4 ASSOCIATION OF OFFICIAL AGRICULTURAL CHEMISTS [Vol. II, No.

T H E SO-CALLED NEUTRALIZATION PRECIPITATE OF COWS'

MILK.

B Y LEROY S. PALMER (Agricultural Experiment Station, Columbia, Mo.).

Van Slyke and Hart, 1 in 1903, called the attention of this association to

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the fact that the filtrate from the acetic acid precipitation of the casein
of cows' milk (by the official association method) gives more or less of a
precipitate on neutralization. They stated that this precipitate usually
amounted to 0.1 per cent of casein. The explanation offered for this phe-
nomenon was as follows: When cows' milk is treated with 1.5 per cent
acetic acid, as in the official method, the precipitate formed is wholly
casein-diacetate. A small portion of this is dissolved by the excess acetic
acid used and reappears when its solution, the filtrate from the casein
precipitation, is neutralized. Whether this neutralization precipitate is
in the form of casein or its acetic acid compound was not stated.
Later work by Van Slyke and Van Slyke2 on the action of dilute acids
upon casein, would indicate that this explanation requires some modifica-
tion. These investigators definitely showed that casein does form acetic
acid, as well as other acid compounds, which are somewhat soluble in an
excess' of the acid used. That these acid compounds are of a definite
character, however, was not borne out by the investigation. They ap-
peared to be adsorption compounds rather than chemical combinations.
It was also shown that the acid-casein compounds, when in solution,
give up their acid on neutralization. It would appear from this that the
neutralization precipitate, if it arises from an acid-soluble casein-acetate,
should be free from acetic acid.
Interest in the so-called neutralization precipitate grew out of its in-
variable appearance, in greater or less amount, in some studies of new
methods for the determination of the heat-coagulable proteins of cows'
milk, in all of which the casein was first removed by acetic acid. It seemed
possible that the protein character of the neutralization precipitate, as
indicated by the previously mentioned work, might have a bearing on the
determination of the casein or the heat-coagulable proteins.
Van Slyke and Van Slyke showed that the precipitate, obtained on care-
ful neutralization of solutions of casein-acid compounds, had all the prop-
erties of casein, being readily soluble in an excess of alkali, and reappearing
again on neutralization. They also found that the precipitation of the
casein on addition of alkali was practically complete before the neutral
point was reached, but could be prevented entirely if the alkali was added
rapidly.
1
Bur. Chem. Bull. 81, p . 91.
2
Am. Chem. J., 1907, 38: 383.
1916] PALMER: NEUTRALIZATION PRECIPITATE OF COWS' MILK 5

A brief study of the properties of the neutralization precipitate obtained

on addition of sodium hydroxid to the casein filtrate showed that it also
possessed the property of being precipitated before neutrality was reached,
and of dissolving again on addition of acid. It was not found possible,
however, to bring about a solution of the precipitate in sodium hydroxid.
This insolubility in sodium hydroxid did not appear to be affected in any

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way either (a) by the manner of adding the alkali, whether rapidly or
drop by drop, or (b) by the amount of sodium hydroxid added, or (c)
by the temperature of the solution. In fact practically no solution
occurred even on long boiling in sodium hydroxid solution. This treat-
ment, however, resulted in rendering some of the precipitate insoluble in
acetic acid.
The question at once arose, is the so-called neutralization precipitate
casein?
As a preliminary investigation, casein was prepared from milk by the
addition of acetic acid, according to the method used by Van Slyke and
Van Slyke. Solutions of this casein and also of Merck's c. p. casein (ac-
cording to Hammarsten) were made in sodium hydroxid and in glacial
acetic acid. These solutions were then neutralized with acetic acid and
sodium hydroxid, respectively, and the casein precipitated from the neutral
solutions by acetic acid under conditions as nearly identical as possible
with those which apparently cause the formation of the neutralization
precipitate in the case of milk. The filtrates were then examined for
neutralization precipitate. None was found in any case.
This result also throws great doubt upon the supposition that the neu-
tralization precipitate is casein. On the other hand, the properties of the
neutralization precipitate already described were so suggestive of. tri-
calcium phosphate that a calcium and phosphorus analysis seemed war-
ranted. For this analysis the neutralization precipitate was isolated from
one liter of skim milk as follows:

