JFS:

Food Chemistry and Toxicology

Hydrolysis of Caseins By Extracts of Cynara

Cardunculus Precipitated by Ammonium Sulfate
S.V. SILVA, R.M. BARROS, AND F.X. MALCATA

Food Chemistry and Toxicology

ABSTRACT: Polyacrylamide gel electrophoresis (in the presence of urea) and gel permeation chromatography
were employed to assess the profile of hydrolysis of caseins and the activity of enzymes contributed by the flowers
of the plant Cynara cardunculus on bovine caseins, after previous precipitation with ammonium sulfate or in a
plain crude aqueous extract. Results indicated that the qualitative degradation profile of bovine caseins by plant
enzymes (cardosins) remains essentially unchanged upon extraction, and quantitative analysis showed that the
precipitated fractions had a higher coagulant-to-proteolytic activity ratio; hence, the results showed that inexpensive precipitation with ammonium sulfate can successfully be used as a purification method in the production
of that plant coagulant in standardized form.
Keywords: milk proteins, thistle, FPLC, urea-PAGE

Introduction

RUDE AQUEOUS EXTRACTS OF FLOWERS OF THE COMMON WILD

thistle, Cynara cardunculus, have been used for ages as a coagulant in the manufacture of several traditional Portuguese
and Spanish cheeses, such as Serra da Estrela, Serpa, and
Azeito ( Vieira de S and Barbosa 1972; Roseiro 1991; Macedo
and Malcata 1997), and Los Pedroches, La Serena, and Flor de
Gua (Fernandez-Salguero and others 1991; Sanjuan and
Fernandez-Salguero 1994). Those flowers are known to contain 2
aspartic proteinases, currently termed cardosins A and B, each
consisting of 2 subunits with apparent molecular weights of 31
and 15 kDa, and 34 and 14 kDa, respectively; it has been claimed
(Pires and others 1994; Verssimo and others 1995) that cardosin
A is similar to chymosin, while cardosin B is similar to pepsin, in
terms of specificity and activity.
Those enzymes are currently purified by a 2-step procedure,
which involves liquid extraction at low pH and separation afterwards by liquid chromatography (gel filtration followed by ionexchange) (Verssimo and others 1995). In addition to being costly, the overall process is rather slow and cumbersome; hence, it is
hardly appropriate when large quantities of those enzymes are
sought. The objective of enzyme purification is to remove nonprotein contaminants, as well as to isolate the enzyme in question from other proteins; the former objective is relatively easy to
achieve, unlike the latter. Precipitation techniques can be used
at an early point, whereas chromatographic procedures (for example, ion-exchange, gel filtrarion, or adsorption chromatography) are usually employed later. Salting-out is the most common
precipitation technique (Picn and others 1994). The salt usually
chosen is ammonium sulfate, (NH4)2SO4, which is highly soluble
in water (Robyt and White 1990) and is known to effectively stabilize proteins (Scopes 1994). Since large amounts of salt will contaminate the precipitated proteins, removal thereof prior to enzyme usage is required; this can easily be achieved by dialysis or
gel filtration (Scopes 1994). In an early approach to this issue,
Sousa and Malcata (1996) have studied the effects of processing
conditions on the caseinolytic activity of crude extracts of C. cardunculus, and found that the maximum specific caseinolytic activity was obtained by grinding the flowers for 36 s, using an ex1746

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traction buffer of pH 5.9 and NaCl content of 0 % (w/w), and homogenizing the ground flower/buffer suspension for 15 min.
The objective of this study was (i) to evaluate the proteolytic
and coagulant activities of said plant enzymes, both in crude
aqueous extract and following precipitation with ammonium sulfate at 2 concentration levels (quantitative action), and (ii) to
characterize the effect of those enzymes on the profile of hydrolysis of bovine s- and -caseins (qualitative action). The working
hypothesis under scrutiny was to ascertain whether salting-out
might replace the (more expensive) classically employed chromatographic separations.

