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acidified through addition of the acidulant glucono-d-lactone (GDL). Our aim
is to understand the underlying mechanisms and to demonstrate how the
microstructure of a food material can be designed by controlling the colloidal
interactions between the casein particles in this model skim milk system.
19.2 Materials and Methods
19.2.1 Solution Preparation
The low-heat skim milk powder (SMP) (Caledonian Cheese Company, UK)
contained 35.6 wt.% protein and had a whey protein nitrogen index of 8 mg of
(undenatured) whey protein nitrogen per gram. Therefore, with respect to whey
protein denaturation, the reconstituted unheated skim milk could be regarded
as a satisfactory model for the behaviour of raw skim milk. The number-
average radius R
n
, the weight-average radius R
w
and the molecular weights, M
n
and M
w
, of the xanthan sample (Keltrol SF/NF; CPKelco, UK) were: R
n
¼
208
nm, R
w
¼
238 nm, M
n
¼
4.65
10
6
g mol
1
and M
w
¼
5.77
10
6
g mol
1
.
These values were determined by high-performance size-exclusion chromatog-
raphy (SEC) coupled with multi-angle laser light scattering (MALLS) and
differential refractometry.
Dispersions of low-heat SMP and XG were separately prepared in distilled
water under magnetic stirring at 501C (SMP) or 801C (XG). Sodium azide was
added to protect against microbial growth in the samples. After storage
overnight in a refrigerator (
51C), distilled water was added to adjust the
final concentration of the dispersions (17.5 wt.% SMP, 0.125 wt.% XG, 0.1
wt.% XG and dilutions thereof). For experiments performed with pre-heated
skim milk, the SMP dispersions (pH 6.54) were heated to 901C (within 4min),
held at 901C for 10 min, and then rapidly cooled to room temperature in a
batch-wise process in a double-walled reactor under constant stirring. The SMP
and XG dispersions were separately equilibrated in a water-bath at 401C for at
least 1 h, and then blended to obtain mixtures containing 14 wt.% SMP
(5 wt.% milk protein) + 0.005-0.025 wt.% XG. Blends of SMP dispersions
with distilled water served as reference (14 wt.% SMP + 0 wt.% XG). The
subsequent experiments were all carried out at 401C.
B
19.2.2 Phase Separation
The blends were produced in culture tubes (Pyrex, Bibby Sterilin, UK), kept in
a water-bath for at least 10 min, and then mixed for 1 min with a Vortex Genie
2 mixer (Scientific Industries, USA). The mixtures were then either loaded into
the temperature-controlled sample cell of the microscope or transferred to an
incubator. Phase separation was observed at the natural pH of the mixtures.
Initial microscopic phase separation was investigated by confocal laser
scanning microscopy (CLSM) with an upright Zeiss Axioplan 2 microscope
equipped with the LSM 510 confocal unit (Carl Zeiss, Germany), operated in
reflection mode at 488 nm with a 40
objective LD Achroplan. The focus
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