CHEM. LIB.
TX
382
V279
B 453517
New York Agricultural Experiment Station.
GENEVA, N. Y.
COMPOSITION AND PROPERTIES OF SOME CASEIN
AND PARACASEIN COMPOUNDS AND THEIR
RELATIONS TO CHEESE.
LUCIUS L. VAN SLYKE AND ALFRED W. BOSWORTH.
EXCELSIOR
PUBLISHED BY THE DEPARTMENT OF AGRICULTURE.

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TECHNICAL BULLETIN No. 26.
▬▬▬
Chemical Library
TX
COMPOSITION AND PROPERTIES OF SOME
CASEIN AND PARACASEIN COMPOUNDS
AND THEIR RELATION TO CHEESE.
LUCIUS L. VAN SLYKE AND ALFRED W. BOSWORTH.
SUMMARY.
1. Object. The work was undertaken to obtain more informa-
tion regarding the compounds formed by casein and paracasein
with bases, especially with Ca. Two compounds have been previ-
ously prepared, one neutral to phenolphthaleïn, containing 1.78
per ct. Ca (2.50 CaO), and the other, neutral to litmus, containing
1.07 per ct. Ca (1.50 CaO). Our main object was to learn if there
were other compounds containing less Ca. Another purpose was
to ascertain the composition of the substance formed in cheese
which is insoluble in water but soluble in 5 per ct. solution of Na Cl.
2. Method of preparing casein.— Casein must be made base-free
for use in such work. Preparations were made containing less than
The usual method was employed in part, pre-
0.1 per ct. of ash.
cipitating separator skim-milk with dilute acetic acid, redissolving
the washed precipitate in dilute NH4OH, continuing precipitation
and solution three or more times. Finally, the remaining calcium
is precipitated from the ammonia solution as oxalate, the precipitate
being removed by centrifuging and filtering, and the filtrate pre-
cipitated with dilute HCl. After washing free from HCl, the casein
is treated with alcohol and ether, and after grinding and partial
drying is dried over H2SO4 under reduced pressure. Analysis of
such casein preparations agrees with the composition generally
accepted, except in the amount of phosphorus and sulphur.
3. Preparation and composition of basic calcium caseinate. The
compound was prepared in two ways, (1) by decomposing CaCO3
with casein and (2) by treating casein with a lime-water solution
and neutralizing the excess with HCl, with phenolphthaleïn as
indicator. The composition of the resulting compound was deter-
mined (1) by weighing the CO₂ expelled from CaCO3, (2) by
determining the Ca in the resulting casein compound and (3) by
analysis of compound formed by treating lime-water solution of
casein with acid until neutral to phenolphthaleïn. The different
results agree closely, showing basic calcium caseinate to contain
about 1.78 per ct. Ca (2.50 CaO), or 1 gram of casein combines
with 9 x 104 gram equivalents of Ca.
[3]
cut
4
50
N
50
N
50
(4) Acid or unsaturated caseinates of ammonium, sodium and
potassium. These compounds were prepared as follows: Ash-free
casein is dissolved in alkali so that 50 cc. of N alkali contain 1 gram
of casein. This is neutralized with HCl, which is added in
small portions, under constant agitation, until a permanent precipi-
tate begins to appear, as shown by centrifuging a portion of the
mixture in a sedimentation tube. This method enables one to
detect the casein precipitated by 0.20 cc. of HCl. The point at
which a permanent precipitate first begins to appear is noted and
addition of acid is continued until all the casein is precipitated,
which point is also noted. Three different casein preparations were
used and numerous determinations were made. It was found that
I gram of casein forms a soluble compound with each of the alkalis
used when combined with amounts somewhere between 1.10 X 104
and 1.15 x 104 gram equivalents of alkali; or, I cc. of alkali
combines with an amount of casein somewhere between 0.87 and
0.91 gram. The proportion of basic element in each compound is
as follows: NH4, 0.20 per ct.; Na, 0.26 per ct.; and K, 0.44 per ct.
Such casein compounds contain the smallest known amount of base
and it is suggested that they be called mono-basic caseinates.
N
10
Special preparations were made of mono-ammonium caseinate,
the compound being isolated and prepared in dry form. This was
found to have the composition called for by the previous results
obtained with the volumetric work.
N
50
(5) Acid or unsaturated caseinates of calcium, strontium and
barium. When a solution of casein in a hydroxide of calcium, etc.,
is treated with an acid, the caseinate is precipitated by the chloride
formed; this difficulty can be overcome by removal of the chloride
through simple dialysis before the amount is sufficient to cause
precipitation. One gram of ash-free casein is dissolved in 250 cc.
of hydroxide solution and HCl is added until the first sign
of a permanent precipitate appears, as shown by centrifuging a
portion. The solution is then dialyzed to remove soluble chloride
and then acid is again added until precipitation again occurs and
another dialysis is made. Alternate addition of acid and dialysis
are continued until finally the dialyzed solution forms a permanent
precipitate with the addition of any acid. The results of many
experiments agree in indicating the formation of two sets of com-
pounds, mono-basic and di-basic, one set containing twice as much
base as the other. In the di-basic compounds, 1 gram of casein
requires between 2.2 x 104 and 2.3 x 104 gram equivalents of
hydroxide to form a compound soluble in water but easily precipi-
table by even a small amount of a soluble chloride of calcium,
strontium or barium. In the di-basic compounds, I gram of casein
combines (a) with 0.44 to 0.46 gram Ca (0.62 to 0.64 CaO), (b) with
0.96 to 1.01 gram Sr (1.14 to 1.19 SrO), and (c) with 1.51 to 1.58
grams Ba (1.69 to 1.76 BaO). In the mono-basic salts, i gram of
N
50
5
casein combines with about 1.1 X 10 gram equivalents of hydroxides
to form insoluble compounds, which are soluble in 5 per ct. solution
of chloride of sodium, ammonium or potassium. This solubility is
due to an exchange of bases; for example, insoluble mono-calcium
caseinate is changed by treatment with solution of NaCl into soluble
mono-sodium caseinate and CaCl2, as shown by special experiments.
Special preparations were made of mono- and di-calcium casein-
ates, each compound being isolated and prepared in dry form.
These were found to have essentially the composition called for by
the previous results obtained with the volumetric work.
(6) Valency of casein molecule and molecular weight of casein.-
On the basis of the composition of the basic calcium caseinate and
mono-calcium caseinate, the former has a valency of 8. These
relations indicate the molecular weight of casein to be 8888
and the equivalent weight IIII.
(7) Method of preparing paracasein.- Separator skim-milk is
heated to 37° C. and treated with 0.12 cc. of rennet-extract (Han-
sen's) per 1,000 cc. of milk. The milk is allowed to stand until
completely precipitated. The resulting curd is broken up by vigorous
stirring, the whey removed and the precipitated paracasein washed
freely with water. It is then dissolved in dilute NH OH, reprecipi-
tated with acid and the operation continued and completed as in
case of casein.
·
(8) Preparation and composition of basic calcium paracaseinate.-
By the same methods of study, paracasein was shown to form with
calcium a paracaseinate similar in composition and properties to
that of basic calcium caseinate.
(9) Acid or unsaturated paracaseinates of ammonium, sodium
and potassium.- In these compounds, I gram of paracasein com-
bines with an amount of alkali somewhere between 2.2 x 104 and
2.3 x 104 gram equivalents in forming soluble compounds with
ammonium, sodium and potassium, which are acid to both litmus
and phenolphthalein. One cc. of alkali combines with 0.435 to
0.455 gram of paracasein. The percentage of basic element in
each compound is, NH4 0.40; Na, 0.52; and K, 0.88. The amount
of each basic element in these paracaseinates is just twice that
present in the corresponding casein compounds.
A preparation of mono-ammonium paracaseinate in dry form gave
results agreeing fairly well in composition with the results obtained
by volumetric work.
(10) Acid or unsaturated paracaseinates of calcium, strontium and
barium. Mono- and di-basic paracaseinates were prepared in the
same manner as the corresponding caseinates and were shown to
differ from them in having just twice as much of the basic element.
In the mono-basic compounds, which are insoluble, I gram of para-
casein combines with about 2.3 x 104 gram equivalents of hydroxide
of calcium, etc.; in the di-basic, which are soluble, with about
6
4.6 x 104 gram equivalents. Their properties resemble those of the
corresponding caseinates.
(11) Valency of paracasein molecule and molecular weight of
paracasein.— The valency and molecular weight are shown to be
one-half those of casein.
(12) Action of rennet-enzym on casein in forming paracasein.—
When casein is treated with rennet-enzym, the casein molecule
appears to be split into two molecules of paracasein.
(13) Composition of brine-soluble compound in cheese. During
the manufacture and ripening of cheddar and many other kinds of
cheese, a protein is always formed which is insoluble in water but
soluble in a 5 per ct. solution of NaCl. Former studies led to erro-
neous conclusions regarding its identity. Extended study shows
that this substance is identical with mono-calcium paracaseinate.
INTRODUCTION.
The uncombined protein, casein, shows the characteristic property
of an acid in combining with bases of the alkalis and alkaline earths.
to form salts and in decomposing their carbonates. The compounds
thus formed, especially those with calcium, have been studied by
numerous investigators, the more important contributions have been
made by the following:
Hammarsten (Zur Kenntniss des Kaseins etc., Upsala, 1877);
Söldner (Landw. Versuchs.-Stat., 35: 351, 1888);
Courant (Pflüger's Arch. Physiol., 50: 109, 1891);
Timpe (Arch. Hyg., 18: 1, 1893);
Béchamp (Bull. Soc. Chim. (3) 11:, 152, 1894);
de Jager (Maly Jahresber. Thierchem., 27: 276, 1897);
Salkowski (Zeitschr. Biol., 37: 415, 1899);
M
Kobrak (Pflüger's Arch. Physiol., 80: 69, 1900);
Osborne (Jour. Physiol., 27: 398, 1901);
Laqueur and Sackur (Beitr. Chem. Physiol. u. Pathol., 3: 193, 1902);
Van Slyke and Hart (Bull. No. 261, N. Y. Agr. Exp. Sta., and Am. Chem. Jour.,
33: 472, 1905);
Long (Jour. Am. Chem. Soc., 28: 72, 1906);
Robertson (Jour. Biol. Chem., 2: 317, 1906);
Robertson (Jour. Physic. Chem., 13: 469, 1909).
