November, 1945
INDUSTRIAL A N D ENGINEERING CHEMISTRY
gests that amino compounds “catalyze” caramelization of the glucose or other aldehyde under milder conditions than when these materials decompose alone. The brown colors obtained from glucose, either by heat or in stronger alkaline solutions, have much the same characteristics as do those obtained in the presence of amino compounds. However, the amines are not true catalysts for such decomposition, since they participate in the reaction. “Humins” prepared in their presence contain varying amounts of nitrogen, depending upon the amount of amine originally present (6). Several investigators have studied the development of brown pigments in heated milk products (IO,18). The reactions are apparently closely related to those which occur during prolonged storage. The color changes that take place in stored foods undoubtedly reflect a number of independent reactions. Joslyn (9) summarized those that are important for fruit products. The parallel development, during storage, of color (reflectance) and fluorescence, when measured after the removal of lipides, is in accord with the results described above for model systems. Dutton and Edwards (4) have shown that carotenoid destruction and the development of brown substances in the lipide fraction of dried egg contribute to the color changes. ACKNOWLEDGMENT
The authors are indebted to F. E. Lindquist for crystalline egg albumin and egg white globulin, to D. K. Mecham for lipovitellin and livetin, t o H. C. Reita for crystalline pepsin, to the Eastern
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Regional Research Laboratory for casein, and t o H. D. Lightbody for helpful suggestions. LITERATURE CITED
(1) Ambler, J. A.,IND. ENO.CHBIM., 21, 47 (1929). (2) Balk A. K., and Swenson, T. L., Food Research, 1, 319 (1936). (3) Chargaff, E., J. Biol. Chem., 142,491 (1942). (4) Dutton, H. J., and Edwards, B. G.. IND. ENQ.CBBIM., 37, 1123 (1945). (6) Enders, C., Biochm. Z . , 312,339 (1942);313,352 (1942-43).
(6) Endera, C., and Sigurdsson, A,, Ber., 76, 560 (1943). (7)Fischer, F. G., and Marschall, A,, Ibid., 64,2825 (1931). (8) Fraenkel-Conrat, H.,Coogcr, M., and Olcott, H. S., J. Am. Cham. Soc., 67,960(1945). (9) Joslyn, M. A.,IND. ENQ.CHIM.,33,308(1941). (10) Kass, J. P., and Palmer, L. S., Ibid., 32,1360 (1940). (11) Kekwiok, R.A.,and Cannon, R. K., BiOchem. J., 30,227 (1936). (12) Loiseleur, J., Compt. r e d . aoc. biol., 196,435(1942). (13) Maillard, W.O.,Ann. chim., [9]5,258 (1916). (14) Mitts, E.,and Hixon, R. M., J . Am. Cham. SOC., 66,483 (1944). (15) Pearoe, J. A., Can. J . Research, D21,gS (1943). (16) Ibid., F22,87 (1944). (17) Pearce, J. A., and Thistle, M. A., Ibid., D20,276 (1942). (18) Ramaey, R.J., Tracy, R. H., and Ruehe, H.A., J. Dairy Soi., 16, 17 (1933). (19) Reeder, W.,and Nelson, V . E.,Proc. SOC.Ezpt. BioE. Med., 45, 792 (1940). (20) Stewart, Q. F.,Beat, L. R., and Lowe, B., Proc. Inat. Food Tech., 1943,77. (21) Stewart, G.F.,and Kline, R. W., Ibid.. 1941,48. (22) Van Slyke, D.D., J . Biol. Chsm., 83,425 (1929). (23) Weast, C. A,, and Maokinney, G., IND.ENG.CHBIM., 33, 1408 (1941).
ROLE OF PHOSPHOLIPIDES AND ALDEHYDES IN DISCOLORATION B. G . EDWARDS AND H. J. DUTTON A brown material from dehydrated whole egg powder, found in both the total ether extract and the cephalin fraction, has been concentrated. Evidence indicates that this material arises from the reaction of a cephalin amino group with aldehydes.
