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PHOSPHORUS IN BUTTER

A THESIS

PRESENTED TO THE FACULTY OF THE GRADUATE SCHOOL
OF CORNELL UNIVERSITY FOR THE DEGREE OF

DOCTOR OF PHILOSOPHY

BY

JAMES THOMAS CUSICK

{Reprinted from Memoir 30, April 1920, of Cornell University Agricultural Experiment
Station.]

CONTENTS

PAGE
ee a WE CME A EMIT G3 ous: iceecs ree ook Ss wee a gs fe aT 159
Experimental and analytical results of the present investigation. .... 164

Distribution of phosphorus among the various products in churning.. 174
Bacteriological investigation as to the cause of fishy flavor in butter.. 179

IE sleirimeni et Tee RAIETIGMEIG So ool Sch. Sica, he maboe Rape arc toeinte sos Rees ee 179
1 CSI FE it Arca © NA ees oe AR AL Ree SPRL GG orl tra 180
TTUEEEL EES LADS SU ie On een eg eiare DE SPUN Parc SAP Rae 183
PRM THNIM See aren ote = On Sate AN eect ee adam PRU, er a er ee 185
Acknowledgments...... A ARCS aN in Rata eae UN a cc OR pt ee 185
ROPER CTU RIARE 027 US Un ws nile a s. 3'0 5 ae aN oo MS A EE mae wie 186
155

PHOSPHORUS IN BUTTER

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PHOSPHORUS IN BUTTER!
Je. Cusick

For a long time manufacturers of butter, and students of its manu-
facture, have been searching for a method that would give a product
capable of withstanding storage conditions and preserving its original
quality. The bulk of the methods investigated have dealt with the way
in which the raw material is treated and handled before churning. While
this earlier treatment does influence the keeping qualities of the churned
product, the chemical changes produced, as evidenced in “ off flavors,”
have been investigated in only a few instances. Bacterial, chemical, and
enzymic action on butter during its period of storage have been studied
almost entirely from the manufacturer’s viewpoint, but the results of the
methods used by the manufacturer have led to no definite conclusions
as to the chemical changes produced by the various manufacturing proc-
esses. It is, therefore, with the view of adding some information to
present knowledge of the chemical changes occurring in butter during
storage, that. the role played by phosphorus is discussed here.. It is
clearly shown that the various methods of handling the raw material, and
the ways in which it is treated by the manufacturer before churning, have
a pronounced influence on the phosphorus in its various forms. One
cannot properly interpret “ off flavors’ and the like without knowing,
first, what constituents undergo change, and secondly, what products of
decomposition are formed and by what agents.

REVIEW OF LITERATURE

Friis (1897)? states that pasteurization of milk or cream appreciably
decreases the content of free fatty acids in the resulting butter. His
experiments show that

the amount of free acids in the butter is greatly decreased when the milk or the cream
is heated previous to the churning; pasteurization of the milk reduces the free fatty acid
content of the fat more than pasteurization of the cream. About half the acidity of fresh
sour-cream butter is due to free fatty acids in the butter, and the other half to the acidity
of the buttermilk remaining in the butter.

1 Also presented to the Faculty of the Graduate School of Cornell University, December, 1919, as a
major thesis in partial fulfillment of the requirements for the degree of doctor of philosophy.

The work here described was done in the Department of Agricultural Chemistry of the New York State
College of Agriculture at Cornell University, under the direction of Professor George W. Cavanaugh.

2 Dates in parenthesis refer tu Literature Cited, page 186.

159

160 J. IT. Cusick

Schmidt (1898) found that pasteurization of the cream at high tem-
peratures, and salting of the butter, lowered the acidity and the number
of bacteria in the butter. Salted butter did not become rancid as soon
as did unsalted butter, in his experiments.

Fishy flavor in butter is due to a bacterium, according to O’Callaghan .

(1902), and is remedied by pasteurization. This theory of the cause of
fishy flavor is denied by many investigators, who claim that the flavor
is due to enzymic or chemical decomposition.

Steiner (1901) states that pasteurization gives as high a loss of albumin
in the butter as 82.5 per cent. The amount of loss depends on the tem-
perature at which the milk is heated and on the time of heating.

Harcourt (1903) found that the amount of nitrogenous matter in butter
does not affect its keeping quality.

A primarily practical viewpoint of the question was taken by Lee
(1909), who conducted pasteurizing experiments on ‘“ farm-skimmed ”’
cream. He concluded that pasteurizing does not affect the body, or
texture, of butter, and does not improve the quality of butter made from
sour ‘‘farm-skimmed”’ cream.

According to Sayer, Rahn, and Farrand (1908), the use of starter in
preparing cream for churning has a decided influence on the butter. Pure
cultures of starter gave butter of a high score, according to these investi-
gators. Lindsey (1908) placed great emphasis on care in the quality of
the starter to be used, and found that the flavor of butter depends primarily
on the cleanliness of the milk, the stage of lactation of the animal, the skill
and care of the butter maker, and, especially, the character of the starter
employed. He concluded also that normal foodstuffs must be con-
sidered of secondary importance in establishing butter flavor.

McKay and Bower (1908) studied the moisture content of butter as it
affects the quality of the product. Their efforts led them to conclude
that high water content between 15 and 16 per cent does not necessarily
mean a low score, nor does less than 13 per cent mean a high score.

Rogers and Gray (1909) made butter from pasteurized and from
unpasteurized cream of varying degrees of acidity, and stored it at tem-
peratures of 32°, 10°, and -10° F. They state:

The butter made from unripened unpasteurized cream always developed a cheesy or
rancid flavor. The butter made from ripened cream, both pasteurized and unpasteurized,
developed cold-storage, fishy, and other flavors typical of storage butter. In all cases the
overripe butter showed marked deterioration. The butter made from pasteurized cream

PHOSPHORUS IN BUTTER 161

without starter usually retained its flavor with little or no change. Even at 32° F. the
sweet-cream butter deteriorated very little .....

Butter made from pasteurized cream with starter added,...... retained its fresh flavor
better than the ripened-cream butter, but was not quite equal in keeping quality to that
made from sweet pasteurized cream.

High acidity reduces the keeping quality, according to these workers.

Melick (1909:281) found that “the use of commercial lactic acid as a

substitute for starter proved advantageous only when used in very rancid

Saponification of the butterfat takes place during storage, according to
Vincent (1910), increasing the insoluble fatty acids. The soluble and
the insoluble fatty acids are formed directly by decomposition. and by
synthesis, respectively. It is evident that the glycerides of the insoluble
acids are changed to a greater degree than are those of the soluble acids.
Vincent found also that glycerol is present in old butter and in old cream,
but not in milk.

During experiments in the manufacture of butter and cheese, Dean
(1910) found that when 10 per cent of starter was added to cream for
making butter, there was a greater loss of fat in the pasteurized than in
the raw cream. This loss increased with the increase of acidity in the
cream at the time of pasteurization. There was little difference in the
quality of fresh butter when pasteurized at different temperatures, but
the keeping quality was improved by pasteurizing the cream at a tem-
perature between 60° and 82° F.

