=476. Kinds of Proteid Bodies in Milk.=—The proteid bodies in milk
are all found in at least partial solution. Some authorities state
that a portion of the casein is present in the form of fine particles
suspended after the manner of the fat globules.[472] The number and
kind of proteid bodies are not known with definiteness. Among those
which are known with certainty are casein, albumin, peptone and fibrin.
The latter body was discovered in milk by Babcock.[473] Lactoglobulin
and lactoprotein are also names given to imperfectly known proteid
bodies in milk. Lactoprotein is not precipitated either by acids or
by heat and is therefore probably a peptone. By far the greater part
of the proteid matters in milk is casein. Casein has been called
caseinogen by Halliburton,[474] and paracasein by Schulze and Röse.[475]
Casein has intimate relations to the mineral matters in milk, and
is probably itself made up of several proteid bodies of slightly
differing properties. In general all that class of proteid matter
contained in milk which is precipitated by rennet or a weak acid, or
spontaneously on the development of lactic acid, is designated by the
term casein, while the albumins and peptones in similar conditions
remain in solution. Casein contains phosphorus, presumably as nuclein.
Fibrin is recognized in milk by the reactions it gives with hydrogen
peroxid or gum guiacum. The decomposition of hydrogen peroxid is not
a certain test for fibrin, inasmuch as pus and many other bodies will
produce the same effect. If the milk decompose hydrogen peroxid,
however, before and not after boiling, an additional proof of the
presence of fibrin is obtained, since boiled fibrin does not act on
the reagent.[476] The gum guiacum test is applied by dipping a strip of
filter paper into the milk and drying. The solution of gum guiacum is
applied to the dried paper and the presence of fibrin is recognized
by the blue color which is produced. The fibrin is probably changed
into some other proteid during the ripening of cream in which the
fibrin is chiefly found. The albumin in milk is coagulated by boiling,
while the casein remains practically unaffected when subjected to that
temperature.
=477. Estimation of Total Proteid Matter.=—The total proteid matter
in milk is determined by any of the general methods applicable to the
estimation of total nitrogen, but the moist combustion method is by far
the most convenient. From the total nitrogen, that which represents
ammonia or other nonproteid nitrogenous bodies, is to be deducted
and the remainder multiplied by an appropriate factor. Practically
all the nitrogen obtained is derived from the proteid matters and,
as a rule, no correction is necessary. The factors employed for
calculating the weight of proteid matter from the nitrogen obtained
vary from 6.25 to 7.04. It is desirable that additional investigations
be made to determine the magnitude of this factor. It is suggested
that provisionally the factor 6.40 be used. In the method adopted by
the Association of Official Agricultural Chemists it is directed that
about five grams of milk be placed in the oxidizing flask and treated
without previous evaporation exactly as described for the estimation
of total nitrogen in the absence of nitrates. The nitrogen obtained is
multiplied by 6.25 to get the total proteid matter.[477] In order to
prevent the too great dilution of the sulfuric acid, the milk may be
evaporated to dryness or nearly so before oxidation. In this laboratory
it is conveniently done by placing the milk first in the oxidizing
flask, connecting this with the vacuum service and placing the flask in
hot water. The aqueous contents of the milk are quickly given off at
a temperature not exceeding 85°, and the time required is only a few
minutes.
The milk may also be dried in dishes made of thin glass or tin foil
and, after desiccation, introduced with the fragments of the dishes
into the oxidizing flask.
The preliminary drying in the oxidizing flask is recommended as the
best.
