BLBS102-c11 BLBS102-Simpson March 21, 2012 13:9 Trim: 276mm X 219mm Printer Name: Yet to Come
226 Part 2: Biotechnology and Enzymology(1970) andCynara humilis(Esteves and Lucey 2002). These
enzymes have been used successfully for many years in Portugal
to make native ripened Serra cheese with excellent flavor and
without bitterness.
In India, enzymes derived fromWithania coagulanshave been
used successfully for cheese making, but commercialization has
been minimal, especially with the development of genetically
derived chymosin.MicrobialSubstitutes from microbial sources have been very successful
and continue to be used. Many act like trypsin and have an op-
timum pH activity between 7 and 8 (Green 1977, Kosikowski
and Mistry 1997). Microorganisms, includingBacillus subtilis,
B. cereus,B. polymyxa,Cryphonectria parasitica(formerlyEn-
dothia parasitica), andMucor pusillusLindt (also known as
Rhizomucor pusillus)andRh. mieheihave been extracted for
their protease enzymes. The bacilli enzyme preparations were
not suited for cheese making because of excessive proteolytic
activity while the fungal-derived enzymes gave good results,
but not without off flavors such as bitter. Commercial enzyme
preparations isolated fromCr. parasiticaled to good quality Em-
mental cheese without bitter flavor, but some cheeses, such as
Cheddar, that use lower cooking temperatures reportedly showed
bitterness. Enzyme preparations ofRh. Miehei, at recommended
levels, produced Cheddar and other hard cheeses of satisfactory
quality without bitter flavor. Fungal enzyme preparations are in
commercial use, particularly in North America. In cottage cheese
utilizing very small amounts of coagulating enzymes, shattering
of curd was minimum in starter-rennet set milk and maximum
in starter–microbial milk coagulating enzyme set milk (Brown
1971).
Enzymes derived fromRh. mieheiandRh. pusillusare not
inactivated by pasteurization. Any residual activity in whey led
to hydrolysis of whey proteins during storage of whey powder.
This problem has been overcome by treating these enzymes with
peroxides to reduce heat stability. Commercial preparations of
Rh. mieheiandRh. pusillusare inactivated by pasteurization.
The heat stability ofCr. parasitica–derived enzyme is similar to
that of chymosin.
These fungal enzymes are also milk-clotting aspartic en-
zymes, and all exceptCr. parasiticaclot milk at the same peptide
bond as chymosin.Cryphonectriaclots kappa-casein at the
104-105 bond.CHYMOSIN ACTION ON MILK
Chymosin produces a smooth curd in milk, and it is relatively
insensitive to small shifts in pH that may be found in milk due
to natural variations, does not cause bitterness over a wide range
of addition, and is not proteolytically active if the cheese sup-
plements other foods. Chymosin coagulates milk optimally at
pH 6.0–6.4 and at 20–30◦C in a two-step reaction, although
the optimum pH of the enzyme is approximately 4 (Kosikowski
and Mistry 1997, Lucey 2003). Optimum temperature for co-
agulation is approximately 40◦C, but milk for cheese makingis coagulated with rennet at 31–32◦C because at this tempera-
ture the curd is rheologically most suitable for cheese making.
Above pH 7.0, activity is lost. Thus, mastitic milk is only weakly
coagulable, or does not coagulate at all.
Chymosin is highly sensitive to shaking, heat, light, alkali,
dilutions, and chemicals. Stability is highest when stored at 7◦C
and pH 5.4–6.0 under dark conditions. Liquid rennet activity is
destroyed at 55◦C, but rennet powders lose little or no activity
when exposed to 140◦C. Standard single-strength rennet activity
deteriorates at about 1% monthly when held cold in dark or
plastic containers.
Single-strength rennet is usually added at 100–200 mL per
1000 kg of milk. It serves to coagulate milk and to hydrolyze ca-
sein during cheese ripening for texture and flavor development.
It should be noted that bovine chymosin has greater speci-
ficity for cow’s milk than chymosin derived from kid or lamb.
Similarly, kid chymosin is better suited for goat milk.
Milk contains fat, protein, sugar, salts, and many minor com-
ponents in true solution, suspension, or emulsion. When milk is
converted into cheese, some of these components are selectively
concentrated as much as ten fold, but some are lost to whey. It
is the fat, casein, and insoluble salts that are concentrated. The
other components are entrapped in the cheese serum or whey, but
only at about the levels at which they existed in the milk. These
soluble components are retained, depending on the degree that
the serum or whey is retained in the cheese. For example, in a
fresh Cheddar cheese, the serum portion is lower in volume than
in milk. Thus, the soluble component percentage of the cheese
is smaller.
In washed curd cheeses such as Edam or Brick, the above rela-
tionship does not hold, and lactose, soluble salts, and vitamins in
the final cheese are reduced considerably. Approximately 90%
of living bacteria in the cheese-milk, including starter bacteria,
are trapped in the cheese curd. Natural milk enzymes and oth-
ers are, in part, preferentially absorbed on fat and protein, and
thus a higher concentration remains with the cheese. In rennet
coagulation of milk and the subsequent removal of much of the
whey or serum, a selective separation of the milk components
occurs, and in the resulting concentrated curd mass, many bio-
logical agents become active, marking the beginning of the final
product, cheese.
Milk for ripened cheese is coagulated at a pH above the iso-
electric point of casein by special proteolytic 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 pro-
duced 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 negatively 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