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Mozzarella Cheese: Impact of Coagulant Type on Chemical Composition and Proteolysis1
Mozzarella Cheese: Impact of Coagulant Type on Chemical Composition and Proteolysis1 J. JOSEPH YUN, DAVID M. BARBANO, and PAUL S. KINDSTEDT2 Northeast Dairy Foods Research Center Department of Food Science Cornell University Ithaca, NY 14853 ABSTRACT
INTRODUCTION
Coagulants for Cheese Making
The impact of different milk c.oagulants on initial chemical compositIOn and proteolytic changes in Mozzarella cheese during refrigerated storage was determined. Three vats of cheese were made in 1 d. each using a different coagulant (i.e.• Endothia parasitica protease, fermentation-derived chymosin. or Mucor miehei protease). Cheese making was replicated on 3 different d using a 3 x. 3 Latin square design. Coagulant type did not affect pH, moisture. protein, or fat contents of cheese. Differences in salt content and fat on a dry weight basis of the cheese were slight. Nitrogen soluble in 12% TeA and in pH 4.6 acetate buffer increased significantly for cheeses made with all coagulants, but the increase was the greatest for cheese made with E. parasitica protease. The as-casein in cheeses made with each of the three coagulants was broken down during storage. Proteolysis of l3-casein was only in the cheese made with E. parasitica protease. The milk coagulant used for Mozzarella cheese manufacture plays an important role in proteolysis during 50 d of storage at 4·C. (Key words: Mozzarella cheese. coagulant. composition. proteolysis) Abbreviation key: CDF by fermentation.
The limited supply and high cost of calf rennet stimulated development of a variety of lower cost milk coagulants from microbial sources (9). However. the use of microbial coagulants from Endothia parasitica and Mucor miehei can significantly affect the development of cheese texture and flavor during storage (6). Functional properties of Mozzarella cheese (20) and cheese yield (3. 6) may also be affected by use of different coagulants for cheese making. All commercial milk coagulants are acid proteases (15). Recent developments in genetic engineering have allowed commercial production of pure chymosin (BC 3.4.23.4) by fermentation technology using a variety of microorganisms (i.e., Escherichia coli. Kluyvermyces lactis, and Aspergillus awamori) (23). Generally. calf rennet and chymosin derived by fermentation (CDF) produce higher cheese yield than do microbial coagulants. The yield and chemical composition of Cheddar cheeses made with calf rennet and CDF were similar (3). Cheeses made with CDF were not distinguishable from those made with calf rennet (23). Role of CoagUlant In Cheese Aging
= chymosin derived
Received November 17. 1992. Accepted August 3. 1993. lUse of trade names. names of ingredients. and identification of specific models of equipment is for scientific clarity and does not constitute any endorsement of product by authors. Cornell University, University of Vermont, or the Northeast Dairy Foods Research Center. 2Department of Animal and Food Science, University of Vermont, Burlington 05405. 1993 J Dairy Sci 76:3648-3656
Residual proteolytic activity of a milk coagulant in cheese depends on its sensitivity to inactivation by temperature and pH during cheese making (15). Enzymes are generally more stable at a pH that is close to their isoelectric point (i.e., near pH 4 for E. parasitica protease, CDF, and M. miehei protease) (15). Evidence of proteolytic activity from residual coagulant during cheese aging was shown in Cheddar (21) and in Gouda cheese (29). The E. parasitica protease is the most heat sensitive milk coagulant (24). However, if
3648
COAGULANT TYPE AND MOZZARELLA COMPOSITION
E. parasitica protease is not inactivated during cheese making, the enzyme is the most proteolytic coagulant in Cheddar (12) and in Gouda cheese (26). Different enzymes produce different patterns of proteolysis products from a sand /3-easeins during cheese aging. Residual coagulant activity in Mozzarella cheese was demonstrated by either a decrease in intact casein content or an increase in soluble nitrogen content (11, 14, 30). Changes in functional properties of Mozzarella cheese during storage (20, 31) may be caused by proteolysis. Creamer (10) observed a slower rate of casein degradation in Mozzarella cheese than in Cheddar or Gouda and suggested that it may have been due to the high temperatures used during stretching. Thus, the extent to which different types of milk coagulants contribute to proteolysis during refrigerated storage of Mozzarella cheese is not clear. The objective of the present study was to determine the impact of three coagulants (i.e., CDF, E. parasitica protease, and M. miehei protease) on initial chemical composition and proteolytic changes in Mozzarella cheese during 50 d of storage at 4°C. MATERIALS AND METHODS CoagUlant Type
Three different coagulants were used: CDF miehei protease (Morcurd Plus®), and E. parasitica protease (Surecurd~). All coagulants were selected from lots of coagulants in commercial use (Pfizer Inc., Milwaukee, WI) at the time of the study. Relative milk-clotting activity of each coagulant used in this study was determined by the Berridge procedure (4, 5) as modified by Ernstrom (13). Relative milk-elotting activity was measured at a research laboratory of a coagulant manufacturer (Pfizer Inc.) where the assay is routinely conducted. Differences in relative milk-clotting activity among coagulants were used to determine the amount of each coagulant to be used per unit of milk for cheese making. (Chymax~), M.
Cheese Making
A "no-brine" Mozzarella cheese-making method (30) was used to produce cheese with homogeneous chemical composition. Raw
3649
skim milk and raw cream were obtained from the Cornell University dairy plant (Ithaca, NY), standardized to 2.3% fat, pasteurized at noc for 16 s, cooled to 4'C, divided into three equal 17o-kg portions, and stored overnight at 4°C. On the next day, the cold, standardized, pasteurized milk (ca. 170 kg per vat) was poured into each of three cheese vats (model 4MX; Kusel Equipment Co., Watertown, WI) and heated to 36°C. Direct-to-vat frozen starter cultures, Streptococcus salivarius spp. thermophilus and Lactobacillus delbrueckii spp. bulgaricus (Thermococcus C120® and Thermorod RI60®, respectively, from RhonePoulenc, Madison, WI) were used (.30 m1Ikg of milk). Milk was ripened for 60 min at 36°C after addition of starter. At the end of ripening, CDF, E. parasitica protease, and M. miehei protease were added to different vats and agitated for exactly I min. Coagulant addition was based on relative milk-clotting activity. The amounts of coagulants used were the following: CDF (Chymax~, double strength), .097 mllkJ of milk; M. miehei protease (Morcurd Plus , double strength), .101 mllkg of milk; and E. parasitica protease (Surecurd®, triple strength), .053 m1Ikg of milk. Following a 30-min set, the milk coagulum was cut with a 1.2-cm wire knife and allowed to heal for 5 min. Next, the curds were stirred gently without heat for 10 min, followed by heating from 36 to 41 'C for 15 min with continuous agitation. Temperature was maintained at 41°C, and agitation continued, until the whey pH reached 6.40 ± .02; then whey was drained, and curd was piled in the center of the vat. Curd slabs were turned over (cheddared) once every 15 min until the curd reached a milling pH of 5.25. The cheese curd was milled and salted at a total rate of 2% (wtJ wt). Salted curd (at pH near 5.20) was stretched in a pilot-scale Mozzarella mixer (model 640; Stainless Steel Fabricating, Columbus, WI) with circulating brine (about 8% salt concentration) at 57°C. During the transit time of 5 to 10 min, the temperature of the curd increased to 55'C. The cheese was then cooled in ice water for I h, vacuum packaged, and stored at 4°C. Six 1.2-kg cylinders of cheese (30 em long x 7.5 cm i.d.) were made per vat. The third and fourth cylinders (sequence of extrusion) were used for chemical analysis. Journal of Dairy Science Vol. 76. No. 12, 1993
3650
YON ET AL.
