BLBS102-c11 BLBS102-Simpson March 21, 2012 13:9 Trim: 276mm X 219mm Printer Name: Yet to Come
11 Chymosin in Cheese Making 227and 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 significantly 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.Milk Coagulation and Protein Hydrolysis
by ChymosinThe process of curd formation by chymosin is a complex process
and involves various interactions of the enzyme with specific
sites on casein, temperature 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 pro-
teolytic enzymes such as pepsin, trypsin, 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 con-
tributed to the explanation of the mechanism of chymosin action
in milk. Waugh and von Hippel (1956) began to unravel the het-
erogeneous nature of casein with their observations on kappa-
casein. Casein comprises 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
genetic variants. Each casein fraction differs in sensitivity to cal-
cium, solubility, amino acid makeup, and electrophoretic mobil-
ity.
An understanding of the dispersion of casein in milk has pro-
voked 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 tech-
niques such as three-dimensional X-ray crystallography, work
on casein micelle modeling is continuing, and additional under-
standing is being gained. The subunit model (Rollema 1992)
is widely accepted. It suggests that the casein micelle is made
up of smaller submicelles that are attached by calcium phos-
phate bonds. Hence, the micelle consists of not only pure ca-
sein (∼93%), but also minerals (∼7%), primarily calcium and
phosphate. Most of the kappa-casein is located on the surface
of the micelle. This casein has few phosphoserine residues and
hence is not affected by ionic calcium. It is located on the sur-
face 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-acetyl neuramic acid, which is
attached to kappa-casein, makes this casein hydrophilic. Thus,
the casein micelle is suspended in milk as long as the prop-
erties of kappa-casein remain unchanged. More recently the
dual-bonding model has been proposed (Horne 1998).
Introducing chymosin to the cheese-milk, normally at about
32 ◦C, destabilizes the casein micelle in a two-step reaction, the
first of which is enzymatic (or primary) and the second, nonen-
zymatic (or secondary) (Kosikowski and Mistry 1997, LuceyFigure 11.1.Destabilization of the casein micelle by introduction of
chymosin.2003). These two steps are separate but cannot be visually distin-
guished: 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. 11.1):
The macropeptide, also known as caseinmacropeptide, con-
tains approximately 30% amino sugar, hence the name glyco-
macropeptide (Lucey 2003). In the ultrafiltration processes of
cheese making, this macropeptide is retained with whey pro-
teins to increase cheese yields significantly. In the conventional
cheese-making process, the macropeptide is found in whey.
The two-step coagulation does not fully explain the presence
of the smooth curd or coagulum. Beau, many years ago, estab-
lished that a strong milk gel arose because the fibrous filaments
of paracasein cross-linked to make a lattice. Bonding at critical
points between phosphorus, calcium, free amino groups, and
free carboxyl groups strengthened the lattice, with its entrapped
lactose and soluble salts. A parallel was drawn between the
gel formed when only lactic acid was involved as in fresh, un-
ripened cheese and when considerable chymosin was involved
as in hard, ripened cheese. Both gels were considered originally
as starting with a fibrous protein cross bonding, but the strictly
lactic acid curd was considered weaker, because not having been
hydrolyzed by a proteolytic enzyme, it possessed less bonding
material. The essential conditions for a smooth gel developing
in either case were sufficient casein, a quiescent environment,
moderate temperature, and sufficient time for reaction.FACTORS AFFECTING CHYMOSIN
ACTION IN MILKRennet coagulation in milk is influenced by many factors that
ultimately have an impact on cheese characteristics (Kosikowski
and Mistry 1997). The cheese maker is usually able to properly
control these factors, some of which have a direct affect on the
primary phase and others on the secondary.