Söldner oxidizes the nitrogen in human milk by boiling ten cubic
centimeters thereof for three hours with twenty-five of sulfuric
acid, fifty milligrams of copper oxid and three drops of a four per
cent platinic chlorid solution, and, after distilling the ammonia,
uses the factor 6.39 for calculating the proteid matter. According to
this author human milk is much less rich in nitrogenous constituents
than is generally supposed, containing not more than one and a half
per cent thereof in average samples collected at least a month after
parturition.[478]
=478. Precipitation of Total Proteids with Copper Sulfate.=—This method
of throwing out the total proteids of milk is due to Ritthausen.[479]
The proteids and fat are precipitated together by the addition of a
measured volume of copper sulfate solution, containing 63.92 grams
of the crystallized salt in one liter. The process, as modified by
Pfeiffer, is conducted as follows:[480]
Ten grams of milk are diluted with ten times that much water, five
cubic centimeters of the copper sulfate solution added and then soda
lye solution drop by drop until the copper is just precipitated. This
is determined by testing a few drops of the filtrate with soda lye,
which, when the copper is precipitated, will give neither a turbidity
nor a blue color.
The mixture is poured into a dry tared filter, the precipitate washed
with hot water, dried to constant weight and weighed. The fat is
removed from the dry pulverized mass by extraction with ether and the
residue dried and weighed.
The quantity of copper oxyhydrate contained in the precipitate is
calculated from the quantity of the copper solution used and amounts
to 0.2026 gram. The casein thus prepared contains not only the copper
compound named, but also some of the sodium sulfate formed on the
addition of the soda lye and other mineral salts present in the milk
and from which it is quite impossible to completely free it. There are
also many other objections to the process, and the product is of such a
nature as to render the data obtained by the method very doubtful.
This method is chiefly valuable on account of its historical interest.
Not only are the drying and weighing of the precipitate rendered
unnecessary by the modern methods of determining nitrogen, but there
are numerous sources of error which seem to throw doubt on the accuracy
of the results. The copper hydroxid does not lose all its water even on
drying at 125°.[481] The method therefore can only be recommended for
practical purposes when all the tedious processes of drying, extracting
and calculating the quantity of copper oxid are abandoned and the moist
washed precipitate used directly for the determination of nitrogen.
=479. Proteid Bodies by Ammonium Sulfate.=—All the proteid bodies
except peptones are precipitated from milk on saturation with ammonium
sulfate. This method has little analytical value because of the
presence of nitrogenous salt in the precipitate. Zinc sulfate may be
substituted for the ammonium salt and thus a determination of proteid
matter other than peptone be obtained. This result subtracted from the
total proteid nitrogen gives that due to peptone.
=480. Total Proteid Matter by Tannic Acid.=—For the determination
of the total proteid matter in milk Sebelien uses the following
process.[482] From three to five grams are diluted with three or four
volumes of water, a few drops of a saline solution added (sodium
phosphate, sodium chlorid, magnesium sulfate, _et similia_), and the
proteid bodies thrown out with an excess of tannic acid solution.
The precipitate is washed with an excess of the precipitant and the
nitrogen therein determined and multiplied by 6.37.
=481. Separation of Casein from Albumin.=—Sebelien prefers magnesium
sulfate or sodium chlorid to acetic acid as the best reagent for
separating casein from lactalbumin. Of the two saline reagents
mentioned, the former is the better. The milk is first diluted
with a double volume of the saturated saline solution and then the
fine powdered salt added until saturation is secured. The casein
is completely thrown out by this treatment, collected on a filter,
washed with the saturated saline solution, and the nitrogen therein
determined. The difference between the total and casein nitrogen gives
the quantity due to the albumin plus the almost negligible quantity due
to globulin.[483]
=482. Van Slyke’s Method of Estimating Casein.=—The casein may be
separated from the other albuminoids in milk by the procedure proposed
by Van Slyke.[484] Ten grams of the fresh milk are diluted with ninety
cubic centimeters of water and the temperature raised to 40°. The
casein is thrown down with a ten per cent solution of acetic acid, of
which about one and a half cubic centimeters are required. The mixture
is well stirred and the precipitate allowed to subside. The whey is
decanted onto a filter, and the precipitate washed two or three times
with cold water, brought finally onto the filter and washed once or
twice with cold water. The filter paper and its contents are used
for the determination of nitrogen in the usual way. The casein is
calculated from the nitrogen found by multiplication by the factor
6.25. Milk may be preserved for this method of determination by adding
to it one part of finely powdered mercuric chlorid for each two
thousand parts of the sample. The method is not applicable to curdled
milk.
