Part Sixth

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.

Since a given amount of milk sugar increases the density of a milk more than the same quantity of casein, it is evident that the formula would not apply to those instances in which the ratio between these two ingredients is greatly disturbed, as for instance, the whey.

The formula of Hehner and Richmond, in its latest form, is expressed as follows: G T = 0.2625 ---- + 1.2_F_, D

in which T represents the total solids, G the reading of the quévenne lactometer, D the specific gravity, and F the fat.

Example.—Let the reading of the lactometer be 31, corresponding to D 1.031, and the percentage of fat be three and five-tenths, what is the percentage of the total solids?

Substituting these values in the formulas we have

31 T = 0.2625 ----- + 1.2 × 3.5 = 12.09. 1.031

To simplify the calculations, Richmond’s formula may be written

G 6_F_ T = ----- + ----- + 0.14. 4 5

Calculated by this shortened formula from the above data T = 12.09, the same as given in the larger formula.

Calculating the solids not fat in the hypothetical case given above by Babcock’s formula, we get t = 8.46, and this plus 3.5 gives 11.96, which is slightly lower than the number obtained by the richmond process.

The babcock formula may be simplified by substituting the number expressing the reading of the quévenne lactometer for that donating the specific gravity, in other words, the specific gravity multiplied by 100 and the quotient diminished by 1000.

The formulas for solids not fat and total solids then become

L L t = ---- + 0.2_F_, and T = ----- + 1.2_F_, 4 4

in which L represents the reading of the lactometer. By the addition of the constant factor 0.14 the results calculated by the formula of Babcock are the same as those obtained by the method of Richmond.

In the following table are given the solids not fat in milks as calculated by Babcock’s formula. To obtain the total solids add the per cent of fat to solids not fat. To obtain total solids according to Richmond’s formula increase that number by 0.14.

TABLE SHOWING PER CENT OF SOLIDS NOT FAT IN MILK CORRESPONDING TO QUÉVENNE’S LACTOMETER READINGS AND PER CENT OF FAT.

Per cent Lactometer reading at 60° F. of fat. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36.

0.0 6.50 6.75 7.00 7.25 7.50 7.75 8.00 8.25 8.50 8.75 9.00 0.1 6.52 6.77 7.02 7.27 7.52 7.77 8.02 8.27 8.52 8.77 9.02 0.2 6.54 6.79 7.04 7.29 7.54 7.79 8.04 8.29 8.54 8.79 9.04 0.3 6.56 6.81 7.06 7.31 7.56 7.81 8.06 8.31 8.56 8.81 9.06 0.4 6.58 6.83 7.08 7.33 7.58 7.83 8.08 8.33 8.58 8.83 9.08 0.5 6.60 6.85 7.10 7.35 7.60 7.85 8.10 8.35 8.60 8.85 9.10 0.6 6.62 6.87 7.12 7.37 7.62 7.87 8.12 8.37 8.62 8.87 9.12 0.7 6.64 6.89 7.14 7.39 7.64 7.89 8.14 8.39 8.64 8.89 9.14 0.8 6.66 6.91 7.16 7.41 7.66 7.91 8.16 8.41 8.66 8.91 9.16 0.9 6.68 6.93 7.18 7.43 7.68 7.93 8.18 8.43 8.68 8.93 9.18 1.0 6.70 6.95 7.20 7.45 7.70 7.95 8.20 8.45 8.70 8.95 9.20 1.1 6.72 6.97 7.22 7.47 7.72 7.97 8.22 8.47 8.72 8.97 9.22 1.2 6.74 6.99 7.24 7.49 7.74 7.99 8.24 8.49 8.74 8.99 9.24 1.3 6.76 7.01 7.26 7.51 7.76 8.01 8.26 8.51 8.76 9.01 9.26 1.4 6.78 7.03 7.28 7.53 7.78 8.03 8.28 8.53 8.78 9.03 9.28 1.5 6.80 7.05 7.30 7.55 7.80 8.05 8.30 8.55 8.80 9.05 9.30 1.6 6.82 7.07 7.32 7.57 7.82 8.07 8.32 8.57 8.82 9.07 9.32 1.7 6.84 7.09 7.34 7.59 7.84 8.09 8.34 8.59 8.84 9.09 9.34 1.8 6.86 7.11 7.36 7.61 7.86 8.11 8.36 8.61 8.86 9.11 9.37 1.9 6.88 7.13 7.38 7.63 7.88 8.13 8.38 8.63 8.88 9.14 9.39 2.0 6.90 7.15 7.40 7.65 7.90 8.15 8.40 8.66 8.91 9.16 9.41 2.1 6.92 7.17 7.42 7.67 7.92 8.17 8.42 8.68 8.93 9.18 9.43 2.2 6.94 7.19 7.44 7.69 7.94 8.19 8.44 8.70 8.95 9.20 9.45 2.3 6.96 7.21 7.46 7.71 7.96 8.21 8.46 8.72 8.97 9.22 9.47 2.4 6.98 7.23 7.48 7.73 7.98 8.23 8.48 8.74 8.99 9.24 9.49 2.5 7.00 7.25 7.50 7.75 8.00 8.25 8.50 8.76 9.01 9.26 9.51 2.6 7.02 7.27 7.52 7.77 8.02 8.27 8.52 8.78 9.03 9.28 9.53 2.7 7.04 7.29 7.54 7.79 8.04 8.29 8.54 8.80 9.05 9.30 9.55 2.8 7.06 7.31 7.56 7.81 8.06 8.31 8.57 8.82 9.07 9.32 9.57 2.9 7.08 7.33 7.58 7.83 8.08 8.33 8.59 8.84 9.09 9.34 9.59 3.0 7.10 7.35 7.60 7.85 8.10 8.36 8.61 8.86 9.11 9.36 9.61 3.1 7.12 7.37 7.62 7.87 8.13 8.38 8.63 8.88 9.13 9.38 9.64 3.2 7.14 7.39 7.64 7.89 8.15 8.40 8.65 8.90 9.15 9.41 9.66 3.3 7.16 7.41 7.66 7.92 8.17 8.42 8.67 8.92 9.18 9.43 9.68 3.4 7.18 7.43 7.69 7.94 8.19 8.44 8.69 8.94 9.20 9.45 9.70 3.5 7.20 7.45 7.71 7.96 8.21 8.46 8.71 8.96 9.22 9.47 9.72 3.6 7.22 7.48 7.73 7.98 8.23 8.48 8.73 8.98 9.24 9.49 9.74 3.7 7.24 7.50 7.75 8.00 8.25 8.50 8.75 9.00 9.26 9.51 9.76 3.8 7.26 7.52 7.77 8.02 8.27 8.52 8.77 9.02 9.28 9.53 9.78 3.9 7.28 7.54 7.79 8.04 8.29 8.54 8.79 9.04 9.30 9.55 9.80 4.0 7.30 7.56 7.81 8.06 8.31 8.56 8.81 9.06 9.32 9.57 9.83 4.1 7.32 7.58 7.83 8.08 8.33 8.58 8.83 9.08 9.34 9.59 9.85 4.2 7.34 7.60 7.85 8.10 8.35 8.60 8.85 9.11 9.36 9.62 9.87 4.3 7.36 7.62 7.87 8.12 8.37 8.62 8.88 9.13 9.38 9.64 9.89 4.4 7.38 7.64 7.89 8.14 8.39 8.64 8.90 9.15 9.40 9.66 9.91 4.5 7.40 7.66 7.91 8.16 8.41 8.66 8.92 9.17 9.42 9.68 9.93 4.6 7.43 7.68 7.93 8.18 8.43 8.68 8.94 9.19 9.44 9.70 9.95 4.7 7.45 7.70 7.95 8.20 8.45 8.70 8.96 9.21 9.46 9.72 9.97 4.8 7.47 7.72 7.97 8.22 8.47 8.72 8.98 9.23 9.48 9.74 9.99 4.9 7.49 7.74 7.99 8.24 8.49 8.74 9.00 9.25 9.50 9.76 10.01 5.0 7.51 7.76 8.01 8.26 8.51 8.76 9.02 9.27 9.52 9.78 10.03 5.1 7.53 7.78 8.03 8.28 8.53 8.79 9.04 9.29 9.54 9.80 10.05 5.2 7.55 7.80 8.05 8.30 8.55 8.81 9.06 9.31 9.56 9.82 10.07 5.3 7.57 7.82 8.07 8.32 8.57 8.83 9.08 9.33 9.58 9.84 10.09 5.4 7.59 7.84 8.09 8.34 8.60 8.85 9.10 9.36 9.61 9.86 10.11 5.5 7.61 7.86 8.11 8.36 8.62 8.87 9.12 9.38 9.63 9.88 10.13 5.6 7.63 7.88 8.13 8.39 8.64 8.89 9.15 9.40 9.65 9.90 10.15 5.7 7.65 7.90 8.15 8.41 8.66 8.91 9.17 9.42 9.67 9.92 10.17 5.8 7.67 7.92 8.17 8.43 8.68 8.94 9.19 9.44 9.69 9.94 10.19 5.9 7.69 7.94 8.20 8.45 8.70 8.96 9.21 9.46 9.71 9.96 10.22 6.0 7.71 7.96 8.22 8.47 8.72 8.98 9.23 9.48 9.73 9.98 10.24

=449. Determination of Ash.=—In the determination of the solid residue obtained by drying milk, it is important to observe the directions already given (=28-32=).

In the direct ignition of the sample, a portion of the sulfur and phosphorus may escape oxidation and be lost as volatile compounds. This loss may be avoided by the use of proper oxidizing agents or by conducting the combustion as heretofore described.[430] In the official method, it is directed to add six cubic centimeters of nitric acid to twenty of milk, evaporate to dryness and ignite the residue at a low red heat until free of carbon.[431] It is doubtful if this precaution be entirely sufficient to save all the sulfur and phosphorus, but the method is evidently more reliable than the common one of direct ignition without any oxidizing reagent whatever.

ESTIMATION OF FAT.

=450. Form of Fat in Milk.=—The fat in milk occurs in the form of globules suspended in the liquid, in other words in the form of an emulsion. Many authorities have asserted that each globule of fat is contained in a haptogenic membrane composed presumably of nitrogenous matter, but there is no convincing evidence of the truth of this opinion. The weight of experimental evidence is in the opposite direction. The supposed action of the membrane and the phenomena produced thereby are more easily explained by the surface tension existing between the fat globules and the menstruum in which they are suspended.

Babcock affirms that the spontaneous coagulation of the fibrin present in milk tends to draw the fat globules into clusters, and this tendency can be arrested by adding a little soda or potash lye to the milk as soon as it is drawn.[432]

The diameter of the fat globules is extremely variable, extending in some cases from two to twenty micromillimeters. In cow milk, the usual diameters are from three to five micromillimeters.

=451. Number of Fat Globules in Milk.=—The number of fat globules in milk depends on their size and the percentage of fat. It is evident that no definite statement of the number can be made. There is a tendency, on the part of the globules, to diminish in size and increase in number as the period of lactation is prolonged. To avoid large numbers, it is convenient to give the number of globules in 0.0001 cubic millimeter. This number may be found within wide limits depending on the individual, race, food and other local conditions to which the animal or herd is subjected. In general, in whole milk this number will be found between 140 and 250.

=452. Method of Counting Globules.=—The number of globules in milk is computed with the aid of the microscope. The most convenient method is the one devised by Babcock.[433] In carrying out this computation, capillary tubes, from two to three centimeters long and about one-tenth millimeter in internal diameter, are provided. The exact diameter of each tube, in at least three points, is determined by the micrometer attachment of the microscope, and from these measurements the mean diameter of the tube is calculated. This known, its cubic content for any given length is easily computed. Ten cubic centimeters of the milk are diluted with distilled water to half a liter and one end of a capillary tube dipped therein. The tube is quickly filled with diluted milk and each end is closed with a little wax to prevent evaporation. Several of these tubes being thus prepared, they are placed in a horizontal position on the stage of the microscope and covered with glycerol and a cover glass. The tubes are left at rest for some time until all the fat globules have attached themselves to the upper surfaces, in which position they are easily counted. The micrometer is so placed as to lie parallel with the tubes, and the number of globules, corresponding to each division of its scale, counted. The mean number of globules corresponding to each division of the micrometer scale is thus determined.

