June, 19501 DAUBNEY AND SEXTON: DETERMINATION OF EGG IN SALAD CREAM 305 The Determination of Egg in Salad Cream Part I BY C. G. DAUBNEY AND G. E. W. SEXTON SYNOPSIS-A method is proposed for determining the amount of egg yolk solids and dry egg solids in salad cream. It is based on a colorimetric deter- mination of the choline derived from egg lecithin by means of acid hydrolysis and conversion of the choline into its pink reineckate, which is soluble in acetone. The choline content of dried whole egg, sugar-dried egg, and dried egg yolk has been determined and shown to be constant in any one type of product . Mustard, a common ingredient of salad cream, causes a complication on account of the choline derived from the sinalbin of white mustard. The method described determines total choline and is applicable only when the nature and amount of any mustard present is known.Satisfactory results were attained ‘for salad creams of known composition. Work is in progress to devise a means of determining egg and mustard separately . THE determination of the egg content of certain foodstuffs is often required for the purpose of quality control and for checking their compliance with Orders issued by the Ministry of Food. The first of these Orders is the Food Standards (Salad Cream and Mayonnaise) Order, 1945 (S.R. & O., 1945, No. 1177), which lays down that in the case of salad cream, mayonnaise and salad dressing, “the product shall contain . . . not less than 1.35 per cent. by weight of egg yolk solids.” The second is the Flour Confectionery (Control and Maximum Prices) (Amendment) Order, 1950 (S.I., 1950, No.87), by which a certain maximum price may be charged for flour confectionery, which includes cakes, puddings, pastry, etc., provided “the combined fat, sugar, and dry egg solids content (or the combined content of any two of these ingredients) . . . is 45 per cent. or more.” The third is the Food Standards (Preserves) (Amendment) Order, 1949 (S.I., 1949, No. 1893), by which fruit curd shall contain not less than 1 per cent. of dried whole egg (or its equivalent as sugar-dried whole egg, liquid or frozen whole egg or shell egg). It will be noticed that in one Order it is “egg yolk solids” that is mentioned and in another “dry egg solids.” None of the Orders specifies a method for the determination of the egg content.It was in consequence of these Government Regulations that the experiments described below on egg and salad cream were carried out ; work is now in progress on flour confectionery and fruit curd, the result of which it is hoped to publish shortly. So far as the analyst is concerned, the term “egg” generally implies that of the hen. In the main, the white of egg is composed of protein and water, whilst the yolk contains, in addition to protein and water, some 30 per cent. of fatty matter and 1.5 per cent. of cholesterol. Of this fatty matter about one-third is phosphatide, chiefly lecithin. The term phosphatide (or phospholipin) refers to a group of substances in which one or more fatty acids and a nitrogenous base are combined with a third constituent, glycero- phosphoric acid.This group can be further subdivided into (a) the lecithins and (b) the cephalins. The compounds in each sub-group conform to the same type of structure but differ in the nature and position of the fatty acid radicals contained in the molecule. The lecithins are represented by the formulae- C q . OOC. R CW.0OC.R I P C H 4 - P - 0 . CH, .CH,. N i (CH,), I OH I CH.0OC.R’ p-e-rorm I CH,-O-ib-O.CHJH,.N l o i (CH& OH I OH a-form306 DAUBNEY AND SEXTON : TH:E DETERMINATION OF [Vol. 75 The cephalins are identical in structure," except that they have /%amino ethanol (cholamine) in place of choline- CH,.OOC.R CH, . OOC. R I I I 0 I II I CH-0-P-0 .CH,.C&.NH, I CH.0OC.R' I OH CH,-O-P-O.CH,.CH, 1 7 .NH, CH,. OOC.R' I OH a-form 8-form These two groups of phosphatides occur in animal tissues in varying proportions, and in egg the /3-form of lecithin is said to predominate. The phosphatides are soluble in most organic solvents except acetone, whilst cephalin is insoluble in alcohol unless water or other lipoid bodies are present. In