A comparison of the formation, rheological

properties and microstructure of acid skim milk

gels made with a bacterial culture or glucono--
lactone
J. A. Lucey,* M. Tamehana, H. Singh & P. A. Munro
Institute of Food, Nutrition and Human Health, Massey University, Palmerston North, New Zealand
Gels were made by acidication of milk with glucono--lactone (GDL) or a
starter culture, at two gelation temperatures. Rheological properties and micro-
structure of these gels were determined using oscillatory rheometry, permeability
and confocal laser scanning microscopy. On addition of GDL to milk the pH
decreased rapidly and it stabilized at pH $4.6. After the addition of starter cul-
ture to milk initially the pH decreased slowly and then decreased steadily but
stabilized at pH $4.0. GDL-induced gels had much shorter gelation times but
higher storage moduli (G
/
), yield stresses and strains, permeability and whey
separation than gels formed by a bacterial culture. Gels formed at 42

C had
shorter gelation times but higher pH at gelation, G
/
, permeability and whey
separation. Loss tangent of all gels increased to a maximum shortly after gelation.
Microstructure of gels formed with a bacterial culture was not greatly aected by
gelation temperature in contrast to GDL-induced gels. #1999 Canadian Institute
of Food Science and Technology. Published by Elsevier Science Ltd. All rights
reserved
Keywords: acid milk gels, yoghurt, rheology, microstructure, permeability, whey
separation, yield stress
INTRODUCTION
Fermented milk products are produced throughout the
world with yoghurt being one of the most popular. Milk
is acidied by bacterial cultures, which ferment lactose
to lactic acid. The manufacture of fermented milks has
previously been reviewed (Tamime and Robinson, 1988;
Robinson and Tamime, 1993; Mulvihill and Gruerty,
1995; Tamime and Marshall, 1997). In the production
of yoghurt the culture consists of Lactococcus del-
brueckii subsp. bulgaricus and Streptococcus salivarius
subsp. thermophilus. It is generally accepted that the
type and character of the starter organisms used in the
production of fermented milks are important factors
determining the overall quality of the nal product
(Tamime and Marshall, 1997). Milk acidication has
been simulated by the use of glucono--lactone (GDL)
where the hydrolysis of GDL to gluconic acid results in
a reduction in pH. GDL has been extensively used to
model the acidication of milk although no detailed
comparison of the rheological, microstructural and
physical properties of gels made by GDL or bacterial
fermentation has been reported.
There have been several reports on the rheological
properties of acid milk gels formed by the use of GDL
(e.g., Arshad et al., 1993; Cobos et al., 1995; van Vliet
and Keetels, 1995; Lucey et al., 1997a,b) or bacterial
fermentation (Biliaderis et al., 1992; Ro nnega rd and
Dejmek, 1993) but very few studies have compared the
Food Research International, Vol. 31, No. 2, pp. 147155, 1998
# 1999 Canadian Institute of Food Science and Technology
Published by Elsevier Science Ltd. All rights reserved
Printed in Great Britain
P I I : S 0 9 6 3 - 9 9 6 9 ( 9 8 ) 0 0 0 7 5 - 1 0963-9969/99/$see front matter
147
*To whom correspondence should be addressed. Tel.: 0064 6
350 5034; fax: 0064 6 350 5655;
e-mail: J.A.Lucey@massey.ac.nz
properties of gels formed by the two methods (van
Marle and Zoon, 1995). The use of GDL in model stu-
dies avoids some of the diculties associated with star-
ter bacteria including variable activity and variation
with type of culture used (e.g., ropy or non-ropy).
Model studies on the formation of acid milk gels with
GDL have normally used a low gelation temperature
(30

