Effect of Ion Exchange Resins on Composition of
Milk and Its Fractions
Milk and Food Research, National Center for Urban and Industrial Health
U. S. Public Health Service, Cincinnati, Ohio
Abstract
Ultraeentrifugation and dialysis studies
were made to determine the distribution of
calcium, phosphorus, and nitrogen in milk
treated with ion exchange resins to remove
radionuclides. In System I, milk was
passed through Dowex 2-X8 ( C l : P : C i t )
resin, acidified to p H 5.35 with citric acid,
passed through IR-120 ( C a : M g : K : N a )
resin, and neutralized to p H 6.8 with K O H .
In System I I , the same cycle was followed,
except that HC1 was used to acidify milk
and the milk was neutralized with IRA-68
(P :Cit :OH) weak base anionic resin.
A comparison of the test results for
treated samples and those for the control
sample showed increased concentrations in
the casein nitrogen and in acid soluble
phosphorus in serum, and a decreased
amount of sedimented casein. The amount
of calcium and acid soluble phosphorus as-
sociated with the sedimented caseinates
also decreased. The extent of these altera-
tions depended upon the type of acid used
to acidify the milk. The effect of these
changes in the ionic balance on the co-
agulation of milk by rennin and ethanol
is briefly discussed.
To remove cationic radionuclides (~°Sr,
"°Ba, and I~Cs) from milk, it is acidified to
p t I 5.3 to 5.4 with eitric or hydrochloric acid
and passed through a column containing Dowex ~
50W-X8 or IR-120 cationic resin in the
C a : M g : K : N a cycle. The milk is neutralized
to p H 6.6 to 6.7 with K O H (4, 11), Dowex
l-X8 ( C I : O H ) , or Dowex 2-X8 ( C I : P : C i t : O H )
anionic resin, depending upon the acidifying
agent (8, 9). To remove ~ I , no pretreatment o f
milk is necessary, but the milk is passed through
a Dowex-2X8 ( C l : P : C i t ) resin column (10).
Previous investigations indicated that the best
possible integrated system for the removal of
all radionuclides was that in which the anionic
resin column preceded the cationic resin col-
umn. In this integrated system, the milk is
Received for publication November 25, 1966.
Mention of commercial products does not imply
endorsement by the Public Health Service.
809
G. K. MURTHY
passed through a Dowex-2X8 ( C l : P : C i t ) col-
umn, acidified to p H 5.3 with 0.75 ~ citric acid,
passed through an IR-120 ( C a : M g : K : N a )
cationic resin, and neutralized to p H 6.6 to 6.8
with K O H (5, 6). I f HC1 is used to acidify
the milk, alternately IRA-68 (P :Cit :OH) weak
base anion exchange resin may be used to neu-
tralize the milk (7). Because of the various
treatments applied to the milk, the nature of
ionic equilibria may be drastically altered. Since
ionic equilibria is one of the important factors
that influence the stability of milk proteins, the
investigation reported here was undertaken to
determine the nitrogen, calcium, and phosphorus
distribution between phases of the milk system.
Experimental Procedure
Materials. Milk samples were obtained on the
Cincinnati market. The cation resin used was
Amberlite IR-120 (H÷), and the anion resins
were Dowex 2-X8 (CI-) and Amberlite IRA-68
I~A-68 (Cl).
Resin regeneration. Methods for the regen-
eration of IR-120 and Dowex 2-X8 resins and
operation of the columns have been described
previously (10, 11). The IRA-68 (C1-) resin
was converted into the P :Cit :OH cycle as fol-
lows: A charging solution was prepared to
contain 5 m ~ of citrate and 100 m ~ of phos-
phate (Natt2PO,'H20) per liter of solution and
the p H of solution was adjusted to 9.0 with
6 ~ NaOH prior to its being made to volume
with deionized water. Approximately 100 ml
of resin was regenerated with 2,500 ml of solu-
tion (8).
Milk treatment. Two systems were consid-
ered. I n System I, milk was passed through
Dowex 2-X8 ( C l : P : C i t ) resin at 0.5 resin bed
volume (rbv) per minute, acidified to p H 5.35
with 0.5 ~¢ citric acid, passed through IR-120
( C a : M g : K : N a ) resin at 0.]25 rbv, and neu-
tralized to p H 6.8 with 1.0 ~[ KOH. In System
I I , the same cycle was followed, except that
1.0 ~ HC1 was used to acidify the milk and
the milk was neutralized to p H 6.8 with I R A -
68 ( P : C i t : O H ) resin by the batch technique
(8, 9).
Centrifugation. Samples of whole milk were
centrifuged at 1,800 rpm for 10 min and al-
810 ~URTHY

