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Effect of Various Salts on the Coagulation of Casein
EFFECT
OF V A R I O U S S A L T S ON T H E
C O A G U L A T I O N OF C A S E I N
DYSON ROSE A~l) It. TESSIER Division of Applied Biology, National Research Council, Ottawa, Canada SUMMARY
Addition of potsssium or sodium chloride (80 or 130 raM) to skimmilk increased the time the milk could be held in frozen storage without development of 4% precipitate by volume. Addition of phosphate or calcium (10 raM) had the opposite effect. Addition of large amounts of sodium chloride (up to 1.0 M) offset the destabilizing effect of phosphate or calcium at 0, 10, and 20 ° F., but addition of similar amounts of potassium chloride stabilized the smnpIes only at 10 and 20 ° F. Addition of potsssium chloride (1.0 M or more) and of phosphate (100 mM or more) induced gelation of milk at room temperature. Addition of sodium chloride and phosphate did not induce gelation. Addition of either potassium or sodium chloride (2.3 M) shifted approximately 20% of the insoluble calcium and 7.5% of the insoluble phosphate to the soluble forms, displaced eMcium from casein, and increased the dissociation of calcimn citrate. A possible explanation of these effects, based on the interlinking of potassium-caIcimn-caseinate micelles by precipitating calcium phosphate, is presented. Varistions in the concentration of certain inorganic salts or ions, particularly calcium, are known to affect the eoagnlation of casein it1 abnormal milk (2, 14), in milk treated with rennet or other coagulating enzymes (10), and in frozen milk (3, 13). The unfrozen portion of milk stored at --7.5 ° C. contains a p p r o x i m a t e l y 0.8 M salt (ten-fold concentration) p r i o r to lactose crystallization (15), and it can be estimated t h a t up to a 30-f~ld concentration develops as lactose crystallizes. Coagulation of the casein occurs as this high concentration of salt develops (17, 18), but it is not known whether total salt concentration or the concentration of specific components induces the coagulation. U n d e r conditions t h a t do not cause salt crystallization, the concentration of a component in the unfrozen portion of frozen milk depends directly upon its initial concentration and the total amount of w a t e r retained in the unfrozen state b y all components at the storage t e m p e r a t u r e used. Therefore, addition of a salt that promotes casein coagulation should decrease the storage life of milk. Addition of a solute that does not pr(mmte coagulation will dilute the other components by retaining more unfrozen water and, thus, should increase storage life. Glycerol, sucrose, and dextrose (1, 13, 20) and hydrotyzed lactose (18) p r o b a b l y act m a i n l y in this manner. This p a p e r presents the results of a s t u d y of the effect of added salts on casein coagulation. MATERIALS
AND Iv]ETHODS
Milk obtained f r o m m o r n i n g milkings at the Central E x p e r i m e n t a l F a r m , Ottawa, was separated and pasteurized at 150 ° F. for 30 rain. Storage tests were carried out as previously described (13), i.e., samples were frozen in 15-ml. vials and, at suitable intervals (depending on the t e m p e r a t u r e ) , single vials were thawed at 40 ° F. overnight and the contents centrifuged at c a . 500 × g. for ]~eeeived for publication December 19, 1958. 989
990
DYSON
I%OSE
AND
H. TESSIER
10 rain. The graphically interpolated time needed to develop 4% precipitate b y volume is r e p o r t e d as the storage life. Analytical methods were: calcium ion in ultrafiltrate (16), total calcium (8), phosphate (9). Ashing was done in platinum crucibles at 550 ° C. for 15 hr. Calcium easeinate suspensions were p r e p a r e d by dispersing 3 g. of casein p r e p a r e d b y V a n Slyke and B a k e r ' s method (19) in a p p r o x i m a t e l y 95 ml. of saturated calcium hydroxide solutions and a d j u s t i n g the p H to' 6.6 to 6.7 with hydrochloric acid. Sufficient quantities of m o l a r solutions of a m m o n i u m phosphate, a m m o n i u m citrate, and magnesium chloride were then added to give concentrations of phosphate, citrate, and magnesium (30, 9, and 5 raM~l, respectively) comparable to those in milk. One per cent glycerol was added to give the freezing point depression of normal milk while avoiding the possibility of lactose crystallization. These p r e p a r a t i o n s were essentially so'dium- and potassium-free. " R e d i s p e r s e d c a s e i n " was casein-calcium-phosphate complex of fresh skimmilk sedimented at 60,000 × g. (Spineo Model L, No. 30 head, 30,000 r.p.m., 1 hr.) and redispersed b y t r i t u r a t i o n into a solution containing calcium, phosphate, citrate, and magnesium, added as the a m m o n i u m and chleride salts, in a p p r o x i m a t e l y the concentrations found in normal milk. One per cent glycerol was added in lieu of 4% lactose. Since samples of milk can differ m a r k e d l y in their storage life when frozen, and in other characteristics, d a t a for single tests are reported in the tables. However, replicate (duplicate or move) tests done u n d e r the stated conditions, a n d tests done u n d e r varied conditions not r e p o r t e d in detail, confirmed the direction a n d relative magnitude of the changes reported. RESULTS
Freezing tests with one salt added. Small additions (up to 40 mM) of calcium or phosphate sharply decreased the sto'rage life of frozen milk at all temperatures studied (Table 1). The destabilizing effect of calcium was greater than that of phosphate, but a p p e a r e d to be relatively independent of temperature. The destabilizing effect of phosphate was less at 0 ° F. (--18 ° C.) than at 20 ° F. ( - 7 ° C.). Similar effects were observed in calcium caseinate and in redisTABLE 1 Effect of added calcium and phosphate on the storage life of frozen skimmilk" Storage life (days) Addition Nolle 2.5 mM Calcium 5.0 mM Calcium 10.0 mM Calcium 2.5 mM Phosphate 5.0 n~/Phosphate 10.0 mM Phosphate All samples adjusted to the pH
20° F.
10 ° F.
35 40 3.5 20 5 4 3 2 35 25 8 18 2 8 of the control, pI~ 6.53, before freezing.
