to boiling, add an excess of phosphotungstic acid, separate the precipitated proteids by filtration and wash with hot water, being careful that the temperature of the solution and wash water shall not be less than 90° C. at any time. Transfer the filter papers containing the tannic acid and phosphotungstic acid precipitates to a Kjeldahl flask and determine nitrogen. The nitrogen so obtained multiplied by 6.25 gives the percentage of proteoses, peptones, and gelatin. (f) MEAT BASES. Deduct from the total nitrogen the sum of the nitrogen obtained in the determination of insoluble proteids, coagulable proteids, proteoses, peptones, and gelatin for the nitrogen of the meat bases. Multiply the result by 3.12 to obtain the percentage of meat bases. 8. Starch. (In chopped meat, sausage, deviled meat, etc.) (a) QUALITATIVE DETERMINATION.-PROVISIONAL. Treat 5 or 6 grams of the sample with boiling water for two or three minutes; cool the mixture, and test the supernatant liquid with iodin solution. In using this test it must be remembered that a small amount of starch may be present as the result of the use of spices. If a marked reaction is given, however, it may be concluded that starch or flour has been added, and a quantitative determination may be made. The above qualitative method may be replaced by a microscopic examination, by which not only the presence of added starch, but also the variety employed may be determined. (b) QUANTITATIVE DETERMINATION. (1) AMBÜHL'S METHOD.-PROVISIONAL. This method is short and convenient, but the results obtained by it are only roughly approximate. Thoroughly macerate 2 grams of the meat under examination with 50 times its weight of water. Boil for thirty minutes and dilute to 100 cc for every gram of meat employed. Cool an aliquot of the liquid, treat with iodin, and compare the depth of color with solutions containing a known amount of the same kind of starch (the variety of starch in the sample is determined microscopically), and boiled for the same length of time. (2) MAYRHOFER'S METHOD & MODIFIED.-PROVISIONAL. Treat from 10 to 20 grams of the sample under examination (depending upon the amount of starch indicated by the iodin reaction) in a porcelain dish or Mallet employs two solutions, one containing 50 and the other 100 grams of crystalline phosphoduodecitungstic acid dissolved in 1 liter of 2.5 per cent hydrochloric acid. He also recommends the addition of sand or pulverized glass to prevent the formation of coagulated proteids in a dense clot. Owing to the liability of "bumping in the presence of such substances, however, during the determination of nitrogen it would seem that such addition should be avoided if possible. Pharm. Centralh., 1881, 22: 478; Abstract Zts. anal. Chem. 1882, 21: 436. Ambühl directs that from 2 to 10 grams be employed, according to the size of the meat particles. If the sample be macerated, however, as directed under the preparation of sample, it is unnecessary to employ a large amount. 4 Forschungsber. für Lebensm., 1896, 3: 141; Ibid., 1897, 4: 47. The casserole with 50 ce of an 8 per cent solution of potassium hydroxid and heat the mixture on the water bath until the meat is entirely dissolved. operation may be hastened by mascerating the larger pieces with a glass rod. Add an equal volume of 95 per cent alcohol, mix thoroughly, filter through an asbestos filter, and wash twice with a hot 4 per cent solution of potassium hydroxid in 50 per cent alcohol. Then wash with 50 per cent alcohol until a small portion of the filtrate does not become turbid upon the addition of acid. Return the precipitate and filter to the original vessel and dissolve, with the aid of heat, in 60 cc of a normal solution of potassium hydroxid. In the case of sausage with a high starch content a somewhat larger volume of alkali may be required. Acidify the filtrate strongly with acetic acid, dilute to a definite volume, thoroughly mix by shaking, filter through a folded filter, and precipitate the starch from an aliquot of the filtrate by means of an equal volume of 95 per cent alcohol. Transfer the precipitate to a filter, thoroughly wash with 50 per cent alcohol, with absolute alcohol, and finally with ether, dry to a constant weight at the temperature of boiling water, and weigh. 9. Glycogen. The determination of glycogen has been suggested as a means of detecting the presence of horse meat. Subsequent results indicate that this determination is of limited value because of the fact that glycogen begins to disappear soon after the death of the animal, and may entirely disappear after a short lapse of time. No definite conclusions can therefore be derived from the results of this determination, but it is of value as a confirmatory test. (a) QUALITATIVE METHOD.