Divalent salts are capable of inducing the cold gelation of preheated whey protein solutions, being in fact much more effective than monovalent salts. The most widely studied divalent salt is calcium, owing to its presence in milk and its health benefits. Calcium has a strong effect on gelation even if large quantities of monovalent cations are present (120 mM) (Kuhn and Foegeding 1991). Calcium cold-set gels are usually manufactured in the laboratory by dialyzing a preheated whey solution against a salt solution at the desired concentration (Barbut and Foegeding 1993). This allows a slow increase in the salt concentration and a more homogeneous network. Long times—about 12 h—are required for the cold-set gels to reach constant mechanical properties (Roff and Foegeding 1996).
Calcium binds strongly with βLg, the amount of which depends on the net negative charge of the protein, and it is well known to promote the isoelectric aggregation of βLg (Zittle and Dellamonica 1956; Zittle et al. 1957), even at low temperatures (Sherwin and Foegeding 1997). Calcium has a strong salting-out effect at low concentrations in native whey proteins, with a maximum aggregation rate around 20–40 mM Ca2+, while at higher concentrations there is resolubilization of the aggre-
gates (salting-in) (Zhu and Damodaran 1994). Calcium has long been employed in heat-induced gelation, resulting in good mechanical properties, such as hardness (Schmidt et al. 1979), compres- sive strength (Mulvihill and Kinsella 1988), and maximum elastic modulus (Gault and Fauquant 1992), at low concentrations, around 10 mM Ca2+; about 10–20 times lower than with monovalent
salts (Mulvihill and Kinsella 1988). The addition of calcium does not significantly alter the circular dichroism spectrum of heated whey protein aggregates in the near-UV; the tertiary structure is already lost during heating (Marangoni et al. 2000). The initial heating step causes a redshift and a significant decrease in intensity in the tryptophan fluorescence as proteins unfold. The addition of calcium causes a small blueshift and a dramatic increase of the fluorescence intensity, to a value 50% higher than for native WPI. Marangoni et al. (2000) argued that the effect of calcium could be caused by an increase of the quantum yield (the efficient fluorescence of the tryptophan) due to charge neutralization after calcium binding and due to aggregation via hydrophobic groups.
The first use of calcium to induce cold gelation was reported by Barbut and Foegeding (1993) in a comparison with heat gelation. Calcium cold-set gels result in different mechanical properties from equivalent calcium heat-set gels; however, the most remarkable is the different microstructure at a constant calcium concentration of 10 mM: fine-stranded in cold gelation against particulate in heat gelation. In fact, calcium heat-set gels are always opaque because of their particulate nature (Mulvihill and Kinsella 1988; Kuhn and Foegeding 1991). Cold-set gels have a much finer structure, even at high calcium concentrations (360 mM), compared to a heat-set gel in 10 mM Ca2+ (Barbut
1995c). This finer structure is due to the fine-stranded nature of the primary aggregates previously formed. The calcium concentration required to induce cold gelation is of the same order as that required to induce heat gelation (~<10 mM), and a bit lower for cold gelation (Kuhn and Foegeding 1991; Barbut and Foegeding 1993; Roff and Foegeding 1996).
Increasing the calcium concentration in cold gelation increases the opacity due to the formation of larger aggregates (Barbut 1995c; Hongsprabhas and Barbut 1996). Above a calcium concentra- tion of 15–30 mM Ca2+, cold-set gels cease to be transparent and become increasingly white (Barbut
1995c; Hongsprabhas and Barbut 1996; Roff and Foegeding 1996; Bryant and McClements 2000a); above 100 nM there are no visual differences in the gels’ clarity (Barbut 1997). The opacity of the gels, caused by the light scatter of large aggregates, is given by the calcium-driven aggregation of the protein aggregates. Figure 10.4 shows a schematic representation of the two types of gels that can be formed depending on the concentration of salt; this representation is independent of the salt used. The rate constant of aggregation increases at 10–30 mM Ca2+ but decreases if the concentration is
increased further (Hongsprabhas and Barbut 1997c; Hongsprabhas et al. 1999; Marangoni et al. 2000). At the molecular level, a fast aggregation of the large aggregates of the preheated protein solution is first observed, followed by a slower aggregation of smaller protein aggregates (Marangoni et al. 2000). Aggregation with calcium is so quick that intermediates are not observed (Croguennec et al. 2004). The quick aggregation at >10 mM Ca2+ results in a sharp decrease of the gelation time
to almost instantaneous values above 25 mM if the calcium and the whey solution are directly mixed (Bryant and McClements 2000a; Wu, Xie, and Morbidelli 2005). In addition, an increase of the gelation temperature from 1 to 24°C results in larger aggregate formation, which decreases the gel strength and water-holding properties (Hongsprabhas and Barbut 1997b). Reheating the cold-set gel at high temperatures (80°C for 30 min) induces rearrangements that result in a harder and less cohesive structure, with higher opacity and larger pore sizes (Hongsprabhas and Barbut 1997a). At low calcium concentrations (10 mM), the increase in aggregate size and network connectivity over
Heat (no salt)
Native protein
Denatured protein Protein aggregates
Fe2+ Low (Fe2+)/(prot)
High (Fe2+)/(prot)
(a) (b)
Fe2+
Filamentous gel Random aggregated gel
FIGURe 10.4 Mechanisms of aggregation and gelation of βLg cold-set gels: (a) random-aggregated gels at high salt concentration and (b) filamentous gels at low salt concentrations. (From Remondetto, G. E., and Subirade, M., Biopolymers, 69, 461–69, 2003.)
