Characterization of the milk-clotting properties of extracts from artichoke (Cynara scolymus, L.) flowers

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International Dairy Journal 17 (2007) 1393–1400 www.elsevier.com/locate/idairyj

Characterization of the milk-clotting properties of extracts from artichoke (Cynara scolymus, L.) flowers Soledad Chazarra1, Lara Sidrach1, Dorotea Lo´pez-Molina, Jose´ Neptuno Rodrı´ guez-Lo´pez Grupo de Investigacio´n de Enzimologı´a, Departamento de Bioquı´mica y Biologı´a Molecular A, Facultad de Biologı´a, Universidad de Murcia, E-30100 Espinardo, Murcia, Spain Received 19 December 2006; accepted 29 April 2007

Abstract Extract of artichoke (Cynara scolymus, L.) flowers have been investigated as a source of enzymes to be used in cheese making as an alternative or in addition to calf rennet. Coagulation activity was highly dependent upon milk pH and temperature. The rennet strength (RS) of this extract increased hyperbolically with increasing concentrations of calcium, and the concentration was saturated at 50 mm. However, the effect of sodium chloride on RS was not significant. Additionally, the properties of individual cynarases obtained from artichoke flowers were also studied. Purification led to a decrease in the specific coagulant activity relative to that of the crude extract in the case of cynarases A and C, whereas cynarase B increased its specific clotting activity. Moreover, whereas the cynarases A and C showed a slight increase in specific peptidase activity relative to the initial extract, the specific peptidase activity of cynarase B was much higher. r 2007 Elsevier Ltd. All rights reserved. Keywords: Artichoke; Cynara scolymus L.; Plant rennet; Cynarases; Milk-clotting

1. Introduction Milk-clotting enzymes are the primary active agents in the manufacture of cheeses. Calf rennet was the first, and still is the most widely used, milk-clotting enzyme preparation. The worldwide increase in cheese production, along with the reduced supply of calf rennet, has led to an increase in the demand for alternatives sources of milk coagulants (Cavalcanti, Teixeira, Lima Filho, & Porto, 2004). Microbial rennet produced by genetically engineered bacteria and moulds have proven suitable substitutes for animal rennet, but increasing attention has been directed toward natural rennet extracted from plants (Tavaria, Sousa, & Malcata, 2001). The use of plant coagulants contributes to improving the nutritional intake of people whose use of animal rennets is restricted (Gupta & Eskin, 1977). Although several plant proteinases are able to coagulate milk, unfortunately, most of the plant rennet Corresponding author. Tel.: +34 968 398284; fax: +34 968 364147. 1

E-mail address: [email protected] (J.N. Rodrı´ guez-Lo´pez). Both authors have contributed equally to the work.

0958-6946/$ - see front matter r 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.idairyj.2007.04.010

obtained has been found to be inappropriate for cheese production due to its excessively proteolytic character, which lowers the final yield of cheese and produces bitter flavors (Lo Piero, 2002). An exception to this general rule is represented by the aqueous extracts of Cynara cardunculus flowers, which have been used for years in the manufacture of several traditional Portuguese and Spanish cheeses (Silva, Barros, & Malcata, 2002), the properties of which have been well characterized (Silva & Malcata, 1998, 1999, 2000a; Silva et al., 2002; Silva & Malcata, 2004; Sousa & Malcata, 1997a, 1997b, 1998, 2002). The extracts of the flowers of two other Cynara species,C. humilis and C. scolymus, have also been claimed to be effective as rennet (Silva & Malcata, 2000b; Verissimo, RamalhoSantos, Faro, & Pires, 1998). Recently, we have purified the proteinases of C. scolymus, a species that contains three proteinases (cynarases A, B and C) with milk clotting activity. All three cynarases are glycoproteins and are composed of one large and one small subunit (Sidrach, Garcı´ a-Ca´novas, Tudela, & Rodrı´ guez-Lo´pez, 2005). This study aimed to characterize the enzymatic action of crude extracts of the C. scolymus and of each cynarase.