To compare the data obtained with each tube they are reduced to a common basis of the number of globules found in a length of fifty divisions of the micrometer scale in a tube having a diameter of 100 divisions, using the formula

10000_n_ N = ---------, _d_²

in which n = the number of globules found in the standard length of tube measured and d = the diameter of the tube. It is not difficult to actually count all the globules in a length of fifty divisions of the scale, but the computation may also be made from the mean numbers found in a few divisions. The usual number of globules found in a length of 0.1 millimeter in a tube 0.1 millimeter in diameter, varies from fifty to one hundred.

Example.—The length of one division of the micrometer scale is 0.002 millimeter, and the internal diameter of the tube 0.1 millimeter. The content of a tube, of a length of 0.002 × 50 = 0.1 millimeter, is therefore 0.0007854 cubic millimeter.

The cubic content of a tube 100 scale divisions in diameter and fifty in length is 0.0031416 cubic millimeter. The number of globules found in fifty divisions of the tubes used is 40. Then the number which would be contained in a tube of a diameter of 100 divisions of the micrometer scale and a length of fifty divisions thereof is

10000_n_ 400000 N = -------- = ------ = 160. _d_² 2500

Since the milk is diluted fifty times, the actual number of globules corresponding to the volume given is 8000. It is convenient to reduce the observations to some definite volume, exempla gratia, 0.0001 cubic centimeter. The equation for this in the above instance is 0.0031416: 0.0001 = 8000: x, whence x = 223, = number of fat globules in 0.0001 cubic millimeter.

In one cubic millimeter of milk there are therefore 2,230,000 fat globules, and in one cubic centimeter 2,230,000,000 globules. In a single drop of milk there are from one to two hundred million fat globules.[434]

=453. Classification of Methods of Determining Fat in Milk.=—The fat, being the most valuable of the constituents of milk, is the subject of a number of analytical processes. An effort will be made here to classify these various methods and to illustrate each class with one or more typical processes. In general the methods may be divided into analytical and commercial, those of the first class being used for scientific and of the other for trade purposes. For normal milk, some of the trade methods have proved to be quite as accurate as the more chronokleptic analytical processes to which, in disputed cases, a final appeal must be taken. When the analyst is called upon to determine the fat in a large number of samples of milk some one of the trade methods may often be adopted with great advantage.

=454. Dry Extraction Methods.=—Among the oldest and most reliable methods of determining fat in milk, are included those processes based on the principle of drying the milk and extracting the fat from the residue by an appropriate solvent. The solvents generally employed are ether and petroleum spirit of low boiling point. The methods of drying are legion.

In extracting with ether, it must not be forgotten that other bodies than fat may pass into solution on the one hand and on the other any substituted glycerid, such as lecithin or nuclein, which may be present may escape solution, at least in part. Perhaps petroleum spirit, boiling at from 45° to 60°, is the best solvent for fat, but it is almost the universal custom in this and other countries to use ether.

=455. The Official Methods.=—In the methods adopted by the Association of Official Agricultural Chemists two processes are recommended.

(1) The Asbestos Process: In this process it is directed to extract the residue from the determination of water by the asbestos method (=447=) with anhydrous pure ether until the fat is removed, evaporate the ether, dry the fat at 100° and weigh. The fat may also be determined by difference, after drying the extracted cylinders at 100°.

(2) Paper Coil Method: This is essentially the method proposed by Adams as modified by the author.[435] Coils made of thick filter paper are cut into strips 6.25 by 62.5 centimeters, thoroughly extracted with ether and alcohol, or the weight of the extract corrected by a constant obtained for the paper. If this latter method be used, a small amount of anhydrous sodium carbonate should be added. Paper free of matters soluble in ether is also to be had for this purpose. From a weighing bottle about five grams of milk are transferred to the coil by a pipette, taking care to keep dry the lower end of the coil. The coil, dry end down, is placed on a piece of glass, and dried at a temperature of boiling water for one hour, or better, dried in hydrogen at a temperature of boiling water, transferred to an extraction apparatus and extracted with absolute ether or petroleum spirit boiling at about 45°. The extracted fat is dried in hydrogen and weighed. Experience has shown that drying in hydrogen is not necessary. The fat may be conveniently dried in partial vacuo.

=456. Variations of Extraction Method.=—The method of preparing the milk for fat extraction is capable of many variations. Some of the most important follow:

(1) Evaporation on Sand: The sand should be pure, dry and of uniform size of grain. It may be held in a dish or tube. The dish may be made of tin foil, so that it can be introduced with its contents into the extraction apparatus after the desiccation is complete. For this purpose, it is cut into fragments of convenient size after its contents have been poured into the extractor. The scissors used are washed with the solvent.

(2) Evaporation on Kieselguhr: Dry kieselguhr (infusorial earth, tripoli) may take the place of the sand as above noted. The manipulation is the same as with sand.

(3) Evaporation on Plaster of Paris,[436] (Soxhlet Method),[437] (4) On Pumice Stone, (5) On Powdered Glass, (6) On Chrysolite:[438] The manipulation in these cases is conducted as with sand and no detailed description is required.

(7) Evaporation on Organic Substances: These variations would fall under the general heading of drying on paper. The following materials have been used; viz., sponge,[439] lint,[440] and wood pulp.[441] In these variations the principal precautions to be observed are to secure the organic material in a dry state and free of any matter soluble in the solvent used.

(8) Dehydration with Anhydrous Copper Sulfate: In this process the water of the sample is absorbed by powdered anhydrous copper sulfate, the residual mass extracted and the butter fat obtained determined by saponification and titration.[442] In the manipulation about twenty grams of the anhydrous copper sulfate are placed in a mortar, a depression made therein in such a manner that ten cubic centimeters of milk can be poured into it without wetting it through to the mortar. The water is soon absorbed when the mass is ground with a little dry sand and transferred to the extractor.

Petroleum spirit of low boiling point is used as a solvent, successive portions of about fifteen cubic centimeters each being forced through the powdered mass under pressure. Two or three treatments with the petroleum are required. The residual butter fat, after the evaporation of the petroleum, is saponified with a measured portion, about twenty-five cubic centimeters, of seminormal alcoholic potash lye. The residual alkali is determined by titration with seminormal hydrochloric acid in the usual manner. From the data obtained is calculated the quantity of alkali employed in the saponification. The weight of butter fat extracted is then calculated on the assumption that 230 milligrams of potash are required to saponify one gram of the fat.

=457. Gypsum Method for Sour Milk.=—In sour milk, extraction of the dry residue with ether is attended with danger of securing a part of the free lactic acid in the extract. This may be avoided, at least in part, by making the milk neutral or slightly alkaline before desiccation. This method is illustrated by a variation of Soxhlet’s method of drying on gypsum proposed by Kühn.[443] The curdled milk is treated with potash lye of forty per cent strength until the reaction is slightly alkaline. For absorbing the sample before drying, a mixture is employed consisting of twenty-five grams of plaster of paris, four of precipitated carbonate of lime and two of acid potassium sulfate. To this mixture ten grams of the milk, rendered alkaline as above noted, are added in a desiccating dish, the excess of moisture evaporated at 100°, the residual mass finely ground and extracted with ether for four hours. A little gypsum may be found in the solution, but in such small quantities as not to interfere seriously with the accuracy of the results obtained.

=458. Estimation of Fat in Altered Milk.=—In altered milk the lactose has usually undergone a fermentation affording considerable quantities of lactic acid. If such milks be treated by the extraction method for fat, the results will always be too high, because of the solubility of lactic acid in ether.

Vizern[444] has proposed to avoid this error by first warming the soured milk for a few minutes to 40°, at which temperature the clabber is easily divided by vigorous shaking. Of the milk thus prepared, thirty grams are diluted with two or three volumes of water and poured onto a smooth and moistened filter. The vessel and filter are washed several times until the filtrate presents no further acid reaction. The filter and its contents are next placed in a vessel containing some fine washed sand. A small quantity of water is added, sufficient to form a paste. With a stirring rod, the filter is entirely broken up and the whole mass thoroughly mixed. Dried on the water bath the material is subjected to extraction in the ordinary way. Several analyses made on fresh milk and on milk kept for several months show that almost identical results are obtained.

In respect of this process there would be danger, on long standing, of the formation of free acids from butter glycerids, and these acids would be removed by the process of washing prescribed. In this case the quantity of fat obtained would be less than in the original sample.

=459. Comparison of Methods.=—An immense amount of work has been done by analysts in comparing the various types of extraction methods outlined above.[445]

The consensus of opinion is that good results are obtained by all the methods when properly conducted, and preference is given to the two methods finally adopted by the Association of Official Chemists. As solvents, pure ether and petroleum spirit of low boiling point are preferred. The direct extraction gravimetric processes are important, since it is to these that all the other quicker and easier methods must appeal for the proof of their accuracy.

=460. Wet Extraction Methods.=—It has been found quite impracticable to extract the fat from milk by shaking it directly with the solvent. An emulsion is produced whereby the solvent itself becomes incorporated with the other constituents of the milk, and from which it is not separated easily even with the aid of whirling. The disturbing element which prevents the separation of the solvent is doubtless the colloid casein, since, when this is previously rendered soluble, the separation of the solvent holding the fat is easily accomplished.

The principle on which the methods of wet extraction are based is a simple one; viz., to secure a complete or partial solution of the casein and subsequently to extract the fat with a solvent immiscible with water. The methods may be divided into three great classes; viz., (1) those in which the solvent is evaporated from the whole of the extracted fat and the residual matters weighed; (2) processes in which an aliquot part of the fat solution is employed and the total fat calculated from the data secured; (3) the density of the fat solution is determined at a definite temperature and the percentage of fat corresponding thereto determined from tables or otherwise. Methods (1) and (2) are practically identical in principle and one or the other may be applied according to convenience or to local considerations. The methods may be further subdivided in respect of the reagents used to secure complete or partial solution of the casein, as, for instance, alkali or acid.[446]

=461. Solution in an Acid.=—A good type of these processes is the method of Schmid.[447] In this process ten cubic centimeters of milk are placed in a test tube of about five times that content, graduated to measure small volumes. An equal quantity of hydrochloric acid is added, the mixture shaken, boiled until it turns dark brown, and cooled quickly. The fat is extracted by shaking with thirty cubic centimeters of ether. After standing some time the ethereal solution separates and its volume is noted. An aliquot part of the solution is removed, the solvent evaporated, and the weight of fat in the whole determined by calculation.

The schmid process has been improved by Stokes,[448] Hill,[449] and Richmond.[450] The most important of these variations consists in weighing instead of measuring the milk employed, thus insuring greater accuracy. Dyer and Roberts affirm that the ether dissolves some of the caramel products formed on boiling condensed milk with hydrochloric acid, and that the data obtained in such cases by the process of Schmid are too high.[451]

Since lactic acid is also slightly soluble in ether, sour milk should not be extracted with that solvent. In these cases petroleum spirit, or a mixture of petroleum and ether, as suggested by Pinette, may be used.[452] Another variation consists in extracting the fat with several portions of the solvent and evaporating all the extracts thus obtained to get the total fat. This method is perhaps the best of those in which the fat is extracted from the residual liquid after the decomposition of the casein by an acid, and may be recommended as both reliable and typical within the limitations mentioned above.

=462. Solution in an Alkali.=—The casein of milk is not so readily dissolved in an alkali as in an acid, but the solution is sufficient to permit the extraction of the fat. Soda and potash lyes and ammonia are the alkaline bodies usually employed. To promote the separation of the emulsions, alcohol is added with advantage. The principle of the process rests on the observed power of an alkali to free the fat globules sufficiently to allow them to dissolve in ether or some other solvent. When the solvent has separated from the emulsion at first formed, the whole or a part of it is used for the determination of fat in a manner entirely analogous to that employed in the process with the acid solutions described above. There are many methods based on this principle, and some of the typical ones will be given below. Experience has shown that extraction from an alkaline solution is more troublesome and less perfect than from an acid and these alkaline methods are, therefore, not so much practiced now as they were formerly.

