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Chemistry of Shell Egg Deterioration: The Deterioration of Separated Components
Chemistry of Shell Egg Deterioration: T h e Deterioration of Separated Components R. E. FEENEY, R. B. SILVA AND L. R. MACDONNELL
Western Regional Research Laboratory,1 Albany, California (Received for publication November 21, 1950)
T
Department of Agriculture.
645
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orative agents or mechanisms arose from recent studies of this'laboratory on the HIS report concerns the initial effects of reducing chemicals on eggs. sources of the deteriorative agents or These recent studies confirmed the earlier mechanisms which cause thinning of the observations of Hoover (1940) that the white and weakening of the yolk during addition of sulfhydryl compounds to egg the storage of eggs. The experiments reported here were designed to answer the white caused thinning (Hoover's observasimple question whether these agents or tions were included in a doctoral dissertamechanisms are inherent in the white and tion and apparently have not received yolk, have their source in the yolks and wide attention). In addition, these recent diffuse into the white or vice versa, or have studies demonstrated that the addition of very small amounts of a variety of reducexternal sources. ing chemicals to eggs not only caused the Previous studies have concerned atthick white to thin but also caused the tempts to identify possible deteriorative yolk to weaken (MacDonnell, Lineweaver agents or mechanisms rather than to deand Feeney, 1950). Chemical reduction termine their sources (Sharp, 1937). thus appeared to be an attractive hyProminent among theories proposed for pothesis for the naturally occurring detethe thinning of white were those subscribriorative mechanisms causing storage ing to an enzyme mechanism and to a deteriorations of the yolk as well as the chemical hydrolysis catalyzed by hywhite. This hypothesis raised the question droxyl ions. However, the enzyme theory of possible sources of reducing substances has been virtually discarded (Balls and or of catalysts accelerating their action. It Hoover, 1940; and Lineweaver et al. was considered that they could originate 1948 and 1949) and no evidence supportfrom the shell or microorganisms on the ing chemical hydrolysis has appeared. shell, that they could diffuse from the Yolk deterioration has been associated yolk into the white or vice versa, or that with the passage of water from the white they could be present in the white and to the yolk and this passage has been concause thinning of the white and weakensidered to be related to the alkalinity of ing of the yolk by direct action at the the white. outer surface of the yolk membrane. An Our interest in the sources of the deteri- obvious way to determine such possible sources would be to study the deteriora1 Bureau of Agricultural and Industrial Chem- tions of the separated components. To istry, Agricultural Research Administration, U. S. this end, a study was attempted of the INTRODUCTION
646
R. E. F E E N E Y , R. B . SILVA AND L. R. M A C D O N N E L L
deteriorations of sterile, broken-out whole eggs, separated whites, and separated yolks. METHODS
Breaking and Separating: These procedures were conducted with strict adherence to the requirements for sterile bacteriological technique. They were carried out in a bacteriological inoculating room and workers wore hair nets and face masks. All materials such as breakers, separators, rubber gloves, etc., were sterilized prior to use and frequently changed during breaking and separating. For breaking, eggs were removed from the alcohol with sterile tongs and the alcohol drained momentarily, ignited, and then allowed to burn off. The sterile egg was then passed to a worker fitted with sterile gloves who completed the breaking operation. Whole eggs were broken directly into sterile, 4-oz., wide-
FIG. 1. Sterile broken-out and separated eggs. From left to right: Whole egg, yolk suspended in a water solution of 1.0% lysozyme and 0.9% sodium chloride, and white. The whole egg and yolk bottles contain a layer of mineral oil to minimize evaporation.
{e.g., 5 % ovalbumin, etc.). N o a t t e m p t was made to remove chalazae adhering to yolk. To reduce evaporation during storage, tubes of white were stoppered and materials in bottles were overlayered with 5 ml. of sterile mineral oil. The mineral oil was particularly desirable for minimizing dry ing of exposed spots of yolks. T h e samples as_ commonly stored are shown in Figure 1 Examination of Quality: White and yolk indices were measured according to
Downloaded from http://ps.oxfordjournals.org/ at Carleton University on June 18, 2015
Preparation of Eggs for Breaking: In order to obtain sterile egg material, it was necessary not only to adequately sterilize the shells b u t also to employ eggs with a low initial incidence of contamination. Reports give the percentages of clean eggs contaminated as 2 to 1 5 % (Haines, 1939), b u t it has been clearly demonstrated that nearly all contamination is caused by penetration of microorganisms through the shell after the egg is laid (Stuart and McNally, 1943). To prevent this entry of microorganisms, clean eggs were collected within 30 minutes of laying and submerged in 7 0 % or 9 5 % ethyl alcohol. The eggs were allowed to remain in the alcohol until just prior to breaking, which was usually 48-72 hours after the eggs were laid. It had been previously reported t h a t little or no alcohol penetrates into the white and yolk (Osborne, 1931). T h e effects of alcoholic penetration were checked during these experiments.
