Handbook of Food Science and Technology 3

1.2.2.
 Protein stability 
Casein micelles and whey proteins differ in their resistance and 
technological stability during the processing of milk. Maintaining the micellar 
structure primarily depends on colloidal calcium phosphate, which acts within 
the micelles. On the other hand, the C-terminal part of 
κ
-casein forms highly 
hydrated and negatively-charged protrusions on the micelle structure, which 
hinder casein micelle self-association. Many technological processes adversely 
affect stability by modifying the physicochemical properties of the micelle 
surface. Figure 1.6
 
shows the impact of the main physicochemical factors on 
the structure and stability of casein micelles.
Figure 1.6.
 Impact of the main physicochemical factors
on casein micelle structure and stability
The stability of whey proteins is governed by a set of low-energy bonds 
(hydrogen bonds, hydrophobic interactions, electrostatic bonds, salt bridges) 
and covalent bonds (disulphide bridges).
C-term casein
Κ
CaHP
O
4

---

20 Handbook of Food Science and Technology 3 
1.2.2.1.
 Effect of temperature 
Temperature affects the solubility of calcium phosphate (inverse solubility 
salt) as well as the state of association of milk proteins. Both refrigeration and 
heat treatment alter the technological properties of the casein micelle, but the 
underlying mechanisms are different.
Partial solubilization of colloidal calcium phosphate (about 10%) during 
the cooling of milk (4°C) is reversible upon heating. In addition, the 
disassociation of 
β
-casein from the micellar structure occurs at low 
temperature due to a reduction in hydrophobic interactions; once in the soluble 
phase, it can be hydrolyzed by plasmin (endogenous milk enzyme), which 
results in a decrease in cheese yield (see section 1.3.4). 
β
-casein and/or 
hydrophobic fragments resulting from its hydrolysis by plasmin associate with 
the micelles during the heating of refrigerated milk. As a consequence, the 
rennet coagulation of such milk is altered. Indeed, it is likely that 
β
-casein, 
mostly located in the center of the native micelle, moves on its surface during 
the refrigeration–heating cycle of milk. Its presence on the micelle surface 
could reduce chymosin site accessibility on 
κ 
casein. 
Unlike whey proteins, casein micelles are relatively stable to heat 
treatment. Heat treatment decreases the solubility of calcium phosphate, which 
either insolubilizes inside the casein micelle or precipitates on the exchanger 
surface. The latter fraction is not recovered during the cooling of milk. If the 
heat treatment is below 95°C for a few seconds, the calcium phosphate 
insolubilized inside the micelle remains in equilibrium with the soluble phase 
of milk and resolubilizes during cooling. For more intense heat treatment (e.g. 
120°C for 20 minutes), irreversible changes take place in the structure and 
distribution of salts between the micelle and the soluble fraction. At 
temperatures above 70°C, whey proteins denature and can interact with each 
other in the soluble phase of milk (formation of soluble aggregates) or with 
κ
-
casein (formation of stable aggregates on the micelle surface). The distribution 
of aggregates between the soluble phase or the micelle surface depends on pH 
and determines the heat stability of milk. The heat treatment of milk with a pH 
above 6.7 promotes the release of 
κ
-casein, which decreases micelle stability. 
When heat treatment is carried out at a pH below 6.6, a large proportion of 
whey proteins remain associated with the casein micelle. Thus, the stability of 
heat-treated milk is greatest when the heat treatment is carried out between pH 
6.6 and 6.7. Aggregation of whey proteins on the casein micelle surface makes 
them stable to chymosin hydrolysis by masking the cleavage site on 
κ
-casein. 