-PROVISIONAL. Boil 50 grams of the macerated sample with 50 cc of water for from fifteen to thirty minutes. Filter the broth through moistened filter paper or fine linen. To a portion of the filtrate in a test tube add a few drops of a mixture of 2 parts iodin, 4 parts potassium iodid, and 100 parts water. In the presence of a large percentage of horse meat the glycogen contained produces a dark-brown color, which is destroyed by heating and reappears on cooling. When starch is present it may be precipitated by two volumes of glacial acetic acid, separated by filtration, and the test for glycogen repeated in the filtrate. (b) QUANTITATIVE METHOD.C-PROVISIONAL. Digest 50 grams of finely macerated meat on the water bath with 200 cc of 2 per cent potassium hydroxid until solution is practically complete. Cool, dilute with water to exactly 200 cc, shake, and filter. Treat 100 cc of the filtrate with 10 grams of potassium iodid and 1 gram of potassium hydroxid and stir until solution is complete. Add 50 cc of 96 per cent alcohol and allow to stand until the following day. Separate the precipitated glycogen by filtration and wash with a solution containing 1 cc of 73 per cent potassium hydroxid, 10 grams of potassium iodid, 100 cc of water, and 50 cc of 96 per cent alcohol. Wash the glycogen with a mixture of two volumes of 96 per cent alcohol and one volume of water (the mixture containing about 7 mg of sodium chlorid per liter), dissolve in water, and remove the remaining traces of proteids by the addition of double iodid of mercury and potassium. It is a Niebel, Zts. angew. Chem., 1895, 620. Courlay and Coremons, Zts. Nahr. Waar., 1896, 10: 173. often found that the proteids are so completely removed that no precipitate is formed with the double iodid, and filtration is not necessary. Add about 2 mg of sodium chlorid per 100 cc of water, precipitate the glycogen again by means of two volumes of 96 per cent alcohol, filter, wash with 96 per cent alcohol, containing about 7 mg of sodium chlorid per liter, then with absolute alcohol, finally with ether, dry to constant weight, and weigh. As a control, invert the precipitated glycogen by boiling for three hours with hydrochloric acid, diluted with 10 parts of water, and determine the reducing sugar by Allihn's method, multiplying the result by 0.9 to obtain the percentage of glycogen. 10. Reducing Sugar.-Provisional. Boil 100 grams of the finely divided meat for fifteen or twenty minutes in a 500 cc graduated flask, with a convenient volume of water. Add a few cubic centimeters of normal lead acetate, cool to room temperature, make up to the mark with water, and filter through a folded filter. Remove the lead and deterufine reducing sugar as dextrose, as described under "VI. General Methods," page 49. 11. Potassium Nitrate. (a) SCHLÖSING-WAGNER METHOD.4-PROVISIONAL. Use a flask of about 250 cc capacity with a rubber stopper having two holes. Through one of them pass the stem of a funnel carrying a glass stopcock; the other carries a delivery tube leading to the receiving vessel. Bend the end of the delivery tube so that it will pass easily under the mouth of the measuring burette and cover with a piece of rubber tubing. Place 50 cc of saturated ferrous chlorid solution and the same quantity of 10 per cent hydrochloric acid in a flask. Prepare the ferrous chlorid solution by dissolving nails or other small pieces of iron in hot hydrochloric acid and keep in glass-stoppered flasks of about 50 cc capacity, entirely filled. The contents of one flask is enough for about twelve determinations, and by using the entire contents of a flask as soon as possible after opening all danger of oxidation is avoided. Boil the contents of the flask until the air is driven off; then place the delivery tube under the measuring tube, which is filled with 40 per cent potassium hydroxid, add a few drops of water, and cover the tube with pieces of filter paper. By a careful and quick inversion the measuring tube can be brought into the vessel receiving it without any danger of air entering. Extract 100 grams of finely macerated meat by boiling repeatedly with successive small portions of water, concentrate the aqueous solution to a small volume, transfer to the funnel, and, with continued boiling, allow it to pass drop by drop into the flask. When the solution has almost all run out, wash the funnel with three 10 cc portions of 10 per cent hydrochloric acid and allow these portions to pass drop by drop into the flask. The temperature of the surrounding water will soon be imparted to the contents of the tube, and the volume of nitric oxid is read with the tube in such a position that the level of the water within and without the tube coincides. The amount of nitric oxid present and the corresponding percentage of nitrate may be calculated in the usual way for the given temperature and barometric pressure, or, to avoid computation, the amount of nitrate may be determined Bieler and Schneidewind, Die agrikultur-chemische Versuchsstation Halle a/S, 1892, p. 50; Wiley, Principles and Practice of Agricultural Analysis, 1897, 2: 228. by comparison of the volume of nitric oxid with that evolved by a definite volume (5 to 10 cc) of normal sodium nitrate solution. (b) PHENOL-SULPHONIC ACID METHOD. PROVISIONAL. Weigh 1 gram of the sample into a 100 cc flask, add from 20 to 30 cc of water, and heat on the water bath for fifteen minutes, shaking occasionally. Add 3 cc of a saturated