C) compared with the usual temperature used for

yoghurt fermentation, which usually ranges from 40 to
45

C.
The objective of the present study was to determine
the eect of two types of acidifying agents (GDL and a
bacterial culture) on the rheological properties, perme-
ability, whey separation and microstructure of acid skim
milk gels. The bacterial culture used for these experi-
ments has been used in previous studies (e.g., Dannen-
berg and Kessler, 1988a,b) and is a fast acidifying,
mixed-strain, non-ropy culture. Two gelation tempera-
tures were used corresponding to the high temperatures
used in yoghurt manufacture and the low temperatures
often used for model studies with GDL or the (slow)
overnight incubation of fermented milks.
MATERIALS AND METHODS
Materials
Low-heat skim milk powder (SMP) was supplied by the
New Zealand Dairy Board (Wellington). The whey
protein nitrogen index of the low-heat SMP was 6.4 mg
undenatured whey protein/g powder. GDL was sup-
plied by Sigma Chemical (St Louis, MO 63178, USA).
Starter culture was supplied by Wiesby Starter Cultures
and Media (D-25899 Niebu ll, Germany).
Reconstitution of skim milk powder and heat treatment
Reconstituted skim milks were prepared by adding 12 g
SMP to 100 g demineralized water. The solutions were
stirred for at least 2 h at 25

C and for the GDL-

induced gels 0.02% (w/w) NaN
3
was added to prevent
bacterial growth. For the whey separation and perme-
ability tests, reconstituted skim milks (>800 ml) were
heated to 85

C in an pilot-scale indirect UHT plant

(Alfa-Laval, Melbourne, Australia) and held for 30
min by transferring the milks to beakers which were
placed in a thermostatically controlled water bath.
The samples were covered with aluminium foil to
prevent evaporation and gently stirred. When smaller
samples (e.g., 100 ml) were required, milks in covered
beakers were placed in water baths at 85

C for 30
min. The time reported included that time required for
the milks to come up to temperature, which was nor-
mally 23 min. After holding for 30 min, milks were
rapidly cooled to the gelation temperature by
immersion in ice-water.
GDL-induced acidication
Heated skim milks were acidied with 1.3% (w/w) GDL
at 30 or 42

C. The pH of the samples was regularly

measured with a combination electrode (Radiometer
model pHC2401, Copenhagen, Denmark) attached to a
model PHM 84 Radiometer pH meter (Copenhagen,
Denmark).
Bacterial fermentation
A mother culture was prepared by adding 7 mg freeze-
dried culture (Joghurt 709, which is a mixture of Lacto-
coccus delbrueckii subsp. bulgaricus and Streptococcus
salivarius subsp. thermophilus), to 100 g sterilized,
reconstituted skim milk. The milk was incubated at
35

C for 16 h, when the pH was 4.6. Heated milk was

inoculated with 2% (w/w) mother culture and incubated
at 30 and 42

C for 15 and 6 h, respectively.

Rheological properties
Acid casein gels are viscoelastic and their low strain
rheological properties can be determined by low ampli-
tude dynamic oscillation, resulting in the measurement
of the storage modulus (G
/
) and loss tangent (tan )
(Roefs et al., 1990a; Lucey et al., 1997a). A controlled
stress, model UDS 200 Physica Rheometer (Physica
Messtechnik GmbH, D-70567 Stuttgart, Germany) was
used. A cup and bob measuring geometry (Z3 DIN)
consisting of two coaxial cylinders (diameters 25 and
27.1 mm) was used. On addition of GDL or mother
culture to the milk, the mixture was stirred for 2 min
and then 17 ml of the mixture was transferred to the
rheometer. The cup and bob of the rheometer were dis-
infected with ethanol (96%) before inoculated milk was
added to the rheometer. Vegetable oil was added on the
milk surface to prevent evaporation. The gap between
the bottom of the bob and the cup was set to 2 mm. An
applied strain of 0.01 was used, which is well within the
linear viscoelastic range of acid milk gels (van Marle
and Zoon, 1995). Samples were oscillated at a frequency
of 0.1 Hz and measurements were taken every 10 min
for 15 h. Gelation was dened as the point when gels
had a G
/
of 51 Pa (van Marle and Zoon, 1995). The
eect of the time scale of deformation on the rheological
properties was determined by a frequency sweep at 6
and 15 h for gels formed at 42 and 30

C, respectively;
frequency was varied from 0.0011.0 Hz.
The large deformation properties of acid milk gels
were also studied by the method described by Lucey et
al. (1997a,b). Gels were made in situ as above and
sheared after ageing for 15 and 6 h, for gels formed at
30 and 42