mouthed bottles. Yolks and whites were separated with sterile, portable trays equipped with a breaking knife and separating cup. Usually sterile gloves and portable breakers were changed after breaking two eggs. T h e whites were collected in sterile beakers or bottles and usually transferred to sterile 20X200 mm. culture tubes. The yolks were washed with sterile saline and collected in bottles containing sterile solutions under study
CHEMISTRY OF SHELL EGG DETERIORATION
RESULTS AND DISCUSSION
Effectiveness of Sterilization Techniques: Sterile egg materials were consistently obtained by the rigorous methods employed. The introduction of various shortcuts, however, caused contaminations to appear. Usually, the contaminants were molds and were not grossly evident until 15-20 days' storage at 35 degrees C. This made it necessary to adhere to the more laborious standard procedures. However, it was possible to sterilize the eggs by means other than alcohol submersion. •Dipping very fresh clean eggs in 5% phenol for 30 seconds, transporting them in sterile jars, and redipping in 5% phenol followed by a sterile water wash before breaking was found equally satisfactory. Although Osborne (1931) reported that little or no alcohol penetrated into an egg submerged in alcohol, in our studies a foreign odor was detectable in the egg contents. However, submersion in alcohol for as long as 10 days caused no measurable changes in broken-out quality and no differences were noted in the extents of deteriorations of broken-out and stored egg contents whether the pre-breaking treatment was by alcohol submersion or phenol dipping. Alcohol submersion not only served for shell sterilization but also prevented the
usual increase in the pH of the white which accompanies the escape of carbon dioxide from the egg. If the eggs were removed from the alcohol within 4-6 hours and then stored unbroken, the pH of the white increased at a rate approximately similar to that of an untreated egg. But, if the eggs were in the alcohol for prolonged periods and at higher temperatures (such as 2 weeks at 35 degrees C.) before they were removed, the pH of the white did not increase, at least during the first 2-3 days of storage. It was not determined whether prolonged storage would cause the pH to rise. It thus appeared that the alcohol effected some change of the egg, possibly of the shell but more probably of the shell membranes. The retention of the original pH of the white prior to the breaking experiments eliminated the alkalinity of the white as an initial variable. In the experiments with separated whites the tubes containing the whites were stored several days (usually in desiccators) before the rubber stoppers were inserted. This allowed an escape of carbon dioxide sufficient to increase the pH of the white to a value more commonly encountered during egg storage (>8.0). Deterioration of Whole Egg Material and Separated Whites: The deteriorations encountered in a series of experiments with whole broken-out eggs and separated whites are listed in Tables 1 and 2. Deteriorations of both the white and yolk are evident with the whole brokenout eggs stored at 35 degrees C , and extensive thinning of sterile, separated white is evident with the white samples stored at 35 degrees C. In both series of experiments these changes were retarded by storage at low temperatures, as is the case with intact eggs in the shell. Only limited information was obtained on the influence of breaking-out and separating on the rates of deterioration.
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Heiman and Carver (1936). The percentage of thick white was determined by the method of Hoist and Almquist (1931). Bacteriological examinations were made by standard plate counts and by direct subculture into thioglycolate broth, into thioglycolate-agar shake-tubes, and into nutrient broth. Because all samples examined had been stored at 35 degrees C , bacteriological tests were also carried out at 35 degrees C. Absence of growth in all tests after 4 days' incubation was termed sterility.
647
648
R. E. FEENEY, R. B. SILVA AND L. R. MACDONNELL TABLE 1.—Deteriorations of asepticatty broken-out eggs* White
Storage Time
Temp.
No. eggs
Days 5
°C. 35
7
27 27
2 35
0 15
35
Exp.
