COMPOSITION

AND PROPERTIES
OF THE
SOLUBLE COMPOUND
IN CHEESE.
BY LUCIUS
(From

the Chemical

L. VAN

SLYKE

Laboratory
ment

ALFRED

W. BOSWORTH.

of the New York State

Station,
Geneva,
N. Y.)

for publication,

February

Agricultural

Experi-

4, 1913.)

During the manufacture and ripening of Cheddar cheese and of

many other kinds of cheese, there is always found a protein that is
soluble in a warm 5 per cent solution of NaCl. The existence of
such a substance in Cheddar cheese was first brought to attention by work done in this 1aboratory.l
The presence of this brinesoluble protein was shown to be associated in some way with the
formation of acid in the cheese and, on the basis of some early
experiments, VanSlyke and Hart were led to conclude erroneously
that the substance consists of a combination of paracasein and lac.tic acid (called by them paracasein mono-lactate), which by the
addition of more lactic acid becomes insoluble in dilute brine solution, forming a compound which they mistakenly regarded as
paracasein di-lactate.
As a result of later work,2 they changed
their first views and came to the conclusion that the so-called paracasein mono-lactate is simply the uncombined protein, paracasein,
and that the so-called paracasein di-lactate is a compound of paracasein and lactic acid (1 gram of paracasein uniting supposedly
with about 0.5 cc. of $$ acid). It may be stated here, in passing,
that it was later shown by L. L. VanSlyke and D. D. VanSlyke3
that the protein casein does not unite with acids to form insoluble
compounds but that the action is simply one of adsorption, by
which more or less acid is taken from the surrounding solution and
concentrated upon the surface of the solid particles of protein;
in other words, it was shown that casein or paracasein mono-lac1 Amer.

2 Ibid.,
3 Ibid.,

Chem. Journ.,.xxviii,
xxxiii, p. 461, 1905.
xxxviii, p. 383, 1907.

p. 411, 1902.

231

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(Received

AND

BRINE-

232

The Brine-Soluble

Compound in Cheese

Technical
6 Technical

Bulletin
Bulletin

No. 4, 1907, New York State Agric. Exp. Sta.

No. 5, 1907, New York State Agric. Exp. Sta.

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tate and di-lactate have no existence as applied to the compound in

question.
It still remained, therefore, to find out what the brinesoluble substance really is, and work was continued along this line
by the writers.* We noticed that calcium is always to be found
associated with the brine-soluble substance when it is separated
from the other cheese constituents by extraction with a solution
of calcium-free NaCl after previous removal of all water-soluble
constituents.
This fact suggested the possibility that the brinesoluble substance might be a combination of paracasein and calcium, containing less calcium than had been previously found in
any combination of this element with paracasein. On the basis
of such a possibility, it could be explained that with the formation
of increased amounts of lactic acid in cheese-making, as a result of
the bacterial decomposition of milk sugar, the acid would combine
with more or less of the calcium contained in calcium paracaseinate, resulting in the production of a paracaseinate containing
less calcium. This suggestion was strengthened by the fact that
in Camembert cheese, the brine-soluble compound is formed during
certain stages of the manufacturing process but soon disappears,
its formation and disappearance being explained as follows, according to Bosworth? The brine-soluble substance is at first formed in
Camembert cheese, as also in the case of Cheddar cheese, but,
owing to the method of making this type of cheese, more acid is
allowed to form in Camembert cheese, and, as a consequence, the
brine-soluble substance loses its calcium and becomes free paracasein, which is insoluble in brine solution.
Therefore, in the manufacture of Camembert cheese, it is found that after the first few
hours the cheese contains no brine-soluble material, and, what is
also significant, all the calcium is found in the water extract.
The relation between the brine-soluble substance and the calcium
found in the brine extract in the two types of cheese is illustrated
in Table 1.
The question necessarily suggests itself whether the calcium
always found in the brine-soluble extract of cheese is not there
incidentally in a mechanical state rather than in a combination with
paracasein. In order to study this question, the following work
was done:

L. L. VanSlyke and A. W. Bosworth

233

TABLE

Comparison

of changes

in

I.

Cheddar and Camembert cheese.

.When curd was cut .......

When curd was cut .......
Ten hours ................
Ten hours ................
Two days.
Two days..
i
Four months..
.!
~__~___
-.- .-~~~______

pm cent

Cheddar
Camembert
Cheddar
Camembert
Cheddar
Camembert
Cheddar

3.13
6.72
96.00
94.00
68.87
4.39
43.09

trace
trace
27.96
17.76
24.47
trace
24.28

In order to throw further light on the character of the brinesoluble compound, a study was made of the solvent effect of several different chlorides. One kilogram of Cheddar cheese was
ground fine, thoroughly mixed, and then 25-gram portions were
ground with sand, placed in bottles and extracted with water in
the manner described in the preceding paragraph.
The residues
were then extracted with solutions of chlorides and the results
given in the following table were obtained.
The solutions of the
salts were used in such strengths that 1000 cc. contained equivalent gram molecules. In the case of the weakest solution, extraction was continued as long as appreciable amounts of protein
were obtained in the extract, 4000 cc. being used; the results in
these cases are given for each 1000 cc. of extract as well as for the
total.

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Twenty-five grams of cheese were ground with sand and extracted with water at about 55C., using 150 cc. portions until the
extract amounted to 1000 cc. The residue, containing the brinesoluble substance, was placed in a dialyzing apparatus and allowed
to dialyze to insure the removal df all soluble calcium.
Sodium
chloride was then added to the contents of the dialyzing tube,
which was then placed in a beaker of water and allowed to remain
four hours. Upon adding ammonium oxalate to some of the water
in the beaker, a precipitate of calcium oxalate appeared.
This
result leads to the belief that the Ca is present in combination in an
insoluble form and that an interchange takes place between it and
Na, when the insoluble compound is treated with NaCl solution.

