lnternatwnal Journal of Food Mwrobwlog3.

6 (1988) 295-307 295

Elsewer

J F M 00209

Microbiological quality and shelf life prediction

of chilled fish
B. Ravn Jorgensen 1, D.M. G i b s o n 2 and H.H. Huss
l Technological Laboratory, Mmtst O, of Ftsherws, Technical Unwerslty, Lyngby, Denmark and : Mtmstry
of Agriculture, Flsherws and Food, Torry Research Statwn, Aberdeen, Scotland, U K
(Received 5 January 1988, accepted 12 February. 1988)

A number of storage experiments have been carried out with whole cod and vacuum-packed cod fillets
stored m ice. The rmcroblologlcal quahty of the fish was determined on the basis of detection time
estimated rapidly by conductance assays in a T M A O - c o n t a l m n g medium at 25 ° C Detection time and
sensory data have been mcorporated mto a predictive hnear model to estimate the r e m m m n g shelf hfe of
the products. It ts concluded that the shelf hfe of iced whole cod can be predicted using this model but
not that of vacuum-packed fillets because of the greater vanablhty of bacterial activity in packaged fish

Key words Fish, chilled, Shelf life, prediction, Conductance measurement

Introduction

Lean, non-fatty marine fish are spoiled primarily by the action of bacteria (see
reviews by Liston (1980), Hobbs and Hodgkiss (1982) and Huss (1983)). Off-odours
and off-flavours are the main symptoms of spoilage and their development fre-
quently coincides with the presence of high microbial numbers. It is usually
assumed that the two factors are related and that bacterial counts in the order of
107/g or higher are necessary to reduce the production of off-odours and off-flavours
(Jay, 1986).
A number of chemical compounds and microbial metabohtes have been sug-
gested as indicators of fish quality (Connell, 1975; Huss, 1983). However, most of
them are found in increasing amounts in the fish only when spodage ~s well
advanced. In other words, they do no more than indicate what is already obvious
(spoilage) and have no pre&ctive value because the fish have no remaimng shelf life.
Therefore, a method to predict the remaining shelf life of fish should be based on an
assessment of the current microbial quality coupled with a precise knowledge of the
possible development and microbial activity under defined environmental condi-

Correspondence address: B.R Jorgensen, Technological Laboratory, Mlmstry of Fisheries, Building 221,
Techmcal University, 2800 Lyngby, Denmark

0168-1605/88/$03 50 © 1988 Elsevier Science Pubhshers B V. (Biomedical Dlvaslon)

296

tlons (temperature, packing, etc.). For such a test to be of any pracucal value, it
would have to be both simple and raptd.
Some rap~d methods for assessing bacteriological quahty have been developed
and are used for various purposes. Both conductance and impedance measurements
have been used to estimate nucrob~ologtcal quahty of fish (G~bson et al., 1984, van
Spreekens and Stekelenburg, 1986), and some workers have attempted to predict
shelf hfe from these data (G~bson, 1985; Gram, 1985; Huss et al, 1987; Gibson and
Ogden, 1987).
Fresh fish ~s contaminated with a mixed bacterial populauon mainly consisting of
psychrotrophac or psychrophihc Gram-negative bacteria. However, these orgamsms
are not equally important in the spoilage process as described by Huss et al. (1974)
who stated that 'total viable count' is a very poor indicator of both quahty and
remalmng shelf hfe of chilled fish.
A count of specific orgamsms revolved m fish spoilage might be expected to be
more useful m predicting the remaining shelf life. Such a count can be made by
convenuonal methods ( G r a m et al., 1987) or by rapid methods using conductance
measurements, if the medm c o m p o s m o n is smtably designed
The present work has been carried out to estabhsh more accurately the rela-
tionship between selected bacterial radices and the sensory quahty of chilled cod
(whole fish in ice and vacuum-packed fillets), and to investigate further the use of
data from conductance measurements for predicting shelf life.

