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Objective analysis of seafood quality
Tom A. Gill
a
a
Canadian Institute of Fisheries Technology, Technical University
of Nova Scotia, P.O. Box 1000, Halifax, Nova Scotia, Canada, B3J
2X4
Version of record first published: 03 Nov 2009.
To cite this article: Tom A. Gill (1990): Objective analysis of seafood quality, Food Reviews
International, 6:4, 681-714
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Food Reviews I nt er nat i onal , 6( 4) , 681-714 (1990)
OBJECTIVE ANALYSIS OF SEAFOOD QUALITY
Tom A. Gi l l
Canadian Institute of Fisheries Technology
Technical University of Nova Scotia
P.O. Box 1000, Halifax
Nova Scotia, Canada
B3J 2X4
ABSTRACT
Canadian seafood quality has traditionally been examined
by the subjective parameters of odor, color, flavor and
appearance. Also, Canadian seafood products have most often
been marketed on a one price system; products of high and
mediocre quality being sold at the same price.
A principal advantage of objective quality assessment is
the ability to assign meaningful numerical scores to raw
material and finished product, thus permitting adjustment of
market prices and providing a cash incentive to fishermen,
processors and retailers to maintain high quality standards.
Development of rapid, inexpensive objective techniques
for seafood quality evaluation has been the focus of research
in our laboratories during the past several years. Rapid
procedures for the analysis of amines, nucleotides and ammonia
are presented and the applicability of each for the overall
evaluation of seafood quality discussed.
681
Copyright 1990 by Marcel Dekker, Inc.
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682 GILL
INTRODUCTION
The unusually rapid rate of deterioration of seafood
quality has been recognized for many years- The rate of
spoilage may be characteristic of individual species or
related to the mode of preservation. For example, the
osmoregulatory substance trimethylamine oxide (TMAO) found in
most marine teleosts is converted to the pungent-smelling
trimethylamine in iced fish, and is often demethylated to form
odorless dimethylamine (DMA) and formaldehyde (FA) in frozen
fish muscle. The latter compound has been implicated in
toughening in frozen seafood.
Because the composition of the muscle varies so widely
among fish species, there are obvious differences in the modes
of spoilage. For example, mackerel which contain from 10-30%
fat in the edible muscle (depending on season of catch), often
undergo extensive oxidative rancidity prior to the onset of
bacterial decomposition. Cod, pollack or hake, which contain
less than 1% fat in the edible tissue, spoil primarily through
the profileration of psychrotrophic bacteria of the genus
Pseudomonas (Altermonas).
It is important to remember that the term "quality
1
means
different things to different people and is a term which must
be defined in association with an individual product type. It
is generally accepted that the best quality (in terms of
edibility) is found in fish which are consumed within the
first few hours postmortem. However, very fresh fish which
have entered rigor mortis are difficult to gut, fillet and
skin because of the distortion and firming of the muscle
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OBJECTIVE ANALYSIS OF SEAFOOD QUALITY 683
during this natural process. Thus, for the processor,
slightly older fish which have passed through the rigor
process, are often more desirable. Truly, quality, like
beauty, is "in the eye of the beholder".
The methods for the assessment of fish quality are
nearly as diverse as the number of fish product types on the
market. The methods of assessment may be conveniently
divided into two broad categories: subjective and objective.
When an individual sits down to an enjoyable dinner, and is
asked to rank his meal with others consumed during the past
week, he (or she) is being asked to make a subjective
comparison. He is not only being asked for a comparison, but
one assumes that he can remember what was consumed over the
past seven days as well as the perceived quality of each
meal. Unfortunately, subjective assessment is often prone to
error and may be easily biased. This does not mean that
subjective quality assessment is not important. After all,
the consumer judges the selection in the grocery store on the
basis of his/her subjective perception of quality, as well as
value. Since the consumer is the ultimate judge of quality,
sensory evaluation of seafoods is often the standard to which
all objective comparisons are made. Thus, sensory evaluation
must be performed scientifically, under carefully controlled
conditions so that effects of environment, personal bias,
etc. may be reduced.
Objective evaluation of seafood dates back to at least
as early as 1936 when, Beatty and Gibbons reported the
separation of a fraction of the volatile nitrogenous bases
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684 GILL
(TVB) from decomposing cod muscle. TVB values are still used
today but appear to correlate with flavor scores for some fish
better than others. The appeal of objective quality
assessment is related to the ability to set quantitative
standards. The establishment of tolerance levels of chemical
spoilage indicators would eliminate the need to base decisions
regarding product quality on personal opinions. Of course, in
most situations, subjective quality evaluation is sufficient
to identify products of very good or very poor quality. Thus,
objective indicators may best be used in resolving issues
involving products of marginal quality. A suitable objective
quality test procedure should possess the following
characteristics :
a) It should be easily performed in a reasonable
period of time by non-technical personnel;
b) It should be inexpensive and therefore applicable
to the screening of large numbers of samples;
c) The test procedure should not require the use of
sophisticated laboratory equipment;
d) It should measure a compound which is either
absent or present in constant amounts in the living
tissue;
e) It should increase (or decrease) in proportion to
the decrease in quality;
f) If the spoilage indicator is to be used for the
quality evaluation of processed fish, the results
should not be affected by the process per se (e.g.,
breakdown of amines in canned products as a result of
the retort process).
