Further Studies on Green or Offcolor Condition
in Precooked Yellowfin Tuna
Marine Biological Laboratory
LIBRA. R-y
APR 2 -1958
WOODS HOLE, MASS.
SPECIAL SCIENTIFIC REPORT- FISHERIES No. 247
UNITED STATES DEPARTMENT OF THE INTERIOR
FISH AND WILDLIFE SERVICE
Explanatory Note
The series embodies results of investigations, usually of restricted
scope, intended to aid or direct management or utilization practices and
as guides for administrative or legislative action. It is issued in limited
quantities for the officialese of Federal, State or cooperating Agencies
and in processed form for economy and to avoid delay in publication.
United States Department of the Interior, Fred A. Seaton, Secretary-
Fish and Wildlife Service, Arnle J. Suomela, Commissioner
FURTHER STUDIES ON GREEN OR OFFCOLOR CONDITION
IN PRECOOKED YELLOWFIN TUNA
By
John J. Naughton, Michael M. Frodyma, and Harry Zeitlin
Chemistry Department, University of Hawaii
Special Scientific Report--Fisheries No. 247
WASHINGTON: November 1957
ABSTRACT
Resulta are reported from a study of the "greening" condition that appears in certain samples of
yellowfin tuna on precooking. Evidence is presented that this is an actual color condition similar to
discoloring processes that occur in other meats, and is caused by an anomalous heme protein oxida-
tion. Related to this tendency to turn green on precooking are the presence of high concentrations of
metnnyoglobin, some denaturation, and a slightly high fat peroxide content in the raw nneat. In addi-
tion, green meat generally has a high flesh pignnent content. Oxygen starvation due to the exhaustion
of the fish that nnight occur in the process of catching does not seem to produce the factors that lead
to greening, but rather a deterioration that goes on even in the frozen state seems to be responsible.
Spectral reflectance was eniployed in much of the work and revealed important in situ changes or
processes in flesh pigments that would have been impossible to note by other means.
CONTENTS
Page
Samples 1
Reflectance 1
Precision of measurements 1
The color of cooked tuna 2
Nature of the pigments in tuna flesh 3
Pigment derivatives present in tuna flesh 4
Pigment changes in raw flesh 4
Oxymyoglobin to metmyoglobin 4
Deoxygenation of oxymyoglobin 4
Pigment denaturation 5
Solubility of metmyoglobin , 5
Pigments in cooked tuna flesh 6
Green pignnent in precooked flesh 6
Direct evidence for green pigment 7
Relation between pigments of raw and precooked tuna flesh 8
Peroxides in fish fats , 9
Further investigations on leaching 10
Fish exhaustion and metmyoglobin content 10
Discussion 11
Sunnmary 11
Literature cited 12
ILLUSTRATIONS
FIGURE Page
1. Chromaticity coordinates for green and normal tuna meat. 2
2. Variation of the 540/640 millimicron peak ratio with absorbancy at Soret peak (410-415
millimicrons), an index of pigment content. 3
3. Separation of aqueous extract of raw tuna into hemoglobin and myoglobin by phosphate
fractionation 3
4. Reflectance curves showing pigment mixtures found in raw tuna flesh. 4
5. A. Simplified absorption curve showing increased concentration of nnethemoglobin with
increasing oxy-he.moglobin. B. Similar simplified absorption curves showing
increased metmyoglobin content after various periods of freezer storage. ... 4
6. Oxidation of flesh pigment on treatment with nitrogen 5
7. Reflectance curves showing denaturation by appearance of hemochrome band at 528 m(i
after reduction with dithionite 5
8. Reflectance curves showing solubility of metmyoglobin and insolubility of oxymyoglobin
in raw tuna flesh 6
9. Reflectance curves showing pigments of cooked tuna flesh. 6
10. Spectral absorbance measured in transmission of extract of precooked green tuna flesh. 7
11. Reflectance curves of acid-acetone and acid-methanol extracted precooked tuna of
decreasing greenness. 8
12. Relation of metmyoglobin content of raw tuna flesh to color rating of the sanne flesh on
precooking. 9
13. Relation of metmyoglobin content of raw fish flesh to color as objectively judged. . . 9
FURTHER STUDIES ON GREEN OR OFFCOLOR CONDITION
IN PRECOOKED YELLOWFIN TUNA
By
John J. Naughton, Michael M. Frodyma, and Harry Zeitlin
Chemistry Department, University of Hawaii
An undesirable "green" color which
develops in the flesh of certain tuna onprecook-
ing prior to canning, results in considerable
economic loss to the fisherman and to the
industry through rejection of such fish. The
cause of this offcolor condition has been the
subject of studies at the Chemistry Department,
University of Hawaii, under contract No. 14-
19-008-2475 with the Pacific Oceanic Fishery
Investigations (POFI), U. S. Fish and Wildlife
Service. The background and the results of
preliminary studies are embodied in a previous
report (Naughton, Frodyma, and Zeitlin, 1956).
The extension of the work herein described
represents an attempt to establish more defi-
nitely the nature and genesis of the pigment or
pigments responsible for the offcolor.
We are most grateful to Mr. Fred Jermann
of Hawaiian Tuna Packers, Ltd., and to Dr.
Albert L. Tester, Director, and other members
of the staff of POFI for suggestions and help in
the investigation.
SAMPLES
The fish used in this study were yellowfin
tuna (Neothunnus nnacropterus) which, for the
most part, were caught on longline by POFI
research vessels in the central equatorial
Pacific in 1955 and 1956. The fish were frozen
on board ship as soon as possible after capture
and were held in a solid frozen condition until
they were needed. Sizes ranging from about
70 to 200 pounds were selected as it had been
noted that the larger fish showed a greater
tendency to turn green on precooking. In addi-
tion small yellowfin (up to 15 pounds) v/ e t e
caught by trolling in local waters by the staff
of the Hawaii Marine Laboratory, University
of Hawaii, and were sampled while fresh.