Gelation and Thickening with Globular Proteins at Low Temperatures 159
time is achieved by clustering of adjacent aggregates, while at high calcium concentrations (120 mM) the increase in aggregate size and connectivity is by enlargement of the aggregates which formed connected paths and filled up interstitial spaces (Hongsprabhas and Barbut 1997d). This also results in an increase of the pore sizes at higher calcium concentrations as a more particulate microstructure is formed, from 60 to 150 nm at 10 mM to 180–250 nm at 120 mM (Hongsprabhas and Barbut 1997a).
The microstructure change from fine-stranded at 10 mM to a particulate-like mixture >30 mM Ca2+ results in deep mechanical differences. From large deformation tests, this transition corre-
lates with a significant increase of the Young’s modulus—gels become more elastic, while there is a decrease of the fracture strain (Barbut 1997; Hongsprabhas and Barbut 1997c; Bryant and McClements 2000a; Marangoni et al. 2000). Higher calcium concentration slightly decreases the Young’s modulus and increases fracture strain, thus at ~30 mM Ca2+ gels are the most rigid and
brittle (Hongsprabhas and Barbut 1997c). Increasing the whey protein concentration of the gels increases the fracture stress and the Young’s modulus at all calcium concentrations (10–150 mM), while the fracture strain is only increased at high concentrations (10 wt%) (Hongsprabhas and Barbut 1997c). The hardness of the gels also increases greatly between 10 and 20 mM Ca2+, and
plateaus at higher concentrations (Ju and Kilara 1998f). The adhesiveness increases between 10 and 40 mM, while the cohesiveness slightly decreases (Ju and Kilara 1998f). Comparing the frac- ture properties of heat- and cold-set gels in the presence of calcium, heat-set gels present a higher increase of the shear stress with the calcium concentration. At low calcium concentrations (10 mM) the shear stress is higher in cold-set than in heat-set gels (Barbut 1995c), but the maximum values found with both methods are comparable, between 30 and 100 mM (Roff and Foegeding 1996). On the other hand, the shear strain at fracture presents different behavior. In cold gelation the shear strain decreases with the salt concentration (e.g., from 2.5 at <5 mM to 1.5 above 50 mM CaCl2),
but increases in heat gelation, becoming constant above 30 mM (Roff and Foegeding 1996). The opposite evolution of the shear stress and strain with the calcium concentration is also observed, surprisingly, in the heat gelation in the presence of NaCl (Kuhn and Foegeding 1991). Finally, the storage modulus increase during calcium cold gelation is much slower than a comparable heat-set gel with calcium (Barbut and Foegeding 1993).
Another major physical difference between heat-set and cold-set gels in the presence of calcium is the WHC. High WHC is observed for fine and homogeneous microstructures, because they are able to retain water better with capillary forces (Barbut 1995c). The different microstructure of both types of gels, particulate vs fine-stranded/mix, results in heat-set gels syneresing at 50 mM CaCl2
while comparable cold-set gels are able to retain water, despite both gels being opaque. This trans- lates into a WHC of ~4.8 g water held/g protein in the latter compared to only ~1.7 in the former (Roff and Foegeding 1996). The microstructure change in cold gelation at higher salt concentra- tions also causes a decrease in WHC (Barbut 1997; Hongsprabhas and Barbut 1997c). However, this decrease is mild compared to that observed in heat gelation, e.g., ~7 g water/g protein at 1 mM CaCl2
compared to ~5 at 100 mM CaCl2 (Roff and Foegeding 1996).