considering the analytical chemistry of this class of substance it must be borne in mind that lecithin and cephalin appear to undergo oxidation and hydrolysis more readily than the fats. Lecithin is hydrolysed on boiling with dilute acid into glycerophosphoric acid, fatty acids and the base choline. Similar decomposition is brought about by the enzyme lecithinase which occurs in animal tissue.l $2 Cephalin decomposes similarily to give cholamine.The methods proposed from time to time for the determination of egg in various foodstuffs have usually depended on a determination of either the phosphatide or the cholesterol ~ o n t e n t . ~ In deciding on a method which was likely to be applicable to salad cream, one based on the determination of the phosphatide fraction of egg seemed to be the most promising, either by way of organically-bound phosphorus, as a measure of the total phosphatides, or by way of choline as a measure of le~ithin.~ Methods6$6 $7 98 19 based on the determination of organically-bound phosphorus require the complete extraction of the phosphatide with suitable solvents and the subsequent deter- mination of phosphorus in the extract. Such methods are, however, liable to give low results owing to the susceptibility of lecithin to decomposition by enzymes or acids, with the con- sequent splitting off of the phosphoric acid ra,dicle as mentioned above.1° Further, the physical nature of salad cream renders solvent extraction methods unsuitable.Methods based on the determination of choline after hydrolysis of the lecithin, on the other hand, present certain advantages. The products of hydrolysis will include any choline previously split off by acid or enzyme action and the experimental technique is readily applicable to salad cream. A disadvantage is that under the conditions of hydrolysis to be described, choline is also derived from white mustard (Sinapis alba) which is often an ingredient of salad cream. White mustard contains the glucoside sinalbin, which was shown by Gadamerll to be- H0.C6H4.CH,.N : C.S.C,H,,O, I O.SO$.O I (CH,), N.CH:,.CH, HO.(OCH,),.C,H,.CH : CH.CO. d This is made up of the non-volatile mustard oil, p-hydroxybenzyl isothiocyanate, linked to glucose and the acid sulphate of the choline ester of sinapinic acid.Black mustard (Sinapis nigra), on the other hand, contains the glucoside sinigrin, which is less complex, and contains no choline. An evaluation of mustard has been described by Viehoever and Nelson12 and by Terry * This formula has recently been disputed; see Hutt, H. H., Malkin, T., Poole, A. G., and Watt, P. R., Nature, 1950, 165, 314.June, 19501 EGG I N SALAD CREAM. PART I 307 and Corran,13 both of whom have based their method on a determination of the liberated sulphate after enzymatic decomposition.The non-volatile mustard oil has been studied by several workers, but it does not lend itself readily to determination. The other important ingredient of salad cream which might give rise to choline is the vegetable 0i1.l~ At the present time there are available to manufacturers various oils all of which, in the crude state, contain small but significant quantities of choline. Examination of the refined oils, as used, has shown that they yield no choline when treated by the method given below. It will be seen, therefore, that any method for the determination of egg in salad cream that depends on the choline content of the egg must also be capable of modification when white mustard is present. The method to be described determines total choline and is only applicable when the nature and amount of any mustard ingredient is known.Experiments are proceeding at the present time with the object of determining the choline from egg lecithin or from sinalbin separately. Choline may be determined in a variety of ways, which include c o l ~ r i m e t r i c , ~ ~ ~ ~ gravimetric,lg j20 volumetric21 and micro-biological methods.22 For the present purpose, a colorimetric method was found to be suitable, in which the choline is precipitated by ammonium reineckate, [Cr(NH,),(SCN),]NH,, the choline reineckate filtered off, dissolved in acetone and the (pink) colour of the