Samples of fresh, prime fillets ("ahi" samples)
of yellowfin caught in the local longline fishery
were purchased from local markets for study.
In order to obtain precooked sannples for
the evaluation of color, frozen fish were thawed
in tanks at Hawaiian Tuna Packers, Ltd., and
cut into loins; one loin from each fish was re-
tained as a raw sample. The remaining loins
were put through the regular commercial
precooking process of that company. Judgment
of color grade of the precooked samples was
made by Mr. Fred Jermann, the company's
food technologist. Specific samples noted to be
characteristic of their types (normal, green,
pink, etc. ) as subjectively judged, were used
in much of the work.
REFLECTANCE
Early in the research on the color of tuna
flesh, an opaque material, the need was recog-
nized for a device for nneasuring spectral
reflectance, thus making possible an objective
evaluation of the actual color of the sannples.
A reflectance attachment to the Beckman DU
spectrophotometer was secured. With the de-
velopment of techniques for reflectance meas -
urement, it became apparent that the method
had unique value as an analytical tool in addi-
tion to its recognized use in the specification
of color. Curves exhibiting optical absorption
peaks could be obtained by spectral reflection
measurements. These were characteristic of
the pigments present in tuna flesh and were
identical with the absorption curves obtained
from transmission measurements made on the
same pigments in solution. Beer's law was
obeyed, thus justifying the use of absorbancy
or optical density in our curve plots. The ob-
vious advantages of being able to make meas -
urements in situ, and on insoluble systems,
such as those encountered in this research,
were points that were vital. A separate com-
munication has been published (Naughton,
Frodyma, and Zeitlin, 1957) which describes
in greater detail the advantages of spectral
reflectance as an analytical tool, especially in
biological systems.
PRECISION OF MEASUREMENTS
The variability of the pigment content
throughout a sample of meat (see below) intro-
duces a difficulty when making reflectance and
pigment-content measurements on a series of
samples, even from the sanne fish. Ground and
thoroughly mixed (homogenized) samples gave
readings that were reproducible to + 0.5 per-
cent reflectance units or within the error of the
measuring instrument. Cn standing, however,
oxidative changes took place in such sannples
with consequent changes in spectral absorption.
Replicate nonhonnogenized samples from a
given fish loin gave an average precision of
+ 1.5 percent reflectance units.
THE COLOR OF COOKED TUNA
A visual examination of different samples
of cooked tuna flesh immediately reveals the
complexity of the color system involved. First,
the variability of the absolute content of pig-
ment in the flesh of various fish should be
recognized. We have encountered variations
from highly bleached meat to a very high pig-
ment concentration with resultant abnornnal
redness. Whether these variations are physio-
logical or due to postmortem changes has not
been determined. Secondly, the condition of
"browning" can be recognized. As usually
understood, this seems to b e a postmortem
and even a postcooking phenomenon which is
due to oxidation on exposure to atmospheric
oxygen. Thirdly, the greening phenomenon ,
which is the main subject of this research, is
observed. This is a color phenomenon that
permeates the flesh of the fish; its character-
istics will be described later in this report.
Also an orange coloration is frequently noted
which seems to occur in much the same manner
as the green color. Finally, it became very
evident after a modicum of work on the pigment
systems of the meat that the distribution of
pigment within a single fish loin was quite vari-
able, although it might seem uniform to the eye.
This was confirmed by nnicroscopic examina-
tion which showed the pignnent to be present in
bands within the meat. Spectral reflectance
measurements gave widely variable estimates
of the amount of pigment present. Uniformity
could be assured only by grinding and thoroughly
mixing all the meat needed in a given sample
sequence. This was not usually done, however,
since finely ground meat was found to change
color even when held in a frozen condition.
At the beginning of this research the reality
of the green of certain offcolor samples of tuna
flesh was questioned by one of the investigators.
The color is of such a subtle shade and exhibits
so many variations that it was supposed that
the so-called greenness might be a lack of pig-
ment. In order to test the reality of the color,
the ICI (International Commission of Illumina-
tion) system of color specification was plotted
for typical cooked green and normal samples.
The results are shown in figure 1. It will be
noted that loci of the coordinates for both sam-
ples are in a region of lightness or brightness
indicative of the general light color of cooked
meat. The green flesh is situated nearer the
0.8
0.4 -I
0.2
~f
1 1 ! I 1 1 1
\tellowish\
-
\ GREEN \ -
\ ^\ ^GREENISH YELLOW
\ /YELLOV^/
\ /gREEn/.^ ^YELLOW
\ j / /a^/v.^yellowish orange
GREEN __Xjy/y\^^ \.
TUNA '^<; \
BRIGHT \ ^\ -
NORMAL \
TUNA ^^^
-
V.-/^'^^^^^!^ , '
0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8
X
Figure 1 . --Chromaticity coordinates for green
and normal tuna meat. ICI systenn.
yellow-green color region. The normal, on the
other hand, has more of the orange component
present. The reality of the green cast of cer-
tain samples seems to be indicated.
Another method used in an attempt to eval-
uate greenness objectively was to compare
absorption in the red and green parts of the
spectrum (640 and 540 millimicrons, respec-
tively) for cooked green and nornnal meat by
reflectance measurements. A green sample
would be expected to show greater absorption
of light in the red end of the spectrum (640
millimicrons) when compared with normal meat.
