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Changes in Egg Proteins Occurring During Cold Storage of Shell Eggs1
Changes in Egg Proteins Occurring During Cold Storage of Shell Eggs1 ROBERT JOHN EVANS, J. A. DAVIDSON AND H E L E N A. BUTTS Departments of Agricultural Chemistry and Poultry Husbandry, Michigan State College, East Lansing, Michigan
ACH year several million cases of eggs are placed in cold storage during the months of highest production and lowest prices to be used at a time of lower production and higher prices. Longer holding times have been somewhat common in recent years because of government purchases of eggs to maintain support prices. Changes occurring in the composition of shell eggs during storage are nutritionally important. That there are chemical changes occurring in shell eggs during storage has been recognized for many years. One of the earliest observations was the increase in the ammonia nitrogen content of stored eggs (Lindit and Hussen, 1917). Other changes early observed were a decrease in the moisture content of the albumen and an increase in the moisture content of the yolk (Jenkins et al., 1920), a rapid increase in the pH of the egg albumen (Sharp and Powell, 1927), and a decrease in the amount of crystallized egg albumin that can be obtained (Sorensen and Hoynip, 1916).
E
The data presented in this paper were obtained as part of an experiment to study changes in the composition of shell eggs taking place during storage. Longer stor1
Published with the approval of the Director of the Michigan Agricultural Experiment Station as Journal Article No. 960 (n.s.)
age periods than would usually be common were used in order to amplify any changes that might take place. These data are concerned with the changes in total protein content and the various protein fractions occurring during storage of the shell eggs. Changes in amino acid content will be reported in a later communication. EXPERIMENTAL During the summer months of each year several cases of eggs from the Michigan State College poultry farm were placed in cold storage. Part of these were stored in the regular egg cases and part in cartons holding one dozen eggs that were kept in the regular cases. Two series of determinations were made. One group of fresh eggs (1 day old) was included in each series. The stored eggs were broken out on the day that they were removed from cold storage. The first series of eggs, broken out in October 1947, consisted of one dozen fresh eggs obtained from the Michigan State College flock, one dozen eggs which had been placed in storage in April, 1946 (18 months storage), and one dozen eggs which had been placed in storage in August, 1945 (26 months storage). Each dozen eggs was divided into three groups. The eggs from each group were broken, the albumen and yolk very carefully separated, and the albumen of the four
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(Received for publication August 9,1948)
PROTEIN CHANGES IN EGGS DURING COLD STORAGE
207
The pH was determined on the diluted albumen and yolk samples with a Beckman pH meter soon after the eggs were broken out. The nitrogen was determined on 5.0 gram samples of the diluted albumen or yolk by Kjeldahl-Gunning-Arnold procedure (A.O.A.C, 1945). The crude protein content was calculated by multiplying nitrogen by 6.25.
Total sulfur was determined by the official sodium carbonate-sodium peroxide fusion procedure (A.O.A.C, 1945). The amounts of the different proteins in egg albumen were determined by a procedure based on methods described by McNally (1934), Longsworth, Cannan, and Mclnnes (1940), and Balls and Hoover (1940). Twenty grams of the di-
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The second series of eggs, broken out in February, 1948, consisted of two dozen fresh eggs obtained from the Michigan State College flock, two dozen eggs placed in storage May, 1947 (9 months), and two dozen which had been placed in storage April, 1946 (22 months). The eggs from each treatment were divided into three groups in the same manner as for the first series, six eggs comprising a group. The yolks and albumen were again carefully separated. This time, however, greater care was taken to be certain that no loss of albumen occurred. As a result some of the albumen adhered to the yolk membrane at times with the second series of eggs. This albumen would have been removed from the yolk and lost by the method used for the first series of eggs. There were no mechanical losses of either yolk or albumen in this series although the yolks were contaminated with some albumen.
The weight of protein per egg albumen or yolk was determined by multiplying the weight of the albumen or yolk by the percentage of protein in it divided by 100. The egg proteins were fractionated by a modification of the official A.O.A.C. method (1945). Twenty grams of diluted egg albumen or yolk were mixed with 100 ml. of H 2 0 and the pH adjusted to 4.0 for the albumen and to 5.2 for the yolks. The mixture was transferred to a 250 ml. volumetric flask, made to volume with H 2 0, and allowed to stand over night and then it was filtered through a dry Whatman No. 42 paper. Fifty ml. of the clear filtrate was transferred to a Kjeldahl flask and nitrogen determined by the Kjeldahl-Gunning-Arnold procedure. This was the water-soluble fraction. The waterinsoluble fraction was determined by difference. One hundred ml. of the filtrate were transferred into a 200-ml. volumetric flask, 15 ml. of a sodium chloride solution (containing 28 gm. of NaCl diluted to 300 mi. volume) were added, and absolute ethanol was added to make to volume. The contents of the flask were mixed and allowed to stand over night, when they were transferred to a 250-ml. centrifuge bottle and centrifuged. One hundred ml. of the supernatant liquid were transferred into a Kjeldahl flask and nitrogen determined on the contents. This was the alcohol-soluble fraction. The "crude albumin" fraction was that portion of the protein that was soluble in water but not soluble in 42 percent ethyl alcohol.
eggs in each group composited to give one sample. The yolks were treated in the same way, giving three samples of albumen and three of yolk for each storage period. An equal weight of 0.9 percent sodium chloride solution was added to each composite sample. The yolk samples were well mixed with a stirring rod, and the albumen samples were mixed in a Waring Blendor in order to break up the firm albumen and make an homogenous suspension. Each composite sample was stored in a stoppered bottle under toluene in the refrigerator until the required determinations could be conducted.