0° F. 50 30 2 2 50 50 3~)
991
E F F E C T OF V A R I O U S SALTS ON T H E C O A G U L A T I O N OF C A S E I N
persed casein when calcium or phosphate was added in excess of the amounts found in normal milk. The converse effect has been observed u p o n removal of calcium (3, 4). L a r g e additions of either calcium or phosphate at near-normal levels of the other could not be studied because of the limited solubility of calcium phosphate salts. Additions (ff either potassium or sodium chloride to skimmilk or to rcdispersed casein had a definite stabilizing effect d u r i n g frozen storage at all temperatures studied (Table 2). The effect of either salt was more marked at high than at low storage temperatures. TABLE 2 Effect of added sodium and potassium chlorides on the storage life of frozen sklmmilk Storage life
Addition Skimmi]k None 130 mM Sodium chloride 80 mM Potassium chloride Redispersed casein None 130 mM Sodium ch]~i~ 80 mM Potassium chloride
(days)
20 ° F.
10 ° F.
0° P.
25 80 58
32 57 41
63 80 80
2 35 29
2 18 12
16 32 17
Freezing tests with two salts added: I f sodium and potassium chlorides exert this stabilizing action b y reducing the concentration of calcium and phosphate in the unfrozen portion of frozen milk, then larger additions of these salts should offset the destabilizing effect of added calcium or phosphate. To test this possibility, u p to 1 M of sodium or potassium chloride was added to milks containing up to 20 mM added calcium and 40 mM added phosphate. The results obtained with sodium chloride indicate that it was possible to offset completely the destabilizing effect of added calcium ( F i g u r e 1A) or phosphate ( F i g u r e 1B), at all storage t e m p e r a t u r e s studied. On the other hand, potassium chloride offset the effect of added calcium at 20 ° F. ( - 8 ° C.), and p a r t i a l l y offset it at 10 ° F. (--12 ° C.), but decreased storage life of two samples and the control, at 0 ° F. (--18 ° C.) ( F i g u r e 2A). Also, potassium chloride offset the effect of added phosphate only p a r t i a l l y at 20 and 10 ° F., and did not stabilize a n y sample with added phosphate at 00 F. ( F i g u r e 2B). The i n a b i l k y of high concentrations of potassium chloride to offset the effects of added calcium or phosphate at 0 ° F. m a y be explained by the assumption t h a t crystal~ lization of the potassium chloride occurred because eutectic conditions were exceeded. I t is a p p a r e n t in F i g u r e s 1 and 2 t h a t there was some tendency for the lowest level (0.1 M) of sodium or potassium chloride to increase the destabilizing effect of added calcium o'r phosphate. A possible explanation of this is given u n d e r Discussion. Tests without freezi~g. P r e l i m i n a r y tests had indicated t h a t addition of potassium chloride and a neutral phosphate solution to milk caused the casein
992
DYSON
ROSE
AND
If. TESSIEI¢
calcium
A-added
150
O°F.
'°°
20"F.
IO°F. /
?o
o LLI
0
--
B-added
I.L
J
0,5
1.0
1
l
,,I
0.5
1.0 0
7: !
0.5
phosphate
f / l-! /j.o2 i_o _..o, I 7/" IO°FJ
150|
I00
!
-
OF
/
oIV//
0.5
~'O'F,
~-I0
~
0
1
1.0
o
lio
o.~°
o l- o.o
LO 0 0,5 LO 0 ADDED SODIUM CHLORIDE, M
0,5
LO
Fro. 1. Effect of adde,] sodium chloride on storage life of frozen skimmilk containing a~lded calcium or phosphate. F i g u r e s on curves indicate concentration, in raM, of added calcium ( A ) or p h o s p h a t e ( B ) . Arrows attached to points Jndict~te t h a t the last sample, taken at tho time shown, had not coagulated. E a c h set of tests (four curves) w~s from a separate sample of milk.
to gel within a few hours at room temperature. The rate of gelation varied between samples of milk, but within any one sample consistent results were obtained. Gelatiou could be iuduced by addition of phosphate to milk containing i M or more added potassium chloride per liter, and gelation time decreased progressively with increasing potassium chloride concentratio~a. When 2.3 M potassium c h l o r i d e w a s present, addition of 240, 160, and 120 mM phosphate per liter caused gelatioli in .5, 2.5, and 4 hr., respectively. Increasing the lactose concentration delayed gelation slightly; increasing citrate delayed it markedly. At a fixed potassium ~hloride and phosphate level, gelation time increased with decreasing p H and with decreasing temperature. A similar gelation was obtained with redispersed casein preparations, provided calcium, phosphate, and potassium chloride were included in the resuspending medium or added to the suspension. Conditions leading to the formation of a calcium phosphate precipitate appeared to be a prerequisite for gel formati(m. I t was also possible to cause gelation in skimmilk by acidifying it
EFFECT
OF VARIOUS
A-added
SALTS ON THE
COAGULATION
~,
20*F,
I 0 °F.
I00
f
5C
/--,o
o-" ---------o
0 _o
993
calcium O°F.
~_
OF CASEIN
I
I
.,J
0
0.5
1.0
/
o
I
I
I
0
0.5
1.0
i 0
r 0.5
J 1.0
.J
B-added
phosphate
o ¢n 150
20°F.
IO'F.
O*F.
--0 tO0
o/° 50
-I0
o - - o ~ o
? 0
-
\40 i 0.5
-
1.0
~°~--~-Z~-~ l 0
e
i
0,5
1.0
20 /
i 0.5
o
1.0
o0
ADDED POTASSIUM CHLORIDE, M F I G . 2. added
Effect
calcium
of added
or phosphate.
potassium Details
chloride
on storage
as in Figure
life of frozen
skimmilk
cont~iaing
1.
to p H 5.2, adding 160 mM phosphate, then neutralizing so as to induce extensive precipitation of calcium phosphate. Gelation could not be induced either in skimmilk or in casein resuspensions by the addition of phosphate and sodium chloride. Addition of sodium chloride to a milk containing 160 mM added phosphate and 1.5 M potassium chloride delayed gelation; aliquots with 0, 0,8, and 1.5 M added sodimn chloride gelled in 100, 180, and 900 rain., respectively. Addition of sodium chloride to' milk caused a greater increase ill acidity than did addition of potassimn chloride (Table 3), but addition of sufficient sodium hydroxide to maintain the original p H did not promote gelatiml in the presence of phosphate and sodium chloride. To determine what effect added sodium and potassium chlorides had on the distribution of salts in milk, ultrafiltrates [obtained as in (16)], and the Supernatant and sedimented solids obtained by centrifuging at 60,000 × g., were analyzed. Analysis of the ultrafiltrates (Table 3) showed that added potassium or sodium chloride eaused a marked increase in calcium ion, total calcium, and total phosphate. Even with sodium chloride, the increase in calcium ion concentration was more than three-fold that caused by addition of hydrochloric acid to give the same pH. Total ultrafiltrable calcium and phosphate appeared to
DYSON ROSE AND H. TESSIER
994
TABLE 3 Analysis of ultrafiltrates from skimmilk with added salt or acid Concentration added
Fi~lal pH
Ca *+
Total calcium
Phosphate
(M ) 0 0.17 0.42 0.92 1.42 1.92
6.67 6.62 6.61 6.60 6.60 6.60
(rail) 2.7 4.4 6.3 7.2 8.0 8.2
(re;g) 8.2 9.5 10.6 11.4 11.1 11.6
( m~ll) 8.6 9.8 9.6 10.2 10.2 10.1
Sodium chloride
0.17 0.42 0.92 1.42 1.92
6.50 6.50 6.42 6.37 6.32
4.9 7.2 8.4 9.0 10.0
9.4 10.7 11.1 11.1 12.0
9.5 10.0 10.7 10.5 10.4
Hydrochloric acid
...... ...... ...... ......