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OBJECTIVE ANALYSIS OF SEAFOOD QUALITY 685
CHEMICAL INDICATORS OF SEAFOOD QUALITY
Amines
Measurement of total volatile base amines (TVB) is one of
the most widely used chemical indicators of seafood quality.
TVB is a general term which includes the measurement of
trimethylamine (TMA), dimethylamine (DMA), ammonia and other
volatile basic nitrogenous compounds associated with seafood
spoilage. Over 65 papers attempting to correlate TVB with
organoleptic quality were reviewed by Frber (196 5).
Thirty-nine suggested a positive correlation, 17 were negative
and 9 were found to have variable results.
Although considered reliable for some types of spoilage
conditions and certain species of fish, the analysis of TVB
may suffer from some disadvantages as a potential fish quality
test. TVB level has been used most commonly as a quality
index for squid (Tanikawa e_t jal., 1956 ). The TVB content of
squid tissue correlated well with the ammonia and ammonia plus
TMA levels in a study of the spoilage of Illex illecebrosus
(LeBlanc and G ill, 198 4). Figure 1 illustrates the autolytic
production of TVB, ammonia and TMA with postmortem age for
squid stored at 2.5C. It was interesting to note that
although TVB, TMA and NH3 data increased with degree of
spoilage, the microbiological quality of the squid tissue
remained constant throughout the 10 day storage experiment
(unpublished data), suggesting that in short-finned squid,
spoilage is predominently due to the enzymatic degradation of
the tissue.
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686
GILL
r
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(
V
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a
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i
100
80
60
40
20
0
O O Ammonia
TVB
A ATMA
2 4 6 8 10
Storage Time at 2.5C (Days)
12
Figure 1. Effect of storage time on production of
ammonia, TVB and TMA in short finned squid (Tllex
illecebrosus).
There are several methods for the determination of TVB in
seafood and it has been shown that the results depend upon the
method of analysis. Botta ^t _al. (1984) found poor agreement
when comparing six different published TVB procedures. Many
of the methods presently used (Billon _et al., 1979; Pearson,
1977; AOAC, 1975; Tomiyama t jal., 1956 and Stansby, 1944)
involve steam distillation of a fish extract and therfore not
convenient for the routine analysis of large numbers of
samples. The microdiffusion approach commonly used in Japan
(Conway, 1962) requires a lengthy incubation period in which
the volatile amines are transported to a boric acid trap by
means of diffusion. Rehbein and Oehlenschlager (1982)
suggested that TVB was unreliable for the measurement of
spoilage during the first 10 days of chilled storage for cod
and several other species.
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OBJECTIVE ANALYSIS OF SEAFOOD QUALITY 687
Ammonia
Ammonia has been found to represent a major proportion of
the volatile amines recovered from several species of spoiling
fish (Rehbein and Oehlenschlager, 1982; Vyncke, 1970; L eBlanc,
1987 and LeBlanc and G ill, 198 4), including cod and squid.
Although ammonia has been identified as a volatile component
of spoiling tissue, few studies have actually reported the
quantitation of this compound. Instead it has most often been
included as a component in the estimation of total volatile
bases. Recently, two convenient methods for the measurement
of ammonia have been reported. The first involves the use of
glutamate dehydrogenase, NADH and alpha-ketoglutarate in the
presence of ammonia. The molar reduction in NH3 thus yields
the production of one mole of glutamic acid and NAD. The
depletion of NADH may then be conveniently monitored by
absorbance measurements at 340 nm. Test kits for ammonia are
now readily available from Sigma (St. L ouis, MO) and
Boehringer Mannheim (Mannheim, W. G ermany). A third type of
diagnostic test kit is available in the form of a paper strip
which is available from Merck (Merckoquant, Darmstadt, W.
G ermany). LeBlanc and Gill (1984) used a modification of the
glutamate dehydrogenase procedure to determine the ammonia
content of postmortem squid without the use of a
spectrophotometer. An indicator dye was coupled to the
oxidation of NADH.
