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
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