lowed to stand at ice-bath temperatures for
30 rain to harden the cream layer. The skim-
milk was removed by siphoning and centrifuged
at 4 C for 120 rain and at 105,500 g with a no.
40 rotor in a Spinco Model L preparative ultra-
centrifuge. The supernatants, free of lipid mate-
rial, were removed with a hypodermic syringe,
and care was taken not to disturb the sedi-
mented syrupy layer above the easeinate frac-
tion. The syrupy layer was carefully removed.
The centrifuge tubes were held inverted, and
the residual serum and lipid materials were re-
moved by rinsing with deionized water. The
caseinate fraction was not washed, but was
dispersed in deionized water with a minimum
of NaOH. All fractions, including the control
and resin-treated skimmilk, were analyzed for
total nitrogen (TN), noneasein nitrogen (NCN),
total phosphorus ( T P ) , acid-soluble phospho-
rus ( A S P ) , and total calcium (Ca). The ob-
served results were corrected for dilution caused
by the addition of acid or alkali, or both.
Dialysis. Sixty milliliters of deionized water
were dialyzed against three liters o£ milk at
4 C for 24 hr (2).
Rennet coagulation. Tests were performed
as described previously (11).
Alcohol coagulation. Tests were made accord-
Lug to Davies and White (3).
Analytical methods. Titratable acidity in
milk was measured by titrating 10-ml portions
against 0.1 ~[ N a O t t and using five drops of
phenolphthalein as indicator; results were ex-
pressed as per cent of lactic acid. Methods
for determination of p i t and A S P are the
same as previously described (10). The TP
on an aliquot of the ash solution (1) was men-
sured by developing the color as of the ASP.
The Ca content of samples was determined by
the oxalate-permanganate titration method (14).
Values for OP were obtained by subtracting
values for A S P from those for TP. The nitro-
gen distribution in milk and its fractions was
determined according to Shahani and Sommer
(15). Values for CN were obtained by sub-
tracting values for NCN from those for TN.
Results and Discussion
Centrifugation results. Data on nitrogen,
calcium, and phosphorus distribution are pre-
sented in Tables 1 and 2.
When compared with the control sample,
treated milk showed no significant differences
in the TN, NCN, TP, and A S P contents, except
that in System I I the Ca and A S P decreased
from 121 and 62.0 mg to 99.2 and 36.8 rag,
respectively. These decreases in the Ca and
A S P resulted from neutralization of acidified
milk with the weak base IRA-68 ( P : C i t : O H )
anionic resin (7), in which Ca is adsorbed as a
citrate complex (8). The CN in serum increased
from 60 to 430 mg (I) and 56 to 90 mg ( I I ) .
When milk is acidified with citric acid, the Ca
ionized from the protein-bound form is com-
plexed with the citric acid and is stabilized as
the p H is increased to 6.8; the caseitt in sermn,
therefore, possibly exists in a complex with
the alkali metals. When milk is acidified with
ttCl, the Ca ionized from the protein-bound
form does not form complexes with the chlo-
rides; consequently, the Ca can bind with the
casein and become stabilized upon neutraliza-
tion to p H 6.8.
When the protein-bound salts are ionized,

