Short Communication: Stability of Chymosin and Cyprosins
Under Milk-Coagulation and Cheese-Ripening Conditions
ABSTRACT
Chymosin was stable under milk-coagulation and
cheese-ripening conditions with a loss of activity not
exceeding 8% of the initial level. Cyprosins lost up to
33% of their activity under milk-coagulation conditions
and less than 6% under cheese-ripening conditions.
Chymosin incubated in the presence of cyprosins lost up
to 94% of its activity under milk-coagulation conditions
and up to 70% under cheese-ripening conditions.
(Key words: cyprosins, chymosin, milk coagulation,
cheese ripening)
INTRODUCTION
Chymosin (EC 3.4.23.4), an acid proteinase present
in the abomasum of young ruminants, shows a highly
specific milk-clotting activity relative to its proteolytic
activity (2). Cyprosins (cynarases) are aspartic protein-
ases present in the aqueous extract of cardoon (Cynara
cardunculus L.) flowers used as milk coagulant for the
manufacture of some Portuguese and Spanish tradi-
tional cheeses. Cyprosins have an activity on K-CN
similar to that of chymosin (7) and a pronounced non-
specific activity on other casein fractions. Plasmin (EC
3.4.21.7), the principal indigenous proteinase in milk,
is involved in the formation and degradation of water-
soluble peptides in cheese (1).
Cheeses made using cardoon coagulant are highly
appreciated. There is a current trend toward partial
substitution of cardoon coagulant by animal rennet in
the manufacture of some traditional cheeses to increase
yield, while maintaining distinct sensory characteris-
tics. Because cyprosins exhibit a strong, nonspecific en-
dopeptidase activity, they might degrade proteinases
present in cheese made with cardoon coagulant once the
most susceptible bonds of caseins have been hydrolyzed,
slowing down the formation of soluble N (4, 6). To study
Received March 15, 1999.
Accepted July 2, 1999.
1
Corresponding author: Manuel Nuñez, Departamento de Tecno-
logı́a de Alimentos, INIA, Carretera de la Coruña Km 7, Madrid,
Spain 28040.
1999 J Dairy Sci 82:2331–2333
A. PICON, J. FERNANDEZ, P. GAYA,
M. MEDINA and M NUÑEZ1
Departamento de Tecnologı́a de Alimentos, Instituto Nacional
de Investigación y Tecnologı́a Agraria y Alimentaria, Madrid, Spain
this phenomenon, we investigated the stability of chy-
mosin and cyprosins under milk-coagulation and
cheese-ripening conditions.
The chymosin used was Maxiren 15L (Gist Brocades
NV, Delft, The Netherlands) with a declared content of
900 μg of chymosin/ml. Cyprosins were extracted from
air-dried, mature cardoon flowers (3). A unit of cyprosin
activity was defined as the activity that produced an
increase of 1 U in the absorbance at 440 nm in 1 h at
37°C using azocasein (10 g/L in 0.1 M phosphate buffer,
pH 7) as substrate (6). The plasmin used (Sigma Chemi-
cal Co., St. Louis, MO) was a lyophilized powder with
a declared activity of 2.5 U/mg of protein.
Milk-coagulation conditions were obtained by incu-
bating solutions of chymosin, cyprosins, or their mix-
ture in pH 6.7 PBS, in a shaker bath at 35°C for 2 h.
Cheese-ripening conditions were obtained by incubat-
ing solutions of chymosin, cyprosins, or their mixture
in 0.01 M citrate buffer, pH 5.0, containing 50 g of
NaCl/L, in a shaker bath at 12°C for 20 h. Enzyme
concentrations in buffer were 15 μg/ml for chymosin
and 0.285 U/ml for cyprosins. When convenient, plas-
min was added at 0.0125 U/ml and ovine casein (Sigma
Chemical Co.) at 20 g/L. The experiments were carried
out in triplicate.
Activity of chymosin and cyprosins before and after
incubation was determined by thrombelastography as
previously described (5). Clotting time of milk was time
in minutes needed by the thrombelastographic curve
to reach an amplitude of 1 mm. Because of the high
specificity of chymosin, it was assumed that chymosin
had no effect on cyprosins when they were incubated
together. Residual chymosin activity after incubation
of the mixture was calculated using a regression equa-
tion of the type t–1 = a + b Ch + c Cy, where t = clotting
time, Ch = chymosin activity, and Cy = cyprosin activity
(5). Results were analyzed by multiple linear regression
using the BMDP1R program (BMDP Statistical Soft-
ware, Los Angeles, CA). Analysis of variance was car-
ried out on the residual activity of chymosin and cypros-
ins, with addition of casein and plasmin as main effects,
and on the residual activity of chymosin with addition
of casein, plasmin, and cyprosins as main effects, using
the BMDP8V program, with P < 0.05.
2331
2332 PICON ET AL.