Chemical Analy.e.
Milk, Whey, and Cheese. Changes in titratable acidity of milk and whey (22) and pH of milk, whey, and cheese were monitored during cheese making (30). Fat contents of milk (1), cream (1), whey (22), and cheese (22) were determined using Babcock tests. All nitrogen determinations were made by the Kjeldahl method (1, 2). Percentage of nitrogen from the analyses of noncasein nitrogen (17) and total nitrogen (1, 2) were multiplied by 6.38 to give milk protein equivalents. Total nitrogen and noncasein nitrogen measurements for milk were performed in triplicate. All other chemical analyses, except for cheese moisture, were performed in duplicate. Cheese moisture was determined gravimetrically in quadruplicate using a forced-air oven (model OV-490A-2; Blue M, Blue Island, IL) at 100°C for 24 h (22). Salt content in cheese was determined by the Volhard procedure (22) and calcium concentration by complexometric titration (18). Proteolysis. Nitrogen soluble in pH 4.6 acetate buffer and in 12% TCA were determined to measure the proteolysis in cheese (7) after 3, 8, 15, 21, 29, and 50 d of storage at 4°C. All soluble nitrogen values were expressed as a percentage of total nitrogen content of cheese. Then, SDS-PAGE (27) was used to monitor the proteolysis of O:s- and ,s-caseins in cheese during refrigerated storage. The amount of O:s- and IS-caseins was determined by scanning gels with a video densitometer (model 620; The I-D Analyst®, BioRad Laboratories, Rockville
Center, NY). Amounts of residual caseins were calculated as the area for zone 1 (O:sl- and O:s2caseins) or zone 2 (,8-casein) as a percentage of the total area of all major bands for each sample (30). Experimental De.lgn and Statl.tical Analysis
Three vats of cheese were made (each using a different coagulant) from one batch of milk. The cheese making was replicated on 3 different d. On each day, the order of cheese making for the three different coagulants was changed so that the effect of day and order of cheese making were blocks in a 3 x 3 Latin square design. The data for initial chemical composition of the cheese were analyzed using PROC ANOVA of SAS (SAS Institute Inc., Cary, NC). The PROC GLM of SAS was used for evaluation of the proteolytic changes during refrigerated storage based on a split-plot design; the whole-plot factor (i.e., effect of coagulant type) was replicated in a 3 x 3 Latin square design in which the effects of day and order were blocks. The factors, degree of freedom, and statistical model are shown in Table 1. The level of significance was P :5: .05 throughout the paper. RESULTS Composition of Milk, Wh.y, and Ch....
The average fat, protein, and casein contents of milks used in three cheese-making trials were 2.31,3.28, and 2.52%, respectively.
TABLE I. Statistical model used for data analyses. Factors Whole-plot factor Coagulant type Day of cheese making Order of cheese making Error Subplot factor Age Age x age Interaction of coagulant type x age Interaction of coagulant type x (age x age) Error
df
Analyzed as
2
Classification
2 2 2
Block Block
1
Quantitative Quantitative Classification x quantitative Classification x quantitative
1
2 2
39 1
IThe degrees of freedom of the error term for the subplot factor error for electrophoresis results were 21 instead of 39 because only four times of aging were used instead of six. Journal of Dairy Science Vol. 76, No. 12, 1993
3651
COAGULANT TYPE AND MOZZARELLA COMPOSITION
Casein as a percentage of total nitrogen was 76.8%. The average fat content of whey at draw was .20%. No significant effect of coagulant type on cheese pH, moisture, fat, protein, and calcium contents was observed (Table 2). Moisture content of cheeses ranged from 44 to 45%, which is slightly lower than the legal minimum (45%) for low moisture, part-skim Mozzarella cheese in the US (8). Salt and fat (on a dry weight basis) were significantly (P < .05) different, depending on the coagulant used (Table 2). However, these differences were too small to be significant in practice. Proteolysis
Amounts of nitrogen soluble in pH 4.6 acetate buffer and in 12% TCA were significantly affected by the differences in coagulant type (Figure I, A and B, respectively; Table 3). Soluble nitrogen increased with refrigerated storage time for all cheeses. The SOS-PAGE patterns of Mozzarella during 50 d of refrigerated storage are shown in Figure 2. Zones 1 and 2 are the two major bands at the top of the gel. Zone I contains asl- and as2-caseins, zone 2 contains ~-casein, and zone 3 contains paraK-casein. Proteolytic breakdown products of a s- and ~-caseins are shown as bands in the area between zones 2 and 3 and below zone 3.
The amount of asl- and as2-caseins as a percentage of total protein decreased significantly for all cheeses during refrigerated storage (Figure 3A; Table 3). No significant effect of coagulant type on asl - and as2-caseins was observed, but a significant coagulant type x age interaction occurred (Table 3). During the same period, ~-casein in cheeses made with COF and M. miehei protease remained relatively constant (Figure 3B). However, ~-casein in cheeses made with E. parasitica protease decreased significantly (Table 3; Figure 3B). The combined amount of a s- and ~-caseins decreased (Table 3; Figure 3C) with storage for all coagulants, but the decrease was the largest with E. parasitica protease. This finding is consistent with the observation that the Mozzarella cheese made with E. parasitica protease had the largest increase in pH 4.6-soluble nitrogen (Figure 1). DISCUSSION
Cheese Making
pH at Drawing Whey. The range of pH at drawing whey in our cheese making was 6.40 ± .02, which may be higher than usual com-
TABLE 2. Initial chemical composition (n = 3) of Mozzarella cheeses made using three different coagulants.
