Food Hydrocolloids 17 (2003) 399–406

www.elsevier.com/locate/foodhyd

Heat-induced gelation of whey proteins observed by rheology, atomic

force microscopy, and Raman scattering spectroscopy
Shinya Ikeda*
Department of Food and Human Health Sciences, Osaka City University, 3-3-138 Sugimoto, Sumiyoshi-ku, Osaka 558 8585, Japan
Received 19 July 2002; revised 21 November 2002; accepted 3 December 2002

Abstract
Rheological and structural transitions during heat-induced gelation of whey proteins were investigated using mechanical spectroscopy,
atomic force microscopy (AFM), and Raman scattering spectroscopy. b-Lactoglobulin aqueous solutions containing 0.1 mol/dm3 NaCl at
pH 7 exhibited solid-like mechanical spectra before gelation. Heating such a solution resulted in the formation of an opaque gel that exhibited
frequency independent tan d values, an indicative of self-similar network structures. Translucent gels were formed in the absence of added
salts at pH 7 and their tan d values were frequency dependent. AFM images of heat-induced gel precursors revealed that these aggregates
were composed of ellipsoidal primary particles, regardless of the concentration of added NaCl, confirming that aggregation occurs in two-
step: the formation of primary aggregates and the subsequent aggregation of the primary aggregates. The size of primary aggregates and the
rate of aggregation increased with increasing NaCl concentrations. Thus, transitions from translucent to opaque gels with increasing ionic
concentrations are likely to be caused mainly by kinetic effects without accompanying fundamental changes in the two-step aggregation
mechanism. Raman scattering spectroscopy allowed discrimination between these two gel types based on secondary structures in denatured
b-lactoglobulin molecules: a decrease in the a-helix structure content was more pronounced in translucent gels, while considerable fractions
of b-sheet structures remained in both types of gels. A significant involvement of hydrophobic interactions in the formation of opaque gels
was suggested by an intense band assigned to hydrophobic side chains.
q 2003 Elsevier Science Ltd. All rights reserved.
Keywords: Globular protein; Heat-denaturation; Sol–gel transition; Viscoelasticity; Atomic force microscopy; Raman scattering spectroscopy

1. Introduction amino acids and potentially functional minor proteins such

Whey protein ingredients such as whey protein concen-
trates (WPCs) and isolates (WPIs) are mixtures of globular
proteins found in bovine milk. Due to mass production of
whey protein ingredients as byproducts from cheese
manufacture, their effective utilization is a continuous
demand in the industry. Extensive investigation has been
conducted primarily on physical functions of whey proteins
such as gelling, foaming, emulsifying, and stabilizing
abilities since physical properties of food products are key
attributes that determine consumer acceptance (Szczesniak,
1998). Currently, physiological functions of whey proteins
are also attracting attention. Whey protein ingredients
contain reasonable amounts of high-sulfur containing
* Tel.: þ81-6-6605-2862; fax: þ 81-6-6605-3086.
E-mail address: ikeda@life.osaka-cu.ac.jp (S. Ikeda).
0268-005X/03/$ - see front matter q 2003 Elsevier Science Ltd. All rights reserved.
doi:10.1016/S0268-005X(03)00033-X
as lactoferrin (Sloan, 2002).