Materials and Methods

Fish

Twelve storage experiments ( A - L ) were carried out with cod (Gadus morhua)
caught by trawl in the Baltic Sea, the Kattegat and the North Sea. The fish were
gutted and ~ced immediately after catching.
One day after catching the fish arrived at the laboratory. In 5 experiments ( A - E )
the fish were stored as whole gutted fish. In 7 experiments ( F - L ) the fish were
filleted and vacuum-packed m portions of 500 g. A Multxvac A G 4 packing machine
and polyamide-polythene film with low oxygen permeabdlty of 2.0 m l / m 2 / 2 4 h at 1
atm., 75% R H and 2 5 ° C were used.
All fish were stored in ice and triplicate pooled samples were removed regularly
over 14 days for sensory, chemical and microbiological examination.

Sensor); assessment

Fillets were heated (80°C, 20 min) m sealed bags and the quahty (cooked
flavour, CF) was assessed by a trained panel of five to seven members. A 10 0
scoring scale was used, tngh scores (10 to 7) indicating high quality fish without any
off-odours or -flavours. A CF score 6 was given to fish wath a flat, neutral
odour/flavour. CF scores below 6 were given to fish with off-odours or off-flavours,
CF below 4 corresponding to unacceptable quality.
 297

Chemical analyses

Total volatile basic nitrogen (TVB-N), trimethylamine nitrogen (TMA) and

trlmethylamine oxide mtrogen (TMAO) were determined by the Conway micro-
diffusion method (Conway and Byrne, 1933). These tests were carried out in
duplicate.
Nucleotide determinations were carried out by remowng a portion of the fish
muscle from behind the head. A 5 g sample was weighed ensuring that it was not
contaminated with any blood, dark muscle or skin. Analyses of the samples were
carried out using High Pressure Liquid Chromatography as described by Murray et
al. (1985).

Microbiological analyses

Samples (25 g) were homogemzed for 30 s m 225 ml of physiological sahne

containing 0.1% peptone using a Colworth Stomacher 400. The homogenate and
suitable dilutions were used for microbiological analyses.
Total viable count and a selecuve count of H2S-producing bacterm were de-
ternuned on Iron agar described by G r a m et al. (1987).

Conductance assays

The conductance assays were carried out at 2 5 ° C using the Malthus Microbial
Growth analyser (Malthus Instruments Ltd., Stoke-on-Trent, England). Conduc-
tance is measured automatically every 6 min and the instrument calculates and
stores the differences in conductance between each consecutive pair of measure-
ments. The detection time (DT) is when three such differences greater than a
preselected value (here 1/~S) occur.
For the conductance assays 2 ml homogenate (10-1 dilution) were added to 10 ml
Malthus cells contaxmng 8 ml of Wood and Baird's (1943) TMAO-broth. The tests
were carried out in duplicate.

Results

Sensory changes

In all trials there was a linear decrease in sensory quality with storage time. The
correlation coefficient was 0.95-0.99 and the standard deviation (SDcF) on the
regression line was approx. 0.5. A statistical t-test at the level of 95% was used to
estimate rejection t~mes (end of maximum shelf life). In this test the standard
deviations on the regression lines were used to determine the quality score which
was significantly lower than CF = 4. The m a x i m u m shelf life was estimated as the
time taken for the fish to reach tlus score.
298

TABLE I
Shelf hfe of whole cod and vacuum-packed cod fillets (0 o C) estimated b5 sensory assessment m relauon
to the lmtlal mmrobtal quality and macroblal and chemical mdmes at the end of shelf hfe

Expert- Shelf Inmal Chermcal and bacteriological values at the end of shelf hfe
ment " hfe bacteriological
TM~ TVB-N Magnitude Ma~mude D~, ~
(days) quahty
(mg N, (mg N ' of TVC of H~S- Ih)
Magmtude DT • 100 g) 100 g) Icfu/g) count
of HzS-count (h} (cfu g)
(cfu/gl
A I0 104 12 4 5-10 15 20 10 ~ 107 10 ~ 5 1 '2
B 12 10 ~ 13 7 - 15 20 10"-10 '~ 10~ 10 ~ 5 1 2
C b 13 10 2 17 4 0- 5 10-15 10 '~ 10- 1(I~ 6
D 13 102 16 3 10-15 20 25 10s-10 ~ 1() ~ 10 ~ 6 l, 2
E 16 101 20 4 5-10 15-20 10"-10 ' 10~-10 '~ 5 1 2
F b 12 10 2 18 6 10 15 20-25 10~-10" 10 e 9
G 15 101 19 7 10-15 20-25 10~-10 ~ 10-' 11
H 13 101 21 0 15 20 25-30 10' 106 l0 > 11
1 14 101 19 6 25-30 30-35 10 ~' 107 10"-10 ~ 12
J 14 102 16 8 25-30 25-30 10~-10" 10~-10 ~ 81 2
K 11 101-102 19 0 30-35 40 45 10"-10 ~' 10" 106 11 1 '2
L 12 101 21 0 40 45 50-55 10" 106 10" 12

