Part 106

The principle of the process is based upon the observation that while starch is easily soluble in an aqueous solution of the alkalies, it is insoluble in an alcoholic solution thereof. The chief constituents of meat, viz., fat and proteid matters, on the other hand, are readily soluble in an alcoholic solution of potash or soda. This renders the separation of the starch easy. The sample is warmed on a water bath with a considerable excess of an eight per cent solution of potassium hydroxid in alcohol whereby the fat and flesh are quickly dissolved. The starch and other carbohydrate bodies, remain in an undissolved state. In order to prevent the gelatinizing of the soap which is formed, the mass is diluted with warm alcohol, the insoluble residue collected upon a filter and washed with alcohol until the alkaline reaction disappears. The residue is then treated with aqueous potassium hydroxid solution, whereby the starch is brought into solution and, after filtration, is treated with alcohol until it is all precipitated. The precipitated starch is collected upon a filter, washed with alcohol and finally with ether, dried and weighed. Starch prepared in this way contains a considerable quantity of potash, the amount of which can be determined by incineration. In order to avoid this trouble, the starch, after separation in the first instance as above mentioned and solution in aqueous potassium hydroxid, is precipitated on the addition of enough acetic to render the solution slightly acid. The precipitated starch, in this instance, is practically free of potash, since potassium acetate is soluble in alcohol.

=543. Detection of Horse Flesh.=—Since horse flesh has become an important article of human food and is often sold as beef and sausage, a method of distinguishing it is desirable. The comparative anatomist is able to detect horse flesh when accompanied by its bones, or in portions sufficiently large for the identification of muscular characteristics. It is well known that horse flesh contains a much higher percentage of glycogen than is found in other edible meats. Niebel has based a method of detecting horse flesh upon this fact, the glycogen being converted into dextrose and determined in the usual way. Whenever the percentage of reducing sugars in the dry fat-free flesh exceeds one per cent, Niebel infers that the sample under examination is horse flesh.

The reaction for horse flesh, proposed by Bräutigam and Edelmann, is preferred by Baumert. In this test about fifty grams of the flesh are boiled for an hour with 200 cubic centimeters of water, the filtered bouillon evaporated to about half its volume, treated with dilute nitric acid and the clear filtrate covered with iodin water. Horse flesh, by reason of its high glycogen content, produces a burgundy red zone at the points of contact of the two liquids. In the case of sausages, if starch have been added, a blue zone is produced, and if dextrin be present, a red zone, both of which obscure the glycogen reaction. The starch is easily removed by treating the bouillon with glacial acetic acid. No method is at present known for separating dextrin from glycogen. The detection of horse flesh is a matter of considerable importance to agriculture as well as to the consumers, especially of sausages. A considerable quantity of horse flesh is annually sent to the market, little of which presumably is sold under its own name. As a cheap substitute for beef and pork in sausages, its use must be regarded as fraudulent, although no objection can be urged against its sale when offered under its own name.

METHODS OF DIGESTION.

=544. Artificial Digestion.=—The nutrient values of cereals and other foods are determined both by chemical analysis and by digestion experiments. The heat forming properties of foods are disclosed by combustion in a calorimeter, but the quantity of heat produced is not in every case a guide to the ascertainment of the nutritive value. This is more certainly shown, especially in the case of proteid bodies, by the action of the natural digestive ferments.

It is probable that the digestion, which is secured by the action of these ferments without the digestive organs, is not always the same as the natural process, but when the conditions which prevail in natural digestion are imitated as closely as possible the effects produced can be considered as approximately those of the alimentary canal in healthy action.

Three classes of ferments are active in artificial digestion, viz., amylolytic ferments, serving to hydrolyze starch and sugars and to convert them into dextrose, maltose and levulose, aliphalytic ferments, which decompose the glycerids and proteolytic ferments, which act on the nitrogenous constituents of foods. When these ferments are made to act on foods under proper conditions of acidity and temperature, artificial digestion ensues, and by the measurement of the extent of the action an approximate estimate of their digestibility can be secured. In artificial digestion, the temperature should be kept near that of the body, viz., at about 40°.

The soluble ferments which are active in the digestion of foods, as has been intimated, comprise three great classes. Among the first class, viz., the amylolytic ferments, are included not only those which convert starch into dextrose, but also those which cause the hydrolysis of sugars in general. Among these may be mentioned ptyalin, invertase, trehalase, maltase, lactase, diastase, inulase, pectase and cyto-hydrolytic ferments which act upon the celluloses and other fibers.

Among the aliphalytic ferments, in addition to those which act also upon proteid matter, may be mentioned a special one, lipase.

In the third class of ferments are found pepsin, trypsin or pancreatin and papain.

For the latest information in regard to the nature of the soluble ferments and their nomenclature, the work of Bourquelot may be consulted.