=463. Method of Short.=—Instead of measuring the volume of the separated fat, Short has proposed a method in which the casein is dissolved in an alkali and the fat at the same time saponified. The soap thus produced is decomposed by sulfuric acid and the volume of the separated fat acids noted. This volume represents eighty-seven per cent of the corresponding volume of fat.[453]

The solvent employed is a mixture of sodium and potassium hydroxids, containing in one liter 125 and 150 grams, respectively, of these alkalies. The sample of milk is mixed with half its volume of the reagent and placed in boiling water for two hours. By this treatment the casein is dissolved and the fat saponified. After cooling to about 60°, the soap is decomposed by the addition of equal parts of sulfuric and acetic acids. The tubes containing the mixture are again placed in boiling water for an hour and they are then filled with boiling water to within one inch of the top. The tubes may either be furnished with a graduation or the column of fat be measured by a scale.

=464. Method of Thörner.=—The process of Short is conducted by Thörner as follows:[454]

Ten cubic centimeters of milk measured at 15° are saponified, in tubes fitting a centrifugal, by the addition of one and a half cubic centimeters of an alcoholic potash lye, containing 160 grams of potassium hydroxid per liter, or one cubic centimeter of an aqueous fifty per cent soda lye. The saponification is hastened by setting the tubes in boiling water, where they remain for two minutes. The soap formed is decomposed with a strong acid, sulfuric preferred, the tubes placed in the centrifugal and whirled for four minutes, when the fat acids will be formed in the narrow graduated part of the tube and the volume occupied thereby is noted after immersion in boiling water. Thörner’s process is not followed in this country, but is used to a considerable extent in Germany.[455]

=465. Liebermann’s Method.=—In this method, fifty cubic centimeters of milk, at ordinary temperatures, are placed in a glass cylinder twenty-five centimeters high and about four and a half internal diameter; five cubic centimeters of potash lye of 1.27 specific gravity are added, the cylinder closed with a well fitting cork stopper and thoroughly shaken.[456] After shaking, fifty cubic centimeters of petroleum spirit, boiling point about 60°, are added. The cylinder is again stoppered and vigorously shaken until an emulsion is formed. To this emulsion fifty cubic centimeters of alcohol of ninety-five per cent strength are added, and the whole again thoroughly shaken. After four or five minutes the petroleum spirit, containing the fat, separates. In order to insure an absolute separation of the fat, however, the shaking may be repeated three or four times for about one-quarter minute, waiting each time between the shakings until the spirit separates.

Of the separated petroleum spirit twenty cubic centimeters are placed in a small weighed flask. The use of the flask is recommended on account of the ease with which the petroleum spirit can be evaporated without danger of loss of fat. Instead of the flask a weighed beaker or other weighed dish may be employed.

The petroleum spirit is carefully evaporated on a water-bath and the residue dried at 110° to 120° for one hour. The weight found multiplied by five gives the content of fat in 100 cubic centimeters of the milk. The percentage by weight can then be calculated by taking into consideration the specific gravity of the milk employed.

The results obtained by this method agree well with those obtained by the paper coil method, when petroleum spirit instead of sulfuric ether is used as the solvent for the fat. Sulfuric ether, however, gives an apparently higher content of fat because of the solution of other bodies not fat present in the milk.

=466. Densimetric Methods.=—Instead of evaporating the separated fat solution and weighing the residue, its density may be determined and the percentage of fat dissolved therein obtained by calculation, or more conveniently from tables. The typical method of this kind is due to Soxhlet, and until the introduction of modern rapid volumetric processes, it was used perhaps more extensively than any other proceeding for the determination of fat in milk.[457] The reagents employed in the process are ether saturated with water and a potash lye containing 400 grams of potash in a liter. The principle of the process is based on the assumption that a milk made alkaline with potash will give up all its fat when shaken with ether and the quantity of fat in solution can be determined by ascertaining the specific gravity of the ethereal solution.

The apparatus is arranged as shown in Fig. 108, whereby it is easy to drive the ethereal fat solution into the measuring vessel by means of the bellows shown. In the bottle, seen at the right of the engraving are placed 200 cubic centimeters of milk, ten of the potash lye and sixty of the aqueous ether. The milk and potash are first added and well shaken, the ether then added, and the contents of the bottle are shaken until a homogeneous emulsion is formed. The bottle is then set aside for the separation of the ethereal solution, which is promoted by gently jarring it from time to time. When the chief part of the solution has separated, a sufficient quantity of it is driven over into the measuring apparatus, by means of the air bulbs, to float the hydrometer contained in the inner cylinder. After a few moments the scale of the oleometer is read and the percentage of fat calculated from the table. All the measurements are made at a temperature of 17°.5. The temperature is preserved constant by filling the outer cylinder of the apparatus with water. If the room be warmer than 17°.5, the water added should be at a temperature slightly below that and vice versa. The oleometer carries a thermometer which indicates the moment when the reading is to be made.

The scale of the oleometer is graduated arbitrarily from 43 to 66, corresponding to the specific gravities 0.743 and 0.766, respectively, or to corresponding fat contents of 2.07 and 5.12 per cent, in the milk, a range which covers most normal milks.

In the use of the table the per cents corresponding to parts of an oleometer division can be easily calculated.

TABLE FOR CALCULATING PER CENT OF FAT IN MILK BY AREOMETRIC METHOD OF SOXHLET.

Reading of Per cent fat Reading of Per cent fat oleometer. in milk. oleometer. in milk.

43 2.07 55 3.49 44 2.18 56 3.63 45 2.30 57 3.75 46 2.40 58 3.90 47 2.52 59 4.03 48 2.64 60 4.18 49 2.76 61 4.32 50 2.88 62 4.47 51 3.00 63 4.63 52 3.12 64 4.79 53 3.25 65 4.95 54 3.37 66 5.12

=467. Application of the Areometric Method.=—Soxhlet’s method, as outlined above, with many modifications, has been extensively used in Europe and to a limited degree in this country, and the results obtained are in general satisfactory, when the sample is a mixed one from a large number of cows and of average composition.

The author has shown that the process is not applicable to abnormal milk and often not to milk derived from one animal alone.[458]

The chief difficulty is found in securing a separation of the emulsion. This trouble can usually be readily overcome by whirling. Any centrifugal machine, which can receive the bottle in which the emulsion is made, may be employed for that purpose.

Since the introduction of more modern and convenient methods of fat determination, the areometric method has fallen into disuse and perhaps is no longer practiced in this country. It is valuable now chiefly from the fact that many of the recorded analyses of milk fat were made by it, and also for its typical character in representing all methods of analysis of fat in milk based on the density of ethereal solutions.

=468. The Lactobutyrometer.=—A typical instrument for measuring the volume of fat in a milk is known as Marchand’s lactobutyrometer. It is based on the observation that ether will dissolve the fat from milk when the casein is wholly or partly dissolved by an alkali, and further, that the fat in an impure form can be separated from its ethereal solution by the action of alcohol. Experience has shown that all the fat is not separated from the ethereal solution by this process, and also that the part separated is a saturated solution in ether. The method cannot be rigorously placed in the two classes given above, but being volumetric demands consideration here chiefly because of its historical interest.[459]

The instrument employed by Marchand is a tube about thirty centimeters long and twelve in diameter, closed at one end and marked in three portions of ten cubic centimeters each. The upper part is divided in tenths of a cubic centimeter. The superior divisions are subdivided so that the readings can be made to hundredths of a cubic centimeter.

The tube is filled with milk to the first mark and two or three drops of a twenty-five per cent solution of soda lye added thereto. Ether is poured in to the second mark, the tube closed and vigorously shaken. Alcohol of about ninety per cent strength is added to the upper mark, the tube closed, shaken and allowed to stand in a vertical position, with occasional jolting, until the separation of the liquids is complete. In order to promote the separation the tube is placed in a cylinder containing water at 40°.

When the separation is complete the milk serum is found at the bottom, the mixture of alcohol and ether in the middle and the fat at the top. The mixture of ether and alcohol contains 0.126 gram of fat, and each cubic centimeter of the separated ether fat 0.233 gram of fat. The total volume of the separated fat, multiplied by 0.233, and the product increased by 0.126, will give the weight of fat in the ten cubic centimeters of milk employed.

Example.—Milk used, ten cubic centimeters of 1.032 specific gravity = 10.32 grams. The observed volume of the saturated ether fat solution is two cubic centimeters. Then the weight of fat is 2 × 0.233 + 0.126 = 0.592 gram. The percentage of fat in the sample is 0.592 × 100 ÷ 10.32 = 5.74.

In the apparatus used in this laboratory the upper division of the graduation is marked 12.6, because this represents the quantity of fat which remains in the ether-alcohol mixture for one liter of milk. From this point the graduation is extended downward to ninety-five, which, for ten cubic centimeters of milk, represents 0.95 gram. After the fat has separated, enough ninety-five per cent alcohol is added to bring the upper surface exactly to the graduation 12.6. The number of grams per liter of milk is then read directly from the scale.

In respect of applicability, the observation made regarding Soxhlet’s areometric method may be repeated.

In practical work in this country the lactobutyrometer is no longer used, but many of the recorded determinations of fat in milk have been made by this method.

=469. Volumetric Methods.=—For practical purposes, the volumetric methods of estimating fat in milk have entirely superseded all the other processes. It has been found that the fat readily separates in a pure state from the other constituents of milk whenever the casein is rendered completely soluble; whereas no process has yet been devised whereby the fat can be easily separated in a pure state from milk which has not been treated with some reagent capable of effecting a solution of the casein. The volumetric methods may be divided into two classes; viz., (1) Those in which the fat is separated by the simple action of gravity, and (2) those in which the natural action of gravity is supplanted by centrifugal motion. Each of these classes embraces a large number of variations and some of the typical ones will be described in the following paragraphs. As solvents for the casein a large number of reagents has been used, including alkalies and single and mixed acids. In practice, preference is given to the least complex and most easily prepared solvents.

=470. Method Of Patrick.=—A typical illustration of the method of collecting the fat after solution of the casein, without the aid of whirling, is found in the process devised by Patrick.[460]

The solvent employed is a mixture of acetic, sulfuric and hydrochloric acids, saturated with sodium sulfate, in the respective volumetric proportions of nine, five and two. The separation is accomplished in a large test tube drawn out near the top into a constricted neck which is graduated to measure the volume of the separated fat or to give direct percentage results.

The tube should have a content of about twenty-five cubic centimeters below the upper mark on the neck. In use 10.4 cubic centimeters of milk and a sufficient quantity of the mixed acids to fill it nearly to the upper mark are placed in the tube, together with a piece of pumice stone, and the mixture boiled. On cooling below 100°, the fat will separate and the volume thereof may be measured in the constricted portion of the tube. The volume of the fat may be converted into weight on multiplying by 0.88 at 60°, or more conveniently the percentage of fat be taken from a table. In practice, the tube is filled with the milk and acid mixture nearly up to the neck, its contents well mixed and additional acid mixture added until the liquid is raised in the tube above the neck. After mixing a second time, the contents are boiled for five minutes and the fat allowed to collect in the expanded part of the tube above the neck. When the fat has collected, the mixture is boiled gently a second time for a few minutes. By this treatment the fat is mixed with the upper portions of the acid liquid and clarified. The clearing of the fat may be hastened by sprinkling over it a little effloresced sodium sulfate. The fat is brought into the graduated neck by opening a small orifice in the belly of the tube, which is closed by means of a rubber band. When the temperature has reached 60°, the space occupied by the fat is noted and the numbers obtained express the percentage of fat in the sample.

This process is illustrative of the principle of analysis, but is no longer used in analytical determinations.

