1746 JOURNAL OF FOOD SCIENCEVol. 67, Nr. 5, 2002 2002 Institute of Food Technologists

Food Chemistry and Toxicology

JFS: Food Chemistry and Toxicology

Hydrolysis of Caseins By Extracts of CynaraCardunculus Precipitated by Ammonium SulfateS.V. SILVA, R.M. BARROS, AND F.X. MALCATA

ABSTRACT: Polyacrylamide gel electrophoresis (in the presence of urea) and gel permeation chromatographywere employed to assess the profile of hydrolysis of caseins and the activity of enzymes contributed by the flowersof the plant Cynara cardunculus on bovine caseins, after previous precipitation with ammonium sulfate or in aplain crude aqueous extract. Results indicated that the qualitative degradation profile of bovine caseins by plantenzymes (cardosins) remains essentially unchanged upon extraction, and quantitative analysis showed that theprecipitated fractions had a higher coagulant-to-proteolytic activity ratio; hence, the results showed that inex-pensive precipitation with ammonium sulfate can successfully be used as a purification method in the productionof that plant coagulant in standardized form.

Keywords: milk proteins, thistle, FPLC, urea-PAGE

Introduction

CRUDE AQUEOUS EXTRACTS OF FLOWERS OF THE COMMON WILDthistle, Cynara cardunculus, have been used for ages as a co-agulant in the manufacture of several traditional Portugueseand Spanish cheeses, such as Serra da Estrela, Serpa, andAzeito (Vieira de S and Barbosa 1972; Roseiro 1991; Macedoand Malcata 1997), and Los Pedroches, La Serena, and Flor deGua (Fernandez-Salguero and others 1991; Sanjuan andFernandez-Salguero 1994). Those flowers are known to contain 2aspartic proteinases, currently termed cardosins A and B, eachconsisting of 2 subunits with apparent molecular weights of 31and 15 kDa, and 34 and 14 kDa, respectively; it has been claimed(Pires and others 1994; Verssimo and others 1995) that cardosinA is similar to chymosin, while cardosin B is similar to pepsin, interms of specificity and activity.

Those enzymes are currently purified by a 2-step procedure,which involves liquid extraction at low pH and separation after-wards by liquid chromatography (gel filtration followed by ion-exchange) (Verssimo and others 1995). In addition to being cost-ly, the overall process is rather slow and cumbersome; hence, it ishardly appropriate when large quantities of those enzymes aresought. The objective of enzyme purification is to remove non-protein contaminants, as well as to isolate the enzyme in ques-tion from other proteins; the former objective is relatively easy toachieve, unlike the latter. Precipitation techniques can be usedat an early point, whereas chromatographic procedures (for ex-ample, ion-exchange, gel filtrarion, or adsorption chromatogra-phy) are usually employed later. Salting-out is the most commonprecipitation technique (Picn and others 1994). The salt usuallychosen is ammonium sulfate, (NH4)2SO4, which is highly solublein water (Robyt and White 1990) and is known to effectively sta-bilize proteins (Scopes 1994). Since large amounts of salt will con-taminate the precipitated proteins, removal thereof prior to en-zyme usage is required; this can easily be achieved by dialysis orgel filtration (Scopes 1994). In an early approach to this issue,Sousa and Malcata (1996) have studied the effects of processingconditions on the caseinolytic activity of crude extracts of C. car-dunculus, and found that the maximum specific caseinolytic ac-tivity was obtained by grinding the flowers for 36 s, using an ex-

traction buffer of pH 5.9 and NaCl content of 0 % (w/w), and ho-mogenizing the ground flower/buffer suspension for 15 min.

The objective of this study was (i) to evaluate the proteolyticand coagulant activities of said plant enzymes, both in crudeaqueous extract and following precipitation with ammonium sul-fate at 2 concentration levels (quantitative action), and (ii) tocharacterize the effect of those enzymes on the profile of hydrol-ysis of bovine s- and -caseins (qualitative action). The workinghypothesis under scrutiny was to ascertain whether salting-outmight replace the (more expensive) classically employed chro-matographic separations.

Material and Methods

Partial purification of enzymesThe source of plant proteases was dry flowers of C. carduncu-

lus: the stigmata and stylets of dry flowers were macerated with0.1 M aqueous citric acid (pH 3.0) at the ratio of 1 gflowers per 10mLbuffer, so as to produce a fluid product. Ammonium sulfate wasthen added to the plant extract up to 30% (w/v) saturation. After30 min, the solution was centrifuged at 10000 rpm for 10 min at4 C; the precipitate was redissolved in water up to approximate-ly twice the pellet volume. Ammonium sulfate was added onceagain to the supernatant up to 70% (w/v) saturation. After 30min, the solution was centrifuged at 10000 rpm at 4 C; as before,the precipitate was redissolved in twice its volume of water. Plantenzyme extracts were dialyzed overnight at 4 C against plain wa-ter, and lyophilized prior to use.