Heating an aqueous solution of globular protein induces
denaturation and aggregation of protein molecules and a
macroscopic gel can be formed at a sufficiently high protein
concentration. A state transition from a sol to a gel is
prominently reflected in rheological transitions. Thus, a
number of gel-forming biopolymers including proteins and
polysaccharides are utilized in the food industry for
structuring products and controlling texture. In the case of
whey proteins, gel network structures and rheological
properties of heat-induced gels can be controlled by varying
gelling conditions such as pH, the ionic strength, species of
co-existing ions, and so forth (Clark, 1998). Whey protein
molecules, the isoelectric points of which are near pH 5, are
net negatively charged at neutral pH. If the ionic strength is
sufficiently low, intermolecular electrostatic repulsion is
dominant. Heat-induced aggregation proceeds relatively
slowly, leading to the formation of a so-called fine-stranded
400 S. Ikeda / Food Hydrocolloids 17 (2003) 399–406
gel, composed of finely stranded nanometer-thick networks,
exhibiting transparent or translucent appearance and
rubbery texture (Clark, 1998; Ikeda & Foegeding, 1999a;
Ikeda, Foegeding, & Hagiwara, 1999; Ikeda & Morris,
2002; Kavanagh, Clark, & Ross-Murphy, 2000b; Langton &
Hermansson, 1992; Stading & Hermansson, 1991). Inter-
molecular repulsion can be screened by shifting pH toward
the isoelectric points or by increasing ionic strength. Heat-
induced aggregation is accelerated in screened conditions
and results in the formation of a white opaque gel composed
of micrometer-sized particulate aggregates (Clark, 1998;
Langton & Hermansson, 1992; Stading & Hermansson,
1991). Gels of this type, often called particulate gels, easily
release liquids and require relatively large stresses to be
deformed. (Ikeda & Foegeding, 1999a; Ikeda et al., 1999;
Stading & Hermansson, 1991). Thus, some phenomenolo-
gical rules have already been established with respect to the
relationships between gelling conditions and structural and
physical features of resulting gels; however, relatively little
knowledge has been accumulated regarding rheological
transitions occurring during gelation (Ikeda, Nishinari, &
Foegeding, 2001b).
Electron microscopy has played a key role in classifying
network structural types in heat-induced globular protein
gels (Clark, 1998; Kavanagh et al., 2000b; Langton &
Hermansson, 1992; Stading & Hermansson, 1991). Electron
microscopy imaging, however, needs to be conducted under
a vacuum, requiring elaborate sample preparation, such as
dehydration, fixing, replication, or staining, in order to
preserve the natural state of the specimen. Alternatively,
hydrated biopolymer samples can be directly imaged in
atmospheric pressure with minimal preparation procedures
using atomic force microscopy (AFM) (Ikeda & Morris,
2002; Ikeda, Morris, & Nishinari, 2001a; Morris, Kirby, &
Gunning, 1999). Samples to be visualized by AFM should
be examined on a substrate that is rigid and molecularly flat.
Biopolymer samples are usually deposited onto freshly
cleaved mica surfaces and scanned in air or under a liquid
with a probe that maintains constant contact force with the
sample surface or constant separation distance between the
sample and the probe (Kirby, Gunning, & Morris, 1996;
Morris et al., 1999). AFM images are generated based on the
vertical movements of the sample or the probe during
scanning, depending on the design of the instruments. Thus,
an AFM image inherently contains quantitative information
on heights of objects in the image.
While AFM has been confirmed to be capable of
resolving even sub-domain structures in native globular
protein molecules (Morris et al., 1999), investigations on
even more detailed molecular structures usually require
spectroscopy. Raman scattering spectroscopy is one of rare
spectroscopy methods that provide information on peptide
backbone conformations in protein molecules at high
protein concentrations sufficient to cause gelation (Li-Chan,
1996). Another advantage of Raman scattering spectroscopy
is that it is directly applicable to aqueous systems due to
weak background scattering from water (Li-Chan, 1996). In
this study, Raman scattering spectroscopy was employed in
order to detect molecular structural changes accompanying
heat-induced gelation of whey proteins. Heat-induced gel
precursors were visualized using AFM and their structures
were investigated. Obtained structural information was
anticipated to give bases for understanding rheological
transitions occurring during the formation of varied types of
heat-induced whey protein gel networks.
