第26巻
259
第5号(1977)
総
説
The 2-Thiobarbituric Acid Reaction, An Objective
Measure of the Oxidative Deterioration
Occurring in Fats and Oils
Russell O.
Department
Sinnhuber
of Food Science
(Corvallis,
and Technology,
Oregon
1 Introduction
More than 30 years ago Kohn and Liver
sedges' noted that aerobic oxidation of brain
tissue produced a compound that when heated
in an acid solution with 2-thiobarbituric acid
(TBA) gave a brilliant red color with an
absorption maximum at 532 nm.
They
postulated that the TBA reactive material was
a carbonyl compound since the reaction was
blocked by semicarbazine or phenylhydrazine.
Bernheim et al.2' isolated an impure red TBA
pigment and suggested that the reaction com
pound was a 3-carbon moiety containing one
oxygen atom. Wilbur et al" proposed that TBA
reaction could be used as an estimation of the
oxidative products of unsaturated fatty acids.
Patton and Kurtz4', interested in the oxidized
flavor of milk fat, tested a number of comV
pounds and based on spectral measurements
suggested that malonaldehyde was the come
pound responsible for the red TBA color test
and the material was present in rancid milk
fat. Patton et al.5' reported that the TBA
reaction material was of low molecular weight,
water soluble, Kries test positive and that
malonaldehyde could be the compound.
Many years earlier, Dox and Plaisance6' a''
demonstrated that furfural and aromatic
aldehydes would react with TBA to yield
highly colored derivatives. Jennings et al.$'
examined the TBA color reaction with milk fat
and the similar color reported by Shepherd"
Technical
Experiment
Corvallis,
Paper
No.
44$5
Station,
Oregon
Oregon 97331.
Oregon
State
Agricultural
University,
and
T.
C. Yu
Oregon
State University
97331, U.S.A)
and by Kohn1" for the reaction of TBA with
sulfadiazine. They concluded that although
both colors gave the same spectral characc
teristics in the visible range that was not
proof that the compound were identical with
malonaldehyde. In a very significant experiment, Patton and Kurtzt s' found that freshly
prepared a or /1unsaturated aldehydes did not
give the TBA test but when autoxidized or
heated with Cu2+a red color developed. They
postulated that a low molecular weight com
pound, possibly malonaldehyde, was respon=
sible for the typical TBA reaction. At that
time, malonaldehyde was not available as
a pure compound because of its extreme
instability
but
l , l ,3, 3-tetraethoxypropane
(TEP), used as intermediate for organic syn=
thesis, was obtainable. TEP hydrolyzes under
mild acid conditions to yield malonaldehyde
and ethyl alcohol. Sinnhuber and Yu'" used
this compound as a source of malonaldehyde
and as a standard for the quantitative determination of malonaldehyde in autoxidized
fishery products using TBA as the reagent.
TBA Pigment
The TBA test has undergone a number of
modifications and refinements since it was
conceived by Kahn and Liversedge" but it
consists basically of treating a lipid containing
material or fraction derived therefrom with an
acid solution of 2-thiobarbituric acid and
heating the mixture to develop the red chro=
mogen. The intensity of the color at 532'535 nm is a measure of the malonaldehyde
content which reflects the degree of lipid
2
260
油
autoxidation
which
has occurred.
In our
laboratory
when
1,1,3,3-tetraethoxypropane
became available
we were able to prepare
the
red pigment in quantity
and compare its che=
mical and physical
properties
with pigments
obtained
from rancid
or autoxidized
salmon
oil and from sulfadiazine,
Sinnhuber
et al.111
In that report,
the three red pigments
were
found to give the identical
absorption
spectra
shown
in Fig.-1.
The main
absorption
a formula
of C11HsN404S2 as shown
Table-1
Elemental
formula
The
absorption
TBA
salmon
pigment
spectra
isolated
of the
from
crystal=
rancid
oil.")
mum is at 532^-535 nm with slight secondary
maxima at 245 nm and 305 nm. The E;,,,
of purified and vacuum dried pigments gave
an average value of 4780. Employing the
molecular extinction coefficient reported by
Sinnhuber and Yu12> of 1.56X10' and the
E,~L the molecular weight of the pigment
may be estimated. The calculated value of
327 agrees quite closely with the molecular
weight of a compound with two molecules of
TBA and one of malonaldehyde.