=471. The Lactocrite.=—One of the earliest methods for fat estimation in milk, depending on the solution of the casein and the collection of the fat by means of whirling, is based on the use of a centrifugal machine known as the lactocrite. This apparatus is modeled very like the machine usually employed for creamery work,[461] and at one time was extensively used, but it has now given place to less troublesome and expensive machines. The acid mixture for freeing the fat of casein is composed of glacial acetic acid carrying five per cent of sulfuric. The samples of milk are heated with the acid mixture in test tubes provided with stoppers and short glass tubes to return the condensation products. The hot mixture is poured into a small metallic cylindrical cup holding about three cubic centimeters. This cup fits by means of an accurately ground shoulder on a metal casing, carrying inside a heavy glass graduated tube of small internal diameter. The excess of the milk mixture escapes through a small aperture in the metallic screw cap of the metal holder. The metal holder is cut away on both sides in order to expose the graduations on the glass tube. The glass tube is held water-tight by means of perforated elastic washers. Thus prepared the tubes are inserted in the radial holes of a revolving steel disk previously heated to a temperature of 60°. The whirling is accomplished in a few minutes by imparting to the steel disk a speed of about 6,000 revolutions per minute. At the end of this operation the fat is found in a clear column in the small glass tube and the number of the divisions it occupies in this tube is noted. Each division of the scale represents one-tenth per cent of fat.

This apparatus is capable of giving accurate results when all its parts are in good working order. In this laboratory the chief difficulty which its use has presented is in keeping the joints in the glass metal tube tight.

This description of the apparatus is given to secure an illustration of the principle involved, a principle which has been worked out in later times into some of the most rapid and practical processes of estimating fat in milk.

=472. Modification of Lindström.=—Many modifications have been proposed for conducting the determination of fat by means of the lactocrite, but they do not involve any new principle and are of doubtful merit. In the modification suggested by Lindström, which has attained quite an extended practical application, the solvent mixture is composed of lactic and sulfuric acids and the butyrometer tubes are so changed as to permit the collection of the fat in the graduated neck after whirling, by means of adding water. The apparatus is also adjusted to secure the congelation of the fat column before its volume is noted.[462] The analyst can read the fat volume at his leisure when it is in the solid state and is not confused by changes of volume during the observation. The best acid mixture has been found to be composed of 100 volumes of lactic, an equal amount of acetic and fifteen volumes of sulfuric acids.[463]

=473. The Babcock Method.=—Among the many quick volumetric methods which have been proposed for the determination of fat in milk, none has secured so wide an application as that suggested by Babcock.[464]

The chief point of advantage in the use of this method is found in effecting the solution of the casein by means of sulfuric acid of about 1.83 specific gravity. By this reagent the casein is dissolved in a few moments without the aid of any other heat than that generated by mixing the milk with the reagent. The bottle in which the separation is made is shown in Fig. 109. The graduations on the neck are based on the use of eighteen grams of milk. To avoid the trouble of weighing, the milk is measured from a pipette graduated to deliver eighteen grams of milk of the usual specific gravity. While it is true that normal milk may vary somewhat in its density, it has been found that a pipette marked at 17.6 cubic centimeters delivers a weight which can be safely assumed to vary but slightly from the one desired. The graduated bottle holds easily thirty-five cubic centimeters of liquid in its expanded portion and the volume of milk just noted is mixed with an equal volume of sulfuric acid, conveniently measured from the lip cylinder shown in the figure. The complete mixture of the milk and acid is effected by gently rotating the bottle until its contents are homogeneous. The final color of the mixture varies from dark brown to black.

While still hot, the bottles are placed in a centrifugal machine and whirled for at least five minutes. The most convenient machine, where it is available, is the one driven by a jet of steam. The revolutions of the centrifugal should be at least 700 per minute for a twenty inch and 1,200 for a twelve inch wheel. After five minutes the bottles are removed and filled to the upper mark or nearly so with hot water, replaced in the machine and whirled for at least one minute. The fat will then be found in a clearly defined column in the graduated neck of the bottle. In reading the scale, the extreme limits between the lowest point marked by the lower meniscus and the highest point marked by the edge of the upper meniscus are to be regarded as the termini of the fat column.

In testing cream by the babcock process, it may either be diluted until the column of fat secured is contained in the graduated part of the neck or specially graduated bottles may be used.

Condensed Milk: In applying the babcock test to condensed milk, it is necessary to weigh the sample and to use only about eight grams.[465] This quantity is placed in the bottle and dissolved in ten cubic centimeters of water and the analysis completed as above. The reading noted is multiplied by eighteen and divided by the weight of the sample taken.

Skim Milk: In determining the fat in skim milk and whey, it is desirable to use a bottle of double the usual capacity, but with the same graduation on the neck. The percentage of fat noted is divided by two.

Cheese: Five grams are a convenient quantity of cheese to employ. To this quantity in the bottle are added fifteen cubic centimeters of hot water and the flask gently shaken and warmed until the cheese is softened. The treatment with acid and whirling are the same as described above. The noted reading is multiplied by eighteen and divided by five.

=474. Solution in Amyl Alcohol and Hydrochloric and Sulfuric Acids.=—Leffmann and Beam have proposed to aid the solution of the casein in sulfuric acid by the previous addition to the milk of a mixture of equal volumes of amyl alcohol and hydrochloric acid.[466] In this process the same graduated flasks may be used as in the babcock process, or a special flask may be employed. In this case the graduation of the neck is for fifteen cubic centimeters of milk, and each one and a half cubic centimeters is divided into eighty-six parts. The quantity of milk noted is placed in the flask, together with three cubic centimeters of the mixture of amyl alcohol and hydrochloric acid, and well shaken. To the mixture, sulfuric acid of 1.83 specific gravity is added until the belly of the flask is nearly full and the contents well mixed by shaking. When the casein is dissolved, the addition of the sulfuric acid is continued until the flask is filled to the upper mark and again the contents mixed. It is well to close the mouth of the flask with a stopper while shaking. The bottle is placed in a centrifugal and whirled for a few moments, when the fat is collected in the graduated neck and its volume noted.

The process is also known in this country as the beimling method.[467] The fat separated in the above process is probably mixed with a little fusel oil, and therefore it is advisable to use the specially graduated bottle instead of one marked in absolute volumes.[468]

The method, when conducted according to the details found in the papers cited, gives accurate results, but is somewhat more complicated than the babcock process and is not now used to any great extent in analytical work.

=475. Method of Gerber.=—The method proposed by Gerber for estimating fat in milk is based on the processes of Babcock, Beimling and Beam already described. The tubes in which the decomposition of the milk and the measurement of the fat are accomplished are of two kinds, one open at only one end for milk and the other open at both ends for cheese. They are closed during the separation by rubber stoppers.[469]

The apparatus have been greatly improved and simplified since the first description of them was published and have come into extensive use in Europe and to a limited extent in this country.[470]

The butyrometer tubes are made of various sizes and shapes, but the most convenient are those noted above as shown in Fig. 110.

Before adding the strong sulfuric acid, one cubic centimeter of amyl alcohol is mixed with the milk in the butyrometer. This admixture serves to clarify the fat and render the reading more easy.

The centrifugal is run by hand, and the required speed of rotation is given it by means of a cord wrapped spirally about its axis, as shown in Fig. 111. The cord in the new machines is replaced by a leather strap working on a ratchet.

The process is more speedy than that of Babcock, and the results have been shown by a large experience to be reliable and accurate.

The sulfuric acid employed is of 1.825 to 1.830 specific gravity. There is no danger of loss by the formation of volatile ethers where the quantity of amyl alcohol used does not exceed one cubic centimeter. In a comparison of the respective merits of the methods of Babcock, Thörner and Gerber, made by Hausamann, the first place is awarded to the Gerber process.[471] In the figure 110, the butyrometers marked 2, 5 and 8 are for milk, and those numbered 1, 3 and 7 are for cream and cheese. In conducting the analysis, ten cubic centimeters of the sulfuric acid are placed in the butyrometer with one cubic centimeter of the amyl alcohol. When mixed, eleven cubic centimeters of the milk are added and the contents of the tube well mixed, the tube stoppered and placed in the centrifugal. The larger tubes, open at both ends, require double the quantities of the reagents mentioned. The measurements are made at about 15°.

Minute directions for conducting the analyses with milk, skim milk, buttermilk, cream, condensed milk, cheese and butter accompany each apparatus.

PROTEID BODIES IN MILK.

=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.

ANALYSIS OF BUTTER.

=494. General Principles.=—The general analysis of butter fat is conducted in accordance with the methods described in the part of this volume devoted to the examination of fats and oils. The methods of sampling, drying, filtering, and of determining physical and chemical properties, are there developed in sufficient detail to guide the analyst in all operations of a general nature. There remain for consideration here only the special processes practiced in butter analysis and which are not applied to fats in general. These processes naturally relate to the study of those properties of a distinctive nature, by means of which butter is differentiated from other fats for which it may be mistaken or with which it may be adulterated. These special studies, therefore, are directed chiefly to the consideration of the peculiar physical properties of butter fat, to its content of volatile acids and to its characteristic forms of crystallization as observed with the aid of the microscope. For dietetic, economic and legal reasons, it is highly important that the analyst be able to distinguish a pure butter from any substitute therefor.

=495. Appearance of Melted Butter.=—Fresh, pure butter, when slowly melted, shows after a short time the butter fat completely separated, of a delicate yellow color and quite transparent. Old samples of butter do not give a fat layer of equal transparency. Oleomargarin, or any artificial butter when similarly treated, gives a fat layer opalescent or opaque. By means of this simple test an easy separation of pure from adulterated butter may be effected. In mixtures, the degree of turbidity shown by the separated fats may be regarded as a rough index of the amount of adulteration. In conducting the work, the samples of butter, in convenient quantities according to the size of the containing vessel, are placed in beakers and warmed slowly at a temperature not exceeding 50°. After a lapse of half an hour the observations are made.

If one part of the melted butter be shaken with two volumes of warm water (40°) and set aside for five minutes the fat is still found as an emulsion, while oleomargarin, similarly treated, shows the fat mostly separated. This process has some merit, but must not be too highly valued.[499]

=496. Microscopic Examination of Butter.=—The microscope is helpful in judging the purity of butter and the admixture of foreign fats, if not in too small quantity to be of any commercial importance, can easily be detected by this means.[500] The methods of preparing butter fat in a crystalline state are the same as those described in paragraphs =307-309=. The crystals of butter fat differ greatly in appearance with the different methods of preparation. When butter is melted, filtered, heated to the boiling point of water and slowly cooled, it forms spheroidal crystalline masses as seen by the microscope, which present a well defined cross with polarized light. This cross is not peculiar to butter fat, but is developed therein with greater distinctiveness than in other fats of animal origin.

Pure, fresh, unmelted butter, when viewed with polarized light through a plate of selenite, presents a field of vision of uniform tint, varying with the relative positions of the nicols. When foreign fats, previously melted, as in rendering, are mixed with the butter the crystallization they undergo disturbs this uniformity of tint and the field of vision appears particolored. Old, rancid or melted butter may give rise to the same or similar phenomena under like conditions of examination. The microscope thus becomes a most valuable instrument for sorting butters and in distinguishing them in a preliminary way from oleomargarin.

=497. Judgment of Suspected Butter or Lard by Refractive Power.=—In discriminating between pure and adulterated butters by the aid of the butyrorefractometer (=301=), the absolute reading of the instrument is of less importance than the difference which is detected between the highest permissible numbers, for any degree of temperature, and the actual reading obtained at that temperature. These differences, within certain limits, do not perceptibly vary with the temperature, and heretofore they have been determined with the aid of a table, and in this respect the observations have been made the more laborious.

Wollny has rendered these tables unnecessary by constructing a thermometer in which the mercury column does not indicate degrees of temperature, but the highest permissible number for butter or lard at the temperature of observation. The scale of the instrument is so adjusted as to include temperatures of from 30° to 40°, which renders it suited to the examination of butter and lard. The oleothermometer is shown in Fig. 112.

The side of the scale B is for butter and that marked S for lard. The use of the instrument is the simplest possible. The sample of fat is placed in the prisms in the usual manner. When the mercury in the thermometer is at rest, the scale of the instrument is read. In the case of a butter, if the reading of the scale give a higher number than that indicated by the thermometer, the sample is pronounced suspicious and the degree of suspicion is proportional to the difference of the two readings.

