776
INDUSTRIAL AND ENGINEERING CHEMISTRY
solvent for both the substance being investigated and the Grignard reagent. From a practical standpoint the complete reaction must take place in a reasonable length of time, which requires that the reaction mixture be so dilute that a precipitate does not interfere by keeping the reactants from each other. Heating at a high temperature or very long standing is not a satisfactory suhstitute for a proper concentration of the alcohol in the reaction mixture. The apparatus described by Kohler and co-workers has been modified as to stopcocks and jacketing and made much more portable, The use of tank nitrogen without further purification is suggested as an inert atmosphere for the Zerewitinoff determination.
Vol. 14, No. 10
Results are given for analyses using methanol, ethanol, 1propanol, tert-butyl alcohol, allyl alcohol, and acetophenone.
Literature Cited ~chim ~ itaz., ~ so, . 11, 53 (1g20).
(1) ciusa, R.,G ( 9 ) Flaschentragcr, 2. phyrioz. Chem.. 146, 219 (1925). (3) Haurowitz, F., Mikrochemie, 7, 88-93 (1929).
~ ~ ~ ~ ~ ~ ~ ~ , " d d c ~ ~ (1904), 7s;;d,0,0;~~~~
(6) Kohler a n d Richtmeyer. J . Am. Chem. SOC.,52, 3737 (1930). (7) Kohler, Stone. and Fuson, Ibid., 49, 3181 (1927). (8) Lieff, Wright, and Hibbert. Ibid., 61, 865 (1939). (') Marian and Marian, Biochem. J * * 746 (193")* (10) Oddo, Ber., 44, 2048 (1911). ( 1 1 ) Zerewitinoff,Ibid,, 40, 2023 (1907). (12) Ibid., 45, 2384 (1912). 241
Determination of Soybean Flour in Sausages and Other Meat Products A Protein Separation Method JOHN BAILEY, Illinois Department of Agriculture, Division of Foods and Dairies, Chicago, Ill.
S
AUSAGE and similar meat food products are prepared from meats and meat by-products that are usually pickled and then cooked and smoked. They may contain a little spice, cereals, starch, milk powder, and eggs (16, 68), and also salt, nitrates, nitrites, sugars, and sodium benzoate (67). The regulations provide that the combined amount of cereal, starch, flour, and milk powder in sausages shall not exceed 3.5 per cent (66), and in certain meat products it is also necessary to know, for label requirements, whether more or less than 5 per cent has been added (66). Soybean flour is now finding a use in these products, and food control officials and members of the industry realize that it will eventually be necessary t o develop a method of analysis for determining the percentage in these products.
Available Methods To analyze such a complex mixture for the amount of soybean flour, some particular constituent of the soybean must be found which will differentiate the soybean from all the other ingredients. This constituent or a definite part of it must withstand the manufacturing process of the sausage, as the cooking and smoking, and also its own manufacturing process in being converted into flour, as the oil expression or extraction. La Wall and Harrisson presented a test based on thp enzyme urease, which may be detected by the liberation of ammonia from urea (36). This is limited to sausage samples where the urease has not been destroyed or inactivated. and later the authors advised ronfirming the test by identifying the characteristic cell structures (87). On the other hand, it has been recommended that, the enzymes of soybean flour be inactivated by heating to a sufficiently high temperature for better processing (18). Kerr recommended these tests as qualitative, pointed out some limitations, and concluded that a quantitative method is urgent (3s). In locating the hourglass or I-shaped rells with the microscope, it is best to examine with polarizcd liAht, under which they take on a certain sheen. The present-day methods of preparing flour make it almost impossible to locate a single cell even with polarized light. Assuming that thcre is little or no nitrogen-free extract in meat, excepting liver products, but a considerable amount in soybean, Hayward reported on a calculating method (14), and also on an immunological method developed by Glynn which is
based on a quantitative precipitin test (II). On the former test no further report was made by the A. 0. A. C., while on the latter a detailed study was recently published (7). The method appears to be specific for soybean but it requires time for the production of a satisfactory serum and the zone of optimal precipitation may shift because of the presenre of other ingredients. A method based on the determination of insoluble nonfermentable sugars was proposed by Hendrey (15). After the sausage is givrn a preliminary treatment to remove moisture and fat, the soluble sugars are removed with 50 per cent alcohol, and the remaining carbohydrates are hydrolyzrd with hydrorhlnric acid and t h e n fermented nith yeast. The sugars remaining after the fermentation are determined and multiplied by a factor. This method was tried out by Lythgoe d a / . (38).who concluded that it is sufficiently accurate for law-enforccment purposes but not specific for eoybcan. For some time the present author has been working on the chemical separation of the soybean protein and has rendered several reports, but has withheld publication because the method was not previously adaptable to liver sausage or t o meat products that contained liver.