TABLE 1
Composition of skimmilk, serum, and sedimented easeinate"--System I
Control Resin-treated
Analyses Milk Serum Caseinate Milk Serum Caseinate
(mg/lO0 ml of milk)
Total nitrogen
(TN) 559 ___12b 181 -+12 -- 549 -+-15 432 __+24 --
Noncasein nitro-
gen (NCN) 121 -+11 -- -- 119 +--11 -- --
Casein nitro-
gen (CN) 438 +-19 60 --+18 390 +-18 430 +-19 313 --+24 130 ___13
Total phosphorus
(TP) 92.0+- 4.0 45.7+- 1.9 48.3___3.0 92.0+- 3.0 77.6-+ 1.3 11.0-+ 4.6
Acid-soluble
phosphorus
(ASP) 62.0--+ 3.0 35.4+- 1.8 27.4-+ 1.5 62.0+- 2.0 53.7-+ 1.3 5.0-+ 4.5
Organic phos-
phorus (OP) 31.0+- 3.0 10.3+- 0.9 20.7+__0.8 31.0-+ 3.0 23.9-+ 2.0 6.1+- 0.6
Total cal-
cium (Ca) 119 +- 4 -- 73.8+- 0.6 116 -----13 -- 16.0-+ 1.3
" Average of seven samples.
b Ranges in the above values represent standard deviations among milk samples.
J. ]>AIRY SCIENCE YOL. 50, NO. 6
 EXCHANGE RESINS AND MILK COMPOSITION 811

the ability of these to recombine with the casein

÷l÷l÷l ÷14141 micelles may depend upon the type of acid
added to the milk, the attainment of equilibrium
between the dissolved and the complexed forms,
and physical restriction• The resulting calcium-
easeinate-phosphate complex will never be the
• ° •
same, however, as that originally present in
milk. Therefore, one may expect the caseinates
in treated milk to react differently to certain
processing conditions than the caseinates in un-
treated milk•
The TP and A S P in serum increased from
45•7 to 77•6 mg and 35•4 to 53.7 rag, respec-
o ~.o.
tively ( I ) . These large increases resulted from
the association of OP and A S P with the in-
creased CN content. I n System I I , however,
these increases were small; increases in TP and
A S P were from 43.0 to 47.9 mg and 33.6 to
36•8 rag, respectively• This was to be expected
since the increase in CN was only from 56.0
to 90.0 rag. Consequently, results for sedi-
mented casein from treated samples showed de-
N N~ 4dd creases of A S P from 27.4 to 5.0 mg ( I ) and
27.6 to 15.4 mg ( I I ) , and decreases of Ca from
73.8 to 16•0 mg ( I ) and 79•1 to 52.8 mg ( I I ) .
The CN in the sediment decreased from 390
to 130 mg (1) and from 407 to 318 mg (II).
¢q 41 4141 2 4,4, Computation of various ratios for the sedinmnted
casein and comparison of results with those for
the control sample will show that under the ex-
perimental conditions of this study less Ca
and A S P are associated with the sedinlented
casein, except that O P / C N values of 0.047
0.004 (I) and 0.049 -+- 0.003 ( I I ) for the treat-
ed samples were comparable to the value for the
o control sample, 0.051 ± 0.001.
The ultracentrifugate fraction of sedimented
syrup increased in the processing treatment.
o
The significance of this fraction can be eluci-
o w.~ dated by further study• F o r System I I , analy-
~9
sis of this fraction showed different composi-
tion than that of the sedimented casein• No
analysis of this fraction was made for System
I, but it is believed that the chemical composi-
tion would differ similarly.
Dialysis studies. To confirm some of the re-
• ~
sults observed with cent~Jfugation, milk samples
that had been treated with anionic and cationic
resins were dialyzed as described under Ex-
perimental Procedure. Preliminary data in-
dicated that passage of milk through Dowex-
2X8 (CI:P :Cit) resin did not significantly af-
fect the p H or the distribution of Ca and A S P
of milk or the dialysate. Therefore, dialysis of
c9 milk treated with anionic resin was discontinued
in subsequent trials• Data in Table 3 show that
in treated milk the dialyzable Ca and A S P in-
creased by 38.8 and 34.9% (I) and 9.8 and
J. D A I R Y S C I E I ~ C E "~OL, 5 0 , N O . 6
812 MURTYIY

