INDUSTRIAL AND ENGINEERING CHEMISTRY
May 1948
ACKNOWLEDGMENT
(6) Dutton. H. J.. and Edwards, B. G., IND.ENQ.CHEM.,38, 347 .
The subject matter of this paper has been undertaken in cooperation with the Committee on Food Research of the Quartermaster Food and Container Institute for the Armed Forces. The opinions or conclusions contained in this report are those of the authors. They are not t o be construed as necessarily reflecting the views or endorsement of the War Department. The authors wish t o thank Armour & Company for its generous financial support of certain of the studies reported here.
(1) Assoc. Official Agr. Chem., ~ f f i c i aand l Tentative Methods of
Analysis, 5th ed. (1940).
(2) Bate-Smith, E. C., Brooks, J., and Hawthorne, J. R., J . Soc.
Chem. I d . , 62,97 (1943). (3) Bate-Smith, E. C., and Hawthorne, J. R., Ibid.,64,297 (1945). (4) Boggs, M.M.,and Fevold, H. L., IND. ENO.CEEM.,38, 1075
(1946). (5) Brooks, J., J . SOC.Chem. Ind., 62,137 (1943).
I
(1946). (7) Frankel, Max, and Xatchalsky, Aaron, Biochem. J., 31, 1595 (1937). (8) Ibid., 32,1904 (1938). (9) Ibid., 35, 1028 (1941). (10)Ibid., 35,1034 (1941). (11) Hawthorne, J. R.,J . SOC.Chem. I d . , 62,135 (1943). (12) Maillard, L.C.,Ann. chim., [9] 5, 258 (1916). ENQ.CHEM.,37, 1119 (13) Olcott. H. 8.. and Dutton. H. J.. IND. (1945). (14) Pearce, J. A., Thistle, M. W., and Reid, M., Can. J. Research, D21,341 (1943). (15) Stewart, G. F., B ~ L,~R., ~and, L ~ B., ~proc.~I , ~ Food ~ ~ Technol., p. 77 (1943). Stewart, F., and Kline, R. w.,Ibid., p. 48 (1941). (17) White, W.H.,and Thistle, M. W., Can. J . Research, D21,211 (1943). RECEIVED January 9, 1947. Journal Paper No. 5-1426,Project No. 942, .
LITERATURE CITED
919
I
.
G+
of the Iowa Agricultural Experiment Station, Ames, Iowa.
Glucose-Protein Reaction in
Dried Egg Albumen R. W. KLINE AND G. F. STEWART Iowa Agricultural Experiment Station, Iowa State College, Ames, Iowa These studies were initiated for purposes of elucidating the mechanism of the insolubility and color-deteriorative reactions in dried egg albumen. Results indicate that the initial reaction between glucose and the egg proteins is followed by others which give rise to fluorescence and insolubility. Added amino acids react preferentially with the glucose, although protein insolubility eventually ensues. Replacing the glucose in egg white with other sugars gives results substantiating the theory that the deterioration is of the Maillard type. Hydroxymethylfurfural does not appear to be an intermediate in the deteriorating reactions.
R
EACTIONS between glucose and the egg white proteins are almost certainly responsible for the changes observed in color, fluorescence, and solubility of dried albumen during storage. The removal of glucose completely inhibits the deterioration. Furthermore, low pH, moisture content, glucose concentration, and storage temperature minimize the rate of change in color and solubility. On the other hand Boggs et al. ( 2 ) , Fevold et al. (6),ahd Stewart, Best, and Lowe (9) have presented evidence indicating that certain of the changes in fluorescence, color, solubility, and palatability occurring in dried whole egg are independent of the glucose-protein reaction. The work of Edwards and Dutton (3, 4)has shown t h a t a reaction between cephalin and a n aldehyde is responsible for the development of fluorescence and color in the lipide fraction of whole egg during storage. Bate-Smith and Hawthorne (1) claim t h a t the addition of certain disaccharides (lactose and sucrose) to whole egg prior to drying inhibits the browning and solubility deteriorations due to glucose. I n view of the results of the other workers just referred to, it is evident that the effects obtained with these sugars are not necessarily attributable t o a n inhibition of the glucose-protein reaction. I n fact, Bate-Smith and Hawthorne themselves present data which show that, in the presence of sucrose and lactose, both amino nitrogen and glucose disappear from dried whole egg undergoing deterioration. Experiments to be reported in this paper were undertaken t o clarify this issue.