Without going into details, it is sufficient for our purpose at this
point to state that results have been reported in which compounds,
formed by treating casein with calcium hydroxide, contain an equiva-
lent of calcium oxide varying all the way from 0.8 to 3 per ct. (equal
to 0.57 to 2.14 per ct. of calcium).
In the chemical laboratory of this Station, the relation of casein
and paracasein to bases has been a subject of continued study for
several years, especially in connection with changes taking place in
the operation of cheese-making. The results here presented include
1
77
a careful revision of work published in previous bulletins of this
laboratory with material extensions of the line of investigation.
Two compounds of casein and calcium have been generally recog-
nized, one containing about 2.50 per ct. CaO (1.78 per ct. Ca), and
the other about 1.50 per ct. CaO (1.07 per ct. Ca). We have found,
in addition, two others, one containing about 0.31 per ct. Cao
(0.22 per ct. Ca), and the other double this amount. Corresponding
compounds are shown in our work to be formed by paracasein with
calcium, and also by both casein and paracasein with ammonium,
sodium, potassium, barium and strontium. A study of the methods
of preparation and of the properties of these compounds is given in
this publication; the ground covered is embraced under the following
outline:
Part I. Casein and some of its compounds.
C
1. Method of preparing ash-free casein.
2. Preparation and composition of basic calcium
caseinate.
3. Preparation and composition of unsaturated or acid
caseinates.
4. Valency of casein molecule and the molecular
weight of casein.
Part II. Paracasein and some of its compounds.
1. Method of preparing ash-free paracasein.
2. Preparation and composition of basic calcium para-
caseinate.
3. Preparation and composition of unsaturated or acid
paracaseinates.
4. Valency of paracasein molecule and the molecular
weight of paracasein.
5. Action of rennet-enzym on casein.
Part III. Composition of brine-soluble compound in cheese.
1. Brine-soluble compound formed in cheese-making.
2. Identity of brine-soluble compound and mono-
calcium paracaseinate.
8
PART I. CASEIN AND SOME OF ITS COMPOUNDS.
METHOD OF PREPARING ASH-FREE CASEIN.
Casein that is to be used in studying its relation to mineral bases
must be free from all such bases. The preparation of really ash-free
casein is much more difficult than has been commonly assumed.
The so-called chemically-pure casein furnished by chemical-supply
houses usually contains 0.6 per ct. of ash. The preparations used
in various investigations in which the ash content has been reported
rarely contain less than 0.2 per ct. of ash and not infrequently as
much as 0.6 per ct.
The principal basic element in casein preparations, as usually
made, is calcium. The calcium in casein preparations is usually due
to the presence of a compound of calcium and casein, containing
0.22 per ct. Ca (equal to 0.31 per ct. CaO), as we shall show later.
This salt is insoluble in water but easily soluble in a 5 per ct. solution
of sodium chloride, while base-free casein is insoluble in both water
and the brine solution.
When casein is carefully precipitated by dilute acids from milk
or from lime-water solutions of casein, the precipitate is apt to
contain more or less of the above-mentioned calcium caseinate
as well as base-free casein. The precipitation of this calcium salt
occurs most readily when the usual precautions in precipitating
casein from milk are most rigidly observed, that is, when excess of
acid is avoided. We have examined casein preparations obtained
from chemical-supply houses and have found that some of them are
soluble in a 5 per ct. solution of sodium chloride to the extent of
50 per ct., or more, of their weight.
After trying different methods of preparing casein so as to contain
a minimum amount of calcium, we have obtained the most satis-
factory results by the method described below. We have been able
to prepare casein containing only 0.06 per ct. of ash, consisting
largely of calcium phosphate, derived from the trace of calcium not
removed and the phosphorus of the casein molecule. The amount
of calcium present in 5 grams of such material was too small to
determine quantitatively.
Our method of preparation is to dilute separator skim-milk with
seven or eight times its volume of distilled water and carefully add
dilute acetic acid (6 cc. of glacial acetic acid diluted to 1 liter) until
the casein separates completely, after which the clear solution is
removed by siphon as soon as the precipitate settles. Distilled
water is then added, the mixture stirred vigorously and the precipi-
tate allowed to settle, after which the wash-water is siphoned off.
More water is then added and the casein is dissolved by adding,
for each liter of milk used, 1 liter of dilute ammonium hydroxide
(6 cc. of strong reagent diluted to 1 liter). When the solution is
complete, the whole is filtered through a thick layer of absorbent
9
G
cotton. The casein is then precipitated again with dilute acetic
acid; the precipitate is allowed to settle, and is then washed, redis-
solved in dilute ammonium hydroxide, and filtered, the process of
precipitation, washing, dissolving, etc., being repeated not less than
four times. Finally an excess of strong ammonium hydroxide
(10 cc.) is added and then 20 cc. of saturated solution of ammonium
oxalate. The mixture is allowed to stand 12 hours or more. Cal-
cium is precipitated as oxalate in very finely divided condition, too
fine to permit its satisfactory removal by ordinary methods of fil-
tration. Better aggregation of the precipitate can, however, be
effected by means of centrifugal force. The centrifuged mixture is
then filtered through double thickness of filter paper. The filtered
solution is next treated with dilute hydrochloric acid (10 cc. of HCl,
sp. gr. 1.20, diluted to 1 liter) until the casein is precipitated. The
precipitate is washed with distilled water until free from chloride
and is then placed on a hardened filter paper in a Buchner funnel,
as much water as possible being now removed from the precipitate
by suction. The mass is next transferred to a large mortar and
thoroughly triturated with 95 per ct. alcohol. The alcohol is then
removed by suction on a Buchner funnel and the casein is then
again placed in a mortar and triturated with absolute alcohol. Most
of the alcohol is removed by filtration and the casein treated twice
with ether in a mortar by trituration, the ether being removed each
time by means of suction on a Buchner funnel. The material is
then placed in a large evaporating dish and spread out in a layer as
thin as possible; it is allowed to stand 12 hours or more in a warm
place; and is finally ground in a mortar until the particles pass a
40-mesh sieve, and is dried two days over sulphuric acid in a
desiccator under diminished pressure.
Three preparations made in this way were found to show an ash
content of 0.10, 0.09 and 0.06 per ct., respectively. These prepa-
rations were insoluble in water and in 50 per ct. alcohol; the first
one was very slightly soluble in a 5 per ct. solution of sodium chloride,
but the two others were not.
N
10
When one gram of these casein preparations was treated with
10 cc. of hydroxide of ammonium, sodium or potassium, and
90 cc. of water, a clear solution was obtained, the casein dissolving
completely. When to this solution a minute amount of a solution
of a barium, calcium or strontium salt was added, there developed
promptly the opalescent appearance characteristic of casein solutions
under such conditions.
Moisture..
In dry substance:
Casein prepared in the manner described was analyzed, with the
following results:
Ash.
Carbon.
Hydrogen.
!
•
•
Per ct.
1.09
0.06
53.50
7.13
In dry substance:
Nitrogen.
Phosphorus.
Sulphur.
Oxygen (by difference)
•
•
•
• •
Per ct.
15.80
0.71
0.72
22.08
10
PREPARATION AND COMPOSITION OF BASIC CALCIUM CASEINATE.
The compound commonly known as basic calcium caseinate con-
tains the largest amount of calcium in combination with casein.
This is the compound that has been most frequently prepared and
studied by investigators, beginning with Söldner (see references on
page 6). Varying results have been obtained by different workers,
the percentage of calcium ranging from 1.66 to 2.13 per ct. (equiva-
lent to 2.32 to 2.98 per ct. CaO).
This compound can be prepared in two different ways: (1) By
decomposing calcium carbonate with casein and (2) by treating
casein with a solution of calcium hydroxide (lime-water).
Preparation of basic calcium caseinate by treating casein with calcium
carbonate. When cascin is treated with calcium carbonate, the
results of the reaction can be measured in two ways: (a) By weigh-
ing the carbon dioxide displaced, and (b) by determining the amount
of calcium in the resulting compound. Both methods were used
by us.
ست
Casein prepared in the manner previously described was placed
in the flask of a Knorr carbon dioxide apparatus and an excess of
calcium carbonate suspended in water was added. The carbon
dioxide formed in the reaction was run into weighed bulbs con-
taining potassium hydroxide and the increase of weight due to
carbon dioxide determined at the end of the reaction. The results
are given in the following table.
Grams.
10.
10.
TABLE I.-- AMOUNTS OF CARBON DIOXIDE EXPELLED FROM CALCIUM CARBONATE
BY CASEIN.
•
•
5.
5.
Average.
•
Gems, matang
•
•
Amount of dry cascin used.
·
·
·
·
►
J
+
•
!
•
•
•
•
Amount of Ca O
Amount of CO2 | (and Ca) for 100
expelled.
grams of casein,
equivalent to CO2.
Grams.
0.1903
0.1980
0.1054
0.1003
Grams.
2.42 (1.73 Ca)
2.52 (1.80 Ca)
2.68 (1.91 Ca)
2.55 (1.81 Ca)
2.54 (1.81 Ca)
For the purpose of measuring the results of the reaction by deter-
mining the amount of calcium in the resulting compound, the casein
was put in a mortar and thoroughly triturated with an excess of
moist calcium carbonate, the excess being removed by filtration at
the end of the reaction. The filtrate was treated with 95 per ct.
11
alcohol, which was free from acid, until the calcium caseinate was
precipitated, after which the precipitate was washed with alcohol
and ether, and dried at 120° C. A weighed portion of this compound
was carefully ignited and the calcium in the resulting ash was deter-
mined, with the following results:
TABLE II. AMOUNT OF Ca COMBINING WITH CASEIN WHEN REACTING WITH CaCO3.


