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Abstract
The present investigation aimed at understanding how the type of cations (NaC and/or Ca2C) influences heat-induced gelation of globular
protein/LM pectin mixtures in comparison to the globular protein only. Two globular proteins (b-lactoglobulin or bovine serum albumin)
were compared. On the basis of viscoelastic and microscopic observations (CLSM), it has been shown that the two cations play a major role
in gelation of the individual biopolymers and, therefore, influence strongly the properties of the mixture. These differences are related to a
competition between gelation of the protein (in absence of Ca2C) or of both biopolymers (if Ca2C is added) and phase separation processes
taking place before the mixed system is entirely gelled.
q 2004 Elsevier Ltd. All rights reserved.
Keywords: Globular protein; LM pectin; Phase separation; Gelation; Rheology; Salts; Confocal laser scanning microscopy
1. Introduction the main protein in whey with a molar mass 18.6 kg molK1
and the same isoelectric point as BSA (Dufour, 2004).
Globular proteins and polysaccharides are two gelling Under heat treatment, globular proteins undergo denatura-
biopolymers used in food industry for their wide range of tion and, depending on medium conditions, a gel can be
textural properties (Dickinson & Mc Clements, 1995). formed (Clark & Lee-Tuffnell, 1986; Gosal & Ross-
When mixed together, a phase separation process often Murphy, 2000). Hydrogen bonding, electrostatic and
occurs because of thermodynamic incompatibility or hydrophobic interactions have been suggested as the
depletion–flocculation mechanisms (Doublier, Garnier, major forces involved in gelation (Kinsella & Whitehead,
Renard, & Sanchez, 2000). If one or both biopolymers 1989). It has also been shown that formation of disulfide
form a gel, mixed gels are formed and a kinetic competition linkages and sufhydryl-disulfide interchange are involved in
between gelation and phase separation takes place. The heat-induced gelation of globular proteins. In addition, salts
control and understanding of the relative rates of these present in the system play a major role in the rate of gelation
processes by way of external factors such as medium and in textural properties of the protein gels (Matsudomi,
conditions can lead to a wide range of microstructures and a Rector, & Kinsella, 1991; Mulvihill & Kinsella, 1988;
large variety of textures (Tolstoguzov, 2003). The present Haque & Aryana, 2002; Jeyarajah & Allen, 1994; Yasuda,
study aimed at evaluating the properties of a model system Nakamura, & Hayakawa, 1986). Depending on the nature of
composed of a globular protein, bovine serum albumin globular protein and on the concentration and valence of
(BSA) or b-lactoglobulin (b-Lg), and an anionic poly- salts added in the medium specific interactions (protein–
saccharide, a low methoxyl pectin (LM pectin). BSA is the ions cross-linkage) or electrostatic interactions (salting in)
most abundant protein in plasma that contributes to colloid can take place and enhance the aggregation process. Above
osmotic blood pressure. Its molar mass is 68 kg molK1 and a critical concentration of added salts, a salting out process
its isoelectric point is around 5.2 (Peters, 1975). b-Lg is can also take place (Relkin, 1996).
Pectins are complex polysaccharides that belong to cell
* Corresponding author. Tel.: C33 240 67 50 45; fax: C33 240 67 50 43.
walls of plant materials. The main chain of pectins is a
E-mail address: [email protected] (C. Garnier). polygalacturonic acid, partially esterified by methoxyl
0268-005X/$ - see front matter q 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.foodhyd.2004.10.019
550 L. Donato et al. / Food Hydrocolloids 19 (2005) 549–556
(Le Pecq, France) with a laser at a wavelength of 543 nm. close to this optimum. It has been suggested that specific
As proteins do not exhibit intrinsic fluorescence at this ionic interactions can occur between calcium and native or
wavelength, proteins were stained by adding rhodamine B pre-heated b-Lg by the formation of intramolecular ion
isothyocyanate (RITC) to the protein solution under bridges between charged groups of b-Lg and Ca2C
magnetic stirring during 1 h. Mixtures of RITC–protein/LM (Matsudomi et al., 1991). Jeyarajah and Allen (1994)
pectin were prepared as described above and poured showed that this interaction induced small structural
between a concave slide and a coverslip, then hermetically changes in the protein that led to increased hydrophobicity.
sealed. The same heat treatment as for rheological The present results, with a dramatic increase in gel strength,
measurements was applied using a thermostated stage are consistent with these descriptions.
(Linkam PE 60). It was verified that labelling did not Addition of 0.85 wt% LM pectin to b-Lg in absence of
change the rheological behaviour of the systems (results not calcium did not change the shape of the G 0 traces. However,
shown). a weaker gel (G 0 Z310 Pa) than for protein only was
yielded. For the mixture with calcium, G 0 was lower during
heat treatment and a larger increase occurred during the
3. Results and discussion cooling step resulting in a stronger gel (G 0 Z510 Pa).
Contribution of LM pectin gel to final gel strength could
3.1. Gelation of globular protein/LM pectin systems explain this result. However, the mechanical spectra (not
in water shown) obtained for the mixtures in presence or not of
calcium ions displayed a pattern similar to that of proteins
3.1.1. b-Lg based systems alone with a slight G 0 (u) and G 00 (u) dependence suggesting
Variations of the storage modulus (G 0 ) as a function of the predominant role of proteins in the viscoelastic behaviour
time for 8 wt% b-Lg and for mixtures in water, in presence (tan dw0.09 for uZ1$ rad. sK1 for mixtures in presence of
or not of calcium ions are shown in Fig. 1. For protein only calcium and w0.11 for protein only in both cases).