Bate-Smith and Hawthorne (1)and Kline and Fox ( 6 ) demonstrated the effects on keeping quality of adding certain amino acids t o egg liquid prior to drying. These compounds exerted a beneficial effect in retaining Qolubility during storage. On the other hand, with the exception of cysteine, they caused a greatly accelerated development of color. When cysteine was added, i t had little effect on color development. The characteristics of the deterioration in stored dried egg albumen and whole egg due to glucose provide strong evidence of the deterioration being the result of reactions of t h e Maillard type. The best evidence for this is to be found in the work of Olcott and Dutton (8). These investigators showed t h a t at le&t four egg proteins (albumin, egg white globulin, livetin, and lipovitellin) react with glucose under conditions simulating those found in dried eggsrundergoing deterioration. (Mixtures of protein and glucose were dried, adjusted to 10% moisture, and stored at 50 C.; the protein-sugar ratios were approximately equivalent to those found in dried egg products.) After storage i t was found t h a t 35 to 45% of the amino nitrogen had disappeared. These changes were paralleled by significant increases in fluorescence and color. Bate-Smith and Hawthorne (2) showed t h a t glucose and amino nitrogen disappear a t equivalent rates during storage of both dried whole egg and albumen. They calculated changes in amino nitrogen on the assumption t h a t one glucose molecule reacts with one amino group and compared these values with those actually found. The agreement between the observed and calculated values was reasonably good, although there were discrepancies. The present studies were initiated in a n effort t o obtain further data as to the nature of the chemical reactions involved in the deterioration of dried albumen. O
MATERIALS AND METHODS
Procedures and analytical methods used in the preparation and analysis of egg samples are the same as those given by Stewart and Kline (10). Hydroxymethylfurfural was prepared from sucrose by heating under pressure in the presence of oxalic acid. It was extracted from the reaction mixture with ethyl acetate. The solvent was evaporated and the resulting product
,
920
INDUSTRIAL AND ENGINEERING CHEMISTRY
3.33
Vol. 40, No. 5
proteins. This is followed by subsequent reactions, one giving rise to fluorescence and 3.00 450 color and another to insolubility. These tKo reactions proceed at different rates, the one 2.67 involving fluorescence being the more rapid. 400 When insolubility develops, fluorescence values decrease because of the fact t h a t the fluores233 350 cent materials are attached to the protein; S t when the latter insolubilizes, they are no 2.00 300 longer extractable. However, fluorescence 5 and color reactions continue in the insoluble 1.67 250 fraction even after this, as is evidenced by W B z m the fact that i t becomes progressively darker when samples are stored for increasing periods L33 200 8 3 of time. LL EFFECTSOH' ADDINGAMINO ACIDS. D a t a 1.00 150 on these tests are given in Table I. These results confirm and elaborate on those ob0.67 100 tained by Bate-Smith and Hawthorne ( 1 ) and Kline and Fox (6). All of the amino acids 0.33 50 tested exerted a powerful effect in retaining the solubility of the dried albumen. With the exception of cysteine, however, their in0.00 0 corporation resulted in tremendously accelerating fluorescence and color development. Figure 1. Chemical Changes in Dried Albumen Undergoing With cysteine, fluorescence developed to about Deterioration at 50' C. the same degree as without. Interesting difGlucose c o n t e n t calculated on a moisture-free basis. Fluorometer reading-quiferences among amino acids with respect to n i n e sulfate, concermtratiou calibration line prepared (100 = 0.5 microgram per milliliter). Fluorescence intensity expressed as concentration of q u i n i n e sulfate X 4. their effect on the rate of change in fluorescence, color, and solubility were noted. Lysine was the most reactive, followed closely by distilled a t 0.5 mm. and 115" t o 120" C. The product gave glycine. Alanine, glutamic acid, and arginine were about a violet color with a-naphthol and concentrated sulfuric acid; equally reactive; they reacted considerably less rapidly and the 2,4-dinitrophenylhydrazone melted at 184" C. extensively than did lysine or glycine, however. Cysteine, although its effects were different from those of the other amino RESULT s acids, was about as reactive as glycine. The comparative COINCIDENTALCHEMICALCHANGESIN DRIED ALBUMEN reactivity of the amino acids is similar to t h a t found by Maillard ( 7 ) for the simple amino acid-glucose reactions. DURING STORAGE. Changes in several of the chemical characteristics of dried egg albumen stored a t 25" and 50' C. were studied. These data are summarized in Figures 1 and 2. Glucose con333 165 tent, pH, and fluorescence intensity changed at rapid rates at 50" C. After a slight delay, 3.00 150 solubility changed rapidly also. This insdubility development was paralleled by a decline 267 135 in fluorescence, a phenomenon noted previously (10). Glucose disappeared completely 233 120 within 4 days; changes in other characteristics S continued until the tenth day. At 25" C. t m the storage changes were considerably slower 200 105 c than at 50" C. I n addition, the rates of 0 W change in the various characteristics of the de3 1.67 90 0 terioration were different. Losses in glucose 0 m ae II: w occurred first; these were followed by simul1.33 7 5 taneous changes in fluorescence and color No significant change in solubility occurred 1.00 during the entire storage period. Other evi60 dence was at hand, however, to show that solubility changes eventually occur at this 0.67 45 temperature (11). T o explain the various manifestations of 0.33 30 the deterioration occurring in dried egg albumen (11), a sequence of reactions is tenta0.00 15 tively postulated. First glucose reacts with the egg proteins, presumably with the formation of simple glucosides with the amino and Figure 2. Chemical Change in Dried Albumen Undergoing possibly other reactive nitrogen groups of the Deterioration at 2 5 O C. 500
2
2
k!
