.5. Physical properties
Rheological behavior, water-holding capacity
(WHC), and graininess of the yogurts were evaluated.
The measurements were performed in triplicate after 1
week of storage at 4 C.
2.5.1. Rheological behavior
The rheological parameters were determined at 10 C,
by means of a control-stress rheometer (model RS1;
Haake, Karlsruhe, Germany), used in harmonic and
stationary modes. The rheometer was equipped with a
cone and plate geometry (60 mm diameter, 2 angle, and
117 mm gap). After gentle mixing by five up and down
movements of a spoon in the yogurt cup, 5 mL of yogurt
775
sample was deposited on the plate of the rheometer by
means of a syringe. The use of the syringe has been
proven as a means to avoid the presence of grains in the
sampled material, which would otherwise greatly disturb
the rheological measurements. Measurements in harmo-
nic mode were carried out using a shear stress of 0.14 Pa
at an oscillation frequency of 1 Hz for 1 min; these
conditions ensure that the applied stress was within the
range of linear viscoelasticity. The complex viscosity, in
Pa s, was calculated by the determination of the mean of
20 measurements performed during the analysis. Mea-
surements in stationary mode were made by applying a
shear rate of 10 s
1. The apparent viscosity in Pa s was
determined after 3 min of shearing. These two comple-
mentary measures were representative of viscoelastic
behavior (where sample structure is not affected) and
flow behavior (where the sample structure is destroyed).
2.5.2. Water-holding capacity
The WHC was determined with a procedure adapted
from GuzmanGonzalez et al. (1999). A sample of about
20 g of native yogurt (NY) was centrifuged for 10 min at
483  g and 20 C. The whey expelled (WE) was
removed and weighed. The WHC was defined as
WHC ð%Þ ¼ 100ðNY
WEÞ=NY:
2.5.3. Graininess
Graininess was measured by image analysis. A sample
of yogurt (1 g) was dispersed in 10 mL of distilled water,
then poured into a Petri dish placed on an illuminated
plate. The diluted sample was visualized by a digital
color camera (JAI M2040, Imasys, France) equipped
with an optical zoom. The image analysis was
performed in gray level mode with Optimas 6.2 software
(Media Cybergenetics, Silver Spring, USA). Grains
having a perimeter >1 mm were enumerated. The
results were expressed as number of grains per gram of
yogurt.
3. Results and discussion
3.1. Physicochemical characteristics of milk bases and
yogurts
Table 3 shows the protein composition of each milk
base and their percentage of denatured whey protein
before and after heating. The protein contents in the
milk bases were close to that expected (45 g kg
1). To
achieve this protein content with SMP addition, it was
necessary to enrich the milk to a higher level of dry
matter (DM) than with caseinates and WPC, because of
its lower protein/DM ratio. The initial percentage of
whey protein denaturation in unheated milk bases
ranged from 1% to 24% of total whey protein. It was
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776 F. Remeuf et al. / International Dairy Journal 13 (2003) 773–782

Table 3
Dry matter, protein composition and whey protein denaturation level of the six milk bases used for yogurt manufacturea. Four contained only one
dairy ingredient: skim milk powder (SMP), Na-caseinate (NaCn), Ca-caseinate (CaCn), or whey protein concentrate (WPC). Two contained a blend
of Ca-caseinate and whey protein concentrate : CaCn and WPC at a ratio of 2:1 (Blend 1); CaCn and WPC at a ratio of 1:2 (Blend 2). Two heat
treatments were applied: 90 C for 1, or 5 min

Milk base SMP NaCn CaCn Blend 1 Blend 2 WPC

1
Dry matter (g kg ) 162.0 132.0 140.0 133.0 134.0 142.0

Protein composition
Protein (g kg
1) 45.2 45.6 46.9 46.3 45.5 44.2
Casein (g kg
1) 37.5 42.3 43.8 37.1 34.4 30.9
Whey protein (g kg
1) 8.0 3.6 3.5 9.3 13.9 17.7
Whey protein-to-casein ratio 0.21 0.08 0.08 0.25 0.40 0.57

Whey denaturation level (g 100g
1 total whey protein)

