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Gelation of Frozen-Defrosted Egg Yolk as Affected by Selected Additives: Viscosity and Electrophoretic Findings
437
INSECTICIDE RESIDUES
producers being careful in selecting and buting feeds that are free of chlorinated insecticide residues, for use in their poultry operations.
sion of Environmental Engineering and Food Protection, W. S. Public Health Service. This article has been accepted as Journal Paper No. 2396 of the Purdue Agricultural Experiment Station.
SUMMARY REFERENCES
ACKNOWLEDGMENT
This investigation was supported in part by grants from the American Poultry and Hatchery Federation, Kansas City, Missouri, The National Egg Council, Kansas City, Missouri, and by PHS Research Grant EF 00049-02 from the Divi-
Draper, C. I., C. Biddulph, D. A. Greenwood, J. R. Harris, W. Binns and M. L. Miner, 1950. Concentration of DDT in tissues of chickens fed varying levels of DDT in the diet. Poultry Sci. 29: 756. Draper, C. E., J. R. Harris, D. A. Greenwood, C. Biddulph, L. E. Harris, F. Mangelson, W. Binns and M. L. Miner, 1952. The transfer of D D T from the food to eggs and body tissues of White Leghorn hens. Poultry Sci. 31:388-393. Ivey, M. C , R. H. Roberts, H. D. Mann and H. U. Claborn, 1961. Lindane residues in chickens and eggs following poultry house sprays. J. Econ. Entomol. 54: 487-488. Liska, B. J., B. E. Langlois, G. C. Mostert and W. J. Stadelman, 1964. Residues in eggs and tissues of chickens on rations containing low levels of DDT. Poultry Sci. 43: 982-984. Naber, E. C , and G. W. Ware, 1961. Lindane in eggs and chicken tissues. J. Econ. Entomol. 54: 675-677. Stemp, A. R., B. J. Liska, B. E. Langlois and W. J. Stadelman, 1964. Analysis of egg yolk and poultry tissues for chlorinated insecticide residues. Poultry Sci. 43: 273-275.
Gelation of Frozen-Defrosted Egg Yolk as Affected by Selected Additives: Viscosity and Electrophoretic Findings DOROTHY D. MEYER AND MARGY WOODBURN Foods and Nutrition Department, Purdue University, Lafayette, Indiana (Received for publication August 17, 1964)
INTRODUCTION
A
T A temperature of — 6°C. or below - egg yolk loses its fluidity. This change is irreversible and has been termed gelation. Several factors affecting this phenomenon have been established. The
mechanism involved, however, remains to be elucidated. This physical alteration in egg yolk may be averted by super-cooling (—11°C.) or freezing in liquid air (—190°C.) and thawing rapidly in mercury at 30° C. (Lopez et al., 1954; Smith,
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1. Supplying the equivalent of 0.1 to 0.15 p.p.m. of lindane, dieldrin, heptachlor and DDT in the feed to laying hens in capsule form for 14 days resulted in some residues in chicken tissues which disappeared rapidly. 2. Supplying the equivalent of 10 to 15 p.p.m. of the same insecticides in the feed for 5 days resulted in significant residues in eggs and abdominal fat which were extremely persistent. 3. Care in feeding residue free feeds to laying flocks is extremely important.
438
D. D . M E Y E R AND M .
Treatments t h a t inhibit or reduce gelation of egg yolk on freezing and thawing are colloidal milling (Lopez et al., 1954), or addition of sugars as arabinose, galactose, glucose, fructose, sucrose, maltose or ramnose (Jordon el al., 1951; Lopez et al., 1954; Marion and Stadelman, 1958; McNally, 1959; Powrie et al., 1963). Feeney and Hill (1960) stated t h a t crotoxin, a lecithinase A, from rattlesnake venom does prevent or result in a reversal of the gelatinous rubbery mass of yolk caused by freezing damage. Other enzymes including papain, bromelin, ficin, lipase, Rhozyme, and trypsin inhibit gelation of frozen and thawed egg yolk (Lopez et al., 1955; Kaloyereas et al., 1962). On the other hand, dilution or concentration of egg yolk did not prevent gelation (Lopez et al., 1954). Water added at a rate of 5 % as an enzyme suspension had no effect on coagulation temperature or the hardening process (gelation at low temperature of 0°F.) of the yolk (Kaloyereas et al, 1962). In contrast, Marion and Stadelman (1958)
reported t h a t addition of water at a rate of 4 % did inhibit gelation. The high density lipoproteins of egg yolk, a- and /3-lipovitellin, are believed to be involved in gelation. Powrie et al. (1963) demonstrated t h a t in crude lipovitellin preparations, proteins form complexes on freezing and thawing. Martin et al. (1963) believed t h a t a- and /3-lipovitellin have a structural integrity approaching t h a t of a protein molecule. Burley (1963) suggested t h a t in the lipovitellins, the proteins and lipids are combined in a structure t h a t is flexible enough to accommodate large amounts of certain other substances and t h a t the process of expansion assists aggregation. The purpose of this study was to investigate the effect of cysteine (hydrochloride and free base forms), an amino acid having a — SH moiety; 2,3 dimercapto 1 propanol, a three-carbon compound with two — SH groups per molecule; water; sodium chloride; or fructose on the inhibition of gelation of egg yolk on freezing and thawing. The compounds with sulfhydryl groups may function as chelating or reducing agents. Sodium chloride m a y exert an ionic effect. Sugars are believed to prevent aggregation and denaturation of proteins although no satisfactory explanation has been offered as to why (Heitefuss et al., 1959; Powrie et al., 1963). Reports in the literature concerning the addition of water on inhibition of egg yolk gelation are contradictory. Because of this and to permit comparison of the effects of the selected compounds added to the yolk in solution, the effect of water, per se, was investigated. EXPERIMENTAL PROCEDURE Methods and Materials. Eggs were obtained from the Purdue Poultry Center on the day of lay. Hens were of a White Leghorn strain, on a breeder laying ration,
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1962). The average freezing point for egg yolk has been reported as — 0.65°C. (Lopez et al., 1954; Smith, 1954; Powrie et al., 1963). Freezing and defrosting rates and storage temperatures and time affect the degree of gelation (Pearce and Lavers, 1949; Lea and Hawke, 1952; Lopez et al., 1954; Marion and Stadelman, 1958; Powrie et al., 1963). On freezing and thawing of yolk, physical changes in the environment may be responsible for molecular reorientation (Lovelock, 1957; Fisher and Gurin, 1964). Water molecules necessary for structural integrity of proteins may be removed in freezing (Lovelock, 1957). Increased ionic strength because of electrolyte concentration has been suggested as a cause but does not appear to harm lipovitellin of egg yolk unless the p H falls below 5.2 (Lovelock, 1957; Smith, 1962).
