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J. Agr. Sci. Tech. (2011) Vol. 13: 567-576

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Influence ofWithania coagulansProtease as a Vegetable

Rennet on Proteolysis of Iranian UF White Cheese

A. Pezeshki1, J. Hesari1*, A. Ahmadi Zonoz1, and B. Ghambarzadeh1

ABSTRACT

Extraction of protease from Withania coagulansfruits and the effect on proteolysis of

Iranian UF white cheese in comparison with pure chymosin and fungi rennet (fromase)

were investigated during ripening. The results indicated that, except for pH which was

significantly (P< 0.05) lower in cheeses made with Withania coagulans, there was no

significant difference observed among the cheeses produced with different rennet

preparations as in moisture, fat and salt contents during ripening. The values of pH 4.6-SN and the Urea-polyacrylamide gel Electrophoresis (PAGE) pattern revealed severe

proteolysis in cheeses produced with Withania coagulans enzyme as compared withanimal and fungi rennets. The caseins s1- and disappeared in cheeses made withWithania coagulans enzyme preparations. Furthermore, a noticeable increase of soluble

nitrogen in 12% trichloroacetic acid (SNTCA) was observed during ripening of cheeses

made with vegetable rennet, probably due to an unspecific proteolitic activity ofWithania

coagulans enzymes as compared to other rennets.

Keywords: Proteolysis, UF white cheese, Vegetable rennet, Withania coagulans.

_____________________________________________________________________________1 Department of Food Science and Technology, Faculty of Agriculture, University of Tabriz, Tabriz,Islamic Republic of Iran.* Corresponding author; e-mail: [email protected]

INTRODUCTION

Rennet is a milk coagulating enzymaticpreparation. The most highly active enzymein animal rennet preparations is chymosin.The principal role of chymosin, in cheesemaking, is to coagulate milk by specifichydrolysis of Phe105-Met106 bond of the k-casein (micelle-stabilising protein), causingmilk coagulation (Fox et al., 2000). Only 015% of the activity of the rennet added tothe milk remains in the curd after cheesemaking (Guinee and Wilkinson, 1992) andthis is one of the main proteolytic factorsinvolved in proteolysis during cheeseripening (Moatsou et al., 2004). In the latterpart of the last century, cheese consumptionincreased while the availability of calfrennet decreased, leading to rennet shortagesand subsequent price increases. In addition,more restrictive ethical concerns associated

with production of such animal rennets ledto a search for suitable rennet substitutes for

cheese making. Several proteases fromanimal, microbial and plant sources wereinvestigated (Drohse and Foltmann, 1989;Guinee and Wilkinson, 1992; Fox et al.,2000). Only a small number of asparticproteinases from plant origin have beenisolated and partially characterised (Sousaand Malcata, 1998; Sousa et al., 2001). Aunique feature shared by most of these plantproteinases is an extra segment of about 100amino acid residues which bears nosequence similarity with proteinases of

mammalian or microbial origins (Faro et al.,1995). Many aspartic and other proteinasesare obtained from plants with some of themhaving been studied as coagulants, i.e.,proteinases from Benincasa cerifera (Sousaet al., 2001), Silybum marianum (Cavalli etal., 2008), Dieffenbachia maculata

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(Padmanabhan et al., 1993), fruit parts ofSodom apple (Aworth and Muller, 1987) andCentaurea calcitrapa (Tavaria et al., 1997).

It is known that, most plant coagulantpreparations possess weak milk clottingactivity while a high proteolytic activity,which results in low cheese yields duringcheese making, and bitter peptidesaccumulation during cheese ripening.Cynara cardunculus coagulant, an exceptionamong the other plant rennet proteases, isobtained from dried flowers of C.cardunculus. This coagulant possesses amilk clotting activity and it has successfullybeen employed, for many centuries, in the