6.55 6.32 6.09 5.86
3.2 4.6 7.2 9.7
8.6 9.3 11.7 12.8
9.7 10.3 12.0 13.7
Compound added Potassium chloride
i n c r e a s e w i t h a d d i t i o n s of u p to 1.0 M of t h e c h l o r i d e s , b u t t h e r e w a s l i t t l e c h a n g e w i t h f u r t h e r a d d i t i o n s . S i n c e t h e i n c r e a s e i n t o t a l c a l c i u m is n o t s u f f i c i e n t to a c c o u n t f o r t h e i n c r e a s e i n c a l c i u m ion, it a p p e a r s t h a t excess s o d i u m o r potassium increased the dissociation of calcium citrate. A n a l y s i s of t h e s e r a a n d solids ( T a b l e 4) s h o w e d t h a t a d d i t i o n of 2.3 M p o t a s s i u m c h l o r i d e s h i f t e d a p p r o x i m a t e l y 2 3 % of t h e i n s o l u b l e c a l c i u m a n d 8 % of t h e i n s o l u b l e p h o s p h a t e i n t o t h e s o l u b l e f o r m ; w h e r e a s , a d d i t i o n of 2.3 M s o d i u m c h l o r i d e s h i f t e d 17 a n d 7 % , r e s p e c t i v e l y . T h e t o t a l ( s o l i d s p l u s s e r u m ) c a l c i u m a n d p h o s p h a t e f o u n d b y t h e s e a n a l y s e s a g r e e d w i t h i n 3 % of t h e v a l u e o b t a i n e d b y a n a l y s i s of t h e o r i g i n a l s k i m m i l k . A s s u m i n g t h a t t h e s h i f t i n p h o s p h a t e r e p r e s e n t s s o l u t i o n of t r i e a l e i u m p h o s p h a t e ( m o l a r C a / P r a t i o , 1.5: 1), 1.5 to 1.8 m M (i.e., 1.5 × a v e r a g e c h a n g e in p h o s p h a t e ) of c a l c i u m w i l l h a v e d i s s o l v e d f r o m t h i s source, a n d t h e r e m a i n i n g 1.4 to' 2.8 m M m u s t h a v e b e e n disTABLE 4 Effect of added sodium and potassium chloride on the composition of milk serum and milk solids ~ Concentration (raM)
Component Calcium in serum Calcium in solids b Totals Calcium in original skimmilk b Phosphate in serum Phosphate in solids b Totals Phosphate in original skimmi]k b
Control 7.9 18.4 26.3 26.9 10.6 14.8 25.4 25.8
After KC1 addition
After NaC1 addition
12.3 14.2 26.5 11.8 13.6 25.4
Change caused by KC1
NaC1
11.6 15.7 27.3"
+4.4 --4.2
+3.7 --2.7
] 1.2 13.5 24.7
+1.2 --1.2
+0.6 --1.3
" 2.3 M salt added, pH 6.61. b Skimmilk and solids analyzed after dry ashing; results expressed as mM in original milk.
EFFECT OF VARIOUS SALTS ON THE COAGULATION OF CASEIN
995
placed from casein by the added sodium or potassium. The greater increase in soluble calcium obtained with added potassium than with added sodium chloride probably indicates a greater displacement of calcium from casein by the smaller (less hydrated) potassium ion, but may be within the experimental error. Addition of 160 mM phosphate precipitated most of the soluble calcium from normal milk and from milk in which the soluble calcium content had been increased by addition of sodium or potassium chloride (Table 5). Therefore, TABLE 5 E f f e c t of a d d e d chlorides a n d p h o s p h a t e on t he c o m p o s i t i o n of m i l k u l t r a f i l t r a t e Concentration Treatment
C a l c i um ion
Control, no a d d i t i o n A d d e d 2.3 M KC1 A d d e d 2.3 M NaC1 A d d e d 160 mM p h o s p h a t e A d d e d 2.3 M KC1 a n d 160 mM p h o s p h a t e A d d e d 2.3 M NaC1 a n d 160 mM p h o s p h a t e
2.4 5.1 7.2 0.4 a 0.4" 0.2 a
('raM) Total calcium 7.1 12.0 11.3 3.4 2.9 1.8
" These v a l u e s are below the r a n g e of a c c u r a c y of the c a l c i u m ion method.
considerably more calcium phosphate must have precipitated when 160 mM phosphate was added to milk containing added potassium or sodium chloride than when the same amount of phosphate was added to skimmilk. DISCUSSION" AN"D CON"CLUSIOI~S The results presented in this paper can be explained on the hypothesis that casein gels and the easily dispersed, floceulent coagula frequently observed during the early stages of frozen storage are developed by the interlinking of mixed calcium-potassium easeinate by precipitating calcium phosphate. The calcium phosphate present in normal milk is presumably insufficient to' induce gelation by itself, but Pyne (11) has shown that phosphate accelerated rennet coagulation of calcium easeinate sols, and decreased the coagulation time of milk at 130 ° C. (12). Fresh calcium phosphate precipitates are known to adsorb on t(~ microorganisms (6, 7) and on to casein (5, 6, 7, Ii, 21), so could conceivably interlink casein micelles. Zittle et al. (21) have shown that phosphate increases the degree of aggregation in calcium caseinate sols at 25 ° C. High ]evels of potassium chloride displaced calcium from casein and citrate, increased the solubility of calcium phosphate (Tables 3 and 4), and increased the amount of fresh calcium phosphate formed on addition of phosphate (Table 5). Sodium chloride behaved similarly as far as could be detected b y chemical analysis, but presumably differences in the physical characteristics (e.g., degree of hydration) of potassium and sodium caseinate prevented gel formation in the presence of excess sodium. In other words, mixed calciumpotassium caseinate readily interlinked to form a gel, but mixed calcium-sodium easeinate did not.
