February, 1925
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Extraction and Estimation of Lipoids in Cereal Products' By Olaf S. RaskZ and Isaac K. Phelpsa FOODCONTROL LABORATORY, BUREAUOF CREMISTRY. WASHINGTON, D . C.
uncooked cereal products has apparently not been deemed necessary, for no report of such application can be found in the literature. One of these methods, hitherto unpublished, 1907 meeting. This method consists of an extraction of a devised in the Bureau of Chemistry by C. R. Smith for depreviously dried sample of the product with anhydrous ether. termining the fat content of baked cereal products, was The extract thus obtained is evaporated to dryness in a tared therefore tried on a number of uncooked cereal products. container, dried at the temMethod for Fat Extraction perature of boiling water, The official method for the determination of fats in unTreat 5 grams of the cooled, and weighed. This cooked cereal products extracts less than one-half of the sample in a 200-cc. Erlenweight is reported either as lipoids or ether-soluble material in macaroni, as measured meyer flask with a mixture ether extract or crude fat, by the amount extracted by the ammoniacal method deof 10 cc. of alcohol (95 per terms commonly regarded scribed in this paper. In the case of egg noodles, the officent), 2 cc. of concentrated as synonymous. Since the cial method extracts about three-fourths of the amount of ammonium hydroxide, and adoption of this method, no lipoids extracted by the ammoniacal alcohol method. 3 cc. of water, keeping the further study of methods The lipoid content of egg noodles, when determined dicontents of the flask a t the for the estimation of fats in rectly by the ammoniacal alcohol method, checks with boiling point for 2 minutes, cereal products has been retheir lipoid content, as computed from the lipoid content preferably on the steam ported, and it has apparof their component materials, also determined by the ambath. After cooling, exently been assumed that moniacal alcohol method. tract the contents of the this method accounts for all If ether-soluble material is combined with other mateflask with three successive fat contained in this class rials, whether carbohydrate or otherwise, so as not to be 25-cc. portions of ethyl of substances. extractable by ether, as believed by Nelson and Taylor in ether, mixing and tamping Recently, Taylor and the case of starch, this combination can be broken by the material thoroughly Nelson6 extracted commeran ammoniacal alcohol solution without hydrolyzing the each time with a glass rod cial starches from various starch, so that apparently all ether-soluble material can flattened a t the end, and sources with the common be extracted by ether in uncooked flour products. decanting the extracts into fat solvents. By hydrolyza 250-cc. beaker. Drain ing with acids the starches t h i s treated, they obtained additional quantities of fat a p out the last of the three 25-cc. portions of ether as compreciably greater than those extracted by fat solvents directly. pletely as possible, and then add another 15-cc. portion of As a result, these investigators conclude that the major part the same ammoniacal alcohol solution to the flask and disof fatty material present in starch cannot be removed by sol- integrate the matted material as thoroughly as possible vents before hydrolysis, and that starches contain fat in com- by means of the flattened glass rod, which may be left in the flask for this purpose. Return the flask to the steam bination with other material, probably carbohydrate. Osborne and Mendele have observed that the fat-soluble bath and repeat the entire procedure, pouring the second vitamin cannot be extracted from food products by ether, set of ether extracts into the beaker containing the first set. but that a preliminary treatment of such products by alcohol In the second treatment with the ammoniacal alcohol mixture, the heating must be more gradual and somewhat and a subsequent extraction with ether are necessary. In a recent, unpublished study of alimentary pastes, the longer than the first, so that the ether remaining in the flask writers observed that the ether extracts of the finished products may be evaporated and the ammoniacal alcohol brought averaged one-half or less of those of their raw materials, to the required boiling point without any mechanical loss whereas they should be the same, for none of the fat origi- to the determination. Evaporate the combined extracts to dryness on the steam nally present in a semolina could possibly have been lost in the process by which it was manufactured into the commercial bath and extract the fat from the residue left in the beaker with paste, macaroni, or spaghetti, These unexpected observa- successive portions (five or six treatments, using about 15 cc. each time) of a mixture of equal volumes of ethyl ether and tions led to this investigation. It is generally known that fats, although soluble in ethyl low boiling petroleum ether (boiling point 40" to 55" C.). or petroleum ether, cannot be extracted completely by these Collect the extracts in a tared platinum dish (do not try to solvents from certain substances, among which are baked filter), and evaporate to dryness on the steam bath. Dry cereal products such as bread and crackers. Special methods the residue in a water-jacketed oven at the temperature of have therefore been adopted for estimating the fat contents boiling water for 30 to 45 minutes, cool in a desiccator, and of such. substances. Application of any of these methods to weigh. This method probably extracts all substances that are sol1 Presented before the Division of Biological Chemistry at the 6 l s t uble in ether, chloroform, carbon tetrachloride, and similar Meeting of the American Chemical Society, Rochester, N . Y.,April 25 to 29, solvents. I n the case of biological material, in general, the 1921. Originally received March 27, 1922; revised manuscript returned by extracted substances will include such widely different comauthors November 11, 1924. * Johns Hopkins University, Baltimore, Md. pounds as glycerides of fatty acids, fatty oils, steroIs, waxes, 8 Wamesit Chemical Co., Lowell, Mass. phospholipins, glycolipins, sulfolipins, and other less familiar 4 Bur. Chemistry, Bull. 107, rev., p. 39. fatlike substances. All these compounds, nevertheless, 8 J.A m . Chem. Soc., 42,1726 (1920). possess certain specific and well-defined properties in common, 8 J . B i d . Chcm., 46, 280 (1921).
