Precipitation of Milk Proteins by Sodium
Ca rboxymethylcellulose
F. J. CLUSKEY, ~ E. L. THOMAS, and S. T. COULTER
Abstract
A study was made to determine opti-
mmn conditions for precipitation of milk
proteins by sodium carboxymethylcellu-
lose and to elucidate the chemical nature of
the resulting gum-protein complex. Car-
boxymethylcellulose precipitated a maxi-
mum of 90 to 96% of the casein from a
simplified casein system and about 71%
of the total protein (principally casein)
from natural skimmilk. Maximum protein
precipitation occurred at p i t 7.5 in sim-
plified casein systems and results indi-
cate a similar relationship in natural
skimmilk. The reaction was calcium-de-
pendent, and protein-calcium-carboxyme-
thylcellulose bridging appears to be in-
volved at p H levels above the isoelectric
point of casein. At the isoelectrie point
(pH 4.6), carboxymethylcellulose sta-
bilizes casein, preventing precipitation.
The typical cellulose-protein complex con-
tained 51.7% protein, 7.3% ash, 0.44%
calcium, and 10 to 17% carboxymethyl-
cellulose.
Millions of pounds of plant po]ysaccharides
and their derivatives, commonly known as
gums, are used in foods yearly. Gums, by vir-
tue of ~ e i r water-binding ability, are used
primarily to control certain physical char-
acteristics of the respective foods. Some gums,
e.g., guar, locust bean gum, carrageenan, and
sodium carboxymethylcellulose (CMC), react
with and precipitate milk proteins. These pro-
reins often cause the curdled melting appear-
ance of ice cream and wheying off of ice
cream mixes and chocolate drinks. This prop-
erty of precipitating milk proteins has been
introduced as a means of isolating milk pro-
teins in their relatively native state. Tyden
(16) in his British patent proposed an im-
proved method of separating "native albumin"
from milk using CMC. Loewenstein (11) in-
Receive4 for publication January 31, 1969.
1 Scientific Journal Series Paper no. 6740, Min-
nesota Agricultural Experiment Station.
2 Present address: United States Army Natick
Laboratories, Natick, Massachusetts 01760.
1181
Department of Food Science and Industries
University of Minnesota, St. Paul 55101
dicated that the product resulting from gum-
milk protein interaction had an exceptional
water-binding ability. Knowledge concerning
the mechanism of milk protein precipitation
was lacking.
Several theories regarding the mechanism
of gum-protein reaction have been proposed.
Gortner (5) and researchers at Hercules (7)
postulated the coacervation theory which in-
volves the mutual precipitation of hydro-
colloids of opposite charge. Researchers at
Seaplant Chemical Corporation (14, 15), in
their work with carrageenan, attributed the
carrageenan-protein interaction to three mecha-
nisms involving ionic bonding, hydrogen bond-
ing, and van der Waals forces. Investigators
at Marine Colloids (12) stated that in the
presence of certain cations, it is possible for
both the proteinate and earrageenan anions
to form a double salt with the cations, and
this may be insoluble and precipitate. Tyden
demonstrated that a CMC (Cellugel F F 100)
concentration of 0.3 to 0.6% gave maximum
gravity protein separation of about 77% of
the total protein. He did not distinguish be-
tween the types of proteins precipitated, but
classified all proteins sedimented as albumin.
Asana (2), with free-boundary electrophoresis,
demonstrated complex formation between a-ca-
sein, fl-casein, and fl-lactoglobulin with CMC
at low p H values (4.7 to 5.6). He attributed
the protein-CMC complex formation to a pro-
tein cation-CMC anion reaction influenced es-
sentially by p H and the isoelectric point of
the protein. Several investigators (1, 2, 6, 12)
have presented some evidence which indicates
the gum reacts with and precipitates only
casein.
The primary objectives of this investigation
were a) to determine the optimum conditions
for milk protein precipitation by CMC, b) to
determine the composition of the precipitated
product, and c) to elucidate the nature of the
CMC-protein reaction.
Experimental Procedure
Source of milk a~d simplified milk systems.
Skimmilk was obtained by separating whole
milk (37 C) from the University herd. Acid
casefll was prepared by precipitating the ca-
1182 C L U S K E ¥ E T AL.
sein with HC1 (37 C), filtering, washing,
freeze drying, and storing at 5 C.
