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Distribution of Milk Clotting Enzymes Between Curd and Whey and Their Survival During Cheddar Cheese Making1, 4
Distribution of Milk Clotting Enzymes Between Curd and Whey and Their Survival During Cheddar Cheese Making 1 ,4 D. G. H O L M E S ~ , J. W. D U E R S C H z , and C. A . E R N S T R O M Department of Nutrition and Food Sciences
Utah State University Logan, UT 84322 ABSTRACT
A linear diffusion test capable of measuring milk clotting enzyme activity at concentrations of 1 x 10 -4 to 1 x 10 -l rennin units per ml is described. The distribution of rennet activity between curd and whey in freshly coagulated milk was measured at 72% in the whey and 31% in the curd at pH 6.6. At pH 5.2, the whey contained 17% and the curd 86%. Total recovery of activity was 102 -+ 5%. Distribution of milk clotting enzymes from Mucor pusillus var. Lindt and Mucor miebei between curd and whey in freshly coagulated milk was independent of pH with approximately 83% in the whey and 17% in the curd. Porcine pepsin was unstable, precluding accurate assessment of enzyme distribution. During the manufacture of Cheddar cheese approximately 35% of the rennet activity was destroyed up to the time the whey was drained, and 6% remained in the cheese following pressing. Neither of the microbial enzymes lost activity during cheese making. Most of the activity remained in the whey while only 2 to 3% was detected in the cheese after pressing. I NT RODUCTI ON
Usual procedures (3, 8) for measuring the activity of milk clotting enzymes are satisfactory for evaluating concentrated solutions such as commercial rennet extract but inadequate for the low enzyme concentrations in
Received January 19, 1977. *Supported in part by a grant from the Whey
Products Institute, Chicago, IL. Foremost Research Center, Dublin, CA. 3Department of Food Science, North Carolina State University, Raleigh, NC. 4Utah Agricultural Experiment Station Journal Article No. 2155.
cheese curd and whey. For this reason there is a dearth of information about the partition of these enzymes between curd and whey and their survival during cheese making. Some workers (2, 19) have attributed an important cheese curing role to milk clotting enzymes while others (1, 14, 16) have considered their curing function to be secondary or insignificant. However, the amount of milk clotting activity remaining in cheese curd has not been reported. Gorini and Lanzavecchia (11) described a sensitive substrate for measuring the milk clotting activity of bacterial proteases. Wang (22) modified this substrate to increase its sensitivity, and, Reyes (18) used it to approximate the residual rennet activity in fresh crud and whey. He accounted for about 90% of the activity added to milk, but the substrate was sensitive to a number of nonrennin substances including the salts in whey and casein in curd slurries. This made i t extremely difficult to standardize the procedure. A passive indirect hemagglutination test and a corresponding inhibition test for measuring residual rennin in cheese were suggested by Elliot and Emmons (6). Clarke and Richards (5) described a rennin assay that was forty times more sensitive than the usual milk clotting procedure. Their test was based on the rate of release of peptides from sodium caseinate under controlled conditions. The released peptides in a perchloric acid filtrate were measured by absorbance at 217 nm. Cheeseman (4) concluded that radial diffusion of concentrated milk clotting enzymes in caseinate-agar gels provided a less satisfactory method for enzyme assay than conventional milk-clotting procedures. However, Lawrence and Sanderson (13) demonstrated that radial diffusion in thin layers of caseinate-agar gels was suitable for quantitative assays. Their procedure required a uniform gel thickness and an enclosed humidified chamber for incubation during diffusion. 862
CLOTTING ENZYMES IN CHEESE AND WHEY The purpose of this study was to simplify the casein-agar diffusion test for milk clotting enzymes and to use it to determine the relative concentrations of these enzymes in curd and whey at different stages during Cheddar cheese making. METHODS AND PROCEDURES Milk Clotting Enzymes
Rennet extracted from veils taken from milk-fed calves slaughtered at 4 to 10 days of age was obtained from the New Zealand Cooperative Rennet Co., Ltd., Ehham, New Zealand. Porcine pepsin, a commercial rennet-pepsin mixture, a commercial microbial protease (Emporaset m) derived from Mucor pusillus var. Lindt (MP protease), and a rennet standard containing 100 rennin units (RU) per milliliter (8) were provided by Dairyland Food Laboratories, Inc., Waukesha, Wisconsin. A commercial microbial protease (Marzymetm)derived from Mucor miebei (MM protease) was obtained from Marschall Division, Miles Laboratories, Inc., Madison, Wisconsin. Casein
Whole casein was prepared from raw skim milk by the method of Van Slyke and Baker (21). Kappa casein was prepared according to Zittle and Custer (23) except that removal of lipids by ethanol extraction was unnecessary. Diffusion Substrate
A casein-agar diffusion substrate described by Lawrence and Sanderson (13) was modified to improve its stability and give a sharper diffusion boundary. Gels containing .54% whole casein, 3.6% sodium acetate, .01% calcium chloride, .7% agar, and adjusted to pH 5.7 with .IN hydrochloric acid sometimes became cloudy during storage because of calcium aggregated casein. This obscured the diffusion boundary and made some gets unusable. This substrate was modified by replacing the whole casein with kappa casein, eliminating the calcium chloride, and adjusting the pH to 5.9. Kappa casein (.6 g) and sodium acetate trihydrate (.6 g) were weighed into a 100 ml beaker. Seventeen milliliters of distilled water were added, and the mixture was stirred with a magnetic stirrer until the kappa casein dis-
863
solved. The pH was adjusted to 5.9 with .1 N hydrochloric acid and the solution brought to 20 g with distilled water. An agar solution was prepared by mixing 6 g sodium acetate trihydrate and .8 g agar (Bacto O140, D I FCO Laboratories) with 80 ml distilled water in a 125 ml Erlenmeyer flask. The pH was adjusted to 5.9 with N hydrochloric acid and brought to 90 g with distilled water. The solution was then autoclaved for 10 rain at 10546 kg/m 2. The agar and kappa casein solutions separately were brought to 75 C in a water bath and mixed. Becton-Dickinson sedimentation and hematocrit tubes No. 6901 (3 mm 1D) were filled ¾ full with the hot kappa casein-agar solution by means of a 10 ml syringe and needle fitted with polyethylene tubing. The tubes were sealed with paraffin wax and stored at 2 C until used.
