BLBS102-c11 BLBS102-Simpson March 21, 2012 13:9 Trim: 276mm X 219mm Printer Name: Yet to Come
224 Part 2: Biotechnology and Enzymologya 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 chy-
mosin 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 under-
gone an interesting transformation over time. It is believed that
in the early days of cheese making, milk coagulation 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 embedded within the
stomach lining diffused into the milk and coagulated it. This
crude process of extracting coagulating enzymes was eventu-
ally finessed into an industry that employed specific methods to
extract and purify the enzyme and develop an extract from the
fourth stomach 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 var-
ious enzymes. Live calves were required for the manufacture
of these products. Because of religious and economic reasons,
another industry also had emerged for manufacturing alternative
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 manufacture were then realized, and in 1990 a new pro-
cess utilizing this technology was approved in the United States
for manufacturing rennet.Rennet ManufactureFor 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 bal-
ance is pepsin. As the calves age and are fed other feeds such
as concentrates, the ratio of chymosin 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 unwashed stomachs
(vells) are preserved for enzyme extraction 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
preservation. Air-dried stomachs give lower yields of chymosin
than frozen stomachs; 12–13 air-dried stomachs 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 ex-
tracting, 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 par-
ticles in suspension are removed by introducing 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 standardized, sothat 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 sus-
pension with sodium chloride or acidifying it with a food-
grade acid. Chymosin precipitates and secondary enzymes such
as pepsin remain in the original suspension. The chymosin-
containing precipitate is dried to rennet powder (Kosikowski
and Mistry 1997).
A method has been developed for manufacturing rennet with-
out 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 com-
mercialized but may be of value where religious practices do not
allow calf slaughter.
Rennet paste, a rich source of lipolytic enzymes, has been a
major factor in the flavor development of ripened Italian cheeses
such as Provolone and Romano. Conceivably, 2100 years ago,
Romans applied rennet paste to cheese milks to develop a typical
“picante” flavor in Romano and related cheeses, for even then
calf rennet was used to set their cheese milks. In more modern
times, many countries, including the United States, applied calf
rennet paste to induce flavor, largely for Italian ripened cheeses.
Farntiam et al. (1964) successfully produced, from goats,
dried preparations of a pregastric-oral nature, also rich in lipoly-
tic enzymes. They applied them to milk for ripened Italian cheese
with excellent results. Since then, lipase powders, of various
character and strength, have largely replaced rennet paste.Chymosin Production by Genetic TechnologyGenetic technology has been used for the commercial produc-
tion of a 100% pure chymosin product from microbes. This type
of chymosin is often called fermentation-produced chymosin.
Uchiyama et al. (1980) and Nishimori et al. (1981) published
early on the subject, and in 1990 Pfizer, Inc., received US Food
and Drug Administration approval to market a genetically trans-
formed product, Chy-Max, with GRAS stature (Duxbury 1990).
Other brands using other microorganisms have also been ap-
proved. Table 11.1 lists some examples of such products.
The microbes used for this type of rennet include nonpa-
thogenic microorganismsEscherichia coliK-12,Kluyveromyces
marxianus var. lactisandAspergillus niger var. awamori.
Prochymosin genes obtained from young calves are transferred
through DNA plasmid intervention into microbial cells. Fermen-
tation follows to produce prochymosin, cell destruction, activa-
tion of the prochymosin to chymosin, and harvesting/producing
large yields of pure, 100% chymosin. This product, transferred
from an animal, is considered a plant product, as microbes are of
the plant kingdom. Thus, they are acceptable to various religious
groups.
These products have been widely studied to evaluate their
impact on the quality and yield of cheese. An initial concern
was that because of the absence of pepsin from these types of