Theory Soybean or soy flour is prepared by finely grinding soybeans, removing the hulls, and debittering, usually by a steaming process. The flour may contain all the fat of the soybean but is generally low in fat, seldom containing above 6 or 7 per cent. Soybean flour may be considered an offspring of the soybean oil meal. Commercial flours in 1915 gave a n average protein content of 42.5 per cent (6S), while oil meals prepared by present-day manufacturing methods give slightly higher figures (71). The chief protein of soybean is a globulin, glycinin, so named by Osborne and Campbell (63). A great part of this protein dissolves in a water solution of the ground seed,. probably owing t o the presence of phosphates, and is precipitated by the addition of a little acid in the form of white spheroids. Some other properties of glycinin are given in Table I. The principle of the method is based on the separation of glycinin or a definite part of it from all the other proteins found in meats and in other binders and vegetable substances included in meat products, Glycinin can be boiled in a water solution with little coagulation and the remaining sol-
ANALYTICAL EDITION
October 15, 1942
uble portion will still be precipitable a t its isoelectric point. On the other hand, the albumins and the animal globulins in general are coagulable by heat, and the uncoagulable water-soluble proteins and the nitrogenous and nonnitrogenous extractives can be filtered through. The glycinin can then be dissolved in a 10 per cent sodium chloride solution, in which it will withstand almost a boiling temperature and a saturation with magnesium sulfate. Any additional gelatin dissolved (4) and perhaps other soluble meat proteins, as well as any casein present (SQ), are expected to precipitate with this saturation. On saturating further with sodium sulfate the glycinin will finally precipitate, possibly with other very soluble proteinate substances, as the solution is strongly concentrated with salts. To obtain this glycinin in a purer state it can again be dissolved and its property of precipitating a t half ammonium sulfate saturation can be utilized. Those proteoses and nitrogenous extractives, which previously had been precipitated along with the glycinin, remain in solution at this relatively low concentration of salt. The precipitated glycinin can again be redissolved and then precipitated as a n insoluble metal proteinate that will lend itself to thorough washing and drying for a final weighing. The vegetable proteins can also be eliminated in the same manner. Among the cereal grains the chief proteins are the prolamins, which are insoluble in salt solutions, and the glutelins, which are also insoluble and coagulable. As may be seen from Table I, there exists only a small amount of globulin in the cereals, wheat, corn, rye, barley, oats, rice, and buckwheat. Flours of tuber origin, like the potato, sweet potato, and tapioca, contain a small amount of protein. The
Source Soybean Wheat flour Embryo Bran Endosperm Corn Rye Barley Oats Rice Buckwheat Potato Sweet potato Tapioca Cottonseed -4dsuki bean
N u n g bean
3 to 7 2.6 16
Tomato seed Peanut meal Pea Cuu pea
Solutions and Materials
...... .....
.........
......... ... .....
......... ......
small amount Small amount
......... .........
......... ......
S&ll amount
.........
.........
...... ...... ......... .........
25
8
..............
Protein
16 ...... Sniall amount
Telvet bean Jack bean
predominating proteins in the oilseeds and legumes are globulins. Cottonseed flour is high in protein, and although its main globulin requires more than half ammonium sulfate saturation for precipitation, it will probably be eliminated earlier in the procedure because of being coagulable. The globulins of the beans and tomato seed are similar. Part of the globulins of peanut and peas have two properties in common with glycinin, uncoagulability and low ammonium sulfate precipitation limits. Because their isoelectric points are higher than that of glycinin (6),they can be expected to filter through when glycinin is precipitated with acid and filtered. Part of the casein can also be eliminated in the same way. I n egg white there are the glycoproteins which along with the nucleoproteins may be entirely neglected because of their presence in insignificant quantities. Likewise, the pumpkin or squash and malt of malted milk contain small amounts of protein. A scheme of analysis can then be worked out to eliminate all t.he proteins and the like save a definite p,wt of the glycinin, which would be a factor of the amount of soybean flour.
IODINE INDICATOR. Dissolve 0.1 gram of iodine and 0.4 gram o:i potassium iodide in 30 cc. of water. ETHYLORANGE INDXATOR (Eastman Kodak No. 122). Dissolve 0.015 gram in 30 cc. of 50 per cent alcohol. METHYLRED INDICATOR. Dissolve 0.03 gram in 30 cc. of 60 per cent alcohol. INDICATOR. Dissolve 0.3 gram in 30 cc. of PHENOLPHTHALEIN 50 per cent alcohol. PHENOLRED INDICATOR. Dissolve 0.03 gram in 30 cc. of water and add 0.1 cc. of N sodium hydroxide. This solution should be just yellow. MALT DIASTASESOLUTION.Digest 2 grams of U. S. P. IX malt d i a s b e TABLEI. REMOVAL OF GLOBULINS AND SIMILAR PROTE:[NS powder in 100 cc. of water at room Isotemperature for 1 or 2 hours and dePrecipitates OIL electric cant the clear solution through a filter, Protein Present Globulin, etc. Coagulation Saturation Point Citation refiltering the first 20 to 40 cc. of filtrate % c. through the same filter. ....... SOLVENTS.Acetone and carbon tetrachloride (technical grades). ................................. 0a Globulin Globulin ................................. SALTS. Sodium chloride, magnesium 2 Globulin ................................... (98) sulfate crystals, sodium sulfate anhydrous Trace Globulin Edestin P a r t coagulable LIgSOa . . (49, 66) powder, ammonium sulfate, and cupric 70 ' . . . . . . . . . . . . . . (46) 0.4 Globulin Maysan . . . . . . . . . Unnamed 62 .............. sulfate crystals (all c. P. grades). P a r t coagulable AIgS04 .. t29i' ' . . . . . . . . . Edestin FILTER PAPER. An open texture crepe Trace Globulin ................................... (44) paper, preferably 15 em., also 12.5 and Trace Globulin ................................... (46) 11 em. Trace Globulin ................................... (41) Trace Globulin . . . . . . . . . . 74-90 , , , , .., .., ., .. (2.9,61) STANDARD SOLUTIONS. Approximately 2 Globulin ................................... (80) N hydrochloric acid and N sodium hyTrace Protein Tuberin 60-80 MgS04 . . (48) 2 Protein ................................... (10) droxide.
Lima bean S a v y bean
777
.........
......... .........
3Iilk solids Egg white
32 ...... Small amount
Squabh
1.3
Protein
Malt
2.8
Globulin
. . . . . . . .