NH3 + alpha-ketoglutarate . Glutamate
NADH NAD + H
+
INT or MTT * Formizan
(INT or MTT)
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688 GILL
en
E
0)
c
o
50
40
30
20
10
O 5CAIR
5C SW, GUTTED
A 5C SW, WHOLE
A 2.5C SW
0 1 2 3 4 5 6 7 8 9
Storage Time (Days)
Figure 2. Effects of time, temperature and contact
medium on the production of ammonia in postmortem
squid (illex illecebrosus). SW = chilled seawater.
so that the ammonia levels in squid extracts could be
determined semi-quantitatively by comparison of either
iodonitrotetrazolium (INT) or 3-[4,5-dimethylthiazol-2-yl] 2,5
diphenyl tetrazolium bromide (MTT) formizans with a set of
color standards. In this study, the postmortem production of
tissue ammonia in squid was found to be a function of the
conditions of storage (Figure 2) as well as storage time.
Increases in tissue ammonia levels in fish during spoilage
have been attributed to several enzymatic processes:
deamination of free amino acids (Soudan, 196 5), degradation of
nucleotides (Tarr, 1966) and oxidation of amines (Richter,
1937). LeBlanc and Gill (1984) reported that a major
proportion of the ammonia produced in squid during the first
24 hours postmortem is generated from conversion of adenosine
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OBJECTIVE ANALYSIS OF SEAFOOD QUALITY
689
360
^ 320
5 280
to
u 240
o
E 200
w
160
120
O
80
< 40
O OMerckoquant NH3
G DH NH3
A ATMA
2 6
O 1 2 3 4 5 6 7 8 9 10 11 12 13 14
Time of Storage (Days)
Figure 3. Production of TMA and ammonia in herring
offal held at 25C. Ammonia was determined by test
strip (Merckoquant) and by the enzymatic method
using glutamate dehydrogenase (G DH).
monophosphate (AMP) to inosine monophosphate (IMP). However,
the ammonia generated by this mechanism only represents a
small proportion of the total NH3 produced in prolonged
chilled storage.
Ammonia has also been found to be useful in evaluating
the quality of fish offal for use in the manufacture of fish
meal (Gill, unpublished report). Figure 3 illustrates the
production of ammonia and TMA in spoiling whole herring
carcasses at 20C. Data for both the glutamate dehydrogenase
and Merckoquant procedures are illustrated for comparison. It
is evident from this study that trimethylamine would be an
unreliable indicator of herring quality since levels changed
only slightly during the storage period.
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690 GILL
CD
CD
O
O
<
24
21
18
15
12
9
6'
3
A M M ON I A LEVEL
A A TOTAL AEROBIC COUNT
4 8 12 16 20
Storage Time on Ice (Days)
360
300 rP
I
240 2
X
180 "o
0>
120 Q.
=)
60 Li-
24
Figure 4. Effect of storage time on ammonia levels and
total aerobic plate counts in iced, gutted cod (Gadus
morhua).
Ammonia levels do increase however in proportion to the
degree of spoilage in cod tissue. Figure 4 illustrates that
the rise in ammonia and bacterial numbers in iced gutted cod
are coincidental.
Trimethylamine/Dimethylamine
Trimethylamine oxide (TMAO) is an osmoregulatory
substance found in many marine teleosts, elasmobrancs and
shellfish. A recent review article gives a comprehensive
overview on TMAO and the trimethylamine (TMA) and
dimethylamine (DMA) which is formed in fish tissue as a result
of the chemical breakdown of this compound (Hebard et al.,
198 2). Rather than citing the hundreds of articles found in
this reference, the important points will be summarized.
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OBJECTIVE ANALYSIS OF SEAFOOD QUALITY 691
The origins of TMA have been established with the
spoilage bacteria associated with Atlantic groundfish (Beatty
and G ibbons, 1936; Laycock and Regier, 1971; Adams e_t al.,
1964; Lerke e_t a^., 1963) althougn in some cases, the
correlation of bacterial numbers and TMA level is poor, (Tarr,
1961; Shewan, 1962; Shaw and Shewan, 1968) presumably because
not all spoilage bacteria associated with certain types of
fish are TMA producers. As mentioned above, TMA is not a
particularly reliable indicator of the edibility of herring
quality. The correlations between TMA level or more
preferably TMA index, where TMA index = log (1+ TMA value)
and eating quality have been shown to be excellent in many
marine fish species (Hoogland, 1958; Hess, 1941 and Wong and
G ill, 198 7).
Dimethylamine (DMA) has been reported to be present in
certain fish species and has most often been associated with
the enzymatic decomposition of TMAO. Since this process
normally takes place in frozen tissues of gadoid (cod, hake,
cusk, pollack) species (Yamada e_t a., 1969; Harada, 1975;
Gill and Paulson, 1982) it is probably of little interest for
the evaluation of fresh fish quality. Since formaldehyde (FA)
and DMA are produced in equimolar quantities, the enzyme,
TMAO-ase has been associated with the toughening of
frozen-stored fish as a result of FA-induced cross-linking of
the myof ibrillar proteins (Gill e_t ajL., 1979).