Larsen, Lund, and Miller (1910) found that the acidity of thoroughly
washed butter at the time of making was less than that of butter that
was washed but little, by the equivalent of 0.3 cubic centimeter of N/10
alkah to 10 grams of butter. At the end of sixteen weeks this difference
had increased to only 0.5 cubic centimeter. Butter made from pasteurized
cream did not increase in acidity as rapidly as did butter made from raw
cream. The average difference between the two samples immediately
after churning was only 0.1 cubic centimeter, but after the butter had
been kept in storage for sixteen weeks this difference had increased to
1.1 cubic centimeters. During the sixteen weeks the acidity of the pasteur-
ized-cream butter had increased only 0.6 cubic centimeter, as compared
with an increased acidity of 1.6 cubic.centimeters in the raw-cream butter.
The acidity in lightly salted butter increased by 4.7 cubic centimeters
in sixteen weeks, as compared with an increase of 1.6 cubic centimeters

162 Jj. L. Custek

for heavily salted butter. The acidity of ripened-cream butter increased
more than did the acidity of sweet-cream butter. The bulletin states
(page 715):

These results indicate that butter from fresh and properly ripened cream not over one day
old keeps better than does butter made from sweet cream. The butter fat from very fresh
cream is apparently in a more stable condition than is the fat in the sour cream over one
day old, and not so predisposed to decomposition. It indicates that butterfat, in the form of
butter, keeps better than does butterfat in the form of cream, even though it be in prop-
erly ripened cream.

It has been found by Rahn, Brown, and Smith (1909) that there is
very little loss of lactose during the storage of butter, and apparently
little or no relation between the water-soluble acids and the amount of
lactose lost. The butter having the highest initial scores showed the
least change in its amino nitrogen content. Butter scoring a little lower
and made from cream of a poor quality showed an increased decomposi-
tion of protein. The butter with the lowest score yielded the largest
amount of protein cleavage products.

The influence of the alkalinity of the wash water was investigated by
Meijeringh (1911). He found that. the water content of butter washed
with alkaline water was higher than that of butter washed with slightly
acid water. He states that the butter in the acid solution clumped
better than did that in the alkaline solution. The effect of bacteria in
the wash water suggested to Melick (1906) the deduction that there is a
direct relation between the bacterial content of the wash water used and
the keeping quality of the butter.

Kooper-Gistrow (1911) states that butter made from sour cream con-
taining iron rust, has a metallic flavor and a marked grayish color inter-
spersed with dark spots. However, a wash water with an iron content
as high as 36 milligrams to 1000 cubic centimeters of water does not
affect the quality of the butter. .

Rogers and Gray (1909) draw the following conclusions from investi-
gations on pasteurized and on unpasteurized cream of varying amounts
of acidity, and on the resulting butter stored at different temperatures:

Butter frequently undergoes marked changes, even when stored at very low temperatures.

These changes are more marked as the acidity of the cream from which the butter is made
is increased,

No bacteria were found in the cream or the butter which could reasonably be expected
to be the cause of the more rapid deterioration of the high-acid butter.

PuHosreHorus IN BurreR 163

The changes in the high-acid butter were not checked by heating the ripened cream, which
shows that they were not brought about by enzymes secreted with or in the cream and
carried into the butter.

Marked changes of an undesirable nature were produced in butter by acidifying pasteurized
cream with various acids. These changes did not take place all at once, but were of a pro-
gressive nature.

The results indicate that the acid developed normally in the cream by the action of the
lactic-acid bacteria, or added directly to the cream in the form of pure acid, brings about or
assists in bringing about a slow decomposition of one or more of the labile compounds of
which butter is largely composed.

Rogers and his associates (1913) found later that copper sulfate pro-
duces fishy flavor in butter. They found also that fishy flavor will
develop in cream ripened while in contact with two sheets of copper.

Mortensen, Gaessler, and Cooper (1914) differ with Lee (1909), claiming
that pasteurization of either sweet or sour cream improves the flavor of
the resulting butter. The protein content of the resulting butter is not
influenced by the pasteurization of sweet cream, according to these investi-
gators, but is decreased by the pasteurization of sour cream.

The decomposition of protein in butter during storage has been studied
by Brown (1915). He finds that the protein of butter is slowly broken
down to amino acids and ammonia. The percentage of total nitrogen in
unsalted butter, from the amino acids and ammonia, was found to be
5.71 at first, and 7.59 after the butter had been stored at -6° C. for two
hundred and forty days. In the salted butter the amino acids and ammonia
increased to 8.19 per cent after a storage period of two hundred and forty
days. Hunziker (1916) also found protein hydrolysis in butter during
storage, as well as amino acids and other products of decomposition.
Hunziker concludes that the initial condition of the cream, the amount of
hydrolyzing agents present, and the temperature, determine the degree
of hydrolysis.

A new protein, soluble in alcohol, has recently been found in milk by
Osborne and Wakeman (1918). It has a phosphorus content, on an
ash-free, water-free basis, of 0.08 per cent P. This protein, while soluble
in alcohol, is insoluble in dilute acids or in salt solution. The results of
the experiments here described seem to show that such a protein exists
in butter. In earlier work on phosphatides in milk, Osborne and Wakeman
(1915) obtained 116 grams of lecithin from 3000 liters of milk. While
they were hydrolyzing a solution of this lecithin with barium sulfate,
some of the lecithin was broken down so that trimethylamine was detected
in the vapors from the hot solution.

164 JT. Cusrex

EXPERIMENTAL AND ANALYTICAL RESULTS OF THE PRESENT
INVESTIGATION

The butter used in all the experiments here discussed was made from
cream containing 30 per cent of butterfat. Seven portions of the cream
were churned separately in a barrel churn, operated by hand. The butter,
washed and worked in the usual manner, was then divided, one half being
packed immediately in porcelain jars, and the other half being salted to
a content of 24 per cent before packing. All samples were then stored in
a refrigerator held at —10° C.

Before churning, the seven portions were treated differently, as
follows: ;

Portion 1: To 5 pounds of sweet raw cream there was added 2 ounces
of starter —a pure culture of lactic-acid-producing bacteria, in skim-
milk. The cream mixture was then held for six hours at room temperature,
after which it was placed overnight in a refrigerator at 0° C. The next
morning it was removed from the refrigerator, and was brought to a tem-
perature of 15.5° C. by immersing the can in warm water. The mixture
had developed an acidity, estimated as lactic acid, of 0.88 per cent. A
few drops of butter color were added and the mixture was then churned.
The temperature during churning was approximately 15.5° C. After
churning, the buttermilk was removed and the butter was washed twice
with water at 15.5° C. Each portion of the wash water was equivalent
in volume to the buttermilk removed from the churn. In washing the
butter, the churn was given only one revolution for each washing. After
washing, the butter was removed from the churn with a wooden ladle
and was worked for about five minutes in a wooden bowl. It was then
divided into two portions, one portion being packed in porcelain jars and
the other portion being salted to a content of 2} per cent. All the samples
were labeled, covered with parchment paper, and stored in a refrigerator
at -102

Portion 2: The second portion consisted of sweet raw cream which was
churned as soon as it was separated from the milk. After churning, the
butter in this sample and that in the following samples was treated the
same as was the butter from portion 1.

Portion 3: Lactic acid of 25 per cent strength was added to sweet raw
cream until the acidity of the cream was 0.38 per cent. The mixture was
then heated to 74° C. and held at that temperature for thirty minutes.

PHOSPHORUS IN BUTTER 165

The flask containing the cream was then suddenly cooled in water to
15.5° C., and after standing for three hours the cream was churned.

Portion 4: Raw cream was allowed to stand at room temperature
until it developed an acidity equivalent to 0.85 per cent, when it was
churned. The sample took three days to develop this degree of acidity.