2. Materials and methods
2.1. Materials and preparation
Three times crystallized b-lactoglobulin (a mixture of
variant A and B, product no. L-0130) was purchased from
Sigma Chemicals (St Louis, MO). WPI was a Bi-Pro grade
product in the form of freeze-dried powders supplied by
Davisco Foods International (LeSuer, MN). Other chemi-
cals were of reagent grade quality. Protein samples were
dissolved in distilled water or 0.1 – 0.3 mol/dm3 NaCl
aqueous solutions. The protein solutions were adjusted to
pH 7, 5.4, or 2 by adding small amounts of hydrochloric acid
or sodium hydroxide. The concentration of b-lactoglobulin
was determined spectrophotometrically at 278 nm using the
extinction coefficient e 278 ¼ 0:955 cm2 =mg (Bell &
McKenzie, 1967) whenever needed.
2.2. Rheological measurements
Degassed sample solutions were placed between a cone
(5 cm in diameter and 0.04 rad in cone angle) and plate test
fixture of the instrument (RFSII, Rheometrics, Inc., NJ) pre-
set at 70 8C and immediately covered with paraffin oil to
prevent water vaporization. Frequency v sweep measure-
ments of the storage modulus G0 and the loss modulus G00
were performed during isothermal heating at 70 8C at either
15 or 30 s intervals in the frequency range of 1– 100 rad/s at
a strain of 0.03, which was within the linear viscoelastic
strain region determined by preliminary experiments. The
examined frequency range was restricted in order to
complete each sweep within ca. 1 min. Values of the
complex modulus Gp ðGp2 ¼ G02 þ G002 ) at a frequency of
1 rad/s and tan d ð¼ G00 =G0 ) at varied frequencies were
calculated and reported.
2.3. Atomic force microscopy
The sample solutions were heated in sealed Pyrex tubes
immersed in a hot water bath preset at 80 8C for pre-
specified times and diluted to protein concentrations of ca.
40 – 44 mg/ml. Aliquots (2 ml) of the diluted sample
solutions were immediately spread onto freshly cleaved
mica sheets and imaged under butanol. AFM images were
produced using an East Coast Scientific (Cambridge, UK)
 S. Ikeda / Food Hydrocolloids 17 (2003) 399–406
manufactured AFM located at the Institute of Food
Research (Norwich, UK). Samples were placed on top of
the piezoelectric scanner, brought into contact with a
V-shaped Nanoprobe cantilever tip (Digital Instruments,
Santa Barbara, CA), and scanned at a preset cantilever
deflection. Topographical images were generated from
vertical movements of the sample during scanning necess-
ary to maintain the preset cantilever deflection. Equivalent
error signal mode images were generated based on the slight
fluctuations of the cantilever deflection around the preset
value during scanning.
2.4. Raman scattering spectroscopy
Heat-induced gels were formed by heating the sample
solutions in sealed containers in a water bath preset at 80 8C
for 60 min, cooled to room temperature, and kept in a 5 8C
cold room overnight. Raman spectra were recorded on a
Raman microscope (System 1000, Renishaw plc, Old Town,
UK) at room temperature (25 8C) with excitation from the
782 nm line of a titanium sapphire crystal laser (Mira model
900-P, Coherent Inc., Santa Clara, CA). A drop (50 ml) of
the sample solution or a gel sample that was cut into a disk
(3 mm in diameter and 2 mm in height) was placed on a
quartz plate. The laser was focused through a £ 50
objective at a power of 50 mW on the sample surface and
back-scattering was collected with the same objective and
captured using a Peltier cooled charge-coupled-device
(CCD) array detector. The accuracy of the wavenumber
was checked daily using the 520 cm21 band of silicon. Each
spectrum was smoothed with the 15 points fifth degree
Savitsky-Golay function using the Grams/386 software
(Galactic Industries Corporation, Salem, NH).
3. Results and discussion
3.1. Rheological measurements
Certain polysaccharides such as agarose, i- or
k-carrageenan, and gellan gum can form a gel on
cooling due to the conformational transition from random
coils to double-helices. Such gelation process can be
monitored rheologically: the mechanical spectrum that is
the frequency v dependence of the storage G0 and loss
moduli G00 exhibits features characteristic of dilute or
semi-dilute solutions before gelation and transforms to a
so-called gel spectrum during gelation (Hossain, Nemoto,
& Nishinari, 1997). However, this was not the case with
heat-induced gelation of whey proteins. Fig. 1 shows the
time evolution of rheological parameters in a 7% w/w
b-lactoglobulin aqueous solution containing 0.1 mol/dm3
NaCl at pH 7, induced by isothermally heating at 70 8C.