Electrophoretic characterization of enzymesPredetermined amounts of each cardosin extract were placed

in separate Eppendorf vials, and 100 mL of 10% (w/w) SDS wasadded to each one. The vials were heated at 90 C for 10 min in aheating block, and then cooled to room temperature. The sepa-ration was carried out in high-density gels: each gel had a stack-ing 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 (Pharma-cia, Uppsala, Sweden) was used to separate the proteins, which

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were stained by Coomassie blue.

Coagulant activityThe clotting activity was determined following the procedure

described by IDF (1992). The reconstituted milk was obtained bydissolving low-heat skim milk (NILACTM) (NIZO, Ede, The Neth-erlands) in 10 mM aqueous CaCl2 (pH 6.5), so as to achieve a con-centration of 0.12 kg/L; the time taken for 0.2 mL of each enzymeextract to coagulate 2 mL of reconstituted milk was measuredand recorded (the coagulation point was determined by manual-ly rotating the test tube periodically, at short time intervals, andchecking for visible clot formation). One rennet unit (R.U.) wasdefined as the amount of protein that coagulates 10 mL of recon-stituted low-heat skim milk powder at 30 C in 100 s (Berridge1945).

Proteolytic activityThe proteolytic activity was determined by the method of

Tomarelli and others (1949), with slight modifications. The lyo-philized enzyme mixture, reconstituted in 0.1 M aqueous sodiumphosphate buffer (pH 6.0), was mixed with 250 L of 2% (w/v)azocasein and incubated at 25 C for 10 min. The reaction wasthen quenched via addition of 0.5 mL of cold 5% (w/v) trichloro-acetic acid, TCA. The solution was centrifuged at 10000 rpm for10 min; 1 mL of supernatant was further mixed with 1 mL of 0.5 Maqueous NaOH to intensify the azo-associated color. The absor-bance of the solution at 440 nm was measured against a blank,prepared in a similar fashion but adding TCA to azocasein priorto addition of the enzyme.

Hydrolysis of substratesCommercial bovine s- and -caseins (Pharmacia) were dis-

solved up to 1 mg/mL in aqueous phosphate buffer (pH 6.5),containing sodium azide (0.05 g/mL) to prevent microbial activi-ty; these solutions were treated independently with cardosinsprecipitated by ammonium sulfate at 30% (w/v) and 70% (w/v)saturation, as well as with the crude enzyme extract, and incu-bated at 30 C; the (enzyme-containing) protein:substrate ratioused in all cases was 1:400 (w/w). At predetermined time inter-vals (0 min, 30 min, 1 h, 2 h, 5 h, 8 h, 12 h, and 24 h), sampleswere taken and the reaction was quenched via addition of dou-ble-concentrated buffer at 50%(v/v) (McSweeney and others1993) in the case of samples for electrophoresis (urea-PAGE), orvia heating at 95 C for 15 min in the case of samples for fast pro-tein liquid chromatography (FPLC).

Electrophoretic characterization of hydrolysatesSamples (0.75 mL) of hydrolysates were obtained, and pre-

pared for urea-PAGE as described above. Urea-PAGE (12.5% forthe separation gel and 4% for the stacking gel; pH 8.9) was per-formed following the procedure of Andrews (1983), with themodifications proposed by Shalabi and Fox (1987). Gels werestained with Coomassie Blue G250 (Bio Rad Laboratories, Her-cules, Calif., U.S.A.) according to the method of Blakesley andBoezi (1977).

FPLC characterization of hydrolyzatesFPLC was conducted according to the following protocol: sep-

aration was performed after injection of 100 L of each hydroly-sed sample in a Superose 12 column HR 10/30 (Pharmacia), withthe aid of an injection valve MV-7 and a UV-MII-detector (operat-ed at 280 nm); the mobile phase was 150 mM of aqueous NaCl in0.05 M aqueous phosphate buffer (pH 7.0), containing 0.2 g/L