The ratio of the absorbancy at the 540/640
nnillimicron absorption peaks therefore should
be greatest for normal and least for green meat
if pigments that absorb in these regions are
involved. In addition, we have been conscious
throughout this work that pigment content must
be a factor affecting the degree or type of alter-
ation that might occur. If we assume that we
can use the height of the nnost characteristic
absorption peak for heme pigments (the Soret
peak, at about 415 millimicrons) as an indica-
tion of the absolute pigment content of the cooked
meat and plot this against the above indicated
greenness index (540/640 absorption peak ratio)
it is possible to observe seversLl significant
relationships. Such a plot has been made in
figure 2, and it will be noted that normal sam-
ples, in general, occupy the portion of the
2.0
1.9 -
«> 16
V.
O
■a-
u
■ 1 1
1* 1
1
1 1
• NORMAL
-
• •
» OFF-COLOR
•• mm
—
• *
•
." ".*
J(
•<
X
•
x<
_
XX •
X
• •
X X
, ,«'
« ,
_
X
X
V
07 08 0.9 10 II 1,2
ABSORBANCY AT SORET PEAK
Figure 2. --Variation of the 540/640 millimicron
peak ratio with absorbancy at Sore t peak
(410-415 millimicrons), an index of pignnent
content.
figure indicative of higher 540/640 peak ratio
as would be expected, and lower pignnent con-
tent. It is noteworthy that both the "albacore
white" color resulting from low pigment content
and the fleshy pink color associated with a high
540/640 peak ratio are accepted as nornnal by
observers, thus emphasizing the complexity of
the systems encompassed by the term "normal. "
Another relationship which can be noted here,
namely the association of offcolor with a high
pigment content, is completely contrary to the
supposition expressed above; that is, that the
appearance of greenness is due to a lack of pig-
ment. It should also be noted in passing that
the overlapping position of some of the points
is due to the uncertainty inherent in the subjec-
tive evaluation of precooked flesh.
NATURE OF THE PIGMENTS IN TUNA FLESH
It is obvious that in an investigation of pig-
ment changes it is necessary fir St to under stand
the nature of the pigment. It is to be expected,
in a muscle system such as tuna flesh, that
muscle hemoglobin (myoglobin^./) would be the
major pigment present and contribute the larg-
est part of the flesh color. We had assumed
this in the investigations herein described, but
certain experiments have cast some doubt on
the validity of the assumption. It was found by
Bowen (1949) and others that the absorption
— Myoglobin is similar to hemoglobin
except for its lower molecular weight.
peaks for mammalian myoglobin were displaced
to longer wave lengths in the case of certain
derivatives of the pigment. No such shift was
observed for fish flesh.
The curves in either transmission or re-
flection for the systenns studied were identical
with those that we have nneasured for hennoglobin
and its derivatives. For verification, the solu-
bility of the extracted pigment in buffered phos-
phate solutions was checked according to the
procedure of Morgan (1936), with the incorpora-
tion of the modifications of Ginger, Watson, and
Schweigert (1954). The absorption curves for
the separated pigments are given in figure 3.
Assuming that these procedures would give
separation of myoglobin from the hemoglobin of
tuna flesh through differences in solubility, it
was found by measurennent of absorption in trans-
mission and reflection that the tuna pigment was
about 95 percent hemoglobin. Myoglobin seemed
to comprise only a minor fraction of the pigment
system. In figure 3 note the residual myoglobin
(curve b) in relation to the total pigment content
of the extract (curve a). It is felt that these
experiments were not exhaustive enough to be
conclusive. As far as color changes are con-
cerned, the division of the pigment between
myoglobin and hemoglobin is not too pertinent
since these changes involve the porphyrin
moiety of the pigment molecule which is identi-
cal for the two types. For convenience, we
shall continue to ascribe the color to myoglobin
1.0 r-i^
IT
o
in
m
<
Ol-^^
400 450 500 550 600
WAVE LENGTH, MJU
650
700
Figure 3. --Separation of aqueous extract of raw
tuna (No. 6) into hemoglobin and nnyoglobin by
phosphate fractionation. (a) Transmission
curve for original extract, (b) Transmission
curve for residual solution after hemoglobin
precipitation with phosphate, (c) Reflectance
curve of phosphate precipitated material.
until the point has been settled. In this connec-
tion, it might be noted that a recent report
(Rossi-Fanelli and Antonini 1955) cites work
in which myoglobin was separated from tuna
flesh giving a connpound differing from the
corresponding compound found in mammalian
flesh. No details of this difference were given.
PIGMENT DERIVATIVES PRESENT
IN TUNA FLESH
As has been previously noted (Naughton,
Frodynna, and Zeitlin, 1956), extraction of raw
flesh gave a colored solution tentatively iden-
tified as being largely met- or ferric myoglobin.
When reflectance measuring techniques were
brought to bear on the problenn, it became evi-
dent that the raw meat used in these studies
contained a mixture of oxy- and metmyoglobin.
Examples of the reflectance curves obtained
can be seen in figure 4.
^^ — \ 1 \
- (A) 11 1
-
(B)
-
- Iw
Ao) .
-
(dK // \y/i
/ \
-
(c) Jv, \ / 1
Ma
' / \\ a!
'^wK \\
\ \l
^^^"^3
^"
^r
-
L,-, ! t 1
I ■ > !
-
500 550 600
WAVE LENGTH, M/J
500 550 600
WAVE LENGTH, M/J
>-
CJ
IT
O
<
450 500 550 600
WAVE LENGTH, M/J
650
700
Figure 4. --Reflectance curves showing pigment
mixtures found in raw tuna flesh, (a) Largely
oxymyoglobin. (b) Largely metmyoglobin.
(c) Mixture of met- and oxymyoglobin.
Figure 5.--A. Simplified absorption curve
showing increased concentration of methe-
moglobin in mixtures with oxyhemoglobin
increasing from curve (a) to (d). (From
Austin and Drabkin 1935-36) B. Similar
simplified absorption curves taken in reflec-
tion for tuna meat after various periods of
freezer storage, showing increased nnetmyo-
globin content, (a) Fresh tuna (sep. sample),
(b) After 24 hours' freezer storage, (c)
After 36 hours' freezer storage, (d) After 84
hours' freezer storage, (e) After complete
conversion to metnnyoglobin by chemical
oxidation.
in a similar fashion the changes in absorption
obtained by Austin and Drabkin (1935-36) for
mixtures of oxyhemoglobin and methemoglobin
of known content.