=498. Estimation of Water, Fat, Casein, Ash and Salt.=—The methods proposed by the author for conducting these determinations, with minor amendments, have been adopted by the Association of Official Agricultural Chemists.[501]

Water.—The sample held in a flat bottom dish is dried to constant weight at about 100°. The weight of the sample used should be proportional to the area of the bottom of the dish, which should be just covered by the film of melted fat. The dish may be previously partly filled with sand, asbestos or pumice stone. The drying may take place in the air, in an inert gas or in a vacuum.

Fat.—The fat in a sample of butter is readily determined by treating the contents of the dish after the determination of water with an appropriate solvent.

The process is conducted as follows:

The dry butter from the water determination is dissolved in the dish with ether or petroleum spirit. The contents of the dish are then transferred to a weighed gooch with the aid of a wash bottle containing the solvent, and washed till free of fat. The crucible and contents are heated at the temperature of boiling water till the weight is constant. The weight of fat is calculated by difference from the data obtained.

The fat may also be determined by drying the butter on asbestos or sand, and subsequently extracting the fat by anhydrous alcohol free ether. The extract, after evaporation of the ether, is dried to constant weight at the temperature of boiling water and weighed.

Casein or Curd and Ash.—The crucible containing the residue from the fat determination is covered and heated, gently at first, gradually raising the temperature to just below redness. The cover may then be removed and the heat continued till the contents of the crucible are white. The loss in weight of the crucible and contents represents casein or curd, and the residue is mineral matter or ash.

Salt.—It is the usual custom in the manufacture of butter in this country to add, as a condiment, a certain proportion of salt. In Europe, the butter offered for consumption is usually unsalted. A convenient method of determining the quantity of salt is found in the removal thereof, from the sample, by repeated washing with hot water and in determining the salt in the wash water by precipitation with silver nitrate. The operation is conducted as follows: From five to ten grams of the sample are placed in a separatory funnel, hot water added, the stopper inserted and the contents of the funnel well shaken. After standing until the fat has all collected on top of the water, the stopcock is opened and the water is allowed to run into an erlenmeyer, being careful to let none of the fat globules pass. Hot water is again added to the beaker, and the extraction is repeated several times, using each time from ten to twenty cubic centimeters of water. The resulting washings contain all but a mere trace of the sodium chlorid originally present in the butter. The sodium chlorid is determined in the filtrate by a set solution of silver nitrate, using a few drops of a solution of potassium chromate as an indicator.

It is evident that the quantity of salt may also be determined from the ash or mineral matter obtained, as above noted, by the same process. If desirable, which is rarely the case, the gravimetric method of estimating the silver chlorid may be used.

=499. Volatile or Soluble Acids.=—The distinguishing feature of butter, from a chemical point of view, is found in its content of volatile or soluble fat acids. Among the volatile acids are reckoned those which are carried over in a current of steam at a temperature only slightly higher than that of boiling water. As soluble acids are regarded those which pass without great difficulty into solution in hot water. These two classes are composed essentially of the same acids. Of these butyric is the most important, followed by caproic, caprylic and capric acids. Small quantities or rather traces of acetic, lauric, myristic and arachidic acids are also sometimes found in butter. Palmitic, stearic and oleic acids also occur in large quantities. The above named acids, in combination with glycerol, form the butter fat.

=500. Relative Proportion of Ingredients.=—The composition of butter fat is given differently by different authorities.[502] A typical dry butter fat may be regarded as having the following composition:

Per cent. Butyrin 7.00 Caproin, Caprylin and Caprin 2.30 Olein 37.70 Palmitin, stearin, etc. 53.00

Pure butter fat consists principally of the above glycerids, some coloring principles, varying in quantity and composition with the food of the animal, and a trace of lecithin, cholesterol, phytosterol and a lipochrome.

=501. Estimation of Volatile or Soluble Acids.=—The volatile or soluble acids in butter fat are estimated by the methods already described (=349, 351=). In practice preference is given to the method of determining volatile acids, based on the principle that under standard conditions practically all the acids of this nature are secured in a certain volume of the distillate. This assumption is not strictly true, but the method offers a convenient and reliable manner of obtaining results which, if not absolute, are at least comparative.

The quantity of acid distilled is determined by titration with tenth normal alkali and for convenience the data are expressed in terms of the volume of the alkali consumed. Five grams of normal butter fat will give a distillate, under the conditions given, requiring about twenty-eight cubic centimeters of tenth normal alkali for complete saturation. This is known as the reichert-meissl number. Occasionally this number may rise to thirty-two or may sink to twenty-five. Cases have been reported where it fell below the latter number, but such samples cannot be regarded as normal butter.

The determination of the reichert-meissl number is the most important of the chemical processes applied to butter fat analysis.

=502. Saponification Value and Reichert Number.=—It may often be convenient to make the same sample of butter fat serve both for the determination of the saponification value and of the reichert number. For this purpose it is convenient to use exactly five grams of the dry filtered fat. The saponification may be accomplished either under pressure or by attaching a reflux condenser to the flask as suggested by Bremer.[503] When the saponification, which is accomplished with alcoholic potash lye containing about 1.25 grams in each ten cubic centimeters of seventy per cent alcohol, is finished, and the contents of the flask are cooled, the residual alkali is titrated with a set sulfuric acid solution, using phenolphthalein as indicator. When the color has almost disappeared, an additional quantity of the indicator is added and the titration continued until the liquid is of an amber tint. A sample of the alkali, treated as above, is titrated at the same time and from the two sets of data obtained, the saponification number is calculated as indicated in paragraph (=345=).

A few drops of the alcoholic lye are added to the contents of the flask and the alcohol removed by evaporation. The residual soap and potassium sulfate are dissolved in 100 cubic centimeters of recently boiled water, some pieces of pumice added, and the volatile acids removed by distillation in the usual way after adding an excess of sulfuric acid. It is important to conduct blank distillations in the same form of apparatus to determine the magnitude of any corrections to be made. The size of the distilling flask and the form of apparatus to prevent mechanical projection of sulfuric acid into the distillate should be the same in all cases.

=503. Modification of the Reichert-Meissl Method.=—Kreis has proposed the use of strong sulfuric acid for saponifying the fats, the saponification and distillation being accomplished in one operation. A source of error of some inconvenience in this method is due to the development of sulfurous acid by the reducing action of the organic matter on the oil of vitriol. Pinette proposes to avoid this difficulty by adding, before the distillation is begun, sufficient potassium permanganate to produce a permanent red coloration. By this means the sulfurous acid is completely oxidized and its transfer to the standard alkali during distillation entirely prevented. The same result is accomplished by Micko by the use of potassium bichromate. The details of the manipulation are as follows:[504]

About five grams of the fused fat (butter or oleomargarin) are placed in a flask of approximately 300 cubic centimeters capacity. After cooling, there are added ten cubic centimeters of sulfuric acid containing three grams of water to each ninety-seven grams of the strongest acid.

The fat and acid are well mixed by a gentle rotatory motion of the flask and placed in a water bath at a temperature of 35° (circa) for fifteen minutes. At the end of this time the flask is removed from the bath and 125 cubic centimeters of water added, little by little, keeping the contents cool. Next are added four cubic centimeters of a four per cent solution of potassium bichromate. The contents of the flask are vigorously shaken and, after five minutes, a solution of ferrous sulfate is added gradually from a burette until the reaction with a drop of potassium ferrocyanid shows a slight excess of the iron salt. The volume of the liquor in the flask is then increased to 150 cubic centimeters by the addition of water and 110 cubic centimeters distilled. After mixing and filtering through a dry filter, the acid in 100 cubic centimeters is determined by standard tenth normal barium hydroxid solution and the number thus obtained increased by one-tenth representing the total acid obtained.

=504. Elimination of Sulfurous Acid.=—Prager and Stern[505] propose to eliminate the sulfurous acid by a stream of air, succeeded by one of carbon dioxid, and proceed as follows: Five grams of the butter fat are brought into a liter flask, ten cubic centimeters of strong sulfuric acid are added and the flask is kept for ten minutes at 30-32° with constant agitation. When the liquid is cold, air is bubbled through it until the odor of sulfurous acid has disappeared. One hundred cubic centimeters of water are added, with precautions against rise of temperature, and carbon dioxid is bubbled through for ten minutes. This is then displaced by a stream of air for another ten minutes, the delivery tube is washed into the flask with fifty cubic centimeters of water and the distillation is effected. The following results are quoted:

Cubic centimeters of tenth normal alkali required by five grams of butter fat:

Reichert-Meissl. Prager-Stern. Sample a 29.86 29.60 ” b 30.23 29.65 ” c 28.34 27.76 ” d 28.20 28.10

The authors do not comment on the possibility of loss of acids other than sulfurous in the stream of air, but they admit that further investigation is requisite to render the suggestion of Kreis serviceable.

=505. Errors Due to Poor Glass.=—The easy solubility of the glass holding the reagents is the cause of some of the difficulties attending the determination of the saponification value. The separated silica tends to carry down, mechanically, a part of the alkali. This is shown by the fact that after the color has been discharged by titration with acid and the flask set aside a reappearance of the red color is noticed, after a time, beginning at the bottom of the flask.[506] In order to avoid difficulties of this nature, either cold saponification should be practiced or the digestion vessels used for moist combustion in sulfuric acid be employed.

Errors may also be easily introduced by the use of uncalibrated burettes and from the employment of varying quantities of the phenolphthalein solution.

=506. Estimation of the Molecular Weight of Butter and Butter Substitutes.=—Garelli and Carono have proposed a method for discriminating between butter and its substitutes by the kryoskopic determination of molecular weights.

The molecular weights of stearin, palmitin and olein are 890, 806 and 884, and of butyrin, caproin and caprylin 303, 386 and 470 respectively. Pure butter, therefore, has a lower mean molecular weight than margarin.

The method and apparatus of Beckmann are used in the determination, fifteen grams of benzol being employed as a solvent.

The constant for the molecular depression of the benzol is found to be 53.

The molecular weight obtained with samples of pure butter varied from 696 to 716, and for oleomargarin from 780 to 883.

The figures obtained with mixtures of twenty, twenty-five, thirty-three and fifty per cent of margarin with butter were 761, 720, 728 and 749 respectively. The method can be relied upon to classify samples as follows:

1. Pure butter.

2. Butter containing margarin.

3. Suspicious butter.[507]

=507. Substitutes and Adulterants of Butter.=—In this country, butter is never adulterated with cocoa or sesame oil, as is sometimes the case in other lands. The common substitute for butter here is oleomargarin, and the most common butter adulterant, neutral lard. The methods of analyses, by means of which these bodies can be identified, have already been sufficiently described. By the use of certain digestive ferments and other bodies, butter may be made to hold an excessive quantity of casein, sugar and water in the form of a somewhat permanent emulsion.[508] This form of adulteration is revealed at once on melting the sample.

=508. Furfurol Reaction with Sesame Oil.=—Olive oil and sometimes butter are mixed with the cheaper body, sesame oil. The latter is detected with certainty, from the red coloration it gives when mixed with furfurol and hydrochloric acid. Instead of furfurol, some body yielding it when subjected to the action of hydrochloric acid, viz., sucrose or a pentose sugar, may be used. It has been found by Wauters, however, that an alcoholic solution of two grams of furfuraldehyd in 100 cubic centimeters of alcohol is the best reagent. One-tenth of a cubic centimeter of this reagent is used for each test.[509]

The test is made as follows: The quantity of the furfuraldehyd solution mentioned above is mixed with ten cubic centimeters of hydrochloric acid, and there are added, without mixing, an equal volume of the suspected oil. On standing, a red coloration is produced at the zone of separation of the two liquids. If the oil be sesame, the coloration is produced instantly. As little as one per cent of sesame in a mixed oil will show the color in two minutes. The manipulation is also varied by shaking together the reagents and the melted butter. Turmeric, which is sometimes used in coloring butter, also gives the rose-red color when treated with hydrochloric acid, but turmeric supplies its own furfuraldehyd. It is easy to distinguish therefore the coloration due to sesame oil, which is developed only when furfuraldehyd is present, from that due to the turmeric, which is produced without the aid of the special reagent.

