248 Part II: Water, Enzymology, Biotechnology, and Protein Cross-linking
CALCIUM
Calcium has a significant impact on rennet curd,
though it does not have a direct effect on the primary
phase of rennet action. Addition of ionic calcium, as
in the form of calcium chloride, for example, re-
duces the rate of rennet clot formation time and also
increases rennet curd firmness. Similarly if the calci-
um content of milk is lowered by approximately
30%, coagulation does not occur. Milks that have a
tendency to form weak curd may be fortified with
calcium chloride prior to the addition of rennet.
MILKPROCESSING
Heating milk to temperatures higher than approxi-
mately 70°C leads to delayed curd formation and
weak rennet curd (Vasbinder et al. 2003). In extreme
cases, there may be no curd at all. These effects are a
result of the formation of a complex involving disul-
phide linkages between kappa-casein and beta-
lactoglobulin under high heat treatment. Under these
conditions the 105-106 bond in kappa-casein is
inaccessible for chymosin action. This effect is gen-
erally not reversible. Under mild overheating condi-
tions, the addition of 0.02% calcium chloride may
help obtain firm rennet curd. Maubois et al. have
developed a process for reversing this effect by the
use of ultrafiltration. Increasing the protein content
by ultrafiltration before or after UHT treatment re-
stores curd-forming ability (Maubois et al. 1972).
According to Ferron-Baumy et al. (1991), such a
phenomenon results from lowering the zeta poten-
tial of casein micelles on ultrafiltration.
Homogenization has a distinct impact on milk
rennet coagulation properties. Homogenized milk
produces softer curd, but when only the cream por-
tion of milk is homogenized, rennet curd becomes
firmer (Nair et al. 2000). This is possibly because of
the reduction of fat globule size due to homogeniza-
tion and coating of the fat globule surface with
casein. These particles then act as casein micelles.
Concentrating milk prior to cheese making is now
a common practice. Techniques include evapora-
tive concentration, ultrafiltration, and microfiltra-
tion. Each of these procedures increases the casein
concentration in milk; thus the rate of casein aggre-
gation during the secondary phase increases. Ren-
net coagulation usually occurs at a lower degree of
kappa-casein hydrolysis, and rennet curd is gener-
ally firmer. For example, in unconcentrated milk
approximately 90% kappa-casein must be hydro-
lyzed by chymosin before curd formation, but in
ultrafiltered milk only 50% must be hydrolyzed.
(Dalgleish 1992). Under high concentration condi-
tions, rennet curd is extremely firm, and difficulties
are encountered in cutting curd using traditional
equipment. In cheeses manufactured from highly
concentrated milks, rennet should be properly mixed
in the milk mixture to prevent localization of rennet
action.GENETICVARIANTSProtein polymorphism (genetic variants) of kappa-
casein has been demonstrated to have an effect on
rennet coagulation (Marzialli and Ng-Kwai-Hang
1986). Protein polymorphism refers to a small varia-
tion in the makeup of proteins due to minor differ-
ences in the amino acid sequence. Examples include
kappa-casein AA, AB, or BB, beta-lactoglobulin
AA, AB, and so on. Milk with kappa-casein BB
variants forms a firmer rennet curd because of in-
creased casein content associated with this variant of
kappa-casein. Some breeds of cows, Jerseys in par-
ticular, have larger proportions of this variant. The
BB variant of beta-lactoglobulin is also associated
with higher casein content in milk.EFFECT OF CHYMOSIN ON
PROTEOLYSIS IN CHEESEOnce rennet curd has been formed and a fresh block
of cheese has been obtained, the ripening process
begins. During ripening, numerous biochemical re-
actions occur and lead to unique flavor and texture
development. During this process residual chymosin
that is retained in the cheese makes important con-
tributions to the ripening process (Kosikowski and
Mistry 1997, Sousa et al. 2001). As the cheese takes
form, chymosin hydrolyzes the paracaseins into
peptides optimally at pH 5.6 and creates a peptide
pool for developing flavor complexes (Kosikowski
and Mistry 1997).
The amount of rennet required to coagulate milk
within 30 minutes can be considerably below that
required to properly break down the paracaseins
during cheese ripening. Consequently, using too
little rennet in cheese making retards ripening, as
discerned by the appearance of the cheese and its