Chapter XVIII: Preface: To Volume Third (18)

The water may also be determined in solid extracts by placing about five grams of the material in a flat bottom tin foil dish about fifty-five millimeters in diameter and twenty millimeters deep. The material is dissolved in enough warm water to fill the dish a little over one-half and the liquid is then absorbed by adding a weighed quantity of fibrous asbestos or of dry fragments of pumice stone. The asbestos is to be preferred because of the fact that it may be subsequently cut into small bits for the determination of the gelatin. The dish thus prepared is dried to constant weight in a steam-bath or vacuum oven. The weight of the dish and of the added absorbent, together with that of the material employed and of the dried dish and its contents, give the data for calculating the percentage of water. The contents of the dish are used as described further on for the determination of gelatin. In liquid extracts the water is determined in an entirely analogous manner, using about twenty grams of the material and omitting the solution in water.

In solid extracts, the part insoluble in cold water is determined separately.

_Ash._—The ash is determined by ignition at the lowest possible temperature, best in a muffle (=28-32=). The ash should be examined qualitively. Where a quantitive analysis is desired, larger quantities of the extract are incinerated and the constituents of the ash determined in the usual way.[389]

_Total Nitrogen._—Since nitrates are not present unless added in the manufacture, the total nitrogen is best determined by moist combustion.[390]

_Nitric Nitrogen._—The extract should be tested for nitrates and if present they are determined in the manner already described.[391]

_Ammoniacal Nitrogen._—When ammonia is present it is determined by distillation with magnesia.[392]

Since boiling with magnesia may cause the distillation of more ammonia than is present as ammonium salts, the plus being due to the decomposition of some other nitrogenous compounds, Stutzer replaces the magnesia with barium carbonate.[393]

_Proteid Nitrogen Insoluble in Sixty-Two Per Cent Alcohol._—The aqueous solution is treated with strong alcohol until the mixture contains about sixty-two per cent of the reagent. The precipitate produced is separated by filtration, washed with sixty-two per cent alcohol and the nitrogen therein determined.

_Albumose Nitrogen._—This is secured by saturating the aqueous solution with zinc or ammonium sulfate. The separated albumoses are skimmed from the surface, thrown in a filter, washed with a saturated solution of zinc sulfate and the nitrogen determined therein by moist combustion. In the filtrate from the above separation, peptone is detected qualitively by adding a few drops of dilute solution of copper sulfate (biuret reaction).

_Kreatin, Kreatinin and Other Flesh Bases._—The clear, aqueous solution of the extract is acidified with sulfuric, mixed with a solution of sodium phosphotungstate and allowed to stand for about six days. The precipitate is collected, washed with a solution of the precipitant, and the nitrogen therein determined. The nitrogen found, less that due to ammonia, represents the total nitrogenous matter precipitated by the phosphotungstic acid. From this quantity is deducted the nitrogen in the proteids, precipitated by sixty-two per cent alcohol and by ammonium or zinc sulfate, and the remainder represents the nitrogen in flesh bases.

The nitrogen thrown out by the phosphotungstic acid is deducted from the total nitrogen, and the remainder represents the nitrogenous bodies not precipitable by the reagent named.

This method of separating the nitrogenous matters in meat extracts is based on the observation that these bodies contain at most only a small quantity of peptones, so small as to be safely negligible.[394]

_Quantities used for Analysis._—In conducting the separations above noted, it will be found convenient to use in each case about five grams of the solid or twenty of the liquid extract. In the nitrogen determinations, the weight of the sample should be inversely proportional to its content of nitrogen.

=417. Preparation of the Phosphotungstic Reagent.=—The phosphotungstic reagent is conveniently prepared as follows:

Dissolve 120 grams of sodium phosphate and 200 of sodium tungstate in one liter of water and add to the solution 100 cubic centimeters of strong sulfuric acid. When the reagent is prepared for general purposes it is customary to acidify with nitric, but in the present instance, inasmuch as the precipitate is used for the determination of nitrogen, it is evident that sulfuric should be substituted for nitric acid. In all cases the analyst must be assured of the strong acidity of the reagent, and in addition to this the solutions of proteid matter to which the reagent is added must first be made strongly acid with sulfuric.

=418. Zinc Sulfate as Reagent for Separating Albumoses from Peptones.=—When the albumoses are separated from the peptones, by precipitation with ammonium sulfate, there may be danger of some of this reagent adhering to the albumose, and in this way the quantity of nitrogen obtained on analysis may be increased. To avoid an accident of this kind Bömer replaces the ammonium by zinc sulfate.[395]

Since the precipitation of the albumoses by saturated saline solutions depends on their hydrolytic power, the substitution of another salt for ammonium sulfate capable of strongly attracting water, may be made if that salt does not possess any objectionable property. Crystallized zinc sulfate will dissolve in less than its own weight of cold water and is therefore well suited for the purpose in view.

In the case of a meat extract, the precipitation is accomplished as follows: Fifty cubic centimeters of the extract, freed from all solid matter by filtration and containing about two grams of the soluble proteids, are saturated in the cold with finely powdered zinc sulfate. The separated albumoses collect on the surface and are skimmed off, poured on a filter and washed with cold saturated zinc sulfate solution. The filter and its contents are used for the determination of nitrogen by moist combustion.[396]

The filtrate from the precipitated albumoses gives no biuret reaction, and, therefore, as in the use of ammonium sulfate, is free of albumin.

The biuret reaction is applied to the zinc sulfate filtrate as follows: The filtrate is greatly diluted with water and freed of zinc by means of a saturated solution of sodium carbonate. The filtrate free of zinc is evaporated on the steam-bath, made strongly alkaline with sodium hydroxid and treated with a few drops of a two per cent copper sulfate solution, added successively.

Another advantage possessed by the zinc sulfate is found in the fact that in the filtrate from the separated albumoses the peptones and other flesh bases can be thrown out by phosphotungstic acid. Before the application of the reagent, the filtrate should be made strongly acid by adding about an equal volume of dilute sulfuric acid (one part of acid to four of water.)

