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International Dairy Journal 18 (2008) 93–98

www.elsevier.com/locate/idairyj

Influence of different amounts of vegetable coagulant from cardoonCynara cardunculus and calf rennet on the proteolysis and sensory

characteristics of cheeses made with sheep milk

E. Galan, F. Prados, A. Pino, L. Tejada, J. Fernandez-Salguero�

Departamento de Bromatologıa y Tecnologıa de los Alimentos, Facultad de Veterinaria, Universidad de Cordoba,

Campus de Rabanales, edificio C-1, Cordoba 14071, Spain

Received 19 April 2006; accepted 8 June 2007

Abstract

Different amounts of powdered vegetable coagulant (PVC) obtained from Cynara cardunculus (normal amount ¼ PVC;

double the normal amount ¼ 2PVC) were compared with calf rennet in cheese made from sheep milk, by determining different

chemical, biochemical, and sensory characteristics throughout of 6 months of ripening. For most of the chemical parameters

studied, no differences were observed between the coagulants assayed. However, significantly higher (po0.05) levels of casein

hydrolysis, measured as soluble nitrogen (SN), non-protein nitrogen (NPN), amino acid nitrogen (AAN) and ammonia-nitrogen

(N.NH3), were observed after 2 days of ripening in cheeses produced with 2PVC compared with those made with normal

amount of PVC. Furthermore, only the levels of SN and NPN were significantly higher (po0.05) in cheeses produced with

PVC than those obtained with calf rennet. The main sensory characteristics were enhanced (po0.05) in cheeses obtained with

vegetable coagulant in comparison to those made with calf rennet. The bitter taste of cheeses produced with 2PVC was not signifi-

cantly stronger (p40.05) than in those produced with a normal amount of vegetable coagulant (PVC). The increased proteolytic

activity of the vegetable enzymes enables manufacturers to produce fully ripened cheeses (especially when the amount of the

vegetable coagulant is doubled) with all the genuine end-product organoleptic characteristics approximately 3 months earlier than if calf

rennet is used.

r 2007 Elsevier Ltd. All rights reserved.

Keywords: Ewes’ milk cheese; Cynara cardunculus; Proteolysis; Accelerated ripening; Vegetable coagulant

1. Introduction

The vast majority of rennet-coagulated cheeses areripened for periods ranging from around 3 weeks(e.g., Mozzarella) to over 12 months (e.g., Cheddar,Manchego, Parmesan). During ripening, a wide range ofmicrobiological, biochemical and chemical events occur, as aresult of which the main constituents of the milk are brokendown into primary and then secondary products. Theseevents generate many different compounds, which inappropriate combinations and concentrations are responsiblefor the flavour of the different cheeses. The ripening of cheese

e front matter r 2007 Elsevier Ltd. All rights reserved.

airyj.2007.06.003

ing author. Tel.: +34957 212010; fax: +34 957 212000.

ess: ao1fecaj@uco.es (J. Fernandez-Salguero).

is a slow and consequently an expensive process that is stillnot fully controllable. Because of the high cost of cheeseripening, the development of an efficient way to reduce theaging time would generate substantial savings in thecheese industry. Various methods, and even the combinedaction of different methods, have been used to reduceripening time and/or enhance flavour formation (Law &Wigmore, 1982), most of these methods have focusedon the addition of enzymes from various sources to themilk (El Soda, 1997). Numerous enzymes have beenstudied, including proteinase, peptidase, lipase, esterase,desulfurilase, demethylase, b-galactosidase, etc.Most of the significant progress in accelerated ripening

has been obtained by adding neutral proteases, mainlyfrom a microbial source. However, the acidity of the cheese

ARTICLE IN PRESSE. Galan et al. / International Dairy Journal 18 (2008) 93–9894

during the initial stages of ripening should reduce enzymeactivity of the neutral proteases during production andripening. Given the results obtained by certain researchers(Kosikowski & Mistry, 1997), another option is toaccelerate the ripening of sheep cheeses by adding acidicproteinases.