=483. Theory of Precipitation.=—Most authorities now agree in supposing
that the state of semisolution in which the casein is held in milk
is secured by the presence of mineral matters in the milk, in some
intimate combination with the casein. Among these bodies lime is of
the most importance. The action of the dilute acid is chiefly on these
mineral bodies, releasing them from combination with the casein, which,
being insoluble in the milk serum, is precipitated.
=484. Factors for Calculation.=—Most analysts still use the common
proteid factor, 6.25, in calculating the quantity of proteids from the
nitrogen determined by analysis. For casein many different factors have
been proposed. According to Makeris the factor varies from 6.83 to
7.04.[485] Munk gives 6.34 for human and 6.37 for cow milk.[486] Sebelien
adopts the latter factor, and Hammersten nearly the same; _viz._,
6.39. The weight of authority, at the present time, favors a factor
considerably above 6.25 for calculating the casein and, in fact, the
total proteids of milk from the weight of nitrogen obtained.
=485. Béchamp’s Method of Preparing Pure Casein.=—The casein in about
one liter of milk is precipitated by adding gradually about three
grams of glacial acetic acid diluted with water. The addition of the
acid is arrested at the moment when litmus paper shows a slightly acid
reaction. The precipitate thus produced, containing all the casein,
the milk globules and the microzymes, is separated by filtration,
being washed by decantation before collecting it on the filter. On
the filter it is washed with distilled water and the fat removed by
shaking with ether. The residue is suspended in water, dissolved in the
least possible quantity of ammonium carbonate, any insoluble residue
(microzymes, globules) separated by filtration and the pure casein
thrown out of the filtrate by the addition of acetic acid. The washing
with distilled water, solution in ammonium carbonate, filtration and
reprecipitation are repeated three or four times in order to obtain
the casein entirely free of other substances. Casein thus prepared is
burned to a carbon free ash with difficulty and contains but little
over one-tenth per cent of mineral matter.[487]
=486. Separation of Casein with Carbon Dioxid.=—The supersaturation of
the lime compounds of casein with carbon dioxid diminishes the solvent
action of the lime and thus helps to throw out the proteid matter. For
this reason Hoppe-Seyler recommends the use of carbon dioxid to promote
the precipitation of the casein.[488] The milk is diluted with about
twenty volumes of water and treated, drop by drop, with very dilute
acetic acid as long as a precipitate is formed. A stream of pure carbon
dioxid is conducted through the mixture for half an hour, and it is
allowed to remain at rest for twelve hours, when the casein will have
all gone down and the supernatant liquid will be clear. Albumins and
peptones are not thrown out by this treatment.
The method of precipitation is advantageously modified by saturating
the diluted milk with carbon dioxid before adding the acetic acid, less
of the latter being required when used in the order just noted.[489]
=487. Separation of Albumin.=—In the filtrate from the casein
precipitate the albumin may be separated by heating to 80°. It may also
be precipitated by tannic acid, in which case it may contain a little
globulin. It may also be thrown out by saturation with ammonium or
zinc sulfates. The latter reagent is to be preferred when the nitrogen
is to be determined in the precipitate. The quantities of albumin and
globulin, especially the latter, present in milk are small compared
with its content of casein.
=488. Separation of Globulin.=—The presence of globulin in milk
is demonstrated by Sebelien in the following manner:[490] The milk
is saturated with finely powdered common salt and the precipitate
produced is separated by filtration. This filtrate in turn is
saturated with magnesium sulfate. The precipitate produced by this
reagent is collected on a filter, dissolved in water and precipitated
by saturation with sodium chlorid. This process is repeated several
times. The final precipitate is proved to be globulin by the following
reactions: When a solution of it is dialyzed the proteid body
separates as a flocculent precipitate, which is easily dissolved in
a weak solution of common salt. The clear solution thus obtained
becomes turbid on adding water, and more so after the addition of a
little acetic acid. A neutral solution of the body is also completely
precipitated by saturation with sodium chlorid. These reactions serve
to identify the body as a globulin and not an albumin. All the globulin
in milk is not obtained by the process, since a part of it is separated
with the casein in the first precipitation.