Enzyme Activity
Milk clotting activities of concentrated enzyme solutions were measured as described (8) in Berridge (3) substrate on a Sommer-Matsen (20) apparatus. Low enzyme concentrations with known activity were prepared from concentrated solutions by quantitative dilution with distilled water. Diffusion tubes were removed from the refrigerator and allowed to come to room temperature. The wax seals were removed and 5 /ll of diluted enzyme solution, whey, or curd extract introduced on top of the gel with a 10 /ll Hamilton syringe. The tubes were resealed with wax and incubated in an upright position for 48 h at 37 C. Enzyme solutions of known concentration were assayed simultaneously with the unknowns. The leading diffusion boundary was marked by a white band of precipitated kappa casein (Fig. 3). The distance from the origin to the leading edge of the diffusion boundary was measured with a transmission densitometer (EC 910, EC Apparatus Corp., St. Petersburg, Florida) and recorded by a millivoh recorder (Model EU20B, Heath Co., Benton Harbor, Michigan). A special plastic tray was built to hold the tubes parallel to the sensor head. Diffusion distances traced by the recorder (Fig. 1) were 1.8 times those in the tubes. All calculations were based on recorder chart distances. Standard curves were prepared by plotting Journal o f Dairy Science Vol. 60, No. 6
864
HOLMES ET AL.
I~--ORIGIN
--ORIGIN
DIFFUSION
I X l O 3 RU/ml
IX 1()2RU/ml
FIG. 1. Densitometer-recorder tracings of diffusion tubes after 48 h diffusion of r e n n e t at 37 C.
the logarithms of known concentrations of each enzyme against diffusion distance. Preparation of Curd Extract
Thirty grams of curd and 450 ml distilled water were blended at slow speed in a Waring blender for 1.5 min. The pH of the curd slurry was adjusted to 6.8 with N sodium hydroxide and allowed to stand at 25 C for 30 min. The slurry was then filtered through student grade coarse filter paper and 5 /al of the filtrate was used for enzyme analysis. In calculating the enzyme concentration in the curd it was assumed that the enzyme was distributed uniformly in the aqueous phase of the curd.
protease are in Fig. 2. Each point on the curve represents the average of 10 replications. Enzyme concentrations are expressed in rennin units per milliliter (RU/ml) based on standardization against rennet (8) in Berridge substrate at pH 6.3. Concentrations as low as 1 × 10 -4 RU/ml were measurable but deviated slightly from linearity. Lower concentrations deviated substantially from linearity, were sometimes undetected, and were considered beyond the range of the test. Concentrations in excess of 1 × 10 -1 RU / m l were measured more easily by the usual milk clotting test (8). Reproducibility of the diffusion test for three different milk clotting enzymes at four concentrations is illustrated in Table 1. Each value represents the standard deviation from the mean for 10 replications. Mean diffusion distances were taken as representing the known concentrations. Deviations from the mean are expressed in concentration units (RU/ml) and indicate variations of 4 to 7% from mean values.
i x 161
E Z
I x t6 z
o
Cheddar Cheese Making
Cheddar cheese was made from 7.6 kg pasteurized milk in 20 cm cubical plastic vats. The procedure outlined by Price and Calbert (17) was used except that 2% starter was employed and ripening of the milk was eliminated. The milk was set with 90 ml of commercial enzyme solution per 454 kg of milk. The curd was cooked at 39 C, and reached pH 5.4 at milling. The curd and whey were recovered quantitatively and measured accurately. Pressing was accomplished in small 10 × 10 × 10 cm plastic boxes with steel weights on top of the curd.
I--
,¢t n.. i.z bJ ¢J Z 0 ro tlJ X >,-
iXl6 3 RENNET ~
N Z I.IJ
,,
MP P R O T E A S E - - *
i x 16 4 I0
20
30
40
50
DIFFUSION DISTANCE (mrM R ESU LTS
Typical standard curves for rennet and MP Journal of Dairy Science Vol. 60, No. 6
FIG. 2. Standard curve for r e n n e t based on 48 h diffusion at 37 C.
and MP protease
CLOTTING ENZYMES 1N CHEESE AND WHEY
865
TABLE 1. Standard deviations 1 of enzyme measurements at four concentrations of rennet, porcine pepsin, and an enzyme from Mucor pusillus var. Lindt. Concentration (RU/ml) Enzyme
1.O0 X 10-4
1.00 X 10- a
1.00 X 10-=
1.00 X 10-1
(Standard deviation) Rennet Porcine pepsin MP protease
.06 × 10-4 .06 X 10-4 .07 X 10-4
.06 × 10- ~ .05 × 10-a .06 X 10-3
.04 X 10-= .04 X I0 -= .04 X 10-~
.05 X 10-1 .05 X 10-1 .05 X 10-1
~N=IO.
Effect of Whey and Salt on Diffusion
S u b s t a n c e s in w h e y a n d curd o t h e r t h a n m i l k c l o t t i n g e n z y m e s can i n t e r f e r e with milk c l o t t i n g tests (18). T h e r e f o r e , t h e e f f e c t o f s o d i u m c h l o r i d e a n d w h e y o n t h e d i f f u s i o n test was d e t e r m i n e d b y d i l u t i n g s t a n d a r d r e n n e t e x t r a c t to a c o n c e n t r a t i o n o f 1 x 1 0 -2 R U / m l w i t h distilled water, h e a t e d (70 C for 30 m i n ) w h e y , a n d .5 a n d 3.0% s o d i u m c h l o r i d e solutions. Statistical analysis o f m e a s u r e d c o n c e n t r a t i o n s revealed n o significant e f f e c t o f t h e s e substances on the measurement of enzyme c o n c e n t r a t i o n b y t h e d i f f u s i o n test. Diffusion t u b e s in t h i s e x p e r i m e n t are s h o w n in Fig. 3.
o f curd were d e t e r m i n e d b y d i f f e r e n c e . E n z y m e c o n c e n t r a t i o n in b o t h t h e curd a n d w h e y was m e a s u r e d b y t h e diffusion test.