The aspartic proteinases extracted from cardoonflowers of various species of the genus Cynara (mainlyC. cardunculus and, to a lesser degree, C. humilis) areamong the few plant-origin enzymes that have been used tomake cheese in Portugal and Spain (Carr, 1981; Fernandez-Salguero & Sanjuan, 1999; Fernandez-Salguero, Tejada, &Gomez, 2002; Freitas, Macedo, & Malcata, 2000; Macedo& Malcata, 1997; Macedo, Malcata, & Oliveira, 1993;Verıssimo et al., 1996). These acidic enzymes are chara-cterized by their high clotting activity, like chymosin,cleaving the Phe105–Met106 peptide bond in bovine andovine k-casein, whereas caprine k-casein is preferentiallycleaved at Lys116–Thr117 (Sousa & Malcata, 1998). Theyalso have a strong proteolytic action that eventually leadsto the extensive breakdown of caseins, producing cheesescharacterized by a soft buttery texture and the developmentof a typical aroma and a slightly piquant and creamyflavour. The specificity of proteinases in vitro fromC. cardunculus using bovine, ovine and caprine caseinsand certain cheeses as substrates has been reviewed bySousa and Malcata (2002). Although these vegetableenzymes are highly proteolytic in terms of substrateproteins and less proteolytic in terms of medium-sizedpeptides (Sousa & Malcata, 1997), they could be used as aproteinase system in the accelerated ripening of certaintypes of cheese made from sheep milk, by adding enzymes(Law & Wigmore, 1982). Cyprosins, on the other hand,display more specific protein hydrolysis in sheepmilk (Cordeiro, Jacob, Puhan, Pais, & Brodelius, 1992).Hence, certain typical cheeses made in Spain andPortugal from ewes’ milk with cardoon-based coagulantdo not have a strong bitter flavour whereas cheesesproduced from cows’ milk and extracts of C. cardunculus

flowers do tend to have a bitter taste (Barbosa, Corradini,& Battistoni, 1981). Certain studies on the specificityof cyprosins suggest that these proteinases may act asboth endopeptidases and exopeptidases (Faro, 1991;Heimgartner et al., 1990) but tend to prefer peptidebonds containing polar amino acids in their hydrolyticattack. The use of these plant proteinases as milkcoagulants is very interesting since they are naturalenzymes and can also be used for producing cheeses aimedat lacto-vegetarian consumers and ecological markets(Gomez et al., 2001). They can also be used for Kosherand Halal products.

The aim of this paper was to examine the effect ofdifferent amounts of powdered vegetable coagulant fromC. cardunculus flowers, in comparison with calf rennet(CR), on changes in the chemical, biochemical and sensorycharacteristic during the ripening process of sheep’s cheeseand as a means of reducing ripening time.

2. Materials and methods

2.1. Cheese making procedure and sampling

The powdered vegetable coagulant (PVC) was preparedin accordance with a previously described method (Tejada& Fernandez-Salguero, 2003).On two different days, raw sheep milk obtained during

one milking day was split into three batches; one batchwas coagulated with powdered vegetable coagulant(PVC, 100%) by adding 21 g for 100L�1 milk; the secondbatch was clotted with double the amount of coagulant(2PVC, 200%); and the third batch was clotted using CRby adding about 2.5 g for 100L�1 milk (commercial CRpowder from Chr. Hansen, Denmark). A commercialstarter culture EZALs from Larbus S.A. (Madrid, Spain)containing a mixture of Lactococcus lactis subsp. lactis,L. lactis subsp. cremoris and L. lactis subsp. lactis var.diacetylactis and Streptococcus thermophilus and specifi-cally designed for traditional milk cheese making wasadded to the milk. Each of the six experimental batches wasmade into seven wheels of cheese. The coagulationtemperature was 2971 1C. For batches coagulated withPVC and CR the coagulation time was approximately55min, and for batches clotted with 2PVC the coagulationtime was approximately 40min. After pressing and saltingin brine (221 Brix at 14 1C for 36 h), the cheeses wereripened at 11 1C and 85% relative humidity and analysedon days 2, 15, 30, 60, 90 120 and 180 (using a whole cheeseper batch on each of the seven analysis days).

