The most common and convenient way to pretreat whey in the industry is by heating. The forma- tion of soluble aggregates during the heating of whey proteins has long been established (Watanabe and Klostermeyer 1976). Early studies verified that the preheated solutions that were later on used in cold gelation experiments corresponded to highly aggregated whey solutions (Kawamura et al. 1993; Ju and Kilara 1998b; Marangoni et al. 2000). For example, high performance liquid chro- matography (HPLC) shows that a typical heat treatment at 80°C for 30 min converts all the native proteins to large aggregates (Ju and Kilara 1998f). Visual confirmation of the formation of large aggregates was provided early on by Nakamura et al. (1995) using transmission electron microscopy (TEM). Heated WPI samples (90°C for 10 min) presented aggregates 30–50 nm large, which were absent in the unheated samples. The soluble aggregates formed are easily observed with nonreduc- ing SDS-polyacrylamide gel electrophoresis (PAGE) (Figure 10.2).
Many heating regimes have been used to form soluble protein aggregates, from 68.5°C for 2 h (Alting et al. 2003a) to 90°C for 30 min (McClements and Keogh 1995). The heating regime must be severe enough, otherwise a gel will not be formed in the second gelation step. For example, a 10 wt% WPI solution heated at 70°C for 10 min does not form a gel in 200 mM NaCl; the heating process
6 5 4 3 2 1 198,000 115,000 93,000 49,800 35,800 29,200 21,300 6400
FIGURe 10.2 SDS-PAGE of different samples: Mw marker (lane 1); native WPI (lane 2); preheated WPI
(pH 7, 80ºC for 1 h) in nonreducing (lane 3) and in reducing conditions (lane 5); preheated WPI which is then incubated with transglutaminase for 10 h at 50ºC, under nonreducing (lane 4) and reducing conditions (lane 6). (From Eissa, A. S., and Khan, S. A., Journal of Agricultural and Food Chemistry, 53, 5010–17, 2005.)
Gelation and Thickening with Globular Proteins at Low Temperatures 151
must be extended to 20 min or the temperature increased to 75°C (Bryant and McClements 2000d). The minimum heating regime to apply is determined by the denaturation and aggregation kinetics of the proteins. In order to maximize the number of proteins involved in the network formation, which will determine the mechanical properties of the final gels, a denaturation and aggregation degree of >95% is usually considered (Alting et al. 2003a). While one heating step is the norm, the effect of two heating steps has also been studied (Glibowski et al. 2006).
The heat-induced denaturation of βLg is a multistep mechanism (Iametti et al. 1996; Qi et al. 1997; Navea, de Juan, and Tauler 2003). The dimeric form, which accounts for most of the proteins at neutral pH, dissociates first to the monomeric form before denaturation, due to the higher thermal stability of the dimers (Apenten and Galani 2000; Apenten, Khokhar, and Galani 2002). An R-type structure is formed around 40–55°C, which differs from the monomer state in only a few confor- mational changes on some chains. Above ~70°C, usually termed the denaturation temperature, βLg presents a molten globule state (de Jongh, Groneveld, and de Groot 2001), defined as “a compact protein conformation that has a secondary structure content like that of the native protein, but poorly defined tertiary structure” (Ewbank and Creighton 1991). Thus, the protein can swell more and the hydrophobic structure has greater accessibility to the solvent (de la Fuente, Singh, and Hemar 2002). The secondary structure is reduced at temperatures above ~60°C, irreversibly above ~70°C, but at 90°C about 25–35% of the β-sheets are still present in the protein structure (Iametti et al. 1996; Qi et al. 1997; de Jongh et al. 2001; Fessas et al. 2001). During the early stages of βLg denaturation by heat, >50°C (Owusu-Apenten and Chee 2004), the free cysteine in βLg is exposed to the solvent (Croguennec et al. 2003), which has a crucial role in aggregation by acting as an initiator of thiol– disulfide exchange reactions with other proteins (Hoffmann and van Mil 1997, 1999). Thiol–disulfide exchange reactions are significantly faster at pHs above neutral (Galani and Apenten 1999).
Once the first oligomers are formed through intermolecular disulfide bridges (usually termed “pri- mary aggregates”), they start to assemble into larger aggregates. This leads to a two-step aggregation process (Vardhanabhuti and Foegeding 1999; Ikeda 2003). These primary aggregates only form above a critical association concentration (>5 g/l) (Baussay et al. 2004). The interactions involved in the growth and aggregation of the primary aggregates are not uniquely established as in the forma- tion of the primary aggregates. Many studies assert that large aggregates are formed almost exclu- sively through intermolecular disulfide bonds (Hoffmann et al. 1997; Hoffmann and van Mil 1997; Schokker et al. 1999), in agreement with the reaction model of Roefs and De Kruif (1994). The critical role of disulfide bonding during aggregation is clearly observed in Figure 10.2; note the dif- ference of polymeric material in nonreducing conditions (lane 3) with reducing conditions (lane 5). Subsequent studies showed that noncovalent interactions are also involved (Anema 2000; Havea, Singh, and Creamer 2001), such as intermolecular β-sheets (Allain, Paquin, and Subirade 1999). The main weak interactions at neutral pH are hydrogen bonding and hydrophobic interactions (Shimada and Cheftel 1988). The formation of hydrophobic interactions requires heating above ~70°C, in order that βLg is sufficiently denatured and the buried hydrophobic amino acids are exposed to the solvent (Galani and Apenten 1999) and high protein concentrations (Havea et al. 1998). Bauer et al. (2000) showed that the formation of primary aggregates was enhanced when increasing the pH from 6.7 to 8.4, as thiol/disulfide exchange reactions are favored, but the formation of larger aggregates was retarded due to the increase of repulsive forces (Hoffmann et al. 1997; Hoffmann and van Mil 1997). A double preheating step has been proposed to maximize the formation and cross-linking of aggre- gates, by first heating at pH 8 to favor the formation of disulfide bridges, followed by a second heating at pH 6–7 to enhance noncovalent interactions (Mleko et al. 2002).
