Internal structure and colloidal behaviour of covalent whey protein microgels
obtained by heat treatment
Christophe Schmitt,
*
a
Christian Moitzi,
b
Claudine Bovay,
a
Martine Rouvet,
a
Lionel Bovetto,
a
Laurence Donato,
a
Martin E. Leser,
a
Peter Schurtenberger
b
and Anna Stradner
b
Covalently cross-linked whey protein microgels (WPM) were produced without the use of a chemical
cross-linking agent. The hierarchical structure of WPM is formed by a complex interplay of heat
denaturation, aggregation, electrostatic repulsion, and formation of disulfide bonds. Therefore, well-
defined spherical particles with a diameter of several hundreds of nanometers and with relatively low
polydispersity are formed in a narrow pH regime (5.8–6.2) only. WPM production was carried out on
large scale by heating a protein solution in a plate-plate heat exchanger. Thereafter, the microgels were
concentrated by microfiltration and spray dried into a powder. The spherical structure of the WPM was
conserved in the powder. After re-dispersion, the microgel dispersions fully recovered their initial
structure and size distribution. Due to the formation of disulfide bonds the particles were internally
covalently cross-linked and were remarkably stable in a large pH range. Because of the pH dependent
charge of the constituents the particles underwent significant size changes upon shifting the pH. Small
angle X-ray scattering experiments were used to reveal their internal structure, and we report on the
pH-induced structural changes occurring on different length scale. Our experiments showed that close
analogies could be drawn to internally cross-linked and pH-responsive microgels based on weak
polyelectrolytes. WPM also exhibited a pronounced swelling at pH values below the isoelectric point
(IEP), and a collapse at the IEP. However, in contrast to classical microgels, WPM are not build up by
simple polymer chains but possess a complex hierarchical structure consisting of strands formed by
clusters of aggregated denatured proteins that act as primary building blocks. They were flexible
enough to respond to changes of the environment, and were stable enough to tolerate pH values where
the proteins were highly charged and the strands were stretched.
Introduction
Polymer-based nanogels and microgels attracted noticeable
research interest during the last decade because of their wide
range of potential applications as for example controlled drug
delivery, immunosensing, protein purification, optics
manufacturing or tissue engineering.
1,2
These colloidal particles
are generally produced by emulsion or dispersion polymerisation
of activated monomers in the presence of a specific solvent or
reaction limiting secondary polymers (e.g. steric stabilizers).
Microgels generally fall in the 100 to 1000 nm diameter size range
as determined by microscopy or scattering techniques.
3
An interesting class of polyampholyte microgels is obtained
when a mixture of polyelectrolytes carrying carboxylic and
amino groups are used as monomers.
4
This leads to stimuli
responsive microgels exhibiting pH- and salt-sensitive colloidal
properties such as aggregation, shrinking or swelling.
5–10
Besides the various synthetic polyelectrolytes available for the
production of polyampholyte microgels, an interesting source are
food grade proteins such as bovine serum albumin (BSA) or egg
albumin.
3
In this respect, it is worth mentioning that some
protein microgels already exist naturally such as the well-known
casein micelles.
11
The latter are cross-linked with calcium-phos-
phate bridges and are therefore acid sensitive. However, they can
be further stabilized by covalent cross-linking using enzymatic
treatment with transglutaminase.
12
Several studies also report on
the use of the bovine whey proteins for production of nanogels
(diameter around 60 nm) using the desolvation method for
delivery purposes.
13,14
Very recently, it was shown that stable dispersions of protein-
based particles were obtained upon fast heating (> 26
C/min) of
a 1 wt% solution of demineralized b-lactoglobulin (b-lg), the
major whey protein in cow milk, in a very narrow pH range of
5.8–6.2.
15
Similar structures were already described when a whey
protein isolate was heated in the same conditions in the pH 6.0–
6.4 range.
16
These microgels were characterized by a hydrody-
namic radius ranging around 100–125 nm, a low polydispersity
index (below 0.2) and a spherical shape.
15,17
They represent an
intermediate aggregation state of b-lg between the already
described particulates (> 1 m m) which are formed at the protein
isoelectric point (IEP) at pH 5.2
18–20
and small fractal aggregates
(< 100 nm) which are obtained for pH values larger than 6.6.