"Expenments A - E represent whole fish m me (0 ° C~

Expenments F L represent vacuum-packed flllet~ 40 ° ( 1
b Whole fish and fillets from same catch
DT = detection time for conductance measurement
DTo = detection time at end of shelf 1,fe

E s t i m a t e d s h e l f h v e s m the v a r i o u s s t o r a g e trials w~th w h o l e c o d a n d v a c u u m -

p a c k e d fillets are s h o w n in T a b l e I. T h e s h e l f h f e v a r i e d f r o m 10 to 16 d a y s .
Stat~sncal analyses have s h o w n that the s t a n d a r d d e w a n o n (SD~) o n a s h e l f life
e s u m a t e ( Y ) a s s e s s e d b y t h e s e n s o r y p a n e l ~s 0.7 d a ySD~ w a s c a l c u l a t e d u s i n g t h e
s t a n d a r d d e v l a h o n SDcr e s t i m a t e d b~ t h e r e g r e s s i o n a n a l y s e s . SD~ = 0.7 d a y is
e q u i v a l e n t t o a 95% c o n f i d e n c e i n t e r v a l o f Y + / - 1 day

Chemical changes

I n all e x p e r i m e n t s w~th w h o l e c o d t h e r e w a s a m o d e r a t e p r o d u c t i o n o f v o l a t i l e
n i t r o g e n b a s e s d u r i n g s t o r a g e . A t t h e r e j e c t i o n u m e < 15 m g N T M A / 1 0 0 g a n d
< 25 m g T V B - N / 1 0 0 g w e r e m e a s u r e d ( T a b l e I).
I n v a c u u m - p a c k e d fillets g e n e r a l l y m o r e m t r o g e n b a s e s w e r e p r o d u c e d a n d m all
experiments there was an increase m TMA during storage, but the number of days
b e f o r e t h e o n s e t o f a n a p p r e c i a b l e T M A p r o d u c t i o n v a r i e d ( F i g . 1). T h e d e v e l o p -
m e n t o f T V B - N f o l l o w e d t h e s a m e p a t t e r n ( d a t a n o t s h o w n ) a n d tt w o u l d a p p e a r
t h a t t h e i n c r e a s e m T V B - N is d u e m a i n l y t o p r o d u c t i o n o f T M A . L e v e l s o f T V B - N
at r e j e c U o n U m e s are s h o w n m T a b l e I.
 299

50

40- J ~

/z
/
30- ,' // /
0 / -x-" ~ = /
0 / / /
7" / ,~
2o- //, ' , / ~

10 1
/"//~k
/ / / /
/~//J / ~

0 i i v i i T w i i i r 1 i i i i

0 2 4 6 8 10 12 14 16 18
Days ~n I c e

Ftg 1 Development of tnmethylarmne (TMA) m vacuum-packed cod fillets stored m ice [], F, ©, G, I ,
H, * , I: III. J, * . K , ~ , L

The nucleotide catabolites were measured in experiments J, K and L. In no case

were adenosine trlphosphate (ATP), adenosine diphosphate (ADP) or adenosine
monophosphate (AMP) detected on the first day of samphng (2-3 days after

7-

6-

an,hne

jjj

0.4 , I I , '