Coagulant Endothia parasitica
Component
protease
CDpl
pH Moisture, % Pal, % FOB,3 % Protein, %
5.15 44.41 22.08 39.7()1 27.83
5.17 44.53 21.92 39.5Qb 27.97 1.59 1.51 ab 3.39ab .804 2.87
M:P'4
1.60
Salt, % S in M,s
%
1.55a 3.49"
Ca,% Ca (%
P),6 %
2.90
.808
of
type
Mucor miehei
protease
SEM
5.16 44.11 22.04 39.40" 28.16 1.57 l.46b 3.31 b .794 2.82
.010 .056 .025 .013 .124 .008 .008 .018 .010 .040
a.b.cMeans within same row not sharing common superscripts are different lChymosin derived by fermentation. 2p = .05. 3Pat content on a dry weight basis. 4Ratio of moisture to protein. sRatio of salt to moisture in the cheese. tlCalcium as a percentage of the protein content of the cheese.
(P
LSI)2 .06
.34 .15 .08 .75 .05 .05 .11 .06
.25
< .05).
Journal of Dairy Science Vol. 76, No. 12, 1993
3652
YUN ET AL.
-"...,
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mercial conditions. The curd pH was about 6.35. A previous study (16) showed that retention of chymosin in cheese increases with decreasing pH at drawing whey, but the retention of M. miehei and Mucor pusillus proteases are not dependent on pH. The high drawing pH (i.e., pH 6.40) in our cheese-making process may have resulted in a lower chymosin retention and less proteolysis than if we had used a lower drawing pH. pH and Temperature During Cooking and Stretching. Throughout the cheese-making process, milk coagulants undergo various heat treatments, and different coagulants exhibit different sensitivities to inactivation by heat (15). The cooking temperature (in the vat) of 4l C at pH 6.5 to 6.4 (whey pH) that was used in our study is not expected to inactivate coagulants. The heat stability of acid proteases D
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Journal of Dairy Science Vol. 76, No. 12, 1993
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Figure 1. Impact of coagulant type on pH 4.6-soluble nitrogen (A; SEM = .26%) and on 12% TeA-soluble nitrogen (B; SEM = .05%) of Mozzarella cheese made using Endolhia parasitica protease ($), chymosin derived by fermentation (II), and Mucor mieMi protease (A).
3653
COAGULANT TYPE AND MOZZARELLA COMPOSITION
is greater at lower pH (15). At milling, curd pH was 5.25, which decreased to 5.20 by the time of stretching. The water temperature at stretching that was used in our cheese making was 57°C (curd temperature, 55·C), which was probably low enough to allow coagulants to survive. particularly at a curd pH of 5.20 (10, 15). Salt Concentration in Cheese at Stretching. The thermostability of aqueous rennet solutions increased at pH 5.6 and at 57°C when the concentration of salt increased (25). During the stretching step in the "no-brine" cheesemaking method, the concentration of salt in the curd is about 1.5%. Thus, the salt concentration in the aqueous phase of the curd is about 3%, which may help make the rennet retained in the curd more heat stable. The final extent of proteolysis caused by the coagulant retained in the cheese is determined
Chymosin derived by fermentation
by the sum of these and other factors, including accessibility of substrate, rate of cooling, and storage time and temperature. Impact of CoagUlant Type
Cheese Composition. The fat, protein, and moisture contents of Mozzarella cheese were unaffected by coagulant type (Table 2). A small but significant difference in salt content existed between cheeses made with E. parasitica protease and M. miehei protease. This difference may be due to differences in curd structure and rate of syneresis during salting. A previous study on Cheddar cheese (3) reported differences in the yields of whey, salt whey, and cheese depending on the coagulant type. Proteolysis. The amounts of nitrogen soluble in pH 4.6 acetate buffer (Figure IA) and in 12% TCA (Figure IB), {3-casein (Figure 3B), and combined (Xs- and {3-caseins (Figure 3C)
Endothia parasitica protease
Mucor miehei protease
storage (d) 3
15
29
50
3
15
29
50
3
15
29
50
Zone Zone
Zone
3~
Figure 2. The SDS-PAGE gel of Mozzarella cheeses made using chymosin derived by fennentation. E1ldothia parasitica protease, and Mucor miehei protease that were sampled at 3. IS. 29. and SO d of storage at 4'C: zone 1 = asl' plus a s2-caseins. zone 2 = P-casein. and zone 3 = para·K-casein. Journal of Dairy Science Vol. 76, No. 12. 1993
3654
YUN ET AL.
were significantly affected by the differences in coagulant type (fable 3). The nitrogen soluble in pH 4.6 acetate buffer (Figure lA) seemed to be more sensitive to differences in coagulant type than nitrogen soluble in 12% TCA (Figure lB). This finding is consistent with 50 r - - - - - - - - - - - - - - - - - , A
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Figure 3. Impact of coagulant type on the relative percentages of O/sl- plus O/s2-caseins (A; SEM 3.8%), flcasein (B; SEM = 1.6%), and the sum of O/sl-' O/s2-, and (3caseins (C; SEM = .8%) as a percentage of all protein in Mozzarella cheese made using Endothia parasitica protease (*), chymosin derived by fermentation (II), and Mucor miehei protease (.A.).