The nitrogen in the precipitate thus obtained is determined by moist combustion in the manner already suggested.

If the proteid matters contain salts of ammonium it is probable that a difficultly soluble double sulfate of zinc and ammonium, (NH₄)₂SO₄.ZnSO₄.6H₂O, will be found in the precipitate. Ammonium salts, if present, should therefore be removed by distillation with magnesia. It is better, however, to throw down the ammonia with the first zinc precipitate, distil this with magnesia and determine the amount of nitrogen derived from the ammonia compounds. In a second sample, the total nitrogen is determined by moist combustion and the difference between the two results gives that due to albumoses.

=419. Examination for Muscular Tissue.=—Some samples of meat extracts contain small quantities of finely ground muscular tissue. For detecting this the extract is treated with cold water and the insoluble residue examined with a microscope. If muscular tissue be found, about eight grams of the extract or twenty-five of the fluid preparation, are treated with cold water, the insoluble matter collected upon a filter, washed with cold water, and the nitrogen determined in the residue. The percentage of nitrogen multiplied by 6.25 gives the quantity of muscle fiber proteids present. The filtrate from the above determination is acidified with acetic, boiled, any precipitate which is formed collected and the nitrogen therein determined. The nitrogen obtained multiplied by 6.25 gives the quantity of coagulable albumin present. An aliquot portion of the filtrate is used for the determination of nitrogen and the percentage therein found, deducted from the total nitrogen of the sample, gives a remainder which may be used as a representative of the whole of the nitrogen present in the form of albumin and muscular tissue.

=420. Estimation of Gelatin.=—The tin foil dish and its contents used for the determination of water, as above described, are cut into small pieces, placed in a beaker and extracted four times with absolute alcohol. After the removal of the alcohol, the residue is extracted with ice water containing ten per cent of alcohol, in which a small piece of ice is kept to avoid a rise of temperature. The beaker should be shaken during the extraction, which should last for about two minutes. Where large numbers of samples are treated at once, any convenient form of shaking machine may be employed. At least two extractions with ice water must be made. The residue is then collected upon a filter and washed with ice water until the washings are completely colorless. The residue on the filter is replaced in the beaker, boiled with water, well washed on the filter with boiling water, the filtrate and washings concentrated and the nitrogen therein determined.

The principle of this determination is based on the fact that gelatin is almost completely insoluble in ice water while serum peptones and albumin peptones are almost completely soluble in that reagent. On the other hand, the flesh bases and the proteids present are almost completely removed by the preliminary treatment with alcohol and ice water or are left undissolved by the hot water. The solution in boiling water, therefore, contains practically nothing but gelatin.[397]

In a later article, Stutzer modifies the method given above as follows:[398]

Of dry and moist extracts from five to seven grams and of liquid extracts from twenty to twenty-five grams are used for the determination and placed in tin foil dishes, as described above. In case of solid extracts, a sufficient quantity of warm water is added to completely dissolve them, the solution being facilitated by stirring. In case the solution is too thin it should be concentrated before going further. It is treated with a sufficient amount of dust-free ignited sand to completely absorb it, and the dish and its contents are then dried to a constant weight. The dried contents of the dish are rubbed up in a mortar, the dish cut into fine bits, and all placed in a beaker. The solid syrphete[399] is extracted four times with 100 cubic centimeters of absolute alcohol, the alcohol in each case being poured through an asbestos filter for the purpose of collecting any matters suspended therein. In a large flask are placed 100 grams of alcohol, 300 grams of ice and 600 grams of cold water, and the flask is placed in a large vessel and packed with finely divided ice. Four beakers marked _b, c, d, e_ are also placed in ice and the beaker containing the syrphete, left after extraction with absolute alcohol as above mentioned, is marked _a_ and also placed in pounded ice. The extraction with cold alcoholic water proceeds as follows:

In beaker _a_ are poured 100 cubic centimeters of the mixture in the large flask, its contents are stirred for two minutes and then the liquid portion poured off into beaker _b_ to which, at the same time, a piece of ice is added. In beaker _a_ are poured again 100 cubic centimeters from the large flask, treated as above described, and the liquid extract poured into beaker _c_. In like manner the extraction in beaker _a_ is continued until each of the beakers has received its portion of the extract. By this time the liquid over the sand in beaker _a_ should be completely colorless. The filtration of the liquid extract is accomplished as follows:

In a funnel of about seven centimeters diameter is placed a perforated porcelain plate about four centimeters in diameter which is covered with asbestos felt with long fiber. Three filters are prepared in this way. On the first filter are poured the contents of beaker _b_. After the liquid has passed through, the sand and other residue in beaker _a_ are transferred to the filter and the beaker and residue washed with the alcoholic ice water from the large flask. The filtration should be accomplished under pressure. On the second filter are poured the contents of beaker _c_. On the third filter the contents of beakers _d_ and _e_. The washing with alcoholic ice water from the large flask is continued in each instance until the filtrate is colorless. At the same time the asbestos filter, which was used in the first instance for filtering the absolute alcohol extract, is washed with the alcoholic ice water mixture from the large flask. At the end the sand remaining in beaker a together with all the asbestos filters are brought together into a porcelain dish, boiled two or three times with water, the aqueous solution filtered and the filtrate concentrated and used for the estimation of the nitrogen. The quantity of nitrogen found multiplied by 6.25 represents the proteid matter in the gelatin of the sample.

The object of the multiple filters, described above, is to accelerate the process, and they are required because the gelatin quickly occludes the filter pores. For this reason the asbestos filters are found to operate better than those made of paper. It should be mentioned that the residue of the peptones insoluble in alcohol may contain, in addition to gelatin, also small quantities of albumoses. From the quantity of albumose nitrogen found, it is understood that the nitrogen in the form of coagulable albumin, determined as described in the first process mentioned above, is to be deducted, since these coagulable albumins are insoluble in alcohol.