Edestin a-Globulin @-Globulin Phaseolin a-Globulin @-Globulin Phaseolin Conphaseolin a-Globulin @-Globulin a-Globulin @-Globulin Canavalin Concanavalin a-Globulin @-Globulin Arachin
Part coagulable MgS 88 97 . . . . . . . . . . . 1.0 (NHa)zSO4 . . (61) 68 ............ 95 ....... 5:i (8,'ibj . . . . . . . . . . . 1.0 ("4) ....... Coagulable . . . . . . . . . . . . . . (43, 70) 95-100 ................... 68-71 +0.5 ( 14 .. ( 70-78 ..... .. 90-100 +0.5 ( 34 .. ( . . . . . . . . . . . 1.0 (NHd2SOa ..... . . . . . . . . . . . 0.6 (NHdzSO4 5 : 5 (6, 32) 74 ................... 96 1.0 (NH,)ZSOI . . (81) Uncoagulable . . . . . . . . . . . . 5 . 3 5 (5, 28,
Conarachin Legumelin Legumin Vicilin Vignin
so Coagulable ...........
Phaseolin Casein Ovomucin Ovomucoid Cur curbitin
........... ........... ........... ...........
.........
95-100 Simijar to legumin
73-95
P a r t coagulable
............
............
..
5.35 5.2
.. 4 :a5
.. .. ..
Method of Analysis Samples must be fresh and free from slime and any discoloration. If it is necessary t o preserve samples for a length of time, run through step 1. STEP1. SOLVENT EXTRACTIONS. Remove the skin, cut off the dried outer layer, and grind the meat finely by passing it from one to four times through a meat chopper of a cutting type. Weigh a 50-gram sample rapidly to avoid loss of moisture, put into a 400-cc. beaker, and add 75 cc. of acetone. Let stand at room temperature for 3 to 4 hours with an occasional brisk stirring to break up meat clusters, decant through a dry 15-cm. filter, and press out as much of the acetone as possible. A 120-ml. (4-ounce) oil sample bottle is very convenient for this pressing because it can be set on the table in an inverted position without loss of particles. If there are a lot of meat particles on the filter, squeeze
778
INDUSTRIAL AND ENGINEERING CHEMISTRY
out the liquid with a flexible spatula and return the solid mass to the beaker. Discard the acetone extract. Add 75 cc. of carbon tetrachloride to the beaker, place in a water bath, and bring the carbon tetrachloride slowly to a boil. Let stand a t room temperature for 3 to 4 hours with occasional stirring, decant through the same filter, and press out as much of the carbon tetrachloride as possible, using the same oil sample bottle. [The final 15 to 25 cc. usually filter very slowly. Set a 7.5-cm. (3-inch) wire triangle, which has been bent inward a little, on the funnel and set the filter in the triangle. The filter aper is sufficiently strong to support liquid almost three-quarters lled. Although this device is not absolutely necessary, it is a great convenience and one can hardly do without it in the succeeding filtrations.] Discard the carbon tetrachloride extract, scrape off whatever particles are on the filter paper and bottle, stir up the meat, and drive off the carbon tetrachloride and acetone by heating in the water bath. Stir occasionally to prevent caking. (May be left overnight or longer.) STEP 2. WATER LEACHING.Add 250 cc. of cold distilled water and mark the beaker a t the water surface. Stir and let soak for about 5 minutes, then add a drop or two of phenolhthalein and with N sodium hydroxide, make pink and then ack to just colorless with N hydrochloric acid, and note the amount of N sodium hydroxide required. Heat slowly with stirring to expel the remaining acetone and carbon tetrachloride, being very careful to avoid loss due to foaming. After danger of foaming is over, boil gently for 5 minutes and scrape down any crust formed on the sides of the beaker. Cool to room temperature, dilute to the 250-cc. mark, and add the equivalent amount of N hydrochloric acid exactly to neutralize the N sodium hydroxide added before the boiling. Add a drop or two of iodine indicator, and if it does not give a blue color or just a trace omit step 2a and proceed with 2b. (a) Add about 5 cc. of freshly prepared malt diastase solution, place in a water bath, and keep with occasional stirring at 40' to 45' C. for about 1 to 5 minutes or until there is no color or just a trace of a blue color with iodine. Cool to room temperature and proceed with 2b. (b) Add N hydrochloric acid in small portions until orange or just faintly pink to ethyl orange added internally. To get the color change let the meat settle and add another dro of indicator. It is necessary to do this after every hydrochiric acid addition and after every stirring; 1.5 t o 5 cc. of N hydrochloric acid are generally required. Let settle and filter through a dry 15-cm. filter into a 250-cc. beaker. In filtering try to retain the meat in the beaker and use the oil sample bottle to press out as much water as possible. Reject filtrate. The filtrate generally comes out yellow or faintly orange. (May be left overnight if kept in a cool place. Fold filter paper so that contents will not dry too much.) STEP 3. SODIUMCHLORIDEEXTRACTION. Using a small wash bottle with 200 cc. of 10 per cent sodium chloride solution, wash the residue on the filter pa er back into the original beaker, and mark the beaker at the sofution surface Avoid throwing in the filter paper. Add a drop or two of phenolphthalein and then make pink with N sodium hydroxide. Keep at room temperature with occasional stirring for 30 minutes and if the phenolphthalein becomes colorless add more N sodium hydroxide, 0.5 to 1 cc. at a time. (There is no harm done if it is accidentally kept 1 hour longer.) Place in a water bath and in a period of 15 to 20 minutes raise Its temperature 85" to 90" C., and then hold for exactly 30 minutes further a t that temperature with occasional stirring. During heating add a drop of phenolphthalein and around 0.5 cc. of N sodium hydroxide occasionally to maintain the red color. Remove from water bath, allow