=545. Amylytic Ferments.=—A very active ferment of this kind is found in the saliva. Saliva may be easily collected from school boys, who will be found willing to engage in its production if supplied with a chewing gum. A gum free of sugar is to be used, or if the chewing gum of commerce is employed, the saliva should not be collected until the sugar has disappeared. A dozen boys with vigorous chewing will soon provide a sufficient quantity of saliva for practical use. The amylolytic digestion is conducted in the apparatus hereinafter described for digestion with pepsin and pancreatin. The starch or sugar in fine powder is mixed with ten parts of water and one part of saliva and kept at about 37°.5 for a definite time. The product is then examined for starch, sucrose, maltose, dextrose, dextrin and levulose by the processes already described. In natural digestion the hydrolysis of the carbohydrates is not completed in the mouth. The action of the ferment is somewhat diminished in the stomach, but not perhaps until half an hour after eating. The dilute hydrochloric acid in the stomach, which accumulates some time after eating, is not active in this hydrolysis. On the contrary the amylolytic ferment of the saliva is somewhat enfeebled by the presence of an acid. The active principle of the saliva is ptyalin.

The diastatic hydrolysis of starch has already been described (=179=). It is best secured at a somewhat higher temperature than that of the human stomach.

=546. Aliphalytic Ferments.=—In the hydrolysis of glycerids in the process of digestion the fat acids and glycerol are set free. Whether the glycerids be completely hydrolyzed before absorption is not definitely known. In certain cases where large quantities of oil have been exhibited for remedial purposes, the fat acids and soaps have been found in spherical masses in the dejecta and have been mistaken for gall stones.

The fat which enters the chyle appears to be mostly unchanged, except that it is emulsified. The aliphalytic ferment can be prepared from the fresh pancreas, preferably from animals that have not been fed for forty hours before killing. It is important to prepare the ferment entirely free of any trace of acid. The fresh glands are rubbed to a fine paste with powdered glass and extracted for four days with pure glycerol, to which one part of one per cent soda solution has been added. The filtered liquor contains aliphalytic, proteolytic and amylytic ferments, and is employed for saponification by shaking with the fat to form an emulsion and keeping the mixture, with occasional shaking, at a temperature of from 40° to 60°. The free acids can be titrated or separated from the unsaponified fats by solution in alcohol.

Heretofore it has not been possible to separate a pure aliphalytic ferment from any of the digestive glands. The digestion of carbohydrates and that of fats are intimately associated, and these two classes of foods seem to play nearly the same rôle in the animal economy.

The aliphalytic ferments, prepared from the fresh pancreas, act also on the glucosids and other ester-like carbohydrate bodies. Since the fats may be regarded as ethers, the double action indicates the similarity of composition in the two classes of bodies. The aliphalytic ferments exist also in plants and have been isolated from rape seed.

=547. Proteolytic Ferments.=—The most important process in artificial digestion is the one relating to the action of the ferments on proteid matters. The hydrolysis of fats and carbohydrates by natural ferments takes place best in an alkaline medium, while in the case of proteids when pepsin is used an acid medium is preferred. Since the acidity of the stomach is due chiefly to hydrochloric, that acid is employed in artificial digestion. The hydrolyte used is uniformly the natural ferment of the gastric secretions, viz., pepsin; but this is often followed by the pancreatic ferment, (pancreatin, trypsin) in an alkaline medium. During the digestion, the proteids are changed into peptones, and the measurement of this change determines the degree of digestion. The total proteid matter is determined in the sample, and after the digestion is completed, the soluble peptones are removed by washing and the residual insoluble proteid matter determined by moist combustion. The difference in the two determinations shows the quantity of proteid matter digested. The investigations of Kühn on the digestion of proteids may be profitably consulted. For a summary of digestion experiments in this country the résumé prepared by Gordon may be consulted. The method followed in this laboratory is fully described by Bigelow and Hamilton.

=548. Ferments Employed.=—Both the pepsins of commerce and those prepared directly from the stomachs of pigs may be used. The commercial scale pepsin is found, as a rule, entirely satisfactory, and more uniform results are secured by its use than from pepsin solutions made from time to time from pig stomachs. In the preparation of the pepsin solution one gram of the best scale pepsin is dissolved in one liter of 0.33 per cent hydrochloric acid. Two grams of the sample of food products, in fine powder, are suspended in 100 cubic centimeters of the solution and kept, with frequent shaking, at a temperature of 40° for twelve hours. The contents of the flask are poured on a wet filter, the residue on the filter well washed with water not above 40°, the filter paper and its contents transferred to a kjeldahl flask and the residual nitrogen determined and multiplied by 6.25 to get the undigested proteid matter. A large number of digestions can be conducted at once in a bath shown in Fig. 117. The quantity of water in the bath should be as large as possible.

=549. Digestion in Pepsin and Pancreatin.=—The digestion of the proteids is not as a rule wholly accomplished by the stomach juices, and, therefore, in order to secure in artificial digestion results approximating those produced in the living organism, it is necessary to follow the treatment with pepsin by a similar one with the pancreas juices. The method employed in this laboratory is essentially that of Stutzer modified by Wilson.