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

Results and Discussion

Characterization of enzymesThe salting-out technique was employed to purify the aspar-

tic proteinases present in the crude aqueous extract of C. cardun-culus; a 1st fraction was precipitated at 30% (w/v) saturation withammonium sulfate and a 2nd one at 70% (w/v) saturation withthe same salt. The protein concentration was 92, 197, and 410mgprotein/ lyophilizate for the crude extract, the 70% (w/v) precipi-tate and the 30% (w/v) precipitate, respectively. SDS-PAGE wasperformed on the purified enzyme fractions, as well as on thecrude aqueous extract (Figure 1). The electrophoretogram re-vealed that precipitates were qualitatively similar to each other,and that the material in all fractions migrated as 4 bands, withmolecular weights 31 and 15 kDa (accounted for by cardosin A),and 34 and 14 kDa (accounted for by cardosin B) (Verssimo andothers 1995). It is worth noting that 2 extra bands, with molecularweights 20 to 26 kDa, were present in the fraction correspondingto the enzymes purified at the lower level of addition of ammoni-um sulfate. These 2 bands may likely be accounted for by con-taminating proteins that co-precipitate at 30% (w/v) saturationof ammonium sulfate, although the possibility that multimericnative proteins dissociate into their monomeric subunits duringthe 1st stage of purification can not be completely ruled out.

Table 1 shows the specific enzymatic activity of the precipi-

Figure 1SDS-PAGE electrophoretogram of fractions pre-cipitated by ammonium sulfate. Lane 1molecular weightmarkers, lane 2crude aqueous extract, lane 3super-natant at 70% (w/v) saturation, lane 4precipitate at 30%(w/v) saturation, lane 5precipitate at 70% (w/v) satura-tion.

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

ulant activity relative to that at 30% (w/v), whereas the oppositeheld for the proteolytic activity. In general, fractionation caused

Figure 2Urea-PAGE electrophoretograms illustrating thedegradation patterns of bovine -casein (-CN) by cardosinspreviously 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 1-8, respectively). Lanes 9 and 10 represent -casein afterincubation for 0 and 24 h, respectively, in the absence ofenzyme.

Figure 3Urea-PAGE electrophoretograms illustrating thedegradation patterns of bovine -casein (-CN) by cardosinspreviously 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 incu-bation for 0 and 24 h, respectively, in the absence of enzyme.

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a substantial decrease of the specific proteolytic activity (P) anda less pronounced decrease in the specific clotting activity (C),both relative to that of the crude extract. A decrease of the totalproteolytic activity of purified fractions of sodom apple (Calotro-pis procera) relative to the initial extract was also reported byAworh and Nakai (1986). Chen and Zall (1986a,b) claimed, inturn, an increase of the clotting activity in a fraction of clam vis-cera extracts fractionated with ethanol. The ratio of coagulant-to-proteolytic activity is a useful indicator of the enzyme appropri-ateness for general use in cheesemaking: a higher C/P ratiousually leads to higher quality cheeses (Chen and Zall 1986b;Harboe 1991; Esteves 1995). The ratio was actually higher for thefractions precipitated with ammonium sulfate, as compared withthe plain, crude aqueous extract; between the precipitated frac-tions, the ratio was much higher for the 30% (w/v) saturation pre-cipitate.

Electrophoretic characterization of hydrolysatesThe progress of hydrolysis of bovine -and s-caseins brought

about by cardosins precipitated at the 2 levels of ammonium sul-fate and by crude aqueous extract is shown in Figure 2 and 3, re-spectively.

Quantitative breakdown of original -casein was observed af-ter as early as 30 min, irrespective of the level of ammonium sul-fate added for precipitation of enzyme, whereas at least 5 h hadto elapse before a similar result was achieved with the crude ex-tract. In the hydrolysates, -I-casein was the 1st peptide to showup in all 3 situations tested, which is presumably a result ofcleavage of Leu192-Tyr193 in bovine -casein; according toSimes (1998), that is the peptide bond in pure bovine -caseinmost susceptible to the action of either cardosin. The peptide (-I-casein) was further hydrolyzed to -II- and -III-casein, ac-cording to the classification proposed by Creamer (1976). Notethat 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 30min, regardless of the level of ammonium sulfate added for theprecipitation of enzyme, whereas at least 2 h had to elapse be-fore an identical situation was produced by the crude extract.One of the fast moving peptide bands was assigned to s1-I-casein, following comparison with the electrophoretic mobility ofbovine s1-I-casein produced via action of chymosin (Mulvihilland Fox 1977); such peptide appeared in all 3 situations after 1min of incubation. Those peptides were subsequently degradedto smaller peptides, which migrated close to each other underthe electric field. Ramalho-Santos and others (1996) have stud-

Table 1Specific coagulant and proteolytic activities, andcorresponding ratio, of ammonium sulfate-precipitatedfractions and crude extract.