=
Journal of Dairy Science Vol. 76, No. 12, 1993
previous reports (21, 28) that coagulants contribute more to the extent of proteolysis, whereas enzymes from starter culture affect the depth of proteolysis in Cheddar and Gouda cheese. The breakdown of a s}- and aS2-caseins during storage was not directly affected by coagulant type in our statistical model (fable 3). However, the interaction of coagulant type x age was significant (fable 3), indicating that the rate of proteolysis with storage was different depending on coagulant type (Figure 3A). The breakdown of a s}- and aS2-caseins during storage of cheese made with CDF was typical of observations in Mozzarella cheese when calf rennet was used as coagulant (30). Proteolysis of l3-casein occurred only in the cheese made with E. parasitica protease (Figure 3B). The difference in substrate specificity that we observed was similar to results of previous studies. Edwards and Kosikowski (12) indicated that E. parasitica protease caused the most proteolysis of l3-casein in Cheddar cheese. Farkye et al. (14) also reported decreased a s- and l3-caseins with storage time in Mozzarella made with E. parasitica protease. Overall, E. parasitica protease was more proteolytic than CDF and M. miehei protease in Mozzarella cheese, as shown by the largest decrease in combined a s- and l3-caseins during storage (Figure 3C). Impact of Age
Proteolysis. The amount of nitrogen soluble in 12% TCA and in pH 4.6 acetate buffer increased significantly (P < .01) from 3 to 50 d of refrigerated storage (Figure 1, A and B; Table 3). Even though linear and quadratic terms of age were significant (P < .01), the mean squares for the linear term of age was greater than the mean squares of the quadratic term of age (fable 3), indicating that soluble nitrogen contents of cheese predominantly tended to increase linearly with age. Also, interactions of coagulant type with linear and quadratic terms of age were significant (P < .01). However, the mean squares of interactions for coagulant type x linear term of age were much greater than the mean squares of coagulant type x quadratic term of age (fable 3). The decrease in a s}- and as2-caseins in the cheese was highly significant during 50 d of
COAGULANT TYPE AND MOZZARELLA COMPOSITION
refrigerated storage (Figure 3A; Table 3). The pattern of decrease in asl- and as2-caseins was linear, not quadratic (fable 3). The decrease in ,s-casein that was due to coagulant type was highly significant during 50 d of refrigerated storage (Figure 3B; Table 3). The major and only decrease in ,s-casein occurred in the cheese made with E. parasitica protease. The amount of ,s-casein in cheeses made with CDP and M. miehei protease remained constant during 50 d of refrigerated storage. The rate of breakdown for the combined a sand ,s-caseins was higher in cheese made with E. parasitica protease than in cheeses made with CDF and M. miehei protease (Figure 3C). This finding is consistent with results from soluble nitrogen analysis. Both types of soluble nitrogen (pH 4.6 acetate buffer and 12% TCA) were higher in cheese made with E. parasitica protease than in cheeses made with CDF and M. miehei protease (Figure 1, A and B). Practical Implications
The extent, rate, and specificity of proteolysis of caseins in Mozzarella cheese can be significantly influenced by the type of coagulant used for cheese making. Each coagulant can be influenced differently by variation in critical cheese-making factors. For example, under the cheese-making conditions used in this study, E. parasitica protease demonstrated higher proteolysis in cheese during refrigerated storage. However, among the three coagulants, E. parasitica protease is the most heat labile (24). Furthermore, the heat sensitivity of E. parasitica protease increased at the pH range outside 4 ± 1 (19). If our experiment was conducted using a high cooking temperature in the cheese vat ~45°C) combined with a pH of curd at drawing whey of 6.35, the cheese made using E. parasitica protease might have the lowest rate of proteolysis during storage relative to CDF and M. miehei protease. Thus, the various combinations of the coagulant type plus the pH and temperature conditions during cheese making can be used to change the rate of proteolysis and, possibly, cheese functionality during refrigerated storage. CONCLUSIONS
Differences in coagulant type did not significantly affect the initial chemical composition. However, proteolysis during the refriger-
3655
ated storage of Mozzarella cheese was affected by coagulant type. The cheese made with E. parasitica protease contained more soluble nitrogen than other cheeses. The as-casein decreased significantly during storage at 4°C for cheeses made with all coagulants. The ,scasein remained constant in the cheeses made with M. miehei protease and CDF but significantly decreased during 50 d of refrigerated storage for cheese made with E. parasitica protease. The coagulant retained in the cheese influences proteolysis during refrigerated storage. Therefore, factors that may influence residual proteolytic activity (i.e., coagulant type, temperature, pH, and amount of coagulant) influence proteolysis and, possibly, functional characteristics of Mozzarella. Development of a better understanding of this system will enable the cheese maker to improve the consistency of the functional properties of Mozzarella cheese. ACKNOWLEDGMENTS
The authors thank Robert Rasmussen, Maureen Chapman, Patricia Nelson, and George Houghton for their technical assistance. Financial support was provided by Northeast Dairy Foods Research Center and Hatch Project 143-444. REFERENCES
1 Association of Official Analytical Chemists. 1990. Official Methods of Analysis. 15th ed. AOAC, Arlington, VA. 2 Barbano, D. M., J. L. Clark, C. E. Dunham, and 1. R. Reming. 1990. Kjeldahl method for determination of total nitrogen content of milk: collaborative study. 1. Assoc. Offic. Anal. Chern. 73:849. 3 Barbano, D. M., and R. R. Rasmussen. 1992. Cheese yield performance of fermentation produced chymosin and other milk coagulants. J. Dairy Sci. 75:1. 4 Berridge, N. J. 1952. Some observations on the determination of the activity of rennet. Analyst (Lond.) 77: 57. 5 Berridge, N. J. 1952. An improved method of observing the clotting of milk containing rennin. J. Dairy Res. 19:328. 6 Birkkjaer, H., and P. Johnk. 1985. Technological suitability of calf rennet substitutes. Page 8 in Int. Dairy Fed. Bull. No. 194. Int. Dairy Fed., Brussels, Belgium. 7 Bynum, D. G., and D. M. Barbano. 1985. Whole milk reverse osmosis retentates for Cheddar cheese manufacture: chemical changes during aging. J. Dairy Sci. 68:1. 8 Code of Federal Regulations. 1991. Food and Drugs. Journal of Dairy Science Vol. 76, No. 12, 1993
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Title 21. Sections 133.156 (Low-moisture mozzarella and scannoza) and 133.158 (Low-moisture part skim mozzarella and scarmoza). Dep. Health and Human Services, Washington, DC. 9 Crawford, R.J.M. 1985. Future developments in rennet and its use in the cheese factory. Page 14 in In!. Dairy Fed. Bull. No. 194. Int. Dairy Fed., Brussels, Belgium. 10 Creamer, L. K. 1976. Casein proteolysis in Mozzarella-type cheese. N.Z. 1. Dairy Sci. Techno!. 11:130. II Di Matteo, M. G., M. G. Chiovitti, and F. Addeo. 1982. Variation in the composition of Mozzarella cheese during storage. Sci. Teen. Latt. Cas. 33:197. 12 Edwards, J. L., and F. V. Kosikowski. 1969. Electrophoretic proteolytic patterns in Cheddar cheese by rennet and fungal rennets: their significance to international classification of cheese varieties. J. Dairy Sci. 52:1675. 13 Emstrom, C. A. 1958. Heterogeneity of crystalline rennin. J. Dairy Sci. 41:1663. 14 Farlcye, N. Y., L. 1. Kiely, R. D. Allshouse, and P. S. Kindstedt. 1991. Proteolysis in Mozzarella cheese during refrigerated storage. J. Dairy Sci. 74:1433. 15 Gamot, P. 1985. Heat stability of milk-clotting enzymes. Page 2 in Int. Dairy Fed. Bull. No. 194. In!. Dairy Fed., Brussels, Belgium. 16 Holmes, D. G., J. W. Duersch, and C. A. Emstrom. 1977. Distribution of milk clotting enzymes between curd and whey and their survival during Cheddar cheese making. J. Dairy Sci. 60:862. 17 International Dairy Federation. 1964. Determination of casein content of milk. Int. Dairy Fed. Stand. 29. In!. Dairy Fed., Brussels, Belgiurn. 18 Kindstedt, P. S., and F. V. Kosikowski. 1985. Improved complexometric determination of calcium in cheese. J. Dairy Sci. 68:806. 19 Larson, M. K., and J. R. Whitaker. 1970. Endothia parasitica protease: parameters affecting stability of the rennin-like enzyme. 1. Dairy Sci. 53:262. 20 Oberg, C. 1., R. K. Merril, R. J. Brown, and G. H. Richardson. 1992. Effects of milk-clotting enzymes on physical properties of Mozzarella cheese. J. Dairy Sci. 75:669.