C, respectively. Gels were subjected to a

constant shear rate (0.00185 s
1
) up to yielding of the
gel, dened as the point when the shear stress started to
decrease (Fig. 1).
148 J. A. Lucey et al.
Measurement of the degree of whey separation
The method recently described by Lucey et al. (1998a)
was used. Gels were made in 250 ml (grade A) glass
volumetric asks (E-mil, UK) that were lled to just
below the base of the neck (&225 g). The asks were
examined after 6 and 15 h for gels formed at 42 and
30

C, respectively, to see if any whey had collected on

the top or around the sides of the gel. Any free whey
was gently poured o and weighed. The gel was allowed
to stand for approximately 1 min and any further sur-
face whey was decanted and weighed. The extent of
whey separation was expressed as a percentage of the
total weight of the milk. Eight asks were used for each
treatment.
Measurement of the permeability coecient
The method previously described by Roefs et al.
(1990a); Lucey et al. (1998c), was used to determine the
permeability coecient (B) of gels as follows
B = ln
(h
o
h
t
2
)
(h
o
h
t
1
)
!
Ha[&g(t
2
t
1
)]Y
where B is the permeability coecient, h
o
is the height
of the whey in the reference tube, h
t
1
is the height of the
whey in the gel tube at time t
1
, h
t
2
is the height of the
whey in the gel tube at time t
2
, is the viscosity of the
whey, H is the length of the gel, & is the density of the
whey and g is acceleration due to gravity. Permeability
measurements were made at the gelation temperature
and when the pH of the samples was 4.6. Gels made
with GDL at 42

C often had cracks or the gel came

away from the sides of the glass tubes; these tubes were
not used to calculate B. The viscosity of the acid whey
at 30 and 42

C was assumed to be 0.95 and 0.77 mPa/s,

respectively. All experiments were done in triplicate and
means and standard deviations are reported where
appropriate.
Confocal scanning laser microscopy (CSLM)
The use of CSLM for evaluating the microstructure of
acid milk gels has recently been reported (Lucey et al.,
1997c, 1998c). The uorescent protein dye, Fast Green
FCF (Merck, 64271 Darmstadt, Germany), was dis-
solved in demineralized water and several drops were
added to 50 ml of milk. The milk was stirred prior to
addition of GDL or culture. After stirring for approxi-
mately 5 min a few drops of the mixture were trans-
ferred to special object glasses with a cavity and a
coverslip placed over the sample. The object glass was
then placed in a petri-dish and held in a temperature
controlled room at 30

C for approximately 16 h. The

gels were examined on a Leica TCS 4D confocal
microscope (Leica Lasertechnik GmbH, Heidelberg,
Germany) with a 100oil immersion objective (numer-
ical aperture=1.4). The CSLM had an air cooled Ar/Kr
laser that was used with an excitation wavelength of 568
nm. All experiments were done in duplicate, many elds
were viewed and typical micrographs are presented.
RESULTS
pH proles
The pH versus incubation time prole of milk acidied
with GDL was dierent from that acidied with bac-
terial cultures (Fig. 2); GDL was rapidly hydrolysed to
gluconic acid, especially at high temperatures, resulting
in a rapid reduction in pH initially. In contrast, after the
addition of starter bacteria to milk at 30

C the pH
changed slowly for 15 ks but thereafter the pH
decreased steadily (Fig. 2(a)); at 42

C starter bacteria
produced acid faster than at 30

C (Fig. 2(b)). The nal

pH (4.6) that was attained in GDL-induced gels was a
function of the amount of the lactone added to milk
whereas starter bacteria continued to produce acid until
a pH of 4.0 was attained when bacteria presumably
became inhibited by the low pH; in practice fermented
milks are cooled when sucient acidity has been devel-
oped. A higher incubation temperature resulted in faster
reduction in pH for both types of gels.
Amice-Quemeneur et al. (1995) compared the pH
proles during the acidication of milk at 42

C by the
addition of 2% starter culture and at 30

C by the addi-
tion of 3% GDL. They also found that on addition of
GDL to milk the pH rapidly decreased but later the pH
decreased slowly. On addition of starter culture to milk
there was a period when the pH did not change very
much, in agreement with the results of Amice-Queme-
neur et al. (1995).
Fig. 1. Shear stress as a function of applied deformation in
constant shear rate (0.00185 s
1
) experiment on acid milk gel.
Milk was heat treated at 85