Weight of
caseinate.
Grams.
Grams.
0.4125
0.0102
0.5134
0.0124
0.3090
0.0077
0.4253
0.0104
Ave. 0.41505 0.010175
Weight of CaO. Weight of Ca. Weight of free CaO) (and Ca) for 100
casein.
grams of cascin.
Weight of
caseinate.
Grams.
1.582
1.471
1.548
Ave. 1.534
Preparation of basic calcium caseinate by treating casein with an
excess of calcium hydroxide.— Weighed portions of casein were dis-
solved in an excess of lime-water. Phenolphthaleïn indicator was
then added to the solution and hydrochloric acid was run in until
the solution became neutral. The solution was then dialyzed to
remove the calcium chloride formed in neutralization. The dialyzed
solution was evaporated to dryness, the residue dried at 120° C.
and weighed. The determination of calcium was made after ignition,
with the following results:
Weight of
CaO.
Grams.
0.0073
0.0089
0.0055
0.0074
0.00726
TABLE III. AMOUNT OF CALCIUM COMBINING WITH CASEIN ON TREATMENT WITH
CALCIUM HYDROXIDE.
Grams.
0.040
0.035
0.038
0.0377
Grams.
0.4052
0.5045
0.3035
0.4179
0.4078
Grams.
2.52 (1.80 Ca)
2.46 (1.76 Ca)
2.54 (1.81 Ca)
2.49 (1.77 Ca)
2.50 (1.78 Ca)

Grams.
0.0286
0.0250
0.0271
0.0269
Weight of Ca. Weight of free CaO (and Ca) for 100
casein.
grams of casein.
Grams.
1.5534
1.4460
1 5209
1.5070
Grams.
2.58 (1.84 Ca)
2.42 (1.73 Ca)
2.50 (1.78 Ca)
2.50 (1.78 Ca)
The three sets of figures presented in Tables I, II, and III indicate
that casein combines with calcium to form a compound containing
about 2.50 per ct. CaO (equal to 1.78 per ct. Ca); the compound in
12
solution is neutral to phenolphthalein. Expressed in another form,
1 gram of casein combines with 9 x 104 gram equivalents of calcium.
This compound is commonly known as basic calcium caseinate.
PREPARATION AND COMPOSITION OF UNSATURATED OR ACID
CASEINATES.
Compounds of casein with bases, in which less base is present than
in the basic calcium caseinate described above, have been reported.
Söldner¹ obtained a compound of casein and calcium containing
1.11 per ct. Ca (equal to 1.55 per ct. CaO); or, expressed in another
form, 1 gram of casein combines with 5.55 x 104 gram equivalents
of calcium. This compound is neutral to litmus but acid to
phenolphthaleïn, and has been commonly known as neutral calcium
caseinate. This compound as prepared by Van Slyke and Hart2
contains about 1.07 per ct. Ca (equal to about 1.50 per ct. CaO),
or 1 gram of casein combines with 5.35 x 104 gram equivalents of
calcium. Courant³ believes that, in addition to the basic and neutral
compounds of casein and calcium, a third exists, in which the calcium
is present in about one-half the amount contained in the neutral
compound and one-third that contained in the basic compound; he
regards them as mono-, di- and tri-calcium caseinates. Timpe¹
reports a compound containing 0.961 per ct. Na (equal to 0.868
per ct. CaO or 0.62 per ct. Ca; or 1 gram of casein combines with
3.1 x 104 gram equivalents of calcium). Long" was able to dissolve
1 gram of casein in just one-half the amount of alkali required for
the phenolphthalein neutralization, and therefore inferred the
existence of acid caseinates containing one-half the amount of base
contained in basic calcium caseinate. The existence of such a
combination is questioned by Robertson."
In the course of our work, we became convinced that casein forms
compounds containing less base than any of those reported by other
workers. While we were at work on this point, an article by Robert-
son' appeared, in which was reported a combination of casein and
sodium hydroxide, 1 cc. of the alkali combining with 0.877 gram
of casein. Our further work confirms Robertson's results, although
we have used a different method of procedure. In addition, we
have been able to prepare and isolate several salts for analysis. Our
study of these individual salts shows that ammonium, sodium and
potassium compounds possess properties of solubility very different
from those of barium, calcium and strontium. As previously stated,
¹Landw. Versuchs.-Stat., 35: 351, 1888.
2 N. Y. Agrl. Expt. Sta. Bull. No. 261, 1905.
³ Pflüger's Archiv. Physiol., 50: 109, 1891.
Arch. Hyg. 18: 1, 1893.
"Jour. Am. Chem. Soc., 28: 372, 1906.
Jour. Biol. Chem., 2: 336, 1906.
Jour. Physical Chem., 13: 469, 1909.
13
I
we have prepared and studied two sets of compounds of casein with
bases, in one of which 1 gram of casein combines approximately
with 1.125 x 104 gram equivalents of base, while in the other
1 gram of casein combines with about 2.25 x 104 gram equivalents
of base.
We will next take up the details of our experimental work in
preparing acid caseinates of the bases of the more common alkalis
and alkaline earths.
N
40
N
b0
N
50
N
συ
The specific object of our work was to ascertain the smallest
quantity of base with which casein combines to form a definite salt.
In the volumetric work our method of procedure was as follows:
In 200 cc. of alkali, we dissolved 5 grams of pure casein as quickly
as possible and then made the volume to 250 cc. Each 50 cc. of
this solution therefore represents 1 gram of casein dissolved in 50 cc.
of alkali. A preliminary or trial determination was next made in
the following manner: Into a 300 cc. Erlenmeyer flask, we measure
50 cc. of the caseinate solution and then add, a drop at a time,
some HCl, until we have used 5 cc., the contents of the flask being
kept in constant agitation in order to prevent premature precipi-
tation of casein. After addition of the 5 cc. of acid, a portion of the
contents of the flask is centrifuged, in order to cause the sedimenta-
tion of precipitated casein, if any, a precipitate serving as an indi-
cator. A sedimentation tube of 50 cc. capacity can be used; the
precipitate collects in the lower V-shaped portion. It is possible in
this manner to detect the casein precipitated by 0.20 cc. of HCl.
In case no casein is precipitated by the first addition of 5 cc. of acid,
another equal amount of acid is added and a portion of the mixture
centrifuged; the process of adding 5 cc. portions of acid and centri-
fuging is continued until a permanent precipitate of casein is
obtained. This shows, within 5 cc. of HCl, how much acid is
required to start definite precipitation of the casein. In order to
ascertain the exact point more closely, another set of determinations
is made, using 50 cc. of the caseinate solution and adding in the
same cautious manner an amount of HCl which is 5 cc. less than
the amount causing the first appearance of a permanent precipitate
in the trial or preliminary determination. The acid is now added
in small amounts with constant agitation of the mixture to prevent
the premature separation of any precipitate, and centrifuged after
the addition of each 0.25 cc. The point at which a permanent
precipitate first appears is noted; the addition of acid is continued
until all the casein is precipitated and this point is also noted. In
our work this method of determination was repeated several times.
with each combination of casein and alkali and three different casein
preparations were used in preparing each caseinate. We will now
present the results of our experimental work in connection with the
unsaturated or acid caseinates of, first, ammonium, sodium and
potassium, and, second, barium, strontium and calcium.
N
50
N
50
14
Acid caseinates of ammonium, sodium and potassium.- In the
manner described above, we made numerous determinations in the
case of preparations of base-free casein dissolved in the hydroxide of
ammonium, sodium and potassium, respectively. Tabulated below,
we give the average results of many such determinations.:
TABLE IV. RELATION OF ALKALI BASES TO CASEIN IN ACID CASEINATES.