The methods used for the determination of TMA and DMA
have evolved through the years. The Dyer (1945) method as
modified by Tozawa e_t _a_l. (1971) has been used for many years
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692 GILL
for the determination of TMA in fish. Other methods of
analysis including gas chromatography (Ruiter, 1973; Miller et
al., 1972; G ruger, 1972; Kuwata et I., 1980; Tokunaga et al.,
1977); ion specific electrode (Chang ^t 1., 1976 ); solid
state gas sensor (Storey et ., 198 4); high performance
liquid chromatography (Gill and Thompson, 1984) and an
enzymatic diagnostic test kit (Wong and G ill, 1987) have been
proposed. The major advantage of the Chromatographie (HPLC)
procedure is specificity. Gill and Thompson (1984) reported
that colorimetric TMA data obtained using the Dyer/Tozawa
procedure were consistently 35% higher than the results
determined by HPL C. These results confirmed the fact that the
colorimetric approach lacked specificity. It has been known
for some time that the picric acid reagent employed in the
Dyer (1945) procedure, reacts with most primary, secondary and
tertiary amines. Recoveries of both DMA and TMA from fish
muscle using ion moderated partition HPLC were 102 and 94%,
respectively. Figure 5 illustrates the recovery curves for
mixtures of both amines which were spiked into minced cod
tissue. However, the high capital cost of equipment has
precluded the general use of Chromatographie techniques for
the routine screening of seafood quality.
Wong and Gill (1987) presented a rapid enzymatic
procedure for the determination of TMA in seafood which was
both reproducible and specific for TMA. The rapid TMA
assay was based upon the oxidation of TMA with phenazine
methosulfate (PMS) in the presence of TMA dehydrogenase. The
reduced PMSH2 formed, converted INT to a red formazan which
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OBJECTIVE ANALYSIS OF SEAFOOD QUALITY
693
C7>
3
O
C
D
O
<
5 10 15 20 25 30
Amount Added (mg%N)
35
Figure 5. Recovery curves for authentic standards of
TMA and DMA added to minced cod. Analyses were performed
by ion moderated partition chromatogrphy.
was monitored spectrophotometrically at 500 nm after a 20
minute incubation period. Figure 6 illustrates the good
agreement among data obtained for TMA in spoiling cod using
three different methods of analysis. The enzymatic method
yielded results similar to both the HPL-C (Gill and Thompson,
1984) and picric acid procedures.
A further advantage of the enzymatic method was that it
could be performed semi-quantitatively without the use of a
spectrophotometer. Inexperienced judges were able to perform
the TMA assay by visual comparison of the unknown samples with
a set of colored standards. Excellent agreement was obtained
between the visual and spectrophotometric determinations both
based on the enzymatic reduction of TMA and the subsequent
production of the red formazan indicator (Fig. 7). The odor
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694
GILL
40
o
32
o
en
E
i
<
O O Picric Acid Method
HPLC Method
A A Enzymatic Method
24
16
8
oooo
0
X , 0.
4 8 12 16
Storage Time on Ice (Days)
20
Figure 6. Comparison of three methods for the
measurement of TMA in iced, gutted cod (Gadus morhua)
The methods were the picric acid procedure TDyer,
1945), the HPLC method of Gill and Thompson (198 4),
and the enzymatic method of Wong and Gill (198 7).
8 16 24 32
TMA - SPEC (mgSK-N)
Figure 7, Performance of the visual color test as
compared to the spectrophotometric procedure for
TMA analysis of fish extracts. Both methods
utilized TMA dehydrogenase.
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OBJECTIVE ANALYSIS OF SEAFOOD QUALITY 695
C D
O
O
LO
O
"O
O
16 24
TMA-N (mg%)
32
Figure 8. Relationship between subjective
evaluation of odor of cod (using experienced
graders) and the TMA level.
of refrigerated cod was assessed by experienced graders and
compared with TMA data obtained using the enzymatic procedure
(Fig. 8 ). The relationship between TMA and subjective
evaluation of odor was approximately linear for a range of 0
to 40 mg TMA nitrogen per 100 g tissue. The importance of
comparing subjective and objective data cannot be
overemphasized and is essential for all forms of quality
determination.
The enzymatic determination of TMA was further refined by
the immobilization of the reagents to a diagnostic test strip
(Wong e^t a^., 198 8 ). The semi-quantitative determination of
TMA in fish press juice was carried out by color comparison in
about 5 minutes. TMA levels in spoiling fish were determined
by test strip and the picric acid procedure of Dyer/Tozawa are
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696 GILL
O 10 20 30 40 50 60 70
TMA Level by Picric Acid (mg TMA-N/iOOg)
Figure 9. Test strip performance compared to the
picric acid method. Each data point represents an
average of 20 determinations.
found to yield similar results (Fig. 9). The major
disadvantage of the enzymatic determination of seafood quality
by TMA analysis is the absence of a commercial source of
trimethylamine dehydrogenase.