Further
confirmation of the molecular composition of
the pigment was obtained by using various
combining ratios since the pigment is highly
insoluble and the yield is quantitative. Various
molecular ratios of TEP to TBA were used
and we found that whenever that ratios
exceeded 1 : 2 the yield approached 100°%,
leading to the conclusion that the pigment was
a condensation of one molecule of malonaldehyde with two molecules of 2-thiobarbituric
acid. Elemental chemical composition of the
pigments prepared from rancid salmon oil,
malonaldehyde (TEP) and sulfadiazine were
all similar and comparable to the theoretical
assuming a molecular weight of 324.35 and
2
in Table-L .
analysis
of TBA
pigments°.
maxi=
Sinnhuber
1Assuming
line
学.
Schmidt1"
in a series of experiments,
employing somewhat
different
conditions,
confirmed
the studies of Sinnhuber
et al.") and the for=
mula of the red TBA pigment.
'From
Fig.-1
化
et al
.13)
a molecular
weight
of 324
.35
and
of C11H3N404S,.
Paper chromatography of the red TBA
pigment, isolated from rancid salmon oil,
sulfadiazine, malonaldehyde (TEP), frozen
tuna and herring meal gave identical RF
values using a solvent system of phenol-iso=
propyl alcohol-formic acid-water (80 : 10 : 10
100 vol/vol).
Fig.-2 shows a photomicrograph of the TB A
reaction product derived from rancid salmon
oil. The crystals are fine purple-black needles
which gave no definite melting point up to
300°C. The pigment is only very slightly
soluble in water, but is readily soluble in dilute
alkaline solutions or pyridine. Alkaline solutions of the pigment show a bathochromic shift
to a longer wave length, 541-547 nm with a
decrease in intensity of spectral absorbance.
The TBA pigment is readily absorbed on
Fig.-2
TBA-malonaldehvde
pigment,
prepared from autoxidized
(280 x )13)
crystals
salmon
oil,
第26巻
261
第5号(1977)
TB:1
Malonaldehvde
Fig.-3
TB.\
Formation
of TBA
pigment
pigment13~
cellulose powder or filter paper pulp from mild
acid solution and may be eluted by weak
alkali, phenol or pyridine solutions.
In Fig.-3, the reaction of TBA with malonal
dehyde and the formation of the TBA pigment
is shown.
It is of interest that crystals of the TBA
pigment prepared in 1957 and stored since
that time were recently subjected to mass
spectrometry, nuclear magnetic resonance and
infrared spectrometry in our laboratory15' and
the results obtained confirm the proposed
formula.
Formation of malonaldehyde in autoxidized
lipids
malonaldehyde, in the free state, does not
appear to be present in large amounts in
autoxidized lipids but seems to exist in a
weakly bound state which is released when the
system is heated with mild acid. Sinnhuber
et al.13' reported that less than 2° of the total
malonaldehyde measured in a highly oxidized
sample of salmon oil was in the free form.
Kenaston et al.16' observed that when linole
nate, linoleate and oleate were oxidized to the
same peroxide level that linolenate produced
60'.- 100 times the TBA chromogens as lino
leate and no color was found with oleate.
Fig.-4
Relationship
between
and
malonaldehyde
during
the oxidation
tetraene,
acid
pentaene
methyl
esters.
peroxide
Autoxidation of linoleate and forma
tion of two a /3 peroxide radicalsl7~.
Dahle et al.''' at the Hormel Institute proposed
a mechanism for the formation of malon
aldehyde in autoxidizing lipids based upon the
well-accepted autoxidation theory of Farmer
and Sutton18'. Employing careful measurement
of oxygen uptake of purified fatty acid esters
combined with the determination of diene
conjugation, TBA assay and peroxide value,
they found that linoleate gave no TBA color
at the early stages of oxidation. They reported
TBA gave a linear color response when reacted
with mildly oxidized triene, tetraene, pentaene
and hexaene fatty acid esters. Fig.-4 depicts
the relationship between peroxide values and
malonaldehyde concentration during oxidation
of the various classes of polyunsaturated fatty
acid esters. From these data they reasoned
that two isomeric peroxide radicals were
possible from autoxidized linoleate as shown in
Fig.-5 and linolenate will yield four as depicted
in Fig.-6. Similarly arachidonate would give
value
concentrations
of diene, triene,
and hexaene
Fig.-5
fatty
Fig.-6
Autoxidation of linolenate and formav
tion of two a /3 peroxide and two ,3 y
peroxide radicals''.
