T h e Amino Acid Content of Fresh
and Stored Shell Eggs. II. Arginine,
Histidine, Lysine, Methionine,
Cystine, Tyrosine, Tryptophan,
Phenylalanine, and Proline*
ROBERT JOHN EVANS, J. A. DAVIDSON, SELMA L. BANDEMER AND HELEN A. BUTTS
Departments of Agricultural Chemistry and Poultry Husbandry, Michigan State College,
East Lansing, Michigan
(Received for publication February 28, 1949)
S OME of the earliest methods of amino
acid determination were for cystine,
tyrosine, tryptophan, and the three basic
amino acids, arginine, histidine, and lysine,
so consequently many data are available
for the content of these amino acids in
egg proteins. Furthermore, values are
also available for methionine and phenyl-
alanine, because of their classification as
essential in the diet and the existence of
suitable methods of determination. How-
ever, few data on the proline content of
egg proteins can be found in the literature.
Although no study has been reported
of changes occurring during storage in the
arginine, histidine, lysine, methionine,
cystine, tryosine, tryptophan, phenyl-
alanine, and proline content of egg pro-
teins, changes in the content of some of
these in eggs during incubation have been
studied (Sendju 1925, 1927; Cho 1926;
Plimmer and Lowndes 1927; Calvery
1929, 1930).
It is the purpose of this paper to present
data on the percentages of the above men-
tioned amino acids in the proteins of egg
* Published with the approval of the Director of
the Michigan Agricultural Experiment Station as
Journal Article No. 1026 (n.s.)
697
albumen, egg yolk, and the whole egg
contents from fresh and stored shell
eggs and to determine if changes in the
total content of these amino acids in
shell eggs occurred during storage.
EXPERIMENTAL
Descriptions of the eggs used, methods
and time of storage, preparation of
samples for analysis, and method of acid
hydrolysis of the proteins have been given
previously (Evans, Davidson, and Butts
1949a, Evans, Butts, Davidson, and
Bandemer 1949b). Amino acids were
determined by microbiological assay.
Lysine, methionine, cystine, and proline
were assayed with Leuconostoc mesenter-
oides P-60, phenylalanine and tryptophan
with Lactobacillus arabinosus 17-5, histi-
dine and tyrosine with Streptococcus
faecalis R, and arginine with Lactobacillus
casei. The media of Sauberlich and
Baumann (1946) were used for arginine,
histidine, lysine, tyrosine, tryptophan,
phenylalanine, and proline assays, and
that of Lyman, et al. (1946) was used for
methionine and cystine. Enzymatic digest
of the egg protein samples were prepared
by hydrolyzing in vitro with pepsin,
trypsin, and erepsin (Evans 1946), and
698 EVANS, DAVIDSON, BANDEMER AND BUTTS

TABLE 1.—The arginine content of egg proteins percentage of methionine, cystine, and
phenylalanine and a lower percentage
Albumen Yolk Whole eggs
of arginine, histidine, lysine, and proline
Age of eggs Per- Per- Per-
mg. mg. mg. than yolk protein; the percentages of
cent cent cent
tyrosine and tryptophan were similar in
Fresh a 6.2 236 7.1 186 6.6 422 yolk and albumen proteins.
Fresh b 6.3 206 7.2 185 6.8 397
9 months 6.0 167 7.2 183 6.7 358 The data presented in Tables 1-9 are
18 months 5.8 180 7.2 171 6.4 351 for the most part in good agreement with
23 months 5.8 179 7.2 187 6.5 369
26 months 5.8 173 7.4 153 6.5 326
TABLE 3.—The lysine content of egg proteins
Literature values: Block and Boiling (1943), 7.0;
Block and Mitchell (1946), 5.8,8.2,6.4; Calvarv and Albumen Yolk Whole eggs
Titus (1934), 5.4, 7.5; Edwards, et al. (1946)', 9.7;
Hess, et al. (1948), 5.6, 7.0, 6.5; Horn, et al. (1948b), Age of eggs Per- Per-
Per- mg. mg.
9.5; Munks, et al. (1945), 5.7,169,6.8,168, 6.4, 344. cent cent mg. cent

