Objective Analysis of Seafood Quality

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Food Reviews In te rna t iona l , 6(4) , 681-714 (1990)

OBJECTIVE ANALYSIS OF SEAFOOD QUALITY

Tom A. G i l l

Canadian Institute of Fisheries TechnologyTechnical University of Nova Scotia

P.O. Box 1000, HalifaxNova 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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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 "quality1 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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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 Färber (1965).

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 Gill, 1984). Figure 1 illustrates the autolytic

production of TVB, ammonia and TMA with postmortem age for

squid stored at 2.5°C. 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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roge

n (

Vol

ati

100

80

60

40

20

0

O O Ammonia• «TVBA ATMA

2 4 6 8 10

Storage Time at 2.5°C (Days)

12

Figure 1. Effect of storage time on production ofammonia, TVB and TMA in short finned squid (Tllexillecebrosus).

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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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; LeBlanc,

1987 and LeBlanc and Gill, 1984), 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. Louis, MO) and

Boehringer Mannheim (Mannheim, W. Germany). A third type of

diagnostic test kit is available in the form of a paper strip

which is available from Merck (Merckoquant, Darmstadt, W.

Germany). 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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enE

0)

co

50

40

30

20

10

O 5°CAIR• 5°C SW, GUTTEDA 5°C SW, WHOLEA 2.5°C SW

0 1 2 3 4 5 6 7 8 9Storage Time (Days)

Figure 2. Effects of time, temperature and contactmedium on the production of ammonia in postmortemsquid (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, 1965), 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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360

^ 320

5 280tou 240oE 200w 160

§ 120O£ 80< 40

O OMerckoquant NH3• »GDH NH3A 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 herringoffal held at 25°C. Ammonia was determined by teststrip (Merckoquant) and by the enzymatic methodusing glutamate dehydrogenase (GDH).

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 20°C. 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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CD

CD

O

O

<

24

21

18

15

12

9

6'

3

• — • A M M O N I A LEVEL

A A TOTAL AEROBIC COUNT

4 8 12 16 20

Storage Time on Ice (Days)

360

300 rPI

240 2X

180 "oí

0>120 Q.

=)60 Li-

24

Figure 4. Effect of storage time on ammonia levels andtotal aerobic plate counts in iced, gutted cod (Gadusmorhua).

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.,

1982). Rather than citing the hundreds of articles found in

this reference, the important points will be summarized.

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The origins of TMA have been established with the

spoilage bacteria associated with Atlantic groundfish (Beatty

and Gibbons, 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

Gill, 1987).

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; Gruger, 1972; Kuwata et ¿I., 1980; Tokunaga et al.,

1977); ion specific electrode (Chang ^t ¿1., 1976); solid

state gas sensor (Storey et ¿¿., 1984); high performance

liquid chromatography (Gill and Thompson, 1984) and an

enzymatic diagnostic test kit (Wong and Gill, 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 HPLC. 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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C7>

3

O

CDO

<5 10 15 20 25 30

Amount Added (mg%N)

35

Figure 5. Recovery curves for authentic standards ofTMA and DMA added to minced cod. Analyses were performedby 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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40

o 32

o

enE

i<

O O Picric Acid Method• «HPLC MethodA A Enzymatic Method

24

16

8

o—o—o—o0X , 0.4 8 12 16

Storage Time on Ice (Days)20

Figure 6. Comparison of three methods for themeasurement of TMA in iced, gutted cod (Gadus morhua)The methods were the picric acid procedure TDyer,1945), the HPLC method of Gill and Thompson (1984),and the enzymatic method of Wong and Gill (1987).

8 16 24 32

TMA - SPEC (mgSK-N)

Figure 7, Performance of the visual color test ascompared to the spectrophotometric procedure forTMA analysis of fish extracts. Both methodsutilized TMA dehydrogenase.

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CD

OO

LO

O"OO

16 24

TMA-N (mg%)

32

Figure 8. Relationship between subjectiveevaluation of odor of cod (using experiencedgraders) 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^., 1988). 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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O 10 20 30 40 50 60 70

TMA Level by Picric Acid (mg TMA-N/iOOg)

Figure 9. Test strip performance compared to thepicric acid method. Each data point represents anaverage 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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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 1982), 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, 1981).

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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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 22-25CC. 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

Uric Acid

Figure 10. Postmortem ATP degradation in fish.Enzymes include: 1. ATP ase; 2. myokinase; 3. AMPdeaminase; 4. IMP phosphohydrolase; 5a. nucleosidephosphorvlase; 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,

1971) as does the hydrolysis of inosine to hypoxanthine. The

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O — OIMP• — •InosineA — A Hypoxanthine

«=•=

4 8 12

Storage Time at 3°C (Days)

16

Figure 11. Changes in IMP, Tno and Hx in sterilecod fillets stored at 3°C.

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,

1965). 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, 1964).

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 degradation of

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W

oE3

O

<

O—OIMP•—•InosineA — A Hypoxanthine

4 8 12

Storage Time at 3°C (Days)

16

Figure 12. Changes in IMP, Tno and Hx in nonsterilecod fillets stored at 3°C.

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, 1987).

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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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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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 20°C 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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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, 1983). 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, 1983).

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706 GILL

Effects of Processing

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

125°C) 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%, 64% 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. 1987).

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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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., 1982). 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,

1982).

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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Objective Analysis of Seafood Quality

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