D
EHYDRATED egg powders tend to acquire a brown color during storage. I n a study of the sources of this discoloration, an ether-soluble brown substance has been traced to the cephalin fraction of the phospholipides. Evidence obtained supports the hypothesis that this substance is the product of a reaction of an aldehyde with an amino constituent of the cephalin fraction. Dehydrated whole egg powder that had darkened markedly during nine months of storage in air a t 37” C. waa used in this inveitigation. From the ether-soluble fraction of the powder a brown product was prepared in concentrated form. It was not removable from the ether extract by water, but after the saponifiable fraction had been strongly acidified with sulfuric acid, the brown material was found in the acid aqueous layer and was no longer soluble in ether. Inorganic salts were largely precipitated and removed from the aqueous solution by addition of absolute ethanol to give a concentration of 80% alcohol. The alcohol, water, and inost of the glycerol were then removed by vacuum distillation. The pressure was reduced to less than 1 mm. of mercury toward the end of the process; the temperature did not exceed 135” C. By this step the water solubility of the brown material was markedly diminished although its ether insolubility was not affected. The material was washed by two
consecutive suspensions in distilled water, followed by centrifugations. From 60 grams of dehydrated whole egg, 86 mg. of brown amorphous material containing 1.1% nitrogen (micro-Kjeldahl) and 1.6% phosphorus (colorimetric method) was obtained. Ita absorption spectrum (obtained with a Beckman spectrophotometer) is given in Figure 1. Since the phosphorus and nitrogen contents of the producb suggested derivation from phospholipides, lecithin and cephalin were separated from the stored dehydrated egg, from freshly lyophilized yolk, and from yolk of fresh shell egg by a modification of the method of Sueyoshi (IO). This method is based on the tendency of phospholipides to precipitate from extracted egg oil, the comparative insolubility in acetone of phospholipides aa compared to fats, and the differences in solubility in aleohol between lecithin and cephalin. In the preparation of the cephalin fraction, the alcohol-insoluble material obtained after separation of the lecithin was further purified by elimination of ether-insoluble matter and subsequent acetone precipitation of the concentrated ether extract. It is well t o mention at this point the qncertainty regarding the purity of phospholipide preparations in general. Complete separation as indicated by total nitrogen, phosphorus, and amino nitrogen values of phospholipide preparations is seldom claimed (6). Such caution has been justified by recent work showing that the brain cephalin fraction is a mixture of phosphatidyl compounds (4) of different solubilities in alcohol. The presence in many preparations of products of partial hydrolysis, as well aa the incompleteness of separation of lecithin and cephalin, permits only qualitative conclusions from experimental results involving the isolated compounds.
INDUSTRIAL AND ENGINEERING CHEMISTRY
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I n a concurrent study under the direction of E. B. Kester on isolation and purification of egg phospholipides, i t was observed that the cephalin-containing fraction from stored dehydrated egg powder appeared as an opaque brown substance in contrast to the transparent yellow of the lecithin. Although both lecithin and cephalin preparations were impure, their marked differences in color were considered significant in view of the practical absence of color from phospholipides similarly prepared from freshly lyophilized yolk and from fresh shell egg. (The cephalin samples average 1.5% nitrogen and 3.5% phosphorus; the lecithins averaged 1.8y0nitrogen and 3.9% phosphorus.)
BROWN DISCOLORATION PRODUCTS IN ACID AQUEOUS FRACTION FROM:
-
Cepholin of Discolored E g g
---- Total Lipids of -.-.-
Discolored Egg
Reactlon Product of Cephai‘in From Fresh E g g with Acetaldehyde
250
300
350
400
450
WAVE LENGTH ( m y )
500
550
Figure 1
When the dissolved cephalin fraction was saponified, acidified, and treated with ether, the brown material remained in the acid aqueous phase, as was true with the total lipide extract of the stored egg powder. (This material differs from the well-known brown substance of aging lecithin and cephalin preparations, which enters the ether phase under the same conditions of treatment.) Spectrophotometric measurements (X = 270 mp) of the acid aqueous layer of the cephalin fraction showed that the brown reaction product in this fraction was concentrated ten to fourteen times its value in the original lipid extract. Moreover, different preparations showed three to seven times as much absorption a t this wave length in the cephalin as in the lecithin fraction. NATURE OF DISCOLORATION
