527. Classification.—In the preceding parts have been set forth the fundamental principles underlying the conduct of agricultural analysis and a résumé of the best practice of the art. The analyst, as a rule, will seldom be required to undertake investigations which are unnoticed in the preceding pages. Cases will arise, however, in which problems are presented which can not be solved by the rules already elucidated. In respect of the great classes of agricultural bodies, it will be observed that dairy products have already received special mention. In respect of foods and fodders in general, it is evident that they are chiefly composed of moisture, ash, carbohydrates, oils and proteid matters. The methods of identifying, separating and estimating these constituents have been fully set forth. It is not necessary, therefore, to study in this part the analytical processes which are applicable to cereals, cattle foods and other food products, further than is necessary to present in the most important cases a working résumé of principles and methods. There remain, however, certain products of importance which require some special modifications of treatment, and it is to these that the present part will be chiefly devoted. Among these are found tobacco, tea and coffee, fruits, fermented and distilled drinks and certain animal products. It is evident that an enumeration of all agricultural products, with a description of their methods of examination, would be impracticable in the available space and undesirable by reason of the repetition which would be required. In each case the analyst, in possession of the methods described, will be able to adapt the means at his disposal to the desired purpose to better advantage than any rigid directions could possibly secure.

In respect of the analytical methods of determining the nutritive value of foods, they may be divided into chemical and physiological. The chemical methods embrace the thermal and artificial digestion investigations, and the physiological include those which are carried out with the help of the animal organisms. In the latter case the digestive process is checked by the analysis of the foods before ingestion and of the excreta of all kinds during and after digestion.

It is evident that a detailed description of this method should be looked for in works devoted to physiological chemistry.

CEREALS AND CEREAL FOODS.

528. General Analysis.—The cereals are prepared for analysis by grinding until the fragments pass a sieve having circular perforations half a millimeter in diameter. The moisture, ash, ether extract, proteids and carbohydrates are determined by some one of the processes already described in detail. In this country the methods of the Association of Official Agricultural Chemists are generally followed.[535] For convenience these methods are summarized below.

Moisture.—Dry from two to three grams of the fine-ground sample for five hours, at the temperature of boiling water, in a current of dry hydrogen. If the substance be held in a glass vessel, the latter should not be in contact with the boiling water.

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

Ether Extract.—Pure ether is prepared by washing the commercial article four or five times with water to free it of the chief part of the alcohol it contains. The residual water is mostly removed by treating the liquid with caustic soda or potash. Any residual alcohol or water is finally removed by the action of metallic sodium. The ether thus prepared is stoppered, after the evolution of hydrogen has ceased, and is kept over metallic sodium. Immediately before use it should be distilled out of contact with moist air.

The residue from the determination of moisture, as described above, is extracted in an appropriate apparatus (39) with the pure ether for sixteen hours. The extract is dried to constant weight. The weight may be checked by drying and weighing the extraction tube and its contents before and after the operation.

Crude Proteids.—Proceed as in the method of determining nitrogen in the absence of nitrates and multiply the weight of nitrogen obtained by 6.25. This factor is a general one, but should not be rigidly applied. In each instance, according to the nature of the cereal, the appropriate factor, pointed out in paragraph 407 should be used, and the factor 6.25 be applied only in those cases where a special factor is not given. The factors for the common cereals are wheat 5.70, rye 5.62, oats 6.06, maize 6.22, barley 5.82 and flaxseed 5.62.

For separating the proteid matters consult paragraphs 392-410. In the case of wheat the methods of Teller may be consulted.[536]

Amid Nitrogen.—The albuminoid nitrogen is determined as directed in paragraph 203 of volume II. The difference between this number and that representing the total nitrogen gives the nitrogen as amids.

Fiber and Carbohydrates.—The methods of analysis are described in detail in Part Third.