=421. Estimation of Nitrogen in the Flesh Bases Soluble in Alcohol.=—About five grams of the dry extract, ten grams of the extract containing water or twenty-five grams of the liquid extract are placed in a beaker and enough water added in each case to make about twenty-five cubic centimeters in all. Usually no water need be added to the liquid extracts. Very thin peptone solutions should be evaporated until the content of water is reduced to seventy-five per cent. The solution, prepared as above indicated, is treated slowly with constant stirring with 250 cubic centimeters of absolute alcohol, the stirring continued for some minutes and the vessel set aside for twelve hours, at the end of which time the precipitate is separated by filtration and washed repeatedly with strong alcohol. Leucin, tyrosin and a part of the flesh bases are dissolved by alcohol. The alcohol is removed by distillation and the residue dissolved in water. Any flocky residue which remains on solution with water is removed by filtration, the nitrogen determined therein and the quantity thereof added to the albumose nitrogen found, as hereafter described.

The volume of the aqueous solution is completed with water to half a liter. One hundred cubic centimeters of this solution are used for the determination of total nitrogen, and another 100 cubic centimeters for the determination of ammoniacal nitrogen by distillation with barium carbonate. A part of the ammonia may have escaped during the preliminary distillation of the alcohol and therefore the amount found may not represent the whole amount originally present. The use of the above determination is principally to ascertain the correction to be made in the amount of total nitrogen found in the first 100 cubic centimeters of the solution.

=422. Treatment of the Residue Insoluble in Alcohol.=—The residue insoluble in alcohol is washed from the filter into the beaker in which the first solution was made. The aqueous mixture is warmed on a water-bath until the alcohol adhering to the precipitate is completely evaporated, when the contents of the beaker are poured upon a filter free of nitrogen. A small part of the albumose, by reason of the treatment with alcohol, tends to remain undissolved, and it is advisable to collect this albumose upon a filter, wash it well with hot water and estimate the nitrogen therein. The quantity of nitrogen thus found is to be added to the albumose nitrogen determined as described later on.

The total filtrate obtained from the last filtration is made up to a volume of half a liter, of which fifty cubic centimeters are used for the determination of total nitrogen, fifty cubic centimeters for the determination of gelatin, albumose and peptone, and 100 cubic centimeters for the residual peptones. The albumose, together with the gelatin and peptones carried down with it, is precipitated with zinc or ammonium sulfate solution, and its per cent calculated from the amount of nitrogen found in the precipitate. The true peptone is determined by subtracting the quantity of nitrogen determined as albumose from the total nitrogen in solution.

The rest of the liquid, _viz._, 300 cubic centimeters, is evaporated to a small volume and tested qualitively for true peptones as follows:

To separate the albumose and gelatin a concentrated liquor is treated with an excess of finely divided ammonium sulfate so that a part of the salt remains undissolved. The separated albumose, gelatin and undissolved ammonium salts are collected on a filter, the filtrate mixed with a few drops of dilute copper sulfate solution and a considerable quantity of concentrated soda or potash lye added. Care should be taken that the quantity of copper is not too great, otherwise the peculiar red coloration will be obscured by the blue color of the copper solution.

=423. Pancreas Peptone.=—The filtrate obtained as described above, by treating the portion of the material insoluble in alcohol with warm water, contains in addition to the albumose and gelatin the whole of the pancreas peptone which may be present. To separate this peptone, 100 cubic centimeters of the aqueous solution are evaporated in a porcelain dish until the volume does not exceed ten cubic centimeters. When cool, at least 100 cubic centimeters of a saturated cooled solution of ammonium sulfate solution are added, the mixture thoroughly stirred, the precipitate collected upon a filter and washed with a cold saturated solution of ammonium sulfate. The contents of the filter are dissolved in boiling water, the filter thoroughly washed and the filtrate and washings evaporated in a porcelain dish with the addition of barium carbonate until, on the addition of new quantities of barium carbonate, no further trace of ammonia can be discovered. The residue is extracted with water, the barium sulfate and carbonate present separated by filtration, well washed and the nitrogen determined in the evaporated filtrate and washings in the usual way and multiplied by 6.25 to determine the quantity of pancreas peptone.

=424. Albumose Peptone.=—A part of the albumose peptone which may be present is determined in conjunction with the other bodies mentioned above. The chief quantity is found in the solution of the residue insoluble in alcohol in the following manner:

Fifty cubic centimeters of the solution of this residue in hot water are mixed with an equal volume of dilute sulfuric acid, one volume of acid to three of water, in the cold, and a solution of sodium phosphotungstate added until it produces no further precipitate. The precipitate is washed with dilute sulfuric acid and the nitrogen determined therein. The nitrogen thus found is derived from the albumose, pancreas peptone and gelatin. The quantity of nitrogen in the pancreas peptone and gelatin, as above described, is subtracted from the total quantity found in the phosphotungstic acid precipitated, and the remainder represents the nitrogen due to the albumose.

=425. Nitrogen in the Form of Flesh Bases Insoluble in Alcohol.=—This is determined by subtracting the quantity of nitrogen, determined by the phosphotungstic acid method already described, from the total quantity of nitrogen found in the precipitate insoluble in alcohol and soluble in water.

AUTHORITIES CITED IN PART FIFTH.

[337] Watts’ Dictionary of Chemistry, new edition, Vol. 4, p. 327.

[338] Vid. op. cit. supra, p. 330.

[339] Barbieri, Journal für praktische Chemie, neue Folge Band 18, S. 114.

[340] Vid. op. cit. 1, p. 339.

[341] Bulletin No. 49, Kansas Experiment Station, May, 1895.

[342] This work, Vol. 2, p. 208.

[343] Vid. op. cit. supra, Vol. 1, p. 570.

[344] Wiley, American Chemical Journal, Vol. 6, No. 5, p. 289.

[345] Obermayer, Chemiker-Zeitung Repertorium, Oct. 1889, S. 269.

[346] Hoppe-Seyler, Handbuch der physiologisch- und pathologisch-chemischen Analyse, S. 269.