solution of silver sulphate for each per cent of sodium chlorid present, then add 10 cc of lead subacetate and 5 cc of aluminia cream, shaking after each addition. Make up to mark with water and filter through a folded filter, returning the filtrate to the filter until it runs clear. Evaporate to dryness 25 cc of the filtrate, add 1 cc of phenol-sulphonic acid, mix thoroughly with a glass rod, add 1 cc of water and 3 or 4 drops of concentrated sulphuric acid, and heat on a steam bath for two or three minutes, being careful not to raise the temperature sufficiently to char the material. Add about 25 cc of water and an excess of ammonium hydroxid. Transfer to a 100 cc flask, add 1 or 2 cc of alumina cream if not perfectly clear, dilute to the mark with water, and filter if necessary. Prepare a number of 50-cc Nessler tubes, preferably the long, narrow tubes, placing in the first 1 cc of the standard nitrate solution, containing 0.01 mg of nitrogen as potassium nitrate in each cubic centimeter, in the second 2 cc, and so on to 10 cc, then 12 cc, 15 cc, 18 cc, and 20 cc. Compare with the standards the solution prepared as directed above. If dilution is necessary to bring within this range, calculate to original concentration, and note in the following table the percentage of potassium nitrate in the original sample. The chemical preservatives commonly used with meat products are borax and boric acid and sulphites. Salicylic and benzoic acids are occasionally used. The methods for the detection of these preservatives are given under “XXVII. Food This method is a modification of the one ordinarily employed for determining potassium nitrate in water. It was adapted to the examination of meat by Mr. Arthur Given. Prepared by mixing 37 cc of concentrated sulphuric acid, 3 cc of distilled water, and 6 grams of phenol. Preservatives," page 179. In general, preservatives may be separated from meat by digesting a few minutes in warm water made slightly acid or slightly alkaline, according as the nature of the preservative is basic or acid. (a) SULPHUROUS ACID. The distillation method for the detection of sulphurous acid (see page 187, under "XXVII. Food Preservatives") may be employed for the examination of meat, using 20 cc instead of 5 cc of the 20 per cent solution of phosphoric acid. Mere traces should be ignored. According to Ostertaga the microscopic examination of meat that has been preserved with sodium or calcium sulphite often discloses the presence of crystals of sodium or calcium sulphate, due to partial oxidation of the sulphite. In the absence of chlorids and nitrates, Kämmerer a employs potassium iodate paper in the following manner: Place the sample of meat on potassium iodate paper and moisten it with dilute sulphuric acid (1:8) free from oxids of nitrogen. In the presence of even minute traces of sulphites a deep blue color is immediately formed, while in the absence of sulphites only a faint blue color is produced, and that after a considerable time. This method is of limited application, since it can not be used with meats containing salt or saltpeter. 13. Detection of Coloring Matter.-Provisional. Sausages and other preparations in which chopped meat is employed rapidly became discolored on exposure to the air. This change does not take place to a marked extent with meat that has been cured in a pickle containing saltpeter. With fresh chopped meat, and sometimes with corned meat, especially that cured without saltpeter, coloring matter is sometimes added to prevent the change of color which would naturally take place. The coloring matter may often be extracted by heating for fifteen or twenty minutes with 50 per cent alcohol, with 50 per cent glycerin slightly acidified, with a mixture of alcohol and glycerin, with ammonium hydroxid, or with a 5 per cent solution of sodium calicylate e in water. Approximately equal weights of meat and solvent may be used. In case the filtered extract by any of these methods is colored red or deep yellow, evaporate it nearly to dryness, slightly acidify with hydrochloric acid, and boil a few minutes after the addition of a thread of fat-free wool. If the wool is dyed, examine it as directed under XXVIII. Coloring Matter," page 190. If the wool is not dyed, examine the solution spectroscopically. 66 If too dilute, the solution may often be concentrated by precipitating the coloring matter as a lake,d that is, allow it to settle, decant the water, dissolve in hydrochloric acid, and make alkaline with ammonium hydroxid. In extracting with 50 per cent alcohol, the proteids of the meat are coagulated, with the formation of a pale, almost white, color. If the meat is not discolored during this extraction, it is probable that some foreign color is present. Marpmann examines sausages microscopically for the presence of coloring matter after dehydrating with alcohol and xylol consecutively, removing the xylol with carbon tetrachlorid, and immersing in cedar oil until the natural colors of the meat have disappeared. a Handbuch der Fleischbeschau, 3d ed., p. 826. & Bremer, Forschungsher für Lebensm., 1897, 4: 45. |