529. Bread.—In general, the same processes are followed in bread analysis as are used with cereals and flours. In addition to the regular analytical processes, breads are to be examined for adulterants, bleaching and coloring matters, and for the purpose of determining the changes which have taken place in their nutrient constituents in the processes of fermentation and cooking.

Temperature of Baking.—The interior of a loaf during the process of baking does not attain the high temperature commonly supposed. This temperature is rarely found to be more than one degree above the boiling point of water.[537] In biscuits and other thin cakes, which become practically dry and which by reason of their thinness are the more readily penetrated by heat, the temperature may go as high as 110°.

Soluble Extract.—The quantity of matters both in flour and bread, soluble in cold water, is determined by extraction in the usual way and drying the extract. Soluble albuminoids, sugars and mineral salts are extracted by this process. When possible, the operation should be conducted both on the bread and the flour from which it is made.

Color.—In baker’s parlance is found an apparent contradiction of terms, since it speaks of bread with “no color” when the loaf is dark brown, while a white loaf is said to have a high color. An ideal color for the interior of a loaf is a light cream tint, which is more desirable than a pure white.[538] The texture, odor and flavor of the loaf are also to be considered, but these are properties of more importance to the technical expert than to the analyst.

Quantity of Water.—It is not possible to set a rule of limitation in respect of the quantity of water a bread should hold. For full loaves, perhaps forty per cent is not too high a maximum, while some authors put it as low as thirty-four per cent. Some flours are capable of holding more water than others, and the loaf should have just enough water to impart to the slice of bread the requisite degree of softness and the proper texture. Most breads will have a content of water ranging from thirty to forty per cent. In biscuits and other thin cakes the moisture is much less in quantity.

Acidity.—The acidity of both bread and flour is determined by shaking ten grams of the sample with 200 cubic centimeters of distilled water for fifteen minutes, pouring the mass on a filter and titrating an aliquot part of the filtrate with tenth-normal alkali. The acidity is reckoned as lactic acid in the case of breads raised by fermentation.

Nature of Nitrogenous Compounds.—The methods of investigation are described in paragraphs 392-410.

530. Determination of Alum in Bread.—The presence of alum in bread may be detected by means of logwood. Five grams of fresh logwood chips are digested with 100 cubic centimeters of amyl alcohol. One cubic centimeter of this decoction and the same quantity of a saturated solution of ammonium carbonate are mixed with ten grams of flour and an equal quantity of water. With pure flour, a slight pink tint is produced. In the presence of alum the color changes to a lavender or blue, which is persistent on heating.

The test may be varied by diluting five cubic centimeters of the reagents mentioned with ninety cubic centimeters of water and pouring the mixture over ten grams of the crumbled bread. After standing for five minutes, any residual liquid is poured off and the residue, washed once with a little water, is dried in a steam bath, when the blue color is developed if alum be present.[539]

531. Chemical Changes Produced by Baking.—Changes of a chemical nature, produced in bread by baking, are found chiefly in modifications of the starch and proteids. The starch is partly converted into dextrin and the albumins are coagulated. The changes in digestion coefficient are determined by the methods which follow. The fermentations which precede the baking are due to the usual decompositions of the carbohydrates under the influence of yeast germs.

FODDERS, GRASSES
AND ENSILAGE.

532. General Principles.—The analyst, in examining the fibrous foods of cattle, is expected to determine moisture, ash, fiber and other carbohydrates, ether extract and albuminoid and amid nitrogen. If a more exhaustive study be required, the sugar and starch are separated from the other non-nitrogenous matters, the carbohydrate bodies yielding furfuraldehyd separately determined and the ash subjected to a quantitive analysis. The processes are conducted in harmony with the principles and methods of procedure fully set forth in the preceding pages.

Green fodders and grasses are easily dried and sampled by comminution in the shredder described on page 9, and roots by that shown on page 10. The moisture is determined by drying a small sample of the shredded mass, while the rest of it is dried, first at about 60° and finally at 100°, or a little above, ground to a fine powder and subjected to analysis by methods already described. The food values as obtained by analysis should be compared, when possible, with those secured by natural and artificial digestion.

