The degree of interactions between the denatured whey proteins and κ-casein on casein micelles depends on various factors such as heating temperatures (Smits & van Brouwershaven, 1980; Jang & Swaisgood, 1990), time (Dalgleish, 1990), heating rate (Oldfield et al., 1998a), concentration of milk components such as salts and whey proteins (Smits & van Brouwershaven, 1980; Dalgleish et al., 1997a; Oldfield, Singh & Taylor, 2005) and pH (Creamer, Berry & Matheson, 1978; Smits & van Brouwershaven, 1980).
The pH at heating plays an important role in the properties of heated milk. The profile of heat coagulation time (i.e. the time taken for the proteins in milk to visibly aggregate) as a function of pH when milk is heated at ~ 140 °C shows an increase in stability up to pH ~6.6, followed by decreasing stability to a minimum at about pH 6.9, then an increase in stability again with further increases in pH (Figure 2.10, Rose, 1961).
Figure 2.10: An example of a heat coagulation time-pH profile of a typical milk sample. Source: Walstra (1984).
The pH at heating was also found to influence the degree of association of denatured whey proteins on the casein micelles (Creamer et al., 1978; Kudo, 1980). When milk was heated at pH < 6.7, a high proportion of the denatured whey proteins associated with the casein micelles whereas heating milk at pH > 6.7 resulted in a high level of soluble complexes between the denatured whey protein and κ- casein (Creamer et al., 1978; Kudo, 1980). Subsequent studies reported that pH at heating also
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influenced the dissociation of κ-casein from the casein micelles with a corresponding increase in the amount of dissociated κ-casein with increasing pH (Singh & Fox, 1985, 1987).
2.8 The heat-induced dissociation of casein proteins and its relation to the
association of denatured whey proteins on casein micelles
The dissociation of casein proteins from casein micelles was thought initially to occur only at temperatures above 90 °C (Morr, 1969; Aoki, Suzuki & Imamura, 1974, 1975; Kudo, 1980; Singh & Fox, 1985). However Anema and Klostermeyer (1997) later found at pH ≥ 6.7, κ-casein dissociated from the micelles as soon as the temperature rose above 20 °C (Figure 2.11A). The level of κ-casein dissociation increased with increasing pH ≥ 6.7. In addition, at each increase in pH, the amount of dissociated κ-caseins increased with increasing temperatures (Anema & Klostermeyer, 1997).
Figure 2.11: Effect of temperature and pH on the percentage of serum proteins. κ-casein (A), αs-casein (B) and β-casein (C) in the serum phase. The proteins were found in the supernatants obtained from 10% total solid reconstituted skim milk samples. ○, pH 6.3, ●, pH 6.5; □, pH 6.7; ■, pH 6.9; △, pH 7.1
(Anema & Klostermeyer, 1997).
αs-Caseins (including both αs1- and αs2-casein) and β-caseins were also found to dissociate on heating milk at pH values ≥ 6.7, although in a different manner compared to the dissociation of κ-caseins (Anema & Klostermeyer, 1997; Anema, 1998). As the temperature increased, the level of dissociated of αs- and β-caseins increased progressively to a maximum at 70 °C and then decreased at higher temperatures (Figure 2.11B and C). Higher levels of κ-casein were dissociated at each temperature when the pH was increased from 6.7 to 7.1 (Anema & Klostermeyer, 1997; Anema & Li, 2000).
This observation can be related to the interactions between denatured β-lactoglobulin and κ-casein. When whey-protein-free milk was heated, the amount of αs- and β-caseins that were dissociated from the casein micelles increased with increasing temperature at pH ≥ 6.7 and this amount was higher than that of heating standard milk at temperatures above 70 °C (Anema & Li, 2000). It was proposed that on heating casein solutions, all the casein proteins dissociated from the casein micelles and on subsequent cooling, κ-caseins were able to stabilise the soluble αs- and β-caseins into small soluble
B
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aggregates. When standard milk was heated at temperatures below 70 °C, all the casein proteins dissociated from the casein micelles. However, at temperatures above 70 °C, the whey proteins became denatured and formed aggregates with κ-casein (Zittle et al., 1962; Jang & Swaisgood, 1990; Corredig & Dalgleish, 1999). The complexes between denatured whey proteins and κ-casein were less efficient in stabilising the soluble αs- and β-caseins, especially in the presence of calcium ions because αs- and β-caseins can associate strongly with each other via calcium (phosphate) bridging (Section 2.3.3). Therefore, on subsequent cooling, the dissociated αs- and β-caseins could re-associate with the casein micelles or form larger aggregates that sediment on centrifugation (Anema & Klostermeyer, 1997; Anema & Li, 2000).
The effect of pH at heating on the interactions between denatured whey proteins and casein micelles has been extensively studied to establish the relation between the dissociation of κ-casein and the association of denatured whey proteins on the casein micelles (Smits & van Brouwershaven, 1980; Singh & Fox, 1991; Anema & Klostermeyer, 1997; Oldfield, Singh, Taylor & Pearce, 2000; Anema & Li, 2003a; Vasbinder & De Kruif, 2003). Most recent studies showed that dissociation of κ-casein from the casein micelles increased linearly with increasing pH on heating milk at pH from 6.5 to 7.1 (Rodriguez Del Angel & Dalgleish, 2006; Anema, 2007). Consequently the dissociation of κ-casein significantly determined the distribution of denatured whey protein between the colloidal and serum phases and the level of soluble proteins increased with the increase of pH at heating (Figure 2.12).
Figure 2.12: Relationship between the denatured whey protein and κ-casein in the serum phase of heated milk. (●), Milk heated at 90 °C for 20 min; (○), 25 min; (▼) and 30 min (Anema, 2007).
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