Factors Influencing Rate of Deterioration in Dried Egg Albumen ~
G. F. STEWART AND R. W. KLINE Iowa Agricultural Experiment S t a t i o n , Iowa S t a t e College, A m e s , Iowa
rate of color and fluorescence development and solubility loss in dried albumen. I t was felt that the results of these studies would be helpful in attaining a better understanding of the characteristics of the deterioration. In addition, precise data on the relative importance of the variables affecting the deterioration would aid materially in designing experiments on the mechanisms of the chemical reactions involved.
T h e quantitative effects of several variables controlling the rate of deterioration of dried kgg albumen have been studied. The reaction or reactions have a large temperature coefficient, greater than that for the coagulation of egg albumen. pH exerts a significant effect on the deterioration rate; decreasing the pH (down to 4.8) decreases the rate. Moisture content has a pronounced effect on deterioration; below 296 deterioration is extremely slow. Glucose content also affects deterioration, as little q causing significant change in color of the albumen as 0.02' during storage.
PROCEDURES AND METHODS
PREPARATION OF SAMPLES.illbumen samples were dried by two methods. One was by drying the liquid in glass pie plates using infrared lamps and a n electric fan. As soon as the surface was firmly crusted, it was cut loose from the pan and inverted. Drying T v a ~then completed without heat. Another ~ ~ by8 drying s under high vacuum from the frozen state (lypholization). 111 most cases the dry material was reduced to a powder by gentle grinding. The moisture Content of the Samples was genCIally adjusted to the desired level by equilibrating them in desiccators Over solutions of the appropriate moisture vapor pressures. Samples were rendered free of glucose either by fermentation with bacteria or by dialysis against tap water. pH adjustments Fere made by slowly adding dilute hydrochloric acid or sodium hydroxide to
egg products now quite generally recognized I)"'"" show Serious deterioration during storage, particularly a t to
temperatures above 20" C. Fortunately, a considerable amount of research has been done on this problem in the last few years. Sllfficient progress has been made so that it is now possible to store these Products for several months nithout encountering serious losses in quality, The beneficial effect of low moisture, @, and storage temperature have been established by the work of Boggs and Fevold (4),Brooks ( 5 ) , Hawthorne (ff), Stewart, Best, and Lowe (151, and White and Thistle (17). The importance of storing whole egg and yolk in the absence of oxygen is shown by the work of Bate-Smith, Brooks, and Hawthorne (8) and Boggs and Fevold (4). The chemical reactions responsible for the storage changes in dried egg products are still somewhat obscure. Bate-Smith and Hawthorne (3),Stewart, Best, and Lowe (I@, and Stewart and Kline (16)have demonstrated that certain of the changes in color and solubility are related to the presence of free glucose in the albumen and whole egg, The work of these and other investigators indicates that storage temperature, PH, and moisture content are factors affecting the rate of these deteriorations due to the presence of glucose. The development of color and insolubility is favored by increased concentrations of glucose, elevated storage temperatures, alkaline reaction, and high moisture contents. In the present experiments it was decided to make some sysiematic studies of the several known variables influencing the
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TlME DAYS
Figure 1. Effect of Storage Temperature on Fluorescenee Development i n Dried Albumen
theANALYTICAL liquid egg METHODS.The moisture content of samples was determined by the A.O.A.C. method (1) modified by using a 1gram sample, eliminating sifting, drying for exactly 5 hours, and cooling the dried samples over fresh phosphorus pentoxide. Fluorescence was determined b the following modification of the method of Pearce, Thistle, anzReid ( 1 4 ): 1 gram of albumen was weighed into 51 test tube, and 20-25 ml. of a 0.1 +yphosphate buffer of p H 4.8 were added. (Buffer is preferred to water because of easier filtering; the buffer had no effect on the fluorescence and solubility values.) This mixture was then thoroughly of a laboratory homogenizer. rt FaS then dispersed by transferred quantitatively to a 100-ml. volumetric flask and made up t o volume with buffer. The solution was then poured into a 250-ml. beaker, and 4 grams of Dicalite Speedflow filter aid were added. After thorough stirring, the mixture was filtered through a 1T7hatman No. 12 filter paper, ~l~~~~~~~~~~ was determined on the clear filtrate, using a Coleman model No. 12 photofluorometer (filters B1 and PC-1). The photofluorometer was adjusted to a reading of 50, using 51 standard solution of quinine sulfate (0.2 mg. per liter) in 0.1 N sulfuric acid (except as noted), Solubility was determined by making a moisture determination on an aliquot portion of the clear filtrate used for the fiuorescence measurement. Values were calculated on the basis of soluble solids. In each case solubility is expressed as percentage of the value obtained for the sample prior to storage. Glucose was determined by a modification of the A.O.A.C. method ( 1 ) : 1 gram of dried albumen was placed in a test tube, and 20-25 ml. of distilled water were added. After the sample was dissolved, i t was thoroughly blended by use of a laboratory homogenizer. The mixture was then transferred quantitatively to a 100-ml. volumetric flask TIME -DAYS, and sufficient distilled Figure 2. Effect of Storage Temperature water added to bring the on Solubility Loss in Dried Albumen volume to approximately