Amount Kind of
of casein
alkali
used
used.
Gram.
1
1
1
NH, OH
Na OH
кон
of
Amount
N
bu
alkali
used.
Cc.
50
50
50
N
Amount of HCI
60
required to cause first
sign of permanent
precipitation.
((
"
Cc.
Cc.
Between 44.25 and 44.50 | 5.5 to 5.75
(
"(
((
"
"
"
"
Amount of alkali left
combined with casein.
"
N
50.
"
((
p
N
10.
Cc.
1.1 to 1.15
((
"
"
<<
Amount
N
of HCI
50
required
to pre-
cipitate
all of the
casein.
Cc.
50
50
50
10
The results in this table indicate that 1 gram of casein forms a
soluble compound with ammonium, sodium and potassium, when
combined with amounts of each somewhere between 1.10 x 104 and
1.15 x 104 gram equivalents of alkali; or, expressed in another
form, 1 cc. of alkali combines with an amount of casein somewhere
between 0.87 and 0.91 gram. The proportion of basic element in
each compound is approximately the following: NH4, 0.20 per ct.;
Na, 0.26 per ct.; and K, 0.44 per ct. Caseinates combining with the
amount of alkali base indicated contain the smallest known amount
of base, according to our present knowledge. It seems proper,
therefore, to suggest that such compounds be called mono-basic
caseinates.
1
N
10
N
10
Preparation of mono-ammonium caseinate. It seemed desirable
that we should carry the work somewhat farther and prepare one
pure compound, at least, in dry form for study. The ammonium
compound was chosen as the one offering least difficulty. The
method of preparation was as follows: In 2 liters of distilled water
containing 250 cc. of NH4OH, 25 grams of base-free casein were
dissolved. After solution, was complete, we slowly added 125 cc.
of HCl, care being taken to agitate the mixture during the addi-
tion of the acid, in order to prevent premature precipitation of any
casein. There was next added very cautiously HCl until a per-
manent precipitate began to appear, as shown by centrifuging the
mixture. The solution was then filtered and measured. The
amount of HCl required to precipitate the casein completely was
determined in an aliquot part. Then one-third of this amount was
added to insure the presence of only mono-basic caseinate. Any
precipitate formed was removed by filtration and the filtrate was
dialyzed until the ammonium chloride that had been formed in the
N
60
N
50
C
15
·
4
reaction was completely removed. The resulting solution, con-
taining mono-ammonium caseinate, was then precipitated by addi-
tion of acid-free alcohol. The precipitate was filtered, washed with
acid-free alcohol and ether and dried at 120° C. In several prepara-
tions thus made the amount of ammonia was determined; the results
are given in the following table:
TABLE V.-COMPOSITION OF MONO-AMMONIUM CASeinate.
Amount Amount of
of
N NH₁ OH
caseinate found.
10
used.
Grams.
5.891
4.870
*4.000
*3.000.
Cc.
6.64
5.38 1
4.30 1
3.16 1
Relation of casein to NH4OH in caseinate.
1 gram of casein to 1.127 x 104 grams equivalents
(6
(6
"
(C
((
((
"1.105 x 104
1.075 x 104
1.053 x 104
((
"
((
((
((
((
((
Percentage
of NH4 in
caseinate.
0.203
0.200
0.194
0.190
*Preparations of caseinates made by Mr. O. B. Winter.
N
40
Acid caseinates of calcium, strontium and barium. In making
preparations of the caseinates of the alkaline earth bases, difficulty
was experienced in obtaining concordant results. The trouble was
finally found to be due to the presence of the chloride formed when
the solution of the caseinate is treated with hydrochloric acid. Such
chlorides tend to cause precipitation of the caseinates either by
decreasing their solubility or, perhaps, by formation of double salts,
consisting of the chloride in combination with the caseinate.¹ The
difficulty of insolubility is readily overcome by removal of the
chloride through simple dialysis before the amount is sufficient to
cause precipitation. To accomplish this, we made use of the fol-
lowing process: In 200 cc. of hydroxide of calcium, strontium or
barium, we dissolved 5 grams of casein and then diluted the solution
to 250 cc. A preliminary or trial determination was made by adding
50 HCI to 50 cc. of the caseinate solution in portions of 5 cc. at a
time, agitating constantly and, after each addition, testing for the
presence of a precipitate by centrifuging a portion, until a precipi-
tate appeared, just as in the case of preparing alkaline caseinates
(p. 13). Then to each of several flasks containing 50 cc. of the
caseinate solution we added an amount of HCl that was 5 cc.
less than the amount causing the first appearance of a permanent
precipitate in the preliminary trial. The contents of the flask were
then placed in dialyzing tubes and, by frequent changes of the sur-
rounding water, most of the soluble chloride that had been formed
was removed. The contents of one tube were then used for another
N
1
N
50
¹ Pfeiffer and Modelski, Ztschr. Physiol. Chem., 81: 329, 1912.
16
preliminary test. An amount of acid less than that required to
produce a precipitate in this second test was then added to all the
tubes and the contents again dialyzed. This operation was con-
tinued in the manner indicated in the following table:
TABLE VI. ILLUSTRATION OF METHOD USED IN PREPARING ACID CASEINATES OF
CALCIUM, STRONTIUM AND BARIUM.

Amount
of casein
in
solution.
Gram.
1
---and
1
1
1
1
1
--
1
1
1
1
1
1
1
1
1
1
1
1
1
parak parmak
1
1
1
1
1
1
1
Amount of
Nhydroxide
solution
used.
50
Cc.
50
50
영영​영영​영영 ​영영​영영 ​영​영영
​50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
Amount of
N
50
HCI
added.
Cc.
30
2009 2030 2
25
30
35
25
30
35
40
25
35
36
38
25
30
35
37
5888888
38
39
25
30
35
37
38
38.5
39
Sign of
precipitation.
Precip.
0
0
Precip.
0
0
Precip.
0
0
0
Precip.
0
0
0
Precip.
0
0
0
Precip.
First trial.
Dialyzed and used for next.
66
((
(<
،،
،،
N
50
Dialyzed and used for next.
(6
((
<<
،،
((
Dialyzed and used for next.
(
<<
((
(C
"
((
((
((
((
<<
Dialyzed and used for next.
"
(6
<<
66
(C
<<
،،
"
((
،،
(6
"
<<
<<
((
(6
Dialyzed and used for next.
،،
(
"
"
(C
((
"
((
،،
((
((
((
((
((
"
((
(C
((
((
In the manner described above, we have made numerous prepara-
tions of calcium, strontium and barium caseinates; the averages of
many results are given in Table VII.
N
We have found that in adding HCI to 50 cc. of a caseinate
solution containing 1 gram of casein dissolved in 50 cc. of solution
of hydroxide of calcium, strontium or barium, it requires less than
60
17
:
N
50 cc. of acid to precipitate the casein completely; the exact amount
is 44.5 cc. of 50 HCl. The remaining amount of base, equal to
5.5 cc. of hydroxide, or 1.1 cc. of hydroxide, appears to be
held in combination in the insoluble compound.
N
50
10
TABLE VII.— CASEINATES OF CALCIUM, STRONTIUM AND BARIUM.