Before heating 4.5 7.9 8.2 1.1 20.7 23.6
After heating at 90 C  1 min 64.5 51.5 49.4 56.9 63.3 63.8
After heating at 90 C  5 min 75.3 80.8 83.5 67.3 77.8 75.4
a
Values presented correspond, for each milk base, to one replication of the preparation of the milk base.

higher in milk base fortified with WPC probably because 360

of a higher level of whey protein denaturation in WPC
than in other dairy ingredients. Thus GuzmanGonzalez 340

et al. (1999) reported values within the range (4–28% of 320

total whey protein) for initial protein denaturation in six
Micelle diameter (nm)

commercial spray-dried WPCs. On average, 58% of 300

whey proteins were denatured after heating at 90 C for

280
1 min, and 77% after 5 min. These levels of whey protein
denaturation were consistent with those obtained 260

by several authors with similar heating conditions

240
(Dannenberg & Kessler, 1988; Mottar et al., 1989;
Law, 1995). 220
The mean diameter of micelles in the unheated and
200
heated milk bases varied from 237 to 330 nm (Fig. 1). SMP NaCn CaCn Blend 1 Blend 2 WPC
(A)
For unheated milk bases, the micelle sizes varied slightly
from 236 nm for the SMP-enriched milk base to 261 nm 1.8
for WPC-enriched milk base. The effect of heat
treatment on average micelle size in the milk base was
Micelle solvatation in yogurt (g.g-1)

1.6
dependent on the type of protein added. In milk bases
enriched with caseinate (Na or Ca) or SMP, the average
micelle size tended to decrease slightly after both mild 1.4

and severe heating. In contrast, for milk enriched with

WPC, or with a blend of Ca caseinate and WPC, a more
1.2
severe heating (90 C/5 min) led to a marked increase of
micelle size (Fig. 1). It has been reported (Singh, 1995;
Tamime & Muir, 1997; Devold, Brovold, Langsrud, & 1.0