996
DYSON ROSE AND I-I. TESSIEt~
D u r i n g freezing, added potassium and sodium chloride a p p a r e n t l y exerted two opposite effects: T h e y i n c r e a s e d c a l c i u m a n d p h o s p h a t e p r e c i p i t a t i o ~ by b r i n g i n g m o r e c a l c i u m into solution, a n d t h e y d e c r e a s e d p r e c i p i t a t i o n by ret a i n i n g a d d i t i o n a l w a t e r i n th e u n f r o z e n state. T h e sh ap e of t h e c u r v e s of F i g u r e s 1 a n d 2 suggests t h a t in th e p r e s e n c e of a d d e d c a l c i u m or p h o s p h a t e t h e first effect, w h i c h r e d u c e s th e s t o r a g e l i f e o'f t h e milk, p r e d o m i n a t e d a t low p o t a s s i u m or s o d i u m levels (0.J M) ; whereas, t h e second or d i l u t i o n effect, w h i c h stabilizes t h e milk, was m o r e i m p o r t a n t at h i g h e r levels (0.5 a n d 1.0 M ) . I n t h e absence of a d d e d c a l c i u m or p h o s p h a t e , t h e d i l u t i o n effect p r e d o m i n a t e d t h r o u g h out ( e x c e p t at 0 ° F . in F i g u r e 2 A ) . H o w e v e r , the a b i l i t y of a d d e d p o t a s s i u m c h l o r i d e to p r o m o t e gel f o r m a t i o n r e d u c e d its s t a b i l i z i n g effect, as c o m p a r e d to t h a t of sodium, a n d the h i g h e r euteetic t e m p e r a t u r e of p o t a s s i u m c h l o r i d e also l i m i t e d the effectiveness of h i g h c o n c e n t r a t i o n s a t 0 ° F . ACKNOWLEDGMENT The authors wish to thank Mr. R. Cyr for technical assistance in this work. REFERENCES (i) BABCOCK, C. J., STROBE~, D. R., YA6~, R. H., AND WINnHAM, E. S. Frozen ~Iomogenlzed Milk. VII. Effect of the Addition of Sucrose and Ascorbie Acid o11 the Keeping Quality of Frozen Homogenized" Milk. J. Dairy Sci., 35" 195. 1952. (2) BOOGA~RT, J. Instability of Milk Due to a High Content of Calcium Ions. Nature, 174:
884. 1954. (3) CHRISTIANSON,G., COULTER, S. T., AND JE.NNESS, R. Two Phenomena Related to Casein Precipitation in Frozen Storage of Milk. J. Dairy Sci., 35 : 481. 1952. (4) DOAN, F. J., AN]) WAma~N, F. G. Observations on the Instability of the Protein Phase of Frozen Concentrated Milk. J. Dairy ScL, 30: 837. 1947. (5) Ev~NHvIS, ~q., AND DEVRIE:S, TH. R. The Condition of Calcium Phosphate in Milk. I. Neth. M~Ik Dairy J., 9: 146. ]955. (6) EVE~HUIS, N., AN]) DI~VRIES, TH. R. The Condition of Calcium Phosphate in Milk. III. h~eth. Mil]~ Dairy J., 10: 101. 1956. (7) GUITIY)NNF~AU,G., AND BEJAMBES, M. Chromoresistance and the Calcium Phosphate Envelopment of Microorganisms in Milk. Lair, 19: 225. 1939. (8) MARIE.R.,J. R., AN]) BOULE.T, M. A. Direct Micro Determination of Calcium in Milk. J. Agr. Food Chem., 4: 720. 1956. (9) POLbEY, J. R. The Mierocolorimetric Determination of Inorganic Phosphate in Plasma and Urine. Ca~. J. Research, E, 27: 265. 1949. (I0) POWI~LL, IV[. E., AN]) PALMER, L. S. Behaviour of Caseinate Sols in a Study of a Hysteresis Phenomenon in the Rennet Coagulation of Milk. J. Dairy Sci., 18: 402. 1935. (11) PYNE., G. T. Rennet ~-Iysteresis and the Calcium Phosphate of Milk. Biochcm. J., 39: 385. 1945. (12) PYNE, G. T. The Heat Coagulation of Milk. II. Variation in Sensitivity of Casein to Calcium Ions. J. Dairy Research, 25: 467. 1958. (13) ROSE., D. Influence of Sugars and Glycerol on Casein Precipitation in Frozen Milk. Can. J. Technol., 34: 145. 1956. (14) SE;I~KLE~S,L., AND S~iE.EiT'S,W. TH. G. M. L'Instabilite du Lair par Suite d'une Augmentation de la Teneur en Ions de Calcium. Lait, 34: 610. 1954. ( 1 5 ) TEISSIER, H., AND ROSE., D. Composition of the Liquid Portion of Frozen Milk. Can. J. Technol., 34: 211. 1956.
EFFECT
OF VARIOUS
SALTS ON THE
COAGULATION
OF CASEIN
997
(16) TESSIER, H., AND I~OSB, D. Calcium Ion Concentration in Milk. J. Dariy Sc~., 41: 351. 1958. (17) T~SSIER, H., ROSE., D., AND LUSENA, C. V. Lactose Crystallization in Frozen Milk. Can. J. Technol., 34: 131. 1956. (18) TUMEIC~[AN,L., F~lVl, H., AND COI~NELY, K. W. The Effect of Lactose Crystallization on Protein Stability in Frozen Concentrated Mi]k. J. Dairy Sci., 37: 830. 1954. (19) VAN SLYKB, L. L., AND BAKEI¢, J. C. The Preparation of Pure Casein. J. Biol. Chem., 38: 127. 1918. (20) WIL])ASIN, H. L., AND DOAN, F. J. Some Additional Influences Affecting the Stability of Concentrated Milk in Frozen Storage. J. Dairy Sci., 34: 438. 1951. (21) ZITTL~, C. A., DEILLA~MONICA, E. S., RUDD, R. i . , AND CUSTEr, 5. I~. Binding of Calcium to Casein: Influence of pH, and Calcium and Phosphate Concentrations. Arch. Biochem. and Biophys., 75: 342. 1958.