Amount
of
casein
used.
Kind of
hydroxide
used.
Grams.
1
Ca (OH)2
1
Sr (OH)2
1 Ba (OH)2
Amount
N
of
50
hydrox-
ide used.
Cc.
50
50
50
Amount
N
of HCI
50
required
to cause
first sign
of perma-
nent pre-
cipitate.
Cc.
38.5 to 39
"
<<
"
"(
"
"
Amount of base com-
bined with casein
in solution.
21888
N
50
Cc.
11 to 11.5
"
"
"
"
((
"
N
10
Cc.
2.2 to 2.3
"(
"(
"
"
"
"
Amount
N
of
50 HCI
required
to pre-
cipitate
all casein.
Cc.
44.5
(
"
Amount of base
in precipitated
casein.
N
50
Cc.
5.5
"C
སྐ
N
10
Cc.
1.1
((
"
These results indicate the formation of two sets of compounds,
when casein is dissolved in a hydroxide of calcium, strontium or
barium and this solution is neutralized with acid under the conditions
of our experiments. One set of compounds contains twice as much
base as the other.
Attention is called to additional details in the following statements:
(1) In the di-basic compounds, as the results show, 1 gram of
casein requires between 2.2 x 104 and 2.3 x 104 gram equivalents
of hydroxide of calcium, strontium or barium to form a compound
which is soluble in water when there is not present any, or more
than a trace of, soluble chloride of any of these elements. The
addition of even a small amount of a soluble salt of any of these
elements to a solution of any of these di-basic caseinates causes the
formation of a precipitate.
(2) In these di-basic compounds, 100 grams of casein combine
(a) with 0.44 to 0.46 gram Ca (equal to 0.62 to 0.64 gram CaO),
(b) with 0.96 to 1.01 gram Sr (equal to 1.14 to 1.19 grams SrO),
or (c) with 1.51 to 1.58 grams Ba (equal to 1.69 to 1.76 grams BaO).
(3) Apparently, with the treatment described above, 1 gram of
casein combines with about 1.1 x 104 gram equivalents of the hydrox-
ide of calcium, strontium or barium to form an insoluble compound,
when an acid is added in amount just sufficient to precipitate the
casein completely. These compounds are regarded as mono-basic.
(4) In these insoluble mono-basic compounds 100 grams of casein
combine approximately (a) with 0.22 gram Ca (equal to 0.31 gram
CaO), (b) with 0.48 gram Sr (equal to 0.57 gram SrO), or (c) with
0.76 gram Ba (equal to 0.85 gram BaO).
(5) These insoluble compounds possess some highly interesting
properties; they are soluble in a 5 per ct. solution of sodium, potas-
18
sium or ammonium chloride. This solubility is due to an exchange
of bases, which, for our purpose, can be represented by the following
reversible reaction:
Ca<
caseinate
2 Na caseinate+ CaCl,
caseinate
(insoluble)
(soluble)
i
That the reaction is a reversible one is supported by the following
experimental evidence: Mono-calcium caseinate was prepared and
freed from soluble calcium salts by washing and dialysis. The
compound was then dissolved in a 5 per ct. solution of calcium-free
sodium chloride. That an interchange of bases had taken place was
shown by the fact that when the caseinate brine solution was dialyzed,
calcium was found in the solution outside the dialyzing tube. This
brine solution of caseinate was then dialyzed until free from cal-
cium and was then filtered. A solution of calcium chloride was
then added to this dialyzed solution and at once a precipitate of
calcium caseinate was produced. That this precipitate is a calcium
salt can be shown in two ways: (1) By washing and dialyzing until
free from soluble chloride and then igniting. Calcium is found in
the ash. (2) By washing and dialyzing until free from soluble
calcium, then redissolving in 5 per ct. solution of calcium-free sodium
chloride and dialyzing. Calcium is found to dialyze out of this
brine solution of caseinate.
+ 2 NaCl
There is another point of interest in connection with this com-
pound which we will briefly refer to here but consider in more detail
in the report of another investigation. When a small amount of
acid is added to milk or is formed in milk by lactic fermentation,
a substance separates on warming which is very stringy and which
easily dissolves in a 5 per ct. solution of sodium chloride. This
substance is probably mono-calcium caseinate.
N
20
N
20
N
20
Preparation of mono- and di-calcium caseinates. In order to study
the composition and properties of these compounds more fully,
preparations of mono- and di-calcium caseinates were made. The
following method was employed: In 800 cc. of Ca(OH)2 there
were dissolved 20 grams of base-free casein. To this solution was
added 400 cc. of HCI; the solution was then dialyzed to remove
most of the resulting calcium chloride. Then HCl was added
very cautiously under constant agitation of the mixture until a
permanent precipitate began to appear, as shown by centrifuge.
The solution was then dialyzed again and then more acid was added
until a precipitate once more began to form. Alternate dialysis and
addition of acid were continued until no more acid could be added
without causing a precipitate. The amount of acid necessary to
precipitate all of the casein was next determined in an aliquot por-
tion, and one-third of this amount of acid was then added. The
19
precipitated casein was filtered out and the filtrate was dialyzed. This
solution contained di-calcium caseinate. The solution was divided,
one portion being used for the preparation of the di-calcium caseinate
and the other for the mono-calcium caseinate.
In completing the preparation of the di-calcium caseinate, the
salt was precipitated by addition of acid-free alcohol, the precipitate
being washed with acid-free alcohol and ether, and then dried at
120° C. The composition of this preparation is given in Table IX.
In preparing the mono-calcium caseinate, the solution of
di-calcium caseinate was treated with enough acid to precipitate
three-fourths of the casein. The resulting precipitate was filtered,
washed with water, acid-free alcohol and ether and then dried at
120° C. The results in Table VIII show the amount of calcium
found in the preparation.
TABLE VIII.- COMPOSITION OF MONO-CALCIUM CASEINATE PREPARATION.
Amount
of com-
pound
used.
Grams.
5
LO
5
LO
5
Average..
Amount
of com-
pound
used.
Grams.
4.2825
4.1215
1
Ave. 4.202
Amount of CaO
found.
Gram.
0.0149 (0.0106 Ca)
0.0141 (0.0101 Ca)
0.0146 (0.0104 Ca)
0.01453 (0.0104 Ca)
Amount of CaO
found.
Grams.
0.0233 (0.0167 Ca)
0.0235 (0.0168 Ca)
0.0234 (0.01675 Ca)
Percentage of CaO
in compound.
TABLE IX.-COMPOSITION OF DI-CALCIUM CASEINATE PREPARATION.
0.298 (0.213 Ca)
0.282 (0.201 Ca)
0.292 (0.209 Ca)
0.291 (0.208 Ca)
Relation of casein to cal-
cium in compound.
1 gram of casein to 1.06
x 104 gram equivalents.
1 gram of casein to 1.01
x 104 gram equivalents.
1 gram of casein to 1.04
x 10 gram equivalents.
0.544 (0.39 Ca)
0.572 (0.41 Ca)
1 gram of casein to 1.04
x 104 gram equivalents.
Percentage of CaO | Relation of casein to calcium
in compound.
in compound.
1 gram of casein to 1.95 x
10-4 gram equivalents.
1 gram of casein to 2.04 x
10-4 gram equivalents.
0.558 (0.40 Ca) 1 gram of casein to 2.00 x
104 gram equivalents.
If we compare the results given in Tables VIII and IX with the
figures given in paragraphs (1), (2), (3) and (4) on page 17, it is
obvious that the results embodied in these tables are lower. The
higher results are obtained by the volumetric method and are believed
20
to be nearer the truth, owing to the difficulty of preparing these
caseinates in pure form. The values by the volumetric method are:
1 gram of casein to 1.10 (to 1.15) x 104 gram equivalents of cal-
cium for the mono-basic caseinate, and 1 gram of casein to 2.2 (to
2.3) x 104 gram equivalents of calcium for the di-basic caseinate.
VALENCY OF CASEIN MOLECULE AND MOLECULAR WEIGHT OF CASEIN.
In the case of the compound of casein and calcium, which is neutral
to phenolphthaleïn, it is found that 1 gram of casein combines with
9 x 104 gram equivalents of calcium. In the case of the mono-
ammonium caseinate, the combination is in the proportion of 1 gram
of casein to a value between 1.1 x 104 and 1.15 x 104 gram equiva-
lents. Since we have one compound of known composition and
another of approximately known composition, it should be possible
by applying the rule of constant proportions to determine the true
composition of the mono-basic caseinate and also the number of
valencies satisfied in the caseinate neutral to phenolphthaleïn.
We have reason to believe that the proportion, 1 gram of casein
to 1.125 x 104 gram equivalents of alkali, is the true value, since,
first, this lies between the two limits (1.10 and 1.15) found in our
volumetric work, and, second, this figure agrees with that found by
assuming a valency of 8 for the basic calcium caseinate, in which
1 gram of casein combines with 9 x 104 gram equivalents of calcium.
Thus, if the valencies satisfied are 8, the proportion becomes 1 gram
of casein to 1.125 x 104 gram equivalents of alkali for mono-basic
caseinates. If, however, we were to assume that the number of
valencies in the basic compound is 7 rather than 8, then the mono-
basic salt would, theoretically, have the composition, 1 gram of
casein to 1.285 x 104 gram equivalents of alkali, a value too high
for our analytical results. If, on the other hand, we were to assume
the number of valencies in the basic compound to be 9, then the
proportion in the mono-basic compound would become 1 gram of
casein to 1 x 10 gram equivalents of alkali, a value too low for
our analytical results obtained with mono-ammonium and other
alkali caseinates. Assuming 8 as the true valency of basic calcium
caseinate gives us the value, 1 gram of casein to 1.125 x 10¹ gram
equivalents of alkali, a result which agrees with the volumetric.
results obtained in case of the mono-alkali caseinates.
If we use the sulphur content as a basis for calculating the mole-
cular weight of casein we have n(320) 100=n4454+. Here the
value of n appears to be 2, and the molecular weight would be 8908,
which is in very close agreement with the value found above,
8888+. This would indicate that there are two atoms of sulphur in
each molecule of casein.
The amount of phosphorus in casein was found to be 0.71 per ct.,
which would lead to the molecular weight, n(310) 100=n4372—.
If the value of n is 2, the molecular weight of casein becomes 8744.
0.7
21
h
1
On the basis of 8 representing the true number of valencies satis-
fied in the basic-calcium caseinate molecule, the molecular weight
of casein is 12x10-4 or 8888+. Robertson¹ reaches similar
results by deducing the molecular weight of casein in several
different ways. This would also make the equivalent weight of
casein equal to 8888 or 1111. This value is in close agreement with
the equivalent weight assigned by other workers to casein prepared
from cow's milk. Laqueur and Sackur2 give about 1135; Mat-
thaiopoulos³ gives 1131.5; Long gives 1124.
As a result of the work here reported, it would seem possible,
theoretically, to prepare a series of not less than eight combinations
of casein with each of the basic elements studied.
According to what we have reason to believe at the present time,
not less than four of these combinations have been prepared.
Using the calcium compounds, we have the following series:
Name of compound.
Mono-calcium caseinate.
Di-calcium caseinate.
Neutral calcium caseinate.
Basic calcium caseinate..
·
Grams Ca for 100 grams of casein.
0.22 (equal to 0.31 CaO)..
0.44 (equal to 0.62 CaO)
1.07 (equal to 1.50 CaO)
1.78 (equal to 2.50 CaO)
•
•
•
Valencies
satisfied.
125∞
8
It is noticeable that in this series compounds are absent repre-
senting valencies of 3, 4, 6 and 7. Whether such compounds can
be prepared no one can say at present.
¹Jour. Physical Chem., 15: 179, 1911.
2Hofmeister's Beiträge, 3: 193, 1902.
³Ztsch. Analyt. Chem., 47: 492, 1908.
4Jour. Am. Chem. Soc., 28: 372, 1906.
5 N. Y. Agr. Exp. Sta. Bull. No. 261, and Am. Chem. Jour., 33: 472, 1905.
PART II. PARACASEIN AND SOME OF ITS COMPOUNDS.
The term paracasein is generally applied to the precipitated
protein compounds formed by treating milk with rennet-extract.
The relations between casein and paracasein are not satisfactorily
understood as yet. Little study has been given to the compounds
formed by paracasein. Van Slyke and Hart' have shown that
paracasein combines with calcium to form a compound neutral to
phenolphthaleïn, containing about 2.40 per ct. CaO (1.71 per ct. Ca),
or 1 gram of paracasein combines with 8.55 x 10 gram equivalents
of calcium. They also found a compound neutral to litmus, in
which there was about 1.50 per ct. CaO (1.07 per ct. Ca), or 1 gram
of paracasein combines with 5.35 x 10 gram equivalents of cal-
cium, According to their results, the compound containing the
22
higher amount of calcium is soluble in water, while the other is
insoluble. It will be later shown by us that both of these com-
pounds are soluble in pure water. In reporting the compound
neutral to litmus to be insoluble in water, the fact was overlooked
that, under the conditions of their experiments, there was always
present in the mixture a considerable amount of calcium chloride,
which was formed by the reaction of the hydrochloric acid upon the
lime-water solution of paracasein, and the presence of this calcium
chloride caused the precipitation of the neutral calcium paracaseinate,
as we shall show later.
We have been able to prepare compounds of paracasein corre-
sponding to the mono- and di-basic caseinates, in which, however,
the proportion by weight of paracasein to base is just one-half that
found in the caseinates.
PREPARATION AND COMPOSITION OF ASH-FREE PARA CASEIN.
Milk from which the fat has been removed as completely as pos-
sible by centrifugal force was heated to 37° C. and rennet-extract
(Hansen's) was added in the proportion of 0.12 cc. per 1,000 cc. of
milk. The milk was allowed to stand until the precipitated para-
caseinate had separated as completely as possible. The resulting
curd was then stirred vigorously in order to break it into small
pieces and hasten the separation of the whey. When the curd had
settled, the supernatant whey was removed by siphon. The para-
caseinate was washed with distilled water several times, and finally
5 liters of water were added for each liter of milk originally used.
Dilute ammonium hydroxide (6 cc. of strong reagent diluted to
1,000 cc.) was then added, as in the case of the preparation of casein
(p. 8), and the mixture stirred until the paracaseinate was dissolved.
The process of precipitating, washing and redissolving was con-
tinued as in the case of casein; the remaining calcium was finally
separated by addition of ammonium oxalate and centrifuging. One
preparation made in this way contained 0.07 per ct. of ash. One
gram gave a clear solution when dissolved in 10 cc. of NH4 OH
and 90 cc. of water. One preparation, with high ash content, gave
the following results on analysis:
N
10
Moisture.
Ash.
In dry substance:
•
Carbon.
Hydrogen..
•
•
Per ct. In dry substance:
1.63
Nitrogen.
Phosphorus.
Sulphur.
0.61
53.50
7.26
→
Oxygen (by difference).
Per ct.
15.80
0.83
0.87
21.13
Another preparation with exceptionally low ash content gave the
following results:
Ash....... 0.07 per ct. Phosphorus.... 0.71 per ct. Sulphur..... 0.72 per ct.
23
PREPARATION AND COMPOSITION OF BASIC CALCIUM PARA CASEINATE.
Like casein, paracasein manifests its acid character by its power
to liberate carbon dioxide from calcium carbonate, forming a calcium
paracaseinate. The results of the reaction were measured by us in
the same manner as in the case of casein (p. 10), and it is, therefore,
not necessary to report any of the details of methods or results.
The average of many determinations indicates that paracasein
unites with calcium to form a paracaseinate which is neutral to
phenolphthalein and has the same general composition; 1 gram of
paracasein combines with 9 x 10 gram equivalents of calcium.
PREPARATION AND COMPOSITION OF UNSATURATED OR ACID
PARA CASEINATES.
In preparing acid paracaseinates of bases, the same volumetric
method of procedure was followed as in case of the casein salts
(p. 12). The appearance of a precipitate in a centrifuged portion
after addition of acid to an alkali solution of paracaseinate was
made to serve as an indicator in regard to the end point of the reaction.
We dissolved 5 grams of the ash-free paracaseinate in 200 cc. of
alkali, made up the solution to 250 cc. and then determined the end
points by careful addition of HCl to 50 cc. portions.
N
40
N
50
Acid paracaseinates of ammonium, sodium and potassium. In the
manner described, determinations were made in the case of base-
free paracasein dissolved in hydroxide of ammonium, sodium and
potassium, with the results tabulated below:
TABLE X.-RELATION OF ALKALI BASES TO PARACASEIN IN ACID PARACASEINATES.
Amount
of para-
casein
used.
Gram.
1
1
1
Kind of
alkali
used.
NH, OH
Na OH
KOH
Amount
N
of
60
alkali
used.
888888888
Cc.
N
Amount of HCI
DU
required to cause first
sign of permanent
precipitate.
Cc.
Between 38.5 and 39
<<
"
"
"
"
"
(C
"
Amount of alkali left
combined with paracasein.
21888
"
N
50
Cc.
11 to 11.5
"
"
"
*