C for 30 min and gelled at 30

C
by the addition of 1.3% glucono--lactone (GDL). The point
when the shear stress started to decrease was taken as the yield
point.
A comparison of the formation, rheological properties and microstructure of acid skim milk gels 149
Rheological properties
High incubation temperature resulted in a reduction in
the gelation time (Table 1). GDL-induced gels had
much shorter (45 fold) gelation times compared with
bacterial gels. For example, at 30

C the gelation times

for GDL and bacterial gels were 100 and 490 min,
respectively. This trend is in agreement with the results
of van Marle and Zoon (1995) who found that GDL-
induced gels had a shorter gelation time compared with
gels formed with a non-ropy bacterial culture. A high
gelation temperature resulted in an increase in the pH of
gelation, in agreement with the results of Heertje et al.
(1985). At 30

C GDL-induced gels had a higher pH at

gelation compared with bacterial gels.
The rheological properties of acid gels as a function
of time are shown in Fig. 3. Low gelation temperature
resulted in slower gel formation but higher values of the
G
/
of the aged gel, in agreement with the results of
Lucey et al. (1997b) for acid casein gels and Arshad et
al. (1993) for acid milk gels. GDL-induced gels had
higher G
/
values than the bacterial gels (1.61.7 fold
higher), for both incubation temperatures (Table 1).
The G
/
of bacterial gels made at 42

C decreased slightly
approximately 4 h (pH44.2) after the inoculation of
milk (Fig. 3(b)). In both types of gels made at 42

C
there was an abrupt change in the slope of G
/
versus
time curve, which occurred soon after gelation; a similar
trend was observed by Ro nnega rd and Dejmek (1993).
At the point of gelation when G
/
started to increase, tan
initially decreased to values <0.5 but then increased
and a ``maximumin tan '' was observed at pH5.1. This
transition occurred between pH 5.3 and 5.0. The value of
this ``maximumin tan '' was higher (and occurred over a
longer period) in gels formed with GDL and at high
incubation temperature (Table 1). A``maximumin tan ''
was also observed just after gelation (pH values 5.25.0)
by Biliaderis et al. (1992), Ro nnega rd and Dejmek (1993);
van Marle and Zoon (1995) for microbially acidied high
heat-treated milk samples. This ``maximum in tan '' in
gels made at 42

Ccoincided with the abrupt change in the

slope of the G
/
versus time curve.
The rheological properties of acid gels depended on
the time scale of the applied deformation. Plotting log
G
/
versus log frequency gave reasonably straight lines
with a slope of 0.15 (Fig. 4(a)); similar slopes were
reported by Lucey et al. (1997a) for acid gels made from
heated milk. Tan decreased slightly with increasing
frequency except for GDL gels made at 42

C where tan
Fig. 2. Changes in pH during the acidication of heated milk
at 30 (a) and 42

C (b). Milk was acidied with (&) 1.3% (w/

w) glucono--lactone (GDL) or (*) 2% (w/w) starter culture.
Heat treatment of milk was 85

C for 30 min. Error bars

represent the standard deviation for three replicates.
Fig. 3. Storage modulus (G
/
)( ) and loss tangent (tan
) () of acid gels made by acidication of heated milk at
30

C (a) and 42

C (b) with (*) 1.3% (w/w) glucono--lactone

(GDL) or (V) 2% (w/w) starter culture. Heat treatment of
milk was 85

C for 30 min. Values are means from triplicate

experiments.
150 J. A. Lucey et al.
had a very low value at low frequencies and increased
with increasing frequency (Fig. 4(b)). Lucey et al. (1997a)
reported that the tan of GDL-induced milk gels made
at 30

C from heated milk decreased slightly with

increasing frequency. A low value of tan at low fre-
quency for GDL-induced sodium caseinate gels formed
at 40

C was recently reported by Lucey et al. (1997b).