WOODBURN
GELATION OF FROZEN EGG YOLK
and L-cysteine (free base). When distilled water only was used, it was added at a rate of 5.5 ml. per 100 grams of yolk. A 30-second mixing period using the portable electric mixer at lowest speed was necessary to thoroughly mix the mass after adding the solution or water. Ninety-five-gram samples for freezing were weighed immediately into glass Petri plates (3|-inch diameter, f-inch depth), covered with Parafilm " M " (Marathon, American Can Co.), and placed on metal shelves in an upright freezer at — 25° C. All frozen samples were analyzed within 20-24 hours. Fresh unfrozen samples were allowed to set 30 minutes at room temperature (22-23°C.) preceding preparation for analysis. Three frozen samples were defrosted simultaneously for 25 minutes in a controlled 35°C. ± 1.0° water bath. Viscosity and pH, Samples for viscosity measurements were placed in 50 ml. glass beakers (inside diameter 40 mm., height 52 mm.) and filled to a height of 50 mm. To determine the viscosity changes of treated yolk quantitatively, the Brookfield Synchro-lectric viscometer, Model RVF, was used at a constant speed of 20 rpm with spindle number 5 for fresh samples and spindle number 7 for frozen defrosted yolk. The temperature of the yolk was 24.5 ± 0.5°C. at the time of measurement. With the exception of one set of aged egg yolk samples, all viscosity measurements were taken 90 + 10 minutes after mixing or removal from water bath. Three readings were taken and averaged. The spindle was allowed to rotate twice before each reading. The same sample was used for the determining of pH of the yolk mixtures using a Beckman Model G pH meter. Paper Electrophoresis. Filter paper electrophoresis was used to separate the lipoproteins in frozen-defrosted and un-
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and from 10-11 months of age. Fresh eggs (1 day) were used for analysis on the day of collection. Eggs were stored for 49 to 54 days (51-day), 63 to 65 days (64-day), and 88-days at 1-4° C. All eggs used were laid during the period from January to April. For each run (three treatments), yolks from three dozen eggs were separated from the whites, washed in distilled water, and dried by rolling gently on paper towels and absorbent tissue. The vitelline membrane was punctured and yolk contents allowed to drain. A 45-second mixing period using a portable electric mixer at the lowest speed was used to insure homogeneity of the mass. The mixture was weighed into 200-gram portions for treatment with additives. The additives were obtained from the following sources: L-cysteine (free base), L-cysteine hydrochloride, and 2,3 dimercapto 1 propanol from Nutritional Biochemicals Corp., Cleveland, Ohio; sodium chloride from J. T. Baker Chemicals, Chicago, Illinois; fructose from Pfanstiehl Laboratories, Waukegan, Illinois. The first three compounds were added at levels of 0.0055 mole in 5.5 ml. distilled water per 100 grams egg yolk. The pH of the cysteine solutions was adjusted with 0.1 N and 5.0 N NaOH to correspond to the pH of the egg yolk. Alkali used for this adjustment was calculated as part of the 5.5 ml. distilled water. The approximate quantity of NaOH used for this purpose was calculated as 0.00004 mole and 0.00567 mole per 100 grams of yolk for L-cysteine (free base) and L-cysteine hydrochloride, respectively. Sodium chloride was added at a level of 0.0426 mole per 100 grams of yolk. Fructose was used on a basis of 0.0286 mole per 100 grams of yolk. The latter two compounds were dispersed in 5.5 ml. distilled water. Difficulty was encountered in dissolving the NaCl
439
440
D. D . M E Y E R AND M . WOODBURN
The electrophoresis cell was of the ridgepole type, Spinco Model R. Phosphate buffer of p H 6.5 and ionic strength of 0.1 was used. A 0.01 ml. diluted sample was applied to filter paper (Schleicher and Schuell, 2043-A mgl, 3.0X30.6 cm.) strips using a micropipette and applicator. Duplicate or triplicate strips were done for each sample. Each electrophoretic run was carried out for 24 hours at 23° C. with constant current of 4 milliamperes. The strips were dried for 30 minutes at 120°C. One paper strip from each sample was stained for lipid by placing it for 18 hours in a saturated solution of Oil Red 0 in 6 0 % ethyl alcohol (Durrum el ah, 1952). These strips were then rinsed for 5 minutes in running t a p water at room temperature and air dried. The remaining strips (one or two from each sample) were stained using an alcoholic bromphenol blue solution (Spinco Dye B—4-1 gram dye per 1 1. methanol); (Beckman Inst. Inc., Stanford Industrial Park, Palo Alto, California). T o study the electrophoretic patterns of proteins and especially of the major lipoproteins, all strips were analyzed using the Beckman Model R B Analytrol at a slit width setting of 2.5. Protein-stained strips were read at 9.1 cm. and lipid-
stained strips at 8.5 cm. Percentages of the mobile and non-mobile fractions were calculated by separating the two major peaks with the aid of a French curve and determining the fractions of each. Migration distance was measured in millimeters from point of origin to front. Moisture. Percent moisture of yolk was determined in a vacuum oven according to the A.O.A.C. method (1957). EXPERIMENTAL RESULTS
Viscosity. With increasing age of egg, decreased viscosity was observed in the untreated control yolk of unfrozen samples (Fig. 1). A slight increase in viscosity was apparent in yolk of the oldest eggs (88-day) as compared with the 64-day
A
\
\ O 20
\
\
\ \
\ CONTROL
' \
/ S O D I U M CHLORIDE
\
^s
x V--CYSTEINE HYDROCHLORIDE
CYSTEINE FREE BASE
AGE OF EGGS (Days)
FIG. 1. Viscosity of unfrozen egg yolk of four ages of eggs allowed to set 30 minutes at room temperature with additive.
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frozen yolk. A six- to ten-gram sample of yolk was diluted with an equal volume of 10% NaCl solution on a weight basis. To obtain a homogeneous mixture, the frozendefrosted samples were mixed using a magnetic stirrer (#384-06 LaPine Scientific) a t room temperature. T h e rheostat control was set at 30 or 40 depending on the size of the stirrer used. Each sample was mixed a minimum of 40 minutes and in many cases, 60 minutes was necessary to facilitate dispersion in saline solution. Unfrozen samples in 1 0 % NaCl solution were stirred by hand using a small glass rod. No problems of dispersion were encountered with the unfrozen yolk.
441
GELATION OF FROZEN EGG YOLK
40 60 AGE OF EGGS (Days!
FIG. 2. Relation between age of eggs and water content of yolk.
Ago of Egg; •
CONTROL
WATER
CYSTEINE HYDROCHLORIDE
l d°y
£3
«4 doyt
HS
88 doys
CYSTEINE FREE BASE
m
FIG. 3. Effect of selected additives on the viscosity of frozen-defrosted egg yolk of eggs of four ages.
which approached that of the untreated control, but the highest viscosity measurement in the frozen-defrosted samples of all additives investigated (Table 1) was found in yolk thus treated. The most extensive change in color of yolk was noted with this compound. A khaki color formed rapidly (within seconds) following the addition of the mercapto propanol solution and increased during mixing and thereafter. A very potent garlic-like odor was present. Electrophoresis. Paper electrophoresis was used to detect differences in electrophoretic mobilities of two major lipoprotein fractions, lipovitellin found in the yolk granules and lipovitellenin in yolk plasma. The distance between the origin or point of application and the front was measured in millimeters of both proteinand lipid-stained electrophoretograms. The distance of migration was greater for the unfrozen egg yolk at all ages (Fig. 4). In the unfrozen samples sodium chloride had the same effect as cysteine-hydrochloride up to the 51-day eggs and then paralleled it very closely through the 88-
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eggs. The percentage of water in the yolk decreased between 64 and 88 days of storage (Fig. 2). This followed the initial transfer of water from white to yolk. The viscosity of untreated and treated frozendefrosted egg yolk decreased with increasing age of egg with the exceptions of one holding period interval each for the sodium chloride and the cysteine hydrochloride treated samples (Fig. 3). No change in pH was observed in yolk on freezing. Yolk of freshly laid eggs (1 day) had an average pH of 6.0, whereas the yolk of eggs that had been stored had a pH range of 6.3 to 6.4. Viscosity changed the least from the unfrozen to the frozen-defrosted state in the sodium chloride treated yolk as indicated by the calculated viscosity ratio data (Table 1) when comparisons among additives were made. Fructose had the greatest effect of any of the additives investigated in that it reduced the viscosity of the unfrozen yolk mixture and inhibited gelation of the frozen-defrosted samples. The addition of 2,3 dimercapto 1 propanol, a strong chelating agent of many metals (Martell and Chaberek, 1959) as well as a reducing agent, resulted in a viscosity reading in unfrozen yolk
442
D . D . M E Y E R AND M .