these digests used for the determination of Fresh a 6.5 245 7.1 186 6.7 431
Fresh b 6.6 216 7.2 184 6.8 400
tryptophan. Cystine was also determined 9 months 6.5 179 7.3 186 6.9 365
chemically by separating as the cuprous 18 months 6.5 202 7.1 169 6.8 371
23 months 6.5 201 7.3 190 6.9 391
mercaptide (Csonka, et al. 1944) and then 26 months 6.7 198 7.1 147 6.9 345
determining the cystine colorimetrically
(Evans 1944). Literature values: Block and Boiling C1943), 6.0;
Block and Mitchell (1946), 6.5, 7.2; Calvery and
Results.—The results obtained are pre- Titus (1934), 5.3, 5.2; Edwards, et al. (194o), 7.8;
Hess, et al. (1948), 4.8, 5.8, 5.1; Horn, et al. (1947),
sented in Tables 1-9. The amino acid con- 7.9; Munks, et al. (1945), 4.9,145, 5.7,141, 5.2, 280.
tents of the egg proteins have been cal-
culated to a basis of 16 percent nitrogen TABLE 4.—The methionine content of egg proteins
in the protein. The amino acid content of
Albumen Yolk Whole eggs
the whole egg is on the basis of milligrams
Age of eggs
of amino acid per egg. Values from the Per-
mg.
Per-
mg.
Per-
mg.
cent cent cent
literature for the amino acid content of
egg proteins are included for comparison. Fresh a 4.0 151 2.6 68 3.4 219
Fresh b 4.1 134 2.1 54 3.2 188
9 months 4.0 110 2.2 56 3.1 166
DISCUSSION 18 months 4.0 125 2.5 60 3.4 185
23 months 4.1 126 2.0 51 3.1 177
Albumen protein contained a higher 26 months 4.0 120 2.6 54 3.5 174

TABLE 2.—The histidine content of egg proteins Literature values: Beach and Teague (1942), 4.1;
Block and Boiling (1943), 3; Block and Mitchell
Albumen Yolk Whole eggs (1946), 4.4, 3.0, 4.1; Edwards, et al. (1946), 3.9;
Hess, et al. (1948), 4.1, 3.6,3.9; Horn, et al. (1946a),
Age of eggs Per- Per- Per- 3.1; Horn, et al. (1946b), 2.7; Munks, et al. (1945),
cent mg. cent mg. cent mg. 6.6,196, 3.0, 74, 5.2, 280.

Fresh a 2.6 97 2.9 75 2.7 172

Fresh b 2.8 89 2.9 74 2.7 157 published values where values are avail-
9 months 2.7 74 2.8 72 2.7 141 able for the amino acid content of the
18 months 2.7 83 2.9 69 2.8 152
23 months 2.6 80 2.8 74 2.7 151 albumen, yolk, or whole egg proteins.
26 months 2.6 78 2.8 57 2.7 135 No appreciable change in the concentra-
Literature values: Block and Boiling (1943), 2.4;
tion of histidine, lysine, methoinine,
Block and Mitchell (1946), 2.2,2.6,2.1; Calverv and cystine, tryptophan, phenylalanine, and
Titus (1934), 1.4, 1.3; Edwards, et al. (1946)", 3.6; proline in egg proteins occurred during
Hess, et al. (1948), 1.2,1.5,1.5; Horn, et al. (1948a),
2.3; Munks, et al. (1945), 1.4, 42, 1.5, 37, 2.0, 108. storage. Albumen protein from stored eggs
 AMINO ACID CONTENT OF EGGS 699

contained less arginine and tyrosine than TABLE 7.—The tryptophan content of egg proteins
that from fresh eggs. Yolk
Albumen Whole eggs
The fewest milligrams per egg albumen
Age of eggs Per- Per- Per-
of most of the amino acids were found in mg. mg. cent mg.
cent cent
the 9-month old eggs, but the fewest milli-
grams per whole egg were in the 26-month Fresh a 2.0 74 47 1.9 121