D a y s in i c e

F~g 2 Changes m the content of mosme (HxR) and hypoxanthme (HX) m vacuum-packed cod fdlets
stored in ice. II, J, *, K, O, k.
300

catching) and mosine monophosphate (IMP) disappeared during the first week of
storage. Production of hypoxanthine (HX) started after 6 - 8 days and the content of
inosine (HxR) was reduced at the same rate as Hx was produced Hx was produced
1-2 days later in experiment J than in experiments K and L (Fig. 2).
By comparing the development of TMA (Fig. 1) and Hx (Fig. 2) in the 3 trials J,
K and L they seem to follow the same pattern, for example being approx. 2 days
delayed in J as compared with K and L

Mwrobtologwal changes

Exponential growth of HzS-producing bacteria was found m all experiments

following a more or less pronounced imtlal lag phase (Figs. 3 and 4).
The increase in total viable count was slow and during storage there was
increasing predominance of H2S-producing bacteria (data not shown). In whole cod
the numbers of HzS-producing bacteria reached 10 v 10 s c f u / g at the end of shelf
life but In vacuum-packed fillets the numbers were 105-10 6 c f u / g only. At that ume
the total viable counts were mainly of H2S-producing bacteria (Table I).
Using direct rmcroscopy, abnormally large cells were seen in vacuum-packed
fillets. This phenomenon may be due to an effect of CO 2 but the sigmficance in the
present situation is unclear.
For the conductance measurements regression analyses have shown that in all
trials there was a linear decrease in DT during storage. The regression equations, the
correlation coefficients and the standard deviations are summarized in Table II. The

2
~ 5
/
~ 4¸
t~
c~
u
3-
D

a_ 2
%
I 1"

0 2 4. 6 8 10 12 14 16 18
D o y s in i c e

Ftg 3 Changes m the number of HzSiproducmg o r g a n i s m s m w h o l e c o d s t o r e d m tce e , A, [2, B, * C,

D,O.E
 301

8-

t~

4¸
u

u 3

0_
i
2

I
1"

0 f r f i i I i I I i i i i

Doys In i c e

F i g 4 C h a n g e s m the n u m b e r of H 2 S - p r o d u c m g o r g a r u s m s m v a c u u m - p a c k e d c o d fillets s t o r e d m Ice

12, F, ©, G , O, H , * , I, II, J, * , K , ~ , L

correlation coefficients vaned from 0.94-0.99 and the standard devlattons on the
estimate from 0.42-1.07 h.
Fig. 5 shows that D T for fish samples correlated very well with the numbers of

T A B L E II
R e l a t ~ o n s l u p b e t w e e n d e t e c t i o n Ume (DT) a n d s t o r a g e t i m e ( t ) o f w h o l e c o d a n d v a c u u m - p a c k e d c o d
fillets m d a y s (0 o C) e s t t m a t e d b y l i n e a r r e g r e s s i o n

Expert- Number of Correlatton Standard Regression

ment a samples coefficient deviation equatton
(n) (r) (SD) (A-dDT/dt t b)
A 6 -0.99 0.42 12 4 - 0 69 t
B 9 -0.99 057 137-069 t
C 7 -0.99 0.75 17 4 - 0 87- t
D 8 -098 096 163-076 t
E 9 - 0 99 0.70 20 4 - 0 97 t
F 7 - 0 99 0 64 18 6 - 0 8 1 - t
G 9 - 0 99 0 63 19 7 - 0 6 3 - t
H 7 - 0 97 0 94 2 1 . 0 - 0.80- t
I 8 - 0 98 0 72 1 9 . 6 - 0 . 6 1 -t
J 9 - 0.95 1.07 16.8 - 0 64 t
K 8 -094 1.05 190-070 t
L 9 -098 0.60 210-077 t

a E x p e r i m e n t s A - E r e p r e s e n t w h o l e fish m ice (0 o C)
E x p e r r m e n t F - L r e p r e s e n t v a c u u m - p a c k e d fdlets m ice (0 o C)
b t ~S the s t o r a g e t~me m d a y s , A is t h e v a l u e o f D T a t t = 0 d a y s
302