Deoxygenation of Oxymyoglobin
PIGMENT CHANGES IN RAW FLESH
Oxymyoglobin to Metmyoglobin
With the continuation of the measurements
on raw meat, it became evident that we were
dealing with apignnent system that was extremely
changeable and evanescent. It was possible to
recognize rather rapid oxidative deterioration
in meat even when held under refrigeration. An
example involving the change from oxymyoglobin
to metmyoglobin can be seen in figure 5. That
the change corresponds to an increase in metmyo-
globin content can be verified by comparison
with the "A" portion of figure 5, which shows
An attempt to bring about the reduction of
oxymyoglobin to nnyoglobin by successive eva-
cuation and purging with inert gas (nitrogen)--a
technique that works well with oxyhemoglobin in
blood--resulted in a more rapid conversion to
metmyoglobin (figure 6). This rather surprising
behavior may result from the very active form
of oxygen released in situ in the flesh, which
readily oxidizes the ferrous iron in the myoglo-
bin to the ferric fornn in metmyoglobin. Lemberg
and Legge (1949, p. 395) have discussed a simi-
lar reaction for hennoglobin in the presence of
proton donors. The implications of this reaction
in the greening of tuna flesh will be discussed
subsequently.
1.4 r—l^
400
450 500 550 600
WAVE LENGTH, M/J
650
700
Figure 6. --Oxidation of flesh pigment on treat-
ment with nitrogen, (a) Reflectance curve
of fresh tana flesh, (b) Reflectance curve
after alternating evacuation and treatment
with nitrogen for 3 hours showing increase in
metmyoglobin content.
Pigment Denaturation
Another change that takes place in raw
flesh pigments is the denaturation of the protein
moiety of the heme pigment molecule. Such
denaturation produces so-called denatured
globin hemichronnes (Lemberg and Legge 1949,
p. 228). These are more easily identified from
their absorption curves in the reduced condition
as the denatured globin hennochromes with pro-
nounced peaks at 528 and 558 nnillimicrons.
Figure 7 shows such spectral reflectance curves
obtained with tuna flesh. Strictly fresh nneat
gave the characteristic single absorption peak
of myoglobin in the green region at 555 milli-
microns (curve a). Mild intentional denatura-
tion of this meat by heating to 50°C. for short
periods (20 minutes in the case illustrated) gave
on reduction the appearance of the distinct hemo-
chrome peak at 528 millimicrons (curve b) .
Similar denaturation can be noted in the absorp-
tion curve (curve c) for raw green tuna flesh,
and, as would be expected, for cooked green
tuna flesh (curve d). It is remarkable that
intense commercial cooking in the last instance
does not seem to nnarkedly increase the denatu-
ration of the pigment when so judged. There is
some indication from a study of a nunnber of
siich raw reduced samples that the degree of
denaturation is related to the amount of
400
450 500 550 600
WAVE LENGTH , M>J
650
700
Figure 7, --Reflectance curves showing denatu-
ration by appearance of hemochrome band at
528 m[i 2ifter reduction with dithionite. (a)
Reduced raw fresh tuna- -myoglobin; (b) Tuna
flesh of curve (a) after denaturation by heat-
ing 20 minutes at 50°C. ; (c) Raw green tuna,
reduced; (d) Commercially precooked green
tuna reduced.
metnayoglobin originally present in the samples
and to the greening tendency.
Solubility of Metmyoglobin
Another example of unusual heme pigment
changes and behavior noted in situ is the unex-
pectedly greater solubility of metmyoglobin
relative to the oxymyoglobin present in tuna
flesh. The phenomenon is illustrated in figure
8. It has been noted previously that aqueous
extraction of tuna flesh gave absorption curves,
nneasured in transmission, that were charac-
teristic of nnetmyoglobin. It was always found
that such extraction left a pigmented residue
whose color showed but slight tendency to
decrease with repetition of the extraction. This
was initially assumed to be due to the inefficiency
of the extraction procedure. Application of
spectral reflectance measurements to the prob-
lem showed that the metmyoglobin in tuna flesh
is characterized by a rather pronounced solubil-
ity, while the oxymyoglobin exhibited a high
degree of insolubility (compare curves a and b,
fig. 8). This suggests the possibility that pig-
ments leaching during thawing and precooking
might be a factor in producing a pale condition
akin to greening in tuna with a high metmyoglobin
400
450 500 550 600
WAVE LENGTH , M/J
650
700
Figure 8. -- Reflectance curves showing solubil-
ity of metmyoglobin and insolubility of oxymyo-
globin in raw tuna flesh, (a) Raw tuna flesh.
(b) Residual flesh after aqueous extraction at
pH 6. 8. Note disappearance of 500 mp. and
630 n-i^i metnnyoglobin absorption peaks.
content. This effect may be one of the complex
of factors related to greening, but it is probably
more closely related to a "washed out" condition
occasionally observed in tuna flesh.
PIGMENTS IN COOKED TUNA FLESH
400
450 500 550 600
WAVE LENGTH, M;U
650
700
Figure 9. --Reflectance curves showing pigments
of cooked tuna flesh. Curves (a) and (b) de-
natured globin hemichrome of cooked tuna flesh.
Curve (c) denatured globin hemochrome of
reduced cooked tuna flesh.
seems to lie in the greater absorption by the
former in the redregion (620 - 660 millimicrons)
for a given absorption in the yellow-green region
(520 - 580 millimicrons). Thus, infigure9, curve
(b) would be characteristic of flesh with a greener
hue than the flesh represented by curve (a).