21–23
The self-limited aggregation of b-lg microgels within this narrow
pH window can be explained by an equilibrium between attrac-
tive hydrophobic forces arising from protein denaturation and
repulsive forces arising from the protein net charge.
15
a
Nestl
e Research Center, Department of Food Science and Technology,
Vers-chez-les-Blanc, P.O. Box 44, CH-1000 Lausanne 26, Switzerland.
E-mail: christophe.schmitt@rdls.nestle.com; Fax: + 41 21 785 85 54;
Tel: + 41 21 785 89 36
b
Adolphe Merkle Institute, University of Fribourg, Route de l’Ancienne
Papeterie, P.O. Box 209, CH-1723 Marly 1, Switzerland
1
Published in
which should be cited to refer to this work.
http://doc.rero.ch
Based on the results obtained with b-lactoglobulin, the aim of
the present study is to explore analogies to synthetic microgels in
order to better understand the complex food colloid. Covalent
protein microgels were synthesised using a commercial whey
protein isolate as the starting material.
24
A powdered ingredient
was produced on large scale by combination of fast heat treat-
ment in a plate-plate heat exchanger, microfiltration and spray
drying. Thereafter, the powdered whey protein microgels (WPM)
were re-dispersed in water and thoroughly characterized for their
colloidal properties as a function of pH, i.e. nature of internal
cross-links, solubility, z-potential, size, and internal structure.
Experimental
Preparation of the whey protein microgel dispersion
A 50 kg batch of WPM powder was produced. This batch was
obtained by heat treatment of a dispersion of whey protein
isolate, WPI (Prolacta 90, Lactalis, Retiers, France) at 4 wt%
protein in softened water (160 mg.L
1
Na
+
) at pH 5.9 0.05
(natural pH 6.48 adjusted with 1 M HCl). The WPI dispersion
was pre-heated to 60
C and then heated to 85
C using a Soja
plate-plate heat-exchanger (PHE) operating at a flow rate of 1000
L.h
1
, followed by a holding time of 15 min in a tubular heat
exchanger and subsequent cooling to 4
C. Under these oper-
ating conditions, the Reynolds number Re was approx. 1,500
ensuring a laminar flow in the PHE. More than 85% of the initial
proteins were converted into WPM (determined by absorbance
measurements at 278 nm after removal of the WPM by centri-
fugation at 26,900g for 20 min). They exhibited a hydrodynamic
radius of 136 7 nm and a polydispersity index of 0.1 (deter-
mined by dynamic light scattering, DLS). Thereafter, the WPM
dispersion was concentrated to 22 wt% by microfiltration using
two Carbosep 0.14 membranes with a total surface of 6.8 m
2
(Novasep Process, Miribel, France) at a temperature of 10
C
and a flow rate of 180 L.h
1
. The liquid concentrate was then
spray dried (feeding rate: 25 kg.h
1
WPM concentrate; inlet air
temperature: 145–150
C; outlet air temperature: 75–77
C;
spraying nozzle Ø: 0.5 mm; spraying pressure 40 bar) using
a GEA Niro SD6.3N spray dryer (Søborg, Denmark) and stored
at 10
C in 2 kg aluminium sealed bags. The WPM powder
contained 97% of the proteins in the form of microgels. Its
composition was (g/100g of wet powder): protein (Nx6.38,
Kjeldhal), 91; moisture, 3.6; lactose, 3; fat, 0.4 and ash, 2.
Mineral composition of the powder was (g/100g of wet powder):
Ca
2+
, 0.320; K
+
, 0.409; Na
+
, 0.468; Mg
2+
, 0.060; Cl
, 0.178 as
determined upon HNO
3
/H
2
O
2
mineralization of the protein
sample and analysis using a Vista MPX simultaneous ICP-AES
spectrometer (Varian Inc. Palo Alto, CA, USA).