OF V A R I O U S S A L T S ON T H E
C O A G U L A T I O N OF C A S E I N
DYSON ROSE A~l) It. TESSIER Division of Applied Biology, National Research Council, Ottawa, Canada SUMMARY
Addition of potsssium or sodium chloride (80 or 130 raM) to skimmilk increased the time the milk could be held in frozen storage without development of 4% precipitate by volume. Addition of phosphate or calcium (10 raM) had the opposite effect. Addition of large amounts of sodium chloride (up to 1.0 M) offset the destabilizing effect of phosphate or calcium at 0, 10, and 20 ° F., but addition of similar amounts of potassium chloride stabilized the smnpIes only at 10 and 20 ° F. Addition of potsssium chloride (1.0 M or more) and of phosphate (100 mM or more) induced gelation of milk at room temperature. Addition of sodium chloride and phosphate did not induce gelation. Addition of either potassium or sodium chloride (2.3 M) shifted approximately 20% of the insoluble calcium and 7.5% of the insoluble phosphate to the soluble forms, displaced eMcium from casein, and increased the dissociation of calcimn citrate. A possible explanation of these effects, based on the interlinking of potassium-caIcimn-caseinate micelles by precipitating calcium phosphate, is presented. Varistions in the concentration of certain inorganic salts or ions, particularly calcium, are known to affect the eoagnlation of casein it1 abnormal milk (2, 14), in milk treated with rennet or other coagulating enzymes (10), and in frozen milk (3, 13). The unfrozen portion of milk stored at --7.5 ° C. contains a p p r o x i m a t e l y 0.8 M salt (ten-fold concentration) p r i o r to lactose crystallization (15), and it can be estimated t h a t up to a 30-f~ld concentration develops as lactose crystallizes. Coagulation of the casein occurs as this high concentration of salt develops (17, 18), but it is not known whether total salt concentration or the concentration of specific components induces the coagulation. U n d e r conditions t h a t do not cause salt crystallization, the concentration of a component in the unfrozen portion of frozen milk depends directly upon its initial concentration and the total amount of w a t e r retained in the unfrozen state b y all components at the storage t e m p e r a t u r e used. Therefore, addition of a salt that promotes casein coagulation should decrease the storage life of milk. Addition of a solute that does not pr(mmte coagulation will dilute the other components by retaining more unfrozen water and, thus, should increase storage life. Glycerol, sucrose, and dextrose (1, 13, 20) and hydrotyzed lactose (18) p r o b a b l y act m a i n l y in this manner. This p a p e r presents the results of a s t u d y of the effect of added salts on casein coagulation. MATERIALS
AND Iv]ETHODS
Milk obtained f r o m m o r n i n g milkings at the Central E x p e r i m e n t a l F a r m , Ottawa, was separated and pasteurized at 150 ° F. for 30 rain. Storage tests were carried out as previously described (13), i.e., samples were frozen in 15-ml. vials and, at suitable intervals (depending on the t e m p e r a t u r e ) , single vials were thawed at 40 ° F. overnight and the contents centrifuged at c a . 500 × g. for ]~eeeived for publication December 19, 1958. 989
990
DYSON
I%OSE
AND
H. TESSIER
10 rain. The graphically interpolated time needed to develop 4% precipitate b y volume is r e p o r t e d as the storage life. Analytical methods were: calcium ion in ultrafiltrate (16), total calcium (8), phosphate (9). Ashing was done in platinum crucibles at 550 ° C. for 15 hr. Calcium easeinate suspensions were p r e p a r e d by dispersing 3 g. of casein p r e p a r e d b y V a n Slyke and B a k e r ' s method (19) in a p p r o x i m a t e l y 95 ml. of saturated calcium hydroxide solutions and a d j u s t i n g the p H to' 6.6 to 6.7 with hydrochloric acid. Sufficient quantities of m o l a r solutions of a m m o n i u m phosphate, a m m o n i u m citrate, and magnesium chloride were then added to give concentrations of phosphate, citrate, and magnesium (30, 9, and 5 raM~l, respectively) comparable to those in milk. One per cent glycerol was added to give the freezing point depression of normal milk while avoiding the possibility of lactose crystallization. These p r e p a r a t i o n s were essentially so'dium- and potassium-free. " R e d i s p e r s e d c a s e i n " was casein-calcium-phosphate complex of fresh skimmilk sedimented at 60,000 × g. (Spineo Model L, No. 30 head, 30,000 r.p.m., 1 hr.) and redispersed b y t r i t u r a t i o n into a solution containing calcium, phosphate, citrate, and magnesium, added as the a m m o n i u m and chleride salts, in a p p r o x i m a t e l y the concentrations found in normal milk. One per cent glycerol was added in lieu of 4% lactose. Since samples of milk can differ m a r k e d l y in their storage life when frozen, and in other characteristics, d a t a for single tests are reported in the tables. However, replicate (duplicate or move) tests done u n d e r the stated conditions, a n d tests done u n d e r varied conditions not r e p o r t e d in detail, confirmed the direction a n d relative magnitude of the changes reported. RESULTS
Freezing tests with one salt added. Small additions (up to 40 mM) of calcium or phosphate sharply decreased the sto'rage life of frozen milk at all temperatures studied (Table 1). The destabilizing effect of calcium was greater than that of phosphate, but a p p e a r e d to be relatively independent of temperature. The destabilizing effect of phosphate was less at 0 ° F. (--18 ° C.) than at 20 ° F. ( - 7 ° C.). Similar effects were observed in calcium caseinate and in redisTABLE 1 Effect of added calcium and phosphate on the storage life of frozen skimmilk" Storage life (days) Addition Nolle 2.5 mM Calcium 5.0 mM Calcium 10.0 mM Calcium 2.5 mM Phosphate 5.0 n~/Phosphate 10.0 mM Phosphate All samples adjusted to the pH
20° F.
10 ° F.
35 40 3.5 20 5 4 3 2 35 25 8 18 2 8 of the control, pI~ 6.53, before freezing.