The cooking of tuna meat results in the
denaturation and coagulation of the proteins
present, with consequent lightening of the meat
color. Denaturation of the heme protein pig-
ments leads to the formation of the hemochrome
(ferrous form), and under oxidative conditions
the hemichrome (ferric form) of the denatured
globin. Characterizing absorption, aside from
the Soret region, occurs at 545 and 575 milli-
nnicrons (fig. 8, curves a and b) in agreement
with the values listed for the denatured globin
hemichrome of hemoglobin (Lemberg and Legge
1949, p. 228). It will be noted by examination
of figure 9 that absorption by this substance is
rather weak and the peaks are ill defined. A
more unique and more easily characterized
curve is obtained for the reduced, or ferrous,
denatured globin hemochrome, as has been dis-
cussed previously. The contrast is evident in
figure 9. The color of the reduced hemochrome
is pink and more desirable from the consumer's
viewpoint. Brown and Tappel (1957) recently
have identified this pink compound as being a
mixed, denatured, globin nicotinamide henno-
chrome.
The difference between the spectral reflec-
tance curves of green and normal fish flesh
GREEN PIGMENT IN PRECOOKED FLESH
It proved to be impractical to extract and
identify the green component in precooked green
flesh, as has been pointed out previously. Another
approach would be to cleave the pyrrole ring
segment from the protein moiety of the pigment
molecule by alcohol--or acetone--hydrochloric
acid treatnnent, and then examine the extract by
spectral transmission, and the residue by spec-
tral reflection. Green pigmentation in meat has
been attributed to an oxidative attack on the
pyrrole ring of the heme pigments, with the
resultant production of choleglobin or verdohemo-
chrome (Watts 1954). Removal of the oxidized
or disrupted ring by the acid-alcohol or acetone
treatnnent, and spectral examination, would be
expected to reveal the nature of the parent pig-
ment. Verdohemochrome, and its postulated
precursor choleglobin, would produce biliverdin-
like compounds (Lemberg and Legge 1949, p.
458).
The acid-methanol or acetone extracts gave
an absorption curve in transmission that showed
no characteristic absorption peaks and was very
similar to that obtained from the leachate of
cooked fish (fig. 10, curve b). Solvent extraction
1.0
1 1
1 ! 1
N ""' \
\ \\
—
\ ', \
(a)
'\^b)
\(c) \
. t 1 1
-::;f:--=.~-^„V^
350 400 450 500 550 600 650 700
WAVE LENGTH, M;J
Figure 10. --Spectral absorbance measured in
transmission of 1:9, 12 N HCl-methanol ex-
tract of precooked green tuna flesh (No. 7) .
(a) Chloroform extract of HCl-acetone ex-
tract, (b) Original HCl-methanol extract .
(c) Chloroform extract of (b).
of this material, however, gave an absorption
curve in transmission with peaks at 382, 510,
540, and 640 millimicrons (fig. 10, curves a
and c). These absorption maxima are compared
in table 1 with the peaks reported for some of
the products of heme protein disruption that
might be identical with the substance found in
precooked tuna flesh.
The coinc. dence of the absorption peaks with
those reported by Lewis (1954) for hemin chlo-
ride in acetone with excess hydrochloric acid
added is particularly striking, and we may con-
clude that this substance is present in the extract.
We should note, however, that we have failed to
find the characteristic hemin chloride platelets
on microscopic exannination of these extracts.
DIRECT EVIDENCE FOR GREEN PIGMENT
The residues left after treatment of the flesh
with acid-acetone and acid-nnethanol were ex-
amined by the spectral reflectance technique .
The curves obtained were very similar to those
reported for acid hematin (Lemberg and Legge
1949, p. 173) and "acid stable hemin" (Lewis
1954). The comparative values are listed in
table 2.
Of far greater interest was the slight but
definite absorption peak that appeared in the red
end of the spectrum. This will be noted in
figure 1 1 at 625 millimicrons for acid-acetone
treated meat (curves a, b, and c), and at 610
millimicrons after acid-methanol treatment
(curves d and e). The curves show a decrease
in this peak height from very green meat (curve
a) to less green (curve b) to nornnal (curve c).
Table 1 . --Characteristic absorption peaks of heme pigment derivatives
A-bsorption maxima, |
Pigment
millimicrons
I
II
ni
IV
1
Muscle pigment in chlorofornn
640
540
510
382
2
Green skeletal pigment of skipjack
in chloroform (Fox and Millott
1954)
660
_
.
380
3
Methanolic ester of biliverdin in
chloroform (Tixier 1945)
665
-
-
384
4
Biliverdin hydrochloride in HCl-
methanol (Lemberg and Legge
1949)
680
_
_
377
5
Protoporphyrin in ether-acetic
acid (Lemberg and Legge 194 9)
632.5
537
502
395
6
Coproporphyrin in chloroform
(Lemberg and Legge 1949)
622.5
533
499
405
7
Hemin chloride and HC 1 (Lewis
1954)
640
540
512
382
Table 2. --Acid-hematin absorption peaks
Pigment
Absorption maxima,
millimicrons
I
III
IV
Soret
1 Reflectance of residue
in precooked tuna meat
after HCl -methanol or
HCI-acetone extraction
2 Acid hematin (Lemberg
and Legge 1949, p. 173)
glacial acetic acid
3 Acid stable hemin (Lewis
1954)
630
630-635
630
540
540
500
410
500
400
400
550 600 650 550 600 650
WAVE LENGTH , M/J
Figure 1 1. --Reflectance curves of acid-acetone
(a, b, c) and acid-methanol (d, e) extracted
precooked tuna of decreasing greenness (a, b
to c; and e to d). The decrease in absorption
with the decrease in greenness in the 610-620
millimicron range is evident.
previously been deduced more or less indirectly.