TABLE 3
Calcium and phosphorus content of milk and dialysate
Resin-treateda Resin-treatedb
Sample Ana/yses Control~ System I System I][
(mg/lO0 ml of mille)
Calcium 118 +-6 114 +-__10 95.8+--1.5
Milk
Acid-soluble phosphorus 67.6+-2.8 63.7+- 6.6 59.7+-3.0
Calcium 40.5+-1.7 83.3___10.0 42.7___0.8
Dialysate
Acid-soluble phosphorus 35.2+-2.5 55.4± 3.9 37.2+-2.0
Average of five samples.
b Average of two samples.

10.0% ( I I ) , respectively. These d a t a agree Table 4 shows t h a t the t r e a t e d milk ( I ) neu-

well with those obtained by c e n t r i f u g a t i o n . The
increased Ca a n d A S P in the serum o f treated
milk a p p e a r to exist in the soluble i n o r g a n i c
f o r m (13).
Coagulatio~ of milk by rennin. I n the coagu-
lation tests, the a m o u n t o f 1 • CaC12 r e q u i r e d to
p r o d u c e eoagulum c o m p a r a b l e to t h a t in control
milk was determined. A f t e r coagulation, the
coagulum was allowed to s t a n d f o r 1 h r b e f o r e
the n a t u r e o f curd a n d whey was determined.
P r e l i m i n a r y e x p e r i m e n t s were made to deter-
mine the effect of neutralizing milk to p H o f
6.4 to 7.0 with K O H u p o n coagulation o f milk.
tralized to p H 6.8 to 7.0 is stable to coagulation
with r e n n i n and alcohol. Coagulation with ren-
n i n at p H 6.4 to 6.6 with less CaCI~ a d d e d
indicates t h a t in spite o f the excess citrate
p r e s e n t , the calcium-citrate complex is less stable
a t p H 6.4 to 6.6 t h a n a t 6.8 to 7.0; t h e r e f o r e ,
some free calcium is present. As p H increased
or acidity decreased, an increased a m o u n t of
CaC12 was required to yield coagulum similar
to that obtained with the control. The coagula-
tion time was p r o l o n g e d logarithmically with
the a m o u n t o f calcium added to the milk (12).
Analysis o f seven milk samples at 30 C showed