Vegarud, 2000) that changes in the size of casein micelles

may be observed in milk when heated to temperatures 0.8
>90 C. The dissociation of casein monomers, more (B) SMP NaCn CaCn Blend 1 Blend 2 WPC
particularly k-casein, from the micelle, could be Fig. 1. Effect of milk supplementation and heating on micelle diameter
responsible for a reduction of average micelle size in in milk bases (A) and micelle solvatation in yogurt (B). Milk bases
milk severely heated (Singh, 1995). On the other hand, were enriched with SMP, NaCn, CaCn, WPC, or blends of CaCn and
the formation of aggregates involving cross-links WPC (blend 1—WPC and CaCn at a ratio of 1:2; blend 2—WPC and
CaCn at a ratio of 2:1). The milk bases were either unheated (’) or
between the casein micelles and denatured whey protein
subjected to one of the following heat treatments: 90 C for 1 min ( )
(Modler, Larmond, Lin, Froelich, & Emmons, 1983; or 90 C for 5 min ( ). The presented data correspond to one
Kessler, 1997) can be responsible for an increase of replication of each combination of milk supplementation and heating
micelle size after heating. The results of this study time.
 ARTICLE IN PRESS
F. Remeuf et al. / International Dairy Journal 13 (2003) 773–782
suggest that this last mechanism is enhanced when the
whey protein/casein ratio increases, as the increase of
mean micelle diameter in heated milk base was roughly
proportional to the quantity of WPC added (Fig. 1).
Formation of large aggregates of whey proteins after
heating is also possible (O’Kennedy, Halbert, & Kelly,
2001; Schorsch, Wilkins, Jones, & Norton, 2001). Heat-
induced formation of aggregates could contribute to the
increase of mean particle size in milk bases highly
enriched with WPC.
Micelle solvation in yogurt varied from 1.05 to
1.44 g g
1 DM according to the type of protein used in
enrichment and the heating time at 90 C (Fig. 1). These
values represented about half of the average values
measured in milk bases (results not shown). They were
consistent with data reported in the literature (Tarodo
de la Fuente & Alais, 1974; Snoeren, Klok, Van
Hooydonk, & Damman, 1984) for fresh or reconstituted
milks that were acidified to pH 4.0 with hydrochloric
acid. Micelle solvation in yogurts made from milk bases
enriched with WPC was 22–28% higher than that in
yogurts obtained from caseinate- or SMP-enriched milk
bases. Two mechanisms might be put forward to explain
this behavior. First, it should be noted that yogurt pH,
i.e. B4.6, is notably lower than the isoelectric pH of
whey protein, i.e. B5.2 (Cayot & Lorient, 1998). Thus,
the yogurt made from WPC-enriched milk base should
contain a relatively high quantity of positively charged
whey proteins and would therefore have a higher water
binding capacity than the protein matrix of yogurt
enriched with SMP. A second explanation lies in the
possible formation of large aggregates of complexed
native micelles and denatured whey proteins in milk
bases fortified with WPC; this hypothesis is supported
by particle size measurements. Such aggregates might be
able to retain a relatively high quantity of water and
thereby contribute to an increase in apparent solvation
of the protein matrix in the gel.
3.2. Yogurt microstructure
SEM micrographs were obtained from yogurts made
with each combination of heat treatment intensity and
type of protein enrichment. They showed that the
organization of the yogurt gel was influenced by the
intensity of heating (results not shown). A denser and
more branched-structured gel was observed when a
severe heating was applied to the milk base, as
compared to a mild heating. Several authors have also
shown a marked effect of milk base heating on the
structure of yogurt and acid gels (Harwalkar & Kalab,
1986; Lucey, Munro, & Singh, 1999). In agreement with
these data, our micrographs showed that the effect of
heating on yogurt microstructure was more pronounced
when milk base was enriched with WPC. This is not
surprising, as the cross-linking capacity of denatured
777
whey proteins plays a key role in yogurt structure after
heating. It contributes to an increase in the degree of
bridging between protein particles (Davies, Shankar,
Brooker, & Hobbs, 1978; Kalab, 1979; Mottar et al.,
1989). In addition, the presence of denatured whey
proteins on the surface of casein micelles would also
sterically restrain the approach of other casein particles
and thereby reduce the formation of dense clusters, as it
is observed in gels from unheated milks (Lucey, Tet Teo,
Munro, & Singh, 1998).
The current results show the comparative effects of
different types of protein enrichment on yogurt micro-
structure, an area which to date has received little
attention. Fig. 2 shows the microstructure of yogurt
obtained from four milk bases (SMP, WPC, CaCn or
NaCn) heated at 90 C for 5 min. In yogurt from SMP-
enriched milk base, the gel organization appeared to be
irregular, with short and individualized casein filaments.
There were numerous pores that were very heteroge-
neous in size. On the other hand, in WPC-enriched
yogurts, the gel exhibited a different structure with a
very fine network, containing numerous very small
pores. Chains of casein micelles were less apparent than
in SMP-enriched yogurts. The acid gel from the Ca