A similar pigment, absorbing between 610 and
620 millimicrons, has been reported in both
transmission measurements on green-colored
extracts and spectral reflectance curves obtained
from discolored beef that had been exposed to
gamma radiationfrom a cobalt-60 source (Ginger,
Lewis, and Schweigert, 1955). The pigment noted
here may be sinnilar, although present in lower
concentration and in an insoluble coagulated form.
The exact nature of the pigment is still under
investigation.
RELATION BETWEEN PIGMENTS OF RAW
AND PRECOOKED TUNA FLESH
The examination of many spectral reflec-
tance curves for the flesh of raw tuna reveals the
tendency for flesh with a high content of metmyo-
globin, characterized by a prominent 630-
millinnicron absorption peak, to give an offcolor
on precooking. Thus, referring to figure 4, one
would usually find that raw meat which gave a
reflectance curve such as (b) would turn green or
gray-green on precooking. The picture is com-
plicated by the presence of oxymyoglobin, which
seems to modify the effect of metmyologbin and,
when present in high concentration, produces
pink nneat that browns rapidly on exposure to air.
Frequently a high concentration of both oxymyo-
globin and metmyoglobin is found. The oxymyo-
globin predominates in effect, and the possible
combinations seem to result in a gamut of gray
to brown to orange pigmentation depending on the
relative amounts of each parent pigment.
where the absorption peak has disappeared. A
similar effect is noted in the acid-methanol
treatment (curve d, normal; curve 3, green
nneat). It is felt that this peak is direct evidence
for the green pigment, the existence of which had
An evaluation of the relation of both sub-
jectively and objectively judged color to metmyo-
globin content is shown in figures 12 and 13. The
height of the absorption peak in the red region at
630 millimicrons, which is characteristic of
+ 2
+ 1
0
-2
p-
o
-3
H
<
-5
-4
_l
<
>
-5
LU
Ul
-6
>
t-
-7
<.)
Ill
—>
-H
m
-)
C/5
-9
l-t-'4
-10 -
\
1 In 1 1
1
\
••» dx
—
\
\ \
•N^ •• • •X
—
\ N
\» ••> •^N
—
S \
\ _ N
N^ • s*8 'MX
-
\ \
\ •»
\
\
-
\ •
N
\
• \
\
-
\ •
-
\
N
\
-
•
•
-
-19
-20-
-1/1-
\
1.0 I.I 1.2 1.3 1.4
630/660 PEAK RATIO
Figure 12. --Relation of metmyoglobin content
of raw tuna flesh (as judged by magnitude of
spectral reflectance at the 630/660 peak ratio)
to the color rating of the same meat on pre-
cooking as subjectively judged.
o 1.7
o
t 1-5
0 NORMAL
A GREEN
a ORANGE 1
'■ 1 1
0
1
1
^j-ORaRlGE □
0
o"--
---'0 -
NORMAL
0
0 -><
~*"--.° ,^""'
0
^°"-°^ ,-
.-0- * -^
""-GREEN ^* i" "
° HIGH
METMYO
630/660 PEAK RATIO
Figure 1 3. --Relation of metmyoglobin content
of raw fishfleshto color as objectively judged
(540/640 ratio). Measurements made from
absorption in spectral reflectance.
metmyoglobin, was used as an indication of the
amount of this pigment in the raw meat and was
measured by means of the ratio of 630/660
nnillimicron absorption. Offcolor of the same
meat on precooking was judged subjectively by
Mr. Jermann of Hawaiian Tuna Packers on a
plus one (excellent); zero (normal); minus value
(for increasing degrees of greenness and off-
color) scale. The results are shown in figure
12. There seems to be a definite relation
between these factors when judged in this nnanner .
Objective evaluation of greenness in pre-
cooked meat was made through the use of the
absorption ratio at 540/640 millimicrons de-
scribed earlier in this report. The comparison
of this color rating with the metmyoglobin con-
tent is shown in figure 13. Again we note the
indication of an interrelation, complicated by
the orange color abnormality that is sometimes
noted. It is rather surprising that this also
seems to be related to a high metmyoglobin
content. The large number of coexisting flesh
pignnent forms and color s(pink, orange, green,
and brown) gives a complicated system in which
correlating factors are readily obscured. None-
theless, the evidence seems to point rather
definitely to metmyoglobin as a factor in the dis-
coloration of tuna flesh.
PEROXIDES IN FISH FATS
The suspicion of oxidation as one factor
related to greening leads one to speculate on the
role of fat peroxides in this phenomenon. The
coupled oxidation of hemoglobin and unsaturated
fats in mammalian meat tissue has been the sub-
ject of much research, summarized by Watts
(1954). The oxidation of unsaturated fats to
peroxides is accelerated by heme compounds
and is accompanied by the oxidation of the h enae.
The resulting fat peroxide may, in turn, play a
role in furthering the deterioration of hemepig-
nnents. It is known that hydrogen peroxide forms
unstable addition compounds with hemoglobin ,
which subsequently deconaposed with destruc-
tion of the heme moiety of the molecule (Keilin
and Hartree 1950). We have observed a green-
ing of tuna meat on treatment with dilute solu-
tions of hydrogen peroxide.
To test the possibility of a correlation
between peroxide formation and oxidation of the
heme pignnent, we have determined the fat con-
tent flesh by a modification of the cold extraction
method of Sperry (1955), carried out in de-
aerated solutions under a blanket of nitrogen.
The peroxide content was determined by titra-
tion of the iodine released fronn an iodide solu-
tion by the peroxide present in the extracted fat
with a standard thiosulfate solution. Appropriate
blank determinations were made. The results
are given in table 3.