Biogenic Amines
Fish muscle has the ability to support the bacterial
formation of a wide variety of amine compounds which result
from the direct decarboxylation of amino acids. Most spoilage
bacteria which produce decarboxylase enzymes do so at acidic
pH. This is presumably so that the organisms may raise the pH
of the growth medium through the production of amines. It is
also interesting to note that some bacteria have the ability
to 'turn off the production of biogenic amines when the pH of
the growth medium becomes too alkaline.
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OBJECTIVE ANALYSIS OF SEAFOOD QUALITY 697
Not only have biogenic amines been used for the
evaluation of quality, but are also of public health concern.
They are vasoactive, resulting in dilation of blood vessels,
and may cause headache, nausea, cramps and a burning sensation
in the mouth.
The decarboxylation products sometimes include cadaverine
from lysine, putrescine from ornithine and histamine from
histidine. Histamine has received most attention since it has
been associated with incidents of scombroid poisoning reported
in conjunction with the ingestion of tuna, mackerel, and
mahi-mahi (dolphin fish). Two excellent review articles have
been presented (Arnold and Brown, 1978; Kimata, 1961 ) on the
relationship between histamine and scrombroid poisoning from
the ingestion of seafood. Although, production of biogenic
amines has been primarily associated with fish from the tuna
family, a possibility exists that they may be potential
quality indicators in other species. Analysis of these
compounds has been accomplished by gas chromatography of
chemical derivatives (Henion et _al., 1981; Staruszkiewicz and
Bond, 1981; Noto et al., 1987; and Wada et a l
M
198 2), HPLC
(Simon and Lemacon, 1987, Walters, 1984; Mietz, 1977; Simpson
ejt al., 1982; Mietz and Karmas, 1978; Skofitsch et al., 1981;
and Gill and Thompson, 1984) and Technicon Autoanalyzer
(Murray and Murray, 198 1).
Perhaps one of the most interesting studies was reported
by Mietz and Karmas (1978) where samples of decomposing
salmon, lobster, shrimp and rockfish were classified' into
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698 GILL
spoilage categories according to sensory evaluation as well as
a spoilage index where:
(ppm putrescine + ppm cadaverine + ppm histamine)
Index =
1 + ppm spermidine + ppm spermine
Good agreement was observed between spoilage index and
sensory scores. However, only a very limited number of
samples were examined. Also, the method involved the
derivitization of the amines with dansyl chloride and was far
too tedious and time-consuming for the routine assessment of
seafood quality.
Recently, Farn and Simms (1987) recommended a gas
Chromatographie procedure developed by Staruszkiewicz and Bond
(1981) for the routine testing of tuna. Although canned tuna
is routinely examined by sensory evaluation in Canada, Farn
and Sims (1987) pointed out that it was highly desirable to
apply objective criteria to support subjective methods of
quality testing. Again, the method proposed by Staruszkiewicz
and Bond (1981) involved the gas chromatography of the
fluoropropionic anhydride derivatives of the amines and, like
the HPLC method proposed by Mietz and Karmas (1978 ), was far
too tedious for efficient analysis of large numbers of
samples. The HPLC procedure of Gill and Thompson (1984) was
capable of separating not only the biogenic amines putrescine,
cadaverine, spermine, and histamine, but also the alkylamines
DMA and TMA directly from perchloric acid extracts of fish
tissue without complicated derivitization procedures.
Unfortunately, detection was carried out by U.V. absorbance at
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OBJECTIVE ANALYSIS OF SEAFOOD QUALITY 699
207 nm and was far too insensitive to measure the ppm levels
detected in tuna using the gas Chromatographie procedures.
Although it has been reported that biogenic amines may be
useful in the detection of the onset of seafood spoilage, it
is important to realize that their absence does not
necessarily reflect a wholesome product. For example,
histamine is only produced by a relatively small group of
spoilage organisms and is generally only produced in the
temperature range of 22-25
C
C. Thus the psychrophilic spoilage
organisms which are generally involved in deterioration of
fish from temperate waters, would not normally produce
histamine and this compound would be unreliable for the
quality determination of such species.
It is clear that more work is required on the development
of rapid procedures for the determination of biogenic amines
in seafood. It is also clear that little is known concerning
the relationship between carboxylation of amino acids and the
eating quality and/or safety of seafood.
