Action of Rennin on Casein. III. Effect of αs- and β-Casein on the Secondary Phase

A c t i o n o f R e n n i n o n Casein. I I I . E f f e c t o f a s- a n d / 3 - C a s e i n on t h e S e c o n d a r y Phase I. KATO and K. ANDO The College of Dairying Nishinopporo 582 Ebetsu, Hokkaido, Japan K. MIKAWA and T. YASUI Department of Animal Science Hokkaido University Sapporo, Hokkaido, Japan ABSTRACT

The action of rennin on K-casein was studied as a function of time, employing turbidity measurements at 610 nm and the release o f nonprotein nitrogen. K-Casein was converted to para-K-casein by the action of rennin. The para-K-casein aggregated to increase turbidity and then precipitated. Turbidity development was enhanced initially and then retarded severely by increasing concentrations of added a s- and 13-casein. The addition of selected amino acids and salts had variable effects on increase of turbidity. INTRODUCTION

There are two phases (primary phase and secondary phase) in coagulation of milk induced by rennin. In the primary phase, the rennin hydrolyzes K-casein. Subsequently, the exposed para-K-casein aggregates in the secondary phase. It is accepted that a s- and /3-casein are degraded gradually over a long time, and this stage has been termed the tertiary phase (1). Nitschmann and Boren (13) determined the increase in 12% trichloroacetic acid (TCA) soluble nonprotein nitrogen (NPN) in the primary phase at intervals during the action of rennin on casein. Tsugo and Yamauchi (16) studied the secondary phase using turbidity development. The turbidity development in the secondary phase has been the topic of (3, 5, 9, 12, 13, 16, 19). Recently, Payens (15) reported the mechanism of the overall rennin reaction, and Hyslop et al. (7) reported the kinetics of coagulation of milk by pepsin. We reported (8) the liberation of 3% TCA

Received February 5, 1979. 1980 J Dairy Sci 63:25--31

soluble NPN from K-casein, by the action of rennin, using Kjeldahl and colorimetric methods, and results were comparable. Another paper (11) showed that the liberation of 3% TCA soluble NPN was strongly inhibited by addition of as- and fl-casein. The current paper describes the effect of a s- and ~-casein on the secondary phase. MATERIALS AND METHODS Casein Preparation

Raw milk was from a single Holstein cow a t the Hokkaido University farm. Acid casein was prepared by the ordinary method of isoelectric precipitation at pH 4.6. K-Casein and as-Casein were prepared by the method of Zittle and Custer (19) from acid precipitated casein. /3-casein was prepared first by the method of H i p p e t al. (6) and then purified further by the Payens and Markwijk method (14). These different caseins were lyophilized after overnight dialysis. Polyacrylamide discgel electrophoresis, including 4.5 M urea and no reducing agent, of /¢-, a s- and [3-casein at pH 8.6 showed no contamination with other caseins. Preparation of Casein Solutions

Freeze dried casein (¢.-, as-, or fi-) .2 g each was dissolved in 100 ml .05 M tris-maleate buffer (pH 6.5). Reaction mixtures contained K-casein in a constant concentration of .2% and a s - or /3-casein in various weight to weight ratios, with a final reaction volume of 4.0 ml. Rennin Preparation

Commercial rennet powder was obtained from Chr. Hansen Laboratories Inc. Rennin was separated by two slow salting out treatments (4) and then purified further b y the diethylaminoethyl (DEAE)-cellulose chromatography 25

26

KATO ET AL.

m e t h o d o f Y o s i n o et al. (18). T h e a c t i v i t y o f r e n n i n was d e t e r m i n e d b y t h e m e t h o d o f Berridge (2). All o t h e r chemicals were o f r e a g e n t grade a n d were used w i t h o u t f u r t h e r p u r i f i c a t i o n .

o/

o~10 Turbidity Measurements

v z

S u b s t r a t e s o l u t i o n s (3 ml o f .2%, K-casein, .05 M tris-maleate b u f f e r at p H 6.5) were k e p t at 25 C in a w a t e r b a t h , were t r a n s f e r r e d i n t o t h e c u v e t o f t h e Hitachi s p e c t r o p h o t o m e t e r m o d e l 2A, a n d were m a i n t a i n e d at 25 C b y circulating water. A f t e r 20 rain, .01 ml o f r e n n i n s o l u t i o n (5 p g N / m l ) was a d d e d , t h e r e a c t i o n m i x t u r e was s h a k e n gently, a n d t u r b i d i t y m e a s u r e d at 610 n m .