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The coagulant activity on milk and the specific peptidase activity of the crude extract and each purified cynarase were also determined. The hydrolysis of caseins by the crude extract and the purified cynarases was characterized by polyacrylamide gel electrophoresis (PAGE) in the presence of sodium dodecyl sulphate (SDS). Although the properties of the crude enzyme preparation may well be derived from a combination of proteins, characterization of the crude extracts was regarded as essential due to their potential use in cheese technology, since in industrial applications, enzyme purity is usually given less importance than cost. Moreover, the artisanal rennet from Cynara flowers usually consists of crude extracts. Therefore, the effects of pH, coagulation temperature, calcium, sodium chloride and enzyme concentrations on the milk-clotting activity of artichoke extracts were investigated, as well as the microbiological characteristics of these extracts.

2.3. Peptidase activity and protein assay Peptidase activity was determined using the synthetic peptide H-Pro-Thr-Glu-Phe-p-ðNO2 Þ-Phe-Arg-Leu-OH (PNPE) (Bachem A.G., Bubendorf, Switzerland) as a substrate. The enzyme preparation was incubated at room temperature with 0:2 mm substrate in 50 mm sodium acetate buffer, pH 5.0, and the rate of hydrolysis of nitrosyl product was monitored at 310 nm in a Perkin-Elmer Lambda-2 UV/Visible spectrophotometer (Waltham, USA). A molar absorption coefficient of 1800 m1 cm1 at 310 nm was used in the calculations (Dunn, Kammerman, & McCurry, 1984). One enzyme unit is defined as the amount of enzyme that hydrolyzed 1 mol min1 of PNPE at 25  C under the above assay conditions. Protein content was determined according to Bradford (1976) using the Bio-Rad reagent (Bio-Rad Laboratories S.A., Madrid, Spain) and bovine serum albumin (Sigma-Aldrich Quı´ mica S.A., Madrid, Spain) as a standard.

2. Materials and methods 2.1. Enzyme source Crude extracts of dried flowers were obtained by homogenization of 15 g of artichoke (C. scolymus L.) stigmas in 50 mL of 50 mm sodium citrate buffer pH 3.0, and centrifugation at 50000  g for 30 min. Three cynarase fractions (A, B and C) were purified from dried flowers of artichokes according to the method described by Sidrach et al. (2005). Artichokes are commercially cultivated in Murcia (Spain) from November until June; purification and studies carried out in our laboratory using material from different seasons and years revealed no evidence of variations in the enzymes activities of the extracts. 2.2. Milk-clotting activity assay The substrate was prepared by dissolving commercial skimmed milk powder (Nestle´ Spain S.A, Barcelona, Spain) in 100 mL of deionized water to a final protein concentration of 13% (w/v). Before use, the samples were incubated for 10 min at 35  C. Sample temperature was controlled using a water bath (Haake D1; Haake Inc., Berlin, Germany). The assay was performed by adding 1 mL of rennet or a cynarase to 10 mL of reconstituted milk. The milk-clotting time was determined by manually rotating the test flask periodically, at short time intervals, and checking for visible clot formation (IDF, 1992; Silva et al., 2002). For each experiment, the clotting time of the sample was measured three times and the average reported as the sample clotting time. The rennet strength (RS) was defined as the number of volumes of coagulated milk clotted by one volume of rennet in 40 min at 35  C. The calculation is: RS ¼ 2400V =tv, where v equals one volume of rennet (mL), V one volume of milk (mL) and t the clotting time in seconds.

2.4. Effect of pH, enzyme concentration, and temperature on milk clotting In order to study the influence of the pH used for cynarase extraction, stigmas (15 g) from dried flowers of C. scolymus L. were homogenized in 50 mL of different buffers, and centrifuged at 50000  g for 30 min. The supernatants were used to study their clotting properties. The buffers employed were 50 mm sodium citrate for pH values 3, 4 and 5; 50 mm sodium phosphate for pH 6.5 and 7.0; 50 mm Tris/HCl for pH 8. For each pH, three enzyme extracts were made. To study the effect of the milk pH on the rennet’s clotting capacity, the pH was adjusted at 35  C by the gradual addition of 0:1 m lactic acid/NaOH during rapid stirring. Standard solutions of each concentration were prepared by diluting the enzyme extract in 50 mm citrate buffer pH 4 (extraction buffer) to maintain the dilution effect resulting from adding the enzyme to the milk samples. Temperature was controlled using a Haake D1 water bath (Haake Inc., Berlin, Germany), measuring the water bath and milk temperatures before and after adding the extract. 2.5. Effects of calcium level and sodium chloride concentration Milk substrates supplemented with different concentrations of calcium were prepared using CaCl2 (3, 7.7, 15.4, 40.3 or 80:6 mm) and calcium lactate (3.2, 6.5, 8.1, 12.9, 16.13, 25.8 or 32:5 mm). Before use, the pH was adjusted to that of milk with added calcium (pH 6.3) and the samples were incubated for 10 min at 35  C. Samples were diluted to maintain a final protein concentration of 13% (w/v). The effect of sodium chloride on rennet’s milk-clotting capacity was studied in reconstituted milk supplemented with this salt to concentrations of 34, 51 or 102 mm.