Material and Methods

Partial purification of enzymes
The source of plant proteases was dry flowers of C. cardunculus: the stigmata and stylets of dry flowers were macerated with
0.1 M aqueous citric acid (pH 3.0) at the ratio of 1 gflowers per 10
mLbuffer, so as to produce a fluid product. Ammonium sulfate was
then added to the plant extract up to 30% (w/v) saturation. After
30 min, the solution was centrifuged at 10000 rpm for 10 min at
4 C; the precipitate was redissolved in water up to approximately twice the pellet volume. Ammonium sulfate was added once
again to the supernatant up to 70% (w/v) saturation. After 30
min, the solution was centrifuged at 10000 rpm at 4 C; as before,
the precipitate was redissolved in twice its volume of water. Plant
enzyme extracts were dialyzed overnight at 4 C against plain water, and lyophilized prior to use.

Electrophoretic characterization of enzymes

Predetermined amounts of each cardosin extract were placed
in separate Eppendorf vials, and 100 mL of 10% (w/w) SDS was
added to each one. The vials were heated at 90 C for 10 min in a
heating block, and then cooled to room temperature. The separation was carried out in high-density gels: each gel had a stacking gel zone (7.5% T, 2% C) and a continuous separating gel zone
(20% T and 2% C); they also contained 30% (v/w) ethylene glycol.
The automated electrophoresis system Phastsystem (Pharmacia, Uppsala, Sweden) was used to separate the proteins, which

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Hydrolysis of caseins by plant enzymes

Coagulant activity
The clotting activity was determined following the procedure
described by IDF (1992). The reconstituted milk was obtained by
dissolving low-heat skim milk (NILACTM) (NIZO, Ede, The Netherlands) in 10 mM aqueous CaCl2 (pH 6.5), so as to achieve a concentration of 0.12 kg/L; the time taken for 0.2 mL of each enzyme
extract to coagulate 2 mL of reconstituted milk was measured
and recorded (the coagulation point was determined by manually rotating the test tube periodically, at short time intervals, and
checking for visible clot formation). One rennet unit (R.U.) was
defined as the amount of protein that coagulates 10 mL of reconstituted low-heat skim milk powder at 30 C in 100 s (Berridge
1945).

Proteolytic activity
The proteolytic activity was determined by the method of
Tomarelli and others (1949), with slight modifications. The lyophilized enzyme mixture, reconstituted in 0.1 M aqueous sodium
phosphate buffer (pH 6.0), was mixed with 250 L of 2% (w/v)
azocasein and incubated at 25 C for 10 min. The reaction was
then quenched via addition of 0.5 mL of cold 5% (w/v) trichloroacetic acid, TCA. The solution was centrifuged at 10000 rpm for
10 min; 1 mL of supernatant was further mixed with 1 mL of 0.5 M
aqueous NaOH to intensify the azo-associated color. The absorbance of the solution at 440 nm was measured against a blank,
prepared in a similar fashion but adding TCA to azocasein prior
to addition of the enzyme.

Hydrolysis of substrates
Commercial bovine s- and -caseins (Pharmacia) were dissolved up to 1 mg/mL in aqueous phosphate buffer (pH 6.5),
containing sodium azide (0.05 g/mL) to prevent microbial activity; these solutions were treated independently with cardosins
precipitated by ammonium sulfate at 30% (w/v) and 70% (w/v)
saturation, as well as with the crude enzyme extract, and incubated at 30 C; the (enzyme-containing) protein:substrate ratio
used in all cases was 1:400 (w/w). At predetermined time intervals (0 min, 30 min, 1 h, 2 h, 5 h, 8 h, 12 h, and 24 h), samples
were taken and the reaction was quenched via addition of double-concentrated buffer at 50%(v/v) (McSweeney and others
1993) in the case of samples for electrophoresis (urea-PAGE), or
via heating at 95 C for 15 min in the case of samples for fast protein liquid chromatography (FPLC).

NaN3 as preservative, for 80 min at a flow rate of 0.4 mL/min. Prior to injection, the sample was passed through a 0.45 m filter
(Nucleopore, Cambridge, Mass., U.S.A.), whereas the buffer was
filtered through 0.22 m paper filter (Nalgene, Rochester, N.Y.,
U.S.A.). The void volume of the column was determined previously using blue dextran. The retention times of the peaks obtained were compared with those of a mixture of molecular
weight standards, namely aldolase (158 kDa), bovine serum albumin (67 kDa), ovalbumin (43 kDa), -lactoglobulin (36 kDa),
-lactalbumin (14.4 kDa), and ribonuclease (13.7 kDa). Each analytical determination was carried out in duplicate.