WOODBURN
TABLE 1.—The effect of selected additives on viscosity change of egg yolkx,i Chemical additive 3
Untreated control Water Cysteine hydrochloride Cysteine (free base) 2,3 dimercapto 1 propanol Sodium chloride5 Fructose 6
No. of samples
Not frozen A4
8 4 2 3 3 1 1
(poises) 27.4±1.29 8.5 + 0.21 8.5 + 0.14 7.0 + 0.70 24.6 + 0.37 36.4 6.0
Frozen - 2 5 ° C . for 20-24 hrs. B4
Viscosity ratio B/A
(poises) 907.9+ 49.24 545.2+ 61.77 453.3+ 4.67 494.4+ 67.20 1,148.7+136.32 200.0 101.2
33 64 53 71 47 5 17
No. of samples
6 4 2 3 3 1 1
1
day eggs. I n like manner, the addition of water had a similar, but lesser, effect on the migrating distance u p to 51-day eggs as did cysteine-hydrochloride and sodium chloride. I n the unfrozen yolk the distance the lipoproteins migrated decreased gradually with age with the exception of the untreated control or where cysteine-free base was the additive (Fig. 4). I n both the unfrozen and frozen-defrosted states of aged eggs, the untreated control showed the greatest migration distance in 51-day eggs and the selected additives tended to show a similar response at 51-days of egg storage in the frozen-defrosted samples. I t was found (Fig. 2) t h a t there was a rapid increase in water content of the yolk from day of lay to 51-days of storage and this may be interpreted as having an effect on the ability of the lipoproteins to migrate in t h a t lipoproteins tend to associate less in the presence of increased water. On the basis of optical density values of eluted dyed proteins, Powrie et al. (1963) indicated t h a t the slowly migrating lipoprotein fraction (lipovitellenin) was altered during freezing and thawing and thereby contributed to the original nonmobile phase. A similar trend was noted
in this study when percentages of mobile and non-mobile phases were calculated using the peaks obtained with an Analytrol Densitometer. Values for the untreated control, water, cysteine hydrochloride and sodium chloride treated yolk for eggs of four ages are presented in Table 2. T h e mobile phase was changed the least from the unfrozen to the frozen-defrosted state when sodium chloride was added. Next to
- CONTROL - WATER - SODIUM CHLORIDE
O
- CYSTEINE HYDROCHLORIDE •
unfrozen frozen-defrosted
- CYSTEINE FREE BASE _J 20
I 1 40 60 AGE OF EGGS IDoysl
1 80
FIG. 4. Average migration distances of protein front by paper electrophoresis of egg yolk at four ages of eggs.
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Yolk of 1-day eggs. Brookfield Synchro-lectric viscometer Model RVF. Temperature of yolk mixtures 24°C. + 0.5°. 3 5.5 ml. water and 0.0055 mole additive per 100 gm. yolk unless stated otherwise. 4 Average + standard deviation. 6 0.0426 mole per 100 gm. yolk. 6 0.0286 mole per 100 gm. yolk. 2
443
GELATION OF FROZEN EGG YOLK TABLE 2.—Calculated mobile and non-mobile protein fractions1 of unfrozen and frozen-defrosted egg yolk as separated by paper electrophoresis2 Treatment and Age Control 1-day 51-day 64-day 88-day
51-day 64-day 88-day Cysteine hydrochloride 1 day 51-day 64-day 88-day Sodium chloride 1-day 51-day 64-day 88-day 1 2 3 4
No.4
Mobile fraction
Non-mobile fraction
%
% 60 70 54 60 47 62 57 78
(49-70) (58-85) (52-56) (58-62)
U F U F U F U F
6 7 2 2 1 1 1 1
40 30 46 40 53 38 43 22
U F U F U F U F
6 4 1 1 1 1 1 1
46 (33-53) 31 (17-41) 38 38 50 20 52 39
54 (47-67) 69 (59-83) 62 62 50 80 48 61
U F TJ F U F U F
4 4 2 2 4 4 2 2
30 24 50 42 50 39 41 36
70 76 50 58 50 61 59 64
U F U F U F U F
1 2 2 2 2 2 2 2
50 45 (44-46) 39 (39-40) 36 (31-41) 44 39 40 (31-50) 39 (37-42)
(30-51) (15-42) (44-48) (38-42)
(25-38) (23-25) (50-51) (43-56) (34-44) (41-42)
(62-75) (75-77) (49-50) (44-57) (56-66) (58-59)
50 55 (53-56) 61 (60-61) 64 (59-69) 56 61 60 (50-69) 61(58-63)
Protein-dyed—Bromphenol blue. Ridgepole type, Spinco Model R. Phosphate buffer, pH 6.5, ionic strength 0.1. U=unfrozen. F=frozen-defrosted. Number of electrophoretograms.
sodium chloride, cysteine-hydrochloride appeared to keep the differences in electrophoretic mobilities between the unfrozen yolk and frozen-defrosted samples to a minimum. Greater differences were apparent for the control and the watertreated yolk than in the two preceding additives discussed. With these calculations no consistent trend is noted and the response of the lipoproteins appears to be dependent upon age of egg as well as the additive. The calculated percentages of mobile and non-mobile fractions for the
lipid-dyed electrophoretograms followed a similar trend as those of the protein-dyed and were inversely proportional as would be expected. Of the additives investigated using frozen-defrosted egg yolk of freshly-laid eggs, the sodium chloride treated samples showed the farthest migration distance of the front on the electrophoretograms, whereas the front of the samples treated with 2,3 dimercapto 1 propanol had the shortest migration distance (Fig. 5). The effect of fructose approached that of
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Water 1-day
Type 3
444
D. D. M E Y E R AND M .
WATER
CYSTEINE CYSTEINE DIMERCAP- SODIUM HYDROFREE TOPRO- CHLORIDE CHLORIDE BASE PANOL
FRUCTOSE
FIG. 5. Eleclrophoretograms of frozen-defrosted and unfrozen egg yolk of one-day eggs.
