International Dairy Journal 51 (2015) 8e15
Contents lists available at ScienceDirect
International Dairy Journal
journal homepage: www.elsevier.com/locate/idairyj
Effect of temperature, calcium and protein concentration on
aggregation of whey protein isolate: Formation of gel-like
micro-particles
Yingzhou Ni a, Lijie Wen a, Lei Wang a, Yali Dang b, Peng Zhou a, Li Liang a, c, *
a
b
c
State Key Lab of Food Science and Technology, School of Food Science and Technology, Jiangnan University, Wuxi, Jiangsu, China
Institute of Health Food of Zhejiang Academy of Medical Sciences, Hangzhou China
State Key Laboratory of Molecular Engineering of Polymers and Department of Macromolecular Science, Fudan University, Shanghai, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 18 March 2015
Received in revised form
2 July 2015
Accepted 2 July 2015
Available online 18 July 2015
Protein aggregation occurs in biological systems and industrial processes, affecting protein solubility and
functional properties. In this study, whey protein isolate (WPI) obtained from bovine milk was used as a
model to study the dependence of aggregation on pre-heating temperature and on protein and calcium
concentrations. WPI solutions (0.1e5.0%, w/v) were heated at 25e85 C for 30 min prior to cooling and
calcium addition. Tryptophan shifted to a more hydrophilic environment as WPI concentrations and preheating temperatures increased. Pre-heated WPI solutions yielded soluble particles, which aggregated to
form porous gel-like particles by addition of calcium chloride. WPI microgel particles could be prepared
by using a cold gelation method and preheated the protein above 65 C. The particle size was monodisperse with sizes of about 190 nm and 255 nm, respectively in solutions pre-heated to 75 or 85 C and
containing 5 mM calcium.
© 2015 Elsevier Ltd. All rights reserved.
1. Introduction
Protein aggregation is a phenomenon that occurs both as a part
of normal biological functions and as an aberrant process in deleterious conditions such as neurodegenerative disease (Morris,
Watzky, & Finke, 2009). It is also a common and troublesome
manifestation of protein instability encountered during fermentation, refolding, shearing, agitation, freeze-thawing, drying, reconstitution and storage processes in the development of proteinbased drugs (Wang, 2005). Studies of protein folding are usually
complicated by aggregation of intermediate and denatured states
(Bondos & Bichnell, 2003). Protein aggregation is a major topic in
the field of food science, and its regulation is believed to affect the
texture of protein-enriched products (De Jongh & Broersen, 2012).
The protein aggregation problem is thus common to biological
systems, scientific research, and medical or industrial applications
(Bondos & Bichnell, 2003). Intrinsic molecular structural factors
and extrinsic factors including concentration, general solution
conditions, container/closure system and surfaces, light and
* Corresponding author. Tel.: þ86 51085197367.
E-mail address: [email protected] (L. Liang).
http://dx.doi.org/10.1016/j.idairyj.2015.07.003
0958-6946/© 2015 Elsevier Ltd. All rights reserved.
irradiation all contribute to protein aggregation (Wang, Nema, &
Teagarden, 2010). It is important to understand the aggregation
process of a variety of proteins to prevent it or exploit it for
applications.
The textural functionality of a protein-based food product is
determined to a large extent by the properties of micro-structural
elements and their mutual interactions. These micro-structural
aspects stem from the aggregation behaviour of the protein,
which in turn is determined by molecular properties and interactions. Bovine milk proteins contain about 80% casein and 20%
whey protein, a mixture of globular proteins. Heat-induced aggregates of k-casein and denatured whey proteins formed in milkbased dairy mixtures (Guyomarc'h, Law, & Dalgleish, 2003). Whey
proteins denatured, aggregated and associated with the casein
micelles during preheating, evaporation and spray drying related to
milk powder manufacture (Oldfield, 1996). In an attempt to increase whey protein levels in cheese, depending on the sizes of
whey protein aggregates, their addition to milk decreased the
strength and contraction capacity of rennet-induced gels but
increased curd moisture content (Giroux, Lanouette, & Britten,
2015).