~z ~,

Measurement of Liberated N PN

R e a c t i o n s o l u t i o n s (40 ml o f .2% K-casein, .05 M tris-maleate b u f f e r s at p H 6.5) were m i x e d w i t h .11 ml o f r e n n i n s o l u t i o n at 25 C. T h e r e a c t i o n was s t o p p e d b y a d d i t i o n o f 15% T C A (final c o n c e n t r a t i o n .3%) at each t i m e . A f t e r 30 m i n , t h e m i x t u r e s were filtered t h r o u g h W h a t m a n No. 2 filter paper. F i l t r a t e .5 ml was a n a l y z e d for NPN b y C u - F o l i n m e t h o d (8).

0/0~

15

/ /

/ /

A/

1.0

// E O

5,(

/

5

/

zx

5 10

36 Reaction Time (rain.)

Figure 1. Rennin activity on K-casein as a function of time. K-casein solution (40 ml of E-casein (.2%), .05 M tris-maleate buffers (pH 6.5) was mixed with rennin (.1 ml, 5 ~gN/mI) and stopped at each reaction time by 15% TCA (final cone. 3%). Then 3% TCA soluble NPN was measured by Cu-Folin method. Turbidity measurement also was similar to that of NPN measurement. Three milliliters of .2% E-casein were transferred into the cuvette of the spectrophotometer and mixed with .01 ml of rennin solution and turbidity development measured. Release of NPN, - - - o - - -; turbidity development, - - - A - _ -

Determination of Rate Constant

B o t h r e a c t i o n s of NPN l i b e r a t i o n a n d t u r b i d i t y d e v e l o p m e n t are e x p r e s s e d g r a p h i c a l l y as a straight line if: log i 0 0 ( N 6 0 -- Nt)lN60 N60 = increase o f NPN at 60 rain (NPN60 -N P N 0 ) ; N t = increase of NPN at 5 m i n (NPN t -NPN0 ); and: log 1 0 0 ( D

-- D t ) / D ~

D~: a b s o r b a n c e (A) at m a x i m u m ; Dt: a b s o r b a n c e at t m i n ) are p l o t t e d against t i m e , a l t h o u g h in t u r b i d i t y d e v e l o p m e n t , o n l y d u r i n g t h e i n d u c t i o n p e r i o d , this is n o t a s t r a i g h t line. RESULTS Comparison of Turbidity Development Curve and NPN Liberation Curve

T h e NPN l i b e r a t i o n a n d t u r b i d i t y d e v e l o p m e n t b y r e n n i n are in Figure 1. In all experi-

Journal of Dairy Science Vol. 63, No. 1, 1980

m e n t s , t h e r e was a rapid initial release of 3% T C A - s o l u b l e NPN a f t e r a d d i t i o n o f r e n n i n . Most NPN f r o m K-casein was l i b e r a t e d w i t h i n 5 rain u n d e r these e x p e r i m e n t a l c o n d i t i o n s , b u t t h e t u r b i d i t y d e v e l o p m e n t caused b y t h e a g g r e g a t i o n of para-K-casein was still increasing a f t e r 15 m i n (Figure 1). T h e r e a c t i o n o f NPN l i b e r a t i o n is a s s u m e d to b e first o r d e r as d e s c r i b e d in t h e r e p o r t s b y N i t s c h m a n n et al. (13) a n d Tsugo et al. (16). Figure 2 shows a linear e x p r e s s i o n o f NPN liberation indicating a pseudo first-order reaction and turbidity development from the curves o f K-casein in Figure 1. T h e i n d u c t i o n p e r i o d at t h e initial stage (lag p h a s e ) o f t u r b i d i t y d e v e l o p m e n t is n o t f i t t e d o n this line. T h e rate c o n s t a n t s f r o m Figure 2 are 3.85 × 10 -3 s -1 for NPN l i b e r a t i o n a n d 2 . 8 4 × 10 -3 s -1 for t u r b i d i t y development. Effect of as-Casein on the Turbidity Development