to cool a little, replace water lost by evaporation, stir up, let settle, and filter as before, finally pressing out as much solution as possible. Reject residue. (May be left overnight but not in an alkaline state. Make just acid with N hydrochloric acid, and upon resumption of Kork again make alkaline with N sodium hydroxide.) SULFATESALTING-OUT.. With N hydroSTEP4. MAGNESIUM chloric acid make just acid to phenolphthalein and measure volume. Add slowly in 3- to 4-gram portions and with gentle stirring 30, grams of magnesium sulfate heptahydrate per 100 cc. of solution. After all the magnesium sulfate has been added and all or nearly all has dissolved, place the beaker in a water bath and in exactly 30 minutes raise the solution gradually to 50" C:, with an occasional slow stirring. (No harm is done if it is accidentally kept 30 minutes longer a t a lower temperature.) Avoid going above 55" C. at any time. Let settle and filter, preferably while warm. Reject residue if any. (May be left overnight.) SALTING-OUT.While the solution STEP5. SODIUMSULFATE is kept at 35' to 40' C. in the water bath, add slowly in 1.5- t,o
E
Vol. 14, No. 10
2-gram portions and with gentle stirring 11 grams of pulverized anhydrous sodium sulfate per 100 cc. of sodium chloride solution used in step 4, disregarding the increase in volume due to the magnesium sulfate and the slight loss due to filtering. After nearly all has dissolved hold at 35" to 40' C. for 75 minutes lon er, and after the precipitate has agglomerated do not stir. PNo harm is done if it reaches 45" C. for a yhile or is kept for 1 t o 2 hours longer at room temperature,) Filtey while warm with as little breaking of the agglomeration as possible. I t is sometimes better to divide the filtering over two separate filter papers, and it is not absolutely necessary for this to filter to complete dryness. Reject filtrate. (Proceed with the next step immediately.) STEP 6. SODIUMHYDROXIDE DISSOLVING.Using a wash bottle, with a strong stream wash the filter with warm water (30" to 40" C.) made pink to henplphthalein with N sodium hydroxide (0.15 to 0.30 cc. of ??sodun hydroxide to 500 cc. of water) into the previous beaker to a. yolume of 150 to 200 cc.; where there is no or hardly any precipitate, 100 cc. will do. If it is necessary to scrape the filter or the volume reaches 250 t o 260 cc., no harm is done. Keep warm and stir a little till all dissolves. Wash sides of beaker down with water. (May be left overnight but not in an alkaline state. Make just acid with N hydrochloric acid, and upon resumption of work again make alkaline with N sodium hydroxide.) STEP7. AMMONIUM SULFATESALTING-OUT.With N hydrochloric acid make just acid to phenolphthalein and measure volume. Add slowly in 2- to 3-gram portions with gentle stirring 22 grams of ammonium sulfate per 100 cc. of solution. After all has been added raise temperature and. keep for 15 minutes at 40" C., towards the end stirring very little and very slowly. (There is no harm done if it is accidentally kept for 1 to 2 hours longer a t room temperature.) Let settle and filter while warm with as little breaking of the agglomeration as' possible. I t is not absolutely necessary for this t o filter t o complete dryness. Reject filtrate. (Proceed with the next step immediately.) STEP 8. SODIUMHYDROXIDE DISSOLVING.Repeat step 6. Cool to room temperature, add a few drops of phenol red, and make just yellow vith N hydrochloric acid. (If necessary may be left overnight.) Measure volume and add slowly in 3-gram portions with gentle stirring 10 grams of magnesium sulfate heptahydrate per 100 cc. of solution. After all has been added and dissolved, add 1 to 3 drops of methyl red, and if solution is not yellow to methyl red also, make it so with A; sodium hydroxide. The solution must be distinctly yellow to both indicators and sometimes it may be necessary to use 0.1 N sodium hydroxide and 0.1 N hydrochloric acid. Filter off the filter paper fuzz and scum if any. (May be left overnight.) STEP 9. CUPRICSULFATEPRECIPITATION. Add 20 cc. of filtered 10 per cent cupric ,sulfate Fentahydrate. and keep at 40' to 45" C. for 60 minutes with occaslonal slow stirring for the first 10 to 20 minutes. Do not stir when it becomes noticeable that the flocculent precipitate starts to agglomerate and settle. (No harm is done if it is kept for 1 to 2 hours longer at room temperature.) Without stirring the precipitate, filter on a moistened tared filter paper, preferably 11 cm., previously dried and weighed in a weighing bottle. In transferring the precipitate from the beaker to the filter use \$arm water, not above 45" C., or it will stick tenaciously to the glass. If it becomes slow in atering owing to a clogged filter, wash the precipitate loose from the filter wjth a stream of hot water, 60" to 75' C. Finally, wash the preci itate 2 or 3 times with hot water, dry, and weigh in a weighing iottle. Calculate, using the factor: 0.035 gram of ppt. = 1.0 per cent of soy flour
Discussion STEP 1. Most sausages are exposed to the smoke of burning woods, which deposit on the surface certain creosotic substances that give a distinctive flavor and a n antiseptic property. Instead of smoking, it is also common practice to treat the meat with crude pyroligneous acid, obtained from the destructive distillation of wood. The small amounts of formaldehyde and tannic acid introduced are both capable of precipitating proteins under certain conditions. Most of these two substances can be removed by cutting off the harder surface, and they can also be extracted from the ground meat with acetone. The carbon tetrachloride is used because the acetone does not remove all the fats, which would interfere in the succeeding two steps.