For the preparation of the WPM dispersions, the WPM
powder was dispersed at 4 wt% (on the protein basis) in Millipore
water (resistivity ¼ 18.2 MU.cm) at room temperature for 2 h
under moderate stirring. Preliminary trials showed that
a homogenisation treatment of the WPM dispersion at 250/50
bar was required to recover solubility and particle size distribu-
tion similar to those of the liquid WPM dispersion before spray
drying. A lab scale Rannie MINI-LAB homogenizer (Kindler
Maschinen AG, Z
€
urich, Switzerland) was used to perform this
processing step. After homogenization, it was checked that the
morphology, hydrodynamic radius and polydispersity index of
the WPM were identical to the values obtained with the liquid
WPM dispersion before microfiltration and spray drying (Fig. 1
and 2). The native pH of the starting 4 wt% WPM dispersion was
6.51.
For pH adjustment, 1M NaOH or HCl of analytical grade
were used (Merck, Darmstadt, Germany). The denaturing
agents, sodium dodecyl sulfate (SDS), urea and dithiothreitol
(DTT) were of analytical grade from Fluka and Merck.
Fig. 1 (a) Negative-staining TEM micrograph from a freshly prepared 4
wt% WPM dispersion. Scale bar is 0.5 mm. (b) SEM micrograph from
a WPM powder granule. Scale bar is 10 mm. (c) TEM micrograph of
a thin-section from the wall of WPM powder granule. Scale bar is 500
nm. (d) Negative-staining TEM micrograph from a 4 wt% WPM
dispersion reconstituted from powder after homogenization at 250/50
bar. Scale bar is 0.5 mm.
Fig. 2 Intensity weighted size distribution from a 0.4 wt% WPM
dispersion measured by DLS at 25
C. Fresh WPM dispersion: open
symbols; WPM dispersion from homogenized powder: filled symbols.
2
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Determination of whey protein microgel morphology in
dispersion and in powder
The WPM dispersion and WPM powder have been investigated
by scanning electron microscopy (SEM) and transmission elec-
tron microscopy (TEM). For TEM, samples were both observed
using cryofixation and embedding in Spurr resin or negative
staining method.
For cryofixation and embedding in Spurr resin, 50 mg of
WPM powder were dispersed in a tube containing 3 mL of a 2.5%
anhydrous glutaraldehyde in methanol solution at 40
C. The
next day, 1 mL of a 20 mg/mL osmic acid in methanol was added
to the tube at 40
C and incubated for 24 h. Tubes were then
transferred at 4
C for 2 h and centrifuged for 5 min at 3500 rpm.
The supernatant was removed and replaced by methanol at 4
C.
After vortexing, samples were left at 4
C for 1 h. A similar
procedure was applied replacing methanol by ethanol (2 times 1
h incubation at room temperature), mix 1 : 1 Spurr resin/ethanol
at 4
C overnight, mix 2 : 1 Spurr resin/ethanol at 4
C48h,
100% Spurr resin at 4
C. After centrifugation, the supernatant
was removed and replaced by freshly prepared Spurr resin and
the tube was placed under vacuum for 2 h. After storage at 4
C
and centrifugation, the bottom phase was dropped in moulds and
covered with 100% Spurr resin. Polymerisation of the resin was
obtained by incubation of the moulds at 70
C for 48 h.
Semi-fine sections were stained by toluidine blue at 0.5% in
Borax buffer 1% and mounted in 45% glycerol. Imaging was
performed using a Zeiss Axioplan II light microscope equipped
with an Axiocam camera. Ultra-thin sections were stained with
uranyl acetate and lead citrate before observation under a Philips
CM12 microscope operating at 80 kV. Images were recorded by
a Gatan Multiscan Camera Model 794.
For the negative staining method, a drop of the WPM
dispersion was deposited onto a carbon support film mounted on
a copper grid. The excess product was removed after 30 s using
a filter paper. A droplet of 1% phosphotungstic acid at pH 7.0
was added for 15 s, any excess being removed as before.
Micrographs were made using a Philips CM12 transmission
electron microscope as described before.
For SEM observation of the powders, the dry products were
mounted on sample holders with double-side adhesive tape. A
part of the sample was cut with a razor blade to reveal the inner
structure of the particles. The samples were examined by SEM
using a FEI Quanta 200F microscope at an accelerating voltage
of 8 kV. The detector used was the LFD (large Field Detector) in
low vacuum mode and no gold was sputtered.