The tan d value gradually decreased with time at first,
followed by slight increase after ca. 2000 s heating, and
then started decreasing rapidly at ca. 2400 s, coinciding
401
Fig. 1. Gp (solid square) and tan d developments in 7% w/w b-lactoglobulin
aqueous solution containing 0.1 mol/dm3 NaCl at pH 7 during isothermal
heating at 70 8C. Values of tan d were determined at 1 rad/s (open circle),
2.5 rad/s (solid triangle), 25 rad/s (open square), and 40 rad/s (solid circle).
with the beginning of increasing Gp values. This point
was thus considered to be the gelation point. The tan d
value was always less than unity even before the gelation
point, suggesting that the sol before gelation behaves as
if it were a solid against linearly small strains. At the
gelation point, tan d values determined at varied
frequencies converged and remained frequency indepen-
dent until ca. 5000 s. Following equations were satisfied
when tan d values were independent of the frequency
G0 , G00 , vn ð1Þ
G00 =G0 ¼ tanðnp=2Þ ð2Þ
These equations have been proposed originally for chemi-
cally crosslinking polymeric systems and claimed to be
valid only at the gelation threshold (Chambon & Winter,
1987; Winter & Chambon, 1986). Such a gel at the gelation
point, referred to as a critical gel, is known to possess self-
similar network structures (Muthukumar, 1989). The value
of the exponent n in Eq. (1) can be related to the fractal
dimension of the self-similar network and the validity of
self-similarity in a wide length scale is guaranteed only
when both equations are satisfied simultaneously (Chambon
& Winter, 1987; Winter & Chambon, 1986). Therefore, the
validity of Eqs. (1) and (2) for heat-induced b-lactoglobulin
gels formed in the presence of 0.1 mol/dm3 NaCl suggests
that backbone structures that transmit the load on the gel
were self-similar. This observation seems to be in line with
the results of previous light-scattering studies conducted at
lower protein concentrations: b-lactoglobulin has been
shown to form self-similar aggregates at the ionic strength
of 0.1 mol/dm3 (Aymard, Gimel, Nicolai, & Durand, 1996;
Gimel, Durand, & Nicolai, 1994). Prolonged heating, longer
402 S. Ikeda / Food Hydrocolloids 17 (2003) 399–406
than ca. 5000 s, expanded differences among tan d values
determined at varied frequencies, suggesting a loss of self-
similarity due to restructuring.
The combined content of b-lactoglobulin and the
second major whey protein a-lactalbumin usually
exceeds 80% w/w of total solids in WPI. Gelation
phenomena of WPI are dominated by b-lactoglobulin
since the heat-stability of a-lactalbumin is even superior
(Kavanagh, Clark, Gosal, & Ross-Murphy, 2000a). Fig. 2
shows the time evolution of rheological parameters
during heating a 7% w/w WPI aqueous solution contain-
ing 0.1 mol/dm3 NaCl at pH 7. Although experimental
conditions were the same as those used for gaining
results shown in Fig. 1, except for using WPI in place
of b-lactoglobulin, dynamic mechanical response was
too low to be detected in an early stage of gelation
(ca. , 1500 s). Slightly before gelation, sufficient mech-
anical response was recovered but solid-like mechanical
characteristics were found to be lost ðtan d . 1Þ:
Frequency independent tan d was observed immediately
after the gelation point. However, no data satisfied both
Eqs. (1) and (2) at the same time. Thus, the presence of
minor whey proteins appeared to disturb the formation of
self-similar gel network structures of b-lactoglobulin.