solution compared with standards similarly prepared. The use of artificial standards of methyl red in a buffer solution has been suggested.EXPERIMENTAL Choline has been determined in the various forms of liquid and dried egg at present available and in salad creams containing known amounts of egg. Where mustard is present the choline content of this ingredient has been determined in the same way, using O G g . of the powder, and due correction made. METHOD- Weigh 0.5 g. of dried egg, 2.0 g. of frozen whole egg or 10 g. of salad cream into a 150-ml. flask containing a few glass beads and add 20 ml. of diluted hydrochloric acid (5 + 3). Warm the mixture on the steam-bath for 5 minutes to reduce subsequent frothing and then boil under a reflux condenser for 1 hour, in apparatus fitted with ground-glass joints. Wash down the condenser with 10 ml. of hot water and transfer the contents of the flask, when cool, to a separator.Extract the aqueous liquid by shaking with 40 ml. of methylated ether in order to remove the bulk of the fatty matter. Allow to separate and run the aqueous layer through a filter into a small conical flask. Wash the ether layer twice with 20-ml. portions of water and run the washings through the same filter. Boil the combined filtrate and washings to expel the ether and reduce the volume. After cooling, add the calculated volume of potassium hydroxide solution to neutralise the acid present, followed by 20 ml. of cold, saturated aqueous baryta solution. Add 1 drop of 1 per cent. alcoholic thymol-phthalein solution followed by glacial acetic acid added dropwise till the blue colour just disappears. Cool the liquid by standing it in a refrigerator for about 1 hour and filter by gentle suction through a filter funnel fitted with a sintered glass plate (No.3 porosity) or a hardened filter- paper. Wash the filter with water and, to the clear combined filtrate and washings, add 6.0ml. of 2 per cent. ammonium reineckate in methyl alcohol. Leave overnight in a refrigerator. Collect the pink precipitate of choline reineckate on a sintered glass filter funnel (No. 3 porosity) with gentle suction, wash twice with 10-ml. portions of ice-cold water and finally three times with 2.5-ml. portions of n-propyl alcohol. When free from solvent, dissolve the precipitate in acetone, collect the solution in a graduated flask and make up to 50ml. with acetone. Transfer to a Nessler tube and match the colour against standards prepared as follows.Dissolve sufficient choline chloride in water to give a solution con- taining 2 per cent. of choline and standardise against 0.1 N silver nitrate.23 From this prepare a dilute solution containing 1 mg. of choline per ml. Add appropriate volumes of this second solution to 150-ml. flasks, dilute to 50 ml. with water, add 6 ml. of 2 per cent. ammonium reineckate in methyl alcohol and proceed as above. In this way prepare the standards that may be necessary containing between 1 and 12 mg. of choline in 1 mg. steps. Fractional standards may be prepared therefrom by dilution with acetone. The colours should be matched within 30 minutes of dissolving the reineckate in acetone as some batches308 DAUBNEY AND SEXTON: THE DETERMINATION OF [Vol.75 of the solvent have been found to cause a marked deterioration in the pink colour on standing. The reineckate precipitates can however be left omn their separate filters after washing with the ut-propyl alcohol and then dissolved when rea'dy for matching. RESULTS Egg-Various types of egg products were examined by the above method. Half a gram was taken for each determination. TABLE :I Product Dried whole egg : Canadian .. Canadian .. Swedish (S quality) Swedish (D quality) Foreign origin . . Sample A . . 11 €3 .. 99 c .. Canadian (grade A) Canadian .. Sample D . . Sugar-dried egg : Choline, mg. per g. .. . . 16.0 ,. . . 14.0 .. . . 15.0 .. . . 14.0 .. . . 17.0 .. . . 16.0 .. . . 15.0 .. . . 16.0 .. . . 10.0 .. , .. 10.0 .. . . 12.0 Product Dried egg yolk: Chinese .. .. Chinese . . .. Foreign origin . . Foreign origin . . Foreign origin . . U.S.A. (sample F) Foreign origin . . Sample E .. Frozen whole egg : Egg albumen: Choline, mg. per g. .. . . 