24-

22~
+
+
20- +
+-,,~ ++
18-
F-
Q r:096
16-
~-~-' % + ~.++ ~,
14-

+ ÷
~ ' ~ +

+ ÷ ~ ÷

0
0
log (CFU/g)
FLg 5 R e l a t i o n s h i p b e t w e e n H . S - p r o d u c m g c o u n t in c o d ( w h o l e f i s h a n d v a c u u m - p a c k e d fillets s t o r e d m
ice) a n d d e t e c t i o n t t m e D T d e t e r r m n e d Ln T M A O - b r o t h at 25 o C T h e c o r r e l a t i o n c o e f f i c i e n t ms 0 96

HzS-producmg bacterxa m the fish. A slightly poorer relationship was seen between
total viable count and D T (Fig. 6). Tins ~s particularly the case at low values of total
wable count This is to be expected as there is an increasing percent of H~S-produc-
mg bacteria during storage.
In whole cod the detectLon time at rejection time (DT0) vaned from 5.5 to 6.5 h
and the number of H2S-producing organisms ranged from 10 7 to 10 ~ cfu/g. For

24

÷
+
20 4-
~+ + ~ + +
18
I,,-
,,,-,
++~x'x + + r = 089
16 ÷ "-~.+,++++
14q

12- +++~+ ÷ +

10-

4
2

o4
0
log (CFU/g)
F i g 6 R e l a t l o n s l u p b e t w e e n t o t a l v t a b l e c o u n t in c o d ( w h o l e f i s h a n d v a c u u m - p a c k e d fillets s t o r e d m ~ce)
and detechon time DT detervmned m TMAO-broth at 25 o C T h e c o r r e l a t i o n c o e f f i c i e n t is 0 89
 303

vacuum-packed fillets more variation was seen. D To varied from 8.5 to 12 h and the
number of H2S-producing organisms from 105 to 107 c f u / g (Table I). Accordingly,
for whole cod a very good correlation between bacterial md~ces and sensory quahty
is found.
This ~s not the case for vacuum-packed fillets. A certain count of H2S-produclng
bacteria may be responsible for very different amounts of metabohtes (TVB-N,
TMA) produced as seen m the batches J and L, for instance (Table I).

Shelf hfe predtctton

Gibson (1985) has suggested a linear model to predict shelf life:

DT- D To
Remaining shelf life (m days) = d D T / d t o o c (1)

where d D T / d t o o c is the daily rate of change in D T for fish stored at 0 ° C and D T o

is the value of D T at cut-off quahty level (at the rejection time).
Since two different nucrobiological spoilage patterns were seen in whole cod and
vacuum-packed fillets, respectively, a predictive model has been established for each
of the two products.
In the experiments with whole cod d D T / d t and D To were almost constant as
shown in Tables I and II. When the remaining shelf life is plotted against D T there
is a linear relationship (Fig. 7). The correlauon coefficient ~s 0.96 and the standard
deviation is as low as 1.1. Therefore, using D T m the predictive model (1) the
remaining shelf life of whole cod can be predicted relatively precisely. A 95%

16- whole hsh in ice

14-

12-

r = 0.96

O -

-- 8- D []

OD D

2- oH
boo
ol 0 ,~
o
,~ ~'8 2'o 24

DT (hours)
Fig 7. R e m a m m g shelf hfe of whole cod stored m ice as a functton of d e t e c t i o n time D T All
e x p e n m e n t s are p o o l e d The c o r r e l a u o n coefficient is 0 96
 304

vacuum-packed fillets ~n ice

1
14~
[]

D O
/
O0 /

r=087

0
[] 7.°
, []

cn

c
64 [] o o o
o

[15
,t t
o o

0 8 12
k
1~ 20 24
DT (hours)
Ftg 8 Remalrung shelf hfe of vacuum-packed cod hllets stored m ice as a function of detecuon time D T
All experiments are pooled. The correlauon coefficient ~s 0 87

confidence interval for the regression hne at for example D T = 14 h is Y + / - 0.5

day.
Applying the predictive model to vacuum-packed fillets by plotting the remaining
shelf hfe against D T there is a linear relationship, but the standard dewation ~s 2.1
(Fig. 8).
This standard devlahon on shelf hfe predlchon found in Fig. 8 ~s assumed to be
due to the variation m D T o, as indicated in Table I. This vanation ~s not due to a
too high varxation on sensory, quality assessment since the standard deviation
between all D T o values is 1.4 with the confidence mterval 0.83 < SD 2 > 9.62 which
~s higher than the standard deviation of shelf life estimation which ~s 0.7.