Portion 5: Commercial lactic acid of 25 per cent strength was added to
the cream until the acidity of the cream was 0.35 per cent. The portion
was thoroughly stirred, quickly brought to a temperature of 15.5° C., and
then churned.

Portion 6: Cream was heated in a flask to 74° C. for thirty minutes, was
- then cooled to 15.5° C., and, after standing for three hours, was churned.

Portion 7: The cream was pasteurized as was portion 6; but instead
of being churned after pasteurization, the sample was cooled, was allowed
to stand at room temperature for four hours after 4 ounces of liquid
starter had been added, and was then placed overnight in a refrigerator
at 0° C. After the cream was removed from the refrigerator the next
morning, its temperature was raised to 15.5° C. and it was churned
immediately. The acidity of the cream at the time of churning was
0.45 per cent. .

As may be séen from the above-described methods of treatment of the
various portions, the manufacturing methods of treatment of cream before
churning were closely followed. The seven methods outlined are the ones
most generally employed in the manufacture of butter, and it was with
the idea of simulating conditions that would be used in any manufacturing
process for butter that these seven portions were treated as described.
The different methods employed seem to have varying influences on the
phosphorus compounds in the milk and the cream, and also on the churned
product during storage.

The phosphorus compounds of milk, which are present also in cream
and in butter, are: casein, lecithin, the new protein found by Osborne and
Wakeman (1918), and inorganic phosphates. To show the effect on these
compounds of the various methods of handling the cream, determinations
were made of the organic and the inorganic phosphorus content of the
respective butters made from the seven portions of cream.

In an attempt to find a method which would give directly the organic
phosphorus in butter, instead of having to obtain it by difference as is
the general custom the experiment was made of incorporating the butter

166 J.D. Cusrom

into calcium sulfate and extracting the organic phosphorus compounds
with alcohol. The method was as follows: 25 grams of butter was inti-
mately mixed with 150 grams of anhydrous calcium sulfate until all the
butter was completely absorbed and the mixture felt dry and powdery to
the touch. In some eases a larger quantity of calcium sulfate is neces-
sary to acquire the desired consistency. The mixture was then placed
in a large Soxhlet apparatus, a filter paper being used instead of the cus-
tomary extraction thimble. About 150 cubic centimeters of 95-per-cent
alcohol was used as the extracting agent, and the apparatus was run for
forty-eight hours. At the end of that time the alcohol was removed,
transferred to an Erlenmeyer flask, and evaporated, and the residue,’
consisting of lecithin, fatty acids, and alcohol-soluble protein, was digested
with nitricacid. From this point, method 2 (b) in the Methods of Analysis

- of the Association of Official Agricultural Chemists * was used. To the

result obtained by the alcoholic extraction is added the per cent of phos-
phorus obtained from the casein. In making the organic phosphorus
determination of the casein, 10 grams of butter was melted and was
filtered on a hot funnel, and the filter was washed with three or four por-
tions, (30 cubic centimeters each) of 80-per-cent alcohol. The filter paper
and its contents were then removed to a 300-cubic-centimeter Erlenmeyer
flask with a glass stopper, and were shaken with 75 cubic centimeters of a
solution of 0.2-per-cent hydrochloric acid. The supernatant liquid in the
flask was then decanted through a dry filter paper, and the extraction
of the casein and the filter paper in the Erlenmeyer flask with the acid
solution was repeated three times. By this method all the phosphorus
compounds of butter, with the exception of the casein, were removed.
The new filter was then removed from the funnel and placed in the Erlen-
meyer flask with the old filter, and phosphorus determination on the whole
was made by the same method as in the case of the alcohol residue. The
combined phosphorus results, from the alcohol residue and the casein
residue, gave the total organic phosphorus content of the butter.

The low results — 0.0035 and 0.0038 per cent of phosphorus (P20;,) —
from the alcoholic residue for total organic phosphorus, seem to indicate that
not all the alcohol-soluble phosphorus compounds were extracted. . Some,

3 A test for phosphates must be made on the residue.

4 Official and provisional methods of analysis, Association of Official Agricultural Chemists, p. 3. U.S.
Bur. Chem., Bul. 107. 1908.

PuHospHorus In Burrer 167

if not all, of the lecithin was extracted, and the alcohol residue showed
the presence of protein also. However, since the accuracy of the method
was not definitely established, its use in obtaining results in this problem
was discontinued; it has nevertheless not been discarded entirely, as
further work on the method is contemplated. The author has not been
able to find any reference to attempts at the direct separation of organic
phosphorus from inorganic phosphorus in butter. Attempts have been
made at the direct determination of organic phosphorus in milk, and a
direct determination of lecithin in milk has been made by Osborne and
Wakeman (1915).

The well-known Hart and Andrews method for determination of soluble
organic and soluble inorganic phosphorus was applied in a general way
to the extraction of the soluble organic phosphorus and the soluble inor-
ganic phosphorus in butter. The method was as follows: 20 grams of
butter was melted in a beaker and washed into a separatory funnel with
50 cubic centimeters of a half-saturated sodium chloride solution which
had been heated to about 45° C. The separatory funnel was shaken vig-
orously, the fat was allowed to separate, and the aqueous part was drawn
off and passed through a filter paper into a beaker. Two other extractions
were similarly made, about 30 cubic centimeters of the salt solution being
used in each case. The salt-water extraction was followed by six washings
in 25 cubic centimeters of a warm solution of 0.2-per-cent hydrochloric
acid. ‘This method extracted all the soluble inorganic phosphorus and an
appreciable amount of the organic phosphorus from the butter. The
different fractions of the solvents having been filtered together, the filtrate
_was then divided into two portions. Total soluble phosphorus was deter-
mined on one portion according to the method of the Association of
Official Agricultural Chemists. Soluble inorganic phosphorus was deter-
mined on the other portion according to the Hart and Andrews method.
This was done by immediately neutralizing the solution to slight alkalinity
with ammonia, precipitating the soluble- inorganic phosphorus with mag-
nesia mixture, allowing it to stand in a cool place for four hours, filtering,
and ashing. The ash was redissolved in a small amount of hydrochloric
acid and hot water, reprecipitated, filtered, ashed, and weighed as mag-

5 Hart, E. B., and Andrews, W. H. The status of phosphorus in certain food materials and animal
py-producte, with special reference to the presence of inorganic forms. Amer. chem. journ. 30: 470-485.
1903.

168 J. T:: Cusicr

nesium pyro-phosphate. The soluble organic phosphorus is then the
difference between the total soluble and the soluble inorganic phosphorus.

The protein residue from the salt-water and hydrochloric-acid extractions
was placed in a 300-cubic-centimeter Erlenmeyer flask and digested with
nitric acid. Total phosphoric acid was determined on this according
to the method of the Association of Official Agricultural Chemists. This
represented the insoluble organic phosphorus in the protein of the butter.

In a few cases the protein residue from the butter, after extraction with
hot salt solution and dilute hydrochloric acid, was extracted on the filter
with several portions of warm 80-per-cent alcohol, and separate deter-
minations were made on the alcoholic filtrate and on the residue left
on the filter. These results are given in table 6 (page 173). They show
_ that some of the protein is soluble in alcohol.

