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.