Coagulant Proteolyticactivity (C) activity (P)

Fraction (R.U./gprotein) (DAbs/gprotein/min) C/P ratioCrude extract 104 4.11 1.53 0.10 67.7 10.3030% (w/v) 90.6 2.01 0.24 0.001 377.5 34.62 saturation70% (w/v) 74.6 2.69 0.79 0.004 94.4 11.64 saturationNote: Abs AbsorbanceValues represent means of 3 replicates confidence interval (p = 95%)Data referred to dry basis

ied the independent action of cardosins A and B on bovines1-casein, and reported that both enzymes have preference forPhe23-Phe24, but are able to cleave bonds Trp164-Tyr165 andPhe153-Tyr154, although to lower extents; Phe23-Phe24 was alsofound by Mulvihill and Fox (1977) to be one of the peptide bondsmost labile to the action of chymosin in the pH range 2.2-7.0.

Consequently, both caseins were fully degraded by the car-dosins, and followed similar degradation patterns in either crudeor partially separated form. However, the rate of hydrolysis by

Figure 4Molecular weight distribution of peptides pro-duced via hydrolysis of bovine -casein by cardosins pre-viously 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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the crude extract was lower than by the partially purified ex-tracts, as expected. Note that the (enzyme-containing)protein:substrate ratio used was the same in the reactions of hy-drolysis of both substrates, but that the amount of enzymepresent was higher in the ammonium sulfate-saturated fractionthan in the crude extract (see Figure 1); this may help explain ourresults, because the lower enzyme content, the lower rate of hy-drolysis. The rate of degradation of s-casein was also slightlyhigher than that of -casein when the crude extract was consid-ered. This finding is in agreement with the reported fact (Mulvi-

hill and Fox 1977) that -casein is less susceptible than s1-caseinto proteolysis by chymosin. Sousa and Malcata (1997) also re-ported that -casein undergoes less degradation than s1-caseinin bovine milk cheeses manufactured with aqueous extracts offlowers of C. cardunculus.

Chromatographic characterization of hydrolysatesThe molecular weight (MW ) distribution of peptides in the

casein hydrolysates is shown in Figures 4 and 5 for - and s-caseins, respectively. For both caseins, hydrolysis was in all casesaccompanied by a decrease in the concentration of high-MWpeptides (20 to 25 kDa) and a concomitant increase in the con-centration of low-MW ones (< 1 kDa and 1 to 2.5 kDa). This obser-vation reflects the expected sequential hydrolysis of longer toshorter peptides. No relevant changes in the concentration ofpeptide material between 2.5 and 15 kDa was noticed over the 24h of hydrolysis in all cases, which indicates somewhat unselec-tive hydrolysis.

As far as -casein is concerned, the family of peptides withMW between 20 and 25 kDa essentially vanished by 2 h of hy-drolysis carried out by the purified extracts (Figure 4a and b).The 1st short peptides to be noticed were those with MW in therange of 1 to 2.5 kDa, after as little as 30 min of reaction; then thepeptide group with MW within 15 to 20 kDa started to build up.The high-MW peptide group was present until 12 h of hydrolysisby the crude extract, but gradually yielded peptides with MW of15 to 20 kDa (Figure 4c).

Regarding s-casein, peptides with MW of 20 to 25 kDa stillshowed up by 2 h of hydrolysis by the crude extract (Figure 5c);however, that group disappeared by 30 min of reaction when thepartially purified extracts were used (Figure 5a and b). In all 3 sit-uations, peptides with MW of 15 to 20 kDa and below 2.5 kDa ap-peared simultaneously with disappearance of the peptide groupwith MW in the range 20 to 25 kDa.

Conclusions

THE DEGRADATION PATTERNS OF S- AND -CASEIN ARE SIMILAR, IR-respective of the extent of purification of the plant extracttested. However, when crude extract is used, the rate of hydroly-sis is lower than when precipitation by ammonium sulfate tookplace previously; the crude extract possesses indeed a lower ratioof coagulant (C) to proteolytic (P) activities. Salting-out of car-dosins is apparently appropriate for the production of more effi-cient coagulants for cheese manufacture in terms of higher C/Pratio, and is thus preferable to classical (more expensive) chro-matographic steps of purification.

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caseins. J Dairy Res 50(1):45-55.Aworh OC, Nakai S. 1986. Extraction of milk clotting enzyme from sodom apple

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Figure 5Molecular weight distribution of peptides pro-duced via hydrolysis of bovine -casein by cardosins pre-viously 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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Sousa MJ, Malcata FX. 1996. Effects of processing conditions on the caseinolyticactivity of crude extracts of Cynara cardunculus L. Food Sci Technol Int 2:255-263.

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

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