T h e e f f e c t o f as-casein o n t u r b i d i t y develop-

ACTION OF RENNIN ON CASEIN

°l\

m e n t is in Figures 3 a n d 6. By t h e a d d i t i o n o f high c o n c e n t r a t i o n s o f as-casein (0ts/t¢>.15), t u r b i d i t y d e v e l o p m e n t was r e t a r d e d . However, it was a c c e l e r a t e d b y t h e a d d i t i o n o f l o w e r c o n c e n t r a t i o n s (%/K <.1).

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Effect of/~-Casein on Turbidity Development

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T u r b i d i t y d e v e l o p m e n t in t h e p r e s e n c e o f ~-casein s h o w e d similar t e n d e n c i e s as t h a t in t h e p r e s e n c e o f CXs-casein (Figures 4 a n d 7). But, t h e i n h i b i t o r y effect o f fl-casein was w e a k in c o m p a r i s o n w i t h ~Xs-casein. T h e a d d i t i o n o f j3-casein t o ~-casein 03h¢>.3) p r o d u c e d a s t r o n g i n h i b i t o r y effect while a small a m o u n t o f fl-casein (/3h¢<.25) a c c e l e r a t e d t h e t u r b i d i t y d e v e l o p m e n t as s h o w n in Figures 4 a n d 7. T h e rate c o n s t a n t s calculated f r o m Figures 6 a n d 7 are in T a b l e 1.

..J

10

20

30

Effect of Amino Acids and Salts on Turbidity Development

40

T h e e f f e c t o f a m i n o acids a n d salts o n t h e t u r b i d i t y d e v e l o p m e n t is in Figures 5 a n d 8. T h e r e was n o c h a n g e in t u r b i d i t y d e v e l o p m e n t w h e n o n e o f t h e f o l l o w i n g was a d d e d : 4 m M glycine, 1 m M p y r o p h o s p h a t e , a n d 4 m M calcium chloride.

Reaction Time (min.)

Figure 2. Linear expression of the NPN liberation and the turbidity development obtained by rennin action on K-casein. This is expressed to calculate the rate constants from Figure 1. Symbols are the same as in Figure 1.

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20

30

40

50

60

70

80

90

100

110

120

Reaction Time (rain.)

Figure 3. Effect of %-casein on turbidity development produced by rennin action on K-casein. For details see legend to Figure 1 and text. The ~s/K ratio of reaction mixture were adjusted as follows: - - - ~ - - - K-casein (control); ~s/K (weight ratio): 1) .O1, 2) .05, 3) .10, 4) .25, 5) .30, 6) .35, 7) .40, 8) .45, 9) .50, 10) .55, 11) .60. Journal of Dairy Science Vol. 63, No. 1, 1980

28

K A T O ET AL.

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Reaction Time (min.) Figure 4. E f f e c t of/3-casein o n t u r b i d i t y d e v e l o p m e n t p r o d u c e d b y r e n n i n a c t i o n o n K-casein. F o r details see l e g e n d to Figure 1 a n d t e x t . T h e j3/~ r a t i o o f r e a c t i o n m i x t u r e w e r e a d j u s t e d as follows: - - - zx - - - K-casein (control); ~/K ( w e i g h t ratio): 1) .01, 2) .05, 3) .10, 4) .25, 5) .30, 6) .35, 7) .40, 8) .45, 9) .50, 10) .55, 11) .60, 12) .65, 13) .70, 14) .75.