Food proteins are used widely in formulated foods since they
have high nutritional value and possess unique functional
Y. Ni et al. / International Dairy Journal 51 (2015) 8e15
9
volume ratio under gentle stirring for 1 h, to obtain a WPI concentration of 0.1, 0.25, 0.5, 1.0, 2.0 or 5.0% and a CaCl2 final concentration of 1.7, 5 or 10 mM. All samples were prepared at least in
duplicate for each subsequent analysis.
properties including gelation, emulsification, foaming, and ligandbinding capacity (Chen, Remondetto, & Subirade, 2006; De Wolf
& Brett, 2000). Their functional properties depend somewhat on
the extent of denaturation or aggregation (Ako, Nicolai, & Durand,
2010; Nicolai, Britten, & Schmitt, 2011). Cold-induced gelation of
globular proteins can be achieved by heat-induced unfolding to
form soluble particles, followed by formation of a network via
cation-mediated or acid-induced aggregation at ambient temperature (Alting, Hamer, De Kruif, & Visschers, 2003; Barbut &
Foegeding, 1993). Pre-heating treatment was required to induce
cold gelation and performed above protein denaturation temperature and below the critical concentration for heat-induced gelation at neutral pH and low ionic strength conditions (Hongsprabhas
& Barbut, 1998; Mudgal, Daubert, & Foegeding, 2011). Formation
and structure of protein self-supporting gels vary with protein and
cation concentrations (Remondetto & Subirade, 2003).
It is interesting to develop nanoparticles (0.5e50 nm in radius)
and microparticles (50e500 nm) for encapsulation and protection
of sensitive and bioactive ingredients, which may reduce the
impact of encapsulated ingredients on food texture and to improve
functional ingredient delivery. The particles based on denatured
whey protein and with the radius ranged from 50 to 150 nm have
been prepared by pH-cycling treatment and use of calcium during
particle formation could control particle compactness (Giroux,
Houde, & Britten, 2010). Cold-set whey protein gels have a
porous inner structure (Kunn, Cavallieri, & da Cunha, 2010). Below a
critical protein concentration of gel formation, stable suspensions
of polydisperse aggregates may be produced (Ako et al., 2010).
Development of high-surface-area protein nano/micro-particles
might provide new opportunities for pulmonary and transdermal
delivery applications (Engstrom, 2007).
In this study, whey protein isolate (WPI) obtained from bovine
milk was used as a model to study the dependence of protein aggregation on pre-heating temperature and on protein and calcium
concentrations. The potential for obtaining cold-set gel-like nano/
micro-particles using this product is discussed. The data obtained
in this study should gain a deeper insight into the WPI aggregation
behaviour and be useful for the development of WPI-based
colloidal particles.
Particle size distribution and z-potential were measured on a
Zetasizer Nano ZS90 particle analyser (Malvern Instruments Ltd,
Malvern, UK) with a He/Ne laser (l ¼ 633 nm). All measurements
were conducted at 25 C and at a scattering angle of 90 . For dynamic light scattering measurement, samples were prepared with
the distilled water (pH 7.0) which was filtered through a 0.22 mm
pore membrane.
2. Materials and methods
2.6. Statistical analysis
2.1. Materials
Data were analysed for significant differences using the online
GraphPad QuikCalcs Free t-test calculator (GraphPad Software Inc.
San Diego, CA, USA).
WPI (BioPRO, ~92% protein) was obtained from Davisco International Inc. (Le Sueur, MN, USA). Pyrene (99.0%, for fluorescence)
and 8-anilino-1-naphthalenesulfonic acid ammonium salt (ANS,
97.0%, for fluorescence) were purchased from Sigma-Aldrich
Corporation (St. Louis, MO, USA). Other reagents were analytical
grade and obtained from SinoPharm CNCM Ltd. (Shanghai, China).