The yield properties of acid milk gels are given in
Table 1. The yield stress of GDL-induced gels formed at
30

C was higher than those of gels formed at a higher

gelation temperature; a similar trend was reported by
Lucey et al. (1997b) for acid casein gels. However, there
were only small dierences in the yield stress or shear
deformation (strain) at yielding of bacterial gels formed
at 30 and 42

C. The yield stress and strain were higher

for gels formed with GDL than bacterial gels.
Structural properties
Confocal laser scanning micrographs of acid milk gels
made at 30 and 42

C using GDL and a bacterial culture

are shown in Fig. 5. GDL-induced gels made at 30

C
appeared to have a branched interlinked type of micro-
structure (Fig. 5(a)), while gels made at 42

C appeared
to have much less branching or cross-linking, thinner
strands and larger pores (Fig. 5(c)). Gels made with a
bacterial culture (Fig. 5(b) and (d)) appeared to have a
more tortuous or clustered type of network with less
obvious cross-linking than GDL-induced gels made at
30

C. Gels made with a bacterial culture appeared to

have thicker strands and clusters of aggregated particles
compared with GDL-induced gels. There were less
Table 1. Properties
a
of acid gels made from milk heated at 85

C for 30 min by acidication with 1.3% (w/w) glucono--lactone (GDL)

or by the addition of 2% (w/w) bacterial culture
GDL Culture
Incubation temperature Incubation temperature
30

C 42

C 30

C 42

C
Gelation
b
time (ks) 5.90 0.2 1.62 0.0 29.47 4.9 7.60 1.0
Gelation
b
pH 5.28 0.04 5.54 0.04 5.05 0.06 5.58 0.04
Storage modulus
c
, G
/
(Pa) 439 31 319 32 257 23 194 17
Maximum in tan
d
0.416 0.003 0.528 0.004 0.395 0.003 0.508 0.005
Fracture
e
strain 0.75 0.08 0.69 0.07 0.36 0.08 0.48 0.03
Fracture
e
stress (Pa) 190 30 124 18 41 14 46 7
Permeability coecient (10
13
m
2
) 1.64 0.15 6.51 2.88 1.07 0.07 3.04 0.83
Whey separation
f
(%) 16.30 1.16 18.48 1.29 7.28 0.66 8.41 0.60
a
Mean and standard deviations of triplicate experiments.
b
Gelation time was dened as the point when gels had a G
/
51 Pa.
c
G
/
value at 6 and 15 h for gels formed at 42 and 30

C, respectively.
d
Maximum in tan was dened as the point after gelation when the value of tan increased to a maximum.
e
Fracture was dened as the point when the shear stress started to decrease when gels were subjected to a constant shear rate
(0.00185 s
1
).
f
Whey separation was determined at 6 and 15 h for gels formed at 42 and 30

C, respectively.
Fig. 4. Storage modulus (G
/
) (a) and loss tangent (tan d) (b) of
acid milk gels as a function of frequency. Gels were made from
heated milk by acidication with glucono--lactone (GDL) at
30 (*) and 42

C (*) or by the addition of 2% (w/w) starter

culture at 30 (!) and 42

C (V). Heat treatment of milk was

85

C for 30 min. Values are means from duplicate experiments.

A comparison of the formation, rheological properties and microstructure of acid skim milk gels 151
obvious dierences in the microstructures of bacterial
gels formed at dierent gelation temperatures (Fig. 5(b)
and (d)). Bacteria were not observed in the micrographs
of bacterial gels possibly because Fast Green FCF may
be less suitable than dyes such as Acridine Orange for
their visualization.
The permeability of acid gels is given in Table 1.
GDL-induced gels had higher B than bacterial gels at
both incubation temperatures. van Marle and Zoon
(1995) also found that GDL-induced gels had a con-
siderably higher permeability compared with bacterial
gels suggesting that the size of the largest pores were
dierent. van Marle (1998) reported that bacteria ll
some of the pores and cavities in yoghurt gels and
reduce their permeability. The B of gels made at 42

C
was higher than those made at 30

C, especially for
GDL-induced gels; a similar trend was reported by
Lucey et al. (1997c) for GDL-induced casein gels.
GDL-induced gels had much higher levels of whey
separation than bacterial gels (Table 1). Whey separa-
tion increased slightly for both types of gels at high
gelation temperature; a similar trend was reported by
Lucey et al. (1998a) for GDL-induced milk gels.
DISCUSSION
The present study clearly showed that most of the
rheological and physical properties of GDL-and bacte-
rially induced gels (e.g., gelation time and pH, G
/
, yield
stress and strain, B, degree of whey separation and gel
microstructure) were dierent. By contrast, van Marle
and Zoon (1995) found, in acid milk gels made at 32