A
b
B • C
Yolk index
PH
Index
Amount thick (average)
0.22
9.1
0.035
46
10"
0.43 0.26
8.7 8.6
0.065 0.029
65 35
9
0.43 0.28
7.7 8.3
0.090 0.060
63 44
40
%
Eggs were broken into bottles as described in text. Figures are averages. Eggs in this lot were several days old before submersion in alcohol. The initial p H of the white was consequently above 8.9 before storage. In the other experiments the initial pH of the white was that of a very fresh egg (7.6-7.7). 0 Overlayered with mineral oil. d Three of these samples were employed for separate bacteriological examinations on the yolks and whites. All samples were negative. b
However, within the limits of the data, no marked differences were noted in the rates of breakdown of separated and unseparated whites. Direct comparisons of rates of deteriorations of shell eggs and broken-out materials were difficult, because extensive microbial deterioration and stuck yolks were encountered with
the shell eggs employed. However, by dipping very fresh, clean eggs in phenol, subsequently handling them under aseptic conditions, and storing them in sterile containers, it was possible to prevent these microbial deteriorations. One group of 4 eggs so handled was from the same lot as Exp. C of Table 2. After 37 days at
TABLE 2.—Thinning of sterile separated egg whites* Storage conditions Exp.
White
No. samples
pH
Amount thick
Time
Temp.
Days 27 27
°C. 2 35
6 8°
8.2 8.5
65 28
B
25 25
2 35
3 6°
8.1 8.5
47 26
C
37 37
2 35
6 11°
D
0 0 26 26
—
5 8° 5 15
A"
2 35
%
d d
7,6 8.4 8.4 8.2
47 23 65 66 58 31
" Sterile egg whites for Experiment A were collected in bottles and stored under mineral oil. Those for Experiments B, C, and D were transferred to tubes which were then stoppered. An undetermined, but small amount, of white was lost in this transfer. Figures are averages. b These whites were from the same lot of eggs used for Experiment B of Table 1. The results are therefore, at least in part, comparable with one another. 0 Microbiological examination of each of these samples after incubation werfe negative. d Undetermined. e These whites were from shell eggs stored 2 days at 2°C. without submersion in alcohol.
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a
CHEMISTRY OF SHELL EGG DETERIORATION
pH during storage with yolks, the rate of decrease was less. In one experiment with lysozyme, 12 yolks were aseptically suspended in sterile 0.5% solutions of the protein in isotonic saline. The initial pH of 7 of these solutions was 9.0 and that of 5 was 7.5. After storage at 35 degrees C. for 5 days, 4 of the former and 3 of the latter were examined. The pH had dropped to an average of 7.2 and 6.8, respectively, but the yolks appeared normal and had an average index of 0.42. However, after storage for 27 days at 35 degrees C , the average pH of the remaining 5 samples was 6.6 and the yolks evidenced deterioration by slight swellings and rupture of 2 of them on attempted measurement. In none of these or similar experiments was a shift in the pH of the yolk contents noted. The average index of the three measurable yolks was 0.35. Two control samples stored at 2 degrees C. had yolks with indices of 0.43 and 0.46. Difficulties in attempting to interpret these results with yolks are easily apparent. Nevertheless, although deteriorations occurred, it might be considered that a remarkable degree of stability was exhibited. The development of an adequate alkaline and isotonic solution in which to store the yolks is necessary in order to determine whether the yolk deteriorates in the absence of the white. If it were then found that the white is the source of the yolk deterioration, it should be possible to employ an adequate solution for testing the individual constituents of the white and to identify the responsible causative agent(s). SUMMARY AND CONCLUSIONS
Sterile, whole broken-out eggs, separated whites and separated yolks were prepared from eggs sterilized by submersion in alcohol and dipping in phenol.