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ROBERT JOHN EVANS, J. A. DAVIDSON AND HELEN A. BUTTS
to about 150 ml., or by heating on the steam bath (75°C.) for 6-8 hours and then evaporating to about 150 ml. The albumin precipitate was filtered, washed well with distilled water, and nitrogen determined on the precipitate and paper. The combined filtrate and washings were evaporated on the steam bath to a sodium sulfate concentration of 32 grams per 100 ml. to precipitate the ovomucoids. The precipitate was collected on a quantitative filter paper, and paper and precipitate were analyzed for nitrogen. RESULTS
The egg contents lost weight during storage, presumably caused mostly by a loss of water from the egg (Fig. 1). This loss occurred mostly in the albumen, although there was some loss in yolk weight
s < a
M O N T H S
. FIG. 1. Changes in weight of contents of shell eggs occurring during storage. E—edible egg contents A—albumen Y—yolk The three points for the fresh eggs on each curve represent the values for the first series of eggs, the second series, and the average. The curve passes through the average point.
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luted egg albumen were mixed with 60 ml. of distilled water and the pH adjusted to 6.9-7.1 with 1.0 normal acetic acid. The precipitated ovomucin was separated by centrifugation, washed with 20 ml. of dilute sodium sulfate solution, transferred to a Kjeldahl flask, and nitrogen determined as previously described. The ovoglobulins were precipitated by adding sodium sulfate to the combined filtrate and washings to give a concentration of 21.3 gm. per 100 ml. The precipitate was filtered onto a No. 42 Whatman paper, washed with 20 ml. of a solution containing 21.3 gm. of sodium sulfate per 100 ml. of distilled water, and nitrogen determined on the paper and precipitate. The albumins were precipitated either by heating the diluted filtrate and washings to boiling and then evaporating on the steam bath
209
PROTEIN CHANGES IN EGGS DURING COLD STORAGE
*
albumens. The protein content of the yolks from eggs stored 9 months was lower than that of either the fresh eggs or the eggs stored twice as long. Figure 3 presents the changes in weight of protein per egg that took place during cold storage. The albumen of the fresh eggs used in the first experiment, because of their larger size, contained 0.5 gram more protein per egg than the albumen of the fresh eggs used in the second experiment (Fig. 1). A loss in the total weight of protein in the albumen of each egg occurred during the first 9 months of storage. The loss in weight of protein per egg yolk was greatest between the 23rd and the 26th months of storage. The results of the protein distribution studies on fresh and stored eggs are presented in Tables 1 and 2. Albumen from
15 -
M EH O
a EH
HI O K
M
10
12
14
16
22
24
MONTHS FIG. 2. Changes in the protein content of shell eggs occurring during storage. E—edible contents A—albumen Y—yolk
26
28
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between the 23rd and the 26th months of storage. The eggs stored for 18 months and those stored for 23 months were from the same lot of eggs but were removed from storage at different times. The smaller loss in weight from the eggs stored the longest was probably the result of decreased mechanical losses because of the precautions taken during the second experiment. The greater yolk weight of the 23-month old eggs was caused by the adherence of some albumen to the yolks. Storage resulted in an increased protein content in the egg albumen (Fig. 2). The protein content of the egg yolks decreased slightly during the 26 months of cold storage, approaching the same value, 16.2 percent, as for the albumens. The protein content of the yolks of eggs stored for 26 months was slightly lower than that of the
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ROBERT JOHN EVANS, J. A. DAVIDSON AND HELEN A. BUTTS
TABLE 1.—Protein distribution, pB, and sulfur content of the albumen of fresh and stored eggs (total protein basis) Months stored Percent water insoluble protein* Percent "crude albumin" Percent alcohol soluble protein pH
9
18
23
26
2.7
3.7
2.5
2.2
1.9
78.3
77.7
81.9
81.9
80.9
19.0
18.5
15.9
15.9
17.2
8.4
8.5
9.1**
8.9
8.9**
1.68
1.72
1.74
1.74
Ave. 0
3.4 2.0 77.8 78.8 18.8 19.1 8.7** 7.7 1.62 1.72
.1.67
* Percent of total protein. ** Measured one day after breaking out.
eggs stored for 9 months contained a larger percentage of water insoluble protein than that from fresh eggs or from eggs stored longer. The percentage of "crude albumin" did not change appreciably. There was a slight decrease in percentage of alcohol soluble protein between the 9th
and 18th months of storage. Apparently a slight increase of "crude albumin" and a decrease of water insoluble protein in egg yolks occurred during the first 9 months of storage. These changes were all slight. The pH of the albumen increased rapidly and reached a maximum after 18
CO
a
«< «
10
12 14 16 MONTHS
FIG. 3. Changes in weight of protein per egg occurring during storage. E—in edible egg contents A—in albumen Y—in yolk
28
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Percent sulfur
0
211
PROTEIN CHANGES IN EGGS DURING COLD STORAGE TABLE 2.—Protein distribution, pB, and sulfur content of the yolks of fresh and stored eggs (total protein basis) Months stored
0
Percent "crude albumin" Percent alcohol soluble protein pH Percent sulfur**
9
18
23
26
78.5
75.9
76.6
75.6
76.6
6.9
10.8
9.1
10.9
8.9
14.5
13.3
14.4
13.6
14.5
6.3
6.9
7.2
7.4
7.2
1.13
1.16
1.14
1.17
1.14
* Percent of total protein. ** Sulfur was determined on a preparation of isolated crude protein.