2.2. Sample preparation
WPI powder was dispersed in distilled water by gentle stirring
for 2 h at room temperature to ensure complete hydration of the
protein, followed by adjusting to pH 7.0 using 0.1 mol L1 NaOH or
HCl. The solution was adjusted to a protein concentration of 6.00%
(w/v), which was calculated without considering the purity of WPI,
and then diluted with the distilled water at pH 7.0 to a WPI concentration of 0.1, 0.25, 0.5, 1.0, 2.0 or 5.0%. The solutions were then
held at a temperature from 25 C to 85 C for 30 min after reaching
the temperature and then cooled and held at room temperature
(~25 C) for 2 h (Leung Sok Line, Remondetto, & Subirade, 2005).
WPI/CaCl2 mixtures were prepared by mixing pre-heated protein
solutions at 0.12, 0.3, 0.6, 1.2, 2.4 or 6.0% and CaCl2 solutions at a 5:1
2.3. Fluorescence measurements
Steady-state fluorescence was measured using a Fluoro Max 4
fluorescence spectrophotometer (Horiba Jobin Yvon Inc., Edison, NJ,
USA), equipped with 10 mm quartz cuvettes. Fluorescence of WPI
was recorded with an excitation wavelength of 295 nm. Fluorescence intensity was normalised relative to the emission maximum
(lmax) peak for 0.1% WPI at 25 C. Pyrene and ANS were added as
fluorescence probes at final concentrations of respectively 1.0 and
19.8 mM. The solutions were then kept at room temperature overnight. Pyrene fluorescence at an excitation wavelength of 335 nm
was recorded from 350 to 450 nm and I1/I3 (ratio of the intensities
of the first and third band of the emission spectra) was calculated.
The fluorescence emission spectrum of ANS excited at 370 nm was
recorded from 390 to 650 nm. The spectral resolutions are 1.5 and
2.0 nm, respectively, for protein intrinsic fluorescence and pyrene
or ANS fluorescence for both excitation and emission.
2.4. Turbidity measurement
Turbidity (% transmittance) of WPI solutions was determined
from the apparent absorbance measured at 500 nm using a Mapada
UV-1200 UVeVisible spectrophotometer (Shanghai Mapada Instruments Co. Ltd, Shanghai, China).
2.5. Size and z-potential measurements
3. Results and discussion
3.1. Impact of whey protein isolate concentration and temperature
3.1.1. Structural sensitivity of whey protein isolate
Intrinsic fluorescence has been used widely to investigate protein tertiary structure and structural transitions (Bhattacharjee &
Das, 2000; Halder, Chakraborty, Das, & Bose, 2012; Liang, Yao, &
Jiang, 2005). Table 1 shows lmax and the lmax intensity of WPI
fluorescence as a function of concentration after pre-heating at
various temperatures. In the case of 25 C treatment and 0.1% WPI, a
lmax was around 335 nm, indicating that tryptophan residues are in
a hydrophobic environment. The lmax changed very little as the
concentration increased, reaching 338 nm at 5.0%. The spectral
intensity increased with concentration up to 0.25% and then began
to decrease. The changes in the fluorescence lmax and intensity
were similar for pre-heating at 25 and 45 C. The overall trend was
similar at 65 C except that intensity at a given concentration was a
bit greater and lmax reached 339 nm at 5.0% WPI. The increases in
10
Y. Ni et al. / International Dairy Journal 51 (2015) 8e15
Table 1
Fluorescence emission maxima (lmax) and their intensity (I) of whey protein isolate at various concentrations in solutions pre-heated at various temperatures.a
Temperature ( C)
Parameter
Whey protein isolate concentration (%)
0.1
25
lmax (nm)
I
45
lmax (nm)
65
lmax (nm)
75
lmax (nm)
85
lmax (nm)
I
I
I
I
a
335
100.00
335
105.10
337
105.34
336
108.33
337
114.56
0.25
± 5.18a
± 9.70a
± 0.70a
± 2.53a
± 0.99aj
335
148.60
336
148.98
335
169.90
337
167.85
338
176.03
0.5
± 0.38b
± 6.66b
± 19.28bg
± 18.97bg
± 3.01g
336
124.36
335
119.04
336
136.31
338
153.60
340
162.56
1.0
± 10.58bc
± 10.91bc
± 20.56bch
± 22.21bch
± 1.04h
335
48.81
336
52.87
336
55.86
339
70.73
342
98.24
2.0
± 1.07d
± 1.23d
± 7.81d
± 13.01d
± 12.94ck
336
10.76
337
11.39
337
12.38
340
24.87
339
39.44
5.0
± 0.10e
± 1.16e
± 0.51e
± 2.38i
± 2.14l
338
7.23 ± 0.18f
338
6.94 ± 0.36f
339
10.06 ± 0.98ef
346
24.78 ± 3.27i
345
27.08 ± 0.14mi
Values for intensity are given as means ± standard deviation; different superscript letters indicate statistically significant differences in mean values.