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35 degrees C , the average pH of the whites was 8.9 and the average amount of thick white was 24%, which value was in close agreement with the amount found in the separated whites. These results show that the shell and its membranes, or microorganisms in the ' shell, are not primary sources of the agents or mechanisms causing these deteriorations of the white and yolk. In like manner, they show that the yolk is not a primary source insofar as the deterioration of the white is concerned and that a principal cause of thinning is inherent in the white itself. Studies with Separated Yolks: The study of separated yolks was seriously hampered by our inability to reproduce the normal pH relationships which exist between the yolk (pH 6.0-6.1) and its environment (the white, pH 7.6-9.3) in the unseparated egg. Solutions for immersion of yolks during storage should maintain this relationship as well as the proper osmotic conditions. In none of the experiments in this study was it possible to maintain the pH of the immersing solutions on the alkaline side and only when protein buffers were employed was it possible to prevent rapid increases in the pH of the yolk contents. When yolks were immersed for only a few hours in alkaline (pH 8.0-9.0), isotonic solutions buffered with carbonate, phosphate, or borate, the pH of the yolks had increased and that of the immersing solution had decreased. The proteins employed as buffers were the three egg-white proteins, ovalbumin (crystalline), ovomucoid, and lysozyme (crystalline). Of these, lysozyme was the most suitable. Sterile saline solutions of either of the other two became acidic within less than 18 hours. The ovalbumin was tested as 1% and 5% solutions and the ovomucoid as a 1% solution. Although the lysozyme solutions also decreased in
649
650
N E W S AND N O T E S
REFERENCES Balls, A. K., and S. R. Hoover, 1940. Behavior of ovomucin in the liquefaction of egg white. Ind. Eng. Chem. 32:594-596. Haines, R. B., 1939. Microbiology in the preservation of the hen's egg. Special Report No. 47, De-
partment of Scientific and Industrial Research, Great Britain. Food Investigation Board. 65 p. Heiman, V., and J. S. Carver, 1936. The albumen index as a physical measurement of observed egg quality. Poultry Sci. 15: 141-148. Hoist, W. F., and H. J. Almquist, 1931. Measurement of deterioration in the stored hen's egg. Hilgardia 6: 49-60. Hoover, S. R., 1940. A physical and chemical study of ovomucin. Ph.D. Thesis, Georgetown University, Washington, D. C , 45 p. Lineweaver, H., H. J. Morris, L. Kline and R. S. Bean, 1948. Enzymes of fresh hen eggs. Arch. Biochem. 16: 443-472. Lineweaver, H., H. Fraenkel-Conrat and R. S. Bean, 1949. Determination of trypsin in the presence of egg white trypsin inhibitor and the demonstration of absence of trypsin from egg white. J. Biol. Chem. 177: 205-207. MacDonnell, L- R-, H. Lineweaver and R. E. Feeney, 1950. Deteriorations of eggs caused by reducing solutions and gases. Poultry Sci. 29: 769. Osborne, W. A., 1931. A note on the permeability of the egg shell. Australian J. Exp. Biol. Med. Sci. 8: 239-240. Sharo, P. F., 1937. Preservation and storage of hen's eggs. Food Research, 2: 470-498. Stuart, L. S., and E. H. McNally, 1943. Bacteriological studies on the shell. U. S. Egg and Poultry Mag. 49: 28-31, 45-47.
News and Notes CORNELL NOTES L. E. Weaver, Poultry Extension Specialist a t Cornell University, Ithaca, New York, retired in April after 30 years of service. He was born on a farm in C h a t a u q u a county, New York and graduated from Cornell in 1918. He t a u g h t agriculture for a time and later became Poultry Extension Specialist in Kentucky. H e managed a hatchery in Kentucky and then returned to Cornell in 1921. I n 1930 he obtained a M.S. degree a t the University of Wisconsin, specializing in genetics. I n 1945, he took oyer the Standard
Official Laying Contest a t Stafford, New York, and a year ago, with D r . J. H . Bruckner, initiated the New York R a n dom Sample Poultry Test at Horseheads. H e will retire to his poultry farm near Ithaca. OHIO NOTES
Professor R. George J a a p of Ohio State University, Columbus, left in J u n e for Edinburgh, Scotland, where he will do research during the 1951-52 academic year under a Fulbright award recently announced by the United States' D e p a r t m e n t of State. A member of the University's Poultry H u s b a n d r y D e p a r t m e n t
{Continued on page 657)
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The deteriorations of these sterile egg components during storage were studied in an a t t e m p t to determine the sources of the agents or mechanisms causing nonmicrobial deteriorations of shell eggs. I t was found t h a t whole, broken-out eggs exhibited the deteriorative weakening of the yolk and thinning of the white which occur in intact eggs in the shell and t h a t sterile, separated whites thinned on storage. Unsatisfactory and inconclusive results were obtained in the studies with separated yolks. I t was concluded t h a t the shells or shell membranes are not significant sources of the agents or mechanisms causing the deteriorations and t h a t a major cause of thinning of the white is inherent in the white itself.