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60 ml. The flask was then immersed in boiling water for 15 minutes (care was taken to shake several times). After the flask and contents were cooled, 1 gram of phosphotungstic acid was added, and the flask was shaken and then allowed to stand for 10 minutes. Ten milliliters of saturated potassium chloride solution were added, and the flask was then made up to volume with distilled water. After thorough mixing, the contents were filtered through a Whatman No. 12 filter paper. Fifty milliliters of the clear filtrate were taken for the glucose determination. Presence or absence of glucose in liquid samples were determined by the following method: 5 ml. of the fermented or dialyzed albumen were pipetted into a 50-ml. beaker which was then placed in a well ventilated air oven at 120" C. for 2 hours. The color of the albumen was then noted. Tests on samples containing varying amounts of glucose showed that, when no color developed (beyond the normal yellow color of dried albumen) during the drying and heating period, the albumen was free of glucose. Color determinations on the albumen samples were made by subjective ratings as well as by determining the transmissivity of a 1% ' albumen solution at 350 mp on a Coleman model 11 Universal sDectroDhotometer. The results of the latter measurements are expressed in terms of concentrations of a solution of potassium ferricyanide of the same absorbing power. p H values were determined by means of a Leeds & Northrup glass electrode p H assembly. RESULTS
Olcot,t and Dutton (IS)have shown that optical density and fluorescence intensity curves for glucose-glycine mixtures undergoing reaction are superimposable. Also Dutton and Edwards (6) have noted a roughly linear relation between negative logarithm of reflectance of ether extracted residues and salt extract fluorescence of dried whole egg samples. This is regarded as evidence for the identity of the color and fluorescent compounds. Results obtained in this laboratory confirm these results and indicate a linear relation between optical density and fluorescence value for deteriorating albumen samples. Since measurement of fluorescence was more sensitive and more convenient to use, most of the data reported in this paper involve
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measurements of this characteristic of the deterioration. It is assumed that the changes in fluorescence are parrtlleled by changes in color. STORAGETEMPERATURE. In order to obtain data on theeffect of temperature on fluorescence and solubility, as well as to establish suitable test conditions, storage studies were made over a wide range of temperature. Conditions of time and temperature were established whereby changes in solubility and fluorescence could be measured without encountering interference from coagulation. These studies were made using both natural and glucose-free albumen samples. It was found t h a t coagulation requir- conditions considerably more severe than those necessary for producing the solubility and fluorescence changes associated with the glucose-protein reaction. However, some care had to be exercised to avoid an overlapping of these two phenomena. Fluorescence development was not observed during coagulation; this made it possible to differentiate between the two phenomena. I n addition., coagulated dried albumen reconstituted as a white mass, in contrast to one of orange or brown color for dried albumen showing typical storage deterioration. The data pertaining to the effects of storage time and temperature are partially summarized in Figures 1 and 2. The temperature coefficients for the reactions involved in fluorescence development and solubility loss are veYy high. At temperatures above 30" C. the fluorescence rose to a maximum, then declined. This decline in fluorescence paralleled insolubility development. (Other data on samples stored at 50", 60°, and 70" C. corroborated these findings; fluorescence values rose to significantly higher levels whenever solubility losses were delayed.) Fluorescence development in samples stored a t 20" and 30" C. were slow but measurable. The color of the dry material as well as the insoluble fraction darkened during storage. Color intensity was found to be dependent also upon storage temperature. (After 45-day storage the samples stored a t 50' C. and above were brownish black, whereas those stored at 40" C. were orange; those at 20' and 30" C. were slightly yellowish orange; the unstored samples had a yellowish cast.) Changes in solubility took place rapidly at temperatures above 50" C. ; they were very much slowed down at 40' C., whereas no significant changes were observed in samples stored at 20 * and 30 O C. High temperature coefficients of reaction have been reported for the amino acid-reducing sugar systems (18). . The present
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Figure 3. Effects of pH on Fluorescence Development in Dried Albumen pH is that of reconstituted sample. Because of a loa6 of carbon dioxide, the pH is higher than that of the original liquid (8.8, 7.0, 6.0, apd 5.0, respectively).
Figure 4.
Effect of pH on Solubility Loss in Dried . Albumen
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TIME- DAYS.