[347] Wiley, American Chemical Journal, Vol. 6, p. 289.

[348] Dragendorff’s Plant Analysis, p. 55.

[349] This work, Vol. 2, pp. 192 et seq.

[350] Chemiker-Zeitung, Band 20, S. 151.

[351] This work, Vol. 2, p. 207.

[352] Landwirtschaftlichen Versuchs-Stationen, Band 17, S. 321: Zeitschrift für analytische Chemie, Band 14, S. 380.

[353] Vid. op. cit. 12, p. 245.

[354] Landwirtschaftlichen Versuchs-Stationen, Band 16, S. 61.

[355] Berichte der deutschen chemischen Gesellschaft, Band 10, Ss. 85, 199; Band 16, S. 312: Chemiker-Zeitung, Band 20, S. 145.

[356] Zeitschrift für analytische Chemie, Band 22, S. 325.

[357] Richardson and Crampton, Berichte der deutschen chemischen Gesellschaft, Band 19, S. 1180.

[358] Maxwell, American Chemical Journal, Vol. 13, p. 470.

[359] Vid. op. cit. supra, Vol. 15, p. 185.

[360] Vid. op. cit. supra, Vol. 13, p. 13: Schulze, Zeitschrift physiologische Chemie, Band 14, S. 491.

[361] This work, Vol. I, p. 411.

[362] Vid. op. cit., 22, Vol. 13, p. 15.

[363] Vid. op. cit. supra, Vol. 15, p. 188.

[364] Hoppe-Seyler, Handbuch der physiologisch- und pathologisch-chemischen Analyse, S. 169.

[365] This work, Vol. 2, p. 225.

[366] Bulletin No. 45, Division of Chemistry, U. S. Department of Agriculture, p. 51.

[367] Osborne and Voorhees, American Chemical Journal, Vol. 15, p. 470.

[368] Vid. op. cit. supra, Vol. 13, p. 385.

[369] Vid. op. cit. supra, p. 412.

[370] Vid. op. cit. supra, Vol. 15, p. 402.

[371] Vid. op. cit. supra, p. 404.

[372] Zeitschrift für analytische Chemie, Band 34, S. 562.

[373] Vid. op. cit. 34, p. 404.

[374] Vid. op. cit. 3, p. 455.

[375] Osborne and Voorhees, vid. op. cit. 34, p. 409.

[376] Vid. op. cit. supra, Vol. 13, p. 464.

[377] Chittenden and Osborne, op. cit. supra, Vol. 14, p. 32.

[378] Vid. op. cit. supra, p. 41.

[379] Vid. op. cit. supra, p. 639.

[380] Vid. op. cit. supra, Vol. 13, p. 399.

[381] Vid. op. cit. supra, pp. 395, 400, 401.

[382] Vid. op. cit. supra, p. 409.

[383] This work, Vol. 1, p. 319.

[384] This work, Vol. 2, pp. 169 et seq.

[385] Vid. op. cit. 47, p. 420.

[386] Osborne, vid. op. cit. 44, p. 410.

[387] Hoppe-Seyler, Handbuch der physiologisch- und pathologisch-chemischen Analyse.

[388] König und Bömer, Zeitschrift für analytische Chemie, Band 34, S. 560.

[389] This work, Vol. 2, pp. 297, 298.

[390] Vid. op. cit. supra, p. 184.

[391] Vid. op. cit. supra, p. 206.

[392] This work, Vol. I, p. 450; Vol. 2, p. 226.

[393] Zeitschrift für analytische Chemie, Band 34, S. 377.

[394] König und Bömer, vid. op. cit. supra, S. 560.

[395] Vid. op. cit. supra, S. 562.

[396] Vid. op. cit. 53, p. 184.

[397] Vid. op. cit. 57, S. 374.

[398] Vid. op. cit. supra, S. 568.

[399] From συρφετος.

PART SIXTH.

DAIRY PRODUCTS.

=426. Introductory.=—The importance of dairy products has led to the publication of a vast amount of literature relating thereto, and it seems almost a hopeless task to present even a typical abstract of the various analytical processes which have been proposed and used in their study. The general principles which have been developed in the preceding parts of this volume are applicable to the study of dairy products, and the analyst who is guided by them can intelligently examine the bodies specially considered in the present part. There have been developed, however, many valuable processes for the special examination of dairy products, which are of such a nature that they could not be properly discussed in the preceding pages. In the present part an effort will be made to present in a typical form the most important of these processes and to state the general principles on which they are based. This subject is naturally subdivided into three parts, _viz._, milk, butter and cheese. The milk sugar industry is not of sufficient importance to receive a special classification.

MILK.

=427. Composition of Milk.=—The composition of milk not only varies with the genus and species of the mammal from which it is derived, but also depends in a marked degree on idiosyncrasy.[400]

Milk is a mixture containing water, proteids, fat, carbohydrates, organic and inorganic acids and mineral salts. There have also been observed in milk in minute quantities ammonia, urea, hypoxanthin, chyme, chyle, biliverdin, cholesterin, mucin, lecithin, kreatin, leucin and tyrosin. In the fermentation which milk undergoes in incipient decomposition there is sometimes developed from the proteid matter, as pointed out by Vaughn, a ptomaine, tyrotoxicon, which is a virulent poison.[401] The presence of these last named bodies is of interest chiefly to the physiologist and pathologist and can receive no further attention here.