Journal of Dairy Science Vo!. 76, No. 12, 1993
210'Keeffe, A. M., P. F. Fox, and C. Daly. 1978. Proteolysis in Cheddar cheese: role of coagulant and starter bacteria. J. Dairy Res. 45:465. 22 Richardson, G. H., ed. 1985. Standard Methods for the Examination of Dairy Products. 15th ed. Am. Pub!. Health Assoc., Inc., Washington, DC. 23 Teuber, M. 1990. Production of chymosin by microorganisms and its use for cheese making. Page 3 in Int. Dairy Fed. Bull. No. 251. Int. Dairy Fed., Brussels, Belgium. 24 Thunell, R. K., J. W. Duersch, and C. A. Emstrom. 1979. Thermal inactivation of residual milk-clotting enzymes in whey. J. Dairy Sci. 62:373. 25 van de Beek, M. J., and S. Y. Gerlsma. 1969. Preservation of the enzymatic activity of rennin during spray drying and during storage, and the effect of sugars and certain other additives. Neth. Milk Dairy J. 23:46. 26 Vanderpoorten, R., and M. Weckx. 1972. Breakdown of casein by rennet and microbial milk-clotting enzymes. Neth. Milk Dairy J. 26:45. 27 Verdi, R. J., D. M. Barbano, M. E. DellaValle, and G. F. Senyk. 1987. Variability in true protein, casein, nonprotein nitrogen and proteolysis in high and low somatic cell milks. J. Dairy Sci. 70:230. 28 Visser, F.M.W. 1977. Contribution of enzymes from rennet, starter bacteria and milk to proteolysis and flavour development in Gouda cheese. 3. Protein breakdown: analysis of the soluble nitrogen and amino acid nitrogen fractions. Neth. Milk Dairy J. 31:210. 29 Visser, F.M.W. 1977. Contribution of enzymes from rennet, starter bacteria and milk to proteolysis and flavour development in Gouda cheese. 4. Protein breakdown: a gel electrophoretical study. Neth. Milk Dairy J. 31:247. 30 Yon, 1. J., D. M. Barbano, and P. S. Kindstedt. 1993. Mozzarella cheese: impact of milling pH on chemical composition and proteolysis. J. Dairy Sci. 76:3629. 31 Yun, J. J., L. J. Kiely, P. S. Kindstedt, and D. M. Barbano. 1993. Mozzarella cheese: impact of milling pH on functional properties. J. Dairy Sci. 76:3639.
INTRODUCTION
Coagulants for Cheese Making
The impact of different milk c.oagulants on initial chemical compositIOn and proteolytic changes in Mozzarella cheese during refrigerated storage was determined. Three vats of cheese were made in 1 d. each using a different coagulant (i.e.• Endothia parasitica protease, fermentation-derived chymosin. or Mucor miehei protease). Cheese making was replicated on 3 different d using a 3 x. 3 Latin square design. Coagulant type did not affect pH, moisture. protein, or fat contents of cheese. Differences in salt content and fat on a dry weight basis of the cheese were slight. Nitrogen soluble in 12% TeA and in pH 4.6 acetate buffer increased significantly for cheeses made with all coagulants, but the increase was the greatest for cheese made with E. parasitica protease. The as-casein in cheeses made with each of the three coagulants was broken down during storage. Proteolysis of l3-casein was only in the cheese made with E. parasitica protease. The milk coagulant used for Mozzarella cheese manufacture plays an important role in proteolysis during 50 d of storage at 4·C. (Key words: Mozzarella cheese. coagulant. composition. proteolysis) Abbreviation key: CDF by fermentation.
The limited supply and high cost of calf rennet stimulated development of a variety of lower cost milk coagulants from microbial sources (9). However. the use of microbial coagulants from Endothia parasitica and Mucor miehei can significantly affect the development of cheese texture and flavor during storage (6). Functional properties of Mozzarella cheese (20) and cheese yield (3. 6) may also be affected by use of different coagulants for cheese making. All commercial milk coagulants are acid proteases (15). Recent developments in genetic engineering have allowed commercial production of pure chymosin (BC 3.4.23.4) by fermentation technology using a variety of microorganisms (i.e., Escherichia coli. Kluyvermyces lactis, and Aspergillus awamori) (23). Generally. calf rennet and chymosin derived by fermentation (CDF) produce higher cheese yield than do microbial coagulants. The yield and chemical composition of Cheddar cheeses made with calf rennet and CDF were similar (3). Cheeses made with CDF were not distinguishable from those made with calf rennet (23). Role of CoagUlant In Cheese Aging
= chymosin derived
Received November 17. 1992. Accepted August 3. 1993. lUse of trade names. names of ingredients. and identification of specific models of equipment is for scientific clarity and does not constitute any endorsement of product by authors. Cornell University, University of Vermont, or the Northeast Dairy Foods Research Center. 2Department of Animal and Food Science, University of Vermont, Burlington 05405. 1993 J Dairy Sci 76:3648-3656
Residual proteolytic activity of a milk coagulant in cheese depends on its sensitivity to inactivation by temperature and pH during cheese making (15). Enzymes are generally more stable at a pH that is close to their isoelectric point (i.e., near pH 4 for E. parasitica protease, CDF, and M. miehei protease) (15). Evidence of proteolytic activity from residual coagulant during cheese aging was shown in Cheddar (21) and in Gouda cheese (29). The E. parasitica protease is the most heat sensitive milk coagulant (24). However, if
3648
COAGULANT TYPE AND MOZZARELLA COMPOSITION
E. parasitica protease is not inactivated during cheese making, the enzyme is the most proteolytic coagulant in Cheddar (12) and in Gouda cheese (26). Different enzymes produce different patterns of proteolysis products from a sand /3-easeins during cheese aging. Residual coagulant activity in Mozzarella cheese was demonstrated by either a decrease in intact casein content or an increase in soluble nitrogen content (11, 14, 30). Changes in functional properties of Mozzarella cheese during storage (20, 31) may be caused by proteolysis. Creamer (10) observed a slower rate of casein degradation in Mozzarella cheese than in Cheddar or Gouda and suggested that it may have been due to the high temperatures used during stretching. Thus, the extent to which different types of milk coagulants contribute to proteolysis during refrigerated storage of Mozzarella cheese is not clear. The objective of the present study was to determine the impact of three coagulants (i.e., CDF, E. parasitica protease, and M. miehei protease) on initial chemical composition and proteolytic changes in Mozzarella cheese during 50 d of storage at 4°C. MATERIALS AND METHODS CoagUlant Type
Three different coagulants were used: CDF miehei protease (Morcurd Plus®), and E. parasitica protease (Surecurd~). All coagulants were selected from lots of coagulants in commercial use (Pfizer Inc., Milwaukee, WI) at the time of the study. Relative milk-clotting activity of each coagulant used in this study was determined by the Berridge procedure (4, 5) as modified by Ernstrom (13). Relative milk-elotting activity was measured at a research laboratory of a coagulant manufacturer (Pfizer Inc.) where the assay is routinely conducted. Differences in relative milk-clotting activity among coagulants were used to determine the amount of each coagulant to be used per unit of milk for cheese making. (Chymax~), M.