C
that the G
/
values of GDL-induced gels (1.95% GDL,
pH 5.35) after ageing for approximately 7 h were
similar to bacterial gels that were aged for 16 h. A likely
Fig. 5. Confocal scanning laser micrographs of acid milk gels formed by acidication of heated milk with (a) 1.3% (w/w) glucono-
d-lactone (GDL) at 30

C, (b) 2% (w/w) starter culture at 30

C, (c) 1.3% (w/w) glucono--lactone (GDL) at 42

C, (d) 2% (w/w)
starter culture at 42

C. Heat treatment of milk was 85

C for 30 min. Scale bar=10 mm. The protein matrix appears white while
pores appear dark.
152 J. A. Lucey et al.
explanation for these contrasting results is the ageing
time used for comparing the G
/
value; the G
/
value of
GDL-induced gels continued to increase even after
incubation for 7 h at 30

C and was still increasing at 16

h (Fig. 3) while the G
/
value for bacterially induced gels
did appear to be starting to atten at approximately 16
h. At the higher GDL concentration and slightly higher
incubation temperature used by van Marle and Zoon
(1995) it is possible that their GDL-induced gels had
reached a plateau at 7 h.
The explanation for the observed dierences between
GDL and bacterial gels presumably lies with the very
dierent mode and rate of acidication for these two gel
systems (Fig. 2). The rate of acidication of milk may
aect the solubilization of colloidal calcium phosphate,
dissociation of caseins, rate of aggregation as well as the
time available for rearrangements of the aggregating
protein particles. It should be noted that there are a
number of other crucial dierences between GDL- and
bacterially induced gels, e.g., starter bacteria or their
exopolysaccharides may contribute to the rigidity of the
gel network. The nal pH of GDL-induced gels is stable
(e.g. 4.6 in the present study) which allows a long
ageing time near the isoelectric point and this may assist
in the continued fusion and rearrangement of casein
particles. In contrast, bacterially induced gels may only
have a limited time near the isoelectric point as they con-
tinue to produce acid until they become inhibited by the
lowpHor the gel is cooled to slowtheir growth. The much
shorter ageing time near the isoelectric point of casein
could partly explain the lower G
/
and yielding properties
of bacterially induced gels compared to GDL gels.
We suggest that the mode of acidication of acid milk
gels may aect the degree of rearrangement that aggre-
gating particles may undergo at an early stage of the
gelation process. It has recently been reported that
GDL-induced casein gels can undergo extensive rear-
rangements at the particle level leading to the formation
of dense clusters during the aggregation stage (Lucey et
al., 1997c; van Vliet et al., 1997). Greater rearrangement
of aggregating particles should lead to a higher perme-
ability, larger pores, lower G
/
and lower yield stress and
strain (Lucey et al., 1997b,c) and indeed this occurred in
GDL-induced gels made at high temperature.
High tan values favour rearrangements of the gel
network (van Vliet et al., 1991). The large value for the
``maximum in tan '' in GDL-induced gels made at high
temperature would also indicate that rearrangements
were occurring during the early stages of the gelation
process. It is possible that bacterially induced gels
undergo considerably less rearrangements even at high
gelation temperatures as indicated by the lower value
for this ``maximum in tan ''.
Lucey et al. (1998b) suggested that the ``maximum in
tan '' reected a transition from an acid milk gel
initially dominated by denatured whey protein-induced
interactions (in particular denatured whey proteins
associated with casein micelles) at a high pH to a net-
work dominated by casein-casein interactions at low pH
values (e.g., 45.0). This transition may alter the
mechanical properties of the network especially as the
pH decreases (Lucey et al., 1998b). Roefs et al. (1990b)
also found that casein gels made by a combination of
acidication and rennet action exhibited a ``maximum
in tan '' at pH 5.2 and they suggested that at this pH
value there was a transition from gels with a rennet-type
character to an acid-type character (i.e., gels with dif-
ferent mechanical properties).
The substantial increase in tan just after the forma-
tion of gels made from heated milks may indicate an
increased susceptibility of bonds to break/relax which
would increase the propensity for structural rearrange-
ments and aect properties like whey separation and
syneresis (Lucey et al., 1998c). Acid gels made from
heated milk with GDL can have visual cracks and
rough surfaces (Lucey et al., 1998c).
It is often considered that faster acidication (e.g., at
high temperatures) would lead to the formation of
coarser networks. At low temperatures, casein particles
will have higher voluminosities and are probably more
deformable than at higher temperature due to less
hydrophobic interactions, which would allow particles
to aggregate with a larger number of protein-protein
bonds between any two particles, thereby causing less
rearrangement of the particles during gel formation at
low temperature (Lucey et al., 1997b). The low G
/
of
acid casein gels formed at high temperatures was
attributed to extensive particle rearrangements during
gel formation, resulting in the formation of dense clus-
ters of aggregated particles, which in turn aggregate to
form a gel (Zoon et al., 1988; Lucey et al., 1997b). The
microstructure of GDL-induced gels formed at high
temperature (Fig. 5(b)) does appear to be less con-
tinuous with many particles hardly contributing to the
rigidity of the network. In addition, GDL-induced gels
formed at high temperature had very high B indicating
the presence of large pores. Gelation temperature had
less pronounced eects on the rheological and micro-
structural properties of bacterial gels.
Many of the properties (e.g., G
/
, yield stress, perme-
ability and microstructure) of bacterially-induced gels
were also aected by gelation temperature but to a les-
ser extent than GDL-induced gels. This is presumably
due to the greater temperature dependence of the
hydrolysis reaction of GDL (Fig. 2). This limitation
with the use of GDL for simulating microbial acidica-
tion has been noted previously (Heertje et al., 1985;
Amice-Quemeneur et al., 1995; Bouzar et al., 1997).
There was an increase in G
/
(or stiness) with time for
all gels except the bacterial gels formed at 42