2.2. Determination of chemical components

Moisture, fat, protein, acidity (as lactic acid) and sodiumchloride were determined by AOAC (1980) methods. Wateractivity (aw), pH and nitrogen fractions, viz. solublenitrogen (SN) at pH 4.6, non-protein nitrogen (NPN) in12% TCA and ammonia nitrogen (NH3N) were deter-mined as described elsewhere (Carmona, Sanjuan, Gomez,& Fernandez-Salguero, 1999; Vioque et al., 2000). Aminoacid nitrogen (AAN) was determined using the methoddescribed by Folkertsma and Fox (1992).

2.3. Sensory evaluation

Certain sensory attributes were determined by 12 trainedpanelists using a 10-point scale with anchor points(from extremely strong to extremely mild) as describedelsewhere (Tejada et al., 2000). Sensory evaluation of thecheeses was carried out on days 60, 90, 120, and 180 of theripening process.

2.4. Statistical treatment

An analysis of variance (ANOVA) was performed on theresults obtained at different ripening stages, using thesoftware package Statistica 5.1 for Windows (Statsoft, Inc.,OK, USA).

ARTICLE IN PRESSE. Galan et al. / International Dairy Journal 18 (2008) 93–98 95

3. Results and discussion

Table 1 shows the mean values and standard deviationsfor moisture, fat, protein, and NaCl (as g 100 g�1 cheese) aswell as pH and aw throughout ripening (on days 2, 15, 30,60, 90, 120 and 180) of cheeses obtained with CR andthe powdered vegetable coagulant, using the normalamount (PVC, 100%) and double the normal amount(2PVC, 200%). The statistical signification between aver-age values within each parameter is indicated by super-scripts. Most of the chemical parameters and pH valueswere very similar in cheeses clotted with animal rennet andvegetable coagulant (PVC) throughout the ripening pro-cess. The moisture levels of cheeses obtained with vegetablecoagulant (PVC) were lower (p40.05) than those producedwith CR. During all ripening, the moisture levels of cheesesmade with 2PVC were significantly different (po0.05) fromthose obtained with CR. Also, except day 180 of ripening,the fat levels of cheeses made with 2PVC were significantlyhigher (po0.05) than those obtained with CR. After day60, the water activity in the cheese obtained with vegetablecoagulant (PVC and 2PVC) was similar, but significantlydifferent (po0.05) from the cheese made with CR.

Fig. 1 shows the average values and standard deviationsfor different soluble nitrogen fractions (SN, NPN, AAN,

Table 1

Average values and standard deviations of moisture, fat, protein and salt (g 100

(CR) and powdered vegetable coagulants (PVC and 2PVC) during the ripenin

Ripening time (days)a

2 15 30 60

Moisture

CR 39.4870.34a 38.8770.14b 37.2170.05de 37.

PVC 38.5570.78bc 37.4570.00d 37.3070.00d 36.

2PVC 38.2870.23c 35.6870.05f 35.6570.16f 34.

Fat

CR 34.0070.00a 34.2570.35a 34.5070.00a 35.

PVC 34.2571.06a 34.5070.71a 34.7570.35a 35.

2PVC 36.5070.71bcd 38.0070.00def 38.5072.83efg 38.

Protein

CR 20.9970.35a 21.2870.17a 21.9570.08bc 23.

PVC 21.4270.59ab 21.9770.09bc 22.3570.18cd 23.

2PVC 21.3270.23a 21.1170.66a 22.8270.43de 22.

NaCl

CR 0.8370.10b 1.0670.01c 1.1170.01cde 1.

PVC 0.6370.01a 1.0670.02c 1.1170.00cde 1.