=509. Butter Colors.=—Where cows are deprived of green food and root crops, such as carrots, and kept on a poorly balanced ration, the butter made from their milk may be almost colorless. To remedy this defect it is quite a common practice to color the product artificially. Almost the sole coloring matter used in this country is anatto.[510] Other coloring matters which are occasionally employed are turmeric, saffron, marigold leaves, yellow wood (Chlorophora tinctoria), carrot juice, chrome yellow (lead chromate) and dinitrocresol.

The use of small quantities of anatto, turmeric or saffron is unobjectionable, from a sanitary point of view, but this is not the case with such a substance as lead chromate. The detection of anatto or saffron in butter may be accomplished by the method of Cornwall.[511] About five grams of the warm filtered fat are dissolved in about fifty cubic centimeters of ordinary ether, in a wide tube, and the solution is vigorously shaken for from ten to fifteen seconds, with from twelve to fifteen cubic centimeters of a very dilute solution of caustic potash or soda in water, only alkaline enough to give a distinct reaction with turmeric paper, and to remain alkaline after separating from the ethereal fat solution. The corked tube is set aside, and in a few hours, at most, the greater part of the aqueous solution, now colored more or less yellow by the anatto, can be drawn from beneath the ether with a pipette or by a stopcock below, in a sufficiently clear state to be evaporated to dryness and tested in the usual way with a drop of concentrated sulfuric acid.

Sometimes it is well to further purify the aqueous solution by shaking it with some fresh ether before evaporating it, and any fat globules that may float on its surface during evaporation should be removed by touching them with a slip of filter paper; but the solution should not be filtered, because the filter paper may retain much of the coloring matter.

The dry yellow or slightly orange residue turns blue or violet blue with sulfuric acid, then quickly green, and finally brownish or somewhat violet this final change being variable, according to the purity of the extract.

Saffron can be extracted in the same way; it differs from anatto very decidedly, the most important difference being in the absence of the green coloration.

Genuine butter, free from foreign coloring matter, imparts at most a very pale yellow color to the alkaline solution; but it is important to note that a mere green coloration of the dry residue on addition of sulfuric acid is not a certain indication of anatto (as some books state) because the writer has thus obtained from genuine butter, free from foreign coloring matter, a dirty green coloration, but not preceded by any blue or violet-blue tint.

Blank tests should be made with the ether.

Turmeric is easily identified by the brownish to reddish stratum that forms between the ethereal fat solution and the alkaline solution before they are intimately mixed. It may be even better recognized by carefully bringing a feebly alkaline solution of ammonia in alcohol beneath the ethereal fat solution with a pipette, and gently agitating the two, so as to mix them partially.

Another method of separating artificial coloring matter has been proposed by Martin.[512]

A method of determining the relative amount of butter color has been worked out by Babcock.[513]

EXAMINATION OF CHEESE.

=510. Composition Of Cheese.=—Pure cheese is made from whole milk by precipitating the casein with rennet. The precipitated casein carries down also the fat of the milk and a little lactose and whey remain incorporated with the cheesy mass. The ingredients of cheese are therefore those of the whole milk less the greater part of the whey, id est, milk sugar, lactalbumin, globulin, soluble mineral matters and water. In the conversion of the crude precipitate noted above into the cheese of commerce, it is subjected to a ripening process which is chiefly conditioned by bacterial action. It is not possible here to enter into a discussion of methods of isolating and identifying the bacteria which promote or retard the ripening process.[514] As a rule, about a month is required for the curing process, before the cheeses are ready for boxing and shipment. The most important changes during ripening take place in the proteid matter, which is so altered as to become more palatable and more digestible as a result of the bacterial activity.

The percentage composition of the principal cheeses of commerce are shown in the following table:[515]

Water, Casein, Fat, Sugar, Ash, Per cent. Per cent. Per cent. Per cent. Per cent. Cheddar 34.38 26.38 32.71 2.95 3.58 Cheshire 32.59 32.51 26.06 4.53 4.31 Stilton 30.35 28.85 35.39 1.59 3.83 Brie 50.35 17.18 25.12 1.94 5.41 Neufchatel 44.47 14.60 33.70 4.24 2.99 Roquefort 31.20 27.63 33.16 2.00 6.01 Edam 36.28 24.06 30.26 4.60 4.90 Swiss 35.80 24.44 37.40 2.36

Full cream, 38.60 25.35 30.25 2.03 4.07 (mean of 143 analyses)

It is evident that the composition of the cheese will vary with the milk from which it is made and the manipulation to which it is subjected. A good American green cheese made from milk of the composition noted below will have the composition which is appended.[516]

TABLE SHOWING MEAN COMPOSITION OF MILK AND CHEESE MADE THEREFROM. Milk. Cheese. Per cent. water 87.38 36.70 ” ” fat 3.73 34.18 ” ” proteids 3.13 23.44 ” ” sugar, ash etc. 5.76 5.68

From the above it is seen that in full milk cheese the ratio of fat to casein is 1.46: 1, and to solids not fat 1.17: 1. This is a point of some importance in judging the purity of a cheese. When the full milk of a mixed herd is used the percentage of fat in a cheese will always be considerably higher than that of casein.

=511. Manipulation of the Milk.=—When sweet milk is received at the cheese factory, a starter of sour milk is added to it in order to hasten its ripening. When it is thought that the proper degree of acidity has been secured, it is subjected to a rennet test. In this test 160 cubic centimeters of the milk are heated to 30° and mixed with five cubic centimeters of the rennet solution made by diluting five cubic centimeters of the rennet of commerce with fifty cubic centimeters of water. The number of seconds required for the milk to curdle is noted. The observation is facilitated by distributing throughout the milk a few fine fragments of charcoal. The contents of the vessel are given a circular motion and, at the moment of setting, the movement of the black particles is suddenly arrested. If coloring matter be added to the milk, it should be done before it becomes sour. The quantity of rennet required is determined by the nature of the cheese which it is desired to make. For a cheese to be rapidly cured, enough rennet should be added to produce coagulation in from fifteen to twenty minutes, and when slow curing is practiced in from thirty to forty-five minutes. When the mass is solid so that it can be cut with a knife, the temperature is raised to 37°, and it is tested on a hot iron until it forms threads an eighth of an inch in length. This test is made by applying an iron heated nearly to redness to the curd. When the curd is in proper condition threads from a few millimeters to two centimeters in length are formed, when the iron is withdrawn. The longer threads indicate, but only to a limited extent, a higher degree of acidity.[517] This test is usually made about two and one-half hours from the time of coagulation. The whey is then drawn off through a strainer and the curd is placed on racks with linen bottoms in order that the residual whey may escape, the curd being stirred meanwhile. In from fifteen to twenty minutes it can be cut into blocks eight or ten inches square and turned over. This is repeated several times in order to facilitate the escape of the whey. When the curd assumes a stringy condition, it is run through a mill and cut into small bits and is ready for salting, being cooled to 27° before the salt is added. From two to three pounds of salt are used for each 100 pounds of curd. The curd is then placed in the molds and pressed into the desired form. The cheeses thus prepared are placed on shelves in the ripening room and the rinds greased. They should be turned and rubbed every day during the ripening, which takes place at a temperature of from 15° to 18°.[518]

=512. Official Methods of Analysis.=—The methods of cheese analysis recommended by the Association of Official Agricultural Chemists are provisional and are not binding on its members. They are as follows:[519]

Preparation of Sample.—Where the cheese can be cut, a narrow wedge reaching from the edge to the center will more nearly represent the average composition than any other sample. This should be chopped quite fine, with care to avoid evaporation of water, and the several portions for analysis taken from the mixed mass. When the sample is obtained with a cheese trier, a plug perpendicular to the surface one-third of the distance from the edge to the center of the cheese more nearly represents the average composition than any other. The plug should either reach entirely or half way through the cheese. For inspection purposes the rind may be rejected, but for investigations where the absolute quantity of fat in the cheese is required the rind should be included in the sample. It is well, when admissible, to secure two or three plugs on different sides of the cheese, and, after splitting them lengthwise with a sharp knife, use portions of each for the different determinations.

Determination of Water.—From five to ten grams of cheese are placed in thin slices in a weighed platinum or porcelain dish which contains a small quantity of freshly ignited asbestos to absorb the fat. The dish is heated in a water oven for ten hours and weighed; the loss in weight is considered as water. If preferred, the dish may be placed in a desiccator over concentrated sulfuric acid and dried to constant weight. In some cases this may require as much as two months. The acid should be renewed when the cheese has become nearly dry.

Determination of Ether Extract.—Grind from five to ten grams of cheese in a small mortar with about twice its weight of anhydrous copper sulfate. The grinding should continue until the cheese is finely pulverized and evenly distributed throughout the mass, which will have a uniform light blue color. This mixture is transferred to a glass tube having a strong filter paper, supported by a piece of muslin, tied over one end. Put a little anhydrous copper sulfate into the tube next to the filter before introducing the mixture containing the cheese. On top of the mixture place a tuft of ignited asbestos, and place the tube in a continuous extraction apparatus and treat with anhydrous ether for fifteen hours. Dry the fat obtained at 100° to constant weight.

Determination of Nitrogen.—The nitrogen is determined by the kjeldahl method, using about two grams of cheese, and multiplying the percentage of nitrogen found by 6.25 for proteid compounds.

Determination of Ash.—The dry residue from the water determination may be used for the ash determination. If the cheese be rich in fat, the asbestos will be saturated therewith. This may be carefully ignited and the fat allowed to burn, the asbestos acting as a wick. No extra heat should be applied during this operation, as there is danger of spurting. When the flame has died out, the burning may be completed in a muffle at low redness. When desired, the salt may be determined in the ash in the manner specified under butter (=498=).

Determination of Other Constituents.—The sum of the percentages of the different constituents, determined as above, subtracted from 100 will give the amount of organic acids, milk sugar etc., in the cheese.

=513. Process of Mueller.=—The process of Müller,[520] as modified by Kruger,[521] is conducted as follows: About ten grams of a good average sample of cheese are rubbed in a porcelain mortar with a mixture of three parts of alcohol and one part of ether. After the mixed liquids have been in contact with the cheese five or ten minutes they are poured upon a weighed filter of from fifteen to sixteen centimeters diameter, and this process is repeated from one to three times, after which the contents of the mortar are brought upon the filter. The filtrate is received in a weighed flask, the alcohol ether driven off by evaporation and the residue dried. Since it is difficult to get all the particles of cheese free from the mortar, it is advisable to perform the above process in a weighed dish which can afterwards be washed thoroughly with ether and alcohol and dried and the amount of matter remaining thereon accounted for. The residue remaining in the flask after drying is treated several times with pure warm ether, and the residue also remaining upon the filter mentioned above is completely extracted with ether. The dried residue obtained in this way from the filter plus the residue in the flask which received the filtrate, plus the amount left upon the dish in which the cheese was originally rubbed up, constitute the total dry matter of the cheese freed of fat. All the material soluble in ether should be collected together, dried and weighed as fat.

By this process the cheesy mass is converted into a fine powder which can be easily and completely freed from fat by ether, and can be dried without becoming a gummy or horny mass.

For the estimation of the nitrogen, about three grams of the well grated cheese are used and the nitrogen determined by moist combustion with sulfuric acid.[522]

For the estimation of ash, about five grams are carbonized, extracted with water, and the ash determined as described below.[523]

Char from two to three grams of the substance and burn to whiteness at the lowest possible red heat. If a white ash cannot be obtained in this manner, exhaust the charred mass with water, collect the insoluble residue on a filter, burn, add this ash to the residue from the evaporation of the aqueous extract and heat the whole to a low redness till the ash is white.