months of cold storage (Table 1). The increase in pH of the egg yolks was slower, reaching a maximum after 23 months of storage (Table 2). The sulfur content of the albumen proteins of the fresh eggs used in the first experiment was lower than that of those used in the second experiment and was significantly lower than the sulfur content of the albumen protein of any of the stored eggs. Similar results were observed for the egg yolk proteins but the differences were not so large. The amounts of the different proteins in the albumen of the second series eggs are presented in Table 3. The higher mucin values for the eggs stored for 9 months corroborate the data presented in Table 1, since the water-insoluble protein is primarily mucin. A slight increase in the ovoglobulin fraction occurred during storage, but it was small. Very little change in TABLE 3.—Protein fractions in the albumen of fresh and stored eggs (total protein basis) Months stored
0
9
23
Percent mucin 2.8 1.4 1.8 Percent ovoglobulin 12.5 13.1 14.7 Percent albumin (Boiling) 76.9 73.7 74.3 Percent albumin (75°C.) (74.6) (49.4) (44.1) Percent ovomucoid 8.3 8.3 8.3 Percent protein not accounted for 0.9 2.1 0.9
the percentage of albumin occurred when albumin was determined by precipitation by boiling. However, the percentage of protein precipitated by heating for several hours at about 75° C. (which should precipitate all of the albumins) decreased with the time of storage from 74.6 percent for fresh eggs to 44.1 percent for eggs stored 23 months. Approximately the same albumin values were obtained for fresh eggs when the albumin was precipitated by boiling as when it was precipitated by heating at 75' C. on the steam bath. DISCUSSION The weight loss from egg albumen during storage and the accompanying concentration of protein in the albumen is not a new observation but agrees with results reported by Jenkins et al. (1920), Romanoff (1940), Szorenyi (1941), and Silva (1946). The small decreased protein con-, tent of the yolk (Fig. 2) may or may not be significant. Jenkins et al. (1920) and Silva (1946), reported a decreased protein content of egg yolks during storage, but Szorenyi (1941), reported no change to occur. The observation that there was an actual loss in weight of edible protein per egg was surprising. The second experiment was conducted to carefully check the
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78.9 78.1 7.0 6.8 14.0 15.1 6.2 6.3 1.10 1.16
Percent water insoluble protein*
Ave. 0
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ROBERT JOHN EVANS, J. A. DAVIDSON AND HELEN A. BUTTS
The eggs stored for 9 months contained fewer grams of protein per egg than eggs stored for 18 months. The yolks also contained a smaller percentage of protein. The 9-months-stored eggs in this respect behaved as though they had been stored longer than they actually were, even though weight losses of the egg contents were what would be expected. All of the eggs, except those which were stored 9 months, were stored in egg cartons containing one dozen eggs each, which cartons were kept in the larger regular egg cases. The eggs stored for 9 months were kept in regular egg cases, as would be done commercially. The egg cases apparently permitted a greater deterioration of the egg contents, which was not related to the loss in moisture, than the smaller cartons. The albumen of the eggs stored 9 months contained the highest percentage of water insoluble protein, which precipitated out to a considerable extent even before dilution of the egg albumen. Difficulty was experienced in the isolation of the water-insoluble protein from the yolks of the eggs stored for 9 months. The protein could not be centrifuged down, and it was filtered out only with difficulty. On standing the
protein rose to the top of the beaker in contrast to the others which settled out. Apparently certain changes took place in the egg yolk proteins that were not measured by the methods of study used. The water insoluble protein fraction of egg albumen can be identified as mucin. There was an increase in this fraction during the first 9 months of storage, followed by a decrease. This observation is of interest in view of the increase in the mucin content of the middle and inner layers of egg albumen observed by Almquist et al. (1934) during 3 months of cold storage. Balls and Hoover (1940), however, found no change in mucin content of egg albumen protein during 18 days of storage at 30°C. Mitchell (1934) obtained an increase in water-soluble nitrogen (which would infer a decrease in mucin) during two months of cold storage. No appreciable difference in the "crude albumin" content of fresh and stored eggs was observed. An attempt was made to separate the ovalbumin and conalbumin by the sodium sulfate fractionation procedure of Kekwick and Cannan (1936). Inconclusive results were obtained because of the difficulty in removing quantitatively the precipitated ovalbumin without changing the salt concentration. The data obtained, however, indicate no conversion of ovalbumin into conalbumin during storage as suggested by Sorensen and Hoyrup (1916) and Bidault (1928) based on the observation that new-laid eggs gave a higher yield of crystallized ovalbumin than eggs which had been kept for a time. By immunological techniques Hekton and Cole (1928, 1929) presented evidence that ovalbumin is not converted to conalbumin, but conalbumin is probably identical with blood albumin. The only very marked difference observed between the behavior of the albu-
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earlier results. A loss in weight of protein was again observed although the loss was not as great because mechanical losses were prevented by using improved techniques. Legaspi (1933), stated that eggs stored under room conditions for 63 days contained practically the same weight of protein as fresh eggs. Mitchell (1934), however, observed a slight decrease in total nitrogen in egg solids during 2 months of cold storage, and Sasaki and Harayawa (1934), found a loss of protein nitrogen after storage of hen's eggs. Romanoff (1940) reported less total nitrogen in the contents of stored eggs than in fresh ones.