Western Regional Research Laboratory,1 Albany, California (Received for publication November 21, 1950)
T
Department of Agriculture.
645
Downloaded from http://ps.oxfordjournals.org/ at Carleton University on June 18, 2015
orative agents or mechanisms arose from recent studies of this'laboratory on the HIS report concerns the initial effects of reducing chemicals on eggs. sources of the deteriorative agents or These recent studies confirmed the earlier mechanisms which cause thinning of the observations of Hoover (1940) that the white and weakening of the yolk during addition of sulfhydryl compounds to egg the storage of eggs. The experiments reported here were designed to answer the white caused thinning (Hoover's observasimple question whether these agents or tions were included in a doctoral dissertamechanisms are inherent in the white and tion and apparently have not received yolk, have their source in the yolks and wide attention). In addition, these recent diffuse into the white or vice versa, or have studies demonstrated that the addition of very small amounts of a variety of reducexternal sources. ing chemicals to eggs not only caused the Previous studies have concerned atthick white to thin but also caused the tempts to identify possible deteriorative yolk to weaken (MacDonnell, Lineweaver agents or mechanisms rather than to deand Feeney, 1950). Chemical reduction termine their sources (Sharp, 1937). thus appeared to be an attractive hyProminent among theories proposed for pothesis for the naturally occurring detethe thinning of white were those subscribriorative mechanisms causing storage ing to an enzyme mechanism and to a deteriorations of the yolk as well as the chemical hydrolysis catalyzed by hywhite. This hypothesis raised the question droxyl ions. However, the enzyme theory of possible sources of reducing substances has been virtually discarded (Balls and or of catalysts accelerating their action. It Hoover, 1940; and Lineweaver et al. was considered that they could originate 1948 and 1949) and no evidence supportfrom the shell or microorganisms on the ing chemical hydrolysis has appeared. shell, that they could diffuse from the Yolk deterioration has been associated yolk into the white or vice versa, or that with the passage of water from the white they could be present in the white and to the yolk and this passage has been concause thinning of the white and weakensidered to be related to the alkalinity of ing of the yolk by direct action at the the white. outer surface of the yolk membrane. An Our interest in the sources of the deteri- obvious way to determine such possible sources would be to study the deteriora1 Bureau of Agricultural and Industrial Chem- tions of the separated components. To istry, Agricultural Research Administration, U. S. this end, a study was attempted of the INTRODUCTION
646
R. E. F E E N E Y , R. B . SILVA AND L. R. M A C D O N N E L L
deteriorations of sterile, broken-out whole eggs, separated whites, and separated yolks. METHODS
Breaking and Separating: These procedures were conducted with strict adherence to the requirements for sterile bacteriological technique. They were carried out in a bacteriological inoculating room and workers wore hair nets and face masks. All materials such as breakers, separators, rubber gloves, etc., were sterilized prior to use and frequently changed during breaking and separating. For breaking, eggs were removed from the alcohol with sterile tongs and the alcohol drained momentarily, ignited, and then allowed to burn off. The sterile egg was then passed to a worker fitted with sterile gloves who completed the breaking operation. Whole eggs were broken directly into sterile, 4-oz., wide-
FIG. 1. Sterile broken-out and separated eggs. From left to right: Whole egg, yolk suspended in a water solution of 1.0% lysozyme and 0.9% sodium chloride, and white. The whole egg and yolk bottles contain a layer of mineral oil to minimize evaporation.
{e.g., 5 % ovalbumin, etc.). N o a t t e m p t was made to remove chalazae adhering to yolk. To reduce evaporation during storage, tubes of white were stoppered and materials in bottles were overlayered with 5 ml. of sterile mineral oil. The mineral oil was particularly desirable for minimizing dry ing of exposed spots of yolks. T h e samples as_ commonly stored are shown in Figure 1 Examination of Quality: White and yolk indices were measured according to
Downloaded from http://ps.oxfordjournals.org/ at Carleton University on June 18, 2015
Preparation of Eggs for Breaking: In order to obtain sterile egg material, it was necessary not only to adequately sterilize the shells b u t also to employ eggs with a low initial incidence of contamination. Reports give the percentages of clean eggs contaminated as 2 to 1 5 % (Haines, 1939), b u t it has been clearly demonstrated that nearly all contamination is caused by penetration of microorganisms through the shell after the egg is laid (Stuart and McNally, 1943). To prevent this entry of microorganisms, clean eggs were collected within 30 minutes of laying and submerged in 7 0 % or 9 5 % ethyl alcohol. The eggs were allowed to remain in the alcohol until just prior to breaking, which was usually 48-72 hours after the eggs were laid. It had been previously reported t h a t little or no alcohol penetrates into the white and yolk (Osborne, 1931). T h e effects of alcoholic penetration were checked during these experiments.