3
?62
油
six, pentaenoate eight and hexaenoate ten
different peroxide radicals. As Dahle et al.'"
significantly points out, two of the four
peroxide radicals "possess unsaturation % y
with respect to the peroxide function. Such
a configuration is significantly absent in the
isomeric radicals of linoleate." They propose
that the /3 y unsaturated peroxide radical
undergoes cyclization to form a 5-membered
ring peroxide which under the acidic conditions
of the TBA test decomposes to form malonalz
dehyde as shown in the equation in Fig.-7
which in turn reacts with TBA to form the
red chromogen. It should be reemphasized
that this particular 5-membered ring peroxide
can occur only during the oxidation of methylc
ene interrupted polyunsaturated systems with
three or more double bonds. As will be shown
in a succeeding section, this makes the TBA
test particularly well suited for the estimation
of oxidative rancidity of fishery products and
fats and oils which contain these highly un
saturated lipids.
化
学
autoxidizing
lipids found that further oxidation
of 2-nonenal
derived
from the oxidation
of
methyl linoleate
would give small amounts
of
the
malonaldehyde-TBA
pigment.
They
postulated
the formation
of
hydroperoxides
from 2-nonenal,
is :
three
isomeric
one of which
Oxidative degradation of this compound would
yield heptanal and malonaldehyde, the latter
which was found after reaction with TBA.
These studies emphasize that while the TBA
test measures malonaldehyde in autoxidizing
lipid systems, it is important to recognize that
it is especially well-suited for the detection of
oxidative rancidity in lipids which are poly
unsaturated and contain three or more double
bonds. Those fats or lipids which contain little
or none of the more polyunsaturated fatty
acids, such as corn or cottonseed, do not give
a strong TBA reaction in the early stages of
oxidative rancidity. The reaction that does
occur is partially due to the secondary oxidation of the primary carbonyl compounds.
TBA Test for Oxidative Rancidity
Fig.-7
Decomposition
cyclic
peroxide
autoxidation
the
conditions
form
of the five-membered
formed
during
the
of
the linolenate
of the
TBA
under
test
to
malonaldehyde'"
In an apparent
contradiction
of the preceding
paragraph was the observation by Biggs and
Bryant1" that in highly oxidizing systems,
even oleate and linoleate gave a positive TBA
color, Tarladgis
and Watts',
Haase and
Dunkley2".
Patton and Kurtz'",
Patton et
al."' and Tauf el and Zimmermann"'
reported
that oxidized 2-alkenals and 2,4-alkadienals
react with TBA to form the red pigment.
Lillard and Day'" in a study of autoxidative
degradation
of the secondary
products
of
4
The TBA test as devised by Kohn and
Liversedge" as an indication of oxidative
reactions in tissues was slightly modified by
Patton and Kurtz" and used to detect milk
fat oxidation. They concluded that the TBA
test was more sensitive than the peroxide value
or Kries test for measuring oxidative deterioc
ration in milk fat and suggested this test could
be applied to a wide variety of fats and fatcontaining foods. As a direct result of their
suggestion, the TBA test was soon adopted by
food scientists as a measure of storage stability
and the quality of foods and fats. A partial
list of the application of the TBA to foods
follows:
Dairy Products:
Patton & Kurtz"
Dunkley & Jennings'"
Biggs & Bryant1"
Sidwell et al.'"