The residue from the pepsin digestion, after washing, is treated for six hours at near 40° with 100 cubic centimeters of pancreas solution, prepared as follows:

Free the pancreas of a healthy steer of fat, pass it through a sausage grinder, rub one kilogram in a mortar with fine sand and allow to stand for a day or longer. Add three liters of lime water, one of glycerol, of 1.23 specific gravity, and a little chloroform and set aside for six days. Separate the liquor by pressure in a bag and filter it through paper. Before using, mix a quarter of a liter of the filtrate with three-quarters of a liter of water and five grams of dry sodium carbonate, or its equivalent crystallized, heat from 38° to 40° for two hours and filter. In order to avoid the trouble of preparing the pancreas solution pure active pancreatin may be used. One and a half grams of pure pancreatin and three grams of sodium carbonate are dissolved in one liter of water and 100 cubic centimeters of this solution are used for each two grams of the sample. In all cases where commercial pepsin and pancreatin are used, their activity should be tested with bodies such as boiled whites of eggs, whose coefficient of digestibility is well known and those samples be rejected which do not prove to have the required activity.

=550. Digestion in Pancreas Extract.=—In order to save the time required for successive digestions in pepsin and pancreatin Niebling has proposed to make the digestion in the pancreas extract alone. This process and also a slight modification of it have been used with success by Bigelow and McElroy. Two grams of the sample are washed with ether and placed in a digestion flask with 100 cubic centimeters of two-tenths per cent hydrochloric acid. The contents of the flask are boiled for fifteen minutes, cooled, and made slightly alkaline with sodium carbonate. One hundred cubic centimeters of the unfiltered pancreas solution, prepared as directed above, are added and the digestion continued at 40° for six hours. The residue is thrown on a filter, washed, and the nitrogen determined. The method is simplified by the substitution of active commercial pancreatin for pancreas extract. The solution of the ferment is made of the same strength as is specified above.

=551. Artificial Digestion of Cheese.=—The artificial digestion of cheese is conducted by Stutzer as follows:

The digestive liquor is prepared from the fresh stomachs of pigs by cutting them into fine pieces and mixing with five liters of water and 100 cubic centimeters of hydrochloric acid for each stomach. To prevent decomposition, two and a half grams of thymol, previously dissolved in alcohol, are added to each 600 cubic centimeters of the mixture. The mixture is allowed to stand for a day with occasional shaking, poured into a flannel bag and the liquid portion allowed to drain without pressing. The liquor obtained in this way is filtered, first through coarse and then through fine paper, and when thus prepared will keep several months without change. It is advisable to determine the content of hydrochloric acid in the liquor by titration and this content should be two-tenths of a per cent. The cheese to be digested is mixed with sand as previously described, freed of fat by extraction with ether, and a quantity corresponding to five grams of cheese placed in a beaker, covered with half a liter of the digestive liquor and kept at a temperature of 40° for forty-eight hours. At intervals of two hours the flasks are well shaken and five cubic centimeters of a ten per cent solution of hydrochloric acid added and this treatment continued until the quantity of hydrochloric acid amounts to one per cent. After the digestion is finished, the contents of the beaker are thrown on a filter, washed with water and the nitrogen determined in the usual way in the residue. By allowing the pepsin solution to act for two days as described above, the subsequent digestion with pancreas solution is superfluous.

=552. Suggestions Regarding Manipulation.=—The filter papers should be as quick working as possible to secure the separation of all undissolved particles. They should be of sufficient size to hold the whole contents of the digestion flask at once, since if allowed to become empty and partially dry, filtration is greatly impeded. The residue should be dried at once if not submitted immediately to moist combustion. After drying, the determination of the nitrogen can be made at any convenient time. Beaker flasks, i. e., lip erlenmeyers with a wide mouth are most convenient for holding the materials during digestion. The flasks are most conveniently held by a crossed rubber band attached at either end to pins in the wooden slats extending across the digestive bath. The bath should be suspended by cords from supports on the ceiling and a gentle rotatory motion imparted to it resembling the peristaltic action attending natural digestion.

=553. Natural Digestion.=—The digestion of foods by natural processes is determined chiefly by the classes of ferments already noted. The principle underlying digestive experiments with the animal organism may be stated as follows: A given weight of food of known composition is fed to a healthy animal under the conditions of careful control and preparation already mentioned. The solid dejecta of the animal during a given period are collected and weighed daily, being received directly from the animal in an appropriate bag, safely secured, as is shown in the accompanying figure. The dejecta are weighed, dried, ground to a fine powder, mixed and a representative part analyzed. The difference between the solid bodies in the dejecta and those given in the food during the period of experiment represents those nutrients which have been digested and absorbed during the passage of the food through the alimentary canal. The urine, containing solid bodies representing the waste of the animal organism, does not require to be analyzed for the simple control of digestive activity outlined above. In a complete determination of this kind the exhalations from the surface of the body and from the lungs are also determined. In the latter case the human animal is selected for the experiment; in the former it is more convenient to employ the lower animals, such as the sheep and cow.

The arrangement of the stalls and of the apparatus for collecting the excreta should be such as is both convenient and effective.