October 15, 1942
ANALYTICAL EDITION
STEP2. When commercial soybean flour extracted with acetone-carbon tetrachloride is boiled with water the glycinin is apparently not coagulated. If the solution is cooled and then acid is added to p H 4.1 to 4.2 the glycinin can be seen precipitating. This is the same isoelectric point as that of petroleum ether-extracted soybean flour (40, 6.2). This phenomenon has the appearance of falling snow. If the p H is a t the higher limits the snow will be heavy, while a t the lower limits the flakes will be fine. If the proper p H has not yet been reached, the solution will be cloudy after the glycinin or snow has stopped falling, a t the exact p H the solution will be clear, and when the isoelectric point has been passed the solution will again be cloudy. I n the presence of the skeletal meats the phenomenon will be the same. After stirring, the meats will settle first and then the snow can be seen. If a little salt, some sugars, or starches are present the behavior will still be the same. With starch the snow is heavier unless removed with malt diastase. K t h hashed or minced liver the picture is different. When the soybean liver mixture is warmed, a break in the cloudiness occurs. Soon the break spreads, a coagulation occurs, and the surrounding liquid becomes brilliantly clear, much before the boiling point is reached. On cooling it becomes evident that the glycinin is not in solution. On long standing in the cold the same thing occurs. If acid is then added to a wide p H range of 4.0 to 4.4 no precipitation occurs. On continuing further with the procedure, a sodium chloride extraction is also brilliantly clear and later there is no precipitation with sodium sulfate. It was found that kidneys also cause the same difficulty but to a lesser degree. Because these two organs are rich in various minerals a n attempt was made to precipitate them with dithizone, &hydroxyquinoline, cupferron, carbonates, etc. Only the carbonates gave promising signs, yet this idea, too, had to be abandoned. Because choline has strong basic properties and its presence may be expected, an exhaustive leaching with alcohol and the addition of potassium triiodide to the water extraction failed to overcome the difficulty. Since the addition of carbonates was somewhat helpful, it occurred to the author that the iron of the hematin may form some positive colloids. A thorough investigation into the hemoglobins was then made. To the hashed liver was added a little sodium nitrite, thoroughly stirred in, and then treated with large quantities of acetone. This produced bright red solutions with acetone, carbon tetrachloride, or alcohol. Exhaustive leaching with acetone was made until the dried liver was almost white and powdery. I t was taken for granted that the red color was due to nitrosohemochromogen, and that in this way all the iron was removed. Again the same difficulty occurred and at times it appeared more pronounced. Long ago Haldane showed that on boiling in solution nitrosohemoglobin is broken down into nitrosohemochromogen and globin ( I S ) . Globin is classified as a histone because of its basic character. It is coagulated by heat. Anson and Mirsky precipitated denatured globin with acetone and then converted a large part of it into a soluble globin which still had the power to combine with heme and form hemoglobin (3). This denatured globin has an isoelectric point of around pH 8.0. A t pH 7 the soluble g:obin may attach itself to acid proteins like glycinin and cause a mutual precipitation. If this is true and it is coagulated by heat, it apparently takes the glycinin down with it forever. If, while the globin is still in the soluble state, alkali is added to pH 8.0 the globin should be discharged and precipitated, and if alkali is added beyond pH 8.0 the globin should take on acidic properties and leave the glycinin alone. Apparently that is what happens, for after this pH adjustment neither liver nor kidney had any effect on the glycinin. STEP 2a. I n the analytical procedure the solution is not adjusted to the optimum p H 4.5 to 5.5 for malt diastase digestion ( I $ ) , because the hydrolysis is accelerated at a higher p H by the presence of soluble proteins (8). The
779
starch must be removed as completely as possible, because globulins enter into combinations with the polysaccharides a t varying experimental conditions (58). The author checked this and found, on not removing the starch in a sample of sausage, two and a half times as much soybean as was actually present. STEP 26. This step is to remove all soluble substances, such as proteins, amino acids, starch, and salts. The ethyl orange just changes color a t the isoelectric point of glycinin. Incidentally, very soluble globulins of different isoelectric points might also be removed a t this point. I n avoiding loss of glycinin the p H of the solution a t filtration is of cardinal importance and is not hard to adjust because of the presence of such buffering substances as gelatin, peptides, amino acids, and sugars. STEP 3. Although the p H of the extracting solution is not uniform, concordant results can be obtained, and this is in accord with the literature (19, 69). STEP 4. I n order to have uniformity in the salting-out, Moir’s method of precipitating casein was followed, in which the solution is made just acid to phenolphthalein before adding the saturating salt (89). The salts lower the p H and bring it within the range of p H 6, 7, or 8, where the same amount of protein is precipitated (60). When the solution is raised to 50” C. the precipitated casein will form a sticky mass that may cling to the beaker or stirring rod or float on top of the solution, while a t a lower temperature it would be almost impossible to filter. STEP 5 . Since the precipitation of colloids depends on whether the precipitating salt is added in one or successive portions (9), the optimum conditions were determined and the same technique followed for both the magnesium and ammonium sulfates. STEP 6. This is a convenient way of bringing the precipitated proteins back into solution. STEP 7. This step was introduced to retain in solution proteins which are more soluble than glycinin and yet were forced out by the double concentration of magnesium and sodium sulfates. STEP8. I n order to avoid the danger of reaching the “tolerance zone” in the next step (84, 64), a desirable p H range and a volume of precipitant were determined. Methyl red was selected so as to be on the alkaline side of the glycinh isoelectric point. Phenol red then keeps the solution a t a p H not high enough to form a metal hydroxide, does not interfere with the color of the methyl red, and yet allows a sufficient p H range to be easily adjusted, since the solution might not always be adequately buffered. Phenolphthalein was entirely unsuitable here, on account of the presence of small amounts of ammonium sulfate. Practically all the gelatin is washed out in the water step, but more will be extracted with the sodium chloride. This small amount can then be salted out with magnesium sulfate a t room temperature (4),while a t a higher temperature some will be retained in solution. This gelatin will then be precipitated with the sodium sulfate and again with the ammonium sulfate. Fortunately, it is not precipitated with the heavy metals, yet it is undesirable in being a very powerful protective colloid. Protective or reversible colloids like gelatin may completely inhibit the precipitating action of electrolytes. Even if the gelatin is present in a minute quantity, it will prevent somewhat, if not entirely, the precipitation of the glycinin by the heavy metal. To prevent this from happening it is necessary to decrease the two stability factors, electrical charge and hydration (56). The addition of the magnesium sulfate will decrease both. A little heat may sometimes be needed to help the dehydration. Although this course may not always be necessary, yet after taking these precautions the precipitations were much better.