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2.6. Hydrolysis of caseins Commercial bovine as -, b- and k-casein (Sigma-Aldrich Quı´ mica S.A.) were dissolved up to 2 mg mL1 in 100 mm sodium phosphate buffer pH 6.2 and incubated independently with cynarase A, B, C or crude enzyme extract or commercial clotting rennet ðRS ¼ 500Þ at 30  C. After 60 min, aliquots were taken and the reaction stopped by adding equal volumes of sample loading buffer (125 mm Tris-HCl pH 6.8, 20% glycerol, 4% SDS, 0.01% bromophenol blue and 1:5 m b-mercaptoethanol) and heated at 100  C for 5 min. Samples were analyzed by SDS-PAGE. Electrophoresis was performed in a vertical gel apparatus (Mini-Protean, Bio-Rad Laboratories S.A.) with 15% precast Tris-Gly gels (Bio-Rad Laboratories S.A.), as described by Laemmli (1970). Proteins were stained with 0.2% (w/v) Coomassie brilliant blue in methanol/acetic acid/water (5:2:5) and destained by repeated washing in a methanol/acetic acid/water (2:1:10) solution. 2.7. Microbiological analysis To study the microbiological characteristics of C. scolymus rennet and the effect of refrigeration, sterile bottles containing 5 mL rennet were kept at 4  C or room temperature for 60 days. When specified, rennet samples were filtered through a HPF Millex Millipore filter (Millipore Iberica S.A., Madrid, Spain). To examine the effect of preservative on the microbiological characteristics, benzoic acid or sorbic acid were added to sterile bottles containing 5 mL rennet prior to incubation to give a final concentration of 0.5% and 1%, respectively. Samples were removed at 0, 1, 2, 4, 6 and 8 weeks to count the bacteria. For this, samples were serially diluted 109 -times and pre-reduced in tryptone water and inoculated onto a range of agars designed to be selective for predominant bacteria. Bacteria were counted on plate count agar (Oxoid S.A., Madrid, Spain) for total mesophilic count, MRS-agar for lactobacilli (Oxoid S.A.), Mackonkey agar for enterobacteriaceae (Difco, Becton Dickinson-Espan˜a, Madrid, Spain), iron sulphite agar for Clostridium spp (Oxoid S.A.) and sabouraud dextrose agar for moulds and yeasts (Oxoid S.A.). Various antibiotics and supplements were added to the agars in order to improve selectivity. The dishes were placed in anaerobic jars at 37  C for Clostridium and Lactobacilli; the rest were incubated aerobically at 37  and colony forming units (cfu) were counted after 48 h of incubation; moulds and yeast were counted after 5 days of incubation at room temperature. All determinations were made in duplicate and the changes in the values of cfu per milliliter were expressed as percentage of increased growth (multiplication factor between 0 and 60 days) with respect to non-treated control rennet. 3. Results and discussion 3.1. Effect of milk temperature To study the influence of temperature on the milkclotting activity of crude extracts of dried flowers of