2PVC 0.5170.06a 1.0870.03cd 1.2270.01efg 1.

awCR 0.98270.00a 0.97370.001bc 0.96570.000de 0.

PVC 0.97870.004ab 0.96870.001cd 0.95970.006f 0.

2PVC 0.97670.006b 0.96570.004def 0.96370.004def 0.

pH

CR 5.1870.04f 5.0970.00de 5.0570.02bc 5.

PVC 5.1170.02e 5.0570.00bcd 5.0070.00a 5.

2PVC 5.3570.00ij 5.1170.00e 5.0870.01cde 5.

aMeans of the same parameter in the same row without a common supersc

and NH3N in g 100 g�1 TN) during the ripening of thecheeses. Soluble nitrogen values in cheeses made withdifferent types of coagulant increased throughout the entireripening process (po0.001). There were significant differ-ences between the SN values of the cheeses clotted with twotypes of coagulants. Although soluble nitrogen is producedby the action of rennet, bacteria in the curd and milkplasmin (Visser, 1977), the high SN levels observed in theearly stages of ripening were caused by the intenseproteolytic action of enzymes in the vegetable coagulants,which displayed virtually maximum enzyme activity atthe pH studied (Heimgartner et al., 1990). On day 2 ofripening, the SN value for the cheese made using PVC(10.52 g 100 g�1 TN) was more than double that ofthe samples obtained with CR (4.25 g 100 g�1 TN), butthe SN value of the cheese obtained with 2PVC(34.27 g 100 g�1 TN) was higher than SN value of cheesemade with CR on day 180 (24.89 g 100 g�1 TN). At the endof ripening period, the cheeses made with PVC(36.70 g 100 g�1 TN) and 2PVC (56.10 g 100 g�1 TN) con-tained approximately 32% and 55% more SN, respectively,than those made with CR. High levels of SN in cheesemade with vegetable coagulant have been observed byother authors (Fernandez-Salguero & Sanjuan, 1999;Nunez, Fernandez del Pozo, Rodrıguez Marın, Gaya, &

g�1 cheese), water activity and pH of the cheeses obtained with calf rennet

g period

90 120 180

1270.07de 35.6470.23f 33.4770.09h 29.6070.00k

7870.00e 34.4070.00g 33.3070.25h 29.5770.15k

2270.04g 32.1470.00i 30.6770.45j 28.2470.20l

0070.00ab 36.5070.71bcd 37.0070.00cde 39.2570.35fg

0070.00ab 35.5070.71abc 36.5070.71bcd 38.0070.00def

5070.71efg 39.0070.00fg 40.0070.00gh 41.0070.00gh

0170.09e 24.4570.05g 25.0970.07h 26.9470.12j

0270.08e 23.8570.16f 25.8370.12i 26.6170.12j

6570.07de 24.3270.25fg 24.6770.49gh 25.7970.16i

2570.07fgh 1.3670.035hi 1.3570.04hi 1.4270.03ij

2070.05def 1.1970.01def 1.3470.00ghi 1.4970.21jk

5170.02jk 1.3570.01hi 1.3770.01hi 1.5870.00k

96070.000ef 0.95370.001g 0.94070.000h 0.94070.000j

94870.002g 0.93770.001hi 0.92970.001j 0.91770.003k

94970.004g 0.93470.004i 0.92770.003j 0.92070.002k

2270.01g 5.3370.03hi 5.6170.02m 5.6670.01n

0170.01ab 5.3070.03h 5.3870.01j 5.4670.01k

1870.01f 5.3570.02ij 5.3570.02ij 5.5470.05l

ript letters (a–n) differ significantly (po0.05).