l_I 4.
INDUSTRIAL AND ENGINEERING CHEMISTRY
May 1948
921
Although. H M F caused c h a n g e s s i m i l a r t o those Glutamic brought about by glucose, Acid Cysteine Arginine Control Glycine A 1ani ne Lysine Time, gross differences were observed. Days Fb S c F S F S F S F S F S F d H M F disappeared rapidly with 0 29 100 34 100 53 100 70 100 34 la0 24 100 39 100 concomitant changes in solu7 120 78 990 100 848 94 1200 100 990 100 97 99 . . . . . . 10 370 99 bility, fluorescence, and color. 14 iib "ii 2 i o o io0 iioo '94 zioo i00 1400 ' $ 5 4ib 9i . . . . . . 25 240 37 2600 98 1600 87 . . . . . 1700 84 350 95 670 81 The fluore'scence and solu50 . , . ., 510 bility changes were consider. . . . 62 .. 72 iio 'ii 2300 ' 8 5 zioo 'is 2iOo 'Qi 1560 '513 300 82 ably slower than those observed PH 9.7 9.3 9.4 8.8 9.2 9.5 9.1 in natural albumen stored Amino acid 0.48 0.52 0.90 under similar circumstances 0.90 0.23 0.27 added, % ' 0.0 (Figure 1). For instance, at 6 Equimolar equivalent plus 10% of the glucose content (0.45 wet 5 Pan-dried, 10% moisture, stored at 50° C. days, losses in solubility were b a p ) added of each amino acid; lysine added as dihydrochloride, cysteine added a8 the hydroohloride. Fluorescence intensity. 83% and 62% for H M F and 0 yo solubility. glucose samples, respectively; at 14 days the value for the TABLE 11. EFFECT OF REPLACING GLUCOSEWITH HYDROXYH M F s a m d e was 68%. and at METHYLFURFURAL ON STORAGE DETERIORATION IN DRIED 15 days the value for the glucose sample Gas 33%. The fluoALBUMEN^ rescence of albumen samples containing glucose rose t o 430 and Time, FluoresSol., Glucose, declined. With H M F the fluorescence rose to only 85, then Days cence % Color b % declined slowly. The color changes in the H M F samples were more rapid than have been observed in those containing glucose. I n addition, the color noted in the H M F samples, a pinkish cast, was of different character. These data indicate that, if H M F is formed as a n intermediate in the deterioration a Sample fermented free of glucose, pH returned to 9.0, and 0.32% HMF of dried albumen, i t is not entirely responsible for the changes added. vacuum-dried 10% moisture stored at 50' C. b Cbncentration 1 K s Fe (CN)e of Lquivalent absorbing power at 350 m p . observed. Fluorescence development particularly must be accounted for by some reaction other than one involving H M F . REPLACING GLUCOSE BY OTHERSUGARSAND SUGAR DERIVATIVES. Inferential data as to the nature of the deteriorative These results indicate t h a t glucose reacts much more rapidly reactions were obtaine'd by making studies of the effect of certain with amino acids than i t does with egg proteins. These reacother substitutions for the naturally occurring glucose in the egg tions lead t o color and fluorescence but not insolubility. T h a t albumen. Results obtained in these tests are shown in Table 111. some reaction between glucose and the egg proteins also ocThe changes found follow exactly what was expected on the curred is evidenced by the fact t h a t some change in solubasis of results obtained in studies on the amino acid-reducing bility was eventually noted (except for samples containing sugar reaction. The results also partially confirm those obtained lysine). Cysteine ahd glucose, in addition to undergoing the by Bate-Smith and Hawthorne (1) with dried whole egg. The typical amino acids-reducing sugar reaction, react t o form the aldo-pentoses reacted most rapidly, the aldo-hexoses were intercolorless thiaeoladine carboxylic acid (6). This accounts for mediate, and the aldo-disaccharides reacted most slowly. It the behavior of the samples containing cysteine. might be argued that a discrepancy ~ c c u r r e din the case of REPLACINQ GLUCOSE WITH HYDROXYMETHYLFURFURAL(5fructose (a keto-hexose). It should be remembered, however, HYDROXYMETHYL-2-FURYLALDEHYDE, H M F ) Haworth has SUgt h a t the tests were run on material which reconstituted to p H gested according t o Bete-Smith and Hawthorne (I), t h