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artichoke, a temperature range from 20 to 60  C was selected. Fig. 1a shows that the clotting time decreased as temperature increased. A progressive reduction in rennet coagulation times as temperature increases from 20 to 40  C has been reported by other authors (Najera, Renobales, & Barron, 2003). However, at high temperatures, the coagulation process slows down (Dybowska & Fujio, 1996). Although changes in salt equilibrium are involved, the factor responsible for the increased rennet clotting time of heated milk is the complex formed between k-casein and b-lactoglobulin or a-lactalbumin (Balcones, Olano, & Calvo, 1996). In the case of artichoke rennet, an increase from 30 to 60  C produces a progressive decrease in clotting time, which is even more pronounced from 20 to 30  C (Fig. 1a). 3.2. Effect of pH The effect of the pH used for cynarase extraction on RS was studied. Crude extracts of dried flowers of artichoke obtained at different pH values were tested for their clotting activity. Maximum activity was observed at pH of around 4, with no significant difference in the studied pH range (3–7). Extraction pH 4 was therefore used for rennet preparation. On the other hand, extractions for individual purified proteases were carried out at pH 3 since the strong purple pigmentation of these extracts was less intense at this pH. The pH also affects both the enzymatic and aggregation phases of the milk coagulation (Castillo, Payne, Hicks, & Lopez, 2000a), although the influence of pH on hydrolysis is less than it is on protein aggregation (Eck, 1989). Fig. 1b shows the influence of milk pH on coagulation. An increase in the pH of the milk was accompanied by a loss of the milk-clotting activity, and at pH 7, 87% of the enzyme activity was lost. Hashem (2000) also observed that an increase in the pH of the reaction mixture was associated with a gradual loss of milk-clotting activity, but at pH 7 the milk-clotting enzyme of Penicillium oxalicum still possessed 38% of its original activity. On the other hand, several authors have observed that clotting time decreases more intensely at the beginning of acidification than later (Castillo et al., 2000a; Eck, 1989; Imafidon & Farkye, 1993; Spreer, 1991). Our result are in accordance with this observation since the increase in rennet clotting time when the pH changed from 6.3 to 7.0 was greater than when pH decreased from 6.3 to 5.5. 3.3. Effect of enzyme concentration Milk-clotting activity is also dependent on the concentration of enzyme, the milk-clotting time decreasing with increasing enzyme concentration (Chitipinityol & Crabbe, 1998). This is a result of the increase in the rate of k-casein proteolysis, although there is no direct proportionality between the values (Lo´pez, Lomholt, & Qvist, 1998). Fig. 1c illustrates the linear relationship between the

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inverse of the enzyme concentration and the milk-clotting activity. Thus, our results are in agreement with the model proposed by van Hooydonk and Walstra (1987) regarding to clotting time as a function of the reciprocal enzyme concentration, and with previous data for extracts from other plant sources (Yousif, McMahon, & Shammet, 1996). On the other hand, there were several models proposed for milk coagulation that correlate clotting time with enzyme concentration (Payens, Wiersma, & Brinkhuis, 1977), the reciprocal clotting time with enzyme concentration (Kopelman & Cogan, 1976), and the clotting time with the reciprocal square root of enzyme concentration (Hyslop, Richardson, & Ryan, 1979). The milk coagulation data were also fitted to these kinetic models, and we found that our experimental data fitted all of the proposed models well. Although a good correlation coefficient was obtained in all cases (results not shown), the experimental data fitted equally well the models proposed by van Hooydonk and Walstra (1987) or by Payens et al. (1977), whereas the poorest regression was obtained with the model of Kopelman and Cogan (1976). The model that relates clotting time with reciprocal enzyme concentration was also the best fit for chymosin (Verı´ ssimo, Esteves, Faro, & Pires, 1995). These results contrast with those observed for the enzymes of C. cardunculus (Silva & Malcata, 2005) where the best fit was obtained with the model of Payens et al. (1977) and the worst fit with the model of Hyslop et al. (1979).

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Fig. 1. Changes in rennet coagulation time with respect to: (a) temperature, (b) pH and (c) crude extract concentration (expressed as protein concentration). For (a) and (b) the assays were performed adding 1 mL of crude extract (2.59 mg protein) to 10 mL of reconstituted milk. For (c) the assays were performed adding 1 mL of crude extract at different concentrations to 10 mL of reconstituted milk. For each experiment, the clotting time of the sample was measured in triplicate and the error bars represent the SD of the data.