From a nutritive point of view, the important components of milk are the fats, proteids and sugar, but especially in the nourishment of the young the value of lime and phosphoric acid must be remembered. The mean composition of the most important milks, as determined by recent analyses, is given below:

Water. Sugar. Proteids. Fat. Ash.
Per cent. Per cent. Per cent. Per cent. Per cent.
Cow 86.90 4.80 3.60 4.00 0.70
Human 88.75 6.00 1.50 3.45 0.30
Goat 85.70 4.45 4.30 4.75 0.80
Ass 89.50 6.25 2.00 1.75 0.50
Mare 90.75 5.70 2.00 1.20 0.35
Sheep 80.80 4.90 6.55 6.85 0.90

The mean composition of milk, as given by Watts and König, is given in the following tables:

WATTS.
Mineral
Water. Solids. Proteids. Fats. Sugar. Salts.
Woman 87.65 12.35 3.07 3.91 5.01 0.17
Ass 90.70 9.30 1.70 1.55 5.80 0.50
Cow 86.56 13.44 4.08 4.03 4.60 0.73
Goat 86.76 13.24 4.23 4.48 3.91 0.62
Sheep 83.31 16.69 5.73 6.05 3.96 0.68
Mare 82.84 17.16 1.64 6.87 8.65

KÖNIG.
Casein and Milk
Water. Fat. albumin. sugar. Ash.
Woman 87.41 3.78 2.29 6.21 0.31
Mare 90.78 1.21 1.99 5.67 0.35
Ass 89.64 1.63 2.22 5.99 0.51
Cow 87.17 3.69 3.55 4.88 0.71

The average composition of 120,540 samples of cow milk, as determined by analysis, extending over a period of eleven years, was found by Vieth to be as follows:[402]

Per cent.
Total solids 12.9
Solids not fat 8.8
Fat 4.1

The quantity of solids and fat in milk is less after longer than after shorter periods between milkings.

The quantity of solids and fat in cow milk is less in the spring than in the autumn.

The chief organic acid naturally present in milk is citric, which exists probably in combination with lime.

The mean content of citric acid in milk is about one-tenth of one per cent.[403]

Citric acid is not found in human milk, and probably exists only in the mammary secretions of herbivores.

Among the mineral acids of milk, phosphoric is the most important, but a part of the phosphorus found as phosphoric acid in the ash of milk may come from pre-existing organic phosphorus (lecithin, nuclein).

The sulfuric acid, which is found in the ash of milk, is derived from the sulfur of the proteid matter during ignition.

Lactic acid is developed from lactose during the souring of milk as the result of bacterial activity.

Gases are also found in solutions of milk, notably carbon dioxid, which gives to freshly drawn milk its brothy appearance.

The ash of milk has the following composition expressed as grams per liter of the original milk:[404]

Grams Probable form Grams
Component. per liter. of combination. per liter.

{ sodium chlorid 0.962
Chlorin 0.90 { potassium chlorid 0.830

{ KH₂PO₄ 1.156
{ K₂HPO₄ 0.853
Phosphoric acid 2.42 { MgHPO₄ 0.336
{ CaHPO₄ 0.671
{ Ca₃(PO₄)₂ 0.806

Potassium 1.80 (as shown above)
and as potassium citrate 0.495

Sodium 0.49 sodium chlorid 0.962

Lime 1.90 (as shown above)
and as calcium citrate 2.133

Magnesia 0.20 MgHPO₄ 0.336

The percentage composition of the ash of milk, according to Fleischmann and Schrott, is expressed as follows:[405]

Per cent.
Potassium oxid, K₂O 25.42
Sodium oxid, Na₂O 10.94
Calcium oxid, CaO 21.45
Magnesium oxid, MgO 2.54
Iron oxid, Fe₂O₃ 0.11
Sulfuric acid, SO₃ 4.11
Phosphoric acid, P₂O₅ 24.11
Chlorin, Cl 14.60
------
103.28
Less Cl as O 3.28
------
100.00

=428. Alterability of Milk.=—The natural souring and coagulation of milk is attributed by most authorities to bacterial action produced by infection from the air or containing vessels.[406] Pasteur, however, shows that fresh milk sterilized at a temperature of 110° may be exposed to the air without danger of souring.[407] After about three days, however, a fermentation is set up which is totally different from that produced by the microzymes naturally present in the milk. This point has been further investigated by Béchamp, who finds that the natural souring of milk is accomplished without the evolution of any gas, while the fermentation produced in sterilized milk by the microbes of the air, is uniformly attended by a gaseous development.[408] As a result of his investigations, he concludes that the souring of milk takes place spontaneously by reason of milk being an organic matter, in the physiological sense of the term, and that this alteration is produced solely by the natural microzymes of the milk.

According to Béchamp, the milk derived from healthy animals is capable of spontaneous alteration, which consists in the development of lactic acid and alcohol, and of curd in those milks which contain caseinates produced by the precipitating action of the acids formed. Oxygen and the germs which are present in the air, according to him, have nothing to do with this alteration in the properties of milk. Milk belongs to that class of organic bodies like blood, which are called organic from a physiological point of view, on account of containing automatic forces which produce rapid changes therein when they are withdrawn from the living organisms.

After milk has become sour by the spontaneous action of the microzymes which it contains, there are developed micro-organisms, such as vibriones and bacteria from a natural evolution from the microzymes.

Milk which is sterilized at a high temperature, _viz._, that of boiling water or above, is no longer milk in the true physiological sense of that term. The globules of the milk undergo changes and the microzymes a modification of their functions, so that in milk thus altered by heat, they are able to produce a coagulation without development of acidity. The microzymes thus modified, however, retain to a large extent their ability to become active. Human milk differs from cow milk in containing neither caseinates nor casein, but special proteid bodies, and also a galactozyme or galactozymase functionally very different from that which exists in cow milk. The extractive matter is also a special kind, consisting of milk globules and microzymes belonging particularly to it and containing three times less phosphate and mineral salts than cow milk. Boiling the milk of the cow or other animals does not render it similar to that of woman. There is no treatment, therefore, of any milk which renders it entirely suited to the nourishment of infants. The composition of the milk of the cow may be represented by three groups:

1. Organic elements in suspension; consisting chiefly of the globules of the milk, which are mostly composed of the fat, of an epidermoid membrane containing mineral matter of special soluble albumins and of microzymes containing also mineral matter.

2. Dissolved constituents; consisting of caseinates, lactalbuminates, galactozymase, holding phosphates in combination, lactose, extractive matter, organic phosphates of lime, acetates, urea and alcohol.