PROTEIN CHANGES IN EGGS DURING COLD STORAGE
tion of sulfur in the egg albumen proteins during storage. SUMMARY
Shell eggs stored at 0°C. for periods of from 9 to 26 months lost weight during storage. This loss occurred in the albumen except for the eggs stored for 26 months, the yolks of which also lost weight. The weight loss from the albumen was accompanied by an increased concentration of protein in the albumen. The stored eggs contained fewer grams of protein per egg than the fresh eggs. The percentage of water-insoluble protein, or mucin, in the albumen protein increased during the first 9 months of storage and then decreased. The percentage of albumin coagulated at 75°C. decreased, but no change in albumin content occurred when determined by precipitation with alcohol or by boiling. There were some other slight changes in protein composition. The sulfur content of the egg albumen proteins increased slightly during storage. No indications that the losses of total protein were due to losses of a particular protein fraction were obtained. REFERENCES
Almquist, H. J., J. W. Givens, and A. Klose, 1934. Transmission of light by egg albumen, Ind. Eng. Chem. 26:847-848. Association of Official Agricultural Chemists, Official Methods of Analysis, Washington, 1945. Balls, A. K., and S. R. Hoover, 1940. Behavior of ovomucin in the liquefaction of egg white. Ind. Eng. Chem. 32:594-596. Bidault, C , 1928, Fresh and cold-storage eggs. (Chem. Abs. 22, 2011) Rev. Hyg. Med. 50: 178-185. Hektoen, L., and A. G. Cole, 1928. The proteins of egg white. The proteins in egg white and their relationship to the blood proteins of the domestic fowl as determined by the precipitation reaction. J. Infect. Disease 42:1-24. Hektoen, L., and A. G. Cole, 1929. The proteins of egg white. 2. On the transformation of crystal-
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men proteins of fresh and stored eggs was the higher temperature required to precipitate the albumins of the stored eggs. Apparently changes occur in the ovalbumin during storage which make it more resistant to heat coagulation. These changes may be associated with the decreased ease of crystallization of the ovalbumin from stored eggs. The data of Tables 1 and 2 do not agree with the observation of Sasaki and Harazawa (1934) that the nitrogen soluble in alcohol increases markedly during storage of the hen's egg or Mitchell's (1934) data indicating an increased content of watersoluble nitrogen and "crude albumin" in egg yolk solids. Sharp and Powell (1927), who studied the changes in pH of egg albumen during storage, observed a pH of about 7.6 for the albumen of freshly laid eggs. This agrees very well with the value of 7.7 for day old eggs obtained in the second experiment. Since the eggs used in the first experiment were allowed to stand overnight after breaking out before making the pH measurement, the relatively high pH is not representative of the freshly broken out eggs. The high pH value of 9.1 is lower than the 9.5 reported by Sharp and Powell (1927). The sulfur values obtained are difficult to explain. The 1.62 percent sulfur contained in the albumen protein of the fresh eggs used in the first experiment was significantly lower than the 1.72 percent for the second experiment. It was also lower than the value for the stored eggs, and indicated a concentration of sulfur in the egg albumen protein during storage. The results of the second experiment did not agree in this respect. An experiment is now being conducted using eggs laid by the same hens at about the same time to determine if there is an actual concentra-
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ROBERT JOHN EVANS, J. A. DAVIDSON AND HELEN A. BUTTS composition of edible shell eggs during storage. J. Assoc. Off. Agr. Chem. 17:506-511. Romanoff, A. L., 1940. Physicochemical changes in unfertilized, incubated eggs of Gallus domesticus. Food Research 5:291-306. Sasaki, R., and H. Harazawa, 1930. Changes of hen's eggs during storage. Univ. of 111., Abst. No. 3290; Nippon Tiksan. Gkw. Ho 4:58-74. Sharp, P. F., and C. K. Powell, 1927. Physico-chemical factors influencing the keeping quality of hen's eggs in storage. Proc. World's Poultry Congress, Ottawa/Canada, pp. 399-402. Silva, H. de T. e, 1946. Fresh and preserved eggs considered from the chemical point of view. (Chem. Abs. 41: 7004.) Bol. ind. Animal (Sao Paulo) 8, No. 3, 91-101. Sorensen, S. P. L., and M. Hoyrup, 1916. Studies on proteins. I. On the preparation of egg-albumin solutions of well defined composition, and on the analytical methods used. Compt. rend. trav. lab., Carlsberg 12:12-67. Szorenyi, F., 1941. The protein and cystine contents of hen eggs. (Chem. Abs. 35: 3301) Kozlemenyek 0sszehasonlit6 Elet-Kortan Korebol 29, 110-111.