Results and Discussion

Characterization of enzymes
The salting-out technique was employed to purify the aspartic proteinases present in the crude aqueous extract of C. cardunculus; a 1st fraction was precipitated at 30% (w/v) saturation with
ammonium sulfate and a 2nd one at 70% (w/v) saturation with
the same salt. The protein concentration was 92, 197, and 410
mgprotein/ lyophilizate for the crude extract, the 70% (w/v) precipitate and the 30% (w/v) precipitate, respectively. SDS-PAGE was
performed on the purified enzyme fractions, as well as on the
crude aqueous extract (Figure 1). The electrophoretogram revealed that precipitates were qualitatively similar to each other,
and that the material in all fractions migrated as 4 bands, with
molecular weights 31 and 15 kDa (accounted for by cardosin A),
and 34 and 14 kDa (accounted for by cardosin B) (Verssimo and
others 1995). It is worth noting that 2 extra bands, with molecular
weights 20 to 26 kDa, were present in the fraction corresponding
to the enzymes purified at the lower level of addition of ammonium sulfate. These 2 bands may likely be accounted for by contaminating proteins that co-precipitate at 30% (w/v) saturation
of ammonium sulfate, although the possibility that multimeric
native proteins dissociate into their monomeric subunits during
the 1st stage of purification can not be completely ruled out.
Table 1 shows the specific enzymatic activity of the precipi-

Electrophoretic characterization of hydrolysates

Samples (0.75 mL) of hydrolysates were obtained, and prepared for urea-PAGE as described above. Urea-PAGE (12.5% for
the separation gel and 4% for the stacking gel; pH 8.9) was performed following the procedure of Andrews (1983), with the
modifications proposed by Shalabi and Fox (1987). Gels were
stained with Coomassie Blue G250 (Bio Rad Laboratories, Hercules, Calif., U.S.A.) according to the method of Blakesley and
Boezi (1977).

FPLC characterization of hydrolyzates

FPLC was conducted according to the following protocol: separation was performed after injection of 100 L of each hydrolysed sample in a Superose 12 column HR 10/30 (Pharmacia), with
the aid of an injection valve MV-7 and a UV-MII-detector (operated at 280 nm); the mobile phase was 150 mM of aqueous NaCl in
0.05 M aqueous phosphate buffer (pH 7.0), containing 0.2 g/L

Figure 1SDS-PAGE electrophoretogram of fractions precipitated by ammonium sulfate. Lane 1molecular weight
markers, lane 2crude aqueous extract, lane 3supernatant at 70% (w/v) saturation, lane 4precipitate at 30%
(w/v) saturation, lane 5precipitate at 70% (w/v) saturation.
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Food Chemistry and Toxicology

were stained by Coomassie blue.

Hydrolysis of caseins by plant enzymes

tates as compared with that of the crude extract. In what pertains
to the ammonium sulfate-precipitated fractions, the precipitation at 70% (w/v) saturation led to a decrease in the specific coag-

ulant activity relative to that at 30% (w/v), whereas the opposite

held for the proteolytic activity. In general, fractionation caused

Food Chemistry and Toxicology

Figure 2Urea-PAGE electrophoretograms illustrating the
degradation patterns of bovine -casein (-CN) by cardosins
previously precipitated with ammonium sulfate at (a) 30%
(w/v) and (b) 70% (w/v) saturation and (c) by crude extract,
after incubation for 0, 0.5, 1, 2, 5, 8, 12 and 24 h (lanes 18, respectively). Lanes 9 and 10 represent -casein after
incubation for 0 and 24 h, respectively, in the absence of
enzyme.
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Figure 3Urea-PAGE electrophoretograms illustrating the

degradation patterns of bovine -casein (-CN) by cardosins
previously precipitated with ammonium sulfate at (a) 30%
(w/v) and (b) 70% (w/v) saturation and (c) by crude extract,
after incubation for 0, 0.5, 1, 2, 5, 8, 12 and 24 h (lanes 18,
respectively). Lanes 9 and 10 represent s-casein after incubation for 0 and 24 h, respectively, in the absence of enzyme.