---

From Milk to Dairy Products 21 
In addition, heat treatment applied to milk (e.g. 95°C for a few minutes) has a 
positive effect on the texture of the gels obtained after slow acidification 
(yoghurt).
On another level, the interaction of lactose with proteins during heat 
treatment (Maillard reaction) may alter their functional characteristics.
1.2.2.2.
 Effect of concentration 
The concentration of milk by evaporation increases the colloidal calcium 
phosphate content of casein micelles. It also increases ionic strength and 
decreases the pH of milk, resulting in the shielding of the negative charges on 
the C-terminal portion of 
κ
-casein. In contrast, the concentration of milk by 
ultrafiltration does not alter the mineral concentration of the soluble phase and 
therefore does not affect the structure and stability of casein micelles.
1.2.2.3.
 Effect of ionic environment
Calcium (generally calcium chloride) is widely used in cheese technology 
to offset the adverse effects of heat treatment and to improve the rheological 
properties of curd. It induces major changes in the distribution of salts between 
the soluble and the colloidal phase. It leads to the formation of calcium 
phosphate (CaHPO
4
), which, given its low solubility, mainly insolubilizes 
inside casein micelles. In addition, some of the calcium ions reduce the zeta 
potential of the micelle and its thermal stability. At the same time, they cause a 
decrease in the level of hydration in the micelle. 
The addition of sodium chloride causes an increase in ionic strength
and a decrease in the activity coefficient of ions in the soluble phase. This 
results in the solubilization of colloidal calcium phosphate. Hydration of the 
casein micelles increases without any change in its size and its surface 
potential.
Citrate is a commonly used complexing agent of calcium. Its addition to 
milk causes a shift in equilibrium, which results in a dissociation of calcium 
phosphate and the solubilization of colloidal calcium phosphate. Depending on 
the amount added, citrate can cause the disintegration of the casein micelles 
and the release of free caseins. Unlike citrate, phosphate addition increases the 
calcium phosphate content of the casein micelles. By reducing the amount of 
ionic calcium, phosphate and citrate increases the thermal stability of milk.

---

22 Handbook of Food Science and Technology 3 
1.2.2.4.
 Effect of acidification 
The acidification of milk causes major physicochemical changes to both 
the casein micelle and serum. Rapid acidification of milk (concentrated 
organic or inorganic acid) causes destabilization of the casein micelle surface 
and flocculation of casein micelles in the form of a precipitate of varying 
granular size dispersed in whey. Slow acidification (lactic acid bacteria, 
glucono-delta-lactone) causes a greater rearrangement of casein micelles 
leading to the formation of a homogeneous gel throughout the entire milk 
volume. During slow acidification (Figure 1.7), the surface potential of casein 
micelles decreases gradually. At the same time, the protonation of citrate and 
phosphate causes the dissociation of soluble calcium salts (mainly calcium 
phosphate and calcium citrate) and a shift in the mineral balance of milk 
resulting in the solubilization of colloidal calcium phosphate and in the release 
of some caseins from the casein micelle. Up to a pH of 5.4, the solubilization 
of colloidal calcium phosphate has little impact on the organization of the 
micelle. At a pH below 5.4, the release of calcium bound to phosphoserines 
causes a gradual disintegration of the micelle, which loses its spherical shape. 
In addition, the amount of soluble caseins (mostly 
β
casein) reaches a 
maximum between pH 5.5 and 5.2 (10 – 30%, depending on temperature). 
When the surface charge of the micelles is zero (pH 5.2), their distribution, 
homogenous until then, becomes inhomogeneous. The disintegrated casein 
micelles form aggregates of a few µm dispersed in the whey, which are 
progressively connected by the solubilized caseins. This results in the 
formation of a gel network containing the entire aqueous phase, which 
contracts continuously when the pH decreases from pH 5.0 to approximately 
4.4 [HEE 85].
1.2.2.5.
 Effect of renneting
Rennet, a mixture of chymosin and pepsin, is the coagulating enzyme of 
casein and is widely used in cheese technology. The destabilization of the 
casein micelle by rennet resulting in the formation of a gel can be divided into 
three stages (Figure 1.8):
– enzymatic hydrolysis of 
κ
-casein; 
– aggregation of hydrolyzed casein micelles; 
– reorganization of the aggregated casein micelles and formation of a gel 
network.

---

From Milk to Dairy Products 23