The Brine-Soluble

234

Compound in Cheese
TABLE

II.

Solvent efrect oj neutral chlorides on the brine-soluble

.__--

1.0
0.8
0.6
0.4

-__

50.47
50.47
45.95
44.52

63.81
43.33
lost
23.57

0
0
0
0

0
0
0
0

1000
1000
1000
1000

47.62
13.33
2.95
trace
--

49.05
10.48
4.10
trace

40.95
13.90
2.00
trace
---___-~

4.00
5.24
4.29

0
0

0
0

4ooo

/ 63.90

63.62

-------~

56.85
___

-..

..---

In connection with the data in the preceding table, attention is

called to certain phases of the results.
1. The chlorides of Ba and Ca have no solvent effect. The
chloride of Mg in strong molecular concentrations acts much like
the chlorides of the alkalies, while in lower molecular concentrations its solvent power is greatly reduced.
2. Sammis and Harts attempted to study the solvent effect of
these salts on the same material, but reached results not concordant
with one another and not in agreement with ours. While we used
solutions of such strength as to show the relation existing between
the solvent action of the salt solution and its molecular concentration, they used solutions containing a uniform percentage by
weight of different salts and extracted-in every case with the same
volume of solution.
By using solutions of different salts having
the same percentage composition by weight, but with a different
molecular concentration,
one would, under the circumstances,
expect to obtain only discordant results, because the solvent
effect of the solution is apparently a result of the mass action
of the salt in solution, as shown by us (this Journal, p. 217). If
Sammis and Hart had in their work continued extraction until
no more solvent effect was appreciable, their results would have
been in satisfactory agreement with ours.
This is strikingly
6 This

JournaZ, vi, p. 181, 1909.

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Tot.al.

cheese.

67.62
65.24
56.43
51.19

1000
1000
1000
1st
2nd
3rd
4th

in

63.57
69.29
56.19
51.43

1L

0.2
0.2
0.2
0.2

.___

compound

L. L. VanSlyke and A. W. Bosworth

235

shown in the above table in the case of the 0.2 N solutions; by

continued extraction the total amounts extracted are found to
be essentially the same as in the more concentrated solutions.
Identity

of the brine-soluble compound of cheese with mono-calcium

paracaseinate.

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We have shown (this Journal, p. 223) that paracasein combines

with Ca to form a compound insoluble in water but soluble in 5
per cent solution of NaCl (Na replacing Ca). In this compound
we have shown that 1 gram .of paracasein is in combination with
2.25X10d4 gram equivalents of Ca. Indications pointed to the
identity of the brine-soluble substance of cheese with this monocalcium paracaseinate, and it remained to ascertain whether the
protein part of the molecule in these two compounds is the same.
In order to accomplish this, a preparation of the protein in the
brine-soluble compound was made from cheese, and its composition
and properties were studied.
One kilogram of Cheddar cheese was ground fine and then extracted with numerous portions of distilled water at about 55C.
in order to remove all soluble compounds.
The residue was then
extracted with many portions of a 5 per cent solution of NaCl
and filtered, first through absorbent cotton and then through
paper. Dilute acetic acid was then added, giving a heavy precipitate, which was washed with water, redissolved in dilute ammonia
and again precipitated with acid. The process was then completed as in the preparation of casein (this JouTnuZ, p. 204). The
preparation
on analysis gave the following results: Moisture,
2.32; ash, 0.25 per cent. In the dry substance, C, 52.97; H, 7.15;
N, 15.82; P, 0.75; S, 0.78; O(by difference), 22.28.
A study of the properties of this substance gave the following
results :
1. The substance is found to act as an acid in combining with
bases.
2. It decomposes CaC03 and gives a compound in which 100
grams of substance combine with the equivalent of 2.52 grams of
CaO (equal to 1.80 grams of Ca), or, 1 gram of substance combines
with 9X10s4 gram equivalents of Ca.

236

The Brine-Soluble Compound in Cheese

SUMMARY.

In many kinds of cheese there is always present a protein soluble

in warm 5 per cent solution of NaCl. Previous efforts to determine
the exact relation of this substance to casein or paracasein have
resulted in erroneous conclusions. An extended study of its properties and composition indicates the substance to be mono-calcium paracaseinate, formed from calcium paracaseinate by removal of part of its Ca through lactic acid produced in the process of
cheese making as a result of the action of lactic acid bacteria upon
the milk sugar.

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3. The solution of this calcium compound is neutral to phenolphthalein.

4. Measured by the volumetric method it was found to form a
compound with ammonium represented by the combination of
1 gram of substance with 2.3 X10T4 gram equivalents, expressed
as hydroxide.
5. With Ca it forms a compound, soluble in 5 per cent solution
of NaCl but insoluble in water, which contains 1 gram of substance combined with 2.3 X 10m4 gram equivalents of Ca.
6. It forms also a compound with Ca that is soluble in water,
containing 1 gram of substance combined with 4.5X10m4 gram
equivalents of Ca.
In view of the marked agreement of the composition and properties of the brine-soluble substance, formed in cheese, with the
compound, mono-calcium paracaseinate, as prepared by us, there
is good reason to believe that the brine-soluble substance is monocalcium paracaseinate, having the composition of 1 gram of paracasein combined with 2.25X10m4 equivalents of Ca.

COMPOSITION AND PROPERTIES OF

THE BRINE-SOLUBLE COMPOUND IN
CHEESE
Lucius L. Van Slyke and Alfred W. Bosworth
J. Biol. Chem. 1913, 14:231-236.

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