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Hydrolysis of caseins by plant enzymes

Table 1Specific coagulant and proteolytic activities, and
corresponding ratio, of ammonium sulfate-precipitated
fractions and crude extract.

Fraction
Crude extract
30% (w/v)
saturation
70% (w/v)
saturation

Coagulant
activity (C)
(R.U./gprotein)

Proteolytic
activity (P)
(DAbs/g protein/min)

104 4.11
90.6 2.01

1.53 0.10
0.24 0.001

67.7 10.30
377.5 34.62

74.6 2.69

0.79 0.004

94.4 11.64

C/P ratio

ied the independent action of cardosins A and B on bovine

s1-casein, and reported that both enzymes have preference for
Phe23-Phe24, but are able to cleave bonds Trp164-Tyr165 and
Phe153-Tyr154, although to lower extents; Phe23-Phe24 was also
found by Mulvihill and Fox (1977) to be one of the peptide bonds
most labile to the action of chymosin in the pH range 2.2-7.0.
Consequently, both caseins were fully degraded by the cardosins, and followed similar degradation patterns in either crude
or partially separated form. However, the rate of hydrolysis by

Food Chemistry and Toxicology

Note: Abs Absorbance

Values represent means of 3 replicates confidence interval (p = 95%)
Data referred to dry basis

a substantial decrease of the specific proteolytic activity (P) and

a less pronounced decrease in the specific clotting activity (C),
both relative to that of the crude extract. A decrease of the total
proteolytic activity of purified fractions of sodom apple (Calotropis procera) relative to the initial extract was also reported by
Aworh and Nakai (1986). Chen and Zall (1986a,b) claimed, in
turn, an increase of the clotting activity in a fraction of clam viscera extracts fractionated with ethanol. The ratio of coagulant-toproteolytic activity is a useful indicator of the enzyme appropriateness for general use in cheesemaking: a higher C/P ratio
usually leads to higher quality cheeses (Chen and Zall 1986b;
Harboe 1991; Esteves 1995). The ratio was actually higher for the
fractions precipitated with ammonium sulfate, as compared with
the plain, crude aqueous extract; between the precipitated fractions, the ratio was much higher for the 30% (w/v) saturation precipitate.

Electrophoretic characterization of hydrolysates

The progress of hydrolysis of bovine -and s-caseins brought
about by cardosins precipitated at the 2 levels of ammonium sulfate and by crude aqueous extract is shown in Figure 2 and 3, respectively.
Quantitative breakdown of original -casein was observed after as early as 30 min, irrespective of the level of ammonium sulfate added for precipitation of enzyme, whereas at least 5 h had
to elapse before a similar result was achieved with the crude extract. In the hydrolysates, -I-casein was the 1st peptide to show
up in all 3 situations tested, which is presumably a result of
cleavage of Leu192-Tyr193 in bovine -casein; according to
Simes (1998), that is the peptide bond in pure bovine -casein
most susceptible to the action of either cardosin. The peptide (I-casein) was further hydrolyzed to -II- and -III-casein, according to the classification proposed by Creamer (1976). Note
that Visser and Slangen (1977) observed that hydrolysis of pure
-casein by chymosin results in the production of 3 peptides,
namely -I-casein (f1-189/192), -II-casein (f1-163/165/167),
and -III-casein (f1-127/139).
The hydrolysis of s-casein was essentially complete by 30
min, regardless of the level of ammonium sulfate added for the
precipitation of enzyme, whereas at least 2 h had to elapse before an identical situation was produced by the crude extract.
One of the fast moving peptide bands was assigned to s1-Icasein, following comparison with the electrophoretic mobility of
bovine s1-I-casein produced via action of chymosin (Mulvihill
and Fox 1977); such peptide appeared in all 3 situations after 1
min of incubation. Those peptides were subsequently degraded
to smaller peptides, which migrated close to each other under
the electric field. Ramalho-Santos and others (1996) have stud-