sodium chloride; cysteine (hydrochloride and free base) and water gave results similar to those of the untreated control. For comparative observation the corresponding electrophoretograms of egg yolk in the unfrozen state are presented below those of the frozen-defrosted (Fig. 5). DISCUSSION Similar effects of the additives on egg yolk were found by viscosity measurements of the frozen-defrosted samples and observation of the electrophoretograms. Sodium chloride and fructose had the greatest effect on gelation as indicated by lower viscosity. From the electrophoretic findings, the ionic effect of sodium chloride appears to play a prominent role. Cysteine
solutions in both forms and water served to reduce the viscosity as compared with the untreated control. However, the presence of sulfhydryl groups at the levels used in this experiment did not appear to affect the migration distances. Since cysteine in the hydrochloride form enhanced the migration distance of lipovitellenin as compared to the free-base form, a beneficial ionic effect is postulated. The greatest viscosity or least fluidity and the least mobility was observed in frozen-defrosted egg yolk with 2,3 dimercapto 1 propanol. Fructose may prevent aggregation of the lipoproteins. The mechanism, however, remains obscure. It is known t h a t fructose complexes with iron and t h a t this
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CONTROL
WOODBURN
GELATION OF FROZEN EGG YOLK
SUMMARY
The effects of water, cysteine-hydrochloride and free base forms, sodium chloride, 2,3 dimercapto 1 propanol, and fructose on the gelation of egg yolk in freezing and thawing were investigated using freshly laid eggs. Of the six additives, the first four were also added to eggs stored for 51, 64 and 88 days. Viscosity, pH, moisture, and electrophoretic mobilities of lipoproteins were determined. Little effect was noted on the pH on freezing of egg yolk. Moisture content of egg yolk increased sharply from 1 day to 51 days and tended to decrease gradually from 64 to 88 days of storage. With increasing age of egg, decreased viscosity was observed in unfrozen yolk up to 64 days. At 88 days of storage, viscosity of yolk tended to increase. In a comparison of the effectiveness of additives, viscosity measurements of unfrozen yolk were similar for the cysteine (both forms) and water-treated samples. Sodium chloride increased yolk viscosity of unfrozen samples above that of the untreated control. In frozen defrosted yolk, cysteine was slightly more effective in gelation inhibi-
tion as measured by viscosity than was water alone. In the stored eggs, sodium chloride inhibited gelation of frozen-defrosted egg yolk to the greatest degree of all four additives used. Fructose, added only to the 1-day egg yolk, reduced the viscosity the most of all additives investigated in both unfrozen and frozendefrosted yolk. Paper electrophoresis was used to study changes in lipoprotein fractions on freezing and thawing. In general, greatest migration distances of the fronts were observed in yolk of the 51-day eggs, which had an increased water content, also. It appeared from the electrophoretograms that ions play an important role in migration of the lipoprotein fraction, lipovitellenin. Fructose serves to promote migration of lipoproteins in an electrophoretic field, also. REFERENCES Association of Official Agricultural Chemists, 1957. Official Methods of Analysis, 8th ed., p. 220, A.O.A.C., Washington, D. C. Burley, R. W., 1963. Interactions of chloroform with a- and 0-lipovitetlin and other egg yolk constituents. Can. J. Biochem. Physiol. 41: 389-395. Charley, P. J., B. Sarkar, C. F. Stitt and P. Saltman, 1963. Chelation of iron by sugars. Biochim. Biophys. Acta, 69: 313-321. Durrum, E. L., M. H. Paul and E. R. B. Smith, 1952. Lipid detection in paper electrophoresis. Sci. 116:428^30. Feeney, R. E., and R. M. Hill, 1960. Protein chemistry and food research. Adv. Food Res. 10: 3 3 43. Fisher, W. R., and S. Gurin, 1964. Structure of lipoproteins: Covalently bound fatty acids. Sci. 143:362-363. Heitefuss, R., D. J. Buchanan-Davidson and M. A. Stahmann, 1959. The stabilization of extracts of cabbage leaf proteins by polyhydroxy compounds for electrophoretic and immunological studies. Arch. Biochem. Biophys. 85: 200-208. Jordon, R., L. E. Dawson and C. J. Echterling, 1952. The effect of selected pretreatments upon the culinary qualities of eggs frozen and stored
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reaction proceeds more rapidly under alkaline than acid conditions (Charley et al., 1963). These investigators emphasized that the proportion of iron to fructose and the concentration of each as well as the pH are important factors. It is suggested, therefore, that fructose may chelate the iron or possibly the copper in egg yolk, thus limiting or reducing the cross-bonding of the protein structure. A more firmly bound physical arrangement as found in egg yolk per se or in egg yolk mixtures where reformation of cross linking had occurred would offer a greater resistance to stress and, therefore, increase the viscosity.
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D. D. MEYER AND M. WOODBTJRN Poultry Sci. 37: 1224. Martell, A. E., and S. Chaberek, 1959. Organic Sequestering Agents. John Wiley and Sons, N. Y. Martin, W. G., N. H. Tattrie and W. H. Cook, 1963. Lipid extraction and distribution studies of egg yolk lipoproteins. Can. J. Biochem. Physiol. 41: 657-666. McNally, E. H., 1959. Observations on the dispersion and precipitation of egg yolk. Poultry Sci. 38: 1227-1228. Pearce, J. A., and C. G. Lavers, 1949. Liquid and frozen eggs. Can. J. Res. 27: 231. Powrie, W. D., H. Little and A. Lopez, 1963. Gelation of egg yolk. J. Food Sci. 28: 38-46. Smith, A. U., 1954. Effects of low temperatures on living cells and tissues. In Harris, R. J. C , Biological Applications of Freezing and Drying, pp. 1-62. Academic Press Inc., N. Y. Smith, A. U., 1962. Biological Effects of Freezing and Supercooling,- p. 480. Williams & Wilkins, Baltimore.
Effect of Vitamin A and Ambient Temperature on Reproductive Performance of White Leghorn Pullets1 B. L. REID, B. W. HEYWANG, 2 A. A. KURNICK, 3 M. G. VAVICH AND B. J. HULETT Departments of Poultry Science and Ag. Biochemistry, University of Arizona, Tucson, Arizona and United States Department of Agriculture (Received for publication August 17, 1964)
W
ARD and Schaible (1963) reported that numerous dietary ingredients failed to affect the incidence of blood spots in chicken eggs, but vitamin A was not included. Bearse el al. (1960) associated a high incidence of blood spotting of egg yolks with vitamin A deficiency in hens. Hill el al. (1961) reported that the minimum vitamin A requirement of lay-
1 Arizona Agricultural Experiment Station Journal Article #903. 2 Southwest Poultry Experiment Station, ARS, Glendale, Arizona. 3 Present Address: The Ray Ewing Company, Div. of Hoffman-LaRoche Inc., Pasadena, Cal.
ing hens for maintenance of body weight and maximum egg production and the minimum incidence of blood spots was 1,200-1,600 U.S.P. units of vitamin A per pound of diet. Heywang (1952) noted that feed consumption of laying and breeding hens decreased during hot weather but the actual vitamin A requirement of the hen did not increase during prolonged periods of high ambient temperature. He also reported that 2,480 U.S.P. units of vitamin A per pound of diet was not adequate for maximum egg production and maintenance of life during hot weather and con-
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in a home-type freezer. II. Sponge cakes. Food Res. 17: 93-99. Kaloyereas, S. A., A. F. Novak and A. B. Watts, 1962. Heat sterilization of liquid eggs after stabilization treatment with various proteinases. Poultry Sci. 16: 284-288. Lea, C. H., and J. C. Hawke, 1952. Lipovitellin. 2. Influence of water on stability of lipovitellin and the effects of freezing and drying. Biochem. J. 52:105-114. Lopez, A., C. A. Fellers and W. D. Powrie, 1954. Some factors affecting gelation of frozen egg yolk. J. Milk Food Technol. 17: 334-339. Lopez, A., C. A. Fellers and W. D. Powrie, 1955. Enzymic inhibition of gelation in frozen egg yolk. J. Milk Food Technol. 18: 77-80. Lovelock, J. E., 1957. The denaturation of lipidprotein complexes as a cause of damage by freezing. Proc. Royal Society (London), 147: 427^33. Marion, W. W., and W. J. Stadelman, 1958. An investigation of the gelation of thawed egg yolk.