caseinate-enriched milk base showed a dense and finely
perforated microstructure, similar to that of yogurt
made with WPC. In contrast, the addition of Na
caseinate to the milk base resulted in a rather coarse
and loose structure, with apparent micelle chains and
large clusters. The micrograph showed a great hetero-
geneity in the pore sizes.
These results can be compared with those of Modler
and Kalab (1983) who observed large and extensively
fused micelles in yogurt when milk was fortified with Na
caseinate. This was responsible for an irregular micro-
structure with large clusters of fused particles. In
contrast, Modler and Kalab (1983) and Puvanenthiran,
Williams, and Augustin (2002) reported that the
addition of WPC led to a different structure where
micelles appeared in the form of individual entities
surrounded with finely flocculated protein and linked
with small whey protein aggregates. This structure
would increase the number of bonds between particles
and thus explain the dense and finely branched network
in yogurt from WPC-enriched milk base. The similar
fine microstructure found in yogurt made from Ca
caseinate-enriched milk base was surprising. The differ-
ence in structure between yogurts fortified with sodium
caseinate or calcium caseinate could arise from differ-
ences in micellar characteristics between the two case-
inates. However, there were little, or no, differences in
micelle size and solvation between milk bases fortified
with NaCn or CaCn. The possible role of cations
must also be emphasized. Ca++ and Na+ ions favor
protein–protein interactions of both whey protein and
casein, through electrostatic shielding and ion-specific
 ARTICLE IN PRESS
778 F. Remeuf et al. / International Dairy Journal 13 (2003) 773–782
SMP
NaCn
Fig. 2. Microstructure of yogurt obtained from milk bases enriched with SMP, WPC, NaCn, and CaCn, and heated at 90 C for 5 min. Bar—20 mm.
hydrophobic interactions (Cayot & Lorient, 1998;
O’Kennedy & Kelly, 2000). In gels obtained from Ca
caseinate-enriched milk base, calcium bridges could
further contribute to a notable increase of the cross-
linking density of the network. They could also sterically
restrain coalescence of the protein particles, leading to a
fine stranded gel structure. In contrast, Na+ ions
brought by enrichment of milk base with Na caseinate
could not lead to the formation of salt bridges. In gel
obtained from Na caseinate-enriched milk bases, inter-
actions between protein particles would be enhanced
without limitation to their fusion. This could be
responsible for a coarser structure of the gel.
3.3. Physical properties of the yogurts
3.3.1. Rheological behavior
The complex viscosity varied between 10 and 110 Pa s,
and the apparent viscosity between 1 and 4 Pa s (Fig. 3).
On average, for all types of protein enrichment, the
increase in apparent and complex viscosities of the
yogurts, when extending the heating time at 90 C from 1
to 5 min, reached 40% and 70%, respectively. These
levels of increase were comparable with those reported
in the literature. For instance, Lucey et al. (1997)
compared acid gels obtained from a milk base heated at
80 C and 85 C for 15 min, which corresponded to
denaturation levels for whey protein of 50% and 80%,
respectively. They noted an increase of 66% of G 0 when
heating temperature was brought from 80 C to 85 C.
However, the influence of heating on rheological
behavior was dependent on the type of milk fortifica-
tion. The yogurts manufactured with milk bases
WPC
CaCn
enriched with caseinates only had a low whey protein-
to-casein ratio (0.08), and were not affected by the heat
treatment. In contrast, extending the heating time at
90 C from 1 to 5 min resulted in higher viscosity in all
yogurts from milk bases having a high whey protein-to-
casein ratio (0.21–0.57).
The milk base containing CaCn and WPC at a ratio
of 1:2 (blend 2) produced yogurts with the highest
viscosity. On the other hand, the yogurt enriched with
SMP was the least viscous (decrease of more than 50%
as compared with blend 2). CaCn, WPC and NaCn
enrichment led to intermediate viscosity values which
were in the order: CaCnoWPCoNaCn. As previously
noted by Guinee et al. (1995), Cho, Lucey, and Singh
(1999) and GuzmanGonzalez et al. (2000), at similar
protein levels, the addition of caseinate instead of SMP
in the milk base, enhanced the yogurt viscosity strongly.
In our study, yogurt enriched with WPC had higher
viscosities than those enriched with SMP. These results
are different than those reported by several authors.
Guinee et al. (1995) obtained similar viscosities for
yogurts made with SMP or WPC (with protein levels of
B750 g kg
1). GuzmanGonzalez et al. (1999) and Cho
et al. (1999) showed higher viscosities when enriching
with SMP than WPC. In all these studies, the levels of
WPC or SMP added were such that the final protein
level was the same. According to Puvanenthiran et al.
(2002), this discrepancy between results from literature
could be related to variation in the WPC preparation,
which strongly influence their functionalities. Guinee
et al. (1995) indicated that for a given protein level, the
influence of WPC on rheological and syneretic proper-
ties of yogurt was very much dependent on the protein
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100
140