One liter of fresh skim milk was diluted with one-half liter of water, and 150 cc.
of 10 per cent acetic acid added, the diluted milk being at a temperature of 40°C.
After cooling, the casein was filtered off. Several nitrations were required to get
a clear filtrate. Sodium hydroxid solution (10 to 20 per cent) was now added to the
filtrate to a faint alkaline reaction (to phenolphthalein). A heavy flocculent pre-
cipitate appeared. It was filtered off, washed somewhat with water, and then sus-
pended in water to which 40 cc. of 10 per cent acetic acid had been added. All but
traces of the precipitate dissolved readily in this solution. The acid solution was
now placed on the steam-bath for an hour and then boiled for several minutes. The
coagulated proteins thus formed were removed by filtration. The filtrate was
treated with 10 to 20 per cent sodium hydroxid solution to faint alkalinity. A
heavy flocculent precipitate came down, most of it before the neutral point was
reached. The neutralization precipitate was filtered off, washed thoroughly on
the paper, and then suspended in water and dissolved by the addition of dilute
6 ASSOCIATION OF OFFICIAL AGRICULTURAL CHEMISTS [Vol. II, No. 1

acetic acid. Sodium hydroxid was added t o this solution, bringing a b o u t r e p r e -

cipitation. This precipitate was washed m a n y times b y decantation. I t w a s
finally dried on a porous plate in t h e electric oven a t 105°C, yielding a fine, p u r e w h i t e
powder, which was dried to constant weight a t 105°C. before analysis. About 1.8
grams of dry material were obtained, no allowance being made for losses in isolation.

The results of the analysis of the neutralization precipitate are here given.

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On ignition at red heat it gave off water and blackened. After burning
off the carbonaceous matter the ash was analyzed for calcium and phos-
phorus.
Per cent
T o t a l loss on ignition 16.92
Water lost on ignition 6.95
Nitrogen in dry substance 0.46
Calcium in ash 36.16
Phosphorus in ash 21.12

The results were found rather difficult to interpret because of the large
loss on ignition. A calculation based on the results obtained, however,
shows that a mixture of 72.5 per cent of Ca 3 (P0 4 )2,24.5 per cent of CaHPQi,
2 per cent of H 2 0 and 2.8 per cent of protein (casein or albumin) would
give results conforming very closely to those obtained. The relation be-
tween the theoretical analysis of such a mixture and the results found is
indicated in the following table:
Theoretical Found for
miit re neulralizatim precipitate
per cent per cent
Calcium ;. 33.55 32.60
Phosphorus '. 19.55 19.10
Water lost on ignition 6.57 6.57
Nitrogen •. 0.46 0.46

If the protein in the precipitate be considered as casein, the relation be-

tween the theoretical results obtained from the ash of such a mixture and
the results obtained for the neutrahzation precipitate would be those given
in the following statement:
ASH ANALYSIS.
Theoretical mixture Neutralization precipitate
per cent per cent
Calcium 36.18 38.45
Phosphorus 21.13 22.58

The greater difference in this case is due to the unexplained loss on

ignition.
These results make untenable the previously advanced explanation of the
origin and character of the so-called neutralization precipitate of cows'
milk. Instead of being a portion of the casein which has been dissolved
by the excess of acid used, it appears to be almost entirely a mixture of di-
and tricalcium phosphates. Whether the protein obtained in the analyses
is a contamination or an essential part of the precipitate is not apparent.
1916] PALMER: NEUTRALIZATION PRECIPITATE OF COWS' MILK 7

It may be stated, however, that the method of isolation would seem to

preclude a protein contamination, which is also borne out by the ready
solubility of the isolated precipitate in acetic acid.
The source of the neutralization precipitate is a question of considerable
interest, especially since some evidence can be presented in regard to it.
Lindet 1 has recently shown that casein, which has been precipitated from