Whey protein dispersions usually form translucent gels
in the absence of added salt at neutral pH. Fig. 3 represents
the time evolution of rheological parameters during heating
14% w/w b-lactoglobulin without added salt. The tan d
value remained below unity at most frequencies at an early
stage of heating (ca. , 1000 s) but increased beyond unity as
the gelation point is approached, which is an indicative of
the progressing formation of linear aggregates. The tan d
Fig. 2. Gp (solid square) and tan d developments in 7% w/w WPI aqueous
solution containing 0.1 mol/dm3 NaCl at pH 7 during isothermal heating at
70 8C. Values of tan d were determined at 1 rad/s (open circle), 16 rad/s
(solid triangle), 25 rad/s (open square), and 40 rad/s (solid circle).
Fig. 3. Gp (solid square) and tan d developments in 14% w/w b-
lactoglobulin aqueous solution at pH 7 during isothermal heating at 70 8C
in the absence of added salt. Values of tan d were determined at 1 rad/s
(open circle), 2.5 rad/s (solid triangle), 6.3 rad/s (open square), and 40 rad/s
(solid circle).
values were highly frequency dependent and critical gel-like
mechanical characteristics (i.e. Eqs. (1) and (2)) were not
observed in the vicinity of the gelation point. Prolonged
heating after gelation narrowed differences in tan d values
determined at varied frequencies. At a late stage of heating,
both Eqs. (1) and (2) were approximately satisfied within the
range of experimental errors, indicating the formation of
self-similar gel networks. According to previous findings
based on light-scattering studies, fine-stranded b-lactoglo-
bulin aggregates possess self-similarity in the length scale
lager than the thickness of strands (Aymard, Nicolai,
Durand, & Clark, 1999).
A WPI dispersion without added salt exhibited tan d
values larger than unity before gelation (Fig. 4). The tan d
value appeared to have a maximum before gelation and
become frequency independent of the gelation point. These
mechanical transitions are similar to those typical of
chemically cross-linking polymer systems (Takahashi,
Yokoyama, Masuda, & Takigawa, 1994). Therefore, it
was considered that, in the heated WPI dispersion, linear
aggregation predominated first, resulting in an increase in
tan d; but effects of extensive branching formation led to a
rapid G0 growth overcoming the developments in G00 : It may
be another point of interest that the value of the exponent n
determined at the gelation point was 0.67, identical with an
ideal value predicted based on the percolation theory
(Takahashi et al., 1994).
3.2. Atomic force microscopy
Complicated rheological transitions during heat-induced
gelation of whey proteins are considered to reflect structural
 S. Ikeda / Food Hydrocolloids 17 (2003) 399–406
Fig. 4. Gp (solid square) and tan d developments in 14% w/w WPI aqueous
solution at pH 7 during isothermal heating at 70 8C in the absence of added
salt. Values of tan d were determined at 1 rad/s (open circle), 2.5 rad/s
(solid triangle), 10 rad/s (open square), and 63 rad/s (solid circle).
developments in protein aggregates. Previous electron
microscopy images of translucent gels formed at neutral
pH without added salt have revealed that the gel networks
are composed of monomer/dimer-thick fine-strands (Lang-
ton & Hermansson, 1992; Stading & Hermansson, 1991).
On the other hand, scattering studies have revealed that
aggregation at neutral pH proceeds in two steps (Aymard
et al., 1996; Gimel et al., 1994). The proposed first step is
the formation of primary aggregates that are globular in
shape and thus designated as globules. Additionally, it has
been found that the second step aggregation among globules
can be suppressed in a dilute system and that, in this case,
the size of globules is always ca. 30 nm in diameter,
insensitive to the ionic strength in the gelling system
(0.003 – 0.1 mol/dm3). Therefore, heat-induced gel precur-
sors were formed at relatively high protein concentrations in
this study and visualized using AFM.