0° F. 50 30 2 2 50 50 3~)
991
E F F E C T OF V A R I O U S SALTS ON T H E C O A G U L A T I O N OF C A S E I N
persed casein when calcium or phosphate was added in excess of the amounts found in normal milk. The converse effect has been observed u p o n removal of calcium (3, 4). L a r g e additions of either calcium or phosphate at near-normal levels of the other could not be studied because of the limited solubility of calcium phosphate salts. Additions (ff either potassium or sodium chloride to skimmilk or to rcdispersed casein had a definite stabilizing effect d u r i n g frozen storage at all temperatures studied (Table 2). The effect of either salt was more marked at high than at low storage temperatures. TABLE 2 Effect of added sodium and potassium chlorides on the storage life of frozen sklmmilk Storage life
Addition Skimmi]k None 130 mM Sodium chloride 80 mM Potassium chloride Redispersed casein None 130 mM Sodium ch]~i~ 80 mM Potassium chloride
(days)
20 ° F.
10 ° F.
0° P.
25 80 58
32 57 41
63 80 80
2 35 29
2 18 12
16 32 17
Freezing tests with two salts added: I f sodium and potassium chlorides exert this stabilizing action b y reducing the concentration of calcium and phosphate in the unfrozen portion of frozen milk, then larger additions of these salts should offset the destabilizing effect of added calcium or phosphate. To test this possibility, u p to 1 M of sodium or potassium chloride was added to milks containing up to 20 mM added calcium and 40 mM added phosphate. The results obtained with sodium chloride indicate that it was possible to offset completely the destabilizing effect of added calcium ( F i g u r e 1A) or phosphate ( F i g u r e 1B), at all storage t e m p e r a t u r e s studied. On the other hand, potassium chloride offset the effect of added calcium at 20 ° F. ( - 8 ° C.), and p a r t i a l l y offset it at 10 ° F. (--12 ° C.), but decreased storage life of two samples and the control, at 0 ° F. (--18 ° C.) ( F i g u r e 2A). Also, potassium chloride offset the effect of added phosphate only p a r t i a l l y at 20 and 10 ° F., and did not stabilize a n y sample with added phosphate at 00 F. ( F i g u r e 2B). The i n a b i l k y of high concentrations of potassium chloride to offset the effects of added calcium or phosphate at 0 ° F. m a y be explained by the assumption t h a t crystal~ lization of the potassium chloride occurred because eutectic conditions were exceeded. I t is a p p a r e n t in F i g u r e s 1 and 2 t h a t there was some tendency for the lowest level (0.1 M) of sodium or potassium chloride to increase the destabilizing effect of added calcium o'r phosphate. A possible explanation of this is given u n d e r Discussion. Tests without freezi~g. P r e l i m i n a r y tests had indicated t h a t addition of potassium chloride and a neutral phosphate solution to milk caused the casein
992
DYSON
ROSE
AND
If. TESSIEI¢
calcium
A-added
150
O°F.
'°°
20"F.
IO°F. /
?o
o LLI
0
--
B-added
I.L
J
0,5
1.0
1
l
,,I
0.5
1.0 0
7: !
0.5
phosphate
f / l-! /j.o2 i_o _..o, I 7/" IO°FJ
150|
I00
!
-
OF
/
oIV//
0.5
~'O'F,
~-I0
~
0
1
1.0
o
lio
o.~°
o l- o.o
LO 0 0,5 LO 0 ADDED SODIUM CHLORIDE, M
0,5
LO
Fro. 1. Effect of adde,] sodium chloride on storage life of frozen skimmilk containing a~lded calcium or phosphate. F i g u r e s on curves indicate concentration, in raM, of added calcium ( A ) or p h o s p h a t e ( B ) . Arrows attached to points Jndict~te t h a t the last sample, taken at tho time shown, had not coagulated. E a c h set of tests (four curves) w~s from a separate sample of milk.
to gel within a few hours at room temperature. The rate of gelation varied between samples of milk, but within any one sample consistent results were obtained. Gelatiou could be iuduced by addition of phosphate to milk containing i M or more added potassium chloride per liter, and gelation time decreased progressively with increasing potassium chloride concentratio~a. When 2.3 M potassium c h l o r i d e w a s present, addition of 240, 160, and 120 mM phosphate per liter caused gelatioli in .5, 2.5, and 4 hr., respectively. Increasing the lactose concentration delayed gelation slightly; increasing citrate delayed it markedly. At a fixed potassium ~hloride and phosphate level, gelation time increased with decreasing p H and with decreasing temperature. A similar gelation was obtained with redispersed casein preparations, provided calcium, phosphate, and potassium chloride were included in the resuspending medium or added to the suspension. Conditions leading to the formation of a calcium phosphate precipitate appeared to be a prerequisite for gel formati(m. I t was also possible to cause gelation in skimmilk by acidifying it
EFFECT
OF VARIOUS
A-added
SALTS ON THE
COAGULATION
~,
20*F,
I 0 °F.
I00
f
5C
/--,o
o-" ---------o
0 _o
993
calcium O°F.
~_
OF CASEIN
I
I
.,J
0
0.5
1.0
/
o
I
I
I
0
0.5
1.0
i 0
r 0.5
J 1.0
.J
B-added
phosphate
o ¢n 150
20°F.
IO'F.
O*F.
--0 tO0
o/° 50
-I0
o - - o ~ o
? 0
-
\40 i 0.5
-
1.0
~°~--~-Z~-~ l 0
e
i
0,5
1.0
20 /
i 0.5
o
1.0
o0
ADDED POTASSIUM CHLORIDE, M F I G . 2. added
Effect
calcium
of added
or phosphate.
potassium Details
chloride
on storage
as in Figure
life of frozen
skimmilk
cont~iaing
1.
to p H 5.2, adding 160 mM phosphate, then neutralizing so as to induce extensive precipitation of calcium phosphate. Gelation could not be induced either in skimmilk or in casein resuspensions by the addition of phosphate and sodium chloride. Addition of sodium chloride to a milk containing 160 mM added phosphate and 1.5 M potassium chloride delayed gelation; aliquots with 0, 0,8, and 1.5 M added sodimn chloride gelled in 100, 180, and 900 rain., respectively. Addition of sodium chloride to' milk caused a greater increase ill acidity than did addition of potassimn chloride (Table 3), but addition of sufficient sodium hydroxide to maintain the original p H did not promote gelatiml in the presence of phosphate and sodium chloride. To determine what effect added sodium and potassium chlorides had on the distribution of salts in milk, ultrafiltrates [obtained as in (16)], and the Supernatant and sedimented solids obtained by centrifuging at 60,000 × g., were analyzed. Analysis of the ultrafiltrates (Table 3) showed that added potassium or sodium chloride eaused a marked increase in calcium ion, total calcium, and total phosphate. Even with sodium chloride, the increase in calcium ion concentration was more than three-fold that caused by addition of hydrochloric acid to give the same pH. Total ultrafiltrable calcium and phosphate appeared to
DYSON ROSE AND H. TESSIER
994
TABLE 3 Analysis of ultrafiltrates from skimmilk with added salt or acid Concentration added
Fi~lal pH
Ca *+
Total calcium
Phosphate
(M ) 0 0.17 0.42 0.92 1.42 1.92
6.67 6.62 6.61 6.60 6.60 6.60
(rail) 2.7 4.4 6.3 7.2 8.0 8.2
(re;g) 8.2 9.5 10.6 11.4 11.1 11.6
( m~ll) 8.6 9.8 9.6 10.2 10.2 10.1
Sodium chloride
0.17 0.42 0.92 1.42 1.92
6.50 6.50 6.42 6.37 6.32
4.9 7.2 8.4 9.0 10.0
9.4 10.7 11.1 11.1 12.0
9.5 10.0 10.7 10.5 10.4
Hydrochloric acid
...... ...... ...... ......