intensity were larger in the higher temperature cases. At 75 C and
85 C, lmax shifted from 336 nm and 337 nm at 0.10% to longer
wavelengths at higher WPI concentrations, reaching about 345 nm
at 5%. The red shift indicates that tryptophan was in a more hydrophilic
environment,
suggesting
protein
unfolding
(Bhattacharjee & Das, 2000). These results indicate that the degree
of WPI unfolding increased as its concentrations and pre-heating
temperatures increased.
The major proteins in bovine whey are b-lactoglobulin (b-LG)
and a-lactalbumin (a-LA), which account, respectively, for about
50% and 20% of the total protein content. Fluorescence emission
spectra of b-LG and a-LA show lmax around 336 and 327 nm,
respectively, in its native state (Liang, Zhang, Zhou, & Subirade,
2013). The fluorescence intensity of b-LG is greater than that of aLA at the same molar concentration (Liang et al., 2013). The thermal
unfolding of these proteins has been investigated thoroughly and
their transition temperatures appear to be respectively about 73 C
and 66 C (Hong & Creamer, 2002), varying slightly with protein
concentration, pH, and ionic composition and concentration
(Relkin, 1996). From the results in Table 1, it can be seen that a
significant change in the WPI fluorescence was observed basically
at the pre-heating temperatures of 75 C and 85 C. The fluorescence characteristics of WPI (Table 1) therefore appear attributable
mainly to b-LG.
The reversibility of the structural transitions in the absence or
presence of stresses is the subject of conflicting reports.
Bhattacharjee and Das (2000) found that b-LG underwent an irreversible change in structure based on ANS fluorescence but a
reversible change based on tryptophan fluorescence when heated
to 90 C followed by gradual cooling or to 70e90 C for 15 min
followed by cooling to 25 C. It is noteworthy that the change in
WPI structure was dependent on concentration when heated to
75 C or 85 C for 30 min followed by cooling to room temperature,
based on tryptophan fluorescence (Table 1). The change in the
fluorescence of WPI was reversible at 0.1% and 75 C and 85 C and
at 0.25% and 75 C but not at higher concentrations.
3.1.2. Whey protein isolate aggregation
The turbidity of a colloidal dispersion at a given wavelength
depends on the particle number concentration, radius and refractive index, as well as the refractive index of the medium (Jeffrey &
Ottewill, 1988). The presence of protein aggregates or particles
often results in an increase in the turbidity of the solution, which
may thus be used to qualitatively assess the extent of aggregation
(Wei & Polozova, 2013). The turbidity of WPI solutions increased
with concentration at certain temperatures in the 25e85 C range
(Fig. 1A). Turbidity was not dependent on temperature, except that
it increased at 85 C and concentrations above 0.25%. These results
suggest the aggregation of WPI increased as the protein concentrations increased and as the preheating temperature increased up
to 85 C. In view of similar results obtained at 25 C and 45 C
Fig. 1. Turbidity of whey protein isolate (WPI) solutions pre-heated at various temperatures, as a function of concentration (A) and the intensity ratio of the first and
third band (I1/I3) of pyrene fluorescence in WPI solutions pre-heated at 25e85, as a
function of protein concentration (B): ( ) 25 C, ( ) 45 C, ( ) 65 C, ( ) 75 C and ( )
85 C. Data are the mean ± standard deviation and different letters (aej) indicate
statistically significant differences in mean values.