Simulated milk ultrafiltrate ( S M U F ) was
prepared by the method of Jenness and Koops
(9). Calcium and magnesium-free ultrafil-
trate was prepared by substituting NaC1 for
CaC12 and MgC12 at levels to maintain a con-
stant ionic strength. Similarly, phosphate-
free ultrafiltrate was prepared by substituting
NaCI for KH2PO e
Analytical. Total protein was calculated
from micro-Kjeldahl values using a conver-
sion factor of 6.38. Total calcium was deter-
mined by the method of Jenness (8) and lac-
tose by the colorimetric method of P e r r y and
Doan (13). Total carbohydrate content was
determined by the colorimetric method of Du-
bois et al. (4). The concentration of CMC
was estimated as the difference between total
carbohydrate and lactose. Total solids and
ash were determined according to methods of
analysis by the AOAC (3).
Polyacrylamide gel electrophoresis. Electro-
phoretic patterns of different fractions were
prepared using an E-C Model 470 vertical
cell according to the procedure recommended
by the manufacturer (E-C A p p a r a t u s Corpo-
ration, Philadelphia, Pennsylvania).
:FIG. 1. Effect of concentration of carboxymethylcellulose in skimmilk on appearance of samples
and percentage of total protein in sedimented portion.
J. DAIRY SCIEI~CE VOL. 52, NO. 8
Protein precipitated ( % ) . Unless otherwise
stated, the following standard procedure was
used: Dry CIVIC (Hercules 7M2XF) was added
slowly to a known quantity of the simulated
or natural milk system. The mixture was
stirred 10 rain with a magnetic stirrer, trans-
ferred to centrifuge tubes, placed in a 5 C
refrigerator for 10 to 14 hr, and centrifuged
at 4,000 × g for 20 rain. The supenlatant
was decanted with a syringe, weighed, and
analyzed for protein. The difference between
the total protein in the original system and
that in the supernatant is considered to be
the amount of protein precipitated. I t is re-
ported as percentage of total protein in sedi-
mented portion. I t is recognized that varying
amounts of serum are included in the sedi-
mented portion, depending upon the degree of
sediment compactness.
Results and Discussion
Protein precipitation from natural skimmilk
by carboxymethylccll~dose. Figure 1 shows the
effect of CMC concentration in skimmilk and
centrifugation on the appearance of the sedi-
ment-containing fraction. Settling of the pre-
cipitate by gravity, shown in F i g u r e 1A, was
complete within four hours in all tubes except
 PROTEIN
those containing 0.6 and 0.7% CMC. The
precipitate in these tubes settled at a slower
rate, presumably due to higher viscosity re-
stilting from the greater CMC concentration.
The data show that a maximum of 71% of
the total protein can be precipitated with
0.5% CMC (Hercules 7M2XF). The quantity
of CMC necessary to cause maximum precipita-
tion was also found to vary with the type of
CMC used. Less was required using the higher
viscosity types (Hercules 7 H F or 7 H S F ) .
Effect of carboxymethylcellulose in simpli-
fied milk systems. Since previous investigators
(1, 2, 6, 12) believed that casein is the princi-
pal protein precipitated by CMC, the effects
of cations, p H , and other factors on the de-
gree of protein precipitated were studied in
a simplified milk system (casein dispersed in
SMU~).
The effect of calcium, Figure 2, indicated
that ealcinm plays an important role in the
precipitation of casein by CYIC. Calcium
chloride was added to a 2.02% casein in cal-
cium- and magnesium-free ultrafiltrate at levels
yielding 0 to 160 mg calcium/100 ml. The
p H was adjusted to p H 6.8 to 6.9 with
NaOH. Variations in the amount of NaOH
necessary were compensated for by the addi-
tion of distilled water. Then CMC was added
and the standard procedure followed.
A t p H 6.8 to 6.9, both casein and CMC
are known to be negatively charged. Under
these conditions, formation of a eoacervate or
IOC
Z
"W 8 ~
if)
7~
Z on percentage of total protein sedimented from
ul
I'- 6C
o
e~ 5 0 J (Mg)WlTH0.4% GMG
~
o
4(1
l Sample description
~ 20
LIL
o !0
,~//~~THOUT GMG
N 0
0 40 80 120 160
Go or Mg (mg/lO0 ml)
FIG. 2. Effect of calcium and magnesium on
percentage of total protein sedimented at pH
6.8 to 6.9 from a 2.02% casein solution with and
without treatment with earboxymethylcellulose.