King'"
第26巻
第5号(1977)
Cereal & Baked Products:
Caldwell & Grogg2"
Meat:
Turner et al.2"
Younathan & Watts3"
Tarladgis et al.31)
Smith et al.32'
Zlpser & Watts33),34)
Yu & Sinnhuber3"
Keskinel et al.36)
Witte et al.37)
Yamauchi3"
Wilson et al.3"
Fish & Marine Products:
Schwartz & Watts4"
Yu & Sinnhuber4"
Sinnhuber & Yu1"
Palmateer et al.4"
Andersson & Danielson43)
Yu & Sinnhuber3"
MacLean & Castell44)
Castell et al.4"
Castell & Spears46)
Greig47'
Botta et al.4"
Fruit-Orange Juice Essence :
Braddock & Petrus4"
Poultry & Egg Products:
Privett et al.5"
Kwon & Norgaard5"
Wilson et al.3"
Wang et al.52'
Fats & Oils :
Sidwell et al.53)
Kenaston et al.1"
Schmidt54)
Tarladgis et al.31)
Jacobson et al.55)
Pohle et al.56)
Brownely & Lachman57'
Pokorny et al.5"
Yu & Sinnhuber5"
Marcuse6"
Asakawa et al.61)
Feedstuffs:
Budde & Wornick6"
Yu & Sinnhuber35)
Nut Meats:
Holland63)
The TBA test for oxidative rancidity in lipid
or fat containing food or tissue has been
adapted by various experimenters to the
particular material under examination. In its
simplest form 100-500 mg of material is
263
heated with the acid TBA reagent for 20-30
min and the intensity of the red color deter
mined in a spectrophotometer at 535 nm. If
the solution is not clear because of an oil
emulsion that may be present, a few ml of
chloroform will generally clear the colored
solution for spectral examination. A reagent
blank should be run simultaneously.
This
procedure has the advantage over the peroxide
or similar tests for oxidative rancidity in that
it may be conducted on the intact sample and
does not require fat extraction. The method
currently used in our laboratory is as follows :
A. Intact sample procedure
Reagents and Equipment
TBA Solution : 1 g of TBA is dissolved in
75 ml of 0.1 N NaOH and diluted to 100 ml
with distilled water.
Trichloroacetic Acid-HC1 Reagent: 50 ml
TCA solution (25%) and 30 ml HCl (0.6 N)
are mixed with 420 ml of distilled water.
Antioxidant Solution: 0.3 g butylated hyv
droxyanisole (BHA) is dissolved in 5.4 g
propylene glycol, and 0.3 g butylated hydroxy
toluene (BHT) is dissolved in 4.0 g of warm
Tween 20. The two solutions are mixed. This
antioxidant preparation is water miscible.
Test tube, screw cap with Teflon liner, 20 mm
O.D., 150 mm length
Pan for boiling water
Test tube basket-4 3/4 X 4 x 51 J4"
Sample size : Oil and fish meal samples :
100200 mg
Fresh or frozen samples
(homogenized) : 200-400 mg
Procedure : A proper quantity of sample is
introduced into a tared test-tube and accurate
ly weighed. Three drops of antioxidant solution
(exact weight is not necessary) and 3 ml of
TBA solution are added to the tube. In case
of homogenized fresh sample, the contents in
the tube are mixed (Vortex Mixer) to disperse
the sample. 17 ml of trichloroacetic acid-HC1
reagent is then added. The tube is flushed with
N2 and then the screw cap is tightly closed.
Another tube containing the same quantity of
reagents, but without the sample is used as a
blank. The tubes are placed in a test tube
basket and heated in a boiling water bath for
30 min. The tubes are cooled to room temc
ro
264
油
perature in tap water, and about 15 ml of the
color solution is transferred into a 50 ml
conical centrifuge tube. Approximately 5 ml of
chloroform is added and the contents mixed
for a few seconds with a Vortex mixer. The
tubes are centrifuged for 10 min at 3,000 rpm.
A part of the aqueous clear color solution is
transferred into a 1 cm cuvet for absorbency
measurement at 532 nm.
The absorbence of a 1 g sample (in 100 ml
reagent) multiplied by the factor 46 is the TBA
number, or the milligram of malonaldehyde per
1,000 g of sample, Sinnhuber et al.12'. 4ccac
sionally instead of the usual bright pink
chromogen a yellow color is encountered due to
interfering substances. These will be discussed
in a later section.
B. Distillation procedure
A distillation method described by Tarladgis
et al.31' has been used in place of the direct
procedure, previously described, usually for
meat samples or those which are highly colored
or which contain considerable carbohydrate
material. The procedure consists of heating
a blended slurry of meat and water in a Kjel=
dahl flask with mild acid and distilling over a
measured volume containing most of the
liberated malonaldehyde. The TBA test is then
conducted on an aliquot of the distillate. The
advantage of this procedure is the larger sample
size and less interfering colors are encountered.