Enzyme Distribution Between Curd and Whey
T h e d i f f u s i o n test was used t o d e t e r m i n e how completely milk clotting enzymes added t o m i l k c o u l d b e a c c o u n t e d f o r in t h e resulting c u r d a n d whey, a n d h o w t h e p H o f t h e milk a f f e c t e d t h e d i s t r i b u t i o n of t h e e n z y m e s bet w e e n t h e curd a n d w h e y . T h e s e s t u d i e s were carried o u t in freshly c o a g u l a t e d m i l k in w h i c h h e a t i n g was a v o i d e d t o p r e v e n t i n a c t i v a t i o n of t h e e n z y m e s as m i g h t o c c u r d u r i n g t h e c o o k i n g o f C h e d d a r cheese. P a s t e u r i z e d w h o l e milk was b r o u g h t t o 22 C a n d t h e pH a d j u s t e d t o t h e desired level with N HCI. It t h e n was weighed a c c u r a t e l y ( 4 5 4 g), w a r m e d to 30 C in a w a t e r bath and a measured amount (equivalent to 90 m l / 4 5 4 kg o f m i l k ) o f c o m m e r c i a l m i l k c l o t t i n g e n z y m e a d d e d . Each s a m p l e was t r a n s f e r r e d i m m e d i a t e l y t o t w o 225 ml plastic c e n t r i f u g e b o t t l e s a n d i n c u b a t e d at 30 C f o r 15 m i n past t h e first sign o f c o a g u l a t i o n . T h e c u r d was b r o k e n b y stirring w i t h a spatula, a n d t h e c u r d a n d w h e y were s e p a r a t e d b y c e n t r i f u g i n g at 3 5 0 0 x g f o r 2 0 m i n . T h e w h e y was d e c a n t e d , m e a s u r e d , a n d weighed. T h e w e i g h t a n d v o l u m e
FIG. 3. Diffusion tubes filled wkh kappa caseinagar gel showing the diffusion of rennet (i × 10 .2 RU/ml) that was diluted with (left to right) distilled water, heat treated whey, .5% NaCI and 3.0% NaCI. Journal of Dairy Science Vol. 60, No. 6
866
HOLMES ET AL.
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~ 8O w
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7Y7 0
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/// 52
60
6.6
pH
64
66
FIG. 4. Effect of pH on the distribution of rennet between curd and whey in freshly coagulated milk.
FIG. 6. Effect of pH on the distribution of MP protease between curd and whey in freshly coagulated milk.
Figures 4, 5, 6, and 7 show the effect of pH on the distribution of rennet, porcine pepsin, MP protease, and MM protease between curd and whey. Vertical marks at the top of the bar graphs represent standard deviations from the mean for 10 replications. At pH 6.6, 31% of the rennet activity was in the curd and 72% in the whey. As the pH of the milk was reduced, more of the rennet remained with the curd until at pH 5.2 86% was in the curd and 17% in the whey. Total rennet accounted for in the curd and whey averaged 102 + 5% of that added to the milk. The pH of the milk also affected the relative amount of curd and whey recovered. At pH 5.2 the curd
averaged 77 g and was quite firm while the curd recovered at pH 6.6 averaged 122 g and was soft. It was impossible to account for all the added pepsin activity in the curd and whey. Pepsin distribution appeared to be pH dependent and follow the same pattern as rennet. However, this enzyme was so unstable that much of it was inactivated during the experiment. To release pepsin f r o m the curd it was necessary to adjust the pH of curd slurries to 6.8. Above pH 6.5, porcine pepsin is extremely unstable and any pepsin in the curd may have been partly inactivated during the assay. Distribution of the two microbial milk
~8
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CURD~
CURD~
.U
II II I/
~! >-40 ~--2O 0
~I-~ 5.2
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6.4
PEPSIN
6.6
pH FIG. 5. Effect of pH on the distribution of porcine pepsin between curd and whey in freshly coagulated milk. Journal of Dairy Science Vol. 60, No. 6
'°H,Ir,ll I! 5.2
6.0
pH
6.4
~M PROTEASE
6.6
FIG. 7. Effect of pH on the distribution of MM protease between curd and whey in freshly coagulated milk.
CLOTFING ENZYMES IN CHEESE AND WHEY
clotting enzymes between curd and whey were similar to each other (Fig. 6 and 7) and independent of pH. Slightly higher activity in the curd at the higher pH can be accounted for by differences in the amount of curd if the increased amount was due to whey carrying the same concentration of enzyme as in the separated whey. Approximately 83% of the enzyme activity from mucor sources was recovered in the whey and 17% in the curd. Total recovery of the enzymes was 99 + 2% for MP protease and 100 -+ 4% for MM protease.
]C)O
oo].
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~ 80 er W ~ 60"
WHEYE:] t~
CURD F'~I
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Distribution and Survival of Milk Clotting Enzyme Activity During Cheddar Cheese Making
Enzyme concentrations in curd and whey were measured at cutting and dipping and in curd after overnight pressing during the manufacture of Cheddar cheese with rennet, porcine, pepsin, MP protease, a commercial mixture of pepsin and rennet (five replications each), and MM protease (four replications). Because of difficulty in measuring weights of curd and whey at cutting without disrupting the cheese making operation, the distribution of the enzyme between curd and whey prepared from freshly coagulated milk adjusted to the pH of the cheese milk at cutting was assumed to be representative of the distribution at cutting. Several assays of curd and whey taken from cheese at cutting indicated that this assumption was valid. Figures 8, 9, 10, 11, and 12 illustrate the
867
O
CUT
r-Q DIP
FIG. 9. Distribution of porcine pepsin between curd and whey and its survival during Cheddar cheese making.
results of these experiments. At dipping 35% of the rennet activity had been lost. The curd contained 7% and the whey 58% of the original activity. After overnight pressing the curd contained only 6% of the original rennet activity (Fig. 8). At dipping only 9% of the original pepsin activity was left in the whey, and none was in the curd. However, if some pepsin had been active in the curd, it most likely was destroyed when the curd slurry was adjusted to pH 6.8 to release the enzyme from the curd. Figure 10 shows the distribution of the
I
8e RENNET
COM. MIX
>
O 60 WHE Y
WHEYI
~40,. ,
I
CURDF-/']
CURD ~ ]
~40>I--
F- 2O
~_200 0
PRESS
CUT
DIP
PRESS
FIG. 8. Distribution of rennet between curd and w h e y and its survival during Cheddar cheese making.
/// /// CUT
DIP
r747 PRESS
FIG. 10. Distribution of e n z y m e activity from a commercial mixture of r e n n e t and porcine pepsin between curd and whey and its survival during Cheddar cheese making. Journal of Dairy Science Vol. 60, No. 6
868
HOLMES ET AL.
._p.-
:801 uJ
~
4% of the MM protease. After overnight pressing the curd contained 3 and 1.8% of the two enzymes. There was no inactivation of either of these enzymes during cheese making.
MP
I
WHEY[
60-
I
CURDIT7}
LLI rY
>- 4 O I-
C--20 l / 1
0
I I I
7-77,,
CUT
DIP
. . I.