3. Mineral matters in solution; consisting of sodium and calcium chlorids, carbon dioxid and oxygen.[409]

It will be noticed from the above classification that Béchamp fails to mention citrate of lime. It is scarcely necessary to add to this brief résumé of the theories of Béchamp that they are entirely at variance with the opinions held by nearly all his contemporaries.

=429. Effects of Boiling on Milk.=—On boiling, the albumin in milk is coagulated and on separating the proteid bodies by saturation with magnesium sulfate no albumin is found in the filtrate. The total casein precipitated from boiled is therefore greater than from unboiled milk. Jager has shown that the casein can be precipitated from boiled milk by rennet, but with greater difficulty than from unboiled.[410] According to this author in 3.75 per cent of proteid in milk there are found 3.15 per cent of casein, 0.35 of albumin and 0.25 of globulin.

=430. Appearance of the Milk.=—The color, taste, odor and other sensible characters of the milk are to be observed and noted at the time the sample is secured. Any variation from the faint yellow color of the milk is due to some abnormal state. A reddish tint indicates the admixture of blood, while a blue color is characteristic of the presence of unusual micro-organisms. Odor and taste will reveal often the character of the food which the animals have eaten. Any marked departure of the sample from the properties of normal milk should at once lead to its condemnation for culinary or dietetic purposes.

=431. Micro-Organisms of the Milk.=—Milk is a natural culture solution for the growth of micro-organisms, and they multiply therein with almost incredible rapidity. Some of these are useful, as, for instance, those which are active in the ripening of cream, and others are of an injurious nature, producing fermentations which destroy the sugars or proteids of the milk and develop acid, alcohol, mucous or ptomaine products. It is not possible here to even enumerate the kinds of micro-organisms which abound in milk and the reader is referred to the standard works on that subject.[411]

For analytical purposes it is important that the sample be kept as free as possible of all micro-organisms, good or bad, which may be accomplished by some of the methods given below.

=432. Sampling Milk.=—It is not difficult to secure for examination representative samples of milk, if the proper precautions be taken. On the other hand, the ease and rapidity with which a milk undergoes profound changes render necessary a careful control of the methods of taking samples. The most rapid changes to which a mass of milk is obnoxious are due to the separation of the fat particles and to the action of bacteria. Even after standing for a few minutes, it will be found that the fat globules are not evenly distributed. Before securing the sample for analysis, it is necessary to well stir or mix the milk. A mean sample may also be secured from a can of milk by the sampling tube devised by Scovell, which will be described below.

In securing samples, a full detailed description of the cow or herd furnishing them is desirable, together with all other data which seem to illustrate in any way the general and particular conditions of the dairy. Samples are to be preserved in clean, well stoppered vessels, properly numbered and securely sealed.

=433. Scovell’s Milk Sampler.=—In sampling large quantities of milk in pails or shipping cans, it is exceedingly inconvenient to mix the milk by pouring from one vessel to another or by any easy process of stirring. In order to get representative samples in such conditions, Scovell has put in use a sampler, by means of which a typical portion of the milk may be withdrawn from a can without either pouring or stirring. The construction of the sampler is shown in Fig. 106, representing it in outline and longitudinal section. The tube _a_, made of brass, is open at both ends and of any convenient dimensions. Its lower end slides in a large tube _b_, closed at the bottom and having three elliptical, lateral openings _c_, which admit the milk as the tube is slowly depressed in the contents of the can. In getting the sample, _a_ is raised as shown in profile. When the bottom of _b_ reaches the bottom of the can _a_ is pushed down as shown in the section. The milk contained in the sampler is then readily withdrawn.

=434. Preserving Milk for Analysis.=—Pasteurizing or boiling the sample is not advisable by reason of the changes produced in the milk by heat. The milk sample may be preserved by adding to it a little chloroform, one part in 100 being sufficient. Boric and salicylic acids may also be used, but not so advantageously as formaldehyd or mercuric chlorid. Rideal has observed that one part of formaldehyd will preserve 10,000 parts of milk in a fresh state for seven days. The formaldehyd sold in the trade contains about one part of formaldehyd in 320 of the mixture. One-half pint of this commercial article is sufficient for about twenty gallons of milk, corresponding to about one part of pure formaldehyd to 45,000 parts of milk. Rideal much prefers formalin (formaldehyd) to borax or boric acid as a milk preservative. No ill effects due to its toxic action have been observed, even when it is consumed in a one per cent solution.[412]

Samples of milk can be kept in this way from four to six weeks by adding about one drop of the commercial formaldehyd to each ounce of sample. The analyst should remember in such cases that the formaldehyd may not all escape on evaporation, on account of forming some kind of a compound with the constituents of the milk, as is pointed out by Bevan.[413]

Bevan suggests that the formaldehyd may not actually be retained in the sample, but that the increase in the apparent amount of total solids is due to the conversion of the lactose into galactose. This point, however, has not been determined.

Richmond and Boseley propose to detect formalin by means of diphenylamin. A solution of diphenylamin is made with water, with the help of just enough sulfuric acid to secure a proper solvent effect. The liquid to be tested, which is supposed to contain formaldehyd, or the distillate therefrom, is added to this solution and boiled. If formaldehyd be present, a white flocculent precipitate is deposited, which is colored green if the acid used contain nitrates. For other methods of detecting formalin and for a partial literature of the subject the paper mentioned above may be consulted.

One gram of fine-ground mercuric chlorid dissolved in 2,000 grams of milk will preserve it, practically unchanged, for several days. One gram of potassium bichromate dissolved in one liter of milk will also preserve it for some time. Thymol, hydrochloric acid, carbon disulfid, ether and other antiseptics may also be employed. No more of the preserving agent should be used than is required to keep the milk until the analysis is completed.

All methods of preservation are rendered more efficient by the maintenance of a low temperature, whereby the vitality of the bacteria is greatly reduced.