N
10
ละ×
Cc:
2.2 to 2.3
ละะ
Amount
N
of
HCI
50
required to
precipitate
all of
paracasein.

Cc.
50
<<
((
These results show that 1 gram of paracasein combines with an
amount of alkali somewhere between 2.2 x 10 and 2.3 x 10¹ gram
equivalents, in forming soluble compounds with ammonium, sodium
and potassium, which are acid to both litmus and phenolphthaleïn.
Expressed in another form, 1 cc. of alkali combines with an amount
of paracasein somewhere between 0.435 and 0.455 gram. The
proportion of basic element in each compound is approximately as
follows: NH4, 0.40 per ct.; Na, 0.52 per ct.; K, 0.88 per ct. The
amount of each basic element in these paracaseinates is just double
that present in the corresponding casein compounds (p. 14).
N
10
24
+1
The amount of acid required to precipitate completely the para-
casein in these compounds is exactly equal to the alkali used to
dissolve the paracasein; this indicates that there is no additional
paracaseinate, in insoluble form, containing less of these basic
elements.
Preparation of mono-ammonium paracaseinate. This compound
was isolated and prepared in dry form, for futher study, in the
manner already described in the preparation of mono-ammonium
caseinate (p. 14). Care must be taken to use a paracasein prepa-
ration free from casein or salts of calcium, strontium, barium, etc.
A determination of the amount of ammonia present in preparations
thus made is given below.
Grams.
4
4
TABLE XI.- COMPOSITION OF MONO-AMMONIUM PARA CASEINATE.