Ensilage is shredded and analyzed in precisely the same way, but in drying, the content of volatile acids formed during fermentation must be considered. In other words, the loss on drying ensilage at 100°, or slightly above, is due not only to the escape of water but also to the volatilization of the acetic acid, which is one of the final products of fermentation which the mass undergoes in the silo.

533. Organic Acids in Ensilage.—In the examination of ensilage, the organic acids which are present may be determined by the processes described in following paragraphs. The acetic acid, formed chiefly by fermentation, is conveniently determined by the method given for tobacco further on. Lactic acid is detected and estimated by expressing the juice from a sample of ensilage, removing the acetic acid by distillation, repeated once or twice, and treating the filtered residue with zinc carbonate in excess, filtering and determining the zinc lactate in the filtrate. The zinc is determined by the method described for evaporated apples and the lactic acid calculated from the weight of zinc found. Crystallized zinc lactate contains 18.18 per cent of water and 27.27 per cent of zinc oxid.[540]

534. Changes due to Fermentation in the Silo.—Silage differs from green fodder in having less starch and sugar, more acetic and lactic acids and alcohol and a higher proportion of amid to albuminoid nitrogen.[541] There is also a considerable loss of nitrogenous substances in ensilage, due probably to their conversion into ammonium acetate, which is lost on drying.

535. Alcohol in Ensilage.—The fermentation which takes place in the silo is not wholly of an alcoholic nature, as the development of lactic acid, noted above, clearly indicates. The alcohol which is formed may escape and but small quantities can be detected in the ripened product. So small is this quantity of alcohol that it appears to be useless to try to secure a quantitive estimation of it. Qualitively, it may be detected by collecting it in a distillate, which is neutralized or made slightly alkaline with soda or potash lye and redistilled. The greater part of the alcohol will be found in the first few cubic centimeters, which are made alkaline with potash lye and as much iodin added as can be without giving a red tint to the solution. Any alcohol which is present will soon separate as iodoform.

536. Comparative Values of Fodder and Ensilage.—In judging of the comparative values of green and dry fodders for feeding purposes, it is necessary to secure representative samples in the green, quickly dried and ensilaged condition. It is quite certain that the greater part of the sugar contained in green fodders is lost both by natural curing and by placing in a silo. When well cured by the usual processes there is but little loss of nitrogenous matters, but in the silo this loss is of considerable magnitude, amounting in some instances to as much as thirty per cent.

The ideal way of preparing green fodders in order to preserve the maximum food value efficiently, is to shred them and dry rapidly by artificial heat, or in the sunlight, until they are in a condition which insures freedom from fermentation. In this condition, when placed in bales, under heavy pressure, the food constituents are preserved in the highest available form. The immense sugar content of the stalks of maize and sorghum could be preserved in this way almost indefinitely.

FLESH PRODUCTS.

537. Names Of Meats.—The parts of the animal from which the meats are taken have received distinctive names, which serve to designate the parts of the carcass offered for sale. These names are not invariable and naturally are quite different in many markets. In this country there is some degree of uniformity among butchers in naming the meats from different parts. The names in scientific use for the parts of mutton, beef and pork are found in the accompanying illustrations.[542]

538. Sampling.—When possible the whole animal should constitute the sample. The relative weights of blood, intestinal organs, hide, hoofs, horns, bones and edible flesh are determined as accurately as possible. The general method of preparing samples of animal products is given in paragraph 5.

The method of sampling employed by Atwater and Woods is essentially that just noted.[543] The sample, as received at the laboratory, is weighed, the flesh (edible portion) is then separated from the refuse (skin, bones etc.) and both portions weighed. There is always a slight loss in the separation, evidently due to evaporation and to small fragments of the tissues that adhere to the hands and to the implements used in preparing the sample. The perfect separation of the flesh from the other tissues is difficult, but the loss resulting from this is small. In sampling the material for analysis, it is finely chopped, either in a tray or in a sausage cutter, and in each case is well mixed.