Cheese Making
A "no-brine" Mozzarella cheese-making method (30) was used to produce cheese with homogeneous chemical composition. Raw
3649
skim milk and raw cream were obtained from the Cornell University dairy plant (Ithaca, NY), standardized to 2.3% fat, pasteurized at noc for 16 s, cooled to 4'C, divided into three equal 17o-kg portions, and stored overnight at 4°C. On the next day, the cold, standardized, pasteurized milk (ca. 170 kg per vat) was poured into each of three cheese vats (model 4MX; Kusel Equipment Co., Watertown, WI) and heated to 36°C. Direct-to-vat frozen starter cultures, Streptococcus salivarius spp. thermophilus and Lactobacillus delbrueckii spp. bulgaricus (Thermococcus C120® and Thermorod RI60®, respectively, from RhonePoulenc, Madison, WI) were used (.30 m1Ikg of milk). Milk was ripened for 60 min at 36°C after addition of starter. At the end of ripening, CDF, E. parasitica protease, and M. miehei protease were added to different vats and agitated for exactly I min. Coagulant addition was based on relative milk-clotting activity. The amounts of coagulants used were the following: CDF (Chymax~, double strength), .097 mllkJ of milk; M. miehei protease (Morcurd Plus , double strength), .101 mllkg of milk; and E. parasitica protease (Surecurd®, triple strength), .053 m1Ikg of milk. Following a 30-min set, the milk coagulum was cut with a 1.2-cm wire knife and allowed to heal for 5 min. Next, the curds were stirred gently without heat for 10 min, followed by heating from 36 to 41 'C for 15 min with continuous agitation. Temperature was maintained at 41°C, and agitation continued, until the whey pH reached 6.40 ± .02; then whey was drained, and curd was piled in the center of the vat. Curd slabs were turned over (cheddared) once every 15 min until the curd reached a milling pH of 5.25. The cheese curd was milled and salted at a total rate of 2% (wtJ wt). Salted curd (at pH near 5.20) was stretched in a pilot-scale Mozzarella mixer (model 640; Stainless Steel Fabricating, Columbus, WI) with circulating brine (about 8% salt concentration) at 57°C. During the transit time of 5 to 10 min, the temperature of the curd increased to 55'C. The cheese was then cooled in ice water for I h, vacuum packaged, and stored at 4°C. Six 1.2-kg cylinders of cheese (30 em long x 7.5 cm i.d.) were made per vat. The third and fourth cylinders (sequence of extrusion) were used for chemical analysis. Journal of Dairy Science Vol. 76. No. 12, 1993
3650
YON ET AL.
Chemical Analy.e.
Milk, Whey, and Cheese. Changes in titratable acidity of milk and whey (22) and pH of milk, whey, and cheese were monitored during cheese making (30). Fat contents of milk (1), cream (1), whey (22), and cheese (22) were determined using Babcock tests. All nitrogen determinations were made by the Kjeldahl method (1, 2). Percentage of nitrogen from the analyses of noncasein nitrogen (17) and total nitrogen (1, 2) were multiplied by 6.38 to give milk protein equivalents. Total nitrogen and noncasein nitrogen measurements for milk were performed in triplicate. All other chemical analyses, except for cheese moisture, were performed in duplicate. Cheese moisture was determined gravimetrically in quadruplicate using a forced-air oven (model OV-490A-2; Blue M, Blue Island, IL) at 100°C for 24 h (22). Salt content in cheese was determined by the Volhard procedure (22) and calcium concentration by complexometric titration (18). Proteolysis. Nitrogen soluble in pH 4.6 acetate buffer and in 12% TCA were determined to measure the proteolysis in cheese (7) after 3, 8, 15, 21, 29, and 50 d of storage at 4°C. All soluble nitrogen values were expressed as a percentage of total nitrogen content of cheese. Then, SDS-PAGE (27) was used to monitor the proteolysis of O:s- and ,s-caseins in cheese during refrigerated storage. The amount of O:s- and IS-caseins was determined by scanning gels with a video densitometer (model 620; The I-D Analyst®, BioRad Laboratories, Rockville
Center, NY). Amounts of residual caseins were calculated as the area for zone 1 (O:sl- and O:s2caseins) or zone 2 (,8-casein) as a percentage of the total area of all major bands for each sample (30). Experimental De.lgn and Statl.tical Analysis
Three vats of cheese were made (each using a different coagulant) from one batch of milk. The cheese making was replicated on 3 different d. On each day, the order of cheese making for the three different coagulants was changed so that the effect of day and order of cheese making were blocks in a 3 x 3 Latin square design. The data for initial chemical composition of the cheese were analyzed using PROC ANOVA of SAS (SAS Institute Inc., Cary, NC). The PROC GLM of SAS was used for evaluation of the proteolytic changes during refrigerated storage based on a split-plot design; the whole-plot factor (i.e., effect of coagulant type) was replicated in a 3 x 3 Latin square design in which the effects of day and order were blocks. The factors, degree of freedom, and statistical model are shown in Table 1. The level of significance was P :5: .05 throughout the paper. RESULTS Composition of Milk, Wh.y, and Ch....
The average fat, protein, and casein contents of milks used in three cheese-making trials were 2.31,3.28, and 2.52%, respectively.