INDUSTRIAL AND ENGINEERING CHEMISTRY
780
STEP9. Precipitation with a heavy metal appeared best for forming an insoluble glycininate so that i t could be washed free from salts, dried, and weighed. Copper is best because other metals may form insoluble sulfates and chlorides. The cupric sulfate solution is sufficiently dilute to avoid reaching the “tolerance zone” if an excess is added and not too dilute t o cause large volumes. The amount of copper in the precipitate should not vary much under such a control, for in similar work, in precipitating milk proteins with copper sulfate, the amount of copper in the precipitate varied from 2.4 to 4.1 per cent (69). Such a variance would not be noticeable. The factor was obtained by analyzing carefully prepared samples and is shown to be proportional to the amount of soybean. Experimental
It was not deemed necessary t o present data on the development of the method. The data shown were obtained by using the method as i t is presented. Table I1 shows the flours used in the succeeding determinations. The work in Table I11 was performed to show that the factor for a particular brand of soy flour is not materially different with the varying percentages, the different kinds of meat, and the different treatments the meat may undergo alone or with the soy flour. The factor was derived by dividing the copper glycininate precipitate by the percentage of soy flour. The meat of samples 1 to 4 was finely ground cured meat for frankfurters, obtained from a small sausage maker. This was mixed with salt and definite amounts of soy flour, stuffed. tight into a 5-cm. (2-inch) casing, and then cooked for about 25 minutes
OF COMMERCIAL SOYFLOURS TABLE 11. ANALYSIS
Brand
Manufacturer
Moisture Fat
% Allied Mills Archer-DanielsMidland Central Soya Glidden Spencer Kellogg
Protein
%
%
Process
Kreemko
5.0
5.5
45.0
ExDeller
Cherry Blossom No. 500 No. 103 Soyalose Soyagrain Special X Soyaflake
7.5 3.0 8.5 4.5 6.5 5.0 4.9
4.0 6.6 4.0 6.8 1.3 7.4 22.1
46.0 52.3 49.9 50.5 52.5 49.6 43.5
Solvent Expeller Solvent Expeller Solvent Expeller Whole fat
TABLE111. ANALYSISOF MEATSWITH KREEMKO Kind of Meat
NO. 1
2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28
Frankfurter meat
Treatment Pickled, cooked Pickled, cooked, smoked
Bologna
............
Meat
Pickled overnight
Veal
............
Meat Veal Meat Veal Pork liver Beef liver Liver sauaage Pork beef lamb kidneys Pork’beard, brains, veal Chicken and beef lungs, beef spleen, cartilage Lamb cheeks, tongue, tripe, ground bone
Raw
..............
Pickled in beaker
............
Raw Pickled in beaker Baked Raw
Soy Flour Added,
Vol. 14, No. 10
at 170’ F. Samples 3 and 4 were smoked by the sausage maker in his usual way. In sam les 5 to 7 U. S. inspected smoked Kosher bologna was groun! and mixed with the soy flour in a beaker. In samples 8 to 11, 5 cc. of a 1 per cent sodium nitrite and 2 grams of sodium chloride in 10 to 15 cc. of water were added to 50 grams of ground meat in beakers, which were placed in the refrigerator overnight. The next day the required amount of soy flour was stirred in. For samples 12 to 14 ground raw meat was weighed and mixed with soy flour. In samples 15 to 18,50 grams of ground raw meat in a beaker were stirred with 5 cc. of 1 per cent sodium nitrite for 4 to 5 minutes, the soy flour was added with a little water, and then the acetone was added. The acetone extract in this case was as red as that of samples 1 to 11. In the next step 2 grams of sodium chloride were added. This same procedure was followed with the other samples that were pickled in the beaker, except that the amount of sodium chloride added in the waterleaching step was varied all the way down to nothing. For sample 21 a chunk of liver was baked for a few minutes until dry, then ground for a sample. The liver sausage of samples 23 and 24 bore the U. S. inspection stamp. Samples 25 to 28 were run because these meats may be present in some meat products, especially dog foods. The method is normal with these present, as shown by the results. In Sam Ies 29 and 30, Table IV, 43 grams of lean hamburger were ickyed in the beaker, and 5 grams of cereal and 2 grams of soy &ur were next stirred in. These two and the next three samples show that starchy materials, even in large amounts, cause no interference when calculated with the factor 0.0251, the known factor for this brand. The meat loaf, obtained from a sausage maker, contained eggs, the usual spices, starch, and no soy flour. The blood sausage was from the same source and contained tongue, starch, and no soy flour. Samples 34 to 38 were prepared similarly, using skim-milk powder in varying proportions. Part of the casein filtered through in step 2, as the filtrates were very cloudy, a large part salted out in step 4 as a large sticky precipitate that fused into a film, and then in step 5 some additional casein precipitated. Although the results for soy flour are not very gratifying with milk solids present, they are somewhat lower than the sum of the milk powder and the soy flour which together make u the total binder used. They appear to be the sum of the soy lour and three fourths of the milk solids. If the amount of milk solids is known, the results can be corrected for their presence. All the meats of Table V were pickled in beakers as before and the factors for other brands were derived. The factor 0.0350 was adopted because i t is almost the average. This factor can be changed from time to time in the light of additional experimental data, and if the source of the soy flour is known an absolute accurate factor can be used. The results calculated with this factor are also shown, and, similarly, in Table IV the presence of milk solids is less of an interference. It is possible to make up other combinations, such as tamales, foreign specialties, etc., for trial, but as this work had to be brought t o a finish, the presence of peas, beans, etc., had to be left with the theoretical explanation.