-
PRESS
FIG. 11. Distribution of MP protease between curd and whey and its survival during Cheddar cheese making.
activity of a commercial rennet-pepsin mixture between curd and whey during the manufacture of five replicate batches of Cheddar cheese. At dipping, only 22 ± 4% of the original activity could be accounted for, 5 -+ 3% in the curd and 17 +- 4% in the whey. After overnight pressing, 4 ± 2% of the activity was left in the curd. Because of the poor stability of porcine pepsin, it was assumed that all the residual activity in the curd was due to rennet. The stability of the microbial proteases during cheese making is illustrated in Fig. 11 and 12. At dipping all enzyme activity was accounted for in curd and whey. At that point the curd contained 6% of the MP protease and
100
I
'"
80
WHEYI
rY ILl
I
C U RDIT7--~
t.)
uJ
MM
protease
r~4o )>2o "~
0
I/ill
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DIP
,..i..,
PRESS
FIG. 12. Distribution of MM protease between curd and whey and its survival during Cheddar cheese making. Journal of Dairy Science Vol. 60, No. 6
DISCUSSION
A linear diffusion test for the quantitative determination of milk clotting enzymes at concentrations of 1 x 10 -4 to 1 × 10 -1 RU/ml proved useful for measurement of residual clotting enzymes in cheese curd and whey. A kappa casein-agar substrate at pH 5.9 gave excellent results when used in small diffusion tubes. The diffusion boundary was always sharp, and its distance from the origin was measured easily with a transmission densitometer. Measurements were not affected by NaCI at concentrations up to 3% in the enzyme solutions, nor were they affected when the enzymes were diluted with whey. All the enzyme activity (except porcine pepsin) added to milk was accounted for in the curd and whey. The distribution of rennet between curd and whey was pH dependent while that of the mucor coagulants was not. It was necessary to raise the pH of acid curd slurries to 6.8 to release the rennet enzymes from the curd so they could be measured. This was not necessary with the mucor enzymes. Porcine pepsin was so unstable in milk that accurate recoveries were impossible even though the results suggested that, like rennet, its affinity for curd increased with decreasing pH. The small percentage of enzyme activity remaining in finished Cheddar cheese, particularly from microbial enzymes, suggests that the role of these enzymes in cheese curing is minor compared to that of microorganisms. This idea is supported further by the fact that reasonably good cheese can be made with porcine pepsin (15) even though it is doubtful that any of this enzyme survives the cheese making process (12). The pH dependence of the affinity of rennet for curd suggests that the more acid the milk at setting, the more rennet activity should remain in the cheese. In these studies little acid had developed by the time the curd was cut and in most instances the pH of the curd was between 6.2 and 6.1 at dipping. Therefore, most of the rennet would have escaped into the whey
CLOTTING ENZYMES IN CHEESE AND WHEY
before the curd was sufficiently acid to " b i n d " much of the enzyme. Few of the hundreds of proteases that have been tried as rennet substitutes have been successful. Significant cheese defects have been associated with the use of too much of the wrong kind of protease for clotting cheese milk (10). It would be of interest to know whether some of these problems might be associated with the amount o f enzyme remaining in the curd. REFERENCES
1 Allen, L. A., and N. R. Knowles. 1934. Studies in the ripening of Cheddar cheese. J. Dairy Res. 5:185. 2 Babcock, S. M., and H. L. Russell. 1902. Curing of Cheddar cheese with especial reference to cold curing. Wisc. Agr. Exp. Sta. Bull. 94. 3 Berridge, N. J. 1952. Some observations on the determination of the activity of rennet. Analyst 77:57. 4 Cheeseman, G. C. 1963. Action of rennet and other proteolytic enzymes on casein in casein-agar gels. J. Dairy Res. 30:17. 5 Clarke, N. H., and E. L. Richards. 1973. An assay for rennin. N. Z. J. Dairy Sci. and Tech. 8:152. 6 Elliot, J. A., and D. B. Emmons. 1971. Rennin detection in cheese with the passive indirect hemagglutination test. Can. Inst. Food Tech. J. 4:16. 7 Emmons, D. B. 1970. Inactivation of pepsin in hard water. J. Dairy Sci. 53:1177. 8 Ernstrom, C. A. 1958. Heterogeneity of crystalline rennin. J. Dairy Sci. 41:1663. 9 Emstrom, C. A. 1961. Milk clotting activity of pepsin and rennin. Milk Prod. J. 52(5):8. 10 Ernstrom, C. A., and N. P. Wong. 1974. Milk clotting enzymes and cheese chemistry. Pages 662 to 771 in B. H. Webb, A. H. Johnson, and J. A. Alford, eds. Fundamentals of Dairy Chemistry,
869
2nd ed. AVI, Westport, CT. 11 Gorini, L., and G. Lanzavecchia. 1954. Recherches sur le mechanisme de production d'une proteinase bacterierme. I. Nouvelle technique de determination d'une proteinase par la coagulation du lair. Biochim. Biophys. Acta 14:407. 12 Green, M. L. 1972. Assessment of swine, bovine and chicken pepsins as rennet substitutes for Cheddar cheese making. J. Dairy Res. 39:261. 13 Lawrence, R. C., and W. B. Sanderson. 1969. A micro-method for the quantitative estimation of rennets and other proteolytic enzymes. J. Dairy Res. 36:21. 14 Marth, E. H. 1963. Microbiological and chemical aspects of Cheddar cheese ripening. A review. J. Dairy Sci. 46: 869. 15 Melachouris, N. P., and S. L. Turkey. 1964. Comparison of the proteolysis produced by rennet extract and the pepsin preparation Metroclot during ripening of Cheddar cheese. J. Dairy Sci. 47:1. 16 Peterson, M. H., M. J. Johnson, and W. V. Price. 1948. Determination of cheese proteinase. J. Dairy Sci. 31:47. 17 Price, W. V., and H. E. Calbert. 1944 (revised 1953). Cheddar cheese from pasteurized milk. Wisc. Agr. Exp. Sta. Bull. 464. 18 Reyes, J. 1971. A procedure for measuring residual rennin activity in whey and curd from freshly coagulated milk. M.S. thesis, Utah State University, Logan. 19 Sherwood, 1. R. 1935. The function of pepsin and rennet in the ripening of Cheddar cheese. J. Dairy Res. 6:407. 20 Sommer, H. H., and H. Matsen. 1935. The relation of mastitis to rennet coagulability and curd strength of milk. J. Dairy Sci. 18:741. 21 Van Slyke, L. L., and J. C. Baker. 1918. The preparation of pure casein. J. Biol. Chem. 35:127. 22 Wang, J. T. 1969. Survival and distribution of rennin during Cheddar cheese manufacture. M.S. thesis, Utah State University, Logan. 23 Zittle, C. A., and J. H. Custer. 1963. Purification and some of the properties of a s casein and g-casein. J. Dairy Sci. 46:1183.