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lized ovalbumin into noncrystallizable conalbumin. J. Infect. Diseases 44: 165-168. Jenkins, M. K., J. S. Hepburn, G. C. Swan, and C. M. Sherwood, 1920. Effects of cold storage on shell eggs. Ice and Refrigeration 58:140-147. Kekwick, R. A., and R. K. Cannan, 1936. The hydrogen ion dissociation curve of the crystalline albumin of the hen's egg. Biochem. J., 30: 227234. Legaspi, M. T., 1933. Changes in chemical composition of Cantonese eggs in holding. Philippine Agr. 22:509-520. Lindit and Hussen, 1917. Changes which occur in eggs, and the sanitary control of the egg business. (Chem. Abs. 11: 2375) Ann. fals. 10: 106111. Longsworth, L. G., R. K. Cannan, and D. A. MacInnes, 1940. An electrophoretic study of the proteins of egg white. J. Am. Chem. Soc, 62: 25802590. McNally, E., 1934. Passage of ovoglobulins through the shell membrane. Proc. Soc. Exptl. Biol. Med. 31: 946-947. Mitchell, L. C , 1934. Progressive changes in the
ACH year several million cases of eggs are placed in cold storage during the months of highest production and lowest prices to be used at a time of lower production and higher prices. Longer holding times have been somewhat common in recent years because of government purchases of eggs to maintain support prices. Changes occurring in the composition of shell eggs during storage are nutritionally important. That there are chemical changes occurring in shell eggs during storage has been recognized for many years. One of the earliest observations was the increase in the ammonia nitrogen content of stored eggs (Lindit and Hussen, 1917). Other changes early observed were a decrease in the moisture content of the albumen and an increase in the moisture content of the yolk (Jenkins et al., 1920), a rapid increase in the pH of the egg albumen (Sharp and Powell, 1927), and a decrease in the amount of crystallized egg albumin that can be obtained (Sorensen and Hoynip, 1916).
E
The data presented in this paper were obtained as part of an experiment to study changes in the composition of shell eggs taking place during storage. Longer stor1
Published with the approval of the Director of the Michigan Agricultural Experiment Station as Journal Article No. 960 (n.s.)
age periods than would usually be common were used in order to amplify any changes that might take place. These data are concerned with the changes in total protein content and the various protein fractions occurring during storage of the shell eggs. Changes in amino acid content will be reported in a later communication. EXPERIMENTAL During the summer months of each year several cases of eggs from the Michigan State College poultry farm were placed in cold storage. Part of these were stored in the regular egg cases and part in cartons holding one dozen eggs that were kept in the regular cases. Two series of determinations were made. One group of fresh eggs (1 day old) was included in each series. The stored eggs were broken out on the day that they were removed from cold storage. The first series of eggs, broken out in October 1947, consisted of one dozen fresh eggs obtained from the Michigan State College flock, one dozen eggs which had been placed in storage in April, 1946 (18 months storage), and one dozen eggs which had been placed in storage in August, 1945 (26 months storage). Each dozen eggs was divided into three groups. The eggs from each group were broken, the albumen and yolk very carefully separated, and the albumen of the four
206
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(Received for publication August 9,1948)
PROTEIN CHANGES IN EGGS DURING COLD STORAGE
207
The pH was determined on the diluted albumen and yolk samples with a Beckman pH meter soon after the eggs were broken out. The nitrogen was determined on 5.0 gram samples of the diluted albumen or yolk by Kjeldahl-Gunning-Arnold procedure (A.O.A.C, 1945). The crude protein content was calculated by multiplying nitrogen by 6.25.
Total sulfur was determined by the official sodium carbonate-sodium peroxide fusion procedure (A.O.A.C, 1945). The amounts of the different proteins in egg albumen were determined by a procedure based on methods described by McNally (1934), Longsworth, Cannan, and Mclnnes (1940), and Balls and Hoover (1940). Twenty grams of the di-
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The second series of eggs, broken out in February, 1948, consisted of two dozen fresh eggs obtained from the Michigan State College flock, two dozen eggs placed in storage May, 1947 (9 months), and two dozen which had been placed in storage April, 1946 (22 months). The eggs from each treatment were divided into three groups in the same manner as for the first series, six eggs comprising a group. The yolks and albumen were again carefully separated. This time, however, greater care was taken to be certain that no loss of albumen occurred. As a result some of the albumen adhered to the yolk membrane at times with the second series of eggs. This albumen would have been removed from the yolk and lost by the method used for the first series of eggs. There were no mechanical losses of either yolk or albumen in this series although the yolks were contaminated with some albumen.
The weight of protein per egg albumen or yolk was determined by multiplying the weight of the albumen or yolk by the percentage of protein in it divided by 100. The egg proteins were fractionated by a modification of the official A.O.A.C. method (1945). Twenty grams of diluted egg albumen or yolk were mixed with 100 ml. of H 2 0 and the pH adjusted to 4.0 for the albumen and to 5.2 for the yolks. The mixture was transferred to a 250 ml. volumetric flask, made to volume with H 2 0, and allowed to stand over night and then it was filtered through a dry Whatman No. 42 paper. Fifty ml. of the clear filtrate was transferred to a Kjeldahl flask and nitrogen determined by the Kjeldahl-Gunning-Arnold procedure. This was the water-soluble fraction. The waterinsoluble fraction was determined by difference. One hundred ml. of the filtrate were transferred into a 200-ml. volumetric flask, 15 ml. of a sodium chloride solution (containing 28 gm. of NaCl diluted to 300 mi. volume) were added, and absolute ethanol was added to make to volume. The contents of the flask were mixed and allowed to stand over night, when they were transferred to a 250-ml. centrifuge bottle and centrifuged. One hundred ml. of the supernatant liquid were transferred into a Kjeldahl flask and nitrogen determined on the contents. This was the alcohol-soluble fraction. The "crude albumin" fraction was that portion of the protein that was soluble in water but not soluble in 42 percent ethyl alcohol.
eggs in each group composited to give one sample. The yolks were treated in the same way, giving three samples of albumen and three of yolk for each storage period. An equal weight of 0.9 percent sodium chloride solution was added to each composite sample. The yolk samples were well mixed with a stirring rod, and the albumen samples were mixed in a Waring Blendor in order to break up the firm albumen and make an homogenous suspension. Each composite sample was stored in a stoppered bottle under toluene in the refrigerator until the required determinations could be conducted.