Iberian Peninsula to produce such traditionalcheeses, as: Serra da Estrela, La Serena,Guia and Los Pedroches (Verissimo et al.,1995; Silva et al., 2003; Silva and Malcata,2005; Tejada and Fernndez-Salguero,2003). Withania coagulans (Solanacea)grows in Pakistan, Afghanistan, India aswell as in southern regions of Iran and hasbeen employed for decades in production oftraditional cheeses from raw cows milk(Dastur, 1948; Singh et al., 1973). Fruits ofthis plant possess milkclotting characteristicand are known as vegetable rennet. Dastur etal. (1948) reported that it is possible toprepare soft cheeses, using rennetpreparations from Withania coagulans.Cheddar cheese manufactured, usingWithania coagulans rennet preparation had agood texture and flavor but gave aperceptible bitter taste which neverthelesscould be reduced to some extent, byprolonging the ripening period. The enzymeis extracted from flowers, through waterextraction and successive precipitation whileadding alcohol and acetone. The enzymaticprecipitate is then dried at room temperature.This enzyme shows an optimum of activitybetween 45 to 60C. The activity of theenzyme decreases rapidly at temperatureshigher than 70C, and it is completelyinactivated at 80C. Enzymes from Withaniacoagulans flowers coagulated boiled milkmore slowly than raw milk but it is possibleto restore its clotting capability by theaddition of calcium to heated milk (Dastur,

1948). There are very few studies maderegarding the proteolytic activity ofWithania coagulans enzymes during cheese

ripening.In the last decades, white-brined cheesefrom ultra-filtered milk has beenmanufactured in several, well organized,dairy plants in Iran. In spite of obviouseconomic advantages, due to highproduction yields as a result of theincorporation of whey proteins in theproduct, UF white-brined cheese usuallyshows slower proteolysis and production ofamino acids during ripening. Moreover itdoes not bear the suitable organoleptic

characteristics (Hesari et al., 2006).The aim of this study was to examine the

effects of vegetable coagulant fromWithania coagulans on proteolysis ofIranian UF white cheese during ripening, anattempt to use this enzyme preparation as alocal new source of rennet in cheese making.

MATERIALS AND METHODS

Extraction of Enzyme

Extraction of enzyme was carried outaccording to the method of Dastur (1948),(Figure 1).

Cheese Making

Experimental UF white cheeses weremade in three trials on separate days. Eachday, from a single batch of UF milk(retentate), three types of cheeses wereproduced: 1-by adding fungi rennet; Rhizomucormiehei

(Renco, Eltham, New Zealand) (30 mgkg

1retentate); 2- pure chymosin (CHR

HANSEN, Denmark) (30 mg kg1

retentate)and 3- Withania coagulansfruits (15 ml of3.3% (w/v) aqueous extract from Withaniacoagulansfruits was added to 450 gretentate). The enzyme activities ofcoagulants were adjusted to reach the rennetcoagulation time of 10 minutes for the threetype cheeses. The cheese making process

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Infuence of Withania Coagulans on UF White Cheese _____________________________

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Fruits ofWithania coagulans

Milling

Separating seeds by sieving

Adding distilled water (10 ml g-1 powder)

Stirring for 1 hour at room temperature

Filtration

Centrifugation (3000 g for 30 minutes at room temperature)

Precipitate Supernatant

Adding ethanol (to get an alcohol concentration of 85%)

Centrifugation (2500 g for 10 minutes at room temperature)

Repeating of adding alcohol to supernatant and centrifugation (two times)

Drying the precipitated enzymes at room temperature

Storing at -18c

Figure 1. Procedure ofenzyme extraction from Withania coagulansfruits.

was repeated on two other days to obtainthree replicates, on three different days, foreach rennet preparation. The retentate wasprepared by Iran Dairy Industry Inc.,PegahCo (Tabriz, Iran) as follows: raw cows milkof high microbial quality was standardizedto 3.5% of fat, and after bactofugationwithin two steps and at 55C, it waspasteurized at 72C for 15 seconds and thenultrafiltered at 50C. The applied membranecartridges were of the spiral wound type (N

o

UFPH20 Invensys APV, Silkeborg,Denmark) with a nominal molecular weightcut-off of approximately 20 kg mol