mouthed bottles. Yolks and whites were separated with sterile, portable trays equipped with a breaking knife and separating cup. Usually sterile gloves and portable breakers were changed after breaking two eggs. T h e whites were collected in sterile beakers or bottles and usually transferred to sterile 20X200 mm. culture tubes. The yolks were washed with sterile saline and collected in bottles containing sterile solutions under study
CHEMISTRY OF SHELL EGG DETERIORATION
RESULTS AND DISCUSSION
Effectiveness of Sterilization Techniques: Sterile egg materials were consistently obtained by the rigorous methods employed. The introduction of various shortcuts, however, caused contaminations to appear. Usually, the contaminants were molds and were not grossly evident until 15-20 days' storage at 35 degrees C. This made it necessary to adhere to the more laborious standard procedures. However, it was possible to sterilize the eggs by means other than alcohol submersion. •Dipping very fresh clean eggs in 5% phenol for 30 seconds, transporting them in sterile jars, and redipping in 5% phenol followed by a sterile water wash before breaking was found equally satisfactory. Although Osborne (1931) reported that little or no alcohol penetrated into an egg submerged in alcohol, in our studies a foreign odor was detectable in the egg contents. However, submersion in alcohol for as long as 10 days caused no measurable changes in broken-out quality and no differences were noted in the extents of deteriorations of broken-out and stored egg contents whether the pre-breaking treatment was by alcohol submersion or phenol dipping. Alcohol submersion not only served for shell sterilization but also prevented the
usual increase in the pH of the white which accompanies the escape of carbon dioxide from the egg. If the eggs were removed from the alcohol within 4-6 hours and then stored unbroken, the pH of the white increased at a rate approximately similar to that of an untreated egg. But, if the eggs were in the alcohol for prolonged periods and at higher temperatures (such as 2 weeks at 35 degrees C.) before they were removed, the pH of the white did not increase, at least during the first 2-3 days of storage. It was not determined whether prolonged storage would cause the pH to rise. It thus appeared that the alcohol effected some change of the egg, possibly of the shell but more probably of the shell membranes. The retention of the original pH of the white prior to the breaking experiments eliminated the alkalinity of the white as an initial variable. In the experiments with separated whites the tubes containing the whites were stored several days (usually in desiccators) before the rubber stoppers were inserted. This allowed an escape of carbon dioxide sufficient to increase the pH of the white to a value more commonly encountered during egg storage (>8.0). Deterioration of Whole Egg Material and Separated Whites: The deteriorations encountered in a series of experiments with whole broken-out eggs and separated whites are listed in Tables 1 and 2. Deteriorations of both the white and yolk are evident with the whole brokenout eggs stored at 35 degrees C , and extensive thinning of sterile, separated white is evident with the white samples stored at 35 degrees C. In both series of experiments these changes were retarded by storage at low temperatures, as is the case with intact eggs in the shell. Only limited information was obtained on the influence of breaking-out and separating on the rates of deterioration.
Downloaded from http://ps.oxfordjournals.org/ at Carleton University on June 18, 2015
Heiman and Carver (1936). The percentage of thick white was determined by the method of Hoist and Almquist (1931). Bacteriological examinations were made by standard plate counts and by direct subculture into thioglycolate broth, into thioglycolate-agar shake-tubes, and into nutrient broth. Because all samples examined had been stored at 35 degrees C , bacteriological tests were also carried out at 35 degrees C. Absence of growth in all tests after 4 days' incubation was termed sterility.
647
648
R. E. FEENEY, R. B. SILVA AND L. R. MACDONNELL TABLE 1.—Deteriorations of asepticatty broken-out eggs* White
Storage Time
Temp.
No. eggs
Days 5
°C. 35
7
27 27
2 35
0 15
35
Exp.