INSECTICIDE RESIDUES
producers being careful in selecting and buting feeds that are free of chlorinated insecticide residues, for use in their poultry operations.
sion of Environmental Engineering and Food Protection, W. S. Public Health Service. This article has been accepted as Journal Paper No. 2396 of the Purdue Agricultural Experiment Station.
SUMMARY REFERENCES
ACKNOWLEDGMENT
This investigation was supported in part by grants from the American Poultry and Hatchery Federation, Kansas City, Missouri, The National Egg Council, Kansas City, Missouri, and by PHS Research Grant EF 00049-02 from the Divi-
Draper, C. I., C. Biddulph, D. A. Greenwood, J. R. Harris, W. Binns and M. L. Miner, 1950. Concentration of DDT in tissues of chickens fed varying levels of DDT in the diet. Poultry Sci. 29: 756. Draper, C. E., J. R. Harris, D. A. Greenwood, C. Biddulph, L. E. Harris, F. Mangelson, W. Binns and M. L. Miner, 1952. The transfer of D D T from the food to eggs and body tissues of White Leghorn hens. Poultry Sci. 31:388-393. Ivey, M. C , R. H. Roberts, H. D. Mann and H. U. Claborn, 1961. Lindane residues in chickens and eggs following poultry house sprays. J. Econ. Entomol. 54: 487-488. Liska, B. J., B. E. Langlois, G. C. Mostert and W. J. Stadelman, 1964. Residues in eggs and tissues of chickens on rations containing low levels of DDT. Poultry Sci. 43: 982-984. Naber, E. C , and G. W. Ware, 1961. Lindane in eggs and chicken tissues. J. Econ. Entomol. 54: 675-677. Stemp, A. R., B. J. Liska, B. E. Langlois and W. J. Stadelman, 1964. Analysis of egg yolk and poultry tissues for chlorinated insecticide residues. Poultry Sci. 43: 273-275.
Gelation of Frozen-Defrosted Egg Yolk as Affected by Selected Additives: Viscosity and Electrophoretic Findings DOROTHY D. MEYER AND MARGY WOODBURN Foods and Nutrition Department, Purdue University, Lafayette, Indiana (Received for publication August 17, 1964)
INTRODUCTION
A
T A temperature of — 6°C. or below - egg yolk loses its fluidity. This change is irreversible and has been termed gelation. Several factors affecting this phenomenon have been established. The
mechanism involved, however, remains to be elucidated. This physical alteration in egg yolk may be averted by super-cooling (—11°C.) or freezing in liquid air (—190°C.) and thawing rapidly in mercury at 30° C. (Lopez et al., 1954; Smith,
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1. Supplying the equivalent of 0.1 to 0.15 p.p.m. of lindane, dieldrin, heptachlor and DDT in the feed to laying hens in capsule form for 14 days resulted in some residues in chicken tissues which disappeared rapidly. 2. Supplying the equivalent of 10 to 15 p.p.m. of the same insecticides in the feed for 5 days resulted in significant residues in eggs and abdominal fat which were extremely persistent. 3. Care in feeding residue free feeds to laying flocks is extremely important.
438
D. D . M E Y E R AND M .
Treatments t h a t inhibit or reduce gelation of egg yolk on freezing and thawing are colloidal milling (Lopez et al., 1954), or addition of sugars as arabinose, galactose, glucose, fructose, sucrose, maltose or ramnose (Jordon el al., 1951; Lopez et al., 1954; Marion and Stadelman, 1958; McNally, 1959; Powrie et al., 1963). Feeney and Hill (1960) stated t h a t crotoxin, a lecithinase A, from rattlesnake venom does prevent or result in a reversal of the gelatinous rubbery mass of yolk caused by freezing damage. Other enzymes including papain, bromelin, ficin, lipase, Rhozyme, and trypsin inhibit gelation of frozen and thawed egg yolk (Lopez et al., 1955; Kaloyereas et al., 1962). On the other hand, dilution or concentration of egg yolk did not prevent gelation (Lopez et al., 1954). Water added at a rate of 5 % as an enzyme suspension had no effect on coagulation temperature or the hardening process (gelation at low temperature of 0°F.) of the yolk (Kaloyereas et al, 1962). In contrast, Marion and Stadelman (1958)
reported t h a t addition of water at a rate of 4 % did inhibit gelation. The high density lipoproteins of egg yolk, a- and /3-lipovitellin, are believed to be involved in gelation. Powrie et al. (1963) demonstrated t h a t in crude lipovitellin preparations, proteins form complexes on freezing and thawing. Martin et al. (1963) believed t h a t a- and /3-lipovitellin have a structural integrity approaching t h a t of a protein molecule. Burley (1963) suggested t h a t in the lipovitellins, the proteins and lipids are combined in a structure t h a t is flexible enough to accommodate large amounts of certain other substances and t h a t the process of expansion assists aggregation. The purpose of this study was to investigate the effect of cysteine (hydrochloride and free base forms), an amino acid having a — SH moiety; 2,3 dimercapto 1 propanol, a three-carbon compound with two — SH groups per molecule; water; sodium chloride; or fructose on the inhibition of gelation of egg yolk on freezing and thawing. The compounds with sulfhydryl groups may function as chelating or reducing agents. Sodium chloride m a y exert an ionic effect. Sugars are believed to prevent aggregation and denaturation of proteins although no satisfactory explanation has been offered as to why (Heitefuss et al., 1959; Powrie et al., 1963). Reports in the literature concerning the addition of water on inhibition of egg yolk gelation are contradictory. Because of this and to permit comparison of the effects of the selected compounds added to the yolk in solution, the effect of water, per se, was investigated. EXPERIMENTAL PROCEDURE Methods and Materials. Eggs were obtained from the Purdue Poultry Center on the day of lay. Hens were of a White Leghorn strain, on a breeder laying ration,
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1962). The average freezing point for egg yolk has been reported as — 0.65°C. (Lopez et al., 1954; Smith, 1954; Powrie et al., 1963). Freezing and defrosting rates and storage temperatures and time affect the degree of gelation (Pearce and Lavers, 1949; Lea and Hawke, 1952; Lopez et al., 1954; Marion and Stadelman, 1958; Powrie et al., 1963). On freezing and thawing of yolk, physical changes in the environment may be responsible for molecular reorientation (Lovelock, 1957; Fisher and Gurin, 1964). Water molecules necessary for structural integrity of proteins may be removed in freezing (Lovelock, 1957). Increased ionic strength because of electrolyte concentration has been suggested as a cause but does not appear to harm lipovitellin of egg yolk unless the p H falls below 5.2 (Lovelock, 1957; Smith, 1962).