For the sannples run, there is an indica-
tion of a high peroxide content in green and
Table 3. --Fat and peroxide values of raw tuna flesh
Percent
Milliequivs. per
Samples
Description
fat
1000 gms. of fat
Green
No. 1
Green
1.29
40
No. 2
Pale green
0.47
14
No. 3
Slightly green
0.63
24
No. 4
Green
0.82
14
0. 79
12
Very pink
No. 1
Abnormally pink
0. 55
27
No. 2
do.
0.92
20
1.07
40
No. 3
Orange
0.46
26
0.44
22
Normal and
pale
Normal
0.50
13
No. 1
No. 2
Washed out
1.57
9
No. 3
Normal
0.59
9
No. 4
do.
0. 58
4
No. 5
do.
0.54
10
0.51
8
offcolor flesh, which is in agreement with the
postulated oxidation of fats along with the oxi-
dation causing greening. The increased perox-
ide is nnost pronounced for abnormally pink or
orange samples, and less so for green.
The investigation of the fat content and
the peroxide values, related as they seem to be
to the oxidation of heme compounds, is particu-
larly pertinent since it is known that fish derive
their fat mainly, if not entirely, from dietary
fat. The fat is deposited in the tissues more or
less unchanged (Shorland 1956). Therefore it is
conceivable that the differences among fish,
which result in greenness being exhibited by
certain specimens, are related to differences
in dietary fat intake. The presence of fats that
are susceptible to oxidation (i.e., linoleic and
linolenic acid fats) would render heme pigments
subject to easy oxidation.
FURTHER INVESTIGATIONS ON LEACHING
It has been noted in previous reports
that on precooking, a solution was drained from
the fish flesh which was found to exhibit a degree
of pigmentation. We have referred to this proc-
ess as "leaching" and to the product as the
"leachate. "
Solutions of all leachate pigments gave
noncharacterizing absorption curves when
measured in transmission that are identical
with curve (b), figure 10. In addition, the resi-
dues left on boiling aqueous extracts of raw flesh
have the same color and give the same type of
absorption curve. As has been indicated pre-
viously, extracts of the hemin of precooked
meat with acetone or methanol- IN hydrochloric
acid (Briickmann and Zondek 1940), gave the
same types of absorption curves on measure-
ment in transmission as those resulting from
similar treatment of raw meat. In view of this,
one would expect to find that heme was the pig-
mented substance cleaved fronnthe myoglobin in
the tuna fie ah and leached out during precooking.
Repeated efforts, however, to produce the
rhomb- shaped crystals of "alpha-hemin"
(chlorohemin, hemin), which are so characteris-
tic of this compound when derived from mam-
malian blood, were not successful (Teichmann
test). The identification of this substance is
particularly pertinent because of the possible
relationship between the phenomenon of leaching
and the pronounced solubility of metmyoglobin
reported above.
FISH EXHAUSTION AND
METMYOGL-OBIN CONTENT
As a result of the accelerated conversion
of oxymyoglobin to metmyoglobin noted in the
absence of oxygen (nitrogen and evacuation), it
was hypothesized that a condition of anoxia, or
10
lack of oxygen, in the muscle of the fish brought
on by exhaustion might lead to a high metmyo-
globin content with consequent increased inci-
dence of greening. Such a condition is probable
for certain fish caught by the longline method
of fishing.
An opportunity to test the theory was
afforded through the utilization of the facilities
of the Coconut Island Marine Laboratory of the
University of Hawaii. A fishing boat was avail-
able with a "live-well" in which it was possible
to maintain small tuna alive. In addition,
small yellowfin tuna abound in local waters in
certain seasons. With the cooperation of the
staff of the Hawaii Marine Laboratory, it was
possible to catch tuna with lures and to kill
certain of these within a few minutes of capture
while maintaining others in a wounded and dying
condition for many hours in the live-well. The
latter were finally killed and the flesh of both
types was analyzed for metmyoglobin by the re-
flectance technique. Two sets of such paired
samples were obtained. Differences in met-
myoglobin content were very slight and seemed
to be randomly distributed between the fish
killed under the two conditions.
DISCUSSION
renders the heme moiety nnore susceptible to
further oxidation, which takes place on precook-
ing, and results in an opening of the porphyrin
ring. This last process may result in the pro-
duction of pigments similar to verdohemo-
chromes, which impart a green color to the
meat. Concomitant browning occurs due to the
formation of denatured globin hennichromes.
Although this picture is hypothetical,
there is some evidence for the steps indicated,
and it is offered as a basis for further work.
SUMMARY
Continued work on the green or offcolor
condition that appears in the flesh of certain
specimens of tuna on precooking has indicated
the following:
1. The greenness is an actual color condition,
rather than a lack of pigment or offcolor.
a. ICI (International Commission on lUunni-
nation) color evaluation of the light
reflected from green meat shows it to be
in the yellow-green region, with some -
what more green color than is present
in normal meat.
Consideration in toto of the experimen-
tal work performed in t h e period covered by
this report reveals that it was guided by the
assumption, based on evidence gathered in
earlier experiments, that there i s an actual
green pigment that produces the undesirable
color in certain samples of precooked tuna. As
a working hypothesis, it has been assumed that
the green color is due to the pignnents that result
from the opening of the porphyrin ring of certain
of the heme proteins of tuna flesh. This process,
which takes place as a result of oxidation, is
familiar to food scientsts in the meat industry.