Nucleotide Catabolites
Much work has been undertaken in studying the pathways of
adenosine 5'-triphosphate (ATP) breakdown in fish and the
application of these time-dependent reactions to freshness
assessment. Unlike TVB and TMA levels which are most
reflective of bacterial spoilage, nucleotide degradation is
believed to be due to both autolytic as well as bacterial
action (Martin t: al_., 1978 ). Early studies with several fish
species (Shewan and Jones, 1957; Saito and Arai, 1957;
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700 GILL
Ur ic Acid
Figure 10. Postmortem ATP degradation in fish.
Enzymes include: 1. ATP ase; 2. myokinase; 3. AMP
deaminase; 4. IMP phosphohydrolase; 5a. nucleoside
phosphorvlase; 5b. inosine nucleosidase; 6,7.
xanthine oxidase.
Creelman and Tomlinson, 1960; Kassemsarn ejt aJN , 1963)
revealed that postmortem nucleotide degradation in fish
involves the multi-step breakdown of ATP yielding uric acid as
a final product (Fig. 10). ATP is dephosphorylated and
deaminated to form inosine monophosphate (IMP) which usually
accumulates quite rapidly after death and whose presence is
normally associated with excellent quality. The conversion of
ATP to IMP is usually complete within one day and is presumed
to be totally autolytic (Jones, 1965; Hiltz e_t al., 1971).
The dephosphorylation rate of IMP to inosine (Ino) varies from
species to species (Jones and Murray, 1964; Dingle and Hines,
1 9 7 1 ) as does the hydrolysis of inosine to hypoxanthine. The
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OBJECTIVE ANALYSIS OFSEAFOOD QUALITY 701
O OIMP
Inosine
A A Hypoxanthine
==
4 8 12
Storage Time at 3C (Days)
16
Figure 1 1 . Changes in IMP, Tno and Hx in sterile
cod fillets stored at 3C.
presence of IMP has often been associated with good quality
fish since this compound reportedly possesses properties as a
flavor enhancer in fish muscle (Oishi t _a_l., 1959; Burt,
1 9 65). The variable rate of IMP dephosphorylation among
species (Spinelli et _al., 1964; Dyer et al., 1966; Creelman
and Tomlinson, 1960) imposes limitations on its use as a
single quality index especially since it reaches basal levels
in many species within the edible storage life (Kassemsarn et
al., 1963; Jones and Murray, 196 4).
Recently, Surette et a^. (1988) clarified the roles of
autolysis and bacterial degradation of IMP and Ino in
postmortem Atlantic cod (Gadus morhua). The rates of
production and breakdown of IMP were the same in both sterile
and nonsterile samples of cod tissue (Figures 11 and 12),
indicating that the catabolic pathway for the degr adation of
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702 GILL
W
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3
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<
OOIMP
I nosi ne
A A Hypoxanthine
4 8 12
Storage Time at 3C (Days)
16
Figure 12. Changes in IMP, Tno and Hx in nonsterile
cod fillets stored at 3C.
ATP through to inosine is entirely due to autolytic enzymes.
The conversion of Ino to hypoxanthine (Hx) was accelerated by
about 2 days for the nonsterile samples, suggesting that
bacterial nucleoside phosphorylase plays a major role in the
postmortem production of hypoxanthine from inosine in
refrigerated cod.
Surette went on to isolate, purify and characterize the
nucleoside phosphorylase from spoilage bacteria recovered from
decomposing fillets (Surette, 198 7).
Saito ejb _a. (1959) proposed using the 'K' value:
[Ino]+[Hx]
K = x 100
[ATP]+[ADP]+[AMP][IMP]+[Ino]+[Hx]
as a quality index. Where ADP and AMP denote adenosine di-
and mono- phosphates, respectively. The index was
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OBJECTIVE ANALYSIS OF SEAFOOD QUALITY 703
subsequently shown to correlate very well with sensory panel
evaluations for a number of fish species (Uchiyama e_t al.,
1970). A simplification of the 'K' value termed the Ki index:
[Ino]+[Hx]
Ki =
[IMP]+[Ino[+[Hx]
was introduced by Karube e_t a_l. (1984) and the concentration
of each compound could be measured enzymatically by an enzyme
sensor which was coupled to an oxygen electrode. These Ki
values correlate very well with Saito's K index since ATP,
ADP, and AMP levels are often negligible shortly after death.
Sample preparation required tissue extraction in perchloric
acid and subsequent pH adjustment. Furthermore, there may be
a problem with the stability of the enzyme-coated membrane,
since it was reported to be stable for only up to 15 days.
Another instrument based on the determination of oxygen
consumption with an electrode for the measurement of
nucleotide catabolites was developed by Ohashi e_t a^., 1984.