in water, G 0 increased strongly during the plateau at 80 8C The corresponding microstructures observed by CLSM
and a smoother increase occurred during the cooling step. are shown in Fig. 2. For protein only in presence or not of
After 1 h at 20 8C, G 0 was nearly constant (G 0 Z2900 Pa) calcium ions, the fluorescence was regularly distributed in
and the system was stable, a strong gel being formed. Same the medium. As gelation has been shown by rheology, it
evolution of G 0 was obtained in presence of calcium ions but could be concluded that the protein network cannot be seen
G 0 was four times higher (G 0 Z11750 Pa) resulting in a at this scale of observation. For the mixtures, inhomogene-
stronger gel. From the literature, it has been shown that ities in the distribution of fluorescence were evidenced by
there is an optimum Ca2C content until the gel is
strengthened. Matsudomi et al. (1991) showed that for 5%
b-Lg gel at pH 8, this optimum in CaCl2 concentration is
between 2 and 5 mM whereas Mulvihill and Kinsella (1988)
found this optimum at 10 mM CaCl2 for 10% b-Lg gel at the
same pH. Therefore, we can suppose that our conditions are
Fig. 1. Variations of G 0 during the thermal process (dashed line: Fig. 2. CLSM micrographs of RITC–b-Lg (A) and RITC–b-Lg/LM pectin
temperature profile) in water for b-Lg (,) and b-Lg/LM pectin mixture mixtures (B) after the thermal treatment, without (first raw) and with
(B) without (empty symbols) and with (filled symbols) 3 mM CaCl2 in (second raw) 3 mM CaCl2 in water at pH 6.8. Proteins appear in bright.
water at pH 6.8. Scale bar is 25 mm.
552 L. Donato et al. / Food Hydrocolloids 19 (2005) 549–556
the G 0 profile was the same as for b-Lg gel but a weaker gel
Fig. 6. CLSM micrographs of RITC–b-Lg (A) and RITC–b-Lg/LM pectin
mixtures (B) at 20 8C after the thermal treatment, without (first raw) and was obtained (G 0 w5!103 Pa instead of w3.5!104 Pa for
with (second raw) 3 mM CaCl2 in 0.1 M NaCl at pH 6.8. Proteins appear in b-Lg). By comparison with Fig. 3, the presence of NaCl
bright. Scale bar is 25 mm. reinforced the BSA gel as it was observed for the b-Lg gel.
Addition of calcium had a slighter strengthening effect than
For the mixed systems in 0.1 M NaCl, fluorescent in water, in opposition with b-Lg. This may suggest that the
particles were dispersed in a continuous matrix (dark total salt concentration in BSA systems is still below the
zones). Again phase separation took place with protein
optimum salt concentration after which the gel is weakened.
microspheres (in bright) dispersed in a LM pectin medium
The G 0 profile for the mixture was similar to the one in
(dark zones). By comparison with Fig. 2B, the presence of
water (Fig. 3) but a weaker gel was obtained in presence of
NaCl in the system seems to decrease the size of protein
NaCl. In presence of calcium, the kinetics was different:
beads (w!2 mm). Addition of calcium to the mixed systems
during the step at 80 8C, G 0 increased slowly but steadily
had the same effect on the structural properties as in water.
and a sharp G 0 increase occurred only during the cooling
The beads tended to aggregate yielding larger dark zones.
step below 60 8C, this being followed by a levelling at
These observations can be related to the rheological
20 8C. Final gel strength (G 0 w400 Pa) was close to that of
properties of the systems particularly from the mechanical
spectra obtained with the final gel. In absence of calcium, the systems without calcium (G 0 w300 Pa) but as already
the viscoelastic behaviour (mechanical spectrum) of the noticed for b-Lg based systems the mechanical spectra were
mixture was similar to that of the protein gel only whereas in different. In these conditions, the gels obtained for the
presence of calcium, the final structure seemed to result mixtures were weaker than for protein gel only but these
from the superposition of LM pectin and protein gels. BSA-based systems were stronger than corresponding b-Lg
Therefore, even if final gel strengths of the mixtures with based systems.
and without calcium are similar, gelation of the system As illustrated in Fig. 8A, similarly to b-Lg gel, the BSA
involved different mechanisms. Without calcium ions network was not visible in 0.1 M NaCl and contrary to b-Lg
added, a protein network is formed with a low connectivity gel, addition of calcium did not clearly change the structure
of protein aggregates likely resulting from a depletion– of the network. A coarsening occurred, which is hardly
flocculation mechanism due to LM pectin chains. When visible here but was identified by image analysis (Donato
calcium is present in the mixture, a mixed gel is formed with et al., 2004a). This may explain the slight increase in G 0
a LM pectin continuous network (evidenced from rheology) showed in Fig. 7.
reinforced by the presence of protein aggregates (visible by The microscopic observations of BSA/LM pectin gels
CLSM observations). (Fig. 8B) were quite similar to the previous one. Without
calcium, the protein beads seemed larger (w!2 mm)
3.2.2. BSA-based systems than in water (w!1 mm). Addition of calcium resulted
The variations of G 0 for BSA and BSA/LM pectin gels in in a decrease in the apparent size of the protein beads
presence or not of CaCl2 are shown in Fig. 7. For BSA only, and the system became slightly more homogeneous.
L. Donato et al. / Food Hydrocolloids 19 (2005) 549–556 555
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