Y. Ni et al. / International Dairy Journal 51 (2015) 8e15
(Table 1 and Fig. 1A), WPI aggregation was not investigated at 45 C
in the experiments discussed below.
The behaviour of WPI in solution was further analysed using
pyrene fluorescence, which has been used widely to monitor the
macromolecular association and micelle formation in solution (Li,
Jiang, & Wu, 1997; Liang, Yao, Gong, & Jiang, 2004). The I1/I3 ratio
was about 1.5 for pyrene in water (Liang, Pinier, Leroux, & Subirade,
2009). In WPI solutions at a concentration of 0.1%, the ratio
decreased to about 1.16 at 25 C and to about 1.14 at pre-heated
temperatures of 65, 75 and 85 C (Fig. 1B), indicating that most
pyrene molecules transferred to a hydrophobic environment and
that WPI aggregated to form colloidal particles. The ratio continued
to decrease with increasing protein concentration, more so at lower
temperatures, reaching 1.02 at 5.0% and 25 C. At this concentration, the I1/I3 ratio was 1.05 at 75 C and 85 C. A slightly greater
ratio suggests that WPI particles were more loosen, possibly due to
thermal unfolding that made the protein form more extended
structure at 75 C and 85 C (Table 1). The pyrene I1/I3 ratios are not
as low as 0.95 measured for bulk polystyrene (Li et al., 1997), suggesting that the core of the WPI particles still contained some hydrophilic groups and/or water molecules.
Fig. 2 shows size distributions of WPI particles at various concentrations in association with different heating treatments. At
0.10% and 25 C, there are two size distributions, a major one
around 64 nm and a minor one around 10 nm. Increases in WPI
concentration did not affect the major size distribution to any
appreciable degree. In the case of heating at 65 C and 0.1%, the size
distribution shows one peak ranging from 17 to 178 nm and averaging around 60 nm. As WPI concentrations increased, the size
distribution did not change significantly, which shifted to about
85 nm at a WPI concentration of 5.0%, while a minor and smaller
size distribution appeared. Only one smaller size distribution exists
if the calculation is based on the number distribution at 25 and
11
65 C (data not shown). At the lower WPI concentrations, the
smaller particles might be not enough to detect by using the distribution by intensity since the intensity of scattering is proportional to the sixth power of the size according to Rayleigh's
approximation. Somewhat similar changes in size distribution were
observed as the WPI concentration was increased at higher temperatures. In the case of heating at 75 C and 0.10%, the size was
around 31 nm, and then a minor and smaller size distribution
around 6 nm appeared and the size distributions basically kept
invariant until 1.0%, after which two peaks were around 10 and
86 nm. At 85 C, the size was around 17 nm at 0.1% WPI and
increased to about 20 nm at 1.0% and then became distributed to
two peaks appearing around 9 and 133 nm at 5.0%. The change in
the size distributions might be attributed to the protein different
structure at various concentrations (Table 1). Together with
turbidity and pyrene fluorescence, these results indicate that WPI
aggregated to form soluble particles with the sizes from nano-to
micro-scales under the investigated conditions.
The z-potential distribution of WPI particles shows a single peak
under the investigated experimental conditions. Fig. 3A shows the
change in the z-potential of WPI particles as a function of temperature. The isoelectric points of b-LG and a-LA are around pH 5.2
and pH 4.5e4.8, respectively (Almecija, Ibanez, Guadix, & Guadix,
2007). The z-potential of WPI particles was negative at pH 7.0. At
25 C, it was 30 mV at 0.1% WPI and decreased as the protein
concentration increased, reaching 21 mV at 5.0%. The z-potential
increased as the pre-heating temperature was increased,
reaching 46 and 30 mV at 0.10% and 5.0% and 85 C, respectively.
Generally, particles with absolute z-potential values above 30 mV
are considered stable (Honary & Zahir, 2013). Negatively charged
groups on the surface of WPI particles provide electrostatic repulsive forces between particles and stabilise the particles in solution.
