2 minutes with 100 cc. of a mixture of 100 grams of alcohol, 300 grams of powdered ice, and 600 grams of ice water, decant onto the filter adding a small piece of ice. Repeat the treatment three times or until the solution. is colorless keeping the decanted portions in ice water during filtration, use two or three Büchner funnels if the filtration is slow and take care that the temperature does not rise above +5° C. Finally boil the mixture of sand and residue, together with the asbestos, with several portions of water, filter, wash with hot water, evaporate the filtrate, and determine nitrogen in an aliquot. Determination of Myosin. König states that myosin may be obtained in the residue after boiling with water by digestion with 15% ammonium chloride solution, filtration and precipitation of the myosin in the filtrate by diluting and boiling or by salting out with sodium chloride or magnesium sulphate. The myosin separated by filtration is dissolved in a definite volume of concentrated sulphuric acid and an aliquot used for nitrogen determination. He considers that the nitrogenous matter remaining undissolved after successive treatment with cold water, boiling water, and 15% ammonium chloride solution is sarcolemma or insoluble muscle fiber. The methods for collagen and myosin are not entirely satisfactory especially in view of our imperfect knowledge of the albuminoids and globulins, present in meat.† Determination of Coagulable Protein (Albumin).-Grindley and Emmett Method.‡—Evaporate 150 cc. of the cold water extract obtained as directed by Emmett to 40 cc. If necessary add carefully very dilute acetic acid or sedium hydroxide solution until faintly acid to litmus paper and boil. Collect the coagulum on a filter, wash with hot water, and determine nitrogen, correcting for any nitrogen that may be present in the paper. NX6.25 coagulable protein or albumin. Trowbridge and Grindley § obtain the maximum results in fresh beef by neutralizing one-fourth of the acidity to phenolphthalein before coagulation. Some analysts filter before neutralization and determine separately * Chemie der Menschlichen Nahrungs- und Genussmittel, 4 Auf. III Bd. 2 Th., 1914, P. 24. † See v. Fürth, Arch. Path. Pharm., 37, 1896, p. 389; Ergebnisse der Physiologie Ab. 1, I, 1902, p. 110; Ibid., Ab. 2, I, 1903, p. 575. ‡ Jour. Amer. Chem. Soc., 27, 1905, p. 665; U. S. Dept. of Agric., Bur. of Chem., Bul. 162, p. 146. § Jour. Am. Chem. Soc., 28, 1906, p. 494. any precipitate that forms on boiling after neutralizing. The second precipitate is known as syntonin or acid albumin. The filtrate from one duplicate may be used for determining proteoses, the filtrate from the other for determining total creatinine. Determination of Proteoses, Peptones, Creatine, Creatinine, Purine Bases, and Total Meat Bases. Follow the methods as described under meat extracts (pp. 253 to 258). No accurate method is available for the determination of total meat bases. The practice of obtaining them from the remainder after subtracting the sum of the coagulable and proteose nitrogen from the total water soluble nitrogen, or after subtracting the proteose nitrogen from the total nitrogen in the filtrate from the coagulable nitrogen as followed by Richardson, does not correct for the peptones or related substances thus determined with the bases. Bigelow and Cook's procedure of calculating the nitrogen in the filtrate from the tannin-salt precipitate, correcting for nitrogen in the reagent and ammonia in the sample, and multiplying by 3.12 to obtain the meat bases is subject to error in that part of the creatine is precipitated by the tannin-salt reagent and that the factor is accurate for only one base, creatine; furthermore, according to Richardson, the method is difficult to handle and obtain concordant results due (in part at least) to nitrogen in the reagent. The estimation of the " peptones" by substracting the sum of the coagulable, proteose, and meat base nitrogen by the tannin-salt method from the total soluble nitrogen and multiplying by 6.25 is likewise unsatisfactory although probably the best procedure now available. Determination of Ash.-Incinerate the residue from the total solids in the original dish at a low red heat. It is usually advantageous, especially in the case of salt meat, to exhaust the charred sample with water, collect the insoluble residue on a filter and ignite. The filtrate is then added, evaporated to dryness, and the whole heated to low redness and weighed. A perfectly white ash is difficult to obtain. Determination of Mineral Constituents.-Determine in the original meat total sulphur and in the ash chlorine, potassium, sodium, phosphoric acid, and other mineral constituents, following the usual methods of analysis. The scheme for ash analysis given on page 310 is applicable to meat ash. Determination of Acidity. The acidity of meat is due largely to d-lactic acid with small amounts of succinic, acetic, and other acids. The results by direct titration are usually calculated in terms of lactic acid. Mondschein found that about one-third of the acid is, held back in the coagulum and proposes the following method: Mondschein Method.