Results of the first series of experiments are given in table 1. These
results show the percentages of total soluble, soluble inorganic, and

TABLE 1. PxHospHorus Content or SALTED SAMPLES OF BuTTER MaApE on JANUARY 11
AND ANALYZED ON JANUARY 28, 1918

Per cent of P.O;

Butter from cream portion

Soluble Total Soluble | In protein Total
inorganic. soluble organic residue he
L584 aS: Bh rT Te es .0129 .0156 .0027 .0123 .0338
Watt: BRA ORO EMCEE ORAS ight sie .0138 .0168 .0030 .0108 .0397 -
aie thy ist ge ee ES et .0100 .0124 0024 .0079 .0249
Aa gee Tea RE erat kei oo ee .0127 .0132 .0005 .0131 .0351
15) SU hee CR Re oe rt me REI ER .0170 .0202 .0032 .0103 .0414
Gis ere EEL oh ye ork maa .0155 -O180 .0025 .0095 .0344
AA SRE er eee ee ae .0108 0124 .0016 .0089 .0300

al

soluble organic phosphorus (P2O;) in the butter (the last-named obtained
by difference), and the percentage of phosphorus in the protein residue.
A separate analysis of the total phosphorus content of the portions of
butter was made in every case also. The numeral preceding each analysis
in the table refers to the portion of cream from which the sample of butter
was made. :
It is shown by the table that the highest percentage of total phosphorus
was retained in the butter when only lactic acid was added and churning

PHOSPHORUS IN BUTTER 169

was begun at once (sample 5). There was little difference between this
sample of butter and that made from sweet raw cream (sample 2), except
that the total soluble phosphorus of sample 5 was greater than that of
sample 2. The soluble organic phosphorus content of both was about
equal, as were their protein residues also. The only difference in the
treatment of the two samples was in the addition of lactic acid to sample 5
before churning. The action of the acid in this case is readily seen in
the higher amount of soluble inorganic phosphorus; either there was some
splitting-off of phosphorus from some organic phosphorus compound which
was heretofore insoluble, or some insoluble phosphates were made soluble
by the lactic acid. Sample 4 retained the next highest amount of total
phosphorus. This butter was made from raw cream ripened without
starter; the acidity developed slowly, at room temperature, at the expense
of the lactose. This sample had the greatest amount of phosphorus in
the protein residue, however, and the smallest amount of soluble organic
phosphorus. The total soluble phosphorus was comparatively very low.

Sample 6, which was made from pasteurized sweet cream, had a total
of 0.0344 per cent of, phosphorus (P20;). Evidently very little of the
organic phosphorus compounds decomposed by heating. The appreciable
amount of organic phosphorus seems to be due to the stable condition of
the protein and the absence of acid.

Sample 1 was made from raw cream ripened with starter. It had a
total phosphorus (P20;) content of 0.0338, and compared very closely
with the pasteurized-sweet-cream sample 6, except that its soluble phos-
phorus was much lower than that of the latter. This difference was made _
up by the protein residue of sample 1, which was greater in this component
by about the same amount as the two samples varied in their soluble
phosphorus.

Sample 7 was made from pasteurized cream which was subsequently
ripened with starter. It showed a marked depreciation in its total and in
its soluble phosphorus. Undoubtedly the heating of the cream during pas-
teurization rendered many of the phosphorus compounds unstable, so that
they were easily broken down by the lactic acid during the long process
of ripening before churning. Contrasted with sample 7 is sample 3, which
was made from pasteurized sweet cream to which lactic acid had been
added. Sample 3 was.com paratively low in total phosphorus, due undoubt-
edly to the high acid content at the time of pasteurization. This proves

170 J: TT: Cusrex

that high temperatures, and especially a pasteurizing temperature, accel-
erate decomposition when acid is present.

The immediate effect of ripening cream with starter showed little
decomposition of the phosphorus compounds in the butter, especially
in its organic phosphorus content. The sweet raw cream, however, had
less splitting-off of phosphorus than had the cream ripened with starter.
Raw cream, which was self-ripened, had little soluble organic phosphorus
left at the time when it was churned, but later, in storage, the soluble
inorganic phosphorus was increased, evidently at the expense of the protein
fraction.

The changes that had taken place in the butter samples designated in
table 1 after they had been in storage for fifteen months, are shown in
table 2. The analyses showed the soluble organic phosphorus to be
approaching or to be almost entirely in the inorganic form. The raw

TABLE 2. PHospHorus CONTENT OF SAME SAMPLES IN ApRIL, 1919

Per cent of P20;

Butter from cream portion
Soluble Total Soluble | In protein

inorganic soluble organic residue Total
tae: CSE te: AE 8 .0158 .0204 0046 .0120
Disc 8, Sere Sede. eer oo Nay eee .0232 .0236 .0004 .0163
er ea ante ecient oe Me eee 0191 .0193 .0002 .0086 | Totals
Bidet WP ERR Me Behe SEA 0157 .O168 0011 .0163 | checked
igo Seen auplest ara in ee Mes 2 .0227 .0242 0015 .0166
DSpace ey Mey ROR A tapered baler cn area S .0201 .0211 .0010 0115

SE EE Ha Ae lA Ree .0148 .0207 0059 0091

cream ripened with starter (sample 1) and the pasteurized cream ripened
with starter (sample 7) had retained the greatest amounts of organic
phosphorus during storage. The phosphorus content of the protein
residue in these samples, however, had not increased, and in this respect
they showed a marked contrast to the remaining samples, in all of which
the phosphorus in the protein residue had increased from a few milligrams
in the pasteurized samples 3 and 6 up to 63 milligrams in the sweet-raw-
cream sample treated with lactic acid (sample 5). As is generally under-
stood, pasteurization of the cream destroys enzymes and leaves but few

PHOSPHORUS IN BUTTER 171

baeteria to survive in the butter and ultimately to produce acid by fer-
menting the lactose; and since sample 5 had both acid added to the cream
from which it was. made, and enzymes and bacteria left undisturbed, it
may be concluded that the high phosphorus content in its protein residue
was due to insoluble phosphorus compounds formed by these agencies.
Another factor which may sustain the theory that high acidity produces
insoluble phosphorus compounds is evidenced in sample 2 (sweet raw
cream), in which a large proportion of the lactose would have been present
in the cream before churning and would therefore have been in reserve
for the formation of acid in the butter. The presence of salt also seems to
have an effect in increasing the insoluble phosphorus compounds during
storage.

Analyses of one unsalted sample are given in table 3, to show the effect
of the absence of salt on the phosphorus content of butter during storage.

TABLE 3. PxHospHorus CoNnTENT OF UNSALTED SAMPLE OF BUTTER FROM CREAM
PoRTION 2

Per cent of P20;

Butter sample

Soluble Total Soluble | In protein Total
inorganic soluble organic residue
Analyzed January 28, 1918..... .0158 .0199 .0041 .0139 .0468
Analyzed in April, 1919........ .0229 .0250 0021 OLA ea reese:

There was a greater amount of soluble organic and soluble inorganic
phosphorus present in this sample, and a larger percentage of phosphorus
in the protein residue, than in the salted sample of the same butter. The
loss of phosphorus compounds in the salted sample, at this stage of the
experiment, was due to the working of the salt into the butter. Later,
in storage, there was an increase in the soluble phosphorus, and an appre-
ciable amount of soluble organic phosphorus was present at the end of
fifteen months. The protein residue, however, increased its phosphorus
content by only a small amount.