The nature of the reactions involving discoloration is suggested in the reports of various workers. Balls and Swenson ( 1 ) and Stewart, Best, and Lowe (9) obtained brown products from glucose-protein interaction. Maillard (6), Weast and Mackinney ( I I ) , and Enders (3) demonstrated brown humin substances from interaction of reducing sugars and amino acids. Olcott and Dutton (7) showed that the reaction product of glucose with the amino groups of proteins was responsible in part for the brown color of stored dried eggs. These reports prompted a study of the reactions of the two phospholipides, cephalin and lecithin, and of ethanolamine and choline with aldehydes. Weighed amounts of cephalin and lecithin preparations from lyophilized fresh egg yolk were reacted with approximately tenfold molar excess of acetaldehyde a t room temperature in sealed glass tubes. The cephalin turned intensely brown within 30 minutes, while the lecithin darkened only slightly. Spectrophotometric measurements (X = 270 mp) of the acid aqueous
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fractions of these samples after removal of acetaldehyde under vacuum showed six times as much brown material in the cephalin as in the lecithin product. More decisive results were obtained when the more nearly pure ethanolamine and choline, which are nitrogenous constituents of cephalin and lecithin, respectively, were reacted with acetaldehyde. Ethanolamine hydrochloride and acetaldehyde gave a deep brown within 24 hours. The choline hydrochloride-acetaldehyde mixture, however, showed only a light translucent brown after 7 months. Figure 1 gives the absorption curves of the brown materials obtained directly from the lipide extract and from the cephalin fraction of discolored dehydrated egg powder, and also from the cephalin fraction of fresh egg upon reaction with acetaldehyde. Lecithin and cephalin from fresh egg and freshly dehydrated egg in the absence of acetaldehyde gave negligible absorption. The curves for brown products from the cephalin of discolored egg and from cephalin reacted with acetaldehyde are practically identical in shape. The absorption curve for the brown material isolated directly from the lipide extract gives less indication of a maximum near 270 mw than do the other two. The presence of other absorbing ether-soluble materials in the brown complex isolated from the whole dehydrated egg powder may account for the more general absorption characteristics observed. The brown reaction products not only yield characteristic absorption curves but have been shown to be fluorcscent. These two optical properties have made possible some analytical methods useful as quality criteria. The methods are now being applied to the study of dehydrated eggs during storage. Since the brown materials have been found concentrated in the cephalin fraction, and since both cephalin and ethanolamine hydrochloride react rapidly with acetaldehyde in contrast to the slight reactivity of lecithin and the nonreactivity of choline hydrochloride, i t seems apparent that at least one brown discoloration product in stored dehydrated egg arises from the reaction of a cephalin amino group with aldehydes. While the aldehydes involved may arise in the course of fat oxidation, their exact origin or character is not known. It is probable, moreover, that lipide amines other than cephalin may contribute to the browning of egg fat. The evidence that cephalin reacts with aldehydes may be significant in connection with its antioxidant activity (8). It is possible that the common failure to detect aldehydes in egg fat is due to the prompt reaction of these compobnds with lipide amines. The cephalin-aldehyde reaction may also be of interest when regard to the masking of amino groups in purified phospholipides as previously reported (8). ACKNOWLEDGMENT
The writers are indebted to E. B. Kester and Julia S. Furlow for the phospholipide preparations used in these studies, and to L. ILL White and G. E. Secor for the nitrogen and phosphorus determinations. LITERATURE CITED
(1) Balls, A. K., and Swenson, T. L., Food Research, 1,319 (1936). (2) Chargaff, E., Ziff, M., and Rittenberg, D.. J . B i d . Chem., 144, 343 (1942). (3) Enders, C.,Biochem. Z., 312,339(1942). (4) Folch, J., J. B i d . Chem., 146,35 (1942). (5) McLean, H.,and McLean, I. S., “Lecithins and Allied Substances”, New York, Longmans, Green and Co., 1927. (6) Maillard, L. C., Ann. chim., [9] 5 , 258 (1916). (7) Oloott, H. S., and Dutton, H. J., IND.ENO.C H ~ M37, . , 1119 (1945). ( 8 ) Olcott, H.S., and Mattill, H. A,, Oil & Soap, 13,98(1936). (9) Stewart, G.F.,Best, L. R., and Lowe, B., Proc. Inst. Food Tech.. 1943,77. (10) Sueyoshi, Y . ,J. Biochem. (Japan), 13, 145-54 (1931). (11) Weast, C. A., and Mackinney, G . , IND.ENG.CHEM,,33, 1408 (1941).