Moreover, due to the presence of steric stabilisation for high-
Fig. 2. Size distributions of whey protein isolate particles obtained at various concentrations of solutions pre-heated at (A) 25 C, (B) 65 C, (C), 75 C, (D) 85 C.
12
Y. Ni et al. / International Dairy Journal 51 (2015) 8e15
molecular-weight molecules, a z-potential of 20 mV may provide
sufficient stabilisation (Honary & Zahir, 2013).
As the WPI concentration increased, the intermolecular distance
in solution decreased, causing an increase in protein molecule interactions (Figs. 1 and 2). It has been reported that formation of blactoglobulin dimers produces fluorescence self-quenching
(Renard, Lefebvre, Griffin, & Griffin, 1998). Tryptophan fluorescence of b-LG was possibly quenched by the proximity of the
Cys66eCys160 disulphide bond, the Arg124 residue, the association site for dimer formation, and lysine amine (ε-NH2) groups
(Bhattacharjee & Das, 2000; Croguennec, Molle, Mehra, &
Bouhallab, 2004). Together with the fluorescence results, it is
thus suggested that WPI aggregation was the principal reason for
the decrease in protein fluorescence intensity above 0.25% (Table 1).
The size and structures of whey protein aggregates were
dependent on the protein concentration and on the heating protocol. Heat denaturation exposed the buried hydrophobic residues
in native state to the solvent phase, which is more pronounced as
WPI concentrations increased (Table 1). WPI aggregated to form
soluble particles containing hydrophilic groups and/or water molecules in the core (Fig. 1) and with the sizes from nano-to microscales (Fig. 2). At neutral pH, b-LG and a-LA interacted to form
mixed oligomers and aggregates by non-covalent bonds and
disulphide bonds, leading to a lower critical association concentration in WPI than in pure b-LG solutions (Nicolai et al., 2011). WPI
particles were stabilised by negatively charged groups on the surface (Fig. 3A).
concentration to 10.0 mM led to the disappearance of two minor
peaks around 10 and 60 nm, suggesting that the salt concentrations
were high enough to interact with total WPI particles to form larger
aggregates. For 75 C, a similar change was observed but with a new
peak appearing around 190 nm at 1.7 mM and disappearance of two
minor peaks at 5.0 mM. For 85 C, a major size distribution around
44 nm was observed with a shoulder around 255 nm at 1.7 mM
CaCl2, while a single 255 nm diameter aggregate category was
observed at 5.0 mM CaCl2. These results suggest that CaCl2 induced
WPI to form larger aggregates in the micro-sized scale at 65e85 C.
The monodisperse microparticles were formed at the highest
concentrations of CaCl2. The size distribution of CaCl2-induced WPI
aggregates at 10 mM CaCl2 was not studied because of precipitation
at 75 C and gel formation at 85 C.
Fig. 3B shows the z-potential of CaCl2-induced WPI aggregates.
The z-potential of aggregates obtained at 1.7 mM CaCl2 is significantly less for WPI particles obtained without CaCl2 (Fig. 3A) for all
pre-heating temperatures tested and continued to decrease with an
increase in the CaCl2 concentration used. In the 25 C and 65 C
cases, the z-potential was about 6 mV when the CaCl2 concentration was increased to 10 mM. For 75 C and 85 C, the z-potential
3.2. CaCl2-induced formation of gel-like whey protein isolate
microparticles
The sensitivity of protein structure to the presence of salts is
well known, and the influence of cations on protein aggregation
during the formation of heat-set and cold-set gel has been studied
widely. Addition of CaCl2 before heat-induced gelation greatly affects the hardness of WPI gel at a protein concentration of 10% (Ju &
Kilara, 1998). Beta-lactoglobulin at 6% formed two different
microstructural gels called “filamentous gel” and “particulate gel”,
depending on the cation concentration (Remondetto & Subirade,
2003). However, Ca2þ-induced aggregation at temperatures
below the denaturation temperature and at protein concentrations
below the critical concentration for cold-set self-supporting gel
formation has not received much attention. In this study, it was
found that addition of CaCl2 caused an increase in WPI solution
turbidity, with the most change at the salt concentrations between
0.5% and 2% (Fig. 4). A protein concentration of 1.0% was selected for
further study of the influence of CaCl2 on WPI aggregate and the
potential of gel-like particle formation. The pyrene fluorescence I1/
I3 ratio at this concentration was about 1.07, regardless of preheating temperature and CaCl2 concentration.