Chemical stability of WPM
Internal bonds responsible for the stability of WPM have been
investigated upon incubation of 1 wt% WPM dispersion in
various denaturing reagents which are specifically affecting:
hydrophobic interactions for SDS, hydrogen bonds for urea and
disulfide bonds for dithiothreitol (DTT).
25,26
SDS and urea
experiments have been performed at room temperature, whereas
DTT incubation required a heat treatment at 70
C for 15 min to
be effective. In addition to the testing of single denaturing agents,
WPM have been exposed to mixtures of these compounds until
the resulting dispersion became transparent and the particle size
determined by DLS was close to that reported for whey protein
soluble aggregates obtained at pH 7.0 in similar heating condi-
tions, i.e. z-average hydrodynamic radius about 20–25 nm.
16,23
In addition, the cross-linking density of the WPM was indi-
rectly evaluated from the loss of soluble proteins after centrifu-
gation of the 4 wt% WPM dispersion at 26 900 g for 20 min at pH
2.0, 6.0 and 8.0. The protein contents in the supernatants were
determined by UV/VIS spectroscopy (molar extinction coeffi-
cient 3
278
¼ 10.1 g
1
.dL.cm
1
as determined experimentally)
using a Nicolet Evolution 100 spectrometre (Sysmex Digitana
SA, Switzerland).
Colloidal stability of WPM
The colloidal stability of the WPM was determined after recon-
stitution at 4 wt% (protein basis) by UV/VIS spectroscopy as
described above. The pH of the WPM dispersion was then
adjusted between 2 and 8 by addition of 1M NaOH or HCl. The
absorbance of the WPM dispersion was measured at 500 nm
using a Nicolet Evolution 100 spectrophotometer (Digitana,
Yverdon-les-Bains, Switzerland) equipped with a 1 cm path-
length cuvette. If necessary, the sample was diluted to obtain an
absorbance value below 1 absorbance unit (where variation of
absorbance is linear with the number of particles for a given size).
The WPM dispersion was then centrifuged at 173g for 5 min
using a Sorvall RC3C Plus centrifuge (Kendro Laboratory
Products, Geneva, CH) and the absorbance of the supernatant
was measured. The protein solubility was expressed as the ratio
between the absorbance at 500 nm before and after centrifuga-
tion normalised by the protein content of the WPM powder. All
experiments were done with two independent samples.
Determination of whey protein microgel size distribution and
z-potential
The hydrodynamic radius R
H
of the WPM was measured by
dynamic light scattering using a Nanosizer ZS instrument
(Malvern Instruments Ltd, Worcestershire, UK). The instrument
was used in the backscattering configuration. Samples were
diluted to a protein concentration of 0.4 wt% and placed in
a square quartz cell (Hellma 282 QS, Germany, pathlength 1 cm).
The hydrodynamic radius and the polydispersity index were
calculated by a cumulant analysis of the autocorrelation func-
tion.
27
The z-potential of the WPM was determined between pH 2.0
and 8.0 by light scattering upon application of an alternating
electrical field into a disposable capillary cell (DTS 1060, Mal-
vern Instruments Ltd, Worcestershire, UK) at a protein
concentration of 0.1 wt%. The Nanosizer ZS apparatus was used
in its z-potential measurement mode at 25
C. The effective
electrical field, E, applied in the measurement cell varied between
50 and 150 V depending on the ionic strength of the samples. The
overall electrophoretic mobility, m, was calculated assuming
spherical particles.
28
The z-potential was then calculated using
the Smoluchoski equation. The accuracy of the measurement of
the z-potential was probed with a standard solution (DTS1050,
Malvern Instruments Ltd, Worcestershire, UK) giving a value
of 50 5 mV.
3
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Determination of the internal structure of whey protein microgels
The small angle X-ray scattering (SAXS) experiments were done
at the Swiss Light Source (Paul Scherrer Institute, Switzerland)
at the cSAXS instrument. Samples of 4 wt% WPM at pH ranging
from 2.0 to 8.0 were measured in 1 mm quartz capillaries at
20
C. The protein concentration was corrected for dilution
induced by pH adjustment below and above pH 6.51 (natural pH
of WPM dispersion). At least fifty 2D images were taken,
azimuthally integrated and averaged according to established
procedures provided by the Paul Scherrer Institute. The q-scale
was calibrated by a measurement of silver behenate. Absolute
calibration was done by measuring the scattered intensity of
water, which depends only on the isothermal compressibility and
on the electron density (I(0)
water,25
C
¼ 0.01633 cm
1
).