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ROBERT JOHN EVANS, J. A. DAVIDSON AND HELEN A. BUTTS
to about 150 ml., or by heating on the steam bath (75°C.) for 6-8 hours and then evaporating to about 150 ml. The albumin precipitate was filtered, washed well with distilled water, and nitrogen determined on the precipitate and paper. The combined filtrate and washings were evaporated on the steam bath to a sodium sulfate concentration of 32 grams per 100 ml. to precipitate the ovomucoids. The precipitate was collected on a quantitative filter paper, and paper and precipitate were analyzed for nitrogen. RESULTS
The egg contents lost weight during storage, presumably caused mostly by a loss of water from the egg (Fig. 1). This loss occurred mostly in the albumen, although there was some loss in yolk weight
s < a
M O N T H S
. FIG. 1. Changes in weight of contents of shell eggs occurring during storage. E—edible egg contents A—albumen Y—yolk The three points for the fresh eggs on each curve represent the values for the first series of eggs, the second series, and the average. The curve passes through the average point.
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luted egg albumen were mixed with 60 ml. of distilled water and the pH adjusted to 6.9-7.1 with 1.0 normal acetic acid. The precipitated ovomucin was separated by centrifugation, washed with 20 ml. of dilute sodium sulfate solution, transferred to a Kjeldahl flask, and nitrogen determined as previously described. The ovoglobulins were precipitated by adding sodium sulfate to the combined filtrate and washings to give a concentration of 21.3 gm. per 100 ml. The precipitate was filtered onto a No. 42 Whatman paper, washed with 20 ml. of a solution containing 21.3 gm. of sodium sulfate per 100 ml. of distilled water, and nitrogen determined on the paper and precipitate. The albumins were precipitated either by heating the diluted filtrate and washings to boiling and then evaporating on the steam bath
209
PROTEIN CHANGES IN EGGS DURING COLD STORAGE
*
albumens. The protein content of the yolks from eggs stored 9 months was lower than that of either the fresh eggs or the eggs stored twice as long. Figure 3 presents the changes in weight of protein per egg that took place during cold storage. The albumen of the fresh eggs used in the first experiment, because of their larger size, contained 0.5 gram more protein per egg than the albumen of the fresh eggs used in the second experiment (Fig. 1). A loss in the total weight of protein in the albumen of each egg occurred during the first 9 months of storage. The loss in weight of protein per egg yolk was greatest between the 23rd and the 26th months of storage. The results of the protein distribution studies on fresh and stored eggs are presented in Tables 1 and 2. Albumen from
15 -
M EH O
a EH
HI O K
M
10
12
14
16
22
24
MONTHS FIG. 2. Changes in the protein content of shell eggs occurring during storage. E—edible contents A—albumen Y—yolk
26
28
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between the 23rd and the 26th months of storage. The eggs stored for 18 months and those stored for 23 months were from the same lot of eggs but were removed from storage at different times. The smaller loss in weight from the eggs stored the longest was probably the result of decreased mechanical losses because of the precautions taken during the second experiment. The greater yolk weight of the 23-month old eggs was caused by the adherence of some albumen to the yolks. Storage resulted in an increased protein content in the egg albumen (Fig. 2). The protein content of the egg yolks decreased slightly during the 26 months of cold storage, approaching the same value, 16.2 percent, as for the albumens. The protein content of the yolks of eggs stored for 26 months was slightly lower than that of the
210
ROBERT JOHN EVANS, J. A. DAVIDSON AND HELEN A. BUTTS
TABLE 1.—Protein distribution, pB, and sulfur content of the albumen of fresh and stored eggs (total protein basis) Months stored Percent water insoluble protein* Percent "crude albumin" Percent alcohol soluble protein pH
9
18
23
26
2.7
3.7
2.5
2.2
1.9
78.3
77.7
81.9
81.9
80.9
19.0
18.5
15.9
15.9
17.2
8.4
8.5
9.1**
8.9
8.9**
1.68
1.72
1.74
1.74
Ave. 0
3.4 2.0 77.8 78.8 18.8 19.1 8.7** 7.7 1.62 1.72
.1.67
* Percent of total protein. ** Measured one day after breaking out.
eggs stored for 9 months contained a larger percentage of water insoluble protein than that from fresh eggs or from eggs stored longer. The percentage of "crude albumin" did not change appreciably. There was a slight decrease in percentage of alcohol soluble protein between the 9th
and 18th months of storage. Apparently a slight increase of "crude albumin" and a decrease of water insoluble protein in egg yolks occurred during the first 9 months of storage. These changes were all slight. The pH of the albumen increased rapidly and reached a maximum after 18
CO
a
«< «
10
12 14 16 MONTHS
FIG. 3. Changes in weight of protein per egg occurring during storage. E—in edible egg contents A—in albumen Y—in yolk
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Percent sulfur
0
211
PROTEIN CHANGES IN EGGS DURING COLD STORAGE TABLE 2.—Protein distribution, pB, and sulfur content of the yolks of fresh and stored eggs (total protein basis) Months stored
0
Percent "crude albumin" Percent alcohol soluble protein pH Percent sulfur**
9
18
23
26
78.5
75.9
76.6
75.6
76.6
6.9
10.8
9.1
10.9
8.9
14.5
13.3
14.4
13.6
14.5
6.3
6.9
7.2
7.4
7.2
1.13
1.16
1.14
1.17
1.14
* Percent of total protein. ** Sulfur was determined on a preparation of isolated crude protein.