TABLE 1. Inactivation of chymosin and cyprosins during incubation under milk-coagulation conditions.1

Residual activity (% initial activity)

Chymosin in the
Casein Plasmin Chymosin Cyprosins presence of cyprosins

(g/L) (U/L) X SD X SD X SD
0 0 93.8 2.1 67.9 6.1 5.8 3.4
20 0 93.4 3.0 82.4 4.7 12.0 3.2
0 12.5 93.9 2.2 67.3 3.9 7.3 3.9
20 12.5 92.4 1.6 78.6 4.8 14.6 2.7
1
Incubation for 2 h at 35°C in PBS, pH 6.7 (experiment in triplicate).

In PBS incubated at 35° for 2 h, chymosin lost 6.2%
of its initial activity (Table 1), because of photo-oxida-
tion or self-digestion. Addition of casein and plasmin
to the incubation medium had no significant effect on
chymosin inactivation under milk-coagulation condi-
tions (Table 1).
Cyprosins incubated in PBS at 35°C for 2 h under-
went self-digestion and lost a third of their initial activ-
ity (Table 1). When casein was added to the incubation
medium, the loss of activity was significantly (P < 0.001)
decreased, approximately by half. As for chymosin, ad-
dition of plasmin showed no significant effect on the
residual activity of cyprosins after incubation.
Most chymosin activity was lost (P < 0.001) after
incubation in PBS at 35°C for 2 h in the presence of
cyprosins (Table 1). Addition of casein to the incubation
medium protected (P < 0.001) chymosin from inactiva-
tion by cyprosins, whereas addition of plasmin had no
significant effect on chymosin residual activity under
milk-coagulation conditions.
Degradation of enzymes during incubation at 35°C
was checked by using HPLC (8) and fast-protein liquid
chromatography (3) techniques for the determination of
chymosin and cyprosins, respectively (data not shown).
The results obtained point to self-digestion of cyprosins
and hydrolysis of chymosin by cyprosins, although the
decreases in the concentration of enzymes did not match
the respective losses of activity.
Inactivation of chymosin after incubation in citrate
buffer at 12°C for 20 h was even lower than under milk-
coagulation conditions (Table 2). Addition of casein and
plasmin to the incubation medium had no significant
effect on the residual activity of chymosin under cheese-
ripening conditions (Table 2).
Cyprosins lost little of their initial activity in citrate
buffer incubated at 12°C for 20 h (Table 2), even though
the pH of the incubation medium was close to their
activity optimum (3). Inactivation of cyprosins under
cheese-ripening conditions, much lower than under
milk-coagulation conditions, was not significantly in-
fluenced by the presence of casein or plasmin in the
incubation medium (Table 2).
Chymosin lost more than 60% of its activity after
incubation in citrate buffer at 12°C for 20 h in the
presence of cyprosins (Table 2). Because chymosin ac-
tivity did not decrease when incubated alone, this loss
was attributed to degradation by cyprosins. Addition of
casein to the incubation medium containing cyprosins
contributed favorably (P < 0.001) to chymosin stability.
Plasmin, which by itself had no apparent effect on chy-
mosin, increased (P < 0.001) the inactivation of this
enzyme in the presence of cyprosins.
Chymosin and cyprosins levels in cheese are much
lower than those used in our experiments; however,
cheese-ripening periods are considerably longer than
were those in our experimental incubation. Residual
caseins in cardoon rennet cheese are at only 10% of
their initial level after 30 d (4). At this stage of ripening,
inactivation of chymosin by cyprosins in cheese made
from milk coagulated with a mixture of chymosin and
cardoon coagulant might occur in the absence of alter-
native substrates for the endopeptidase activity of cy-

TABLE 2. Inactivation of chymosin and cyprosins during incubation under cheese-ripening conditions.1
Residual activity (% initial activity)
Chymosin in the
Casein Plasmin Chymosin Cyprosins presence of cyprosins

(g/L) (U/L) X SD X SD X SD
0 0 97.3 2.5 95.2 4.6 38.6 5.8
20 0 98.8 2.2 96.4 3.3 57.2 8.9
0 12.5 98.0 1.8 94.7 3.1 30.2 5.5
20 12.5 95.8 2.2 97.9 2.6 45.7 4.3
1
Incubation for 20 h at 12°C in citrate buffer, pH 5.0, containing 50 g of NaCl/L (experiment in triplicate).

Journal of Dairy Science Vol. 82, No. 11, 1999

 SHORT COMMUNICATION: STABILITY OF CHYMOSIN AND CYPROSINS
prosins. However, self-digestion of cyprosins does not
seem to be a feasible event in light of the small loss of
activity recorded for cyprosins incubated under cheese-
ripening conditions.
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Journal of Dairy Science Vol. 82, No. 11, 1999