HE present officiaI method for determining fats in raw or uncooked cereal products was adopted by the Association of Official Agricultural Chemists4 a t its
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which justify the scheme of placing them in one general class. These properties are greasy or fatlike feel, solubility in the solvents mentioned, and insolubility in water. The term “ether extract” cannot be used for either this class or for the product extracted by the foregoing procedure, because this term implies a product extracted by ether in a manner similar or equivalent to that specified in the official method. Neither are the terms “fat” or “crude fat” wholly satisfactory. The use of these terms in this sense would be inconsistent with their more generally accepted meaning, according to which “fat” denotes glycerides of fatty acids and “crude fat” the same substance contaminated with impurities which may or may not be of a fatlike nature. For these reasons the word “lipoids” seems more appropriate. In this article, unless otherwise specified, the term “lipoids” denotes products obtained by the ammoniacal alcohol method, and “ether extract” denotes products of the A. 0. A. C. method for fats in cereals. Results
On samples of semolina and the macaroni into which it had been manufactured, the results of this method compared as follows with those of the official method: Ether extract Lipoids
Semolina Per cent 1 02 1.83
Macaroni Per cent 0.47 1.85
On the following uncooked cereal products the results of this method compared as follows with those of the official method: Sther extract Per cent
Lipoids Per cent
Patent flour Straight flour Red dog Wheat Cornstarch
It is worthy of notice that this method of extracting lipoids does not hydrolyze or even gelatinize starch. A microscopic examination of the residue showed the starch grains intact. Tests on Egg Noodles
In order to obtain suitable material for a further and more systematic study of the extraction and estimation of fats in cereal products, two types or kinds of egg noodles were prepared. These were manufactured in a commercial macaroni and noodle factory under the obscrvation of a representative of the Bureau of Chemistry, who collected samples of the raw materials as well as of the finished products. One of these noodles was composed of commercial, desiccated, whole eggs and durum semolina. The other noodle was manufactured from commercial desiccated egg yolk and the same semolina as was used in the whole egg noodle. These noodles were manufactured according to the following formula: semolina, 190 pounds; egg matter, 8.5 pounds; salt, 2.5 pounds. These values are given on a moisture-free percentage basis in Table I. ts in F i n i s h e d Noodles, C a l c u l a t e d t o M o i s t u r e F r e e Basis Whole egg noodle Yolk noodle Per cent Per cent Semolina 94.1 94 04 Whole egg solids 4.46 4 54 1 41 1 41 Salt
Table I1 gives the composition of the raw materials and finished products on the moisture-free basis in terms of those constants which are commonly determined in this class of products by control laboratories and food analysts. Ash was determined by the so-called calcium acetate method, according to which 10 grams are charred in a tared platinum dish, cooled, treated with 5 cc. of a calcium acetate solution containing 5 mg. of calcium oxide, evaporated t o dryness, and incinerated to a light gray ash. After cooling in a desiccator the dish was weighed, and in computing the ash percentage a correction was made for the calcium oxide. Total P,Oj was found by determining this constituent in the ash by the so-called acidimetric method. Total nitrogen was determined by the A. 0. A. C. modification of the Kjeldahl method, using a 1-gram sample. Water was determined by the A. 0. A. C. method, using a 3-gram sample. LecithinPZOSwas determined by the Juckenack method, using a 30gram sample. These noodles served in this investigation as composite products prepared according to known formulas from