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0

10

20

30

40

50

60

0

5

10

15

20

25

30

0 30 60 90 120 150 180

0 30 60 90 120 150 180

0 30 60 90 120 150 180

0 30 60 90 120 150 180

2PVC

PVC

CR

PVC

CR

2PVCNPN

SN

0

1

2

3

4

5

6

0

1

2

3

4

5

PVC

CR

2PVC

2PVC

CR

PVC

NH3N

AAN

Days of ripening

Days of ripening

Days of ripening

Days of ripening

g 1

00

g-1

of

TN

g 1

00

g-1

of

TN

g 1

00

g-1

of

TN

g 1

00

g-1

of

TN

Fig. 1. Average values and standard deviations of the nitrogen fractions:

soluble-N (SN), non-protein-N (NPN), amino acid-N (AAN) and

ammonia-N (NH3N) as g 100 g�1 of TN of the cheeses obtained with

calf rennet (CR), powdered vegetable coagulant (PVC, 100%) or double

the normal amount of powdered vegetable coagulant (2PVC, 200%)

during 180 days of the ripening.

E. Galan et al. / International Dairy Journal 18 (2008) 93–9896

Medina, 1991; O’Mahoney, Sousa, & McSweeney, 2003;Prados, Pino, & Fernandez-Salguero, 2007; Sousa &Malcata, 1997; Tejada & Fernandez-Salguero, 2003).

The NPN fraction increased steadily and significantlythroughout the ripening in cheese made with both types of

coagulants. Significant difference (po0.05) was observed inrelation to NPN, but it was only slightly higher in cheeseobtained with PVC than in cheese made using CR. TheNPN of cheeses made with 2PVC was significantly higherthan those produced with PVC throughout the ripeningperiod. As previously demonstrated (Fernandez-Salgueroet al., 2003; Sousa & Malcata, 1997), the increasedproteolytic activity in the breakdown of caseins in cheesesmade with PVC led to the production of more substrates(casein polypeptides) which is used by lactic bacteria andother enzymes (McSweeney & Fox, 1993) to producehigher amounts of low molecular weight component(NPN) than in cheese made with CR. The other twonitrogen fractions (AAN and NH3N) also increasedsteadily during the ripening process and the amounts ofthese nitrogen components were higher in cheese made withPVC than in cheeses obtained with CR. The cheeses madewith 2PVC displayed significantly higher (po0.05) levels ofamino acids and ammonia nitrogen in comparison withcheeses obtained with CR and PVC.Table 2 shows the mean values and standard deviations

for sensory characteristics on days 60, 90, 120, and 180, incheeses made with CR and powdered vegetable coagulant(PVC and 2PVC). Also, the statistical significance betweenaverage values within each attribute is indicated bysuperscripts. The odour and colour increased (po0.05;ripening time) with the age of cheeses. For these twoattributes, significant differences (po0.05) were observedin cheeses made with vegetable coagulant in comparison tothose produced with CR and the evaluation of the judgingpanel were higher for cheeses made with 2PVC incomparison to those obtained with PVC. A positivecorrelation (po0.05) was found between odour, colourand taste intensity values for cheeses made with CR, PVCand 2PVC and their respective amounts of SN and NPN.In relation to the attributes hardness and creaminess,cheeses produced with vegetable coagulant (2PVC andPVC) were significantly (po0.05) softer and more butterythan those made with CR. In cheeses obtained withvegetable coagulant, (PVC and 2PVC) a negative correla-tion (po0.05) was found between hardness values and therespective amounts of SN and NPN. The higher proteolyticactivity in the breakdown of caseins and the firstdegradations products in cheeses made with vegetablecoagulant led to a softer and creamier texture (po0.05)than in those obtained with CR (Table 2). The soft texture,slightly piquant aroma and creamy flavour of the cheesesmade with PVC are highly valued by consumers.The taste intensity of cheese produced with vegetable

coagulant was significantly higher (po0.05) than with CRexcept on day 180; even on day 60, the cheese made with2PVC (7.0270.51) had more taste intensity than the cheeseproduced with CR on day 180 (6.9870.34). Generally, thecheeses made with 2PVC and PVC were more bitter andacidic than cheeses made with CR but no constantsignificant differences were observed. The general accep-tance of the cheese made with both types of coagulant was