When glutamic acid or aspartic acid was added at 4 mM, turbidity development was accelerated markedly as compared with the control as in Figures 5 and 8. Turbidity develop-

~/÷ .//

2,

ment was not affected by the addition of 10 mM glutamic acid or aspartic acid but was inhibited by addition of 20 mM glutamic acid or aspratic acid or o f 10 mM pyrophosphate.

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26

3o

4'o

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Reaction Time (min.) Figure 5. E f f e c t o f a m i n o acids a n d salts o n turb i d i t y d e v e l o p m e n t p r o d u c e d b y r e n n i n a c t i o n on Kcasein. S e l e c t e d salts and a m i n o acids were m i x e d as follows: ( - - - A - - - ) K-casein ( c o n t r o l ) , ( - - - o - - - ) l m M and ( - • - ) 20 m M p y r o p h o s p h a t e , ( - - - v - - - ) 4 mM glycine, (---o---) 4 m M and ( - • - ) 10 m M CaC12, ( - - - e - - - ) 4raM,(-+-) 10 r e m a n d ( - × - ) 2 0 2 0 m M g l u t a m i c acid, (- - - • - - -) 4 0 m M a s p a r t i c acid. J o u r n a l o f D a i r y Science Vol. 63, No. 1, 1 9 8 0

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20

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R e a c t i o n T i m e (min,) Figure

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expression

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the

turbidity

development by addition of %-casein. This is e x p r e s s e d to c a l c u l a t e the rate c o n s t a n t s f r o m Figure 3. N u m b e r s are the s a m e as in Figure 3 - - - n - - - K-casein (control).

ACTION OF RENNIN ON CASEIN

29

TABLE 1. Comparison of rate constants obtained by addition of a s- or ~3-casein for rennin action on K-casein.a

No.

%-/K" (ratio)

1 2 3 4 5 6 7

.01 .05 .10 ,15 .20 .25 .

k(" 10 -3 s "1 ) 4.04 5.48 4.26 2.40 1.67 1.70

.

.

.

.

.

.

.

~3-/K (ratio)

k(. 10 -3 s-1 )

.01 .05 .10 .25 .30 .35 40

4.79 7.61 5.12 4,52 1.83 1.79 1.00

aThe ratio of CZs/Kor [31t¢ are weight ratios and are the same as in Figures 6 and 7. The rate constant (k) of K-casein (control) was calculated at 2.84 × 10 -3 s -z .

Rate c o n s t a n t s calculated f r o m Figure 8 are in Table 2.

DISCUSSION

U n d e r the c o n d i t i o n s in t h e s e e x p e r i m e n t s , m o s t o f t h e NPN in K-casein was liberated in the first 5 min while t h e t u r b i d i t y began to increase rapidly only a f t e r 15 min (Figure 1). The rate o f NPN liberation is faster t h a n t u r b i d i t y d e v e l o p m e n t (Figure 2). Significant i n h i b i t i o n o f t u r b i d i t y developm e n t b y large a m o u n t s o f a s- and ¢3-casein is in Figures 6, 7 and Table 1. T h e r e f o r e , the rate o f t u r b i d i t y d e v e l o p m e n t n o t o n l y decreased w i t h t h e increase o f a s- and/3-casein (for a s / K > . 1 5 ,

/3/K>.3), b u t also the t i m e in w h i c h t u r b i d i t y d e v e l o p m e n t began was d e l a y e d (Figures 3 a n d 4). While small a m o u n t s o f as- and /3-casein (as/K <.1, 13/K< . 2 5 ) accelerated turbidity d e v e l o p m e n t and s h o w e d rate c o n s t a n t s o f 1.4 to 2.6 times the c o n t r o l (Table 1), and also s h o r t e n e d t h e t i m e r e q u i r e d for t h e process to occur, L a w r e n c e and C r e a m e r (9) s h o w e d t h a t as- and t3-casein e x e r t e d a m a r k e d i n h i b i t o r y e f f e c t on the s e c o n d a r y phase, and t u r b i d i t y d e v e l o p m e n t was d e l a y e d a l t h o u g h t h e maxim u m t u r b i d i t y was e v e n t u a l l y r e a c h e d . Mackenzie (10) also p o i n t e d o u t t h a t the aggregation o f n e w l y f o r m e d para-K-casein is p r e v e n t e d b y u n c h a n g e d K-casein.

