242 Part II: Water, Enzymology, Biotechnology, and Protein Cross-linking
of flavor and texture. All this is the result of the action
of the enzyme chymosin (Andren 2003).
CHYMOSIN
Chymosin, rennet, and rennin are often used inter-
changeably to refer to this enzyme. The latter, ren-
nin, should not be confused with renin, which is an
enzyme associated with kidneys and does not clot
milk. Chymosin is the biochemical name of the
enzyme that was formerly known as rennin. It be-
longs to the group of aspartic acid proteinases, EC
3.4.23, that have a high content of dicarboxylic and
hydroxyamino acids and a low content of basic
amino acids. Its molecular mass is 40 kDa (Andren
2003, Foltmann 1993).
Rennet is the stomach extract that contains the
enzyme chymosin in a stabilized form that is usable
for cheese making (Green 1977). While the amount
of chymosin that is required for cheese making is
very small, this enzyme industry has undergone an
interesting transformation over time. It is believed
that in the early days of cheese making, milk coagu-
lation occurred either by filling dried stomachs of
calves or lambs with milk or by immersing pieces
of such stomachs in milk (Kosikowski and Mistry
1997). The chymosin enzyme imbedded within the
stomach lining diffused into the milk and coagulated
it. This crude process of extracting coagulating en-
zymes was eventually finessed into an industry that
employed specific methods to extract and purify the
enzyme and develop an extract from the fourth stom-
ach of the calf or lamb. Extracts of known enzyme
activity and predictable milk clotting properties then
became available as liquids, concentrates, powders,
and blends of various enzymes. Live calves were
required for the manufacture of these products. Be-
cause of religious and economic reasons, another
industry also had emerged for manufacturing alter-
native milk clotting enzymes from plants, fungi, and
bacteria. These products remain popular and meet
the needs of various kosher and other religious needs.
Applications of genetic technology in rennet manu-
facture were then realized, and in 1990 a new
process utilizing this technology was approved in the
United States for manufacturing rennet.
RENNETMANUFACTURE
For the manufacture of traditional rennet, calves,
lambs, or kids that are no more than about 2 weeks old
and fed only milk are used (Kosikowski and Mistry
1997). As calves become older and begin eating other
feeds, the proportion of bovine pepsin in relation to
chymosin increases. Extracts from milk-fed calves
that are 3 weeks old contain over 90% chymosin, and
the balance is pepsin. As the calves age and are fed
other feeds such as concentrates, the ratio of chy-
mosin to pepsin drops to 30:70 by 6 months of age. In
a full-grown cow there are only traces of chymosin.
Milk-fed calves are slaughtered, and the unwash-
ed stomachs (vells) are preserved for enzyme extrac-
tion by emptying their contents, blowing them into
small balloons and drying them. The vells may also
be slit opened and dry salted or frozen for preserva-
tion. Air-dried stomachs give lower yields of chy-
mosin than frozen stomachs; 12–13 air-dried stom-
achs make 1 L of rennet standardized to 1:10,000
strength, but only 7–8 frozen stomachs would be
required for the same yield.
Extraction of chymosin and production of rennet
begin by extracting, for several days, chopped or
macerated stomachs with a 10% sodium chloride
solution containing about 5% boric acid or glycerol.
Additional salt up to a total of 16–18% is introduced
followed by filtration and clarification. Mucine and
grass particles in suspension are removed by intro-
ducing 1% of potash alum, followed by an equal
amount of potassium phosphate. The suspension is
adjusted to pH 5.0 to activate prochymosin (zymo-
gen) to chymosin, and the enzyme strength is stan-
dardized, so that one part coagulates 15,000 or
10,000 parts of milk. Sodium benzoate, propylene
glycol, and salt are added as preservatives for the
final rennet. Caramel color is also usually added.
The finished rennet solutions must be kept cold and
protected from light.
Powdered rennet is manufactured by saturating a
rennet suspension with sodium chloride or acidify-
ing it with a food-grade acid. Chymosin precipitates
and secondary enzymes such as pepsin remain in the
original suspension. The chymosin-containing pre-
cipitate is dried to rennet powder (Kosikowski and
Mistry 1997).
A method has been developed for manufacturing
rennet without slaughtering calves. A hole (fistula)
is surgically bored in the side of live calves, and at
milk feeding time, excreted rennet is removed. After
the calf matures, the hole is plugged, and the animal
is returned to the herd. This method has not been
commercialized but may be of value where religious
practices do not allow calf slaughter.