=435. Freezing Point of Milk.=—By reason of its content of sugar and other dissolved solids, the freezing point of milk is depressed below 0°. A good idea of the purity of whole milk is secured by subjecting it to a kryoscopic test. The apparatus employed for this purpose is that used in general analytical work in the determination of freezing points. Pure full milk freezes at about 0°.55 below zero, and any marked variation from this number shows adulteration or abnormal composition.[414] A simple apparatus, especially adapted to milk, is described by Beckmann.[415] The kryoscopic investigation may also be extended to butter fat dissolved in benzol.

=436. Electric Conductivity of Milk.=—The electric conductivity of milk may also be used as an index of its composition. The addition of water to milk diminishes its conductivity.[416] This method of investigation has at present but little practical value.

=437. Viscosity Of Milk.=—The viscosity of milk may be determined by the methods already described. Any variation from the usual degree of fluidity is indicated either by the abstraction of some of the contents of the milk, the addition of some adulterant or the result of fermentation.

=438. Acidity and Alkalinity of Milk.=—Fresh milk of normal constitution has an amphoteric reaction. It will redden blue and blue red litmus paper. This arises from the presence in the milk of both neutral and acid phosphates of the alkalies. A saturated alkaline phosphate, _i. e._, one in which all the acid hydrogen of the acid has been replaced by the base has an alkaline reaction while the acid phosphates react acid. When fresh milk is boiled its reaction becomes strongly alkaline and this arises chiefly from the escape of the dissolved carbon dioxid. By the action of micro-organisms on the lactose of milk, the alkaline reaction soon becomes acid, and delicate test paper will show this decomposition long before it becomes perceptible to the taste. It is advisable to test the reactions of the milk as soon as possible after it is drawn from the udder, both before and after boiling.

=439. Determination of the Acidity of Milk.=—In the determination of the acidity of milk it is important that it first be freed of the carbon dioxid it contains.[417] Van Slyke has found that too high results are obtained by the direct titration of milk for acidity, and when the milk is previously diluted the results are also somewhat too high.[418] Good results are got by diluting the milk with hot water and boiling for a short time to expel the carbon dioxid. Twenty-five cubic centimeters of milk are diluted with water to about a quarter of a liter, as above, two cubic centimeters of a one per cent alcoholic phenolphthalien added and the titration accomplished by decinormal alkali. This variation of the methods of procedure, suggested by Hopkins and Powers, appears to be the best process at present known for the determination of acidity. The reader is referred to the paper cited above for references to other methods which have been proposed.

=440. Opacity Of Milk.=—The white color and opacity of milk are doubtless due to the presence of the suspended fat particles and to the colloid casein. On the latter it is probably principally dependent since the color of milk is not very sensibly changed after it has passed the extractor, which leaves not to exceed one-tenth of one per cent of fat in it. Some idea of the quality of the milk, however, may be obtained by determining its opacity. This is accomplished by the use of a lactoscope. The one generally employed was devised by Feser and is shown in Fig. 107.

The instrument consists of a cylindrical glass vessel of a little more than 100 cubic centimeters content, in the lower part of which is set a cone of white glass marked with black lines. Into this part are placed four cubic centimeters of milk. A small quantity of water is added and the contents of the vessel shaken. This operation is repeated until the black lines on the white glass just become visible. The graduations on the left side show the volume of water which is necessary to bring the dark lines into view, while those on the right indicate approximately the percentage of fat present.

Among the other lactoscopes which have been used may be mentioned those of Donné, Vogel, Hoppe-Seyler, Trommer, Seidlitz, Reischauer, Mittelstrass, Hénocque, and Heusner.[419] Since the invention of so many quick and accurate methods of fat estimation these instruments have little more than a historical interest.

=441. Creamometry.=—The volume of cream which a sample of milk affords under arbitrary conditions of time and temperature is sometimes of value in judging the quality of milk. A convenient creamometer is a small cylinder graduated in such a way that the volume of cream separated in a given time can be easily noted. There are many kinds of apparatus used for this purpose, a typical one being shown in Fig. 107.

The usual time of setting is twenty-four hours. A quicker determination is secured by placing the milk in strong glass graduated tubes and subjecting these to centrifugal action. The process is not exact and is now rarely practiced as an analytical method, even for valuing the butter making properties of milk.

=442. Specific Gravity.=—The specific gravity of milk is uniformly referred to a temperature of 15°. Generally no attempt is made to free the milk of dissolved gases beforehand. This should not be done by boiling but by placing the sample in a vacuum for some time. Any of the methods described for determining specific gravity in sugar solutions may be used for milk (=48-59=). The specific gravity of milk varies in general from 1.028 to 1.034. Nearly all good cow milk from herds will show a specific gravity varying from 1.030 to 1.032. In extreme cases from single cows the limits may exceed those first given above, but such milk cannot be regarded as normal.

Increasing quantities of solids not fat in solution, tend to increase the specific gravity, while an excess of fat tends to diminish it. There is a general ratio existing between the solids not fat and the fat in cow milk, which may be expressed as 9: 4. The removal of cream and the addition of water in such a manner as not to affect the specific gravity of the sample disturbs this ratio.

The determination of the specific gravity alone, therefore, cannot be relied upon as an index of the purity of a milk.

=443.= =Lactometry.=—A hydrometer especially constructed for use in determining the density of milk is called a lactometer. In this country the one most commonly used is known as the lactometer of the New York Board of Health. It is a hydrometer, delicately constructed, with a large cylindrical air space and a small stem carrying the thermometric and lactometric scales. It is shown held in the creamometer in Fig. 107. The milk is brought to a temperature of 60° F. and the reading of the lactometer scale observed. This is converted into a number expressing the specific gravity by means of a table of corresponding values given below. Each mark on the scale of the instrument corresponds to two degrees and these marks extend from 0° to 120°. The numbers of this scale can be converted into those corresponding to the direct reading instrument, described in the next paragraph, by multiplying them by 0.29.