Amount Amount of
of para-N NH OH | Relation of paracasein to NH OH in paracaseinate.
caseinate found.
used.
10
Cc.
8.201 1 gram of paracasein to 2.05 x 104 gram equivalents
7.98 1
2.00 x 10-4
<<
((
(6
(C
(6
Percent-
age of
NH₁ in
para-
caseinate.
0.37
0.36
These results show the difficulty of making a pure compound, but
they indicate that the percentage of ammonium is double that found
in the corresponding mono-ammonium caseinate.
Acid paracaseinates of calcium, strontium and barium.- In pre-
paring paracasein salts of calcium, strontium and barium, the pres-
ence of their chlorides causes much more trouble in respect to pre-
cipitation than in case of the casein salts. Special care must be
taken to prevent the accumulation of chlorides of these elements.
By sufficiently frequent dialysis it was possible to obtain the results
reported below (Table XIII). Another point in connection with
paracasein is the fact of its slow rate of solution in the hydroxides.
of calcium, strontium and barium; on this account we used 400 cc.
of hydroxide to dissolve 5 grams of paracasein, making the volume
up to 500 cc. with water.
N
40
Trial or preliminary determinations were made in the same manner
N HCI
as with casein (p. 15), in order to determine the amount of 60
required to precipitate the paracasein in the absence of chlorides of
calcium, strontium and barium. The specific details employed and
results obtained are indicated in the following table:
:
2,5
TABLE XII. ILLUSTRATION OF METHOD OF PREPARING ACID PARACASEINATES OF
CALICUM, STRONTIUM AND BARIUM.

Amount
of para-
casein in
solution.
Grams.
1
1
1
1
1
1
HHAT
−−−
1
1
1
1
1
HAH——
1
1
1
1
1
1
1
1
1
1
1
Jorda
Amount
of para-
casein.
Gram.
1
1
Amount of
N hydroxide
50 solution
used.
Cc.
Kind of
hydrox-
ide.
100
Ca (OH)2
Sr (OH)2
Ba (OH)2
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
毚
​Amount of
N
50
HCI
added.
Amount
N
of 50
hydrox-
Cc.
Cc.
100
100
100
28 POF
75
76
76.5
77
75
76
76.5
77
77.5
75
76
76.5
77
77.25
77.50
of
Amount
N
HCI
50
required
to cause
ide used. first sign of
Precipita-
tion.
permanent
precipitate.
Precip.
0
Precip.
0
0
Precip.
0
0
0
Precip.
0
0
Precip.
0
0
0
Precip.
First trial.
Dialyzed and used for next.
The results obtained by the method of procedure indicated above
are given in the following table for calcium, strontium and barium.
TABLE XIII.- PARACASEINATES OF CALCIUM, STRONTIUM AND BArium.
BARIUM.
Dialyzed and used for next.
"
((
((
((
(C
N
50
Dialyzed and used for next.
(<
((
،،
((
"
((
((
(C
((
((
Dialyzed and used for next.
(C
((
(6
"
<<
((
((
((
(<
(C
((
Amount of base left
combined with para-
casein in solution.
Dialyzed and used for next.
(C
"
(C
،،
"
(
((
((
((
((
(
((
((
"(
(6
((
((
N
10
(
Cc.
Cc.
Cc.
77.25 to 77.5 22.5 to 22.75 4.5 to 4.55
77.25 to 77.5 22.5 to 22.75 4.5 to 4.55
77.25 to 77.5 22.5 to 22.75 4.5 to 4.55
❤
(C
(6
of
*
required
to pre-
cipitate
all para-
casein.
،،
،،
Amount Amount of base
N
in precipitated
paracasein.
50 HCI
Cc.
88.5
88.5
88.5
((
((

N
50
Cc.
11.5
11.5
11.5
N
10
Cc.
2.3
2.3
2.3
26
These results indicate the formation of two sets of compounds
when paracasein is dissolved in a hydroxide of calcium, strontium.
or barium and this solution is neutralized with acid under the condi-
tions of our experiments. One set of compounds contains twice as
much base as the other, corresponding to the two sets of casein
compounds. Additional details are discussed below:
-4
(1) In the di-basic compounds, the results show that 1 gram of
paracasein requires between 4.5 x 10 and 4.55 x 10 gram equiv-
alents of hydroxide of calcium, strontium or barium to form a com-
pound which is soluble in pure water. These compounds are easily
precipitated from their water solutions by a minute amount of a
soluble salt of calcium, strontium or barium.
(2) In these di-basic compounds, 100 grams of paracasein com-
bine, approximately, (a) with 0.90 gram Ca (equal to 1.26 grams
CaO), (b) with 1.97 grams Sr (equal to 2.33 grams SrO), or (c) with
3.09 grams Ba (equal to 3.45 grams BaO).
(3) It is indicated that, with the treatment described above,
1 gram of paracasein combines with about 2.3 x 10 gram equiva-
lents of the hydroxide of calcium, strontium or barium to form an
insoluble compound. These compounds are regarded as mono-basic
paracaseinates.
(4) In these insoluble mono-basic paracaseinates, 100 grams of
paracasein combine, approximately, (a) with 0.46 gram Ca (equal
to 0.64 gram CaO), (b) with 1.01 grams Sr (equal to 1.19 grams
SrO), or (c) with 1.58 grams Ba (equal to 1.76 grams BaO).
(5) Mono-basic paracaseinates of calcium, strontium and barium
are completely soluble in warm 5 per ct. solution of ammonium,
sodium or potassium chloride. This solubility is due to interchange
of bases, just as in the case of caseinates (p. 18); the reaction was
studied experimentally with paracaseinates and the same results
obtained as in case of the caseinates.
(6) A comparison of the composition of the caseinates and para-
caseinates shows that twice as much base is present in paracaseinates
as in the corresponding caseinates. This is easily seen in the fol-
lowing table.
TABLE XIV. COMPARISON OF COMPOSITION OF CASEINATES AND PARACASEINATES.
Amount of basic element combined with 100 grams of casein and paracasein.
Basic
element.
Ca...
Sr..
Ba.
•
Mag