*-Suspend 50 grams of the finely ground sample in 60 to 80 cc. of water, coagulate by boiling. filter by suction, and wash with boiling water three times or until the reaction is no longer acid. Titrate the filtrate with N/10 alkali using phenolphthalein as indicator. Calculate as free lactic acid. Remove the matted coagulum from the filter, stir up in a beaker with 50 cc. of water, add 10 cc. of 10% sodium hydroxide solution, and boil, taking care that the liquid does not froth over, thus liquefying the mass except for a few particles. Add 100 cc. of saturated sodium chloride solution, heat to boiling, and saturate at boiling heat with solid sodium chloride. Filter the precipitate thus formed with the aid of suction, wash with a hot saturated sodium chloride solution, and add to the filtrate sulphuric acid to faint acid reaction, thus precipitating proteins. Boil, add 5 cc. of sulphuric acid, make up to 500 cc., and filter through a dry paper. Heat 250 cc. of the filtrate to boiling and add N/10 potassium permanganate solution until the lactic acid is split up into acetaldehyde, carbon dioxide, and water.† Add an equal bulk of potassium bisulphite solution (12 grams per liter), the strength of which has been determined against N/10 iodine solution, mix, and after 15 minutes titrate with N/10 iodine solution 1 cc. of which equals 0.005 gram lactic acid.‡ Mondschein also describes a more complicated method for use in separating d-lactic from 8-hydroxybutyric acid, which however does not appear to have practical application in meat analysis. Determination of Starch. In the following methods it is assumed that glycogen is not present in sufficient amount to appreciably affect the results. If microscopic examination shows the presence of starch and horse meat or liver is suspected, follow the Mayrhofer- Polenske method (p. 234). Mayrhofer Method. The first paragraph of the description of the Mayrhofer-Polenske method is essentially the Mayrhofer method as originally devised for starch. Mayrhofer-Price Method. §-Heat on a boiling water bath 10 grams of finely-divided meat with 75 cc. of 8% potassium hydroxide in 95% alcohol * Biochem. Zeits., 42, 1912, pp. 91, 105. † v. Fürth and Charnass, Biochem. Zeits, 26, 1910, p. 199. § U. S. Dept. of Agric. Bur. of Anim. Ind. Circ., 203, 1912. until all the meat is dissolved (30 to 40 min.). Add an equal volume of 95% alcohol, cool, and after 1 hour decant carefully on a Gooch crucible with a thin layer of asbestos. Wash carefully by decantation twice with 4% potassium hydroxide in 50% alcohol and twice with warm 50% alcohol. Add to the residue and crucible with contents 40 cc. of water and then with constant stirring 25 cc. of concentrated sulphuric acid. After 5 minutes add 40 cc. of water and heat just to boiling with constant stirring. Transfer to a 500-cc. graduated flask, add 2 cc. of 20% phosphotungstic acid solution, cool, make up to the mark, mix, and filter through starchfree filter paper. Neutralize an aliquot portion of the filtrate and determine dextrose by one of the methods described in Chapter XIV. Price recommends Low's method.* Identification of Horse Flesh.-Although certain authorities have found distinguishing characteristics in color, consistency, odor, etc., between horse flesh on the one hand, and beef and pork on the other, it is extremely difficult, by its physical properties, to detect horse flesh when mixed with other meat, especially when the mixture is chopped. Horse flesh has a much coarser texture and is darker in color than beef. The muscle fibers are, as a rule, shorter in horse flesh. On treating horse flesh with formaldehyde, Ehrlich † has found that a very characteristic odor is developed within forty-eight hours, suggestive of roasted goose flesh. Certain of the constants of the fat of horse meat differ from those of beef and pork, notably the iodine value and the refractometer readings. These constants are compared as follows: The fact that glycogen usually exists to a much larger extent in horseflesh than in other meat, and that a considerable amount remains after that of other meat has disappeared, renders it possible in some cases to detect horse flesh, when present in the mixture. The following table prepared by Bujard shows the relative amount of glycogen in various kinds of meat and sausages: In beef Bujard found 0.073 and 0.74 per cent of glycogen calculated in terms of dried substance, and, in sausages made exclusively from horse meat, amounts of glycogen ranging from 0.05 to 5.34, the sample in the latter case being made from the liver. It was formerly thought possible to detect as small an amount as 5% of horse flesh in mixture, but later investigation showed that after the death of the animal, glycogen, though present at first in considerable quantity, decomposes more or less rapidly, going over into muscle sugar (dextrose). Hence, while the presence of much glycogen is suspicious, its absence is by no means proof that horse flesh was not used. Niebel did not consider the failure of the glycogen test as sufficiently conclusive to establish the absence of horse flesh, on account of the tendency toward decomposition of the glycogen. In the absence of starch, he regards the presence of more than 1% of dextrose in the fat-free meat, after conversion of the carbohydrates, to be proof of the presence of horseflesh. Detection of Glycogen.-From the well-known reaction produced by iodine on glycogen, horse flesh can often be detected, when present in sausages, unless obscured by the presence of starch or dextrin. Brautigam and Edelmann * proceed as follows: 50 grams of the finely divided meat are boiled with 200 cc. of water for an hour, and, after cooling, dilute nitric acid is added to the broth to precipitate the proteins and to decolorize. The broth is then filtered, and a portion of the filtrate is treated in a test-tube with a freshly prepared, saturated, aqueous solution of iodine, or, better, with a mixture of 2 parts iodine to 4 parts potassium iodide and Ico parts water, the reagent being carefully added so as not * Pharm. Central., 1898, p. 557. |