Fig. 5 shows the size distribution of CaCl2-induced aggregates of
WPI at various temperatures. At 25 C and 1.7 mM CaCl2, there were
three size categories, the major one being around 190 nm, which is
larger than the size of pure WPI particles. Further aggregation was
observed as the CaCl2 concentration increased, with two major size
distributions around 122 and 600 nm at 5.0 mM and around 106 and
1281 nm at 10.0 mM. At the same time, minor size distributions,
which are smaller than that of pure WPI particles, were also
observed. These results suggest that CaCl2 induced simultaneous
dissociation and aggregation of WPI particles. The phenomenon
was also observed for milk concentration proteins added calciumchelating salts (Yang, Liu, & Zhou, 2012). In the case of heating at
65 C, adding 1.7 mM or 5.0 mM CaCl2 resulted in the appearance of a
size distribution around 295 nm, suggesting the salts induced the
aggregation of a portion of WPI particles. Increasing the CaCl2
Fig. 3. z-potential of (A) whey protein isolate (WPI) nanoparticles as a function of
protein concentration ( , 0.1%; , 0.25%; , 0.5%; , 1.0%; , 2.0%; , 5.0%) and (B)
CaCl2-induced WPI aggregates as a function of calcium concentration ( , 1.7 mM; ,
5 mM; , 10 mM), obtained from solutions pre-heated at 25e85 C,. Data are the
mean ± standard deviation.
Y. Ni et al. / International Dairy Journal 51 (2015) 8e15
was about 11 mV at 5 mM CaCl2. These results suggest that adding
CaCl2 neutralised negative charges on the WPI particle surface
(Fig. 3A) thereby allowing intermolecular cross-linking and hence
aggregation (Remondetto & Subirade, 2003). Moreover, it has been
reported that ion-induced conformational changes and electrostatic shielding are involved in salt-induced protein aggregation
(O'Kennedy & Mounsey, 2009).
ANS is a well-known fluorescence probe used for measuring
protein surface hydrophobicity and characterising structure
porosity during unfolding and aggregation (Bhattacharjee & Das,
2000; Kundu & Guptasarma, 2002). It has a low fluorescence
quantum yield at lmax ~525 nm in aqueous solution (data not
shown). When ANS was added to WPI solution in the absence of
CaCl2, the lmax shifted to 470 nm in the cases of 25 C and 65 C
treatments and to 466 nm for 75 C and 85 C, accompanied by a
significant increase in fluorescence intensity (Fig. 6), indicating that
ANS was bound to the hydrophobic regions of the whey protein
molecules. For these four temperatures, the fluorescence intensity
of ANS in WPI solution was about 237, 209, 198 and 248 times that
of ANS in aqueous solution. Adding CaCl2 did not affect the lmax but
caused a change in fluorescence intensity. At 25 C, a slight increase
was observed but stayed constant as the calcium concentration
increased from 1.7 to 10 mM. For 65 C, the fluorescence intensity
increased gradually with CaCl2 concentration. A similar change
with an overall more pronounced effect was observed at 75 and
85 C. For the four temperature treatments, the ANS fluorescence
intensity at 5 mM CaCl2 was respectively about 1.07, 1.15, 1.48, 1.84
times that in WPI solution, suggesting that CaCl2-induced WPI
aggregates have more hydrophobic surface exposed for access by
ANS molecules. The surface area of a particle generally increases as
size decreases. Considering the results obtained for CaCl2-induced
aggregation of whey protein (Fig. 4), it may be concluded that the
aggregates are porous. This result is consistent with the formation
of a honeycomb inner structure in calcium-induced soy-proteinisolate nanoparticles by using a cold gelation method (Zhang, Liang,
Tian, Chen, & Subirade, 2012). It is therefore conceivable that CaCl2
caused the aggregation of WPI particles to form cold-set gel-like
particles.