29
Results and discussion
Structure and physicochemical interactions in whey protein
microgels
The morphology of whey protein microgels (WPM) was inves-
tigated before and after spray drying using transmission and
scanning electron microscopy (Fig. 1). Fig. 1a and 1d show that
WPM exhibit a spherical shape and a quite high density as esti-
mated from the strong contrast between the particles and the
background upon staining. The particles had the tendency to
aggregate on the observation grid, but they remained stable in
solution. Interestingly, the morphology of the WPM was not
markedly affected by the spray drying treatment and homoge-
nisation at 250/50 bar. Based on TEM pictures, the diameter of
the WPM ranged between 120 to 300 nm which was slightly
lower compared to the light scattering data, but might be due to
a slight dehydration of the WPM during the sample treatment for
TEM experiments. These values are slightly larger than those
reported for pure b-lg microgels obtained at 1 wt% under similar
heating conditions,
15,17
but the difference could be due to the
fourfold higher protein concentration used here as well as due to
the use of an industrial plate heat exchanger rather than a water
bath for performing the heat treatment step as well as to the
mineral composition of the whey protein isolate.
24
The appear-
ance and size of the WPM were similar to those of the casein
micelles held together by calcium phosphate bridges or internally
cross-linked with the transglutaminase enzyme,
12,35
but also to
that of synthetic microgels such as copolymers of poly-
(methacrylic acid) and poly(2-(diethylamino)ethyl methacrylate)
(PMAA/PDEA)
36
or lightly cross-linked sterically stabilized
poly(2-vinylpiridine) latexes.
37
The analysis of the WPM powder by SEM revealed that
microgels were packed into spherical hollow particles having
a diameter of 30–40 m m formed by water evaporation during the
spray-drying step. This led to a granular internal surface of the
powder granules (Fig. 1b). At a larger magnification using resin
embedding/sectioning and TEM, the internal structure of the
powder particle could be better resolved (Fig. 1c). WPM were
densely packed within the wall of the powder granule, but they
were keeping their globular shape without exhibiting noticeable
fusion. The volume fraction occupied by the whey protein
microgels within the powder granule was about 60–70%, which
corresponds to the expected packing given by random close
packing of hard spheres.
The stability of the whey protein microgels to spray drying and
subsequent homogenization treatment was also investigated by
dynamic light scattering on dilute dispersions (Fig. 2). It could be
seen that the size distribution obtained for the fresh WPM
dispersion before spray drying matched with the one of the WPM
dispersion prepared by homogenization of the spray dried
powder. Calculation of the z-average hydrodynamic radii resul-
ted in 136 7 nm for the ‘‘fresh’’ WPM (polydispersity index:
0.1) and 142 3 nm for the powdered and re-dispersed ones
(polydispersity index: 0.07), which agreed with the radii esti-
mated from the TEM micrographs.
In a next step, the bonds responsible for the stability of the
WPM were probed in presence of a series of denaturing agents:
sodium dodecyl sulfate (SDS), urea and dithiothreitol (DTT).
Combined urea and SDS were not able to dissociate WPM,
revealing that not only hydrogen and hydrophobic bonds were
present. However, when urea or SDS were combined with DTT,
an agent which is reducing disulfide bonds, WPM could be
dissociated, resulting in transparent stable solutions. For
example, the treatment of 1 wt% WPM with 50 mM DTT and 4
M urea led to the formation of so-called soluble aggregates
16
exhibiting a z-average hydrodynamic radius of about 40 nm
(Fig. 3). These soluble aggregates most likely correspond to the
building blocks of the WPM under the denaturating conditions
that were used in this study.
17
Based on the above experiments we propose the following
mechanism for the formation of WPM. Upon heat denaturation
the unfolded whey proteins expose hydrophobic regions which
associate depending on the external physicochemical conditions,
pH and protein concentration used. The quick formation of
small aggregates is followed by aggregation which leads to the
formation of particles that are primarily maintained by hydro-
phobic and hydrogen bonds (as already described for whey
protein gels formed under neutral pH conditions).
