BLBS102-c11 BLBS102-Simpson March 21, 2012 13:9 Trim: 276mm X 219mm Printer Name: Yet to Come
228 Part 2: Biotechnology and EnzymologyChymosin ConcentrationChymosin concentration affects gel firmness (Lomholt and Qvist
1999), and enzyme kinetics have been used to study these effects.
An increase in the chymosin concentration (larger amounts of
rennet added to milk) reduces the total time required for rennet
clotting, measured by the appearance of the first curd particle. As
a result, the secondary phase of rennet action will also proceed
much earlier, with the net result of an increase in the rate of
increase in gel firmness. This property of chymosin is sometimes
used to control the rennet coagulation in concentrated milks,
which have a tendency to form firmer curd because of the higher
protein content. Lowering the amount of rennet reduces the rate
of curd firming. It should also be noted that with an alteration in
the amount of rennet added to milk, there also will be a change
in the amount of residual chymosin in the cheese.TemperatureThe optimal condition for curd formation in milk with chy-
mosin is 40–45◦C, but this temperature is not suitable for cheese
making. Rennet coagulation for cheese making generally oc-
curs at 30–35◦C for proper firmness. At lower temperatures,
rennet clotting rate is significantly reduced, and at refrigeration
temperatures, virtually no curd is obtained.pHChymosin action in milk is optimal at approximately pH 6. This
is obtained with starter bacteria. If the pH is lowered further,
rennet clotting occurs at a faster rate, and curd firmness is re-
duced. Lowering pH also changes the water-holding capacity,
which will have an impact on curd firmness.CalciumCalcium 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, reduces the rate of rennet clot formation time and also
increases rennet curd firmness. Similarly, if the calcium 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.Milk ProcessingHeating milk to temperatures higher than approximately 70◦C
leads to delayed curd formation and weak rennet curd (Vas-
binder 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 disulphide 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 generally not reversible. Under mild over-
heating conditions, the addition of 0.02% calcium chloride may
help obtain firm rennet curd. Maubois et al. have developed aprocess for reversing this effect by the use of ultrafiltration. In-
creasing the protein content by ultrafiltration before or after UHT
treatment restores curd-forming ability (Maubois et al. 1972).
According to Ferron-Baumy et al. (1991), such a phenomenon
results from lowering the zeta potential of casein micelles on
ultrafiltration.
Homogenization has a distinct impact on milk rennet coagu-
lation properties. Homogenized milk produces softer curd, but
when only the cream portion 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 homogenization 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 evaporative concentration, ultrafil-
tration, and microfiltration. Each of these procedures increases
the casein concentration in milk; thus, the rate of casein aggrega-
tion during the secondary phase increases. Ren-net coagulation
usually occurs at a lower degree of kappa-casein hydrolysis, and
rennet curd is generally firmer. For example, in unconcentrated
milk, approximately 90% kappa-casein must be hydrolyzed by
chymosin before curd formation, but in ultrafiltered milk only
50% must be hydrolyzed. (Dalgleish 1992). Under high concen-
tration conditions, 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 local-
ization of rennet action.Genetic VariantsProtein 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 variation in the makeup of proteins due to mi-
nor differences 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 increased casein content associated with this
variant of kappa-casein. Some breeds of cows, Jerseys in partic-
ular, 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 reactions occur and lead to unique flavor
and texture development. During this process, residual chymosin
that is retained in the cheese makes important contributions
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).