539. Methods of Analysis.—The general methods for the analyses of food products are applicable to meats and animal products in general. In the separation of the nitrogenous constituents the methods described in paragraphs 411-414 are followed. It is not safe to estimate as proteids the total nitrogen multiplied by 6.25, since the flesh bases have much higher percentages of nitrogen than are found in proteid matters. As indicated in paragraph 280 the complete extraction of dried meats by ether is difficult of accomplishment. After a few hours it may be assumed that the total extract will represent the fat, although additional soluble matters are obtained by continuing the process. The heat producing power may be calculated from the analytical data secured. The methods which have been described in the preceding pages will be found sufficient for guidance in the examination of animal products, and the analyst will find them, when modified to suit particular cases, adapted to the isolation and estimation of proximate food principles.

The methods of analyses followed by Atwater and Woods are given below:[544]

Water and Water-Free Substance.—The drying is done in ordinary water ovens at a temperature of nominally 100°, but actually at 96° and 98°. For each analysis of animal tissues (flesh) one or more samples of from fifty to one hundred grams of the freshly chopped substance are weighed on a small plate, heated for from twenty-four to forty-eight hours, cooled, allowed to stand in the open air for about twenty-four hours, weighed, ground, sifted through a sieve with circular holes one-half millimeter in diameter, bottled and set aside for analysis. In case of fat samples which cannot be worked through so fine a sieve, either a coarser sieve is used or the substance crushed as finely as practicable and bottled without sifting.

For the complete desiccation, about two grams of material are dried for three hours. It is extremely difficult to get an absolutely constant weight, though it is found that this is in most cases approximately attained in four hours.

Nitrogen, Protein, Albuminoids etc.—The nitrogen is determined in the partly dried substance by the method of Kjeldahl. The protein is calculated by multiplying the percentage of nitrogen by 6.25. The nitrogenous matters in meats and fish, i. e., in the materials which have practically no carbohydrates, are also estimated by subtracting the sum of ether extract and ash from the water-free substance, or the sum of water, ether extract and ash from the fresh substance, the remainder being taken as proteids, albuminoids etc., by difference. While this is not an absolutely correct measure of the total nitrogenous matter, it is doubtless more nearly so than the product of the nitrogen multiplied by 6.25.

Fat (Ether Extract).—The fat is extracted with ether in the usual manner. The point at which the extraction is complete is not always easy to determine. For the most part, the extraction is continued for such time as experience indicates to be sufficient, and then the flask is replaced by another and the extraction repeated until the new flask shows no increase in weight.

According to experience, the fat of many animal tissues is much more difficult to extract than that of most vegetable substances. In general, the greater the percentage of fat in a substance the more difficult is the removal of the last traces. Dried flesh is frequently so hard that the fineness of the material to be extracted seems to be a very important matter.

Ash.—Ash is determined by the method recommended by the Association of Official Agricultural Chemists.

Food Value—Potential Energy.—The food materials are not necessarily burned in the calorimeter, but the fuel value of a pound of each of the foods, as given in the tables, is obtained by multiplying the number of hundredths of a pound of protein and of carbohydrates by 18.6 and the number of hundredths of a pound of fat by 42.2, and taking the sum of these three products as the number of calories of potential energy in the materials.

More reliable results are obtained by using the factors obtained by Stohmann; viz., 5731 calories for proteids, 9500 calories for common glycerids, 9231 calories for butter fat, 3746 calories for pentose sugars, 3749 calories for dextrose and levulose and 3953 calories for sucrose and milk sugar.[545]

540. Further Examination of Nitrogenous Bodies.—It is evident that both of the methods proposed above for the examination of the nitrogenous constituents of meats are unreliable. If the total nitrogen be determined and multiplied by 6.25 the product does not by any means represent the true quantity of nitrogenous matter since the flesh bases contain in some instances more than twenty-five per cent of nitrogen.