Fig. 5 shows examples of WPI gel precursors formed in
an 11% w/w WPI solution at pH 7 without added salts by
heating at 80 8C for 60 min. Visualized aggregates were
polydisperse in size and shape although the observed shapes
of the aggregates are not necessarily representatives of their
three-dimensional shapes in a sol due to probable orien-
tation, flow, or extension occurring on spread on mica
surfaces. However, the aggregates were generally composed
of fairly regular globular particles, confirming that the heat-
induced gelation of WPI at neutral pH also occurs in two
steps. An estimated average height of individual primary
aggregates was 11 nm, much shorter than what is expected
from the results of previous scattering studies made for pure
b-lactoglobulin systems. These results may indicate the
aggregation of b-lactoglobulin was more or less obstructed
403
Fig. 5. Topographical (a) and equivalent error signal mode AFM image (b)
of WPI aggregates formed in 11% w/w WPI aqueous solution at pH 7 by
heating at 80 8C for 60 min in the absence of added salt. Image size is
5 mm £ 5 mm.
in the presence of other whey proteins. It is also likely that at
a high protein concentration the growth of primary
aggregates is forced to cease by the secondary aggregation
among growing primary aggregates.
In the presence of 0.1 mol/dm3 NaCl at pH 7, heating a
10% w/w WPI solution at 80 8C for only a few minutes
normally results in the formation of an opaque gel (Ikeda &
Foegeding, 1999b). Thus, gel precursors were formed by
heating a 2% w/w solution at 80 8C for 30 min and imaged
using AFM (Fig. 6). The heated solution before dilution
appeared to be white opaque, indicating intensive aggrega-
tion despite the much lower protein concentration, com-
pared to the case without added NaCl. Observed aggregates
were once again composed of elementary globular aggre-
gates. Measured heights of these primary particles were ca.
18 nm, larger than those formed in the absence of NaCl.
These results suggest that well-known transitions in gel
network types from fine-stranded to particulate with
increasing ionic strength at neutral pH do not involve
fundamental changes in the two-step aggregation mechan-
ism but result from accelerated aggregation.
Translucent fine-stranded gels of WPI can also be formed
at acidic pH below the isoelectric points of the proteins.
Fig. 6. Topographical (a) and equivalent error signal mode AFM image (b)
of WPI aggregates formed in 2% w/w WPI aqueous solution containing
0.1 mol/dm3 NaCl at pH 7 by heating at 80 8C for 30 min. Image size is
1.5 mm £ 1.5 mm.
404 S. Ikeda / Food Hydrocolloids 17 (2003) 399–406
Fig. 7. Topographical (a) and equivalent error signal mode AFM image (b)
of WPI aggregates formed in 2% w/w WPI aqueous solution containing
0.1 mol/dm3 NaCl at pH 2 by heating at 80 8C for 180 min. Image size is
3 mm £ 3 mm.
Fig. 7 shows WPI gel precursors formed at pH 2 in the
presence of 0.1 mol/dmd3 NaCl by heating at 80 8C for
180 min. NaCl was added in order to avoid possible partial
precipitation of protein molecules (Aymard et al., 1999), but
the heated solution remained clear to the eye. Fig. 7 reveals
that finely stranded aggregates were formed in the heated
solution. The width and height of the fibrous aggregates
were fairly uniform. The measured heights were ca. 10 nm.
Since the radius of native b-lactoglobulin molecules is ca.
1.9 nm in an aqueous solution (Aymard et al., 1999), 10 nm-
thick strands cannot be just linearly connected monomers of
b-lactoglobulin. Previous rheological studies on b-lacto-
globulin and a-lactalbumin blends (Kavanagh et al., 2000a)
have concluded that these whey proteins can form coupled
gel network at pH 3 and 7. Moreover, it is known in the field
of biomedical research, where a possible link between the
formation of fibrous protein deposits and physiological
malfunctioning is an important subject, that various globular
proteins with no structural similarities can form fibrous
aggregates when protein molecules are only partially
denatured and slowly aggregate (Guijarro, Sunde, Jones,
Campbell, & Dobson, 1998). Surface probe microscopy is
anticipated to serve as a tool in future studies for clarifying
Fig. 8. Effects of heat-induced gelation of 15% w/v b-lactoglobulin on Raman scattering peak intensity at 940 cm21 normalized using peak intensity at
1005 cm21 (a), and on peak wavenumber around 1240 cm21 (b) and 1665 cm21 (c). Open and solid columns represent unheated solutions and gels formed by
heating at 80 8C for 60 min, respectively. The numbers below pH values represent the NaCl concentration in mol/dm3.