6.55 6.32 6.09 5.86
3.2 4.6 7.2 9.7
8.6 9.3 11.7 12.8
9.7 10.3 12.0 13.7
Compound added Potassium chloride
i n c r e a s e w i t h a d d i t i o n s of u p to 1.0 M of t h e c h l o r i d e s , b u t t h e r e w a s l i t t l e c h a n g e w i t h f u r t h e r a d d i t i o n s . S i n c e t h e i n c r e a s e i n t o t a l c a l c i u m is n o t s u f f i c i e n t to a c c o u n t f o r t h e i n c r e a s e i n c a l c i u m ion, it a p p e a r s t h a t excess s o d i u m o r potassium increased the dissociation of calcium citrate. A n a l y s i s of t h e s e r a a n d solids ( T a b l e 4) s h o w e d t h a t a d d i t i o n of 2.3 M p o t a s s i u m c h l o r i d e s h i f t e d a p p r o x i m a t e l y 2 3 % of t h e i n s o l u b l e c a l c i u m a n d 8 % of t h e i n s o l u b l e p h o s p h a t e i n t o t h e s o l u b l e f o r m ; w h e r e a s , a d d i t i o n of 2.3 M s o d i u m c h l o r i d e s h i f t e d 17 a n d 7 % , r e s p e c t i v e l y . T h e t o t a l ( s o l i d s p l u s s e r u m ) c a l c i u m a n d p h o s p h a t e f o u n d b y t h e s e a n a l y s e s a g r e e d w i t h i n 3 % of t h e v a l u e o b t a i n e d b y a n a l y s i s of t h e o r i g i n a l s k i m m i l k . A s s u m i n g t h a t t h e s h i f t i n p h o s p h a t e r e p r e s e n t s s o l u t i o n of t r i e a l e i u m p h o s p h a t e ( m o l a r C a / P r a t i o , 1.5: 1), 1.5 to 1.8 m M (i.e., 1.5 × a v e r a g e c h a n g e in p h o s p h a t e ) of c a l c i u m w i l l h a v e d i s s o l v e d f r o m t h i s source, a n d t h e r e m a i n i n g 1.4 to' 2.8 m M m u s t h a v e b e e n disTABLE 4 Effect of added sodium and potassium chloride on the composition of milk serum and milk solids ~ Concentration (raM)
Component Calcium in serum Calcium in solids b Totals Calcium in original skimmilk b Phosphate in serum Phosphate in solids b Totals Phosphate in original skimmi]k b
Control 7.9 18.4 26.3 26.9 10.6 14.8 25.4 25.8
After KC1 addition
After NaC1 addition
12.3 14.2 26.5 11.8 13.6 25.4
Change caused by KC1
NaC1
11.6 15.7 27.3"
+4.4 --4.2
+3.7 --2.7
] 1.2 13.5 24.7
+1.2 --1.2
+0.6 --1.3
" 2.3 M salt added, pH 6.61. b Skimmilk and solids analyzed after dry ashing; results expressed as mM in original milk.
EFFECT OF VARIOUS SALTS ON THE COAGULATION OF CASEIN
995
placed from casein by the added sodium or potassium. The greater increase in soluble calcium obtained with added potassium than with added sodium chloride probably indicates a greater displacement of calcium from casein by the smaller (less hydrated) potassium ion, but may be within the experimental error. Addition of 160 mM phosphate precipitated most of the soluble calcium from normal milk and from milk in which the soluble calcium content had been increased by addition of sodium or potassium chloride (Table 5). Therefore, TABLE 5 E f f e c t of a d d e d chlorides a n d p h o s p h a t e on t he c o m p o s i t i o n of m i l k u l t r a f i l t r a t e Concentration Treatment
C a l c i um ion
Control, no a d d i t i o n A d d e d 2.3 M KC1 A d d e d 2.3 M NaC1 A d d e d 160 mM p h o s p h a t e A d d e d 2.3 M KC1 a n d 160 mM p h o s p h a t e A d d e d 2.3 M NaC1 a n d 160 mM p h o s p h a t e
2.4 5.1 7.2 0.4 a 0.4" 0.2 a
('raM) Total calcium 7.1 12.0 11.3 3.4 2.9 1.8
" These v a l u e s are below the r a n g e of a c c u r a c y of the c a l c i u m ion method.
considerably more calcium phosphate must have precipitated when 160 mM phosphate was added to milk containing added potassium or sodium chloride than when the same amount of phosphate was added to skimmilk. DISCUSSION" AN"D CON"CLUSIOI~S The results presented in this paper can be explained on the hypothesis that casein gels and the easily dispersed, floceulent coagula frequently observed during the early stages of frozen storage are developed by the interlinking of mixed calcium-potassium easeinate by precipitating calcium phosphate. The calcium phosphate present in normal milk is presumably insufficient to' induce gelation by itself, but Pyne (11) has shown that phosphate accelerated rennet coagulation of calcium easeinate sols, and decreased the coagulation time of milk at 130 ° C. (12). Fresh calcium phosphate precipitates are known to adsorb on t(~ microorganisms (6, 7) and on to casein (5, 6, 7, Ii, 21), so could conceivably interlink casein micelles. Zittle et al. (21) have shown that phosphate increases the degree of aggregation in calcium caseinate sols at 25 ° C. High ]evels of potassium chloride displaced calcium from casein and citrate, increased the solubility of calcium phosphate (Tables 3 and 4), and increased the amount of fresh calcium phosphate formed on addition of phosphate (Table 5). Sodium chloride behaved similarly as far as could be detected b y chemical analysis, but presumably differences in the physical characteristics (e.g., degree of hydration) of potassium and sodium caseinate prevented gel formation in the presence of excess sodium. In other words, mixed calciumpotassium caseinate readily interlinked to form a gel, but mixed calcium-sodium easeinate did not.