Journal of Dairy Science Vol. 60, No. 6
Utah State University Logan, UT 84322 ABSTRACT
A linear diffusion test capable of measuring milk clotting enzyme activity at concentrations of 1 x 10 -4 to 1 x 10 -l rennin units per ml is described. The distribution of rennet activity between curd and whey in freshly coagulated milk was measured at 72% in the whey and 31% in the curd at pH 6.6. At pH 5.2, the whey contained 17% and the curd 86%. Total recovery of activity was 102 -+ 5%. Distribution of milk clotting enzymes from Mucor pusillus var. Lindt and Mucor miebei between curd and whey in freshly coagulated milk was independent of pH with approximately 83% in the whey and 17% in the curd. Porcine pepsin was unstable, precluding accurate assessment of enzyme distribution. During the manufacture of Cheddar cheese approximately 35% of the rennet activity was destroyed up to the time the whey was drained, and 6% remained in the cheese following pressing. Neither of the microbial enzymes lost activity during cheese making. Most of the activity remained in the whey while only 2 to 3% was detected in the cheese after pressing. I NT RODUCTI ON
Usual procedures (3, 8) for measuring the activity of milk clotting enzymes are satisfactory for evaluating concentrated solutions such as commercial rennet extract but inadequate for the low enzyme concentrations in
Received January 19, 1977. *Supported in part by a grant from the Whey
Products Institute, Chicago, IL. Foremost Research Center, Dublin, CA. 3Department of Food Science, North Carolina State University, Raleigh, NC. 4Utah Agricultural Experiment Station Journal Article No. 2155.
cheese curd and whey. For this reason there is a dearth of information about the partition of these enzymes between curd and whey and their survival during cheese making. Some workers (2, 19) have attributed an important cheese curing role to milk clotting enzymes while others (1, 14, 16) have considered their curing function to be secondary or insignificant. However, the amount of milk clotting activity remaining in cheese curd has not been reported. Gorini and Lanzavecchia (11) described a sensitive substrate for measuring the milk clotting activity of bacterial proteases. Wang (22) modified this substrate to increase its sensitivity, and, Reyes (18) used it to approximate the residual rennet activity in fresh crud and whey. He accounted for about 90% of the activity added to milk, but the substrate was sensitive to a number of nonrennin substances including the salts in whey and casein in curd slurries. This made i t extremely difficult to standardize the procedure. A passive indirect hemagglutination test and a corresponding inhibition test for measuring residual rennin in cheese were suggested by Elliot and Emmons (6). Clarke and Richards (5) described a rennin assay that was forty times more sensitive than the usual milk clotting procedure. Their test was based on the rate of release of peptides from sodium caseinate under controlled conditions. The released peptides in a perchloric acid filtrate were measured by absorbance at 217 nm. Cheeseman (4) concluded that radial diffusion of concentrated milk clotting enzymes in caseinate-agar gels provided a less satisfactory method for enzyme assay than conventional milk-clotting procedures. However, Lawrence and Sanderson (13) demonstrated that radial diffusion in thin layers of caseinate-agar gels was suitable for quantitative assays. Their procedure required a uniform gel thickness and an enclosed humidified chamber for incubation during diffusion. 862
CLOTTING ENZYMES IN CHEESE AND WHEY The purpose of this study was to simplify the casein-agar diffusion test for milk clotting enzymes and to use it to determine the relative concentrations of these enzymes in curd and whey at different stages during Cheddar cheese making. METHODS AND PROCEDURES Milk Clotting Enzymes
Rennet extracted from veils taken from milk-fed calves slaughtered at 4 to 10 days of age was obtained from the New Zealand Cooperative Rennet Co., Ltd., Ehham, New Zealand. Porcine pepsin, a commercial rennet-pepsin mixture, a commercial microbial protease (Emporaset m) derived from Mucor pusillus var. Lindt (MP protease), and a rennet standard containing 100 rennin units (RU) per milliliter (8) were provided by Dairyland Food Laboratories, Inc., Waukesha, Wisconsin. A commercial microbial protease (Marzymetm)derived from Mucor miebei (MM protease) was obtained from Marschall Division, Miles Laboratories, Inc., Madison, Wisconsin. Casein
Whole casein was prepared from raw skim milk by the method of Van Slyke and Baker (21). Kappa casein was prepared according to Zittle and Custer (23) except that removal of lipids by ethanol extraction was unnecessary. Diffusion Substrate
A casein-agar diffusion substrate described by Lawrence and Sanderson (13) was modified to improve its stability and give a sharper diffusion boundary. Gels containing .54% whole casein, 3.6% sodium acetate, .01% calcium chloride, .7% agar, and adjusted to pH 5.7 with .IN hydrochloric acid sometimes became cloudy during storage because of calcium aggregated casein. This obscured the diffusion boundary and made some gets unusable. This substrate was modified by replacing the whole casein with kappa casein, eliminating the calcium chloride, and adjusting the pH to 5.9. Kappa casein (.6 g) and sodium acetate trihydrate (.6 g) were weighed into a 100 ml beaker. Seventeen milliliters of distilled water were added, and the mixture was stirred with a magnetic stirrer until the kappa casein dis-
863
solved. The pH was adjusted to 5.9 with .1 N hydrochloric acid and the solution brought to 20 g with distilled water. An agar solution was prepared by mixing 6 g sodium acetate trihydrate and .8 g agar (Bacto O140, D I FCO Laboratories) with 80 ml distilled water in a 125 ml Erlenmeyer flask. The pH was adjusted to 5.9 with N hydrochloric acid and brought to 90 g with distilled water. The solution was then autoclaved for 10 rain at 10546 kg/m 2. The agar and kappa casein solutions separately were brought to 75 C in a water bath and mixed. Becton-Dickinson sedimentation and hematocrit tubes No. 6901 (3 mm 1D) were filled ¾ full with the hot kappa casein-agar solution by means of a 10 ml syringe and needle fitted with polyethylene tubing. The tubes were sealed with paraffin wax and stored at 2 C until used.
Enzyme Activity
Milk clotting activities of concentrated enzyme solutions were measured as described (8) in Berridge (3) substrate on a Sommer-Matsen (20) apparatus. Low enzyme concentrations with known activity were prepared from concentrated solutions by quantitative dilution with distilled water. Diffusion tubes were removed from the refrigerator and allowed to come to room temperature. The wax seals were removed and 5 /ll of diluted enzyme solution, whey, or curd extract introduced on top of the gel with a 10 /ll Hamilton syringe. The tubes were resealed with wax and incubated in an upright position for 48 h at 37 C. Enzyme solutions of known concentration were assayed simultaneously with the unknowns. The leading diffusion boundary was marked by a white band of precipitated kappa casein (Fig. 3). The distance from the origin to the leading edge of the diffusion boundary was measured with a transmission densitometer (EC 910, EC Apparatus Corp., St. Petersburg, Florida) and recorded by a millivoh recorder (Model EU20B, Heath Co., Benton Harbor, Michigan). A special plastic tray was built to hold the tubes parallel to the sensor head. Diffusion distances traced by the recorder (Fig. 1) were 1.8 times those in the tubes. All calculations were based on recorder chart distances. Standard curves were prepared by plotting Journal o f Dairy Science Vol. 60, No. 6
864
HOLMES ET AL.