-1and a

surface area of 16.9 m2. The ultrafiltration

unit was operated at an inlet pressure of 5.3and an outlet pressure of 1.7 bars. Before

making of the cheese, the retentate waspasteurized at 78C for 60 seconds and thencooled to 35C. A mixture of mesophilic (G3mix, Lc. cremoris and Lc. lactis) andthermophilic (Joghurt 709, Str. thermophilusand Lb. delbrueckii subsp. bulgaricus)cultures (Laboratorium Visby, Tender Aps,Denmark) in the ratio of 7:1 was used asstarter. Subsequently, following the additionof rennet preparations, the milk was pouredinto containers (450 g). The samples wereallowed to coagulate at 30-32C of roomtemperature for 20 minutes. A parchmentpaper was placed on top of the coagulumand dry salt (3%) added. The containerswere sealed with aluminum foil. Saltgradually adsorbed moisture from curd and a

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Table I. Physicochemical parameters of 1-day-old experimental ultrafiltered (UF) Iranian white cheeseswith three different sources of coagulant. (Results are expressed as average of three independent replicatetrials standard deviations).

cheeses pH NaCl% moisture% Protein% Fat %

Withania

coagulans4.690.03 3.140.12 65.780.8 11.270.88 17.850.35

Chymosin 4.480.02 3.040.14 64.730.36 12.250.77 17.920.41Rhizomucor

miehei4.540.08 3.100.11 63.450.23 12.350.51 18.250.42

Fvalue * NS NS NS NS

NS: Not significant, * Significant at P < 0.01.

layer of brine formed around cheese samplesin the containers. Cheese packs were held at2628C for 24 hours and then transferred to

a cool room (8C). Cheeses were ripened for45 days. One cheese sample of each trialwas sampled out at 1, 15, 30, and 45 days ofripening.

Analytical Methods

Chemical Composition

Cheeses at 1, 15, 30 and 45 days ofripening were analysed for moisture (ovendrying method at 1022C; IDF, 1982), salt

(potentiometric method; Fox, 1963), fat(Gerber method; Marshal, 1992), totalnitrogen (TN), soluble nitrogen in pH 4.6(SN) and in 12% trichloroacetic acid(SNTCA) (macro-Kjeldahl method; IDF,1964), total protein (NT6.38). The pH ofthe cheese was measured by direct insertionof an electrode (PHC3031-9, RadiometerAnalytical, Copenhagen, Denmark) intocheese (Marshal, 1992). All analyses wereperformed in triplicate and results reportedas meanstandarddeviation.

Assessment of ProteolysisThe nitrogen fractions of the cheese

samples, including pH 4.6soluble nitrogen

(SN) and soluble nitrogen in 12%TCA(SNTCA) were obtained modifying theprocedure of Kuchroo and Fox (1982) asdescribed by Sousa and McSweeney (2001).Urea-polyacrylamide gel electrophoresis(PAGE) of the pH 4.6-insoluble fraction ofthe cheese was performed using a Protean II

XI vertical slabgelunit (Bio-RadLaboratories Ltd., Watford, UK) accordingto the method of Andrews (1983) modified

by Shalabi and Fox (1987). Gels werestained directly with Coomassie BrillantBlue G250, as described by Blakesley andBoezi (1977).

Statistical Analysis

Statistical analysis of the experimentaldata were done using a split plot modelbased on, a randomised complete blockdesign (CRB) (MSDATC and SPSS Version

15 for Windows 2003; SPSS Inc., Chicago,IL, USA). The model incorporated threetreatments (cheese samples made withWithania coagulans, cheeses made withanimal rennet and samples made with fungirennet) and three blocks (trials). Thesignificance of differences was estimatedusing 1-way ANOVA.