Several other samples, duplicates of those designated in table 1 in every
respect except that they were not salted, were analyzed and gave results

ive? Jc. Custcn

comparable with those shown in table 3. There were only slight increases
in the phosphorus content of the protein residues. This seems to prove
that salt is a contributing factor in the production of insoluble phosphorus
compounds during the storage of butter. There was in almost every case
a larger amount of total phosphorus in the unsalted samples than in the
salted ones. As noted above, during the process of salting, the butter
loses a small quantity of liquid which carries away with it an appreciable
amount of phosphorus compounds.

Another series of experiments, conducted in exactly the same manner as
the foregoing, was made about a month later than the first series. The
results were almost completely in accord with those of the earlier experi-
ments. These results are given in tables 4 and 5:

TABLE 4. PuospHorus Content or Butrer Mave on JANUARY 31 AnD ‘-

ANALYZED ON Fresruary 3, 1918

Per cent of P.O;

Butter from cream portion
. Soluble Total Soluble | In protein

inorganic | soluble organic residue Total
1 ea 9 arta ee ei tar WN .0125 0152 0027 0133 .0335
DAR RE A URIER pron pih Mente EAR at Coat .0130 .0164 0034 0100 .0392
SRE ee eee Pee en eee .0106 01388 00382 .0079 .0318
FS ea ne BS URE, nd ec .0102 0146 0044 .0122 .0357
RAS Rr Sac Leek he A AE .0138 0169 0031 .0076 .0398
Gisele ae eects oe eee .0137 .0167 0030 .0102 .0386

Are A Bee Ree Oe ont Tee 0078 .0105 .0027 .0069 0243

TABLE 5. PxHospHorus ConTENT OF THRSE OF THE SAME SAMPLES IN APRIL, 1919

Per cent of P2O;

Butter from cream portion |
Soluble Total Soluble | In protein! Total

inorganic soluble | organic residue
Sie en eC Sah ire, Wie 0191 .0190 0000 , -0092 Total
cE eearig S ipaetie rad Ret, 0199 0199 0000). 5. DESTR Wi onee

Mish welts Gat gen GON aaa tea 0163 0170 0007 ‘oceg | checked

PHOSPHORUS IN BUTTER 173

As the protein residue seemed to be affected by the methods of handling
the cream and by pasteurization, it was thought advisable to determine
whether all the residue was casein, or whether the casein had adsorbed some
phosphorus compound, or a portion thereof, which it released only during
pasteurization. ‘To determine this point, the samples of butter were suc-
cessively treated with half-saturated sodium chloride solution and a solution
of 0.2-per-cent hydrochloric acid, as inthe extraction method previously
described (page 167). The protein residue on the filter was removed in
» the filter paper to an Erlenmeyer flask and shaken with 50 cubic centimeters
of 80-per-cent alcohol. The alcohol was then decanted through a dry
filter into a clean beaker, and two similar extractions were made on the
residue in the Erlenmeyer flask. Finally, the residue and the filter were
washed with a small quantity of ether. The filter paper was removed to
the Erlenmeyer flask and total phosphorus was determined on this. This
was the casein portion. The alcohol-ether filtrate was likewise placed in
an Erlenmeyer flask and the total contents were evaporated to dryness.
Total phosphorus was then determined on this portion also. The results
are given in: table 6. Samples 2 and 5 (table 1) were made from cream
portions 2 and 5, respectively. They were one month old when these

TABLE 6. PuHospHorus ConTENT oF RESIDUE AS SHOWN BY SEPARATION OF ALCOHOL-
SOLUBLE PROTEIN FROM CASEIN

Per cent of P.O;

Butter from cream portion Protein
residue Ga
(alcohol- oe
thor residue
extraction)
Ey (GDELTCS TDNS gh SS SO SI Tea on ae daa 0030 .0077
E (Glide 16 EW aS e Per eG a eae pede 0030 0077
“(Caleta aD Goad Wlek Ba S Raee Cre s a meee gna .0040 .0075

results were obtained. Sample 2 (table 4) was made from cream portion 2
in the second series, and was three days old when analyzed. All three
samples were unsalted.

The results given in table 6 show that an appreciable amount of organic
phosphorus ¢an be extracted from the supposed casein residue by alcohol.

174 Pi Pied beats Gi afsh(e2 <2

This phosphorus con.pound did not give the tests for lecithin, nor was
it soluble in weak salt solution. No further work was done on this alcohol-
soluble phosphorus compound except to obtain a positive test for protein.
According to the work of Osborne and Wakeman (1918), it appears to
be the new protein found in milk by them.

DISTRIBUTION OF PHOSPHORUS AMONG THE VARIOUS PRODUCTS
IN CHURNING

A study was made of the distribution of phosphorus among the various
products in the manufacture of butter. Results obtained from churnings
in which the cream was not pasteurized and was ripened with starter, are
shown in tables 7, 8, and 9.

The history of the sample used for the data given in table 7 was as
follows: To 20 pounds of sweet cream from milk recently skimmed, a

TABLE 7. DistriputTion oF PHospHORUS ConTENT IN BuTTER Mabe on Frsruary 22,
1919. ANALYSES STARTED ON THE SAME DaTE

Per cent of PsO;

Total Soluble | Soluble

soluble | inorganic ; organic Total
IVE eta ee tin © ee orem. 1387 -1178 .0209 .2161
Cres ree Xia at ARE ee ee .1033 .0902 0131 .1619
Bultiternatl oe Gen ee eee . 1222 .1143 .0079 PAGS)
bist wash=waters. cs. ae eee es .0037 .0031 .0006 .0115
weconad- wash wavelet oct cine eee .0000 .0000 .0000 .0004
Saltwater? se: cb 2-3 oeaces ceo hee ee .0263 .0251 .0012 .0340
Butter se ok. Berean ak eS: .O191 .0154 .0037 .0485
Washed putter: ssc soca once eee el scars oh ee era eee Al Oc aa 0089
Extracted butter sc steatie oon oe ee eens bee ote ||| a cee ae .00388

* Only 100 cubic centimeters of salt water was expressed from the butter.

pint of liquid starter was added. The mixture was allowed to stand at
room temperature for three hours and was then placed in a refrigerator
overnight. The next morning it was removed from the refrigerator and
was found to have developed an acidity of 0.48 per cent. It was then
churned in the usual manner, was salted, and was stored at a temperature
of 0° C. The fat content of the cream at the time of churning was 24.5
per cent. After five months in storage the sample scored 91, which is
considered a high score for storage butter.

PHOSPHORUS IN BUTTER 175

There was 49.5 per cent of fat in the cream from which the butter
ased for the data given in table 8 was made. The cream was fairly fresh
and was ripened with starter. The acidity of the cream at the time of

TABLE 8. DistrrputTion oF PHospHoRvs Content IN Butter Mape on Aprit 5, 1919.
ANALYSES STARTED ON THE SAME Date

Per cent of P:O;

Total Soluble Soluble

soluble inorganic organic Total
(CHARI 5 oo go OES BU Se Se eee .0689 .0599 .0090 .0905
Biathermilics..sccss 2k Sy es Ae eis Bee .1025 .0960 .0065 . 1657
LOTR SES SSE PE eg cn a .0076 .0058 .0018 .0170
DECORA WASNEWALED< 0.5.0. ciue cessed ssis a tins .0038 .0028 .0010 -0050
SAU ATER tae ae seers Segoe ei tase ss Se OlGs .0153 (O010\| ee eee
[EXWUGET Ea Geese Sine OI eR .0280 .0205 .0075 .0325

* Only 10 cubic centimeters of salt water was expressed from the butter.