I~--ORIGIN
--ORIGIN
DIFFUSION
I X l O 3 RU/ml
IX 1()2RU/ml
FIG. 1. Densitometer-recorder tracings of diffusion tubes after 48 h diffusion of r e n n e t at 37 C.
the logarithms of known concentrations of each enzyme against diffusion distance. Preparation of Curd Extract
Thirty grams of curd and 450 ml distilled water were blended at slow speed in a Waring blender for 1.5 min. The pH of the curd slurry was adjusted to 6.8 with N sodium hydroxide and allowed to stand at 25 C for 30 min. The slurry was then filtered through student grade coarse filter paper and 5 /al of the filtrate was used for enzyme analysis. In calculating the enzyme concentration in the curd it was assumed that the enzyme was distributed uniformly in the aqueous phase of the curd.
protease are in Fig. 2. Each point on the curve represents the average of 10 replications. Enzyme concentrations are expressed in rennin units per milliliter (RU/ml) based on standardization against rennet (8) in Berridge substrate at pH 6.3. Concentrations as low as 1 × 10 -4 RU/ml were measurable but deviated slightly from linearity. Lower concentrations deviated substantially from linearity, were sometimes undetected, and were considered beyond the range of the test. Concentrations in excess of 1 × 10 -1 RU / m l were measured more easily by the usual milk clotting test (8). Reproducibility of the diffusion test for three different milk clotting enzymes at four concentrations is illustrated in Table 1. Each value represents the standard deviation from the mean for 10 replications. Mean diffusion distances were taken as representing the known concentrations. Deviations from the mean are expressed in concentration units (RU/ml) and indicate variations of 4 to 7% from mean values.
i x 161
E Z
I x t6 z
o
Cheddar Cheese Making
Cheddar cheese was made from 7.6 kg pasteurized milk in 20 cm cubical plastic vats. The procedure outlined by Price and Calbert (17) was used except that 2% starter was employed and ripening of the milk was eliminated. The milk was set with 90 ml of commercial enzyme solution per 454 kg of milk. The curd was cooked at 39 C, and reached pH 5.4 at milling. The curd and whey were recovered quantitatively and measured accurately. Pressing was accomplished in small 10 × 10 × 10 cm plastic boxes with steel weights on top of the curd.
I--
,¢t n.. i.z bJ ¢J Z 0 ro tlJ X >,-
iXl6 3 RENNET ~
N Z I.IJ
,,
MP P R O T E A S E - - *
i x 16 4 I0
20
30
40
50
DIFFUSION DISTANCE (mrM R ESU LTS
Typical standard curves for rennet and MP Journal of Dairy Science Vol. 60, No. 6
FIG. 2. Standard curve for r e n n e t based on 48 h diffusion at 37 C.
and MP protease
CLOTTING ENZYMES 1N CHEESE AND WHEY
865
TABLE 1. Standard deviations 1 of enzyme measurements at four concentrations of rennet, porcine pepsin, and an enzyme from Mucor pusillus var. Lindt. Concentration (RU/ml) Enzyme
1.O0 X 10-4
1.00 X 10- a
1.00 X 10-=
1.00 X 10-1
(Standard deviation) Rennet Porcine pepsin MP protease
.06 × 10-4 .06 X 10-4 .07 X 10-4
.06 × 10- ~ .05 × 10-a .06 X 10-3
.04 X 10-= .04 X I0 -= .04 X 10-~
.05 X 10-1 .05 X 10-1 .05 X 10-1
~N=IO.
Effect of Whey and Salt on Diffusion
S u b s t a n c e s in w h e y a n d curd o t h e r t h a n m i l k c l o t t i n g e n z y m e s can i n t e r f e r e with milk c l o t t i n g tests (18). T h e r e f o r e , t h e e f f e c t o f s o d i u m c h l o r i d e a n d w h e y o n t h e d i f f u s i o n test was d e t e r m i n e d b y d i l u t i n g s t a n d a r d r e n n e t e x t r a c t to a c o n c e n t r a t i o n o f 1 x 1 0 -2 R U / m l w i t h distilled water, h e a t e d (70 C for 30 m i n ) w h e y , a n d .5 a n d 3.0% s o d i u m c h l o r i d e solutions. Statistical analysis o f m e a s u r e d c o n c e n t r a t i o n s revealed n o significant e f f e c t o f t h e s e substances on the measurement of enzyme c o n c e n t r a t i o n b y t h e d i f f u s i o n test. Diffusion t u b e s in t h i s e x p e r i m e n t are s h o w n in Fig. 3.
o f curd were d e t e r m i n e d b y d i f f e r e n c e . E n z y m e c o n c e n t r a t i o n in b o t h t h e curd a n d w h e y was m e a s u r e d b y t h e diffusion test.
Enzyme Distribution Between Curd and Whey
T h e d i f f u s i o n test was used t o d e t e r m i n e how completely milk clotting enzymes added t o m i l k c o u l d b e a c c o u n t e d f o r in t h e resulting c u r d a n d whey, a n d h o w t h e p H o f t h e milk a f f e c t e d t h e d i s t r i b u t i o n of t h e e n z y m e s bet w e e n t h e curd a n d w h e y . T h e s e s t u d i e s were carried o u t in freshly c o a g u l a t e d m i l k in w h i c h h e a t i n g was a v o i d e d t o p r e v e n t i n a c t i v a t i o n of t h e e n z y m e s as m i g h t o c c u r d u r i n g t h e c o o k i n g o f C h e d d a r cheese. P a s t e u r i z e d w h o l e milk was b r o u g h t t o 22 C a n d t h e pH a d j u s t e d t o t h e desired level with N HCI. It t h e n was weighed a c c u r a t e l y ( 4 5 4 g), w a r m e d to 30 C in a w a t e r bath and a measured amount (equivalent to 90 m l / 4 5 4 kg o f m i l k ) o f c o m m e r c i a l m i l k c l o t t i n g e n z y m e a d d e d . Each s a m p l e was t r a n s f e r r e d i m m e d i a t e l y t o t w o 225 ml plastic c e n t r i f u g e b o t t l e s a n d i n c u b a t e d at 30 C f o r 15 m i n past t h e first sign o f c o a g u l a t i o n . T h e c u r d was b r o k e n b y stirring w i t h a spatula, a n d t h e c u r d a n d w h e y were s e p a r a t e d b y c e n t r i f u g i n g at 3 5 0 0 x g f o r 2 0 m i n . T h e w h e y was d e c a n t e d , m e a s u r e d , a n d weighed. T h e w e i g h t a n d v o l u m e
FIG. 3. Diffusion tubes filled wkh kappa caseinagar gel showing the diffusion of rennet (i × 10 .2 RU/ml) that was diluted with (left to right) distilled water, heat treated whey, .5% NaCI and 3.0% NaCI. Journal of Dairy Science Vol. 60, No. 6
866
HOLMES ET AL.