months of cold storage (Table 1). The increase in pH of the egg yolks was slower, reaching a maximum after 23 months of storage (Table 2). The sulfur content of the albumen proteins of the fresh eggs used in the first experiment was lower than that of those used in the second experiment and was significantly lower than the sulfur content of the albumen protein of any of the stored eggs. Similar results were observed for the egg yolk proteins but the differences were not so large. The amounts of the different proteins in the albumen of the second series eggs are presented in Table 3. The higher mucin values for the eggs stored for 9 months corroborate the data presented in Table 1, since the water-insoluble protein is primarily mucin. A slight increase in the ovoglobulin fraction occurred during storage, but it was small. Very little change in TABLE 3.—Protein fractions in the albumen of fresh and stored eggs (total protein basis) Months stored
0
9
23
Percent mucin 2.8 1.4 1.8 Percent ovoglobulin 12.5 13.1 14.7 Percent albumin (Boiling) 76.9 73.7 74.3 Percent albumin (75°C.) (74.6) (49.4) (44.1) Percent ovomucoid 8.3 8.3 8.3 Percent protein not accounted for 0.9 2.1 0.9
the percentage of albumin occurred when albumin was determined by precipitation by boiling. However, the percentage of protein precipitated by heating for several hours at about 75° C. (which should precipitate all of the albumins) decreased with the time of storage from 74.6 percent for fresh eggs to 44.1 percent for eggs stored 23 months. Approximately the same albumin values were obtained for fresh eggs when the albumin was precipitated by boiling as when it was precipitated by heating at 75' C. on the steam bath. DISCUSSION The weight loss from egg albumen during storage and the accompanying concentration of protein in the albumen is not a new observation but agrees with results reported by Jenkins et al. (1920), Romanoff (1940), Szorenyi (1941), and Silva (1946). The small decreased protein con-, tent of the yolk (Fig. 2) may or may not be significant. Jenkins et al. (1920) and Silva (1946), reported a decreased protein content of egg yolks during storage, but Szorenyi (1941), reported no change to occur. The observation that there was an actual loss in weight of edible protein per egg was surprising. The second experiment was conducted to carefully check the
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78.9 78.1 7.0 6.8 14.0 15.1 6.2 6.3 1.10 1.16
Percent water insoluble protein*
Ave. 0
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ROBERT JOHN EVANS, J. A. DAVIDSON AND HELEN A. BUTTS
The eggs stored for 9 months contained fewer grams of protein per egg than eggs stored for 18 months. The yolks also contained a smaller percentage of protein. The 9-months-stored eggs in this respect behaved as though they had been stored longer than they actually were, even though weight losses of the egg contents were what would be expected. All of the eggs, except those which were stored 9 months, were stored in egg cartons containing one dozen eggs each, which cartons were kept in the larger regular egg cases. The eggs stored for 9 months were kept in regular egg cases, as would be done commercially. The egg cases apparently permitted a greater deterioration of the egg contents, which was not related to the loss in moisture, than the smaller cartons. The albumen of the eggs stored 9 months contained the highest percentage of water insoluble protein, which precipitated out to a considerable extent even before dilution of the egg albumen. Difficulty was experienced in the isolation of the water-insoluble protein from the yolks of the eggs stored for 9 months. The protein could not be centrifuged down, and it was filtered out only with difficulty. On standing the
protein rose to the top of the beaker in contrast to the others which settled out. Apparently certain changes took place in the egg yolk proteins that were not measured by the methods of study used. The water insoluble protein fraction of egg albumen can be identified as mucin. There was an increase in this fraction during the first 9 months of storage, followed by a decrease. This observation is of interest in view of the increase in the mucin content of the middle and inner layers of egg albumen observed by Almquist et al. (1934) during 3 months of cold storage. Balls and Hoover (1940), however, found no change in mucin content of egg albumen protein during 18 days of storage at 30°C. Mitchell (1934) obtained an increase in water-soluble nitrogen (which would infer a decrease in mucin) during two months of cold storage. No appreciable difference in the "crude albumin" content of fresh and stored eggs was observed. An attempt was made to separate the ovalbumin and conalbumin by the sodium sulfate fractionation procedure of Kekwick and Cannan (1936). Inconclusive results were obtained because of the difficulty in removing quantitatively the precipitated ovalbumin without changing the salt concentration. The data obtained, however, indicate no conversion of ovalbumin into conalbumin during storage as suggested by Sorensen and Hoyrup (1916) and Bidault (1928) based on the observation that new-laid eggs gave a higher yield of crystallized ovalbumin than eggs which had been kept for a time. By immunological techniques Hekton and Cole (1928, 1929) presented evidence that ovalbumin is not converted to conalbumin, but conalbumin is probably identical with blood albumin. The only very marked difference observed between the behavior of the albu-
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earlier results. A loss in weight of protein was again observed although the loss was not as great because mechanical losses were prevented by using improved techniques. Legaspi (1933), stated that eggs stored under room conditions for 63 days contained practically the same weight of protein as fresh eggs. Mitchell (1934), however, observed a slight decrease in total nitrogen in egg solids during 2 months of cold storage, and Sasaki and Harayawa (1934), found a loss of protein nitrogen after storage of hen's eggs. Romanoff (1940) reported less total nitrogen in the contents of stored eggs than in fresh ones.