A hypothetical picture of the processes
occurring in the greening of precooked meat
follows. Certain samples of raw tuna flesh con-
tain a high concentration of metmyoglobin in
proportion to the oxymyoglobin content. There
is evidence that such tuna are prone to green-
ness on precooking. The high concentration of
metmyoglobin is a result of the decomposition
of oxymyoglobin which releases oxygen in an
active form capable of oxidizing the ferrous iron
of myoglobin, and also capable of producing a
denaturation of the protein moiety of the heme
protein molecule. This oxidation takes place
largely during storage after the death of the
fish, and may be catalyzed by the presence of
certain fats ingested as a result of peculiarities
of the fish diet. The denaturation of the pigment
b. Study of spectral reflection shows gen-
erally a greater relative absorption of
light in the red region of the spectrum
for green meat--hence the green color--
and indicates a higher concentration of a
conripound or mixture of compounds ab-
sorbing in this region (thegreen pigments).
c. Evidence is offered for a green pigment
in precooked meat that has been exposed
to hemin cleavage by chemical means.
Spectral reflectance studies reveal oxymyo-
globin and metmyoglobin as the chief pig -
ments of raw tuna flesh. Mixtures are usually
present with a high concentration of oxymyo-
globin in the freshest meat, and increasing
concentrations of metmyoglobin in older
stored meat. Metmyoglobin content increases
even on freezer storage.
The reactions of the pigments in situ in the
raw tuna flesh were studied by reflectance
methods and revealed the following:
a. Interconversion from oxy- to met- to
myoglobin, and reverse were readily
achieved by chennical means.
b. Metmyoglobin was found to be very soluble
in aqueous media while oxymyoglobin was
11
not easily extracted into the same
media.
Katsuwonus pelamis (Linnaeus).
Experientia 10(4): 185-187.
c. Oxymyoglobin was converted to metmyo-
globin on de-aeration.
d. Stored meat showed evidence of progres-
sive denaturation of the pigment protein.
4. The characteristics of the pigments of cooked
tunaflesh were investigated. One of the pig-
ments, denatured globin hemichrome, was
readily reduced to the hemochrome by chemi-
cal means.
5. Evidence is presented showing a relation
between high metmyoglobin in the raw flesh
and a tendency towards discoloration on pre-
cooking,
6. Data are cited indicating a low fat peroxide
content in normal meat relative to meat
inclined to become offcolor on precooking.
It is felt that fat oxidation and heme pigment
oxidation are interrelated.
The conclusion reached is that greening
in tuna flesh is similar to the greening process
that occurs in other meats, and is due to an
anomalous heme protein oxidation. A hypothe-
tical pathway for the production of the green
condition in tuna flesh is outlined.
LITERATURE CITED
GINGER, 1. E., G. D. WILSON, and
B. S. SCHWEIGERT
1954. Biochemistry of myoglobin. Quanti-
tative determination in beef and pork
muscle. Agri. and Food Chem. 2(20):
1037-1040.
GINGER, I. E., U. J. LEWIS, and
B. S. SCHWEIGERT
1955. Changes associated with irradiating
meat and meat extracts with gamma
rays. Agri. and Food Chem. 3(2):
156-159.
KEILIN, D., and E. F. HARTREE
1950. Reaction of methaemglobin with hydro-
gen peroxide. Nature 166(422 1): 513-
514.
LEMBERG, R. , and J. W. LEGGE
1949. Hennatin compounds and bile pigments.
New York: Interscience Publishers,
Inc. 748 p.
LEWIS, U. J.
1954. Acid cleavage of heme proteins. Jour.
Biol. Chem. 206(1): 109-120.
MORGAN, V. E.
1936. Studies on myoglobin. I. The solu-
bility of myoglobin in concentrated
ammoniunn sulfate solutions. Jour.
Biol. Chem. 112(2): 557-563.
AUSTIN, J. H. , and D. L. DRABKIN
1935-36. Spectrophotometric studies. III.
Methemoglobin. Jour. Biol. Chem.
112(1): 67-88.
BOWEN, W. J.
1949. The absorption spectra and extinction
coefficients of myoglobin. Jour. Biol.
Chem. 179(1): 235-245.
BROWN, W. DUANE, and A. L. TAPPEL
1957. Identification of the pink pigment of
canned tuna. Food Research 22(2) :
214-221.
BRUCKMANN. G. , and S. G. ZONDEK
1940. An improved method for the deter-
mination of nonhemin iron. Jour.
Biol. Chem. 135(1): 23-30.
FOX, D. L. , and M. MILLOTT
1954. A biliverdin-like pigment in the skull
and vertebrae of the ocecin skipjack.
NAUGHTON, J. J., M. M. FRODYMA, and
H. ZEITLIN
1956. Nature of green or offcolor condition
in precooked yellowfin tuna. U. S.
Fish and Wildlife Service, Spec. Sci.
Rept. — Fish. No. 197, 7 p.
1957. Spectral reflectance applied t o the
study of heme pigments. Science
125(3238): 121-122.
ROSSI-FANELLI, A,, and E. ANTONINI
1955. Myoglobin and hemoglobin of sea
fishes. Crystallization and some
biochenaical properties, Congr.
intern, biochem. Resumes connmuns.
3 Congr. Brussels. (Chemical
Abstracts 50(5): 3659e. 1956).
SHORLAND, F. B.
1956. New trends in fats research. The
Austral. Jour, of Sci. 18(4A): 49-62.
12
SPERRY, W. M.
1955. Lipide analysis. In: Methods of bio-
chem. anal. 2:83-112. New York:
Interscience Publishers, Inc.
TIXIER, R.
1945. Contribution a I'etude de quelques
pignnents pyrroliques naturels des
coquilles de moUusques, de I'oeuf
d'emeu et du squellette du corail bleu
(Helioporacaerulea). Annales Insti.
Oceanographique 22(5): 343-397.
WATTS, BETTY M.
1954. Oxidative rancidity and discoloration
in meat. In: Advances in food re-
search 5:1-52. New York: Academic
Press.
13
5 WHSE 01182
Live Music Archive
Librivox Free Audio
Metropolitan Museum
Cleveland Museum of Art
Internet Arcade
Console Living Room
Books to Borrow
Open Library
TV News
Understanding 9/11