The minimum density for whole milk at 60° F. is fixed by this instrument at 100°, corresponding to a specific gravity of 1.029. The instrument is also constructed without the thermometric scale. The mean density of many thousand samples of pure milk, as observed by the New York authorities, is 1.0319.

The specific gravity is easily secured, and while not of itself decisive, should always be determined. The specific gravity of milk increases for some time after it is drawn and should be made both when fresh and after the lapse of several hours.[420]

TABLE SHOWING SPECIFIC GRAVITIES CORRESPONDING TO DEGREES OF THE NEW YORK BOARD OF HEALTH LACTOMETER. TEMPERATURE 60° F.

Degree. Sp. gr. Degree. Sp. gr.
90 1.02619 106 1.03074
91 1.02639 107 1.03103
92 1.02668 108 1.03132
93 1.02697 109 1.03161
94 1.02726 110 1.03190
95 1.02755 111 1.03219
96 1.02784 112 1.03248
97 1.02813 113 1.03277
98 1.02842 114 1.03306
99 1.02871 115 1.03335
100 1.02900 116 1.03364
101 1.02929 117 1.03393
102 1.02958 118 1.03422
103 1.02987 119 1.03451
104 1.03016 120 1.03480
105 1.03045

=444.= =Direct Reading Lactometer.=—A more convenient form of lactometer is one which gives the specific gravity directly on the scale. The figures given represent those found in the second and third decimal places of the number expressing the specific gravity. Thus 31 on the scale indicates a specific gravity of 1.031. This instrument is also known as the lactometer of Quévenne. For use with milk, the scale of the instrument does not need to embrace a wider limit than from 25 to 35, and such an instrument is capable of giving more delicate readings than when the scale extends from 14 to 42, as is usually the case with the quévenne instrument.

Langlet has invented a lactoscope with a scale, showing the corrections to be applied for temperatures other than 15°. A detailed description of this instrument, as well as the one proposed by Pinchon, is unnecessary.[421]

=445. Density of Sour Milk.=—Coagulated milk cannot be used directly for the determination of the specific gravity, both because of its consistence and by reason of the fact that the fat is more or less completely separated. In such a case, the casein may be dissolved by the addition of a measured quantity of a solvent of a known specific gravity, the density of the resulting solution determined and that of the original milk calculated from the observed data. Ammonia is a suitable solvent for this purpose.[422]

=446. Density of the Milk Serum.=—The specific gravity of the milk serum, after the removal of the fat and casein by precipitation and filtration, may also be determined. For normal cow milk the number is about 1.027.

=447. Total Solids.=—The direct gravimetric determination of the total solids in milk is attended with many difficulties, and has been the theme of a very extended periodical literature. A mere examination of the many processes which have been proposed would require several pages.

The most direct method of procedure is to dry a small quantity of milk in a flat-bottom dish to constant weight on a steam-bath. The surface of the dish should be very large, even for one or two grams of milk; in fact the relation between the quantity of milk and the surface of the dish should be such that the fluid is just sufficient in amount to moisten the bottom of the dish with the thinnest possible film. The dish, during drying, is kept in a horizontal position at least until its contents will not flow. The water of the sample will be practically all evaporated in about two hours. The operation may be accelerated by drying in vacuo.

The drying may also be accomplished by using a flat-bottom dish containing some absorbent, such as sand, pumice stone, asbestos or crysolite. The milk may also be absorbed by a dried paper coil and dried thereon (=26=).

It is convenient to determine the water in the sample subsequently to be used for the gravimetric determination of the fat, and this is secured by the adoption of the paper coil method, as suggested by the author, or by the use of a perforated metal tube containing porous asbestos, as proposed by Babcock.[423]

The process is conveniently carried out as follows:

Provide a hollow cylinder of perforated sheet metal sixty millimeters long and twenty millimeters in diameter, closed five millimeters from one end by a disk of the same material. The perforations should be about 0.7 millimeter in diameter and as close together as possible. Fill loosely with from one and a half to two and a half grams of dry woolly asbestos and weigh. Introduce a weighed quantity of milk (about five grams). Dry at 100° for four hours. During the first part of the drying the door of the oven should be left partly open to allow escape of moisture. Cool in a desiccator and weigh. Repeat the drying until the weight remains constant. Place in an extractor and treat with anhydrous ether for two hours. Evaporate the ether and dry the fat at 100°. The extracted fat is weighed and the number thus obtained may be checked by drying and weighing the cylinder containing the residue.

The asbestos best suited for use in this process should be of a woolly nature, quite absorbent, and, previous to use, be ignited to free it of moisture and organic matter. A variety of serpentine, crysolite is sometimes used instead of asbestos. When the content of water alone is desired, it is accurately determined by drying in vacuo over pumice stone (page 33).

The methods above mentioned are typical and will prove a sufficient guide for conducting the desiccation, either as described or by any modification of the methods which may be preferred.

=448. Calculation of Total Solids.=—By reason of the ease and celerity with which the density of a milk and its content of fat can be obtained, analysts have found it convenient to calculate the percentage of total solids instead of determining it directly. This is accomplished by arbitrary formulas based on the data of numerous analyses. These formulas give satisfactory results when the samples do not vary widely from the normal and may be used with advantage in most cases.

Among the earliest formulas for the calculation may be mentioned those of Fleischmann and Morgen,[424] Behrend and Morgen,[425] Claus, Stutzer and Meyer,[426] Hehner,[427] and Hehner and Richmond.[428] Without doing more than citing these papers it will be sufficient here to give the formulas as corrected by the most recent experience.

In the formula worked out by Babcock the specific gravity of the sample is represented by _S_, the fat by _F_, and the solids not fat by _t_. The formula is written as follows:[429]

100_S_ - _FS_
_t_ = ( ----------------- - 1)(250 - 2.5 _F_).
100 - 1.0753_FS_

In this formula it is assumed that the difference between the specific gravity of the milk serum and that of water is directly proportional to the per cent of solids in the serum, but this assumption is not strictly correct. Even in extreme cases, however, the error does not amount to more than 0.05 per cent.