PROTEIN CHANGES IN EGGS DURING COLD STORAGE
tion of sulfur in the egg albumen proteins during storage. SUMMARY
Shell eggs stored at 0°C. for periods of from 9 to 26 months lost weight during storage. This loss occurred in the albumen except for the eggs stored for 26 months, the yolks of which also lost weight. The weight loss from the albumen was accompanied by an increased concentration of protein in the albumen. The stored eggs contained fewer grams of protein per egg than the fresh eggs. The percentage of water-insoluble protein, or mucin, in the albumen protein increased during the first 9 months of storage and then decreased. The percentage of albumin coagulated at 75°C. decreased, but no change in albumin content occurred when determined by precipitation with alcohol or by boiling. There were some other slight changes in protein composition. The sulfur content of the egg albumen proteins increased slightly during storage. No indications that the losses of total protein were due to losses of a particular protein fraction were obtained. REFERENCES
Almquist, H. J., J. W. Givens, and A. Klose, 1934. Transmission of light by egg albumen, Ind. Eng. Chem. 26:847-848. Association of Official Agricultural Chemists, Official Methods of Analysis, Washington, 1945. Balls, A. K., and S. R. Hoover, 1940. Behavior of ovomucin in the liquefaction of egg white. Ind. Eng. Chem. 32:594-596. Bidault, C , 1928, Fresh and cold-storage eggs. (Chem. Abs. 22, 2011) Rev. Hyg. Med. 50: 178-185. Hektoen, L., and A. G. Cole, 1928. The proteins of egg white. The proteins in egg white and their relationship to the blood proteins of the domestic fowl as determined by the precipitation reaction. J. Infect. Disease 42:1-24. Hektoen, L., and A. G. Cole, 1929. The proteins of egg white. 2. On the transformation of crystal-
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men proteins of fresh and stored eggs was the higher temperature required to precipitate the albumins of the stored eggs. Apparently changes occur in the ovalbumin during storage which make it more resistant to heat coagulation. These changes may be associated with the decreased ease of crystallization of the ovalbumin from stored eggs. The data of Tables 1 and 2 do not agree with the observation of Sasaki and Harazawa (1934) that the nitrogen soluble in alcohol increases markedly during storage of the hen's egg or Mitchell's (1934) data indicating an increased content of watersoluble nitrogen and "crude albumin" in egg yolk solids. Sharp and Powell (1927), who studied the changes in pH of egg albumen during storage, observed a pH of about 7.6 for the albumen of freshly laid eggs. This agrees very well with the value of 7.7 for day old eggs obtained in the second experiment. Since the eggs used in the first experiment were allowed to stand overnight after breaking out before making the pH measurement, the relatively high pH is not representative of the freshly broken out eggs. The high pH value of 9.1 is lower than the 9.5 reported by Sharp and Powell (1927). The sulfur values obtained are difficult to explain. The 1.62 percent sulfur contained in the albumen protein of the fresh eggs used in the first experiment was significantly lower than the 1.72 percent for the second experiment. It was also lower than the value for the stored eggs, and indicated a concentration of sulfur in the egg albumen protein during storage. The results of the second experiment did not agree in this respect. An experiment is now being conducted using eggs laid by the same hens at about the same time to determine if there is an actual concentra-
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ROBERT JOHN EVANS, J. A. DAVIDSON AND HELEN A. BUTTS composition of edible shell eggs during storage. J. Assoc. Off. Agr. Chem. 17:506-511. Romanoff, A. L., 1940. Physicochemical changes in unfertilized, incubated eggs of Gallus domesticus. Food Research 5:291-306. Sasaki, R., and H. Harazawa, 1930. Changes of hen's eggs during storage. Univ. of 111., Abst. No. 3290; Nippon Tiksan. Gkw. Ho 4:58-74. Sharp, P. F., and C. K. Powell, 1927. Physico-chemical factors influencing the keeping quality of hen's eggs in storage. Proc. World's Poultry Congress, Ottawa/Canada, pp. 399-402. Silva, H. de T. e, 1946. Fresh and preserved eggs considered from the chemical point of view. (Chem. Abs. 41: 7004.) Bol. ind. Animal (Sao Paulo) 8, No. 3, 91-101. Sorensen, S. P. L., and M. Hoyrup, 1916. Studies on proteins. I. On the preparation of egg-albumin solutions of well defined composition, and on the analytical methods used. Compt. rend. trav. lab., Carlsberg 12:12-67. Szorenyi, F., 1941. The protein and cystine contents of hen eggs. (Chem. Abs. 35: 3301) Kozlemenyek 0sszehasonlit6 Elet-Kortan Korebol 29, 110-111.
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lized ovalbumin into noncrystallizable conalbumin. J. Infect. Diseases 44: 165-168. Jenkins, M. K., J. S. Hepburn, G. C. Swan, and C. M. Sherwood, 1920. Effects of cold storage on shell eggs. Ice and Refrigeration 58:140-147. Kekwick, R. A., and R. K. Cannan, 1936. The hydrogen ion dissociation curve of the crystalline albumin of the hen's egg. Biochem. J., 30: 227234. Legaspi, M. T., 1933. Changes in chemical composition of Cantonese eggs in holding. Philippine Agr. 22:509-520. Lindit and Hussen, 1917. Changes which occur in eggs, and the sanitary control of the egg business. (Chem. Abs. 11: 2375) Ann. fals. 10: 106111. Longsworth, L. G., R. K. Cannan, and D. A. MacInnes, 1940. An electrophoretic study of the proteins of egg white. J. Am. Chem. Soc, 62: 25802590. McNally, E., 1934. Passage of ovoglobulins through the shell membrane. Proc. Soc. Exptl. Biol. Med. 31: 946-947. Mitchell, L. C , 1934. Progressive changes in the