The main disadvantage is that the distillation
is an emperical procedure requiring the collec=
Lion of a specified volume of distillate. Since
the distillation is not complete, a correction
factor is derived, based on blank determinac
tions and distillation of a malonaldehyde
standard (TEP). Approximately 70°'s of the
added malonaldehyde is recovered.
Bryant1"
Interfering Pigments in TBA-Malonaldehyde
Reaction
Fig.-8
With certain foods and tissues, instead of
the typical pink color, an orange or yellow
color results with a secondary absorption peak
about 450'-460 nm. If the color is of sufficient
intensity, it may overlap the TBA-malonal
dehyde absorption causing an erronously high
value. The compounds responsible include
carbohydrates, Wilbur et al.3' ; Biggs and
6
; formic
acid,
Schmidt'"
化
学
; furfural
compounds,
Keeney
and Bassette6"
; glyoxal,
Smith
et at.'";
glycolic
aldehyde,
glyceric
aldehyde
and dihydroxy
acetone,
Landucci
et al.6" ; epihydrin
aldehyde
and glyceralzdehyde, Patton'"
Caldwell and Grogg23', in a study of rancidity
of cereal and baked
products,
removed
the
yellow interfering
color by selective absorption
on a cellulose
column.
An acid distillation
technique
was employed
by Sidwell et al.5"
and Tarladgis
et al."' to separate
malonalde
hyde from other interfering
food materials.
Yu and Sinnhuber36'
developed
a chromatoc
graphic procedure
for separating
and purifying
the pink and interfering
pigments.
Using this
method,
a correction
factor may be derived for
the particular
product
tested
which obviates
the need for the separation
procedure in success
sive determinations.
This procedure
has been
demonstrated
to be effective in products
which
contain sugar or carbohydrate
material such as
beef liver, oysters,
and dehydrated
ham and
other food products,
e.g., frozen tuna pie, dog
food, dry fish food. Fig.-8
depicts
the absorption
spectra of the 450 and 532 pigments
in dehydrated
ham and the separation
that
can be achieved.
Absorption
spectra
ants in dehydrated
of the
ham.
TBA
react
A purified malonaldehyde pigment (o) was
obtained by chromatographic separation of
the interfering yellow pigment (0) from the
original orange-red solution (0)35
Asakawa
et al."' outlined a procedure
using
sodium
sulfite with the TBA reagent
when
the 450 nm pigments
are present.
This modi
fication
appears
promising
in eliminating
第26巻
2651
第5号(1977)
interfering pigments without affecting the
malonaldehyde determination.
Tarladgis et al.67' questioned the validity of
the TBA test when conducted on the intact
sample stating that TUBAundergoes a number
of side reactions when heated with acid to
produce interfering pigments which give erv
roneous TBA values. Yu and Sinnhuber66' in
a series of experiments refuted the work of
Tarladgis et al.67'. They found that if the
reagents were pure and free from contaminat
ing substances, no decomposition of TBA
occurred to produce interfering colors even
when TBA was heated with acids, oxidizing
agents or hydroperoxides. In conclusion Yu
and Sinnhuber6" contend that the only com=
pound responsible for the red chromogen with
an absorption maximum at 532'.'535 nm is a
condensation product of malonaldehyde with
TBA.
Ho and Brown69' demonstrated that impure
solvents such as petroleum ester, acetic acid,
diethyl ether and residual distillate fractions
of ethanol and methanol often contain TBA
reacting substances which can be removed by
purification. They suggested refluxing the
solvents with TBA which effectively trapped
the TBA reacting material, thus rendering the
distillate from such systems free of the chro=
mogen.
In a recent study Marcuse and Johansson7o~
examined the TBA reaction in oxidized lipids
systems and with various aldehydes and im
plied that the conditions for TBA test are not
clearly defined and proof was lacking that red
pigment with an absorption at 530 nm was
due to malonaldehyde. Patton71' responded
with evidence from a number of experimenters
which prove that the red pigment at 532 is
indeed the reaction product of malonaldehyde
and TBA. Patton further stated that the TBA
test has proven to be a highly sensitive and
useful method of monitoring lipid oxidation in
many systems. He added, however, that the
test results require careful interpretation and
should be compared with organoleptic findings
and comparisons with other chemical tests.