RESULTS AND DISCUSSION

Chemical Composition

Macro nutrients and physicochemicalparameters of 1-day old experimentalIranian UF white brined cheeses arepresented in Table 1. Except for pH that wassignificantly (P< 0.05) lower in cheesesmade with Withania coagulans as coagulantthan those in cheeses with animal or fungirennets, there were no significant differences

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0

10

20

30

40

50

60

70

1 15 30 45

Ripening days

%

SN/TN withania coagulans

Fungi rennet

pure chymosin

Figure 2. Evolution during ripening of pH 4.6-soluble nitrogen fraction (pH 4.6-SN) in UF Iranian white

cheeses produced with fungi, animal and vegetable rennet.

observed among cheeses in moisture, fat,protein and salt contents. The use ofdifferent rennet preparations did not have a

significant effect on the main grosscomposition of cheeses as reported by otherworkers (Sousa and Malcata 1997; Wium etal., 1998; Mara and Kelly, 1999).

Indices of Proteolysis: pH 4.6-SN and

SNTCA/TN

Levels of pH 4.6-SN content inexperimental UF white cheeses duringripening are shown in Figure 2. The levels of

%pH 4.6-SN increased gradually until 45days of ripening in all the cheeses. It isnoticed that pH 4.6-SN levels differedsignificantly (P< 0.01) among the cheesescoagulated using the three types of rennet.Non casein nitrogen contents of cheesemade with vegetable rennet were higher thanthose in the samples made with purechymosin and fungi rennets (

Rhizomucor miehei)

during ripening. After 15 days of ripening,the content in SN compounds of cheesesmade with vegetable rennet was more than

twice that in the samples produced usinganimal or fungi rennet. The estimation ofmean SN values for the samples analysed at1, 15, 30 and 45 days after cheese making,showed that cheese made with vegetablerennet contained about 100% more solublenitrogen than that made with animal rennet.

The higher levels of SN found in cheesesproduced, using vegetable rennet, suggest amore intense proteolytic activity ofWithania

coagulans protease than microbial andanimal proteases. Similar results werereported by other authors in relation withvarious cheeses. Galan et al. (2007) andPrados et al. (2006) showed that in ewesmilk cheese the SN and SNTCA contentswere significantly higher in cheesesproduced with vegetable coagulant thanthose made, using calf rennet. Duringripening, the levels of SN and SNTCAfractions of Los Pedroches cheese produced,using either Cynara cardunculus L.

proteinases or animal rennet, were foundhigher in samples made with vegetablerennet and were similar to the levels of LaSerena cheese (Sanjun et al., 2002).Malcata and Freitas (1995), in a study aboutthe influence of cheese making conditionson the characteristics of Picante da BeiraBaixa cheese, concluded that vegetablerennet increased proteolysis as comparedwith animal rennet.

The pH 4.6-SN fraction used as an indexof ripening (Sousa et al., 2001), includes

peptides of medium to small molecularweight, proteoso peptones, whey proteinsand free amino acids. These nitrogencompounds are the main results of rennetand plasmin proteolytic activity and/ormicroorganism peptidases (Okeefe et al.,1978). The proteinases of milk clotting

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0

5

10

15

20

25

30

35

40

45

50

1 15 30 45

Ripening days

%NP

N/TN

withaniacoagulans

Fungi rennet

pure chymosin

Figure 3. Evolution during ripening of non-protein nitrogen (SNTCA/TN) in UF Iranian white

cheeses produced with fungi rennet, animal and vegetable rennet.

preparations are mainly responsible for theprimary proteolysis of the caseins in cheese.Their action modifies the cheese texture by

slowly degrading s1- and, to a lesser extent,-caseins. This proteolytic process leads tothe framework of cheese matrix, andproduces precursors of sapid compounds(Hayaloglu etal., 2004) but it contributes tominority and indirectly to the formation offree amino acids (Okeefe et al., 1978).