a t H M F 9.5. Under such alkaline conditions the fructose molecule might be an intermediate in the insolubilization of dried albuundergoes rearrangement which results in the formation of men during storage. However, Bate-Smith and Hawthorne (1) glucose and mannose. Since these aldo-hexoses reacted readily found no increase in the rate of deterioration of t h e dried prodwith the egg proteins, the results observed can be reconciled. uct when glucose-free albumen was exposed to H M F vapor. Samples containing the nonreducing sugars sucrose, trehalose, Few details were given; consequently it seemed desirable to and raffinose showed no evidence of reaction, as indicated by investigate this matter further. The data obtained are shown changes in fluorescence, color, and solubility. Similarly, no in Table 11. effects were noted when sorbitol replaced glucose, . These results
TABLEI. EFFECTOF ADDEDAMINOACIDSON DETERIORATION IN DRIEDALBUMEND
.
TABLE 111. EFFECTO F REPLACINQ GLUCOSE WITH SUGAR PRODUCTS ~
i Hr.
0 6 8
12 18 24 32 48 85 120 25 days 50 days
~Control ~ FbSC 20 100
, Xylose
F
S
540 100 .. .. .. .. .. . 310 89 .....
Arabinose Galactose F S F S 385 100 95 100 . . . . 97 . . iso ' 9 9 510 290 59 152 67
ON
STORAGE DETERIORATION I N DRIED ALBUMEN'
Glucose Mannose Fructose Lactose ----___---F S F S F S F S 47 100 30 100 15 100 29 100
Maltose F S 23 100
Sucrose Trehalose Raffinose Sorbitol F S F S F S F 9 20 100 20 100 20 100 20 100
.......................... . . . . . . . . . .......... I 86 . . iOo ::: :: :: : : . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 155 43 ............................... ....................... 150 95 . . . . . . . g 4 68 iio .................... ............................. 93 99 .................... .............................. Si . 400 . . . . . . . . . . . . I . . 100 83 28 65 32 56 35 58 45 245 79 95 325 : : : ,, 100 . . 100 . . io0 : : io0 21 ... 47 22 68 27 21 22 33 32 81 60 260 81 290 100 315 100 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 62 280 7 8 470 89 . . . . . . . . . . . . . . . . . . . . 35 iOO ::: ::: . . . . . . . . . . . . . . . . . . . . . ii b6 24 28 15 28 27 46 100 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ii i6a 46 io6 46 i0b 46 it% I
,
a Vacuum-dried, 10% moisture, pH 92-9.7; stored at 50' C. Fermented to remove natural glucose, pH brought back to normal, sugar added: pentoses at 0.38% level, hexoses a t 0.45%, and disaccharides a t 0.85% (all wet basis).
b Fluorescence intensity.
' % solubility.
I N D U S T R I A L A N D E N G I N E E R I N G CHEMISTRY
922
indicate t h a t only those sugars containing the free aldehyde group will react with egg proteins to produce deteriorative changes. This is contradictory to the conclusions drawn by Bate-Smith and HaTythorne ( 1 ) in studies on the effect of sucrose and lactose on the retention of solubility in whole egg. It is to be recalled, however, that reactions besides those involving glucose are known to be responsible for changes in the solubility of whole egg (9). ' T h e beneficial effects of sucrose and lactose on solubility retention cannot be explained on the basis of their effects on the glucose-protein reaction; the rekults obtained in the present studies specifically rule out this possibility. ACKNOWLEDGMEYT
The subject matter of this paper has been undertaken in cooperation with the Committee on Food Research of the Quartermaster Food and Container Institute for the Armed Forces. The opinions or conclusions contained in this report are those of the authors. They are not t o be construed as necessarily reflecting the views or endorsement of the War I3epar tment .
Vol. 40, No. 5
LITERATURE CITED
Bate-Smith, E. C., a n d Hawthorne, J. It.,J SOC.Chem Ind , 64, 297 (1945). Boggs, M. M.,D u t t o n , H. J., Edwards, B. G. and Fevold, H. L., IKD. EKG.CHEM.,38,1082 (1946). D u t t o n , H. J., a n d Edwards, B. G., IND.EXC.CHEM. ANAL. ED.,18, 38 (1946). Edwards, B. G., and Dutton, H. J , IND.ENC.CHEM.,37, 1121 fN4.i). \--
- - ?