Relationship of TBA Value to Peroxide Number
of fats and oils comparing the TBA and per
oxide determinations by Sidwell et aL5° showed
that higher TBA values were obtained for
soybean oil than for cottonseed oil at com
parable peroxide values. A partial explanation
for this difference may be found in the experi
ments of Kenaston et al.'° when they demon=
strated that oxidizing methyl linolenate gave
higher TBA and peroxide values than linoleate
and no TBA values were found in oxidizing
oleate. Dahle et al.17' in a series of experiments
reported a linear relationship between TBAA
value and peroxide value for the polyun
saturated fatty acids. However, pure diene,
linoleate, even when oxidized to a peroxide
value of 2,000 failed to give a TBA reaction.
A linear relationship between peroxide value
and malonaldehyde content was also found
(see Fig.-9) during the autoxidation of men
Fig.-9
Relationship
during
oil59a.
the
of
POV
autoxidation
to
MA
values
of menhaden
haden oil by Yu and Sinnhuber59'.
Prior to
using the TBA procedure as a measure of
oxidative rancidity in fats or foodstuffs, it is
important that the fatty acid composition of
these products be known. Oxidative rancidity
in lipids containing little or no fatty acids of
the linolenate or higher unsaturation would not
be expected to show significant TBA values
even though these lipids gave a high peroxide
value. The slight TBA color which occurs in
systems containing only oleate or linoleate can
be explained as the further autoxidation of a,
/t unsaturated aldehydes, initially formed from
the decomposition of hydroperoxides,
Pattonn
and Kurtz4', Patton et al.2°, Lillard and Day.24;,
Early studies of the oxidative degradation
τ
油
266
Relationship of the TBA Value to Oxidized
Flavor in Food Systems
While the peroxide value has been well
correlated with the oxidation of fats and oils
and the accompanying off-odors and flavors,
there are many difficulties associated when the
peroxide test is applied to food systems which
are low in fat content. Extraction of unstable
hydroperoxide, without decomposition, in suf
ficient quantity for determination presents
many problems. The suggestion by Wilbur
et aL3' that the TBA test could be used as an
estimation of oxidative products of unsaturat
ed fatty acids soon received the attention of
scientists who were searching for an additional
method for measuring lipid oxidation in foods,
particularly if it could be related to the develc
opment of oxidized or rancid flavors. Dunkley' 2',
Dunkley and Jennings25', Patton and Kurtz4'
found a good correlation between TBA values
and oxidized flavor in milk.
Biggs and
Bryant19' applied the TBA test to butter,
-cheese and whole milk powder and reported
that the test is capable of detecting several
degrees of oxidation below the level of organo
leptic sensitivity. Sidwell et a1.26'improved the
TBA procedure for dried milk products by
distillation and stated that their results corn
related well with sensory evaluations. Turner
et al.2" applied the TBA test to frozen pork
and found it to be a more reliable index of
age and quality than other chemical tests for
fat rancidity.
They reported a significant
positive correlation between TBA results and
taste test acceptability scores for wieners and
pork patties.
From these reports it is evident that the
TBA procedure has been widely accepted as
a sensitive method for the measurement of
malonaldehyde in foods and the results can be
correlated with the development of off-odors
and flavors. It should be recognized that the
fatty acid composition of the food to be ex
amined is an important factor in the applica=
tion of the TBA test.
S
学
mogen with an absorption
maximum
at
532 nm is a rapid, highly sensitive and reliable
procedure for the measurement of oxidative
rancidity in foods and lipids and especially if
these systems contain fatty acids of greater
unsaturation
than linoleate.
Correlation with
taste panels is exceptionally
good in these
systems. The possibility of application directly
to the material to be tested without lipid
extraction is a decided advantage over other
chemical tests. Procedures have been devised
to overcome the effects of interfering sub=
stances which are sometimes encountered.
Acknowledgements
The authors wish to thank Dr. Noboru Matsuo
of Seikei University, and the Japan Oil Chemists'
Society for their generous support and sponsors
ship of this review.
(Received March 8, 1977)
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9