WOODBURN
GELATION OF FROZEN EGG YOLK
and L-cysteine (free base). When distilled water only was used, it was added at a rate of 5.5 ml. per 100 grams of yolk. A 30-second mixing period using the portable electric mixer at lowest speed was necessary to thoroughly mix the mass after adding the solution or water. Ninety-five-gram samples for freezing were weighed immediately into glass Petri plates (3|-inch diameter, f-inch depth), covered with Parafilm " M " (Marathon, American Can Co.), and placed on metal shelves in an upright freezer at — 25° C. All frozen samples were analyzed within 20-24 hours. Fresh unfrozen samples were allowed to set 30 minutes at room temperature (22-23°C.) preceding preparation for analysis. Three frozen samples were defrosted simultaneously for 25 minutes in a controlled 35°C. ± 1.0° water bath. Viscosity and pH, Samples for viscosity measurements were placed in 50 ml. glass beakers (inside diameter 40 mm., height 52 mm.) and filled to a height of 50 mm. To determine the viscosity changes of treated yolk quantitatively, the Brookfield Synchro-lectric viscometer, Model RVF, was used at a constant speed of 20 rpm with spindle number 5 for fresh samples and spindle number 7 for frozen defrosted yolk. The temperature of the yolk was 24.5 ± 0.5°C. at the time of measurement. With the exception of one set of aged egg yolk samples, all viscosity measurements were taken 90 + 10 minutes after mixing or removal from water bath. Three readings were taken and averaged. The spindle was allowed to rotate twice before each reading. The same sample was used for the determining of pH of the yolk mixtures using a Beckman Model G pH meter. Paper Electrophoresis. Filter paper electrophoresis was used to separate the lipoproteins in frozen-defrosted and un-
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and from 10-11 months of age. Fresh eggs (1 day) were used for analysis on the day of collection. Eggs were stored for 49 to 54 days (51-day), 63 to 65 days (64-day), and 88-days at 1-4° C. All eggs used were laid during the period from January to April. For each run (three treatments), yolks from three dozen eggs were separated from the whites, washed in distilled water, and dried by rolling gently on paper towels and absorbent tissue. The vitelline membrane was punctured and yolk contents allowed to drain. A 45-second mixing period using a portable electric mixer at the lowest speed was used to insure homogeneity of the mass. The mixture was weighed into 200-gram portions for treatment with additives. The additives were obtained from the following sources: L-cysteine (free base), L-cysteine hydrochloride, and 2,3 dimercapto 1 propanol from Nutritional Biochemicals Corp., Cleveland, Ohio; sodium chloride from J. T. Baker Chemicals, Chicago, Illinois; fructose from Pfanstiehl Laboratories, Waukegan, Illinois. The first three compounds were added at levels of 0.0055 mole in 5.5 ml. distilled water per 100 grams egg yolk. The pH of the cysteine solutions was adjusted with 0.1 N and 5.0 N NaOH to correspond to the pH of the egg yolk. Alkali used for this adjustment was calculated as part of the 5.5 ml. distilled water. The approximate quantity of NaOH used for this purpose was calculated as 0.00004 mole and 0.00567 mole per 100 grams of yolk for L-cysteine (free base) and L-cysteine hydrochloride, respectively. Sodium chloride was added at a level of 0.0426 mole per 100 grams of yolk. Fructose was used on a basis of 0.0286 mole per 100 grams of yolk. The latter two compounds were dispersed in 5.5 ml. distilled water. Difficulty was encountered in dissolving the NaCl
439
440
D. D . M E Y E R AND M . WOODBURN
The electrophoresis cell was of the ridgepole type, Spinco Model R. Phosphate buffer of p H 6.5 and ionic strength of 0.1 was used. A 0.01 ml. diluted sample was applied to filter paper (Schleicher and Schuell, 2043-A mgl, 3.0X30.6 cm.) strips using a micropipette and applicator. Duplicate or triplicate strips were done for each sample. Each electrophoretic run was carried out for 24 hours at 23° C. with constant current of 4 milliamperes. The strips were dried for 30 minutes at 120°C. One paper strip from each sample was stained for lipid by placing it for 18 hours in a saturated solution of Oil Red 0 in 6 0 % ethyl alcohol (Durrum el ah, 1952). These strips were then rinsed for 5 minutes in running t a p water at room temperature and air dried. The remaining strips (one or two from each sample) were stained using an alcoholic bromphenol blue solution (Spinco Dye B—4-1 gram dye per 1 1. methanol); (Beckman Inst. Inc., Stanford Industrial Park, Palo Alto, California). T o study the electrophoretic patterns of proteins and especially of the major lipoproteins, all strips were analyzed using the Beckman Model R B Analytrol at a slit width setting of 2.5. Protein-stained strips were read at 9.1 cm. and lipid-
stained strips at 8.5 cm. Percentages of the mobile and non-mobile fractions were calculated by separating the two major peaks with the aid of a French curve and determining the fractions of each. Migration distance was measured in millimeters from point of origin to front. Moisture. Percent moisture of yolk was determined in a vacuum oven according to the A.O.A.C. method (1957). EXPERIMENTAL RESULTS
Viscosity. With increasing age of egg, decreased viscosity was observed in the untreated control yolk of unfrozen samples (Fig. 1). A slight increase in viscosity was apparent in yolk of the oldest eggs (88-day) as compared with the 64-day
A
\
\ O 20
\
\
\ \
\ CONTROL
' \
/ S O D I U M CHLORIDE
\
^s
x V--CYSTEINE HYDROCHLORIDE
CYSTEINE FREE BASE
AGE OF EGGS (Days)
FIG. 1. Viscosity of unfrozen egg yolk of four ages of eggs allowed to set 30 minutes at room temperature with additive.
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frozen yolk. A six- to ten-gram sample of yolk was diluted with an equal volume of 10% NaCl solution on a weight basis. To obtain a homogeneous mixture, the frozendefrosted samples were mixed using a magnetic stirrer (#384-06 LaPine Scientific) a t room temperature. T h e rheostat control was set at 30 or 40 depending on the size of the stirrer used. Each sample was mixed a minimum of 40 minutes and in many cases, 60 minutes was necessary to facilitate dispersion in saline solution. Unfrozen samples in 1 0 % NaCl solution were stirred by hand using a small glass rod. No problems of dispersion were encountered with the unfrozen yolk.
441
GELATION OF FROZEN EGG YOLK
40 60 AGE OF EGGS (Days!
FIG. 2. Relation between age of eggs and water content of yolk.
Ago of Egg; •
CONTROL
WATER
CYSTEINE HYDROCHLORIDE
l d°y
£3
«4 doyt
HS
88 doys
CYSTEINE FREE BASE
m
FIG. 3. Effect of selected additives on the viscosity of frozen-defrosted egg yolk of eggs of four ages.
which approached that of the untreated control, but the highest viscosity measurement in the frozen-defrosted samples of all additives investigated (Table 1) was found in yolk thus treated. The most extensive change in color of yolk was noted with this compound. A khaki color formed rapidly (within seconds) following the addition of the mercapto propanol solution and increased during mixing and thereafter. A very potent garlic-like odor was present. Electrophoresis. Paper electrophoresis was used to detect differences in electrophoretic mobilities of two major lipoprotein fractions, lipovitellin found in the yolk granules and lipovitellenin in yolk plasma. The distance between the origin or point of application and the front was measured in millimeters of both proteinand lipid-stained electrophoretograms. The distance of migration was greater for the unfrozen egg yolk at all ages (Fig. 4). In the unfrozen samples sodium chloride had the same effect as cysteine-hydrochloride up to the 51-day eggs and then paralleled it very closely through the 88-
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eggs. The percentage of water in the yolk decreased between 64 and 88 days of storage (Fig. 2). This followed the initial transfer of water from white to yolk. The viscosity of untreated and treated frozendefrosted egg yolk decreased with increasing age of egg with the exceptions of one holding period interval each for the sodium chloride and the cysteine hydrochloride treated samples (Fig. 3). No change in pH was observed in yolk on freezing. Yolk of freshly laid eggs (1 day) had an average pH of 6.0, whereas the yolk of eggs that had been stored had a pH range of 6.3 to 6.4. Viscosity changed the least from the unfrozen to the frozen-defrosted state in the sodium chloride treated yolk as indicated by the calculated viscosity ratio data (Table 1) when comparisons among additives were made. Fructose had the greatest effect of any of the additives investigated in that it reduced the viscosity of the unfrozen yolk mixture and inhibited gelation of the frozen-defrosted samples. The addition of 2,3 dimercapto 1 propanol, a strong chelating agent of many metals (Martell and Chaberek, 1959) as well as a reducing agent, resulted in a viscosity reading in unfrozen yolk
442
D . D . M E Y E R AND M .