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cows' milk by rennet, will yield CaO and P 2 O s on treatment with dilute
acetic acid, equivalent to 3.45 per cent Ca3(P04)2 and an additional 1.84
per cent CaO. Since this result is obtained merely by washing with
dilute acid, it would seem that the explanation of the origin of the cal-
cium phosphate of the neutralization precipitate is very simple. It is
possible, of course, that some of the calcium phosphate, especially the
dicalcium phosphate, may result from preexisting phosphates in the milk
serum. In view of Lindet's results and the fact that it was not found
possible to bring about a further liberation of calcium phosphate (neu-
tralization precipitate) from casein which had been thrown down by acetic
acid, it would seem to be a very easy matter to demonstrate whether the
source of the neutralization precipitate is the caseinogen of the milk or
preexisting phosphates of the serum, or whether it arises from both of these
sources. According to Lindet's results, casein precipitated by rennet
should yield a neutralization precipitate after treatment with acetic acid;
and if this is the only source of the neutralization precipitate, the filtrate
from the rennet coagulation should yield no neutralization precipitate.
In the acetic acid coagulation of casein, the filtrate from the coagulation
of the albumin and globulin by heat will usually show the neutralization
precipitate as readily as the filtrate from the casein coagulation.
No difficulty was experienced in showing the absence of neutralization
precipitate in the filtrate from the rennet coagulation of casein, either be-
fore or after coagulation of the albumin and globulin with heat. On the
other hand, neither was it found possible to show that rennet-coagulated
casein will yield a neutralization precipitate after treatment with acetic
acid. In this study practically the same procedure was followed as in the
previous study of the acid-coagulated casein, with the exception that
ammonium, hydroxid2 was used for the alkaline solvent in place of sodium
hydroxid. Calcium phosphate being somewhat soluble in ammonium ace-
tate solution, it was necessary in this case to remove the ammonia by
distillation in the presence of fixed alkali, before testing for the neutrali-
zation precipitate.
The explanation of this result is not apparent. Possibly the condi-
tions were not right, or the calcium phosphates obtained in the usual
1
Proc. 8th Intern. Cong. Appl. Chem., 1912, 19: 199.
_ i Lindet states that rennet-coagulated casein dissolves in ammonium hydroxid
without liberation of calcium phosphate.
8 ASSOCIATION OF OFFICIAL AGRICULTURAL CHEMISTS [Vol. II, No. 1

neutralization precipitate are the result of a chemical reaction and not of a

mere washing with dilute acetic acid. Even then, it is not apparent
why a repetition of Lindet's work did not yield a precipitate of calcium
phosphate on neutralization.

CONCLUSIONS.

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(1) The so-called neutralization precipitate obtained from the filtrate
of the acetic acid coagulation of cows' milk appears to be largely a mix-
ture of di- and tricalcium phosphates. The precipitate isolated in this
study was mixed with a small proportion of protein, probably casein
or albumin, either as a contamination or as an essential part of the
precipitate.
(2) The origin of the calcium phosphates of the neutralization precipi-
tate would presumably be largely the caseinogen of the milk, but attempts
to show this fact, using casein coagulated by rennet, have so far been
unsuccessful.
(3) The very small amount of protein contained in the neutralization
precipitate precludes its being an important factor in the determination
of casein or the heat-coagulable proteins.

REPORT ON DAIRY PRODUCTS.

B Y LEWIS I. NURENBERG (State Board of Health, Boston, Mass.), Referee.1
The following recommendations were made for collaborative work:
(1) That the copper sulphate method of preparing milk serum be studied with a
view of its adoption as an optional provisional method.
(2) That the Harding-Parkin method for fat determination be studied in com-
parison with the present official and provisional methods.
(3) That in U. S. Bur. Chem. Bull. 107, Rev., p. 122, after the paragraph on
Cream, the following paragraph be inserted under the heading "Condensed Milk
(Unsweetened)": "Dilute 40 grams of the homogeneous material with 60 grams of
distilled water, and proceed as directed under 'Milk', and correct the results for
dilution"; and that the word "Sweetened" be inserted before the word "Condensed"
in the subsequent heading.
(4) That subjects now under consideration be given further study.

No work on the copper sulphate method has been reported to the asso-
ciation since 1909. In the study of the copper sulphate method it was the
referee's idea to collect and compile figures from all available sources.
Twenty-two letters were sent out to the various food and dairy depart-
ments, asking for figures on known purity milk by the copper refraction
1
Presented by H. C. Lythgoe.