(Hercules 7M2XF.) Protein sedimented using
1,000 × g for one hour.
PRECIPITATION 1183
any ionic precipitate is not probable. There-
fore, it was postulated that CMC and casein
were cross-linking with a third reactant., pos-
sibly calcium.
To determine whether this reaction was spe-
cific for calcium or another divalent cation, a
similar experiment using magnesium (Figure
2) was performed. The basic difference was
that magnesium chloride was added in place
of calcium chloride. The precipitate obtained
using magnesium was slimy and not compact;
that obtained with calcium was in the form
of a compact gel. Because of the different
types of precipitate and the great difference
in the yields~ it appeared that the reaction
was primarily calcinm-dependent.
The dependency on calcium was further
demonstrated in skimmilk. Disodinm and tet-
rasodium E D T A were added to skimmilk be-
fore addition of CMC in amounts calculated
to sequester all the calcium. These results
(Table 1) d e a r l y show that no precipitate
occurred when EDTA_ was added, whereas
68% of the total protein was precipitated in
the absence of EDTA. This is considered as
further evidence that the CMC-protein reac-
tion is calcium-dependent.
The effect of p i t on the precipitation of
casein in a simplified system was also deter,
mined. Carboxymetbylcellulose was added as
usual, the mixture at p H 5.5 was separated
and adjusted to p H 6.5, 7.5, 8.5, and 10.0
with ~ NaOH. The dilution factor was equal-
ized with distilled water. The solutions at each
p H were equally divided. One set served as
a control while 120 mg Ca/100 z111in the form
of CaC12. 2 t t 2 0 was added to the second set.
TABLE 1. Effect of ethylenediaminetetraacetate
skimmilk treated with carboxymethylcellulose.
Protein
sedimented a
(% of total)
Skimmilk 0
Skimmilk plus E D T A b 0
Skimmilk plus CMC e 68
Skimmilk plus E D T A b
plus CMC c 0
a Protein sedimented at 1,000 X g for 20
rain after quiescent storage at 5 C for 17 hr.
b 0;50 g disodium E D T A and 0.80 g tetra-
sodium EDTA/100 g skimmilk.
e Carboxymethylcellulose (Hercules 7M2XF)
concentration of 0.5%.
J . DAIRY SOrEI~TOE ~¢~0I~. 52, NO. 8
1184
100
v-
Z
I.g
~=
90
80
xJx
J
Iit 7o
~ 6o
o 5O
4O
~ 30
o x-|20 rag, G a . / I O O rrl. ADDED
u..
o oo NO Ca. ADDED
5,5 6.5 "Z5 ~85
pH
FIG. 3. Effect of pH and calcium on percentage
of total protein sedimented from a 2.18% casein
solution treated with 0.4% carboxymethylcellulose
(Hercules 7M2XF). Protein sedimented at 4,000
× g for 20 rain.
Results in Figure 3 indicate that p H 7.5 is
the optimum p H for casein precipitation.
Changes in the milk salt system due to varia-
tions in pH, as reported by Jenness and Pat-
ton (10), suggest an explanation for these
findings. Their data show that as the hydrogen
ion activity decreases, the ionic calcimn con-
centration decreases. Therefore, as the ionic
calcium concentration decreases, less calcium
bridging should occur, and thus less protein
would be precipitated. Another factor that
Acknowledgment
should be considered is the charge on the pro-
tein. As the p i t is increased, negativity of
Industrial Research Fellowship granted by the
the protein also increases. The force of electro-
Dairy and Food Industries Supply Association,
static repulsion might be large enough to pre- Inc. to the senior author.
vent calcium bridging.
The centrifugal force required to recover References
the precipitate was investigated. I n varying
the force from 500 to 4,000 X g for 20 min
with 0.4% CMC in a 1.70% casein system,
no essential difference in the percentage of
total protein precipitated occurred. A force
of 500 X g for 20 min was sufficient for maxi-
mum protein sedimentation.
The effect of the method and order of add-
ing CMC, CaC12" 2 It20 , and the casein solu-
tion on the precipitation of casein was inves-
tigated. The following alternatives did not
seem to alter the amount of total protein pre-
cipitated: a) carboxymethylcellulose added dry
or in solution, or b) calcium chloride added
before or after mixing CMC with the ca-
sein solution, or c) a mixture of CMC and
calcium chloride added to the casein solution.