3.4. Effects of calcium level and sodium chloride concentration on rennet coagulating activity To test the effect of calcium on milk coagulation, milk samples were supplemented with calcium at different concentrations of calcium lactate and CaCl2 . Because the milk pH decreased when the calcium was added, adjustment to the pH of milk without calcium supplement was carried out. Fig. 2 shows that the RS of this extract increases hyperbolically with increasing concentrations of calcium. In view of these results, a concentration 50 mm was considered sufficient to increase the RS to an effective level. Other authors have reported that the addition of CaCl2 to milk increases the overall enzymatic coagulation rate, although at lower calcium concentrations (Bencini, 2002; Lagaude, Fernandez, Cuq, & Marchesseau, 2004; Najera et al., 2003). Since the pH was adjusted after the addition of calcium was made, the variations observed with calcium concentration would has been due to a direct effect on the aggregation and firming rates (Castillo, Payne, Hicks, Laencina, & Lopez, 2002b). On the other hand, no significant differences were observed between the clotting times obtained with both salts. The addition of NaCl to milk promotes the dissociation of calcium and phosphate from within casein micelles and into solution (Gatti & Pires, 1995; Gaucheron, Le Graet, & Briard, 2000). Besides its effect on the colloidal state of milk, NaCl also affects the action of the coagulant

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Fig. 3. Rennet strength and peptidase activities of purified cynarases and the initial crude extract. The specific activity data per mg of protein are expressed as percentage of the maximum value for each activity. For each sample, both activities were determined in triplicate and the error bars represent the SD of the data.

3.6. Casein hydrolysis (Famelart, Le Graet, & Raulot, 1999; Zoon, van Vliet, & Walstra, 1989). In the current work, the effect of sodium chloride on the milk clotting process was studied by incubating reconstituted milk supplemented with this salt at concentrations of 0, 34, 51 or 102 mm. The effect of sodium chloride at these concentrations on RS was not significant (RS ¼ 387, 387, 422, 422, respectively), a finding that is in contrast with previous data for other rennet solutions, where clotting time increased with increasing amounts of added NaCl (Abd El-Salam, Alichanidis, & Zerfiridis, 1993; Awad, 2007; Famelart et al., 1999; Zoon et al., 1989). The discrepancy in these results could be the consequence of working with a lower range of NaCl concentrations, although Famelart et al. (1999) described that rennet clotting time increased from 500 to 1100 s as NaCl concentration increased from 0 to 120 mm, a similar working range to that used in our study.

When a potential rennet substitute is studied, it is important to evaluate the degradation patterns of the caseins because of their effect on the yield, consistency, and flavor of the final cheese (Fox, 1989). The hydrolysis of bovine a-, b- and k-casein brought about by three cynarases (A, B and C) and by the crude extract is depicted in Fig. 4. All three cynarases present in artichoke cleave k-casein at the same peptide bond as the calf stomach rennet. However, in the hydrolysis of a- and b-casein the electrophoretic patterns produced by the three cynarases differed. The degradation patterns produced via the action of cynarases A and C on a- and b-casein were similar to each other and different from those observed with calf rennet, whereas cynarase B produced an electrophoretic pattern more similar to that obtained with calf rennet. 3.7. Microbiology studies

3.5. Clotting and peptidase activities Clotting activity was determined for crude extracts and pure enzymes, which is an important parameter when evaluating rennets for cheesemaking. The clotting activity is plotted in Fig. 3. Purification led to a decrease in the specific coagulant activity relative to that of the crude extract in the case of cynarases A and C, whereas the specific clotting activity of cynarase B increased. In addition, peptidase activity with PNPE was also assayed for crude extracts and purified enzymes. Fig. 3 shows the specific peptidase activities obtained using the synthetic peptide as a substrate. In this case, too, cynarase B performed best. Whereas the cynarases A and C showed a slight increase in specific activity relative to the initial extract, the specific activity of cynarase B was much higher.

The use of coagulants has been shown repeatedly to preserve the original and distinctive flavors of traditional cheeses (Irigoyen, Izco, Iba´n˜ez, & Torre, 2002) but may cause additional microbial contamination of milk (Flo´rez, Herna´ndez-Barranco, Marcos, & Mayo, 2006). For this reason, we studied the changes occurring in the main bacterial groups to examine the effect of refrigeration and the addition of preservatives on the microbiological characteristics of rennet. Fig. 5 shows the percentage increase of growth of the microbial groups examined in the rennet extracts. The non-filtered controls at both 4  C and room temperature showed similar microbial growth (data not shown). However, the filtered controls maintained at 4  C for two months showed less microbial growth than the rennet at room temperature, suggesting