\.

10

20

30

40

50

60

70

I0 Reaction Time (min.)

Figure 7. Linear expression of the turbidity development by addition of p-casein. This is expressed to calculate the rate constant from Figure 4. Numbers are the same as in Figure 4 - - - zx_ _ _ K-casein (control).

20

30

40 50 60 Reaction Time (rain.)

70

Figure 8. Linear expression of the turbidity development by addition of salts and amino acids. This is expressed to calculate the rate constant from Figure 5. Symbols are the same as in Figure 5 - - - I' - - g-casein (control). Journal of Dairy Science Vol. 63, No. 1, 1980

30

KATO ET AL.

TABLE 2. Comparison of the rate constants obtained by addition of salts or amino acids for rennin action on K-casein. Salt or amino acid

mM

k(.10-3 s-1 )a

Pyrophosphate Pyrophosphate Glycine Calcium (CaC12 ) Calcium (CaC12 ) Glutamic acid Glutamic acid Glutamic acid Aspartic acid

1 20 4 4 10 4 10 20 4

2.84 1.02 3.49 2.95 2.33 4.80 2.84 1.92 4.80

aThe rate constant (k) of K-casein (control) was calculated at 2.84.10 -v s-1 .

Payens (15) r e p o r t e d t h e overall process o f t h e f o r m a t i o n of para-x-casein a n d its subseq u e n t c l o t t i n g in 1979. He c o n c l u d e d t h a t r e t a r d a t i o n o f c l o t t i n g o f para-r-casein, caused b y t h e a d d i t i o n o f a s- or /3-casein, was d u e to f o r m a t i o n o f soluble c o m p l e x e s w i t h t h e para-g-casein. O u r results agree w i t h t h e l i t e r a t u r e c o n c e r n ing t h e effect o f large a m o u n t s o f ~ - a n d fl-casein o n t u r b i d i t y d e v e l o p m e n t . T h e e n h a n c ing effect o f small q u a n t i t i e s o f t h e s e p r o t e i n s , s h o w n for t h e first t i m e , m a y b e p a r t l y d u e t o charge effects, as in Figures 5 a n d 8. This e f f e c t m a y involve also h y d r o p h o b i c b o n d i n g . A c i d i c a m i n o acids ( g l u t a m i c acid a n d a s p a r t i c acid) at low c o n c e n t r a t i o n s (4 m M ) a c c e l e r a t e d t u r b i d i t y d e v e l o p m e n t w h i l e at high c o n c e n t r a t i o n s (20 m M ) i n h i b i t i o n was o b t a i n e d (Figures 5, 8, a n d T a b l e 2). No e f f e c t o n t u r b i d i t y d e v e l o p m e n t was o b s e r v e d b y a d d i t i o n o f n e u t r a l a m i n o acids (glycine) o r n e u t r a l salts (CaCI2). It is a s s u m e d t h a t negatively charged a m i n o acids i n t e r a c t w i t h para-g-casein, p o s s i b l y d u e t o e l e c t r o s t a t i c or h y d r o p h o b i c i n t e r a c t i o n s . T h e result o f t h e s e i n t e r a c t i o n s m a y b e t h e s u p p r e s s i o n o f charge r e p u l s i o n b e t w e e n para-~-casein m o l e c u l e s leading to a c c e l e r a t e d turbidity development. However, the mechanism o f aggregation is n o t clear, a n d a d e q u a t e e x p l a n a t i o n for t h e s e results c o u l d n o t b e d e v e l o p e d f r o m t h e p r e s e n t s t u d y alone. As a result, f u r t h e r w o r k is t o e l u c i d a t e t h e phenomena. Journal of Dairy Science Vol. 63, No. 1, 1980