OO OOOO Ov 00 00
Fresh b 2.0 65 46 1.9 111
9 months 1.8 49 46 1.8 95
TABLE 5.—The cystine content of egg proteins 18 months 1.9 58 44 1.9 102
23 months 1.7 53 47 1.8 100
26 months 1.9 55 37 1.8 92
Albumen Yolk Whole eggs
Age of eggs Literature values: Block and Boiling (1943), 1.6;
Per- Per- Per-
cent mg. cent mg. mg. Block and Mitchell (1946) 1.6,1.6,1.5; Celvery and
cent
Titus (1934), 1.5, 1.3; Edwards, et al. (1946), 1.6;
Fresh a 2.6 98 1.6 43 2.2 141 Fiirth and Lieben (1921), 2.0, 2.1, 2.1; Hess, et al.
Fresh b 2.4 80 1.8 45 2.1 125 (1948), 1.4, 1.2, 1.4; McFarlane, et al. (1930), 1.5,
9 months 2.3 63 1.9 48 2.1 111 1.5; Munks, et al. (1945), 1.4, 42, 1.4, 34, 1.4, 75.
18 months 2.5 76 1.7 40 2.1 116
23 months 2.4 73 1.8 47 2.1 120 TABLE %.—The phenylalanine content of egg proteins
26 months 2.6 76 1.7 35 2.2 109
Albumen Yolk Whole eggs
Literature values: Block and Boiling (1943), 2.1;
Block and Mitchell (1946), 2.3,1.9, 2.4; Calvery and Age of eggs Per- Per- Per-
Titus (1934), 2.1, 1.6; Hess, et al. (1948), 2.5, 1.8, cent mg. cent mg. cent mg.
2.3; McFarlane, el al. (1930), 2.1,1.7; Munks, et al.
(1945), 1.9, 56, 2.2, 54, 2.2, 118. Fresh a 4.6 5.4 345
6.0 226 119
Fresh b 6.2 202 4.5 114 5.4 316
TABLE 6.—The tyrosine content of egg proteins 9 months 6.1 167 4.6 118 5.4 285
18 months 6.1 189 4.6 110 5.4 299
23 months 6.1 184 4.7 122 5.4 310
Albumen Yolk Whole eggs 26 months 6.0 179 4.5 93 5.4 272
Age of eggs Per- Per- Per-
cent mg. cent mg. cent mg. Literature values: Block and Boiling (1943), 5.6;
Block and Mitchell (1946), 5.5, 5.7, 6.3; Edwards
Fresh a 4.7 179 4.5 118 4.6 297 et al. (1946), 6.1; Grau (1947), 7.6; Hess, et al.
Fresh b 4.5 147 4.6 118 4.5 265 (1948), 5.4, 3.7, 4.5; Horn, et al. (1948), 7.3; Moto-
9 months 4.3 119 4.6 119 4.5 238 mura (1929), 1.6; Munks, et al. (1945), 6.2,184, 4.4,
18 months 4.2 129 4.4 106 4.3 235 108, 5.8, 312.
23 months 4.5 137 4.6 120 4.5 257
26 months 4.2 126 4.6 95 4.4 221 TABLE 9.— The proline content of egg proteins