components of known composition. The percentage of any constituent of such composite products can be computed from the percentages of those constituents in the component substances. Values obtained in this manner can be used as a check on the methods employed in obtaining those values by direct analysis. This was done in the case of the methods used for determining ether extracts and lipoids of these noodles and the relative merits of these two methods were judged by this procedure. The results are given in Table 111, which also contains the values for the same constituents as obtained by direct analysis. The percentage which the amount by direct analysis constitutes of that computed from amounts found in component materials is given for each constituent in Table 111. T a b l e 111-Values a n d C o m p u t a t i o n s 0btaine.d b y Direct Analysis WHOLEEGGNOODLE YOLKNOODLE PERCENTAGE Computed Computed FOUND B Y DIfrom afrom aRECT ANALYSIS By direct mounts in By direct mounts in Whole CONSTIT- analysis components analysis components egg Yolk LENT Per cent Per cent Per cent Per cent noodle noodle Nitrogen 2.68 2.63 2.55 2.54 101.9 100.4 Total PzOa 0,5102 0.5084 0.540 0.5463 100.3 98.8 Ether extract 2,17 2.90 3.06 3.60 74.5 85.0
The differences between the nitrogen and total PzOscontents, as found by direct analysis and as computed from the amounts found in the components, are so small as to be within the range of experimental error of the analytical procedures employed. The corresponding differences in the case of the ether extracts are too great to be explained in this manner. They indicate that ether does not extract the same substance or amount of substance from the finished noodles as it does from the flour and egg matter. The lipoid content of the finished noodles and of their component materials is given in Table IV. For convenience of comparison, ether extracts and the percentage of the lipoids which these ether extracts represent are also given.
T a b l e I-Ingredien
T a b l e IV-Lipoid
Semolina
--Whple egg YOLK Whole egg noodle
Yolk noodle T a b l e 11-Percentage C o m p o s i t i o n of F i n i s h e d Noodles a n d T h e i r C o m p o n e n t M a t e r i a l s (Dry Basis) Whole Whole egg Yolk Semolina egg Yolk noodle noodle Ash (salt-free) 0.845 ... ... 1,16 0,820 Total nitrogen 2.436 7.61 5.50 2.68 2.55 2.17 3.06 Ether extract 1.223 42.38 53.95 Lecithin-PzOa 0.0289 1.273 1.745 0.057 0.0684 Total Pa06 0.448 1.951 2.754 0,5102 0.540
Vol. 17. Xo. 2
C o n t e n t and E t h e r E x t r a c t s Lipoids Per cent 2.17
50.2
0J.i
4.25 5.00
Ether extract Per cent 1,223 42.38 53,95 2.17 3.06
Per cent ether extract in lipoids 56.4 84.4 82.1 51.08 61.2
The lipoid content of the finished noodles, as computed from the lipoid content of their component materials, is: whole egg noodles, 4.28 per cent; yolk noodles, 5.02 per cent. The lipoid content of these products, when determined directly, constitutes, in case of the whole egg noodle, 99.3 per cent, and, in case of the yolk noodle, 99.6 per cent of the
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February, 1925
values given. The corresponding ether extracts constitute 50.7 and 61.0 per cent of the same values. These relationships show that ether extracts from these materials do not constitute all of the fatty or fatlike matter contained in them. The close agreements in lipoid content of composite substances (finished noodle products), as determined directly and as
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computed from that contained in their component materials, indicate that the ammoniacal alcohol method makes complete extraction of all fatlike substances in cereal products. Results of this method may be applied in calculating the formulas used in the manufacture of composite products whose component substances contain known amounts of lipoids.
Estimation of Lipoid Phosphorus in Cereal Products’ By Olaf S. Rask2 and Isaac K. Phelps3 FOOD CONTROL LABORATORY. BUREAU OF CHEMISTRY, WASHINGTON, D. C.