ACKNOWLEDGMENTS

We a c k n o w l e d g e t h e s u p p o r t o f Kogo Yusa, College o f Dairying, a n d t h e e d i t o r i a l assistance o f F. Wolfe, D e p a r t m e n t o f F o o d Science, University o f A l b e r t a . REFERENCES

1 Alais, C-, G. Mocquot, H. Nitschmann, and P. Zahler. 1953. Lab und seine wirkung und das casein der milch. VII. Uber die abspaltung yon nicht-protein stickstoff (NPN) aus casein durch lab und ihre bezichung zue primarreacktion der lab gerinnung der milch. Heir. Chem. Acta. 36:1955. 2 Berridge, N. J. 1945. The purification and crystallization of rennin. Biochem. J. 39:179. 3 Bingham, E. W. 1974. Action of rennin on K-casein. J. Dairy Sci. 58:13. 4 Foltmann, B. 1959. On the crystallization, stability and proteolytic activity of rennin. Acta Chem. Scand. 13:1927. 5 Gizelta, K.-P., and T. Sanner. 1973. Comparison of the specificity and kinetic properties of 3 milkclotting enzymes. J. Dairy Res. 40:263. 6 Hipp, N. J., M. L. Groves, J. H. Custer, and T. L. McMeekin. 1952. Separation of ~-,/3- and 3,-casein. J. Dairy Sci. 35:272. 7 Hyslop, D., T. Richardson, and D. Ryan. 1979. Kinetics of pepsin-initiated coagulation of K-casein. Biochim. Biophys. Acta. 566:397. 8 Kato, I., K. Mikawa, Y. K. Kim, and T. Yasui. 1970. Action of rennin on casein. I. Effect of salts on the primary phase. Memoirs Fac. Agric. Hokkaido Univ. 7:477. 9 Lawrence, R. C., and L. K. Creamer. 1969. The action of calf rennet and other proteolytic enzymes on K-casein. J. Dairy Res. 36:11. 10 McKenzie, H. A. 1967. Milk proteins. Adv. Protein Chem. 22:55. 11 Mikawa, K., I. Kato, and T. Yasui. 1973. Action of rennin on casein. II. Effect of a s- and-/3-casein on the primary phase. Memoirs Fac. Agr. Hokkaido Univ. 9:110. 12 Mullin, W. J., and F. H. Wolfe. 1973. Disc gel electrophoresis of caseins treated with proteolytic and glycolytic enzymes. J. Dairy Sci. 57:9. 13 Nitschmann, H., and H. V. Bohren. 1955. Das lab und seine wirkung auf das casein der milch. X) Eine methode zur direkten bestimmung der geschwindigkeit der primarreaktion der labgerinnung der milch. Heir. Chim. Acta. 38:1953. 14 Payens, T.A.J., and B. W. Van Markwijk. 1963. Some features of the association of beta casein. Biochim. Biophys. Acta. 71 : 517. 15 Payens, T.A.J. 1976. On the enzyme-triggered clotting of casein: a preliminary account. Netherlands Milk Dairy J. 30:55. 16 Tsugo, T., and K. Yamauchi. 1960. Specific liberation of non-protein nitrogen from ~-casein fraction by rennin and pepsin. Bull. Agric. Chem. Soc. Japan 24:96. 17 Tuszynski, W. B. 1971. A kinetic model of the clotting of casein by rennet. J. Dairy Res. 38:115. 18 Yosino, U., N. Nakatani, Y. Tokoro, and K.

ACTION OF RENNIN ON CASEIN Yamauchi. 1966. Purification o f rennet by DEAE - cellulose chromatography. Nippon Nogeikagaku Kaishi. 40: 52.

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19 Zittle, C. A., and J. H. Custer. 1963.Purification and some of the properties of%-casein and K-casein. J. Dairy Sci. 4 6 : 1 1 8 3 .

Journal of Dairy Science Vol. 63, No. 1, 1980