A
b
B • C
Yolk index
PH
Index
Amount thick (average)
0.22
9.1
0.035
46
10"
0.43 0.26
8.7 8.6
0.065 0.029
65 35
9
0.43 0.28
7.7 8.3
0.090 0.060
63 44
40
%
Eggs were broken into bottles as described in text. Figures are averages. Eggs in this lot were several days old before submersion in alcohol. The initial p H of the white was consequently above 8.9 before storage. In the other experiments the initial pH of the white was that of a very fresh egg (7.6-7.7). 0 Overlayered with mineral oil. d Three of these samples were employed for separate bacteriological examinations on the yolks and whites. All samples were negative. b
However, within the limits of the data, no marked differences were noted in the rates of breakdown of separated and unseparated whites. Direct comparisons of rates of deteriorations of shell eggs and broken-out materials were difficult, because extensive microbial deterioration and stuck yolks were encountered with
the shell eggs employed. However, by dipping very fresh, clean eggs in phenol, subsequently handling them under aseptic conditions, and storing them in sterile containers, it was possible to prevent these microbial deteriorations. One group of 4 eggs so handled was from the same lot as Exp. C of Table 2. After 37 days at
TABLE 2.—Thinning of sterile separated egg whites* Storage conditions Exp.
White
No. samples
pH
Amount thick
Time
Temp.
Days 27 27
°C. 2 35
6 8°
8.2 8.5
65 28
B
25 25
2 35
3 6°
8.1 8.5
47 26
C
37 37
2 35
6 11°
D
0 0 26 26
—
5 8° 5 15
A"
2 35
%
d d
7,6 8.4 8.4 8.2
47 23 65 66 58 31
" Sterile egg whites for Experiment A were collected in bottles and stored under mineral oil. Those for Experiments B, C, and D were transferred to tubes which were then stoppered. An undetermined, but small amount, of white was lost in this transfer. Figures are averages. b These whites were from the same lot of eggs used for Experiment B of Table 1. The results are therefore, at least in part, comparable with one another. 0 Microbiological examination of each of these samples after incubation werfe negative. d Undetermined. e These whites were from shell eggs stored 2 days at 2°C. without submersion in alcohol.
Downloaded from http://ps.oxfordjournals.org/ at Carleton University on June 18, 2015
a
CHEMISTRY OF SHELL EGG DETERIORATION
pH during storage with yolks, the rate of decrease was less. In one experiment with lysozyme, 12 yolks were aseptically suspended in sterile 0.5% solutions of the protein in isotonic saline. The initial pH of 7 of these solutions was 9.0 and that of 5 was 7.5. After storage at 35 degrees C. for 5 days, 4 of the former and 3 of the latter were examined. The pH had dropped to an average of 7.2 and 6.8, respectively, but the yolks appeared normal and had an average index of 0.42. However, after storage for 27 days at 35 degrees C , the average pH of the remaining 5 samples was 6.6 and the yolks evidenced deterioration by slight swellings and rupture of 2 of them on attempted measurement. In none of these or similar experiments was a shift in the pH of the yolk contents noted. The average index of the three measurable yolks was 0.35. Two control samples stored at 2 degrees C. had yolks with indices of 0.43 and 0.46. Difficulties in attempting to interpret these results with yolks are easily apparent. Nevertheless, although deteriorations occurred, it might be considered that a remarkable degree of stability was exhibited. The development of an adequate alkaline and isotonic solution in which to store the yolks is necessary in order to determine whether the yolk deteriorates in the absence of the white. If it were then found that the white is the source of the yolk deterioration, it should be possible to employ an adequate solution for testing the individual constituents of the white and to identify the responsible causative agent(s). SUMMARY AND CONCLUSIONS
Sterile, whole broken-out eggs, separated whites and separated yolks were prepared from eggs sterilized by submersion in alcohol and dipping in phenol.