if each individual aggregate of whey proteins is composed
of individual or mixed species of globular proteins.
3.3. Raman scattering spectroscopy
Recent Fourier transform infrared (FT-IR) spectroscopy
studies on heat-induced gelation of b-lactoglobulin have
concluded that fine-stranded gels are formed by extensively
denatured protein molecules and that the formation of
particulate gels involves only minor modification of the
conformation of the protein (Lefèvre & Subirade, 2000).
This model seems to be worth revisiting since the b-barrel
structure in b-lactoglobulin is known to be heat-resistant
and the molecular size has been found to increase only ca.
10% by heating at normal gelling temperatures in dilute
conditions (Panick, Malessa, & Winter, 1999). In the
present study, Raman scattering spectroscopy was
employed to detect secondary structural changes accom-
panying heat-induced gelation of b-lactoglobulin. Com-
parisons were made between two types of gel networks:
translucent fine-stranded b-lactoglobulin gels formed at pH
7 and 2 without added salt and opaque particulate gels
formed at pH 7 with 0.1 or 0.3 mol/dm3 NaCl and at pH 5.4.
The intensity of the band around 940 cm21 is considered
to be proportional to the a-helix content (Clark, Saunderson,
& Suggett, 1981; Li-Chan, 1996). Recorded intensities of
this a-helix band were normalized using 1005 cm21 band
intensities, according to the conventional procedure
(Li-Chan, 1996), and reported in Fig. 8(a). Relatively
weak intensities of this band is perhaps due to the low a-
helix content of b-lactoglobulin: only about one tenth of the
total residues are involved in the a-helix structures
(Fogolari, Ragona, Zetta 1998). The intensity at pH 2 was
substantially low even before heating, which is probably
due to effects of protonation of carboxyl side groups and
ionization of amino side groups in the a-helix portion and/or
unfolding of a-helix structures although b-lactoglobulin is
known as an exceptionally acid-stable protein (Fogolari
et al., 1998). The band intensity decreased by heating at pH
 S. Ikeda / Food Hydrocolloids 17 (2003) 399–406
7 with or without 0.1 mol/dm3 NaCl and at pH 2 but was not
totally lost. In the case of particulate gels formed at pH 5.4
or 7 with 0.3 mol/dm3 NaCl, the intensity remained almost
unchanged. The a-helix fraction in b-lactoglobulin is
known to totally unfold when heated to ca. 70 8C in a dilute
solution (Qi Holt, Mc Nulty, Clarke, Brownlow, Jones
1997). Therefore, the results shown in Fig. 8(a) may indicate
that heat-resistance of a-helix structures can be improved at
a relatively high protein concentration. It is also possible
that non-aggregated and renatured molecules on cooling
contributed to the band intensity of heated samples.
The Raman scattering intensity peak representing
b-sheet and disordered structures is expected to appear
near 1230 –1240 and 1245 –1270 cm21, respectively (Clark
et al., 1981; Li-Chan, 1996). The peak position at ca.
1242 cm 21 before heating (Fig. 8(b)) is typical of
predominant contributions from b-sheet structures and
consistent with primarily b-sheet pleated structures of
b-lactoglobulin (Fogolari et al., 1998). This peak is known
to shift to a higher wavenumber when b-sheet structures are
unfolded (Frushour & Koenig, 1975) but remained almost at
the same position after heat-induced gelation except for the
case at pH 2 (Fig. 8(b)). Therefore, b-sheet structures
appeared not to unfold significantly during heat-induced
gelation. In the formation of fine-stranded gels at pH 2, this
b-sheet peak shifted to a lower wavenumber, which is an
indicative of strongly hydrogen bonded b-sheets. Similar
peak shifts accompanying the formation of fine-stranded
gels at acidic pH have been observed in the previous FT-IR
studies (Lefèvre & Subirade, 2000).