120
Complex viscosity (Pa.s)

Water holding capacity (%)

95
100

80
90
60

40
85
20

0
(A) SMP NaCn CaCn Blend 1 Blend 2 WPC 80
(A) SMP NaCn CaCn Blend 1 Blend 2 WPC
5
300

4
250
Apparent viscosity (Pa.s)

Graininess (grains.g-1)
3 200

2 150

100
1

50
0
(B) SMP NaCn CaCn Blend 1 Blend 2 WPC
0
Fig. 3. Effect of milk supplementation and heating on complex (A) (B) SMP NaCn CaCn Blend 1 Blend 2 WPC
and apparent (B) viscosities of yogurts made with different milk bases.
Fig. 4. Effect of milk supplementation and heating on WHC (A) and
Milk bases were enriched with SMP, NaCn, CaCn, WPC, or blends of
graininess (B) of yogurts made with different milk bases. Milk bases
CaCn and WPC (blend 1—WPC and CaCn at a ratio of 1:2; blend 2—
were enriched with SMP, NaCn, CaCn, WPC, or blends of CaCn and
WPC and CaCn at a ratio of 2:1). The milk bases were subjected to one
WPC (blend 1—WPC and CaCn at a ratio of 1:2; blend 2—WPC and
of the following heat treatments: 90 C for 1 min ( ) or 90 C for 5 min
CaCn at a ratio of 2:1). The milk bases were subjected to one of the
( ). The presented data correspond to one replication of each
following heat treatments: 90 C for 1 min ( ) or 90 C for 5 min ( ).
combination of milk supplementation and heating time.
The presented data correspond to one replication of each combination
of milk supplementation and heating time.