The preheating should be performed at low salt concentrations and at pH away from the pI, preferably at neutral conditions, in order that these aggregates do not aggregate further, and eventu- ally form a heat-set gel, but remain soluble. Heat-set gels become increasingly easy to form at high protein concentrations, as covalent and noncovalent interactions are facilitated. In practice, there is a maximum protein concentration where a large amount of aggregates can be formed without forming a gel, about 10–12 wt% at neutral pH in the absence of salts, usually referred to as the
critical gelation concentration (Kavanagh, Clark, and Ross-Murphy 2000b). The addition of salts and acidification decrease this gelation concentration markedly (Mehalebi, Nicolai, and Durand 2008a, 2008b). Salts reduce the interprotein repulsion and facilitate interactions (Kitabatake, Wada, and Fujita 2001; Croguennec, O’Kennedy, and Mehra 2004), which is a desired event in the final gelation step but not during the pretreatment.
The typical whey protein aggregates formed in the preheating step, at low salt concentrations and pH close to neutral, are formed from primary aggregates of about 100 βLg monomers (Durand, Gimel, and Nicolai 2002), curved strands of 10×50 nm according to TEM (Pouzot et al. 2005), or ~20–30 nm with AFM (Elofsson et al. 1997; Ikeda and Morris 2002). Figure 10.3c and d show the typical morphology of heat-induced βLg aggregates. These globular aggregates subsequently interact, suggested to be head-to-tails (Pouzot et al. 2005), to form elongated aggregates (Durand et al. 2002) with a fractal dimension of 1.7 at low ionic strength (Baussay et al. 2004; Mehalebi et al. 2008b) and of 2 at >30 mM (Le Bon, Durand, and Nicolai 2002; Pouzot et al. 2005). A pH above
100 nm (a) (c) (b) (d) 100 nm 100 nm 100 nm
FIGURe 10.3 Cry-TEM micrographs of dispersions of aggregates heated at different protein concentrations.
(a) 2.5 wt% ovalbumin, 78ºC for 22 h; (b) 5 wt% ovalbumin, 78ºC for 22 h; (c) 3 wt% WPI, 68.6ºC for 24 h; (d) 9 wt% WPI, 68.5ºC for 2 h. (From Alting, A. C., Weijers, M., De Hoog, E. H. A., van de Pijpekamp, A. M., Stuart, M. A. C., Hamer, R. J., De Kruif, C. G., and Visschers, R. W., Journal of Agricultural and Food Chemistry, 52, 623–31, 2004.)
Gelation and Thickening with Globular Proteins at Low Temperatures 153
5.8 does not affect the structure of the aggregates at small length scales (Mehalebi et al. 2008b), nor does the ionic strength below 100 mM (Pouzot et al. 2005).
The use of pure βLg, preferred in academic studies, is economically unsound for any other applications. Cheaper whey mixtures, such as WPI and whey protein concentrate (WPC), can only be used in the industry. The aggregation behavior of bovine serum albumin (BSA) and αLa, the other major whey proteins, and mixtures with βLg is briefly discussed. BSA can form good heat- induced gels (Tobitani and Ross-Murphy 1997), but not αLa on its own (Matsudomi et al. 1992; Dalgleish, Senaratne, and Francois 1997). Gels formed from BSA/βLg mixtures have intermediate properties depending on the protein ratio (Gezimati, Singh, and Creamer 1996). In typical whey mixtures, the observed behavior will be that of βLg, the most abundant. In heated αLa/βLg mix- tures, αLa is incorporated in the βLg aggregates and enhances the gelation process (Dalgleish et al. 1997; Kavanagh et al. 2000a; Schokker, Singh, and Creamer 2000), probably because αLa has four internal disulfide bonds susceptible to interact with the free thiol group in βLg and BSA (Havea et al. 2001). Therefore, all major whey proteins present positive interactions in the formation of aggregates at neutral pH (Hines and Foegeding, 1993). If preheating is conducted at very acidic pH, such as 2, when βLg aggregates in amyloid-like fibrils (Bromley, Krebs, and Donald 2005), αLa and BSA are not incorporated in these fibrils and there are no mixed aggregates (Bolder et al. 2006b), and therefore, would not be part of the stress-bearing gel network (Bolder et al. 2006a). However, such low pH conditions are highly atypical in the formation of soluble aggregates for cold gelation applications; at neutral pH, the information collected from pure βLg studies is usually directly applicable to typical whey mixtures, e.g., microstructure of WPI and βLg aggregates and gels (Langton and Hermansson 1992; Ikeda and Morris 2002; Mahmoudi et al. 2007).