The peak position around 1665 – 1672 cm21 reflects
relative amounts of secondary structures: a-helix bands
exhibit a peak around 1660 cm21 and b-sheet and
disordered structure bands have a peak around 1670 cm21
(Carew, Stanley, Seidel, & Gergely, 1983). Thus, the
general peak shift from ca. 1665 to 1672 cm21 due to heat-
induced gelation (Fig. 8(c)) suggests a general decrease in
a-helical structures with an increase in disordered and/or b-
sheet structures. No obvious difference was recognized
between different gel network types.
Particulate gels formed at pH 7 with 0.3 mol/dm3 NaCl
and at pH 5.4 showed an intense and broad band centered
around 1345 cm21 (not shown) that is considered to reflect
significant changes in hydrophobic environments around
aliphatic and aromatic side chains (Li-Chan, 1996). There-
fore, particulate gels are likely to be formed via hydro-
phobic interactions among only slightly denatured protein
molecules. At the secondary structural level, fine-stranded
gels can be characterized by a more prominent loss in
a-helical structures than particulate gels but b-sheet
structures appeared to be preserved well in both types of
gels. Thus, the shift from two-step aggregation at neutral pH
to fine-stranded aggregation at acidic pH appeared not to
involve drastic changes in secondary structures. It is often
pointed out that the involvement of the disulfide exchange
reaction that occurs at neutral pH but not at pH 2 should be
405
responsible for the two-step aggregation at neutral pH
(Aymard et al., 1999). Further Raman scattering spec-
troscopy studies are desirable since some Raman scattering
intensity bands are known to reflect conformational states of
disulfide bonds in protein molecules (Li-Chan, 1996).
4. Conclusion
Heat-induced aggregation of b-lactoglobulin and WPI
was confirmed to be a two-step process at neutral pH,
consisting of the formation of granular primary aggregates
as the first step and the subsequent aggregation of these
primary aggregates, regardless of the ionic concentration.
The growth and aggregation of primary particles were found
to be concurrent processes, which seems to cause rheolo-
gical transitions during heat-induced gelation of whey
proteins quite different from those of ordinary gelling
systems. Heat-induced gels formed by b-lactoglobulin
alone in the presence of 0.1 mol/dm3 NaCl exhibited
rheological features of self-similar networks, while the
presence of other whey proteins disturbed aggregation of
b-lactoglobulin more or less, preventing from the formation
of self-similar networks from WPI. In the absence of added
salt, an increase in tan d values is more pronounced at the
early stage of gelation, which is an indicative of more linear
aggregation of primary particles. Heat-induced aggregation
at acidic pH below the isoelectric point resulted in the
formation of finely stranded aggregates with only a few
molecular thickness. Secondary structural changes due to
heat-induced gelation reflected more clearly transitions in
gel network types with an increase in the ionic concentration
at neutral pH rather than the shift from two-step aggregation
at neutral pH to fine-stranded aggregation at acidic pH.
Acknowledgements
The author is grateful to Professor Katsuyoshi Nishinari
of Osaka City University, Osaka, Japan and Professor
E. Allen Foegeding of North Carolina State University,
Raleigh, USA for supporting rheological studies, Dr V. J.
Morris of Institute of Food Research, Norwich, UK for
instructing AFM, Professor E. C. Y. Li-Chan of The
University of British Columbia, Vancouver, Canada for
instructing Raman scattering spectroscopy, and Professor
Shuryo Nakai of The University of British Columbia,
Vancouver, Canada for valuable discussions on biophysical
functions of proteins.
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