level of the WPC. This could indicate a possible
protective effect of increased lactose concentration
towards in situ denaturation of whey protein (Jelen &
Rattray, 1995). Furthermore, Cho et al. (1999) reported
an increase of elastic modulus from 13 to 80 Pa for
yogurt gel enriched with two kinds of WPC, having low
or high level of denaturation. The two heat treatments
applied to the milk base in our study were high, with a
level of denaturation of whey protein >50%. This
should induce a high bridging capacity of whey proteins
resulting in a viscosity-increasing effect similar to, or
higher than that, of caseinate.
The viscosity of stirred yogurts enriched with blends
of caseinates and WPC was only studied by Guzman-
Gonzalez et al. (2000). These authors found a strong
influence of the whey protein-to-casein ratio on yogurt
viscosity. Our results confirmed their hypothesis, show-
ing that yogurt viscosity was different between the two
blends tested. The viscosity obtained for yogurts
enriched with the blends of caseinates and WPC was
generally higher than the viscosity reached for yogurts
fortified with only CaCn or WPC. This suggested a
synergistic effect of casein and whey protein on yogurt
viscosity, and the probable occurrence of one or more
optimum values for the whey protein-to-casein ratio.
Furthermore, the properties of native whey protein and
casein were clearly different than those obtained after
membrane separation (WPC) or chemical treatment
(CaCn). With a similar whey protein-to-casein ratio
(0.21–0.25), the complex viscosity of the yogurts
enriched with the blend 1 was 1.5–5 times higher than
those of yogurts enriched with SMP.
3.3.2. Water-holding capacity
Milk fortification and heating had an effect on the
WHC of yogurts which varied from 84% to 92% (Fig.
4). The higher WHC was obtained for yogurts made
using milks fortified with SMP or with blends of
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780 F. Remeuf et al. / International Dairy Journal 13 (2003) 773–782
whey proteins and caseinate. The separate use of
caseinate or WPC to enrich milk gave yogurts with
lower WHC (less than 90%). The high DM content in
milk fortified with SMP could explain the high WHC in
yogurts made from this milk base (Harwalkar & Kalab,
1986). Our data showed a slight enhancement of the
WHC as the heating time of the milk at 90 C was
increased from 1 to 5 min; a similar trend was reported
by several authors (Dannenberg & Kessler, 1988;
Mottar et al., 1989; Augustin, Cheng, & Clarke, 1999).
It could be related to the higher solvation of the micellar
system and to a more branched yogurt microstructure,
less susceptible to losing water when submitted to
centrifugal forces.
3.3.3. Graininess
Enrichment of milk with SMP (Fig. 4) gave a very low
graininess in yogurts (B5 grains g
1). On the other
hand, the use of WPC or blend 2 led to a high level of
graininess, i.e. 50–250 grains g
1. Enrichment with case-
inate or blend 1 led to intermediate graininess
(o50 grains g
1 in yogurts manufactured with these
ingredients). Graininess increased considerably when
heating time was raised to 5 min, e.g., the graininess in
WPC yogurt increased from B75 to 250 grains g
1 on
extending the heating time at 90 C from 1 to 5 min.
According to these results, graininess seemed to be
linked to the amount of denatured whey protein in the
yogurt. One explanation of this phenomena could be
that acid gels made from severely heated milks involve a
two-step coagulation (O’Kennedy & Kelly, 2000).
Gelation of whey protein occurs as pH reaches B5.2
(Cayot & Lorient, 1998). Then, when pH decreases
below B4.8, gelation of casein particles begins, leading
to some rearrangements and local stresses in the protein
network, and to the appearance of small fractures in the
gel. Such fractures would provide grains in yogurt after
stirring (Lucey & Singh, 1998). This mechanism should
be greatly enhanced when the ratio of denatured whey
protein-to-casein increases, as was observed in the
present study.
3.4. Relations between yogurt microstructure and
physical properties
The degree of micelle aggregation or fusion in acid gels
is an important factor in determining the physical
properties of yogurt (Modler & Kalab, 1983; Mottar
et al., 1989). Micelle fusion is influenced by the whey
protein-to-casein ratio, and through the formation of
k-casein/b-lactoglobulin complexes. Our results suggest
that the higher degree of micelle solvation in yogurt from
WPC-enriched milk bases could retard micelle fusion, via
the absorption of whey protein at the micelle surface.
That could explain the fine, highly branched, and
homogenous microstructure observed in these gels.
In contrast, gels from NaCn- and SMP-enriched milk
bases had a coarse structure in which the degree of casein
micelles solvation was low. Thus, micelle fusion was more
marked in these gels, and more particularly in gel from
NaCn-enriched milk bases which were characterized by a
low whey protein-to-casein ratio. Consequently, these
gels exhibited a very coarse microstructure with thick
casein chains and a low WHC. A similar observation was
reported by Modler and Kalab (1983). The formation of
a fine and homogenous microstructure in yogurt from
milk enriched with CaCn could be ascribed to the
possible role of calcium bridges in limiting micelle fusion
(Augustin, 2000).
This relationship between the degree of micelle fusion
in an acid gel and its physical properties is also
supported by the observations made in this study
concerning the particle size in milk base. All milks
enriched with WPC, and highly heated, showed a
parallel increase in average micelle size, yogurt micelle
solvation and yogurt graininess. All these parameters
changed roughly in proportion to the quantity of WPC
added. The formation of large, and highly hydrated,
protein aggregates in the milk base enriched with WPC,
and highly heated, could contribute to restrain micelle
fusion during milk acidification. Thus, casein micelles
would be embedded in a denatured whey protein
network, limiting their possibilities of interaction and
fusion. The presence of two stages in the gel formation
(i.e. initial gelation of whey proteins followed by
coagulation of casein) is also suggested by the formation
of a high quantity of grains after stirring in yogurts
made from milk base enriched with WPC. Furthermore,
the results of the present study showed that average
micelle size in milk base might be used as a predictive
indicator of graininess in yogurt.
4. Conclusion
In the current work, the addition of SMP for milk
base preparation was considered as the reference process
for yogurt preparation. For the same total protein
content, five different types of protein enrichment gave
yogurts with higher viscosity than those from SMP-
enriched milk base. In contrast, the enrichment of milk
base with caseinates or WPC was responsible for the
development of some defects in yogurt, principally a low
WHC with caseinate addition, and a high graininess
with WPC addition. An interaction between heating
intensity and type of protein added was emphasized.
Some relationships between microstructure and physical
properties in yogurt could be inferred from our results.
However, in spite of similar microstructural properties,
yogurt from milk base enriched with NaCn showed
much higher values of viscosities and lower WHC than
yogurt from SMP-enriched milk base. This discrepancy
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F. Remeuf et al. / International Dairy Journal 13 (2003) 773–782
suggests that yogurt texture might also be influenced
by factors other than the ratio of casein to whey
protein.
Finally, our results showed that it is possible to
improve the viscosity of yogurt without causing graini-
ness defects by adding a blend of CaCn and WPC to
milk, instead of SMP. But further work seems necessary
to optimize milk fortification for yogurt manufacture,
and for a better understanding of the structure–function
relations underlying changes in yogurt texture.
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