TABLE I. Statistical model used for data analyses. Factors Whole-plot factor Coagulant type Day of cheese making Order of cheese making Error Subplot factor Age Age x age Interaction of coagulant type x age Interaction of coagulant type x (age x age) Error
df
Analyzed as
2
Classification
2 2 2
Block Block
1
Quantitative Quantitative Classification x quantitative Classification x quantitative
1
2 2
39 1
IThe degrees of freedom of the error term for the subplot factor error for electrophoresis results were 21 instead of 39 because only four times of aging were used instead of six. Journal of Dairy Science Vol. 76, No. 12, 1993
3651
COAGULANT TYPE AND MOZZARELLA COMPOSITION
Casein as a percentage of total nitrogen was 76.8%. The average fat content of whey at draw was .20%. No significant effect of coagulant type on cheese pH, moisture, fat, protein, and calcium contents was observed (Table 2). Moisture content of cheeses ranged from 44 to 45%, which is slightly lower than the legal minimum (45%) for low moisture, part-skim Mozzarella cheese in the US (8). Salt and fat (on a dry weight basis) were significantly (P < .05) different, depending on the coagulant used (Table 2). However, these differences were too small to be significant in practice. Proteolysis
Amounts of nitrogen soluble in pH 4.6 acetate buffer and in 12% TCA were significantly affected by the differences in coagulant type (Figure I, A and B, respectively; Table 3). Soluble nitrogen increased with refrigerated storage time for all cheeses. The SOS-PAGE patterns of Mozzarella during 50 d of refrigerated storage are shown in Figure 2. Zones 1 and 2 are the two major bands at the top of the gel. Zone I contains asl- and as2-caseins, zone 2 contains ~-casein, and zone 3 contains paraK-casein. Proteolytic breakdown products of a s- and ~-caseins are shown as bands in the area between zones 2 and 3 and below zone 3.
The amount of asl- and as2-caseins as a percentage of total protein decreased significantly for all cheeses during refrigerated storage (Figure 3A; Table 3). No significant effect of coagulant type on asl - and as2-caseins was observed, but a significant coagulant type x age interaction occurred (Table 3). During the same period, ~-casein in cheeses made with COF and M. miehei protease remained relatively constant (Figure 3B). However, ~-casein in cheeses made with E. parasitica protease decreased significantly (Table 3; Figure 3B). The combined amount of a s- and ~-caseins decreased (Table 3; Figure 3C) with storage for all coagulants, but the decrease was the largest with E. parasitica protease. This finding is consistent with the observation that the Mozzarella cheese made with E. parasitica protease had the largest increase in pH 4.6-soluble nitrogen (Figure 1). DISCUSSION
Cheese Making
pH at Drawing Whey. The range of pH at drawing whey in our cheese making was 6.40 ± .02, which may be higher than usual com-
TABLE 2. Initial chemical composition (n = 3) of Mozzarella cheeses made using three different coagulants.
Coagulant Endothia parasitica
Component
protease
CDpl
pH Moisture, % Pal, % FOB,3 % Protein, %
5.15 44.41 22.08 39.7()1 27.83
5.17 44.53 21.92 39.5Qb 27.97 1.59 1.51 ab 3.39ab .804 2.87
M:P'4
1.60
Salt, % S in M,s
%
1.55a 3.49"
Ca,% Ca (%
P),6 %
2.90
.808
of
type
Mucor miehei
protease
SEM
5.16 44.11 22.04 39.40" 28.16 1.57 l.46b 3.31 b .794 2.82
.010 .056 .025 .013 .124 .008 .008 .018 .010 .040
a.b.cMeans within same row not sharing common superscripts are different lChymosin derived by fermentation. 2p = .05. 3Pat content on a dry weight basis. 4Ratio of moisture to protein. sRatio of salt to moisture in the cheese. tlCalcium as a percentage of the protein content of the cheese.
(P
LSI)2 .06
.34 .15 .08 .75 .05 .05 .11 .06
.25
< .05).
Journal of Dairy Science Vol. 76, No. 12, 1993
3652
YUN ET AL.
-"...,
"• "0 Cl., 0• •
000 Ci V V V
000 0
V'vv
mercial conditions. The curd pH was about 6.35. A previous study (16) showed that retention of chymosin in cheese increases with decreasing pH at drawing whey, but the retention of M. miehei and Mucor pusillus proteases are not dependent on pH. The high drawing pH (i.e., pH 6.40) in our cheese-making process may have resulted in a lower chymosin retention and less proteolysis than if we had used a lower drawing pH. pH and Temperature During Cooking and Stretching. Throughout the cheese-making process, milk coagulants undergo various heat treatments, and different coagulants exhibit different sensitivities to inactivation by heat (15). The cooking temperature (in the vat) of 4l C at pH 6.5 to 6.4 (whey pH) that was used in our study is not expected to inactivate coagulants. The heat stability of acid proteases D
r---
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Journal of Dairy Science Vol. 76, No. 12, 1993
.~
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Figure 1. Impact of coagulant type on pH 4.6-soluble nitrogen (A; SEM = .26%) and on 12% TeA-soluble nitrogen (B; SEM = .05%) of Mozzarella cheese made using Endolhia parasitica protease ($), chymosin derived by fermentation (II), and Mucor mieMi protease (A).
3653
COAGULANT TYPE AND MOZZARELLA COMPOSITION
is greater at lower pH (15). At milling, curd pH was 5.25, which decreased to 5.20 by the time of stretching. The water temperature at stretching that was used in our cheese making was 57°C (curd temperature, 55·C), which was probably low enough to allow coagulants to survive. particularly at a curd pH of 5.20 (10, 15). Salt Concentration in Cheese at Stretching. The thermostability of aqueous rennet solutions increased at pH 5.6 and at 57°C when the concentration of salt increased (25). During the stretching step in the "no-brine" cheesemaking method, the concentration of salt in the curd is about 1.5%. Thus, the salt concentration in the aqueous phase of the curd is about 3%, which may help make the rennet retained in the curd more heat stable. The final extent of proteolysis caused by the coagulant retained in the cheese is determined
Chymosin derived by fermentation
by the sum of these and other factors, including accessibility of substrate, rate of cooling, and storage time and temperature. Impact of CoagUlant Type
Cheese Composition. The fat, protein, and moisture contents of Mozzarella cheese were unaffected by coagulant type (Table 2). A small but significant difference in salt content existed between cheeses made with E. parasitica protease and M. miehei protease. This difference may be due to differences in curd structure and rate of syneresis during salting. A previous study on Cheddar cheese (3) reported differences in the yields of whey, salt whey, and cheese depending on the coagulant type. Proteolysis. The amounts of nitrogen soluble in pH 4.6 acetate buffer (Figure IA) and in 12% TCA (Figure IB), {3-casein (Figure 3B), and combined (Xs- and {3-caseins (Figure 3C)
Endothia parasitica protease
Mucor miehei protease
storage (d) 3
15
29
50
3
15
29
50
3
15
29
50
Zone Zone
Zone
3~
Figure 2. The SDS-PAGE gel of Mozzarella cheeses made using chymosin derived by fennentation. E1ldothia parasitica protease, and Mucor miehei protease that were sampled at 3. IS. 29. and SO d of storage at 4'C: zone 1 = asl' plus a s2-caseins. zone 2 = P-casein. and zone 3 = para·K-casein. Journal of Dairy Science Vol. 76, No. 12. 1993
3654
YUN ET AL.