Discussion

Thas work has shown a very good correlation between number of H2S-producmg
organisms and shelf hfe for whole fish stored in ~ce. It has also confirmed that the
number of H2S-producmg orgamsms can be accurately estimated using a raptd
method such as conductance measurement. As both detection time ( D T ) and fish
quahty are shown to decrease linearly with time a predictive model for remaimng
shelf life can be estabhshed for this product as already suggested by Gibson (1985).
This situation is qmte different with vacuum-packed fillets, also stored at 0 ° C.
Fillets generally spoil faster than whole fish at the same temperature, and vacuum
packaging of fillets generally extends their shelf life. In the experiments described
here, the vacuum-packaged fillets had the same shelf life as whole fish, but much
lower numbers of spoilage bacteria were responsible for larger amounts of bacterial
 305

metabolites and the counts at the end of shelf life were much lower than those
found with whole cod. Similar results were demonstrated by Jensen et al. (1980).
All the fish used in the packaging experiments were of good imtial quality as
shown by the low numbers of H2S-producing organisms (~< 102 cfu/g). However,
when fish of poor microbiological quality were packaged, a higher count may be
found at rejection time. When fresh fillets were inoculated with H2S-produclng
organisms at a level of 105 c f u / g and vacuum packaged, a count of 108 c f u / g was
recorded at the end of shelf life (data not shown).
As the bacterial growth in vacuum-packed fillets is mainly related to H2S-pro-
ducmg orgamsms and as these orgamsms constttute the dominating part of the flora
at the termination of shelf life it is most likely that they are also responsible for
spoilage. Further, in 3 expenments the flora at the rejection rime was identified and
tested for spoilage potential. Only the HzS-producmg orgamsms were classified as
'spoders' using the production of off-odours when the strains were grown in sterile
fish juice as an indication of spoilage potential (data not included).
Under condinons in which the bacterial growth is inhibited, the shelf hfe of
chilled fish including vacuum-packed fillets may be deterrmned by autolync changes
(Bremner et al., 1987). However, the present work, supports the conclusions of both
Jones (1965) and Cohen (1986) that the spoilage bacteria are also acttvely revolved
in the final break down of inosine to hypoxanthine. Further, at the rejecnon time
large amounts of TVB-N and T M A were produced by bacteria. Thus, in the
vacuum-packed fillets it seems to be qmte clear, that they do spoil as a result of
bacterial actLvlty.
The difference in bacterial activity nonced in the expenments with vacuum-packed
fish may be explained by a variation m spoilage potentml among strains of
H2S-producing organisms or a variation in intrinsic parameters between fish used m
the vanous experiments. However, no s~gnificant differences in these factors were
noted m model systems using raw sterile-filtered fish juice made from fish of the
same batches used m three of the experiments as substrate for pure cultures of
HzS-producmg bacteria (data not included).
The exclusion of O z is not inhibitory to growth of Shewanella putrefactens, one of
the main spoilage bacteria for fish stored at 0 o C, since ~t ts able to utilize T M A O as
a terminal electron acceptor (Easter et al., 1983; Rango et al., 1984). The inhibition
of growth (or cell division) in the vacuum packs may on the other hand be due to
accumulation of CO z. It is well documented that CO 2 has a specific inhibitory effect
on HzS-producing organisms (Jensen et al., 1980; Lannelongue et al., 1982;
Woyewoda et al., 1984). In the present work, head space analyses were made after
12 days of storage. At this time all O z had been used and replaced by varying
amounts of CO z and N z (data not included).
Although the correlation between the numbers of H2S-producing organisms m
vacuum-packed fillets and D T is as good as for whole cod, the poorer correlation
between the numbers of HzS-producers and shelf life makes the same predictive
model as used for iced whole fish too unrehable. For accurate predictions of quality,
all the extrinsic factors prevailing in vacuum-packaged fish, including CO 2 tension,
should be incorporated in the model.
306

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