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35 degrees C , the average pH of the whites was 8.9 and the average amount of thick white was 24%, which value was in close agreement with the amount found in the separated whites. These results show that the shell and its membranes, or microorganisms in the ' shell, are not primary sources of the agents or mechanisms causing these deteriorations of the white and yolk. In like manner, they show that the yolk is not a primary source insofar as the deterioration of the white is concerned and that a principal cause of thinning is inherent in the white itself. Studies with Separated Yolks: The study of separated yolks was seriously hampered by our inability to reproduce the normal pH relationships which exist between the yolk (pH 6.0-6.1) and its environment (the white, pH 7.6-9.3) in the unseparated egg. Solutions for immersion of yolks during storage should maintain this relationship as well as the proper osmotic conditions. In none of the experiments in this study was it possible to maintain the pH of the immersing solutions on the alkaline side and only when protein buffers were employed was it possible to prevent rapid increases in the pH of the yolk contents. When yolks were immersed for only a few hours in alkaline (pH 8.0-9.0), isotonic solutions buffered with carbonate, phosphate, or borate, the pH of the yolks had increased and that of the immersing solution had decreased. The proteins employed as buffers were the three egg-white proteins, ovalbumin (crystalline), ovomucoid, and lysozyme (crystalline). Of these, lysozyme was the most suitable. Sterile saline solutions of either of the other two became acidic within less than 18 hours. The ovalbumin was tested as 1% and 5% solutions and the ovomucoid as a 1% solution. Although the lysozyme solutions also decreased in
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REFERENCES Balls, A. K., and S. R. Hoover, 1940. Behavior of ovomucin in the liquefaction of egg white. Ind. Eng. Chem. 32:594-596. Haines, R. B., 1939. Microbiology in the preservation of the hen's egg. Special Report No. 47, De-
partment of Scientific and Industrial Research, Great Britain. Food Investigation Board. 65 p. Heiman, V., and J. S. Carver, 1936. The albumen index as a physical measurement of observed egg quality. Poultry Sci. 15: 141-148. Hoist, W. F., and H. J. Almquist, 1931. Measurement of deterioration in the stored hen's egg. Hilgardia 6: 49-60. Hoover, S. R., 1940. A physical and chemical study of ovomucin. Ph.D. Thesis, Georgetown University, Washington, D. C , 45 p. Lineweaver, H., H. J. Morris, L. Kline and R. S. Bean, 1948. Enzymes of fresh hen eggs. Arch. Biochem. 16: 443-472. Lineweaver, H., H. Fraenkel-Conrat and R. S. Bean, 1949. Determination of trypsin in the presence of egg white trypsin inhibitor and the demonstration of absence of trypsin from egg white. J. Biol. Chem. 177: 205-207. MacDonnell, L- R-, H. Lineweaver and R. E. Feeney, 1950. Deteriorations of eggs caused by reducing solutions and gases. Poultry Sci. 29: 769. Osborne, W. A., 1931. A note on the permeability of the egg shell. Australian J. Exp. Biol. Med. Sci. 8: 239-240. Sharo, P. F., 1937. Preservation and storage of hen's eggs. Food Research, 2: 470-498. Stuart, L. S., and E. H. McNally, 1943. Bacteriological studies on the shell. U. S. Egg and Poultry Mag. 49: 28-31, 45-47.
News and Notes CORNELL NOTES L. E. Weaver, Poultry Extension Specialist a t Cornell University, Ithaca, New York, retired in April after 30 years of service. He was born on a farm in C h a t a u q u a county, New York and graduated from Cornell in 1918. He t a u g h t agriculture for a time and later became Poultry Extension Specialist in Kentucky. H e managed a hatchery in Kentucky and then returned to Cornell in 1921. I n 1930 he obtained a M.S. degree a t the University of Wisconsin, specializing in genetics. I n 1945, he took oyer the Standard
Official Laying Contest a t Stafford, New York, and a year ago, with D r . J. H . Bruckner, initiated the New York R a n dom Sample Poultry Test at Horseheads. H e will retire to his poultry farm near Ithaca. OHIO NOTES
Professor R. George J a a p of Ohio State University, Columbus, left in J u n e for Edinburgh, Scotland, where he will do research during the 1951-52 academic year under a Fulbright award recently announced by the United States' D e p a r t m e n t of State. A member of the University's Poultry H u s b a n d r y D e p a r t m e n t
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The deteriorations of these sterile egg components during storage were studied in an a t t e m p t to determine the sources of the agents or mechanisms causing nonmicrobial deteriorations of shell eggs. I t was found t h a t whole, broken-out eggs exhibited the deteriorative weakening of the yolk and thinning of the white which occur in intact eggs in the shell and t h a t sterile, separated whites thinned on storage. Unsatisfactory and inconclusive results were obtained in the studies with separated yolks. I t was concluded t h a t the shells or shell membranes are not significant sources of the agents or mechanisms causing the deteriorations and t h a t a major cause of thinning of the white is inherent in the white itself.