=489. Other Precipitants of Milk Proteids.=—Many other reagents besides
those mentioned have been used for precipitating milk proteids, wholly
or in part. Among these may be mentioned the dilute mineral acids,
lactic acid, rennet, mercuric iodid in acetic acid, phosphotungstic
acid, acid mercuric nitrate, lead acetate and many others.
It has been shown by the author that many of these precipitants do not
remove all the nitrogen but that among others the mercury salts are
effective.[491] When nitrogen is to be subsequently determined the acid
mercuric nitrate cannot be employed.
=490. Precipitation by Dialysis.=—Since the casein is supposed to be
held in solution by the action of salts it is probable that it may be
precipitated by removing these salts by dialysis.
=491. Carbohydrates in Milk.=—The methods of determining lactose in
milk, both by the copper reduction and optical processes, have been
fully set forth in foregoing paragraphs (=243, 244, 259, 262=). In
general, the optical method by double dilution is to be preferred
as practically exact and capable of application with the minimum
consumption of time.[492] For normal milks a single polarization is
entirely sufficient, making an arbitrary correction for the volume
occupied by the precipitated proteids and fat. This correction is
conveniently placed at six and a half per cent of the volume of milk
employed.
The polarimetric estimation of lactose in human milk is likely to give
erroneous results by reason of the existence in the serum of polarizing
bodies not precipitable by the reagents commonly employed for the
removal of proteids.[493] The same statement may be made in respect of
ass and mare milk. The use of acetopicric acid for removing disturbing
bodies, as proposed by Thibonet[494] does not insure results free from
error. With the milks above mentioned, it is safer to rely on the data
obtained by the alkaline copper reagents.
=492. Dextrinoid Body in Milk.=—In treating the precipitate, produced
in milk by copper sulfate, with alcohol and ether for the purpose
of removing the fat, Ritthausen isolated a dextrin like body quite
different from lactose in its properties.[495] The alcohol ether extract
evaporated to dryness leaves a mass not wholly soluble in ether, and
therefore not composed of fat. This residue extracted with ether,
presents flocky particles, soluble in water and mostly precipitated
therefrom by alcohol. This body has a slight reducing effect on
alkaline copper salts and produces a gray color with bismuth nitrate.
The quantity of this material is so minute as to lead Ritthausen to
observe that it does not sensibly affect the fat determinations when
not separated. It is not clearly demonstrated that it is a dextrinoid
body and the analyst need not fear that the optical determination of
milk sugar will be sensibly affected thereby.
Raumer and Späth assume that certain discrepancies, observed by them
in the data obtained for lactose by the copper and optical methods,
are due to the presence of this dextrinoid body, but no positive proof
thereof is adduced.[496]
=493. Amyloid Bodies in Milk.=—Herz has observed in milk a body
having some of the characteristics of starch.[497] Observed by the
microscope, these particles have some of the characteristics of the
starch grains of vegetables, with a diameter of from ten to thirty-five
micromillimeters. They are colored blue by iodin. When boiled with
water, however, these particles differ from starch in not forming
a paste. The particles are most abundant in the turbid layer found
immediately beneath the ether fat solution in the areometric process of
Soxhlet.
The amyloid particles may be collected from cheese and butter by
boiling with water, when they settle and can be observed on the
sediment after freeing of fat by ether.
Some of the statements regarding the adulteration of dairy products
with starch may have been made erroneously by reason of the natural
occurrence of these particles.
As in the case of the dextrin like body mentioned above this starchy
substance, if it really exist, occurs in too minute a quantity to
influence the results of any of the analytical methods heretofore
described.
In connection with the supposed presence of an amyloid body in milk,
it should be remembered that certain decomposition nitrogenous bodies
give practically the same reactions as are noted above.[498] Among these
may be mentioned chitin, which occurs very extensively in the animal
world. The proof of the existence of dextrinoid and amyloid bodies in
milk rests on evidence which should be thoroughly revised before being
undoubtedly accepted.