WOODBURN
TABLE 1.—The effect of selected additives on viscosity change of egg yolkx,i Chemical additive 3
Untreated control Water Cysteine hydrochloride Cysteine (free base) 2,3 dimercapto 1 propanol Sodium chloride5 Fructose 6
No. of samples
Not frozen A4
8 4 2 3 3 1 1
(poises) 27.4±1.29 8.5 + 0.21 8.5 + 0.14 7.0 + 0.70 24.6 + 0.37 36.4 6.0
Frozen - 2 5 ° C . for 20-24 hrs. B4
Viscosity ratio B/A
(poises) 907.9+ 49.24 545.2+ 61.77 453.3+ 4.67 494.4+ 67.20 1,148.7+136.32 200.0 101.2
33 64 53 71 47 5 17
No. of samples
6 4 2 3 3 1 1
1
day eggs. I n like manner, the addition of water had a similar, but lesser, effect on the migrating distance u p to 51-day eggs as did cysteine-hydrochloride and sodium chloride. I n the unfrozen yolk the distance the lipoproteins migrated decreased gradually with age with the exception of the untreated control or where cysteine-free base was the additive (Fig. 4). I n both the unfrozen and frozen-defrosted states of aged eggs, the untreated control showed the greatest migration distance in 51-day eggs and the selected additives tended to show a similar response at 51-days of egg storage in the frozen-defrosted samples. I t was found (Fig. 2) t h a t there was a rapid increase in water content of the yolk from day of lay to 51-days of storage and this may be interpreted as having an effect on the ability of the lipoproteins to migrate in t h a t lipoproteins tend to associate less in the presence of increased water. On the basis of optical density values of eluted dyed proteins, Powrie et al. (1963) indicated t h a t the slowly migrating lipoprotein fraction (lipovitellenin) was altered during freezing and thawing and thereby contributed to the original nonmobile phase. A similar trend was noted
in this study when percentages of mobile and non-mobile phases were calculated using the peaks obtained with an Analytrol Densitometer. Values for the untreated control, water, cysteine hydrochloride and sodium chloride treated yolk for eggs of four ages are presented in Table 2. T h e mobile phase was changed the least from the unfrozen to the frozen-defrosted state when sodium chloride was added. Next to
- CONTROL - WATER - SODIUM CHLORIDE
O
- CYSTEINE HYDROCHLORIDE •
unfrozen frozen-defrosted
- CYSTEINE FREE BASE _J 20
I 1 40 60 AGE OF EGGS IDoysl
1 80
FIG. 4. Average migration distances of protein front by paper electrophoresis of egg yolk at four ages of eggs.
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Yolk of 1-day eggs. Brookfield Synchro-lectric viscometer Model RVF. Temperature of yolk mixtures 24°C. + 0.5°. 3 5.5 ml. water and 0.0055 mole additive per 100 gm. yolk unless stated otherwise. 4 Average + standard deviation. 6 0.0426 mole per 100 gm. yolk. 6 0.0286 mole per 100 gm. yolk. 2
443
GELATION OF FROZEN EGG YOLK TABLE 2.—Calculated mobile and non-mobile protein fractions1 of unfrozen and frozen-defrosted egg yolk as separated by paper electrophoresis2 Treatment and Age Control 1-day 51-day 64-day 88-day
51-day 64-day 88-day Cysteine hydrochloride 1 day 51-day 64-day 88-day Sodium chloride 1-day 51-day 64-day 88-day 1 2 3 4
No.4
Mobile fraction
Non-mobile fraction
%
% 60 70 54 60 47 62 57 78
(49-70) (58-85) (52-56) (58-62)
U F U F U F U F
6 7 2 2 1 1 1 1
40 30 46 40 53 38 43 22
U F U F U F U F
6 4 1 1 1 1 1 1
46 (33-53) 31 (17-41) 38 38 50 20 52 39
54 (47-67) 69 (59-83) 62 62 50 80 48 61
U F TJ F U F U F
4 4 2 2 4 4 2 2
30 24 50 42 50 39 41 36
70 76 50 58 50 61 59 64
U F U F U F U F
1 2 2 2 2 2 2 2
50 45 (44-46) 39 (39-40) 36 (31-41) 44 39 40 (31-50) 39 (37-42)
(30-51) (15-42) (44-48) (38-42)
(25-38) (23-25) (50-51) (43-56) (34-44) (41-42)
(62-75) (75-77) (49-50) (44-57) (56-66) (58-59)
50 55 (53-56) 61 (60-61) 64 (59-69) 56 61 60 (50-69) 61(58-63)
Protein-dyed—Bromphenol blue. Ridgepole type, Spinco Model R. Phosphate buffer, pH 6.5, ionic strength 0.1. U=unfrozen. F=frozen-defrosted. Number of electrophoretograms.
sodium chloride, cysteine-hydrochloride appeared to keep the differences in electrophoretic mobilities between the unfrozen yolk and frozen-defrosted samples to a minimum. Greater differences were apparent for the control and the watertreated yolk than in the two preceding additives discussed. With these calculations no consistent trend is noted and the response of the lipoproteins appears to be dependent upon age of egg as well as the additive. The calculated percentages of mobile and non-mobile fractions for the
lipid-dyed electrophoretograms followed a similar trend as those of the protein-dyed and were inversely proportional as would be expected. Of the additives investigated using frozen-defrosted egg yolk of freshly-laid eggs, the sodium chloride treated samples showed the farthest migration distance of the front on the electrophoretograms, whereas the front of the samples treated with 2,3 dimercapto 1 propanol had the shortest migration distance (Fig. 5). The effect of fructose approached that of
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Water 1-day
Type 3
444
D. D. M E Y E R AND M .
WATER
CYSTEINE CYSTEINE DIMERCAP- SODIUM HYDROFREE TOPRO- CHLORIDE CHLORIDE BASE PANOL
FRUCTOSE
FIG. 5. Eleclrophoretograms of frozen-defrosted and unfrozen egg yolk of one-day eggs.