C when G
/
started to decrease once the pH of the gel approached
4.0. The increase in G
/
of gels can be attributed to
increased fusion of particles and clusters (and casein
molecules inside particles) due to rearrangement of both
A comparison of the formation, rheological properties and microstructure of acid skim milk gels 153
inter- and intra-molecular forces (Roefs et al., 1990a;
Lucey et al., 1997b). It may also be possible that the
slow increase in G
/
of acid gels after gelation reects the
incorporation of additional protein clusters into the gel
network (Lucey and Singh, 1997). The decrease in G
/
at
very low pH may be due to an increase in net positive
charge between casein molecules resulting in an increase
in electrostatic repulsion.
Factors that aect the yielding properties of gels
include the number of bonds per cross-section of the
strand, the strength of each bond (van Vliet et al., 1991)
and the tortuosity of the gel network (Bremer et al.,
1990). The method and temperature of gel formation
alters the microstructure of acid gels (Fig. 5), which
would aect the mechanical properties. GDL-induced
gels made at 30

C appeared to have numerous cross-

links and a high degree of interconnectivity (Fig. 5(a)),
which is in agreement with the very high stiness (G
/
)
and yield stress for this gel. Bacterial gels were much
more tortuous with less apparent cross-linking than
GDL-gels. Gelation temperature had a large eect on
the microstructure of GDL-induced gels but not bac-
terial gels. The reduction in the yield stresses and strains
in gels formed with GDL at high temperature may
make these gels more brittle and susceptible to rearran-
gements (after gel formation), compared with gels
formed at low temperature. The larger pores observed
in GDL gels made at 42

C (Fig. 5(c)) could provide

weak spots in the gel and lessen the yield stress as well
as increasing the permeability and whey separation,
compared with gels formed at low temperature.
The results of the present study suggest that there
are dierences in the rheological and physical proper-
ties of acid gels made with GDL or by bacterial fer-
mentation. Since GDL is often used to mimic
bacterial fermentation these results suggest that con-
clusions from model studies should be veried in
experiments using bacterial cultures. A limitation of
this study is that only one (non-ropy) strain was used;
further work could be performed to conrm these
conclusions with other strains.
ACKNOWLEDGEMENTS
This work was supported by the Foundation for
Research, Science and Technology, New Zealand. The
authors wish to thank Crop and Food Research, Pal-
merston North, for the use of their Physica Rheometer
and Miche le Lonnee for assistance with some of the
experimental work.
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(Received 11 May 1998; accepted 8 September 1998)
A comparison of the formation, rheological properties and microstructure of acid skim milk gels 155