Literature values: Abderhalden and Kempe Albumen Yolk Whole eggs

(1907), 1.8; Block and Boiling (1943), 5.0; Block and
Mitchell (1946), 4.8, 5.3, 4.5; Calvery and Titus Age of eggs Per- Per- Per-
(1934), 4.0, 5.4; Grace (1947), 4.1; Gunness, et al. cent mg. cent mg. cent mg.
(1946), 4.1; McFarlane, et al. (1930), 5.0, 5.1;
Munks, et al. (1945), 4.5, 133, 4.7, 116, 4.8, 258. Fresh a 4.1 157 4.3 113 4.2 270
Fresh b 3.8 126 4.5 114 4.1 240
9 months 3.9 107 4.5 116 4.2 223
old ones. The one exception was methi- 18 months 4.3 133 4.6 111 4.4 244
onine, which occurred in smaller amounts 23 months 4.0 123 4.5 118 4.2 241
26 months 4.2 124 4.5 93 4.3 217
in the 9-month old eggs. The loss from the
26-month old eggs was from the yolk Literature value: Motomura (1929), 3.6.
rather than from the albumen.
Only serine, threonine (Evans, et al. protein of eggs which had been stored for
1949b), arginine, and tyrosine were found 18 months, the proline level was high
in smaller amounts in the 18- than in the compared with fresh eggs and with those
9-month old eggs. In contrast with the stored for other lengths of time. This is
low level of tyrosine and arginine in the reflected in a comparatively large number
700 EVANS, DAVIDSON, BANDEMER AND BUTTS
of milligrams of proline in both the al-
bumen and the yolk of the 18-month old
eggs. There was more phenylalanine in the
18- than in the 9-month old eggs, although
it is very similar structurally to tyrosine
differing only in the absence of the hy-
droxyl group. There was no evidence that
tyrosine was converted to phenylalanine.
Yolks of the 23-month old eggs con-
tained more of each of the amino acids
except methionine than yolks of any other
stored eggs. There was less methionine
in the yolks of 23- than of 26-month old
eggs.
The 4.0-4.1 percent methionine in egg
albumen protein (Table 4) agrees well
with literature values except for that of
Munk etal. (1945) who estimated methio-
nine by difference, subtracting the cystine
sulfur, determined by precipitation as
the cuprous mercaptide, from the total
organic sulfur. Values presented in Table
4 for the methionine content of yolk
protein are lower than most reported in
the literature.
Good agreement was obtained between
chemical and microbiological methods for
the determination of cystine. The average
values determined colorimetrically and
microbiologically respectively were for
albumen protein 2.44 and 2.45, yolk
protein 1.76 and 1.74, and whole egg
protein 2.15 and 2.14 percent.
Except for the tryptophan data re-
ported by Fiirth and Lieben (1921),
the value of 1.9 percent tryptophan in
whole egg protein presented in Table 7 is
the highest coming to the authors' atten-
tion. Other investigators obtained values
of 1.4 to. 1.6 percent. The use of enzymatic
digestion in vitro for hydrolyzing the
proteins previous to making the trypto-
phan determinations rather than the usual
alkaline hydrolysis, may have been,
partially at least, responsible for the
high tryptophan values obtained in the
present investigation.
Nutritionally it is doubtful if any of
the eggs except those that were stored for
9 months need be given consideration,
because shell eggs are not usually kept
in storage for a longer time. The greatest
losses of essential amino acids from the
9-month old eggs were of methionine and
tryptophan, each of which decreased by
18 percent, and the least were of arginine
and lysine with 12 percent decreases.
Losses of most of the essential amino
acids were not much greater from the
26- than from the 9-month old eggs,
except that arginine and threonine losses
from the 26-month old eggs were nearly
twice those from the 9-month old ones.
20 percent of the arginine and 30 percent
of the threonine (Evans etal. 1949b) were
lost.
Of the essential amino acids only
threonine occurred in lower concentration
in the protein of 9-month old eggs than
in that of fresh ones, and the decrease was
so small as to be unimportant nutritionally
(4 percent of the threonine).
The differences of amino acid concentra-
tion in yolk and albumen proteins show
the importance of eating the whole egg
in order to supply a well balanced protein.
Such is especially true for the two sulfur-
containing amino acids, cystine and
methionine, because egg albumen protein
contains 1.6 times as high a concentration
as egg yolk protein. Mitchell and Block
(1946) consider whole eggs to be the most
ideal protein source nutritionally and have
used the amino acid composition of whole
egg protein as a basis of comparison for
other proteins and protein concentrates.
According to their standards the nutritive
value of egg yolk protein would be limited
by the low concentration of cystine plus
methionine, because it contains only 4.1
percent compared to 5.7 percent in whole
egg protein. When one considers that
 AMINO ACID CONTENT OF EGGS
egg albumen protein contains 6.6 percent
of cystine plus methionine, the necessity
of eating the egg albumen along with the
yolk is indicated if the most economical
utilization of the egg protein is to be
obtained. Otherwise more protein must be
consumed to meet the body's need for
cystine and methionine.
SUMMARY
Arginine, histidine, lysine, methionine,
cystine, tyrosine, tryptophan, phenyl-
alanine, and proline contents of fresh eggs
and of eggs stored for 9, 18, 23, and 26
months were determined by microbiologi-
cal assay. Cystine was also determined
colorimetrically, and good agreements
were obtained between the two methods.
Higher values for tryptophan were ob-
tained by employing enzymatic hydrolysis
in vitro than had previously been reported
with alkaline hydrolysis. Arginine and
tyrosine were the only ones of these amino
acids present in albumen protein of stored
eggs in a lower concentration than in
albumen protein of fresh eggs. Egg
albumen protein contained 1.6 times as
high a concentration of cystine plus
methionine as yolk protein. The con-
centrations of the basic amino acids and
proline in egg yolk protein were higher
than in the albumen protein.
Albumens of eggs stored for 9 months
had fewer milligrams of each of the amino
acids than did those from fresh eggs or
eggs stored longer. Yolks from 26-month
old eggs contained fewer milligrams of
each of the amino acids, except methionine
than the other eggs, but the yolks of the
23-month old eggs had the least methio-
nine. Of these amino acids, only tyrosine
and arginine were present in larger
amounts in 9- than in 18-month old eggs.
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