996
DYSON ROSE AND I-I. TESSIEt~
D u r i n g freezing, added potassium and sodium chloride a p p a r e n t l y exerted two opposite effects: T h e y i n c r e a s e d c a l c i u m a n d p h o s p h a t e p r e c i p i t a t i o ~ by b r i n g i n g m o r e c a l c i u m into solution, a n d t h e y d e c r e a s e d p r e c i p i t a t i o n by ret a i n i n g a d d i t i o n a l w a t e r i n th e u n f r o z e n state. T h e sh ap e of t h e c u r v e s of F i g u r e s 1 a n d 2 suggests t h a t in th e p r e s e n c e of a d d e d c a l c i u m or p h o s p h a t e t h e first effect, w h i c h r e d u c e s th e s t o r a g e l i f e o'f t h e milk, p r e d o m i n a t e d a t low p o t a s s i u m or s o d i u m levels (0.J M) ; whereas, t h e second or d i l u t i o n effect, w h i c h stabilizes t h e milk, was m o r e i m p o r t a n t at h i g h e r levels (0.5 a n d 1.0 M ) . I n t h e absence of a d d e d c a l c i u m or p h o s p h a t e , t h e d i l u t i o n effect p r e d o m i n a t e d t h r o u g h out ( e x c e p t at 0 ° F . in F i g u r e 2 A ) . H o w e v e r , the a b i l i t y of a d d e d p o t a s s i u m c h l o r i d e to p r o m o t e gel f o r m a t i o n r e d u c e d its s t a b i l i z i n g effect, as c o m p a r e d to t h a t of sodium, a n d the h i g h e r euteetic t e m p e r a t u r e of p o t a s s i u m c h l o r i d e also l i m i t e d the effectiveness of h i g h c o n c e n t r a t i o n s a t 0 ° F . ACKNOWLEDGMENT The authors wish to thank Mr. R. Cyr for technical assistance in this work. REFERENCES (i) BABCOCK, C. J., STROBE~, D. R., YA6~, R. H., AND WINnHAM, E. S. Frozen ~Iomogenlzed Milk. VII. Effect of the Addition of Sucrose and Ascorbie Acid o11 the Keeping Quality of Frozen Homogenized" Milk. J. Dairy Sci., 35" 195. 1952. (2) BOOGA~RT, J. Instability of Milk Due to a High Content of Calcium Ions. Nature, 174:
884. 1954. (3) CHRISTIANSON,G., COULTER, S. T., AND JE.NNESS, R. Two Phenomena Related to Casein Precipitation in Frozen Storage of Milk. J. Dairy Sci., 35 : 481. 1952. (4) DOAN, F. J., AN]) WAma~N, F. G. Observations on the Instability of the Protein Phase of Frozen Concentrated Milk. J. Dairy ScL, 30: 837. 1947. (5) Ev~NHvIS, ~q., AND DEVRIE:S, TH. R. The Condition of Calcium Phosphate in Milk. I. Neth. M~Ik Dairy J., 9: 146. ]955. (6) EVE~HUIS, N., AN]) DI~VRIES, TH. R. The Condition of Calcium Phosphate in Milk. III. h~eth. Mil]~ Dairy J., 10: 101. 1956. (7) GUITIY)NNF~AU,G., AND BEJAMBES, M. Chromoresistance and the Calcium Phosphate Envelopment of Microorganisms in Milk. Lair, 19: 225. 1939. (8) MARIE.R.,J. R., AN]) BOULE.T, M. A. Direct Micro Determination of Calcium in Milk. J. Agr. Food Chem., 4: 720. 1956. (9) POLbEY, J. R. The Mierocolorimetric Determination of Inorganic Phosphate in Plasma and Urine. Ca~. J. Research, E, 27: 265. 1949. (I0) POWI~LL, IV[. E., AN]) PALMER, L. S. Behaviour of Caseinate Sols in a Study of a Hysteresis Phenomenon in the Rennet Coagulation of Milk. J. Dairy Sci., 18: 402. 1935. (11) PYNE., G. T. Rennet ~-Iysteresis and the Calcium Phosphate of Milk. Biochcm. J., 39: 385. 1945. (12) PYNE, G. T. The Heat Coagulation of Milk. II. Variation in Sensitivity of Casein to Calcium Ions. J. Dairy Research, 25: 467. 1958. (13) ROSE., D. Influence of Sugars and Glycerol on Casein Precipitation in Frozen Milk. Can. J. Technol., 34: 145. 1956. (14) SE;I~KLE~S,L., AND S~iE.EiT'S,W. TH. G. M. L'Instabilite du Lair par Suite d'une Augmentation de la Teneur en Ions de Calcium. Lait, 34: 610. 1954. ( 1 5 ) TEISSIER, H., AND ROSE., D. Composition of the Liquid Portion of Frozen Milk. Can. J. Technol., 34: 211. 1956.
EFFECT
OF VARIOUS
SALTS ON THE
COAGULATION
OF CASEIN
997
(16) TESSIER, H., AND I~OSB, D. Calcium Ion Concentration in Milk. J. Dariy Sc~., 41: 351. 1958. (17) T~SSIER, H., ROSE., D., AND LUSENA, C. V. Lactose Crystallization in Frozen Milk. Can. J. Technol., 34: 131. 1956. (18) TUMEIC~[AN,L., F~lVl, H., AND COI~NELY, K. W. The Effect of Lactose Crystallization on Protein Stability in Frozen Concentrated Mi]k. J. Dairy Sci., 37: 830. 1954. (19) VAN SLYKB, L. L., AND BAKEI¢, J. C. The Preparation of Pure Casein. J. Biol. Chem., 38: 127. 1918. (20) WIL])ASIN, H. L., AND DOAN, F. J. Some Additional Influences Affecting the Stability of Concentrated Milk in Frozen Storage. J. Dairy Sci., 34: 438. 1951. (21) ZITTL~, C. A., DEILLA~MONICA, E. S., RUDD, R. i . , AND CUSTEr, 5. I~. Binding of Calcium to Casein: Influence of pH, and Calcium and Phosphate Concentrations. Arch. Biochem. and Biophys., 75: 342. 1958.