%
Factor
1 4
0.0255 0.0250
Summary
3 5 0 4 9
0.0248 0.0245
A gravimetric method for the determination of soybean flour in meat products is based on determining a certain fraction of glycinin, the chief soybean protein. The meat sample is subjected to a series of operations in which the other proteins and interfering substances are removed by solvent extractions, different salt concentrations, coagulation temperature, and an isoelectric and heavy metal precipitation. Provision is made for removing starch. The method is adaptable to the various meats and meat by-products, whether cooked, smoked, or pickled, and whether the meat product contains flours, starches, cereals, a known amount of milk powder, or tomatoes. Theoretically the method should also be adaptable to meat specialties that contain beans, peas, etc. The analytical procedure is divided into steps, each step eliminating some substances or else bringing the glycinin back into solution. All conditions, such as pH, time, temperatures, concentrations, reagents, etc., were worked out and standardized. Although the method is time-consuming,
0
2 0
2 0 3 4 3 6 8 7 5 3 4 4 0
4
....
0.0305 0.0263
....
0.0249
....
0.0245
....
0.0252 0.0258 0,0246 0.0255 0.0260 0.0255 0.0245 0.0195 0.0254 0.0248
....
Raw Pickled in beaker
4
4
0.0261 0.0250 0.0253
Raw
4
0.0232
Pickled in beaker
4 0.0245 Av. 0.0251
October 15, 1942
ANALYTICAL EDITION
TABLE IV. ANALYSIS OF MEATS WITH SEVERAL BINDERS SOV
FGur
No. Kind of Meat
Other Bindera Added
% 10 Barley, buckwheat oatmeal, and corn’ 30 10 Noodles, wheat flour, corn and potato starches 31 7 Rye bread and rice 3 Dried tomatoes 32 Meat loaf Starch present 33 Blood sausage Starch present 34 Meat 3 Mjlk powder 35 2 Milk powder 36 6 Milk powder 37 2 Milk powder 38 3 Milk powder 29
Hamburger
TABLE V. No.
Kind of Meat
SOY
Added Flour Found (Kreem- Factor Factor ko) 0.0251 0.0350
%
%
%
4
3.71
2.67
4
4.05
2.91
0 0 0
0.01 0.00 0.00
0.01 0.00 0.00
3 4 3 0
2.39
0
2.93 4.70 8.27 4.00
ANALYSISOF MEATSWITH SOYFLOURS Factor
Soy Flour Found (Factor 0.0350)
0.0345 0.0355 0.0406
3.93 4.05 4.63
0.0401 0.0363 0.0333 0.0336 0.0251 Av. 0.0349
4.59 4.14 3.81 3.84 2.87
Soy Flour Added Brand of Soy Flour
% 39 40 41 42 43 44 45 1-28
Meat
2.10 3.37 5.93 2.87 1.73
No.500
No. 103
Cherry Blossom
%
4 4 4
Soyalose 4 Soyagrain 4 Special X 4 Soyaflake 4 Kreernko (calculated on 4)
i t is very simple and requires no special skill nor special apparatus. The determination is made by multiplying a copper proteinate precipitate by a n empirical factor.
Acknowledgment Grateful acknowledgment is made to Lester Aronberg, Lake Chemical Co., Chicago, for his cooperation and assistance throughout the duration of this work. Appreciation for assistance is expressed to the five companies which were originally interested in this work: Allied Mills, Inc., Peoria, Ill.; American Soya Products Corp., Evansville, Ind. (out of business) ; Shellabarger Grain Products Co. (now Spencer Kellogg & Sons), Decatur, Ill.; A. E. Staley Mfg. Co., Decatur, Ill.; and Stein-Hall Mfg. Co., Chicago, Ill. Assistance is also acknowledged from the Archer-Daniels-Midland Co., Minneapolis, Minn.; Central Soya Co., Decatur, Ind.; and The Glidden Co., Chicago, Ill.
Literature Cited (1) Agcaoili, F., Philippine J . Sci., 11A, 91-100 (1916). (2) Ammann, M. P., Compt. rend., 170, 1333-4 (1920). (3) Anson, M.L., and Mirsky, A. E.. J . Gen. Phy~iol.,13, 469-76 (1930). (4) Bogue, R. H., Chem. & Met. Eng., 23, 105-9 (1923). (5) Csonka, F. A., Murphy, 3. C., and Jones, D. B., J . Am. Chem. SOC.,48, 783-8 (1926). (6) Eicholz, A., J . Physiol., 23, 163-77 (1898). (7) Ferguson, C. S., Racicot, P. A., and Rane, L., J . Assoc. Oflcial Agr. Chem., 25, 533-7 (1942). (8) Filipowicz, B., Biochem. J., 25, 1874-84 (1931). (9) Freundlich, H., 2. physik. Chem., 44, 129-60 (1903). (10) Galang, F. G., Philippine J . Agr.. 3, 91-104 (1932). (11) Glynn, J . H., Science, 89, 444 (1939). (12) Gore, H. C., J . Am. Chem. SOC., 47, 281-3 (1925). (13) Haldane, J., J. Hyg., 1, 115-22 (1901). (14) Hayward, J. W., J . Assoc. Official Agr. Chem., 22, 552-4 (1939). (15) Hendrey, W. B., IND.EIG. CHEM.,ANAL. ED., 11, 611-13 (1939).