If, on the other hand, the water, ash and fat in a meat sample be determined and the sum of their per cents be subtracted from 100, the difference represents the nitrogenous bodies plus all undetermined matters and errors of analysis. The assumption that meats are free of carbohydrates is not tenable since glycogen is constantly found therein and in horse flesh in comparatively large amounts. In a thoroughly scientific analysis of meats, the nitrogenous bodies should be separated and determined by groups, according to the principles developed in paragraphs 411-414. This process requires a great amount of analytical work and in general it will be sufficient to make a cold water extract to secure the flesh bases and a hot water extract to secure the gelatin. The nitrogen is then determined in each of these portions separately. The nitrogen in the cold water extract is multiplied by four, in the hot water extract by six and in the residue by 6.25. The sum of these products represents approximately the total nitrogenous matter in the sample.

Aqueous extracts containing nitrogen are easily prepared for moist combustion by placing them in the digestion flasks, connecting the latter with the vacuum service and evaporating the contents of the flask nearly to dryness. The sulfuric acid is then added and the nitrogen converted into ammonia and determined in the usual manner.

541. Fractional Analysis of Meats.—A better idea of the composition of a meat is obtained by separating its constituents into several groups by the action of different solvents. This method has been elaborated by Knorr.[546]

The separation of the meats in edible portion and waste and the determination of moisture and fat are conducted as already described. The residue from the fat extraction is exhausted with alcohol, and in the extract are found the nitrogenous bases kreatin, kreatinin, sarkin and xanthin, and urea, lactic, butyric, acetic and formic acids, glycogen and inosit. In the residue from the alcohol extraction, the proteid nitrogen is determined in a separate sample.

A separate portion of the sample is ground to a fine paste and repeatedly rubbed up with cold water, which is poured through a tared filter. When the extraction is complete, the filter and its contents are dried and the dry residue determined. This residue represents the nitrogenous constituents of the muscle fibers and their sheaths together with any other bodies insoluble in cold water. The filtrate from the cold water extraction is heated to boiling to precipitate the albuminous matters which are collected, dried and weighed, or the nitrogen therein determined and the albuminous matters calculated by multiplying by the usual factor. The filtrate from the coagulated albuminous bodies is evaporated to dryness and weighed. It consists essentially of the same materials as the alcoholic extract mentioned above. The ash and nitrogen in the aqueous extract are also determined.

The mean content of the edible parts of common meats, expressed as per cents in groups as mentioned, follow:

542. Estimation of Starch in Sausages.—Starchy substances are sometimes added to sausages for the purpose of increasing their weight. The presence of starch in a sausage is easily detected by iodin. The quantity may be determined by the following process:[547]

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.[548]

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.[549]

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.[550]

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[551] and have been mistaken for gall stones.

The fat which enters the chyle appears to be mostly unchanged, except that it is emulsified.[552] 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.[553]

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.[554] The aliphalytic ferments exist also in plants and have been isolated from rape seed.[555]

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.[556] For a summary of digestion experiments in this country the résumé prepared by Gordon may be consulted.[557] The method followed in this laboratory is fully described by Bigelow and Hamilton.[558]

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.[559] 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.[560]

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.[561] In order to avoid the trouble of preparing the pancreas solution pure active pancreatin may be used.[562] 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.[563]

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.[564] This process and also a slight modification of it have been used with success by Bigelow and McElroy.[565] 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:[566]

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.[567]

The method of constructing a bag for attachment to a sheep is shown in Fig. 118. It is made according to the directions given by Gay, of heavy cloth and in such a way as to fit closely the posterior parts of the animal.[568] When attached, its appearance is shown in Fig. 119.