=514. Separation of Fat from Cheese.=—It is often desirable to secure a considerable quantity of the cheese fat for physical and chemical examination without the necessity of effecting a complete quantitive separation. In this laboratory this is accomplished by the method of Henzold.[524] The cheese, in quantities of about 300 grams, is cut into fragments about the size of a pea and treated with 700 cubic centimeters of potash lye, which has previously been brought to a temperature of about 20°. The strength of the lye should be such that about fifty grams of the caustic potash are contained in each liter of the solution.

The treatment is conveniently conducted in a wide neck flask and the solution of the casein is promoted by vigorous shaking. After from five to ten minutes, it will be found that the casein is dissolved and the fat is found swimming upon the surface of the solution in the form of lumps. The lumps of fat are collected in as large a mass as possible by a gentle shaking to and fro. Cold water is poured into the flask until the fat is driven up into the neck, whence it is removed by means of a spoon.

The fat obtained in this way is washed a few times with as little cold water as possible in order to remove the residue of potash lye which it may contain. Experience shows that the fat by this treatment is not perceptibly attacked by the potash lye. In a short time, by this procedure, the fat is practically all separated and is then easily prepared for chemical analysis by filtering and drying in the manner already described (=283=). The fat may also be separated, but with less convenience, by partially drying the sample, reducing it to a finely divided state and applying any of the usual solvents. The solvent is removed from the extract by evaporation and the residual fat is filtered and prepared for examination as usual.

=515. Filled Cheese.=—The skim milk coming from the separators is unfortunately too often used for cheese making. The abstracted fat is replaced with a cheaper one, usually lard. These spurious cheeses are found in nearly every market and are generally sold as genuine. The purchasers only discover the fraud when the cheese is consumed. Many of the States have forbidden by statute the manufacture and sale of this fraudulent article. Imported cheeses may also be regarded with suspicion, inasmuch as the method of preparing filled cheese is well known and extensively practiced abroad. A mere determination of the percentage of fat in the sample is not an index of the purity of the cheese. It is necessary to extract the fat by one of the methods already described and, after drying and filtering, to submit the suspected fat to a microscopic and chemical examination. A low content of volatile fat acid and the occurrence of crystalline forms foreign to butter will furnish the data for a competent judgment.

When the reichert-meissl number falls below twenty-five the sample may be regarded with suspicion. The detection of the characteristic crystals of lard or tallow is reliable corroborating evidence (=308=).

It is stated by Kühn[525] that the margarin factory of Mohr, at Bahrenfeld-Altona, has made for many years a perfect emulsion of fat with skim milk. This product has been much used in the manufacture of filled cheese which is often found upon the German market.

=516. Separation of the Nitrogenous Bodies in Cheese.=—The general methods of separation already described for proteid bodies (=417-425=) are also applicable to the different nitrogenous bodies present in cheese, representing the residue of these bodies as originally occurring in the milk, and also the products which are formed therefrom during the period of ripening. For practical dietary and analytical purposes, these bodies may be considered in three groups:

(a) The useless (from a nutrient point of view) nitrogenous bodies, including ammonia, nitric acid, the phenylamido-propionic acids, tyrosin, leucin and other amid bodies.

(b) The albumoses and peptones, products of fermentation soluble in boiling water.

(c) The caseins and albuminates, insoluble in boiling water.

The group of bodies under (a), according to Stutzer, may be separated from the groups (b) and (c) by means of phosphotungstic acid. For this purpose a portion of an intimate mixture of fine sand and cheese (100 cheese, 400 sand) corresponding to five grams of cheese, is shaken for fifteen minutes with 150 cubic centimeters of water. After remaining at rest for another fifteen minutes 100 cubic centimeters of dilute sulfuric acid (one acid, three water) are added, followed by treatment with the phosphotungstic acid as long as any precipitate is produced. The mixture is thrown on a filter and the insoluble matters washed with dilute sulfuric acid until the filtrate amounts to half a liter. Of this quantity an aliquot part (200 cubic centimeters) is used for the determination of nitrogen. From the quantity of nitrogen found, that representing the ammonia, as determined in a separate portion, is deducted and the remainder represents the nitrogen present in the cheese as amids.[526]

Albumoses and Peptones.—Albumoses and peptones are determined in cheese by the following method:[527] A quantity of the sand mixture already described, corresponding to five grams of the cheese, is treated with about 100 cubic centimeters of water, heated to boiling, and the clear liquid above the sand poured into a flask of half a liter capacity. The extraction is continued with successive portions of water in like manner until the volume of the extract is nearly half a liter. When cold, the volume of the extract is completed to half a liter with water, the liquor filtered, 200 cubic centimeters of the filtrate treated with an equal volume of dilute sulfuric acid (one to three) and phosphotungstic acid added until no further precipitate takes place. The nitrogen is determined in the precipitate after filtration and washing with dilute sulfuric acid.

Casein and Albuminates.—The quantity of casein and albuminates in cheese is calculated by subtracting from the total nitrogen that corresponding to ammonia, amids, that in the indigestible residue and that corresponding to the albumose and peptone. In three samples of cheese, viz., camembert, swiss, and gervais, Stutzer found the nitrogen, determined as above, distributed as follows:[528]

Camembert. Swiss. Gervais. N as ammonia 13.0 3.7 1.6 N as amids 38.5 9.0 5.2 N as albumose peptone 30.5 8.6 15.5 N indigestible 4.0 2.4 8.6 N as casein, albuminates 14.0 76.3 69.1

Ammoniacal Nitrogen.—The ammoniacal nitrogen is determined by mixing a quantity of the sand-cheese corresponding to five grams of cheese, with 200 cubic centimeters of water, adding an excess of barium carbonate and collecting the ammonia by distillation in the usual way.

Digestible Proteids.—The digestible proteids in cheese are determined by the process of artificial digestion, which will be described in the part of this volume treating of the nutritive value of foods.

These data show the remarkable changes which the proteids undergo where the ripening is carried very far as in the camembert cheese.

=517. Koumiss.=—Fermented mare milk has long been a favorite beverage in the East, where it is known as koumiss. In Europe and this country cow milk is employed in the manufacture of fermented milk, although it is less rich in lactose than mare milk. The process of manufacture is simple, provided a suitable starter is at hand. A portion of a previous brewing is the most convenient one, the fermentation being promoted by the addition of a little yeast. After the process of fermentation is finished the koumiss is placed in bottles and preserved in a horizontal position in a cellar, where the temperature is not allowed to rise above 12°.

=518. Determination of Carbon Dioxid.=—The carbon dioxid in koumiss is conveniently estimated by connecting the bottle by means of a champagne tap with a system of absorption bulbs.[529] The exit tube from the koumiss bottle passes first into an erlenmeyer, which serves to break and retain any bubbles that pass over. The water is next removed by means of sulfuric acid. The koumiss bottle is placed in a bath of water which is raised to the boiling point as the evolution of the gas is accomplished. The arrangement of the apparatus is shown in Fig. 113. At the end of the operation any residual carbon dioxid in the apparatus is removed by aspiration after removing the tap and connecting it with a soda-lime tube to hold the carbon dioxid in the air. A large balance suited to weighing the koumiss bottle is required for this determination. The carbon dioxid may also be determined, but less accurately, by loss of weight in the koumiss bottle after adding weight of water retained in the apparatus.

=519. Acidity.=—Although koumiss may contain a trace of acetic acid, it is best to determine the acid as lactic. The clarification is most easily accomplished by mixing the koumiss with an equal volume of ninety-five per cent alcohol, shaking and filtering. The first filtrate will usually be found clear. If not it is refiltered. In an aliquot part of the filtrate the acidity is determined by titration with tenth-normal sodium hydroxid solution, using phenolphthalein as indicator. The necessary corrections for dilution and volume of the precipitated casein are to be made. A linen filter may be used when paper is found too slow.

=520. Alcohol.=—Half a liter of koumiss, to which 100 cubic centimeters of water have been added, is distilled until the distillate amounts to 500 cubic centimeters.

If the distillate be turbid 100 cubic centimeters of water are added and the distillation repeated. The alcohol is determined by the processes described hereafter.

=521. Lactose.=—The milk sugar may be determined by any of the methods described, but most conveniently by double dilution and polarization (=86=).

=522. Fat.=—Evaporate twenty grams of the sample to dryness and extract with pure ether or petroleum spirit in the manner already described (=455=).

The analysis is more quickly accomplished by the volumetric method of Babcock or Gerber (=473-475=).

=523. Proteids.=—The total proteids are most easily estimated by the official kjeldahl method.[530] The separation of the proteid bodies is accomplished by the methods described in paragraphs =475-489=.

In addition to the methods already described for separating the soluble and suspended proteid bodies in milk, and which may be used also for koumiss, the following should also be mentioned as of especial worth:

Separation by Filtration through Porous Porcelain.—A purely physical method, and one which is to be recommended by reason of the absence of any chemical action upon the different proteid matters, is that proposed by Lehmann, depending upon the principle that when milk is forced through porous porcelain, the albumin passes through together with the milk, sugar and other soluble constituents as a clear filtrate, while the casein and fat are perfectly retained.[531]

By this method it is quite certain that the albumin and other perfectly soluble proteids of milk may be obtained in the purest form.

Separation by Precipitation with Alum.—Probably the best chemical method of separating the two classes of proteid matters is that proposed by Schlosmann, which is effected by means of precipitating the casein with a solution of alum.[532]

The principle of this separation rests upon the fact that a solution of potash alum, when added to milk diluted with four or five times its volume of water, will completely separate the casein without affecting the albumin or globulin. The operation is conducted as follows:

Ten cubic centimeters of the milk are diluted with from three to five times that quantity of water and warmed to a temperature of about 40°. One cubic centimeter of a concentrated solution of potash alum is added, the mixture well stirred and the coagula which are formed allowed to subside. If the coagulation of the casein does not take place promptly, a small addition of the alum solution is made, usually not exceeding half a cubic centimeter, until the precipitation is complete. The temperature during the process should be kept as nearly as possible 40°. After a few minutes, the mixture is poured upon a filter and the filtrate, if not perfectly clear, is poured back until it is secured free of turbidity. In difficult cases the filtration may be promoted by the addition of some common salt or calcium phosphate, the latter acting mechanically in holding back the fine particles of casein. The precipitate is washed with water at a temperature of 40°, and afterwards with alcohol, not allowing the alcohol wash water to flow into the filtrate. When the water has been chiefly removed from the precipitate by washing with alcohol, the fat of the precipitated casein is removed with ether and the residue used for the determination of nitrogen in the usual way. The albumin is removed from the filtrate by a tannin solution in the manner already described (=480=). If it be desired to separate the albumin and globulin, the methods described in paragraph =399= may be used.

=524. Mercurial Method.=—A volumetric method for determining the total proteid matter in milk has lately been proposed by Deniges.[533] It is based upon the observation that in the precipitation of proteid matter by mercury salts, a definite quantity of mercury in proportion to the amount of proteid, is carried down therewith. The precipitation is made with a mercurial salt of known strength and the excess of the mercurial salt in the filtrate is determined by titration. For the details of the manipulation, the paper cited above may be consulted.

=525. Water and Ash.=—From two to five grams of the koumiss are dried to constant weight in a flat platinum dish over ignited sand, asbestos or pumice stone, and the dried residue incinerated.

=526. Composition of Koumiss.=—The composition of koumiss varies with the character of the milk used and the extent of the fermentation. Some of the data obtained by analysts are given below:[534]

COMPOSITION OF KOUMISS.

Carbon Kind Water, Sugar, Alcohol, Fat, Proteid, dioxid, Acidity, of Per Per Per Per Per Per Per milk. cent. cent. cent cent. cent. cent. cent. Cow 89.32 4.38 0.76 2.08 2.56 0.83 0.47 Probably 3.95 1.38 0.88 2.89 0.82 cow skim’d Mare 91.87 0.79 2.89 1.19 1.91 1.04

From the above it is seen that koumiss is made either from whole or skim milk, and that the percentage of alcohol may vary within large limits, its proportion being inverse to that of the milk sugar.

Koumiss is a beverage which is very palatable, easily digested and one which is not appreciated in this country in proportion to its merits, especially for the use of invalids.

AUTHORITIES CITED IN PART SIXTH.