sodium chloride; cysteine (hydrochloride and free base) and water gave results similar to those of the untreated control. For comparative observation the corresponding electrophoretograms of egg yolk in the unfrozen state are presented below those of the frozen-defrosted (Fig. 5). DISCUSSION Similar effects of the additives on egg yolk were found by viscosity measurements of the frozen-defrosted samples and observation of the electrophoretograms. Sodium chloride and fructose had the greatest effect on gelation as indicated by lower viscosity. From the electrophoretic findings, the ionic effect of sodium chloride appears to play a prominent role. Cysteine
solutions in both forms and water served to reduce the viscosity as compared with the untreated control. However, the presence of sulfhydryl groups at the levels used in this experiment did not appear to affect the migration distances. Since cysteine in the hydrochloride form enhanced the migration distance of lipovitellenin as compared to the free-base form, a beneficial ionic effect is postulated. The greatest viscosity or least fluidity and the least mobility was observed in frozen-defrosted egg yolk with 2,3 dimercapto 1 propanol. Fructose may prevent aggregation of the lipoproteins. The mechanism, however, remains obscure. It is known t h a t fructose complexes with iron and t h a t this
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CONTROL
WOODBURN
GELATION OF FROZEN EGG YOLK
SUMMARY
The effects of water, cysteine-hydrochloride and free base forms, sodium chloride, 2,3 dimercapto 1 propanol, and fructose on the gelation of egg yolk in freezing and thawing were investigated using freshly laid eggs. Of the six additives, the first four were also added to eggs stored for 51, 64 and 88 days. Viscosity, pH, moisture, and electrophoretic mobilities of lipoproteins were determined. Little effect was noted on the pH on freezing of egg yolk. Moisture content of egg yolk increased sharply from 1 day to 51 days and tended to decrease gradually from 64 to 88 days of storage. With increasing age of egg, decreased viscosity was observed in unfrozen yolk up to 64 days. At 88 days of storage, viscosity of yolk tended to increase. In a comparison of the effectiveness of additives, viscosity measurements of unfrozen yolk were similar for the cysteine (both forms) and water-treated samples. Sodium chloride increased yolk viscosity of unfrozen samples above that of the untreated control. In frozen defrosted yolk, cysteine was slightly more effective in gelation inhibi-
tion as measured by viscosity than was water alone. In the stored eggs, sodium chloride inhibited gelation of frozen-defrosted egg yolk to the greatest degree of all four additives used. Fructose, added only to the 1-day egg yolk, reduced the viscosity the most of all additives investigated in both unfrozen and frozendefrosted yolk. Paper electrophoresis was used to study changes in lipoprotein fractions on freezing and thawing. In general, greatest migration distances of the fronts were observed in yolk of the 51-day eggs, which had an increased water content, also. It appeared from the electrophoretograms that ions play an important role in migration of the lipoprotein fraction, lipovitellenin. Fructose serves to promote migration of lipoproteins in an electrophoretic field, also. REFERENCES Association of Official Agricultural Chemists, 1957. Official Methods of Analysis, 8th ed., p. 220, A.O.A.C., Washington, D. C. Burley, R. W., 1963. Interactions of chloroform with a- and 0-lipovitetlin and other egg yolk constituents. Can. J. Biochem. Physiol. 41: 389-395. Charley, P. J., B. Sarkar, C. F. Stitt and P. Saltman, 1963. Chelation of iron by sugars. Biochim. Biophys. Acta, 69: 313-321. Durrum, E. L., M. H. Paul and E. R. B. Smith, 1952. Lipid detection in paper electrophoresis. Sci. 116:428^30. Feeney, R. E., and R. M. Hill, 1960. Protein chemistry and food research. Adv. Food Res. 10: 3 3 43. Fisher, W. R., and S. Gurin, 1964. Structure of lipoproteins: Covalently bound fatty acids. Sci. 143:362-363. Heitefuss, R., D. J. Buchanan-Davidson and M. A. Stahmann, 1959. The stabilization of extracts of cabbage leaf proteins by polyhydroxy compounds for electrophoretic and immunological studies. Arch. Biochem. Biophys. 85: 200-208. Jordon, R., L. E. Dawson and C. J. Echterling, 1952. The effect of selected pretreatments upon the culinary qualities of eggs frozen and stored
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reaction proceeds more rapidly under alkaline than acid conditions (Charley et al., 1963). These investigators emphasized that the proportion of iron to fructose and the concentration of each as well as the pH are important factors. It is suggested, therefore, that fructose may chelate the iron or possibly the copper in egg yolk, thus limiting or reducing the cross-bonding of the protein structure. A more firmly bound physical arrangement as found in egg yolk per se or in egg yolk mixtures where reformation of cross linking had occurred would offer a greater resistance to stress and, therefore, increase the viscosity.
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D. D. MEYER AND M. WOODBTJRN Poultry Sci. 37: 1224. Martell, A. E., and S. Chaberek, 1959. Organic Sequestering Agents. John Wiley and Sons, N. Y. Martin, W. G., N. H. Tattrie and W. H. Cook, 1963. Lipid extraction and distribution studies of egg yolk lipoproteins. Can. J. Biochem. Physiol. 41: 657-666. McNally, E. H., 1959. Observations on the dispersion and precipitation of egg yolk. Poultry Sci. 38: 1227-1228. Pearce, J. A., and C. G. Lavers, 1949. Liquid and frozen eggs. Can. J. Res. 27: 231. Powrie, W. D., H. Little and A. Lopez, 1963. Gelation of egg yolk. J. Food Sci. 28: 38-46. Smith, A. U., 1954. Effects of low temperatures on living cells and tissues. In Harris, R. J. C , Biological Applications of Freezing and Drying, pp. 1-62. Academic Press Inc., N. Y. Smith, A. U., 1962. Biological Effects of Freezing and Supercooling,- p. 480. Williams & Wilkins, Baltimore.
Effect of Vitamin A and Ambient Temperature on Reproductive Performance of White Leghorn Pullets1 B. L. REID, B. W. HEYWANG, 2 A. A. KURNICK, 3 M. G. VAVICH AND B. J. HULETT Departments of Poultry Science and Ag. Biochemistry, University of Arizona, Tucson, Arizona and United States Department of Agriculture (Received for publication August 17, 1964)
W
ARD and Schaible (1963) reported that numerous dietary ingredients failed to affect the incidence of blood spots in chicken eggs, but vitamin A was not included. Bearse el al. (1960) associated a high incidence of blood spotting of egg yolks with vitamin A deficiency in hens. Hill el al. (1961) reported that the minimum vitamin A requirement of lay-
1 Arizona Agricultural Experiment Station Journal Article #903. 2 Southwest Poultry Experiment Station, ARS, Glendale, Arizona. 3 Present Address: The Ray Ewing Company, Div. of Hoffman-LaRoche Inc., Pasadena, Cal.
ing hens for maintenance of body weight and maximum egg production and the minimum incidence of blood spots was 1,200-1,600 U.S.P. units of vitamin A per pound of diet. Heywang (1952) noted that feed consumption of laying and breeding hens decreased during hot weather but the actual vitamin A requirement of the hen did not increase during prolonged periods of high ambient temperature. He also reported that 2,480 U.S.P. units of vitamin A per pound of diet was not adequate for maximum egg production and maintenance of life during hot weather and con-
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in a home-type freezer. II. Sponge cakes. Food Res. 17: 93-99. Kaloyereas, S. A., A. F. Novak and A. B. Watts, 1962. Heat sterilization of liquid eggs after stabilization treatment with various proteinases. Poultry Sci. 16: 284-288. Lea, C. H., and J. C. Hawke, 1952. Lipovitellin. 2. Influence of water on stability of lipovitellin and the effects of freezing and drying. Biochem. J. 52:105-114. Lopez, A., C. A. Fellers and W. D. Powrie, 1954. Some factors affecting gelation of frozen egg yolk. J. Milk Food Technol. 17: 334-339. Lopez, A., C. A. Fellers and W. D. Powrie, 1955. Enzymic inhibition of gelation in frozen egg yolk. J. Milk Food Technol. 18: 77-80. Lovelock, J. E., 1957. The denaturation of lipidprotein complexes as a cause of damage by freezing. Proc. Royal Society (London), 147: 427^33. Marion, W. W., and W. J. Stadelman, 1958. An investigation of the gelation of thawed egg yolk.