The levels of non-protein nitrogen(SNTCA), soluble in 12% TCA, as apercentage of total nitrogen (TN), inexperimental UF white cheeses duringripening are shown in Figure 3. The extent

of secondary proteolysis, in terms ofSNTCA or its main components (peptides,aminoacids and ammonia), weresignificantly higher (P< 0.01) in cheesesproduced using vegetable rennets than thesamples made with the use of pure chymosinand fungi rennet ( Rhizomucor miehei) at 1, 15, 30and 45 days of ripening. In cheesesproduced using vegetable rennet, the levelsof SNTCA/TN increased from 22% at thebeginning of ripening to 46% at the end ofripening (45 days). On the contrary, in

cheeses made with animal or fungi rennet,the SNTCA/TN content improved moreslowly during ripening (Figure 3). Althoughlactic acid bacteria and other enzymes arethe principal agents for the production ofSNTCA in cheese, the higher breakdown of

caseins in cheeses made with plantcoagulants suggest that these cheesescontain more substrate (casein polypeptides)

that contribute to the secondary proteolysis(Tejada et al., 2008). Hesari et al. (2006)showed that rennet had indirect butimportant role on the secondary proteolysis.In fact the omission of rennet or starterduring cheese making causes an obviousreduction in the levels of peptide and freeamino acids produced during ripening ofIranian UF white cheese. Prados et al.(2006) showed that % SNTCA/TN washigher in Mancheco type cheese,manufactured with powdered vegetable

rennet of Cynara cardunculus as comparedwith animal rennet. On the contrary differentstudies on ewes milk cheeses (Fernndez-Salguero and Sanjun, 1999) and LosPedroches cheese (Sanjun et al., 2002)showed that SNTCA values and theirchanges, during ripening, were closelysimilar in cheeses made with vegetable oranimal rennet.

Urea-PAGE of the pH 4.6: Insoluble

Fraction

Urea-PAGE electrophoretograms of thepH 4.6-insoluble nitrogen fraction ofexperimental UF white cheeses (samples oftrial 1) at different ripening stages are shown

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Figure 4. Urea polyacrylamide gel electrophoretograms of experimental UF Iranian whitecheeses after 1, 15, 30 and 45 days of ripening.

in Figure 4. Results of the other twoexperimental trials were similar (data notshown). Electrophoretic profile indicatesthat initial proteolysis of caseins inexperimental Iranian UF white cheeses wascarried out mainly by coagulant enzymes.There were notable differences in

electrophoretic patterns among the threecheese typologies. In samples made withanimal rennet, -casein degraded moreslowly than s1-casein, but in cheesesamples produced with Withaniacoagulansenzyme, the hydrolysis of boths1- and -casein occurred more rapidly andintensely with the intact caseins disappearedapparently. Tejada et al. (2008) showed inMurcia al vino cheeses made with plantcoagulant that s1-casein degradation duringripening was more intense than that incheeses made with animal rennet, whereas-casein hydrolysis was very similar in bothcases. Similar observations were reported byMoatsou et al. (2002) for Feta cheese.Sanjun et al. (2002) in their study showedthat in Los Pedroches cheese during ripeningthe proteolytic process was more intense incheeses made with vegetable rennet ascompared with cheeses made through animalrennet. Many authors (OMahony et al.,2003; Sarantinopoulos et al., 2002;

Hayaloglu et al., 2004) have reported theresistance of-casein to hydrolysis duringripening of many cheese varieties.Furthermore, in UF type cheese, the rates ofdegradation ofs1-casein and particularly of-casein are lower than in conventionalcheeses. The inhibition of plasmin (EC

3.4.21.7) and rennet enzymes by wheyproteins has also been suggested as reasonfor the slow proteolytic process of UFcheeses (Hesari et al., 2006). Theseexperimental results show the high ability ofenzymes from Withania coagulans in s1-and -casein degradation in UF cheeses, thisbehavior being possible to be used inaccelerating the proteolysis of UF cheeseduring ripening at a suitablelevel.

CONCLUSIONS

The experimental results indicated thatrennet enzymes are the principal ripeningagent in primary proteolysis of UF whitecheese, but they also have notable effects onthe secondary proteolysis and insolublepeptide profiles of UF white cheeses. Theassessment of proteolytic process showed anintense proteolysis in cheese produced withWithania coagulansenzyme compared to

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animal and fungi rennets. This behaviorcould be foreseen in the use of Withaniacoagulans enzyme in cheese making to

accelerate the UF white cheese ripeningprocess.

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