246 Part II: Water, Enzymology, Biotechnology, and Protein Cross-linking
Milk for ripened cheese is coagulated at a pH
above the isoelectric point of casein by special pro-
teolytic enzymes, which are activated by small
amounts of lactic acid produced by added starter
bacteria. The curds are sweeter and more shrinkable
and pliable than those of fresh, unripened cheeses,
which are produced by isoelectric precipitation.
These enzymes are typified by chymosin that is found
in the fourth stomach, or abomasum, of a young calf.
The isoelectric condition of a protein is that at
which the net electric charge on a protein surface is
zero. In their natural state in milk, caseins are nega-
tively charged, and this helps maintain the protein in
suspension. Lactic acid neutralizes the charge on the
casein. The casein then precipitates as a curd at pH
4.6, the isoelectric point, as in cottage or cream
cheese and yogurt. For some types of cheeses this
type of curd is not desirable because it would be too
acid, and the texture would be too firm and grainy.
For ripened cheeses such as Cheddar and Swiss, a
sweeter curd is desired, so casein is precipitated as a
curd near neutrality, pH 6.2, by chymosin in 30–45
minutes at 30–32°C. This type of curd is significant-
ly different than isoelectric curd and much more
suitable for ripened cheeses. Milk proteins other
than casein are not precipitated by chymosin. The
uniform gel formed is made up of modified casein
with fat entrapped within the gel.
MILKCOAGULATION ANDPROTEIN
HYDROLYSIS BYCHYMOSIN
The process of curd formation by chymosin is a
complex process and involves various interactions
of the enzyme with specific sites on casein, temper-
ature and acid conditions, and calcium. Milk is
coagulated by chymosin into a smooth gel capable
of extruding whey at a uniformly rapid rate. Other
common proteolytic enzymes such as pepsin, try-
psin, and papain coagulate milk too, but may cause
bitterness and loss of yield and are more sensitive to
pH and temperature changes.
An understanding of the structure of casein in
milk has contributed to the explanation of the mech-
anism of chymosin action in milk. Waugh and von
Hippel (1956) began to unravel the heterogeneous
nature of casein with their observations on kappa-
casein. Casein is comprised of 45–50% alpha-
s-casein, 25–35% beta-casein, 8–15% kappa-casein,
and 3–7% gamma-casein. Various subfractions of
alpha-casein have also been identified, as have gen-
etic variants. Each casein fraction differs in sensitiv-
ity to calcium, solubility, amino acid makeup and
electrophoretic mobility.
An understanding of the dispersion of casein in
milk has provoked much interest. Several models of
casein micelles have been proposed including the
coat-core, internal structure, and subunit models.
With the availability of newer analytical techniques
such as three-dimensional X-ray crystallography,
work on casein micelle modeling is continuing, and
additional understanding is being gained. The sub-
unit model (Rollema 1992) is widely accepted. It
suggests that the casein micelle is made up of small-
er submicelles that are attached by calcium phos-
phate bonds. Hence, the micelle consists of not only
pure casein (approximately 93%), but also minerals
(approximately 7%), primarily calcium and phos-
phate. Most of the kappa-casein is located on the
surface of the micelle. This casein has few phospho-
serine residues and hence is not affected by ionic
calcium. It is located on the surface of the micelle
and provides for stability of the micelle and protects
the calcium-sensitive and hydrophobic interior, in-
cluding alpha- and beta- caseins from ionic calcium.
These two caseins have phosphoserine residues and
high calcium-binding affinity. The carbohydrate, N-
acetly neuramic acid, which is attached to kappa-
casein, makes this casein hydrophilic. Thus, the
casein micelle is suspended in milk as long as the
properties of kappa-casein remain unchanged. More
recently the dual-bonding model has been proposed
(Horne 1998).
Introducing chymosin to the cheese-milk, nor-
mally at about 32°C, destabilizes the casein micelle
in a two-step reaction, the first of which is enzymat-
ic (or primary) and the second, nonenzymatic (or
secondary) (Kosikowski and Mistry 1997, Lucey
2003). These two steps are separate but cannot be
visually distinguished: only the appearance of a curd
signifies the completion of both steps. The primary
phase was probably first observed by Hammersten
in the late 1800s (Kosikowski and Mistry 1997) and
must occur before the secondary phase begins.
In the primary phase, chymosin cleaves the
phenylalanine-methionine bond (105-106) of kappa-
casein, thus eliminating its stabilizing action on
calcium-sensitive alpha-s- and beta-caseins. In the
secondary phase, the micelles without intact kappa-
casein aggregate in the presence of ionic calcium in
milk and form a gel (curd). This mechanism may be
summarized as follows (Fig. 10.1):