Figure 4Molecular weight distribution of peptides produced via hydrolysis of bovine -casein by cardosins previously precipitated with (a) 30% (w/v) and (b) 70% (w/v)
saturation of ammonium sulfate and by (c) crude extract,
after incubation for 0, 0.5, 1, 2, 5, 8, 12 and 24 h.
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Hydrolysis of caseins by plant enzymes

the crude extract was lower than by the partially purified extracts, as expected. Note that the (enzyme-containing)
protein:substrate ratio used was the same in the reactions of hydrolysis of both substrates, but that the amount of enzyme
present was higher in the ammonium sulfate-saturated fraction
than in the crude extract (see Figure 1); this may help explain our
results, because the lower enzyme content, the lower rate of hydrolysis. The rate of degradation of s-casein was also slightly
higher than that of -casein when the crude extract was considered. This finding is in agreement with the reported fact (Mulvi-

hill and Fox 1977) that -casein is less susceptible than s1-casein
to proteolysis by chymosin. Sousa and Malcata (1997) also reported that -casein undergoes less degradation than s1-casein
in bovine milk cheeses manufactured with aqueous extracts of
flowers of C. cardunculus.

Chromatographic characterization of hydrolysates

Food Chemistry and Toxicology

The molecular weight (MW ) distribution of peptides in the

casein hydrolysates is shown in Figures 4 and 5 for - and scaseins, respectively. For both caseins, hydrolysis was in all cases
accompanied by a decrease in the concentration of high-MW
peptides (20 to 25 kDa) and a concomitant increase in the concentration of low-MW ones (< 1 kDa and 1 to 2.5 kDa). This observation reflects the expected sequential hydrolysis of longer to
shorter peptides. No relevant changes in the concentration of
peptide material between 2.5 and 15 kDa was noticed over the 24
h of hydrolysis in all cases, which indicates somewhat unselective hydrolysis.
As far as -casein is concerned, the family of peptides with
MW between 20 and 25 kDa essentially vanished by 2 h of hydrolysis carried out by the purified extracts (Figure 4a and b).
The 1st short peptides to be noticed were those with MW in the
range of 1 to 2.5 kDa, after as little as 30 min of reaction; then the
peptide group with MW within 15 to 20 kDa started to build up.
The high-MW peptide group was present until 12 h of hydrolysis
by the crude extract, but gradually yielded peptides with MW of
15 to 20 kDa (Figure 4c).
Regarding s-casein, peptides with MW of 20 to 25 kDa still
showed up by 2 h of hydrolysis by the crude extract (Figure 5c);
however, that group disappeared by 30 min of reaction when the
partially purified extracts were used (Figure 5a and b). In all 3 situations, peptides with MW of 15 to 20 kDa and below 2.5 kDa appeared simultaneously with disappearance of the peptide group
with MW in the range 20 to 25 kDa.

Conclusions
HE DEGRADATION PATTERNS OF S- AND -CASEIN ARE SIMILAR, IR-

respective of the extent of purification of the plant extract

tested. However, when crude extract is used, the rate of hydrolysis is lower than when precipitation by ammonium sulfate took
place previously; the crude extract possesses indeed a lower ratio
of coagulant (C) to proteolytic (P) activities. Salting-out of cardosins is apparently appropriate for the production of more efficient coagulants for cheese manufacture in terms of higher C/P
ratio, and is thus preferable to classical (more expensive) chromatographic steps of purification.

References

Figure 5Molecular weight distribution of peptides produced via hydrolysis of bovine -casein by cardosins previously precipitated with (a) 30% (w/v) and (b) 70% (w/v)
saturation of ammonium sulfate and by (c) crude extract,
after incubation for 0, 0.5, 1, 2, 5, 8, 12 and 24 h.
1750

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MS 20010529 Submitted 9/25/01, Accepted 12/4/01, Received 12/4/01
Financial support for authors Silva and Barros was provided by PhD fellowships (BD/18479/
98 and BD/16037/98, respectively), issued by program PRAXIS XXI (FCT, Portugal).

Authors are with the Escola Superior de Biotecnologia, Univ. Catlica

Portuguesa, Rua Dr. Antnio Bernardino de Almeida, P-4200-072 Porto, Portugal. Direct inquiries to author Malcata (E-mail: xmalcata@esb.ucp.pt).

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Hydrolysis of caseins by plant enzymes