25,38
Subse-
quently, intra-particle disulfide bonds are formed, leading to
a covalent stabilisation of the structure.
39–41
The sum of all these
Fig. 3 Variation of the z-average hydrodynamic radius from a 1 wt%
WPM dispersion after incubation with an increasing content of DDT in
the presence of 4 M urea at 70
C for 15 min.
4
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interactions explains the remarkable stability of the whey protein
microgels against physical treatments such as spray drying and
homogenization. Further measurements by UV/VIS spectro-
photometry revealed that only about 6.2% of the total protein
content were released from the microgels at pH 2.0, 6.0 or 8.0,
confirming that the cross-linking density within the WPM was
high.
42
Colloidal stability and internal structure of whey protein
microgels as a function of pH
The stability of 4 wt% WPM dispersion was investigated between
pH 2.0 and 8.0. Whey protein microgels were forming stable
dispersions for most of the pH values tested with the exception of
the range 4.0 < pH < 5.5, where they were found to be unstable,
leading to precipitation (dashed area on Fig. 4). In order to shed
more light on the pH-dependent stability, the variation of the z-
potential of the microgels was measured as a function of pH.
Fig. 4 shows that the WPM exhibited a polyampholyte character
around a critical pH value of 4.82 (isoelectric point; IEP) for
which the overall surface charge was zero. Below this pH value,
WPM were positively charged, above they were negatively
charged. The samples were stable against aggregation/precipita-
tion when the z-potential exceeded an absolute value of 20 mV.
43
This limit was reached at pH values below 4.0 and above 5.5.
From these results, it could be concluded that the colloidal
stability of the whey protein microgels was mainly controlled by
their overall charge. This charge resulted from the balance
between the dissociation of carboxylic and amino groups of the
whey protein side chains as a function of pH. In the pH region
between 4.0 and 5.5, electrostatic repulsion between individual
microgels was too low to provide colloidal stability. Thus, WPM
aggregation and precipitation occurred. Such pH-sensitive
behaviour is typical for polyampholyte microgels and was
recently described for synthetic polyampholyte microgels of 40%
poly(methacrylic acid) and 60% poly(2-(diethylamino)ethyl
methacrylate) (PMAA/PDEA) having an isoelectrical pH of
about 5.0.
36
A similar pH-behaviour was also reported for 150–
280 nm human serum albumin microgels with an IEP of 5.0 that
were produced by desolvation in ethanol and cross-linking using
glutaraldehyde.
13
The internal structure of whey protein microgels was investi-
gated by means of SAXS. Despite the relatively large protein
concentration, we did not see significant interaction effects. This
could be explained by the relatively large polydispersity of WPM
and by the limited q-range accessible by SAXS. The experimental
scattering curves at three selected pH values are shown in Fig. 5a.
Overall the data exhibited no dramatic changes upon variation of
the pH, highlighting the internal stability of the microgels
between pH 2.0 and 8.0. Nevertheless, changes in the position of
a clearly visible shoulder at approx. 0.05 nm
1
and in the slope of
the scattering curve for q $ 0.1 nm
1
were observed. A closer
look at the high-q behaviour furthermore revealed the presence
Fig. 4 Variation of the z-potential from a 0.1 wt% WPM dispersion at
25
C as a function of pH. The vertical line indicates z-potential value of
zero. Vertical bars indicate standard deviation. The dashed area is indi-
cating the pH regime where WPM aggregate. In the stable regions the
WPM solubility is larger than 90% while in the aggregated regime it is
below 5%.
Fig. 5 (a) SAXS curves obtained from 4 wt% WPM dispersions at
selected pH values after subtraction of the solvent scattering. The curves
are shifted vertically for better visibility. The fitting model consisted of
a lognormal distribution of homogeneous spheres plus a power law decay
of the intensity due to the internal fractal structure. The samples close to
the IEP show an upturn of the scattered intensity at very small q-values
due to aggregation. The fitted curve deviates from the experimental data
in this regime. (b) Enlargement of the high q region of the SAXS curves
showing the presence of a correlation peak at q ¼ 0.67 nm
1
.
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