In mono-basic In mono-basic
caseinates. paracaseinates.
Gram.
0.22
0.48
0.76
Grams.
0.46
1.01
1.58
In di-basic
caseinates.
Grams.
0.44 to 0.46
0.96 to 1.01
1.51 to 1.58'
In di-basic
paracaseinates.
Grams.
0.90
1.97
3.09
27
Preparation of mono- and di-calcium paracaseinates. In order to
study the composition and properties of these compounds further,
preparations of the mono- and di-calcium paracaseinates were made.
The first steps in making these compounds are the same.
An excess
of ash-free paracasein is agitated with lime-water until a saturated
solution is formed, the undissolved paracasein being removed by
filtration. To the solution HCl is added until a permanent pre-
cipitate begins to appear. The solution is again filtered and then
dialyzed. Alternate addition of acid and dialysis are continued
until no more acid can be added after dialysis without causing
precipitation. The amount of HCl required to precipitate all
the paracasein is next determined in an aliquot portion, and one-
third that amount of acid is added. The solution is then filtered
and dialyzed. This solution contains di-calcium paracaseinate.
This solution is divided into two portions; in one the di-calcium
paracaseinate is precipitated by addition of acid-free alcohol, the
precipitate being washed with acid-free alcohol and ether and dried
at 120°C. This preparation was found to contain between 4.2 x 10¹
and 4.6 x 10 gram equivalents of calcium for 1 gram of paracasein.
N
50
50
In the second portion of di-calcium paracaseinate solution, enough.
N HCl is very slowly added to precipitate three-fourths of the para-
casein in solution. The precipitate is mono-calcium paracaseinate;
this is filtered, washed with acid-free alcohol and ether, and dried
at 120° C. Before being washed with alcohol, the precipitate is
completely soluble in 5 per ct. solution of sodium chloride. This
compound, mono-calcium paracaseinate, is identical in its properties.
with the brine-soluble compound formed in cheddar cheese, to which
attention was first called by Van Slyke and Hart under the expres-
sion, "salt-soluble compound." Attention will again be called to
this compound later. An analysis of this preparation showed it to
contain between 2 x 104 and 2.3 x 10 gram equivalents of calcium
for 1 gram of paracasein.
N
20
VALENCY OF PARA CASEIN MOLECULE AND MOLECULAR WEIGHT OF
PARA CASEIN.
In the case of basic calcium paracaseinate, the compound that is
neutral to phenolphthaleïn, it is found that 1 gram combines with
9 x 10 gram equivalents of calcium, while in the case of mono-
ammonium paracaseinate the combination is in the ratio of 1 gram
of paracasein to a value between 2.2 x 104 and 2.3 x 10 gram
equivalents. According to the rule of constant proportions, the
number of valencies satisfied in the first compound would be between
22 and 2 or 4.
or 4. The molecular weight of paracasein would
therefore be 2 or 4444+.
1
Our results indicate that the
2.26 x 104
molecular weight of casein, 8888, is just twice that of paracasein.
4444. Using the sulphur content as a basis for calculating the
molecular weight of paracasein, we have n(32) 100= n 4454+.
9
2.
2.3
72
28
"
31.04
The percentage of phosphorus would give n(31) 100n 4372-.
The value of n appears to be 1 and each molecule of paracasein
would contain one atom each of sulphur and phosphorus.
Theoretically, it should be possible to make a series of four salts.
of paracasein with bases. We have prepared three, those in which
one, two and four valencies are satisfied.
ACTION OF RENNET ENZYM ON CASEIN IN FORMING PARA CASEIN.
N
50
N
bu
The action of the principal enzym contained in rennet-extract in
splitting casein into two molecules of paracasein is further shown
by the following experiment: Five grams of casein are dissolved
in 250 cc. of KOH. Using the volumetric method given on page 13,
it was found that 44.5 cc. of HCl could be added to 50 cc. of the
caseinate solution, containing 1 gram of casein, before a permanent
precipitate begins to appear. To another 50 cc. of caseinate solu-
tion a few drops of neutral rennet-extract are added. Under the
conditions of the experiment, no precipitate or curd is produced by
the action of the rennet-enzym. After a few minutes HCl is
added and it is found that a permanent precipitate begins to form
as soon as we add only 39 cc. HCI.
N
50
N
60
We have in hand a more extended investigation relating to the
action of rennet-enzym upon casein, the results of which will be
published later.
PART III. COMPOSITION OF THE BRINE-SOLUBLE COM-
POUND IN CHEESE.
1
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 ct. solution of sodium chloride. The exis-
tence of such a substance in cheddar cheese was first brought to
attention by work done at this Station. The presence of this
brine-soluble 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, Van Slyke and Hart were led to conclude erroneously
that the substance consists of a combination of paracasein and
lactic 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 work2 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
1 N. Y. Agr. Exp. Sta. Bul. No. 214, 1902.
2 N. Y. Agr. Exp. Sta. Bul. No. 261, 1905.
29
N
10
2
lactic acid (1 gram of paracasein uniting supposedly with about
0.5 cc. acid). It may be stated here, in passing, that it was later
shown by L. L. Van Slyke and D. D. Van Slyke¹ 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 on
the surface of the solid particles of protein; in other words, it was
shown that casein or paracasein mono-lactate or di-lactate have no
existence as applied to the compounds in question. It still remained,
therefore, to find out what the brine-soluble 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 sodium chloride after
previous removal of all water-soluble constituents. This fact sug-
gested the possibility that the brine-soluble 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 para-
caseinate 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 the cheese, and, as a consequence,
the brine-soluble substance loses its calcium and becomes free para-
casein, which is insoluble in brine solution. Therefore, in the manu-
facture of Camembert cheese, it is found after the first few hours
that the cheese contains no brine-soluble material and, what is also
significant, all the calcium is found in the water extract. The rela-
tion between the brine-soluble substance and the calcium found in
the brine extract in the two types of cheese is illustrated in
Table XV.
M
The question necessarily suggests itself as to whether the calcium
always found in the brine-soluble extract of cheese is not there
incidentally in a mechanical state rather than in combination with
paracasein. In order to study this question the following work
¹N Y. Agr Exp. Sta. Tech. Bul. No. 3, 1906.
2 N. Y. Agr. Exp. Sta. Tech. Bul. No. 4, 1907.
3 N. Y. Agr. Exp. Sta. Tech. Bul. No. 5, 1907.
30
TABLE XV.- COMPARISON OF CHANGES IN CHEDDAR AND CAMEMBERT CHEESE.

Age of cheese.
When curd was cut..
10 hours.
2 days.
4 months.
·
•
•
•
Kind of cheese.
Cheddar.
7 Camembert.
S Cheddar..
Camembert.
Cheddar..
Camembert.
Cheddar.
•
•
+
•
•
•
Total nitrogen Total calcium
in the form of found in
brine-soluble brine-soluble
compound. compound.
Per ct.
3.13
6.72
96.00
94.00
68.87
4.39
43.09
Per ct.
trace.
trace.
27.96
17.76
24.47
trace.
24.28
was done: Twenty-five grams of cheese were ground with sand and
extracted with water at about 55° C., using 150 cc. portions, until
the extract amounted to 1,000 cc. The residue, containing the
brine-soluble substance, was placed in a dialyzing apparatus and
allowed to dialyze to insure the removal of 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 4 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 calcium is present in com-
bination in an insoluble form and that an interchange takes place
between it and sodium, when the insoluble compound is treated
with sodium chloride solution.
In order to throw further light on the character of the brine-
soluble 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 strength that 1,000 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,
4,000 cc. being used; the results in these cases are given for each
1,000 cc. of extract, as well as for the total.
In connection with the data in Table XVI, attention is called to
certain phases of the results.
(1) The chlorides of barium and calcium have no solvent effect.
The chloride of magnesium in strong molecular concentrations acts
much like the chlorides of sodium, ammonium and potassium,
while in lower molecular concentrations its solvent power is
greatly reduced.
31
TABLE XVI.-SOLVENT ACTION OF NEUTRAL CHLORIDES ON THE BRINE-SOLUBLE
COMPOUND IN CHEESE.

Strength of
solution.
Gram
equivalents
per 1,000.
1.0.
0.8.
0.6.
0.4..
0.2
0.2
0.2
0.2
·
•
•
•
·
•
•
Total..
·
•
Amount of
extract.
1st 1,000
2nd 1,000
3rd 1,000
4th 1,000
4,000
Percentage of total nitrogen in water-insoluble residue
of cheese extracted by
Na Cl. NH, Cl. KCI.
Cc.
1,000 68.57 67.62
1,000 69.29 65.24
1,000 56.19 56.43
1,000
51.43 51.19
47.62
13.33
2.95
trace
63.90
50.47
50.47
45.95
44.52
49.05
40.95
10.48
13.90
4.10
2.00
trace
trace
63.63 56.85
Mg Cl2
63.81
48.33
lost
23.57
4.00
5.24
4.29
Ba Cl₂
0
0
0
0
0
Ca Cl₂
0
0
0
0
(2) Sammis and Hart' undertook 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 concentra-
tion, 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 (p. 18).
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 shown in
the above table in the case of the 0.2 normal solutions; by continued
extraction, the total amounts extracted are found to be essentially
the same as in case of the more concentrated solutions.
G
IDENTITY OF THE BRINE-SOLUBLE COMPOUND OF CHEESE WITH MONO-
CALCIUM PARA CASEINATE.
We have shown (p. 26) that paracasein combines with calcium to
form a compound insoluble in water but soluble in 5 per ct. solution
of sodium chloride (sodium replacing calcium). In this compound,
we have shown, 1 gram of paracasein is in combination with
¹Jour. Biol. Chem., 6: 181, 1909.

32
2.25 x 10 gram equivalents of calcium. Indications pointed to the
identity of the brine-soluble compound of cheese with this mono-
calcium 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 55° C. in order
to remove all soluble compounds. The residue was then extracted
with many portions of a 5 per ct. solution of sodium chloride 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 (p. 9). The preparation on analysis gave the
following results, which are probably high, owing to the ash content:
Moisture.
Ash.
In dry substance:
Carbon..
Hydrogen.
•
Per ct.
2.32
0.25
52.97
7.15
In dry substance:
Nitrogen.
Phosphorus.
Sulphur..
•
•
•
Oxygen (by difference) . . .
•
•
Per ct.
15.82
0.75
0.78
22.28
A study of the properties of this substance resulted as follows:
(1) The substance acts as an acid in combining with bases.
(2) It decomposes calcium carbonate and gives a compound in
which 100 grams of substance combines with the equivalent of 2.52
grams CaO (equal to 1.80 grams Ca), or 1 gram of substance com-
bines with 9 x 10 gram equivalents of calcium.
(3) The solution of this calcium compound is neutral to
phenolphthaleïn.
-
(4) Measured by the volumetric method, it was found to form a
compound with ammonia represented by the combination of 1 gram
of substance with 2.3 x 104 gram equivalents.
(5) With calcium it forms a compound soluble in 5 per ct. solution
of sodium chloride but insoluble in water, which contains 1 gram of
substance combined with 2.3 x 10 gram equivalents of calcium.
-4
(6) It forms also a compound with calcium that is soluble in water
containing 1 gram of substance combined with 4.5 x 10
equivalents.
gram
In view of the marked agreement of the composition and prop
erties of the brine-soluble substance, formed in cheese, with the
compound, mono-calcium paracaseinate, as prepared by us, there i
good reason to believe that the brine-soluble substance is mono
calcium paracaseinate, having the composition of 1 gram of para
casein combined with 2.25 x 10 gram equivalents of calcium.

THE UNIVERSI
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UNIVERSITY OF MICHIGAN
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1
121
i