churning was 0.52 per cent. Thesalt content of the butter was 3.8 per cent.
Only 10 cubic centimeters of salt water was expressed from this butter in
working it. The analysis of the first wash water from this churning is
given in table 9:

TABLE 9. ANaAtysis oF First WasH WATER FROM BuTTeR CHURNING
(From butter used for data given in table 8)

Substance Per cent
AC LOSCMMET ran Are ranenr Senne kT Ree TE, MLD Peat. Oe Shake Uae. Sead, 0.45
Tari TE etoile lets Bee Cohn ee oi has cack RE we Ae nS ee PORE Fae grate ee aif 0.602
Bs i ert mn ates Saye Lek oh nl eto Mite boos) haucy's Suaudeyeeate Wdhe a 0.050
ILEWEUO PELL STUN pe ecenonities Sohne ROE Ee On IG Ie Nien men A Pe Lae Cae Present
IL pBeRA UY OOUTTETT Gs at or esd dice ac RSE Se ce ae Ch ny ee ee None

Experiments were made on several portions of the butter used for the
data in table 7, to determine the effect of varying amounts of salt on the
keeping qualities of the product. The portions were salted so as to give
the following salt content: 0.5 per cent, 1 per cent, 2 per cent, 3 per cent,
5 per cent, 7 per cent, 10 per cent, and 15 per cent. A fresh (unsalted)
sample also was included in the series. The samples were stored in porce-

176 | J. T. Cusick

lain jars in a refrigerator held at 0° C., and were scored every two weeks
for quality. The effect of the various salt concentrations on the soluble
phosphorus compounds during the storage period of the butter is shown
in table 10:
TABLE 10. InriueNce oF SAtt CONCENTRATION CN SOLUBILITY OF THE PHOSPHORUS
Compounps IN BuTTER
(Butter made on February 22, analyzed on May 25, 1919)

Per cent of P:O;

Salt concentration |
Total Soluble Soluble

soluble organic | inorganic
(Winaaltedsce = epee ie: le eee oe reo ee ee 0200 .0041 .0159
DORGET CONE. BUTANE coopers ee ters, = oe ici = te Pa eens .0280 0031 .0249
Super cent: Macs es see des ae Ok 5d BOR Rie tig sR Soe .0274 .0018 0256
LO er (CR Re eA Se pete tetera ia aha ore 0255 0000 -0255
{5 percentaces 2 ote ee te ee a eee 0242 .0000 .0239

The samples of washed and extracted butter shown in table 7 were
treated by eliminating from them as far as possible all the salt-soluble
compounds, in order to show what would be the effect on the keeping
qualities. For the washed butter, 100 grams of the butter was treated
on the work board with small amounts of a saturated salt solution until
about 300 cubic centimeters of the solution had been used. The salt
solution was thoroughly mixed with the butter by means of a wooden
ladle, and then drained off. The fat was not melted nor removed from the
board at any time until it was finally put into the storage jar. It will be
seen that in this sample of butter the phosphorus content was reduced,
by washing, from 0.0485 per cent (P2O0;) to 0.0089 per cent. However,
it must be admitted that insoluble matter was carried away in the salt
solution by this method.

In the case of the extracted butter, the sample was first treated in the
same manner as was the washed butter. It was then melted in an evaporat-
ing dish, and the protein and the fat were separated by means of a hot
saturated salt solution in a separatory funnel. The salt-water-insoluble
residue was caught on a hard filter paper, and the salt water from the
repeated. salt-water extractions of the fat was used to wash the filter.
Finally, the protein residue was scraped from the filter paper and mixed

PHosPHORUS IN BUTTER . 177

in a jar with the extracted fat. Enough saturated salt solution was then
added to equal the water content of the original butter, and the mixture
was stirred, hardened, and stored.

Both these samples, the washed and the extracted, were prepared to
demonstrate the possibilities that may occur when butter is overworked,
and also to show the effect of the absence of salt-water-soluble com-
pounds on the keeping qualities of butter. The only comment made
on either of these samples by the scorers, was that the butter was
“tallowy.” This was of course due to handling.

No fishy flavor was developed in the samples of butter used for tables
7 and 10, despite the fact that salt is a contributing factor in the develop-
ment of fishy flavor in butter during storage. As lecithin is soluble in
salt solution, the reason for washing and extracting the butter samples
was to remove this phosphorus compound and note whether fishy flavor
developed in the samples treated in this manner. The samples included
in table 10 were used to ascertain whether butter samples of varying salt
content would develop fishy flavor more quickly in one salt concentration
than in another. These samples were used also to test the influence of
the varying salt concentrations on the soluble phosphorus compounds in
the butter. No flavor which in any way resembled fishy flavor was
detected in any of the samples, but table 10 shows the influence of: the
various salt concentrations on the soluble phosphorus compounds.

It is the author’s opinion, however, that fishy flavor in butter is caused
by the decomposition or partial decomposition of the lecithin before
churning, and that through the solvent agency of the salt solution the
products of decomposition are further broken down in storage to tri-
methylamine. Trimethylamine may in turn be broken down, or it may
be completely adsorbed by some other compound. This latter theory
is advanced from the fact that fishy flavor is transitory in some butter.

As evidence of the breaking-down of lecithin in butter during storage,
it is only necessary to compare the soluble organic phosphorus content of
the butter in table 1 with the soluble organic phosphorus content of the
same butter analyzed fifteen months later as shown in table 2. The long
period of storage reduced the soluble organic phosphorus almost entirely
to the inorganic form in all the samples except 1 and 7, which had a pure
lactic acid culture added. Samples 1 and 7 held their soluble organic
phosphorus during storage, and there was no decomposition of the organic

178 Jj; T:-Cusiex

phosphorus which became soluble during storage. | The lactic-acid bacteria
seem to protect the soluble organic phosphorus from decomposition.

Lecithin is soluble in sodium chloride solution, and it is claimed: that
fishy flavor in butter is caused by the breaking-down of this compound
while in solution with salt. The handling of the milk and cream before
churning influences the solubility of the lecithin, and its decomposition,
both before and after churning, is clearly shown by the records in tables
1 and 2. Its decomposition in butter during storage necessarily follows,
as the records show also. Lewkowitsch (1914:802) says, in referring to
the lecithin content of butter: ‘! Lecithin has been stated by various
observers to occur in butter fat to the extent of 0.017 or even 0.15 to 0.17
per cent (calculated from phosphoric acid). Wrampelmeyer stated 0.007
to 0.033 per cent of lecithin. Jaeckle, however, showed that butter fat
contains no compound of phosphorus.’ These results seem to supply
additional proof that lecithin is a variable compound in butter, and give
emphasis to the author’s conclusions that the methods of handling the
milk and cream before churning determine the quantity of lecithin in the
resulting butter. Also, the rate at which the lecithin is decomposed
during storage is determined by the condition and the handling of the
milk and cream before churning. However, the réle played by Bacterium
lactis acidi in protecting the soluble organic phosphorus from decomposi-
tion is not clearly understood. That some organisms have a specific
action on phosphorus compounds has been observed, and a summary
of the investigation of this subject is contained herein.