--'-'-+-
~OOT I
t WHEYF-1 CURDEZ~
I °°
WHEY!
> 6O
CURD~
>120 .1:=
~- 20
vA 6.0
pH
6.4
I
9 lxJ
a: >- 40 F-
5.2
_.+--
~ 8O w
4O
RENNET
I
7Y7 0
rAP pr'~ease
/// 52
60
6.6
pH
64
66
FIG. 4. Effect of pH on the distribution of rennet between curd and whey in freshly coagulated milk.
FIG. 6. Effect of pH on the distribution of MP protease between curd and whey in freshly coagulated milk.
Figures 4, 5, 6, and 7 show the effect of pH on the distribution of rennet, porcine pepsin, MP protease, and MM protease between curd and whey. Vertical marks at the top of the bar graphs represent standard deviations from the mean for 10 replications. At pH 6.6, 31% of the rennet activity was in the curd and 72% in the whey. As the pH of the milk was reduced, more of the rennet remained with the curd until at pH 5.2 86% was in the curd and 17% in the whey. Total rennet accounted for in the curd and whey averaged 102 + 5% of that added to the milk. The pH of the milk also affected the relative amount of curd and whey recovered. At pH 5.2 the curd
averaged 77 g and was quite firm while the curd recovered at pH 6.6 averaged 122 g and was soft. It was impossible to account for all the added pepsin activity in the curd and whey. Pepsin distribution appeared to be pH dependent and follow the same pattern as rennet. However, this enzyme was so unstable that much of it was inactivated during the experiment. To release pepsin f r o m the curd it was necessary to adjust the pH of curd slurries to 6.8. Above pH 6.5, porcine pepsin is extremely unstable and any pepsin in the curd may have been partly inactivated during the assay. Distribution of the two microbial milk
~8
hi
WHEY[
WHEY~-7
CURD~
CURD~
.U
II II I/
~! >-40 ~--2O 0
~I-~ 5.2
6.0
6.4
PEPSIN
6.6
pH FIG. 5. Effect of pH on the distribution of porcine pepsin between curd and whey in freshly coagulated milk. Journal of Dairy Science Vol. 60, No. 6
'°H,Ir,ll I! 5.2
6.0
pH
6.4
~M PROTEASE
6.6
FIG. 7. Effect of pH on the distribution of MM protease between curd and whey in freshly coagulated milk.
CLOTFING ENZYMES IN CHEESE AND WHEY
clotting enzymes between curd and whey were similar to each other (Fig. 6 and 7) and independent of pH. Slightly higher activity in the curd at the higher pH can be accounted for by differences in the amount of curd if the increased amount was due to whey carrying the same concentration of enzyme as in the separated whey. Approximately 83% of the enzyme activity from mucor sources was recovered in the whey and 17% in the curd. Total recovery of the enzymes was 99 + 2% for MP protease and 100 -+ 4% for MM protease.
]C)O
oo].
PEPSIN
~ 80 er W ~ 60"
WHEYE:] t~
CURD F'~I
40" >p>-- 2 0 p-
Distribution and Survival of Milk Clotting Enzyme Activity During Cheddar Cheese Making
Enzyme concentrations in curd and whey were measured at cutting and dipping and in curd after overnight pressing during the manufacture of Cheddar cheese with rennet, porcine, pepsin, MP protease, a commercial mixture of pepsin and rennet (five replications each), and MM protease (four replications). Because of difficulty in measuring weights of curd and whey at cutting without disrupting the cheese making operation, the distribution of the enzyme between curd and whey prepared from freshly coagulated milk adjusted to the pH of the cheese milk at cutting was assumed to be representative of the distribution at cutting. Several assays of curd and whey taken from cheese at cutting indicated that this assumption was valid. Figures 8, 9, 10, 11, and 12 illustrate the
867
O
CUT
r-Q DIP
FIG. 9. Distribution of porcine pepsin between curd and whey and its survival during Cheddar cheese making.
results of these experiments. At dipping 35% of the rennet activity had been lost. The curd contained 7% and the whey 58% of the original activity. After overnight pressing the curd contained only 6% of the original rennet activity (Fig. 8). At dipping only 9% of the original pepsin activity was left in the whey, and none was in the curd. However, if some pepsin had been active in the curd, it most likely was destroyed when the curd slurry was adjusted to pH 6.8 to release the enzyme from the curd. Figure 10 shows the distribution of the
I
8e RENNET
COM. MIX
>
O 60 WHE Y
WHEYI
~40,. ,
I
CURDF-/']
CURD ~ ]
~40>I--
F- 2O
~_200 0
PRESS
CUT
DIP
PRESS
FIG. 8. Distribution of rennet between curd and w h e y and its survival during Cheddar cheese making.
/// /// CUT
DIP
r747 PRESS
FIG. 10. Distribution of e n z y m e activity from a commercial mixture of r e n n e t and porcine pepsin between curd and whey and its survival during Cheddar cheese making. Journal of Dairy Science Vol. 60, No. 6
868
HOLMES ET AL.
._p.-
:801 uJ
~
4% of the MM protease. After overnight pressing the curd contained 3 and 1.8% of the two enzymes. There was no inactivation of either of these enzymes during cheese making.
MP
I
WHEY[
60-
I
CURDIT7}
LLI rY
>- 4 O I-
C--20 l / 1
0
I I I
7-77,,
CUT
DIP
. . I.