This device measures the consumption of oxygen in an enclosed
cell in which trichloroacetic acid extracts of fish are placed
and the appropriate enzymes added. Values for the K index are
obtained by following one reaction which contains nucleoside
phosphorylase (NP) and xanthine oxidase (XO), thus giving a
numerical value for hypoxanthine + inosine. A second reaction
containing XO, NP and alkaline phosphatase will give a value
for the denominator of the K index. Readings can be performed
in 3 minutes, but sample preparation is time-consuming and the
initial instrument costs are high.
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704 GILL
Although nucleotide levels may be very useful for certain
fish species, they may be unreliable for others. For example,
cod fillet data illustrated in Figures 11 and 12 reached a
maximum K value in 3 to 4 days and then remained constant
throughout the duration of the study, regardless of the
presence or absence of spoilage organisms on the tissue. In a
study of previously frozen yellowfin tuna (Thunnus albacares),
Gill t _al. (1987) found little change in the K value even for
prolonged storage at 20C although the measurement of
hypoxanthine alone was far more sensitive to the changes in
quality.
Rapid visual methods for hypoxanthine determination have
been developed for use in field tests (Burt ej: _al., 1969;
Jahns et al.
f
1976 ). The method of Burt e_t 1. (1969) used
the redox indicator, 2,6-dichlorophenol to allow visual
monitoring of uric acid production from hypoxanthine in the
presence of xanthine oxidase. Jahns j2t _al. (1976) immobilized
xanthine oxidase and resazurin to a test strip. In both tests,
the colors of strips prepared from unknown fish were compared
with standards consisting of known concentrations of Hx or a
standard color chart. Unfortunately, most experts agree that
in many cases, Hx, alone, is not a particularly reliable
indicator of freshness.
The concept of using immobilized enzymes, coupled with a
redox indicator dye has recently been applied to the
manufacture of a "freshness testing paper" or FTP which
determines the K value. FTP is the registered trademark of
EAC Corporation, 1-1, Higashi-Ikebukuro 3-chome, Toshima,
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OBJECTIVE ANALYSIS OF SEAFOOD QUALITY 705
Japan. Recently, Nordin (1987) examined the use of the strip
for the evaluation of chum salmon quality. It was concluded
from the study that because of the high standard deviations,
the FTP strip gives only a rough estimate of the postmortem
age of the fish and appears to be most useful within the first
few days of storage.
Ethanol
The use of ethanol as an objective quality indicator for
seafood has been reported by several authors (Iida et al.,
1981 a,b; Tokunga e_t 1., 1982; Lerke and Huck, 1977; Khayat,
1979; Crosgrove, 1978; Human and Khayat, 1981; Hollingworth
and Throm, 1982, 1983; and Kelleher and Zall, 198 3). Since
ethanol can be derived from carbohydrates via anaerobic
fermentation (glycolysis) and/or deamination and
decarboxylation of amino acids such as alanine, it is a common
metabolite of a variety of bacteria. It was used as an
indicator of decomposition in mackerel, salmon and sardines
(Holaday, 1939) and in tuna as well as other foodstuffs
(Hillig, 1958 ).
To date, the simplest and most reliable means of
measuring ethanol in fish tissue is the use of the commercial
test kits which utilize alcohol dehydrogenase and a simple
spectrophotometric technique. Such kits are presently
available from Boehringer Mannheim (Germany) or Diagnostic
Chemicals (Charlottetown, P.E.I., Canada). This technique was
used successfully for several species of Atlantic groundfish
(Kelleher and Zall, 198 3).
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706 GILL
Effects of Pr ocessing
Thermal processing of canned fish may lead to the
destruction or alteration of objective quality indicators such
as nucleotide catabolites and amines, so that special
consideration must be given to the evaluation of products
which may have been subjected to high temperature (115 to
125C) for several minutes to several hours.
To investigate the thermal stability of nucleotides, Gill
et _al. (1987) canned tuna which had been spiked with authentic
nucleotide standards. Recoveries averaging 50%, 75%, 6 4% and
92% were measured for AMP, IMP, Ino and Hx, respectively. The
relatively low recovery rate for AMP suggested that this
compound was the least heat stable of all compounds tested,
although thermal destruction of ATP and ADP would be expected
to exceed even AMP since authentic standards of these
compounds decompose even at room temperature (Gill,
unpublished observation).
Generally speaking, it should be possible to estimate the
quality of raw material by examination of finished canned
products by analysis of nucleotides. However, such
extrapolation would only be possible if the extent of thermal
treatment were accurately known (Gill e_t al. 198 7).
Alkylamines such as DMA and TMA as well as the common
precursor TMAO would be expected to decompose at high
temperature although the thermal stability of histamine and
other biogenic amines is believed to be good and are presently
being used in the U.S. and Canada for the determination of
canned tuna quality.
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OBJECTIVE ANALYSIS OF SEAFOOD QUALITY 707
Despite the thermal destruction of alkyl amines and TMAO,
it has been shown in some cases that the DMA/TMA ratio may be
(used to reflect pre-process spoilage in certain fish
Tokunaga, 1975; Tokunaga ^t _al., 198 2). Nevertheless, the
reliability of thermally-labile amines for the estimation of
canned fish quality should be questioned.