781
(16) Hoagland. R., U. S. Dept. Agr., Circ. 230, 1-9 (1932). (17) Hoffman, W. F., and Gortner, R. A., Cereal Chem., 4, 221-9 (1927). (18) Horvath, A. A., Food Industries, 7, 15-16 (1935). (19) Howe, P. E., J. Biol. Chem., 61, 493-522 (1924). (20) Johns, C. O., and Chernoff, L. H., Ibid., 34, 439-45 (1918). (21) Johns, C. 0.. and Gersdorff, C. E. F., Ibid., 51,439-52 (1922). (22) Johns, C. 0.. and Jones, D. B., Ibid., 28, 77-87 (1916). (23) Johns, C. O., and Waterman, H. C., Ibid., 42, 59-69 (1920). (24) Ibid., 44, 308-17 (1920). (25) Jones, D. B., and Csonka. F. A., Ibid., 64, 673-83 (1925). (26) Ibid., 97, xxix-xxx (1932). (27) Jones, D. B., Finks, A. J., and Gersdorff, C. E. F., Ibid., 51, 103-14 (1422). -..~ (28) Jones, D. B., and Gersdorff, C. E. F., Ibid., 58, 117-31 (1923). (29) Ibid., 74, 415-26 (1927). (30) Jones, D. B., Gersdorff, C. E. F., Johns, C. O., and Finks, A. J., Ibid,., 53, 231-40 (1922). (31) Jones, D. B., and Horn, M. J., J . Agr. Research, 40, 673-82 (1930). (32) Jones, D. B., and Johns, C. O., J . Biol. Chem., 28, 67-75 (1916). (33) Kerr, R. H., J . Assoc. Oficial Agr. Chem., 19, 409-11 (1936). (34) Kodama, K., J . Biochem. (Japan), 2, 505-24 (1923). (35) Kruyt, H. R., and Jong, H. G. B. de, Kolloidchem. Beihefte, 28, 1-54 (1929). (36) La Wall, C. H., and Harrisson, J. W. E., J . Assoc. Ogicial AUT. C h m . , 17, 329-34 (1934). (37) Ibid., 18, 644 (1935). (38) Lythgoe, H. C., Ferguson, C. S., and Racicot, P. A., Ibid., 24, 799-800 (1941). (39) Moir, G. M., Analyst, 56, 228-35 (1931). (40) Monaghan-Watts, B., IND.ENG.CHEM.,29, 1009-11 (1937). (41) Osborne, T. B., Am. Chem. J.,13, 385-417 (1891); 14, 212-24 11842). \ - - - - I -
(42) Ibid., 14, 683-9 (1892). (43) Osborne, T. B., J . Am. Chem. Soc., 16, 633-43, 703-12, 757-64 (1894). (44) Ibid., 17, 429-48 (1895). (45) Ibid., 17, 539-67 (1895). (46) Ibid., 19, 525-32 (1897). (47) Osborne, T. B., and Campbell, G. F., Ibid., 18, 542-58 (1896). (48) Ibid., 18, 575-82 (1896). (49) Ibid., 18, 623 (1896). (50) Ibid., 19, 494-500 (1897). (51) Ibid., 19, 509-13 (1897). (52) Ibid., 20, 348-62 (1898). (53) Ibid.. 20. 419-28 (1898). (54j %id.; 22; 379-413‘(1900). (55) Ibid., 22, 422-50 (1900). (56) Osborne, T. B., and Voorhees, C. G., Am. Chem. J., 15, 392-471 (1893). (57) Osborne, T. B., and Voorhees, C. G., J . Am. Chem. Soc., 16, 778-85 (1894). (58) Przylecki, St. J. von, Mystkowski, E., and Niklewski, B., Biochem. Z., 262, 260-71 (1933). (59) Ritchie, W. S., J. Assoc. OflCial Agr. Chem., 12, 411-15 (1929). (60) Ritchie, W. S., and Hogan, A. G., J . Am. Chem. SOC.,51, 880-6 (1929). (61) Kosenheim, O., and Kajiura, S.. J. Physiol., 36, liv-lv (1908). (62) Smith, A. K., and Circle, S. J., IND.ENG. CHEM.,30, 1414-18 (1938). (63) Street, J. P., and Bailey, E iM.,Ibid., 7, 853-8 (1915). (64) Thomas, A . W., and Norris, E. R., J . Am. Chem. SOC.,47, 50113 (1925). (65) U. S. Dept. Agr., Bur. Animal Industry, Order 211, (Revised), p. 37 (1922). (66) Ibid., Amend. 3 (1925). (67) Ibid., Amend. 4 (1925). (68) U. S. Dept. A n . , Food Drug Admin., Service Regulatory Announcements, No. 2, Rev. 5 (1936). (69) Vandevelde, A. J. J., Lait, 3, 437-47 (1923). (70) Waterman, H. C.. Johns, C. O., and Jones, D. B., J. Biol.Chem., 5 5 , 93-104 (1923). (71) Wilgus, H . S., Jr., Norris, L. C., and Heuser, G. F., IND.ENO. CHEM., 28, 586-8 (1936).
CORRECTION.In the article entitled “Fractionation of Colloidal Systems”, by Fancher, Oliphant, and Houssiere [IND.ENG. CHEM.,ANAL.ED., 14, 552 (1942)], the last term in Equation 1 should read
Xi - R: 4