-
PRESS
FIG. 11. Distribution of MP protease between curd and whey and its survival during Cheddar cheese making.
activity of a commercial rennet-pepsin mixture between curd and whey during the manufacture of five replicate batches of Cheddar cheese. At dipping, only 22 ± 4% of the original activity could be accounted for, 5 -+ 3% in the curd and 17 +- 4% in the whey. After overnight pressing, 4 ± 2% of the activity was left in the curd. Because of the poor stability of porcine pepsin, it was assumed that all the residual activity in the curd was due to rennet. The stability of the microbial proteases during cheese making is illustrated in Fig. 11 and 12. At dipping all enzyme activity was accounted for in curd and whey. At that point the curd contained 6% of the MP protease and
100
I
'"
80
WHEYI
rY ILl
I
C U RDIT7--~
t.)
uJ
MM
protease
r~4o )>2o "~
0
I/ill
CUT
DIP
,..i..,
PRESS
FIG. 12. Distribution of MM protease between curd and whey and its survival during Cheddar cheese making. Journal of Dairy Science Vol. 60, No. 6
DISCUSSION
A linear diffusion test for the quantitative determination of milk clotting enzymes at concentrations of 1 x 10 -4 to 1 × 10 -1 RU/ml proved useful for measurement of residual clotting enzymes in cheese curd and whey. A kappa casein-agar substrate at pH 5.9 gave excellent results when used in small diffusion tubes. The diffusion boundary was always sharp, and its distance from the origin was measured easily with a transmission densitometer. Measurements were not affected by NaCI at concentrations up to 3% in the enzyme solutions, nor were they affected when the enzymes were diluted with whey. All the enzyme activity (except porcine pepsin) added to milk was accounted for in the curd and whey. The distribution of rennet between curd and whey was pH dependent while that of the mucor coagulants was not. It was necessary to raise the pH of acid curd slurries to 6.8 to release the rennet enzymes from the curd so they could be measured. This was not necessary with the mucor enzymes. Porcine pepsin was so unstable in milk that accurate recoveries were impossible even though the results suggested that, like rennet, its affinity for curd increased with decreasing pH. The small percentage of enzyme activity remaining in finished Cheddar cheese, particularly from microbial enzymes, suggests that the role of these enzymes in cheese curing is minor compared to that of microorganisms. This idea is supported further by the fact that reasonably good cheese can be made with porcine pepsin (15) even though it is doubtful that any of this enzyme survives the cheese making process (12). The pH dependence of the affinity of rennet for curd suggests that the more acid the milk at setting, the more rennet activity should remain in the cheese. In these studies little acid had developed by the time the curd was cut and in most instances the pH of the curd was between 6.2 and 6.1 at dipping. Therefore, most of the rennet would have escaped into the whey
CLOTTING ENZYMES IN CHEESE AND WHEY
before the curd was sufficiently acid to " b i n d " much of the enzyme. Few of the hundreds of proteases that have been tried as rennet substitutes have been successful. Significant cheese defects have been associated with the use of too much of the wrong kind of protease for clotting cheese milk (10). It would be of interest to know whether some of these problems might be associated with the amount o f enzyme remaining in the curd. REFERENCES
1 Allen, L. A., and N. R. Knowles. 1934. Studies in the ripening of Cheddar cheese. J. Dairy Res. 5:185. 2 Babcock, S. M., and H. L. Russell. 1902. Curing of Cheddar cheese with especial reference to cold curing. Wisc. Agr. Exp. Sta. Bull. 94. 3 Berridge, N. J. 1952. Some observations on the determination of the activity of rennet. Analyst 77:57. 4 Cheeseman, G. C. 1963. Action of rennet and other proteolytic enzymes on casein in casein-agar gels. J. Dairy Res. 30:17. 5 Clarke, N. H., and E. L. Richards. 1973. An assay for rennin. N. Z. J. Dairy Sci. and Tech. 8:152. 6 Elliot, J. A., and D. B. Emmons. 1971. Rennin detection in cheese with the passive indirect hemagglutination test. Can. Inst. Food Tech. J. 4:16. 7 Emmons, D. B. 1970. Inactivation of pepsin in hard water. J. Dairy Sci. 53:1177. 8 Ernstrom, C. A. 1958. Heterogeneity of crystalline rennin. J. Dairy Sci. 41:1663. 9 Emstrom, C. A. 1961. Milk clotting activity of pepsin and rennin. Milk Prod. J. 52(5):8. 10 Ernstrom, C. A., and N. P. Wong. 1974. Milk clotting enzymes and cheese chemistry. Pages 662 to 771 in B. H. Webb, A. H. Johnson, and J. A. Alford, eds. Fundamentals of Dairy Chemistry,
869
2nd ed. AVI, Westport, CT. 11 Gorini, L., and G. Lanzavecchia. 1954. Recherches sur le mechanisme de production d'une proteinase bacterierme. I. Nouvelle technique de determination d'une proteinase par la coagulation du lair. Biochim. Biophys. Acta 14:407. 12 Green, M. L. 1972. Assessment of swine, bovine and chicken pepsins as rennet substitutes for Cheddar cheese making. J. Dairy Res. 39:261. 13 Lawrence, R. C., and W. B. Sanderson. 1969. A micro-method for the quantitative estimation of rennets and other proteolytic enzymes. J. Dairy Res. 36:21. 14 Marth, E. H. 1963. Microbiological and chemical aspects of Cheddar cheese ripening. A review. J. Dairy Sci. 46: 869. 15 Melachouris, N. P., and S. L. Turkey. 1964. Comparison of the proteolysis produced by rennet extract and the pepsin preparation Metroclot during ripening of Cheddar cheese. J. Dairy Sci. 47:1. 16 Peterson, M. H., M. J. Johnson, and W. V. Price. 1948. Determination of cheese proteinase. J. Dairy Sci. 31:47. 17 Price, W. V., and H. E. Calbert. 1944 (revised 1953). Cheddar cheese from pasteurized milk. Wisc. Agr. Exp. Sta. Bull. 464. 18 Reyes, J. 1971. A procedure for measuring residual rennin activity in whey and curd from freshly coagulated milk. M.S. thesis, Utah State University, Logan. 19 Sherwood, 1. R. 1935. The function of pepsin and rennet in the ripening of Cheddar cheese. J. Dairy Res. 6:407. 20 Sommer, H. H., and H. Matsen. 1935. The relation of mastitis to rennet coagulability and curd strength of milk. J. Dairy Sci. 18:741. 21 Van Slyke, L. L., and J. C. Baker. 1918. The preparation of pure casein. J. Biol. Chem. 35:127. 22 Wang, J. T. 1969. Survival and distribution of rennin during Cheddar cheese manufacture. M.S. thesis, Utah State University, Logan. 23 Zittle, C. A., and J. H. Custer. 1963. Purification and some of the properties of a s casein and g-casein. J. Dairy Sci. 46:1183.
Journal of Dairy Science Vol. 60, No. 6