Per cent of P20;

Butter from cream portion NaCl NaCl HCl Protein Butter-
extract, extract, | extract of pean fat
inorganic soluble protein Se residue
extraction
eae Ms Er Nek ae eM RIE A 5 .0107 0153 .0051 .0120 - .0022
aA EEN: Oe teat a, ce a, See OR .0179 .0183 .0053 .0163 None
Bit. Ee ee Re eee .0145 .0147 0046 .0086 Trace
AR Dg ee ere .0096 0107 .0061 .0163 Trace
Lyte yy neha eats ice aes hie ork | CES .0189 .0204 .0038 .0089 Trace
(Ree ee te oe ee ene saan Coe yaaa S .0148 .0158 .0053 0115 Trace

CCEA Ra AA Hae ote ee eee 0087 .O148 0059 0091 0030

PHOSPHORUS IN BUTTER 179°

Since it may be of interest to some readers to know what amounts of
phosphorus were extracted separately by the various extracting agents,
‘ table 2 is given in detail on the opposite page.

BACTERIOLOGICAL INVESTIGATION AS TO THE CAUSE OF FISHY FLAVOR
IN BUTTER

Since butter contains lecithin — a mononitrogenous monophosphatid —
which on decomposition yields glycerophosphoric acid and choline,— the
latter further decomposing to methylamine, ammonia, carbon dioxide,
and methane,— it can naturally be inferred that the decomposition prod-
ucts of choline are the cause of the fishy flavor found in some butter.
Trimethylamine, which tastes and smells like decomposed fish and which
is a decomposition product of choline, is believed to cause fishy flavor in
butter. And, since both fishy odor and fishy flavor have been produced
in butter when the cream from which the butter was made was inoculated,
it can only be assumed that the phosphorus compound which decomposes
to trimethylamine is broken down or used as pabulum by the bacteria.
The bacterium used in the author’s experiment was the one isolated by
Hammer (1917) from a can of fishy-flavored milk and named Bacterium
ichthyosmius.

PLAN OF THE EXPERIMENT

Cream containing 30 per cent of butterfat was treated in the following
ways:

Portion D: 750 cubic centimeters of sweet cream was pasteurized at
63° C. for thirty minutes, cooled to 9° C., inoculated with 1 cubic centimeter
of an aqueous solution of Bacteriwm ich'hyosmius, and, churned at once.

After churning, the butter in this case and in all the following treatments
was divided, one-half being salted and the other half left unsalted. A check
sample (uninoculated and salted) was made also, and all three samples
were stored at —10° C.

Portion E: Sweet raw cream was inoculated in the same way as was
portion D, incubated at 37° C. for three hours, cooled to 15° C., and
churned. .

Portion. K: Sweet cream was inoculated, and was then incubated
at 37° C. for three hours. At the end of that period the acidity was
neutralized to phenolpthalein with sodium hydroxide, and the sample
was cooled to 15.5° C. and then churned.

. 180 J.P: JCesick

Portion N: Cream was inoculated, incubated, and neutralized as
was portion K. After neutralizing, one ounce of liquid starter was added,
and the mixture was allowed to stand at 15.5° C. for three hours and
then churned.

Portion R: Cream was inoculated, one ounce of starter was added,
and the cream was allowed to stand at room temperature for three hours
to ripen. It was then neutralized and churned.

Portion X: Cream was inoculated, incubated at 37° C. for three
hours, and cooled to 15.5 C., and one ounce of starter was added. After
three hours the cream was churned.

Each portion of cream was thus represented by an unsalted sample and
a salted sample of butter inoculated with Bact. ichthyosmius, and a check
sample (salted) which was not inoculated. These were all stored at
-10° C. Every two weeks during a period of eight months, a count
was taken of the bacteria in both the inoculated samples from each portion.
All three samples from each portion were scored frequently for quality
(flavor).

RESULTS

None of the check samples (uninoculated) were scored as fishy. None
of the unsalted samples (inoculated) were scored as fishy. The salted
sample from portion D (inoculated), made from fresh cream which was
pasteurized and then churned at once, was not scored as fishy by any
of the four judges. The bacteria count decreased rapidly both in the
salted and in the unsalted sample of portion D.

Samples from portion E were not scored as fishy by the judges. The
bacteria count on the salted inoculated sample was 120,000,000 at the
time when it was churned, and at the end of six months the count was
460,000. The unsalted sample had a count of only a few bacteria at
the end of four months.

The score for portion K was as follows:

Judge Unsalted sample Salted sample
Lia bere er RAS aR oie, Sa.CR bs eeu cto akan No comment............| Metallic
INEZ 5 stage ister ee eet as ape No comment............| No comment
INOS oo Se tr ht Re ee ae NG Re IAs Notcommente--2 56. «2.02 Fishy
IN ORAS Oriya oN Eat ee sa coer Noicommenti tic acer: Fishy

PuHospHoRuUS IN BUTTER 181

The only difference between the treatment of the cream in this portion
and that in portion E was in the neutralization of the cream after
incubation.

In portion N there was no comment on the quality of the sample of
unsalted butter. The salted inoculated.sample was accidentally destroyed
at the beginning of the fourth morfth in storage. The writer, however,
had scored the sample as fishy a short time before it was destroyed.

Portion R scored as follows:

Judge Unsalted sample Salted sample
J GE lhe os crete Bee eC iaa li a No comment............| Fishy
INlay, AS os 6 Sale Sas op tet SRR Se eee ae eee eee ae Woycomment7 22 <0. Oily
ING) Dip Wide dk ES Re Re oer e EOne Noreomment 22. 4-2 nen Fishy
Td de sc) gee eR Novcommente:: «2b se Fishy

After four months in storage the salted inoculated sample of this portion
had a strong fishy odor. At the end of seven months this odor had to
some extent disappeared.

The samples from portion X scored as follows:

Judge Unsaited sample Salted sample
Ripe leeerr a nee re rk em No comment............| Metallic
TSG ES Sens Oty an 0 ee ean Oba nae oa ea No jconiument: 4090) ..5 5 Metallic
Ores Geter: cians cities: Sy 4c) ee ee eee No comment............ Tallowy
I Gye ads Getta ois by coca SIS jer ee a No comment............| Fishy

The bacteria in the salted sample of portion X numbered several millions
even after the sample had been in storage for some months.

In the samples of butter just described, fishy flavor was produced
by inoculating the cream with Bacterium ichthoysmius. The handling of
the cream before churning affected the growth of the bacteria differently
in every portion. The sweet-cream butter from portion D decreased
in its bacteria count very rapidly, and retained a good flavor after being
. stored for eight months. Portion D compares fairly closely with portion 6
of the earlier experiments as to the way in which it was treated before

182 J) Ts Custer

churning. The sample from portion 6 retained a large proportion of its
total phosphorus, and the soluble organic phosphorus suffered little
decomposition, if any. The butter from portion E may be compared
with that from portion 2 of the earlier experiments. The total phosphorus
content in sample 2 was very high and the amount of soluble organic
phosphorus was large. The samples that were scored as fishy in these -
experiments (from portions K, R, and X) underwent treatment similar
to that of sample 4 in table 1. Sample 4 showed a very small amount
of organic phosphorus in the soluble form, and it may be inferred that
some of its soluble organic phosphorus had been decomposed. All three
samples (K, R, and X) prove that where there is a loss of soluble organic
phosphorus, fishy flavor develops in the butter. Sample 4 in table 1
also developed a fishy flavor in storage.