22.0 .. . . 22.0 .. . * 22.0 .. . . 24.0 .. . . 20.0 .. . , 21.0 .. . . 4.5 . . . . nil NOTE-Samples A-F supplied by manufacturers of salad creams as typical of egg ingredient used. Salad cram-Various commercial products. Ten grams of sample were taken for each determination. TABLE 111 Mustard Egg yo choline/g. mg./g. % % /--Jc-, Choline in (-*- Sample Egg component Present, mg. of sample, Added, Found,* 1 Dried whole egg, 16 mg. of choline/g. -- 0.18; 0.19 1.42 1.1; 1.2 2 --I 0.51; 0.55 3.25 3.2; 3.4 -- 0.90 5-62 5.6 -- 1.20 7.64 7.5t 3 4 1.5 4-4 0-55 3.1 3.0 ** 5.3 7.8 1.07 3-6 4.1 5 6 7 Dried egg yolk, 22 mg.of choline/g. 3.0 8.0 0-65 2.2 1.9 8 3.0 8.0 0.65 2.2 1-9 9 3.0 8.0 0.70 1.87 2.1 10 3.0 8.0 0.65 1-87 1.9 21 mg. of choline/g. . . 5.3 7-8 0.88 2.3 2.2 ** 5.3 7.8 1-27 3.9 4.0 11 12 13 5.3 7.8 0.90 2.3 2.3 14 Frozen whole egg, 4.5 mg. of choline/g.** 3-0 8.0 0-6 10.5 8-0 15 ** 3-0 8.0 0-66 10.5 9.1 16 5.5 9.0 0.90 9.6 9.1 17 5-5 9.0 0.75 6.2 5.7 (approx. 1 18 Sugar-dried egg, 12 mg, of cholini/g. 1.0 5.6 0.70 6.0 5.4 NOTE-Samples 1-4 specially prepared by B.F.M. I.R.A. * Corrected for mustard content. t 7.75% found by B.F.M.I.R.A. using the same method. * * Presumed choline content of egg ingredient. DISCUSSION As described, the choline is determined by matching colours in Nessler tubes. It is possible to use a Spekker absorptiometer, in which method the graph of the optical density of the solution plotted against choline is a straight line passing through the point of origin (private communication from C.L. Hinton). The method has been applied to a number of samples of dried and liquid .egg from different sources including bulk shipments and manufacturers' supplies. The results given in Table I show that the choline content of any one type of product is virtually constant,June, 19501 EGG IN SALAD CREAM. PART I For the purpose of calculation the following values may be taken- Dried whole egg . . . . 16 mg. of choline per g. Dried egg yolk . . . . 22 3) Frozen whole egg . , .. 4.5 >> Sugar-dried egg . . a . 11 > > 309 These results are in conformity with the known relationship in chemical composition between the various products.In applying the method to the determination of egg in salad cream, products prepared on a laboratory scale as well as proprietary brands have been examined. The method can be applied directly to preparations that do not contain mustard, but in the presence of mustard it is necessary to know the nature and proportion of this ingredient in order to apply a correction. A sample of white mustard yields some 9mg. of choline per gram when hydrolysed in the manner described, but most commercial flours were found to contain a lower proportion on account of blending. Work is in hand to devise a method for determining egg and mustard separately. Table I1 sets out the results obtained and satisfactory agreement is shown between the declared egg used and the amount found.It must be borne in mind that the majority of samples were commercial products and in many of them no sample of the actual egg ingredient used was available. In determining the choline, matching was carried out to the nearest 0.5 mg. standard. cream. 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. The authors wish to thank the Government Chemist for permission to publish this paper and Miss B. Askew for assistance with much of the experimental work. Their thanks are also due to the British Food Manufacturing Industries Research Association, and to Messrs. Crosse and Blackwell, Heinz, Ocean Preserving Co., Rayner and Sutton, for supplies of salad REFERENCES King, E.’J., Biochem.J., 1931, 25, 799. “Methods of Analysis of the Association of Official Agricultural Chemists,” 1946 Edition, Nottbohm, F. E., and Mayer, F., 2. Unters. 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Winton, “The Analysis of Foods,” 1945 Edition, New York, p. 552. Hodson, A. Z., J . Biol. Chem., 1945, 157, 383. “U.S. Pharmacopoeia,” XI11 Edition, p. 756. -, Ibid., 1934, 23, 476. Washington, D.C., p. 348. Washington, D.C., p. 347. GOVERNMENT CHEMIST’S DEPARTMENT DUDLEY HOUSE ENDELL STREET, W.C.2 December, 1949