TABLE 4
Effect of radionuclide removal process on titratable acidity and coagulation of milk by ethanol and
rennin
Titra- Rennin coagulation
Acid Neutra- table Eth-
treat- lizing acid- anol Coag.
Sample ment agent pl=[ ity conc CaCh time pl=[
(ml 1.0 ~¢
(% per
Lavtic) (%) liter) (see)
1 Control None None 6.89 0.14 85 0.0 402 ......
2 Control None None 6.90 0.14 85 0.0 356
3 Control None None 6.82 0.14 82 0.0 385 6.70
1 Treated (1) Citric KOIt 6.38 0.26 56 4.0 166 ......
2 Treated (1) Citric KOtt 6.40 0.23 70 4.0 144
3 Treated (1) Citric KOH 6.39 O.26 54 4.0 119 6.15
1 Treated (1) Citric KOH 6.60 0.20 90 6.0 162 ......
2 Treated (1) Citric KOIt 6.62 0.20 90 5.0 202
3 Treated (1) Citric KOH 6.60 0.23 80 5.0 141 6.20
1 Treated (1) Citric KOH 6.80 0.16 None 7.0 202 ......
2 Treated (1) Citric KO]:[ 6.8O 0.16 None 7.0 269
3 Treated (1) Citric KOIt 6.80 0.19 None 6.0 189 6.40
1 Treated (1) Citric KOH 7.02 ().13 None 9.0 327 ......
2 Treated (1) Citric KOtI 7.00 0.16 None 8.0 360
3 Treated (1) Citric KOH 7.00 0.15 None 9.0 319 6.50
l Control None None 6.80 0.16 .... 0.0 395 ......
2 Control None None 6.80 0.14 .... 0.0 435 .....
3 Control None None 6.80 0.16 .... O.0 385 ......
IRA-68
1 Treated ( I I ) tIC1 ( P : C i t :OH) 6.80 0.16 .... 3.0 323 ......
[R.A-68
2 Treated ( I I ) tIC1 (P :Cit :OH) 6.80 0.16 .... 2.5 280 ......
IRA-68
3 Treated ( I I ) tIC1 ( P : C i t :OH) 6.80 0.14 .... 3.0 367 ......

J. DAIRY SCIENCE VOL.: 50, NO. 6

 EXCHANGE RESINS AND MILK COMPOSITION
t h a t 5.8 ___ 0.5 ml of 1 ~I CaCl~ was r e q u i r e d to
o b t a i n s a t i s f a c t o r y coagulum. The coagulation
times f o r the control a n d the t r e a t e d milks (3)
were 585 ± 85 a n d 380 + 30 sec, respectively.
The citric acid c o n t e n t of t r e a t e d milk is 3.85
to 4.0 g p e r liter, as c o m p a r e d to 1.75 to 1.85 g
p e r liter f o r u n t r e a t e d m i l k ; therefore, on a (4)
m o l a r basis, 1 mM of Ca m u s t be added f o r
every 2 mM excess citric acid p r e s e n t in milk.
S p o t checks on the p H of the coagulum in- (5)
dicated a d r o p of 0.3 to 0.4 unit, because of
acidic reaction o f CaCI~; whereas, the p H d r o p
with the control s a m p l e was a b o u t 0.1 to 0.2.
(6)
Linlited results were o b t a i n e d f r o m System
I I . W h e n r e n n i n was added to milk, the co-
a g u l a t i o n time was prolonged, the coagulum
was slushy, a n d the whey was highly turbid. (7)
A d d i t i o n of 2.5 to 3.0 ml of 1 ~ CaCl~ p e r liter
of milk was needed to yield a c o a g u l u m com-
p a r a b l e to t h a t of the control sample. This is
a t t r i b u t e d to depletion of calcium ( t 5 to 2 0 % )
(8)
in milk d u r i n g n e u t r a l i z a t i o n w i t h I R A - 6 8
(P:Cit:0H) resin ( 7 ) . (9)
The above i n v e s t i g a t i o n s revealed t h a t the
nitrogen, calcium, a n d p h o s p h o r u s d i s t r i b u t i o n
between casein micelle a n d the s e r u m p h a s e s (10)
is drastically altered d u r i n g p r o c e s s i n g of milk
to remove radionuclides. Because of these al-
terations, the caseinates are stable to co- (11)
a g u l a t i o n with r e n n i n a n d alcohol ( I ) . I f milk
is to be used f o r m a k i n g cheeses or o t h e r milk
products, its composition o r the processing con- (12)
ditions, or both, m u s t be modified to o b t a i n a
s a t i s f a c t o r y end p r o d u c t .
Acknowledgment
The author is grateful to Dr. J. E. Campbell
for his interest in the problem and to L. Kaylor (13)
for his technical help.
References
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1955. Official Methods of Analysis. 8th ed.
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(2) Davies, D. T., and White, J. C. D. 1960. (15)
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~]3
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J. DAIRY SCIENCE VOL. 50, NO. 6