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Fig. 4. SDS-PAGE electrophoretograms illustrating the degradation of bovine a- (a), b- (b) and k-casein (c) by the crude extract and by the purified cynarases. Lane 1 contains molecular weight protein markers (the same for the three gels). Lanes 2 and 8 contain the corresponding casein: a- (a); b- (b) and k-casein (c). Lanes 3–7 correspond to the hydrolysis of caseins by the following coagulants: (3) crude extract of artichoke flowers; (4) purified cynarase A; (5) purified cynarase B; (6) purified cynarase C; (7) calf stomach rennet. Coagulants were used at catalytic concentrations and, therefore, did not produce detectable protein bands after Coomassie staining.

that the filtering process eliminated total and potentially pathogenic bacteria. The filtered samples treated with sorbic acid (0.5%) prevented increases in all microbial populations, whether maintained at 4  C or at room temperature, and even showed less microbial growth than the filtered controls.

Fig. 5. Changes in the values of colony forming units per milliliter expressed as percentage of increased growth with respect to a non-filtered control rennet (multiplication factor between 0 and 60 days) of different bacterial populations in rennet extracts of Cynara scolymus flower stigmas, after keeping for 60 days at (a) room temperature or (b) 4  C, filtered (CF), with benzoic acid (B), with benzoic acid and filtered (BF), with sorbic acid (S), with sorbic acid and filtered (SF). All determinations were made in duplicate and the error bars represent the SD of the data. Enterobacteriaceae, Mesophylic, Moulds and yeast, Lactobacilli, Costridium spp.

However, the rennet treated with this additive but nonfiltered and kept at 4  C showed an increased load of total mesophilic bacteria, enterobacteria, moulds and yeasts, and a lower count of clostridium and lactobacilli. However when the rennet was stored at room temperature the population of all the microbial groups examined increased. The rennets treated with benzoic acid, filtered and stored at 4  C or at room temperature represented an improvement

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over their respective controls. However, the samples treated with benzoic acid but non-filtered had a lower bacterial population when stored at 4  C than at room temperature. At the lower temperature only the moulds and yeasts increased, whereas the rest of the microbial groups decreased. At room temperature, the population of total mesophilic bacteria, enterobacteria, moulds and yeasts increased but clostridium and lactobacilli did not. We can conclude that both benzoic acid and sorbic acid are good additives for preserving rennet when filtered, in which case it can be kept for at least two months at room temperature. However, if it is not filtered, the rennet must be kept at 4  C. In this case, the rennet treated with the additive benzoic acid (1%) presented the better results with respect to bacterial contamination. 4. Conclusions Numerous attempts have been made to replace calf rennet because of limited supply and increasingly high prices. Moreover, there is a renewed interest in using enzymes from plant sources. Characterization of plant rennet from the genus Cynara has been extensively performed, however, this characterization has been focused mainly on the species C. cardunculus. The knowledge on the properties of other members of this family such us humilis and scolymus is more scarce. The results obtained in this study on the properties of artichoke rennet are of importance, and indicate the possibility of the use of this rennet in the cheese manufacture. Moreover, it could represent an alternative to improving the nutritional input of vegetarian and other people whose use of animal rennets is restricted. Acknowledgments This research was supported by Consejerı´ a de Economı´ a, Industria e Innovacio´n (Regio´n de Murcia, Spain), project 2I04SU004 to J.N.R.-L. L.S. is contracted by the programme Torres-Quevedo from the Ministerio de Educacio´n y Ciencia (Spain). References Abd El-Salam, M. H., Alichanidis, E., & Zerfiridis, G. K. (1993). Domiati and Feta type cheese. In P. F. Fox (Ed.), Cheese: Physical, chemical, and biological aspects (Vol. 2) (2nd ed.) (pp. 301–336). London, UK: Chapman & Hall. Awad, S. (2007). Effect of sodium chloride and pH on the rennet coagulation and gel firmness. Lebensmittel Wissenschaft und Technologie—Food Science and Technology, 40, 220–224. Balcones, E., Olano, A., & Calvo, M. M. (1996). Factors affecting the rennet clotting properties of ewe’s milk. Journal of Agriculture and Food Chemistry, 44, 1993–1996. Bencini, R. (2002). Factors affecting the clotting properties of sheep milk. Journal of the Science of Food and Agriculture, 82, 705–719. Bradford, M. M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of proteindye binding. Analytical Biochemistry, 72, 248–254.

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