[400] Wiley; Proceedings of the Society for the Promotion of Agricultural Science, 1889, p. 84. (Omit “food” before idiosyncrasy.)

[401] Pharmaceutical Journal and Transactions, Series 3, Vol. 18, p. 479.

[402] The Analyst, 1892, p. 85.

[403] Henkel; Wiener Landwirtschaftliche Zeitung, 1888, S. 401: Bulletin No. 24, Division of Chemistry, U. S. Department of Agriculture, p. 155.

[404] Die Landwirtschaftlichen Versuchs-Stationen, Band 35, S. 351: Bulletin No. 24, Division of Chemistry, U. S. Department of Agriculture, p. 151.

[405] Baumeister; Milch und Molkerei-Producte, S. 16.

[406] Bulletins Nos. 9 and 25 of the Office of Experiment Stations, U. S. Department of Agriculture: Farmers’ Bulletins Nos. 9 and 29, U. S. Department of Agriculture.

[407] Annales de Chimie et de Physique, 3e Série, Tome 64, p. 61.

[408] Bulletin de la Société Chimique de Paris, 3ᵉ Série, Tome 15-16, p. 248.

[409] Vid. op. cit. supra, p. 453.

[410] Central-Blatt für medicinische Wissenschaft, Band 34, S. 145.

[411] Conn; Farmers’ Bulletins 9 and 25, Office of Experiment Stations, U. S. Department of Agriculture: Farmers’ Bulletins 9 and 29, Department of Agriculture: Les Microbes et leur Rôle dans la Laiterie Freudenreich: Langlois, Le Lait, pp. 95 et seq.

[412] The Analyst, Vol. 20, p. 157.

[413] Vid. op. cit. supra, p. 152.

[414] Forschungs-Berichte über Lebensmittel etc., Band 2, S. 368.

[415] Vid. op. cit. supra, Band 1, S. 422.

[416] Vid. op. cit. supra, S. 372.

[417] Hopkins and Powers; Bulletin No. 47, Division of Chemistry, U. S. Department of Agriculture, p. 127.

[418] Bulletin No. 38, Division of Chemistry, U. S. Department of Agriculture, p. 118.

[419] Becke; Die Milchprüfungs-Methoden, S. 45: Rouvier; Le Lait, p. 45.

[420] The Analyst, 1890, Vol. 16, p. 170.

[421] Rouvier; Le Lait, p. 35.

[422] Central-Blatt für Nahrungs und Genussmittel Chemie, Band 13, S. 277.

[423] Bulletin No. 46, Division of Chemistry, U. S. Department of Agriculture, p. 36.

[424] Journal für Landwirtschaft, 1882, S. 293; 1885, S. 251.

[425] Vid. op. cit. supra, 1879, S. 249.

[426] Forschungen auf dem Gebiete der Viehhaltung, 1879, S. 265.

[427] The Analyst, Vol. 7, p. 129.

[428] Vid. op. cit. supra, Vol. 13, p. 26.

[429] Bulletin No. 47, Division of Chemistry, U. S. Department of Agriculture, p. 123.

[430] This work, Vol. 1, page 411.

[431] Bulletin No. 16, Division of Chemistry, U. S. Department of Agriculture, p. 36.

[432] Sixth Annual Report Wisconsin Agricultural Experiment Station, p. 64.

[433] Fourth Annual Report New York (Geneva) Agricultural Experiment Station, p. 298.

[434] Woll; Seventh Annual Report Wisconsin Agricultural Experiment Station, p. 238.

[435] The Analyst, 1885, p. 46: Bulletin No. 13, Part 1, Division of Chemistry, U. S. Department of Agriculture, p. 86.

[436] Haidlen; Die Milchprüfungs-Methoden, S. 12.

[437] Dingler’s polytechnisches Journal, Band 232, S. 461.

[438] Macfarlane; The Analyst, Vol. 18, p. 73.

[439] Duclaux; Le Lait, p. 176.

[440] Abraham; The Analyst, Vol. 9, p. 22.

[441] Gantter; Zeitschrift für analytische Chemie, Band 26, S. 677.

[442] Morse, Piggot and Burton; American Chemical Journal, Vol. 9, pp. 108 and 222.

[443] Chemiker-Zeitung Repertorium, 1889, S. 228.

[444] Journal de Pharmacie et de Chimie, 1890, p. 460.

[445] Richmond; The Analyst, Vol. 17, p. 48: Bulletins 28, 31, 35, 38, 43, and 46, Division of Chemistry, U. S. Department of Agriculture.

[446] Bulletin No. 28, Division of Chemistry, U. S. Department of Agriculture, p. 31.

[447] Zeitschrift für analytische Chemie, Band 27, S. 464.

[448] Chemical News, Nov. 1889.

[449] The Analyst, Vol. 16, p. 67.

[450] Vid. op. cit. supra, Vol. 18, p. 53.

[451] Vid. op. cit. supra, Vol. 17, p. 81.

[452] Chemiker-Zeitung, Band 15, S. 1833.

[453] Journal of Analytical Chemistry, 1888, Vol. 2, p. 371: Fifth Annual Report Wisconsin Agricultural Experiment Station.

[454] Molkerei Zeitung, 1892, No. 1; Chemisches Central-Blatt, 1892, Band 2, S. 429.

[455] Chemiker-Zeitung, Band 18, S. 1816; Band 19, S. 348.

[456] Zeitschrift für analytische Chemie, Band 32, S. 168.

[457] Zeitschrift des Landwirtschaftlichen Vereins in Bayern, 1880; Zeitschrift für analytische Chemie, Band 20, S. 452.

[458] Bulletin No. 13, Division of Chemistry, U. S. Department of Agriculture, p. 92.

[459] Instruction sur l’Emploi du Lactobutyrometer, Paris, 1856 et 1878: Becke; Die Milchprüfungs-Methoden, S. 66.

[460] Bulletin No. 8, Iowa Agricultural Experiment Station, p. 295.

[461] Dingler’s polytechnisches Journal, Band 261, S. 219.

[462] Milch Zeitung, Band 21, S. 496.

[463] Op. cit. supra, Band 22, S. 85.

[464] Bulletin No. 24, Wisconsin Agricultural Experiment Station.

[465] Bulletin No. 31, Wisconsin Agricultural Experiment Station.

[466] The Analyst, Vol. 17, p. 83.

[467] Bulletin No. 21, Vermont Agricultural Experiment Station.

[468] Vid. op. cit. 67, Vol. 17, p. 144; Vol. 18, p. 130; Vol. 19, p. 62.

[469] Chemiker-Zeitung, Band 16, S. 1839.

[470] Vid. op. cit. supra, Band 19, S. 348; Band 18, S. 1816.

[471] Vid. op. cit. supra, Band 19, S. 348.

[472] Comptes rendus, Tome 107, p. 772; Hoppe-Seyler’s Handbuch der Physiologisch- und Pathologisch-Chemischen Analyse, S. 479.

[473] Proceedings of the Society for the Promotion of Agricultural Science, 1888, p. 13.

[474] Journal of Physiology, Vol. 11, p. 459.

[475] Die Land wirtschaftlichen Versuchs-Stationen, Band 31, S. 131.

[476] Sixth Annual Report of the Wisconsin Agricultural Experiment Station, p. 64.

[477] Bulletin No. 46, Division of Chemistry, U. S. Department of Agriculture, p. 36.

[478] Zeitschrift für Biologie, Band 33, S. 43.

[479] Journal für praktische Chemie, {2}, Band 15, S. 329.

[480] Vid. op. cit. 79, Band 33, {Neue Folge, 15}, S. 55.

[481] Stenberg; Zeitschrift für physiologische Chemie, Band 13, S. 138.

[482] Vid. op. cit. supra, S. 137.

[483] Vid. op. cit. supra, S. 160.

[484] Journal of the American Chemical Society, Vol. 15, p. 644.

[485] Handbuch der Physiologisch- und Pathologisch-Chemischen Analyse, S. 285. (Read, Makris instead of Makeris.)

[486] Zeitschrift für Biologie, Band 23, S. 64.

[487] Bulletin de la Société Chimique de Paris, 3ᵉ Série, Tome 11, p. 152.

[488] Vid. op. cit. 86, S. 487.

[489] Zeitschrift für Nahrungsmittel-Untersuchung, Band 10, S. 104.

[490] Zeitschrift für physiologische Chemie, Band 9, S. 445.

[491] American Chemical Journal, Vol. 6, p. 289.

[492] Journal of the American Chemical Society, Vol. 18, p. 428.

[493] Journal de Pharmacie et de Chimie, 6e Série, Tome 4, p. 65.

[494] Contribution à l’Étude des Lactoses, Thèse pour le diplôme supérieure de Pharmacie, Paris, 1892. (Read Thibault instead of Thibonet.)

[495] Journal für praktische Chemie, Neue Folge, Band 15, S. 348.

[496] Zeitschrift für angewandte Chemie, 1896, S. 72.

[497] Chemisches Central-Blatt, 1892, Band 2, S. 1028.

[498] Vid. op. cit. supra, Band 21, S. 753.

[499] Vid. op. cit. 90, S. 86.

[500] Bulletin No. 13, Division of Chemistry, U. S. Department of Agriculture, pp. 29 et seq.

[501] Vid. op. cit. supra, pp. 73-75: Bulletin No. 46, Division of Chemistry, U. S. Department of Agriculture, p. 26.

[502] Benedikt and Lewkowitsch; Oils, Fats and Waxes, p. 490.

[503] Forsuchungs-Berichte über Lebensmittel, 1895, Band 2, S. 424; Chemiker-Zeitung Repertorium, 1896, Band 20, S. 15.

[504] Revue Internationale des Falsifications, Mai, 1893, p. 157.

[505] Chemiker-Zeitung, 1893, Band 17, S. 468.

[506] Zeitschrift für angewandte Chemie, 1896, S. 177.

[507] Vid. op. cit. 90, Aug. 26, 1894, S. 219; Le Stazioni Sperimentali Agrarie Italiane, 1893, pp. 25-77.

[508] Farmers’ Bulletin No. 12, U. S. Department of Agriculture.

[509] Bulletin de l’Association Belge des Chimistes, Tome 9, p. 279.

[510] Vid. op. cit. 101, p. 26.

[511] Vid. op. cit. supra, p. 27: Chemical News, Vol. 55, p. 49.

[512] Vid. op. cit. 111, p. 28.

[513] Vid. op. et. loc. cit. supra.

[514] Russell; Dairy Bacteriology.

[515] Woll; Dairy Calendar, p. 223.

[516] Van Slyke; Bulletin 82, New Series, New York Agricultural Experiment Station, p. 654.

[517] Babcock; Twelfth Annual Report Wisconsin Agricultural Experiment Station, p. 133.

[518] Woll; Dairy Calendar, 1895, p. 220.

[519] Bulletin No. 46, Division of Chemistry, U. S. Department of Agriculture, p. 37.

[520] Landwirtschaftliches Jahrbuch, 1872, part 1.

[521] Molkerei Zeitung, 1893, Nos. 20, 22.

[522] This work, Vol. 2, p. 204.

[523] Vid. op. cit. 120, p. 24.

[524] Milch Zeitung, 1895, Band 24, S. 729: Chemiker-Zeitung Repertorium, Band 19, S. 372.

[525] Chemiker-Zeitung, 1895, S. 554.

[526] Zeitschrift für analytische Chemie, Band 35, S. 497.

[527] Vid. op. cit. supra, S. 499.

[528] Vid. op. cit. supra, S. 502.

[529] Bulletin No. 13, Division of Chemistry, U. S. Department of Agriculture, pp. 118, 293.

[530] This work, Vol. 2, p. 204.

[531] Pflüger’s Archiv, Band 56, S. 558.

[532] Hoppe-Seyler’s Zeitschrift für physiologische Chemie, Band 22, S. 213.

[533] Bulletin de la Société Chimique de Paris, Tomes 15-16, p. 1126.

[534] American Chemical Journal, Vol. 8, p. 200: Bulletin 13, Division of Chemistry, U. S. Department of Agriculture, p. 120.

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