Apparently, ethanol is not altered in any way by thermal
processing in hermetically-sealed containers and its level
correlates well with subjective quality assessment of canned
sockeye, pink, coho and chum salmon (Hollingworth and Throm,
198 2).
Summary
Throughout the published literature, one common theme
emerges regarding the use of objective methods for fish
quality evaluation - it is unlikely that one method of quality
evaluation will be found which accurately relfects the
edibility of all fish species. This is perhaps not surprising
when one considers the wide diversity with regard to
composition, enzymes present and modes of spoilage. By the
same reasoning, certain types of fish have been known to
decompose by different means according to the environment or
conditions of storage. In these circumstances, it is unlikely
that a single test for quality could provide an accurate
picture even for one specie of fish. The complexity of
seafood spoilage has frustrated scientists for years and is
likely to continue to provide challenges for those who will
seek future methods for the simplified assessment of fish
quality.
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REFERENCES
1. S.A. Beatty and N.E. G ibbons, J. Fish. Res. Bd. Can. 3, ll
(1936 ).
2. L. Farber, in "Fish as Food" (G. Borgstrom, ed.), Academic
Press, NY, 1965.
3. E. Tanikawa, T. Morohiro and K. Tomioka, Bull. Fac. Fish.,
Hokkaido Univ. 7, 165 (1956 ).
4. R.J. LeBlanc and T.A. G ill, Can. Inst. Food Sci. Technol.
J. 17, 195 (198 4).
5. J.R. Botta, J.T. Lauder and M.A. Jewer, J. Food Sci. 49,
734 (198 4).
6. J. Billon, N. Ollieuz and S.H. Tao, Rev. Technique
Veterinaire de l'Alimentation 18, 13 (1979).
7. D. Pearson, "The Chemical Analysis of Food", Chem. Publ.
Co. Inc., NY, 1977.
8. A.O.A.C. "Offical Methods of Analyses", 12th ed., Assoc.
Off. Anal. Chem., Washington, DC, 1975.
9. T. Tomiyama, A.A. deCosta and J.A. Stern, Food Technol.
10, 614 (1956 ).
10. M.E. Stansby, R.W. Harrison, J. Dassow and M. Slater,
Ind. and Eng. Chem. (Analytical Ed.) 16, 593 (1944).
11. W.J. Conway, "Microdiffusion Analysis and Volumetric
Error", Crosby Lockwood, London, 1962, p. 98 .
12. H. Rehbein and J. Oehlenschlager, Arch, fur
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13. W. Vyncke, Mededelingen Fakulteit Landbouwwetenschappen
G ent. 35, 1033 (1970).
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OBJECTIVE ANALYSIS OF SEAFOOD QUALITY 709
14. P.J. L eBlanc, "Approaches to the Study of
Nucleotide Catabolism for Fish Freshness Evaluation,"
M.Sc. Thesis, Technical University of Nova Scotia, 1987.
15. F. Soudan, "La Conservation par le Froid des Poissons,
Crustaces et Mollusques", J.B. Bailliere, Paris, 1965.
16. H.L.A. Tarr, J. Food Sci. 31, 846 (196 6 ).
17. D. Richter, Biochem. J. 31, 2022 (1937).
18. C.E. Hebard, G.J. Flick and R.E. Martin, in "Chemistry and
Biochemistry of Marine Food Products" (R.E. Martin, G.J.
Flick, C.E. Hebard and D.R. Ward, eds.), AVI Publishing
Co., Westport, CN, 1982, p. 149.
19. R.A. Laycock and L.W. Regier, J. Fish. Res. Bd. Can. 28 ,
305 (1971).
20. R. Adams, L. Parber and P. L erke, Appl. Microbiol. 12, 277
(196 4).
21. P. L erke, R. Adams and L. Farber, Appl. Microbiol. 11, 458
(196 3).
22. H.L.A. Tarr, Cdn. Fish. Rep. Sept. 1961, p. 27.
23. J.M. Shewan in "Recent Advances in Food Science" (J.
Hawthorn and J.M. Leitch, eds.), Butterworths, London,
1962, p. 167.
24. B.G. Shaw and J.M. Shewan, J. Appl. Bacteriol. 31, 89
(196 8 ).
25. P.L. Hoogland, J. Fish. Res. Bd. Can. 15, 717 (1958 ).
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6 1. N.R. Jones, in "The Technology of Fish Utilization" (R.
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74. M. Ohashi, N. Arakawa, T. Ashahara and S. Sakamoto,
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90. F. Hillig, J.A.O.A.C. 41, 776 (1958 ).
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