OKSIDERABLE significance is attached to the phospholipin content of food products on account of the important role that phospholipins play in nutrition and in many other vital phenomena. The lipoid phosphate content of many food and medicinal products has an additional significance, because it is regarded by food and drug officials as an index to the egg or lecithin content of such products. This is particularly true of egg noodles, in which case the lipoid phosphate is arbitrarily designated as lecithin-phosphoric acid. The large amount of investigational work on phospholipins has resulted in the development of a number of methods for their extraction, isolation, and estimation. A review of these methods shows that most of them are applications of the principle first observed by Beyer, that phospholipins require alcohol as well as the common fat solvents for their extraction. Almost all of these methods are too long and complicated to be applicable to routine work. The only excevtion is Juckenack’s methodI5which was developed primarily for determining the lecithin-phosphoric acid content of egg noodles, to which its use appears to have been confined. A recent study of this method by the Food Control Laboratory shows that it fails to make a complete extraction of the lecithin-phosphoric acid in egg noodles. Therefore, for the want of a rapid and otherwise satisfactory method for the determination of lipoid phosphate, the development of such a method was undertaken. I n this work the substances semolina, whole egg or egg yolk, and noodles, used in the authors’ study of lipoids,6were employed.
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showed that this method fails to make a complete extraction of phospholipins in cereal products. If the ammoniacal alcohol method, described in the authors’ study of lipoids in cereal productsI6makes a complete extraction of all lipoids, in general, then all phospholipins of a substance will be contained in the lipoids extracted from it by this method and may be found by determining total phosphates in the total lipoids thus obtained. This postulate accords with the general principle of Beyer on which most methods for the determination of phospholipins are based. , therefore determined Total phosphorus, calculated as P z O ~was in the lipoids extracted from the substances listed in Table I and the amounts thus obtained were computed to percentages of the original substance. The results of this procedure and the percentages which the Juckenack values constitute of the authors’ values are given in the last two columns of Table I. The Juckenack determinations were made in an extractor designed especially for lecithin-P205 determinations in egg noodles (Figure 1). Resul ,s obtained by its use averaged 21.4 yer cent higher than those obtainec by the use of the Soxhlet in a seris :s of lecithin-P205 determinations on egg noodles.
The results of the first three columns of Table I confirm those of the earlier study of the Juckenack method, which
Note -Although this extractor failed to solve t h e problem of determining lipoid PzOs, i . seems probable that it will have corresp mding advantages over the Soxhlet extract >r in other extraction procedures. These r dvantages may be attributed to the high ar d uniform temperature a t which the extract on is made. The vapors of the solvent ar they ascend to the condenser completely envelop the siphon tube which contains t i e material t o be extracted. As a result, :he extraction is made a t the boiling point o ‘ t h e solvent, which insures a high and constai t temperature. I n most extractions a high temperature is desirable. Where temper%tureenters as a factor, constant temperature is also important, because i t insures uniform conditions without which uniforri and comparable results are impossibl 1.
1 Presented under the title, “Estimation of Phospholipins in Cereal Products” before the Division of Biological Chemistry a t the 61st Meeting April 25 t o 29, 1921 of the American Chemical Society, Rochester, N. Y., Originally received March 27, 1922, revised manuscript returned by authors November 11, 1924. 3 Johns Hopkins University, Baltimore, Md. 8 Wamesit Chemical Co., Lowell, Mass. 4 Landw. Vers. Sta., 14, 161 (1871). 6 2. Nahr. Genussm., 3, 1 (1900); Juckenack and Pasternack, I b i d . , 8 , 94 (1904). 0 See page 187 of this issue.
It is reasonable to assume, therefore, ;hat the lecithin-Pz05 content of the 3e materials by the Juckenack F i g u r e 1-Extractor method would have been somewhat Lecithin-PZOb D e t e r mfor ilower than those by this method n a t i o n s had the Soxhlet extractor been used. Table I1 gives a comparison of the lecithin-P,Oj content of the finished noodles, as computed from the total PZOScontained in the lipoids of their component materials, and the
Values on the M o i s t u r e - F r e e Basis (b) (a) Computed Determined from amounts directly in components (Juckenack’s (Juckenack’s (c) method) method) Percentage From lipoids Percentage SUBSTANCE Per cent Per cent ( a ) is of (. 6.) Per cent (.a.) is of (c) Semolina 0.0289 0.0548 52.7 Whole egg 1.273 ... ... 1.380 92.3 ... ... 1.745 100,o Yolk 1.745 Whole egg noodle 0.0574 0.0840 68.3 0.1125 51.0 Yolk noodle 0.0684 0.1065 64.2 0.1290 53.0 T a b l e I-Lecithin-PzO6
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