J. DAIRY SCIEI~CE VOL. 52, NO. 8
C L U S K E Y E T AL.
Continuous agitation using an Erbach hori-
zontal reciprocating agitator hastened precipi-
tation, ttolding for ]5 rain and agitating
permitted recovery of 95.5% of the protein;
whereas, without agitation, only 88.2% was
recovered. I t is, therefore, conceivable that
under optimum conditions the process could be
adapted to a continuous operation.
The sedimented portion, shown in Figure 1,
using 0.5% CIVIC contained 51.7% protein,
7.3% ash, 23.9% lactose, 0.44% calcium, and
10 to 17% CMC. Since CMC stabilizes casein
at p H 4.6, the Rowland distribution was not
determined. To verify that the precipitate
was basically casein, electrophoretic patterns
were prepared from the sedimented portion
~.0 previously analyzed. The mobility and density
of the bands of the sedimented portion were
essentially the same as those of an acid casein
standard. These data indicate that CMC pre-
cipitated the casein micelle and not any one
casein fraction. Other electrophoretie patterns
showed that a small quantity of whey pro-
teins, probably occluded, was also present in
the sedimented portion.
The method investigated appears to be a
relatively efficient method for precipitating ca-
sein from skimmilk. The mechanism of pre-
cipitation is largely the cross-linking through
calcium of casein and CMC. However, we
realize that this is not the only mechanism
since both nonionie (gnat) and anionic gums
(~-carrageenan and CMC) precipitate milk
proteins.
This work was supported in part by a Dairy
(1) Andersen, G. ]962. Interactions between
carrageenins and milk proteins. Milchwis-
senschaft, 17: 75.
(2) Asano, Y. 1966. The interaction between
milk proteins and carboxymethylcellulose
in fruits-flavoured milk. Sonderdruck. Sec-
tion F5: 695.
(3) Association of Official Agricultural Chem-
ists. 1960. Official Methods of Analysis.
9th ed. Association Official Agricultural
Chemists, Washington, D.C.
(4) Dubois, M., K. H. Gilles, J. K. Hamilton,
P. H. Rebers, and F. Smith. 1956. Colori-
metric method for determination of sugars
and related substances. Anal. Chem.,
28 : 350.
(5) Gortner, R. A. 1949. Outlines of Biochem-
istry. John Wiley and Sons, Inc., New
York.
 PROTEIN PRECIPITATION
(6) Hansen, P. M. T. 1966. Distribution of
carrageenin stabilizers in milk. J. Dairy
Sci., 49 : 698.
(7) Hercules Incorporated. ]966. Chemical and
physical properties of sodium carboxy-
methylce]lulose. Wilmington, Delaware.
(8) Jenness, R. 1953. Titration of calcium and
magnesium in milk and milk fractions in
ethylenediamine tetraacetate. Anal. Chem.,
25 : 966.
(9) Jenness, R., and J. Koops. 1962. Prepara-
tion and properties of a salt solution
which simulates milk ultrafiltrate. Neth-
erlands Milk Dairy J., 16: 153.
(10) Jenness, R., and S. Patton. 1959. Prin-
ciples of Dairy Chemistry. J o h n Wiley
and Sons, Inc., New York.
1185
(11) Loewenstein, M. 1961. Protein-hydroeol-
loid powder and process for producing the
same. U.S. Pat. 3,001,876.
(12) Marine Colloids, Inc. 1965. The action of
potassium ions on earrageenan water gel
systems. Colloid-O-Scope, 11 : 1.
(13) Perry, N. A., and F. J. Doan. 1950. A
picric acid method for the simultaneous
determination of lactose and sucrose in
dairy products. J. Dairy Sci., 33: 176.
(14) Seaplant Chemical Corporation. 1956.
Protein reactions. SeaKem Extracts, 3: 1.
(15) Seaplant Chemical Corporation. 1957. Sea-
Kern Irish moss extractives (Ca~'rageenan).
SeaKem Extracts, Technical Issue, Winter.
(16) Tyden, C. H. 1962. Improved method of
separating native albumin from milk. Brit.
Pat. 947,976.
J. DAIRY SCIENCE VOL. 52, NO. 8