Since it seemed necessary to identify the trimethylamine directly,
a sample of butter which had been scored as fishy was selected and the
trimethylamine was extracted and identified. The method used was as
follows: 100 grams of fishy butter was extracted in a separatory funnel
with several portions (about 50 cubic centimeters each) of hot water.
The washings were separated from the fat and placed in a liter flask.
About 25 cubic centimeters of 1:1 caustic potash solution was added
to the contents of the flask, the flask was attached to a condenser, and
the solution was heated. The distillate was caught in about 50 cubic
centimeters of N/10 sulfuric acid solution. The distillation was continued —
until the volume of the distillate measured about 200 cubic centimeters.
The distillate was then evaporated, on a water bath, to dryness. The
residue was taken up with a small quantity of 95-per-cent alcohol and
again evaporated to dryness. Dehydrated alcohol was then added to
the residue, and the extractions were passed through a filter paper into
a clean evaporating dish. Several extractions with alcohol were made,
after which the new filtrate was evaporated to dryness on a water bath
in a covered hood. The new residue, consisting of trimethylamine sulfate,
was dissolved in a small quantity of warm water and the contents of the
evaporating dish were- washed into a 300-cubic-centimeter Erlenmeyer
flask. About 5 cubic centimeters of 1:1 caustic potash solution was added
to the contents of the flask, and the whole was distilled into 50 cubic
centimeters of N/50 hydrochloric acid. A few drops of the distillate
were carefully mixed on a glass slide with a solution of platinic chloride,

PuospHorvs IN BurrEeR 183

and orange-colored crystals, octahedral in shape, were produced. Blank
checks on the reagents and the water used showed no evidence of con-
tamination with ammonium or potassium, both of which yield erystals of
this type. To check the results, a solution of trimethylamine hydro-
chloride was prepared for comparison, and this gave results identical to
those of the trimethylamine salt obtained from the fishy-flavored butter.

As the experimental data show, when cream is handled in certain ways
the organic phosphorus compounds which are soluble in salt solution
are decomposed more or less before churning or soon after the resulting
butter is placed in storage. The organic phosphorus compound lecithin,
which is soluble in sodium chloride solution, represents almost entirely,
if not entirely, the soluble phosphorus in the organic form as shown
in table 1.©This soluble organic phosphorus gradually suffers decom-
position, as may be seen in table 2. With the exception of two cases,
samples 1 and 7, all the samples that had a significant amount of soluble
organic phosphorus suffered decomposition of this component during
storage; and in the experiment with Bacterium ichthyosmius, the butter
which, from the method of handling the cream before churning, could be
compared to samples 1 and 7, did not develop the fishy flavor. The
butter that did decrease in its soluble organic phosphorus content as
shown in tables 1 and 2, may be compared to those samples that became
fishy in the inoculation experiment.

This publication deals only with the phosphorus in butter, in its various
compounds. However, it is an appreciated fact that there are com-
pounds of other salts, and of calcium especially, which have a marked
influence on the keeping qualities of butter. Lactic acid also must play
no small part in bringing about chemical changes in butter. Further
studies are being made on the keeping qualities of butter as affected by
calcium compounds and lactic acid.

SUMMARY

For the experiments described in the foregoing pages, butter was made
from cream treated in the following ways: sweet cream ripened with
starter; sweet cream churned without starter; lactic acid added to cream
and the mixture pasteurized; raw cream self-ripened; lactic acid added
to cream and churning begun at once; sweet cream pasteurized and then
churned immediately; pasteurized sweet cream ripened with starter.

184 J. T. CustcKx

All the methods of handling had some influence on the phosphorus com-
pounds in the cream and subsequently on the phosphorus compounds
in the stored butter. Pasteurization had the most decided influence.
When cream containing acid was pasteurized, an appreciable amount of
phosphorus was rendered soluble and lost in the buttermilk and the
wash waters. Much of this phosphorus evidently came from the insoluble
protein residue. Pasteurized sweet cream suffered but little phosphorus
loss except in the protein residue. More phosphorus was lost when
pasteurized sweet cream was subsequently ripened with starter. In the
unpasteurized samples, sweet-raw-cream butter retained the largest
amount of phosphorus, butter made from raw cream ripened without
starter was next, while butter made with starter ranked third.

After fifteen months the samples were again analyzed, and the analyses
showed that the phosphorus in the organic compounds had broken down
to the inorganic form. Exceptions to this were in the butter made from
raw cream ripened with starter, and in the butter made from pasteurized
sweet cream subsequently ripened with starter. With the exception of
these two, all the samples increased in the phosphorus content of the
protein residue. All samples, without exception, increased in soluble
inorganic phosphorus during storage. The organic phosphorus compounds
in the unsalted samples were slower to break down than were the organic
phosphorus compounds in the corresponding salted samples.

There seems to be plenty of evidence that an alcohol-soluble protein
containing phosphorus exists in butter and is closely related to casein.

From the results obtained with the samples of butter containing varying
amounts of sodium chloride, it can be inferred that salt has an accelerating
action on the solubility of insoluble organic phosphorus compounds.

About two-thirds of the total phosphorus of the cream is retained
in the buttermilk, and the remaining one-third is shared by the wash
waters, the salt exudates, and the butter. The butter finally retains
about one-quarter of the phosphorus originally present in the cream.

After fifteen months in storage, all the phosphorus compounds in the
fat could be extracted by shaking in a separatory funnel with half-saturated
sodium chloride solution.® A 0.2-per-cent solution of hydrochloric acid was
found necessary to extract the soluble phosphorus in the protein residue.

6 Extraction with the aid of centrifugation was not tried on the samples used for table 1.

PuospHorvs IN BurreR 185

The substance that produces fishy flavor in butter, is undoubtedly
preformed in the cream by the breaking-down of the lecithin. It may
be assumed that through the solvent action of salt water and lactic acid,
trimethylamine (the constituent giving fishy flavor) is formed from one
of these broken-down fractions.

_ CONCLUSIONS

1. In churning, about one-fourth of the total phosphorus of the cream
is retained in the butter made therefrom. The remaining three-fourths
is lost in the buttermilk, wash waters, and exudates during the salting
process.

2 The methods of treatment of milk and cream before churning have
an influence on the amount and the form of phosphorus retained in the
butter.

3. In storage the soluble organic phosphorus compounds break down,
“giving inorganic phosphorus compounds.

4. The methods of treatment of milk and cream before churning deter-
mine how soon after storage organic phosphorus compounds will assume
the inorganic form.
| 5 Salt in butter has a marked effect in bringing about protein decom-
position during storage, even at a temperature of —10° C.

6. The new protein of milk which is soluble in alcohol exists also in
butter.

7 Under certain conditions, bacteria are the controlling factors in bring-
ing about chemical changes in the phosphorus compounds of butter.

8. The breaking-down of lecithin and the forming of trimethylamine
is the cause of fishy flavor in butter. ;

9. When fishy flavor develops in butter there is always an appreciable
loss of soluble organic phosphorus.

ACKNOWLEDGMENTS

The author wishes to express his thanks to the Department of Dairy
Industry of the New York State College of Agriculture for the donation
of cream and other material, and to Professor E. S. Guthrie and Mr.
H. C. Jackson for the scoring of samples of butter.

186 J.T. Custcx

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Memoir 27, The Influence of Low Temperature on Soil Bacteria, the third preceding number in this
series of publications, was mailed on November 15, 1919.

Memoir 28, Cooperative Marketing in the Chautauqua-Erie Grape Industry, the second preceding number
in this series ‘of publications, was mai iled on November 2C
_ Memoir 29, The Lecithin Conient af Butter and Its Posable ih eens to the Fishy Flavor, the Loca
ing number in this series of publications, was mailed on Lecember 23, 1919.

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