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Table 2

Average values and standard deviations of some attributes in cheeses

obtained with calf rennet (CR) and powdered vegetable coagulant

(PVC and 2PVC) during the ripening period

Days of ripeninga

60 90 120 180

Odour

CR 4.3470.41a 5.1871.12bc 4.5770.63ab 6.2570.72def

PVC 5.5970.71cd 5.2870.84bc 5.7670.91cde 6.6670.90f

2PVC 6.1971.17def 6.4470.83ef 6.1370.59 def 7.9770.31g

Colour

CR 4.3370.31a 4.9670.42b 5.2871.26bc 6.4470.53e

PVC 5.3170.33bc 5.6770.40cd 6.1070.70de 6.9070.46f

2PVC 5.8670.57d 6.1270.36de 6.1470.61de 7.5970.41g

Hardness

CR 5.9070.62d 6.3270.56d 7.1670.69e 8.2970.19f

PVC 3.1570.32b 3.3770.58bc 3.8070.31c 3.7971.22c

2PVC 3.2770.40bc 2.1270.10a 2.3970.29a 2.3370.30a

Creaminess

CR 2.9670.55a 3.8471.41b 4.6370.32c 4.2970.73bc

PVC 6.1970.73d 6.8270.52ef 7.1370.55efg 7.8070.67hi

2PVC 6.7270.36de 7.4171.16fgh 7.6670.71ghi 8.0470.44i

Taste intensity

CR 5.0670.62a 5.3970.92ab 5.0970.90a 6.9870.34d

PVC 5.7370.65bc 6.1770.46c 5.7670.88bc 6.9270.55d

2PVC 7.0270.51d 6.2370.84c 6.9470.75d 7.4070.43d

Bitter taste

CR 2.8870.61a 2.7770.80a 3.3870.36ab 3.2870.76a

PVC 2.9870.64a 2.9170.79a 4.1570.54cd 4.6670.59cd

2PVC 3.0670.68a 4.2370.76cd 4.0470.37bc 4.7671.24d

Acid taste

CR 4.3970.93b 5.3870.45bcd 5.7670.59cde 6.2670.87def

PVC 4.9070.73bc 5.7170.21cde 5.9971.04cde 7.2370.93fg

2PVC 2.5970.58a 6.4270.41def 6.5270.93ef 8.0770.73g

General acceptance

CR 5.4370.52abc 6.3071.01c 5.2970.85abc 7.3970.54d

PVC 4.4770.67a 5.5770.46bc 4.7470.46ab 5.2270.92ab

2PVC 4.8870.33ab 5.1770.50ab 5.8871.38bc 6.2370.61c

aMeans of the same attribute in the same row without a common

superscript letter (a–i) differ significantly (po0.05).

E. Galan et al. / International Dairy Journal 18 (2008) 93–98 97

similar, except on day 180, when the acceptance of cheesesobtained with CR was significantly different (po 0.05)from those produced with 2PVC and PVC.

4. Conclusions

Both the proteolysis (extent and intensity of the break-down) and the sensorial characteristics of the cheeses madewith the powdered vegetable coagulant were higher thanthose made with calf rennet. The cheeses obtained usingdouble the normal amount of powdered vegetable coagu-lant underwent a considerable increase in proteolysis; andorganoleptic characteristic of fully ripened cheese wereachieved after a ripening period of approximately 3months. Furthermore, the specificity of C. cardunculus

enzymes in ovine casein led to the production of a normal

bitter taste in cheeses produced with vegetable coagulant.Therefore, it is possible to accelerate the ripening of cheesemade from ewe’s milk cheese, while still retaining all theorganoleptic properties of this type of cheese, by addingdifferent amounts of cardoon C. cardunculus enzymes.

Acknowledgement

The authors acknowledge their thanks to the Ministry ofEducation and Science for their help through the projectAGL2002-02752.

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