Heat-induced microgels with a radius varied between 75 and
150 nm were previously prepared using b-LG, a-LA/b-LG mixtures
(20/80, w/w), and whey protein isolate at a protein concentration of
4% and heating at 85 C for 15 min at pH 5.7e6.2 (Schmitt, Bovay,
Vuilliomenet, Rouvet, & Bovetto, 2011). Cold-set WPI gel particles
below 50 mM in radius were prepared by an emulsification/internal
gelation technique while cold-set WPI gel beads with the radius of
0.5e1 mm were produced by the external method involving
dropwise addition of denatured WPI into a CaCl2 solution (Egan,
Jacquier, Rosenberg, & Rosenberg, 2013). In this study, WPI
microgel particles with a monodisperse size at 190 and 255 nm
were prepared by preheated the protein respectively at 75 and
85 C and by using a cold gelation method. The cold-set WPI
microgels could be used as an effective carrier of heat-sensitive and
120
120
A
1.7 mM
5.0 mM
10.0 mM
80
60
40
B
100
Turbidity (%)
Turbidity (%)
100
1.7 mM
5.0 mM
10.0 mM
80
60
40
20
20
0
0
0.1
0.2
0.5
1
2
5
0.2
0.1
WPI concentration (%)
C
100
Turbidity (%)
Turbidity (%)
1
2
5
WPI concentration (%)
80
60
40
D
1.7 mM
5.0 mM
10.0 mM
80
60
40
20
20
0
0.5
120
120
100
13
0
0.1
0.2
0.5
1
2
WPI concentration (%)
5
0.1
0.2
0.5
1
2
5
WPI concentration (%)
Fig. 4. Turbidity of whey protein isolate (WPI) solutions pre-heated at (A) 25 C, (B) 65 C, (C) 75 C and (D) 85 C in the presence of ( ) 1.7 mM, ( ) 5 mM and ( ) 10 mM CaCl2, as a
function of concentration. Data are the mean ± standard deviation.
14
Y. Ni et al. / International Dairy Journal 51 (2015) 8e15
Fig. 5. Size distributions of whey protein isolate particles obtained at various CaCl2 concentrations from solutions pre-heated at (A) 25 C, (B) 65 C, (C) 75 C and (D) 85 C.
Fig. 6. Fluorescence emission spectra of 8-anilino-1-naphthalenesulfonic acid ammonium salt (ANS) in suspensions of Ca2þ-induced whey protein isolate aggregates at various Ca2þ
concentrations (
, 0 mM;
, 1.7 mM;
, 5 mM;
, 10 mM) at (A) 25 C, (B) 65 C, (C) 75 C and (D) 85 C.
Y. Ni et al. / International Dairy Journal 51 (2015) 8e15
bioactive ingredients and be added to food systems where their
incorporation would be acceptable from a texture perspective.
4. Conclusions
The aggregation of WPI was dependent on pre-heating temperature and on protein and calcium concentrations. Pre-heated WPI
solutions yielded soluble particles containing hydrophilic groups
and/or water molecules in the core and stabilised by negatively
charged surface groups. The WPI aggregation resulted in a decrease
in the tryptophan fluorescence intensity at WPI concentrations
above 0.25%. Calcium ions caused further aggregation of whey
proteins, yielding particles that were porous and having a gel-like
inner structure when formed from solutions pre-heated above
65 C. These particles were mono-dispersed with a micro-scale size
when the calcium concentration was 5 mM and the pre-heating
temperature was 75 C or 85 C. It is thus concluded that WPI
micro-particles could be prepared by using a cold gelation method.
Acknowledgements
This work received support from the National Natural Science
Foundation of China (NSFC Projects 31201291 and 31101344) and
from the Free Exploration Project Program of State Key Laboratory
of Food Science and Technology, Jiangnan University (No. SKLFZZA-201506).
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