Fevold, H. L., Edwards, B. G., Dimick, 4.L. and Boggs, & AII. ., Zbid., 3 8 , 1079 (1946). Kline, R. W., and Fox, S. W., Iowa State Coil. J . S c i . , 20, 265
(1946).
Maillard, L. C., Ann. chim., [9] 5, 258 (1916). Olcott, H. S.,a n d D u t t o n , H. J . , IKD.ENG.CHEM.,3 7 , 1118
(1945).
Stewart; G. F., Best, L. R., a n d Lowe, B., Proc. Inst. Food Technol., 77 (1943). ENG.C H m f . , 40, 916 (lo) Stewart, G . F.,a n d Kline, R. W., IXD.
(1948). G. F., and Kline, R. W., Proc. Inst. Food Technol., (11) Stcwait, 48 (1941). RECEIVED January
9, 1947. Journal Paper NO. J-1425,Project K O 942, of t h e Iowa Agricultural Experiment Station, .4mes, Iowa.
Thiophene-Formaldehyde Condensation P. D. CAESAR AND A. N. SACHANEN Socony-Vacuum Laboratories, Paulsboro, N . J .
Thiophene has been condensed with formaldehyde under mildly acidic conditions to form heat-reversible and irreversible resins. Bases will not catalyze the reaction. Yet thiophene-phenol cocondensation products with formaldehyde, containing up to 60 parts of thiophene t o 40 parts of phenol, will thermoset at an alkaline pM in the presence of hexamethylenetetramine. A mechanism for the condensation of thiophene and phenol with formaldehyde is proposed, based on the analogous reactivity of their rings.
T
HE condensation of aromatic compounds with formaldehyde
was discovered by Baeyer ( 3 ) in 1872. Benzene was found t o condense with formaldehyde in the presence of considerable amounts of concentrated sulfuric acid to produce diphenylmethane and powders or viscous liquids of considerably higher molecular weight. Nastyulrov and Malyarov ( 1 1 ) applied the reaction t o petroleum oils and fractions containing aromatic hydrocarbons. The condensation products, formolites, were viscous liquids or powders, and were formed, apparently, by reaction of the formaldehyde with the aromatic hydrocarbons present in the oil. Concentrated sulfuric acid was used as catalyst. These resinous products have found little practical application. The solids are brittle, and neither the liquid nor solid resins can be fused into tough, mechanically strong thermoplastic or thermoset resins. I n 1872 Baeyer (5)discovered also that phenol and formaldehyde could be condensed to form resins. I n 1909 Baekeland ( 1 ) developed this reaction and started the now-famous commercial Bakelite processes. Phenol-formaldehyde condensations, unlike those of benzene and other aromatic hydrocarbons, take place in the presence of dilute acids and a t moderate temperatures. The degree of condensation can be varied accurately
and widely, and the final products are extremely tough, mechanically strong thermoset resins. The practical applications of the various products are extensive. It has been discovered in this laboratory that thiophene condenses with formaldehyde to form resins. This might be expected since thiophene, ever since its isolation from coal tar benzene by Meyer (9) in 1883, has been considered analogous to benzene in its physical and chemical properties. However, the conditions necessary to promote the condensation of thiophene and benzene with formaldehyde and the properties of the resins formed differ considerably. It has been found in this laboratory that the chemical reactivity of thiophene and phenol rings is of the same order in alkylation and acylation reactions, and that this analogy can be furthered by a comparison of their reaction with formaldehyde. For example, less than 2y0sulfuric acid by weight of total charge is sufficient t o catalyze the condensation of thiophene, phenol, or a mixture of both, with aqueous formaldehyde a t temperatures of the order of 100 O C. Xot only are the reaction conditions similar, but the products themselves offer a direct comparison. The primary products of acidic condensation of thiophene with formaldehyde are viscous liquids or plastic solids. They could well be classified as thiophene Novolacs. These thiophene Novolacs, unlike those of phenol, cannot be thermoset at an alkaline pH. Since the thiophene ring positions alpha to the sulfur atom are, according to Steinkopf (16),more reactive than the beta positions, this resistance to further condensation could be predicted from the work of Holmes and Megson (5) on phenol and its homologs. These authors found that ortho- or parasubstituted phenols, those having but two active ring positions, would readily form Novolacs, which could be thermoset only under conditions sufficiently severe to force methylene linkages at the meta positions. Similar conditions are necessarv to force