were significantly affected by the differences in coagulant type (fable 3). The nitrogen soluble in pH 4.6 acetate buffer (Figure lA) seemed to be more sensitive to differences in coagulant type than nitrogen soluble in 12% TCA (Figure lB). This finding is consistent with 50 r - - - - - - - - - - - - - - - - - , A
""
40
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~
20
I <0.
10
10
20 30 Storage Time (d)
40
50
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----~------"
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20 30 Storage Time (dl
40
50
Figure 3. Impact of coagulant type on the relative percentages of O/sl- plus O/s2-caseins (A; SEM 3.8%), flcasein (B; SEM = 1.6%), and the sum of O/sl-' O/s2-, and (3caseins (C; SEM = .8%) as a percentage of all protein in Mozzarella cheese made using Endothia parasitica protease (*), chymosin derived by fermentation (II), and Mucor miehei protease (.A.).
=
Journal of Dairy Science Vol. 76, No. 12, 1993
previous reports (21, 28) that coagulants contribute more to the extent of proteolysis, whereas enzymes from starter culture affect the depth of proteolysis in Cheddar and Gouda cheese. The breakdown of a s}- and aS2-caseins during storage was not directly affected by coagulant type in our statistical model (fable 3). However, the interaction of coagulant type x age was significant (fable 3), indicating that the rate of proteolysis with storage was different depending on coagulant type (Figure 3A). The breakdown of a s}- and aS2-caseins during storage of cheese made with CDF was typical of observations in Mozzarella cheese when calf rennet was used as coagulant (30). Proteolysis of l3-casein occurred only in the cheese made with E. parasitica protease (Figure 3B). The difference in substrate specificity that we observed was similar to results of previous studies. Edwards and Kosikowski (12) indicated that E. parasitica protease caused the most proteolysis of l3-casein in Cheddar cheese. Farkye et al. (14) also reported decreased a s- and l3-caseins with storage time in Mozzarella made with E. parasitica protease. Overall, E. parasitica protease was more proteolytic than CDF and M. miehei protease in Mozzarella cheese, as shown by the largest decrease in combined a s- and l3-caseins during storage (Figure 3C). Impact of Age
Proteolysis. The amount of nitrogen soluble in 12% TCA and in pH 4.6 acetate buffer increased significantly (P < .01) from 3 to 50 d of refrigerated storage (Figure 1, A and B; Table 3). Even though linear and quadratic terms of age were significant (P < .01), the mean squares for the linear term of age was greater than the mean squares of the quadratic term of age (fable 3), indicating that soluble nitrogen contents of cheese predominantly tended to increase linearly with age. Also, interactions of coagulant type with linear and quadratic terms of age were significant (P < .01). However, the mean squares of interactions for coagulant type x linear term of age were much greater than the mean squares of coagulant type x quadratic term of age (fable 3). The decrease in a s}- and as2-caseins in the cheese was highly significant during 50 d of
COAGULANT TYPE AND MOZZARELLA COMPOSITION
refrigerated storage (Figure 3A; Table 3). The pattern of decrease in asl- and as2-caseins was linear, not quadratic (fable 3). The decrease in ,s-casein that was due to coagulant type was highly significant during 50 d of refrigerated storage (Figure 3B; Table 3). The major and only decrease in ,s-casein occurred in the cheese made with E. parasitica protease. The amount of ,s-casein in cheeses made with CDP and M. miehei protease remained constant during 50 d of refrigerated storage. The rate of breakdown for the combined a sand ,s-caseins was higher in cheese made with E. parasitica protease than in cheeses made with CDF and M. miehei protease (Figure 3C). This finding is consistent with results from soluble nitrogen analysis. Both types of soluble nitrogen (pH 4.6 acetate buffer and 12% TCA) were higher in cheese made with E. parasitica protease than in cheeses made with CDF and M. miehei protease (Figure 1, A and B). Practical Implications
The extent, rate, and specificity of proteolysis of caseins in Mozzarella cheese can be significantly influenced by the type of coagulant used for cheese making. Each coagulant can be influenced differently by variation in critical cheese-making factors. For example, under the cheese-making conditions used in this study, E. parasitica protease demonstrated higher proteolysis in cheese during refrigerated storage. However, among the three coagulants, E. parasitica protease is the most heat labile (24). Furthermore, the heat sensitivity of E. parasitica protease increased at the pH range outside 4 ± 1 (19). If our experiment was conducted using a high cooking temperature in the cheese vat ~45°C) combined with a pH of curd at drawing whey of 6.35, the cheese made using E. parasitica protease might have the lowest rate of proteolysis during storage relative to CDF and M. miehei protease. Thus, the various combinations of the coagulant type plus the pH and temperature conditions during cheese making can be used to change the rate of proteolysis and, possibly, cheese functionality during refrigerated storage. CONCLUSIONS
Differences in coagulant type did not significantly affect the initial chemical composition. However, proteolysis during the refriger-
3655
ated storage of Mozzarella cheese was affected by coagulant type. The cheese made with E. parasitica protease contained more soluble nitrogen than other cheeses. The as-casein decreased significantly during storage at 4°C for cheeses made with all coagulants. The ,scasein remained constant in the cheeses made with M. miehei protease and CDF but significantly decreased during 50 d of refrigerated storage for cheese made with E. parasitica protease. The coagulant retained in the cheese influences proteolysis during refrigerated storage. Therefore, factors that may influence residual proteolytic activity (i.e., coagulant type, temperature, pH, and amount of coagulant) influence proteolysis and, possibly, functional characteristics of Mozzarella. Development of a better understanding of this system will enable the cheese maker to improve the consistency of the functional properties of Mozzarella cheese. ACKNOWLEDGMENTS
The authors thank Robert Rasmussen, Maureen Chapman, Patricia Nelson, and George Houghton for their technical assistance. Financial support was provided by Northeast Dairy Foods Research Center and Hatch Project 143-444. REFERENCES
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