Figure 5. Effect of Moisture Content on Loss in Solubility during Storage in Dried Albumen
data indicate that the reactions responsible for fluorescence are different from and have higher temperature coefficients than those responsible for changes in solubility (compare Figures 1 and 2). pH. The effects of p H are summarized in Figures 3 and 4. Lowering the p H from 9.5 to 9.0 slowed down the rate of change in solubility considerably. Further decreases in p H changed the rate only moderately. Lowering the p H from 9.5 to 9.0 and t o 7.3 did not much change the rate of fluorescence development. The fluorescence intensity rose to higher values before declining at p H 9.0, 7.3, and 4.8. At p H 4.8 the initial changes in fluorescence were delayed, although once begun the development of fluorescence proceeded a t a rate roughly equal to t h a t found under alkaline conditions. I n studies which have been made on the amino acid-reducing sugar reaction, alkaline conditions have been found to exert a n accelerating effect ( I d ) . However, Frankel and Katchalsky (7, 8) have shown that for both amino acids and peptides there is a n optimum p H (about 8.0) for both the rate and extent of reaction. It has been suggested that the reactions are qualitatively different above p H 9.0 (9, 10). Results obtained in the present experiments do not lend themselves readily to interpretation in t e r m of the literature just referred to. They do lend support to the hypothesis that the reactions giving rise to
Vol. 40, No. 5
fluorescence development are qualitatively different from those resulting in solubility loss. MOISTURECONTENT.Previous work from this laboratory demonstrated that moisture content exerts a pronounced effect on the deteriorat,ive reactions in dried egg albumen (15). These results are substantiated and elaborated by the data shown in Figures 5 and 6. At moisture contents below 5yo the changes were slow, although significant changes were noted at 1.3% moisture. These data make evident the much greater importance of moisture content over p H in controlling the rate of change in solubility and color in dried egg albumen. GLCCOSECONTENT. The effect of low levels of glucose on deterioration rates are summarized in Table I. These results indicate that glucose concentration affects the rate tremendously. As little as 0.02% is sufficient to cause an appreciable fluorescence development. No change in solubility was noted. However, 0.05% glucose caused a definite loss in solubility. The importance of glucose level is further indicated by some observations made on color changes in samples held a t 120" C. It has been repeatedly noted that as little as 0.02% causes color (from yellow t o orange or brown) to develop during holding a t this temperature. This observation led to the development of the rapid test already described for glucose. OTHER FACTORS. Results from replicated storage tests on albumen samples of different origin but which were carefully controlled as to moisture content, pH, glucose concentration, and storage temperature showed considerable variation in the rates at which fluorescence and insolubility develop during storage. This is interpreted to mean that there are additional variables which affect the rate of deterioration. Thus far, no clue as t o their nature has been discovered. SUMMARY
The quantitative effects of several factors affecting the deterioration rate in dried albumen were studied. Storage temperature, glucose and moisture contents, and p H were found to influence the rate of deterioration. The temperature coefficients for the reactions responsible for deterioration are great, being higher than for coagulation. At 40 a C. solubility decreases steadily with time while fluorescence rises sharply to a maximum, then declines. This decline seems to be related to insolubility, since the insoluble fraction is highly colored (and presumably highly fluorescent) and initiates simultaneously with the first changes in solubility. Acidification decreases the rate of solubility changes occurring in dried albumen. The greatest decrease in insolubility development resulted from a change from p H 9.5 t o 9.0, which change had little effect on the rate of fluorescence development. At pH 4.8 fluorescence and insolubility development were both delayed. Moisture content has a pronounced effect on the rate of deterioration in dried albumen. At moisture contents below 2Y0 the rate is slow. The variations in moisture content produced proportionately greater effects than those in pH. Glucose concentration exerts a powerful effect on the deterioration rate in dried albumen. Concentrations of 0.029Z0 and higher cause appreciable changes in fluorescence and color, whereas concentrations of 0.05% and above give rise to measurable changes in solubility. Other factors still unknown affect the rate of ddterioration in driedalbumen.
OF GLUCOSE COSTEKTON DETERIORATION OF TABLE I. EFFECT DRIED -4LBU41ENa
Elapsed Time, Days
Figure 6. Effect of Moisture Content on Color Development during Storage of Dried Albumen
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Dry samples rated subjectively; in range 8 to 8, 0 = normal yellowish white, 8 brownish black
0.0670
Fluorescence Intensity
0.0270
glucose
0.10%
glucose
0.05%
glucose
100 100 100 31 29 ... 92 l/a 1 81 240 480 94 100 1 '/2 73 210 ... 220 210 2 76 98 175 210 3 47 93 165 220 4 43 83 96 160 210 5 37 a Vacuum-dried, 10% moisture, pH 9.6-9.7, stored a t 60' C. 0
DAYS.
Solubility a t 0.10%
glucoseb glucose
... ...
...
...
.*.
... ... ...
...
Glucose concentration in liquid albumen expressed on wet basis.
0.02%
glucose 20
.. .. ..
.. .. ..
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ACKNOWLEDGMENT
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(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.
(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
(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).
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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
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